VE-PTP controls blood vessel development by balancing Tie-2 activity

Vascular endothelial protein tyrosine phosphatase (VE-PTP) is an endothelial-specific receptor-type tyrosine phosphatase that associates with Tie-2 and VE-cadherin. VE-PTP gene disruption leads to embryonic lethality, vascular remodeling defects, and enlargement of vascular structures in extraembryonic tissues. We show here that antibodies against the extracellular part of VE-PTP mimic the effects of VE-PTP gene disruption exemplified by vessel enlargement in allantois explants. These effects require the presence of the angiopoietin receptor Tie-2. Analyzing the mechanism we found that anti–VE-PTP antibodies trigger endocytosis and selectively affect Tie-2–associated, but not VE-cadherin–associated VE-PTP. Dissociation of VE-PTP triggers the activation of Tie-2, leading to enhanced endothelial cell proliferation and enlargement of vascular structures through activation of Erk1/2. Importantly, the antibody effect on vessel enlargement is also observed in newborn mice. We conclude that VE-PTP is required to balance Tie-2 activity and endothelial cell proliferation, thereby controlling blood vessel development and vessel size

The Impact of Intermittent Umbilical Cord Occlusions on the Inflammatory Response in Pre-Term Fetal Sheep

Fetal hypoxic episodes may occur antepartum with the potential to induce systemic and cerebral inflammatory responses thereby contributing to brain injury. We hypothesized that intermittent umbilical cord occlusions (UCOs) of sufficient severity but without cumulative acidosis will lead to a fetal inflammatory response. Thirty-one chronically instrumented fetal sheep at ∼0.85 of gestation underwent four consecutive days of hourly UCOs from one to three minutes duration for six hours each day. Maternal and fetal blood samples were taken for blood gases/pH and plasma interleukin (IL)-1β and IL-6 levels. Animals were euthanized at the end of experimental study with brain tissue processed for subsequent counting of microglia and mast cells. Intermittent UCOs resulted in transitory fetal hypoxemia with associated acidemia which progressively worsened the longer umbilical blood flow was occluded, but with no cumulative blood gas or pH changes over the four days of study. Fetal arterial IL-1β and IL-6 values showed no significant change regardless of the severity of the UCOs, nor was there any evident impact on the microglia and mast cell counts for any of the brain regions studied. Accordingly, intermittent UCOs of up to three minutes duration with severe, but limited fetal hypoxemia and no cumulative acidemia, do not result in either a systemic or brain inflammatory response in the pre-term ovine fetus. However, fetal IL-1B and IL-6 values were found to be well correlated with corresponding maternal values supporting the placenta as a primary source for these cytokines with related secretion into both circulations. Female fetuses were also found to have higher IL-1β levels than males, indicating that gender may impact on the fetal inflammatory response to various stimuli

IP-10 detection in urine is associated with lung diseases

<p>Abstract</p> <p>Background</p> <p>blood cytokines and chemokines have been proposed as biomarkers for tuberculosis (TB). Recently, some immune mediators found in the urine of patients with renal dysfunctions have also been suggested as potential biomarkers. Finding biomarkers for TB in urine would present several advantages over blood in terms of collection and safety. The objective of this study was to investigate the presence of cytokines and chemokines in the urine of patients with pulmonary TB at the time of diagnosis. In a subgroup, the evaluation was also performed during TB treatment and at therapy completion. Patients with lung diseases other than TB, and healthy subjects were also enrolled.</p> <p>Methods</p> <p>urine samples from 138 individuals, after exclusion of renal dysfunctions, were collected during an 18 month-period. Among them, 58 received a diagnosis of pulmonary TB, 28 resulted having lung diseases other than TB, and 34 were healthy subjects. Moreover, 18 TB patients, 9 of whom were tested 2 months after AFB smear sputum reversion and 9 of whom were cured of TB were also included. Cytokines and chemokines in urine were evaluated using a Cytometric-Bead-Array-Flex-Set. IP-10 detection in 49 subjects was also carried out in parallel by using an Enzyme Linked ImmunoSorbent Assay (ELISA).</p> <p>Results</p> <p>IFN-γ, TNF-α, IL-2, IL-8, MIP-1α, MIP-1β and RANTES were poorly detected in all urine samples. Conversely, IP-10 was consistently detected in urine and its level was significantly increased in patients with lung disease compared to healthy subjects (p < 0.001). Increased IP-10 levels were found in both pulmonary TB and lung diseases other than TB. Moreover lower IP-10 levels were found in cured-TB patients compared to the levels at the time of diagnosis, and this difference was close to significance (p = 0.06). Interestingly, we demonstrated a significant correlation between the data obtained by flow cytometry and ELISA (r²0.82, p < 0.0001).</p> <p>Conclusions</p> <p>IP-10, in contrast to IFN-γ, TNF-α, IL-2, IL-8, MIP-1α, MIP-1β and RANTES, is detectable in the urine of patients with pulmonary diseases in the absence of renal dysfunctions. Moreover, the IP-10 level in cured-TB patients is comparable to that found in healthy subjects. More studies are needed to further investigate the clinical utility of these findings.</p

A Combined CXCL10, CXCL8 and H-FABP Panel for the Staging of Human African Trypanosomiasis Patients

The actual serological and parasitological tests used for the diagnosis of human African trypanosomiasis (HAT), also known as sleeping sickness, are not sensitive and specific enough. The card agglutination test for trypanosomiasis (CATT) assay, widely used for the diagnosis, is restricted to the gambiense form of the disease, and parasitological detection in the blood and cerebrospinal fluid (CSF) is often very difficult. Another very important problem is the difficulty of staging the disease, a crucial step in the decision of the treatment to be given. While eflornithine is difficult to administer, melarsoprol is highly toxic with incidences of reactive encephalopathy as high as 20%. Staging, which could be diagnosed as early (stage 1) or late (stage 2), relies on the examination of CSF for the presence of parasite and/or white blood cell (WBC) counting. However, the parasite is rarely found in CSF and WBC count is not standardised (cutoff set between 5 and 20 WBC per µL). In the present study, we hypothesized that an early detection of stage 2 patients with one or several proteins in association with clinical evaluation and WBC count would improve staging accuracy and allow more appropriate therapeutic interventions

The Association between Intrauterine Inflammation and Spontaneous Vaginal Delivery at Term: A Cross-Sectional Study

BACKGROUND:Different factors contribute to the onset of labor at term. In animal models onset of labor is characterized by an inflammatory response. The role of intrauterine inflammation, although implicated in preterm birth, is not yet established in human term labor. We hypothesized that intrauterine inflammation at term is associated with spontaneous onset of labor. METHODS/RESULTS:In two large urban hospitals in the Netherlands, a cross-sectional study of spontaneous onset term vaginal deliveries and elective caesarean sections (CS), without signs of labor, was carried out. Placentas and amniotic fluid samples were collected during labor and/or at delivery. Histological signs of placenta inflammation were determined. Amniotic fluid proinflammatory cytokine concentrations were measured using ELISA. A total of 375 women were included. In term vaginal deliveries, more signs of intrauterine inflammation were found than in elective CS: the prevalence of chorioamnionitis was higher (18 vs 4%, p = 0.02) and amniotic fluid concentration of IL-6 was higher (3.1 vs 0.37 ng/mL, p<0.001). Similar results were obtained for IL-8 (10.93 vs 0.96 ng/mL, p<0.001) and percentage of detectable TNF-alpha (50 vs 4%, p<0.001). CONCLUSIONS:This large cross-sectional study shows that spontaneous term delivery is characterized by histopathological signs of placenta inflammation and increased amniotic fluid proinflammatory cytokines

Limited Relationship between Cervico-Vaginal Fluid Cytokine Profiles and Cervical Shortening in Women at High Risk of Spontaneous Preterm Birth

Objective: to determine the relationship between high vaginal pro-inflammatory cytokines and cervical shortening in women at high risk of spontaneous preterm labor and to assess the influence of cervical cerclage and vaginal progesterone on this relationship. Methods: this prospective longitudinal observational study assessed 112 women with at least one previous preterm delivery between 16 and 34 weeks’ gestation. Transvaginal cervical length was measured and cervico-vaginal fluid sampled every two weeks until 28 weeks. If the cervix shortened (<25 mm) before 24 weeks’ gestation, women (cases) were randomly assigned to cerclage or progesterone and sampled weekly. Cytokine concentrations were measured in a subset of cervico-vaginal fluid samples (n = 477 from 78 women) by 11-plex fluid-phase immunoassay. Results: all 11 inflammatory cytokines investigated were detected in cervico-vaginal fluid from women at high risk of preterm birth, irrespective of later cervical shortening. At less than 24 weeks’ gestation and prior to intervention, women destined to develop a short cervix (n = 36) exhibited higher cervico-vaginal concentrations than controls (n = 42) of granulocyte-macrophage colony-stimulating factor [(GM-CSF) 16.2 fold increase, confidence interval (CI) 1.8–147; p = 0.01] and monocyte chemotactic protein-1 [(MCP-1) 4.8, CI 1.0–23.0; p = 0.05]. Other cytokines were similar between cases and controls. Progesterone treatment did not suppress cytokine concentrations. Interleukin (IL)-6, IL-8, granulocyte colony-stimulating factor (G-CSF), interferon (IFN)-γ and tumour necrosis factor (TNF)-α concentrations were higher following randomization to cerclage versus progesterone (p<0.05). Cerclage, but not progesterone treatment, was followed by a significant increase in cervical length [mean 11.4 mm, CI 5.0–17.7; p<0.001]. Conclusions: although GM-CSF and MCP-1 cervico-vaginal fluid concentrations were raised, the majority of cervico-vaginal cytokines did not increase in association with cervical shortening. Progesterone treatment showed no significant anti-inflammation action on cytokine concentrations. Cerclage insertion was associated with an increase in the majority of inflammatory markers and cervical length

Chemoattractant Receptor Homologous to the T Helper 2 Cell (CRTH2) Is Not Expressed in Human Amniocytes and Myocytes

BACKGROUND: 15-deoxy-Δ 12,14- Prostaglandin J2 (15dPGJ2) inhibits Nuclear factor kappa B (NF-κB) in human myocytes and amniocytes and delays inflammation induced preterm labour in the mouse. 15dPGJ2 is a ligand for the Chemoattractant Receptor Homologous to the T helper 2 cell (CRTH2), a G protein-coupled receptor, present on a subset of T helper 2 (Th2) cells, eosinophils and basophils. It is the second receptor for Prostaglandin D2, whose activation leads to chemotaxis and the production of Th2-type interleukins. The cellular distribution of CRTH2 in non-immune cells has not been extensively researched, and its identification at the protein level has been limited by the lack of specific antibodies. In this study we explored the possibility that CRTH2 plays a role in 15dPGJ2-mediated inhibition of NF-κB and would therefore represent a novel small molecule therapeutic target for the prevention of inflammation induced preterm labour. METHODS: The effect of a small molecule CRTH2 agonist on NF-κB activity in human cultured amniocytes and myocytes was assessed by detection of p65 and phospho-p65 by immunoblot. Endogenous CRTH2 expression in amniocytes, myocytes and peripheral blood mononuclear cells (PBMCs) was examined by PCR, western analysis and flow cytometry, with amniocytes and myocytes transfected with CRTH2 acting as a positive control in flow cytometry studies. RESULTS: The CRTH2 agonist had no effect on NF-κB activity in amniocytes and myocytes. Although CRTH2 mRNA was detected in amniocytes and myocytes, CRTH2 was not detectable at the protein level, as demonstrated by western analysis and flow cytometry. 15dPGJ2 inhibited phospho-65 in PBMC'S, however the CRTH2 antagonist was not able to attenuate this effect. In conclusion, CRTH2 is not expressed on human amniocytes or myocytes and plays no role in the mechanism of 15dPGJ2-mediated inhibition of NF-κB

Pro-inflammatory profile of preeclamptic placental mesenchymal stromal cells: new insights into the etiopathogenesis of preeclampsia.

The objective of the present study was to evaluate whether placental mesenchymal stromal cells (PDMSCs) derived from normal and preeclamptic (PE) chorionic villous tissue presented differences in their cytokines expression profiles. Moreover, we investigated the effects of conditioned media from normal and PE-PDMSCs on the expression of pro-inflammatory Macrophage migration Inhibitory Factor (MIF), Vascular Endothelial Growth Factor (VEGF), soluble FMS-like tyrosine kinase-1 (sFlt-1) and free β-human Chorionic Gonadotropin (βhCG) by normal term villous explants. This information will help to understand whether anomalies in PE-PDMSCs could cause or contribute to the anomalies typical of preeclampsia. METHODS: Chorionic villous PDMSCs were isolated from severe preeclamptic (n = 12) and physiological control term (n = 12) placentae. Control and PE-PDMSCs’s cytokines expression profiles were determined by Cytokine Array. Control and PE-PDMSCs were plated for 72 h and conditioned media (CM) was collected. Physiological villous explants (n = 48) were treated with control or PE-PDMSCs CM for 72 h and processed for mRNA and protein isolation. MIF, VEGF and sFlt-1 mRNA and protein expression were analyzed by Real Time PCR and Western Blot respectively. Free βhCG was assessed by immunofluorescent. RESULTS: Cytokine array showed increased release of pro-inflammatory cytokines by PE relative to control PDMSCs. Physiological explants treated with PE-PDMSCs CM showed significantly increased MIF and sFlt-1 expression relative to untreated and control PDMSCs CM explants. Interestingly, both control and PE-PDMSCs media induced VEGF mRNA increase while only normal PDMSCs media promoted VEGF protein accumulation. PE-PDMSCs CM explants released significantly increased amounts of free βhCG relative to normal PDMSCs CM ones. CONCLUSIONS: Herein, we reported elevated production of pro-inflammatory cytokines by PE-PDMSCs. Importantly, PE PDMSCs induced a PE-like phenotype in physiological villous explants. Our data clearly depict chorionic mesenchymal stromal cells as central players in placental physiopathology, thus opening to new intriguing perspectives for the treatment of human placental-related disorders as preeclampsia

Microbial Prevalence, Diversity and Abundance in Amniotic Fluid During Preterm Labor: A Molecular and Culture-Based Investigation

BACKGROUND: Preterm delivery causes substantial neonatal mortality and morbidity. Unrecognized intra-amniotic infections caused by cultivation-resistant microbes may play a role. Molecular methods can detect, characterize and quantify microbes independently of traditional culture techniques. However, molecular studies that define the diversity and abundance of microbes invading the amniotic cavity, and evaluate their clinical significance within a causal framework, are lacking. METHODS AND FINDINGS: In parallel with culture, we used broad-range end-point and real-time PCR assays to amplify, identify and quantify ribosomal DNA (rDNA) of bacteria, fungi and archaea from amniotic fluid of 166 women in preterm labor with intact membranes. We sequenced up to 24 rRNA clones per positive specimen and assigned taxonomic designations to approximately the species level. Microbial prevalence, diversity and abundance were correlated with host inflammation and with gestational and neonatal outcomes. Study subjects who delivered at term served as controls. The combined use of molecular and culture methods revealed a greater prevalence (15% of subjects) and diversity (18 taxa) of microbes in amniotic fluid than did culture alone (9.6% of subjects; 11 taxa). The taxa detected only by PCR included a related group of fastidious bacteria, comprised of Sneathia sanguinegens, Leptotrichia amnionii and an unassigned, uncultivated, and previously-uncharacterized bacterium; one or more members of this group were detected in 25% of positive specimens. A positive PCR was associated with histologic chorioamnionitis (adjusted odds ratio [OR] 20; 95% CI, 2.4 to 172), and funisitis (adjusted OR 18; 95% CI, 3.1 to 99). The positive predictive value of PCR for preterm delivery was 100 percent. A temporal association between a positive PCR and delivery was supported by a shortened amniocentesis-to-delivery interval (adjusted hazard ratio 4.6; 95% CI, 2.2 to 9.5). A dose-response association was demonstrated between bacterial rDNA abundance and gestational age at delivery (r(2) = 0.42; P<0.002). CONCLUSIONS: The amniotic cavity of women in preterm labor harbors DNA from a greater diversity of microbes than previously suspected, including as-yet uncultivated, previously-uncharacterized taxa. The strength, temporality and gradient with which these microbial sequence types are associated with preterm delivery support a causal relationship

Distamycin A Inhibits HMGA1-Binding to the P-Selectin Promoter and Attenuates Lung and Liver Inflammation during Murine Endotoxemia

Background: The architectural transcription factor High Mobility Group-A1 (HMGA1) binds to the minor groove of AT-rich DNA and forms transcription factor complexes (“enhanceosomes”) that upregulate expression of select genes within the inflammatory cascade during critical illness syndromes such as acute lung injury (ALI). AT-rich regions of DNA surround transcription factor binding sites in genes critical for the inflammatory response. Minor groove binding drugs (MGBs), such as Distamycin A (Dist A), interfere with AT-rich region DNA binding in a sequence and conformation-specific manner, and HMGA1 is one of the few transcription factors whose binding is inhibited by MGBs. Objectives: To determine whether MGBs exert beneficial effects during endotoxemia through attenuating tissue inflammation via interfering with HMGA1-DNA binding and modulating expression of adhesion molecules. Methodology/Principal Findings: Administration of Dist A significantly decreased lung and liver inflammation during murine endotoxemia. In intravital microscopy studies, Dist A attenuated neutrophil-endothelial interactions in vivo following an inflammatory stimulus. Endotoxin induction of P-selectin expression in lung and liver tissue and promoter activity in endothelial cells was significantly reduced by Dist A, while E-selectin induction was not significantly affected. Moreover, Dist A disrupted formation of an inducible complex containing NF-κB that binds an AT-rich region of the P-selectin promoter. Transfection studies demonstrated a critical role for HMGA1 in facilitating cytokine and NF-κB induction of P-selectin promoter activity, and Dist A inhibited binding of HMGA1 to this AT-rich region of the P-selectin promoter in vivo. Conclusions/Significance: We describe a novel targeted approach in modulating lung and liver inflammation in vivo during murine endotoxemia through decreasing binding of HMGA1 to a distinct AT-rich region of the P-selectin promoter. These studies highlight the ability of MGBs to function as molecular tools for dissecting transcriptional mechanisms in vivo and suggest alternative treatment approaches for critical illness