Biomedical subjects

Roger F Searle

Publications and source records attributed to Roger F Searle.

7 recordsLinked to original sources

PubMed2006

Effector activity of decidual CD8+ T lymphocytes in early human pregnancy.

Although CD8+ T lymphocytes are present in human decidua throughout pregnancy, albeit as a minor population in early pregnancy, their role in normal pregnancy is largely unknown. The present study aimed to characterize their effector phenotype, including cytolytic activity, cytokine profile, and capacity to affect placental invasion. CD8+ lymphocytes were positively selected from normal early pregnancy decidua (7-14 wks gestational age). Decidual CD8+ T lymphocytes were studied using standard and redirected chromium release assays to investigate natural killer cell-sensitive cytotoxicity and cytotoxicity that requires T-cell receptor signal transduction respectively, multiplex cytokine analysis to analyze cytokine production, and a placental explant invasion model to assess the effect of soluble products of decidual CD8+ T lymphocytes on trophoblast invasion. Decidual CD8+ T lymphocytes exhibited cytolytic ability against P815 target cells (mean % Specific Chromium Release at effector:target ratio of 32:1 [SCR(32)] of 32.7 +/- 5.8) and against K562 target cells (mean SCR(32) of 20.3 +/- 0.5). Phytohemagglutinin-P (PHA-P)-stimulated decidual CD8(+) T lymphocytes produced high levels of both interferon gamma and interleukin (IL) 8, and low levels of granulocyte-macrophage colony-stimulating factor (CSF2), IL1B, IL2, IL6, IL10, IL12, and tumor necrosis factor; these did not vary with gestational age. IL4 was undetectable. Decidual CD8+ T lymphocyte supernatants increased the capacity of extravillous trophoblast cells to invade through Matrigel compared with the PHA-P control. These findings suggest that decidual CD8+ T cells can display cytolytic activity, do not evoke a predominant local intrauterine Th2 type cytokine environment, and may act to regulate invasion of extravillous trophoblast cells into the uterus, a crucial process for normal uteroplacental development.

CD8-Positive T-Lymphocytes↗

PubMed2006

Expression of angiogenic growth factors by uterine natural killer cells during early pregnancy.

Remodeling of uterine spiral arteries is critical for the continuation of a successful pregnancy. Uterine natural killer (uNK) cells are the predominant leukocyte population in the early pregnant decidua, and a role for these cells in spiral artery remodeling in pregnancy has been suggested. Angiogenic growth factors were measured in isolated uNK and total (unseparated) decidual cells (8-10 or 12-14 weeks gestation, n=5 each gestational age) after culture for 48 h. Angiopoietin (Ang)1, placental growth factor, transforming growth factor-beta1 (TGF-beta1), and vascular endothelial growth factor (VEGF)-C were measured by enzyme-linked immunosorbent assay. Angiogenin, Ang2, fibroblast growth factor basic, intercellular adhesion molecule (ICAM)-1, keratinocyte growth factor (KGF), platelet-derived growth factor-BB, and VEGF-A were measured using a FASTQuant angiogenic growth factor multiplex protein assay. Levels of Ang2, ICAM-1, and KGF, secreted by the total decidual fraction, decreased with increasing gestational age. uNK levels of Ang2 and VEGF-C also decreased with increasing gestational age. At 8-10 weeks gestation, there was no difference in the level of Ang1, Ang2, TGF-beta1, and VEGF-C secreted by uNK cells and the total decidual fraction. At 12-14 weeks, uNK cells secreted significantly lower levels of VEGF-C than the total decidual fraction. Early pregnancy decidua is a major source of angiogenic growth factors whose levels decrease with increasing gestational age, suggesting that they may play a role in spiral artery remodeling. uNK cells appear to be a prominent source of Ang1, Ang2, TGF-beta1, and VEGF-C within the placental bed.

Angiogenic Proteins↗

PubMed2006

Comparison of three multiplex cytokine analysis systems: Luminex, SearchLight and FAST Quant.

Multiplex cytokine analysis technologies have become readily available in the last five years. Two main formats exist: multiplex sandwich ELISA and bead based assays. While these have each been compared to individual ELISAs, there has been no direct comparison between the two formats. We report here the comparison of two multiplex sandwich ELISA procedures (FAST Quant and SearchLight) and a bead based assay (UpState Luminex). All three kits differed from each other for different analytes and there was no clear pattern of one system giving systematically different results than another for any analyte studied. We suggest that each system has merits and several factors including range of analytes available, prospect of development of new analytes, dynamic range of the assay, sensitivity of the assay, cost of equipment, cost of consumables, ease of use and ease of data analysis need to be considered when choosing a system for use. We also suggest that results obtained from different systems cannot be combined.

CD8-Positive T-Lymphocytes↗

PubMed2006

Interferon-gamma inhibits extravillous trophoblast cell invasion by a mechanism that involves both changes in apoptosis and protease levels.

BACKGROUND: Extravillous trophoblast cell (EVT) invasion of decidua and inner third of the myometrium is critical for a successful pregnancy. Many decidual factors are likely to play a role in regulating this process, including uterine natural killer (uNK) cell-derived cytokines. HYPOTHESES: 1) uNK cells are a major source of IFN gamma (IFN-gamma) and 2) IFN-gamma inhibits EVT invasion via an increase in EVT apoptosis and/or a decrease in active protease levels. METHODS: Total decidual and uNK cells from 8-10 wk and 12-14 wk gestational age were cultured. IFN-gamma mRNA (real-time RT-polymerase chain reaction) and protein levels (FastQuant multicytokine analysis) were determined. EVT invasion in the presence of IFN-gamma or anti-IFN-gamma-neutralizing antibodies was assessed. Trophoblast apoptosis and proliferation was assessed in explants by immunohistochemistry for M30 and Ki67. Substrate zymography was performed to determine levels of secreted MMP2, MMP9, and uPA. RESULTS: mRNA and protein for IFN-gamma was detected in both total decidual and uNK cell fractions. Trophoblast invasion was inhibited by IFN-gamma. The level of M30-positive EVT was increased in the presence of IFN-gamma whereas levels of secreted MMP2 were decreased. CONCLUSIONS: uNK cells are a source of IFN-gamma within early human pregnancy decidua. Mechanisms of IFN-gamma inhibition of EVT invasion include both increased EVT apoptosis and reduced levels of active proteases.

Apoptosis↗

PubMed2005

Low oxygen concentrations inhibit trophoblast cell invasion from early gestation placental explants via alterations in levels of the urokinase plasminogen activator system.

Extravillous trophoblast cell (EVT) invasion in early pregnancy occurs in a relatively low-oxygen environment. The role of oxygen in regulation of EVT invasion remains controversial. We hypothesized that 1) culture in 3% oxygen inhibits EVT invasion compared with culture at 8% or 20% oxygen and 2) inhibition of invasion is due to alterations in levels of components of the urokinase plasminogen activator (PLAU, uPA) system rather than through increased apoptosis and/or decreased proliferation. Placental samples (8-10, 12-14, and 16-20 wk gestation) were obtained from women undergoing elective surgical termination of pregnancy or after cesarean section delivery (term) at the Royal Victoria Infirmary, Newcastle upon Tyne, U.K. EVT invasion from placental explants cultured at 3%, 8%, or 20% oxygen was assessed using Matrigel invasion assays. Invasion was assessed on Day 6, explants were harvested for analysis of apoptosis and proliferation, and medium was stored for analysis of PLAU system components by ELISA and casein zymography. Culture at 3% oxygen inhibited EVT invasion. PLAU receptor and plasminogen activator inhibitor-2 protein levels were increased and PLAU activity decreased in these cultures. There was no difference in the proliferation in explants cultured at the three different oxygen concentrations. Apoptosis, assessed by M30 immunostaining, was increased in EVT at both 3% and 8% oxygen. The reduction in the invasive capacity of EVT cultured at 3% oxygen appears to be mediated both by a general inhibition of the PLAU system and a decrease in the number of cells available to invade.

Amyloid beta-Protein Precursor↗

PubMed2005

Inhibition of trophoblast cell invasion by TGFB1, 2, and 3 is associated with a decrease in active proteases.

Invasion of extravillous trophoblast cells into the uterus in human pregnancy is tightly regulated. The transforming growth factor-beta (TGFB) family has been suggested to play a role in controlling this process. We hypothesized that TGFB1, 2, and 3 would inhibit the invasive capacity of extravillous trophoblast cells. We also studied trophoblast apoptosis and proliferation and secreted protease levels as potential mechanisms by which these cytokines may act. Inhibition of endogenous TGFB1, 2, and 3 with neutralizing antibodies increased the invasive capacity of extravillous trophoblast cells derived from placental explants. Similarly, addition of exogenous TGFB1, 2, and 3 inhibited the invasive capacity of these cells in a dose-dependent manner. Proliferation of trophoblast in the placental explants did not alter in response to any of the cytokines tested. Apoptosis of villous and extravillous trophoblast did not alter in response to TGFB1, 2, and 3. There was a reduction in secreted levels of matrix metalloproteinase (MMP) 9 and urokinase plasminogen activator in response to all three cytokines. MMP2 and tissue inhibitor of metalloproteinase 1 and 3 levels were not altered. These results suggest that TGFB1, 2, and 3 inhibit trophoblast invasion by a mechanism dependent on reduced protease activity.

Apoptosis↗

PubMed2005

Macrophages and not granulocytes are involved in cervical ripening.

To clarify the role of leucocytes in human cervical ripening and dilatation, cervical biopsies were obtained from six non-pregnant women, eight women undergoing early termination of pregnancy, 18 pregnant women undergoing elective Caesarean section at term (both with and without a ripe cervix as determined by Bishop score) and 11 women after term vaginal delivery. Leucocytes were localised by immunohistochemistry labelling and quantified in subepithelial and deep stromal areas. CD45+ leucocytes were more numerous in the subepithelial area of the cervix than in the deep stroma in all groups (P<0.01). CD14+ macrophages and CD15+ granulocytes were increased in both the subepithelial and deep stromal areas only in the vaginal delivery group (P<0.01). The number of macrophages in the ripening cervix (Bishop score above 4) was higher than in the unripe cervix (Bishop score 4 or less; P<0.05) with no differences in other leucocyte populations. CD3+ CD8+ T cells in the subepithelial area were reduced in late pregnancy and after vaginal delivery (P<0.01), but showed no relationship to Bishop score. Macrophages and granulocytes may be involved in the process of cervical dilatation, but macrophage infiltration into the ripening cervix before labour suggests their role in the ripening process. Reduced numbers of CD3+ CD8+ T-lymphocytes in late pregnancy and after vaginal delivery suggests that local immunity is down-regulated in the late pregnancy period. Regional differences in leucocyte subpopulations in the cervix indicate that leucocyte infiltration is likely to be regulated by local factors.

Antigens, CD↗