Biomedical subjects

Harry A Otun

Publications and source records attributed to Harry A Otun.

5 recordsLinked to original sources

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

Spatial and temporal expression of the myometrial mitogen-activated protein kinases p38 and ERK1/2 in the human uterus during pregnancy and labor.

OBJECTIVE: We have recently identified a novel putative spliced variant of the activating transcription factor 2 (ATF2) in the human myometrium during pregnancy and labor. This protein, termed ATF2-sm like full-length ATF2, acts as a potent transactivator of cyclic adenosine monophosphate response element (CRE)-containing promoter reporter genes. Similarly, employing microarray gene profiling in myometrial cells, we have shown ATF2-sm to affect the expression of several specific myometrial genes associated with regulating uterine activity during pregnancy and labor. At some point after conception this transcription factor becomes spatially expressed within the body of the uterus, with significantly higher levels detected in the upper (corpus) compared to the lower uterine segment. Because ATF2 species are the primary substrate for phosphorylation by the mitogen-activated protein kinases (MAPKs) p38 and ERK1/2, the purpose of the current investigation was to define the expression levels of these kinases in upper and lower segment myometrium during pregnancy and labor to see if they also correlated with expression of ATF2-sm. METHODS: Paired myometrial samples were collected from the upper (corpus) and lower uterine segments from term nonlaboring and spontaneously laboring women undergoing elective and emergency cesarean deliveries, respectively. Non-pregnant myometrial samples were collected from premenopausal women having hysterectomies for benign gynecologic disorders. The MAPKs p38 and ERK1/2 present in individual myometrial homogenates were resolved using sodium dodecyl sulfate polacrylamide gel electropheresis (SDS-PAGE) with subsequent Western blotting with specific antibodies and scanning densitometry. Expression of the individual MAPKs in myometrial tissues was confirmed in situ using immunohistochemistry. RESULTS: In non-pregnant tissues, p38 and ERK1/2 expression was uniform throughout the uterus. In term pregnant nonlaboring and spontaneously laboring samples expression of p38 and ERK1 was significantly elevated in the upper uterine segment compared to the lower segment, respectively. In contrast, there was no difference in ERK2 expression. CONCLUSION: The data from this study indicate that both p38 and ERK1 are spatially regulated in different uterine regions during pregnancy/labor and suggest that they may be involved in regulating the activity of ATF2 isoforms and their subsequent effects on myometrial function.

Activating Transcription Factor 2↗