Educational Impact of a Pulsatile Human Cadaver Circulation Model for Endovascular Training.

OBJECTIVES: The face and construct validity of a novel pulsatile human cadaver model (PHCM) was recently demonstrated for endovascular training. This study aimed to assess the model's educational impact. METHODS: Twenty-four endovascular novices were recruited and split into two equal training groups: PHCM and virtual reality simulator (VRS). Each candidate performed eight consecutive training attempts of endovascular renal artery catheterisation on their designated model, and a final crossover attempt on the alternate model. Performances were video recorded and scored using a validated scoring tool by two independent endovascular experts, blinded to the candidate's identity and attempt number. Each participant was given a task specific checklist score (TSC), global rating score (GRS), and overall procedure score (OPS). RESULTS: In the PHCM group average OPS improved gradually from 19.42 (TSC 8.58, GRS 10.83) to 39.50 (TSC 15.00, GRS 24.5) over eight attempts (p < .0005). In the VRS group OPS improved from 20.54 (TSC 10.29, GRS 10.25) to 36.04 (TSC 14.21, GRS 21.88) between the first and eighth attempts (p < .0005), with limited improvement after the second attempt. PHCM training significantly improved OPS on their VRS crossover attempt (p ≤ .0001), achieving a similar OPS to candidates who had completed VRS training (p = .398). VRS training significantly improved OPS on PHCM (p < 0.05); however, OPS was significantly worse than candidates who had completed PHCM training (p ≤ .001). CONCLUSIONS: PHCM training has a longer learning curve, with gradual improvement, reflecting the enhanced difficulty of a more realistic model. These results support the use of PHCM preceded by VRS training, prior to performing endovascular surgery on patients.

A Pulsatile Fresh Frozen Human Cadaver Circulation Model for Endovascular Training: A Trial of Face Validity.

BACKGROUND: The authors have published their design of a pulsatile fresh frozen human cadaver circulation model (PHCM) for endovascular training the face validity of the PHCM for training endovascular practitioners was subsequently assessed. METHODS: Twelve endovascular clinicians performed the same 2 procedures (catheterization of the left renal artery and left subclavian artery) on PHCM and Simbionix angiomentor virtual reality simulator (SVR). They were randomized to begin on either the PHCM or SVR. A pretrial questionnaire determined participants' endovascular experience. After training, participants rated statements relating to their experience on a numerical scale from 1 to 5, with 1 representing the strongest agreement with the statement. RESULTS: When participants were asked to compare the realism of training modalities with live patients, PHCM scored significantly higher than SVR on statements regarding "realism of vascular access" (P = 0.002), "guide-wire manipulation" (P = 0.001), and "vessel catheterization" (P = 0.004). Candidates again favored PHCM as "a valuable learning exercise" (P = 0.016) and strongly favored PHCM as a "useful training model" compared to SVR (P = 0.004). CONCLUSIONS: This is the first published trial in world literature to assess the validity of a PHCM for training endovascular practitioners. The PHCM demonstrates good face validity when compared to both real patients and the SVR model and holds exciting potential.

Evaluating the Construct Validity of a Pulsatile Fresh Frozen Human Cadaver Circulation Model for Endovascular Training.

BACKGROUND: We recently described a pulsatile fresh frozen human cadaver model (PHCM) for training endovascular practitioners. This present study aims to assess the construct validity of PHCM; its ability to differentiate between participants of varying expertise. METHODS: Twenty-three participants with varying endovascular experience (12 novice, 4 intermediate, and 7 expert) were recruited. Each attempted catheterization of the left renal artery on PHCM within 10 min under exam conditions. Performances were video recorded and scored using a validated scoring tool by 2 independent endovascular experts, blinded to performer status. Each participant was given a task-specific checklist score (TSC), global rating score (GRS), and overall procedure score (OPS). Finally, examiners were asked whether they would be happy to supervise the participant in theater, with each participant graded as "fail", "borderline," or "pass". RESULTS: All expert and intermediate participants completed the index procedure within the allotted 10 min; however, only one of the 12 novice participants achieved this (P < 0.0005). Endovascular novices had significantly lower TSC, GRS, and OPS than both intermediate participants and endovascular experts. There were no significant differences in TSC, GRS, or OPS between intermediate participants and endovascular experts. When participants were graded as "fail", "borderline," or "pass," there were significant differences between groups (P = 0.001). All of the intermediate and expert participants received a pass. Out of the 12 novice participants, 2 received a pass, 6 received a borderline, and 4 were failed. CONCLUSIONS: The PHCM demonstrates construct validity. Further work is required to determine its educational impact in endovascular training.

Design of a Pulsatile Fresh Frozen Human Cadaver Circulation Model for Endovascular Training.

BACKGROUND: The objective of this project was to create a model capable of training endovascular skills using a freshly frozen human cadaver (HC). We present the results of our experience creating a cadaveric model for endovascular skills training. We undertook a unique cadaver laboratory-based research project. METHODS: We favor a minimally invasive surgical technique with inflow into the right common carotid artery and outflow through the left common femoral and right superficial femoral arteries. Endovascular access was through the right common femoral artery. RESULTS: Through this technique, the arch, thoracic, abdominal, and iliac vessels are all accessible. We perfuse the model through an open pulsatile flow circuit at varying rates to maximize angiographic image capture while minimizing cadaveric edema thus expanding the models longevity. CONCLUSIONS: A fresh frozen pulsatile human cadaver training model is a feasible and credible training model that has exciting potential for endovascular skills training.

Role of SimMan in teaching clinical skills to preclinical medical students.

BACKGROUND: Simulation training has potential in developing clinical skills in pre-clinical medical students, but there is little evidence on its effectiveness. METHODS: Twenty four first year graduate entry preclinical medical students participated in this crossover study. They were divided into two groups, one performed chest examination on each other and the other used SimMan. The groups then crossed over. A pretest, midtest and post-test was conducted in which the students answered the same questionnaire with ten questions on knowledge, and confidence levels rated using a 5 point Likert scale. They were assessed formatively using the OSCE marking scheme. At the end of the session, 23 students completed a feedback questionnaire. Data was analyzed using one-way ANOVA and independent t-test. RESULTS: When the two groups were compared, there was no significant difference in the pretest and the post-test scores on knowledge questions whereas the midtest scores increased significantly (P< 0.001) with the group using SimMan initially scoring higher. A significant increase in the test scores was seen between the pre-test and the mid-test for this group (P=0.009). There was a similar albeit non significant trend between the midtest and the post-test for the group using peer examination initially.Mean confidence ratings increased from the pretest to midtest and then further in the post-test for both groups. Their confidence ratings increased significantly in differentiating between normal and abnormal signs [Group starting with SimMan, between pretest and midtest (P= 0.01) and group starting with peer examination, between midtest and post-test (P=0.02)]. When the students' ability to perform examination on each other for both groups was compared, there was a significant increase in the scores of the group starting with SimMan (P=0.007). CONCLUSIONS: This pilot study demonstrated a significant improvement in the students' knowledge and competence to perform chest examination after simulation with an increase in the student's perceived levels of confidence. Feedback from the students was extremely positive. SimMan acts as a useful adjunct to teach clinical skills to preclinical medical students by providing a simulated safe environment and thus aids in bridging the gap between the preclinical and clinical years in medical undergraduate education.

Anatomy teaching with portable ultrasound to medical students.

BACKGROUND: Medical students as future clinicians will apply their anatomy knowledge in medical imaging. There are various radiological resources available for the medical students to learn anatomy and contextualise it to the clinical setting. Ultrasound is a safe and non- invasive imaging procedure commonly used in clinical practice. This study aimed to use portable ultrasound and evaluate its impact as an adjunct to cadaveric anatomy teaching together with cross sectional anatomy images and line diagrams. METHODS: Ultrasound teaching was incorporated into upper limb and lower limb anatomy practical dissecting room sessions. The number of medical students who participated was 121 students from the year 2008 - 2009 and 94 students from the year 2009- 2010. The students were divided into groups of 15-20. Initially ultrasound demonstration was carried out on a volunteer and then the students were given the opportunity to use the ultrasound and identify normal anatomical structures visualized on images. For the students in the year 2009- 2010, ultrasound teaching was supplemented with cross sectional anatomy images and line diagrams. Questionnaires were distributed with seven questions rated using four point Likert scale and free text. Qualitative data was analysed using 2- proportion Z test and Fischer's exact test. RESULTS: The number of students in the 2009-2010 year group who were confident in interpreting ultrasound images increased significantly when compared to the 2008-2009 year group of students. The majority of students were able to identify structures like bone, muscles and blood vessels on ultrasound images. There was a significant increase in the number of students who found the ultrasound teaching useful and also those who regarded ultrasound to have improved understanding of anatomy considerably. CONCLUSIONS: Ultrasound acts as a useful adjunct to teach anatomy in a clinical context to medical students. The use of cross sectional anatomy images and line diagrams together can aid ultrasound image orientation of structures during these sessions. Early exposure to this imaging technology may prime students for later encounters with ultrasound during clinical practice.

Interaction between uterine natural killer cells and extravillous trophoblast cells: effect on cytokine and angiogenic growth factor production.

BACKGROUND: Uterine natural killer (uNK) cells are a major source of cytokines and angiogenic growth factors (AGFs), with AGF levels decreasing and cytokine levels increasing with gestational age. The factors that regulate AGF and cytokine secretion are unclear but may involve interactions between uNK cells and extravillous trophoblast (EVT) cells. We hypothesize that uNK cell interaction with EVT cells alters their cytokine and AGF secretion. METHODS: Ex vivo co-cultures of uNK cells with either EVT (irradiated or fresh) or villous cytotrophoblast (CTB; control cell type) cells isolated from the same patients at 8-10 or 12-14 weeks gestational age (n = 10 each group) were established. Co-cultures were established with either direct contact between the different cell types or with the cells separated by a 0.4 µm filter. AGFs and cytokines were measured in cell culture supernatants using multiplex analysis (FAST Quant) or ELISA. RESULTS: Secretion of angiopoietin-1 (P < 0.006) and vascular endothelial growth factor-C (P < 0.001) by uNK cells was lower when these cells were co-cultured, either directly or indirectly, with both trophoblast cell types at both gestational ages tested compared with when cultured alone. In contrast, interleukin (IL)-6 (P < 0.0001), IL-8 (P < 0.0001) and transforming growth factor-ß1 (P < 0.002) were decreased only in direct uNK/EVT and uNK/CTB co-culture conditions at 8-10 and 12-14 weeks gestational age. CONCLUSIONS: AGF and cytokine secretion was reduced after co-culture of uNK cells and both EVT and CTB cells. It remains unclear whether uNK cell AGF and cytokine production was reduced after co-culture with trophoblast cells (EVT or CTB) or whether trophoblast cell (EVT or CTB) AGF and cytokine production was reduced after co-culture with uNK cells. Local production of AGFs and cytokines in the placental bed may be lowered when uNK cells come in direct contact with EVT cells.

Regulation of extravillous trophoblast invasion by uterine natural killer cells is dependent on gestational age.

BACKGROUND: Extravillous trophoblast (EVT) cell invasion of uterine decidua and the inner third of myometrium is critical for successful pregnancy. Many decidual factors are likely to play a role in regulating this process. We have previously shown that cytokines, known to be produced by uterine natural killer (uNK) cells, such as TNF-alpha, TGF-beta1 and IFN-gamma inhibit EVT invasion. We therefore hypothesized that supernatants from purified uNK cells would inhibit EVT invasion. METHODS AND RESULTS: Total unfractionated decidual cell supernatants from 8 to 10 weeks gestation increased EVT invasion from placental villous explants, although uNK cell supernatants from 8 to 10 weeks gestation had no effect. In contrast, both total decidual and uNK cell supernatants from 12 to 14 weeks gestation stimulated EVT invasion. MMP-2, uPA, PAI-1 and PAI-2 levels did not differ under any of the conditions tested, whereas MMP-9 levels were increased in the presence of both total decidual and uNK cell supernatants from both gestational age groups. There was a decrease in the level of EVT apoptosis in the presence of uNK cell supernatant from 12 to 14 weeks, but not 8-10 weeks, gestation. CONCLUSIONS: Decidual uNK cell supernatants from 12 to 14 weeks gestational age stimulated EVT invasion, potentially by increasing MMP9 levels and reducing apoptosis. Total decidual cell isolates stimulated EVT invasion at both gestational ages investigated, potentially reflecting the complex nature of these cell culture supernatants.

Localization of matrix metalloproteinase (MMP)-2, MMP-9 and tissue inhibitors for MMPs (TIMPs) in uterine natural killer cells in early human pregnancy.

BACKGROUND: Invasion by extravillous trophoblast into uterine decidua and myometrium with remodeling of spiral arteries is essential for normal human pregnancy and is tightly regulated. Uterine natural killer (uNK) cells appear to be a major maternal regulator of placentation through the secretion of growth factors, cytokines and proteinases. METHOD: Matrix metalloproteinase (MMP)-2 and MMP-9 activity in placental bed biopsies was studied using in situ gelatin zymography. Expression by uNK cells of MMP-2, MMP-9 and their tissue inhibitors, TIMP-1, TIMP-2 and TIMP-3, was localized in the placental bed by immunohistochemistry. Levels of MMP-2, MMP-9, TIMP-1, TIMP-2 and TIMP-3 secreted into 24 h cell culture supernatants of isolated uNK and unseparated (total) decidual cells (8-10 and 12-14 weeks gestation, n = 10 each group) were determined by gelatin gel zymography or western blot as appropriate. RESULTS: Gelatinase activity in situ appeared greater in decidua than myometrium. uNK cells showed strong immunostaining for MMP-2 and TIMP-2. MMP-9 activity was lower in uNK cells than total decidual supernatants (8-10 weeks: P = 0.0003; 12-14 weeks: P = 0.0012). In contrast, there was no difference in MMP-2 secreted by either uNK cell or total decidual cultures. CONCLUSIONS: uNK cells from early human pregnancy decidua possess innate protease activity, especially MMP-2, providing further evidence for a role for these cells in regulation of trophoblast invasion and spiral artery remodeling in early placentation.

Altered decidual leucocyte populations in the placental bed in pre-eclampsia and foetal growth restriction: a comparison with late normal pregnancy.

Alterations in the balance of leucocyte populations in uterine decidua may lead to the generation of an unfavourable cytokine environment that is associated with unsuccessful pregnancy. Single and double immunohistochemical labelling was used to examine leucocyte populations in decidua from normal third trimester, foetal growth-restricted and pre-eclamptic pregnancies. Placental bed biopsies from 12 women undergoing elective Caesarean section with no hypertension or foetal growth restriction (FGR), 8 women with FGR without maternal hypertension and 12 women with pre-eclampsia (PE) were used to quantify decidual CD56+ uterine NK cells, CD14+ macrophages, CD3+T-lymphocytes and CD8+ lymphocytes. CD3+CD56+, CD8+CD56+ and CD161+CD3+ double-labelled cells in decidua were compared in PE and control decidua. Decidual CD3+T-lymphocytes (P<0.01), CD8+ cytotoxic T-lymphocytes (P<0.05), CD14+ macrophages (P<0.0001) and CD56+ uterine natural killer (uNK) cells (P=0.01) were decreased in placental bed biopsies from women with PE compared with control third trimester decidua. By contrast, only CD56+ uNK cells were decreased in FGR decidua (P<0.05). Double-positive CD8+CD56+ cells were also decreased in PE compared with control third trimester decidua (P<0.05). The reduction in specific leucocyte subset numbers in PE and uNK cells in FGR suggests that altered local cytokine balance may be important in defective trophoblast invasion and spiral artery transformation in these pathological pregnancies.

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.

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.

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.

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.

Student Views on the Use of 2 Styles of Video-Enhanced Feedback Compared to Standard Lecture Feedback During Clinical Skills Training.

BACKGROUND: Feedback plays an important role in the learning process. However, often this may be delivered in an unstructured fashion that can detract from its potential benefit. Further, students may have different preferences in how feedback should be delivered, which may be influenced by which method they feel will lead to the most effective learning. The aim of this study was to evaluate student views on 3 different modes of feedback particularly in relation to the benefit each conferred. METHODS: Undergraduate medical students participating in a surgical suturing study were asked to give feedback using a semi-structured questionnaire. Discrete questions using a Likert scale and open responses were solicited. Students received either standard lecture feedback (SLF), individualized video feedback (IVF), or enhanced unsupervised video feedback (UVF). RESULTS: Students had a strong preference for IVF over UVF or SLF. These responses correlated with their perception of how much each type of feedback improved their performance. However, there was no statistical difference in suturing skill improvement between IVF and UVF, which were both significantly better than SLF. CONCLUSION: Students have a strong preference for IVF. This relates to a perception that this will lead to the greatest level of skill improvement. However, an equal effect in improvement can be achieved by using less resource-demanding UVF.

Randomized trial to assess the effect of supervised and unsupervised video feedback on teaching practical skills.

BACKGROUND: Feedback is a vital component of the learning process; however, great variation exists in the quality, quantity, and method of delivery. Video feedback is not commonly used in the teaching of surgical skills. The aim of this trial was to evaluate the benefit of 2 types of video feedback-individualized video feedback (IVF), with the student reviewing their performance with an expert tutor, and unsupervised video-enhanced feedback (UVF), where the student reviews their own performance together with an expert teaching video-to determine if these improve performance when compared with a standard lecture feedback. METHODS: A prospective blinded randomized control trial comparing lecture feedback with IVF and UVF was carried out. Students were scored by 2 experts directly observing the performance and 2 blinded experts using a validated pro forma. Participants were recorded on video when performing a suturing task. They then received their feedback via any of the 3 methods before being invited to repeat the task. RESULTS: A total of 32 students were recruited between the 3 groups. There was no significant difference in suturing skill performance scores given by those directly observing the students and those blinded to the participant. There was no statistically significant difference between the 2 video feedback groups (p = 1.000), but there was significant improvement between standard lecture feedback and UVF (p = 0.047) and IVF (p = 0.001). CONCLUSION: Video feedback can facilitate greater learning of clinical skills. Students can attain a similar level of surgical skills improvement with UVF as with teacher-intensive IVF.

Hydrothermal vent fields and chemosynthetic biota on the world's deepest seafloor spreading centre.

The Mid-Cayman spreading centre is an ultraslow-spreading ridge in the Caribbean Sea. Its extreme depth and geographic isolation from other mid-ocean ridges offer insights into the effects of pressure on hydrothermal venting, and the biogeography of vent fauna. Here we report the discovery of two hydrothermal vent fields on the Mid-Cayman spreading centre. The Von Damm Vent Field is located on the upper slopes of an oceanic core complex at a depth of 2,300 m. High-temperature venting in this off-axis setting suggests that the global incidence of vent fields may be underestimated. At a depth of 4,960 m on the Mid-Cayman spreading centre axis, the Beebe Vent Field emits copper-enriched fluids and a buoyant plume that rises 1,100 m, consistent with >400 °C venting from the world's deepest known hydrothermal system. At both sites, a new morphospecies of alvinocaridid shrimp dominates faunal assemblages, which exhibit similarities to those of Mid-Atlantic vents.

Effect of tumour necrosis factor-α in combination with interferon-γ on first trimester extravillous trophoblast invasion.

Successful pregnancy is dependent upon invasion of the uterine tissues by extravillous trophoblast cells (EVT). The mechanisms that control trophoblast invasion are unclear, but several cytokines and growth factors appear to be involved. We have previously demonstrated that IFN-γ inhibits EVT invasion via a mechanism partially dependent on an increase in EVT apoptosis and decreased secretion of matrix metalloproteinase (MMP)-2. In the current study we show that TNF-α, both alone and in combination with IFN-γ, inhibits EVT invasion via a mechanism associated with increased trophoblast apoptosis, decreased trophoblast proliferation and/or altered production of active proteases. TNF-α and its receptors, TNF-αRI and TNF-αRII, were immunolocalised in the placental bed. Uterine natural killer (uNK) cells, EVT and villous cytotrophoblast were shown to all produce TNF-α, and TNF-α receptors were primarily immunolocalised to EVT in the placental bed. TNF-α increased EVT apoptosis, decreased villous cytotrophoblast proliferation and increased expression of pro-MMP-9 (but not active MMP-9), urokinase plasminogen activator (uPA) and plasminogen activator inhibitor (PAI)-1 by EVT. The combination of TNF-α and IFN-γ inhibited EVT via a mechanism associated with increased EVT apoptosis, reduced proliferation, reduced pro-MMP-2 secretion and increased secretion of uPA. TNF-α is one of several decidua-derived factors with the capacity to inhibit EVT invasion. The mode of activity of TNF-α was modified by the presence of IFN-γ, suggesting that the local cytokine milieu may be critical in determining spatial and/or temporal changes in EVT invasion.

Secretion of cytokines by villous cytotrophoblast and extravillous trophoblast in the first trimester of human pregnancy.

Cytokines are proposed to play roles in regulation of trophoblast invasion, spiral artery remodeling and immunoregulation during early pregnancy. Secretion of 12 cytokines (interleukin (IL)-1ß, IL-2, IL-4, IL-5, IL-8, IL-10, IL-12p70, IL-13, IFNγ, GM-CSF, MCP-1 and RANTES) by first trimester extravillous trophoblast and villous cytotrophoblast cells was examined using multiplex cytokine array technology. Seven (IL-1ß, IL-8, IL-12p70, IL-13, GM-CSF, MCP-1 and RANTES) of the 12 cytokines examined were detectable in the samples studied (n=10 each group). Villous cytotrophoblast production of IL-1ß and IL-8 increased with gestational age. Extravillous trophoblast production of IL-8, IL-13 and RANTES increased with gestational age. At 12-14 weeks gestation extravillous trophoblast cells secreted higher levels of IL-8, IL-13 and RANTES than villous cytotrophoblast cells.

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.