Quantification of spiral artery remodelling using an Adobe Photoshop-based technique.

Uterine spiral arteries undergo remodelling in normal pregnancy, with replacement of the musculoelastic arterial media by fibrinoid containing extravillous trophoblast cells. Deficient spiral artery remodelling is associated with several adverse pregnancy outcomes. Although there are distinct components of spiral artery remodelling, assessment is subjective and often based on an overall impression of morphology. We aimed to develop a quantitative approach for assessment of uterine spiral artery remodelling. Placental bed biopsies were immunostained using smooth muscle markers, digital images of spiral arteries were captured and Adobe Photoshop was used to analyse positive immunostaining. The method was then used to investigate variation in the same vessel at different levels within a paraffin block, and the effect of parity, pre-eclampsia or miscarriage on vascular smooth muscle cell content. Results were also compared with a more subjective morphology-based assessment system. There was good intra- and interobserver agreement and the method correlated well with the more subjective assessment system. There was an overall reduction in vascular smooth muscle, as detected by caldesmon 1 (h-caldesmon) immunopositivity, with increasing gestational age from 8 weeks to term. A previous pregnancy did not affect the amount of spiral artery smooth muscle. Comparison of pre-eclampsia and late miscarriage samples with controls of the appropriate gestational age demonstrated increased medial smooth muscle in pathological samples. This technique provides a simple, rapid, reproducible and inexpensive approach to quantitative assessment of spiral artery remodelling in normal and pathological human pregnancy, a process which although fundamental for successful pregnancy, is still incompletely understood.

Detection of alloantigens during preimplantation development and early trophoblast differentiation in the mouse by immunoperoxidase labeling.

An immunoperoxidase-labeling technique allowing visualization of antibody binding to the cell surface at the electron microscopical level has been employed an an analysis of H-2 and non-H-2 alloantigen expression on the early mouse embryo. The presence of non-H-2 antigenic determinants has been confirmed on eight-cell, morula, and blastocyst stages of development. Contrary to previous reports, however, low levels of H-2 antigen have also been detected on the blastocyst. This is the earliest stage at which H-2 has been shown to be expressed on the fertilized mouse egg and may reflect the greater resolution of the immunoperoxidase technique. Using two different models to study the critical peri-implantation stages, those of experimentally induced blastocyst activation and blastocyst outgrowth in vitro, it has been demonstrated that antigen loss occurs on the trophectoderm at the time of implantation, and that this is not necessarily dependent upon maternal influence. It is suggested that the loss may be an important factor in the prevention of maternal immune rejection during the establishment of the fetal allograft. The two major components of the early postimplantation conceptus display a striking differential in antigenic status. The embryonic sac shows a high degree of peroxidase labeling, while the ectoplacental cone trophoblast is unlabeled. These findings add support to the concept of antigenic neutrality of the early trophoblast and its role in the maintenance of a normal fetomaternal immunological equilibrium.

Effect of low oxygen concentrations on trophoblast-like cell line invasion.

The applicability of trophoblast-like cell lines to the study of trophoblast function has been widely debated. The present study investigated the effect of oxygen on the invasiveness, apoptosis, proliferation and secreted proteases of four different trophoblast cell lines; HTR-8/SVneo, SGHPL-4, JEG3 and JAR. All experiments were performed at 20% and 3% oxygen for 24, 48 and 72h. Immunostaining for integrins alpha1, alpha6 and beta3, cytokeratin 7 and HLA-G was used to determine the phenotype of the different cell lines. Invasion was assessed using the Matrigel invasion assay. Immunostaining for M30 and Ki67 determined levels of apoptosis and proliferation, respectively. Gelatin and casein/plasminogen zymography were performed on conditioned media to determine levels of secreted matrix metalloproteinase (MMP) 2 and MMP9 and urokinase plasminogen activator (uPA), respectively. None of the cell lines immunostained for all markers normally expressed by extravillous trophoblast cells. Invasiveness of HTR-8/SVneo and JEG3 cells cultured in 3% oxygen was increased after 24h but was inhibited by 72h in culture. Invasion of SGHPL-4 cells was inhibited after culture in 3% oxygen for 24h. Invasion by JAR cells was not affected by changes in oxygen concentration. The different cell lines also displayed different responses to culture period in 3% oxygen with respect to apoptosis, proliferation and secreted proteases. Care should be taken before results obtained using cell lines as a model for EVT are extrapolated to extravillous trophoblast cell behaviour in vivo.

Interleukin-17 expression in the human placenta.

Interleukin (IL)-17 is a proinflammatory cytokine with pleiotropic activities including inducing neovascularization and production of proangiogenic molecules. As pregnancy outcome depends on the balance of Th1-like/Th2-like cytokines and an increased blood supply to the fetoplacental unit, the expression of IL-17 mRNA and protein in human placental tissues was investigated. IL-17 mRNA was expressed by purified cytokeratin-positive term placental trophoblast cells, HLA-G+ extravillous trophoblast cells and placental macrophages (Hofbauer cells). IL-17 localized in both cyto- and syncytiotrophoblasts of normal term pregnancy, spontaneous miscarriage and in molar pregnancy. In spontaneous miscarriage and molar pregnancy extravillous trophoblast cells were consistently immunoreactive for IL-17. IL-17 expression in human placenta may play a key role in angiogenesis and/or immunoregulation in the establishment of pregnancy.

A technique for liver transplantation in the inbred mouse.

The technique of liver grafting to the kidney of inbred mice was used to characterise ultrastructurally liver rejection under controlled conditions. Electron microscopy showed that liver rejection was initiated by intimate contract between the hepatocytes and the infiltrating large lymphoid cells and brought about by means of narrow cytoplasmic processes extended from the latter. This was prevented by ATS-immunosuppressive treatment.

Differential expression of class II major histocompatibility complex and Thy 1.2 antigens on mouse decidua.

A differential expression of class II major histocompatibility complex (MHC) and Thy 1.2 antigens was detected on two morphologically distinct cell populations in short-term cultures of murine decidual tissue. Stromal type decidual cells expressed Thy 1.2, albeit transiently, and consistently lacked class II antigens. By contrast decidual macrophages expressed class II antigens and lacked Thy 1.2 antigens. Stromal type decidual cells, after culture in the presence of indomethacin, displayed no evidence of prostaglandin-mediated modulation of class II expression. These findings suggest that class II positive decidual macrophages are responsible for the antigen-presenting capacity of decidua.

Placental Fas and Fas ligand expression in normal early, term and molar pregnancy.

To clarify the Fas and Fas-ligand status of normal and molar trophoblast, the expression of Fas and FasL by placental trophoblast populations in partial and complete hydatidiform moles was compared with that in normal first trimester and term pregnancies using an avidin-biotin peroxidase technique on frozen and formalin-fixed paraffin-embedded placental tissues with both monoclonal and polyclonal antibodies. The TUNEL technique was used to detect apoptotic cells in the same tissues. The immunoreactivity for Fas and Fas-ligand was comparable with both monoclonal and polyclonal antibodies on frozen as well as paraffin-embedded sections. In normal early and molar pregnancy there was strong FasL expression by villous cytotrophoblast and syncytiotrophoblast. However, there were significant differences in FasL expression by trophoblast subpopulations in both early and term normal pregnancy and between the same trophoblast subpopulation at different gestations, with FasL staining generally being weaker at term. Strong FasL staining by cytotrophoblast cells in the distal parts of cell columns contrasted with unstained cytotrophoblast in the proximal part of columns. Distinct trophoblast subpopulations in partial hydatidiform mole also differentially expressed FasL with reduced FasL expression in proliferating syncytiotrophoblast. In contrast there was no differential FasL expression in complete hydatidiform mole, all trophoblast subpopulations strongly expressing FasL. Unlike the differential expression of FasL there were no differences in Fas expression by trophoblast populations in normal early or term placental tissues. Fas expression was reduced in villous cytotrophoblast at term. Differential expression of Fas by different trophoblast subpopulations was noted in partial and complete hydatidiform mole. In complete mole villous cytotrophoblast and syncytiotrophoblast stained strongly compared with proliferating trophoblast. Using TUNEL labelling apoptosis was rarely detected in placental trophoblast. Differential Fas and FasL expression by trophoblast subpopulations in normal and pathological pregnancy does not appear to be related to apoptosis of trophoblast.

Detection of human and murine trophoblast-specific antigens and an assessment of their species specificity.

The distribution and species specificity of human and mouse trophoblast-specific surface antigens detected by heterologous anti-mouse ectoplacental cone (anti-EPC) trophoblast and anti-human first-trimester trophoblast plasma membrane (anti-TrPM) antisera were assessed using several immunochemical and immunolabelling assays. The findings with immunofluorescence assays using anti-EPC antiserum on monolayer cultures indicate that the expression of mouse trophoblast-specific antigens is restricted to the major trophoblast components of the mouse placenta, as well as pre- and early post-implantation trophoblast, and does not occur on fetal, amnion or tumour tissues. The anti-EPC antiserum does not cross-react with human chorionic villous trophoblast. The human trophoblast-specific antigens also displayed species specificity. The anti-TrPM antiserum showed no evidence of cross-reactivity with rhesus monkey, rabbit, guinea pig or mouse trophoblast by immunodiffusion, crossed immunoelectrophoresis, immunofluorescence or immunoperoxidase labelling.

Ia antigen-bearing decidual cells and macrophages in cultures of mouse decidual tissue.

The ontogeny of Ia antigen expression on cells within the decidua basalis of the placenta and on decidual cells differentiating in vitro was investigated in the mouse. The results indicate that Ia antigen expression is temporarily restricted and can be detected in short-term cultures of decidual tissue only during the final third of gestation. The Ia antigen positive cells, which are non-phagocytic, non-specific esterase negative and lack Fc receptors, appear to be true decidual cells on the basis of their fine structural characteristics. In contrast, morphologically identical dendritic decidual cells arising from differentiation in vitro of endometrial cells do not express Ia antigens. A population of small rounded cells was also present in cultures of both decidual tissue and in vitro decidualized endometrial cells. These rounded cells possess the macrophage-like properties of Fc receptors, non-specific esterase and phagocytic activity. The possible function of these cell populations within the decidual tissue is discussed.

Morphological changes in cultured human periodontal ligament cells exposed to dental materials.

The cytotoxicity of a range of dental restorative cements was assessed by continuous observation of cultures with inverted microscopy and by light microscopic study of fixed preparations, using an in vitro model with cultured test cells derived from human periodontal ligament. The sequential morphological changes observed over a seven day period showed different degrees of cell loss and patterns of injury in response to different restorative materials, reflecting primarily either nuclear or cytoplasmic damage. Attempts at recovery were frequently identified as the culture period was extended and were characterized by recolonization of denuded areas of the culture well. It was concluded that differing dental cements damage cells through a variety of mechanisms and that the test cells exhibit differing degrees of susceptibility to injury. Assays based on short-term cultures may overestimate cytotoxicity by not allowing for cell recovery from reversible injury or repopulation of monolayers by proliferation of resistant cells.

Ia antigen expression on the developing mouse embryo and placenta.

The expression of I region controlled antigens on the mouse embryo and placenta has been investigated using immuno-peroxidase labelling and a mixed haemadsorption assay. The results indicate that Ia antigens are absent from selected pre- and post-implantation embryonic and trophoblastic tissues and from the trophoblast of the definitive placenta. In contrast, cells derived from the peri-placental maternal decidual tissues are Ia antigen-positive. The findings are discussed in relation to the allograft status of the conceptus and the identity and functional significance of the Ia-bearing maternal cells at the foeto-maternal interface.

Alloantigen presenting capacity of human decidual tissue.

The capacity of first trimester human decidua-derived cells to serve as accessory cells for the presentation of alloantigens to unprimed T lymphocytes was assessed using a mixed lymphocyte culture (MLC) between accessory cell-depleted responder and stimulator peripheral blood lymphocytes (d PBL). In all of the seven experiments performed decidua-derived cells achieved significant reconstitution of the lymphoproliferative response between autologous responder and stimulator dPBL. Reconstitution indices (RIs) ranged between 3.1 and 22.2 and were significant in all cases. In six out of the seven experiments the level of lymphoproliferation exceeded that between untreated responder PBLs co-cultured with stimulator d PBL. Substitution of recombinant interleukin 1 (rIL-1) alpha or beta for the decidua-derived cells resulted in significantly lower RIs than those achieved by the decidua-derived cells. These findings suggest that decidua-derived cells can serve as antigen presenting cells for the presentation of membrane-bound alloantigens in a primary lymphoproliferative response. This may have implications for maternal recognition of fetal antigens during first pregnancy.

The role of prostaglandins in the immunosuppressive effects of supernatants from adherent cells of murine decidual tissue.

Supernatants from short-term cultures of murine decidual tissue (DS) were assessed for their regulatory effects on T cell lymphoproliferation and cytotoxic T lymphocyte (CTL) activity. DS non-specifically suppressed antigen- and mitogen-induced lymphoproliferation, spontaneous thymocyte proliferation, the mixed lymphocyte reaction (MLR) and CTL generation, but had no effect on CTL lytic activity. The immunosuppressive activity was lost after dialysis (14 kDa cut off). Supernatants from indomethacin-treated decidual tissue cultures (indomethacin-DS) lacked suppressive activity in the MLR, mitogen and thymocyte proliferation assays. Indomethacin-DS also showed markedly reduced or no suppressive effects on CTL generation. These findings suggest that prostaglandin production by the decidual component of the placenta could play a role in materno-fetal cellular interactions by regulating T cell lymphoproliferative responses and CTL generation.

Antigen presenting capacity of murine decidual tissue in vivo.

Antigen presenting cells (APC) within murine decidual tissue in vivo have been shown to process the soluble antigen ovalbumin after intravenous administration and to present it in a form recognizable by immune T lymphocytes. In vivo antigen pulsed decidual APC stimulated T cell proliferation as efficiently as splenic APC and in an MHC restricted manner. In addition, anti-class II antibody plus complement treatment significantly reduced decidual antigen presenting capacity in vitro. These findings show that class II positive cells within the decidua can present antigen effectively in vivo and may therefore serve as APC for the presentation of fetal antigens to the maternal immune system during pregnancy.

Antigen presenting capacity of human decidua: no evidence for human decidual antigen presenting cell mediated immunoregulation.

Decidual antigen presenting cell (APC) mediated maternal immunoregulation has been reported. In the present study the ability of villous chorion as well as fetal cell pulsed early human pregnancy decidual APC to generate selectively antigen non-specific and MHC class II unrestricted CD8 positive T suppressor cells was reassessed in view of the fact that placental trophoblast, unlike the fetus, constitutes the fetal tissue of major contact at the maternal-fetal interface. Neither fetal cell nor villous chorion pulsed decidual APC generated maternal T cells with the ability to immunosuppress PHA-, Con A- and PWM-induced autologous or allogeneic lymphoproliferation. In only 2 out of 45 assays with villous chorion pulsed decidual APC was significant inhibition of mitogen induced lymphoproliferation detected and on no occasion with fetal cell pulsed decidual APC. No change in CD4/CD8 ratio of the maternal putative regulatory cells was detected by FACS analysis compared with control cultures. These findings suggest that decidual APC mediated immunoregulation plays no role in directing the maternal immune response.

Detection of macrophages and the characterization of Fc receptor-bearing cells in the mouse decidua, placenta and yolk sac using the macrophage-specific monoclonal antibody F4/80.

A mouse macrophage-specific rat monoclonal antibody, F4/80, has been used to detect directly macrophages in short term cultures of mouse decidua, fetal placenta and yolk sac and to investigate the identity of Fc receptor (FcR) bearing cells in these tissues. We find that a significant proportion of FcR positive cells in decidual, placental and yolk sac tissues are macrophages as defined by the expression of the macrophage marker, F4/80 antigen. Macrophages may act as immunocompetent cells near to the maternal-fetal interface and play a significant role in the mechanism of the transfer of passive immunity from mother to fetus across the mouse yolk sac.

Decidual T lymphocyte activation in hydatidiform mole.

AIM: To quantify and compare decidual leucocyte subpopulations in complete and partial hydatidiform molar pregnancy with those in normal early pregnancy. METHODS: Decidual leucocytes were characterised using an avidin-biotin technique and a panel of monoclonal antibodies in formalin fixed, paraffin embedded decidual tissues from 10 normal first trimester pregnancy terminations and from 13 partial and 13 complete hydatidiform moles. Immunostained cells were fully quantitated and differences between subject groups were analysed using the Mann-Whitney test. T lymphocyte populations were further characterised using double immunohistochemical labelling. RESULTS: The numbers and percentages of CD3+ and CD4+ T cells were significantly increased in complete hydatidiform moles compared with partial moles and normal early pregnancy decidua. No differences were found in the number or percentage of CD8+ T cells. The CD8+ to CD4+ T cell ratio decreased significantly in complete mole compared with partial mole and normal decidua. The numbers and percentages of CD45RO+ cells increased significantly in both partial and complete hydatidiform mole compared with normal first trimester decidua. Double labelling confirmed that 50% of CD3+ T cells in complete and partial molar pregnancy coexpressed CD45RO, compared with 30% in normal pregnancy. Other leucocyte populations (eGLs, macrophages, B cells, and classical natural killer cells) did not differ between complete and partial mole and normal pregnancy decidua. CONCLUSIONS: The presence of an increased population of activated decidual CD45RO+ T cells in complete and partial molar pregnancy suggests altered maternal immune responses against molar trophoblast.

Immunohistochemical characterization of stromal leukocytes in ovarian endometriosis: comparison of eutopic and ectopic endometrium with normal endometrium.

OBJECTIVE: To compare stromal leukocyte subpopulations in different phases of the menstrual cycle in eutopic and ectopic endometrium from women with ovarian endometriosis and in control endometrium. DESIGN: Retrospective immunohistochemical study. SETTING: Department of Pathology, Royal Victoria Infirmary, Newcastle-upon-Tyne, United Kingdom. PATIENTS: Paraffin-embedded tissue blocks from 30 patients with endometriosis and 30 control blocks from patients undergoing hysterectomy for nonendometrial pathology were retrieved from archive files. MAIN OUTCOME MEASURE: Quantitative assessment of defined stromal leukocyte subpopulations in eutopic, ectopic and control endometrium at different stages of the menstrual cycle. RESULTS: In the proliferative and early secretory phases, ectopic endometrium contained elevated numbers of CD45+, CD3+, and CD43+ cells but reduced percentages of CD68+ macrophages. The proportions of granulated cells were reduced in ectopic endometrium throughout the cycle. No differences were noted between eutopic endometrium from women with endometriosis and control endometrium. CONCLUSION: Differences between eutopic and ectopic leukocyte subpopulations with the exception of large granular lymphocytes may be due to the lack of cyclicity demonstrated by endometriotic lesions.

Antigen-presenting capacity of mouse decidual tissue and placenta.

The antigen-presenting capacity of cells within mouse decidual tissue and the fetal placenta was assessed using the antigen-presenting cell assay to measure immune T cell proliferation stimulated by a second presentation of antigen. Cells within decidual tissue bind and present the soluble antigen dinitrophenylated ovalbumin in an antigen-specific and major histocompatibility complex-restricted manner. Antigen-presenting cells (APCs) were detected in decidual tissue from day 8 until day 15 of pregnancy, in deciduoma and in fetal placental tissues. These findings show that cells within decidual tissue may serve as APCs with the ability to process and present fetal antigens to the maternal immune system and may therefore determine the direction of the maternal immune response of pregnancy.