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Biomedical subjects

G Dohr

Publications and source records attributed to G Dohr.

At least 19 recordsLinked to original sources

Apoptosis affects integration frequency: adult stem cells injected in blastocysts show high caspase-3 activity.

Chimeric organisms are commonly generated by injecting stem cells into blastocysts. Embryonic stem cells injected into the blastocoel cavity participate in the further development of the embryo. Adult stem cells have also been used in injection experiments to study their potential plasticity. In this study we focused on the early fate of injected human adult hematopoietic stem cells (HSCs). HSCs were followed immunohistochemically 1-19 h after injection into murine blastocysts. We found that they only rarely attached and integrated into the blastocysts. The high rate of loss of injected cells after prolonged in vitro culture of the chimeras can be explained by apoptosis. Our findings are consistent with previous studies reporting a low rate of integration of adult cells injected to produce chimeric embryos, but this is the first demonstration that the low efficiency of adult stem cell injections into blastocysts is influenced by apoptosis.

Adult↗

Insulin control of placental gene expression shifts from mother to foetus over the course of pregnancy.

AIMS/HYPOTHESIS: The human placenta is a complex organ situated at the interface between mother and foetus that separates maternal from foetal blood. The placental surfaces exposed to the two bloodstreams are different, i.e. trophoblasts and endothelial cells are in contact with the maternal and foetal circulation, respectively. Both cell types produce high insulin receptor levels. The aim of the present study was to test the hypothesis that spatio-temporal changes in insulin receptor expression in trophoblasts from first trimester to the endothelium at term shift the control of insulin-dependent processes from mother to foetus. METHODS: Global microarray analysis of primary trophoblasts from first trimester and term human placentas and endothelial cells from term human placentas cultured under hyperinsulinaemic and control conditions identified different sets of regulated genes in trophoblasts and endothelial cells. RESULTS: Insulin effects on placental gene expression underwent developmental changes from trophoblasts in the first trimester to endothelial cells at term that were paralleled by changes in levels of activated insulin receptors. The changes in gene regulation were both quantitative (i.e. magnitude of effect) and qualitative (i.e. specific genes affected and direction of regulation). CONCLUSIONS/INTERPRETATION: This spatio-temporal shift in insulin sensitivity throughout pregnancy allows maternal and foetal insulin to regulate different processes within the placenta at different gestational stages, facilitated by compartmentalisation of the insulin response. Thus, by altering the levels and function of insulin receptors in space and time, control of insulin-dependent processes in the human placenta will change from mother to foetus throughout gestation. This will be of particular interest in conditions associated with altered maternal or foetal insulin levels, i.e. diabetes mellitus or intrauterine growth restriction.

Blood Circulation↗

The soluble pool of HLA-G produced by human trophoblasts does not include detectable levels of the intron 4-containing HLA-G5 and HLA-G6 isoforms.

In the context of implantation and pregnancy, several immunomodulating functions have been attributed to the different HLA-G isoforms. Increasing attention is now being addressed to the actively secreted soluble forms, because they might have a systemic function or could be useful as diagnostic tools. However, the cellular source of secretion, even during pregnancy, where HLA-G expression level is known to be highest, is still under debate. To elucidate the conflicting results, we investigated the isoform distribution in human first trimester and term placentas in situ and in vitro. Results obtained by applying immunohistochemistry, western blot, enzyme-linked immunosorbent assay (ELISA) and RT-PCR show that (1) all of the alpha1 domain-containing HLA-G isoforms are restrictedly expressed in the extravillous cytotrophoblasts (EVCTs) and very few first-trimester syncytiotrophoblasts, which directly cover cell columns, whereas mesenchymal cells of the villous chorion do not express HLA-G; (2) as demonstrated in western blots, trophoblasts express only the HLA-G1 isoform; (3) HLA-G5 and -G6 transcripts could be detected in human term placenta and isolated first-trimester trophoblasts but levels are extremely low; and (4) conditioned media of primary first-trimester trophoblasts, and the chorion laeve-derived trophoblastic cell line AC1-M59 do contain HLA-G1 fragments shed from the cell surface. Our data provide substantial evidence that none of the intron 4-containing isoforms, the so-called actively secreted, soluble HLA-G5 or -G6, are produced by human trophoblasts in situ or in vitro.

Alternative Splicing↗

Paraffin-embedded manipulated blastocysts: a tool to demonstrate stem cell plasticity?

One of the big question marks in current stem cell research is whether there is true plasticity of adult progenitor cells (APC) or if cell fusion is the principle source of the supposed plasticity. The generation of chimeras by injecting adult progenitor cells into blastocysts is not new. This paper describes an efficient embedding technique for murine blastocysts injected with human APC. This method could help in establishing a novel tool to analyse the process of plasticity, if it truly exists. If this is the case, this technology could be of great help to characterize surface markers of stem cells in great detail. On the other hand, fusion of cells could also be investigated. A system of embedding blastocysts was set up using paraffin for further analysis by means of light microscopy and immunohistochemistry. The embedding of the chimaeras consists of fixing them first with paraformaldehyde in phosphate-buffered saline (PFA/PBS), embedding them in gelatine, fixing the gelatine block with PFA/PBS and finally fixing the gelatine block in a Petri dish by embedding it in paraffin. Using this protocol, the morphology of the blastocysts is well preserved.

Animals↗

Expression of insulin, insulin-like growth factor I and glucocorticoid receptor in rat uterus and embryo during decidualization, implantation and organogenesis.

The significance of insulin, insulin-like growth factor I (IGF-I) and glucocorticoids to the early mammalian embryo is clear in that they are key regulators of both mitogenic and metabolic effects during development. In the present study, the temporal sequence of expression of the respective receptor proteins was investigated for the first time in the developing rat utero-embryonic unit between conception and day 12 of gestation using immunocytochemistry. Insulin, IGF-I and glucocorticoid receptor were expressed in embryonic tissues after the start of implantation, and were co-localized in the primary ectoderm, extraembryonic ectoderm as well as in the ectoplacental cone. The parietal endoderm was devoid of glucocorticoid receptor staining, whereas IGF-I receptor was absent in visceral endoderm. After completion of basic organogenesis, the neural tube, notochord, otic placode, Wolffian duct, mesonephros and intestinal tube expressed insulin, IGF-I and glucocorticoid receptor. The glucocorticoid receptor was not expressed in heart tube and dorsal aortae. Considerable amounts of insulin receptor were detected in trophoblast-derived giant cells. In the uterus, luminal epithelium, endometrial stromal and myometrial smooth muscle cells immunoreacted with antisera against insulin, IGF-I and glucocorticoid receptor. Endometrial glands remained negative for the glucocorticoid receptor throughout the gestational period investigated. Uterine hormone receptor expression reached a peak at days 4 and 5 of gestation in endometrial stromal cells and decidua, respectively. In conclusion, the demonstrated ontogenetic pattern of insulin, IGF-I and glucocorticoid receptor expression indicates the potential sites of biological action of the respective ligands, providing supportive evidence for their critical importance during the course of embryogenesis in rats.

Animals↗

Localization of indoleamine 2,3-dioxygenase in human female reproductive organs and the placenta.

Indoleamine 2,3-dioxygenase (IDO) has been implicated in regulation of feto-maternal tolerance and protection against intracellular and extracellular pathogens. We have studied the expression of IDO in the human female reproductive tract and the placenta by immunohistochemistry. Endometrial glandular and surface epithelial cells showed increasing IDO expression during the course of the menstrual cycle. In term placenta, IDO was irregularly localized to the mesenchymal core and found in isolated areas of the syncytiotrophoblast. In first trimester pregnancy, IDO was not present in placental villi, but was present in glandular epithelium of the decidua, and there were distinctly positive cells scattered in the connective tissue, sometimes in conjunction with lymphoid aggregates. The endothelium of spiral arteries and of capillaries showed some, albeit no generalized, reactivity. IDO was also present in the epithelium of cervical glands and of Fallopian tubes. Specificity of antibody binding was confirmed by Western blot analysis. IDO mRNA was detected in first trimester decidua as determined by RT-PCR. IDO is secreted, as determined by analysis of cervical mucus by high pressure liquid chromatography for the presence of the tryptophan metabolite L-kynurenine, indicating IDO activity. Our results support the concept of IDO providing a mechanism of innate immunity protecting against ascending infections in the female reproductive tract.

Female↗

Prooxidant activity of melatonin promotes fas-induced cell death in human leukemic Jurkat cells.

The antioxidant activity of melatonin (MEL) has been considered to constitute part of its physiological as well as pharmacological effects. However, as described herein we found a profound prooxidant activity of micro- to millimolar concentrations of MEL in the human leukemic Jurkat cell line. This prooxidant effect was increased in glutathione-depleted cells and counteracted by antioxidants. As a consequence MEL promoted fas-induced cell death. These data therefore indicate that MEL may be a modulator of the cellular redox status, but does not necessarily act as an intracellular antioxidant.

Antioxidants↗

Myeloperoxidase-dependent generation of hypochlorite-modified proteins in human placental tissues during normal pregnancy.

Myeloperoxidase (MPO), which is released from cytoplasmic granules of activated phagocytes by a degranulation process, reacts with H(2)O(2) (generated during the oxidative burst) and chloride ions to generate hypochlorous acid/hypochlorite (HOCl/OCl(-)). HOCl, a strong oxidant, in turn reacts with proteins to form HOCl-modified proteins. The presence of these cytotoxic chloramines during inflammatory conditions, eg, atherosclerosis and glomerular and tubulointerstitial injury, suggested that chloramines are powerful oxidants that can have profound biologic effects. In the present study, immunoreactive MPO was identified in fetal membranes and the basal plate and in maternal and fetal blood cells of human placental tissues. Monocytes/macrophages represent the major cell source for MPO in human placental tissues. Immunohistochemical findings revealed that HOCl-modified proteins are present in normal human term placenta but not during the first trimester of pregnancy (Weeks 7 to 12). HOCl-modified proteins were localized in areas formed by fetally derived cells as well as maternal decidual tissues, ie, areas where fetal extravillous trophoblast cells invade the maternal tissue and stimulate the maternal immune system. HOCl-modified proteins, products of the MPO-H(2)O(2)-chloride system in vivo, were not present intracellularly, but immunoreactivity for HOCl-modified proteins was cell-associated and/or present in the extracellular matrix. Extravillous trophoblast cells, which may also exert phagocytic activities, showed no intracellular immunoreactivity for MPO or HOCl-modified proteins. The present findings indicate that the generation of HOCl-modified proteins during normal pregnancy is a physiologic rather than a pathophysiologic process.

Chorionic Villi↗

Uterine natural killer cells in a three-dimensional tissue culture model to study trophoblast invasion.

The high numbers of CD56(+) cells with natural killer (NK) functions present in the uterine mucosa during the late secretory phase of the menstrual cycle and during early pregnancy have been considered to be implicated in implantation and in the regulation of trophoblast invasion. A three-dimensional organ culture model was used to study the interactions of these uterine NK cells with Jeg-3 and BeWo choriocarcinoma cells as a model of the invasive trophoblast. For this purpose, fragments of endometrial and decidual tissue were put in close contact with multicellular spheroids of choriocarcinoma cells in small silicon funnels. After the formation of stable contacts, the confrontation cultures were transferred to spinner flasks, cultivated for up to 6 days, and prepared for immunohistochemistry. During 2 days of cocultivation, the first cells started to move forward into the stromal component of the confrontation culture as demonstrated by staining of the choriocarcinoma cells using anti-human cytokeratin. Invasion advanced until, after a total of 6 days, some choriocarcinoma cells had already penetrated deeply into the host tissue. After a cultivation period of 1 week, both the endometrial and decidual tissue fragments still contained several CD56(+) uterine NK cells, and some of them expressed the proliferation-associated marker Ki-67 without any exogenous activation. A few CD56(+) cells were found directly at the invasion front, as well as between the choriocarcinoma cells. These cells also contained the cytolytic granule protein perforin indicating a migration of NK cells with cytolytic potential toward the potentially invasive cells. In conclusion, this human system closely resembles the in vivo conditions during trophoblast invasion and provides an appropriate in vitro model for studying dynamic processes involving various cell types during trophoblast invasion at the experimental level. Moreover, it enables us to study the effects of cytokines and growth factors that possibly regulate trophoblast invasion.

Choriocarcinoma↗

Differential mitogenic responses of human macrovascular and microvascular endothelial cells to cytokines underline their phenotypic heterogeneity.

A variety of growth factors promote the complex multistep process of angiogenesis. The mitogenic activity of vascular endothelial growth factors (VEGFs) and placental growth factors (PlGFs), known as cytokines acting predominantly on endothelial cells, was tested on human umbilical vein endothelial cells (HUVEC) and microvascular endothelial cells (MIEC) and compared with the potency of the universally acting basic fibroblast growth factor (FGF-2). The cells were seeded at different cell numbers and incubated with various doses of growth factors for a period of 24-72 h in culture medium +/- serum. Proliferation was determined by measuring the optical density after staining the cells with the tetrazolium salt WST-1. VEGF121 and VEGF165 increased the number of HUVEC and MIEC at low and high seeding densities various doses and incubation times. The efficiency of FGF-2 was less pronounced at high seeding densities of the cells under serum-free conditions. PlGF-1 and PlGF-2 stimulated mitogenesis on HUVEC only at low cell numbers and after a short incubation time by 125 +/- 3% and 102 +/- 5% (P < 0.001), respectively. Longer incubation times with the lower seeding density in the absence of FCS did not induce a significant stimulatory effect of the PlGFs. MIEC responded stronger to all growth factors. In particular under serum free conditions, PlGF-1 and PlGF-2 effectively stimulated cell proliferation by 247 +/- 54% (P < 0.01) and 288 +/- 40% (P < 0.05) at low cell numbers, and by 81 +/- 13% (P < 0.05) and 49 +/- 13% (P < 0.01), respectively, at high cell numbers. The addition of fetal calf serum caused a reduced proliferative response of all growth factors on both cell types related to the controls. In conclusion, MIEC and HUVEC differ in their proliferative response to VEGFs, PlGFs and FGF-2.

Capillaries↗

Role of various tissues in apo(a) fragmentation and excretion of fragments by the kidney.

BACKGROUND: Lipoprotein(a) [Lp(a)] is an atherothrombotic plasma lipoprotein with unknown function. Little is known about the catabolism of this lipoprotein, in particular the steps related to apolipoprotein(a) [apo(a)] fragmentation and excretion by the kidney. MATERIAL AND METHODS: High plasma levels (up to 9 mg dL(-1)) of the N-terminal fragment of apo(a) were expressed in mice by adenovirus mediated gene transfer. Plasma of such N-apo(a) mice was injected into acceptor mice and the fragmentation and urinary secretion of N-apo(a) were followed by immunochemical techniques. RESULTS: Mice transduced with N-Ad expressed apo(a)-fragments with 3-11 kringle-IV (KIV) repeats. Injection of N-apo(a)-plasma from donor mice into acceptor mice resulted in fragmentation of N-apo(a)s with 3-11 KIVs yielding smaller peptides down to 2 KIVs. Secretion of N-apo(a)-fragments with 2 to maximally 6 KIVs into urine occurred as early as 2 min after injection. Immunohistochemical studies of kidney suggested filtration as a mechanism of apo(a)-fragment excretion. When N-apo(a) was incubated in vitro with various tissues from perfused mice, skeletal muscle and kidney followed by liver and spleen contributed to fragmentation. Tissues from unperfused organs, or the addition of normal mouse plasma, caused marked reduction in N-apo(a) fragmentation. EDTA, and not aprotinin or leupeptin, prevented apo(a) cleavage. CONCLUSION: Here we provide evidence that apo(a) is cleaved by metalloproteinases located on skeletal muscle, kidney and other organs. Small apo(a)-fragments up to a size of 6 KIVs are excreted into urine, yet a major portion of apo(a) fragments is removed from circulation extrarenally.

Adenoviridae↗

From maternal glucose to fetal glycogen: expression of key regulators in the human placenta.

The present study investigated the expression of glycogenin, the protein primer for glycogen synthesis, and the high affinity glucose transporter isoform GLUT3 as a further potential regulator of cellular glycogen metabolism, in first trimester and term human placenta using immunohistochemistry and Western blotting. At term, glycogenin was most abundant in the endothelium of fetal vessels. Trophoblast as well as basal decidual cells were moderately stained. The glycogenin distribution pattern in first trimester placentae resembled that at term, but reactivity was generally less intense. Extravillous trophoblast and villous cytotrophoblast were the major sites of GLUT3 expression. Endothelial cells were also strongly labelled with the GLUT3 antiserum. Western blotting identified both free and glucosylated glycogenin, as well as a 48 kDa band reacting with GLUT3 antiserum in placental villous tissue. Glycogenin immunoreactivity remained unaffected by amylolytic glycogen digestion, although preceding electron microscopical examination demonstrated the presence of glycogen. These data may indicate that placental glycogenin can be recycled from the immature glycogen or that it is located on the surface of the glycogen molecule. In conclusion, the co-expression of glycogenin with GLUT3 might enable glycogen-storing cells to exchange glucose quite effectively according to prevailing metabolic demands of glycogen synthesis or degradation.

Cells, Cultured↗

Glucose transporter expression in rat embryo and uterus during decidualization, implantation, and early postimplantation.

Efficient transfer of glucose from the mother to the embryonic compartment is crucial to sustain the survival and normal development of the embryo in utero, because the embryo's production of this primary substrate for oxidative metabolism is minimal. In the present study, the temporal sequence of expression of the sodium-independent facilitative glucose transporter isoforms GLUTs 1, 3, 4, and 5 was investigated in the developing rat uteroembryonic unit between conception and Gestational Day 8 using immunohistochemistry. The GLUTs 1, 3, and 4 were expressed in the embryonic tissues after the start of implantation, being colocalized in the parietal endoderm, visceral endoderm, primary ectoderm, extraembryonic ectoderm, and the ectoplacental cone. In the uterus, a faint GLUT1 labeling emerged, but not until Gestational Day 3, in the luminal epithelium, endometrial stroma, and decidual cells. The intensity of GLUT1 staining increased in the latter population with progressing decidualization. Endometrial glands and myometrial smooth muscle cells stained neither for GLUT1 nor for GLUT3 until postimplantation. During all developmental stages examined, GLUT4 was visualized throughout the pregnant rat uterus, as was GLUT3 (with the above-mentioned exceptions). The density of GLUT5 was generally less than the sensitivity of the immunohistochemical detection method in all tissues investigated. In conclusion, the data point to a significant expression of the high-affinity glucose transporters GLUTs 1, 3, and 4 in the rat uteroembryonic unit, providing supportive evidence for an important role of facilitative glucose diffusion during peri-implantation development.

Animals↗

HLA Class I protein expression in the human placenta.

The maternal tolerance to the semiallogeneic fetus is still a central theme in reproductive immunology. During placentation, fetally-derived, genetically dissimilar tissue and cells come into close contact with maternal tissue and cells, thus forming the so-called feto-maternal interface. The most extensive contact between fetally-derived and maternal blood cells is formed by the villous trophoblastic barrier, where the syncytiotrophoblast surface permanently floats in maternal blood. Further contact is made by some extravillous cytotrophoblast cells, either located at villous tips, in so-called cell islands, or the endovascular trophoblast population within the uteroplacental spiral arteries. The third contact zone is the so-called junctional zone within the decidua where the invading extravillous trophoblast cells encounter all maternal tissue leukocytes, which are mainly NK cells, macrophages and T cells; this junctional zone extends at the edge of the placenta to the amnio-chorionic membranes where the chorionic laeve trophoblast has intimate contact with decidua tissue. It is worth mentioning that evidence has shown that even in healthy pregnancies fetal and maternal lymphoid cells are able to transgress the trophoblastic barrier, which, anatomically, seems completely impermeable. Because of this intimate contact of foreign cells to the foreign immune system it is important to define the antigenic status of the placental cells, in particular with respect to antigens of the Major Histocompatibility complex. The role of the highly polymorphic classical class I molecules HLA -A, -B, -C, which are expressed on almost all somatic cells, is the induction of a specific immune response by presenting peptide antigens to T cells. In contrast, the non-classical HLA class I molecules HLA-G and HLA-E are thought to be involved in the induction of immune tolerance by acting as ligands for inhibitory receptors present on NK cells and macrophages. The non-classical HLA-E is also expressed ubiquitously, but HLA-G expression is characterized by a unique tissue expression mainly in the human placenta. A further feature of HLA-G is that its mRNA has undergone alternative splicing, resulting in at least 6 different isoforms, encoding different proteins: 4 membrane-bound and 2 soluble forms, which could simultaneously maintain different functions depending on their molecular structure. In our immunohistochemical study we investigated the expression of classical and non-classical HLA class I proteins in human placenta using various mAbs, which were kindly provided by the groups of A. Ziegler, D. Geraghty, O. Genbacev, MT. McMaster, A. King, YW. Loke and Ph. Le Bouteiller. For HLA-A,-B detection we used the antibody LA45; for detection of HLA-C,-B mAbs Tü149 and HC10. HLA-C expression alone was detected with mAb L31. HLA-G expression was studied using antibodies 4H84, G233, 87G, 16G1 and BFL.1. For HLA-E staining we used antibodies DT9 and V16. The classical HLA class I proteins are expressed in all non-trophoblastic cells including the fetal and maternal cells. Comparison of HLA-A and HLA-B staining intensities within the villous stroma indicates that during first trimester of pregnancy the fetal HLA-B proteins are expressed before HLA-A appears. Among the trophoblast populations, the syncytiotrophoblast does not show any HLA class I staining, but the extravillous cells express high amounts of HLA-G together with HLA-C. King and co-workers have shown recently, using methods other than immunohistochemistry, that first trimester extravillous trophoblast cells are also likely to express HLA-E. By contrast we did not detect HLA-E in any trophoblasts with antibodies DT9 and V16. There is still an ongoing and also controversial discussion about which kinds of cells in the placenta, other than extravillous trophoblast, express which kind of the HLA-G isoforms. Depending on the antibodies and the different immunohistochemical techniques used, different results have been described: Antibody 16G1 specific for soluble HLA-G labels syncytiotrophoblast, antibody BFL.1 endothelial cells of chorionic fetal blood vessels and antibody 87G Hofbauer cells. All these HLA-G labelings, apart from extravillous trophoblasts, are in complete contrast to the reaction pattern (merely extravillous trophoblast) given by antibody 4H84, which recognizes all HLA-G isoforms -including the soluble ones- through an epitope located on the a 1-domain of HLA-G. Future studies employing isoform-specific antibodies, which are not yet available for all of the possible isoforms, will elucidate the function and expression pattern of HLA-G in the human placenta.

Antibody Specificity↗

Hyperglycaemia-induced subcellular redistribution of GLUT1 glucose transporters in cultured human term placental trophoblast cells.

AIMS/HYPOTHESIS: We have recently shown that hyperglycaemia down-regulates the GLUT1 glucose transport system of term placental trophoblast. The reduction in GLUT1 protein alone was, however, not sufficient to explain the decrease in net glucose uptake, suggesting additional mechanisms. Therefore, we hypothesised that hyperglycaemia in vitro leads to a GLUT1 translocation from the trophoblast surface to intracellular sites. METHODS: This was tested in our study by determining the subcellular distribution of GLUT1 in human term placental trophoblast (n = 5 placentas) cultured for 48 h with 5 compared with 25 mmol/l D-glucose in vitro using immunogold labelling. RESULTS: Electron microscopic examination of cell profiles showed that 73% of total GLUT1 molecules reside in the trophoblast plasma membrane under basal conditions. The reduced GLUT1 expression (-20%; p < 0.05) after culture of the cells with 25 mmol/l glucose was accompanied by an internalisation of plasma membrane GLUT1, resulting in a loss of 40% (p < 0.05) in cell surface transporter labelling. Western blotting identified a characteristically broad band between 55-65 kDa, confirming the specificity of the GLUT1 antiserum. CONCLUSION/INTERPRETATION: We postulate that in addition to down-regulating human GLUT1 protein concentrations, glucose exerts its autoregulatory effect on hexose transport in term placental trophoblast by altering GLUT1 partitioning between the plasma membrane and intracellular sites in favour of the latter.

Cell Survival↗

Expression of Fas-ligand in first trimester and term human placental villi.

The expression of Fas-ligand (FasL) on trophoblast cells is thought to play a role in immune regulation during human pregnancy. However, there are some discrepancies in the published data concerning the cell types expressing FasL in the placental villi. Therefore, we examined the expression of FasL on cryosections of first trimester and term placental tissue with three different anti-sera against FasL, which are in common use. By immunohistochemistry, all three anti-sera principally gave the same staining result. In the first trimester of pregnancy, villous cytotrophoblast cells underlying the syncytium, as well as all extravillous trophoblast cells of cell columns and cell islands, gave a clear, mainly membrane-located staining, whereas the syncytiotrophoblast, which forms the borderline to the maternal blood flow, only gave a spot-like reaction in distinct areas. The same result was obtained with term placental villi; however, in this tissue, the staining of the villous cytotrophoblast cells was less pronounced. From our results, we suggest that in placental villi, an important role of FasL in immune regulation is not very conclusive because this molecule is mainly expressed on trophoblast with no access to maternal blood or tissue. This is in contrast to the uterine part of the placenta, where FasL expressing trophoblast cells are in close contact with apoptotic maternal leukocytes.

Chorionic Villi↗

Reaction patterns of monoclonal antibodies to HLA-G in human tissues and on cell lines: a comparative study.

We compared the immunohistochemical reaction patterns of HLA-G-specific antibodies 87G, 4H84, G233, 16G1, and BFL.1 on human placentas under three different preparative conditions and on cryosections of other human tissues. Human and murine cell lines, either naturally expressing or transfected with HLA-G, were analyzed for their reaction patterns by immunocytochemistry and flow cytometry. Antibodies HCA2, TP25.99, W6/32 to classical HLA class I, anti-beta(2)-m and various non-HLA-G expressing cell lines were used as controls. The binding ability of the antibodies depends on the histotechnical procedure used. 4H84 and HCA2 bind to HLA-G despite aldehyde fixation and also paraffin embedding. 87G does not bind HLA-G in studies involving fixation with aldehydes. G233 labels HLA-G in aldehyde fixed but not paraffin embedded tissues. By immunocytochemistry HLA-G2 is merely detected with antibodies 4H84 and HCA2. MAb 16G1 binds to HLA-Gsol transfected cell lines only. The HLA-G specificity of mAb BFL.1 was considered as doubtful because it failed to react with most of the HLA-G transfected cell lines. Binding of 87G to the surface of monocytes or U-937 cells stimulated with IFN-gamma and GM-CSF is an Fc-receptor mediated phenomenon.

Animals↗