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Apoptosis (programmed cell death) is an active process that plays a critical role in multiple biologic processes from embryologic development, to lymphocyte development and selection, and homeostasis. The two major mechanisms of cell death are referred to as the intrinsic and extrinsic pathways. These pathways lead to a cascade of events that ultimately converge to the activation of an effector enzyme, caspase-3. Caspase-3 is a cysteine protease with aspartic specificity and a well-characterized effector of apoptosis or programmed cell death signaling. The pro-form of caspase-3 (p32 caspase-3) is sequestered as a zymogen, where upon proteolysis at a conserved DEVD sequence, is converted to the active (p17 caspase-3) enzyme capable of disassembling the cell. Cell death can become disregulated under various conditions and multiple disease states (e.g., viral infection, carcinogenesis, and metastasis). Sensitive and reproducible detection of active caspase-3 is critical to advance the understanding of cellular functions and multiple pathologies of various etiologies. Here, we provide two simple and reproducible methods to measure active caspase-3 in multiple cell types and conditions using a flow cytometric-based analysis. [ABSTRACT FROM AUTHOR]

Most authors currently quantify the frequency of apoptotic cells in a given phenotypically defined population after calculating the apoptotic index (AI), that is, the percentage of apoptotic cells displaying a specific lineage antigen (LAg) within a population of cells that remain unfragmented and retain the expression of the LAg. However, this approach has two major limitations. First, apoptotic cells fragment into apoptotic bodies that later disintegrate. Second, apoptotic cells frequently lose, partially or even completely, the cell surface expression of the LAg used for the identification of specific cell subsets. This chapter will describe a flow cytometry method to calculate the apoptotic rate (AR) that takes into account both cell fragmentation and loss of LAg expression on measurement of apoptosis using flow cytometry ratiometric cell enumeration that emerges as a more accurate method of measurement of the occurrence of apoptosis in normal and tumoral cell cultures. [ABSTRACT FROM AUTHOR]

Gene expression analysis provides an insight into the unique and defining biomolecular characteristics of a given cell type. However, heterogeneous cellular compositions hinder gene analysis studies from most tissue samples. The laser microdissection (LMD) technique allows for the unambiguous isolation of a desired cell population. However, preserving RNA integrity can be challenging because of the deliberately limited amount of starting material, sometimes as little as a single cell. General laboratory procedures for reducing ribonuclease (RNase) activity, both in reagents and in the laboratory environment, are required for successful downstream RNA isolation and quantitation. Quality RNA can be extracted from sections made from flash-frozen and paraffin-embedded tissue. The standard histological stains such as hematoxylin and eosin (H&E), or toluidine blue, can provide visualization of the cells of interest. Following LMD, validation of RNA integrity should precede downstream analysis. [ABSTRACT FROM AUTHOR]

The extrinsic apoptosis pathway is activated when certain members of the tumor necrosis factor (TNF) receptor superfamily (TNFRSF) are oligomerized by their cognate ligands that are members of the TNF superfamily (TNFSF). The apoptosis-inducing capacity of a member of the TNFRSF relies on the presence of a death domain (DD) in the intracellular portion of the receptor protein. Such receptors are also referred to as death receptors. Binding of a TNFSF ligand to a TNFRSF receptor that is expressed on the surface of a cell results in the formation of a receptor proximal protein complex. This protein complex is the platform for further signaling events within the cell. In case of death receptors like TNF-related apoptosis-inducing ligand receptor 1 (TRAIL-R1/DR4), TRAIL-R2 (KILLER/APO-2/DR5/TRICK), CD95 (Fas, APO-1), or TNF receptor 1 (TNF-R1), this complex is termed death-inducing signaling complex (DISC). The compositions of the various DISCs have been intensively studied in the last 12 years. For the CD95 and the TRAIL-R1/R2 DISCs, it is now clear that the adaptor protein Fas-associated DD protein (FADD) forms part of these complexes and is necessary for recruitment of the pro-apoptotic signaling molecules caspase-8 and caspase-10. Recruitment of these proteases allows for their activation at the DISC and subsequent induction of apoptosis. The caspase-8 homologous cellular FLICE-like inhibitory protein (cFLIP) can also be recruited to the DISC. cFLIP acts as an anti-apoptotic regulator by interfering with activation of caspases 8 and 10 at the DISC. Interestingly, treatment of TRAIL-resistant tumor cells with conventional chemotherapeutic drugs or with proteasome inhibitors renders these cells sensitive for TRAIL-induced apoptosis. By applying the methodology of the biochemical analysis of the TRAIL DISC described here, we were able to show that this sensitization is mainly due to changes in the CIP[-9.5]From: Methods in Molecular Biology, vol. 414: Apoptosis and Cancer Edited by: G. Mor and A. B. Alvero © Humana Press Inc., Totowa, NJ biochemical composition of the DISC as the apoptosis-initiating protein complex of the extrinsic pathway. [ABSTRACT FROM AUTHOR]

Caspases are central to the execution of apoptosis. Their proteolytic activity is responsible for the demise of cells in many physiological and pathological states. Great advances in understanding caspases have been made using recombinant caspase expression and enzymatic characterization. Assays to measure caspase activity in apoptotic cell extracts and the development of a reconstituted cell-free assay were also critical in establishing the hierarchy in the caspase activation cascade and comprehend how caspase-9 is activated by the apoptosome. More recently, new tools such as activity-based probes allowed us to detect caspase activation in their working environment providing readout of the system with minimal interference. This chapter describes some of the methods used by our group to study the activation mechanisms of caspases and their activity. [ABSTRACT FROM AUTHOR]

Here we describe a simple protocol that uses positively charged nylon membrane dot blot to profile miRNA expression. A library of 515 antisense oligodeoxynucleotides of human and mouse mature miRNAs was synthesized and spotted on GeneScreen Plus membrane using a dot-blot equipment. Total RNA or enriched small molecular weight RNAs (smwRNAs) were enzymatically radiolabeled by poly (A) polymerase and then hybridized to the nylon membrane oligo arrays. The spot signal intensity on the membrane was analyzed using phosphorimaging. This method offers a convenient and economic way to simultaneously detect the expression of hundreds of miRNAs. [ABSTRACT FROM AUTHOR]

Protein degradation is mediated predominantly through the ubiquitin-proteasome pathway. The importance of the proteasome in regulating degradation of proteins involved in cell-cycle control, apoptosis, and angiogenesis led to the recognition of the proteasome as a therapeutic target for cancer (1-4). The proteasome is also essential for degrading misfolded and aberrant proteins, and impaired proteasome function has been implicated in diseases such as Parkinson's and Alzheimer's citech13:bib05. The importance of the proteasome for general cell homeostasis has been established, and the 2004 Nobel Prize for Chemistry honored the researchers that discovered the ubiquitin-proteasome pathway. Robust, sensitive assays are essential for monitoring proteasome activity and for developing inhibitors of the proteasome. Peptide-conjugated fluorophores are widely used as substrates for monitoring proteasome activity, but fluorogenic substrates can exhibit significant background and can be problematic for screening because of cellular autoflorescence or fluorescent library compounds. To address these issues, we developed a homogeneous, bioluminescent method that combines peptide-conjugated aminoluciferin substrates and a stabilized luciferase. We have developed homogeneous, bioluminescent assays for all three proteasome activities, the chymotrypsin-like, trypsin-like, and caspase-like, using purified proteasome. We have also applied this technology to a cellular assay using the substrate for the chymotrypsin-like activity in combination with a selective membrane permeabilization step (patent pending). The proteasome assays are designed in a simple "add and read" format and have been tested in 96- and 384-well plates. The bioluminescent, coupled-enzyme format enables sensitive and rapid protease assays ideal for inhibitor screening. [ABSTRACT FROM AUTHOR]

Multiplexed assay chemistries provide for multiple measurements of cellular parameters within a single assay well. This experimental practice not only is more cost efficient but provides more informational content about a compound or treatment. For instance, multiplexed caspase activity assays can help establish the kinetics and magnitude of initiator and effector caspase induction by candidate compounds or treatments. The ability to combine the activity profiles within the same sample provides a level of normalization not possible with parallel assays. Furthermore, multiplexing caspase activity assays with viability and/or cytotoxicity assays can support conclusions regarding cytotoxic mechanism and provide normalization that may help correct for differences in cell number. [ABSTRACT FROM AUTHOR]

Caspase activity assays in multi-well plate formats represent powerful tools for understanding experimental modulation of the apoptotic response. These assays are configured to exploit functional, biochemical, and temporal differences in substrate specificity and selectivity, which are useful in defining the magnitude and mechanism of a compound or treatment effect. New advances in fluorescent and luminescent chemistries now enable single addition "add-mix-measure" determinations of caspase activity directly in the sample plate with unprecedented sensitivity. Unlike other more cumbersome or laborious techniques, caspase activity induction or inhibition measures are quantifiable and definitive. The highlighted techniques in this chapter are cost efficient and allow for the rapid exploration of thousands of combinations and conditions. [ABSTRACT FROM AUTHOR]

Apoptosis detection methodology is an ever evolving science. The caspase family of cysteine proteases plays a central role in this environmentally conserved mechanism of regulated cell death. New methods that allow for the improved detection and monitoring of the apoptosis-associated proteases are key for further advancement of our understanding of apoptosis-mediated disease states such as cancer and Alzheimer's disease. From the use of membrane permeant fluorescent-labeled inhibitors of caspases (FLICA) probe technology, we have demonstrated their successful use as tools in the detection of apoptosis activity within the in vitro and in vivo research setting. In this chapter, we provide detailed methods for performing in vitro apoptosis detection assays in whole living cells, using flow cytometry, and 96-well fluorescence plate reader analysis methods. Furthermore, novel flow cytometry-based cytotoxicity assay methods, which incorporate the FLICA probe for early apoptosis detection, are described. Inclusion of this sensitive apoptosis detection probe component into the flow-based cytotoxicity assay format results in an extremely sensitive cytotoxicity detection mechanism. Lastly, in this chapter, we describe the use of the FLICA probe for the in vivo detection of tumor cell apoptosis in mice and rats. These early stage in vivo-type assays show great potential for whole animal apoptosis detection research. [ABSTRACT FROM AUTHOR]

Since the discovery of the key role of cytochrome C in the activation of caspase 9, intense interest has focused on the role of mitochondria in apoptosis/programmed cell death. Mitochondria undergo two major alterations during apoptosis. The first is the permeabilization of the outer mitochondrial membrane. This event is tightly regulated by members of the Bcl-2 family and involves the conformational change of pro-apoptotic family members such as Bax. Second, the electrochemical gradient that is normally present across the inner mitochondrial membrane is lost (membrane depolarization). This event is sometimes mediated by the permeability transition pore (PTP). The order in which these events occur and whether one causes the other has been hotly debated in the literature. Nonetheless, the majority of reports suggest that mitochondria outer membrane permeabilization (MOMP) precedes membrane depolarization. In this chapter, methods that examine membrane depolarization and the conformational change in Bax are described. [ABSTRACT FROM AUTHOR]

Apoptotic markers include proteins from the intrinsic and extrinsic pathways. These cascades include both pro-apoptotic and anti-apoptotic elements. The expression levels of these elements can be assessed by immunohistochemistry (IHC) and can indicate general trends in pro- versus anti-apoptotic tendencies of the cells. IHC is particularly useful when studying large cohorts of paraffin-embedded specimens. Advances in tissue microarray (TMA) technology have facilitated evaluation of large cohorts of specimens, as cores from hundreds of patients can be represented on a single glass slide and stained in a uniform fashion. In this chapter, we discuss construction and staining methods of TMAs and present examples of assessment of apoptotic marker expression in malignant and benign cells using a novel method of automated, quantitative analysis of in situ protein expression. [ABSTRACT FROM AUTHOR]

The immediate assessment of response to therapy is most beneficial to ovarian cancer patients. This study shows the correlation of drug-induced caspase activation determined by western blot analysis and by Caspase-Glo™ assay. Our findings demonstrate that the use of the Caspase-Glo™ assay allows a simple, fast, and sensitive alternative for the evaluation of in vitro response to chemotherapy. [ABSTRACT FROM AUTHOR]

The ChemoFx® Assay is an ex vivo assay designed to predict the sensitivity and resistance of a given patient's solid tumor to a variety of chemotherapy agents. A portion of a patient's solid tumor, as small as a core biopsy, is mechanically disaggregated and established in primary culture where malignant epithelial cells migrate out of tumor explants to form a monolayer. Cultures are verified as epithelial and exposed to increasing doses of selected chemotherapeutic agents. The number of live cells remaining post-treatment is enumerated microscopically using automated cell-counting software. The resultant cell counts in treated wells are compared with those in untreated control wells to generate a dose-response curve for each chemotherapeutic agent tested on a given patient specimen. Features of each dose-response curve are used to score a tumor's response to each ex vivo treatment as "responsive," "intermediate response," or "non-responsive." Collectively, these scores are used to assist an oncologist in making treatment decisions. [ABSTRACT FROM AUTHOR]

Tumors are heterogeneous structures that contain different cell populations. Laser capture microdissection (LCM) can be used to obtain pure cancer cells from fresh-frozen cancer tissue and the surrounded environment, thus providing an accurate snapshot of the tumor and its microenvironment in vivo. We describe a new approach to isolate pure cancer cell population and evaluate protein expression. The process includes immunocytochemistry, laser microdissection, and western blot analysis. Using this technique, we can detect proteins such as X-linked inhibitor of apoptosis protein (XIAP) and Fas ligand (FasL) with as little as 1000 cells. [ABSTRACT FROM AUTHOR]

A number of methods have been developed to identify the cells that undergo apoptosis by analyzing the morphological, biochemical, and molecular changes that take place during this universal biological process. The best recognized biochemical hallmark of both early and late stages of apoptosis is the activation of cysteine proteases (caspases). Detection of active caspase-3 in cells and tissues is an important method for apoptosis induced by a wide variety of apoptotic signals. Most common assays for examining caspase-3 activation include immunostaining, immunoblotting for active caspase-3, colorimetric assays using fluorochrome substrates, as well as employing the fluorescein-labeled CaspaTag pan-caspase in situ detection kit. [ABSTRACT FROM AUTHOR]

Interference with the innate apoptotic activity is a hallmark of neoplastic transformation and tumor formation. Modulation of the apoptotic cascade has been proposed as a new approach for the treatment of cancer. In this chapter, we discuss the role of apoptosis in ovarian cancer and the use of phenoxodiol as a model for the regulation of apoptosis and potential use as chemosensitizer for chemoresistant ovarian cancer cells. [ABSTRACT FROM AUTHOR]

During implantation and pregnancy, the invading trophoblast population is within close contact to maternal immune cells, particularly macrophages. During this period, a low level of trophoblast cell death occurs as part of the normal process of tissue renewal. Macrophage engulfment of apoptotic trophoblast cells prevents the release of potentially pro-immunogenic intracellular contents and prevents a maternal immune response. Furthermore, the uptake of apoptotic cells suppresses macrophages from secreting pro-inflammatory cytokines and promotes the release of anti-inflammatory cytokines, thus creating a microenvironment that will promote trophoblast survival and a successful pregnancy. The present chapter will describe some of the approaches used to study the interaction between macrophages and trophoblast cells. [ABSTRACT FROM AUTHOR]

The loss of replicative potential with each cell division has been attributed to the progressive shortening of telomeres. This "mitotic clock" occurs because most normal human cells are telomerase-negative. Telomerase is a multicomponent enzyme that prevents loss of telomeric DNA associated with normal cell division. Transfection of cells with vectors expressing the catalytic subunit of human telomerase (hTERT) is often sufficient for immortalization. In this article, we will address this approach in the establishment of immortalized endometrial cells and its value in facilitating in vitro studies. [ABSTRACT FROM AUTHOR]

Pregnancy loss is the one of the most common obstetrical complications. The majority of pregnancy losses are random or isolated incidences that in many cases are related to genetic abnormalities. However, 2-5% of reproductive age women experience recurrent miscarriages.1,2 Recurrent pregnancy loss is typically defined as two or three or more consecutive pregnancy losses. Genetic, hormonal, metabolic, uterine anatomical, infectious, environmental, occupational and personal habits, thrombophilia, or immune disorders were reported as possible etiologies.3-6 Despite the many etiologies, a majority of women with recurrent miscarriage have no discernible cause. It has been postulated that immunologic aberrations may be the cause in many of such cases. Immunopathological evaluation of placenta from women with recurrent pregnancy losses of immune etiologies often demonstrate increased inflammatory cell infiltration at the implantation site and increased fibrin deposition on deciduas and/or perivillous placental membrane.7 In addition, thromboembolism has been noticed in 33.9% of the decidual vessels of the placenta from these women.7 These findings and others have suggested that inflammation and coagulation play a role in recurrent pregnancy loss. [ABSTRACT FROM AUTHOR]

Infertility and pregnancy wastage affect one of every nine couples in Western Europe and in the United States. The molecular events of embryonic attachment to the endometrial epithelium and subsequent invasion and nidation into the stroma have long been of interest, scientifically to reproductive biologists and clinically to couples with infertility or habitual abortion and to the physicians caring for them. In order to achieve a successful pregnancy in the human, two major conditions have to be fulfilled: during the 4-5 days of transport through the fallopian tube, the embryo must undergo a series of complex maturation processes and, in the same time, a receptive endometrium must have developed. Human endometrium undergoes characteristic cyclic changes of proliferation and secretion and, without embryonic implantation, the endometrium is shed and the menstrual bleeding occurs. Uterine endometrium therefore is the anatomic prerequisite for the continuation of our species and its main purpose during the reproductive age is to communicate with, receive, nourish and protect the implanting blastocyst.1 [ABSTRACT FROM AUTHOR]

Parturition is the act of giving birth.1 It is a complex process that includes the anatomical, physiological, biochemical and immunological events taking place in the mother, placenta and fetus. These events are responsible for: (1) the preparation of the uterus for labor; (2) labor per se, leading to the delivery of the fetus and placenta; and (3) post-partum uterine involution, breast feeding and adaptation of maternal behavior. [ABSTRACT FROM AUTHOR]

During pregnancy some cells traffic between the fetus and mother and recent studies indicate low levels persist in the respective hosts decades later. Microchimerism (Me) refers to a small population of cells or DNA harbored by one individual that derive from a genetically distinct individual. Persistent Me can also arise from cell transfer between twins in utero or after a blood transfusion. Because women are preferentially affected by autoimmune disease, often with an increased incidence in post-reproductive years, fetal Me has been investigated in diseases such as systemic sclerosis (SSc), autoimmune thyroiditis, primary biliary cirrhosis, Sjögren's syndrome and systemic lupus erythematosus. Maternal Me has been investigated in SSc, myositis and neonatal lupus. Evidence implicating fetal Me is strongest in SSc where quantitatively higher levels of fetal Me have been found and particular human leukocyte antigen (HLA) relationships of mother and child are associated with increased risk of subsequent SSc in the mother. Maternal Me is implicated in myositis and neonatal lupus. It is unknown how Me might be involved in autoimmune disease. Me could play a role in the effector arm of immune responses either directly or indirectly. Microchimeric cells could be targets of an immune response, an intriguing possibility suggested by a recent study in which maternal cells identified in hearts of infants with neonatal lupus congenital heart block were predominantly cardiac myocytes. Alternatively microchimeric cells could be recruited secondarily to diseased tissues and function in tissue repair. The long-term consequences of naturally acquired Me deriving from pregnancy are not yet known. Because persistent fetal and maternal Me are not uncommon in healthy individuals it seems likely that beneficial effects may also accrue to the host. Recent advances in this active frontier of scientific research are discussed. [ABSTRACT FROM AUTHOR]

Inherited thrombophilias are a heterogeneous group of conditions which have been associated with a variety of pregnancy complications, including early and late fetal loss, intrauterine growth restriction, abruptio placentae, and preeclampsia.1 As the functional significance of the burgeoning list of thrombophilic conditions is better understood, more rational and thoughtful approach to their detection and usefulness in clinical practice will likely emerge. While dominant conditions, such as antithrombin deficiency are rarely present without clinical manifestations, other less thrombogenic mutations, such as factor V Leiden, often are not associated with obvious pregnancy complications, as noted by the finding that the presence of heterozygous factor V Leiden is associated with a 0.2% risk of maternal thromboembolism. Emerging data suggests the quality and quantity of thrombophilic conditions, in addition to genetic and environmental influences create a ‘threshold milieu' for the clinical manifestation of these heterogeneous prothrombotic conditions. This review will summarize the current knowledge of thrombophilic conditions and their association with first trimester pregnancy outcome. [ABSTRACT FROM AUTHOR]

Medawar, in the early 1950s, recognized for the first time, the unique immunology of the maternal-fetal interface and its potential relevance for transplantation. In his original work, he described the "fetal allograft analogy" whereby the fetus may be viewed as a semi-allogeneic conceptus that has evaded rejection by the maternal immune system. Although numerous hypotheses have been proposed to prove this observation, none have demonstrated that the maternal immune system is antagonist to the invading trophoblast. In the present manuscript we have reviewed recent studies demonstrating the expression and function of TLRs in trophoblast cells and based on this data we propose an alternative view for maternal-fetal immune interactions. [ABSTRACT FROM AUTHOR]

The immunological paradox of mammalian pregnancy is the acceptance of the fetus, a semiallogeneic allograft that normally should provoke an immune response of the maternal T cells leading to fetal rejection. In this chapter the current understanding of decidual T-cell immunobiology is reviewed and discussed. Previous and current results about decidual γδT-, αβT-, NKT- and regulatory-T cells are summarized. The significance of thymic involution in pregnancy and extrathymic T-cell differentiation in decidua as mechanisms creating maternal tolerance towards the fetus is considered. Available evidence regarding T-cell contribution to the immune tolerance is integrated into a hypothetical model. An outline of recent findings supporting the increasing role for γδT-, NKT- and innate regulatory T cells in the maintenance of pregnancy is given. We conclude that decidual T cells play a central role in pregnancy and a greater knowledge of their immunobiology is of crucial importance in the understanding of fetal tolerance. [ABSTRACT FROM AUTHOR]

All sexually reproducing organisms produce gametes that must be protected from immune challenge. Recent data indicates that the majority of the carbohydrate sequences that coat the murine zona pellucida are also upregulated on activated lymphocytes, and some participate in gamete binding. This overlap indicates that there may be a "species recognition system" (SRS) that is employed to identify both immune cells and gametes in the context of "species" rather than "self". In eutherians, histoincompatible progeny must also be protected from the maternal immune response. The composite data indicates that several glycoconjugates produced in the pregnant can modify immune responses in vitro. We have previously referred to these collective factors as the "eutherian fetoembryonic defense system" or eu-FEDS. Based on the available results, it is very likely that these glycoconjugates are utilizing specific carbohydrate sequences as functional groups to mediate these activities. Many different persistent pathogens and tumor cells also either mimic or acquire these carbohydrate functional groups, indicating that they may be able to evoke similar effects. We outline new data that clearly implicate glycobiological subterfuge in the pathological effects associated with infection with lentiviruses like HIV-1 and simian immunodeficiency virus (SIV). In addition, recent data showing that the uterine mucin CA125 and the major surface glycoprotein of HIV-1 (gpl20) express the same N-glycans suggests a linkage to the pregnant uterus. Complete integration of this protective effect associated with eu-FEDS could also explain why the natural hosts of SIV (sooty mangabees and African green monkeys) are able to completely accommodate this virus without pathological effects. In summary, the requirements for accommodation of the germ cells and different stages of developing progeny could provide a very substantial "Achilles heel" for exploitation by pathogens and tumorcells. These more recent finding should provide a great impetus to further investigate the eu-FEDS hypothesis and its strong linkages to pathogenesis. [ABSTRACT FROM AUTHOR]

Maternal immune tolerance to paternal allo-antigens expressed by the fetus is a precondition of successful pregnancy in viviparous mammals. This occurs despite exposure of the maternal immune system to potentially immunogenic fetal tissue. Local immune evasion mechanisms are thought to prevent maternal immune aggression towards the fetus. Nevertheless, maternal lymphocytes capable of mediating anti-fetal immune aggression are detectable throughout gestation. This review focuses on the role played by regulatory T cells in suppressing alloreactive immune responses, thereby promoting materno-fetal tolerance. The characteristics of naturally occurring regulatory T cells, their function in mouse and human pregnancy and the implications for infertility, premature abortion syndromes, transplantation and autoimmunity are discussed in detail. [ABSTRACT FROM AUTHOR]

The mechanisms of acceptance of the fetus by the maternal immune system are mediated in part by immunomodulatory proteins expressed by placental cells. The recent discovery of novel members of the B7 family of immunomodulators has prompted much excitement among the scientific community because of their role in immunological tolerance. Many members of the B7 family are now known to be expressed in the placenta, suggesting that they contribute to the modification of the surrounding immunological milieu such that the fetal allograft is tolerated. In this chapter, we will summarize recent findings regarding the expression and possible functions of B7 proteins in the placenta. [ABSTRACT FROM AUTHOR]

Embryo implantation is critically dependent on a supportive uterine environment. Uterine receptivity is the culmination of a cellular and molecular transformation mediated locally by paracrine signals under the governance of ovarian steroid hormones, with cells and cytokines of the immune system playing integral roles in this process. Semen is now recognised as contributing to endometrial preparation for embryo implantation, through the agency of specific factors in the seminal plasma fraction of the ejaculate. Transforming growth factor-β (TGFβ) and other immunoactive moieties derived from male accessory glands interact with epithelial cells in female reproductive tissues to induce pro-inflammatory cytokine expression and initiate an inflammatory cascade. The consequences are recruitment and activation of macrophages, granulocytes and dendritic cells which have immune-regulatory and tissue remodelling roles. The cytokines elicited by seminal activation also exert embryotrophic effects and contribute to optimal preimplantation embryo development. This review summarises our current understanding of the molecular and cellular basis of interactions between seminal plasma and the female reproductive tract, and explores the potential mechanisms through which seminal plasma influences the establishment of pregnancy. [ABSTRACT FROM AUTHOR]

The mechanisms of protection of the allogeneic fetus from the maternal immune response during pregnancy remain mysterious more than fifty years after the paradox of maternal tolerance was first raised by Peter Medawar. Preimplantation embryos express paternal antigens early in development. After implantation, placental tissue is derived from both maternal tissue and paternal-antigen expressing fetal tissue that is in intimate association with and bathed in maternal blood. There appears to be a key role for an unusual subset of major histocompatibility complex (MHC) Class I proteins of both maternal and paternal origin in the mediation of tolerance at the maternal/fetal interface and in the control of preimplantation embryonic growth rate. This subset of MHC products is composed of two nonclassical MHC Class Ib proteins, HLA-E and HLA-G in combination with a classical MHC Class la protein, HLA-C. This chapter reviews the history of the discovery of the major histocompatibilty complex Class I genes and the elucidation of the biological role of the proteins encoded by these genes in the immune response and in reproduction. MHC genes have also been implicated in reproductive choice and nurturing behaviors. We hypothesize that the vertebrate immune system derived from ancestral recognition systems driven by reproductive requirements, and was later coopted for immune recognition under additional evolutionary pressures. The complex interactions of MHC Class I proteins with components of both the innate and the adaptive immune systems in the context of the preimplantation embryo and the trophoblast of early pregnancy are described in detail, as are the difficulties inherent in studying these systems. Finally, potential future directions of research and the need for new model systems to study both preimplantation embryos and the maternal/fetal placental interface are discussed. [ABSTRACT FROM AUTHOR]

The unusual tolerance against fetal antigens is still one of the greatest miracles of pregnancy. Dealing with reproductive immunology, the question arises as to how the maternal immune system handles the foreign fetal antigens leading to that tolerance. Focussing on the various subsets of immunocompetent cells playing in concert with the human immune system, tolerance induction nowadays is often accredited to a newcomer, at present the "hottest star" for immunologists is the dendritic cell (DC). [ABSTRACT FROM AUTHOR]

Viviparity remains an immunological paradox despite increased knowledge of immuno-logical processes that occur during mammalian pregnancy. The maternal immune system protects both mother and fetus from invading pathogens during gestation, but also has to maintain immunological tolerance towards the fetus. Medawar proposed three ways in which this paradox might be resolved: physical segregation of maternal and fetal tissues, immunologic immaturity of fetal tissues, and immunological anergy (tolerance) of the maternal immune system towards fetal alloantigens.1 [ABSTRACT FROM AUTHOR]

The hypothalamic neuropeptide corticotropin-releasing hormone (CRH), as well as its receptors, have been identified in several reproductive organs, including the endometrial glands, the decidualized endometrial stroma and the placental trophoblast, synctiotrophoblast and decidua.1−9 "Reproductive" CRH is a form of "tissue" CRH (CRH found in peripheral tissues), analogous to the "immune" CRH detected in peripheral inflammatory sites.10 "Immune" CRH possesses potent proinflammatory properties, influencing both innate and acquired immune processes. Intrauterine CRH may participate in local immune phenomena associated with embryo implantation (Table 1). [ABSTRACT FROM AUTHOR]

Natural killer cells are found in large numbers in the endometrium and decidua, and data suggest that NK cell functions and interactions with fetal-derived trophoblasts can have a profound impact on pregnancy. Altered NK cell numbers and activity have been associated with a variety of clinical conditions such as endometriosis, recurrent pregnancy loss, and preeclampsia. Uterine NK cells have a unique phenotype compared to blood NK cells and this is likely due to the specific tissue environment in which they reside. Specific chemokines produced by human endometrium and trophoblasts have been identified that may be responsible for recruitment of NK cells. Uterine NK cells can produce cytokines and may be an important part of vascular remodelling during placenta! development. This chapter summarizes current knowledge of NK cells in the uterus and their role in pregnancy and reproductive disorders. [ABSTRACT FROM AUTHOR]

Although the etiology of intrauterine growth restriction (IUGR) and preeclampsia (PE) remains unclear, most investigators attribute the initial "insult" to poor utero-placental perfusion due to defective trophoblast invasion that ultimately compromises fetal well-being.1-3 The resultant hypoxia curtails the remodeling of uterine vessels by invasive cytotrophoblasts in the second trimester.1,2 Our results suggest that mediators of fetal stress [i.e., glucocorticoids (GC)] may in fact alter placental gene expression and contribute to the destruction of the placental villous network in pregnancies with IUGR/PE. We will present a molecular model through which GC, induced in response to fetal stress, promotes the placental villous damage observed in pregnancies associated with IUGR/PE. This model incorporates the roles of trophoblast plasminogen activator inhibitor (PAI)-l, mesenchymal extracellular matrix (ECM) proteins, and their regulation by transforming growth factor (TGF)-β. We will employ the term "IUGR/PE" to describe those pregnancies with severely growth-restricted fetuses may also complicated by maternal PE These conditions frequently coexist, and a review of the literature suggests that this placental pathology may be associated with both IUGR and PE. Furthermore, considerable attention has been given to the role of exogenously administered and stress-induced endogenous increases in fetal GC and the development of IUGR. There is mounting evidence that aberrant elevations in GC during fetal life and/or IUGR may result in fetal programming of chronic diseases of adulthood such as diabetes, coronary artery disease, and hypertension. [ABSTRACT FROM AUTHOR]

During implantation, apoptosis is critical for the appropriate tissue remodeling of the maternal decidua and invasion of the developing embryo. Yet the regulation of apoptosis is also imperative for a successful pregnancy. The quick and effective removal of apoptotic cells by tissue macrophages represents an essential process, which prevents the release of self-antigens and, in the case of pregnancy, paternal alloantigens. Recent studies have shown that the process of apoptotic cell clearance is not a neutral event, but rather an active one which induces macrophage production of anti-inflammatory cytokines and survival factors. Therefore, apoptotic cell clearance is necessary for the resolution of inflammatory conditions, which during pregnancy could have lethal consequences. The function of the maternal immune system during implantation and throughout pregnancy is, thus, an important area of investigation. This review will discuss the role of decidual macrophages in apoptotic cell clearance during pregnancy. [ABSTRACT FROM AUTHOR]

Apoptosis occurs in the villous trophoblast of normal placentas throughout pregnancy, but with higher frequency near term in comparison to the first trimester. In pregnancies complicated by preeclampsia or intrauterine growth restriction (IUGR), a greater incidence of villous and extravillous trophoblast apoptosis has been observed, suggesting that the deregulation of trophoblast apoptosis may contribute to pathological conditions. Apoptosis may be initiated by one of two known pathways, the mitochondrial (instrinsic) pathway and the death receptor-mediated (extrinsic) pathway, or in response to exogenous stimuli such as cytokines or hypoxia. The central executioners of apoptosis are the caspase family of cysteine proteases, which cleave numerous vital cellular proteins to affect the apoptotic cascade. By controlling caspase activation, several endogenous inhibitors, including FLIP, IAPs and anti-apoptotic Bcl-2 family members prevent further propagation of the death signal. This review focuses on the molecular mechanisms by which normal trophoblast apoptosis occurs and how it is regulated to avoid excessive trophoblast apoptosis and pregnancy complications. [ABSTRACT FROM AUTHOR]

Successful embryo implantation requires the synchronization of embryo development and uterine preparation. The embryo must have developed to the blastocyst stage and the endometrium must be in a receptive phase. Wilcox et al1 have estimated that 65% of conceptions end in unrecognized losses. These failures can be divided into failure to implant (20%), initial apposition but no adhesion or invasion (28%), and failure to develop immediately after implantation (17%). After implantation, approximately 15% of human conceptions end in a clinically detected spontaneous abortion. Furthermore, approximately 31% of pregnancies are lost after detection using sensitive assays for the beta subunit of human chorion gonadotropin (β-hCG). Therefore, implantation failure and pregnancy loss are the most common complications of pregnancy. Understanding the molecular factors involved in each phase of implantation and early pregnancy is critical to understanding the mechanism controlling reproduction. [ABSTRACT FROM AUTHOR]

Soon after the principles of nonself immunological recognition were discovered, it was realized that the state of pregnancy seemingly presents a paradox. In an outbreed population, half of the fetal genes are paternal, thus the fetus may be considered a semi-allograft. Yet, unlike the outcome of a surgical tissue graft, the mother tolerates and nurtures the fetus. [ABSTRACT FROM AUTHOR]

The maternal-fetal interface represents an immunologically unique site that must promote tolerance to the allogenic fetus, whilst maintaining host defense against a diverse array of possible pathogens. Clinical studies have shown a strong association between certain pregnancy complications and intrauterine infections. Therefore, innate immune responses to microorganisms at the maternal-fetal interface may have a significant impact on the success of a pregnancy. There is growing evidence that trophoblast cells are able to recognize and respond to pathogens through the expression of Toll-like receptors, a system characteristic of innate immune cells. This review will discuss the role of Toll-like receptors at the maternal-fetal interface, the potential for trophoblast cells to function as components of the innate immune system and the impact TLR-mediated trophoblast responses may have on a pregnancy. [ABSTRACT FROM AUTHOR]