Academic literature on the topic 'Placental proteins'

Introduction: Dysfunction of placental development is involved in early pregnancy loss. Senescent changes have been seen in missed miscarriage, one type of pregnancy loss. Extracellular vesicles (EVs) have been widely implicated in the pathogenesis of diseases. In this study, we investigated the protein profiles in placental EVs derived from missed miscarriage in comparison with healthy pregnancy. We also investigated whether cargos packed into EVs are involved in the dysfunctional development of the placenta seen in missed miscarriage. Methods: Proteomic analysis of placental EVs derived from healthy and missed-miscarriage placentae was performed. Three senescence-repair-associated proteins, replication protein A-70 (RPA-70), proteasome activator subunit-4 (PMSE-4), and protein activated kinase-2, (PAK-2) were examined in placental EVs and placentae, and in placental explants that had been treated with or without GW4869, by western blotting and immunohistochemistry. Results: The total number of proteins associated with placental EVs was not different between the two groups. However, there were 106 and 151 abundantly expressed proteins associated with placental micro- or nano-EVs from missed miscarriage in comparison with EVs from controls. Of these abundant proteins, 59 and 81 proteins in placental micro- or nano-EVs, respectively, are associated with DNA damage/repair and cell death/survival. We further found higher levels of three senescence-repair-associated proteins (RPA-70, PMSE-4, and PAK-2) associated with placental EVs, but lower levels of these proteins in missed-miscarriage placentae. Regarding inhibition of EV formation or release by GW4869, we found that the expression of these three proteins was higher in GW4869-treated placental explants from missed miscarriage. Discussion: Our data may suggest that “inadvertently” sorting of cargos and exporting proteins associated with senescence-repair by placental EVs may be associated with the dysfunction of placental development seen in missed miscarriage.

Preeclampsia, characterised by maternal endothelial cell activation, is triggered by toxic factors, such as placental extracellular vesicles (EVs) from a dysfunctional placenta. The increased oxidative stress seen in the preeclamptic placenta links to endoplasmic reticulum (ER) stress. The ER regulates protein folding and trafficking. When the ER is stressed, proteins are misfolded, and misfolded proteins are toxic. Misfolded proteins can be exported from cells, via EVs which target to other cells where the misfolded proteins may also be toxic. Melatonin is a hormone and antioxidant produced by the pineal gland and placenta. Levels of melatonin are reduced in preeclampsia. In this study we investigated whether melatonin treatment can change the nature of placental EVs that are released from a preeclamptic placenta. EVs were collected from preeclamptic (n = 6) and normotensive (n = 6) placental explants cultured in the presence or absence of melatonin for 18 h. Misfolded proteins were measured using a fluorescent compound, Thioflavin-T (ThT). Endothelial cells were exposed to placental EVs overnight. Endothelial cell activation was measured by the quantification of cell-surface ICAM-1 using a cell-based ELISA. EVs from preeclamptic placentae carried significantly (p < 0.001) more misfolded proteins than normotensive controls. Incubating preeclamptic placental explants in the presence of melatonin (1 µM and 10 µM) significantly (p < 0.001) reduced the misfolded proteins carried by EVs. Culturing endothelial cells in the presence of preeclamptic EVs significantly increased the expression of ICAM-1. This increased ICAM-1 expression was significantly reduced when the endothelial cells were exposed to preeclamptic EVs cultured in the presence of melatonin. This study demonstrates that melatonin reduces the amount of misfolded proteins carried by EVs from preeclamptic placentae and reduces the ability of these EVs to activate endothelial cells. Our study provides further preclinical support for the use of melatonin as a treatment for preeclampsia.

Low birth weight is associated with an increased risk for developing type 2 diabetes and metabolic diseases. The placental capacity to supply nutrients and oxygen to the fetus represents the main determiner of fetal growth. However, few studies have investigated the effects of maternal diet on the placenta. We explored placental adaptive proteomic processes implicated in response to maternal undernutrition. Rat term placentas from 70% food-restricted (FR30) mothers were used for a proteomic screen. Placental mitochondrial functions were evaluated using molecular and functional approaches, and ATP production was measured. FR30 drastically reduced placental and fetal weights. FR30 placentas displayed 14 proteins that were differentially expressed, including several mitochondrial proteins. FR30 induced a marked increase in placental mtDNA content and changes in mitochondrial functions, including modulation of the expression of genes implicated in biogenesis and bioenergetic pathways. FR30 mitochondria showed higher oxygen consumption but failed to maintain their ATP production. Maternal undernutrition induces placental mitochondrial abnormalities. Although an increase in biogenesis and bioenergetic efficiency was noted, placental ATP level was reduced. Our data suggest that placental mitochondrial defects may be implicated in fetoplacental pathologies.

BackgroundMore than 50 human placental proteins were isolated and physico-chemically characterized in the 70–80s by Hans Bohn and co-workers. Many of these proteins turned to have important role in placental functions and diagnostic significance in pregnancy complications. Among these proteins was membrane-associated placental protein 4 (MP4), for which identity or function has not been identified yet. Our aim was to analyze the sequence and placental expression of this protein in normal and complicated pregnancies including miscarriage, preeclampsia and HELLP syndrome.MethodsLyophilized MP4 protein and frozen healthy placental tissue were analyzed using HPLC-MS/MS. Placental tissue samples were obtained from women with elective termination of pregnancy (first trimester controls,n= 31), early pregnancy loss (EPL) (n= 13), early preeclampsia without HELLP syndrome (n= 7) and with HELLP syndrome (n= 8), late preeclampsia (n= 8), third trimester early controls (n= 5) and third trimester late controls (n= 9). Tissue microarrays were constructed from paraffin-embedded placentas (n= 81). Slides were immunostained with monoclonal perlecan antibody and evaluated using light microscopy and virtual microscopy. Perlecan was also analyzed for its expression in placentas from normal pregnancies using microarray data.ResultsMass spectrometry-based proteomics of MP4 resulted in the identification of basement membrane-specific heparan sulfate proteoglycan core protein also known as perlecan. Immunohistochemistry showed cytoplasmic perlecan localization in syncytiotrophoblast and cytotrophoblasts of the villi. Perlecan immunoscore decreased with gestational age in the placenta. Perlecan immunoscores were higher in EPL compared to controls. Perlecan immunoscores were higher in early preeclampsia without and with HELLP syndrome and lower in late preeclampsia than in respective controls. Among patients with preeclampsia, placental perlecan expression positively correlated with maternal vascular malperfusion and negatively correlated with placental weight.ConclusionOur findings suggest that an increased placental perlecan expression may be associated with hypoxic ischaemic injury of the placenta in miscarriages and in early preeclampsia with or without HELLP syndrome.

Bioethical limitations impair deeper studies in human placental physiology, then most studies use human term placentas or murine models. To overcome these challenges, new models have been proposed to mimetize the placental three-dimensional microenvironment. The placental extracellular matrix plays an essential role in several processes, being a part of the establishment of materno-fetal interaction. Regarding these aspects, this study aimed to investigate term mice placental ECM components, highlighting its collagenous and non-collagenous content, and proposing a potential three-dimensional model to mimetize the placental microenvironment. For that, 18.5-day-old mice placenta, both control and decellularized (n = 3 per group) were analyzed on Orbitrap Fusion Lumos spectrometer (ThermoScientific) and LFQ intensity generated on MaxQuant software. Proteomic analysis identified 2317 proteins. Using ECM and cell junction-related ontologies, 118 (5.1%) proteins were filtered. Control and decellularized conditions had no significant differential expression on 76 (64.4%) ECM and cell junction-related proteins. Enriched ontologies in the cellular component domain were related to cell junction, collagen and lipoprotein particles, biological process domain, cell adhesion, vasculature, proteolysis, ECM organization, and molecular function. Enriched pathways were clustered in cell adhesion and invasion, and labyrinthine vasculature regulation. These preserved ECM proteins are responsible for tissue stiffness and could support cell anchoring, modeling a three-dimensional structure that may allow placental microenvironment reconstruction.

Preeclampsia (PE) is one of the most common causes of maternal-fetal morbidity and mortality world-wide. While the underlying causes of PE remain elusive, aberrant trophoblast differentiation and function are thought to cause an imbalance of secreted angiogenic proteins resulting in systemic endothelial dysfunction and organ damage in the mother. The placental dysfunction is also characterized by a reduction of the transcription factor, peroxisome proliferator activated receptor γ (PPARγ) which normally promotes trophoblast differentiation and healthy placental function. This study aimed to understand how placental activation of PPARγ effects the secretion of angiogenic proteins and subsequently endothelial function. To study this, healthy and PE placental tissues were cultured with or without the PPARγ agonist, Rosiglitazone, and a Luminex assay was performed to measure secreted proteins from the placenta. To assess the angiogenic effects of placental activation of PPARγ, human umbilical vein endothelial cells (HUVECs) were cultured with the placental conditioned media and the net angiogenic potential of these cells was measured by a tube formation assay. This is the first study to show PPARγ’s beneficial effect on the angiogenic profile in the human preeclamptic placenta through the reduction of anti-angiogenic angiopoietin-2 and soluble endoglin and the upregulation of pro-angiogenic placental growth factor, fibroblast growth factor-2, heparin-binding epidermal growth factor, and follistatin. The changes in the angiogenic profile were supported by the increased angiogenic potential observed in the HUVECs when cultured with conditioned media from rosiglitazone-treated preeclamptic placentas. The restoration of these disrupted pathways by activation of PPARγ in the preeclamptic placenta offers potential to improve placental and endothelial function in PE.

The vasculature of the placenta plays a crucial role during the course of pregnancy in order to maintain the growing need of the fetus. Abnormal placental structure and function significantly increase the risk of stillbirth. Various growth factors and cytokines play an important role in the vasculogenesis and angiogenesis of placenta. These processes are stimulated by various pro-angiogenic factors. The activities of these factors are also stimulated by hypoxia. In some of the physiological phenomenon like ovulation, embryogenesis as well as in wound healing intense blood vessel growth can be seen similar to that seen in placenta. Therefore, factors that induce and maintain placental vascular growth and function are of considerable developmental and clinical significance. The total arterial architecture may also depend upon the pro-angiogenic factors. Hormones and other growth factors are other contributors of this vasculogenesis and angiogenesis. Any dysfunction of factors can lead to foetal hypoxia and related complications. This review describes the major growth factors and their significant role in vasculogenesis and angiogenesis of placenta.

Extracellular glutathione peroxidase (eGPX) is a selenoglycoprotein distinct from cellular glutathione peroxidase (cGPX). The cDNA for eGPX has recently been cloned from human placenta. To determine whether human placenta makes both cGPX and eGPX and secretes eGPX, we used specific immunoprecipitations of 75Se metabolically labeled proteins from full-term placental explants in culture and perfused placental lobules. Placental explants and metabolically active, dually perfused placental lobules synthesized and contained both cGPX and eGPX and secreted eGPX. Perfused tissue secreted eGPX into the maternal but not into the fetal perfusate. In situ hybridizations using antisense and sense eGPX riboprobes were performed on sections of first-, second-, and third-trimester placentas. In the first-trimester placenta, transcripts were localized predominantly to cytotrophoblast cells, whereas in the full-term placenta syncytiotrophoblast cells and stromal cells but not fetal endothelial cells expressed eGPX mRNA. It is concluded that human placenta synthesizes both cGPX and eGPX and secretes eGPX into the maternal circulation, consistent with the location of the eGPX mRNA.

Vascularity is increased in placentas from high- compared with low-altitude pregnancies. An angiogenic response to hypoxia may protect an organ from further hypoxic insult by increasing blood flow and oxygen delivery to the tissue. We hypothesized that increased placental vascularity is sufficient to adapt to high altitude. Therefore, indexes of hypoxic stress would not be present in placentas from successful high-altitude pregnancies. Full-thickness placental biopsies were 1) collected and frozen in liquid nitrogen within 5 min of placental delivery and 2) fixed in formalin for stereologic analyses at high (3,100 m, n = 10) and low (1,600 m, n = 10) altitude. Hypoxia-inducible transcription factor (HIF-1) activity was analyzed by ELISA. Western blot analyses were used to evaluate HIF-1α, HIF-1β, HIF-2α, von Hippel-Lindau protein, VEGF, Flt-1, enolase, and GAPDH. Magnetic resonance spectroscopy was used to evaluate endogenous metabolism. The ratio of placental capillary surface density to villous surface density was 70% greater at high compared with low altitude. HIF-1 activity and HIF-1-associated proteins were unchanged in placentas from high- vs. low-altitude pregnancies. Placental expression of HIF-1-mediated proteins VEGF, Flt-1, enolase, and GAPDH were unchanged at high vs. low altitude. Succinate, GSH, phosphomonoesters, and ADP were elevated in placenta from high compared with low altitude. Placentas from uncomplicated high-altitude pregnancies have greater vascularity and no indication of significant hypoxic stress at term compared with placentas from low altitude.

Abstract Placentally derived CRH plays a major role in the mechanisms controlling human pregnancy and parturition. In this study, we sought to investigate the signal transduction mechanisms of CRH Type-1 receptors in the feto-placental unit. To clarify the signal transduction components in placenta and fetal membranes, we investigated the expression of G proteins and adenylate cyclase. Using the nonhydrolysable photoreactive analog [α-32P] GTP-azidoanilide and peptide antisera raised against G proteinα -subunits, we studied coupling of CRH receptors to G proteins in both placental and fetal membranes. Treatment of placental membranes with human CRH (100 nm) increased the labeling of Gq, Go, and Gz but not Gi and Gs. Treatment of fetal membranes with human CRH (100 nm) increased the labeling of Go and Gq but not Gi, Gs, and Gz. These results were supported by experiments that showed that CRH failed to activate adenylate cyclase in these tissues, but induced an increase in inositol phosphates instead. These findings provide new insights into the components of the signal transduction machinery in both fetal and placental membranes and suggest that CRH Type-1 receptors can couple to different G proteins in different tissues. The physiological significance of these observations remains to be elucidated.

UVOD: Hipertenzivna oboljenja u trudnoći predstavljaju heterogenu grupu bolesti koja se javljaju kod 3-8% trudnica opšte populacije. Najteže forme ovih oboljenja preeklampsija, eklampsija i HELLP sindrom su vodeći uzročnici morbiditeta i mortaliteta majke i ploda u odnosu na sve druge komplikacije u trudnoći. Etiopatogeneza ovih oboljenja je još uvek nedovoljno razjašnjena ali se smatra da placenta ima ključnu ulogu u nastanku ovih komplikacija, odnosno da placentalna insuficijencija, koja nastaje kao posledica nedovoljne adaptacije decidualnih i intramiometrijalnih delova spiralnih arterija već u prvih nekoliko nedelja trudnoće, dovodi do redukcije utero-placentalne cirkulacije i lokalne placentalne hipoksije, što se nepovoljno održava i na majku i na plod. U cilju razjašnjenja patofizioloških mehanizama nastanka hipertenzivnih oboljenja u trudnoći i pronalaska dovoljno senzitivnih makera koji bi pomogli u ranom predviđanju nastanka najtežih formi ovih oboljenja, do sada su ispitivani brojni proteini koji učestvuju u procesima stvaranja i razvoja placentalne vaskularne mreže kao što su vaskularni endotelni faktor rasta (VEGF-A), placentalni faktor rasta (PlGF) i solubilni receptor fms-like tirozin kinaza receptor (sFlt-1). CILJ: Uporediti serumske koncentracije (sFlt-1, PlGF, VEGF-A, PAPP-a, freeßhCG, glukoze, ukupnog holesterola, HDL holesterola, LDL holesterola, triglicerida, apo-AI, apoB, AST, ALT, GGT, kreatinina, ureje, mokraćne kiseline, hsCRP, Na, K, Cl, P, Mg i Ca između grupe trudnica sa preeklampsijom, hroničnom i gestacijskom hipertenzijom i kontrolne grupe trudnica u prvom trimestru trudnoće između 11 i 14. nedelje gestacije. Ispitati da li se vrednosti odabranih laboratorijskih parametara (sFlt-1, VEGF-A i PLGF) kod ispitivanih trudnica statistički značajno razlikuju u odnosu na gestacijsku nedelju u trenutku porođaja, težinu i dužinu i APGAR skor bodovanja novorođenčeta. Ispitati da li se vrednosti angiogenih proteina:sFlt-1, VEGF-A, PlGF značajno razlikuju kod ispitivanih trudnica u odnosu na broj prethodnih trudnoća i starosti trudnica. MATERIJAL I METODE: Istraživanje je sprovedeno kao prospektivno analitička studija u Kliničkom centru Vojvodine, u periodu od juna 2012. do februara 2015. godine. U istraživanje je uključeno ukupno 143 trudnice starosti od 18 – 43 godine. Sve trudnice uključene u istraživanje podeljene su na dve ispitivane i jednu kontrolnu grupu. Prvu ispitivanu grupu činilo je 43 trudnice koje su po definisanim kriterijuma razvile preeklampsiju u aktuelnoj trudnoći. Drugu ispitivanu grupu činilo je 46 trudnica kojima je dijagnostikovana ili potvrđena hronična ili gestacijska hipertenzija u aktuelnoj trudnoći. Kontrolnu grupu činilo je 54 zdravih trudnica sa verifikovanim fiziološkim ishodom trudnoće u terminu, bez maternalnih i fetalnih komplikacija. Prilikom regrutovanja trudnica (između 11+0 i 13+6 nedelja gestacije) za učešće u istraživanju, uzeti su anamnestički podaci o faktorima rizika za pojavu hipertenzivnih oboljenja u trudnoći, i u okviru kliničkog i akušerskog pregleda urađena su antropometrijska merenja, merenje krvnog pritiska, i specijalizovani ultrazvučni pregled ploda radi utvrđivanja gestacijske starosti ploda i određivanja rizika za pojavu hromozomskih anomalija ploda. Trudnicama je nakon uzimanja anamnestičkih podataka i kliničkog i akušerskog pregleda i potpisanog pisanog pristanka pacijenta o dobrovoljnom učestvovanju u istraživanju izvađena krv radi određivanja odabranih laboratorijskih parametara. Serumske koncentracije sFlt1, VEGF-A i PIGF određivane su kvantitativnom ELISA tehnikom (R&D Systems Europe Ltd. Abingdon, UK), dok su: glukoza, ukupni holesterol, HDL holesterol, LDL holesterol, trigliceridi, apo-AI I apoB, AST, ALT, GGT, kreatinin, ureja, mokraćna kiselina, hsCRP, Na, K, Cl, Mg, P, Ca određivani na automatizovanim analizatorskim sistemima. Sve trudnice su kategorisane u 2 ispitivane i kontrolnu grupu na osnovu pojave ili isključenja hipertenzivnih oboljenja u aktuelnoj trudnoći. Statistička obrada podataka urađena je u statističkom programu STATISTICA 12 (StatSoft Inc.,Tulsa, OK, USA). Podaci su predstavljeni tabelarno i grafički, nivo statističe značajnosti p, je tumačen statistički značajnim ukoliko su vrednosti p<0,05. REZULTATI: Vrednosti serumskih koncentracija sFlt-1 se statistički značajno razlikuju u sve tri grupe ispitanica i značajno su više u grupama sa hipertenzivnim oboljenjima u odnosu na zdravu grupu ispitanica, p<0,001. Serumske koncentracije VEGF-A su značajno niže u grupi trudnica sa preeklampsijom u odnosu na zdrave trudnice kontrolne grupe (p<0,001), dok se nivoi serumskih koncentracija PlGF statistički značajno razlikuju između sve tri grupe trudnica tako da su najniže vrednosti uočene u grupi sa preeklampsijom (p<0,001) u odnosu na preostale dve grupe ispitanica. Nije uočeno postojanje statistički značajne razlike u nivoima PAPP-A, biohemijskih parametara (glukoze, AST, ALT, GGT kreatinina, ureje, mokraćne kiseline), lipidskih parametara (uk. holesterol, LDL, apo A-I, apo B), parametara inflamatornog (kompletna krvna slika, fibrinogen), hemostaznog (D-dimer, vWF-antigen) i elektrolitskog statusa (Na, K, Cl, P, Mg), p>0,05. Nivoi free ßhCG i HDL holesterola su značajno niže, dok su vrednosti hsCRP i triglicerida značajno više u grupi trudnica sa preeklampsijom u odnosu na grupu bez hipertenzivnih poremećaja u trudnoći. Serumske koncentracije sFlt-1 preko 865 pg/ml imaju senzitivnost od 93% i specifičnost od 81,5% u predviđanju nastanka preeklampsije, dok serumske koncentracije PlGF ispod 60 pg/ml senzitivnost od 88,4% i specifičnost od 79,6% u predviđanju pojave preeklampsije. Serumske koncentracije sFlt-1, VEGF-A i PlGF ne pokazuju statistički značajnu razliku u odnosu na godine života trudnice i broja prethodnih trudnoća p>0,05. Serumske koncentracije sFlt-1 i PlGF se značajno razlikuju u odnosu na telesnu težinu novorođenčeta, tako da su niže vrednosti oba proteina detektovane u grupi novorođenčadi sa porođajnom težinom ispod 1500 gr. u odnosu na telesnu masu između 2800-3300 gr, p<0,001. Takođe su nađene niže vrednosti sFlt-1 i PlGF u grupi trudnica koje su se porodile pre 33. nedelje gestacije u odnosu na nedelju gestacije u trenutku porođaja preko 37 nedelje gestacije, p<0,001. Serumske koncentracije sFlt-1 i PlGF se značajno razlikuju u odnosu na indeks telesne mase majke tako da su više vrednosti sFlt-1 i niže vednosti PlGF nađene u grupi trudnica sa indeksom telesne mase ispod 25 u odnosu na grupu trudnica sa indeksom telesne mase preko 30 kg/m2, p<0,001. Serumske koncentracije sFlt-1 u prvom trimestru trudnoće su značajno povezane sa parametrima inflamacije (hsCRP), vrednostima dijastolnog krvnog pritiska i nivoima free ßhCG. Takođe se uočava značajna povezanost koncentracije PlGF sa indeksom telesne mase, vrednostima sistolnog krvnog pritiska i koncentracijom hsCRP u prvom trimestru trudnoće. ZAKLJUČAK: Nivoi antiangiogenog proteina sFlt-1 su više u grupi trudnica sa preeklampsijom u odnosu na grupu sa hroničnom i gestacijskom hipertenzijom i grupu trudnica bez hipertenzivnih poremećaja trudnoći. Nivoi proangiogenog proteina VEGF-A su značajno niže u grupi trudnica sa preeklampsijom i hroničnom i gestacijskom hipertenzijom u odnosu na grupu trudnica bez hipertenzivnih poremećaja u trudnoći. Serumske koncentracije proangiogenog proteina PlGF su niže u grupi trudnica sa preeklampsijom u odnosu na grupu sa hroničnom i gestacijskom hipertenzijom i grupu trudnica bez hipertenzivnih poremećaja trudnoći. Serumske koncentracije placentalnog proteina free ßhCG i HDL holesterola su značajno niže, dok su vrednosti hsCRP i triglicerida značajno više u grupi trudnica sa preeklampsijom u odnosu na grupu bez hipertenzivnih poremećaja u trudnoći. Između trudnica sa hipertenzivnim poremećajima u trudnoći i zdravih trudnica nije uočeno postojanje značajne razlike u vrednostima placentalnog proteina PAPP-A, biohemijskih parametara (glukoze, AST, ALT, GGT kreatinina, ureje, mokraćne kiseline), lipidskih parametara (uk. holesterol, LDL, apo A-I, apo B), parametara inflamatornog (kompletna krvna slika, fibrinogen), hemostaznog (D-dimer, vWF-antigen) i elektrolitskog statusa (Na, K, Cl, P, Mg). Serumske koncentracije sFlt-1 i PlGF se značajno razlikuju u odnosu na gestacijsku starost na porođaju i telesnu masu novorođenčeta i niže su kod trudnica koje su se prevremeno porodile kao i kod novorođenčati sa manjom porođajnom težinom. Serumske koncentracije sFlt-1 se značajno razlikuju u odnosu telesnu dužinu i APGAR skor novorođenčeta, tako da su više vrednosti sFlt-1 udružene sa većom telesnom dužinom novorođenčeta i boljim APGAR skorom. Serumske koncentracije sFlt-1, VEGF-A i PlGF se ne razlikuju značajno u odnosu na godine života trudnice i broja prethodnih trudnoća. Nivoi proteina angiogeneze sFlt-1 i PlGF predstavljaju dobre prediktore u predviđanju nastanka preeklampsije u prvom trimestru trudnoće.
INTRODUCTION: Hypertensive disorders in pregnancy are a heterogeneous group of diseases that occur in 3-8% of all pregnancies. The most difficult forms of these diseases: preeclampsia, eclampsia and HELLP syndrome are the leading causes of maternal and fetal morbidity and mortality in relation to all other pregnancy complications. Etiopathogenesis of these diseases is still insufficiently understood but it is thought that the placenta plays a key role in the development of these complications, and that placental insufficiency, which occurs as a result of insufficient adaptation of decidual intramiometrial and parts of the spiral arteries in the first few weeks of pregnancy, leading to a reduction of utero- placental circulation and local placental hypoxia, which adversely affects the mother and the fetus. In order to elucidate the pathophysiological mechanisms of hypertensive disorders in pregnancy and to find sufficiently sensitive makers for early prediction of the most severe forms of these diseases, so far have been investigated a number of proteins involved in the processes of creation and development of placental vascular network such as vascular endothelial growth factor (VEGF-A), placental growth factor (PlGF) and soluble fms-like receptor tyrosine kinase receptor (sFlt-1). OBJECTIVE: The aim of the study was to compare serum concentration of sFlt-1, PlGF, VEGF-A, PAPP-A, freeßhCG, glucose, total cholesterol, HDL cholesterol, LDL cholesterol, triglycerides, apo-AI, apo B, AST, ALT, GGT, creatinine, urea, uric acid, hsCRP, Na, K, Cl, P, Mg and Ca between the group of pregnant women with preeclampsia, chronic and gestational hypertension and the control group of pregnant women in the first trimester of pregnancy between 11 and 14 weeks gestation. Also the aim was to examine whether the value of selected laboratory parameters (sFlt-1, VEGF-A and PlGF) differ in relation to gestational week at the time of birth, weight, length and APGAR scoring system of newborns. The aim was to examine whether the value of angiogenic proteins: sFlt-1, VEGF-A and PlGF differ significantly in relation to the number of previous pregnancies and age of the pregnant woman. MATERIALS AND METHODS: The study was conducted as a prospective analytical study in the Clinical Center of Vojvodina, in the period from June 2012 to February 2015. The study included a total of 143 pregnant women aged 18 - 43 years. All pregnant women included in the study were divided into two study and one control group. The first study group consisted of 43 pregnant women who developed preeclampsia during the current pregnancy. The second study group consisted of 46 pregnant women who are newly diagnosed or confirmed chronic or gestational hypertension during the current pregnancy. The control group consisted of 54 healthy pregnant women with verified physiological outcome of pregnancy at term without maternal and fetal complications. Patients were included in the study between 11 + 0 and 13 + 6 weeks of gestation. All patients had data about risk factors for developing hypertensive disorders in pregnancy. After clinical and obstetric examination all patients underwent anthropometric measurements, measurement of blood pressure, and specialized ultrasound examination to determine precise gestational age of the fetus and to determine the risk for fetal chromosomal abnormalities. All patients signed a written consent of the patient's voluntary participation in the study. Serum levels of sFlt1, VEGF-A and PlGF were determined by quantitative ELISA (R & D Systems Europe Ltd., Abingdon, UK), while glucose, total cholesterol, HDL cholesterol, LDL cholesterol, triglycerides, apo-AI, apo B, AST, ALT, GGT, creatinine, urea, uric acid, hsCRP, Na, K, Cl, P, Mg, Ca were determined on automated analyzer systems. All pregnant women were categorized into 2 study and a control group on the basis of presence of hypertensive disorders in the current pregnancy. Statistical analysis was performed in 12 statistical program STATISTICA (StatSoft Inc., Tulsa, OK, USA). The data are presented in tables and graphs, the level of significance p is interpreted statistically significant if the p value was less than <0.05. RESULTS: Serum concentrations of sFlt-1 are statistically significantly different in all study groups and significantly higher in the groups with hypertensive disorders compared to healthy subjects p <0.001. Serum levels of VEGF-A are significantly lower in the preeclampsia group compared to healthy control group (p <0.001), while the levels of serum concentration of PlGF statistically significantly different between all groups so that the lowest values are observed in the preeclampsia group (p <0.001) compared to the other two study groups. There is no statistically significant differences in the levels of PAPP-A, biochemical parameters (glucose, AST, ALT, GGT creatinine, urea, uric acid), lipid parameters (total cholesterol, LDL, apo AI, apo B), inflammatory parameters (complete blood count, fibrinogen), hemostatic (D-dimer, vWF-antigen) and electrolyte status (Na, K, Cl, P, Mg, Ca), p> 0.05. Levels of free ßhCG and HDL cholesterol levels are significantly lower, while the value of hsCRP and triglycerides significantly higher in the group of women with preeclampsia compared to the healthy control group. Serum concentrations of sFlt-1 over 865 pg/ml have a sensitivity of 93% and specificity of 81.5% in predicting preeclampsia, while serum PlGF concentration below 60 pg/ml, a sensitivity of 88.4% and a specificity of 79.6% in predicting preeclampsia. Serum concentrations of sFlt-1, VEGF-A and PlGF do not show a statistically significant difference compared to the age of pregnant women and the number of previous pregnancies p> 0.05. Serum concentrations of sFlt-1 and PlGF are significantly different in relation to the weight of the newborn, so that the lower values of both proteins are in the group of infants with birth weight below 1500 gr. in relation to the body weight between 2800-3300 gr., p <0.001. There is also lower concentrations of sFlt-1 and PlGF in a group with deliveries before 33 weeks of gestation compared to the deliveries after 37 week of gestation, p <0.001. Serum concentrations of sFlt-1 and PlGF are significantly different in relation to the mother's body mass index so that the lower values of sFlt-1 and PlGF are in the group of women with a body mass index below 25 in relation to a group with a body mass index over 30 kg/m2, p <0.001. Serum concentrations of sFlt-1 in the first trimester of pregnancy were significantly associated with the parameters of inflammation (hsCRP), diastolic blood pressure and levels of free ßhCG. It is also observed a significant correlation between PlGF with a body mass index, systolic blood pressure and hsCRP concentration in the first trimester of pregnancy. CONCLUSION: The levels of anti-angiogenic protein sFlt-1 are higher in the group of pregnant women with preeclampsia than in the group with chronic and gestational hypertension and the control healthy group. Levels of proangiogenic VEGF-A protein are significantly lower in the preeclampsia group and group with gestational and chronic hypertension compared to the control group. Serum levels of proangiogenic PlGF protein are significantly lower in the preeclampsia group than in the group with chronic and gestational hypertension and the control group. Serum concentrations of placental protein free ßhCG and HDL cholesterol are significantly lower, while the value of hsCRP and triglycerides significantly higher in the preeclampsia group compared to the control group. Among pregnant women with hypertensive disorders in pregnancy and healthy pregnant women there are no significant differences in the values of placental PAPP-A protein, biochemical parameters (glucose, AST, ALT, GGT creatinine, urea, uric acid), lipid parameters (total cholesterol, LDL, apo AI, apo B), inflammatory parameters (complete blood count, fibrinogen), hemostatic (D-dimer, vWF-antigen) and electrolyte status (Na, K, Cl, P, Mg, Ca). Serum concentrations of sFlt-1 and PlGF are significantly different in relation to gestational age at delivery and newborn body weight and are lower in group with preterm delivery and newborns with lower birth weight. Serum concentrations of sFlt-1 are significantly different compared to body length and Apgar score, so that the higher values of sFlt-1 are associated with better outcome of newborns (greater body length and better APGAR score). Serum concentrations of sFlt-1, VEGF-A and PlGF are not different significantly with respect to age of pregnancy and the number of previous pregnancies. The levels of sFlt-1 and PlGF represents helpful markers in prediction of preeclampsia in the first trimester of pregnancy.

The placenta is responsible for mediating fetal growth and development, thereby influencing health across the lifespan. Physical activity (PA) confers benefits to mother and baby during pregnancy, but little is known about its impact on the placenta. There were two purposes of this study: i) to determine if maternal PA during pregnancy influences placenta mitochondrial protein content and function, and ii) to determine if there were differences in placenta mitochondrial protein content and function in different regions of the placenta, namely proximal or distal to the centre of the placenta. Healthy women between 12-28 weeks gestation were recruited, and free-living PA was objectively assessed at multiple time points during pregnancy using an accelerometer. Participants were grouped by minutes of moderate-to-vigorous PA (MVPA) per day. Placenta tissue samples were collected from central and distal placental regions immediately post-birth and were used for two separate analyses. Half of the samples were flash frozen in liquid nitrogen and used for western blot analysis of mitochondrial complex I-V proteins. Fresh mitochondria were isolated from the other half of the samples, and high-resolution respirometry was used to measure placental mitochondrial respiration. There were significant positive correlations between maternal PA and mitochondrial protein content in peripheral tissue samples, but protein content was significantly higher in central tissue compared to peripheral tissue samples. In addition, state 3 respiration was higher in central tissue samples of placentas from participants with high MVPA compared to participants with low MVPA. Finally, complex I protein was higher in central tissue samples of placentas from female offspring compared to placentas of male offspring. However, many of these results are underpowered and further study is warranted. This study provides new avenues to explore the relationship between PA and placenta mitochondria in healthy populations.

Apesar de sua importância clínica e epidemiológica, a fisiopatologia da préeclâmpsia ainda não foi completamente compreendida. Sabe-se que a doença constitui-se de uma fase pré-clínica e um estágio clínico. Durante a última década muito esforço tem se concentrado na identificação precoce da doença, ainda em sua fase pré-clínica. A literatura científica tem demonstrado claramente um desequilíbrio na regulação da angiogênese das gestantes com pré-eclâmpsia, marcado por níveis elevados do fator antiangiogênico soluble fms-like tyrosine kinase-1 (sFlt-1) e níveis diminuídos do fator pró-angiogênico placental growth fator (PlGF). Embora um número crescente de estudos em populações de alto risco tenha avaliado o papel desses biomarcadores no diagnóstico de pré-eclâmpsia, dados sobre sua utilização para a predição de pré-eclâmpsia superajuntada, cujo diagnóstico pode ser particularmente difícil, permanecem relativamente escassos e controversos. Com o presente estudo pretendemos avaliar o desempenho de medidas seriadas dos níveis maternos circulantes dos fatores sFlt-1 e PlGF, bem como da razão sFlt-1/PlGF, para predição de pré-eclâmpsia superajuntada e compará-lo ao seu desempenho na predição de pré-eclâmpsia em sua forma \"pura\", não superajuntada. Para este propósito, estudamos uma coorte prospectiva composta de dois braços, um de gestantes com hipertensão arterial crônica e outro de gestantes normotensas, e avaliamos os níveis séricos de sFlt-1 e de PlGF e a razão sFlt-1/PlGF nas idades gestacionais de 20, 26, 32 e 36 semanas, tendo como desfecho principal o diagnóstico de pré-eclâmpsia. Um total de 97 gestantes foram acompanhadas, 37 normotensas e 60 com hipertensão arterial crônica. Entre elas, 4 (10,8%) desenvolveram pré-eclâmpsia e 14 (23,3%) desenvolveram pré-eclâmpsia superajuntada. Para predição de pré-eclâmpsia, a análise ROC (Receiver Operating Characteristics) apresentou área sob a curva (AUC - area under curve) de 0,83 (IC 95% = 0,68-0,99, P = 0,035) para dosagem de PlGF com 20 semanas e AUC = 0,92 (IC 95% = 0,81 - 1,00, P = 0,007) para a razão sFlt-1/PlGF com 26 semanas de gestação. A variação percentual dos níveis de PlGF entre 26 e 32 semanas de gestação apresentou AUC = 0,96 (IC de 95% = 0,89-1,00, P = 0,003). Para a predição de pré-eclâmpsia superajuntada, a razão sFlt-1/PIGF na idade gestacional de 32 semanas apresentou AUC = 0,69 (IC de 95% = 0,53-0,85, P = 0,039). Entre 20 e 26 semanas de gestação, a variação percentual do PIGF e da razão sFlt-1/PlGF apresentaram, respectivamente, AUC = 0,74 (IC de 95% = 0,58-0,90, P = 0,018) e AUC = 0,71 (IC 95% = 0,52-0,91, P = 0,034). Por nossos resultados podemos concluir que, embora os níveis de PlGF e a razão sFlt-1/ PlGF tenham apresentado bons desempenhos na predição de pré-eclâmpsia, é preciso ter cuidado ao usá-los para a predição de pré-eclâmpsia superajuntada. Nessas gestantes, a dosagem dos fatores angiogênicos apresenta capacidade de predição menor e mais tardia. Avaliações seriadas dos fatores podem melhorar o desempenho dos testes para predição de pré-eclâmpsia superajuntada em idades gestacionais mais precoces
Despite being a major public health problem, the pathophysiology of preeclampsia is incompletely understood. Preeclampsia progression comprises a pre-clinical stage and a clinical stage. During the last decade much work has focused on identifying the pre-clinical stage of preeclampsia. Many researchers have clearly demonstrated an anti-angiogenic imbalance that is marked by higher levels of soluble fms-like tyrosine kinase-1 (sFlt-1) and lower levels of placental growth factor (PlGF) in the subjects who develop preeclampsia compared with those who do not. Although a growing number of studies in the high-risk population have shown the role of these biomarkers in diagnosing preeclampsia, superimposed preeclampsia, which can be a challenging diagnosis, remains partially understudied and the literature regarding this subject continues to be relatively scarce as well as controversial. By this study, we aimed to evaluate the performance of serial measurements of maternal circulating sFlt-1 and PlGF levels for the prediction of superimposed preeclampsia in chronic hypertensive subjects and to compare it to the prediction of preeclampsia in normotensive control subjects. For this purpose, we evaluated a two-armed prospective cohort of women with normotensive and chronic hypertensive pregnancies and assessed the serum levels of sFlt-1 and PlGF and the sFlt-1/PlGF ratio at gestational ages of 20, 26, 32 and 36 weeks, having preeclampsia as the primary outcome to be predicted. A total of 97 women were followed-up, 37 in the normotensive group and 60 in the chronic hypertensive group. Among them, 4 (10.8%) women developed preeclampsia and 14 (23.3%) developed superimposed preeclampsia. For predicting preeclampsia, PlGF at 20 gestational weeks presented an AUC=0.83 (CI 95% = 0.68 - 0.99, P=0.035) and the sFlt-1/PlGF ratio at 26 gestational weeks presented an AUC=0.92 (CI95% = 0.81 - 1.00, P=0.007). The percent change of the PlGF levels between 26 and 32 gestational weeks presented an AUC=0.96 (CI 95% = 0.89 - 1.00, P=0.003). For predicting superimposed preeclampsia, the sFlt-1/PlGF ratio at 32 gestational weeks presented an AUC=0.69 (CI 95% = 0.53 - 0.85, P=0.039). Between 20 and 26 gestational weeks, the percent change of PlGF and the sFlt-1/PlGF ratio presented, respectively, an AUC=0.74 (CI 95% = 0.58 - 0.90, P=0.018) and an AUC=0.71 (CI 95% = 0.52 - 0.91, P=0.034). By our results, we concluded that, although the PlGF level and the sFlt-1/PlGF ratio present good performances in the prediction of preeclampsia, caution is required when using them for the prediction of superimposed preeclampsia. Sequential assessments slightly improve the test performances for predicting superimposed preeclampsia at earlier gestational ages

Placenta is a transient association of the fetal and maternal tissues, that develops during pregnancy, in most viviparous animals. The evolution of placenta ensured the development of the fetus inside the womb of the mother, providing a protected environment for the development of the fetus, and preventing the loss of progeny due to unfavorable environmental conditions. Because it is strategically poised at the maternal and fetal interface, the placenta is ideally suited to carry out alimentary, respiratory and excretory functions for the developing fetus. In addition, it serves as an immunological barrier preventing the rejection of the fetal semi-allograft, by the maternal immune system. Furthermore, the placenta elaborates a variety of protein, polypeptide and steroid hormones. These include growth factors, growth factor receptors, neuropeptides, opioids, progesterone and estrogen, whose secretion is dependent on the gestational age of the placenta and its differentiation status. The human placenta, adapts itself remarkably to cater to the changing requirements of the developing fetus. For instance, during the first trimester of pregnancy, the placenta is an actively dividing, a highly invasive and a rapidly differentiating organ; while near term, it represents a fully differentiated and a non-invasive unit. Furthermore, the placenta of the first trimester and that at term differ in their hormone profiles, extents of apoptosis, expression of several transcription factors, etc. This dramatic change in the phenotype of the human placenta can be considered to be the outcome of an intrinsically programmed pattern of differentiation, which may be referred to as the functional differentiation of the placenta. It may be hypothesized therefore, that this functional differentiation could be brought about by the differential expression of genes in the first trimester and the term placenta. The objectives of the present study were: 1. To gain an insight into this process of " functional differentiation” by investigating the differential expression of genes in the two developmentally distinct stages during gestation, viz. during the first trimester and at term. 2. To understand the functional relevance of the differentially expressed genes. A general introduction of the human placenta, describing the importance of differential expression in modulating placental function, is discussed in chapter 1. The functions of the human placenta along with a brief description of its development and differentiation are also briefly described. A Differential Display RT-PCR-based (DD RT-PCR) approach, using total RNA from the first-trimester and term placental villi, was employed to display the differentially expressed genes in the first trimester and the term placenta. The display so generated was used to identify a few differentially expressed cDNAs. This study was aimed at understanding the functional significance of the transcripts which were identified from the display, rather than just concentrate on documenting the differences in the gene expression patterns in the first trimester and the term placental tissue. A detailed description of the methodology adopted for performing DD-PCR using placental tissue, discussing the advantages and disadvantages of using differential display PCR, is described in chapter2. The use of DD-PCR for studying differential gene expression in the human placenta was validated by the finding that one of the cDNAs that was differentially expressed in the first trimester placental tissue, is a fragment of β-hCG cDNA. It is well documented that the differential expression of the β-subunit of hCG (human chorionic gondatrophin) during the first eight weeks of gestation is the rate limiting step in the synthesis and secretion of the functional hormone, which comprises the α and the β-subunits. Furthermore, the use of the model system viz., the first trimester and term placental tissue, was also validated for carrying out DD-PCR by ensuring that all placental samples used for DD analysis were free of endometrial contamination. A detailed description of optimization and validation of DD-PCR in human placental tissues is given in chapter 2. Cloning and sequencing of yet another cDNA from the first trimester differential display revealed that it is T-Plastin. T-Plastin is a member of a family of proteins that are involved in actin-bundling. Northern blot analysis and immunohistochemical studies using an antibody generated to a peptide corresponding to human T-Plastin, confirmed its differential expression and localization in the first trimester placenta. Considering the fact that several carcinomas show enhanced expression of T-Plastin, we tested the hypothesis that its differential expression is correlated with the proliferative potential of the first-trimester placenta It was observed that the first-trimester tissue expressed high levels of beta-actin as compared to the term placental tissue. This is in agreement with the up-regulation of beta-actin following mitogenic stimulation/proliferation and during neoplastic transformation or transformation-associated invasive behaviour of cells, two characteristic features shared by the early placenta with cancerous tissues. Based on our studies and available information in the literature, it is proposed that T-Plastin expression in the first trimester placenta is a growth-associated phenomenon which is partially responsible for the tumor-like phenotype of the first trimester tissue. Studies carried out with the partial T-Plastin cDNA clone that was isolated from the first trimester differential display, are presented in chapter 3. Sequencing of yet another cDNA clone identified from the term placental differential display, T-18 revealed that it had no homology to any known sequence in the nucleotide or est databases. The sequence corresponding to this clone was submitted to the GenBank and was assigned an accession number- AF089811. The differential expression of T-18 was confirmed by Northern blot analysis and RT-PCR analysis. Attempts were made to isolate the full-length cDNA corresponding to T-18 from a commercially available library from Clontech. However, repeated trials to identify the clone corresponding to T-18 did not yield any positive results. However, a genome database search revealed that T-18 was a portion of a large contig contained in chromosome 15. Analysis of the annotated gene sequences in and around the region in which T-18 is located in chromosome 15, revealed that there are very few ests reported in this contig and quite a few repeat sequences reported. Interestingly, it was observed that 6 kb downstream of the region in which T-18 is located, there was an est that had homology to a Bcl-2 precursor protein (an evolutionarily conserved, anti-apoptotic protein, capable of conferring protection against death-inducing signals) and the death adaptor protein, CRADD {Caspase and RIP adapter with death domain). Further updating of the ests in the database might probably be of help in the identification of the full-length cDNA corresponding to T-18 and confirm as to whether T-18 is a part of the gene/gene cluster that comprises the afore-mentioned est. An account of the identification and cloning of T-18 from the term placenta and the attempts to isolate the full-length cDNA clone corresponding to T-18 from a term placental cDNA library, is described in chapter 4. In the absence of any information on the identity of T-18, a study to understand the functional significance of T-18 expression was carried out. Since it was not possible to carry out studies pertaining to the temporal expression of T-18 throughout gestation on the human placenta for ethical reasons, alternate animal/organ models were employed to study T-18 expression. Rat placenta and rat Corpus Luteum (CL) were chosen as alternate models for studying T-18 expression as these two organs/tissues underwent dynamic changes in their function throughout pregnancy. For instance, it is well known that CL is the primary source of progesterone for maintaining pregnancy in the rat and that the progesterone secreting capacity of the luteal cells peak on day 16 of gestation and decline thereafter. Interestingly, a common feature among all the tissues that were chosen for investigating the regulation of T-18 expression, is the fact that they underwent apoptosis with increase in gestational age. The expression of T-18, in tissues exhibiting increased incidence of apoptosis suggested that T-18 maybe an apoptosis-associated gene. Using an explant culture model it was demonstrated that placental villi when cultured in vitro underwent spontaneous apoptosis and that the levels of T-18 message increased, under these conditions. Furthermore, this spontaneous induction of apoptosis in explant cultures could be blocked when villi were cultured in the presence of superoxide dismutase, a free radical scavenging enzyme. In addition, the expression of T-18 was shown to be modulated following treatment with SOD, or in response to oxidative stress. These studies clearly indicate a role for T-18 in placental apoptosis and moreover, implicate the usefulness of explant culture to examine the molecular mechanisms involved in placental apoptosis. Furthermore, the expression of the anti- and pro-apoptotic genes, bcl-x and bax respectively, were investigated, in an attempt to elucidate the signalling pathway(s) that led to the activation of an important downstream protease, caspase-3, in placental apoptosis. The present study revealed that induction of apoptosis in the placenta in vitro involved a bcl/bax independent, caspase-3 dependant pathway. The validation of an explant culture model for studying placental apoptosis and data pertaining to the role of T-18, bcl-x, bax and CPP32 in placental apoptosis, in response to oxidative stress, are presented in chapter 5. The last section titled general discussion summarizes the work carried out in this study and proposes a model for the apoptotic mechanism(s) that may be operating in placenta In conclusion, the present study has led to the identification of two developmentally-regulated factors, T-Plastin and T-18 in the first trimester and term placenta, respectively. The differential expression of these genes, in addition to several other molecular players, is proposed to be responsible for the overall functional differentiation of the placenta through the course of gestation.

The placental type plasminogen activator inhibitor (PAI-2) has been purified from extracts of human placenta and from a histiocytic lymphoma cell line. It is mainly an uPA inhibitor but it also inhibits the two-chain form of tPA.In order to determine the factors regulating PAI-2 gene expression and thereby clarify the physiological role of PAI-2 we have undertaken the molecular cloning of PAI-2 cDNA. A λgt11 expression library prepared from placental mRNA, was screened, immunologically using a monoclonal antibody probe developed against PAI-2 purified from human placenta. When 1.7×105 recombinant phages were screened six positive clones were obtained. Hybridization experiments and comparison of restriction enzyme cleavege pattern revealed that the DNA inserts of the six clones were, related. To identify the clones as coding for PAI-2, a lysogen made from one of them was induced, and the proteins were separated by SDS-PAGE. In immuno-blotting wxperiments the recombinant fusion protein and purified PAI-2 were recognized by the monoclonal antibody and a monospecific polyclonal antibody against PAI-2, revealing an immunological similarity. The nucleotide sequence of the largest cDNA was determined. It was found to code for a protein with extensive sequence homology with members of the serine protease inhibitor family (serpins) Alignment of the active center region with other serpins indicates that PAI-2 is an arg-serpin, as expected for an inhibitor of plasminogen activators.

Placental function including oxygen delivery and nutrient transport are critical determinants of fetal growth, moderating the risks of obesity and metabolic diseases later in life. Previously, we demonstrated in a mouse model that parental diet and exercise play important roles in placental lipid content and inflammation. Transient receptor potential canonical channel 1 (TRPC1) is a Ca2+-permeable integral membrane protein. We have demonstrated that TRPC1 increases total body adiposity in mice by decreasing the efficacy of exercise to limit adipose accumulation under a high fat (HF) diet. Importantly, intracellular calcium may regulate total body adiposity and increased total body adiposity could promote placental lipid accumulation. Similarly, intracellular calcium regulates membrane lipid content via the activation of the protein kinase C. Membrane lipids such as sphingomyelin are key regulators of cell signaling. Maternal HF diets increase placental tissue lipid concentrations resulting in compromised nutrient transport to fetus. However, the specific lipid species that accumulate due to the absence of the placental TRPC1 gene under maternal HF diet feeding is not yet known. We hypothesized that placental tissue response to a maternal HF diet is disrupted in TRPC1 mice fed a maternal HF diet resulting in greater cellular sphingomyelin concentrations. Results showed placentae from TRPC1 KO mice fed high fat diet (45% en, HF) had increased sphingomyelin concentrations compared to control diet (16% en, NF). Placentae from WT mice fed HF diet exhibited diet-dependent increases in ceramide concentration with no concomitant increase in sphingomyelins compared to NF fed WT mice. Additionally, 11 placental triacylglycerol (TAG) species were different based on diet, 16 based on genotype, and 5 were affected by both diet and genotype. These results suggest that during a HF diet, loss of TRPC1 function reduces placental sphingomyelin hydrolysis into ceramide and that placental TAG concentrations respond in diet- and genotype-dependent manner.

An anticoagulant protein was purified from the soluble fraction of human placenta by ammonium sulfate precipitation and column chromatography on DEAE-Sepharose, Sephadex G-75, and mono S (Pharmacia). Approximately 30 mg of the protein was purified from one placenta. The purified protein gave a single band on SDS polyacrylamide gel and had a molecular weight of 36,500. This protein inhibited both kaolin and thromboplastin induced clotting times of normal human plasma. It also inhibited the clotting time of platelet-rich plasma induced by factor Xa, but did not affect thrombin activity of fibrinogen-fibrin conversion. The protein neither bound factor Xa nor inhibited the amidase activity of factor Xa. This protein specifically bound to phospholipid vesicles prepared from a mixture of phosphatidylserine and phosphatidylcholine (20 to 80 weight ratio) in the presence of calcium ions. The purified protein was digested with cyanogen bromide and the resulting fragments were separated by FPLC. Partial amino acid sequences of the cyanogen bromide fragments showed that this protein was composed of at least three repeats that were homologous to the four repeats found in lipocortin I and II. Lipocortins are known to inhibit the phospholipase A2 activity, probably by binding to the phospholipid substrate. These results indicate that the placental anticoagulant protein is a member of the family of lipocortins and probably inhibits coagulation by binding to phospholipid vesicles. Supported in part by grants HL 16919 and HL 18645 from National Institute of Health.

Endothelium is important to maintain blood fluidity preventing coagulation. Glycosaminoglycan in the endothelial cell plasma membrane has been thought to prevent activation of blood coagulation. Heparin-like compound, which is a potent anticoagulant activity, has been localized on the surface of the cultured endothelial cells. Anticoagulant action associated with thrombomodulin, which is present in endothelial cells, is another mechanism to provide hemostatic nature of endothelial cells.We wondered whether any other intracellular protein(s) is involved in coagulation. We looked for such a protein(s) in cultured bovine aortic endothelial cells. We soon found an anticoagulant activity in the soluble fraction of endothelial cells and it was partially purified. This activity was adsorbed to DEAE-Sepharose and eluted from a gel filtration column in a molecular weight range of 30,000-40,000. However, limited amounts of the cells made it difficult to purify this activity. We then chose human placenta as a substitute source of this protein and have continued the purification of this anticoagulant activity.In this communication, we describe the isolation and characterization of a placental anticoagulant protein, called "PAP", which is silmilar or possible same as the endothelial anticoaguant protein. PAP was purified from the soluble fraction of human placenta by ammonium sulfate precipitation and column chromatography on DEAE-Sepharose, Sephadex G-75, and mono S (Pharmacia). Approximately 20 mg of the protein was purified from one placenta. The purified protein gave a single band by SDS polyacrylamide gel electrophoresis with a molecular weight of 36,500. This protein inhibited both kaolin- and thromboplastin-induced partial thromboplastin times of normal human plasma. It also inhibited the clotting time of platelet-rich plasma induced by factor Xa, but did not affect the thrombin activity of fibrinogen-fibrin conversion. The purified protein completely inhibited the prothrombin activation by reconstituted prothrombinase. The protein neither inhibited the amidolytic activity of factor Xa nor bound factor Xa. This protein specifically bound to phospholipid vesicles (20% phosphatidylserine and 80% phosphatidylcholine) in the presence of calcium ions. These results indicate that PAP inhibits coagulation through the binding to phospholipid vesicles. The study on the amino acid sequence of PAP is in progress in our laboratory. Surprisingly, the sequence analysis of the cyanogen bromide fragments revealed that PAP is a new member of the lipocortin or calpactin family. The sequences of several cyanogen bromide fragments of PAP aligns with the sequences of lipocortin I and II with over 50% identity.Since PAP interacts directly with phospholipid rather than factor Xa, other activation steps in the coagulation cascade, in which phospholipid is involved, are pro^|bly affected by PAP. These reactions are the activation of factor X by a complex of factor IXa-factor VIIIa-phospholipid-Ca++ and the activations of factor X and factor IX by a tissue factor-factor VIIa-Ca++ complex.Reutelingsperger et. al,, have reported the isolation of a novel inhibitor from arteries of human umbilical cord. This protein inhibited the prothrombin activation by prothrombinase. The authors proposed that the inhibition mechanism of this inhibitor was a competition with factor Xa for binding to phospholipid. This protein is very similar to PAP as to the mode of inhibition. The molecular weight of this inhibitor is 32,000, which is slightly smaller than PAP. With the limited chemical characterization of this protein, presently it is difficult to identify this inhibitor with PAP.At the present time, the physiological role and origin of PAP is not known. PAP may originate from the endothelium of placenta, because we have detected a PAP-like anticoagulant activity in bovine aortic endothelial cells. This activity and PAP were quite alike in the purification up to the gel filtration step. If PAP antibody recognizes the antigen in the endothelial cells, it is interesting to see whether PAP localizes on the surface or inside the cells. Nevertheless, if PAP is present in the endothelial cells, it may play an important role to maintain the hemostatic nature of endothelium. PAP may bind phospholipid components at injured sites, before coagulation factors come in contact with lipid components and initiate thrombolytic events.

Interferences of plasmin inhibitors are a main drawback in the functional assay of PAI in plasma. To overcome them, an acidification step, prior to residual tPA assay has been frequently used.A simple chromogenic method, based on the inhibition of purified tPA by plasma prediluted in PAI-depleted plasma, is described. The residual tPA was subsequently measured in the presence of fibrinogen fragments used as Stimulator, plasminogen and chromogenic substrate (CBS 30.41). The assay can discriminate between PAI and plasmin inhibitors since the concentration of these proteins remains constant because brought in excess by the PAI-depleted plasma. Parallel dose-response curve were obtained when PAI-depleted plasma was used instead of buffer, for the preparation of the reference curve and for the dilution of samples. The residual tPA activity measured was directly proportional to the PAI activity of the sample using a single concentration of tPA. Samples containing 0 to 30 IU/ml of PAI could be measured using a 1:40 dilution.Values for 27 normal volunteers at rest were 5.1 IU/ml (range : 0 to 11.8 IU/ml). PAI activity was decreased in afternoon samples (4.6 IU/ml) compared to those of the morning (5.8 IU/ml). After stasis, when a concomitant release of tPA was observed, the values obtained (3.1 IU/ml) were significantly lower than the values obtained before (4.1 IU/ml).The activity measured is probably related to PAI-1 (endothelial ceil type PAI), but PAI-2 related activity (placental PAI) can be also estimated since pregnancy plasma showed high levels of inhibitor (mean : 15.1 IU/ml, range : 9.5 to 28.1 IU/rni).The present method is simple and rapid to perform. It can be routinely used in the screening of various thrombotic disorders.

Thrombomodulin(TM) is a cell surface protein found on endothelial cell that binds thrombin and increases thrombin's ability to activate protein C(PC). In the present study, we examined hematological characteristics and behavior of plasma PC level in pregnant women and localization of TM in the placental villous tissue. The results obtained are reported here. Cubital venous blood of 20 normal pregnants, 8 purperants and 60 non-pregnants. PC antigen(PC:Ag) was measured by the Laurell's technique using Assera plate-proteinC. Localization of TM was determined in such a way that the villous tissue was fixed in formalin, cut into paraffin sections, and stained by ABC method using anti-TM antibody. Coagulation-related factors of the pregnant blood, i.e. fibrinogen, FV, FVIII, FX, FXII and prekallikren showed statistically higher values compared with the control group, and AT-III showed almost simillar value to the control group or tended to decrease to scxne extent compared therewith. As for fibrinolysis-related factors, on the other hand, plasminogen, α1-antitripsin showed higher values with the progress of pregnancy, and α2-macro_ globulin showed slightly lower values in both 3rd trimester and puerperal stage. PC:Ag increased in the 2nd and 3rd trimester (p<0.01). Comparative examinations made of PC:Ag level between pre- and post-taking oral centraceptives revealed a significant (p<0.05) decrease frcm 131.0%( pre-taking) to 117.0%(post-taking). At the 11th week of pregnancy, TM was confirmed to be highly localized in syncytiotrophoblast in the villous tissue, especially in microvilli. At the 40th week, TM was also confirmed in the same site, but with weaker stainability. It was suggested that thrombosis and hemostasis-related factors were in a state of co-existing overproduction and consumption. Moreover we supposed that the existence of TM and the increase in PC might be just appropriate for the maintenance of anti-thrombogenesis in the uteroplacental circulation.

It has been reported that the tissue factor(TF) of monocytes and leukemic leukocytes is one of the trigger substances of disseminated intravascular coagulation(DIC) in leukemia patients. To assess the properties of TF of leukemia cells, their TF was analyzed by the method of Western blotting. Placenta TF was purified using Concanavalin-A affinity chromatography. Briefly, human placenta TF was extracted from placenta acetone powder using Triton X-100 extraction and purified by Concanavalin-A affinity chromatography and SDS-preparative PAGE. Hie final product of the purified placenta TF-apoprotein exhibited a molecular weight (MW) approximately 46 kD in non-reduced condition and 47 kD in reduced condition and showed single band on analytical SDS-PAGE. Activity of purified placenta apo-TF after relipidation with placenta phospholipid was 4500 units/mg and was 6300 times purified from the starting material. Serum IgG fraction was separated from rabbits after 8 times weekly injections of purified placenta TF. This anti-TF-IgG fraction showed single precipitin line against purified placenta apo-TF and inhibited procoagulant activity of saline extract of the placenta as well as those of homogenates of the endotoxin(LPS)-stimulated Molt-4 cells and U-937 cells. Using this antibody, analysis of TF of cultured leukemia cells(Molt-4 and U-937) and acute promyelocytic leukemia(APL) cells was done by the Western blotting technique as follows: LPS-stimulated Molt-4, U-937 or APL cells were homogenized, delipidated and solubilized with 1% SDS-PAGE. Thereafter, Western blotting was done by the method of Towbin and TF of the blotted protein was immunologically identified. The nitrocellulose paper was soaked with anti-TF-IgG for 18 hours’, then peroxidase-conjugated anti-rabbit-goat IgG was used as an indicator of the antibody and o-dianisidine as substrate. In either case of APL cells, LPS-stimulated Molt-4 or U-937, single band which showed procoagulant activity was detected and the MW of the protein was approximately 48 kD in non-reduced condition. These results indicate that TF of all these leukemic leukocytes has common antigenic determinant and equal MW to placenta TF.

The activation of protein C (PC) by thrombin requires the presence of an endothelial membrane cofactor, thrombomodulin (TM). Activated PC (APC) exerts its anticoagulant activity by degrading factors (F) Va and Villa in the presence of phospholipids and of a vitamin K-dependent cofactor, protein S. Tissue factor (TF) is the essential cofactor of factor Vll/VIIain the activation of factor X. TF is synthetized by several cell lines including endothelial cells. Using a specific TM assay, up to 0.85 units of TM activity could be detected in commercial thromboplastin (TP) preparations from human placenta or rabbit or porcine lung, when the amount of TP was adjusted to contain 1 unit of TF activity. Preparations from brain contained very low amounts, if any, of this activity (< 0.02 TM units). In order to evaluate the effects of the presence of TM activity in some TP preparations, the stability of F V and VIII activities was examined after activation of the coagulation system by these TP in various plasmas. PC deficient plasmas, plasmas lacking F V, VIII or IX and immunoadsorbed PC deficient plasma supplemented with purified human PC (5 Ug/ml) were used. After activation with placenta or lung TP, F V and VIII activities were markedly reduced ( ∼ 90 % reduction) in normal and hemophiliac plasmas, whereas they remained high after activation with brain TP. F V and VIII activities were preserved in protein C deficient plasma after activation by all TP preparations. The same decrease of F V and VIII activities was observed after activation of immunoadsorbed PC deficient plasma supplemented with purified PC with placenta or lung TP only. Preincubation of TP from human placenta with antibodies to human TM raised in laying hens abolished the capacity of this preparation to destroy F V activity of PC containing plasmas. These results establish the presence of TM activity in crude thromboplastin preparations from placenta or from lung. Surprisingly, this anti-coagulant activity seems to be absent from brain. TM from placenta or lung extracts is responsible for the degradation of F V and VIII.

Central problems in sheep and dairy cattle production are reproductive failure due to embryonic/fetal mortality and low birth weights, especially in prolific breeds, and reduced milk yields which adversely affect neonatal survival and economy of production. The sheep placenta expresses lactogenic (ovine placental lactogen, oPL) and somatogenic (ovine placental growth hormone, oGH) hormones. Our research has focused on the biological roles of oPL and oGH in function of the uterine endometrium during gestation and the mammary gland during pregnancy and lactation. Major conclusions were that: ( 1 ) immunization of prepubertal ewes against oPL resulted in increased birth weights of their lambs and their milk production during lactation; (2) neither oPL nor oGH had an antiluteolytic effect on uterine endometrium to affect lifespan of the corpus luteum; (3) only sequential exposure of the progesterone stimulated uterus to oIFNt and oPL or oGH increased endometrial gland proliferation and secretory protein gene expression; (4) oPL signals through a homodimer of ovine prolactin receptor (PRL-R) and heterodimer of oPRL-R and growth hormone receptor (GH-R); (5) exogenous recombinant oPL and oGH stimulated mammogenesis and milk yield during lactation; and (6) mutation of oPL and oGH was used to define specific biological effects and a rational basis for design of a specific receptor agonists or antagonists. This project was very productive in elucidating basic biological effects of oPL and oGH on intracellular signal transduction pathways, uterine development and secretory function, as well as mammogenesis and lactogenesis. We determined that immunization of prepubertal ewes against roPL increased birth weights of their lambs, especially those born as twins and triplets, as well as enhanced lactational performance. These studies significantly extended our knowledge of uterine and fetal-placental physiology and provided a foundation for new strategies to enhance reproductive and lactation efficiency. Based on these results, the major achievements were: 1) creation of a practical and cost effective management tool for producers to increase reproductive performance, neonatal survival, and milk yield of ewes in commercial flocks; and 2) define, for the first time, biological effects of oPL on endometrial functions and gene expression by uterine gland epithelium.

Original Objectives 1. To determine the Brucella genes that lead to chronic macrophage infection. 2. To identify Brucella genes that contribute to infection. 3. To confirm the importance of Brucella genes in macrophages and placental cells by mutational analysis. Background Brucella spp. is a Gram-negative facultative intracellular bacterium that infects ruminants causing abortion or birth of severely debilitated animals. Brucellosis continues in Israel, caused by B. melitensis despite an intensive eradication campaign. Problems with the Rev1 vaccine emphasize the need for a greater understanding of Brucella pathogenesis that could improve vaccine designs. Virulent Brucella has developed a successful strategy for survival in its host and transmission to other hosts. To invade the host, virulent Brucella establishes an intracellular niche within macrophages avoiding macrophage killing, ensuring its long-term survival. Then, to exit the host, Brucella uses placenta where it replicates to high numbers resulting in abortion. Also, Brucella traffics to the mammary gland where it is secreted in milk. Missing from our understanding of brucellosis is the surprisingly lillie basic information detailing the mechanisms that permit bacterial persistence in infected macrophages (chronic infection) and dissemination to other animals from infected placental cells and milk (acute infection). Microarray analysis is a powerful approach to determine global gene expression in bacteria. The close genomic similarities of Brucella species and our recent comparative genomic studies of Brucella species using our B. melitensis microarray, suqqests that the data obtained from studying B. melitensis 16M would enable understanding the pathogenicity of other Brucella organisms, particularly the diverse B. melitensis variants that confound Brucella eradication in Israel. Conclusions Results from our BARD studies have identified previously unknown mechanisms of Brucella melitensis pathogenesis- i.e., response to blue light, quorum sensing, second messenger signaling by cyclic di-GMP, the importance of genomic island 2 for lipopolysaccharide in the outer bacterial membrane, and the role of a TIR domain containing protein that mimics a host intracellular signaling molecule. Each one of these pathogenic mechanisms offers major steps in our understanding of Brucella pathogenesis. Strikingly, our molecular results have correlated well to the pathognomonic profile of the disease. We have shown that infected cattle do not elicit antibodies to the organisms at the onset of infection, in correlation to the stealth pathogenesis shown by a molecular approach. Moreover, our field studies have shown that Brucella exploit this time frame to transmit in nature by synchronizing their life cycle to the gestation cycle of their host succumbing to abortion in the last trimester of pregnancy that spreads massive numbers of organisms in the environment. Knowing the bacterial mechanisms that contribute to the virulence of Brucella in its host has initiated the agricultural opportunities for developing new vaccines and diagnostic assays as well as improving control and eradication campaigns based on herd management and linking diagnosis to the pregnancy status of the animals. Scientific and Agricultural Implications Our BARD funded studies have revealed important Brucella virulence mechanisms of pathogenesis. Our publication in Science has identified a highly novel concept where Brucella utilizes blue light to increase its virulence similar to some plant bacterial pathogens. Further, our studies have revealed bacterial second messengers that regulate virulence, quorum sensing mechanisms permitting bacteria to evaluate their environment, and a genomic island that controls synthesis of its lipopolysaccharide surface. Discussions are ongoing with a vaccine company for application of this genomic island knowledge in a Brucella vaccine by the U.S. lab. Also, our new technology of bioengineering bioluminescent Brucella has resulted in a spin-off application for diagnosis of Brucella infected animals by the Israeli lab by prioritizing bacterial diagnosis over serological diagnosis.

Aim 1: To determine levels of this second messenger in: a) B. melitensiscyclic-dimericguanosinemonophosphate-regulating mutants (BMEI1448, BMEI1453, and BMEI1520), and b) B. melitensis16M (wild type) and mutant infections of macrophages and immune competent mice. (US lab primary) Aim 2: To determine proteomic differences between Brucelladeletion mutants BMEI1453 (high cyclic-dimericguanosinemonophosphate, chronic persistent state) and BMEI1520 (low cyclicdimericguanosinemonophosphate, acute virulent state) compared to wild type B. melitensisto identify the role of this second messenger in establishing the two polar states of brucellosis. (US lab primary with synergistic assistance from the Israel lab Aim 3: Determine the level of Brucellacyclic-dimericguanosinemonophosphate and transcriptional expression from naturally infected placenta. (Israel lab primary with synergistic assistance from the US lab). B. Background Brucellaspecies are Gram-negative, facultative intracellular bacterial pathogens that cause brucellosis, the most prevalent zoonosis worldwide. Brucellosis is characterized by increased abortion, weak offspring, and decreased milk production in animals. Humans are infected with Brucellaby consuming contaminated milk products or via inhalation of aerosolized bacteria from occupational hazards. Chronic human infections can result in complications such as liver damage, orchitis, endocarditis, and arthritis. Brucellaspp. have the ability to infect both professional and non-professional phagocytes. Because of this, Brucellaencounter varied environments both throughout the body and within a cell and must adapt accordingly. To date, few virulence factors have been identified in B. melitensisand even less is known about how these virulence factors are regulated. Subsequently, little is known about how Brucellaadapt to its rapidly changing environments, and how it alternates between acute and chronic virulence. Our studies suggest that decreased concentrations of cyclic dimericguanosinemonophosphate (c-di-GMP) lead to an acute virulent state and increased concentrations of c-di-GMP lead to persistent, chronic state of B. melitensisin a mouse model of infection. We hypothesize that B. melitensisuses c-di-GMP to transition from the chronic state of an infected host to the acute, virulent stage of infection in the placenta where the bacteria prepare to infect a new host. Studies on environmental pathogens such as Vibrio choleraeand Pseudomonas aeruginosasupport a mechanism where changes in c-di-GMP levels cause the bacterium to alternate between virulent and chronic states. Little work exists on understanding the role of c-di-GMP in dangerous intracellular pathogens, like Brucellathat is a frequent pathogen in Israeli domestic animals and U.S. elk and bison. Brucellamust carefully regulate virulence factors during infection of a host to ensure proper expression at appropriate times in response to host cues. Recently, the novel secondary signaling molecule c-di-GMP has been identified as a major component of bacterial regulation and we have identified c-di-GMP as an important signaling factor in B. melitensishost adaptation. C. Major conclusions, solutions, achievements 1. The B. melitensis1453 deletion mutant has increased c-di-GMP, while the 1520 deletion mutant has decreased c-di-GMP. 2. Both mutants grow similarly in in vitro cultures; however, the 1453 mutant has a microcolony phenotype both in vitro and in vivo 3. The 1453 mutant has increased crystal violet staining suggesting biofilm formation. 4. Scanning electron microscopy revealed an abnormal coccus appearance with in increased cell area. 5. Proteomic analysis revealed the 1453 mutant possessed increased production of proteins involved in cell wall processes, cell division, and the Type IV secretion system, and a decrease in proteins involved in amino acid transport/metabolism, carbohydrate metabolism, fatty acid production, and iron acquisition suggesting less preparedness for intracellular survival. 6. RNAseq analysis of bone marrow derived macrophages infected with the mutants revealed the host immune response is greatly reduced with the 1453 mutant infection. These findings support that microlocalization of proteins involved in c-di-GMP homeostasis serve a second messenger to B. melitensisregulating functions of the bacteria during infection of the host.

The goal of the one-year feasibility study was to identify specific cytotoxic T-lymphocyte (CTL) epitopes to Neosporacaninum in the natural bovine host in order to make progress toward developing an effective peptide-based vaccine against bovine neosporosis. We tested the hypothesis that: N. caninum SRS2 peptides contain immunogenicCTLepitope clusters cross-presented by multiple bovine MHC-I and MHC-IIhaplotypes. The specific objectives were: (1) Map bovine CTLepitopes of N. caninum NcSRS-2 and identify consensus MHC-I and class-II binding motifs; and (2) Determine if subunit immunization with peptides containing N. caninum-specificCTLepitopes cross-reactive to multiple bovine MHChaplotypes induces a CTL response in cattle with disparate MHChaplotypes. Neosporosis is a major cause of infectious abortion and congenital disease in cattle, persisting in cattle herds via vertical transmission.5 N. caninum abortions are reported in Israel; a serological survey of 52 Israeli dairy herds with reported abortions indicated a 31% infection rate in cows and 16% infection rate in aborted fetuses.9,14 Broad economic loss due to bovine neosporosis is estimated at $35,000,000 per year in California, USA, and $100,000,000 (Australian) per year in Australia and New Zealand.13 Per herd losses in a Canadian herd of 50 cattle are estimated more conservatively at $2,305 (Canadian) annually.4 Up to date practical measures to reduce losses from neosporosis in cattle have not been achieved. There is no chemotherapy available and, although progress has been made toward understanding immunity to Neospora infections, no efficacious vaccine is available to limit outbreaks or prevent abortions. Vaccine development to prevent N. caninum abortion and congenital infection remains a high research priority. To this end, our research group has over the past decade: 1) Identified the importance of T-lymphocyte-mediated immunity, particularly IFN-γ responses, as necessary for immune protection to congenital neosporosis in mice,1,2,10,11 and 2) Identified MHC class II restricted CD4+ CTL in Neosporainfected Holstein cattle,16 and 3) Identified NcSRS2 as a highly conserved surface protein associated with immunity to Neospora infections in mice and cattle.7,8,15 In this BARD-funded 12 month feasibility study, we continued our study of Neospora immunity in cattle and successfully completed T-lymphocyte epitope mapping of NcSRS2 surface protein with peptides and bovine immune cells,15 fulfilling objective 1. We also documented the importance of immune responses NcSRS2 by showing that immunization with native NcSRS2 reduces congenital Neospora transmission in mice,7 and that antibodies to NcSRS2 specifically inhibition invasion of placental trophoblasts.8 Most importantly we showed that T-lymphocyte responses similar to parasite infection, namely induction of activated IFN-γ secreting Tlymphocytes, could be induced by subunit immunization with NcSRS2 peptides containing the Neospora-specificCTLepitopes (Baszler et al, In preparation) fulfilling objective 2. Both DNA and peptide-based subunit approaches were tested. Only lipopeptide-based NcSRS2 subunits, modified with N-terminal linked palmitic acid to enhance Toll-like receptors 2 and 1 (TLR2-TLR1), stimulated robust antigen-specific T-lymphocyte proliferation, IFN-γ secretion, and serum antibody production across different MHC-IIhaplotypes. The discovery of MHC-II cross-reactive T-cellinducing parasite peptides capable of inducing a potentially protective immune response following subunit immunization in cattle is of significant practical importance to vaccine development to bovine neosporosis. In addition, our findings are more widely applicable in future investigations of protective T-cell, subunit-based immunity against other infectious diseases in outbred cattle populations.