Expression of insulin-like growth factor (IGF) family members in porcine pregnancy.

Prenatal mortality is a prime concern for commercial swine industry in North America. Fetal losses occur throughout gestation but cluster in early (~day20) and mid (~day50) pregnancy. Adequate vascularization of the attachment site has emerged as a key factor contributing to fetal success. Since Insulin-Like Growth Factor (IGF) family members regulate angiogenesis in addition to promoting fetal development and growth, we hypothesized that conceptus success is governed by members of the IGF family. Using quantitative real time PCR, we analyzed expression of IGF family members (IGF-I, IGF-II, IGF-I Receptor (IGF-IR), IGF-IIR and their binding proteins, IGFBPs) in matched maternal and fetal tissues of healthy and arresting conceptuses at gestation days (gd) 20 and 50. IGF-II transcripts were 100 fold increased in both maternal and fetal tissues compared to IGF-I, but receptor transcripts were found in similar abundance irrespective of health status and gestation point. IGFBP3 was the most abundantly transcribed of the binding proteins. Using immunohistochemistry we confirmed the expression of IGF family members in maternal luminal and glandular epithelial cells, the endothelium of blood vessels and some scattered stromal cells. Our results suggest that IGF-I and II and their receptors are differentially expressed at the maternal and fetal components of the attachment site.

Expression of angiogenic basic fibroblast growth factor, platelet derived growth factor, thrombospondin-1 and their receptors at the porcine maternal-fetal interface.

BACKGROUND: Commercial swine breeds in North America undergo two waves of spontaneous fetal loss; one during peri-attachment and another during mid-gestation. Although an exact mechanism for this loss is not known, deficits in vasculature at the attachment sites appear to be a major cause. We hypothesized that a balance between pro-angiogenic and anti-angiogenic factors is needed at the maternal-fetal interface for successful conceptus development. Six selected members of the pro-angiogenic fibroblast growth factor (FGF) and platelet derived growth factor (PDGF) families and anti-angiogenic factor thrombospondin-1 (TSP-1) and its receptor CD36 were quantified and localized at the porcine maternal-fetal interface at early and midgestation time points. METHODS: Mesometrial endometrium was collected from non-pregnant gilts (n = 8). Endometrial and chorioallantoic membrane samples were collected from healthy and arresting conceptus attachment sites at gestation day (gd) 20 (n = 8) and gd 50 (n = 8). At gd20 arresting conceptus attachment sites were distinguished by decreased vasculature of the placental membranes and decreased conceptus size. At gd50 arresting conceptuses attachment sites were identified by smaller conceptus length and weight measurements. Quantitative real time PCR was used to determine relative transcript levels of genes of interest, and cellular localization was determined by immunohistochemistry in paraffin embedded endometrial sections. RESULTS: At gd20, endometrial samples from arresting conceptuses had elevated transcripts for bFGF, and PDGF-bb than healthy sites (p < 0.05). At gd50, bFGF, FGFR2, and CD36 were more abundant at arresting than at healthy conceptus attachment sites (p < 0.05). Chorioallantoic membrane from arresting conceptus attachment sites at gd20 had elevated transcripts for bFGF, FGFR1, FGFR2 and CD36 compared with healthy sites (p < 0.05). FGFR2 transcripts were more abundant in chorioallantoic membrane from arresting conceptuses at gd 50 (p < 0.05). Immunohistochemical localization of selected pro- and anti-angiogenic factors and receptors revealed their abundance in the luminal epithelium, uterine glands and perivascular areas of endometrium at gd20 and gd50. CONCLUSIONS: We provide comprehensive analysis of pro and anti-angiogenic factors at the porcine maternal fetal interface during early and mid-pregnancy. At mRNA levels, the majority of pro-angiogenic factors investigated were elevated at the sites of fetal arrest. These observations contrast with our previous findings of decreased Vascular Endothelial Growth Factor (VEGF) family members at arresting sites, and suggest that the bFGF family functions as a compensatory survival mechanism when major angiogenic proteins are decreasing at the sites of fetal arrest.

NKT cell costimulation: experimental progress and therapeutic promise.

Invariant natural killer T (iNKT) cells are innate lymphocytes with unique specificity for glycolipid antigens and remarkable immunomodulatory properties. The role of costimulatory interactions in iNKT cell responses has recently come under scrutiny. Although iNKT cells and their prototype glycolipid agonist α-galactosylceramide (α-GalCer) have shown promise in several clinical trials conducted in patients with cancer or viral diseases, current iNKT cell-based therapies are far from effective. The concomitant targeting of T cell receptors (TCRs) and costimulatory molecules on iNKT cells represents an exciting new opportunity to optimize such therapeutic approaches. Here, we review recent advances in our understanding of iNKT cell costimulation and discuss potential treatment modalities based on the responsiveness of iNKT cells to disease-tailored glycolipids and select costimulatory ligands.

Expression of chemokine decoy receptors and their ligands at the porcine maternal-fetal interface.

Successful pregnancy requires coordinated maternal-fetal cross-talk to establish vascular connections that support conceptus growth. In pigs, two waves of spontaneous fetal loss occur and 30-40% of conceptuses are lost before parturition. Previous studies associated these losses with decreased angiogenic and increased inflammatory cytokines. Chemokines, a sub-category of cytokines, and decoy receptors control leukocyte trafficking, angiogenesis and development. The availability of chemokines is regulated by three non-signalling decoy receptors: chemokine decoy receptor (D6), Duffy antigen receptor for chemokines (DARC) and Chemocentryx decoy receptor (CCX CKR). We hypothesized that the expression of these receptors and their chemokine ligands regulate the porcine pregnancy success or failure. Here, we describe for the first time the transcription and translation of all three decoy receptors and several chemokine ligands in endometrium and trophoblast associated with healthy and arresting conceptuses at gestation day (gd) 20 and gd50. Among decoy receptors, transcripts for DARC were significantly reduced in endometrium, whereas that for CCX CKR were significantly increased in endometrium and trophoblast at gd50 arresting compared with healthy sites. However, western blot analysis revealed no differences in decoy receptor expression between healthy and arresting tissues. Transcripts for decoy receptor ligands CCL2, CCL3, CCL4, CCL5, CCL11, CCL19, CCL21, CXCL2 and CXCL8 were stable between healthy and arresting littermates. Quantification by SearchLight chemiluminescent protein array confirmed ligand expression at the protein level. These data indicate that decoy receptors and ligands are expressed at the porcine maternal-fetal interface and dysregulation of decoy receptor (DARC and CCX CKR) transcripts occurs at sites of fetal arrest.

Angiogenic DC-SIGN(+) cells are present at the attachment sites of epitheliochorial placentae.

Spontaneous early and mid-gestation fetal losses occur in swine. At both stages, endometrial lymphocytes associated with smaller, paler conceptuses have fewer pro-angiogenic and more pro-inflammatory cytokine transcripts compared with robust conceptuses. We hypothesized that similar differences occur in conceptus-associated dendritic cells (DCs). Using laser capture-microdissection, dendritic cell-specific intercellular adhesion molecule-grabbing non-integrin (DC-SIGN)(+) cells were isolated from attachment sites of healthy and arresting conceptuses at gestation day (gd)20 and 50. DC-SIGN(+) cells were screened using real-time PCR for vascular endothelial cell growth factor (Vegf), its receptors, semaphorins (Sema) and plexins (Plxn), and for toll-like receptor (Tlr) transcripts to address potential activation pathways. Homogenized endometrial and trophoblast biopsies were quantified for type 1/type 2 cytokine transcripts/proteins. DC-SIGN(+) cells from healthy and arresting conceptuses had more Vegf transcripts at early than mid gestation whereas transcripts for Vegfr1 and Vegfr2 were stable. In gd20 arresting site DC-SIGN(+) cells, Neuropilin-2 transcripts were elevated, whereas at gd50 arresting sites, Plxn-A2 increased and Sema3A transcripts were lost. Tlr-1, Tlr-4 and Tlr-6 transcript abundance was independent of conceptus health. At gd20, type 1 cytokines were prevalent, whereas at gd50 type 2 cytokines predominated in endometrium and trophoblast. Thus, gestational features, characteristic of haemochorial placentation, are present in species with distinctly different placentation.

Cytokine array comparisons of plasma from cycling fertile women on cycle day 5 and ovulation.

PROBLEM: To identify plasma immuno-regulatory molecules up or down regulated between the follicular phase and ovulation of the human menstrual cycle. METHOD OF STUDY: RayBio cytokine arrays were used to screen 174 immuno-regulatory molecules in plasma collected during the follicular phase at menstrual cycle day 5 and at ovulation from five healthy, non-smoking, fertile women of reproductive age not using hormonal contraception. RESULTS: A total of 23 differentially expressed molecules were found: 10 molecules were differentially up-regulated and 13 down-regulated at ovulation compared with that at the follicular phase (alpha = 0.05, false discovery rate of 0.45). CONCLUSION: Circulating immuno-regulatory molecules fluctuate over the menstrual cycle in healthy women. The combination of differentially expressed molecules suggests roles in cyclical regulation of angiogenesis and immune cell trafficking.

CD56bright cells increase expression of {alpha}4 integrin at ovulation in fertile cycles.

Leukocyte content of human endometrium changes rapidly after ovulation, particularly as a result of gains in CD56(bright) uterine NK (uNK) cells. We have proposed that uNK precursor cells are found within the blood CD56(bright) pool and are recruited to decidualizing endometrium through functional changes in their adhesion molecules and chemokine receptors. This study sought to quantify alterations in adhesion molecules, cytokines, chemokines, and receptors induced in circulating CD56(+) cells of fertile and infertile women by ovulation. Blood was drawn from 12 fertile volunteers and six female-infertility patients at Menstrual Cycle Day (d) 5 and on the day following the preovulatory surge of luteinizing hormone (LH). CD56(bright), CD56(dim), and CD56(+)CD3(+) cell subsets were isolated and evaluated by flow cytometry, quantitative PCR, or Western blotting. In CD56(bright) cells from fertile but not infertile women, alpha(4) integrin increased between d5 and the preovulatory LH surge. CD56(dim) and NKT cells did not show a change in alpha(4) integrin but differed significantly between fertile and infertile donors, and infertile donors had reduced homing molecule expression in CD56(dim) and NKT cells, and at ovulation, their NKT cells showed elevated cytokine production. None of the circulating CD56(+) cell subsets had transcripts for receptors for estrogen, progesterone, LH, or prolactin. Thus, immunological events associated with the LH surge induce alterations in all subsets of CD56(+) cells, and the unique induction of alpha(4) integrin in CD56(bright) cells of fertile women constitutes a potential method to promote uterine homing.

CD56+ cells are recruited to the uterus in two waves: at ovulation and during the first 2 weeks after missed menses.

PROBLEM: Uterine natural killer (uNK) cells are enriched in the post-ovulatory uterus and during pregnancy. Whether these cells arise from blood pre-cursors or from stem cells in the uterus is undefined. To support a hypothesis that precursors of uNK cells are recruited from blood, adhesive function of blood CD56+ subsets were assessed during one cycle and during pregnancy. METHOD OF STUDY: Fifteen women of proven fertility provided serial blood samples during one menstrual cycle and thirty women with a history of implantation failure or recurrent spontaneous abortion provided serial samples during infertility treatment. RESULTS: CD56(bright) cells, but not CD56(dim) cells or NKT cells, increased in ligand-binding capacity during ovulation in fertile cycles only and during the first 2 weeks from date of missed menses. CONCLUSION: Enhanced adhesive function at ovulation in CD56(bright) cells in fertile cycles and during early gestation supports a hypothesis of recruitment of pre-uNK cells from the blood CD56(bright) subset.

Decline in number of elevated blood CD3(+) CD56(+) NKT cells in response to intravenous immunoglobulin treatment correlates with successful pregnancy.

PROBLEM: Patients with elevated blood natural killer (NK) cells may be offered intravenous immunoglobulin (IVIG) treatment, but there is controversy about the utility of blood NK cell testing. Human CD56(+) NK cells include several subpopulations that include the putatively cytotoxic CD56(+) CD16(+) subset. In mouse models of pregnant failure, NKT cells appear to be important. However, a mouse model may only be pertinent to a subset of patients, as recurrent pregnancy failure is a heterogenous group. METHOD OF STUDY: An ethics-approved observational study was done to observe the effect of treatment on total blood lymphoid cells, and subsets of CD56(+) blood lymphocytes including CD56(+) CD3(+) NKT cells determined by flow cytometry, and to correlate with pregnancy outcome. Fifteen fertile women with a history of successful pregnancy and thirty-one women suffering from repeated implantation failure or recurrent spontaneous abortion provided serial blood samples during one menstrual cycle or prior to and during treatment. IVIG was administered to the latter group with or without heparin/aspirin. RESULTS: Eight of thirty infertile women presented with high numbers of CD56(+) CD3(+) NKT cells, which declined after treatment with IVIG. The elevated NKT cell group with or without concomitant autoimmunity achieved a significantly higher successful pregnancy rate over the course of the study, as compared to women with average numbers of NKT cells and no evidence of autoimmunity (P = 0.018). Elevated NKT levels alone was an independent predictor of success on treatment (P = 0.003). CONCLUSION: Elevated NKT cells in recurrent pregnancy loss or implantation failure can be ameliorated with IVIG treatment, and result in successful pregnancy. Assay of NKT cell numbers may identify patients who are more likely to benefit from IVIG therapy and merits further examination in randomized phase II studies.

Defibrinated bovine plasma inhibits retroviral transcription by blocking p52 activation of the NFkappaB element in the long terminal repeat.

Bovine leukemia virus (BLV) induces a persistent but latent infection in cattle. Viral latency is invoked by a protein known as plasma blocking factor (PBF) that is found in both bovine and human plasma. We report here on pathways that mediate latency in the presence of PBF. Reporter-gene constructs driven by the promoters of 6 retroviruses were used to measure the production of chloramphenicol acetyl transferase (CAT) in cell lines cultured with or without defibrinated bovine plasma. Plasma inhibited CAT production only in constructs containing an NFkappaB-binding element proximal to the initiation site (BLV, human immunodeficiency virus, and human T-cell leukemia virus). The promoters of Bovine immunodeficiency virus, Feline immunodeficiency virus, or Feline leukemia virus were not inhibited in the presence of bovine plasma. Using gel mobility shift assays, we demonstrated that activation of viral transcription upon stimulation with phorbol esters and ionomycin was mediated through the NFkappaB element and that this was abrogated in the presence of plasma. Furthermore, analysis of individual NFkappaB proteins in nuclear extracts of mononuclear cells or Jurkat cells showed that all 5 members of the NFkappaB family were upregulated in response to stimulation, but only p52 was significantly downregulated in the presence of bovine plasma. Thus, we infer that plasma effects are mediated through interference with either p52 translocation to the nucleus or p52 synthesis.

Uterine natural killer cells: a specialized differentiation regulated by ovarian hormones.

In adult females of many species, a transient population of natural killer (NK) cells appears in cycles within the uterine endometrium (lining). Appearance of these lymphocytes coincides with specific phases of the ovarian hormone cycle and/or early pregnancy. Studies in rodents, women, and pigs dominate the literature and suggest the uterine (u)NK cells are an activated subset sharing many but not all features with circulating or lymphoid organ-residing NK cells. During successful murine pregnancy, uNK cells appear to regulate initiation of structural changes in the feed arterial systems that support maternal endometrial tissue at sites of implantation and subsequent placental development. These changes, which reverse after pregnancy, create a higher volume arterial bed with flaccid vessels unresponsive to vasoactive compounds. These unique pregnancy-associated arterial changes elevate the volume of low-pressure, nutrient-rich, maternal arterial blood available to conceptuses. Regulation of the differentiation, activation, and functions of uNK cells is only partially known, and there is lively debate regarding whether and how uNK cells participate in infertility or spontaneous abortion. This review highlights the biology of uNK cells during successful pregnancy.

A review of trafficking and activation of uterine natural killer cells.

PROBLEM: Enrichment of uterine natural killer (uNK) cells occurs during pregnancy in many species. However, functions of uNK cells and regulation of their uterine homing are not fully defined. In mice and women, uNK cells contribute to angiogenesis, a role reviewed here and now addressed in a mammal with an alternative placental type. METHODS OF STUDY: To address lymphocyte functions, RNA from murine or porcine endometrium and lymphocytes purified from endometrium were analyzed using real-time or reverse transcription PCR. To address homing potential, human blood CD56(+) lymphocytes were evaluated using both RNA and functional adhesion to endothelium presented under shear force in frozen sections of gestation day 7 C57Bl/6J implantation sites. Women were serially sampled over a menstrual cycle or a clinical preparatory cycle for embryo transfer. RESULTS: Activation of murine uNK cells is associated with much greater increases in transcription for Eomes than for T-bet (Tbx21). Lymphocytes from normal porcine implantation sites transcribe vascular endothelial growth factor, placental growth factor, interferon-gamma and hypoxia-inducible factor (HIF)-1alpha. In fertile women, increases in L-selectin- and alpha4-integrin-mediated interactions between CD56(+) cells and endothelium occur at luteinizing hormone (LH) surge (cycling women) to oocyte pick up or embryo transfer, then return to pre-LH levels. CONCLUSIONS: Uterine lymphocytes may universally promote pregnancy-associated endometrial angiogenesis. Recruitment of uNK precursor cells from blood appears to occur in a window promoted by rising plasma estrogen and LH and limited by rising progesterone.

Purified bovine plasma blocking factor decreases Bovine leukemia virus p24 expression while increasing protein synthesis and transcriptional activity of peripheral blood mononuclear cells in short-term culture.

Bovine leukemia virus (BLV) induces a persistent infection in the B-cells causing polyclonal expansion of B-cells in one-third of infected cattle and lymphosarcoma in less than 5% of infected cattle. While BLV is difficult to detect in vivo, it is readily produced by cultured lymphocytes and is diminished when supplemented by bovine plasma. This phenomenon is attributed to a poorly characterized plasma blocking factor (PBF). We assessed the effects of bovine plasma on cell viability and BLV p24 expression, and the effects of purified PBF on protein synthesis and gene expression of short-term cultures of bovine lymphocytes. The addition of 25% plasma or semi-purified PBF to cultures had no significant effect on cell viability but caused significant decreases in BLV p24 production and significantly increased de novo protein synthesis. Utilizing a human microarray, the RNA messages of 83 genes involved in cell division, cell metabolism, and gene regulation were up-regulated.

Trafficking of circulating pro-NK cells to the decidualizing uterus: regulatory mechanisms in the mouse and human.

Natural Killer (NK) lymphocytes, strongly expressing CD56, become abundant in the human uterus three to five days after the mid-menstrual cycle surge in pituitary-derived luteinizing hormone (LH). The primary functions of LH are to initiate final oocyte maturation/ovulation and to contribute to decidualization of the uterine stroma. Decidualization is the transformation of estrogen-primed uterine stromal fibroblasts into large hormone-producing cells under the influence of progesterone (P4). Decidual CD56bright (dNK) cells are a distinct, transient, tissue-specific NK cell subset that undergoes proliferation, terminal differentiation, and then death prior to menses. If pregnancy occurs, dNK cells increase during first trimester, then decline and are virtually absent in late pregnancy. In mouse models, pregnancy-associated uterine NK (uNK) cells appear coincident with onset of decidualization during embryonic implantation. Murine uNK cells traffic from the circulation to the antimesometrial side of the uterus and migrate to the mesometrial side of each implantation site. Here they proliferate and are implicated in regulation of midgestation structural changes to major arteries supplying the placenta, before dying in late gestation. Emerging data indicate that interactions between lymphocytes and endothelial cells within the uterine microenvironment are mediated by classical molecules associated with lymphocyte trafficking in immune surveillance and in response to inflammation. Here, we review factors influencing NK cell trafficking to decidualizing murine and human uteri and the differentiation and functions of these cells within the uterus.

Isolation of a bovine plasma fibronectin-containing complex which inhibits the expression of bovine leukemia virus p24.

Bovine leukemia virus (BLV) is a deltaretrovirus that infects cattle worldwide. In agriculturally intensive regions, approximately 30% of dairy cows are BLV infected. Like the human T-cell leukemia virus (HTLV), there is a lengthy period of viral quiescence after initial infection with BLV. Unlike HTLV, BLV resides predominantly in B cells. Lymphoma is observed in less than 10% of BLV-infected adult cattle. Although viremia is undetectable in vivo, BLV-infected peripheral blood mononuclear cells readily become productive when cultured in vitro. Productivity is markedly diminished when cultures are supplemented with bovine plasma. This inhibitory activity of bovine plasma has been attributed to the "plasma blocking factor" (PBF). Here, we describe the purification of a PBF whose activity was resistant to heating to 65 degrees C for 10 min and was attributable to a fibronectin-containing complex of approximately 320 kDa under nonreducing conditions. By use of two-dimensional polyacrylamide gel electrophoresis and matrix-assisted laser desorption ionization-time of flight (mass spectrometry), a protein with a size of 220 kDa and a pI of 5.4 was identified as a member of the fibronectin group of molecules. Both the purified protein and the commercially available bovine fibronectin inhibited BLV production in naturally infected peripheral blood mononuclear cells, although the fibronectin was less biologically active.

Periovulatory increases in tissue homing potential of circulating CD56(bright) cells are associated with fertile menstrual cycles.

CD56(bright) lymphocytes appear in the uterus 3-5 d after ovulation coincident with the onset of stromal cell decidualization. Although the source of these uterine immune cells is not defined, a subset of blood CD56(bright) cells exhibits enhanced capacity to adhere to decidual vascular endothelium during the periovulatory period of menstrual cycles. In this study, the effects of early pregnancy on the adhesive capacity of CD56(bright) cells to bind uterine substrates were examined in a time-course study of 18 infertile women undergoing natural cycles before transfer of frozen/thawed embryos and 18 infertile women undergoing controlled ovarian stimulation. There were three pregnancies in the natural cycle group and seven in the hormone-stimulated cohort. Hormone levels, and number and quality of transferred embryos were similar between pregnant and nonpregnant cycles. However, the adhesive function of CD56(bright) cells increased before ovulation in hormone-treated women who became pregnant and before embryo transfer in naturally cycling women who became pregnant. This pattern of incremental adhesion, which was less frequently observed in unsuccessful cycles, suggests a role for NK cells in implantation. These results support the idea that temporal control of NK cell homing to the uterine microenvironment is a prerequisite to pregnancy.

Menstrual cycle hormones induce changes in functional interactions between lymphocytes and decidual vascular endothelial cells.

During the secretory phase of the menstrual cycle, a natural killer (NK) cell subset expressing cluster of differentiation (CD)56bright appears in the decidualizing uterus and remains until onset of menses. If pregnancy occurs, decidual NK cells increase to become the predominant uterine lymphocytes of early pregnancy. To elucidate mechanisms of CD56bright cell recruitment to the uterus, an in vitro adhesion assay was used to assess the effect of the menstrual cycle, as well as cycle-associated hormones on adhesive properties of human lymphocytes. Adhesion of human peripheral blood lymphocytes to pregnant mouse lymph nodes and Peyer's Patches tissue sections was constant throughout the cycle. When uterine tissue was used as the substrate, adhesive CD56+ cells were found only in decidua basalis. Adhesion increased at the LH surge. Adhesion was mediated through both L-selectin and alpha4-integrin-dependent mechanisms. Furthermore, we observed increased adhesive function in CD56+ cells from male donors which had been cultured with estradiol or LH compared with cell aliquots cultured without additives. Lymphocytes adherent to mouse uterine tissue were predominantly CD56bright, suggesting that peripheral NK cells may be actively recruited to the uterus in an important, brief endocrine-regulated fashion at the time of ovulation to establish the decidual NK population of early pregnancy.