A True Positive and a False Negative? The Dilemma of Negative Colonoscopy After a Positive Fecal Occult Blood Test.

INTRODUCTION: Many colonoscopies following a positive fecal immunochemical test (FIT) will not identify a probable cause for fecal blood, and missed neoplasia is a concern. The study determined whether the absence of neoplasia at a FIT positive diagnostic colonoscopy was due to a missed lesion and whether the initial FIT hemoglobin (f-Hb) concentration could predict missed lesions. METHODS: This was a retrospective audit of patients who had undergone diagnostic colonoscopy after FIT screening (2 sample ≥ 20 µg Hb/g feces). Probable bleeding lesions including cancer, advanced adenoma, colitis, and angiodysplasia were considered a "positive colonoscopy outcome." For those with a negative outcome, findings at the subsequent colonoscopy were assessed. RESULTS: There were 1087 good quality colonoscopies within 12 months of a positive FIT. In total, 171 (15.7%) patients had a positive outcome at the diagnostic colonoscopy. Subsequent colonoscopies of negative outcome cases (n = 418, median of 3.1y later) were reviewed; of these, there were 57 (13.6%) cases with a positive outcome. This included CRC in 0.5% (n = 2) and advanced adenoma in 11.7% (n = 49). High f-Hb and having both FIT samples ≥ 20 µg/g feces were associated with a positive outcome at the original diagnostic colonoscopy (p < 0.05). However, f-Hb was not predictive for a positive outcome at the subsequent colonoscopy by either maximum f-Hb (p = 0.768), total f-Hb (p = 0.459), or both FIT samples ≥ 20 µg/g (p = 0.091). CONCLUSION: A small proportion of "false" positive FIT results had cancer or advanced adenoma found at the subsequent colonoscopy. A missed lesion could not be predicted by the initial FIT f-Hb.

IFPA Senior Award Lecture: Reproductive immunology in perspective--reprogramming at the maternal-fetal interface.

Involvement of the maternal and fetal immune systems in the events of pregnancy was generally overlooked by reproductive biologists until the mid-twentieth century when many landmark explorations were reported. Now, more than half a century later, it is well understood that with the initiation of pregnancy, immune cells in mammalian uteri are reprogrammed, losing their cytotoxic potential and providing an immunosuppressive environment suitable for harboring the genetically different fetus. We propose that it is the placenta that is mainly responsible for this conversion and maintenance throughout pregnancy. Studies in our laboratory indicate that trophoblast-derived soluble HLA-G has a subtle but well defined role in programming uterine placental macrophages, a potentially destructive immune cell population. Thus, placental HLA-G plays a critical role in assuring that the developing fetus emerges unscathed at parturition.

Immunomodulatory molecules are released from the first trimester and term placenta via exosomes.

The semiallogenic fetus is tolerated by the maternal immune system through control of innate and adaptive immune responses. Trophoblast cells secrete nanometer scale membranous particles called exosomes, which have been implicated in modulation of the local and systemic maternal immune system. Here we investigate the possibility that exosomes secreted from the first trimester and term placenta carry HLA-G and B7 family immunomodulators. Confocal microscopy of placental sections revealed intracellular co-localization of B7-H1 with CD63, suggesting that B7-H1 associates with subcellular vesicles that give rise to exosomes. First trimester and term placental explants were then cultured for 24 h. B7H-1 (CD274), B7-H3 (CD276) and HLA-G5 were abundant in pelleted supernatants of these cultures that contained microparticles and exosomes; the latter, however, was observed only in first trimester pellets and was nearly undetectable in term explant-derived pellets. Further purification of exosomes by sucrose density fractionation confirmed the association of these proteins specifically with exosomes. Finally, culture of purified trophoblast cells in the presence or absence of EGF suggested that despite the absence of HLA-G5 association with term explant-derived exosomes, it is present in exosomes secreted from mononuclear cytotrophoblast cells. Further, differentiation of cytotrophoblast cells reduced the presence of HLA-G5 in secreted exosomes. Together, the results suggest that the immunomodulatory proteins HLA-G5, B7-H1 and B7-H3, are secreted from early and term placenta, and have important implications in the mechanisms by which trophoblast immunomodulators modify the maternal immunological environment.

Novel HLA-G-binding leukocyte immunoglobulin-like receptor (LILR) expression patterns in human placentas and umbilical cords.

Human placentas are sources of cytokines, hormones and other substances that program receptive cells. One of these substances is HLA-G, which influences the functioning of both leukocytes and endothelial cells. In this study, we investigated the possibility that these and/or other types of cells in extraembryonic fetal tissues might respond to HLA-G by interacting with one or another of the leukocyte immunoglobulin-like receptors (LILR). LILRB1 is expressed by most leukocytes and LILRB2 is expressed primarily by monocytes, macrophages and dendritic cells. Analysis of term placentas by immunohistochemistry and Real Time PCR demonstrated that LILRB1 and LILRB2 protein and specific messages are produced in the mesenchyme of term villous placenta but are differently localized. LILRB1 was abundant in stromal cells and LILRB2 was prominent perivascularly. Neither receptor was identified in trophoblast. Further investigation using double label immunofluorescence indicated that placental vascular smooth muscle but not endothelia exhibit LILRB2. Term umbilical cord exhibited the same LILRB2 patterns as term placenta. Samples obtained by laser capture dissection of vascular smooth muscle in umbilical cords demonstrated LILRB2 mRNA, and double label immunofluorescence showed that cord vascular smooth muscle but not endothelium exhibited LILRB2 protein. The presence of LILRB1 in placental stromal cells and LILRB2 in vascular smooth muscle strongly suggest that HLA-G has novel functions in these tissues that could include regulation of placental immunity as well as development and function of the extraembryonic vasculature.

A commentary on gestational programming and functions of HLA-G in pregnancy.

HLA-G is an HLA class Ib gene that is highly expressed in human trophoblast cells. The single HLA-G mRNA is alternatively spliced to generate at least seven transcripts, three of which encode soluble isoforms. Many studies have shown that high levels of soluble antigens are associated with successful implantation and graft acceptance. To study expression, regulation and functions of two of the soluble isoforms, HLA-G5 and HLA-G6, we generated recombinant proteins in eukaryotic cells and developed monoclonal antibodies specific for each of the two proteins. In addition, we investigated the olive baboon Paan-AG gene as a potential functional correlate of HLA-G. Here, we present summaries of the studies that have been conducted in our laboratory using these tools and discuss the results within the context of the research on this topic that is ongoing in ours and other laboratories worldwide. Collectively, the data indicate that soluble HLA-G is a critical contributor to immune privilege in pregnancy and imply that this placenta-derived substance may impact other pathways leading to successful reproduction.

FLICE-inhibitory protein: expression in early and late gestation human placentas.

The apoptosis cascade that plays a central role in normal and pathological processes is strictly controlled, in part by FLIP (Fas-associated death domain-like interleukin-1beta-converting enzyme-inhibitory protein), an inhibitor of caspase-8. Here, we report the expression of long and short isoforms of FLIP mRNAs and proteins in early and late gestation human placentas, term cytotrophoblast cells and two choriocarcinoma cell lines, JEG-3 and Jar. Reverse transcriptase polymerase chain reaction identified mRNAs derived from the FLIP gene in all samples. Analysis by immunoblotting revealed that both long and short forms of FLIP proteins are present in early and late gestation human placentas with increasing levels over gestation and that FLIP proteins are present in normal and transformed trophoblast cells. Immunohistochemical experiments performed on paraformaldehyde-fixed tissue sections taken from early and late stages of pregnancy demonstrated that FLIP proteins are present in caspase-8-expressing cells and that expression patterns of FLIP differed according to cell lineage and stage of cell differentiation. The results of this study are consistent with the postulate that FLIP proteins have critical roles in placental cell survival and suggest that FLIP may protect normal and transformed trophoblast cells from cell death.

Antigen presenting cells and HLA-G--a review.

Maternal antigen presenting cells, which are macrophages and dendritic cells, are scattered throughout human decidualized endometrium during all stages of pregnancy. These powerful, multi-functional leukocytes reside in close proximity to uterine glandular epithelium, uterine blood vessels, and HLA-G-producing invasive cytotrophoblast cells. Macrophages and dendritic cells, which express the HLA-G receptors, ILT2 and ILT4, play major roles in driving innate and adaptive immune responses, altering the behavior of local stromal cells, shaping the cytokine microenvironment, and protecting the tissue from infection. Therefore, encounters between decidual antigen presenting cells and HLA-G molecules are likely to influence uterine and placental homeostasis as well as local maternal immune responses to the fetus during pregnancy.

The olive baboon (Papio anubis): a potential animal model to study the function of human leukocyte antigen-G (HLA-G).

The human class Ib major histocompatibility complex (MHC) molecule, HLA-G, is unique in its limited polymorphism, high expression in the placenta and generation of multiple transcripts by alternative splicing. The proteins encoded by these transcripts are believed to modulate maternal-fetal immunological relationships during pregnancy. The baboon placenta expresses Paan-AG, a novel MHC molecule that is evolutionarily related to the MHC-A locus but shares unique characteristics with HLA-G. In this brief review, we present evidence suggesting that Paan-AG may be the functional homologue of HLA-G, and propose that the baboon would compromise an excellent animal model for functional studies of HLA-G proteins in human pregnancy.

Immunogenicity of the soluble isoforms of HLA-G.

Soluble class Ib HLA-G glycoproteins synthesized in the placenta are abundant in the pregnant uterus and circulate in maternal blood throughout pregnancy. To establish immunogenicity of these proteins, we tested sera from 64 women with at least one successful pregnancy (multigravid), 21 women who had never been pregnant, and 54 males for antibodies to epitopes present on recombinant sHLA-G isoforms (sHLA-G1, sHLA-G2) derived from HLA 6.0 cDNA (HLA-G*0101 allele). By indirect enzyme-linked immunosorbent assay, antibodies to sHLA-G isoforms were identified in six sera, all from multigravid women; all other sera were negative (P = 0.0083). Immunoblots showed that two of the positive sera reacted exclusively with sHLA-G1 and -G2 whereas four reacted to both sHLA-G and pooled HLA class I antigens. To establish potential relationships between anti-sHLA-G and exposure to foreign paternal alleles (*0101, *0103, *0104, *0106), all multigravid women and their partners were genotyped. No relationship between allelic disparity and antibody production was identified. Taken together, these results indicate that (i) tolerance to HLA-G is the usual condition as antibodies to HLA-G were not detected in 91% (58/64) multigravid women, and (ii) pregnancy stimulates loss of tolerance in 9% (6/64) of multigravid women. All six women delivered healthy babies, demonstrating that maternal antibodies to epitopes on sHLA-G do not abrogate pregnancy.

Investigation of the role of nitric oxide synthase 2 in pregnancy using mutant mice.

Nitric oxide (NO) has been implicated as a signalling molecule in many cellular processes. As nitric oxide synthase 2 (NOS-2) is the main isoform expressed in mouse decidua and metrial gland, mice with a targeted disruption of the gene encoding NOS-2 were used to determine the potential roles of this enzyme during pregnancy. Reproductive success and the morphology of implantation sites throughout pregnancy were compared in NOS-2 deficient (NOS-2-/-) and wild-type (WT) mice. Although there were no significant differences in the duration of gestation or birth weight, NOS-2-/- mice had significantly fewer viable embryos at mid-gestation and delivered smaller litters than did WT mice. Histological sections of uteroplacental units from WT and NOS-2-/- mice were compared to establish the mechanisms underlying the loss of fetuses. No morphological differences were observed on day 6 or day 8 of gestation, indicating that implantation and early development of implantation sites were unaffected by the absence of NOS-2. However, by mid-gestation, decidua of NOS-2-/- mice had reduced cellularity and their decidual arteries had abnormally thickened walls. These observations were quantified by morphometric measurements, which showed a significant reduction in decidual cellular area and a significant increase in the blood vessel wall:lumen ratio in NOS-2-/- mice. The increase in the thickness of the blood vessel walls was not due to abnormal cellular infiltration or to altered expression of alpha-actin in vascular smooth muscle. These results indicate that NOS-2 has a functional role in the maintenance of decidual cellular integrity and development of appropriate uterine vasculature, and may play a supportive role in promoting embryo survival.

B7 family molecules: novel immunomodulators at the maternal-fetal interface.

The placenta utilizes both active and passive mechanisms to evade rejection by the maternal immune system. Recently, the mRNA for two newly cloned members of the B7 family of immunomodulatory cell-associated proteins have been identified in the human term placenta. In this article, we review the current knowledge of the B7 family member B7-H1, and discuss how it may participate in modulation of the maternal immune system at the maternal-fetal interface. B7-H1 has been found to possess immunostimulatory or immunoinhibitory properties, and immunohistological examination of first trimester and term placenta has revealed that this protein is abundant in the placenta. B7-H1 is highly expressed by both the syncytiotrophoblast and extravillous cytotrophoblast, both of which lie in direct contact with maternal blood and tissue. Further, treatment of the choriocarcinoma cell line, JEG-3, with recombinant human interferon (IFN)-gamma resulted in a dose-dependent increase in the abundance of the message for B7-H1, suggesting that IFN-gamma could regulate expression of B7-H1 by the trophoblast. These studies document that the positioning of B7-H1 at the maternal-fetal interface is such that it could participate in suppression of activated maternal leukocytes.

Death-inducing tumour necrosis factor (TNF) superfamily ligands and receptors are transcribed in human placentae, cytotrophoblasts, placental macrophages and placental cell lines.

Human placentae and two of the cell types in placentae (cytotrophoblasts and macrophages) were examined by RT-PCR for transcripts of the eight TNF superfamily ligands known to induce death of activated immune cells, tumour cells, and virus-infected cells (TNFalpha, LT alpha, LT beta, FasL, TRAIL, TWEAK, LIGHT, 4-1BBL). Transcripts for all ligands were detected in term placenta but LT alpha and 4-1BBL were not detected in first trimester placenta. Although term cytotrophoblasts contained mRNAs specific for TNF alpha, LT alpha, TWEAK, and 4-1BBL, messages encoding LT beta, FasL, TRAIL, and LIGHT were absent. In term placental macrophages, messages for all ligands except 4-1BBL were present. Transcripts for the 14 receptors to which the ligands bind, six of which contain death-domains (TNFR1, Fas, DR3, DR4, DR5, DR6), were also identified using RT-PCR. Term and first trimester placentae contained transcripts for all receptors except 4-1BB. Although term cytotrophoblasts lacked receptor mRNA encoding 4-1BB and OPG, term placental macrophages lacked DcR1 and OPG. Detection of nearly all the death-inducing TNF superfamily ligands and their receptors in human placentae implies that these powerful cytokines contribute to programmed or activated cell death in this organ.

rbcL data reveal two monophyletic groups of filmy ferns (Filicopsida: Hymenophyllaceae).

The "filmy fern" family, Hymenophyllaceae, is traditionally partitioned into two principal genera, Trichomanes s.l. (sensu lato) and Hymenophyllum s.l., based upon sorus shape characters. This basic split in the family has been widely debated this past century and hence was evaluated here by using rbcL nucleotide sequence data in a phylogenetic study of 26 filmy ferns and nine outgroup taxa. Our results confirm the monophyly of the family and provide robust support for two monophyletic groups that correspond to the two classical genera. In addition, we show that some taxa of uncertain affinity, such as the monotypic genera Cardiomanes and Serpyllopsis, and at least one species of Microtrichomanes, are convincingly included within Hymenophyllum s.l. The tubular- or conical-based sorus that typifies Trichomanes s.l. and Cardiomanes, the most basal member of Hymenophyllum s.l., is a plesiomorphic character state for the family. Tubular-based sori occurring in other members of Hymenophyllum s.l. are most likely derived independently and more than one time. While rbcL data are able to provide a well-supported phylogenetic estimate within Trichomanes s.l., they are inadequate for resolving relationships within Hymenophyllum s.l., which will require data from additional sources. This disparity in resolution reflects differential rates of evolution for rbcL within Hymenophyllaceae.

Horsetails and ferns are a monophyletic group and the closest living relatives to seed plants.

Most of the 470-million-year history of plants on land belongs to bryophytes, pteridophytes and gymnosperms, which eventually yielded to the ecological dominance by angiosperms 90 Myr ago. Our knowledge of angiosperm phylogeny, particularly the branching order of the earliest lineages, has recently been increased by the concurrence of multigene sequence analyses. However, reconstructing relationships for all the main lineages of vascular plants that diverged since the Devonian period has remained a challenge. Here we report phylogenetic analyses of combined data--from morphology and from four genes--for 35 representatives from all the main lineages of land plants. We show that there are three monophyletic groups of extant vascular plants: (1) lycophytes, (2) seed plants and (3) a clade including equisetophytes (horsetails), psilotophytes (whisk ferns) and all eusporangiate and leptosporangiate ferns. Our maximum-likelihood analysis shows unambiguously that horsetails and ferns together are the closest relatives to seed plants. This refutes the prevailing view that horsetails and ferns are transitional evolutionary grades between bryophytes and seed plants, and has important implications for our understanding of the development and evolution of plants.

Is that what I said?: witnesses' responses to interviewer modifications.

Modifications occur when interviewers contradict statements made by witnesses or imply that witnesses provided information that they (interviewers) did not provide. Because of their suggestive nature, modifications threaten the reliability of investigative interviews. This study investigated developmental differences in witnesses' responses to modifications during interviews as well as in inclusion of modified misinformation in subsequent answers. Preschool, elementary school, and college students were interviewed about a video presentation. In the experimental conditions, the interviewer contradicted information about the video provided by the participants. Participants then answered two sets of follow-up questions: one immediately following the interview and another 6-8 days later. Results indicated that participants were more likely to ignore modifications than to correct or agree with them. Adult participants were most likely to disagree with modifications. Preschoolers were most likely to incorporate modified misinformation into subsequent answers. Implications of these findings for investigative interviews are discussed.

Three polymorphisms in the 3' UTR of the TRAIL (TNF-related apoptosis-inducing ligand) gene.

Screening of the TRAIL (TNF-related apoptosis inducing ligand/Apo-2L) gene revealed three single nucleotide polymorphisms (SNPs) in the 3' UTR at nucleotides 1525G/A, 1588G/A, and 1591C/T. Over 50 individuals from each of two populations, Caucasian and African Americans, were genotyped for these three polymorphisms and allele frequencies were determined.

Eosinophils are cellular targets of the novel uteroplacental heparin-binding cytokine decidual/trophoblast prolactin-related protein.

The uterus and placenta of the mouse and rat produce a member of the prolactin (PRL) family referred to as decidual/trophoblast PRL-related protein (d/tPRP). This cytokine/hormone has been hypothesized to regulate decidual cell activities needed for the establishment and maintenance of gestation. An alkaline phosphatase (AP)-tagging strategy was used to identify d/tPRP target cells. AP-d/tPRP bound to virtually all cells and tissues to which it was exposed, consistent with our earlier evidence that d/tPRP binds to heparin-containing molecules. Moreover, we found that co-incubation with heparin or pretreatment with heparitinase greatly decreased the binding of AP-d/tPRP to tissue sections. In addition, we observed that the AP-d/tPRP probe bound to the surface of Chinese hamster ovary (CHO) cells but not to heparan sulfate-deficient CHO-pgsD-677 cells. Potential unique non-heparin d/tPRP binding sites within mouse and rat uteroplacental tissues were identified by consecutively incubating sections with AP-d/tPRP followed by heparin. This strategy led to the identification of d/tPRP target cells associated with the uterus and the labyrinth zone of the chorioallantoic placenta. Within the uterus, d/tPRP specifically bound to eosinophils. d/tPRP-binding and eosinophil peroxidase activity were co-localized and showed similar patterns of distribution during the estrous cycle, pregnancy, and following hormonal manipulation. d/tPRP interactions with eosinophils were further demonstrated in the lung and intestine, with eosinophils isolated from the peritoneum, and in mice with generalized tissue eosinophilia. Collectively, these findings suggest that intercellular d/tPRP targeting is mediated through associations with heparin-containing molecules which help direct d/tPRP to specific interactions with eosinophils within the uterus and with the labyrinthine compartment of the chorioallantoic placenta.

Soluble HLA-G circulates in maternal blood during pregnancy.

OBJECTIVE: Soluble isoforms of the HLA class Ib gene HLA-G have been identified at the maternal-fetal interface. Because soluble forms of other HLA class I antigens modulate T-cell reactivity and induce cellactivated apoptosis, our goal was to determine whether soluble HLA-G circulates in maternal or fetal blood and to identify the specific isoform. STUDY DESIGN: Capture enzyme-linked immunosorbent assays with mouse monoclonal antibodies directed toward an epitope present on all isoforms of soluble HLA-G were constructed to identify soluble HLA-G in 44 serum samples from nonpregnant control subjects, 129 serum samples from pregnant women, and 10 samples of term cord blood. Distinguishing between soluble HLA-G1, which is composed of heavy chains complexed with light chains (beta(2)-microglobulin), and soluble HLA-G2, which consists only of heavy chains, was achieved by substituting a monoclonal antibody that requires beta(2)-microglobulin for binding (W6/32) in the capture phase of the enzyme-linked immunosorbent assay. RESULTS: Capture enzyme-linked immunosorbent assays with mouse anti-soluble HLA-G showed that soluble HLA-G was present at all stages of gestation and that levels of soluble HLA-G were statistically significantly higher in serum samples from pregnant women than in serum samples from nonpregnant women. In contrast, W6/32 failed to detect soluble HLA-G in serum samples from pregnant women. Cord serum samples did not contain detectable soluble HLA-G. CONCLUSION: Collectively, the data indicate that pregnancy is characterized by the presence of soluble HLA-G circulating in maternal blood and strongly suggest that the major isoform is soluble HLA-G2.