Objectives: To predict miscarriage before 10 weeks of gestation in IVF pregnancies. Methods: Calc... more Objectives: To predict miscarriage before 10 weeks of gestation in IVF pregnancies. Methods: Calculation of gestational age (GA); ultrasound evaluation of Crown-rump length (CRL), mean gestational sac diameter (GSD) and volume (GSV), mean yolk sac diameter (YSD) and volume (YSV); fetal heart rate (FHR), mean uterine arteries pulsatility index (UtA-PI) and maternal blood placental protein-13 (PP13) levels. Patients were divided into three groups by GA; and early miscarriage versus ongoing pregnancy after GA 10 weeks. Settings: tertiary university-affiliated medical centre (April 2017-June 2018). Results: Patients: Cohort of 115 healthy IVF patients with a singleton viable embryo in early first trimester. Early fetal loss occurred in 14.8% of patients; miscarriage group had higher discrepancy between calculated and measured GA (p < 0.001), lower GSD and GSV (P = 0.005 and P = 0.02, respectively), significantly different YSD and YSV, lower GSD/YSD and GSV/YSV ratios (P = 0.001 and P = 0.003, respectively). UtA-PI/CRL ratio was higher in patients with miscarriage at GA 46-48 days and at GA >48 days (P = 0.034 and P = 0.026, respectively). PP13/CRL ratio was higher in patients with miscarriage at GA >48 days (P = 0.041). Conclusions: In IVF pregnancies with live embryo at first ultrasound scan, high UtA-PI/CRL and maternal blood PP13/CRL ratios may indicate impaired placentation preceded early pregnancy loss. A larger cohort is needed to further verify these predictions.
Workshops are an important part of the IFPA annual meeting. At the IFPA meeting 2008 diverse topi... more Workshops are an important part of the IFPA annual meeting. At the IFPA meeting 2008 diverse topics were discussed in 12 themed workshops. Topics covered included: immunology of placentation; galectins and trophoblast invasion; signaling in implantation and invasion; markers to identify trophoblast subpopulations; placental pathology; placental toxicology; stereology; placental transport of fatty acids; placental mesenchymal stem cells; comparative placentation; trophoblast and neoplasia; trophoblast differentiation. This report is a summary of the various topics covered.
Penaeoid shrimp oocytes nearing the completion of oogenesis are enveloped in an acellular vitelli... more Penaeoid shrimp oocytes nearing the completion of oogenesis are enveloped in an acellular vitelline envelope and possess extracellular cortical rods (CRs) that extended into the cortical cytoplasm. These cortical specializations are precursors of the jelly layer (JL) of the egg. In searching for highly expressed mRNAs during oogenesis in the marine shrimp (Penaeus semisulcatus), two related cDNAs have been isolated that encode a mature protein of 250 amino acid residues. The deduced amino acid sequences revealed the presence of repeated cysteine-rich domains that are related to the chitin-binding domains of insect intestinal peritrophins. Similar cysteine-rich domains were reported in insect intestinal mucin, crustacean tachycitin, and invertebrate chitinases. The shrimp ovarian peritrophin (SOP) is glycosylated and can bind chitin when extracted from CRs. Its apparent molecular mass in SDS-PAGE is 29-35 kDa and 33-36 kDa, under nonreducing or reducing conditions, respectively. SOP is a major protein of CRs and the JL, and was immunodetected in ovaries; purified CRs; fertilized eggs that were surrounded by a JL matrix; and in the cloudy, whitish flocculent material appearing in sea water immediately after spawning. Immunolocalization in tissue sections determined that SOP was present in oocyte cytoplasm and in extraoocytic CRs. Shrimp expressed SOP mRNA in ovaries at all oocyte developmental stages, whereas expression in the hepatopancreas was restricted to vitellogenic stages. SOP mRNA was abundant in the shrimp ovary and was detected before the presence of the corresponding protein. This is the first demonstration that a protein with similar features to insect intestinal peritrophins is a component of CRs and is therefore a main precursor of the JL of spawned shrimp eggs.
(Y. Li). Background: To evaluate the quantity and function changes of CD8+T cells in peripheral b... more (Y. Li). Background: To evaluate the quantity and function changes of CD8+T cells in peripheral blood and endometrium of women with repeated implantation failure (RIF). Methods: 37 patients with RIF and 19 normal controls were enrolled in this study, peripheral blood and endometrium were collected during the mid-luteal phase. The percentage of peripheral CD8+T subsets and the expression levels of perforin and granzyme B of peripheral CD8+T cells were assessed by flow cytometric analysis. The percentage of endometrial CD8+T cells was analyzed by IHC, the production of perforin and granzyme B of endometrial CD8+T cells was analyzed by IF. Results: Compared to the control group, the percentage of peripheral CD8+T cells in patients with RIF was not significantly different (37.22% vs 37.15%, P > 0.05). However, the porportion of endometrial CD8+T cells was higher in RIF group than in Control group (1.99% vs 3.77%, P < 0.001). The expression levels of perforin and granzyme B in peripheral and endometrial CD8+T cells in patients with RIF were similar with control group. Conclusions: Compared to the control group, the percentage of endometrial CD8+ T were largely upregulated in patients with RIF. However, the cytotoxicity of CD8+T cells was similar between control and RIF group.
We evaluated the role of placental protein 13 (PP13; galectin 13) in the process of trophoblast i... more We evaluated the role of placental protein 13 (PP13; galectin 13) in the process of trophoblast invasion and decidual necrosis. Immunohistochemical analysis for PP13, immune cells, human placental lactogen, cytokeratin, and apoptosis markers was performed on 20 elective pregnancy termination specimens between 6 and 15 weeks of gestation. Placental protein 13 was localized to syncytiotrophoblasts in the chorionic villi and to occasional multinucleated luminal trophoblasts within converted decidual spiral arterioles. Cytotrophoblasts, anchoring trophoblasts, and invasive trophoblasts did not stain for PP13. Extracellular PP13 aggregates were found around decidual veins associated with T-cell-, neutrophiland macrophage-containing decidual zones of necrosis (ZONEs). We hypothesize that PP13 is secreted into the intervillus space, drains through the decidua basalis veins, and forms perivenous PP13 aggregates which attract and activate maternal immune cells. Thus, syncytiotrophoblast-deri...
Introduction: The importance of biomarkers for pharmaceutical drug development and clinical diagn... more Introduction: The importance of biomarkers for pharmaceutical drug development and clinical diagnostics is more significant than ever in the current shift toward personalized medicine. Biomarkers have taken a central position either as companion markers to support drug development and patient selection, or as indicators aiming to detect the earliest perturbations indicative of disease, minimizing therapeutic intervention or even enabling disease reversal. Protein biomarkers are of particular interest given their central role in biochemical pathways. Hence, capabilities to analyze multiple protein biomarkers in one assay are highly interesting for biomedical research. Areas covered: We here review multiple methods that are suitable for robust, high throughput, standardized, and affordable analysis of protein biomarkers in a multiplex format. We describe innovative developments in immunoassays, the vanguard of methods in clinical laboratories, and mass spectrometry, increasingly implemented for protein biomarker analysis. Moreover, emerging techniques are discussed with potentially improved protein capture, separation, and detection that will further boost multiplex analyses. Expert commentary: The development of clinically applied multiplex protein biomarker assays is essential as multi-protein signatures provide more comprehensive information about biological systems than single biomarkers, leading to improved insights in mechanisms of disease, diagnostics, and the effect of personalized medicine.
P-selectin (CD62P) is a Ca2+-dependent endogenous lectin that can be expressed by vascular endoth... more P-selectin (CD62P) is a Ca2+-dependent endogenous lectin that can be expressed by vascular endothelium and platelets. The major ligand for P-selectin on leukocytes is P-selectin glycoprotein ligand-1 (PSGL-1). P-selectin can also bind to carcinoma cells, but the nature of the ligand(s) on these cells is unknown. Here we investigated the P-selectin binding to a breast and a small cell lung carcinoma cell line that are negative for PSGL-1. We report that CD24, a mucin-type glycosylphosphatidylinositol-linked cell surface molecule on human neutrophils, pre B lymphocytes, and many tumors can promote binding to P-selectin. Latex beads coated with purified CD24 from the two carcinoma cell lines but also neutrophils could bind specifically to P-selectin-IgG. The binding was dependent on divalent cations and was abolished by treatment with O-sialoglycoprotein endopeptidase but not endoglycosidase F or sialidase. The beads were stained with a monoclonal antibody (MoAb) to CD57 (HNK-1 carbohy...
Objective To investigate the value of maternal serum placental protein 13 (PP-13) measurement and... more Objective To investigate the value of maternal serum placental protein 13 (PP-13) measurement and uterine artery Doppler during first-trimester screening in the prediction of early pre-eclampsia.
Nectadrin, the cell surface glycoprotein recognized by the novel mAb 79, was found to be immunolo... more Nectadrin, the cell surface glycoprotein recognized by the novel mAb 79, was found to be immunologically identical to the heat-stable antigen (HSA). It is a glycoprotein with a polypeptide core of only 30 amino acids and a very high carbohydrate content (Wenger, R. H., M. Ayane, R. Bose, G. Kthler, and P. J. . Eur. J. lmmunol. 21:1039-1046. Immunocytological studies using cultured splenic B-lymphocytes, neuroblastoma cells, and cerebellar cells indicated that nectadrin is preferentially expressed at sites of cell-cell contact. Purified nectadrin and monoclonal nectadrin antibody 79, but not other monoclonal nectadrin antibodies, inhibited the aggregation of B-lymphocytes by 70%, suggesting that nectadrin may act as a cell adhesion molecule. Nectadrin
Proceedings of the National Academy of Sciences, 2003
Brachydactyly (BD) type A2 is an autosomal dominant hand malformation characterized by shortening... more Brachydactyly (BD) type A2 is an autosomal dominant hand malformation characterized by shortening and lateral deviation of the index fingers and, to a variable degree, shortening and deviation of the first and second toes. We performed linkage analysis in two unrelated German families and mapped a locus for BD type A2 to 4q21-q25. This interval includes the gene bone morphogenetic protein receptor 1B (BMPR1B), a type I transmembrane serinethreonine kinase. In one family, we identified a T599 3 A mutation changing an isoleucine into a lysine residue (I200K) within the glycine͞serine (GS) domain of BMPR1B, a region involved in phosphorylation of the receptor. In the other family we identified a C1456 3 T mutation leading to an arginine-to-tryptophan amino acid change (R486W) in a highly conserved region C-terminal of the BMPR1B kinase domain. An in vitro kinase assay showed that the I200K mutation is kinase-deficient, whereas the R486W mutation has normal kinase activity, indicating a different pathogenic mechanism. Functional analyses with a micromass culture system revealed a strong inhibition of chondrogenesis by both mutant receptors. Overexpression of mutant chBmpR1b in vivo in chick embryos by using a retroviral system resulted either in a BD phenotype with shortening and͞or missing phalanges similar to the human phenotype or in severe hypoplasia of the entire limb. These findings imply that both mutations identified in human BMPR1B affect cartilage formation in a dominant-negative manner.
Proceedings of the National Academy of Sciences, 1987
Antibodies to the synthetic peptide (carriercoupled) corresponding to amino acids 210-223 of the ... more Antibodies to the synthetic peptide (carriercoupled) corresponding to amino acids 210-223 of the primary sequence of eel Na channel (Cl+ peptide) were generated. The antipeptide antibodies were used to identify functional roles as well as the accessibility from the external membrane surface of the C1 domains. Rabbit antipeptide antibodies bound specifically to the Cl' synthetic peptide and to an eel membrane fraction bearing a high density of Na channels. When applied
In the field of preeclampsia, enormous efforts are ongoing to identify biomarkers predicting the ... more In the field of preeclampsia, enormous efforts are ongoing to identify biomarkers predicting the syndrome already in the first trimester of pregnancy. At the same time, there is the need for in vitro models to test such biomarkers prior to their use in clinical trials. In addition, in vitro models may accelerate the development and evaluation of the benefit of any putative therapeutics. Therefore, in vitro systems have been established to evaluate the release of biomarkers and measure the effect of putative therapeutics using placental villous explants as well as the choriocarcinoma cell line BeWo. For explants, a cryogenic method to freeze, transport and thaw villous explants was developed to use such tissues for a multi-site tissue culture evaluation.
Preeclampsia is one of the leading causes for maternal and fetal morbidity. Attempts to prevent p... more Preeclampsia is one of the leading causes for maternal and fetal morbidity. Attempts to prevent preeclampsia have already been made using low-dose aspirin, low-molecular-weight heparin (LMWH), and calcium supplementation. Magnesium sulphate is used at the time of disease to prevent eclampsia. Here we investigated the effect of these agents on PP13 release from placental explants. Placentas harvested after C-section of term or preterm control and preeclampsia cases or first trimester terminations were used to obtain explants. Explants were incubated for 24h with/without respective agents, harvested, weighed and subjected to PP13 determination in the culture medium and the explant. LDH was used to determine viability. Dose response curves were obtained for each drug. P &lt; 0.05 was considered significant. Exposure to magnesium (0.7-7g/day) slightly decreased PP13 release from controls, and slightly increased it in preeclampsia and first trimester termination. Calcium (0. 3-6g/day) showed a tendency to decrease the release in control and preeclampsia, whereas in first trimester release was increased in a bell-shaped manner. Aspirin (0-250 mg/day) tended to decrease the release in controls but increased it in a bell-shaped manner in first trimester and preeclampsia. LMWH showed no effect from 0 to 80 mg/day in controls but tended to decrease PP13 release in preeclampsia and first trimester. This data might point to a beneficial effect of aspirin and calcium supplementation in the first trimester of pregnancy and aspirin at the time of disease, although the interaction with the maternal system still needs to be elucidated.
To compare the distribution of placental protein 13 (PP13) in fetal and maternal blood and amnion... more To compare the distribution of placental protein 13 (PP13) in fetal and maternal blood and amnionic fluid and to correlate it with PP13 protein and mRNA in the placenta. Umbilical arterial serum, amnionic fluid, maternal venous serum and placental tissues were collected from normal outcome pregnancies (N = 63) (GA&gt;37), early onset preeclampsia (PE) (N = 12, GA: 26-33), and HELLP syndrome (N = 5, GA: 27-29). Because PE and HELLP cases delivered preterm, cases of preterm delivery (PTD) (N = 6, GA: 31-36) served as additional control. PP13 was determined by ELISA, Western blot, and immunohistochemistry. PP13 mRNA was measured by PCR (RT-PCR). Continuous parameters were compared by t-test, P &lt; 0.05 was considered significant. In women with normal pregnancy outcome significantly higher PP13 levels were found in maternal serum compared to amnionic fluid and negligible amount was found in fetal serum. A similar pattern was identified in cases of PTD with concentrations similar to term control. In PE and HELLP cases PP13 levels in amnionic fluid level were more than twice compared to maternal serum (P &lt; 0.001). Umbilical cord level was negligible in PE but high in HELLP corresponding to the much higher level of PP13 in this patient group compared to all others. In the placenta PP13 level in term controls was higher compared to PTD. In PE and HELLP (similar early delivery time as PTD) the level was significantly higher (P &lt; 0.01) compared to PTD or term controls. PP13 mRNA levels in term control and PTD were similar while PP13 mRNA levels in PE and HELLP placentas were significantly lower compared to term controls or PTD or the two combined. Syncytiotrophoblast labeling appeared stronger in PE and HELLP compared to term controls and PTD. In all cases but HELLP, PP13 in fetal blood is very low indicating that routing of PP13 to fetal blood is limited and that the fetus is unlikely to generate PP13. PP13 mRNA is lower in the third trimester at the time of disease while protein level accumulates and become higher creating an unparallel change in the level of the mRNA and the corresponding protein.
Fish eggs contain carotenoids, retinals (retinal and dehydroretinal) and retinols (retinol, dehyd... more Fish eggs contain carotenoids, retinals (retinal and dehydroretinal) and retinols (retinol, dehydroretinol and retinyl-esters) that are utilized during embryonic development, after fertilization. The carotenoids (mainly astaxanthins) are transported in the plasma by the low density lipoproteins, high density lipoproteins, and very high density lipoproteins (VHDL) and were found to be associated also with serum albumin. Retinals were found to be associated vitellogenin (VTG), a component of the plasma VHDL fraction that is internalized by oocytes during vitellogenesis. However, the transport of retinols and retinyl-esters that were located in the oil droplet fraction of homogenized eggs, has yet to be elucidated. Retinols are more abundant in freshwater fish eggs than in eggs of marine fish species. Since retinol is transported in the plasma of vertebrates in association with retinol binding protein (RBP), recent studies on the molecular characterization and expression sites of RBP, could contribute to determining the involvement of RBP in transporting retinol to developing oocytes in vertebrates.
The heterodimeric ol4 integrins c~4~7 lymphocyte Peyer's patch adhesion molecule ([LPAM]-I) and o... more The heterodimeric ol4 integrins c~4~7 lymphocyte Peyer's patch adhesion molecule ([LPAM]-I) and ol431 (very late antigen-4) are cell surface adhesion molecules involved in lymphocyte trafficking and lymphocyte-cell and matrix interactions. Known cellular ligands include vascular cell adhesion molecule (VCAM)-I, which binds to ~431 and ol437, and the mucosal addressin cell adhesion molecule (MAdCAM)-I, which binds to ol437. Here we show that the o~4 chain of these integrins can itself serve as a ligand. The c~4 chain, immunoaffinity purified and immobilized on glass slides, binds thymocytes and T lymphocytes. Binding exhibits divalent cation requirements and temperature sensitivity which are characteristic of integrin-mediated interactions, and is specifically inhibited by anti-cx4 integrin antibodies, which exert their effect at the cell surface. Cells expressing exclusively c~4/~7 (TK-1) or c~431 (L1-2) both bound avidly, whereas ol4-negative cells did not. A soluble 34-kD ol4 chain fragment retained binding activity, and it inhibited lymphocyte adhesion to ~4 ligands. It has been shown that c~4 integrin binding to fibronectin involves an leucine-aspartic acid-valine (LDV) motif in the HeplI/IIICS region of fibronectin (CS-1 peptide), and homologous sequences are important in binding to VCAM-1 and MAdCAM-1. Three conserved LDV motifs occur in the extracellular sequence of c~4. A synthetic LDV-containing c~4-derived oligopeptide supports c~4-integrin-dependent lymphocyte adhesion and blocks binding to the 34-kD ol4 chain fragment. Our results suggest that r and r integrins may be able to bind to the ol4 subunit on adjacent cells, providing a novel mechanism for ol4 integrin-mediated and activation-regulated lymphocyte interactions during immune responses.
Objectives: To predict miscarriage before 10 weeks of gestation in IVF pregnancies. Methods: Calc... more Objectives: To predict miscarriage before 10 weeks of gestation in IVF pregnancies. Methods: Calculation of gestational age (GA); ultrasound evaluation of Crown-rump length (CRL), mean gestational sac diameter (GSD) and volume (GSV), mean yolk sac diameter (YSD) and volume (YSV); fetal heart rate (FHR), mean uterine arteries pulsatility index (UtA-PI) and maternal blood placental protein-13 (PP13) levels. Patients were divided into three groups by GA; and early miscarriage versus ongoing pregnancy after GA 10 weeks. Settings: tertiary university-affiliated medical centre (April 2017-June 2018). Results: Patients: Cohort of 115 healthy IVF patients with a singleton viable embryo in early first trimester. Early fetal loss occurred in 14.8% of patients; miscarriage group had higher discrepancy between calculated and measured GA (p < 0.001), lower GSD and GSV (P = 0.005 and P = 0.02, respectively), significantly different YSD and YSV, lower GSD/YSD and GSV/YSV ratios (P = 0.001 and P = 0.003, respectively). UtA-PI/CRL ratio was higher in patients with miscarriage at GA 46-48 days and at GA >48 days (P = 0.034 and P = 0.026, respectively). PP13/CRL ratio was higher in patients with miscarriage at GA >48 days (P = 0.041). Conclusions: In IVF pregnancies with live embryo at first ultrasound scan, high UtA-PI/CRL and maternal blood PP13/CRL ratios may indicate impaired placentation preceded early pregnancy loss. A larger cohort is needed to further verify these predictions.
Workshops are an important part of the IFPA annual meeting. At the IFPA meeting 2008 diverse topi... more Workshops are an important part of the IFPA annual meeting. At the IFPA meeting 2008 diverse topics were discussed in 12 themed workshops. Topics covered included: immunology of placentation; galectins and trophoblast invasion; signaling in implantation and invasion; markers to identify trophoblast subpopulations; placental pathology; placental toxicology; stereology; placental transport of fatty acids; placental mesenchymal stem cells; comparative placentation; trophoblast and neoplasia; trophoblast differentiation. This report is a summary of the various topics covered.
Penaeoid shrimp oocytes nearing the completion of oogenesis are enveloped in an acellular vitelli... more Penaeoid shrimp oocytes nearing the completion of oogenesis are enveloped in an acellular vitelline envelope and possess extracellular cortical rods (CRs) that extended into the cortical cytoplasm. These cortical specializations are precursors of the jelly layer (JL) of the egg. In searching for highly expressed mRNAs during oogenesis in the marine shrimp (Penaeus semisulcatus), two related cDNAs have been isolated that encode a mature protein of 250 amino acid residues. The deduced amino acid sequences revealed the presence of repeated cysteine-rich domains that are related to the chitin-binding domains of insect intestinal peritrophins. Similar cysteine-rich domains were reported in insect intestinal mucin, crustacean tachycitin, and invertebrate chitinases. The shrimp ovarian peritrophin (SOP) is glycosylated and can bind chitin when extracted from CRs. Its apparent molecular mass in SDS-PAGE is 29-35 kDa and 33-36 kDa, under nonreducing or reducing conditions, respectively. SOP is a major protein of CRs and the JL, and was immunodetected in ovaries; purified CRs; fertilized eggs that were surrounded by a JL matrix; and in the cloudy, whitish flocculent material appearing in sea water immediately after spawning. Immunolocalization in tissue sections determined that SOP was present in oocyte cytoplasm and in extraoocytic CRs. Shrimp expressed SOP mRNA in ovaries at all oocyte developmental stages, whereas expression in the hepatopancreas was restricted to vitellogenic stages. SOP mRNA was abundant in the shrimp ovary and was detected before the presence of the corresponding protein. This is the first demonstration that a protein with similar features to insect intestinal peritrophins is a component of CRs and is therefore a main precursor of the JL of spawned shrimp eggs.
(Y. Li). Background: To evaluate the quantity and function changes of CD8+T cells in peripheral b... more (Y. Li). Background: To evaluate the quantity and function changes of CD8+T cells in peripheral blood and endometrium of women with repeated implantation failure (RIF). Methods: 37 patients with RIF and 19 normal controls were enrolled in this study, peripheral blood and endometrium were collected during the mid-luteal phase. The percentage of peripheral CD8+T subsets and the expression levels of perforin and granzyme B of peripheral CD8+T cells were assessed by flow cytometric analysis. The percentage of endometrial CD8+T cells was analyzed by IHC, the production of perforin and granzyme B of endometrial CD8+T cells was analyzed by IF. Results: Compared to the control group, the percentage of peripheral CD8+T cells in patients with RIF was not significantly different (37.22% vs 37.15%, P > 0.05). However, the porportion of endometrial CD8+T cells was higher in RIF group than in Control group (1.99% vs 3.77%, P < 0.001). The expression levels of perforin and granzyme B in peripheral and endometrial CD8+T cells in patients with RIF were similar with control group. Conclusions: Compared to the control group, the percentage of endometrial CD8+ T were largely upregulated in patients with RIF. However, the cytotoxicity of CD8+T cells was similar between control and RIF group.
We evaluated the role of placental protein 13 (PP13; galectin 13) in the process of trophoblast i... more We evaluated the role of placental protein 13 (PP13; galectin 13) in the process of trophoblast invasion and decidual necrosis. Immunohistochemical analysis for PP13, immune cells, human placental lactogen, cytokeratin, and apoptosis markers was performed on 20 elective pregnancy termination specimens between 6 and 15 weeks of gestation. Placental protein 13 was localized to syncytiotrophoblasts in the chorionic villi and to occasional multinucleated luminal trophoblasts within converted decidual spiral arterioles. Cytotrophoblasts, anchoring trophoblasts, and invasive trophoblasts did not stain for PP13. Extracellular PP13 aggregates were found around decidual veins associated with T-cell-, neutrophiland macrophage-containing decidual zones of necrosis (ZONEs). We hypothesize that PP13 is secreted into the intervillus space, drains through the decidua basalis veins, and forms perivenous PP13 aggregates which attract and activate maternal immune cells. Thus, syncytiotrophoblast-deri...
Introduction: The importance of biomarkers for pharmaceutical drug development and clinical diagn... more Introduction: The importance of biomarkers for pharmaceutical drug development and clinical diagnostics is more significant than ever in the current shift toward personalized medicine. Biomarkers have taken a central position either as companion markers to support drug development and patient selection, or as indicators aiming to detect the earliest perturbations indicative of disease, minimizing therapeutic intervention or even enabling disease reversal. Protein biomarkers are of particular interest given their central role in biochemical pathways. Hence, capabilities to analyze multiple protein biomarkers in one assay are highly interesting for biomedical research. Areas covered: We here review multiple methods that are suitable for robust, high throughput, standardized, and affordable analysis of protein biomarkers in a multiplex format. We describe innovative developments in immunoassays, the vanguard of methods in clinical laboratories, and mass spectrometry, increasingly implemented for protein biomarker analysis. Moreover, emerging techniques are discussed with potentially improved protein capture, separation, and detection that will further boost multiplex analyses. Expert commentary: The development of clinically applied multiplex protein biomarker assays is essential as multi-protein signatures provide more comprehensive information about biological systems than single biomarkers, leading to improved insights in mechanisms of disease, diagnostics, and the effect of personalized medicine.
P-selectin (CD62P) is a Ca2+-dependent endogenous lectin that can be expressed by vascular endoth... more P-selectin (CD62P) is a Ca2+-dependent endogenous lectin that can be expressed by vascular endothelium and platelets. The major ligand for P-selectin on leukocytes is P-selectin glycoprotein ligand-1 (PSGL-1). P-selectin can also bind to carcinoma cells, but the nature of the ligand(s) on these cells is unknown. Here we investigated the P-selectin binding to a breast and a small cell lung carcinoma cell line that are negative for PSGL-1. We report that CD24, a mucin-type glycosylphosphatidylinositol-linked cell surface molecule on human neutrophils, pre B lymphocytes, and many tumors can promote binding to P-selectin. Latex beads coated with purified CD24 from the two carcinoma cell lines but also neutrophils could bind specifically to P-selectin-IgG. The binding was dependent on divalent cations and was abolished by treatment with O-sialoglycoprotein endopeptidase but not endoglycosidase F or sialidase. The beads were stained with a monoclonal antibody (MoAb) to CD57 (HNK-1 carbohy...
Objective To investigate the value of maternal serum placental protein 13 (PP-13) measurement and... more Objective To investigate the value of maternal serum placental protein 13 (PP-13) measurement and uterine artery Doppler during first-trimester screening in the prediction of early pre-eclampsia.
Nectadrin, the cell surface glycoprotein recognized by the novel mAb 79, was found to be immunolo... more Nectadrin, the cell surface glycoprotein recognized by the novel mAb 79, was found to be immunologically identical to the heat-stable antigen (HSA). It is a glycoprotein with a polypeptide core of only 30 amino acids and a very high carbohydrate content (Wenger, R. H., M. Ayane, R. Bose, G. Kthler, and P. J. . Eur. J. lmmunol. 21:1039-1046. Immunocytological studies using cultured splenic B-lymphocytes, neuroblastoma cells, and cerebellar cells indicated that nectadrin is preferentially expressed at sites of cell-cell contact. Purified nectadrin and monoclonal nectadrin antibody 79, but not other monoclonal nectadrin antibodies, inhibited the aggregation of B-lymphocytes by 70%, suggesting that nectadrin may act as a cell adhesion molecule. Nectadrin
Proceedings of the National Academy of Sciences, 2003
Brachydactyly (BD) type A2 is an autosomal dominant hand malformation characterized by shortening... more Brachydactyly (BD) type A2 is an autosomal dominant hand malformation characterized by shortening and lateral deviation of the index fingers and, to a variable degree, shortening and deviation of the first and second toes. We performed linkage analysis in two unrelated German families and mapped a locus for BD type A2 to 4q21-q25. This interval includes the gene bone morphogenetic protein receptor 1B (BMPR1B), a type I transmembrane serinethreonine kinase. In one family, we identified a T599 3 A mutation changing an isoleucine into a lysine residue (I200K) within the glycine͞serine (GS) domain of BMPR1B, a region involved in phosphorylation of the receptor. In the other family we identified a C1456 3 T mutation leading to an arginine-to-tryptophan amino acid change (R486W) in a highly conserved region C-terminal of the BMPR1B kinase domain. An in vitro kinase assay showed that the I200K mutation is kinase-deficient, whereas the R486W mutation has normal kinase activity, indicating a different pathogenic mechanism. Functional analyses with a micromass culture system revealed a strong inhibition of chondrogenesis by both mutant receptors. Overexpression of mutant chBmpR1b in vivo in chick embryos by using a retroviral system resulted either in a BD phenotype with shortening and͞or missing phalanges similar to the human phenotype or in severe hypoplasia of the entire limb. These findings imply that both mutations identified in human BMPR1B affect cartilage formation in a dominant-negative manner.
Proceedings of the National Academy of Sciences, 1987
Antibodies to the synthetic peptide (carriercoupled) corresponding to amino acids 210-223 of the ... more Antibodies to the synthetic peptide (carriercoupled) corresponding to amino acids 210-223 of the primary sequence of eel Na channel (Cl+ peptide) were generated. The antipeptide antibodies were used to identify functional roles as well as the accessibility from the external membrane surface of the C1 domains. Rabbit antipeptide antibodies bound specifically to the Cl' synthetic peptide and to an eel membrane fraction bearing a high density of Na channels. When applied
In the field of preeclampsia, enormous efforts are ongoing to identify biomarkers predicting the ... more In the field of preeclampsia, enormous efforts are ongoing to identify biomarkers predicting the syndrome already in the first trimester of pregnancy. At the same time, there is the need for in vitro models to test such biomarkers prior to their use in clinical trials. In addition, in vitro models may accelerate the development and evaluation of the benefit of any putative therapeutics. Therefore, in vitro systems have been established to evaluate the release of biomarkers and measure the effect of putative therapeutics using placental villous explants as well as the choriocarcinoma cell line BeWo. For explants, a cryogenic method to freeze, transport and thaw villous explants was developed to use such tissues for a multi-site tissue culture evaluation.
Preeclampsia is one of the leading causes for maternal and fetal morbidity. Attempts to prevent p... more Preeclampsia is one of the leading causes for maternal and fetal morbidity. Attempts to prevent preeclampsia have already been made using low-dose aspirin, low-molecular-weight heparin (LMWH), and calcium supplementation. Magnesium sulphate is used at the time of disease to prevent eclampsia. Here we investigated the effect of these agents on PP13 release from placental explants. Placentas harvested after C-section of term or preterm control and preeclampsia cases or first trimester terminations were used to obtain explants. Explants were incubated for 24h with/without respective agents, harvested, weighed and subjected to PP13 determination in the culture medium and the explant. LDH was used to determine viability. Dose response curves were obtained for each drug. P &lt; 0.05 was considered significant. Exposure to magnesium (0.7-7g/day) slightly decreased PP13 release from controls, and slightly increased it in preeclampsia and first trimester termination. Calcium (0. 3-6g/day) showed a tendency to decrease the release in control and preeclampsia, whereas in first trimester release was increased in a bell-shaped manner. Aspirin (0-250 mg/day) tended to decrease the release in controls but increased it in a bell-shaped manner in first trimester and preeclampsia. LMWH showed no effect from 0 to 80 mg/day in controls but tended to decrease PP13 release in preeclampsia and first trimester. This data might point to a beneficial effect of aspirin and calcium supplementation in the first trimester of pregnancy and aspirin at the time of disease, although the interaction with the maternal system still needs to be elucidated.
To compare the distribution of placental protein 13 (PP13) in fetal and maternal blood and amnion... more To compare the distribution of placental protein 13 (PP13) in fetal and maternal blood and amnionic fluid and to correlate it with PP13 protein and mRNA in the placenta. Umbilical arterial serum, amnionic fluid, maternal venous serum and placental tissues were collected from normal outcome pregnancies (N = 63) (GA&gt;37), early onset preeclampsia (PE) (N = 12, GA: 26-33), and HELLP syndrome (N = 5, GA: 27-29). Because PE and HELLP cases delivered preterm, cases of preterm delivery (PTD) (N = 6, GA: 31-36) served as additional control. PP13 was determined by ELISA, Western blot, and immunohistochemistry. PP13 mRNA was measured by PCR (RT-PCR). Continuous parameters were compared by t-test, P &lt; 0.05 was considered significant. In women with normal pregnancy outcome significantly higher PP13 levels were found in maternal serum compared to amnionic fluid and negligible amount was found in fetal serum. A similar pattern was identified in cases of PTD with concentrations similar to term control. In PE and HELLP cases PP13 levels in amnionic fluid level were more than twice compared to maternal serum (P &lt; 0.001). Umbilical cord level was negligible in PE but high in HELLP corresponding to the much higher level of PP13 in this patient group compared to all others. In the placenta PP13 level in term controls was higher compared to PTD. In PE and HELLP (similar early delivery time as PTD) the level was significantly higher (P &lt; 0.01) compared to PTD or term controls. PP13 mRNA levels in term control and PTD were similar while PP13 mRNA levels in PE and HELLP placentas were significantly lower compared to term controls or PTD or the two combined. Syncytiotrophoblast labeling appeared stronger in PE and HELLP compared to term controls and PTD. In all cases but HELLP, PP13 in fetal blood is very low indicating that routing of PP13 to fetal blood is limited and that the fetus is unlikely to generate PP13. PP13 mRNA is lower in the third trimester at the time of disease while protein level accumulates and become higher creating an unparallel change in the level of the mRNA and the corresponding protein.
Fish eggs contain carotenoids, retinals (retinal and dehydroretinal) and retinols (retinol, dehyd... more Fish eggs contain carotenoids, retinals (retinal and dehydroretinal) and retinols (retinol, dehydroretinol and retinyl-esters) that are utilized during embryonic development, after fertilization. The carotenoids (mainly astaxanthins) are transported in the plasma by the low density lipoproteins, high density lipoproteins, and very high density lipoproteins (VHDL) and were found to be associated also with serum albumin. Retinals were found to be associated vitellogenin (VTG), a component of the plasma VHDL fraction that is internalized by oocytes during vitellogenesis. However, the transport of retinols and retinyl-esters that were located in the oil droplet fraction of homogenized eggs, has yet to be elucidated. Retinols are more abundant in freshwater fish eggs than in eggs of marine fish species. Since retinol is transported in the plasma of vertebrates in association with retinol binding protein (RBP), recent studies on the molecular characterization and expression sites of RBP, could contribute to determining the involvement of RBP in transporting retinol to developing oocytes in vertebrates.
The heterodimeric ol4 integrins c~4~7 lymphocyte Peyer's patch adhesion molecule ([LPAM]-I) and o... more The heterodimeric ol4 integrins c~4~7 lymphocyte Peyer's patch adhesion molecule ([LPAM]-I) and ol431 (very late antigen-4) are cell surface adhesion molecules involved in lymphocyte trafficking and lymphocyte-cell and matrix interactions. Known cellular ligands include vascular cell adhesion molecule (VCAM)-I, which binds to ~431 and ol437, and the mucosal addressin cell adhesion molecule (MAdCAM)-I, which binds to ol437. Here we show that the o~4 chain of these integrins can itself serve as a ligand. The c~4 chain, immunoaffinity purified and immobilized on glass slides, binds thymocytes and T lymphocytes. Binding exhibits divalent cation requirements and temperature sensitivity which are characteristic of integrin-mediated interactions, and is specifically inhibited by anti-cx4 integrin antibodies, which exert their effect at the cell surface. Cells expressing exclusively c~4/~7 (TK-1) or c~431 (L1-2) both bound avidly, whereas ol4-negative cells did not. A soluble 34-kD ol4 chain fragment retained binding activity, and it inhibited lymphocyte adhesion to ~4 ligands. It has been shown that c~4 integrin binding to fibronectin involves an leucine-aspartic acid-valine (LDV) motif in the HeplI/IIICS region of fibronectin (CS-1 peptide), and homologous sequences are important in binding to VCAM-1 and MAdCAM-1. Three conserved LDV motifs occur in the extracellular sequence of c~4. A synthetic LDV-containing c~4-derived oligopeptide supports c~4-integrin-dependent lymphocyte adhesion and blocks binding to the 34-kD ol4 chain fragment. Our results suggest that r and r integrins may be able to bind to the ol4 subunit on adjacent cells, providing a novel mechanism for ol4 integrin-mediated and activation-regulated lymphocyte interactions during immune responses.
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Papers by M. Sammar