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Biomedical subjects

R Sager

Publications and source records attributed to R Sager.

At least 19 recordsLinked to original sources
PubMed2005

C-reactive protein production in term human placental tissue.

OBJECTIVE: C-reactive protein (CRP) is a marker of systemic inflammation. Recently, it has been shown that CRP is present in amniotic fluid and fetal urine, and that elevated levels are associated with adverse pregnancy outcome. However, the precise source of amniotic fluid CRP, its regulation, and function during pregnancy is still a matter of debate. The present in vivo and in vitro studies were designed to investigate the production of CRP in human placental tissues. MATERIAL AND METHODS: Ten paired blood samples from peripheral maternal vein (MV), umbilical cord artery (UA) and umbilical vein (UV) were collected from women with elective caesarean sections at term. The placental protein accumulation capacity of hCG, hPL, leptin and CRP was compared with the dual in vitro perfusion method of an isolated cotyledon of human term placentae and quantified by ELISA. Values for accumulation (release) were calculated as total accumulation of maternal and fetal circuits normalized for tissue weight and duration of perfusion. For gene expression, RNA was extracted from placental tissue and reverse transcribed. RT-PCR and real-time PCR were performed using specific primers. RESULTS: The median (range) CRP level was significantly different between UA and UV [50.1 ng/ml (12.1-684.6) vs. 61 ng/ml (16.9-708.1)]. The median (range) difference between UV and UA was 9.3 ng/ml (2.2-31.6). A significant correlation was found between MV CRP and both UA and UV CRP levels. Median (range) MV CRP levels [2649 ng/ml (260.1-8299)] were 61.2 (6.5-96.8) fold higher than in the fetus. In vitro, the total accumulation rates (mean+/-SD) were 31+/-13 (mU/g/min, hCG), 1.16+/-0.19 (microg/g/min, hPL), 4.71+/-1.91 (ng/g/min, CRP), and 259+/-118 (pg/g/min, leptin). mRNA for hCG, hPL and leptin was detectable using conventional RT-PCR, while CRP mRNA could only be demonstrated by applying real-time RT-PCR. In the perfused tissue the transcript levels for the four proteins were comparable to those detected in the native control tissue. CONCLUSIONS: Our results demonstrate that the human placenta produces and releases CRP mainly into the maternal circulation similarly to other analyzed placental proteins under in vitro conditions. Further studies are needed to explore the exact role of placental CRP during pregnancy.

Adult↗
PubMed2003

Asymmetrical transport of glucose across the in vitro perfused human placenta.

The transplacental flux of glucose together with the consumption by the tissue was studied in human term placenta using the dual in vitro perfusion of an isolated cotyledon. The effect of different transplacental glucose gradients going either from the maternal to the foetal side or in the opposite direction was tested. A linear correlation between uptake from the donor circuit as well as transplacental flux and concentration difference of glucose between the two sides was found in both directions. At comparable gradients both uptake and flux were significantly higher with the gradient going from the maternal to the foetal side as compared to the other direction. For the non-metabolizable 2-deoxy-analog of D -glucose no asymmetry of flux was seen. The large fraction of glucose uptake, which is metabolized by placental tissue together with the difference in membrane transport capacity across the microvillous as compared to the basal membrane of the syncytiotrophoblast could be an explanation for the asymmetry in transplacental glucose flux.

Adult↗
PubMed2001

Capacity for hormone production of cultured trophoblast cells obtained from placentae at term and in early pregnancy.

PROBLEM: There is an increased doubt about the identity of isolated cytotrophoblast cells at term. Therefore, we compared pregnancy serum levels of three hormones [human placental lactogen (hPL), human chorionic gonadotropin (hCG), and leptin] with the capacity for hormone production of early placentae [EP; 8-13 weeks of gestation (WG)] and term placentae (TP; 38-42 WG). METHODS: Serum levels of these hormones were determined in 15 paired maternal (7-41 WG) and fetal (37-41 WG) samples. Cytotrophoblast cells were isolated from term (TP; 38-42 weeks) and early (EP; 8-13 weeks) placentae by enzymatic digestion and subsequent purification on a Percoll gradient. These cells were cultured for 6 days. The production of the hormones hPL, hCG, and leptin was determined as release during culture + cell content after culture - cell content before culture. RESULTS: Serum levels (mean +/- SD; n = 15) at 7-12 and 37-41 WG were 89,652 +/- 21,431 and 13,620 +/- 5854 mIU/ml for hCG, 400 +/- 182 and 7088 +/- 2030 ng/ml for hPL, and 12,675 +/- 4266 and 32,236 +/- 10,961 pg/ml for leptin, respectively. For cultured cells from EP and TP, hCG and hPL showed different patterns of release during the first 2-3 days. While the release of these two hormones by EP cytotrophoblast cells continued during 6 days in culture, their concentrations reached a plateau for TP cytotrophoblasts between 4 and 6 days. Leptin was undetectable (< 15 pg/ml) in TP cell cultured media, while for EP all three hormones showed the same release profiles. Production calculated for 30,000 TP trophoblast cells cultured for 6 days (n = 8) was 2-31 mIU for hCG and 0.5-2 ng for hPL. For EP (n = 11), it was 50-1070 mIU for hCG, 15-323 ng for hPL, and 137-580 pg for leptin. Net synthesis of hCG and hPL for TP was > 10-fold and < 1-fold, respectively. In contrast, the production of all three hormones for EP was at least 100 times the initial cell content. CONCLUSIONS: These results demonstrate that trophoblasts from early pregnancy show much higher production rates of hCG, hPL, and leptin than at term. However, the in vitro findings are difficult to be reconciled with the different serum concentrations of the two hormones hPL and leptin observed during the course of pregnancy.

Cells, Cultured↗
PubMed2001

Effect of hypoxia, oxidative stress and lipopolysaccharides on the release of prostaglandins and cytokines from human term placental explants.

Placental hypoxia, ischaemia, reperfusion and resultant oxidative stress, with the release of various factors into the maternal vasculature acting as mediators of endothelial cell dysfunction, play an important role in the development of pre-eclampsia. Human term placental tissue explants were exposed to different stressors, e.g. hypoxia, oxidative stress and lipopolysaccarides, and the effect on the release of prostanoids and cytokines was determined. The hypoxic environment consisted of 2 per cent O2, 5 per cent CO2and 93 per cent N2. Oxidative stress was induced by addition of xanthine together with xanthine oxidase to the incubation medium. As a third experimental variable, lipopolysaccharide was added to the medium. Prostaglandins (8-iso-PGF(2alpha), or 6-keto-PGF(1alpha)and TXB(2)as stable metabolites of prostacyclin and thromboxane, respectively) and cytokines (TNF-alpha, IL-1alpha, IL-1beta, IL-6) were measured using commercial ELISA assays. Under control conditions, the production of prostaglandins in ng/24 h (mean +/- s.d.) was 6 +/- 3 for 8-iso-PGF(2alpha), 19 +/- 9 for 6-keto-PGF(1alpha)and 5 +/- 2 for TXB2. The production of cytokines was 13 +/- 6 pg for TNF-alpha, 7 +/- 2 pg for IL-1alpha, 5 +/- 3 pg for IL-1beta and 18 +/- 9 ng for IL-6. Under hypoxia the production of prostaglandins remained unchanged and of the cytokines only IL-1beta showed a 15-fold increase. Oxidative stress resulted in an increase in the release of prostaglandins and of cytokines of 4- to 15- and 3- to 130-fold, respectively. Lipopolysaccharides and oxidative stress had a similar effect on the production of prostaglandins, whereas the stimulatory effect of lipopolysaccharides on cytokines was significantly higher than that of oxidative stress.

Adult↗
PubMed2000

Reduced mammary tumor progression in WAP-TAg/WAP-maspin bitransgenic mice.

Maspin is a unique serpin involved in the suppression of tumor growth and metastasis. To investigate whether increased levels of maspin protect against tumor progression in vivo, we established a transgenic model in which maspin is targeted to mammary epithelial cells by the Whey Acidic Protein (WAP) promoter for overexpression. We crossed these WAP-maspin transgenic mice with the WAP-TAg mouse model of tumor progression. Maspin overexpression increased the rate of apoptosis of both preneoplastic and carcinomatous mammary epithelial cells. Maspin reduced tumor growth through a combination of reduced angiogenesis and increased apoptosis. The number of pulmonary metastases was reduced in the presence of maspin overexpression. These data demonstrate that targeted overexpression of maspin can inhibit tumor progression in vivo, likely through a combination of increased apoptosis, decreased angiogenesis, and inhibition of tumor cell migration.

Adenocarcinoma↗
PubMed2000

The surface of prostate carcinoma DU145 cells mediates the inhibition of urokinase-type plasminogen activator by maspin.

Maspin is a novel serine protease inhibitor (serpin) with tumor suppressive potential in breast and prostate cancer, acting at the level of tumor invasion and metastasis. It was subsequently demonstrated that maspin inhibits tumor invasion, at least in part, by inhibiting cell motility. Interestingly, in cell-free solutions, maspin does not inhibit several serine proteases including tissue-type plasminogen activator and urokinase-type plasminogen activator (uPA). Despite the recent biochemical evidence that maspin specifically inhibits tissue-type plasminogen activator that is associated with fibrinogen or poly-L-lysine, the molecular mechanism underlying the tumor-suppressive effect of maspin remains elusive. The goal of this study was to investigate the effect of maspin on cell surface-associated uPA. In our experimental system, we chose prostate carcinoma DU145 cells because these cells mediate plasminogen activation primarily by uPA, as shown by two different colorimetric enzyme activity assays. Purified recombinant maspin produced in baculovirus-infected Spodoptera frugiperda Sf9 insect cells [rMaspin(i)] binds specifically to the surface of DU145 cells, inhibits the DU145 cell surface-bound uPA, and forms a stable complex with the uPA in DU145 cell lysate. The inhibitory effect of rMaspin(i) on cell surface-bound uPA was similar to that of an uPA-neutralizing antibody and was reversed by a polyclonal antibody against the reactive site loop sequence of maspin. The Ki value for rMaspin(i) in cell surface-mediated plasminogen activation was 20 nM, which was comparable to the Ki values for plasminogen activator inhibitor 1 and plasminogen activator inhibitor 2, respectively. Furthermore, the proteolytic inhibitory effect of rMaspin(i) was quantitatively consistent with its inhibitory effect on the motility of DU145 cells in vitro. Our data demonstrate an important role for the prostate carcinoma cell surface in mediating the inhibitory interaction between rMaspin(i) and uPA. Thus, future maspin-based therapeutic strategies may prove useful in blocking the invasion and metastasis of uPA-positive prostate carcinoma.

Antineoplastic Agents↗
PubMed2000

Linking gene expression patterns to therapeutic groups in breast cancer.

A major objective of current cancer research is to develop a detailed molecular characterization of tumor cells and tissues that is linked to clinical information. Toward this end, we have identified approximately one-quarter of all genes that were aberrantly expressed in a breast cancer cell line using differential display. The cancer cells lost the expression of many genes involved in cell adhesion, communication, and maintenance of cell shape, while they gained the expression of many synthetic and metabolic enzymes important for cell proliferation. High-density, membrane-based hybridization arrays were used to study mRNA expression patterns of these genes in cultured cells and archived tumor tissue. Cluster analysis was then used to identify groups of genes, the expression patterns of which correlated with clinical information. Two clusters of genes, represented by p53 and maspin, had expression patterns that strongly associated with estrogen receptor status. A third cluster that included HSP-90 tended to be associated with clinical tumor stage, whereas a forth cluster that included keratin 14 tended to be associated with tumor size. Expression levels of these clinically relevant gene clusters allowed breast tumors to be grouped into distinct categories. Gene expression fingerprints that include these four gene clusters have the potential to improve prognostic accuracy and therapeutic outcomes for breast cancer patients.

Adult↗
PubMed2000

Maspin--a novel protease inhibitor with tumor-suppressing activity in breast cancer.

Maspin (mammary serpin) is a novel serine protease inhibitor related to the serpin family with a tumor-suppressing function in breast cancer. Maspin was originally identified from normal mammary epithelium by subtractive hybridization and might function as a class II tumor-suppressor gene. Maspin's decreased expression with increased level of malignancy and its loss in metastatic cells is regulated at the transcriptional level. Cytosin methylation and heterochromatinization in the promoter region might account for this down-regulation of maspin. Transfection of tumor cells with maspin cDNA inhibits invasion and motility and decreases tumor growth and metastatic ability in nude mice. Maspin interacts with the p53 tumor-suppressor pathway and function as an inhibitor of angiogenesis in vitro and in vivo. The progressive loss of expression of maspin during tumor progression makes this new protein an interesting diagnostic and prognostic marker. The re-expression of maspin by pharmacological intervention potentially offers a promising approach as a therapeutic option in breast cancer therapy.

Animals↗
PubMed2000

Production of protein hormones by cultured trophoblast cells isolated from term and early placentae.

PROBLEM: To compare the capacity of de novo hormone synthesis by cultured trophoblast cells isolated from early and term placenta as cytotrophoblast, and to determine the ability of these cells to proliferate in culture. METHOD OF STUDY: Cytotrophoblast cells were isolated from term (TP, 38-42 weeks) and early placentae (EP, 8-13 weeks) by enzymatic digestion and subsequent purification on a percoll gradient. The net synthesis of the hormones human placental lactogen (hPL) and human chorionic gonadotropin (hCG) was determined as the release during culture + cell content after culture - cell content before culture. Proliferation was determined using a dedicated colorimetric reagent (CellTiter 96). RESULTS: Using a percoll gradient we were able to isolate three cell bands with densities of 1.051, 1.058, and 1.063 g/mL, which were predominantly cytotrophoblast cells as shown by immunocytochemical analysis. The cytotrophoblast cells with the highest density (1.063 g/mL) were used because they were found to release the highest amount of hormones and have shown the lowest rate of cell death after 6 days in culture. Both hCG and hPL showed different patterns of release during the first 2-3 days of culture between TP and EP. While the release by EP cytotrophoblast cells continued during 6 days of culture (n = 4), the concentrations for TP cytotrophoblast (n = 4) reached a plateau between 4 and 6 days. Net de novo synthesis calculated for 3 x 10(4) TP trophoblast cells cultured for 6 days (mean +/- SD, n = 4) was 8.65 +/- 9.05 mU for hCG and 0.95 +/- 0.45 ng for hPL. For EP, it was 395.5 +/- 265.5 mU for hCG and 148.8 +/- 84.2 ng for hPL. Net synthesis of hCG was > 10-fold (TP) and > 70-fold (EP) higher than the initial cell content. While at term, hPL synthesis was only a fraction of the initial cell content, production by EP cytotrophoblast was 106 times the initial cell content. The extent of cell death after 6 days in culture was significantly (P < 0.02) higher for term (30-40%) than for early trophoblast (10-20%). Using a proliferation detection agent during the first 3 days of culture with first trimester cytotrophoblast cells, we did not find any changes in the proliferative activity. CONCLUSIONS: There are differences in the functional activity between trophoblast cells obtained from first and third trimester. The in vitro findings are difficult to reconcile with the different patterns of plasma concentrations of the two hormones observed in vitro during the course of pregnancy.

Cells, Cultured↗
PubMed2000

Leptin production and release in the dually in vitro perfused human placenta.

There is clear evidence that the placenta produces leptin. However, it is still unclear to what extent leptin is released into the maternal and the fetal circulation. The aim of our study was to determine placental leptin release rates into these 2 compartments. In 10 term placentas, using dual in vitro perfusion of an isolated cotyledon, concentrations of leptin, hCG, and human placental lactogen (hPL) were determined in perfusates and in the tissue before and after perfusion. With perfusions lasting 270-840 min, total leptin production was 225 pg/g x min [median; interquartile range (IQR), 76-334 pg/g x min]. The release into the fetal circulation was very low (median, 2.5; IQR, 1.1-5.9 pg/g x min) compared with the release into the maternal circulation (median, 203; IQR, 79-373 pg/g x min) corresponding to 1.6% and 98.4% of net release. Only 0.05% of hPL and hCG were released into the fetal circulation and 99.95% into the maternal circulation, confirming previous results. Release into the fetal circulation correlated significantly with release into the maternal circulation for leptin (r = 0.648; P < 0.05) and hPL (r = 0.721; P < 0.05). Furthermore, release of leptin into the fetal circulation was positively correlated with release of fetal hCG (r = 0.661; P < 0.05). Most of the leptin produced by the placenta is released into the maternal circulation, but compared with other placental hormones (hCG and hPL), a considerably higher proportion of leptin is released into the fetal circulation. These findings may at least partially explain the marked increase in maternal serum leptin levels in pregnancy. The rapid postnatal decrease in leptin levels in both the mother and the neonate is also consistent with the concept of placental origin.

Chorionic Gonadotropin↗
PubMed1999

Maspin plays an important role in mammary gland development.

Maspin is a unique member of the serpin family, which functions as a class II tumor suppressor gene. Despite its known activity against tumor invasion and motility, little is known about maspin's functions in normal mammary gland development. In this paper, we show that maspin does not act as a tPA inhibitor in the mammary gland. However, targeted expression of maspin by the whey acidic protein gene promoter inhibits the development of lobular-alveolar structures during pregnancy and disrupts mammary gland differentiation. Apoptosis was increased in alveolar cells from transgenic mammary glands at midpregnancy. However, the rate of proliferation was increased in early lactating glands to compensate for the retarded development during pregnancy. These findings demonstrate that maspin plays an important role in mammary development and that its effect is stage dependent.

Animals↗
PubMed1999

Re-expression of SPR1 in breast cancer cells by phorbol 12-myristate 13-acetate (PMA) or UV irradiation is mediated by the AP-1 binding site in the SPR1 promoter.

BACKGROUND: Invasive tumor cells are characterized by multiple phenotypic changes as a result of the large number of cDNAs being differentially expressed in tumor cells compared to normal progenitors. Expression genetics focuses on changes at the RNA level with the aim of identifying functionally important genes whose aberrant expression in cancer cells is regulated at the level of transcription. These genes were named class II genes and are distinguished from class I genes, which are characterized by genomic mutations, deletions, or other alterations. Reversal of the tumor cell phenotype accompanying normalization of the expression of such genes may be exploited therapeutically if gene expression can be specifically modulated by drugs or other treatments. Considering that genes are coordinately regulated in complex networks, it is likely that the expression of multiple genes can be simultaneously modulated in tumor cells by drugs acting on the signal transduction pathway that regulates their expression. The SPR1 gene is associated with differentiation and its expression is down-regulated or inactivated in malignant cells. Analysis of the SPR1 promoter showed that down-regulation of SPR1 expression in breast tumor cells occurs at the level of transcription. SPR1 presents an example of class II genes, since its expression was up-regulated in tumor cells by phorbol 12-myristate 13-acetate (PMA) or by ultraviolet (UV) irradiation. MATERIALS AND METHODS: The SPR1 gene was identified by differential display on the basis of its reduced or absent expression in human breast tumor cell lines compared to normal mammary epithelial cell strains. Differential expression was confirmed by Northern blot analysis employing multiple normal and tumor cell lines. The promoter region -619 to +15 of the SPR1 gene was sequenced and analyzed by CAT assays, deletion analysis, and mutagenesis. Up-regulation of SPR1 expression by PMA and UV irradiation was monitored by Northern analysis and analyzed by CAT assays. RESULTS: The mechanism of down-regulation of SPR1 expression in breast tumor cells was investigated. It was found that the -619 to +15 upstream promoter region is sufficient for SPR1 expression in normal breast cells, but it is transcriptionally silent in most breast tumor cell lines. By deletion analysis and mutagenesis, two upstream cis-acting promoter elements were identified. Our data indicate that the AP-1 element located between -139 and -133 acts as a major enhancer of SPR1 transcription only in normal mammary epithelial cells but not in corresponding tumor cells, whereas the sequences flanking the AP-1 site do not affect its promoter enhancing activity. In addition, a transcriptional repressor was identified that binds unknown factor(s) and is active in both normal and tumor breast cells. Inhibitor function was mapped to a 35-bp element located from -178 to -139 upstream of the human SPR1 mRNA start site. The expression of SPR1 could be induced in the 21MT-2 metastatic breast tumor cell line by PMA treatment or by short UV irradiation via a transcriptional mechanism. AP-1 is the cis element mediating the transcriptional activation of SPR1 by PMA, which induces the expression of AP-1 factors in 21MT-2 cells. Mutation of the AP-1 site abolishes the induction of SPR1 expression by PMA. CONCLUSIONS: Our results demonstrate that loss of SPR1 expression in breast tumor cells results from impaired transactivation through the AP-1 site in the SPR1 promoter, as well as from the presence of a negative regulatory element active in both normal and tumor cells. Furthermore, our results provide a basis for therapeutic manipulation of down-regulated genes, such as SPR1, in human cancers.

Base Sequence↗
PubMed1998

maspin suppresses the invasive phenotype of human breast carcinoma.

The recently discovered tumor suppressor gene maspin has been shown to inhibit tumor cell motility, invasion, and metastasis in breast cancer by our laboratories. Nonetheless, the exploitation of maspin as a potential diagnostic and/or therapeutic tool has remained limited due to the lack of knowledge concerning its molecular and biological mechanism(s) of action. The work reported here demonstrates that recombinant maspin (rMaspin) has the ability to induce higher cell surface levels of alpha5- and alpha3-containing integrins and reduced levels of alpha2-, alpha4-, alpha6-, alpha(v)-, and some beta1-containing integrins in the metastatic human breast carcinoma cell line MDA-MB-435 concomitant with its ability to inhibit the invasive process in vitro. Furthermore, treatment of MDA-MB-435 cells with rMaspin results in the selective adhesion of the cell to a fibronectin matrix and conversion from a fibroblastic to a more epithelial-like phenotype. In addition, the ability of rMaspin to inhibit the invasive process can be abrogated with a blocking antibody to the alpha5beta1 integrin, which diminishes the ability of the cells to invade through a fibronectin matrix-containing barrier in vitro. Taken together, these data address the hypothesis that rMaspin reduces the invasive phenotype of MDA-MB-435 cells by altering their integrin profile, particularly alpha5, which in turn converts these cells to a more benign epithelial phenotype, with less invasive ability. These data provide new insights into the biological significance of this tumor suppressor gene found in normal mammary epithelium and may form the basis of novel therapeutic strategies in the management of breast carcinoma.

Antibodies, Blocking↗
PubMed1998

Tissue-type plasminogen activator is a target of the tumor suppressor gene maspin.

The maspin protein has tumor suppressor activity in breast and prostate cancers. It inhibits cell motility and invasion in vitro and tumor growth and metastasis in nude mice. Maspin is structurally a member of the serpin (serine protease inhibitors) superfamily but deviates somewhat from classical serpins. We find that single-chain tissue plasminogen activator (sctPA) specifically interacts with the maspin reactive site loop peptide and forms a stable complex with recombinant maspin [rMaspin(i)]. Major effects of rMaspin(i) are observed on plasminogen activation by sctPA. First, rMaspin(i) activates free sctPA. Second, it inhibits sctPA preactivated by poly-D-lysine. Third, rMaspin(i) exerts a biphasic effect on the activity of sctPA preactivated by fibrinogen/gelatin, acting as a competitive inhibitor at low concentrations (< 0.5 microM) and as a stimulator at higher concentrations. Fourth, 38-kDa C-terminal truncated rMaspin(i) further stimulates fibrinogen/gelatin-associated sctPA. rMaspin(i) acts specifically; it does not inhibit urokinase-type plasminogen activator, plasmin, chymotrypsin, trypsin, or elastase. Our kinetic data are quantitatively consistent with a model in which two segregated domains of maspin interact with the catalytic and activating domains of sctPA. These complex interactions between maspin and sctPA in vitro suggest a mechanism by which maspin regulates plasminogen activation by sctPA bound to the epithelial cell surface.

Animals↗
PubMed1998

Expression genetics: a different approach to cancer diagnosis and prognosis.

Expression genetics is a new approach to the identification of cancer-related genes. Instead of studying gene mutations at the genome level, it focuses on the investigation of heredity at the RNA level. By isolating genes whose expression is up or down regulated in cancers, expression geneticists study their function in the context of gene regulation. A major goal of expression genetics in cancer is to correct gene expression in tumors by the application of potential therapeutic agents.

Gene Expression Regulation, Neoplastic↗
PubMed1998

Transport of proteins across the human placenta.

PROBLEM: The transport of various proteins across the human placenta was investigated by comparing maternal and fetal concentrations of tetanus antigen (TT-AG), anti-tetanus (TT)-immunoglobulin G (IgG) (following maternal vaccination), IgA, human chorionic gonadotropin (hCG), human placental lactogen (hPL), and alpha-fetoprotein (AFP) at term. METHOD OF STUDY: The concentrations of the six proteins were determined using enzyme-linked immunosorbent assay in serum of maternal venous and umbilical (fetal) vein samples obtained at delivery from uncomplicated term pregnancies (n = 16). RESULTS: The ratios (mean +/- standard deviation) of fetal (umbilical) to maternal level were 1.41 +/- 0.33 (anti-TT-IgG), 0.91 +/- 0.37 (TT-AG), 0.002 +/- 0.001 (IgA), 0.003 +/- 0.001 (hCG), and 0.008 +/- 0.004 (hPL), while the maternal:fetal concentration ratio of AFP was 0.002 +/- 0.002. IgA, hCG, hPL, and AFP showed a close correlation between maternal and fetal levels varying between r2 = 0.47 to 0.73 (P < 0.004-0.0001). Because AFP is produced by the fetus while IgA originates in the mother, the appearance of small amounts of these two proteins in the maternal or fetal compartment, respectively, suggests a slow rate of diffusion following a high concentration gradient. The detection of hCG and hPL in fetal serum is also interpreted as diffusion from the maternal into the fetal blood. Anti-TT-IgG has a significantly higher concentration in the fetal as compared with the maternal serum, which is in line with the well-documented active transfer of IgG. Fetal TT-antigen levels were similar to maternal concentrations, showing a close correlation (r2 = 0.74, P < 0.0001) between the two proteins. CONCLUSIONS: The correlation between maternal and fetal concentrations of various proteins like IgA (150,000 Da), hCG (42,000 Da), and hPL (21,000 Da) suggests passive diffusion of these macromolecules across the placenta from the maternal to the fetal side, albeit at a slow rate. A similar process is postulated for AFP (70,000 Da) diffusing in the opposite direction from the fetus to the mother. There was no significant difference between the transplacental fetomaternal gradient of IgA and hCG and the maternal-fetal gradient of AFP. In view of the substantially larger volume of circulating maternal as compared with fetal blood, a significantly higher rate of crossing of AFP as compared with the other proteins must be assumed. It is uncertain whether a difference in the rate of transplacental transfer in the two directions or an additional source of AFP production in the maternal compartment explains the high maternal level. Anti-TT-IgG concentration is significantly higher in fetal than in maternal serum suggesting active transfer from the mother to the fetus. Furthermore, there is considerable transfer of TT-AG and a close correlation of fetal:maternal ratios of anti-TT-IgG (150,000 Da) and TT-AG (150,000 Da) could be an indication for a specific transfer of the antigen antibody complex.

Biological Transport↗
PubMed1998

Identification and verification of differential display cDNAs using gene-specific primers and hybridization arrays.

An accurate and streamlined approach to differential display (DD) band identification and verification is described. To minimize false positives, the strategy avoids the use of impure Northern blot probes obtained from PCR-amplified DD bands. To increase throughput, the cloning of DD bands is replaced by a gene-specific primer approach, and hybridization arrays are used in place of Northern blots. In summary, DD bands obtained with long primers were directly sequenced to allow the design and synthesis of gene-specific primers, which were then used to PCR-amplify homogeneous probes for the verification of expression patterns by hybridization array analysis. Differential expression of 60 of the 63 genes tested was confirmed. Thus, false positives are not inherent to DD. The results demonstrate the power of DD used with hybridization arrays to rapidly generate information on expression patterns of differentially expressed genes.

Biomarkers, Tumor↗