Human Cord Serum Research Articles

Endocrine disruptors in the maternal and fetal compartments

SUSAN LASHLEY, ANTONIA CALAFAT, DANA BARR, THOMAS LEDOUX, PAROMITA HORE, MARIAN LAKE, MARK ROBSON, JOHN SMULIAN, UMDNJRobert Wood Johnson Medical School/Robert Wood Johnson University Hospital, Obstetrics, Gynecology and Reproductive Sciences, New Brunswick, New Jersey, Centers for Disease Control and Prevention (CDC), Division of Laboratory Sciences, National Center for Environmental Health, Atlanta, Georgia, New Jersey Department of Environmental Protection, Risk Assessment & Toxicology Section, Division of Science and Research, Trenton, New Jersey, University of Medicine and Dentistry of New Jersey, School of Public Health, Dept. of Environmental & Occupational Health, Piscataway, New Jersey OBJECTIVE: Phthalates (plasticizers) are known endocrine disruptors in the animal kingdom and are associated with developmental toxicities. This study was designed to describe the relationship between levels of phthalate metabolites in human maternal serum and cord serum. STUDY DESIGN: Cord blood and maternal blood were collected from 50 maternal-fetal pairs at the time of elective cesarean section between July 2003May 2004. The collection materials were tested prior to obtaining samples and the collection procedure was designed to minimize the potential for phthalate contamination. The Centers for Disease Control and Prevention analyzed the maternal and fetal sera, using automated solid phase extraction-isotope dilutionhigh performance liquid chromatography-tandem mass spectrometry, for nine metabolites of phthalates. Correlation of phthalates in maternal and fetal sera was assessed based on spearman rank correlation coefficient (r). RESULTS: Phthalate metabolites were detected in 100% of maternal and cord serum samples. However, the frequency of detection for each specific metabolite differed between the two compartments. Seven of the nine phthalate metabolites were more frequently detected in cord serum than maternal serum. For example, mono-2-ethylhexyl phthalate (MEHP) was detected in all of the cord samples but in only 69% of maternal samples (P ! .001). The concentration of MEHP, mono-(2-ethyl-5-hydroxyhexyl) phthalate, and mono-(2-ethyl-5-oxohexyl) phthalate were higher in cord serum then the maternal serum. When metabolites were present in both compartments, a correlation between levels in maternal and fetal sera was found for mono-n-butyl phthalate P = .03 (r =.54), mono-benzyl phthalate P = .04 (r =.40), mono-ethyl phthalate P = .007 (r =.41). CONCLUSION: This study confirms that phthalates metabolites are present in the human fetal compartment. The metabolites levels in cord serum were generally higher than in maternal serum. These results may indicate that the mechanism of fetal clearance of phthalates may predispose to trapping of phthalates in the fetal compartment.

Organ culture for preservation of the cornea: human umbilical cord serum versus fetal bovine serum

To evaluate the changes of porcine corneal endothelium, the morphology, histology, ultrastructure, enzymes activity and metabolism of the cornea induced by organ culture with two different media containing fetal bovine serum (FBS) or human umbilical cord serum (HCS). Fifty pairs of porcine corneas were preserved at 31 degrees C for 7, 14, 21, 28 days. One cornea of each pair was cultivated in medium I containing 10% FBS (group 1); the other one was stored in medium II containing 10% HCS (group 2). Thirteen fresh porcine corneas served as controls. All stored corneas were dehydrated for 24 hours. Twelve corneas from each group were evaluated each week, including the morphology, histology and enzyme histochemical staining of the cornea. Scanning electron microscopy was performed on one cornea from each group at 14 and 28 days and compared with the fresh cornea. pH value, glucose and lactate concentration of the culture media before and after culture were examined. Microbiological evaluation was also performed. Endothelium evaluation did not differ statistically between the two groups of porcine corneas. The morphological endothelium study showed some alterations such as pleomorphism. After 28 days of cultivation, the mean cell losses of endothelium were 10.98% and 10.85% in medium I and medium II stored corneas, respectively. There were no statistical differences of the histology, ultrastructure and enzymes activity of corneas between the two groups. The histological study showed corneal swelling and epithelial sloughing after preservation. Scanning electron microscopy showed an intact endothelial layer in all corneas. Enzyme histochemical staining showed vigorous enzyme activity in the corneal epithelium and endothelium. Enzyme activity in stroma decreased with preservation time. Corneas showed good glucose metabolism. Incidence of contamination was 6% for storage medium. The corneal endothelium can maintain a good viability for 4 weeks in these two organ culture media. HCS can replace FBS in the organ culture medium.

Human fetal and cord serum thyroid hormones: developmental trends and interrelationships.

Thyroid hormone is essential for fetal and neonatal development in particular of the brain, but little is known about regulation of fetal thyroid hormone levels throughout human gestation. The purpose of this study was to clarify developmental trends and interrelationships among T(4), free T(4) (FT4), thyroxine-binding globulin (TBG), TSH, T(3), rT(3), and T(4) sulfate (T4S) levels in cord and fetal blood sera (n = 639, 15-42 wk gestation) and correlate infant levels (23-42 wk gestation) to maternal values (n = 428, 16-45 yr) and those of nonpregnant women (n = 233, 16-46 yr). In cord and fetal serum, T(4), T(3), and TBG levels increase with gestation until term; TSH, FT4, T4S, and rT(3) levels increase and peak in the late second/early third trimester and then decline to term; T(4)/TBG ratios increase until late second trimester and plateau to term. Term cord sera TSH, TBG, and all iodothyronine levels, except T(3), are higher than nonpregnant women. In the third trimester, cord serum FT4, TSH, rT(3), and T4S levels are also higher than corresponding maternal levels, but T(4), T(3), and TBG levels are lower than maternal values. The late second/early third trimester is a critical transition period in fetal thyroid hormone metabolism, which may be interrupted by preterm birth and contribute to postnatal thyroid dysfunction.

The sensitivity ofEscherichia coli strains with K1 surface antigen and rods without this antigen to the bactericidal effect of serum

The susceptibility of Escherichia coli strains with K1 surface antigen (K1+) and rods without this antigen (K1-) to the bactericidal action of normal bovine serum and human normal cord serum was determined. Seventy E. coli strains (35 K1+ and 35 K1-) were isolated from urine obtained from children with urinary tract infections. The strains investigated showed variable sensitivity to the bactericidal action of the sera. E. coli K1+ strains were characterized by lower sensitivity to bactericidal effect of the sera in comparison with K1- rods. The role of the particular mechanisms of complement activation in the process of killing of the E. coli strains was also determined.

Meconium Is a Potent Activator of Complement in Human Serum and in Piglets

Meconium aspiration syndrome (MAS) is a clinical condition in the newborn infant with a significant morbidity and mortality. The complex pathophysiology of MAS, leading to both pulmonary and systemic complications, is characterized by an incompletely understood inflammatory reaction. Treatment is symptomatic, mainly limited to airway cleaning and ventilatory support. In this study, we show for the first time that meconium is a potent activator of complement, a key mediator of inflammation. In vitro, meconium activated the alternative complement pathway in human umbilical cord serum as judged by a substantial increase in the alternative pathway convertase C3bBbP. The activation proceeded through C3 (C3bc) and the terminal C5-9 pathway (terminal SC5b-9 complement complex), whereas the classical and lectin pathways were not activated (C1rs-C1-inhibitor complexes and C4bc). The lipid fraction, containing, e.g. free fatty acids, and the water fraction, containing, e.g. bile acids, contributed equally to the complement activation. A blocking antibody to factor D (alternative pathway) completely inhibited the meconium-induced complement activation, whereas blocking antibodies to mannose-binding lectin (lectin pathway) and C2 (classical and lectin pathway) had no effect. In vivo, meconium induced systemic complement activation in a piglet model of MAS, paralleling the increase in lung dysfunction. In conclusion, meconium is a potent activator of the complement system both in vitro and in vivo. Complement may be important in the pathogenesis of MAS, and specific complement inhibition might be a possible treatment approach in MAS.

Relationship between serum 5′-nucleotide phosphodiesterase activity and thrombophilia: enzyme activity in fetus and in patients with acute coronary syndrome

Inhibition of ADP-induced platelet aggregation by a placental substance was first reported by Myatt et al. in 1977 [1]. This substance was assumed to play an important role in maintaining fetal–placental circulation under extremely low oxygen pressure. However, the principle behind this activity has not been fully characterized as yet. Hutton et al. [2] reported in 1980 that this activity was derived from placental heat stable alkaline phosphatase. Sakura et al. [3] proposed that placental 5Vnucleotide phosphodiesterase (PDEase) is involved in the inhibition of the platelet aggregation mentioned above. 5V-Nucleotide phosphodiesterase is an ectoenzyme, and hydrolyzes phosphodiester bonds of DNA and RNA releasing nucleoside 5V-monophosphate. This enzyme also hydrolyzes pyrophosphate bond of ATP, ADP and NAD [4,5]. Enzyme activity in serum is elevated in hepatic disease [6,7]. The physiological function of the enzyme in umbilical cord serum remains to be clarified. We have shown that the enzyme purified from human umbilical cord serum hydrolyzed ADP and suppressed ADP-induced platelet aggregation depending on the enzyme concentration in platelet-rich plasma [8]. These data suggest that serum PDEase is a potent inhibitor of platelet aggregation. In neonates and infants, thromboembolic events have been shown to be rare, possibly due to low vitamin K-dependent coagulation factors and poor ATP storage in neonate platelet [9–12]. Serum PDEase

An expression of IGF-binding proteins in normal and pre-eclamptic human umbilical cord serum and tissues.

Pre-eclampsia, is the most common, pregnancy-associated pathological syndrome accompanied by a significant increase in collagen and sulphated glycosaminoglycans (GAGs) contents in the umbilical cord arteries (UCAs). Insulin-like growth factor-I (IGF-I) is expressed in most foetal tissues and it is involved in anabolic effects. It stimulates protein (mainly collagen) and GAG biosynthesis, cell proliferation and differentiation. Previously, we have found that pre-eclampsia is associated with an increase of IGF-I concentration in the umbilical cord blood. A family of IGF-I-binding proteins (BPs) modulates the activity of IGF-I. We demonstrated qualitative differences between BPs of normal and pre-eclamptic human umbilical cord (UC) serum and UC-tissues (UCA-wall and Wharton's jelly) by Western immunoblot analysis. All examined sera and tissues contained BP-1 and BP-5 as well lower molecular weight materials. The BP-2 was recovered from both control and pre-eclamptic sera, while it was not detected in the UC-tissues. Instead, lower molecular weight forms of BP-2 were found as judged by the anti-BP-2 antibody. The BP-3 was detected in sera, UCA and Wharton's jelly. The most distinct expression of BP-3 was found in the UCA. The pre-eclamptic UCA and Wharton's jelly contained additional BP-3-reactive material of lower molecular weight. The BP-4 was strongly expressed in pre-eclamptic UC-serum and the expression was decreased in pre-eclamptic UC-tissues, compared to respective controls. Ligand binding assay revealed that most of IGF-I was bound to 46 kDa region (typical for BP-3) in both control and pre-eclamptic sera and tissues. However, distinctly less IGF-I was bound in pre-eclamptic serum, distinctly more in pre-eclamptic UCA and no differences were found in pre-eclamptic Wharton's jelly, compared to controls. We demonstrated that both normal and pre-eclamptic UC-sera and tissues are able to degrade 46 kDa IGF-I-BP. The degradation may result in a decrease of IGF-I binding, contributing to increase in free IGF-I that may stimulate the cells to produce extracellular matrix (ECM) components. The specific BPs and their proteolytic modification in UC tissues may be important modulators of IGF-I action during foetal development.

Binding of Polyreactive Antibodies to Self Versus Foreign Antigens

Aside from their ability to bind to multiple antigens, the classic hallmark of polyreactive antibodies is their autoreactivity. Because of their ability to bind a number of common autoantigens, it has long been speculated that polyreactive antibodies are involved in the clearance of self-antigens. However, it has been demonstrated more recently that polyreactive antibodies are also capable of binding to some foreign and synthetic antigens. Although data regarding the relative reactivity of polyreactive antibodies with self versus foreign antigens is lacking, it is generally thought that both activities may play an important biological role. In this study, the relative reactivity of polyclonal human polyreactive IgM with human proteins and tissue extracts versus foreign (xenogeneic) proteins and tissue extracts was probed. The binding of affinity purified anti-ssDNA IgM from adult human serum and the binding of polyreactive IgM in human cord serum and in human adult serum were evaluated. Using competitive and direct binding assays, human polyreactive IgM were found to be generally more reactive with foreign (xenogeneic) proteins than with self or allogeneic proteins. These data shed light on the fundamental nature of polyreactive antibodies, and may provide additional insight into their putative biological roles.

Human α-fetoprotein as a Zn 2+-binding protein. Tight cation binding is not accompanied by global changes in protein structure and stability

The binding of zinc to human α-fetoprotein (AFP) isolated from human umbilical cord serum was studied by fluorimetric Zn 2+-titration. We found that the total number of strong binding sites for zinc on this protein was 5: AFP has one very strong (dissociation constant, K d<10 −8 M) and at least four lower affinity zinc binding sites ( K d<10 −5 M). Fourier transform infrared (FTIR) analysis revealed that aspartate and histidine residues could be involved in the strong coordination of zinc. Intriguingly, binding of zinc to the protein does not induce structural changes that can be detected by circular dichroism, FTIR, intrinsic fluorescence or (1,1′)-bi-(4-anilino)naphthalene-5,5′-disulfonic acid (bis-ANS) binding. Finally, scanning microcalorimetry measurements showed that stability of the protein is also unaffected by zinc binding in spite of the strength of the coordination. Such strong interactions without major structural consequences are highly unusual, and AFP may therefore be the first characterized representative of a new class of ligand-binding proteins.

Evidence for polyreactive xenoreactive antibodies in the repertoire of human anti-swine antibodies: the ‘next’ humoral barrier to xenotransplantation?

The xenoreactive nature of anti-Galα1–3Gal antibodies, and to a lesser extent, polyreactive antibodies, has been characterized by a number of investigators. With the advent of therapies that avoid hyperacute xenograft rejection due to anti-Galα1–3Gal antibodies coupled with the possible development of Galα1–3Gal deficient swine, the Galα1–3Gal antigen may soon cease to be a barrier to xenotransplantation. With this in mind, the potential xenoreactive nature of polyreactive antibodies was investigated using several approaches. The levels of polyreactive antibodies from the serum of newborn ( n=2) and adult ( n=4) baboons undergoing pulmonary xenotransplantation were evaluated. Depletion of 95% and 94% of total serum IgM, without any decrease in albumin levels, was observed in the newborn baboons. This finding indicates that the IgM present at birth and germ line polyreactive IgM was adsorbed by the xenografts. The depletion of polyreactive antibodies (43–83% reduction of anti-DNP IgM) from adult baboons was also observed following pulmonary xenotransplantation or immunoadsorption therapy plus pulmonary xenotransplantation. Additional experiments using human cord serum indicated that most human polyreactive IgM were adsorbed by pig lung homogenate and that the human polyreactive IgM bound approximately two-fold more to immobilized pig lung antigens than to immobilized human lung antigens. These findings indicate that germline polyreactive antibodies are, for the most part, xenoreactive. These data suggest that polyreactive antibodies, although autoreactive, may be more xenoreactive than autoreactive.

Epidermal growth factor enhances lipopolysaccharide-induced procoagulant activity on the cell surface of endothelial cells.

Endotoxin [lipopolysaccharide (LPS)] and some cytokines are known to induce tissue factors with the expression of procoagulant activity (PCA) on the cell surface of endothelial cells. We established a modified quantitative procedure for PCA expressed on the cell surface, and studied the effects of serum and epidermal growth factor (EGF) on the LPS-induced activation of PCA on cultured human umbilical vein endothelial cells. Meanwhile, human umbilical cord serum elevated PCA more than either fetal bovine serum (FBS) or human adult serum. Generally, the existence of LPS raises the concentration of EGF in the blood, since monocytes and other types of cells express EGF and release it into the vessel by LPS stimulation. Although 10 ng/ml EGF did not induce PCA even in the presence of 10% FBS, it enhanced LPS-induced PCA under the same conditions. In the presence of 10% FBS, PCA induced both by LPS alone and by LPS + EGF was reduced the same amount by the protein kinase C (PKC) inhibitors H7 and H8. These results suggest that the serum contains some cofactor other than EGF, and that EGF enhanced this LPS-induced PCA independent of the cofactor through PKC-involved signaling.

The recombinant third domain of human alpha-fetoprotein retains the antiestrotrophic activity found in the full-length molecule

Previous studies have shown that alpha-fetoprotein (AFP) interferes with estrogen (E 2)-stimulated growth, including E 2-stimulated breast cancer growth. In an effort to localize the antiestrotrophic portion of the molecule, the C-terminal one-third (200 amino acids) of human AFP, known as Domain III, was produced in a baculovirus expression system as a fusion protein containing an amino terminal histidine tag. The histidine tag was included to facilitate purification by metal ion affinity chromatography. The purified recombinant Domain III fusion protein was functionally similar to full-length natural AFP isolated from human cord sera or from cultured human hepatoma cells (HepG2) in that they all produced significant and quantitatively similar inhibition of E 2-stimulated growth of immature mouse uterus. Furthermore, the dose–response profiles of the recombinant Domain III AFP and natural full-length AFP were similar. Preincubation of either protein in a molar excess of E 2 lowered the minimally effective antiestrotrophic dose and produced a difference spectrum consistent with a change in conformation. These findings indicate that the antiestrotrophic activity of AFP is contained within the third domain of the molecule, and they have obvious implications for the production of biologically active peptides derived from this portion of the AFP molecule.

The suitability and efficiency of human follicular fluid as a protein supplement in human in vitro fertilization programs

Objective: To evaluate the suitability and efficiency of human follicular fluid (HFF) as a protein supplement in human IVF programs. Design: Comparative study of the effects of HFF and other protein supplements on the in vitro development of mouse oocytes and on the pregnancy rate in human IVF programs. Setting: In Vitro Fertilization Center, Hanna Women’s Clinic, Seoul, Korea. Patient(s): Three hundred twenty-seven patients (388 cycles) who were down-regulated with GnRH agonist and stimulated with hMG. Intervention(s): The suitability was evaluated with the results of animal studies and the efficiency of HFF was investigated with the results of human IVF programs. Main Outcome Measure(s): Meiotic maturation of mouse oocytes, development of mouse embryos, morphological grades of human embryos, pregnancy rate in human IVF programs, and electrophoresis. Result(s): Human follicular fluid significantly stimulated meiotic resumption in mouse oocytes, even in the presence of meiotic inhibitors, and enhanced the developmental potential of mouse embryos in vitro. Compared with human fetal cord serum, human follicular fluid also improved the morphological grade of human embryos by reducing cytoplasmic fragmentation. In conventional IVF cycles of human IVF programs, use of HFF significantly increased the clinical PR (109/234 cycles, 46.5%; P < .05), compared with use of human fetal cord serum (14/52 cycles, 26.9%). However, in intracytoplasmic sperm injection cycles, there was no difference in PRs between use of HFF (38/85 cycles, 44.7%) and use of human fetal cord serum (7/17 cycles, 41.1%). When the protein compositions of human fetal cord serum and HFF were investigated by electrophoresis, a protein of 21 kD was detected specifically in HFF. Conclusion(s): Human follicular fluid is suitable for use as a protein supplement and is effective in increasing the pregnancy rate in human IVF programs.

α-Fetoprotein structure depends on the protein purification procedure: further evidence on the structure forming role of the ligands

Abstract: α.-Fetoprotein samples isolated from human cord serum by metal affinity chromatography and immunoaffi.nity method were compared by circular dichroism, fluorescence spectroscopy and scanning microcalorimetry. The isolation procedures were quite distinguished by their influence on native protein structure (and, consequently, by the effectiveness of natural ligand release from a protein molecule). As a result, structural properties and conformational stability of a.-fetoprotein in the samples were found to be rather different.

Resistance of Acanthamoeba Species to Complement Lysis

Acanthamoeba species were evaluated for susceptibility to complement lysis as determined by release of radiolabeled uridine. The 3 Acanthamoeba species tested, A. culbertsoni (ATCC 30171), A. castellanii (ATCC 30010), and A. polyphaga (ATCC 30461), depleted hemolytic complement activity from normal human serum (NHS), yet were resistant to its lytic effects. Examination of microtiter plates containing amoebae incubated in NHS demonstrated formation of a pellet in the wells. Pellet formation was not observed when amoebae were incubated in human cord serum, heat-inactivated serum, or C1q-deficient serum. Ultrastructural examination of serum-treated amoebae revealed the presence of a finely granular substance that surrounded the amoebae. Treatment of amoebae with enzymes or metabolic inhibitors prior to incubation in NHS was performed to investigate the mechanism of complement resistance. Cycloheximide or cytochalasin D pretreatment increased the susceptibility of A. culbertsoni and A. castellanii to complement lysis. Cytochalasin D treatment also increased the susceptibility of A. polyphaga to complement lysis. Inhibition of serine protease activity by phenylmethylsulfonylfluoride increased complement susceptibility of all 3 species of Acanthamoeba. Enzymatic removal of surface components from A. polyphaga or A. castellanii, with trypsin, neuraminidase, or phosphatidylinositol-specific phospholipase C (PIPLC), did not affect serum resistance. In contrast, PIPLC treatment of A. culbertsoni significantly increased lysis by complement. The ability of Acanthamoeba species to activate the alternative complement pathway yet resist complement-mediated cellular lysis can be attributed to both the release of a transport-dependent extracellular matrix as well as the presence of complement inhibitory surface proteins.

Human sperm capacitation induced by biological fluids and progesterone, but not by NADH or NADPH, is associated with the production of superoxide anion.

Recent evidence indicated that human sperm capacitation is associated with an increased production of superoxide anion (O2.-). To further study the role and importance of O2.- in capacitation, we investigated whether the O2.- generation is a general feature of capacitating spermatozoa, irrespective of the inducer used, and is correlated with capacitation levels and increased tyrosine phosphorylation of two sperm proteins (p105/p81). We also studied the time courses of O2.- production and action. Percoll-washed human spermatozoa were incubated in Ham's F-10 medium, supplemented or not supplemented with various capacitation inducers and in the presence or absence of superoxide dismutase (SOD). Sperm capacitation was measured by induction of the acrosome reaction with lysophosphatidylcholine, O2.- production was measured by chemiluminescence, and tyrosine phosphorylation was measured by immunodetection after electrophoresis and western blotting of sperm proteins. Progesterone and ultrafiltrates of human fetal cord serum, follicular fluid, and seminal plasma individually promoted sperm generation of O2.-, tyrosine phosphorylation of p105/p81, and capacitation. Fetal cord serum ultrafiltrate triggered a fivefold higher O2.- production than the other inducers (1,700 +/- 300 and 300 to 400 mV/10s/8 x 10(6) cells, respectively), a phenomenon possibly associated with the higher potency of this fluid to promote sperm hyperactivation. The production of O2.- by spermatozoa was rapid and transient. SOD prevented sperm capacitation triggered by the inducers mentioned above, but only when SOD was added at the beginning of incubation, and not after 30 minutes, indicating that the O2.- initiates a chain of early events leading to sperm capacitation. NADH and NADPH (5 mM) triggered sperm capacitation and phosphorylation of p105/p81, but these processes were not prevented by SOD or catalase, nor were they associated with an increased O2.- production. Therefore, these cofactors appeared to act by mechanisms different from those used by the other inducers studied. The sperm enzyme responsible for the O2.- generation may be very different from the NADPH oxidase of neutrophils.

In vitro stimulation of cumulus-cell expansion by human cord serum in mouse oocyte-cumulus complexes.

The aim of this study was to test whether human cord serum (HCS) containing gonadotropins has an effect on the expansion of oocyte-cumulus complexes (OCCs). The concentration of follicle stimulating hormone (FSH) and luteinizing hormone (LH) was measured in HCS by radioimmunoassay (RIA). After short-term culture (4 hr) with or without OCCs, medium containing 0.4% bovine serum albumin (BSA) as control or 10% HCS was collected and analyzed for its concentration of estradiol, progesterone, and testosterone. The FSH concentration was at the basal level, but the LH level was as high as 142.4 mIU/ml in both natured and denatured serum. Undetectable levels of steroids were observed in control media with or without OCCs. In contrast, a moderate amount of steroid hormones was detected in culture medium containing HCS. OCCs secreted a minute amount of steroid hormones in response to HCS. Similar patterns of cumulus expansion were observable by treatment with HCS, human chorionic gonadotropin (hCG), or HCS plus hCG after 4, 8, or 22 hr of culture. However, no cumulus expansion was observed in controls. These results suggest that LH in HCS induces cumulus expansion but does not affect the secretion of steroid hormones by OCCs during culture.

Similarity between natural and recombinant human alpha-fetoprotein as inhibitors of estrogen-dependent breast cancer growth.

Alpha-fetoprotein (AFP) isolated from rodent amniotic fluid or human cord sera, upon incubation with a molar excess of estradiol, is converted to a form which inhibits estrogen-stimulated tissue growth. The purpose of this study was to determine whether recombinant human AFP produced in an E. coli expression system retained this function. The recombinant protein was similar to the natural protein isolated from pooled human cord sera in all functional aspects evaluated. It was detected by monoclonal and polyclonal antibodies to the natural protein. Following exposure to estradiol, it was converted to an inhibitor of estrogen-stimulated growth of immature mouse uterus yielding a dose/response curve similar to that of the natural protein. It inhibited the growth of estrogen-dependent (MCF-7) but not estrogen-independent (MDA-MB-231) breast cancer xenografts with the same schedule dependency and resultant histological changes as the natural protein. Availability of large quantities of homogeneous, biologically active recombinant human AFP will facilitate further studies of structure/function, mechanism, and therapeutic potential of this agent as a regular of breast cancer growth.

The effects of storage conditions and trophic supplementation on the survival of fetal mesencephalic cells

It is estimated that only 5–10% of dopamine (DA) neurons implanted into the striatum of patients undergoing fetal-nigral transplantation as a treatment for Parkinson's Disease survive. Because it is often necessary to store fetal tissue prior to transplantation, we evaluated various storage parameters that could influence DA neuron viability in rostral mesencephalic tegmentum (RMT) cultures using tyrosine hydroxylase immunoreactive (THir) cell counts as an index of DA neuron survival. A high K + hibernation media (HM) was used in all studies. We found that RMT cell viability and THir cell counts decreased as storage duration increased (up to 120 h). Storage at 37°C in HM killed all cells, while storage at 10°C yielded higher survival rates than 4°C. In comparison to trypsinization, mechanical dissociation of tissue increased cell viability. Neutral pH and a storage density of at least 1 × 10 6 cells/mL were found to be optimal, while striatal coculture of RMT cells with striatal feeder layers increased THir viability up to 16-fold in comparison to monocultures. The nurturing effect of striatal coculture may be explained by the release of autotrophic factors, and we tested this hypothesis by supplementing the HM with human placental cord serum (HPCS, 8%), glial-derived neurotrophic factor (GDNF; 10 μg/mL), and brain-derived neurotrophic factor (BDNF; 10 μg/mL). GDNF and HPCS supplements increased RMT cell viability by 10–15%, while GDNF, BDNF, and HPCS increased viability of THir cells by approximately 40% at all time points studied. As Klenow enzyme labeling technique indicated that 33% of stored RMT cells were undergoing apoptosis, we found that GDNF, BDNF, and HPCS reduced apoptosis by 50%. DNA laddering and DAPI nuclear stain confirmed the presence of apoptosis in hibernated RMT cells, leading us to postulate that the high viability counts seen with trypan blue exclusion are misleading.

The effects of storage conditions and trophic supplementation on the survival of fetal mesencephalic cells.

It is estimated that only 5-10% of dopamine (DA) neurons implanted into the striatum of patients undergoing fetal-nigral transplantation as a treatment for Parkinson's Disease survive. Because it is often necessary to store fetal tissue prior to transplantation, we evaluated various storage parameters that could influence DA neuron viability in rostral mesencephalic tegmentum (RMT) cultures using tyrosine hydroxylase immunoreactive (THir) cell counts as an index of DA neuron survival. A high K+ hibernation media (HM) was used in all studies. We found that RMT cell viability and THir cell counts decreased as storage duration increased (up to 120 h). Storage at 37 degrees C in HM killed all cells, while storage at 10 degrees C yielded higher survival rates than 4 degrees C. In comparison to trypsinization, mechanical dissociation of tissue increased cell viability. Neutral pH and a storage density of at least 1 x 10(6) cells/mL were found to be optimal, while striatal coculture of RMT cells with striatal feeder layers increased THir viability up to 16-fold in comparison to monocultures. The nurturing effect of striatal coculture may be explained by the release of autotrophic factors, and we tested this hypothesis by supplementing the HM with human placental cord serum (HPCS, 8%), glial-derived neurotrophic factor (GDNF; 10 microg/mL), and brain-derived neurotrophic factor (BDNF; 10 microg/mL). GDNF and HPCS supplements increased RMT cell viability by 10-15%, while GDNF, BDNF, and HPCS increased viability of THir cells by approximately 40% at all time points studied. As Klenow enzyme labeling technique indicated that 33% of stored RMT cells were undergoing apoptosis, we found that GDNF, BDNF, and HPCS reduced apoptosis by 50%. DNA laddering and DAPI nuclear stain confirmed the presence of apoptosis in hibernated RMT cells, leading us to postulate that the high viability counts seen with trypan blue exclusion are misleading.