Alpha-Fetoprotein Subtractions in Germ Cell Tumors Identified by Con A or LCH Crossed-Line Affinity Immunoelectrophoresis

ABSTRACT: Using concanavalin A (Con A) crossed-line affinity immunoelectrophoresis and lentil lectin (LCH) crossed-line affinity immunoelectrophoresis, alpha-fetoprotein (AFP) subfractions were studied in sera including three sera from nude mice heterotran-splanted with human yolk sac tumor of the ovary and three sera from patients with yolk sac tumor, mature solid teratoma, or immature solid teratoma of the ovary. In sera of nude mice bearing yolk sac tumor or from a patient with yolk sac tumor, subfractions from yolk sac and those from fetal liver were identified. Since AFP subfractions from yolk sac and fetal liver can be differentiated according to the carbohydrate moieties, our findings indicate that AFP produced by yolk sac tumor and fetal yolk sac are to some extent differently glycosylated. We also found that AFP in both mature and immature solid teratoma was composed of two subfractions ontogenetically originating from yolk sac or fetal liver. All these findings indicate that more than two different factors are responsible for the AFP synthesis in germ cell tumor of the ovary.     

Concanavalin A-affinity Molecular Variants of Rat AFP: Differences Among Amniotic Fluid, Fetal Serum, Newborn Serum and Maternal Serum

The concanavalin A-affinity molecular variants of rat alpha-fetoprotein were measured in amniotic fluid, fetal serum, newborn serum and maternal serum. During the last 6 days of gestation, 54% ± 2% (mean ± S.E.M.) of the amniotic fluid alpha-fetoprotein does not react with concanavalin A. At 17 days gestation, 54% ± 4% of the fetal serum alpha-fetoprotein does not react with concanavalin A. Between 18 and 21 days gestation the percentage of the concanavalin A-nonreactive molecular variant of fetal serum alpha-fetoprotein decreases to 43% and remains at 43% ± 0.4% through the 28-day newborn period studied. Following day 18 of gestation the proportions of the alpha-fetoprotein concanavalin A-affinity molecular variants in serum and in amniotic fluid are different (p<0.001). On day 21 of gestation, 41% of the alpha-fetoprotein present in maternal serum does not react with concanavalin A. These results suggest that during late gestation yolk sac and fetal liver synthesize different patterns of alpha-fetoprotein concanavalin A-affinity molecular variants and that on day 21 of gestation the major portion of the maternal serum alpha-fetoprotein is derived from fetal serum.     

Microheterogeneity of Alpha-fetoprotein in Patient Serum as Demonstrated by Lectin Affino-electrophoresis

Microheterogeneity of alpha-fetoprotein (AFP) present in the sera of 76 patients was studied by lectin affino-immunoelectrophoresis. Seventeen patients had benign liver disorders and the remaining 59 patients were treated for primary or secondary liver cancer or yolk sac tumour. By means of Con A, AFP was divided into two variants, while lentil lectin (LCA) made it possible to separate AFP in three variants. In some patients the relative concentrations of Con A and LCA AFP variants were similar; these patients were believed to produce AFP of the same 'profile'. Fourteen AFP profiles were observed by estimation of the area enclosed by precipitates corresponding to respective AFP variants. It was also possible to estimate the AFP profile on the basis of a simple visual analysis of the electrophoretic plates. The obtained results indicate that the AFP profiles of patients with cancer were variable. In spite of variations of the AFP profile in cancer patients, in most cases it was possible to differentiate primary liver cancer from yolk sac tumour and from liver metastases of cancer. In addition, in two-thirds of hepatoma patients the AFP profile was different from the profile observed in patients with benign liver disorders.     

Mechanisms of Fetal Demise in Pregnant Mice Immunized to Murine Alpha-Fetoprotein

ABSTRACT: Nya: NYLAR mice were immunized to murine alpha-fetoprotein (AFP) by active immunization with rat AFP in Freund's adjuvant emulsion before mating or by passive immunization with a high or low dose of whole rabbit antimouse AFP serum or rabbit anti-AFP IgG at 8–20 days of gestation. In the passively immunized group, anti-AFP serum or purified anti-AFP IgG administered at the end of the second week of gestation produced abortion after 24 hours of 41 and 48% of fetuses, respectively. Although abortion did not occur in the low-dose group, the anti-AFP serum produced fetal death in 32%, as did the anti-AFP IgG in 26%, in 72 hours. In the actively immunized group rat AFP produced developmental arrest, but not abortion, in mothers bearing autologous antibodies to mouse AFP. Histopathologic analysis revealed that fetal death resulted from separation of fetal and maternal tissues of the placenta due to subplacental hemorrhages. Immunofluorescent localization of the rabbit IgG implicated both the chorioallontois and yolk sac placenta as target tissues.     

Alpha fetoprotein levels in maternal serum and in amniotic fluid from early normal pregnancies

Alpha fetoprotein (AFP) was determined in serum from 241 women in the 8th to 43rd week of gestation by quantitative radioimmunoelectrophoresis, and in 103 amniotic fluid samples from the first half of pregnancy by rocket immunoelectrophoresis. Both serum and amniotic fluid samples were obtained from a larger material, but samples were included only from pregnant women whose infants were normal at birth. The 90% normal AFP range was calculated both for maternal serum and for amniotic fluid.     

A new mutation in the AFP gene responsible for a total absence of alpha feto-protein on second trimester maternal serum screening for Down syndrome

Alpha feto-protein (AFP) is a major plasma protein produced by the yolk sac and the liver during the fetal period. During the second trimester of pregnancy, APF and betahCG serum concentrations are commonly used for screening Down syndrome. AFP deficiency is rare (estimated to be 1/105,000 newborns) and only one sequence alteration has previously been reported in the AFP gene. We report a new mutation in exon 5 of the AFP gene, leading to a total absence of AFP on 2nd-trimester maternal serum screening for Down syndrome, confirmed on the amniotic fluid. Despite this, fetal development and birth were normal. After PCR-amplification, the whole AFP gene was sequenced. The new mutation was a guanine to adenine transition in position 543 creating a premature stop codon in position 181. In order to search for eventual modifications of the amniotic fluid profile, proteins were separated by electrophoresis and compared with 10 normal amniotic fluids sampled at the same developmental age (18 weeks). In the amniotic fluid of our patient albumin rate was reduced whereas alpha1 and beta protein fractions were increased, suggesting that AFP deficiency may modify the distribution of protein fractions. This observation emphasizes the complex molecular mechanisms of compensation of serum protein deficiency. Studies on other families with AFP deficiency are necessary to confirm this observation.     

Protein and steroid levels in embryonic cavities in early human pregnancy

Biochemical analysis including concentrations of urea, creatinine,human chorionic gonadotrophin (HCG), oestradiol, progesterone,and -fetoprotein (AFP), twodimensional gel electrophoresis,and the affinity of AFP for Concanavalin A (Con A)—Sepharosewere performed on samples of exocoelomic and amniotic fluidsretrieved by transvaginal puncture and maternal serum from 25normal pregnancies between 5 and 12 weeks of gestation. Biochemicalassays showed that during this period of gestation no differencesin urea concentration were found between fluids from the threecompartments, whereas creatinine concentration decreased significantly(P < 0.001) from maternal serum to amniotic fluid. The exocoelomicfluid contained significantly (P < 0.001) higher concentrationsof oestradiol, progesterone and HCG than both maternal serumand amniotic fluid. AFP concentration was similar in amnioticand exocoelomic fluids and significantly (P < 0.001) lowerin maternal serum. Between the second and the third months ofgestation, urea concentration decreased significantly (P <0.05) and oestradiol, HCG and AFP increased significantly inmaternal serum (P < 0.05, P < 0.05, P < 0.001, respectively).During the same period of gestation, exocoelomic fluid concentrationsof urea and HCG decreased significantly (P < 0.005, P <0.001, respectively). Comparison of the two-dimensional gelpatterns obtained from maternal serum with those from exocoelomicamniotic fluids revealed no significant qualitative differences,except for several small proteins. These results suggest thatprotein pathways across materno-embryonic membranes are notsimply passive transfers. Con A affinity molecular variantsof AFP demonstrated that both exocoelomic and amniotic fluidAFP were mainly of yolk sac origin and that maternal serum AFPwas mainly of fetal liver origin, suggesting that the humansecondary yolk sac has both absorptive and excretory functions.     

The effect of heterologous antisera on embryonic development. XIII. Lack of effect of antisera to alpha fetoprotein

This investigation was carried out to determine whether heterologous antisera to alpha fetoprotein (AFP) are embryotoxic to developing rat embryos. Homogeneous rat AFP was isolated and antisera directed against this glycoprotein were produced in rabbits, horse and goat. The effect of the antisera on embryonic development was examined by injecting the antisera intraperitoneally into pregnant rats on the ninth, eleventh and thirteenth days of gestation. The results demonstrated that there was no evidence of increased incidence of fetal abnormalities in 472 surviving fetuses of 42 injected rats. There was no evidence of increase embryonic death or retardation of intrauterine growth following administration of the antisera on the ninth, eleventh and thirteenth days of gestation. The localization of the injected antisera was examined by the indirect immunofluorescent method. The results showed that the heterologous AFP antibodies localized specifically in the visceral yolk sac placenta. No antibody localization was observed in the embryo proper or the chorioallantoic placenta. It is speculated that the localization of AFP antibodies in the visceral yolk sac does not interfere with the embryotrophic function of the visceral yolk sac placenta.     

Distribution of acute-phase alpha 2-macroglobulin in rat fetomaternal compartments

Rat acute-phase alpha 2-macroglobulin (AP alpha 2M) concentration was measured by radioimmunoassay in maternal serum, fetal plasma, maternal liver, fetal liver, and amniotic fluid as a function of gestational and neonatal age. The concentration profiles of AP alpha 2M in maternal serum and fetal plasma displayed two peaks, one in early gestation and another during late gestation. Synthesis of AP alpha 2M was confirmed by the immunoprecipitation of [35S]methionine incorporated into cultures of selected tissues. The following observations were made. 1) Maternal serum concentrations of AP alpha 2M were higher than those observed in fetal plasma in early gestation. This was attributable to a high level of maternal AP alpha 2M synthesis in metrial gland which was absent in liver and moderate in yolk sac. 2) In late gestation fetal plasma concentrations of AP alpha 2M greatly exceeded those observed in maternal serum. This could be explained by the pronounced synthesis of AP alpha 2M in fetal liver that was not apparent in maternal liver or yolk sac. 3) During labor, a transient increase in AP alpha 2M concentration was observed in maternal serum and fetal plasma. 4) During lactation a moderately elevated maternal serum concentration of AP alpha 2M was maintained. 5) Amniotic fluid concentration of AP alpha 2M was very low throughout gestation, which indicated that the fetal glomerulus was relatively impermeable to this large protein. It is concluded that in early gestation a principal maternal source of AP alpha 2M appears to be the metrial gland, whereas in late gestation fetal liver is a major source of AP alpha 2M appearing in fetal plasma from where some of this macroglobulin is speculated to be transported to the maternal circulation.     

Rat Yolk Sac Tumor: Biological Aspects and Microheterogeneity of AFP

α-Fetoprotein (AFP)-producing yolk sac tumors were established in rats as transplantable tumor lines growing in either solid or ascitic form. AFP levels in the ascites and in the serum of recipients were quite high, but higher in castrated female rats than male rats. The tumor cells were cultured also in vitro and showed synthesis of AFP and albumin. AFP produced by the yolk sac tumors was investigated comparatively with AFP produced by a hepatoma. There was no remarkable difference between two AFPs in behavior in gel filtration; however, AFP of yolk sac tumors migrated more slowly than that of hepatoma in immunoelectrophoresis. However, the mobilities of two AFP preparations became same after desialization by neuraminidase treatment. This fact suggests that AFP of yolk sac tumors contains sialic acids in smaller amount than that of hepatomas.     

Serum alpha-fetoprotein and its lectin reactivity in liver diseases: a review.

Increased serum concentration of alpha-fetoprotein (AFP) can be found in benign and malignant liver diseases, in yolk sac tumors, and in several nonhepatic neoplasms at advanced stage. The frequency and level of elevated serum AFP are highest in hepatocellular carcinoma (HCC) and yolk sac tumors. Most levels of serum AFP in HCC are greater than 500 ng per mL, whereas the serum AFP in most of the benign liver diseases is only moderately elevated and is transient in nature. Determination of lectin reactivity of serum AFP is helpful for the differentiation of HCC from other diseases associated with elevated serum AFP. Determination of Len culinaris agglutinin (LCA) reactivity of serum AFP is useful for the differentiation of HCC from benign liver diseases, and for early detection of hepatoma. Determination of concanavalin A (Con A) nonreactive AFP variant is useful for the differentiation of HCC from yolk sac tumors and may also allow for the differentiation of HCC from nonhepatic neoplasms. However, reaction with several lectins may be required if differentiation among various nonhepatic neoplasms is needed.     

Hepcidin and iron species distribution inside the first-trimester human gestational sac

We have investigated factors affecting iron distribution in the first-trimester gestational sac, by the measurement of transferrin, non-transferrin-bound iron (NTBI) and pro-hepcidin (Hep) in maternal serum, coelomic fluid (CF) and amniotic fluid (AF) and by immunostaining for Hep in villous and secondary yolk sac biopsies. These samples were obtained from 15 first-trimester pregnancies at 8-11 weeks gestation. Transferrin concentrations were significantly lower in fetal (0.56 mg/ml) than maternal serum (1.71 mg/ml), with very low concentrations in CF and AF (0.09 mg/ml). In contrast, transferrin saturations were significantly higher in fetal (77%) than maternal serum (33%). NTBI was present in fetal serum, CF and AF, presumably as a consequence of low transferrin concentrations in these compartments. Pro-Hep was present at lower levels in fetal (140.0 ± 11.1) than maternal serum (206.2 ± 9.2) and at low concentrations in CF (19.4 ± 3.1) and AF (21.8 ± 5.2). Immunostaining with Hep antibody was found in the syncytiotrophoblast of first-trimester placenta as well as in mesothelial and endodermal layers of the secondary yolk sac at 10 weeks. The presence of Hep in syncytiotrophoblast cells of first-trimester placenta as well as in mesothelial and endodermal layers of the secondary yolk sac suggest a key regulatory role for this protein in iron transfer to the first-trimester fetus. The low transferrin concentrations and the presence of NTBI in CF and AF suggest that transferrin-independent iron transfer is important in early gestation.     

Alpha-feto-protein during development and in disease.

An alpha-feto-protein (AFP) is present in many mammals, in birds, and in sharks during development. The AFP present in different species have similar physicochemical properties and often have common antigenic determinants. Their study, both in health and disease, has provided a useful model for the understanding of other phase-specific antigens and the activation of the genes which control their synthesis. In the human fetus, the level of AFP falls with increasing maturity. The more sensitive methods of detection have disclosed that this fetal protein persists in trace amounts throughout life and its level increases in maternal blood during pregnancy. The principal sites of synthesis are the fetal liver and in some mammals, the yolk sac splanchnopleur. In humans as well as in mice and cows, it is notable that the synthesis of AFP is increased in liver cancer cells and that high levels of this protein are present in serum. Elevated values of AFP have also been detected in human subjects with undifferentiated tumours of the testis and ovary. A fall to normal levels has been noted in cases of complete remission after surgery and a return to high levels in patients who develop metastases. In some patients with hepatitis a temporary rise in the level of AFP has also been observed. In recent years, the detection of high levels of AFP in amniotic fluid has proved to be of great value for the prenatal diagnosis of neural-tube defects. Abnormal levels have also been found in the amniotic fluid or in maternal serum in cases of spontaneous abortion. Such measurements are now being assessed as a methodof monitoring abnormal pregnancy.     

The amniotic, allantoic and yolk sac epithelia of the cat: SEM and TEM studies

Summary The epithelial layers of the feline allantoamnion and yolk sac between the 23rd day and full term were studied using SEM, TEM, and light microscopical histochemistry. Amniotic, allantoic and yolk sac fluid were analysed. The attenuated and relatively inert amniotic epithelium sloughs off completely around the 54th day, the amniotic cavity is then lined by fibrous connective tissue only. The exocoelomic epithelium is regarded as the source of the abundant macrophages, rich in lysosomal enzymes, that are seen in the allantoamniotic membrane after obliteration of the exocoelomic cleft. The allantoic epithelium has been mistaken for the amniotic one by many authors. Its cuboidal, glycogen-rich cells develop coral-like luminal outgrowths after the 45th day, which contain almost all kinds of organelles. The basal cell poles form large interdigitations, and the nuclei become heavily lobulated. The changes are accompanied by drastic alterations in the ionic composition of the allantoic fluid. The yolk sac endoderm differs greatly from the allantoic endoderm. It is the most active epithelium of the accessory fetal membranes. The yolk sac mesothelium retains many long microvilli over the whole gestational period. The nutritional value of the yolk sac fluid is only higher than that of the amniotic fluid in its glycerol and cholesterol values.     

Alpha-fetoprotein levels in different strains of mice during development.

The levels of alpha-fetoprotein (AFP) were measured at birth and at 7 days of age in plasma from Q, C3H/He-mg/Crc, BALB/c/Crc, A/Crc, CBA/Ca/Crc and C57BL/Mcl mice, and in adult blood samples and amniotic fluid at 14 days' gestation from Q, C3H and BALB/c mice. The AFP levels in amniotic fluid and neonatal plasma were significantly higher in BALB/c mice than in the other strains tested, but by postnatal day 7, C57BL and C3H mice had the highest plasma levels. It seems therefore that the genetic mechanisms controlling the synthesis of AFP prenatally may be distinct from those concerned with its reduction after birth. At 6 weeks of age the level in C3H mice was somewhat higher than in BALB/c, and more than double that in Q mice. An attempt to increase AFP levels in response to galactosamine-induced liver damage was successful only in Q adult mice. When amniotic fluids and day 0 plasma from several strains of mice were tested by polyacrylamide gel electrophoresis, no differences in AFP mobility were detected. Preliminary studies were also carried out to see if the administration of sodium butyrate, claimed to delay the timing of the fetal to adult haemoglobin switch in some mammalian species, could also affect the rate of synthesis of AFP during fetal life.     

Lens culinaris agglutinin-reactive alpha-fetoprotein, an alternative variant to alpha-fetoprotein in prenatal screening for Down's syndrome.

BACKGROUND: Three serum tests, alpha-fetoprotein (AFP), human chorionic gonadotrophin and unconjugated oestriol, are now widely used for screening for Down's syndrome. Lens culinaris agglutinin-reactive alpha-fetoprotein (AFP-L3) is a variant of alpha-fetoprotein with alpha1-->6 fucose appended to the reducing terminal N-acetylglucosamine. It is the most prominent AFP detected in the serum of patients with hepatocellular carcinoma. METHODS: We investigated microheterogeneities of the carbohydrate chain on AFP in fetal liver tissues, amniotic fluids and maternal sera obtained from pregnancies with Down's syndrome using lectin affinity electrophoresis with four lectins. The percentages of AFP-L3 in maternal sera from 22 Down's syndrome and 227 unaffected pregnancies were determined. RESULTS: Unlike the case with AFP concentration, the percentage of AFP-L3 in maternal serum and amniotic fluid was similar, and apparently not influenced by membrane permeability. Knowing the percentage of AFP-L3 in maternal serum was effective for discriminating between Down's syndrome-affected pregnancies and unaffected pregnancies. The percentage of AFP-L3 in maternal serum identified 55% of Down's syndrome cases with a 5% false-positive rate. CONCLUSIONS: AFP-L3 should be an effective replacement for AFP in prenatal Down's syndrome screening.     

Ontogeny of plasma cells in the early rat yolk sac

The present study investigated the development of plasma cells in the early rat yolk sac (days 10-16 of gestation) by light microscopy, transmission electron microscopy, immunoelectron microscopy, and indirect immunofluoresce techniques. Cells delineating the morphology of plasma cells in the yolk sac were observed as early as 12 days of embryonic life. As for positive immune staining for the intra-cytoplasmic immunoglobulin (Ig) production (IgA, IgM and IgG), the intensity of the immune staining was very weak on days 10 and 11 of gestation, while it turned very dense on day 12 of gestation. At 14 days of gestation, the number of positive cells was markedly reduced. Immunoelectron microscopy visualized products of the immune reaction in cisterns of the rough endoplasmic reticulum. Conventional electron microscopic examination of 12, 13, and 16-day yolk sacs confirmed the development and differentiation of plasma cells with their well-known ultrastructural features, making this the first study to demonstrate these in the early rat yolk sac. The development of plasma cells in the early yolk sac implies the ability of the yolk sac to effect a humoral immune response at this stage of fetal life. The probable role of plasma cells in the yolk sac is also discussed.     

羊水甲胎蛋白浓度与胎儿畸形的关系

目的了解羊水甲胎蛋白（AFP）浓度与胎儿畸形的关系。方法对2007年1月至2012年5月在本院进行羊膜腔穿刺的孕17～23w孕妇5262例,运用酶联免疫吸附法对羊水进行AFP检测,根据其2．5倍中位数倍数（MoM）值进行分组,观察孕妇的妊娠结局,分析羊水AFP升高与胎儿畸形的关系。结果1．各孕周羊水AFP的2．5MoM值分别为：孕17w 41150ng／ml,18w38395ng／ml,19w 34995ng／ml,20w 31700ng／ml,21w 29655ng／ml,22w 26940ng／ml,23w 21800ng／ml。2．羊水AFP≥2．5MoM时发生率明显升高的胎儿畸形为：神经管缺陷,颈部淋巴管瘤,死胎。结论羊水AFP≥2．5MoM时,临床诊断除考虑神经管缺陷,还应警惕是否有颈部淋巴管瘤或死胎可能。羊水AFP≥2．5MoM对于诊断神经管缺陷的特异度及阴性预测价值较高,但灵敏度及阳性预测价值一般,需结合系统彩超排除相关畸形。     

7076例孕中期唐氏综合征产前筛查结果临床分析

目的 探讨孕中期唐氏综合征（DS）等筛查对检出胎儿出生缺陷和不良妊娠结局的实用价值.方法 用时间分辨荧光分析法对日照市7076名15 ～20孕周孕妇的血清甲胎蛋白（AFP）和游离绒毛膜促性腺激素（F-βHCG）进行检测,结合孕妇年龄、体重、孕周等因素,利用配套孕期胎儿唐氏综合征产前筛查分析软件,分析胎儿患DS、开放性神经管缺陷（NTD）、18-三体综合征的发病风险率,并对高风险孕妇行羊水胎儿细胞染色体核型分析或B超跟踪检查.结果 7076名孕妇中筛出高风险孕妇396例,阳性率为5.6％,在接受羊水检查的56名DS高风险孕妇中,检出唐氏胎儿3例,死胎3例,其它异常3例;对57例神经管缺陷高危孕妇进行B超检查,发现1例无脑儿;在43例18-三体高危的孕妇中,6例做羊水染色体检查,结果均正常.结论 孕中期以唐氏综合征（Ds）产前筛查作为对胎儿先天缺陷,尤其是胎儿染色体异常的筛查是行之有效方法,筛查结果呈高危孕妇须进行羊水染色体核型分析或B超检查.     

AFP阳性胃癌的组织形态及其分型研究

目的：探讨ＡＦＰ阳性胃癌的病理组织形态及其分型。方法：应用光镜、电镜、组化和免疫组织化学方法,对８７例ＡＦＰ生胃癌中临床病理资料较完整的６３例进行病理组织形态态观察,并用２例３个月妊娠的胚胎胃肠组织以同样方法行相关的对比研究。结果：ＡＦＰ阳性胃癌的发生泫为６．２％（８７／１３９５例）。６３例中依据其病理组织形态学特点和肿瘤组织内癌细胞ＡＦＰ染色阳性表达,并对比３个月妊娠胎胃肠组织学的改变,将其组织