Immunosuppressive characteristics of human AFP: effect on tests of cell mediated immunity and induction of human suppressor cells.

Murine AFP has been reported to be immunosuppressive in a variety of systems. However, the extent and degree of inhibition has varied in different species and laboratories. Therefore, we have examined the potential suppressive effect of purified human AFP on several in vitro tests of cellular immunity and the potential mechanism of its action. AFP purified from fetal and liver cancer sera significantly inhibited mitogen and antigen-induced proliferative responses but had no effect on lymphocyte E rosetting, MIF production or mitogen induced T cell cytotoxicity to Chang target cells. Purified human AFP induced human suppressor cell activity, capable of suppressing a one-way mixed lymphocyte reaction (MLC). In contrast to Con A induced suppressor cells, AFP induced suppressor cell activity was overcome by mitogen augmentation of the proliferative response in MLC. These data suggest that the inhibition of lymphocyte proliferation by human AFP may be mediated by the induction of a subpopulation of human suppressor cells. Furthermore, mitogen induced cell mediated cytotoxicity was partially inhibited by primary liver cancer serum and completely inhibited by newborn cord serum, in contrast to purified fetal or tumor AFP which had no effect. These data suggest that there are other immunosuppressive factors in fetal and tumor serum which require further characterization. These other serum factors may be responsible for some of the immunosuppressive effects attributed to AFP. Although AFP is unlikely to play a major immunosuppressive role physiologically in vivo, its selective effect on proliferative responses, apparently mediated by suppressor cells, may prove to be a useful pharmacologic probe of the mechanism of these in vitro lymphocyte responses and biological interactions.     

Immunosuppressive Characteristics of Human AFP: Effect on Tests of Cell Mediated Immunity and Induction of Humam Suppressor Cells

Murine AFP has been reported to be immunosuppressive in a variety of systems. However, the extent and degree of inhibition has varied in different species and laboratories. Therefore, we have examined the potential suppressive effect of purified human AFP on several in vitro tests of cellular immunity and the potential mechanism of its action AFP purified from fetal and liver cancer sera significantly inhibited mitogen and antigen-induced proliferative responses but had no effect on lymphocyte E rosetting, MIF production or mitogen induced T cell cyto-toxicity to Chang target cells. Purified human AFP induced human suppressor cell activity, capable of suppressing a one-way mixed lymphocyte reaction (MLC). In contrast to Con A induced suppressor cells, AFP induced suppressor cell activity was overcome by mitogen augmentation of the proliferative response in MLC. These data suggest that the inhibition of lymphocyte proliferation by human AFP may be mediated by the induction of a subpopulation of human suppressor cells. Furthermore, mitogen induced cell mediated cytotoxicity was partially inhibited by primary liver cancer serum and completely inhibited by newborn cord serum, in contrast to purified fetal or tumor AFP which had no effect. These data suggest that there are other immunosuppressive factors in fetal and tumor serum which require further characterization. These other serum factors may be responsible for some of the immunosuppressive effects attributed to AFP. Although AFP is unlikely to play a major immunosuppressive role physiologically in vivo, its selective effect on proliferative responses, apparently mediated by suppressor cells, may prove to be a useful pharmacologic probe of the mechanism of these in vitro lymphocyte responses and biological interactions.     

The effect of normal and rheumatic pregnancy sera on intracellular cathepsin B activity in human monocytes.

In a prospective study of pregnant patients with rheumatic disease and healthy pregnant women, the effect of pregnancy serum on human blood monocytes was investigated. Intracellular activity of the collagen degrading enzyme cathepsin B was found to be significantly depressed in monocyte cultures exposed to pregnancy and cord sera, independent of the presence or absence of an inflammatory state in the pregnant woman. Enzyme inhibition developed in a dose-dependent fashion over 3 days in culture. In contrast to cord and pregnancy sera, non-pregnant serum and serum from oral contraceptive users displayed enzyme stimulating activity. The response of monocytes to the stimulating agent carrageenan was unaffected by inhibitory sera. The nature of possible inhibitory factors in pregnancy and cord serum is discussed.     

Enzymoimmunoassay of human alpha-fetoprotein.

A non-competitive method for the determination of human alpha-fetoprotein (AFP) in serum, using a pure specific antibody linked to glucose oxidase is described. When applied to human AFP, this method gives reproducible results in the range 0.7 to 15 ng/ml, in a relatively short time (6 hr). AFP sera levels of healthy human adults, pregnant women and adults with liver diseases, were tested both by enzymoimmunoassay and radioimmunoassay. In all cases, good agreement was noted between the two methods.     

Immunosuppressive activity of isolated rat and human fetoproteins.

Alpha-fetoprotein (AFP) was purified from rat hepatoma sera and pooled human cord sera. Both AFP-rich fractions prepared by consecutive ion exchange and gel filtration chromatography and pure rat AFP prepared by liquid phase immunoabsorption lacked in vitro suppressive activity. Human AFP purified by affinity chromatography was suppressive, but so was similarly purified human cord albumin. Alteration of ionic conditions was shown to affect the activity of both human AFP and cord albumin. The primary mixed lymphocyte response, the generation of the secondary response, and the memory cell, per se, were all found to be sensitive to active human AFP under the appropriate culture conditions.     

A sensitive blotting system for detection of alpha-fetoprotein variants with monoclonal and polyclonal antibodies

Human alpha-fetoprotein (AFP) variants from cord sera were separated by isoelectric focusing in agarose gels under native conditions, transferred to nitrocellulose paper and detected with polyclonal and monoclonal antibodies (Moabs). Rabbit anti-AFP recognized up to 9 individual electrophoretic variants in the range of pH 4.5 to pH 5.2. The reactivity of 8 Moabs ranged from weak to strong and showed variability in the pattern of AFP bands recognized. Moabs were separated into 3 groups according to the number of bands detected: group 1 detected 6 to 7 bands; group 2 recognized only one band; and group 3 recognized 4 bands. The sensitivity of the system with polyclonal antibodies was 0.15 ng of AFP in complete cord serum and varied between 300 and 0.2 ng with Moabs.     

A population of human cord blood mononuclear cells with surface alpha fetoprotein.

Suspensions of mononuclear cells from adult peripheral blood (PBL) and mononuclear cells from cord blood (CBL) were examined for the presence of surface alpha-fetoprotein (AFP) using a fluoresceinated F(ab')2 fragment of rabbit IgG anti-human AFP. The mean proportion of CBL with AFP was increased (10%) when compared with PBL (1%) although some CBL specimens did not demonstrate such an increase (range 0--15%). The presence of AFP on CBL could be either due to cytophilic AFP attached to a unique surface receptor or intrinsic AFP synthesis. The following observations could not distinguish between these two possibilities: (1) After treatment with trypsin, only minor reappearance of surface AFP could be observed in AFP-free medium in contrast to the larger numbers observed in medium containing AFP. Such selective reappearance depending on the media could be related to either cytophilic attachment of heterologous or homologous AFP or preferential stimulation of intrinsic AFP synthesis. (2) The reappearance of AFP positive CBL following trypsin treatment and incubation in media with or without AFP containing sera was inhibited by cyclohexamide. Such inhibition could be due to inhibition of synthesis of an AFP surface receptor or intrinsic AFP. (3) The shedding of surface AFP observed at 2--4 degrees C could be due to release of exogenous cytophilic AFP or the continued "turnover" of intrinsic AFP without concomitant AFP synthesis due to the cold temperature. Finally, the removal of AFP positive cells via selective depletion of B cells using bead columns coated with IgG-anti-IgG and the absence of depletion of AFP positive cells after successive gradient centrifugation of E-rosettes and cells with IgG-Fc receptors are consistent with the identity of AFP positive CBL as cells without IgG-Fc receptors or lymphocytes without conventional T-cell markers as defined by E-rosettes.     

Maternal Immunocompetence II. Proliferative Responses of Maternal Lymphocytes In Vitro and Inhibition by Serum from Pregnant Rats

Peripheral blood and splenic lymphocytes from interstrain (L X BN) or intrastrain (L X L) primigravida rats were equivalent to those from virgin L females in their in vitro DNA synthetic responses to paternal strain cells (BN), to unrelated allogeneic cells (ACI), and to the mitogen phytohemagglutinin (PHA). Heat-inactivated serum from pregnant L rats, when compared to serum from virgin or postpartum L rats, regularly suppressed the in vitro response of L lymphocytes to paternal and allogeneic cells. The response of L cells to PHA was not suppressed. The degree of inhibition was related to the final concentration of pregnant serum in culture, concentrations above 2% producing more than 80% inhibition of the mixed lymphocyte reaction. The inhibiting sera were not cytotoxic by a sensitive 51Cr release assay. Histoincompatibility between mother and fetus is not required for production of this inhibitory effect, since it is consistently present in intrastrain (L X L) pregnant rats. Among interstrain pregnant rats, the degree of inhibition is influenced by, but not specific for paternal-strain alloantigens. Thus, the proliferative function of thymus-derived (T) lymphocytes from pregnant rats is intrinsically normal, but the response to allogeneic cells can be altered by factors present in pregnant serum.     

Reference interval for maternal serum alpha-fetoprotein

Maternal serum alpha-fetoprotein (AFP) concentration was measured in 237 samples taken throughout the pregnancies of 28 normal pregnant women who all gave birth to normal infants. The patterns from these 28 individuals curves were compared graphically with an earlier published non-parametric 90% reference interval based upon single serum samples from other normal, pregnant women. The two "reference areas" were found to be quite similar. Maternal serum AFP values before and after induced abortion in a case of acrania and spina bifida demonstrated the value of serial maternal serum AFP quantitations in cases of suspected neural tube defects (NTD).     

Immunosuppressive Activity of Isolated Rat and Human Fetoproteins

Alpha-fetoprotein (AFP) was purified from rat hepatoma sera and pooled human cord sera. Both AFP-rich fractions prepared by consecutive ion exchange and gel filtration chromatography and pure rat AFP prepared by liquid phase immunoabsorption lacked in vitro suppressive activity. than ATP purified by affinity chromatography was suppressive, but so was similarly purified human cord albumin. Alteration of ionic conditions was shown to affect the activity of both human AFP and cord albumin. The primary mixed lymphocyte response, the generation of the secondary response, and the memory cell, per se, were all found to be sensitive to active human AFP under the appropriate culture conditions.     

A Population of Human Cord Blood Mononuclear Cells with Surface Alpha Fetopfotein

Suspensions of mononuclear cells from adult peripheral blood (PBL) and mononuclear cells from cord blood (CBL) were examined for the presence of surface alpha-fetoprotein (AFP) using a fluoresceinated F(ab')₂ fragment of rabbit IgG anti-human AFP. The mean proportion of CBL with AFP was increased (10%) when compared with PBL (1%) although some CBL specimens did not demonstrate such an increase (range 0-15%). The presence of AFP on CBL could be either due to cytophilic AFP attached to a unique surface receptor or intrinsic AFP synthesis. The following observations could not distinguish between these two possibilities: (1) After treatment with trypsin, only minor reappearance of surface AFP could be observed in AFP - free medium in contrast to the larger numbers observed in medium containing AFP. Such selective reappearance depending on the media could be related to either cytophilic attachment of heterologous or homologous AFP or preferential stimulation of intrinsic AFP synthesis. (2) The reappearance of AFP positive CBL following trypsin treatment and incubation in media with or without AFP containing sera was inhibited by cyclohexamide. Such inhibition could be due to inhibition of synthesis of an AFP surface recpetor or intrinsic AFP. (3) The shedding of surface AFP observed at 2-4°C could be due to release of exogenous cytophilic AFP or the continued “turnover” of intrinsic AFP without concomitant AFP synthesis due to the cold temperature. Finally, the removal of AFP positive cells via selective depletion of B cells using bead columns coated with IgG-anti-IgG and the absence of depletion of AFP positive cells after successive gradient centrifugation of E-rosettes and cells with IgG-Fc receptors are consistent with the identity of AFP positive CBL as cells without IgG-Fc receptors or lymphocytes without conventional T-cell markers as defined by E-rosettes.     

Immunosuppressive Activity of Isolated Rat and Human Fetoproteins

Alpha-fetoprotein (AFP) was purified from rat hepatoma sera and pooled human cord sera. Both AFP-rich fractions prepared by consecutive ion exchange and gel filtration chromatography and pure rat AFP prepared by liquid phase immunoabsorption lacked in vitro suppressive activity. than ATP purified by affinity chromatography was suppressive, but so was similarly purified human cord albumin. Alteration of ionic conditions was shown to affect the activity of both human AFP and cord albumin. The primary mixed lymphocyte response, the generation of the secondary response, and the memory cell, per se, were all found to be sensitive to active human AFP under the appropriate culture conditions.     

BK virus specific IgM responses in cord sera,young children and healthy adults detected by RIA

Summary An IgM capture solid-phase radioimmunoassay (MACRIA) for BK virus (BKV) specific IgM is described. This test was found to be more sensitive in detecting BKV specific IgM than both haemagglutination inhibition and immune electron microscopy with serum fractions from sucrose density gradients. The use of this specific assay allowed large numbers of sera to be examined with ease so that the distribution of BKV specific IgM in different populations could be studied more fully.BKV specific IgM was detected in 11/300 sera from London blood donors, in 24/114 sera from children aged between 2 and 11 years admitted to a paediatric unit and 14/79 sera taken from children aged between 2 and 5 years for the investigation of anti-streptolysin 0 titres. BKV specific IgM was not detected in 404 cord sera examined to investigate the transplacental transfmission of BK virus.With 3 Figures     

Antinuclear autoantibodies in healthy nonpregnant and pregnant women and their offspring.

Antinuclear autoantibodies have previously been detected in sera of healthy women although less frequently than in sera of women with autoimmune disorders. The effect of pregnancy on antinuclear autoantibody production in healthy women is as yet debatable. We present four studies in which, by employing the ELISA method, we evaluated the presence of six antinuclear autoantibodies (anti-ds DNA, anti-ss DNA, anti-poly(I), anti-cardiolipin, anti-Sm, and anti-RNP) in the sera of more than 1,000 healthy pregnant and nonpregnant women, including 196 pairs of matched maternal and cord blood sera. In all four studies healthy pregnant women did not demonstrate significantly higher prevalence rates of various serum antinuclear autoantibodies as compared to healthy non-pregnant women. All detected autoantibodies were of the IgM isotype. In only one infant (born to a healthy seronegative mother) was an autoantibody (IgM anti-ss DNA) detected. This may indicate that in certain circumstances the fetus is capable of self-production of autoantibodies.     

Investigation on the Maternal-Infantile Infection with Human Parvovirus B19

With the investigations on pregnant women and newbornsinfected withToxoplasma, rubella virus, cytomegalovirus,herpes simplex virus (TORCH), it was found that humanparvovirus B19 (B19 virus), which belongs to the familyParvoviridae and the genus Erythrovir…     

Fetal and Maternal Red Cell Immune Adherence (RCIA) Receptors*

ABSTRACT: The addition of autologous serum to mixtures containing human red cells, from pregnant and nonpregnant females, and sheep red cells resulted in the formation of mixed aggregates containing both human and sheep red cells. In contrast, no aggregate formation occurred when autologous cord serum was addded to mixtures containing cord red cells and sheep red cells. Heat inactivation of the adult serum or the presence of 0.15 M EDTA prevented the formation of mixed aggregates. These observations indicated that the mixed aggregates occurred through the complement-dependent red cell immune adherence (RCIA) phenomenon. The addition of untreated cord serum to mixtures containing inactivated adult serum restored the formation of mixed aggregates, indicating that the cord serum contained sufficient complement for RCIA. Natural antibody against sheep red cells was present in adult sera but was absent in cord sera. Using the RCIA receptor assay, the RCIA receptor activity of cord red cells was found to exceed significantly that of the adult pregnant cells (p < 0.0025). It is postulated that this may represent an aspect of immune adaptation between mother and fetus.     

The Immunosuppressive Role of Alpha-Fetoprotein During Pregnancy

Cell-mediated and humoral immune responses were assessed in mice at mid-term (day 10) in pregnancy. A significant but selective suppression of the primary in vivo antibody (plaque-forming cell) response to SRBC was observed, with the most pronounced effect being on the gammaA response. Similar results were obtained for secondary in vitro antibody synthesis by antigen-primed spleen cells from pregnant mice, demonstrating the intrinsic nature of the inhibition. Pregnant mouse serum (PMS) was shown to suppress primary in vitro antibody synthesis, and the inhibitory effect was abrogated by the selective removal of alpha-fetoprotein (AFP) using affinity chromatography. Normal mouse serum became similarly suppressive in vitro when purified AFP of fetal origin was added to it in concentrations approximating that found in PMS. Spleen cells from pregnant mice showed a suppressed mitogenic response to phytohemagglutinin, a lowered response to concanavalin. A, and a normal response to lipopolysaccharide. In contrast, the allogeneic response of these animals as measured in the one-way mixed lymphocyte culture was enhanced. PMS suppressed both allogeneic and mitogen-induced lymphocyte transformation by spleen cells from nonpregnant mice, and the effect was eliminated by the selective removal of AFP. These findings indicate an important functional role for AFP in normal embryological development.     

Serological investigation of BK papovavirus infection in pregnant women and their offspring.

Paired sera from 150 pregnant women and 387 umbilical cord sera were tested for BK virus (BKV) antibodies. The hemagglutination inhibition, neutralization, and indirect immunofluorescence tests were employed for the detection of antibodies. Treatment of serum with anti-gamma Fc and tests of immunoglobulin M (IgM) fractions for antibodies were utilized as required to detect and validate the presence of virus-specific IgM. The BKV antibody prevalence in the sera collected at the time of the first prenatal visit was 75% by hemagglutination inhibition and 91% by neutralization tests. A total of 95% of the women had antibodies by at least one of the three serological tests. Five of 100 women with normal pregnancies exhibited BKV activity during pregnancy as evidenced by a greater than fourfold rise in BKV hemagglutination inhibition antibody titers and acquisition of BKV-specific IgM. The antibody rise occurred in the younger women and appeared to be a result of reactivation of the virus rather than of primary infection. Two instances of possible recent BKV infections were identified. BKV-specific IgM was not detected in any of the 387 umbilical cord sera which included three specimens from infants born to mothers with definite or probable BKV activity during pregnancy and 50 specimens with IgM levels of > 20 mg/100 ml. The results indicate that few women in the child-bearing age are nonimmune to BKV and that, although reactivation of infection occurs in pregnancy, congenital transmission of the virus either does not occur or is rare.     

Detection of aberrant p16INK4A methylation in sera of patients with liver cirrhosis and hepatocellular carcinoma

Hepatocellular carcinomas (HCCs) show genomic alterations, including DNA rearrangements associated with HBV DNA integration, loss of heterozygosity, and chromosomal amplification. The genes most frequently involved are those encoding tumor suppressors. The p16INK4A tumor suppressor gene frequently displays genetic alteration in HCC tissues. The present study was performed to examine the incidence of methylated p16INK4A in the sera of liver cirrhosis (LC) and HCC patients, and to evaluate its role as a tumor marker of HCC. The sera of 23 LC patients and 46 HCC patients were examined in this study. The methylation status of p16INK4A was evaluated by methylation-specific PCR of serum samples. Methylated p16INK4A was detected in 17.4% (4/23) of LC patients and in 47.8% (22/46) of HCC patients. No association was demonstrated between p16INK4A methylation and serum AFP level. As the status of p16INK4A methylation was not associated with serum AFP level, it may have a role as a tumor marker of HCC.     

Alpha-1 Antitrypsin (AAT) and Alpha-fetoprotein (AFP) in Sera of Patients with Germ Cell Neoplasm

Serum alpha-fetoprotein (AFP) and serum alpha-1 antitrypsin (AAT) was determined in 24 patients with germ cell neoplasms of the gonads and extra-gonadal sites.
Although when serial determinations are performed a correlation between AAT and AFP can be seen, 71 determinations of AAT as well as AFP in the serum of the patients with germ cell tumours gave the following results: when the AFP-concentration was below 20 ng/ml, the AAT values were always below the upper limit of the normal range (4.0 g/l); with AFP-values well above 20 ng/ml, the AAT concentration was elevated (>4.0 g/l) in only 33% of the cases; in all the sera with an elevated AAT-level, the AFP-concentration was—mostly considerably—enhanced; and in 45% of the sera with normal (or lowered) AAT, there was a diagnostically meaningful elevation of the AFP-concentration. In addition data from 9 patients with tumours not of germ cell origin undergoing therapy showed considerable variations of AAT-values at normal AFP concentrations.
It is therefore considered that although there is evidence that AAT is produced by EST, serum AAT is not a useful monitor of disease activity in these patients, especially when compared with serum AFP.