Autoantibodies against homocysteinylated protein in a mouse model of folate deficiency-induced neural tube defects.

BACKGROUND: Periconceptional supplementation with folic acid results in a significant reduction in the incidence of neural tube defects (NTDs). Nonetheless, NTDs remain a leading cause of perinatal morbidity and mortality worldwide, and the mechanism(s) by which folate exerts its protective effects are unknown. Homocysteine is an amino acid that accumulates under conditions of folate-deficiency, and is suggested as a risk factor for NTDs. One proposed mechanism of homocysteine toxicity is its accumulation into proteins in a process termed homocysteinylation. METHODS & RESULTS: Herein, we used a folate-deficient diet in pregnant mice to demonstrate that there is: (i) a significant inverse correlation between maternal serum folate levels and serum homocysteine; (ii) a significant positive correlation between serum homocysteine levels and titers of autoantibodies against homocysteinylated protein; and (iii) a significant increase in congenital malformations and NTDs in mice deficient in serum folate. Furthermore, in mice administered the folate-deplete diet before conception, supplementation with folic acid during the gestational period completely rescued the embryos from congenital defects, and resulted in homocysteinylated protein titers at term that are comparable to that of mice administered a folate-replete diet throughout both the pre- and postconception period. These results demonstrate that a low-folate diet that induces NTDs also increases protein homocysteinylation and the subsequent generation of autoantibodies against homocysteinylated proteins. CONCLUSION: These data support the hypotheses that homocysteinylation results in neo-self antigen formation under conditions of maternal folate deficiency, and that this process is reversible with folic acid supplementation.

The diagnostic utility of folate receptor autoantibodies in blood.

Folate supplementation reduces the risk of neural tube defect (NTD) pregnancy, and folinic acid has been used to correct cerebral folate deficiency (CFD) in children with developmental disorders. In the absence of systemic folate deficiency, the discovery of autoantibodies (AuAbs) to folate receptor α (FRα) that block the uptake of folate offers one mechanism to explain the response to folate in these disorders. The association of FRα AuAbs with pregnancy-related complications, CFD syndrome, and autism spectrum disorders and response to folate therapy is highly suggestive of the involvement of these AuAbs in the disruption of brain development and function via folate pathways. The two types of antibodies identified in the serum of patients are blocking antibody and binding antibody. The two antibodies can be measured by the specific assays described and exert their pathological effects either by functional blocking of folate transport as previously shown or hypothetically by disrupting the FR by an antigen-antibody-mediated inflammatory response. We have identified both IgG and IgM AuAbs in these conditions. The predominant antibodies in women with NTD pregnancy belong to the IgG1 and IgG2 isotype and in CFD children, the IgG1 and IgG4 isotype. This review describes the methods used to measure these AuAbs, their binding characteristics, affinity, cross-reactivity, and potential mechanisms by which folate therapy could work. Because these AuAbs are associated with various pathologies during fetal and neonatal development, early detection and intervention could prevent or reverse the consequences of exposure to these AuAbs.

Lack of association between folate receptor autoantibodies and conotruncal congenital heart defects.

Conotruncal cardiac defects are partially prevented by maternal folic acid supplementation. However, the biochemical mechanism is unknown. Maternal autoantibodies to folate receptors, previously associated with increased risk for neural tube defects, also may account for this effect. This study aimed to examine the titers of folate receptor-blocking autoantibodies in mothers of children with conotruncal congenital heart defects and to compare them with those in the general population. Serum samples were obtained from 22 women whose pregnancies were complicated by conotruncal congenital heart malformations. Groups of samples were analyzed for autoantibodies against [(3)H] folic acid-labeled folate receptors, quantitative amounts of immunoglobulin G (IgG) and IgM autoantibodies to the folate receptor, and for ability to block-bind folic acid to receptors. No elevated levels of antibodies binding to [(3)H] folic acid-labeled folate receptors were found. No difference was found in antifolate receptor alpha-IgG or IgM median levels between cases (261 vs. 240 µg/mL) and control subjects (773 vs. 924 µg/mL). There was no increased blocking of folic acid binding between cases [0.69 ng/mL; 95 % confidence interval (CI), 0.006-0.01] and control subjects (0.69 ng/mL; 95 % CI, 0.003-0.013). Although epidemiologic evidence suggests that periconceptual folic acid may prevent many conotruncal congenital heart defects, the current study suggests that this effect is unlikely to be explained by the presence of maternal autoantibodies to folate receptor. These data suggest that a strategy of screening women for such autoantibodies will not identify a high-risk group of women to target for supplemental folic acid to prevent congenital heart defects.

Lack of association between folate-receptor autoantibodies and neural-tube defects.

BACKGROUND: A previous report described the presence of autoantibodies against folate receptors in 75% of serum samples from women with a history of pregnancy complicated by a neural-tube defect, as compared with 10% of controls. We sought to confirm this finding in an Irish population, which traditionally has had a high prevalence of neural-tube defects. METHODS: We performed two studies. Study 1 consisted of analysis of stored frozen blood samples collected from 1993 through 1994 from 103 mothers with a history of pregnancy complicated by a neural-tube defect (case mothers), 103 mothers with a history of pregnancy but no complication by a neural-tube defect (matched with regard to number of pregnancies and sampling dates), 58 women who had never been pregnant, and 36 men. Study 2, conducted to confirm that the storage of samples did not influence the folate-receptor autoantibodies, included fresh samples from 37 case mothers, 22 control mothers, 10 women who had never been pregnant, and 9 men. All samples were assayed for blocking and binding autoantibodies against folate receptors. RESULTS: In Study 1, blocking autoantibodies were found in 17% of case mothers, as compared with 13% of control mothers (odds ratio, 1.54; 95% confidence interval [CI], 0.70 to 3.39), and binding autoantibodies in 29%, as compared with 32%, respectively (odds ratio, 0.82; 95% CI, 0.44 to 1.50). Study 2 showed similar results, indicating that sample degradation was unlikely. CONCLUSIONS: The presence and titer of maternal folate-receptor autoantibodies were not significantly associated with a neural-tube defect-affected pregnancy in this Irish population.

The reduced GM-CSF priming of ROS production in granulocytes from patients with myelodysplasia is associated with an impaired lipid raft formation.

Patients with myelodysplasia (MDS) show an impaired reactive oxygen species (ROS) production in response to fMLP stimulation of GM-CSF-primed neutrophils. In this study, we investigated the involvement of lipid rafts in this process and showed that treatment of neutrophils with the lipid raft-disrupting agent methyl-beta-cyclodextrin abrogates fMLP-induced ROS production and activation of ERK1/2 and protein kinase B/Akt, two signal transduction pathways involved in ROS production in unprimed and GM-CSF-primed neutrophils. We subsequently showed that there was a decreased presence of Lyn, gp91(phox), and p22(phox) in lipid raft fractions from neutrophils of MDS. Furthermore, the plasma membrane expression of the lipid raft marker GM1, which increases upon stimulation of GM-CSF-primed cells with fMLP, was reduced significantly in MDS patients. By electron microscopy, we showed that the fMLP-induced increase in GM1 expression in GM-CSF-primed cells was a result of de novo synthesis, which was less efficient in MDS neutrophils. Taken together, these data indicate an involvement of lipid rafts in activation of signal transduction pathways leading to ROS production and show that in MDS neutrophils, an impaired lipid raft formation in GM-CSF-primed cells results in an impaired ROS production.

Persistence of parvovirus B19 IgM antibodies and DNA in pure red cell aplasia resulting in myelodysplasia--a case report.

Human erythrovirus B19 (B19), previously known as parvovirus B19, is a small spherical, non-enveloped single stranded DNA virus. It has been shown to cause a wide spectrum of clinical conditons including various hematological disorders. We report here for the first time from Inida a case of pure red cell aplasia in a 45-year-old female for last 7 years due to chronic persistent B19 infection leading to myelodysplasia after 4 years. Her sera were positive for two times 4 months apart for B19 IgM and B19 DNA at the initial stage. Presently the patient is on repeated blood transfusion on every 15-20 days.

Autoantibodies against folate receptors in women with a pregnancy complicated by a neural-tube defect.

BACKGROUND: In the absence of clinical folate deficiency, periconceptional supplementation with folic acid reduces a woman's risk of having an infant with a neural-tube defect. Since antiserum to folate receptors induces embryo resorption and malformations in rats, we hypothesized that autoantibodies against folate receptors in women may be associated with pregnancy complicated by a neural-tube defect. METHODS: Serum from 12 women who were or had been pregnant with a fetus with a neural-tube defect and from 24 control women (20 with current or prior normal pregnancies and 4 who were nulligravid) was analyzed for autoantibodies by incubation with human placental folate receptors radiolabeled with [3H]folic acid. The properties of these autoantibodies were characterized by incubating serum and the autoantibodies isolated from serum with placental membranes, ED27 cells, and KB cells, which express the folate receptors. RESULTS: Serum from 9 of 12 women with a current or previous affected pregnancy (index subjects) and 2 of 20 control subjects contained autoantibodies against folate receptors (P<0.001). The autoantibodies blocked the binding of [3H]folic acid to folate receptors on placental membranes and on ED27 and KB cells incubated at 4 degrees C and blocked the uptake of [3H]folic acid by KB cells when incubated at 37 degrees C. CONCLUSIONS: Serum from women with a pregnancy complicated by a neural-tube defect contains autoantibodies that bind to folate receptors and can block the cellular uptake of folate. Further study is warranted to assess whether the observed association between maternal autoantibodies against folate receptors and neural-tube defects reflects a causal relation.

The immunohistochemical profile of the tethered filum terminale.

The embryopathy underlying tethering of the filum terminale is poorly understood. Knowledge of normal filum development is the foundation upon which to compare normal fila to fila from patients with tethered cord syndrome. Thirty-four fila from patients with tethered cord syndrome were immunostained with caudal neural tube developmental markers H4C4 (CD44), VIN-IS-53, AC4, FP3 and NOT1, and a panel of mature neuroglial, neural crest, epithelial and mesenchymal markers. H4C4 (CD44) and NOT1 exhibited significant alterations in immunoexpression in tethered fila compared to controls. The change in expression may be indicative of altered cell identity in the filum and constitute the predisposition to tethering.

Amniotic fluid soluble human leukocyte antigen G is markedly decreased in offspring with neural tube defects.

Pregnancies affected by a neural tube defect show changes in thymus morphology, neonatal and maternal T-cell repertoire. Amniotic fluid levels of soluble human leukocyte antigen G (HLA-G) (an immuno-modulatory protein) were found to be significantly lower as compared to controls. This may reflect a diminished cell-mediated immunity in neural tube defects.

Bcl10 is a positive regulator of antigen receptor-induced activation of NF-kappaB and neural tube closure.

Bcl10, a CARD-containing protein identified from the t(1;14)(p22;q32) breakpoint in MALT lymphomas, has been shown to induce apoptosis and activate NF-kappaB in vitro. We show that one-third of bcl10-/- embryos developed exencephaly, leading to embryonic lethality. Surprisingly, bcl10-/- cells retained susceptibility to various apoptotic stimuli in vivo and in vitro. However, surviving bcl10-/- mice were severely immunodeficient and bcl10-/- lymphocytes are defective in antigen receptor or PMA/Ionomycin-induced activation. Early tyrosine phosphorylation, MAPK and AP-1 activation, and Ca2+ signaling were normal in mutant lymphocytes, but antigen receptor-induced NF-kappaB activation was absent. Thus, Bcl10 functions as a positive regulator of lymphocyte proliferation that specifically connects antigen receptor signaling in B and T cells to NF-kappaB activation.

Allergic reactions to latex in myelodysplasia: a review of the literature.

Current research has identified clinically relevant allergens in natural latex. Children with myelodysplasia are especially considered to be at risk for the development of immunoglobulin E-mediated hypersensitivity, which can lead to life-threatening intraoperative anaphylaxis. A careful medical history is mandatory to identify patients who might be predisposed to anaphylactic reactions. Preventive measures involve primarily the avoidance of latex contact at home and in hospitals for all patients with myelodysplasia. The effectiveness of additional pharmacologic prophylaxis has to be determined further.

CD1+ cells in mothers of stillborn infants with neural tube defects.

In healthy individuals, CD1+ cells are found in thymic- tissue, but not in peripheral blood. The thymus, as a key organ of the neuroendocrine system, frequently shows gross abnormalities in infants with neural tube defects. In order to study the immunologic significance of fetal thymic findings, maternal T-lymphocyte subpopulations were investigated. In 10 mothers of healthy newborns, 5 mothers of stillborn infants who had no gross abnormalities, and 5 mothers of stillborn infants with neural tube defects, CD1+, CD3+, CD4+, and CD8+ cells were studied. Only the mothers of the infants with open neural tube defects showed CD1+ cells in their peripheral blood.

Neonatal lymphocyte subpopulations of the cases with neural tube defects.

Neural tube defects (NTDs) are among the most common congenital malformations of central nervous system and are frequently associated with other organ system abnormalities. In order to evaluate the immunological changes in such patients, we examined the lymphocyte subpopulations of 13 newborns with NTDs and 32 healthy newborns. Leukocyte counts, absolute neutrophil and lymphocyte counts were lower in the babies with NTDs. Also the lymphocyte subpopulations such as percentages and absolute counts of T-cells, CD4+ cells, and CD45+ cells showed statistically significant decreases. These findings suggest that the affected babies are immunosuppressed.

Maternal lymphocyte subsets in the cases with neural tube defects.

Neural tube defects (NTDs) are among the most prevalent of congenital malformations. Organ system anomalies and certain changes in the thymus gland have also been reported. Lymphocyte subset changes can be expected in infants with NTD as a result of these thymic anomalies. We investigated lymphocyte subpopulations in twenty-one mothers of NTD cases in order to evaluate the maternal immunological alterations. The mothers of twenty-one healthy infants were chosen as a control group. Pregnancy is known to be associated with a depression in cellular immunity. We found, however, some additional changes in cellular immunity in the mothers of NTDs. The CD4/CD8 ratios were significantly lower than those of control mothers (p = 0.044). This difference was related to the increased percentage of CD8 cells found in mothers of NTDs (p = 0.004). These results suggest that immunosuppression is more evident in the mothers of babies with NTDs.

Neutral-red uptake and expression of monocytic antigens in amniotic-fluid mononuclear phagocytes: evaluation of a novel approach for prenatal diagnosis of neural-tube defects.

In cases of fetal neural-tube defects macrophages are present in the amniotic fluid. We found that these viable phagocytic cells take up neutral-red and are easily identified as "red cells" by microscopic examination. This method is suitable for the rapid identification and counting of amniotic-fluid macrophages in suspension. We have studied 298 amniotic fluid samples. In the 226 normal cases studied, 0 to 1,200 macrophages per milliliter amniotic fluid have been found. In contrast, we found 1,250 to 99,000 macrophages per milliliter amniotic fluid in our 70 open neural tube defect (ONTD) cases. Statistical evaluation was performed to estimate the normal and pathologic ranges. Specificity and sensitivity of the neutral-red test and predictive value of positive and negative results have been calculated and presented in comparison with alpha-fetoprotein (AFP) determinations and ultrasonic methods. In 5 cases of anencephaly and 7 normal cases amniotic fluid cells were studied by immunocytochemistry: mononuclear cells present in the abnormal cases showed intense immunoreactivity for the Mo1 and Mo2 surface antigens of the phagocytic cell lineage.

Parental HLA compatibility, fetal wastage and neural tube defects: evidence for a T/t-like locus in humans.

To test the hypothesis that a locus in or near the human major histocompatibility complex (HLA) contributes to both involuntary fetal loss and neural tube defects (NTD), we evaluated sharing of antigens of the HLA-A, HLA-B, or HLA-DR loci of couples who had three or more first-trimester spontaneous abortions or who had a child with an NTD (myelomeningocele or anencephaly). HLA-A antigen sharing was increased in couples with three or more spontaneous abortions and in couples who had an anencephalic fetus, when compared with couples who had three or more pregnancies and no fetal loss. Increased sharing of antigens at the HLA-A and B loci was not seen in the entire group of couples with children with myelomeningocele, but was found in the subgroup of 36 couples whose child had a lumbar myelomeningocele. An increase in HLA-DR sharing was not seen in any group or subgroup when compared with the control couples. Among the aborting couples, increased sharing was not restricted to the couples who had no term pregnancies, but was also found in the couples whose fetal losses occurred after one or more normal term pregnancies. These results are consistent with the hypothesis that a locus on the HLA-A side of the HLA-DR locus contributes to some fetal loss and susceptibility to NTD. This model is proposed as an alternative to the hypothesis that the maternal immune response to paternal major histocompatibility complex (MHC) antigens is the basis for increased HLA sharing in couples with fetal wastage.

[immunoglobulins in amniotic fluid in normal pregnancy and fetal malformation (author's transl)]. / Immunoglobuline im Fruchtwasser bei normaler Schwangerschaft und fötaler Missbildung.

In 20 cases of normal pregnancy and 9 cases of foetal monstrosities the immunoglobulins G, A, M and albumin C3C and alpha 2MG were determined in the amniotic fluid. In neural tube defects the IgG, IgA and albumin concentrations were clearly above those found in normal pregnancies. IgM was not found in any of the cases, whereas alpha 2MG occurred in two cases of foetal monstrosity. There was no difference in C3C concentrations of both groups.

Effect of human cord and postoperative serum on experimental inflammation in the rat.

Human cord and postoperative serum depressed the oedema provoked by mediators of the inflammatory reaction such as bradykinin, histamine, serotonin and prostaglandin E2 and also the experimental inflammation caused by carrageenin. Normal human and pregnancy serum did not have such an effect. In two cases of open neural tube defect, one of anencephaly and another of spina bifida, human amniotic fluid also had a strongly depressing effect on the experimental oedema provoked by serotonin. Human amniotic fluid from normal pregnancies did not inhibit this experimental inflammation. A protein-fraction of mol. wt 30,000--100,000 has been isolated from the inhibiting sera and shows the anti-inflammatory activity to be dose-related towards all the oedema-provoking substances used. Immunological studies showed that the inhibiting factor could be a protein in the pre-albuminic region, while alpha-foetoprotein did not appear to be responsible for the anti-inflammatory activity. Our conclusion is that human serum contains a protein of foetal origin with an acute-phase character and strong anti-inflammatory activities analogous to rat alpha 2-macrofoetoprotein.