Generation of nanobodies targeting the human, transcobalamin-mediated vitamin B12 uptake route.

Cellular uptake of vitamin B12 in humans is mediated by the endocytosis of the B12 carrier protein transcobalamin (TC) via its cognate cell surface receptor TCblR, encoded by the CD320 gene. Because CD320 expression is associated with the cell cycle and upregulated in highly proliferating cells including cancer cells, this uptake route is a potential target for cancer therapy. We developed and characterized four camelid nanobodies that bind holo-TC (TC in complex with B12 ) or the interface of the human holo-TC:TCblR complex with nanomolar affinities. We determined X-ray crystal structures of these nanobodies bound to holo-TC:TCblR, which enabled us to map their binding epitopes. When conjugated to the model toxin saporin, three of our nanobodies caused growth inhibition of HEK293T cells and therefore have the potential to inhibit the growth of human cancer cells. We visualized the cellular binding and endocytic uptake of the most potent nanobody (TC-Nb4) using fluorescent light microscopy. The co-crystal structure of holo-TC:TCblR with another nanobody (TC-Nb34) revealed novel features of the interface of TC and the LDLR-A1 domain of TCblR, rationalizing the decrease in the affinity of TC-B12 binding caused by the Δ88 mutation in CD320.

Cellular uptake of vitamin B12: Role and fate of TCblR/CD320, the transcobalamin receptor.

Cellular uptake of vitamin B12 (cobalamin, Cbl) is mediated by a cell surface receptor (TCblR/CD320) that binds transcobalamin (TC) saturated with Cbl. TC is secreted by the vascular endothelium, has a relatively short half-life, binds Cbl with high affinity and presents the vitamin to the receptor for cellular uptake. Here we show binding and internalization of the TC-Cbl complex along with its' receptor (TCblR) in several human cell lines. The expression of TCblR is linked to the cell cycle with highest expression in actively proliferating cells. Upon binding TC-Cbl, the receptors appear to segregate on the plasma membrane and are internalized over the course of 30-60 min. Subsequently, the receptors appear to be destroyed along with the TC, which results in the release of free Cbl in the lysosome. The appearance of TCblR on the cell surface is limited to newly synthesized protein without contribution from recycling of the receptor. Therefore, Cbl uptake into cells is fully dependent on the expression of newly synthesized TCblR that is up-regulated in actively proliferating cells. The cell cycle-associated up-regulation of TCblR in cancers provides a route for targeted drug delivery.

Neuropathology of vitamin B12 deficiency in the Cd320-/- mouse.

In humans, vitamin B12 deficiency causes peripheral and CNS manifestations. Loss of myelin in the peripheral nerves and the spinal cord (SC) contributes to peripheral neuropathy and motor deficits. The metabolic basis for the demyelination and brain disorder is unknown. The transcobalamin receptor-knockout mouse ( Cd320-/-) develops cobalamin (Cbl) deficiency in the nervous system, with mild anemia. A decreased S-adenosylmethionine: S-adenosylhomocysteine ratio and increased methionine were seen in the brain with no significant changes in neurotransmitter metabolites. The structural pathology in the SC presented as loss of myelin in the axonal tracts with inflammation. The sciatic nerve (SN) showed increased nonuniform, internodal segments suggesting demyelination, and remyelination in progress. Consistent with these changes, the Cd320-/- mouse showed an increased latency to thermal nociception. Further, lower amplitude of compound action potential in the SN suggested that the functional capacity of the heavily myelinated axons were preferentially compromised, leading to loss of peripheral sensation. Although the metabolic basis for the demyelination and the structural and functional alterations of the nervous system in Cbl deficiency remain unresolved, the Cd320-/- mouse provides a unique model to investigate the pathologic consequences of vitamin B12 deficiency. -Arora, K., Sequeira, J. M., Alarcon, J. M., Wasek, B., Arning, E., Bottiglieri, T., Quadros, E. V. Neuropathology of vitamin B12 deficiency in the Cd320-/- mouse.

N-homocysteinylation of tau and MAP1 is increased in autopsy specimens of Alzheimer's disease and vascular dementia.

The pathomechanisms that associate a deficit in folate and/or vitamin B12 and the subsequent hyperhomocysteinemia with pathological brain ageing are unclear. We investigated the homocysteinylation of microtubule-associated proteins (MAPs) in brains of patients with Alzheimer's disease or vascular dementia, and in rats depleted in folate and vitamin B12, Cd320 KO mice with selective B12 brain deficiency and H19-7 neuroprogenitors lacking folate. Compared with controls, N-homocysteinylated tau and MAP1 were increased and accumulated in protein aggregates and tangles in the cortex, hippocampus and cerebellum of patients and animals. N-homocysteinylation dissociated tau and MAPs from ß-tubulin, and MS analysis showed that it targets lysine residues critical for their binding to ß-tubulin. N-homocysteinylation increased in rats exposed to vitamin B12 and folate deficit during gestation and lactation and remained significantly higher when they became 450 days-old, despite returning to normal diet at weaning, compared with controls. It was correlated with plasma homocysteine (Hcy) and brain expression of methionine tRNAsynthetase (MARS), the enzyme required for the synthesis of Hcy-thiolactone, the substrate of N-homocysteinylation. Experimental inactivation of MARS prevented the N-homocysteinylation of tau and MAP1, and the dissociation of tau and MAP1 from ß-tubulin and PSD95 in cultured neuroprogenitors. In conclusion, increased N-homocysteinylation of tau and MAP1 is a mechanism of brain ageing that depends on Hcy concentration and expression of MARS enzyme. Its irreversibility and cumulative occurrence throughout life may explain why B12 and folate supplementation of the elderly has limited effects, if any, to prevent pathological brain ageing and cognitive decline. Copyright © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Inherited disorders of cobalamin metabolism disrupt nucleocytoplasmic transport of mRNA through impaired methylation/phosphorylation of ELAVL1/HuR.

The molecular mechanisms that underlie the neurological manifestations of patients with inherited diseases of vitamin B12 (cobalamin) metabolism remain to date obscure. We observed transcriptomic changes of genes involved in RNA metabolism and endoplasmic reticulum stress in a neuronal cell model with impaired cobalamin metabolism. These changes were related to the subcellular mislocalization of several RNA binding proteins, including the ELAVL1/HuR protein implicated in neuronal stress, in this cell model and in patient fibroblasts with inborn errors of cobalamin metabolism and Cd320 knockout mice. The decreased interaction of ELAVL1/HuR with the CRM1/exportin protein of the nuclear pore complex and its subsequent mislocalization resulted from hypomethylation at R-217 produced by decreased S-adenosylmethionine and protein methyl transferase CARM1 and dephosphorylation at S221 by increased protein phosphatase PP2A. The mislocalization of ELAVL1/HuR triggered the decreased expression of SIRT1 deacetylase and genes involved in brain development, neuroplasticity, myelin formation, and brain aging. The mislocalization was reversible upon treatment with siPpp2ca, cobalamin, S-adenosylmethionine, or PP2A inhibitor okadaic acid. In conclusion, our data highlight the key role of the disruption of ELAVL1/HuR nuclear export, with genomic changes consistent with the effects of inborn errors of Cbl metabolisms on brain development, neuroplasticity and myelin formation.

Transcellular transport of cobalamin in aortic endothelial cells.

Cobalamin [Cbl (or B12)] deficiency causes megaloblastic anemia and a variety of neuropathies. However, homeostatic mechanisms of cyanocobalamin (CNCbl) and other Cbls by vascular endothelial cells are poorly understood. Herein, we describe our investigation into whether cultured bovine aortic endothelial cells (BAECs) perform transcytosis of B12, namely, the complex formed between serum transcobalamin and B12, designated as holo-transcobalamin (holo-TC). We show that cultured BAECs endocytose [57Co]-CNCbl-TC (source material) via the CD320 receptor. The bound Cbl is transported across the cell both via exocytosis in its free form, [57Co]-CNCbl, and via transcytosis as [57Co]-CNCbl-TC. Transcellular mobilization of Cbl occurred in a bidirectional manner. A portion of the endocytosed [57Co]-CNCbl was enzymatically processed by methylmalonic aciduria combined with homocystinuria type C (cblC) with subsequent formation of hydroxocobalamin, methylcobalamin, and adenosylcobalamin, which were also transported across the cell in a bidirectional manner. This demonstrates that transport mechanisms for Cbl in vascular endothelial cells do not discriminate between various ß-axial ligands of the vitamin. Competition studies with apoprotein- and holo-TC and holo-intrinsic factor showed that only holo-TC was effective at inhibiting transcellular transport of Cbl. Incubation of BAECs with a blocking antibody against the extracellular domain of the CD320 receptor inhibited uptake and transcytosis by ∼40%. This study reveals that endothelial cells recycle uncommitted intracellular Cbl for downstream usage by other cell types and suggests that the endothelium is self-sufficient for the specific acquisition and subsequent distribution of circulating B12 via the CD320 receptor. We posit that the endothelial lining of the vasculature is an essential component for the maintenance of serum-tissue homeostasis of B12.-Hannibal, L., Bolisetty, K., Axhemi, A., DiBello, P. M., Quadros, E. V., Fedosov, S., Jacobsen, D. W. Transcellular transport of cobalamin in aortic endothelial cells.

Maternofetal transport of vitamin B12: role of TCblR/CD320 and megalin.

Vitamin B12 deficiency causes megaloblastic anemia and neurologic disorder in humans. Gene defects of transcobalamin (TC) and the transcobalamin receptor (TCblR), needed for cellular uptake of the TC-bound B12, do not confer embryonic lethality. TC deficiency can produce the hematologic and neurologic complications after birth, whereas TCblR/CD320 gene defects appear to produce mild metabolic changes. Alternate maternofetal transport mechanisms appear to provide adequate B12 to the fetus. To understand this mechanism, we evaluated the role of TC, TCblR/CD320, and megalin in maternofetal transport of B12 in a TCblR/CD320-knockout (KO) mouse. Our results showed high expression of TCblR/CD320 in the labyrinth of the placenta, embryonic brain, and spinal column in wild-type (WT) mice. Megalin expression was about the same in both WT and KO mouse visceral yolk sac, brain, and spinal column. Megalin mRNA was down-regulated in the KO embryonic spinal cord (SC) and kidneys. Megalin expression remained unaltered in adult WT and KO mouse brain, SC, and kidneys. Injected dsRed-TC-B12 and TC-57CoB12 accumulated in the visceral yolk sac of KO mice where megalin is expressed and provides an alternate mechanism for the maternofetal transport of Cbl during fetal development.-Arora, K., Sequeira, J. M., Quadros, E. V. Maternofetal transport of vitamin B12: role of TCblR/CD320 and megalin.

Folate receptor autoantibodies in pregnancy related complications.

BACKGROUND: Folate receptor autoantibodies in women have been associated with neural tube pregnancy and in children with cerebral folate deficiency syndrome and autism. These autoantibodies have been implicated in blocking folate transport to the fetus and to the brain in infants. METHODS: We report a woman with multiple pregnancy related complications who was diagnosed with autoantibodies to the folate receptor alpha. RESULTS: A treatment strategy with folate supplementation and reducing the antibody titer proved effective in normal pregnancy outcome. CONCLUSION: This long-term follow up of a subject with folate receptor autoantibodies is a first report of its kind and describes treatment strategy to prevent pregnancy related complications due to folate receptor autoantibodies.

Mapping the functional domains of TCblR/CD320, the receptor for cellular uptake of transcobalamin-bound cobalamin.

The membrane receptor TCblR/CD320 binds transcobalamin (TC) saturated with vitamin B12 [cobalamin (Cbl)] and mediates cellular uptake of the vitamin. The specificity of TC for Cbl and of the receptor for TC-Cbl ensures efficient uptake of Cbl into cells. The high-affinity interaction of TCblR with TC-Cbl (Ka=10 nM(-1)) was investigated using deletions and mutations of amino acid sequences in TCblR. Only the extracellular region (aa 32-229) is needed for TC-Cbl binding, but the N-glycosylation sites (N126, N195, and N213) are of no importance for this function. Deleting the cysteine-rich region (aa 95-141) that separates the two low-density lipoprotein receptor type A (LDLR-A) domains does not affect TC-Cbl binding (Ka = 19-24 nM(-1)). The two LDLR-A domains (aa 54-89 and 132-167) with the negatively charged acidic residues involved in Ca(2+) binding are critical determinants of ligand binding. The cytoplasmic tail is apparently crucial for internalization of the ligand. Within this region, the RPLGLL motif and the PDZ binding motifs (QERL/KESL) appear to be involved in initiating and completing the process of ligand internalization. Mutations and deletions of these regions involved in binding and internalization of TC-Cbl are likely to produce the biochemical and clinical phenotype of Cbl deficiency.

The transcobalamin receptor knockout mouse: a model for vitamin B12 deficiency in the central nervous system.

The membrane receptor (TCblR/CD320) for transcobalamin (TC)-bound cobalamin (Cbl) facilitates the cellular uptake of Cbl. A genetically modified mouse model involving ablation of the CD320 gene was generated to study the effects on cobalamin homeostasis. The nonlethal nature of this knockout and the lack of systemic cobalamin deficiency point to other mechanisms for cellular Cbl uptake in the mouse. However, severe cobalamin depletion in the central nervous system (CNS) after birth (P<0.01) indicates that TCblR is the only receptor responsible for Cbl uptake in the CNS. Metabolic Cbl deficiency in the brain was evident from the increased methylmalonic acid (P<0.01-0.04), homocysteine (P<0.01), cystathionine (P<0.01), and the decreased S-adenosylmethionine/S-adenosyl homocysteine ratio (P<0.01). The CNS pathology of Cbl deficiency seen in humans may not manifest in this mouse model; however, it does provide a model with which to evaluate metabolic pathways and genes affected.

Folate deficiency in rat pups during weaning causes learning and memory deficits.

Folate is essential for fetal development, and its deficiency during gestation causes behavioural deficits in the offspring. The present study investigated its influence during weaning on brain function in the pups of rats that were put on a folate-deficient (FD) diet on postnatal day (PND) 1. Systemic folate deficiency in pups on the FD diet (n 15) was evident from the dramatically lower hepatic folate concentrations (median 23·7, range 8·1-48·4 ng/mg protein) and higher homocysteine concentrations (median 27·7, range 14·7-45·5 pmol/mg protein), respectively, compared with those of pups on the normal diet (ND; n 9) (median 114·5, range 64·5-158·5 ng/mg protein and median 15·5, range 11·6-18·9 pmol/mg protein) on PND 23. Brain folate concentrations although low were similar in pups on the FD diet (median 10·5, range 5·5-24·5 ng/mg protein) and ND (median 11·1, range 7·1-24·2 ng/mg protein). There was a high accumulation of homocysteine in the brain of FD pups, mostly in the hippocampus (median 58·1, range 40·8-99·7 pmol/mg protein) and cerebellum (median 69·1, range 50·8-126·6 pmol/mg protein), indicating metabolic folate deficiency despite normal brain folate concentrations. Developmental deficits or autistic traits were more frequent in the FD group than in the ND group and repetitive self-grooming occurred, on average, three times (range 1-8) v. once (range 0-3) during 5 min, respectively. Long-term memory or spatial learning and set-shifting deficits affected 12 to 62% of rats in the FD group compared with none in the ND group. Post-weaning folic acid supplementation did not correct these deficits. These observations indicate that folate deficiency during weaning affects postnatal development even when gestational folate supply is normal.

Binding Folate Receptor Alpha Autoantibody Is a Biomarker for Leucovorin Treatment Response in Autism Spectrum Disorder.

Autism spectrum disorder (ASD) affects up to 1 in 36 children in the United States. It is a heterogeneous neurodevelopmental disorder with life-long consequences. Patients with ASD and folate pathway abnormalities have demonstrated improved symptoms after treatment with leucovorin (folinic acid), a reduced form of folate. However, biomarkers for treatment response have not been well investigated and clinical trials are lacking. In this retrospective analysis, a cohort of prospectively collected data from 110 consecutive ASD clinic patients [mean (SD) age: 10.5 (6.2) years; 74% male] was examined. These patients all underwent testing for folate receptor alpha autoantibodies (FRAAs) and soluble folate binding proteins (sFBPs) biomarkers and were treated with leucovorin, if appropriate. Analyses examined whether these biomarkers could predict response to leucovorin treatment as well as the severity of ASD characteristics at baseline. The social responsiveness scale (SRS), a measure of core ASD symptoms, and the aberrant behavior checklist (ABC), a measure of disruptive behavior, were collected at each clinic visit. Those positive for sFBPs had more severe ASD symptoms, and higher binding FRAA titers were associated with greater ABC irritability. Treatment with leucovorin improved most SRS subscales with higher binding FRAA titers associated with greater response. Leucovorin treatment also improved ABC irritability. These results confirm and expand on previous studies, underscore the need for biomarkers to guide treatment of folate pathways in ASD, and suggest that leucovorin may be effective for children with ASD.

Clinical recognition and aspects of the cerebral folate deficiency syndromes.

We characterized cerebral folate deficiency (CFD) as any neuro-psychiatric condition associated with low spinal fluid (CSF) N5-methyltetrahydrofolate (MTHF) but normal folate status outside the central nervous system (CNS). The commonest cause underlying CFD syndromes is the presence of serum autoantibodies of the blocking type directed against folate receptor-α (FRα) attached to the plasma-side of choroid plexus epithelial cells. Blocking FR antibodies inhibit MTHF transport across the choroid plexus. Serum titers of FR antibodies may fluctuate significantly over time. Less frequent causes of CFD are FOLR-1 mutations, mitochondrial disorders and inborn errors affecting folate metabolism. Maternal FR antibodies have been associated with neural tube defects while the presence of FR antibodies in either one or both parents increases the risk of an offspring with infantile autism. Recognizable CFD syndromes attributed to FR-antibodies in childhood are infantile-onset CFD presenting 4-6 months after birth, infantile autism with neurological deficits, and a spastic ataxic syndrome from the age of 1 year, while progressive dystonic or schizophrenic syndromes develop during adolescence. FR autoantibodies are frequently found in autism spectrum disorders, in an Aicardi-Goutières variant and in Rett syndrome. The heterogeneous phenotype of CFD syndromes might be determined by different ages of onset and periods when FR autoantibodies are generated with consequent CNS folate deficiency. Folate deficiency during various critical stages of fetal and infantile development affects structural and functional refinement of the brain. Awareness of CFD syndromes should lead to early detection, diagnosis and improved prognosis of these potentially treatable group of autoimmune and genetically determined conditions.

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.

Folate and Other B Vitamins in Brain Health and Disease.

B vitamins as a group play essential roles in a multitude of metabolic reactions involved in cellular replication, energy production, the synthesis of intermediary compounds, and neurotransmitters [...].

Brain Uptake of Folate Forms in the Presence of Folate Receptor Alpha Antibodies in Young Rats: Folate and Antibody Distribution.

In a rat model, following exposure to rat folate receptor alpha antibodies (FRαAb) during gestation, FRαAb accumulates in the placenta and the fetus and blocks folate transport to the fetal brain and produces behavioral deficits in the offspring. These deficits could be prevented with folinic acid. Therefore, we sought to evaluate folate transport to the brain in young rat pups and determine what effect FRαAb has on this process, to better understand the folate receptor autoimmune disorder associated with cerebral folate deficiency (CFD) in autism spectrum disorders (ASD). When injected intraperitoneally (IP), FRαAb localizes to the choroid plexus and blood vessels including the capillaries throughout the brain parenchyma. Biotin-tagged folic acid shows distribution in the white matter tracts in the cerebrum and cerebellum. Since these antibodies can block folate transport to the brain, we orally administered various folate forms to identify the form that is better-absorbed and transported to the brain and is most effective in restoring cerebral folate status in the presence of FRαAb. The three forms of folate, namely folic acid, D,L-folinic acid and levofolinate, are converted to methylfolate while L-methylfolate is absorbed as such and all are efficiently distributed to the brain. However, significantly higher folate concentration is seen in the cerebrum and cerebellum with levofolinate in the presence or absence of FRαAb. Our results in the rat model support testing levofolinate to treat CFD in children with ASD.

Transcobalamin receptor gene polymorphisms and mutation in an elderly population.

BACKGROUND & AIMS: Cellular uptake of the essential nutrient vitamin B12 (cobalamin) occurs via the transcobalamin receptor (TCblR/CD320), a ubiquitous membrane receptor. Polymorphisms in the receptor exist, though the effect of such variants across patient populations is unknown. METHODS: We determined CD320 genotype in 377 randomly selected elderly individuals. RESULTS: Three polymorphisms and a codon deletion were identified in the exon 2 region. Haplotype variants had significantly higher holotranscobalamin (holo-TC) values and a higher holo-TC/total cobalamin ratio. TCblR haplotype explained 46% of the variability in holo-TC values. CONCLUSIONS: This has significant implications for the clinical utility of the 'combined indicator' of B12 status since it is based on a standard rate of intracellular flux via the TC-Cbl receptor. Modification of the model may be required to account for CD320 haplotype.

FTY720 requires vitamin B12-TCN2-CD320 signaling in astrocytes to reduce disease in an animal model of multiple sclerosis.

Vitamin B12 (B12) deficiency causes neurological manifestations resembling multiple sclerosis (MS); however, a molecular explanation for the similarity is unknown. FTY720 (fingolimod) is a sphingosine 1-phosphate (S1P) receptor modulator and sphingosine analog approved for MS therapy that can functionally antagonize S1P1. Here, we report that FTY720 suppresses neuroinflammation by functionally and physically regulating the B12 pathways. Genetic and pharmacological S1P1 inhibition upregulates a transcobalamin 2 (TCN2)-B12 receptor, CD320, in immediate-early astrocytes (ieAstrocytes; a c-Fos-activated astrocyte subset that tracks with experimental autoimmune encephalomyelitis [EAE] severity). CD320 is also reduced in MS plaques. Deficiency of CD320 or dietary B12 restriction worsens EAE and eliminates FTY720's efficacy while concomitantly downregulating type I interferon signaling. TCN2 functions as a chaperone for FTY720 and sphingosine, whose complex induces astrocytic CD320 internalization, suggesting a delivery mechanism of FTY720/sphingosine via the TCN2-CD320 pathway. Taken together, the B12-TCN2-CD320 pathway is essential for the mechanism of action of FTY720.

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.

Down-regulation of transcobalamin receptor TCblR/CD320 by siRNA inhibits cobalamin uptake and proliferation of cells in culture.

The clinical phenotype of cobalamin (Cbl) deficiency is dictated by the essential role of this vitamin in two key enzymatic reactions. Multiple proteins and receptors participate in the absorption, transport and delivery of this vitamin to tissue cells. Cellular uptake of Cbl is mediated by transcobalamin (TC), a plasma protein and a transmembrane receptor (TCblR) with high affinity for TC saturated with Cbl. Knockdown of TCblR with siRNA results in decreased TC-Cbl uptake. The ensuing Cbl deficiency leads to an increase in doubling time and decreased proliferation of these cells. The study confirms the seminal role of this receptor in the cellular uptake of Cbl and its down-regulation as a potential strategy to inhibit proliferation of cancer cells.