Organometallic DNA-B12 Conjugates as Potential Oligonucleotide Vectors: Synthesis and Structural and Binding Studies with Human Cobalamin-Transport Proteins.

The synthesis and structural characterization of Co-(dN)25 -Cbl (Cbl: cobalamin; dN: deoxynucleotide) and Co-(dN)39 -Cbl, which are organometallic DNA-B12 conjugates with single DNA strands consisting of 25 and 39 deoxynucleotides, respectively, and binding studies of these two DNA-Cbl conjugates to three homologous human Cbl transporting proteins, transcobalamin (TC), intrinsic factor (IF), and haptocorrin (HC), are reported. This investigation tests the suitability of such DNA-Cbls for the task of eventual in vivo oligonucleotide delivery. The binding of DNA-Cbl to TC, IF, and HC was investigated in competition with either a fluorescent Cbl derivative and Co-(dN)25 -Cbl, or radiolabeled vitamin B12 (57 Co-CNCbl) and Co-(dN)25 -Cbl or Co-(dN)39 -Cbl. Binding of the new DNA-Cbl conjugates was fast and tight with TC, but poorer with HC and IF, which extends a similar original finding with the simpler DNA-Cbl, Co-(dN)18 -Cbl. The contrasting affinities of TC versus IF and HC for the DNA-Cbl conjugates are rationalized herein by a stepwise mechanism of Cbl binding. Critical contributions to overall affinity result from gradual conformational adaptations of the Cbl-binding proteins to the DNA-Cbl, which is first bound to the respective ß domains. This transition is fast with TC, but slow with IF and HC, with which weaker binding results. The invariably tight interaction of the DNA-Cbl conjugates with TC makes the Cbl moiety a potential natural vector for the specific delivery of oligonucleotide loads from the blood into cells.

Organometallic B12-DNA conjugate: synthesis, structure analysis, and studies of binding to human B12-transporter proteins.

Design, synthesis, and structural characterization of a B12-octadecanucleotide are presented herein, a new organometallic B12-DNA conjugate. In such covalent conjugates, the natural B12 moiety may be a versatile vector for controlled in vivo delivery of oligonucleotides to cellular targets in humans and animals, through the endogenous B12 transport systems. Binding of the organometallic B12 octadecanucleotide to the three important human proteins of B12 transport was studied, to examine its structural suitability for the task of eventual in vivo oligonucleotide delivery. Binding was efficient with transcobalamin (TC), but not so efficient with the homologous glycoproteins intrinsic factor and haptocorrin. Binding of the B12 octadecanucleotide to TC suggests the capacity of the B12 moiety to serve as a natural vector for specific transport of single stranded, organometallic oligonucleotide loads from the blood stream into cells.

4-ethylphenyl-cobalamin impairs tissue uptake of vitamin B12 and causes vitamin B12 deficiency in mice.

Coß-4-ethylphenyl-cob(III) alamin (EtPhCbl) is an organometallic analogue of vitamin B12 (CNCbl) which binds to transcobalamin (TC), a plasma protein that facilitates the cellular uptake of cobalamin (Cbl). In vitro assays with key enzymes do not convert EtPhCbl to the active coenzyme forms of Cbl suggesting that administration of EtPhCbl may cause cellular Cbl deficiency. Here, we investigate the in vivo effect of EtPhCbl in mice and its ability, if any, to induce Cbl deficiency. We show that EtPhCbl binds to mouse TC and we examined mice that received 3.5 nmol/24h EtPhCbl (n=6), 3.5 nmol/24h CNCbl (n=7) or NaCl (control group) (n=5) through osmotic mini-pumps for four weeks. We analyzed plasma, urine, liver, spleen, submaxillary glands and spinal cord for Cbl and markers of Cbl deficiency including methylmalonic acid (MMA) and homocysteine (tHcy). Plasma MMA (mean±SEM) was elevated in animals treated with EtPhCbl (1.01±0.12 µmol/L) compared to controls (0.30±0.02 µmol/L) and CNCbl (0.29±0.01 µmol/L) treated animals. The same pattern was observed for tHcy. Plasma total Cbl concentration was higher in animals treated with EtPhCbl (128.82±1.87 nmol/L) than in CNCbl treated animals (87.64±0.93 nmol/L). However, the organ levels of total Cbl were significantly lower in animals treated with EtPhCbl compared to CNCbl treated animals or controls, notably in the liver (157.07±8.56 pmol/g vs. 603.85±20.02 pmol/g, and 443.09±12.32 pmol/g, respectively). Differences between the three groups was analysed using one-way ANOVA and, Bonferroni post-hoc test. EtPhCbl was present in all tissues, except the spinal cord, accounting for 35-90% of total Cbl. In conclusion, treatment with EtPhCbl induces biochemical evidence of Cbl deficiency. This may in part be caused by a compromised tissue accumulation of Cbl.

Vitamin B12 dependent changes in mouse spinal cord expression of vitamin B12 related proteins and the epidermal growth factor system.

Chronic vitamin B12 (cobalamin) deficiency in the mammalian central nervous system causes degenerative damage, especially in the spinal cord. Previous studies have shown that cobalamin status alters spinal cord expression of epidermal growth factor (EGF) and its receptor in rats. Employing a mouse model of cobalamin-depletion and loading, we have explored the influence of Cbl status on spinal cord expression of cobalamin related proteins, as well as all four known EGF receptors and their activating ligands. Following four weeks of osmotic minipump infusion (n=7 in each group) with cobinamide (4.25nmol/h), saline or cobalamin (1.75nmol/h) the spinal cords were analyzed for cobalamin and for the mRNA levels of cobalamin related proteins and members of the EGF system using quantitative reverse transcription PCR. The median spinal cord cobalamin content was 17, 32, and 52pmol/gr of tissues in cobinamide, saline, and cobalamin treated animals, respectively. Both cobinamide and cobalamin induced a significant decrease in the expression of the lysosomal membrane cobalamin transporter. All four EGF receptors and their activating ligands, except for EGF, were expressed in the spinal cord. Notably, the expression of one of the EGF receptors, HER3, and the ligands heparin-binding EGF-like growth factor, transforming growth factor-α, and neuregulins 1α was increased in cobalamin treated mice. Our studies show that four weeks treatment of mice with cobinamide induces spinal cord cobalamin depletion and that cobalamin loading induces an altered expression pattern of the EGF system thus confirming a spinal cord cross talk between Cbl and the EGF system.

Maximal load of the vitamin B12 transport system: a study on mice treated for four weeks with high-dose vitamin B12 or cobinamide.

Several studies suggest that the vitamin B12 (B12) transport system can be used for the cellular delivery of B12-conjugated drugs, also in long-term treatment Whether this strategy will affect the endogenous metabolism of B12 is not known. To study the effect of treatment with excess B12 or an inert derivative, we established a mouse model using implanted osmotic minipumps to deliver saline, cobinamide (Cbi) (4.25 nmol/h), or B12 (1.75 nmol/h) for 27 days (n = 7 in each group). B12 content and markers of B12 metabolism were analysed in plasma, urine, kidney, liver, and salivary glands. Both Cbi and B12 treatment saturated the transcobalamin protein in mouse plasma. Cbi decreased the content of B12 in tissues to 33-50% of the level in control animals but did not influence any of the markers examined. B12 treatment increased the tissue B12 level up to 350%. In addition, the transcript levels for methylenetetrahydrofolate reductase in kidneys and for transcobalamin and transcobalamin receptor in the salivary glands were reduced. Our study confirms the feasibility of delivering drugs through the B12 transport system but emphasises that B12 status should be monitored because there is a risk of decreasing the transport of endogenous B12. This risk may lead to B12 deficiency during prolonged treatment.

Mast cell infiltration discriminates between histopathological phenotypes of chronic obstructive pulmonary disease.

RATIONALE: COPD is a complex disease with heterogeneous manifestations. Attempts have been made to define different phenotypes that could guide toward better disease understanding. We described before that smokers can develop either panlobular (PLE) or centrilobular emphysema (CLE). The latter has worse small airways remodeling and narrowing, which account for the airflow obstruction similar to asthma. OBJECTIVES: Because of the small airways involvement in CLE similar to asthma, we hypothesized a role for mast cells in CLE but not in PLE. Hence, we investigated mast cell infiltration, along with overall inflammation, and their relation with hyperreactivity and emphysema type in COPD. METHODS: We studied lung function, emphysema type, mast cells, and overall inflammation in small airways and alveolar walls, along with alveolar wall thickening in 67 subjects undergoing lung resection (59 smokers, 8 nonsmokers). MEASUREMENTS AND MAIN RESULTS: Twenty-seven smokers had CLE, 24 had PLE, and 8 had no emphysema. Mast cells were significantly increased in CLE compared with PLE and control subjects. Especially relevant was the mast cell increase in airway smooth muscle in CLE, which related significantly to airway hyperreactivity. CD4(+)T cells, neutrophils, and macrophages, but not eosinophils and CD8(+)T cells, were significantly higher in CLE than PLE. Alveolar wall thickness was increased in all smokers, but significantly more in CLE. CONCLUSIONS: The pathological phenotypes of COPD CLE and PLE show important differences in their overall inflammation with a protagonism of mast cells, which are related to airway reactivity. These findings highlight the distinctness of these COPD phenotypes and the role of mast cells in the pathophysiology of COPD.

Cobalamin (vitamin B(12)) regulation of PrP(C), PrP(C)-mRNA and copper levels in rat central nervous system.

The pathogenesis of cobalamin (Cbl)-deficient (Cbl-D) neuropathy is not clear, nor is the role of prions (PrP(C)) in myelin maintenance. However, as it is known that Cbl deficiency damages myelin by increasing tumor necrosis factor (TNF)-α and decreasing epidermal growth factor (EGF) levels in rat spinal cord (SC), and that TNF-α and EGF regulate PrP(C) expression in vitro, we investigated whether Cbl deficiency modifies SC PrP(C) and PrP(C)-mRNA levels in Cbl-D rats. PrP(C) levels had increased by the time myelin lesions appeared. This increase was mediated by excess myelinotoxic TNF-α and prevented by EGF, which proved to be as effective as Cbl in preventing Cbl deficiency-induced lesions. There were no significant changes in hepatic PrP(C) levels of Cbl-D rats. Anti-octapeptide repeat (OR) region antibodies normalized SC myelin morphology. Cbl deficiency greatly reduced SC PrP(C)-mRNA levels, which were subsequently increased by Cbl and EGF. Cbl deficiency-induced excess OR is myelin-damaging, but new PrP(C) synthesis is a common effect of different myelinotrophic agents.

The octapeptide repeat PrP(C) region and cobalamin-deficient polyneuropathy of the rat.

INTRODUCTION: Cobalamin (Cbl) deficiency affects the peripheral nervous system (PNS) morphologically and functionally. We investigated whether the octapeptide repeat (OR) region of prion protein (PrP(C)) (which is claimed to have myelinotrophic properties) is involved in the pathogenesis of rat Cbl-deficient (Cbl-D) polyneuropathy. METHODS: We intracerebroventricularly administered antibodies (Abs) against the OR region (OR-Abs) to Cbl-D rats to prevent myelin damage and maximum nerve conduction velocity (MNCV) abnormalities, and PrP(C)s to normal rats to reproduce PNS Cbl-D-like lesions. We measured nerve PrP(C) levels and MNCV. RESULTS: The OR-Abs normalized myelin ultrastructure, MNCV values, and tumor necrosis factor (TNF)-α levels in the sciatic and tibial nerves of Cbl-D rats. PrP(C) levels increased in Cbl-D nerves. The nerves of the PrP(C)-treated rats showed typical Cbl-D lesions, significantly decreased MNCV values, and significantly increased TNF-α levels. CONCLUSIONS: OR-Abs prevent the myelin damage caused by increased OR regions, and excess TNF-α is involved in the pathogenesis of Cbl-D polyneuropathy.

Cobalamin deficiency-induced changes of epidermal growth factor (EGF)-receptor expression and EGF levels in rat spinal cord.

We investigated the effect of cobalamin (Cbl) deficiency on epidermal growth factor receptor (EGFR) mRNA levels in the spinal cord (SC) and liver of rats made Cbl-deficient (Cbl-D) by means of total gastrectomy or a Cbl-D diet, and simultaneously measured the levels of the epidermal growth factor (EGF). Both methods of inducing Cbl deficiency decreased EGFR expression in the SC and liver. Cbl replacement treatment normalized or nearly so most of the abnormalities in EGFR expression in the totally gastrectomized (TGX) rats at different times. The EGFR-immunostaining intensity decreased in the SC white matter of the Cbl-D rats and significantly increased in that of the TGX, Cbl-treated rats. EGF levels significantly increased in liver of TGX rats and in SC of 4-month TGX rats, and the increases returned to almost normal levels after a postoperative 2-month administration of Cbl to TGX rats. These findings demonstrate that Cbl deficiency dysregulates the EGFR-EGF dyad in these tissues.

Loss of epidermal growth factor regulation by cobalamin in multiple sclerosis.

We investigated whether the physiological regulation of cerebrospinal fluid (CSF) levels of tumor necrosis factor (TNF)-alpha, epidermal growth factor (EGF), and nerve growth factor (NGF) by cobalamin (Cbl) that is observed in rat and human central nervous system (CNS) is retained in the CSF of patients with multiple sclerosis (MS). The study involved 158 MS patients grouped on the basis of the different clinical courses (relapsing-remitting (RR), secondary-progressive (SP), and primary-progressive (PP)), and 76 gender- and age-matched control patients with other non-inflammatory and non-neoplastic neurological diseases. The MS patients were therapy-free at the time of lumbar puncture. CSF Cbl and EGF were blindly measured by means of radioimmunoassays, and CSF TNF-alpha, and NGF by means of highly sensitive enzyme-linked immunosorbent assays. Serum EGF was also measured in 38 of the MS patients and 20 healthy controls. CSF Cbl levels were significantly higher (RR patients 27.9+/-9.7 pg/ml, p<0.0001 vs. C; SP patients 25.4+/-8 pg/ml, p<0.02 vs. C), and CSF TNF-alpha and EGF levels significantly lower in the patients with the RR (TNF-alpha 28.3+/-23.4 x 10(-3) pg/ml, p<0.0001 vs. C; EGF 129.9+/-44.8 pg/ml, p<0.02 vs. C) or SP (TNF-alpha 20.5+/-20.5 x 10(-3) pg/ml, p<0.001 vs. C; EGF 116.5+/-24.8 pg/ml, p<0.05 vs. C) clinical course than in controls (Cbl 21+/-4.6 pg/ml; TNF-alpha 75.6+/-34.7 x 10(-3) pg/ml; EGF 170.2+/-54.8 pg/ml). There were no differences in CSF NGF or serum EGF levels between any of the MS clinical courses and controls. Our results indicate that: (a) the positive Cbl-mediated regulation of myelino- and oligodendrocyte-trophic EGF is lost in the CSF of RR- or SP-MS patients; (b) the decrease in EGF levels in the CSF may be one factor impeding CNS remyelination in MS; and (c) the PP clinical course may have different pathogenetic mechanism(s) also on the basis of the molecules investigated in this study.

Cobalamin deficiency-induced changes in magnetic resonance imaging of cerebrospinal fluid volume in the cervical tract in the rat.

We wanted to verify the magnetic resonance imaging (MRI) abnormalities that occur in the central nervous system (CNS) of cobalamin-deficient (Cbl-D) rats. The rats were made Cbl-D by means of total gastrectomy or feeding a Cbl-D diet. MR images of the cervical tract of the vertebral canal were recorded using a vertical spectrometer, and the volume of cerebrospinal fluid (CSF) in this part of the vertebral canal was calculated. The findings of the present study demonstrate that: (i) there was a significant decrease in cervical tract CSF volume regardless of the way in which the vitamin deficiency was induced; (ii) this volume normalized in the totally gastrectomized rats after chronic Cbl treatment; (iii) no blood-brain or blood-CSF barrier lesions were found in Cbl-D rats, using either MRI with a paramagnetic contrast agent or calculating the albumin CSF/serum concentration quotient. Cbl deficiency decreases CSF volume in the cervical tract of the vertebral canal of the rat, without apparently impairing the blood-brain barrier.

Experimental and clinical evidence of the role of cytokines and growth factors in the pathogenesis of acquired cobalamin-deficient leukoneuropathy.

Our experimental and clinical studies have highlighted the non-coenzyme functions of cobalamin (Cbl; vitamin B12). The neuropathy of the rat central nervous system (CNS) due to Cbl deficiency is associated with increases in CNS tissue and/or cerebrospinal fluid (CSF) levels of some neurotoxic molecules, and decreases in local and/or CSF levels of some neurotrophic molecules. The increased molecules are nerve growth factor (NGF), tumor necrosis factor (TNF)-alpha, and the soluble (s)CD40:sCD40 Ligand dyad; the decreased molecules are epidermal growth factor (EGF) and interleukin-6. The morphological lesions of the CNS white matter in Cbl-deficient (Cbl-Df) rats can be prevented to the same extent by treatments replacing Cbl or the deficient neurotrophic molecules, or treatment with agents that antagonize the excess neurotoxic molecules. Patients with neurological and/or hematological manifestations of severe Cbl deficiency also have high TNF-alpha levels and low EGF levels in CSF and serum. Cbl replacement treatment corrects cytokine and growth factor abnormalities in Cbl-Df patients and Cbl-Df rats, and so Cbl-Df CNS neuropathy is also due to an imbalance in local cytokine/growth factor networks. TNF-alpha and NGF levels are also increased in Cbl-Df rat liver, which is morphologically unaffected by Cbl deficiency. The increases in TNF-alpha and NGF levels increase nuclear factor-kappaB activity levels in both the CNS and liver, and this indirect regulation supports the idea that Cbl may modulate the expression of some cytokine/growth factor genes in rat CNS and other tissues. Finally, we have tried to harmonize our pathogenetic theory of cytokine and growth factor dysregulation with the biochemical interpretation.

Indirect down-regulation of nuclear NF-kappaB levels by cobalamin in the spinal cord and liver of the rat.

We used electrophoretic mobility shift assays to investigate the effects of cobalamin (Cbl) deficiency on the levels of activated nuclear factor-kappa B (NF-kappaB) in the spinal cords (SCs) and livers of rats made Cbl-deficient (Cbl-D) by total gastrectomy or a Cbl-D diet. We chose the SC and liver because they are severely or scarcely affected, respectively, by Cbl deficiency in terms of histological damage. We found permanently increased NF-kappaB levels (particularly the p50 and p65 subunits) in the SCs and livers of both types of Cbl-D rats, and Western blot analysis demonstrated increased p65 levels. NF-kappaB and p65 protein levels normalized when the totally gastrectomized (TGX) rats were treated with Cbl replacement. As we have previously demonstrated that Cbl deficiency increases tumor necrosis factor (TNF)-alpha and nerve growth factor (NGF) levels in the SC (each of which is a known NF-kappaB activator), we redetermined NF-kappaB levels in the SCs and livers of TGX rats treated with anti-TNF-alpha or anti-NGF antibodies and found that NF-kappaB levels normalized in both tissues after either treatment. These results demonstrate that: (1) Cbl physiologically and indirectly down-regulates NF-kappaB levels in rat SC and liver, and (2) NF-kappaB is an important signaling molecule after Cbl deficiency injury.

Cobalamin deficiency-induced down-regulation of p75-immunoreactive cell levels in rat central nervous system.

We investigated immunoreactivity for p75 neurotrophin receptor (NTR) in the spinal cord white matter and septum of rats made cobalamin-deficient (Cbl-D) by means of total gastrectomy or a Cbl-D diet. Cbl deficiency down-regulates p75NTR-immunoreactive cell levels in spinal cord white matter and septum with different time courses. On the whole, the spinal cord white matter seems to be more affected in terms of p75NTR-immunoreactive cells, most of which are astrocytes. The p75NTR-immunoreactive cell levels in the spinal cord white matter and septum normalized in rats treated with Cbl (scheme b) and killed 4 months after total gastrectomy. However, Western blot analysis of p75NTR in the spinal cords of Cbl-D rats shows increased p75NTR protein levels, which are resistant to Cbl replacement. These findings demonstrate that a neurotrophic vitamin (Cbl) positively regulates the levels of a neurotrophic receptor (p75NTR) (at least in terms of immunohistochemistry) in rat central nervous system, although the underlying mechanism(s) are still unknown.

Cobalamin-mediated regulation of transcobalamin receptor levels in rat organs.

Total gastrectomy (TG) causes cobalamin (Cbl) deficiency followed by increases in tumor necrosis factor (TNF)-alpha levels in the spinal cord (SC) of the rat. In order to understand how Cbl deficiency may influence cell Cbl transport, we have measured by immunoblotting protein levels of the receptor for the Cbl-transcobalamin (TC) complex (TC-R) in both animal and cell models. TC-R protein levels were elevated in the total membranes of duodenal mucosa, kidneys, liver, and SC of rats made Cbl-deficient (Cbl-D) by means of TG or feeding with a Cbl-D diet. Postoperative Cbl-replacement treatment normalized the TC-R protein levels in each of the tested organs, regardless of whether this treatment was given during the first two post-TG or during the third and fourth post-TG mo. In Caco-2 cells, progressively increasing TNF-alpha concentrations supplemented to culture medium induced an up-regulation of TC-R protein levels. We provide the first evidence of the regulation of a Cbl-specific receptor by the vitamin itself in some rat organs.

New pathogenesis of the cobalamin-deficient neuropathy.

Subacute combined degeneration (SCD) is considered the neurological counterpart of pernicious anaemia because it is the paradigmatic neurological manifestation of acquired vitamin B12 (cobalamin (Cbl)) deficiency in adulthood. Hitherto, the theories advanced to explain the pathogenesis of SCD have postulated a causal relationship between SCD lesions and the impairment of either or both of two Cbl-dependent reactions. We have identified a new experimental model, the totally gastrectomised (TGX) rat, to reproduce the key morphological features of the disease, and found new mechanisms responsible for the pathogenesis of SCD. We have demonstrated that the neuropathological lesions in TGX rats are not only due to mere vitamin withdrawal but also to the overproduction of the myelinolytic tumour necrosis factor (TNF)-alpha, nerve growth factor, the soluble(s) CD40:sCD40 ligand dyad, and the reduced synthesis of the neurotrophic agents, epidermal growth factor and interleukin-6. Cbl replacement treatments normalised all of these abnormalities.

Increased levels of the CD40:CD40 ligand dyad in the cerebrospinal fluid of rats with vitamin B12(cobalamin)-deficient central neuropathy.

The levels of the soluble (s) CD40:sCD40 ligand (L) dyad, which belongs to the tumor necrosis factor (TNF)-alpha:TNF-alpha-receptor superfamily, are significantly increased in the cerebrospinal fluid (CSF), but not the serum of cobalamin (Cbl)-deficient (Cbl-D) rats. They were normalized or significantly reduced after treatment with Cbl, transforming growth factor-beta1 or S-adenosyl-L-methionine, and the normal myelin ultrastructure of the spinal cord was concomitantly restored. The concomitance of the two beneficial effects of these treatments strongly suggests that the increases in CSF sCD40:sCD40L levels may participate in the pathogenesis of purely myelinolytic Cbl-D central neuropathy in the rat. In keeping with this, an anti-CD40 treatment prevented myelin lesions.

Increased spinal cord NGF levels in rats with cobalamin (vitamin B12) deficiency.

We have recently demonstrated that the neuropathological morphological alterations caused by cobalamin (Cbl) deficiency in the rat central nervous system are related to the vitamin's inability to modulate the synthesis of some neurotoxic and neurotrophic agents in opposite directions. In the present study, we measured nerve growth factor (NGF) levels in the spinal cord (SC) and cerebrospinal fluid (CSF) of rats made Cbl-deficient (Cbl-D) by means of total gastrectomy (TG) or a Cbl-D diet. In both cases, Cbl deficiency increased SC and CSF NGF levels after the appearance of myelinolytic lesions in the SC white matter (SCWM) (i.e. after the second post-TG month), and these changes were normalised by Cbl treatment in the 4-month-totally-gastrectomised (TGX) rats. Intracerebroventricular (i.c.v.) anti-NGF-antibody treatment prevented the onset of the myelinolytic SCWM lesions in the 2-month-TGX rats (i.e. when SC and CSF NGF levels are still normal) and normalised the ultrastructure of the SCWM in the 4-month-TGX rats, which was however worsened by the i.c.v. administration of NGF. These findings demonstrate that: (i) Cbl deficiency increases SC and CSF NGF levels; and (ii) endogenous NGF seems to play a noxious role in the progression of rat Cbl-D central neuropathy.

New basis of the neurotrophic action of vitamin B12.

Over the last few years we have reproduced all of the key morphological and biochemical features of human subacute combined degeneration in the central nervous system and peripheral nervous system of rats made cobalamin-deficient by means of total gastrectomy or a chronic cobalamin-deficient diet. We have also recently clarified the pathogenesis of experimental subacute combined degeneration induced in the rat by cobalamin deprivation. The results of our studies strongly support the notion that cobalamin plays a pivotal role in regulating the balance of the network of cytokines and growth factors in the central nervous system of the rat. We have demonstrated that cobalamin tightly regulates the central nervous system synthesis and/or the cerebrospinal fluid level of two cytokines, tumor necrosis factor-alpha and interleukin-6, and a growth factor, epidermal growth factor. Of these neuroactive agents, one, tumor necrosis factor-alpha, is neurotoxic, whereas the others are neurotrophic. Therefore, it becomes clear that cobalamin-deficient central neuropathy is caused not by the withdrawal of the vitamin, but reflects a locally increased production of neurotoxic agents, combined with the locally decreased production of neurotrophic agents.

Cobalamin (vitamin B12)-deficiency-induced changes in the proteome of rat cerebrospinal fluid.

We studied the changes in the proteome of CSF (cerebrospinal fluid) in two animal models of Cbl (cobalamin) deficiency: TGX (totally gastrectomized) rats and rats fed a Cbl-D (Cbl-deficient) diet. Two-dimensional PAGE was used to detect qualitative and quantitative variations in proteins in the CSF samples. The peak increase in total CSF protein concentration was observed 4 months after TG (total gastrectomy) and after 6 months of eating a Cbl-D diet. There is a specific increase 4 months after TG in the spots corresponding to alpha1-antitrypsin and the de novo presence of thiostatin and haptoglobin beta. Cbl-replacement treatment in 4-month-TGX rats corrected these alterations in the CSF proteome. However, most of the CSF proteome alterations attenuated in Cbl-untreated 8-month-TGX rats and in rats fed a Cbl-D diet for 16 months. Transthyretin concentration varied slightly in the CSF of both types of Cbl-D rat, whereas the relative abundance of prostaglandin D synthase rose sharply in the CSF of the rats fed a Cbl-D diet for 16 months. We have demonstrated previously that the histological and ultrastructural CNS (central nervous system) damage in both types of Cbl-D rat appears within 2-3 months of Cbl deficiency, and thus appears to precede the alterations in the CSF proteome. The CSF proteome patterns of rats in which phlogosis was induced in or outside the CNS are quite different from those of the CSF of Cbl-D rats. All these findings demonstrate that the alterations in the CSF proteome of Cbl-D rats are specifically linked to Cbl deficiency.