TET family dioxygenases and the TET activator vitamin C in immune responses and cancer.

Vitamin C serves as a cofactor for Fe(II) and 2-oxoglutarate-dependent dioxygenases including TET family enzymes, which catalyze the oxidation of 5-methylcytosine into 5-hydroxymethylcytosine and further oxidize methylcytosines. Loss-of-function mutations in epigenetic regulators such as TET genes are prevalent in hematopoietic malignancies. Vitamin C deficiency is frequently observed in cancer patients. In this review, we discuss the role of vitamin C and TET proteins in cancer, with a focus on hematopoietic malignancies, T regulatory cells, and other immune system cells.

Vitamin D in pediatric health and disease.

Several scientific societies established that vitamin D (VD), in its metabolized form 25(OH)D, levels higher than 20 ng/mL are sufficient to ensure optimal bone health, while 25(OH)D levels higher than 30 ng/mL are needed to favor VD extraskeletal actions. However, it has been estimated that approximately 30% of children and 60% of adults worldwide are VD deficient and insufficient, respectively. This is the reason why it is important to provide a practical approach to VD supplementation for infants, children, and adolescents. It is the pediatrician's role to evaluate the modifiable lifestyle risk factors for deficiency, particularly a reduced sun exposure, following an evidence-based approach, and to suggest VD supplementation only when there is a rational reason to support its use.

Ascorbic acid deficiency in patients with lichen planus.

OBJECTIVES: Recent studies have highlighted the role of oxidative stress in the pathogenesis of lichen planus (LP). In the present study, the interest of the authors is focused on the investigation of ascorbic acid status in patients with LP and identification of parameters that might influence the level of this vitamin. MATERIAL AND METHOD: We analyzed the level of urinary ascorbic acid (reflectometric method) in 77 patients with LP (cutaneous LP (CLP)-49 cases; oral LP (OLP)-28 cases) and 50 control subjects. The evaluation of all participants included clinical examination and laboratory and imaging tests. RESULTS: Compared to the control group (19.82 mg/dl) the level of ascorbic acid was significantly lower both in patients with CLP (8.47 mg/dl, p = 0.001) and in those with OLP (8.04 mg/dl, p = 0.001). In patients with LP it was found that the deficiency of ascorbic acid increases with age (r = -0.318, p = 0.032). The urinary concentrations of ascorbic acid were significantly lower in patients with LP associated with infections compared to patients with LP without infections. CONCLUSIONS: The urinary ascorbic acid level may be a useful parameter in identifying patients with LP who are at risk of developing viral or bacterial infections.

Dietary vitamin C deficiency depressed the gill physical barriers and immune barriers referring to Nrf2, apoptosis, MLCK, NF-κB and TOR signaling in grass carp (Ctenopharyngodon idella) under infection of Flavobacterium columnare.

This study explored the effects of vitamin C on the physical barriers and immune barriers, and relative mRNA levels of signaling molecules in the gill of grass carp (Ctenopharyngodon idella) under infection of Flavobacterium columnare. The results indicated that compared with optimal vitamin C supplementation, vitamin C deficiency (2.9 mg/kg diet) (1) increased reactive oxygen species, malondialdehyde and protein carbonyl (PC) contents (P < 0.05), decreased the copper/zinc superoxide dismutase, manganese superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase activities and mRNA levels (P < 0.05), and glutathione and vitamin C contents (P < 0.05), down-regulated NF-E2-related factor 2 mRNA level (P < 0.05), and up-regulated Kelch-like ECH-associating protein (Keap) 1a (rather than Keap1b) mRNA level (P < 0.05) in the gill of grass carp under infection of F. columnare, suggesting that vitamin C deficiency induced oxidative injury in fish gill; (2) up-regulated caspase-3, -7, -8, -9, Fas ligand, B-cell lymphoma protein 2 associated X protein, apoptotic protease activating factor-1 mRNA levels (P < 0.05), and down-regulated inhibitor of apoptosis protein and B-cell lymphoma-2 (rather than myeloid cell leukemia-1) mRNA level (P < 0.05) in the gill of grass carp under infection of F. columnare, suggesting that vitamin C deficiency aggravated cell apoptosis in fish gill; (3) up-regulated pore-forming TJs Claudin-12, 15a, -15b, and related signaling molecules myosin light chain kinase, p38 mitogen-activated protein kinase (rather than c-Jun N-terminal kinases) mRNA levels (P < 0.05), and down-regulated barrier-forming TJs Occludin, zonula occludens (ZO) 1, ZO-2, Claudin-c, -3c, -7a, -7b mRNA levels (P < 0.05) in the gill of grass carp under infection of F. columnare, suggesting that vitamin C deficiency disrupted tight junctional complexes in fish gill; (4) decreased lysozyme and acid phosphatase (ACP) activities, and complement 3 (C3), C4 and IgM contents (P < 0.05), down-regulated the mRNA levels of antimicrobial peptides liver expressed antimicrobial peptide (LEAP) 2A, LEAP-2B, Hepcidin, ß-defensin mRNA levels (P < 0.05) in the gill of grass carp under infection of F. columnare, suggesting that vitamin C deficiency decrease fish gill immune function; (5) down-regulated the mRNA levels of anti-inflammatory cytokines-related factors interleukin 10 (IL-10), IL-11, transforming growth factor (TGF) ß1, TGF-ß2, inhibitor of κBa and eIF4E-binding protein 1 (4E-BP1) (rather than 4E-BP2) (P < 0.05), and up-regulated pro-inflammatory cytokines-related factors interferon γ2, IL-1ß, IL-6, IL-8, IL-12 P35, IL-12 P40, nuclear factor κB (NF-κB) p65 (rather than NF-κB p52), IκB kinases (IKK) (only IKKα and IKKγ), target of rapamycin and ribosomal protein S6 kinase 1 mRNA levels (P < 0.05) in the gill of grass carp under infection of F. columnare, suggesting that vitamin C deficiency aggravated fish gill inflammation. In conclusion, vitamin C deficiency disrupted physical barriers and immune barriers, and regulated relative mRNA levels of signaling molecules in fish gill. The vitamin C requirement for against gill rot morbidity of grass carp (264-1031 g) was estimated to be 156.0 mg/kg diet. In addition, based on the gill biochemical indices (antioxidant indices MDA, PC and vitamin C contents, and immune indices LA and ACP activity) the vitamin C requirements for grass carp (264-1031 g) were estimated to be 116.8, 156.6, 110.8, 57.8 and 134.9 mg/kg diet, respectively.

Suppressive effects of vitamin C-treated induced-regulatory T cells on heart allograft rejection under vitamin C-deficient or -sufficient conditions.

Foxp3 stability of vitamin C-treated induced-regulatory T cells (V-iTregs) is superior to that of conventional iTregs (C-iTregs). However, the role of V-iTregs in allograft rejection under vitamin C-deficient conditions, such as those seen in humans, remains unclear. We aimed to elucidate the role of vitamin C treatment on generation and maintenance of iTregs from gulo knockout (Gulo-KO) mice as well as wild type (WT) mice, and in vitro and in vivo suppressive effects of V-iTregs on heart allograft rejection in either Gulo-KO or WT recipient mice. Conversion efficiency of iTregs was similar between C- and V-iTregs in both WT and Gulo-KO mice. V-iTregs from WT or Gulo-KO mice showed better in vitro Foxp3 stability than C-iTregs, although there was no difference between WT V-iTregs and Gulo-KO V-iTregs. Furthermore, V-iTregs from WT or Gulo-KO mice suppressed in vitro T cell proliferation better than C-iTregs. Heterotrophic heart transplantation from BALB/c mice to WT or vitamin C-deficient Gulo-KO C57BL/6J mice was performed following adoptive transfer of C- or V-iTregs. V-iTregs as well as C-iTregs prolonged heart allograft survival in WT and Gulo-KO mice. However, there was no difference between the C- and V-iTreg groups. Supplementation of low- or high-dose vitamin C did not induce significant changes in heart allograft survival in Gulo-KO recipients that had received V-iTregs. In conclusion, V-iTregs do not exert better suppressive effects on heart allograft survival than C-iTregs in either WT or vitamin C-deficient recipients.

Ascorbate sustains neutrophil NOS expression, catalysis, and oxidative burst.

Previous studies from this lab have demonstrated that in vitro ascorbate augments neutrophil nitric oxide (NO) generation and oxidative burst. The present study was therefore undertaken in guinea pigs to further assess the implication of ascorbate deficiency in vivo on neutrophil ascorbate and tetrahydrobiopterin content, NOS expression/activity, phagocytosis, and respiratory burst. Ascorbate deficiency significantly reduced ascorbate and tetrahydrobiopterin amounts, NOS expression/activity, and NO as well as free radical generation in neutrophils from scorbutics. Ascorbate and tetrahydrobiopterin supplementation in vitro, though, significantly enhanced NOS catalysis in neutrophil lysates and NO generation in live cells, but could not restore them to control levels. Although phagocytic activity remained unaffected, scorbutic neutrophils were compromised in free radical generation. Ascorbate-induced free radical generation was NO dependent and prevented by NOS and NADPH oxidase inhibitors. Augmentation of oxidative burst with dehydroascorbate (DHA) was counteracted in the presence of glucose (DHA uptake inhibitor) and iodoacetamide (glutaredoxin inhibitor), suggesting the importance of ascorbate recycling in neutrophils. Ascorbate uptake was, however, unaffected among scorbutic neutrophils. These observations thus convincingly demonstrate a novel role for ascorbate in augmenting both NOS expression and activity in vivo, thereby reinforcing oxidative microbicidal actions of neutrophils.

Ascorbate deficiency results in impaired neutrophil apoptosis and clearance and is associated with up-regulation of hypoxia-inducible factor 1alpha.

Some cells, including neutrophils, accumulate high intracellular ascorbate concentrations, which suggests that they have an important function in these cells. In this study we have used L-gulono-gamma-lactone oxidase (Gulo)-/- mice, which are unable to synthesize ascorbate, to generate ascorbate-deficient neutrophils and have used these to investigate the effect of ascorbate on neutrophil function. Peritoneal neutrophils from ascorbate-deficient animals had normal morphology and respiratory burst activity but failed to undergo spontaneous apoptosis, determined by morphology and the surface expression of phosphatidylserine. Initially, there was increased cell survival, but death eventually occurred by necrosis within 48 h. Neutrophils persisted in thioglycollate-induced inflammation in Gulo-/- mice with the later appearance of necrotic cells, suggesting that apoptosis was also affected in vivo. Also, ascorbate-deficient neutrophils were not recognized by macrophages in an in vitro assay for phagocytosis, providing further evidence for defective apoptosis and clearance. Neutrophils from Gulo-/- mice had elevated levels of hypoxia-inducible factor (HIF)-1alpha, a transcription factor regulated by Fe2+-dependent hydroxylases which require ascorbate for optimal activity. HIF-1alpha has been shown previously to inhibit neutrophil apoptosis under hypoxic conditions. Our results suggest that in ascorbate deficiency, up-regulation of HIF-1alpha blocks neutrophil apoptosis under normoxic conditions and that this represents a novel and important function for vitamin C in inflammatory cells.

Vitamin C and Immune Function.

Vitamin C is an essential micronutrient for humans, with pleiotropic functions related to its ability to donate electrons. It is a potent antioxidant and a cofactor for a family of biosynthetic and gene regulatory enzymes. Vitamin C contributes to immune defense by supporting various cellular functions of both the innate and adaptive immune system. Vitamin C supports epithelial barrier function against pathogens and promotes the oxidant scavenging activity of the skin, thereby potentially protecting against environmental oxidative stress. Vitamin C accumulates in phagocytic cells, such as neutrophils, and can enhance chemotaxis, phagocytosis, generation of reactive oxygen species, and ultimately microbial killing. It is also needed for apoptosis and clearance of the spent neutrophils from sites of infection by macrophages, thereby decreasing necrosis/NETosis and potential tissue damage. The role of vitamin C in lymphocytes is less clear, but it has been shown to enhance differentiation and proliferation of B- and T-cells, likely due to its gene regulating effects. Vitamin C deficiency results in impaired immunity and higher susceptibility to infections. In turn, infections significantly impact on vitamin C levels due to enhanced inflammation and metabolic requirements. Furthermore, supplementation with vitamin C appears to be able to both prevent and treat respiratory and systemic infections. Prophylactic prevention of infection requires dietary vitamin C intakes that provide at least adequate, if not saturating plasma levels (i.e., 100-200 mg/day), which optimize cell and tissue levels. In contrast, treatment of established infections requires significantly higher (gram) doses of the vitamin to compensate for the increased inflammatory response and metabolic demand.

Immunocompetence and oxidant defense during ascorbate depletion of healthy men.

To determine nonscorbutic effects of moderate vitamin C deficiency we measured immune function and oxidative damage in eight healthy men (25-43 y) who consumed 5-250 mg/d of ascorbic acid over 92 d on a metabolic unit. During ascorbic acid intakes of 5, 10, or 20 mg/d, subjects attained a state of moderate ascorbic acid deficiency as ascorbic acid concentrations in plasma, leucocytes, semen, and buccal cells dropped to less than 50% of baseline with no scorbutic symptoms observed. No changes in cell proliferation, erythrocyte antioxidant enzymes, and DNA strand breaks were observed; however, blood levels of glutathione and NAD(P) decreased during ascorbic acid deficiency, as did delayed hypersensitivity responsiveness. Concentrations of the oxidatively modified DNA base, 8-hydroxydeoxyguanosine in sperm DNA and fecapentaenes, ubiquitous fecal mutagens, were increased during ascorbic acid depletion. Moderate vitamin C deficiency, in the absence of scurvy, results in alteration of antioxidant chemistries and may permit increased oxidative damage.

Cell-mediated cytotoxicity and humoral immune response in ascorbic acid-deficient guinea pigs.

Guinea pigs were divided into three dietary groups: ascorbic-acid deficient, pair-fed, and ad libitum control. Two weeks later guinea pigs were immunized intradermally with 5 x 10(8) chicken erythrocytes in Freund's complete adjuvant. Hemagglutinating antibody titers to chicken erythrocytes 2 weeks after immunization were comparable in all three dietary groups. In vitro 51Cr release from labeled chicken erythrocyte target cells incubated with lymphoid cells from spleens of ascorbic acid-deficient guinea pigs was significantly less than with spleen cells from pair-fed and ad libitum control guinea pigs. The percentage of splenic lymphoid cells that formed rosettes with rabbit erythrocytes, a T cell marker, was the same in all three dietary groups. The defect of ascorbic acid deficiency may reflect an impairment of T lymphocytes function in cell-mediated cytotoxicity or a change in number or function of another cell type.

Human T-cell function in experimental ascorbic acid deficiency and spontaneous scurvy.

Studies in animal models suggest that ascorbic deficiency impairs T-cell-mediated immunity. We studied five normal volunteers hospitalized on a metabolic unit and consuming a strictly controlled diet deficient in ascorbic acid I) after a 5-wk control period of ascorbic acid supplementation (75 mg/day) and 2) after a 9-wk period of no supplementation. Three of the subjects were restudied after a 5-wk period of ascorbic acid supplementation after the deficient period. At the end of both control periods ascorbic acid levels in plasma ranged from 0.9 to 1.3 mg/dl and in leukocytes from 19 to 30 microgram/10(8) cells. At the end of the deficient period levels of ascorbic acid in plasma ranged from 0.09 to 0.15 mg/dl and in leukocytes from 6.2 to 10 microgram/10(8) cells, levels at or below those frequently found in frank scurvy. None of the T-cell parameters tested including mitogen responsiveness to phytohemagglutinin and percentage of T-cells bearing receptors for IgM (helper cells) and IgG (suppressor cells) was different in the deficient period compared to the control periods. One patient with spontaneous scurvy (plasma ascorbic acid 0.07 mg/dl, leukocytic ascorbic acid 4.9 microgram/10(8) cells) was studied at the time of admission and after vigorous ascorbic acid repletion. All T-cell parameters after repletion were unchanged from admission. We conclude that in man ascorbic acid deficiency, even at the scorbutic level, does not alter T-cell numbers or impair in vitro T-cell function.

Macrophage function in vitamin C-deficient guinea pigs.

Guinea pigs were fed a vitamin C-deficient diet and at various time periods thereafter their peritoneal cells were tested for biological activity. The serum levels of vitamin C in the deficient animals indicated a progressive state of ascorbic acid deficiency with time and this correlated well with clinical signs and symptoms of scurvy. Fewer macrophages were obtained from the peritoneal cavities of deficient animals and in structural appearance under the phase contrast and light microscope they were smaller in size. They showed no significant impairment in phagocytosis of bacterial cells. The macrophages, however, exhibited significantly reduced migration on glass as compared to the normal cells. In vitro addition of vitamin C partially reversed this reduced migration.

Micronutrients and immune function: some recent developments.

Micronutrient deficiencies probably have few direct effects on the functioning of immune cells. The main effect appears to be a reduction in cell mass that may indirectly affect immune cell function, particularly where T helper cell numbers are reduced. Results of many human studies are contradictory. Some of this contradiction may be accounted for by the fact that disease may lower concentrations of micronutrients in plasma that may be misinterpreted as deficiency. Low plasma vitamin A concentrations however appear to impair immune responsiveness and have deleterious effects on membrane integrity and mucosal function. Zinc may have similar effects on gut integrity and appears to be particularly useful in the treatment of acute diarrhoea. Low concentrations of other nutrients such as ascorbate and iron, may not necessarily impair immune function. Low plasma ascorbate may assist the removal of iron from plasma and low iron concentrations appear to increase the cytotoxicity of macrophages.

Modulation of interleukin production by ascorbic acid.

We studied the influence of ascorbate (vitamin C) on peripheral blood mononuclear cells (PBMC) of pigs with hereditary deficiency in ascorbate synthesis. Groups of animals were depleted of, or supplemented with dietary ascorbate for up to 5 weeks. B lymphocytes and T lymphocyte subsets differed in the two experimental groups only marginally and transiently as determined by analysis of cell surface markers. The proliferative response of PBMC to B and T lymphocyte mitogens was lower in depleted as compared to supplemented animals. Interleukin (IL)-2 and IL-6 were determined by bioassays and were secreted within few hours after mitogenic activation of PBMC which contained normal physiological concentrations of ascorbate. IL-2 production peaked at about 24 h of in vitro culture after Con A activation, but it lasted for 2-3 days after PWM activation. The production of IL-2 and IL-6 were compared during systemic depletion and supplementation with ascorbate. Depleted PBMC produced IL-2 which accumulated in cultures instead of being rapidly consumed by IL-2 dependent cell growth. This suggests that cellular ascorbate influences the production of IL-2. Secretion of IL-6 by mitogen activated PBMC was also affected by prolonged dietary ascorbate depletion. The results suggest that ascorbate levels exert an early effect on immune homeostasis via reactive oxygen intermediates (ROI)-dependent expression of interleukin genes, since the transcription factor NF-kappa B is sensitive to ROI and regulates the expression of interleukin genes.

Effect of marginal ascorbic acid deficiency on saliva level of cortisol in the guinea pig.

Male guinea pigs subjected to prolonged marginal ascorbic acid deficiency developed moon facies and oedema, features of functional adrenal hypercorticism. Compared with age- and sex-matched controls fed an adequate diet for a similar period, ascorbate deficiency had no effect on submandibular gland weight but elicited a significant (p < 0.005) reduction in stimulated whole-saliva flow rate. Plasma cortisol concentration (nmol/L) was significantly increased (p < 0.005) in the deficient animals (998.21 +/- 57.19 compared to 254.66 +/- 15.62 for the controls). Associated with marked hypercortisolaemia in the deficient animals was a significant (p < 0.01) but less prominent increase in the whole-saliva cortisol level, resulting in a mean saliva/plasma cortisol ratio of 46% for this group compared to 72% for the controls. Increased corticosteroid levels suppress immunological and inflammatory responses, particularly neutrophil function, impair production of some cytokines, inhibit collagen synthesis, and impair wound healing and bone matrix formation. Numerous conditions such as ageing, stress, smoking, ionizing radiation, ingestion of drugs, protein malnutrition, diabetes, and several other pathological states, which are among the risk factors for xerostomia and periodontal/oral mucosal lesions, promote tissue depletion of ascorbate. This study suggests that increased salivary and blood levels of glucocorticoids in these conditions may be important in reducing the ability of the host to mount an effective immune response to oral pathogens.

The effect of vitamin C deficiency on complement systems and complement components.

The changes in the complement systems and complement hemolytic activities through classical and alternative pathways, and the individual components, C1, C4, C2 and C3 were demonstrated in the course of vitamin C deficiency in guinea pigs. During the course of vitamin C deficiency during the first week after the start of the experiment, all components except C1 slightly decreased, gradually increasing in the following weeks. This indicates that this period is important in the formation of an immune defence system in the host. At the time the symptoms of vitamin C deficiency appeared, C1, C2 and CH50 started to decrease. C3 increased when vitamin C deficiency became severe and showed a completely different pattern from those of the other components. The lowered C1 will be due to a collagen-like region in the characteristic of Clq subcomponent, since insufficient vitamin C state produces impaired collagen formation. The activity of the alternative pathway did not produce any change in this course, even in the severe stage. This indicates that the increase in C3 will contribute to maintaining the level of the alternative pathway and maintaining the body defence system in the vitamin D-deficient state, and that the complement system will be supported through the alternative pathway.

[Ascorbic acid level and the indicators of cellular immunity in patients with hepatitis A during pathogenetic therapy]. / Obespechennost' askorbinovoi kislotoi i pokazateli kletochnogo immuniteta u bol'nykh virusnym gepatitom A v protsesse patogeneticheskoi terapii.

Ascorbic acid content was assayed in plasma and neutrophils of peripheral blood in 260 patients with virus hepatitis A during acute period of the disease. The data obtained have evidenced C-hypovitaminosis in these patients, that was more pronounced in the winter-spring period, according to the disease severity 300 mg of ascorbic acid for 1.5 g of galascorbin/day have proved to be the most optimal dose for vitamin C deficiency elimination. A significant decrease in T-lymphocyte level and an increase in 0-cell content were recorded in the acute period of virus hepatitis A. Ascorbic acid drugs promoted more rapid recovery of T-lymphocyte content in the peripheral blood that has confirmed the necessity of vitamin C administration in combined pathogenetic therapy of patients with virus hepatitis A.

In vivo consequence of vitamin C insufficiency in liver injury: vitamin C ameliorates T-cell-mediated acute liver injury in gulo(-/-) mice.

AIM: l-ascorbic acid (vitamin C) insufficiency is considered one of the major risk factors for the development of liver disease. However, its specific effects and related mechanisms in vivo are largely unknown. The objective of this study was to investigate the in vivo protective role of vitamin C and its related mechanisms in liver injury with Gulo(-/-) mice that cannot synthesize vitamin C like humans due to the lack of l-gulonolactone-γ-oxidase (Gulo), an essential enzyme for vitamin C synthesis. RESULTS: When liver injury was induced in Gulo(-/-) mice by injection of concanavalin A (Con A), there was greater extensive liver damage accompanied by an increased number of apoptotic hepatocytes in vitamin C-insufficient Gulo(-/-) mice. Additionally, the plasma and hepatic levels of the proinflammatory cytokines, such as TNF-α and IFN-γ, were much higher in the vitamin C-insufficient Gulo(-/-) mice than in the control mice. Moreover, increased numbers of liver-infiltrating T-cells in the vitamin C-insufficient Gulo(-/-) mice were related to the increased hepatic levels of IFN-inducible factor (IP-10). Although the vitamin C-insufficient Gulo(-/-) mice had higher amounts of interleukin-22 (IL-22), a hepatoprotective cytokine, a defect in IL-22Rα expression and its downstream STAT3 activation in hepatocytes were found. INNOVATION: We first demonstrate the novel in vivo action mechanisms of vitamin C on the prevention of disease development in the liver, through the regulation of excessive immune activation and maintenance of the IL-22Rα signaling pathways. CONCLUSION: These results suggest that severe liver damage induced by inflammation could be prevented by sufficient supplementation with vitamin C.