Dr. Cícero Galli Coimbra, fundador e Presidente do Instituto de Investigação e Tratamento de Autoimunidade, medico neurologista, Phd., M.D., professor na Universidade Federal de São Paulo

cool-photography-sun-surf-surfing-Favim.com-138479Arquivos em Objeto Dignidade

Dr. Cícero Galli Coimbra, fundador e Presidente do Instituto de Investigação e Tratamento de Autoimunidade, medico neurologista, Phd., M.D., professor na Universidade Federal de São Paulo

 

·       Algumas perguntas sobre o Hormônio-Vitamina D

———-

Como funciona e qual é a relação entre vitamina D e proteção ao câncer

Vitamina D – Sem Censura – Dr. Cicero Galli Coimbra e Daniel Cunha

https://www.youtube.com/watch?v=cIwIWim4hNM&list=PLeqEGmvbpULN2NfNfnLU6bYse4fp9alQS&index=44

Altos níveis do Hormônio-Vitamina D reduzem o risco de 19 tipos de câncer – Dr William Grant

https://www.youtube.com/watch?list=PLeqEGmvbpULN2NfNfnLU6bYse4fp9alQS&v=n0HmGAF8Dr4&feature=player_detailpage

 Vitamina D e Câncer – Considerações Finais – Vitamin D and Cancer – Closing Remarks

http://www.youtube.com/watch?feature=player_detailpage&v=o2fzGJoNBk4

 

Como funciona e qual é a relação entre vitamina D e proteção ao câncer

 

Autor(a):       Rita de Cássia Borges de Castro

Data:             09/11/2012 15:10:00

http://www.nutritotal.com.br/perguntas/?acao=bu&id=695&categoria=16
Como funciona e qual é a relação entre vitamina D e proteção ao câncer?
A vitamina D tem sido associada com a prevenção de várias doenças crônicas, incluindo diversos tipos de câncer, como colorretal, mama, próstata e pele, além da relação com a manutenção da homeostase do cálcio  a saúde óssea.A exposição solar, através dos raios UVB (ultravioleta B), induz na pele a síntese vitamina D pela conversão de 7-deidrocolesterol em vitamina D3 (colecalciferol ou pré-vitamina D3). A vitamina D pode ser obtida pela dieta através do colecalciferol, de origem animal e o ergocalciferol (vitamina D2), de origem vegetal. As principais fontes naturais de vitamina D são peixes como salmão, atum e cavala, seguido de fígado, gema de ovo, queijo e cogumelos. Existem também diversos alimentos fortificados com vitamina D sintética, incluindo leite e produtos lácteos, suco de laranja, cereais matinais, barras de cereais, fórmulas infantis e margarinas.

A vitamina D proveniente da dieta é absorvida no intestino delgado na forma lipossolúvel incorporada a quilomícron. No fígado, esse complexo se liga a uma proteína-ligante de vitamina D e é metabolizado, juntamente com a vitamina D3 sintetizada pela pele. A vitamina D sofre hidroxilação na posição C-25 pela enzima hepática 25-hidroxilase, resultando na formação da 25-hidroxicolecalciferol (25(OH)D3 ou calcidiol), principal forma circulante da vitamina D. Nos rins ocorre uma segunda hidroxilação na posição C-1 do calcidiol através da enzima 1-alfa-hidroxilase (CYP27B1), formando o 1,25(OH)2D3, a forma mais ativa da vitamina D. Os níveis séricos de 25(OH)D é a principal forma circulante de vitamina D, pois sua meia vida é consideravelmente mais longa do que a da 1,25(OH)2D3 (15 dias contra 15 horas).

Diversos mecanismos moleculares têm sido propostos para os efeitos protetores da vitamina D no câncer. Muitos desses mecanismos estão relacionados com a produção de 1,25(OH)2D3 por tecidos que possuem a enzima CYP27B1, como a próstata, cólon, mama e pâncreas. Nas células destes tecidos, a 1,25(OH)2D3 liga-se ao receptor da vitamina D (VDR) em que, no núcleo celular formam um complexo para influenciar a expressão de genes envolvidos na regulação da inflamação, apoptose de células tumorais, diminuição da proliferação, diferenciação celular e imunomodulação. Estudos têm verificado que uma maior expressão do receptor de vitamina D está diretamente relacionada na modulação da proliferação e diferenciação celular, bem como na indução de apoptose em células tumorais.

Diversos estudos in vitro, in vivo e estudos epidemiológicos demonstram um papel importante da vitamina D especialmente na redução da incidência do câncer colo-retal. Pesquisadores verificaram em uma metanálise que indivíduos com os níveis séricos de 25(OH)D3 ≥ 82 nmol/l tiveram uma incidência 50% menor de câncer colorretal do que aqueles com níveis ≤ 30 nmol/l.

A relação entre os níveis séricos de vitamina D e o risco de desenvolver alguns tipos de câncer foi revisada em 2008 por Holick, no qual o autor sugere uma redução de 30 a 50% do risco de desenvolver câncer colorretal, mama e próstata, caso haja um aumento de vitamina D, através da ingestão diária de 1000 UI ao dia ou da exposição solar, elevando os níveis séricos de 25(OH)D3 para mais de 30 ng/mL.

Outro estudo verificou que a ingestão de alimentos enriquecidos com vitamina D, totalizando 400 UI/dia, foi associada à redução do risco de desenvolver câncer de mama. Pesquisas sugerem que os níveis séricos de 25(OH)D3 ou 1,25 (OH)2D3 parecem menores em pacientes com câncer de mama avançado ou metastático em relação àquelas pacientes com doenças em estágio inicial. Além disso, existem evidências que baixos níveis sérios de 25(OH)D3 ao diagnóstico estejam relacionados a um pior prognóstico, isto é, maior chance de recidiva da doença.

Em resumo, as evidências são bastante convincentes de que existe uma relação entre níveis inadequados de vitamina D com o aumento do risco de câncer e/ou a progressão do tumor. Entretanto, ainda são necessários mais estudos para essa afirmação e não é recomendada suplementação com altas doses de vitamina D com o intuito de prevenir o câncer. Os pesquisadores sugerem que a ingestão de vitamina D seja em torno de 600 UI (15 mcg) por dia, conforme as novas DRI (Dietary Reference Intakes), publicadas em 2010.

Leia mais

Novas recomendações para cálcio e vitamina D

Suplementação de vitamina D com cálcio diminui mortalidade em idosos

Quais pacientes podem se beneficiar do uso de suplementos de cálcio e vitamina D?

Bibliografia (s)Davis CD, Milner JA. Nutrigenomics, vitamin D and cancer prevention. J Nutrigenet Nutrigenomics. 2011;4(1):1-11.

Fleet JC, DeSmet M, Johnson R, Li Y. Vitamin D and cancer: a review of molecular mechanisms. Biochem J. 2012;441(1):61-76.

Toner CD, Davis CD, Milner JA. The vitamin D and cancer conundrum: aiming at a moving target. J Am Diet Assoc. 2010;110(10):1492-500.

McCullough ML, Bostick RM, Mayo TL. Vitamin D gene pathway polymorphisms and risk of colorectal, breast, and prostate cancer. Annu Rev Nutr. 2009;29:111-32.

Serum 25-Hydroxyvitamin D Levels and Risk of Multiple Sclerosis

0 Serum 25-Hydroxyvitamin D Levels and Risk of Multiple Sclerosis 

Original Contribution | December 20, 2006

Serum 25-Hydroxyvitamin D Levels and Risk of Multiple Sclerosis FREE

 

Kassandra L. Munger, MSc; Lynn I. Levin, PhD, MPH; Bruce W. Hollis, PhD; Noel S. Howard, MD; Alberto Ascherio, MD, DrPH

[+-] Author Affiliations

Author Affiliations: Departments of Nutrition (Ms Munger and Dr Ascherio) and Epidemiology (Dr Ascherio), Harvard School of Public Health, and Channing Laboratory, Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School (Dr Ascherio), Boston, Mass; Division of Preventive Medicine, Walter Reed Army Institute of Research, Silver Spring, Md (Dr Levin); Departments of Pediatrics, Biochemistry, and Molecular Biology, Medical University of South Carolina, Charleston (Dr Hollis); and Department of the Navy, Secretary of the Navy Council of Review Boards, Washington, DC (Dr Howard).

More Author Information

JAMA. 2006;296(23):2832-2838. doi:10.1001/jama.296.23.2832.

 

ABSTRACT

Context  Epidemiological and experimental evidence suggests that high levels of vitamin D, a potent immunomodulator, may decrease the risk of multiple sclerosis. There are no prospective studies addressing this hypothesis.

Objective  To examine whether levels of 25-hydroxyvitamin D are associated with risk of multiple sclerosis.

Design, Setting, and Participants  Prospective, nested case-control study among more than 7 million US military personnel who have serum samples stored in the Department of Defense Serum Repository. Multiple sclerosis cases were identified through Army and Navy physical disability databases for 1992 through 2004, and diagnoses were confirmed by medical record review. Each case (n = 257) was matched to 2 controls by age, sex, race/ethnicity, and dates of blood collection. Vitamin D status was estimated by averaging 25-hydroxyvitamin D levels of 2 or more serum samples collected before the date of initial multiple sclerosis symptoms.

Main Outcome Measures  Odds ratios of multiple sclerosis associated with continuous or categorical levels (quantiles or a priori–defined categories) of serum 25-hydroxyvitamin D within each racial/ethnic group.

Results  Among whites (148 cases, 296 controls), the risk of multiple sclerosis significantly decreased with increasing levels of 25-hydroxyvitamin D (odds ratio [OR] for a 50-nmol/L increase in 25-hydroxyvitamin D, 0.59; 95% confidence interval, 0.36-0.97). In categorical analyses using the lowest quintile (<63.3 nmol/L) as the reference, the ORs for each subsequent quintile were 0.57, 0.57, 0.74, and 0.38 (P = .02 for trend across quintiles). Only the OR for the highest quintile, corresponding to 25-hydroxyvitamin D levels higher than 99.1 nmol/L, was significantly different from 1.00 (OR, 0.38; 95% confidence interval, 0.19-0.75; P = .006). The inverse relation with multiple sclerosis risk was particularly strong for 25-hydroxyvitamin D levels measured before age 20 years. Among blacks and Hispanics (109 cases, 218 controls), who had lower 25-hydroxyvitamin D levels than whites, no significant associations between vitamin D and multiple sclerosis risk were found.

Conclusion  The results of our study suggest that high circulating levels of vitamin D are associated with a lower risk of multiple sclerosis.

Figures in this Article

a1

Multiple sclerosis (MS) is among the most common neurological diseases in young adults, affecting 350 000 individuals in the United States and 2 million worldwide.1 Prevailing thought is that MS is an autoimmune disorder whereby an unknown agent or agents triggers a T cell–mediated inflammatory attack, causing demyelination of central nervous system tissue.2

A striking feature of the global distribution of MS is a multifold increase in incidence with increasing latitude, both north and south of the equator.3 Genetic predisposition contributes to this variation,4 but the change in MS risk with migration among people of common ancestry5 strongly supports a role for environmental factors. One potential factor may be vitamin D,6– 9 a potent immunomodulator that in its hormonal form can prevent experimental autoimmune encephalomyelitis (EAE), an animal model of MS.10 Because food provides little vitamin D, the major source for most people is through skin exposure to sunlight.11 At latitudes of 42° or more (eg, Boston, Mass), in winter most UV-B radiation is absorbed by the atmosphere, and even prolonged sun exposure is insufficient to generate vitamin D.12 As a result, seasonal vitamin D deficiency is common.11

A protective effect of vitamin D on MS is supported by the reduced MS risk associated with sun exposure13– 14 and use of vitamin D supplements,15 but evidence remains inconclusive. In the present study, we examined prospectively for the first time whether high blood levels of 25-hydroxyvitamin D, a good marker of vitamin D availability to tissues,11 predict a lower risk of MS.

METHODS

This study has been approved by the institutional review boards of the Harvard School of Public Health and the Walter Reed Army Institute of Research, both of which waived the need for informed consent to use archived blood products and medical records.

Study Population

The study population includes more than 7 million active-duty US military personnel who have at least 1 serum sample stored in the Department of Defense Serum Repository (DoDSR). Since 1985, the DoDSR has collected and stored more than 30 million serum samples leftover from routine human immunodeficiency virus and worldwide deployment-related blood tests.16 Personnel generally provide 1 sample at entry into the military and, on average, every 2 years thereafter. All samples are cataloged and stored at −30°C.16

Case and Control Ascertainment

Multiple sclerosis case ascertainment within the military has been previously described.17 Briefly, active-duty personnel in the US Army and the US Navy (which includes the Marines) who were evaluated by their respective Physical Evaluation Boards for a diagnosis of MS between 1993 and 2004 (Army) or 1992 and 2004 (Navy) were identified by searching the Physical Evaluation Boards’ databases for members with the Veterans Administration Schedule for Rating Disabilities code for MS (code 8018). This search identified 515 potential MS cases. Medical records of the potential cases were reviewed and abstracted by 2 trained study personnel.

Cases included in this study were classified as either definite or probable MS. A case was definite if the final diagnosis in the medical record was made by a neurologist and specified as definite, clinically definite, or laboratory-supported definite MS,18 or if there was a history of 2 or more neurological attacks, a magnetic resonance imaging result consistent with MS, and a diagnosis of MS made by a neurologist. A case was considered probable if there was a neurologist’s diagnosis of probable, clinically probable, or laboratory-supported probable MS18 or at least 2 of the following: clinical history of 2 or more attacks, magnetic resonance imaging findings consistent with MS, and a diagnosis of MS made by a neurologist. Of the 515 cases reviewed, 315 had definite (n = 237) or probable (n = 78) MS and had at least 1 serum sample collected prior to their date of onset (the date of first neurological symptoms attributable to MS noted in the medical record)19; 83 of these 315 cases were included in our previous study on Epstein-Barr virus (EBV) and MS among Army personnel.17 For each case, we obtained up to 4 serum samples: 3 before the date of onset (the earliest and latest available, as well as a third sample collected between those 2) and 1 after the date of MS onset (the earliest available).

Controls were randomly selected from the DoDSR population, and 2 controls were matched to each case by age (±1 year), sex, race/ethnicity (non-Hispanic white, non-Hispanic black, Hispanic, or other), dates of sample collection (±30 days, except for the sample collected after the date of MS onset), and branch of military service (Army, Navy, or Marines). Controls had to be on active duty on the date of onset of the matched case. Appropriate controls could not be found for 10 cases, which were therefore excluded from further analysis. Race/ethnicity status was provided by the Army Medical Surveillance Activity, based on categories defined by the Department of Defense independent from the investigators. Cases and controls were matched on race/ethnicity to control for confounding—blacks have a lower risk of MS than whites, likely because of genetic factors,20 and lower 25-hydroxyvitamin D levels, mostly because of darker skin pigmentation, which decreases UV-B–induced subcutaneous production of vitamin D.21

Covariates

In addition to the matching factors, information was collected on latitude of place of residence at time of entry into the military. As in previous studies, latitude was attributed as follows: northern latitudes were states approximately 41° to 42° latitude or higher; middle latitudes, states between 37° and 41° latitude; southern latitudes, states approximately 37° latitude or lower; and outside of the continental United States (including Alaska, Hawaii, and Puerto Rico).22 We also created a UV index variable from the state of residence at entry into the military using the average UV index by state for 1995 (the earliest available year) from the National Oceanic and Atmospheric Administration23 and categorized as less than 5, 5 to less than 6, and 6 or higher. For consistency with the latitude variable, Alaska, Hawaii, and Puerto Rico were not included in the UV index.

Although the latitude gradient in MS risk could be a result of a protective effect of vitamin D, latitudes at birth or in early childhood also correlate with socioeconomic status and age at infection with common viruses, which are potential risk factors for MS24 and, thus, could confound the association of 25-hydroxyvitamin D and MS. In our study population, place of birth or residence in childhood was not generally available. However, 25-hydroxyvitamin D levels reflect recent UV exposure, and adjustment for latitude at entry into the military would be expected to remove any correlation that may exist between 25-hydroxyvitamin D levels and latitude at earlier ages.

The validity of the information on latitude of residence at entry into the military is supported by its expected correlation with 25-hydroxyvitamin D levels in samples collected prior to entry into the military (that is, at the time of application or initial screening), when the service member was likely to be residing in his/her state of entry. In these samples, among white controls (n = 87), mean 25-hydroxyvitamin D levels increased from 74.4 nmol/L in the northern latitudes to 81.4 nmol/L in the middle latitudes and to 90.6 nmol/L in the southern latitudes and from 71.3 nmol/L to 79.7 nmol/L and 89.9 nmol/L for UV index ratings of less than 5, 5 to less than 6, and 6 or higher, respectively. All the analyses presented were therefore adjusted for latitude of residence at entry. Adjusting for UV index as either a categorical or a continuous variable did not materially change the results.

Laboratory Analyses

25-Hydroxyvitamin D levels were measured in the laboratory of B.W.H., as previously described.25 Briefly, 25-hydroxyvitamin D was extracted from each serum sample using acetonitrile, and a radioimmunoassay with an iodine I 125–labeled tracer was used to measure the amount of 25-hydroxyvitamin D.25 The serum samples were randomly sorted within each matched case-control triplet, and the laboratory was blinded to the case/control status of the samples. The intra-assay coefficient of variation, determined from blind quality control samples included with the study samples, ranged from 4.5% to 7.9% in different batches.

Statistical Analyses

All analyses were stratified by race/ethnicity because, as expected, whites had much higher 25-hydroxyvitamin D levels than blacks (see “Results” section). Because of small numbers, we combined Hispanic and other race/ethnicity determinations into 1 group. To remove extraneous variation in 25-hydroxyvitamin D due to season of blood collection and other sources, we regressed the 25-hydroxyvitamin D levels on the periodic function −sin(2ΠX/12)−cos(2ΠX/12), where X is month of sample collection,26 age at sample collection, sex, and laboratory assay batch. The residuals from this model were added to the sex-specific 25-hydroxyvitamin D means derived from the model to create an adjusted 25-hydroxyvitamin D measurement.

To obtain an integrated measure of long-term, preclinical 25-hydroxyvitamin D level for each individual, we calculated the average of these adjusted 25-hydroxyvitamin D levels from all the available samples, except for those collected after the onset of MS among cases. Because a single measurement of serum 25-hydroxyvitamin D may not fully reflect long-term vitamin D status, the analyses were restricted to the 257 cases and 514 matched controls who had at least two 25-hydroxyvitamin D measurements before MS onset.

Conditional logistic regression analysis, adjusting for latitude of residence at entry into the military, was used to estimate odds ratios (ORs).27 We modeled 25-hydroxyvitamin D level both as a continuous variable, to estimate its association with MS risk under a linear assumption, and in quantiles, to explore the dose-response relationship. Quintiles among whites and tertiles (because of the smaller sample size) among blacks were determined based on the distributions of average 25-hydroxyvitamin D levels among their respective controls. In tests for trend, the medians of the quintiles or tertiles were modeled as continuous variables.

We also conducted analyses classifying individuals into 5 a priori–defined categories of 25-hydroxyvitamin D by 25-nmol/L increments (<25, 25 to <50, 50 to <75, 75 to <100, or ≥100 nmol/L). However, because of small sample sizes in the lower 25-hydroxyvitamin D categories among the white and Hispanic/other groups, the first 3 categories were collapsed and used as the referent in those analyses, and because few blacks had 25-hydroxyvitamin D levels higher than 75 nmol/L, the category of 50 to less than 75 nmol/L was used as the referent in the black race–specific analysis. Repeated-measures linear models were used to compare changes in 25-hydroxyvitamin D level over time among cases. The statistical significance level was set at P<.05 for 2-tailed tests.

Epstein-Barr virus antibody titers were strongly associated with risk of MS in analyses conducted in a subset of 83 cases and 166 controls from this population17 but were not correlated with 25-hydroxyvitamin D levels (data not shown) and, thus, are unlikely to confound the association between 25-hydroxyvitamin D levels and MS. For this reason and because EBV serologic results were unavailable for most cases and controls in the present study, EBV antibody titers are not included in this report. Results of analyses restricted to definite cases are materially identical to those including all cases and also are not shown.

Analyses were conducted using SAS software, version 9.1 (SAS Institute Inc, Cary, NC).

RESULTS

The main characteristics of the cases and controls are shown in the Table. Multiple sclerosis cases were, on average, 28.5 years old (age range, 18-48 years) at symptom onset. The initial disease course was relapsing-remitting in 73%, primary progressive in 7%, and uncertain in the remaining 20%. The average time between collection of the first and last samples before MS symptom onset was 4.4 years (range, <1-11.8 years) and between the first sample and MS symptom onset was 5.3 years (range, <1-13 years). The average serum 25-hydroxyvitamin D level among whites (mean [SD], 75.2 [28.1] nmol/L) was 29.7 nmol/L higher than that among blacks (mean [SD], 45.5 [21.2] nmol/L; P<.001), and was 8.6 nmol/L higher than that of the Hispanic/other group (mean [SD], 66.6 [25.4] nmol/L; P<.001). The mean for each group is consistent with levels in the general US population.28

Table Grahic Jump Location Table. Selected Characteristics of Multiple Sclerosis Cases and Matched Controls*

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White Race/Ethnicity

Among whites, there was a 41% decrease in MS risk for every 50-nmol/L increase in 25-hydroxyvitamin D (OR, 0.59; 95% confidence interval [CI], 0.36-0.97; P = .04), and there was no significant difference by sex (men: OR, 0.60; 95% CI, 0.33-1.10; women: OR, 0.53; 95% CI, 0.22-1.29; P = .90 for interaction). In analysis by quintiles, MS risk was highest among individuals in the bottom quintile and lowest among those in the top quintile of 25-hydroxyvitamin D levels (OR for top vs bottom quintile, 0.38; 95% CI, 0.19-0.75; P = .006). Risks in quintiles 2 through 4 were intermediate, and the overall trend across quintiles was significant (Figure). Results based on the a priori–defined categories of 25-hydroxyvitamin D were similar: using individuals with 25-hydroxyvitamin D levels of less than 75 nmol/L as the reference (69 cases and 114 controls) there was a nonsignificant reduction in risk among those with 25-hydroxyvitamin D levels of 75 to less than 100 nmol/L (62 cases and 124 controls; OR, 0.83; 95% CI, 0.54-1.29; P = .41) and a significant 51% reduction among those with 25-hydroxyvitamin D levels of 100 nmol/L or higher (17 cases and 58 controls; OR, 0.49; 95% CI, 0.27-0.91; P = .02).

Figure. Odds Ratios of MS by Quantile of Serum 25-Hydroxyvitamin D Among Whites and Blacks

Error bars indicate 95% confidence intervals.

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Adolescence appears to be a crucial exposure period for MS.5 Therefore, we further examined whether serum 25-hydroxyvitamin D concentrations before age 20 years predict MS risk. One of 39 cases and 16 of 76 controls (2 of the 78 matched controls were 20 years old at time of blood collection and were excluded) had 25-hydroxyvitamin D levels of 100 nmol/L or higher, resulting in an OR of 0.09 (95% CI, 0.01-0.75; P = .03) compared with levels less than 100 nmol/L.

We also were concerned that our results could reflect an effect of MS on 25-hydroxyvitamin D levels rather than an effect of 25-hydroxyvitamin D levels on MS risk. Multiple sclerosis could affect 25-hydroxyvitamin D levels either by some as yet unknown effect on vitamin D metabolism or, more likely, by changes in behavior—because heat commonly exacerbates MS symptoms, individuals with MS tend to avoid sun exposure and, thus, may have lower 25-hydroxyvitamin D levels than healthy individuals.29 If heat intolerance and sun avoidance preceded the neurological symptoms recognized as the first onset of MS, higher serum levels of 25-hydroxyvitamin D would spuriously appear to be protective. To address this possibility, we examined the temporal relationship between serum 25-hydroxyvitamin D concentrations and the date of onset of MS symptoms among white cases. Average 25-hydroxyvitamin D levels among individuals who developed MS were stable during the years preceding symptom onset (P = .42 for trend) but significantly decreased after onset of symptoms (P = .002). Mean 25-hydroxyvitamin D levels were 71.8 nmol/L more than 6 years before symptom onset (51 cases), 71.6 nmol/L between 4 and 6 years (51 cases), 73.5 nmol/L between 2 and 4 years (87 cases), 70.3 nmol/L between 1 and 2 years (136 cases), and 63.3 nmol/L after symptom onset (128 cases). These results argue against the possibility that the low preclinical 25-hydroxyvitamin D levels among individuals with MS are a consequence rather than a cause of the disease, although this possibility cannot be completely excluded.

Black and Hispanic Race/Ethnicity

Among blacks, the overall association between 25-hydroxyvitamin D levels and MS risk was not significant (OR for 50-nmol/L increase in 25-hydroxyvitamin D, 0.66; 95% CI, 0.24-1.78; P = .41), and there was no significant interaction by sex (P = .70). The OR for MS did not appreciably change by 25-hydroxyvitamin D tertile (Figure). Because there were no black cases or controls with 25-hydroxyvitamin D levels of 100 nmol/L or higher and all but 1 case and 5 controls had levels less than 75 nmol/L, we could not assess whether high levels of 25-hydroxyvitamin D in blacks are associated with reduced MS risk.

Among Hispanics and those of other race/ethnicity, the OR associated with a 50-nmol/L increase of 25-hydroxyvitamin D was 0.97 (95% CI, 0.28-3.33; P = .96). Because this group is small, we did not conduct a quantile analysis; in categorical analyses, the OR among individuals with 25-hydroxyvitamin D levels of 100 nmol/L or more (3 cases and 8 controls) compared with individuals with levels of less than 75 nmol/L (18 cases and 39 controls) was 0.61 (95% CI, 0.13-2.93; P = .54).

COMMENT

In this large prospective study, we found that the risk of MS decreased with increasing serum levels of 25-hydroxyvitamin D. Although this association was not seen among blacks, their smaller sample size and substantially lower 25-hydroxyvitamin D levels may have reduced the power to detect an association in this group.

Our results converge with a growing body of evidence supporting a protective role for vitamin D in MS development. Vitamin D is a potent immunomodulator,10 and several studies have shown that administration of the biologically active hormone 1,25-dihydroxyvitamin D prevents EAE onset and progression in mice.30– 31 The exact mechanisms of this protection are unknown, but evidence suggests an indirect effect, possibly mediated by regulatory T cells.10,32 Of interest, regulatory T cells have been shown to be suppressed in individuals with MS.33 Physiological blood levels of 1,25-dihydroxyvitamin D, however, are tightly regulated and are not measurably affected by exposure to sunlight or dietary vitamin D.34

In contrast, circulating levels of 25-hydroxyvitamin D are sensitive to both factors. Therefore, an important question is whether 25-hydroxyvitamin D has a role in regulating immune responses. Serum levels of 25-hydroxyvitamin D were recently shown to control the Toll-like receptor–mediated generation of the microbicide cathelicidin by human monocytes and macrophages in response to Mycobacterium tuberculosis challenge, suggesting that nutritional vitamin D status could be key in innate immune response.35 An inhibitory effect of levels of 25-hydroxyvitamin D in autoimmune reactions is consistent with the accelerated onset of EAE31 and experimental type 1 diabetes in vitamin D–deficient mice.36 This effect could be mediated by local synthesis of 1,25-dihydroxyvitamin D from 25-hydroxyvitamin D by activated macrophages expressing 1-α-hydroxylase. If sufficient 1,25-dihydroxyvitamin D is produced, it may exert paracrine effects on surrounding T lymphocytes, thereby regulating the tissue-specific immune responses.10 Some support for this hypothesis comes from recent experiments showing that mice fed diets high in vitamin D had significantly fewer clinical and pathological signs of EAE than mice fed a vitamin D–deficient diet.37 Central nervous system levels of 1,25-dihydroxyvitamin D, but not blood levels, were higher in supplemented mice than in vitamin D–deficient mice and correlated inversely with disease severity.

Although the results of our study support a direct role of vitamin D in MS prevention, other potential explanations should be considered. Although unlikely, a genetic predisposition to both MS and circulating low 25-hydroxyvitamin D levels could appear as a protective effect of vitamin D on MS in our study. Additionally, we cannot exclude the possibility that some other effect of exposure to UV light, rather than vitamin D production, contributes to protection. Serum levels of 25-hydroxyvitamin D largely reflect differences in exposure to UV radiation from sunlight. Whole-body UV light exposure has been shown to suppress EAE in mice38; it also enhances regulatory T-cell function and increases production of the immunosuppressive cytokines interleukin 4 and interleukin 10.39 The relative importance of direct vs vitamin D–dependent effects of UV light at the level of exposure typical of human populations is uncertain,40 but our previous finding of a lower MS risk among women taking vitamin D supplements15 supports a specific role for vitamin D.

In most migration studies, the change in MS risk among migrants is stronger when migration occurs in childhood and tends to decrease with increasing age at migration.5 These results suggest that vitamin D levels earlier in life may be critical in conferring protection for MS and our finding of a strong protective effect of 25-hydroxyvitamin D levels of 100 nmol/L or higher before age 20 years supports this view. Vitamin D supplementation in infancy seems to exert a strong protective effect against the autoimmune disease type 1 diabetes,41 and vitamin D levels in early childhood could also have an impact on the risk of MS. Although there are no data on vitamin D levels earlier in life and risk of MS, the strong inverse association between MS risk and 25-hydroxyvitamin D levels at ages 16 to 19 years suggests that levels in late adolescence are likely to be important.

A key question is whether it may be possible to reduce the incidence of MS in populations at high risk by increasing circulating levels of 25-hydroxyvitamin D. Almost half of white and two thirds of black adults in the United States have 25-hydroxyvitamin D levels below 70 nmol/L.28 Although levels above 25 nmol/L have traditionally been considered normal and almost everyone in this study had measurements above this level, much higher levels may be required for bone mineralization and prevention of fractures. According to a recent review, the best serum 25-hydroxyvitamin D concentrations are between 90 and 100 nmol/L.42 Adolescents have somewhat higher levels than adults, but few have levels higher than that associated with a reduced risk of MS in our study.43 If the association reported here reflects a true protective effect of vitamin D, increasing the vitamin D levels of adolescents and young adults could result in an important reduction in MS incidence. Such an increase could be achieved by using vitamin D supplements.44– 45 Although the current Institute of Medicine adequate intake of vitamin D is 200 U/d for adults younger than 50 years,46 and the highest dose that is considered safe is 2000 U/d,46 adverse effects have been reported only at intakes several-fold higher.45

A broad recommendation for a several-fold increase in vitamin D intake among adolescents and young adults requires stronger evidence than that provided by observational studies alone. First-degree relatives of individuals with MS are at a higher risk of developing MS,47 and a prevention trial among this population would be possible and timely. Meanwhile, use of vitamin D supplements for MS prevention should not be undertaken until efficacy is proven.

 

AUTHOR INFORMATION

 

Corresponding Author: Alberto Ascherio, MD, DrPH, Harvard School of Public Health, 655 Huntington Ave, Third Floor, Boston, MA 02115 (aascheri@hsph.harvard.edu).

Author Contributions: Dr Ascherio had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Study concept and design: Levin, Ascherio.

Acquisition of data: Munger, Levin, Howard, Ascherio.

Analysis and interpretation of data: Munger, Levin, Hollis, Ascherio.

Drafting of the manuscript: Munger, Hollis, Ascherio.

Critical revision of the manuscript for important intellectual content: Munger, Levin, Hollis, Howard, Ascherio.

Statistical analysis: Munger, Ascherio.

Obtained funding: Munger, Levin, Ascherio.

Administrative, technical, or material support: Munger, Levin, Howard, Ascherio.

Study supervision: Howard, Ascherio.

Financial Disclosures: Dr Hollis is a consultant for Diasorin. No other disclosures were reported.

Funding/Support: This work was supported by grants NS46635 and NS042194 from the National Institute of Neurological Diseases and Stroke and by a pilot grant from the National Multiple Sclerosis Society.

Role of the Sponsors: The sponsors had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; or preparation, review, or approval of the manuscript.

Disclaimer: The views expressed are those of the authors and should not be construed to represent the positions of the Department of the Army, the Department of the Navy, or the Department of Defense.

Acknowledgment: We thank Mark Rubertone, MD, MPH, Army Medical Surveillance Activity, US Army Center for Health Promotion and Preventive Medicine, Washington, DC, and Remington Nevin, MD, MPH, US Army Medical Corps, Center for Biosurveillance, Army Medical Surveillance Activity, Washington, DC, for control and sample identification and retrieval; Charles Peck, Jr, MD, and David Armitage, MD, JD, US Army Physical Disability Agency, Washington, DC, for MS case identification in the US Army; Walter Willett, MD, MPH, DrPH, and Meir Stampfer, MD, DrPH, Departments of Nutrition and Epidemiology, Harvard School of Public Health, and Channing Laboratory, Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, Mass, for their valuable input, and Leslie Unger and Eilis O’Reilly, MSc, Department of Nutrition, Harvard School of Public Health, Boston, Mass, for technical assistance.

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High Doses of Vitamin D Cut MS Relapses

High Doses of Vitamin D Cut MS Relapses

Study Shows Vitamin D May Help Reduce Relapse Rate of Multiple Sclerosis
By

WebMD Health News

Reviewed by Louise Chang, MD
d_improves_ms

April 28, 2009 (Seattle) — High doses of vitamin D dramatically cut the relapse rate in people with multiple sclerosis, a study shows.

Sixteen percent of 25 people with multiple sclerosis (MS) given an average of 14,000 international units (IU) of vitamin D a day for a year suffered relapses, says Jodie Burton, MD, a neurologist at the University of Toronto. In contrast, close to 40% of 24 MS patients who took an average of 1,000 IU a day — the amount recommended by many MS specialists — relapsed, she says.

Also, people taking high-dose vitamin D suffered 41% fewer relapses than the year before the study began, compared with 17% of those taking typical doses.

People taking high doses of vitamin D did not suffer any significant side effects, Burton tells WebMD.

The findings were presented at the annual meeting of the American Academy of Neurology.

In contrast to many vitamins, no recommended dietary allowance (RDA) has been established for vitamin D. Instead, the Institute of Medicine has set a so-called adequate intake level; the recommendations are 200 IU daily for people under 50, 400 IU daily for people 50 to 70, and 600 IU for those over 70.

John Hooge, MD, an MS specialist at the University of British Columbia in Vancouver who was not involved with the research, says he recommends MS patients take at least 1,000 IU and “probably 2,000 IU” day.

“This is an impressive study that shows that even higher doses are probably safe and even more effective. Maybe next year, I’ll be recommending higher doses,” he tells WebMD.

Vitamin D vs. Relapsing MS

Most of the people in the study had the relapsing form of MS, characterized by repeated relapses with periods of recovery in between. They suffered from the disease for an average of eight years.

“They had very mild disease, with an average score of 1.25 on the Extended Disability Status Scale, where zero corresponds to normal and 10 to death,” Burton says. Participants suffered one relapse every other year, an average.

People in the high-dose group were given escalating doses of vitamin D in the form of a concentrate that could be added to juice for six months, to a maximum of 40,000 IU daily. Then doses were gradually lowered over the next six months, averaging out to 14,000 IU daily for the year.

The rest of the participants were allowed to take as much vitamin D as they and their doctors thought was warranted, but it averaged out to only 1,000 IU daily.

Everyone also took 1,200 milligrams of calcium a day. Vitamin D is essential for promoting calcium absorption in the gut and together with calcium, helps promote bone health.

1|2

Vitamin D vs. Relapsing MS continued…

Vitamin D appears to suppress the autoimmune responses thought to cause MS, Burton says. In MS, haywire T lymphocytes — the cellular “generals” of the immune system — order attacks on the myelin sheaths that surround and protect the brain cells.

In people given high-dose vitamin D in the study, T cell activity dropped significantly. That didn’t happen in people who took lower doses.

The researchers also measured the concentration of 25-hydroxyvitamin D [25(OH)D], also known as calcidiol, in the blood. The Institute of Medicine says that is the best indicator of a person’s vitamin D status.

There’s no ideal level, although concentrations of less than 50 nanomoles per liter of blood are considered inadequate for good health. In the study, it appeared MS patients did best if levels reached 100 nanomoles per liter, Burton says.

People with MS should talk to their doctors about whether they might benefit from vitamin D supplements, she says.

“Too much vitamin D can be harmful for people with certain medical conditions such as kidney disease,” Burton says. “Also doctors can monitor your blood levels of 25(OH)D.”

http://www.webmd.com/multiple-sclerosis/news/20090428/high-doses-vitamin-d-cut-ms-relapses

—-

Immunomodulatory Effects Of Vitamin D In Multiple Sclerosis

dc2

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Dr Cicero Galli Coimbra e Daniel Cunha – Vitamina D Sem Censura

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—————-

vitamin

Immunomodulatory Effects Of Vitamin D In Multiple Sclerosis

http://brain.oxfordjournals.org/content/132/5/1146.full

This Article

Brain (2009) 132 (5): 1146-1160. doi: 10.1093/brain/awp033 First published online: March 24, 2009

April 2013 136 (4)

Oxford Journals

Medicine

Brain

Volume 132, Issue 5

Pp. 1146-1160.

Brainbrain.oxfordjournals.org

Brain (2009) 132 (5): 1146-1160. doi: 10.1093/brain/awp033 First published online: March 24, 2009

IMMUNOMODULATORY EFFECTS OF VITAMIN D IN MULTIPLE SCLEROSIS

Jorge Correale,

María Célica Ysrraelit and

María Inés Gaitán

+ Author Affiliations

Department of Neurology, Institute for Neurological Research Dr. Raúl Carrea, FLENI, Buenos Aires, Argentina

Correspondence to: Jorge Correale, MD, Raúl Carrea Institute for Neurological Research, FLENI, Montañeses 2325, (1428) Buenos Aires, Argentina E-mail: jcorreale@fleni.org.ar; bairesla@fibertel.com.ar

Received July 22, 2008.

Revision received December 29, 2008.

Accepted January 22, 2009.

SUMMARY

Although Vitamin D is best known as a modulator of calcium homeostasis, it also has immune modulating potential. A protective effect of Vitamin D on multiple sclerosis is supported by the reduced risk associated with sun exposure and use of Vitamin D supplements. Moreover, high circulating levels of Vitamin D have been associated with lower risk of multiple sclerosis. In this study, we measured 1,25 (OH)2 Vitamin D and 25 (OH) Vitamin D levels in multiple sclerosis patients separated into different clinical subgroups according to disease status. In addition, direct effects of 1,25 (OH)2 Vitamin D on ex vivo CD4+ T cells and myelin-peptide specific T cell lines were investigated to gain more insight into putative regulatory mechanisms in the disease pathogenesis. One hundred and thirty-two Hispanic patients with clinically definite multiple sclerosis were studied, 58 with relapsing remitting multiple sclerosis during remission, 34 during relapse and 40 primary progressive multiple sclerosis cases. Sixty healthy individuals matched with respect to place of residence, race/ethnicity, age and gender served as controls. Levels of 25(OH)D3 and 1,25(OH)2D3, measured by ELISA were significantly lower in relapsing–remitting patients than in controls. In addition, levels in patients suffering relapse were lower than during remissions. In contrast, primary progressive patients showed similar values to controls. Proliferation of both freshly isolated CD4+ T cells and MBP-specific T cells was significantly inhibited by 1,25(OH)2D3. Moreover, activated Vitamin D enhanced the development of IL-10 producing cells, and reduced the number of IL-6 and IL-17 secreting cells. Notably, Vitamin D receptor expression was induced by 1,25(OH)2D3 in both activated and resting cells. Interestingly, T cells were able to metabolize 25(OH)D3 into biologically active 1,25(OH)2D3, since T cells express α1-hydroxylase constitutively. Finally, 1,25(OH)2D3 also increased the expression and biological activity of indoleamine 2,3-dioxygenase, mediating significant increase in the number of CD4+CD25+ T regulatory cells. Collectively, these data suggest that 1,25(OH)2D3 plays an important role in T cell homeostasis during the course of multiple sclerosis, thus making correction of its deficiency may be useful during treatment of the disease.

INTRODUCTION

Several lines of evidence support the hypothesis that autoimmunity plays a major role in multiple sclerosis development (McFarland and Martin, 2007). It is generally accepted that autoimmune diseases arise from a complex interactions between genetic susceptibility and environmental factors. Geographical variations in the incidence and prevalence of this disease, as well as studies of populations emigrating from areas of low to areas of high risk, indicate that the environment is also a key factor in its pathogenesis (Ascherio and Munger, 2007; Pugliatti et al., 2008).

Vitamin D is best known as a calcium homeostasis modulator; however both experimental and clinical observations provide evidence that Vitamin D is also one of several important environmental factors that can affect multiple sclerosis prevalence. A protective effect has been supported by reduced risk of the illness associated with sunlight exposure and use of Vitamin D supplements (Munger et al., 2004; van der Mei et al., 2001). A recent longitudinal study conducted among American patients showed decreased risk in white patients with increasing serum levels of 25(OH) Vitamin D (Munger et al., 2006). The evidence obtained from these studies supports the role of Vitamin D as an immunomodulatory molecule.

Vitamin D, ingested orally or formed in the skin following exposure to sunlight is hydroxilyzed to the major circulating form, 25 (OH) Vitamin D in the liver, levels of which are sensitive to both Vitamin D intake and sun exposure and are markers of Vitamin D availability to tissues, best reflecting Vitamin D status of the patient. However 25(OH) Vitamin D is inert and requires additional hydroxylation, mainly in the kidney, by the 25-hydroxy Vitamin D-1α hydroxylase (1α-hydroxylase) to 1,25 (OH)2 Vitamin D. This is the biologically active metabolite, with most biological effects mediated through binding to the Vitamin D receptor. Expression of Vitamin D receptor and 1-α hydroxylase have been also described in immune cells. For instance, the presence of both molecules has been demonstrated in dendritic cells, macrophages (Overbergh et al., 2000; Veldman et al., 2000), B cells (Chen et al., 2007), and activated T cells (Veldman et al., 2000; Norman, 2006). In vitro 1,25(OH)2 Vitamin D inhibits the differentiation of monocytes into dendritic cells, and suppresses IL-12 secretion as well as surface expression of co-stimulatory molecules (D’Ambrosio et al., 1998; Penna and Adorini, 2000; Adorini, 2002). In addition, evidence supports a model in which 1,25 (OH)2 Vitamin D mediates a shift of CD4+ T cells to an anti-inflammatory profile (Adorini, 2002; Meehan and DeLuca, 2002; Smolders et al., 2008). Moreover, B cell proliferation, plasma differentiation and immunoglobulin production are inhibited in vitro by 1,25 (OH)2 Vitamin D (Chen et al., 2007). These immunomodulatory effects are consistent with the accelerated onset of experimental allergic encephalomyelitis (EAE), an animal model for multiple sclerosis, in Vitamin D-deficient mice (Cantorna et al., 1996). In contrast, injection of 1,25 (OH)2 Vitamin D was found to prevent clinical and pathological signs of disease, a phenomenon associated with profound reduction in autoreactive T cells, peptide-specific proliferation and Th1 cell development (Lemire and Archer, 1991).

Tryptophan is an essential amino acid that is catabolized by the rate-limiting enzyme indoleamine 2,3-dioxygenase (IDO) to generate kynurenin. IDO is expressed in different immune cells, such as dendritic cells and activated macrophages (Munn et al., 2002). Data from recent studies indicate that dendritic cells expressing high levels of IDO play an important role in the induction of regulatory T cell responses, and in maintenance of peripheral tolerance (Munn et al., 2002; Meisel et al., 2004). Defects in IDO-mediated tryptophan metabolism have been associated with tolerance impairment in experimental animal models such as non-obese diabetic mice, in which autoreactive T cells persist during disease progression (Grohmann et al., 2003).

In this study, we measured 1,25 (OH)2 Vitamin D and 25 (OH) Vitamin D levels in patients with multiple sclerosis, separated into different subgroups according to clinical status. In addition, the direct effects of 1,25 (OH)2 Vitamin D and IDO on ex vivo CD4+ T cells and myelin-peptide specific T cell lines were investigated, to gain more insight into putative regulatory mechanisms of both molecules in multiple sclerosis pathogenesis.

 

MATERIAL AND METHODS

Patients and control subjects

One hundred and thirty-two Hispanic patients with diagnosis of clinically definite multiple sclerosis according to Poser’s and McDonald’s criteria were studied. Clinical course of disease was defined following Lublin and Reingold (1996) criteria, and patients were subdivided into three groups: (i) relapsing remitting multiple sclerosis in remission (n = 58); (ii) relapsing remitting multiple sclerosis during acute exacerbation (n = 34); and (iii) primary progressive multiple sclerosis (n = 40). Exacerbations were defined as development of new symptoms or worsening of a pre-existing one, confirmed on neurological examination and lasting at least 48 h, in the absence of fever, and preceded by stability or improvement lasting at least 30 days. Patients with inflammatory, endocrine or major psychiatric disorders were excluded from the study. No patients had received steroids for at least 6 months prior to study entry, nor inmunomodulatory, immunosuppressive drugs, or dietary supplements. All patients were living in the city of Buenos Aires (latitude 34°S, longitude 58°W). Sixty healthy individuals, selected to match patients with respect to place of residence, race/ethnicity, age and gender, served as controls. Because relapsing remitting and primary progressive patients differ significantly in age, two control groups were included in the study, one similar to primary progressive patients (control group 1) and another to relapsing remitting ones (control group 2). Thorough clinical and neurological examination, as well as standard chemical and haematological laboratory examinations ruled out presence of underlying disorders in these subjects. Control individuals were not receiving any regular medication, or dietary supplements. Samples from healthy controls were collected during the same months of the year as samples from multiple sclerosis patients. Demographic and clinical characteristics of patients and controls are shown in Table 1.

Table 1

PPMS Controls 1 RRRMS RREMS Controls 2
N

40

30

58

34

30

Age (years)

49.5 ± 12.7

49.0 ± 5.0

36.8 ± 10

37.5 ± 8.4

33.9 ± 5.9

Female:male

18:22

16:14

41:17

29:5

23:7

EDSS

4.3 ± 1.6

1.3 ± 1.1

2 ± 1.4

Disease duration (years)

4.1 ± 4.7

3.9 ± 4.9

4.3 ± 5.7

Demographic and clinical characteristics of the study populationa

Determination of serum 25 (OH) Vitamin D and 1,25 (OH)2 Vitamin D levels

Serum 25 (OH) Vitamin D and 1,25 (OH)2 Vitamin D levels were measured using commercially available ELISA kits (Immunodiagnostik, Bensheim, Germany) according to manufacturer’s instructions. Assay sensitivity levels were: 10 ng/ml and 6.0 pg/ml, respectively. Intra- and interassay variation coefficients were <7.5 and 6.8%, respectively for both assays.

Antigen preparation

MBP83–102, MBP143–168 and MOG63-87 peptides were synthesized using an automated peptide synthesizer, and purity was assessed using high-pressure liquid chromatography analysis.

CD4+ cell enrichment and generation of MBP- and MOG-peptide reactive T cell lines

Peripheral blood mononuclear cells (PBMC) were isolated by Ficoll-Hypaque density gradient centrifugation (Pharmacia LKB Biotechnology, Piscataway, NJ), and CD4+ T cells were positively selected using Dynabeads M-450 CD4 beads (Dynal Biotech ASA, Oslo, Norway). Separation was monitored using flow cytometry analysis, demonstrating over 97% purity. MBP- and MOG-peptide specific T cell lines were expanded from peripheral blood as previously described (Correale et al., 1995). Briefly, 5 × 106 PBMC were stimulated with optimal concentrations of MBP or MOG peptides (10–20 μg/ml). After 5–7 days, cells were re-cultured in fresh medium containing 50 U/ml of recombinant human IL-2 (rIL-2; R&D Systems, Minneapolis) for an additional week. Re-stimulation cycles with autologous irradiated PBMCs (3000 Rads) as antigen presenting cells plus peptide, followed by expansion with rIL-2, were repeated weekly. After four cycles of restimulation and expansion, T-cell lines were evaluated using standard proliferation assays. Cutoff value for a positive response was set at stimulation index >3. All MBP- and MOG-reactive T-cell lines were >93% CD4+.

T helper cell polarization

Human CD4+ T cells were purified from PBMCs of relapsing remitting patients and healthy controls as described above, and cultured for 5 days with PHA (1 μg/ml; Sigma-Aldrich), and rhIL-2 (50 U/ml; R&D Systems) in neutral o polarizing conditions as described (Hannier et al., 2002): Th1, IL-12 (2 ng/ml; BD Biosciences, San Diego, CA), plus anti-IL-4 mAb (100 ng/ml; BD Biosciences) and Th2, IL-4 (5 ng/ml; Sigma) plus anti-IL-12 (2 μg/ml; BD Biosciences). For Th-17 differentiation, naïve CD4+ T cells were stimulated for 5 days (Bettelli et al., 2006) with plate bound anti-CD3 (5 μg/ml; ATCC) and soluble anti-CD28 (1 μg/ml; BD Biosciences) in the presence of TGF-β (3 ng/ml), IL-6 (20 ng/ml) and IL-23 (20 ng/ml, all from R&D Systems), and neutralizing antibodies anti IL-4 (10 μg/ml), and anti- IFN-γ (10 μg/ml), both from BD Biosciences. Cultures were supplemented with rhIL-2 (50 U/ml; R&D Systems) on Days 2 and 4.

Generation of dendritic cells

Dendritic cells were generated as previously described (Correale and Farez, 2007). Briefly, monocytes were positively selected from PBMCs isolated from relapsing remitting patients and healthy controls using anti-CD14 coated magnetic beads (Miltenyi Biotec, Bergisch Glandbach, Germany). Cells were cultured in the presence of 100 ng/ml of GM-CSF (R&D Systems), and 50 ng/ml of rhIL-4 (R&D Systems). On Days 2 and 4, half the culture medium was replaced maintaining the same GM-CSF and IL-4 concentrations. After 5 days, 100 ng/ml of LPS were added to stimulate dendritic cell maturation. Contamination with CD3+ T cells was verified by flow cytometry and found to be <0.4%.

Stimulation of CD4+ T cells and peptide-specific T-cell lines, and proliferation assays

For peptide Antigen (Ag)-specific stimulation, T-cell lines were cultured at a density of 5 × 104 cells/well in the presence of 5 × 103 adherent irradiated autologous PBMC as the source of antigen presenting cells, and 10 μg/ml of appropriated peptide, in the presence or absence of 1,25 (OH)2 Vitamin D, or the analogues 24,25 (OH)2 Vitamin D and 25,26 (OH)2 Vitamin D (all from Sigma-Aldrich, St Louis; MO). For non-specific stimulation, peptide-specific T-cell lines (5 x 104 cells/well) were cultured in the presence of 1 μg/ml of immobilized anti-CD3 mAb (OKT3; American Type Culture Collection, Manassas, VA), in the presence or in the absence of 1,25 (OH)2 Vitamin D. Irradiated PBMC were not included under these conditions. Meanwhile, purified CD4+ T cells were plated at a density of 5 × 104 cells/well and stimulated with 1 μg/ml of PHA (Sigma-Aldrich). After stimulation, T cell proliferation was examined in a standard 60-h assay measuring [3H]thymidine (ICN Biomedicals, Irvine, CA) incorporation, as previously described (Correale et al., 1995).

1,25 (OH)2 Vitamin D and its analogues were dissolved in absolute ethanol at a stock concentration of 0.01 M, and further diluted in RPMI 1640 medium to the tested concentrations. Additional controls included viability studies using tryphan blue dye exclusion, and the addition of ethanol diluted to concentrations equivalent to those used for 1,25 (OH)2 Vitamin D or its analogues dilutions. In all experiments, 1,25 (OH)2 Vitamin D was used at the pharmacological dose of 10 nM. This dose is ∼82- to 550-fold higher than circulating levels in healthy individuals.

Real-time quantitative RT-PCR analysis

For quantitative assessment of relative mRNA levels, total RNA was prepared using TRIzol LS reagent (Invitrogen, Carlsbad, CA) following manufacturer instructions. RNA was reverse transcribed using a M-MLV RT reverse transcription kit with random hexamer primers (Invitrogen). Relative levels of VDR, 1-α hydroxylase, and IDO mRNAs were determined by real-time PCR (ABI 7000 sequence detection system; Applied Biosystems, Foster City, CA), using a SYBR green PCR mix. Values obtained were normalized to the amount of GAPDH. Primer sequences used were as follows: GAPDH: forward 5′-GAAGGTGAAGTCGGAGTC-3′, reverse 5′-GAAGATGGTGATGGGATTTC-3′; VDR: forward 5′-CTTCAGGCGAAGCATGAAGC, reverse 5′-CCTTCATCATGCCGATGTCC-3′; 1-α hydroxylase: forward 5′-TTGGCAAGCGCAGCTGTAT-3′, reverse 5′-TGTGTTAGGATCTGGGCCAAA-3′; IDO: forward 5′-ACTGGAGGCACTGATTTA-3′, reverse 5′-ATTAGTTTGTGGCTCTGTTA-3′. Each specific primer was used at a final concentration of 250 nM.

Quantification of secreted cytokines

The number of CD4+ T cells or MBP- and MOG-peptide specific T cells secreting IL-4, IL-6, IL-10, IL-17 and IFN-γ was evaluated using commercially available kits for single-cell resolution enzyme-linked immunospot (ELISPOT) assays following manufacturer instructions (R&D Systems, Minneapolis, MN, USA), in the presence and in the absence of 1,25 (OH)2 Vitamin D. The number of cytokine secreting cells was calculated by subtracting the numbers of spots obtained in control cultures without Ag stimulation, from the number of spots obtained in cultures exposed to stimulating Ag. Results are reported as number of spots per 105 PBMC. To assess the role of IL-10 as an autocrine factor, a neutralizing anti IL-10 receptor mAb (10 μg/ml; Pharmingen, San Diego, CA) was added to culture in some experiments.

T1MSD

Polarized T helper cells were stimulated with 1 μg/ml of plate-bound anti-CD3 monoclonal antibody (ATCC). Supernatants were removed at 36 (IL-4), or 72 h (IFN-γ, and IL-17), and cytokines measured using commercially available ELISA kits, purchased from R&D Systems.

Measurement of IDO activity

CD4+ T cells, as well as MBP and MOG peptide-specific T-cell lines were cultured in the presence or in the absence of 1,25 (OH)2 Vitamin D. Seventy-two hours after culture, supernatants were collected and kynurenine concentrations were measured with HPLC using a reverse-phase column as previously described (Widner et al., 1997). Kynurenine was detected using UV detection at 360 nm wavelength, and values referred to a calibration curve previously constructed with defined kynurenine concentrations.

Evaluation of CD4+CD25+FoxP3+ regulatory T cells

To evaluate the role of 1,25 (OH)2 Vitamin D on regulatory T cell induction, 5 × 104, PBMC were stimulated with soluble anti-CD3, and soluble anti-CD28 (BD Biosciences) antibodies, both at 5 μg/ml concentration, in the presence or the absence of 1,25 (OH)2 Vitamin D. After 7 days, the number of CD4+CD25+FoxP3+ T cells was evaluated by flow cytometry, using commercially available regulatory T cell staining kits following the manufacturer instructions (eBioscience, San Diego, CA). To evaluate the role of IDO in regulatory T cell induction, the IDO inhibitor 1-methyl-d-tryptophan (1-MT; Sigma-Aldrich) was added to the cultures in some experiments at a concentration of 1000 μM.

To evaluate 1,25 (OH)2 Vitamin D activity induced by regulatory T cells, CD4+ CD25− and CD4+CD25high populations were isolated by FACS sorting. Sort gates were restricted to the population of lymphocytes by forward and side scatter properties. Large, activated T cells were excluded. To ensure purity of isolated populations, a portion of each sample was re-analysed, and purity was determined to be >95%. Variable numbers of CD4+ CD25+ cells were added to a constant number of CD4+CD25- indicator cells (3 × 103 cells/well), in order to achieve CD4+CD25+:CD4+CD25− ratios of 10:1, 5:1, 2.5:1 and 1:1, respectively. Stimulation was provided by the addition of soluble anti-CD3, and soluble anti-CD28 (BD Biosciences), both at 5 μg/ml concentrations in the presence of 3 × 104/well irradiated PBMC, depleted of CD3+ T cells as a source of antigen presenting cells. Proliferation was determined on Day 6, with [3H] thymidine added during the last 18 h of culture. Mean counts per minute ± SEM were calculated for triplicate measurements. To measure IFN-γ production, supernatants were removed from each well before [3H] thymidine addition, and analysed using commercially available ELISA kits (R&D systems).

Statistical analysis

Differences in the levels of 25 (OH) Vitamin D and 1,25 (OH)2 Vitamin D between distinct populations were analysed with a two-tailed Student’s t-test. Differences in immunological variables were tested for significance using Mann–Whitney U-test. In all cases, P-values below 0.05 were considered statistically significant.

RESULTS

Relapsing remitting multiple sclerosis patients have significantly reduced levels of 25 (OH) Vitamin D and 1,25 (OH)2 Vitamin D

25 (OH) Vitamin D serum levels were significantly lower in relapsing remitting patients (47.3 ± 9.0 ng/ml during remission and 38.5 ± 8.7 ng/ml during exacerbations) than in healthy controls (61.2 ± 5.6 ng/ml; Fig. 1A). Additional analysis demonstrated significantly lower levels of 25 (OH) Vitamin D in patients during exacerbations compared with patients in remission (Fig. 1A). In contrast, 25 (OH) Vitamin D levels in primary progressive patients (52.2 ± 6.5 ng/ml) were not statistically different from those observed in healthy controls (52.7 ± 8.2 ng/ml; Fig. 1B). Likewise, 1,25 (OH)2 Vitamin D serum level were also significantly lower in relapsing remitting patients (29.2 ± 5.6 pg/ml during remission, and 23.0 ± 5.6 pg/ml during exacerbations) than in healthy controls (35.1 ± 6.6 pg/ml; Fig. 1C). Once again, 1,25 (OH)2 Vitamin D levels were significantly lower in patients with relapsing remitting symptoms during exacerbations compared with patients in remission (Fig. 1C). In contrast, 1,25 (OH)2 Vitamin D serum levels in patients with primary progressive multiple sclerosis (32.5 ± 5.6 pg/ml) were not statistically different from levels in normal controls (31.3 ± 6.1 pg/ml; Fig. 1D). Overall, these results indicated that relapsing remitting patients, had decreased levels of 25 (OH) Vitamin D and 1,25 (OH)2 Vitamin D, particularly during exacerbations. In subsequent experiments impact of Vitamin D on T cell function was examined.

 

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Figure 1

Patients with relapsing remitting multiple sclerosis have decreased 25(OH) Vitamin D and 1,25 (OH)2 Vitamin D levels. Serum levels of 25(OH) Vitamin D and 1,25 (OH)2 Vitamin D were measured by ELISA. (A) 25(OH) Vitamin D levels in relapsing remitting patients during remission (n = 58) and exacerbations (n = 34) compared with normal controls (n = 30). (B) 25(OH) Vitamin D levels in patients with primary progressive multiple sclerosis (n = 40) compared with normal controls (n = 30). (C) 1,25 (OH)2 Vitamin D levels in relapsing remitting patients during remission (n = 58) and exacerbations (n = 34) compared with normal controls (n = 30). (D) 1,25 (OH)2 Vitamin D levels in primary progressive patients (n = 40) compared with normal controls (n = 30). Each circle represents average Vitamin D levels from a single individual. Horizontal lines indicate mean group values.

1,25 (OH)2 Vitamin D inhibits proliferation of T cells

1,25 (OH)2 Vitamin D capacity to inhibit T cell proliferation was assessed in comparison with the analogues 24,25 (OH)2 Vitamin D and 25,26 (OH)2 Vitamin D used as controls. None of the molecules affected CD4+ T cell survival, as measured by tryphan blue dye exclusion. All three compounds were tested in a PHA-induced proliferation assay using ex vivo CD4+ T cells and in CD4+ MBP-peptide specific T cell lines stimulated with the cognate Ag, isolated from both relapsing remitting patients during remission and from healthy controls. As illustrated in Fig. 2A and B, both ex vivo CD4+ T cells and MBP-peptide specific T cell proliferation were inhibited by 1,25 (OH)2 Vitamin D in concentration-dependent manner. In contrast, the analogues 24,25 (OH)2 Vitamin D and 25,26 (OH)2 Vitamin D did not inhibit CD4+ T cell proliferation (Fig. 2C and D), even after testing at concentrations 100-fold higher than those used for 1,25 (OH)2 Vitamin D. Percentages of inhibition mediated by 1,25 (OH)2 Vitamin D did not differ between relapsing remitting patients and healthy controls.

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T2 MSD

Figure 2

Vitamin D inhibits the proliferation of human CD4+ T cells and MBP peptide-specific T-cell lines. (A–B) Inhibition of CD4+ T cells (open circles) and MBP peptide-specific T-cell lines (full circles) is dose dependent. Results are presented as mean ± SEM values from 20 relapsing remitting multiple sclerosis patients and 20 control subjects. Purified CD4+ T cells (C), and MBP peptide-specific T-cell lines (D) were cultured in the presence or in the absence of 1,25 (OH)2 Vitamin D (10 nM), or the Vitamin D analogues 25,26 (OH)2 Vitamin D and 24,25 (OH)2 Vitamin D. After stimulation, T cell proliferation was examined in a standard 60-h assay measuring [3H]thymidine incorporation. Data represent mean ± SEM values from 20 relapsing remitting multiple sclerosis patients and 20 control subjects. **P < 0.0001 compared with untreated cultures. Percentages of inhibition mediated by 1,25 (OH)2 Vitamin D were similar in both relapsing remitting patients and healthy controls.

VDR is induced by T cell activation and 1,25 (OH)2 Vitamin D

VDR expression has been described in monocytes and activated lymphocytes (Veldman et al., 2000; Norman, 2006). We therefore chose to examine the expression of VDR on CD4+ T cells, isolated from relapsing remitting patients and healthy controls, using quantitative PCR. Both freshly isolated CD4+ T cells, and resting MBP-peptide specific T cells, expressed very low levels of VDR mRNA (Fig. 3). After activation, a 5- to 10-fold increase in CD4+ T cells and MBP peptide specific T-cell lines VDR expression was observed (P < 0.0001). Moreover, 1,25 (OH)2 Vitamin D caused a significant increase in VDR expression (3- to 7-fold; P < 0.0001) on both un-stimulated and activated CD4+ T cells, as well as on un-stimulated and activated MBP peptide-specific T-cell lines (Fig. 3A and B). 1,25 (OH)2 Vitamin D increased VDR expression in both relapsing remitting patients and healthy controls.

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Figure 3

T3MSZD

VDR is upregulated on activated T cells, and after exposure to 1,25 (OH)2 Vitamin D. Purified CD4+ T cells and MBP peptide-specific T-cell lines were cultured in resting state and after activation in the presence or in the absence of 1,25 (OH)2 Vitamin D (10 nM). Total RNA was extracted after 3 days in culture, and gene expression was detected by quantitative RT-PCR. Data are expressed as VDR mRNA relative to GAPDH, mean values ± SEM of mRNA expression in ex vivo CD4+ T cells isolated from 25 relapsing remitting multiple sclerosis patients, and 20 healthy controls (A), as well as from 35 myelin peptide-specific T-cell lines isolated from 20 relapsing remitting patients, and 30 myelin peptide-specific T-cell lines isolated from 20 healthy subjects (B). No significant differences were observed between relapsing remitting patients and healthy controls.

T cells metabolize 25 (OH) Vitamin D to 1,25 (OH)2 Vitamin D

Synthesis of 1,25 (OH)2 Vitamin D from the major circulating form of Vitamin D, 25 (OH) Vitamin D, is catalyzed by 1α hydroxylase, an enzyme recently found to be present in different tissues (Hewison et al., 2007). Thus, we study expression and regulation of 1α-hydroxylase mRNA by CD4+ T cells. As shown in Fig. 4A and B, both resting ex vivo CD4+ T cells and MBP-peptide specific T cells constitutively expressed 1α-hydroxylase mRNA. Levels of expression were significantly up-regulated following activation, but not further induced by 1,25 (OH)2 Vitamin D. To determine whether this up-regulation had functional consequences, two different sets of experiments were performed. First, 1α-hydroxylase activity in purified CD4+ T cells was measured. In this assay, cells were stimulated with PHA for different time periods, [3H]-25(OH) Vitamin D was included for a further 4 h, and conversion to [3H]-1,25(OH)2 Vitamin D measured using thin-layer chromatography. As shown in Fig. 4C, activity of 1α-hydroxylase was induced 8- to 10-fold after 24 h of incubation, increasing 25- to 33-fold after 48 h. In a second group of experiments, different concentrations of the precursor 25 (OH) Vitamin D were added to purified CD4+ T cells stimulated with PHA, and the proliferation of CD4+ T cells examined as read out of the assay. As illustrated in Fig. 4D, 25 (OH) Vitamin D decreased the proliferation of CD4+ T cells in a concentration-dependent manner. Collectively, these results indicate that CD4+ T cells are capable of metabolizing 25 (OH) Vitamin D to 1,25 (OH)2 Vitamin D, which in turn inhibits T cell function.

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Figure 4

T4MSD

CD4+ and autoreactive T cells can metabolize 25(OH) Vitamin D into 1,25 (OH)2 Vitamin D. Ex vivo CD4+ T cells (A) and MBP-peptide specific T cells (B) constitutively expressed 1α-hydroxylase mRNA, upregulated by activation, but not further induced by 1,25 (OH)2 Vitamin D. Cells were stimulated with PHA or the cognate peptide after 5 days in culture, 1α-hydroxylase mRNA expression was determined by quantitative RT-PCR and expressed relative to GAPDH. The mean ± SEM of eight independents experiments is shown. (C) Regulation of 1α-hydroxylase. Cells were stimulated with or without 1,25 (OH)2 Vitamin D (10 nM) for 12, 24, 48 and 72 h. [3H]-25(OH) Vitamin D was included for the final 4 h. Data shown correspond to 1,25 (OH)2 Vitamin D production (1α-hydroxylase activity). Results are expressed as mean ± SEM of triplicate cultures, represent seven independent experiments. (D) Ex vivo CD4+ T cells from relapsing remitting multiple sclerosis patients and healthy controls were stimulated with PHA (1 μg/ml) in the presence of different concentrations of 25 (OH) Vitamin D ranging from 0 to 1000 nM. T cell proliferation was examined in a 60-h assay measuring [3H]-thymidine uptake. Data represent mean ± SEM values from 10 relapsing remitting patients and 10 healthy control subjects. Percentages of inhibition were similar in both relapsing remitting patients and healthy subjects.

Effects of 1,25 (OH)2 Vitamin D on cytokine secretion by CD4+ T cells

The impact of 1,25 (OH)2 Vitamin D on cytokines production by T cells was assessed using purified CD4+ T cells as well as MBP-peptide specific T cell lines, stimulated with immobilized anti-CD3 mAb, in the presence and in the absence of 1,25 (OH)2 Vitamin D. T cells were assessed under these conditions for their ability to secrete IFN-γ, IL-4, IL-6, IL-10, and IL-17. Data on Fig. 5A and B show changes in cytokine-secreting cell numbers following culture with 1,25 (OH)2 Vitamin D relative to vehicle-treated cells in relapsing remitting patients and healthy controls. 1,25 (OH)2 Vitamin D led to increased number of IL-10 producing T cells and decreased numbers of IL-6 and IL-17 producing cells. No changes were observed in numbers of T cells producing IL-4 or IFN-γ. Similar results were achieved when T cells were stimulated either with PHA or the cognate peptide in the presence of antigen presenting cells (data not shown). These findings were similar in both relapsing remitting patients and control subjects.

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Figure 5

T55555555555555555555555555555555555555555555555555 B

1,25 (OH)2 Vitamin D increases the development of IL-10 secreting cells and down-regulates the number of IL-6 and L-17 secreting cells. Purified CD4+ T cells (A) and MBP peptide-specific T-cell lines (B) isolated from both relapsing remitting multiple sclerosis patients and healthy controls, were stimulated with 1 μg/ml of plate-bound anti-CD3 mAb in the presence or in the absence of 1,25 (OH)2 Vitamin D (10 nM). After 72 h in culture the number of cytokine secreting cells was evaluated using ELISPOT assays. In some experiments CD4+ T cells (C) or MBP peptide-specific T-cell lines (D) were incubated with IL-10 (ng/ml) alone or in combination with 1,25 (OH)2 Vitamin D. In other groups of experiments neutralizing anti IL-10 receptor mAb (10 μg/ml) was simultaneously added to culture, and the number of IL-10 secreting cells evaluated using ELISPOT assays. Results of 16 experiments are shown. *P = 0.01, §P = 0.001, **P < 0.0001, compared with untreated cells.

Because IL-10 is a positive autocrine factor enhancing development of IL-10 producing T cells, we assessed the role of IL-10 in this process. Using an antigen presenting cell free system, where T cells were stimulated with anti-CD3 mAb we found that the combination of 1,25 (OH)2 Vitamin D and IL-10 led to enhanced numbers of human IL-10 producing T cells, compared with the effects of 1,25 (OH)2 Vitamin D alone (Fig. 5C and D). In addition, the number of IL-10 producing T cells was significantly abrogated when a neutralizing anti-IL-10 receptor mAb was simultaneously added to cultures. Isotype control mAb had no effect (Fig. 5C and D). These results confirm that IL-10 is a positive autocrine factor that acts directly on T cells in the absence of antigen presenting cells, enhancing the action of 1,25 (OH)2 Vitamin D. Similar results were observed in both relapsing remitting patients and healthy controls.

To evaluate 1,25 (OH)2 Vitamin D effects on significant number of cytokine producing cells, experiments using polarized Th1, Th2, and Th17 effector cells generated in short-term cultures of purified CD4+ T cells from both relapsing remitting patients and healthy controls were performed. Polarized phenotypes were confirmed by analysing IFN-γ, IL-4 and IL-17 producing CD4+ T cell percentage as well as IFN-γ, IL-4 and IL-17 secretion levels. In addition, transcription factor T-bet (for Th1 cells), GATA-3 (for Th2 cells) and RORγt (for Th17 cells) were also measured (data not shown). As shown on Fig. 6, 1,25 (OH)2 Vitamin D significantly inhibited IL-17 production in both relapsing remitting patients and healthy controls compared with vehicle treated polarized Th17 cells, compared with 1,25 (OH)2 Vitamin D, which had no effect on IL-4 production by Th2 polarized cells. VDR agonists have been found to directly inhibit Th1 cytokines such as IL-2 and IFN-γ (Adorini and Penna, 2008). To assess the relationship between 1,25 (OH)2 Vitamin D and IFN-γ secretion, we used Th1 polarized cells. Although difference observed were not statistically significant, 1,25 (OH)2 Vitamin D treatment resulted in a trend of reduced IFN-γ production, both in relapsing remitting patients and healthy controls (P = 0.055; Fig. 6)

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Figure 6

T6MDD

1,25 (OH)2 Vitamin D downregulated IL-17 production in Th17 polarized cells. Purified CD4+ T cells isolated from relapsing remitting multiple sclerosis patients and healthy controls were polarized as described in Materials and Methods to obtain Th1, Th2 and Th17 cells. Polarized T helper cells were stimulated with plate bound anti-CD3 mAb, and with supernantant collected at 36 (IL-4), and 72 h (IFN-γ and IL-17). Cytokine secretion was measured using ELISA. Data represent mean ± SEM values from 20 relapsing remitting patients and 20 control subjects. **P < 0.0001, ‡P = 0.055, compared with untreated cells.

1,25 (OH)2 Vitamin D induces CD4+CD25+ FoxP3+ T cells through an IDO-mediated pathway

IDO is a key enzyme in tryptophan metabolism, catalysing its degradation to generate kynurenine. IDO induced tryptophan catabolism suppresses in turn T cell responses and promotes immune tolerance in autoimmunity, mammalian pregnancy and allergic inflammation. RT-PCR analysis indicated IDO mRNA presence in dendritic cells derived from both relapsing remitting patients and healthy controls. Likewise, both resting ex vivo CD4+ T cells, and MBP-peptide specific T cells from both populations constitutively expressed IDO mRNA, although at significantly lower levels (Fig. 7A; P < 0.0001). Expression levels were significantly increased after 1,25 (OH)2 Vitamin D exposure (Fig. 7B; P < 0.0001). Culture of MBP-peptide specific T cells in complete medium containing tryptophan in the presence of 1,25 (OH)2 Vitamin D, induced production of significantly higher levels of kynurenine, compared with those observed in the absence of 1,25 (OH)2 Vitamin D. Interestingly, the addition of IDO inhibitor 1-MT significantly abrogated kynurenine production (Fig. 7C). These data suggest that IDO expressed by CD4+ T cells is functionally active. IDO has a role in regulatory T cell generation under both physiological and pathological conditions. Likewise, 1,25 (OH)2 Vitamin D and its analogues enhance CD4+CD25+ regulatory T cell numbers, inhibiting autoimmune diseases and graft rejection in different experimental models. On the basis of this information, we studied IDO and 1,25 (OH)2 Vitamin D regulatory T cell generating capacity, for which PBMC were cultured in the presence and in the absence of 1,25 (OH)2 Vitamin D and the IDO inhibitor 1-MT. As shown in Fig. 7D culture of PBMC in the presence of 1,25 (OH)2 Vitamin D significantly increased CD4+CD25+FoxP3+ T cell percentage. The addition of 1-MT to cultures restored the of CD4+CD25+FoxP3+ T cell percentage to levels observed prior to adding 1,25 (OH)2 Vitamin D (Fig. 7D), suggesting that Vitamin D induces CD4+CD25+FOxP3+ regulatory T cells through an IDO dependent pathway. Cell viability when cultured in presence of 1-MT did not differ from that of cells cultured in medium alone. We also analysed IDO mRNA expression on CD4+CD25-, and CD4+CD25+ T cells, in both relapsing remitting patients and healthy controls, and found no differences between either cell population (Fig. 8A).

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Figure 7

 

(A) Dendritic cells, and CD4+ T cells from relapsing remitting multiple sclerosis patients, and healthy controls constitutively expressed IDO. (B) IDO expression was up-regulated by 1,25 (OH)2 Vitamin D. Cells were cultured for 3 days in the presence and in the absence of 1,25 (OH)2 Vitamin D, IDO mRNA expression was determined by quantitative RT-PCR and expressed relative to GADPH. (C) Ex vivo CD4+ derived from relapsing remitting patients and healthy controls, were cultured in medium supplemented with additional tryptophan with or without 1,25 (OH)2 Vitamin D for 48 h. Kynurenine in the culture supernatant was measured by HPLC. The addition of the IDO inhibitor 1-MT abrogated the 1,25 (OH)2 Vitamin D effect. (D) Culture of PBMC in the presence of 1,25 (OH)2 Vitamin D significantly increased the percentage of CD4+CD25+FoxP3+ regulatory T cells as measured by flow cytometry. Once again the addition of the IDO inhibitor 1-MT abrogated 1,25 (OH)2 Vitamin D effect. Data represent mean ± SEM values from 20 relapsing remitting patients and 18 control subjects. **P < 0.0001.

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Figure 8

 

T8 MSF

(A) Both CD4+CD25− and CD4+ CD25+ T cells constitutively expressed similar amounts of IDO mRNA. (B) CD4+CD25+FoxP3+ regulatory T cells induced by 1,25 (OH)2 Vitamin D mediated suppression of proliferation induced by anti-CD3/anti CD-28 in CD4+CD25− target T cells, titrating from high to low ratios of CD4+CD25+: CD4+CD25−. Data represent mean values ± SEM of 16 experiments. (C) CD4+CD25+FoxP3+ regulatory T cells induced by 1,25 (OH)2 Vitamin D were potent suppressors of IFN-γ secretion induced by anti-CD3/anti-CD28 in CD4+CD25− target cells. Data represent mean values ± SEM of 16 experiments. (D) CD4+CD25+Foxp3+ T cells induced by 1,25 (OH)2 Vitamin D were co-cultured with CD4+CD25− T cells at a ratio 10:1, and stimulated with anti-CD3 and soluble anti-CD28 mAbs, both at 5 μg/ml concentrations in the presence of irradiated PBMC, depleted of CD3+ T cells as a source of antigen presenting cells; 1 MT was added at a concentration of 1000 μM. Data represent mean ± SEM values from 18 relapsing remitting multiple sclerosis patients and 16 healthy controls. (E) Culture conditions were similar to those described for panel (D). To measure IFN-γ, supernatant was removed from each well and analyzed using commercially available ELISA kits. Data represent mean ± SEM values from 18 relapsing remitting patients and 16 healthy controls. (F) MBP- and MOG-peptide-specific T cell lines from relapsing remitting patients and control subjects were cultured as described in Materials and methods section, in the presence and in the absence of 1,25 (OH)2 Vitamin D, and 1 MT at 1000 μM concentration. Data represent mean ± values from 18 relapsing remitting patients and 18 healthy controls. Similar results were observed using ex vivo CD4+ T cells. **P < 0.0001 and §P = 0.001 compared with 1,25 (OH)2 Vitamin D treated cultures.

Regulatory properties of CD4+CD25+ FoxP3+ T cells isolated after 1,25 (OH)2 Vitamin D exposure were further investigated by testing ability of these cells to suppress proliferative responses and IFN-γ secretion by CD4+CD25− cells. To do this, CD4+CD25− T cells were stimulated with anti-CD3 and anti-CD28 mAb, while increasing numbers of autologous CD4+CD25+ cells were added. As illustrated in Fig. 8B CD4+CD25+FoxP3+ cells generated by exposure to 1,25 (OH)2 Vitamin D were able to suppress the proliferation of indicator CD4+CD25− T cells, titrating from high to low ratios of CD4+CD25+:CD4+CD25−. Furthermore, CD4+D25+FoxP3+ cells suppressed the proliferation of CD4+CD25− T cells in response to PHA (66 ± 18%, at ratio 10:1), and to plate-bound anti-CD3 stimulation (75 ± 20%, at ratio 10:1), suggesting that the suppressive effect of CD4+D25+FoxP3+ is independent of antigen presenting cells. Moreover, prevention of cell contact abolished the regulatory function that CD4+CD25+ Treg cells exert on CD4+CD25− T cells (data not shown). In addition, CD4+CD25+FoxP3+ T cells generated after 1,25 (OH)2 Vitamin D exposure were also capable of suppressing the production of IFN-γ by CD4+CD25− T cells activated by anti CD3/CD28 mAbs (Fig. 8C). Overall, these data suggest that 1,25 (OH)2 Vitamin D induced CD4+CD25+FoxP3+ T cell development through an IDO-mediated pathway while retaining immunosuppressive activity and may thus be considered bona fide regulatory T cells. Addition of 1MT to culture media did not affect the suppressive action of CD4+CD25+ Treg cells on CD4+ T cell proliferation (Fig. 8D), or IFN-γ secretion (Fig. 8E); suggesting that IDO has impact on Treg cell development induced by Vitamin D, but does not affect regulatory properties of these same cells. In contrast, addition of IDO inhibitors to either CD4+ T cells or MBP-peptide specific T cell cultures abrogated 1,25 (OH)2 Vitamin D mediated inhibitory effects on T cells proliferation (Fig. 8F). This effect was significantly higher in cultures from healthy subjects, suggesting differences in IDO-mediated tryptophan deprivation.

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Discussion

In this study, we show that relapsing remitting patients, particularly during exacerbations, have reduced serum levels of 25 (OH) Vitamin D, and 1,25 (OH)2 Vitamin D compared with healthy subjects, suggesting that in this group of patients Vitamin D-dependent T cell regulation may play an important role in maintaining T cell homeostasis. Recently, high circulating levels of 25 (OH) Vitamin D have been associated with a lower risk of MS (Munger et al., 2006). The results of this study showed serum levels of 25 (OH) Vitamin D, and 1,25 (OH)2 Vitamin D in primary progressive patients were similar to those observed in healthy individuals. Consistent with these observations, a growing body of evidence indicates that distinct pathogenic processes mediate brain damage in different groups of multiple sclerosis patients. Active focal inflammatory demyelinating lesions in the white matter are mainly present in patients with relapsing remitting multiple sclerosis for example, while diffuse injury of normal-appearing white matter, cortical demyelination and signs of oligodendrocyte dystrophy are mainly hallmarks of the primary progressive disease (Lucchinetti et al., 2000; Kutzelnigg et al., 2005). Moreover, while immunomodulatory and immunosuppressive treatments are beneficial during acute and relapsing stages of the disease, they have no effect in primary progressive patients (Noseworthy et al., 2000; Goodin et al., 2002). Overall, these findings would appear to indicate that pathological features additional to classic inflammatory demyelinating lesions might be important in primary progressive pathogenesis.

Serum concentration of 25 (OH) Vitamin D is a reflection of the balance between Vitamin D intake in food, its synthesis from pro-vitamins in the skin under the influence of UV light, and its catabolism by 24-hydroxylase. Thus, either a decrease in synthesis or an increase in 25 (OH) Vitamin D degradation could explain low serum levels observed in multiple sclerosis patients. Some studies associating higher dietary intake of Vitamin D with protection against autoimmune diseases have been published (Hyppönen et al., 2001; Merino et al., 2004), and inverse correlation between multiple sclerosis prevalence and sunlight exposure has been observed both in early ecological studies, as well as in recent case–control ones (Acheson et al., 1960; van der Mei et al., 2003; Islam et al., 2007). Likewise, seasonal changes in Vitamin D levels have also been reported as low during winter months and elevated in summer ones. Furthermore, correlation of seasonality has also been found between low Vitamin D levels and multiple sclerosis flares (Bamford et al., 1983). Despite the fact that both patients and control subjects included in this investigation lived in the same area, and that samples from both groups were collected during the same months of the year, it is important to consider that patients frequently have difficulty exercising or moving about and therefore spend less time outdoors and are less exposed to sunlight. Measurement of sunlight exposure in patients compared with control subjects using specific questionnaires is currently under investigation. Likewise, influence of 24-hydroxylase on 25 (OH) Vitamin D levels, not assessed in this study, is now under examination in ongoing studies at our laboratory.

1,25 (OH)2 Vitamin D effects are mediated primarily through interaction with intracellular VDR (Adorini and Penna, 2008), although recent reports have highlighted rapid non-genomic effects of 1,25 (OH)2 Vitamin D (Norman, 1998). VDR is present in different tissues including cells of the immune system such as monocytes, dendritic cells and B cells (Veldman et al., 2000; Norman, 2006; Chen et al., 2007). We demonstrated that VDR mRNA is constitutively expressed in CD4+ T cells, and is upregulated following activation and 1,25 (OH)2 Vitamin D exposure. Likewise, 1-α hydroxylase, mainly found in renal tubule cells, was recently described in a wide variety of tissues (Hewison et al., 2007), and cloning studies have revealed the same mRNA for 1-α hydroxylase in renal and extra-renal tissues (Monkawa et al., 1997). However, although the enzyme present in immune cells is identical to the renal form, its regulation seems to be under a different control system, mediated mainly by immune signals, such as antigenic stimulators or inflammatory mediators like IFN-γ (Hewison et al., 2003; Overbergh et al., 2006). Interestingly, we found that 1-α hydroxylase mRNA was also expressed in resting CD4+ T cells at low levels, and increased its expression significantly after stimulation, but not following 1,25 (OH)2 Vitamin D exposure. Moreover, we found that 25 (OH) Vitamin D could be metabolized to 1,25 (OH)2 Vitamin D by CD4+ T cells, representing local production of active Vitamin D, as was previously demonstrated for macrophages, dendritic cells and thyroid tissue.

Activation of the VDR is known to alter transcription, proliferation and differentiation of immune cells (Dong et al., 2005; Muthian et al., 2006; Adorini and Penna, 2008). In this study, we demonstrated that activated Vitamin D has different immunoregulatory effects on CD4+ T cells, namely it (i) inhibits CD4+ T cell proliferation; (ii) enhances IL-10 secreting cell development, and inhibits IL-6 and IL-17 producing cell development; and (iii) induces CD4+CD25+ FoxP3+ regulatory T cells through an IDO-mediated pathway.

1,25 (OH)2 Vitamin D inhibition of cell proliferation has been reported in lymphocytes and different cancer cell lines (Munker et al., 1996; Li et al., 2004; Chen et al., 2007; Smolders et al., 2008). Proliferation inhibition has been linked to different mechanisms, such as inducing gene transcription of CDK inhibitors p21 and p27, which inhibit cell cycle progression (Liu et al., 2002; Li et al., 2004; Chen et al., 2007). Alternatively, 1,25 (OH)2 Vitamin D may help to limit uncontrolled proliferation through apoptosis induction, since dendritic cells stimulated with 1,25 (OH)2 Vitamin D or Vitamin D analogues induce T cell apoptosis (van Halteren et al., 2004). Interestingly, in a recent study apoptosis induction of activated CD4+ T cells was observed in the CNS and spleen of EAE animals (Pedersen et al., 2007).

Active Vitamin D is known to inhibit the activation and action of different transcriptions factors involved in cytokine gene regulation (Alroy et al., 1995; D’Ambrosio et al., 1998; Takeuchi et al., 1998). A combination of 1,25 (OH)2 Vitamin D and dexamethasone has been shown to induce human and mouse naïve CD4+ T cells to differentiate in vitro into IL-10 secreting cells (Barrat et al., 2002). In the present study, upregulation of IL-10 by 1,25 (OH)2 Vitamin D stimulation was also present in CD4+ T cells from MS patients in the absence of dexamethasone. Similar results have been previously shown in patients with Crohn’s disease (Bartels et al., 2007). Our data also showed that IL-10 itself had synergistic action with 1,25 (OH)2 Vitamin D. These observations are supported by experiments in the EAE model, where 1,25 (OH)2 Vitamin D significantly inhibits the disease in wild-type animals, but not in IL-10 or IL-10 receptor knockout strains, indicating that the IL-10/IL-10 receptor pathway is essential for 1,25 (OH)2 Vitamin D-mediated EAE inhibition (Spach et al., 2006). Aside from up-regulated IL-10 production, we observed significant down-regulation of IL-6, and IL-17 secreting T cells. Investigators have found TGF-β, and IL-6 are critical factors for Th17 development (Weaver et al., 2007). These data are in agreement with the known inhibition of IL-17 production by VDR agonists during the course of experimental autoimmune prostatitis (Penna et al., 2006). In contrast to other groups, we observed no significant effect on IFN-γ production. Even when CD4+ T cells were polarized to a Th1 phenotype, 1,25 (OH)2 Vitamin D treatment resulted in a trend of reduced IFN-γ production, although the level of reduction was not statistically significant. 1,25 (OH)2 Vitamin D capacity to inhibit T cell IFN-γ production has been previously reported, but could not be confirmed by other studies (Cantorna et al., 1998; Nashold et al., 2001; Mahon et al., 2003; Muthian et al., 2006). Overall, these data support the notion that 1,25 (OH)2 Vitamin D plays an important role in shaping the development of T cell responses, inducing T cells with immunosuppressive properties. It is important to note that in these experiments 1,25 (OH)2 Vitamin D was used at pharmacological doses ∼82- to 525-fold higher than circulating levels found in healthy individuals. It appears likely that increasing Vitamin D levels could reduce the risk of multiple sclerosis. If so, it is important to establish optimal Vitamin D levels, and to verify whether Vitamin D supplements administered at these levels contribute to prevention without producing side effects.

An important effect of 1,25 (OH)2 Vitamin D is the induction of T cells with regulatory properties (Adorini and Penna, 2008). Regulatory T cells are induced by the modulation of antigen presenting cells, particularly dendritic cells. Notably, in vitro treatment of dendritic cells with 1,25 (OH)2 Vitamin D leads to down-regulated expression of co-stimulatory molecules and reduced IL-12 production, with acquisition of a tolerogenic phenotype (Adorini, 2003). These dendritic cells could favour peripheral tolerance not only by inhibiting effector T cell activation, but also by inducing the differentiation of CD4+CD25+FoxP3+ regulatory T cells able to arrest the development of autoimmune responses. In our study the percentage of CD4+CD25+FoxP3+ was significantly increased when PBMC were cultured in the presence of 1,25 (OH)2 Vitamin D. In the present study we show that this process is mediated by an IDO-dependent pathway.

IDO has been widely investigated for the induction of immunological tolerance, and its expression associated with an increased number of circulating CD4+CD25+FoxP3+ T cells in physiological and pathological conditions (Munn et al., 1998; Uyttenhove et al., 2003). This effect could be explained through different mechanisms: (i) expansion of CD4+CD25+FoxP3+; (ii) increased apoptosis of CD4+CD25− over CD4+CD25+ T cells; and (iii) conversion of CD4+CD25− into CD4+CD25+ T cells (Curti et al., 2007). Different models support the notion that microenvironments containing reduced tryptophan concentration and high kynurenine concentration favor the emergence of CD4+CD25+FoxP3+ T cells by conversion from CD4+CD25- T cells (Fallarino et al., 2006; Curti et al., 2007). Thus, the role of IDO has shifted from that of a metabolic regulator of tryptophan availability to one that is central to immune homeostasis. Indeed, it has been recently proposed that IDO acts as a bridge between dendritic cells and CD4+ regulatory T cells (Puccetti and Grohmann, 2007).

Overall, 1,25 (OH)2 Vitamin D affects the immune system at different levels through different mechanisms, conferring on the whole an immunosuppressive effect. Correction of 1,25 (OH)2 Vitamin D deficiency may be useful to suppress autoimmune disorders such as MS. Nevertheless, before conclusions can be drawn, further investigations are needed to determine optimal dose. Similarly, studies are warranted to compare efficacy and safety of different Vitamin D analogues with less hypercalcemic effects.

Abbreviations:

Ag

Antigen

EAE

experimental allergic encephalomyelitis

IDO

indoleamine 2,3-dioxygenase

PBMC

peripheral blood mononuclear cells

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April 2013 136 (4)

OXFORD OPEN

PUBMEDCENTRAL

THE NATURAL HISTORY OF MULTIPLE SCLEROSIS: A GEOGRAPHICALLY BASED STUDY: I. CLINICAL COURSE AND DISABILITY

Vitamin D Deficiency

Vitamin D Deficiency

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If you shun the sun, suffer from milk allergies, or adhere to a strict vegetarian diet, you may be at risk for vitamin D deficiency. Known as the sunshine vitamin, vitamin D is produced by the body in response to sunlight. It is also occurs naturally in a few foods — including some fish, fish liver oils, and egg yolks — and in fortified dairy and grain products.

Vitamin D is essential for strong bones because it helps the body use calcium from the diet. Traditionally, vitamin D deficiency has been associated with rickets, a disease in which the bone tissue doesn’t properly mineralize, leading to soft bones and skeletal deformities. But increasingly, research is revealing the importance of vitamin D in protecting against a host of health problems.

See More About the Benefits of Vitamin D

 

Symptoms and Health Risks of Vitamin D Deficiency

Symptoms of bone pain and muscle weakness can mean you have a vitamin D deficiency. However, for many people, the symptoms are subtle. Yet even without symptoms, too little vitamin D can pose health risks. Low blood levels of the vitamin have been associated with the following:

  • Increased risk of death from cardiovascular disease
  • Cognitive impairment in older adults
  • Severe asthma in children
  • Cancer

Research suggests that vitamin D could play a role in the prevention and treatment of a number of different conditions, including type1 and type 2 diabetes, hypertension, glucose intolerance, and multiple sclerosis.

 

Causes of Vitamin D Deficiency

Vitamin D deficiency can occur for a number of reasons:

You don’t consume the recommended levels of the vitamin over time. This is likely if you follow a strict vegetarian diet, because most of the natural sources are animal-based, including fish and fish oils, egg yolks, cheese, fortified milk, and beef liver.

Your exposure to sunlight is limited.Because the body makes vitamin D when your skin is exposed to sunlight, you may be at risk of deficiency if you are homebound, live in northern latitudes, wear long robes or head coverings for religious reasons, or have an occupation that prevents sun exposure.

You have dark skin. The pigment melanin reduces the skin’s ability to make vitamin D in response to sunlight exposure. Some studies show that older adults with darker skin are at high risk of vitamin D deficiency.

Your kidneys cannot convert vitamin D to its active form. As people age their kidneys are less able to convert vitamin D to its active form, thus increasing their risk of vitamin D deficiency.

Your digestive tract cannot adequately absorb vitamin D. Certain medical problems, including Crohn’s disease, cystic fibrosis, and celiac disease, can affect your intestine’s ability to absorb vitamin D from the food you eat.

You are obese. Vitamin D is extracted from the blood by fat cells, altering its release into the circulation. People with a body mass index of 30 or greater often have low blood levels of vitamin D.

 

Further Reading:

Vitamin D Deficiency

(continued)

 

Tests for Vitamin D Deficiency

The most accurate way to measure how much vitamin D is in your body is the 25-hydroxy vitamin D blood test. A level of 20 nanograms/milliliter to 50 ng/mL is considered adequate for healthy people. A level less than 12 ng/mL indicates vitamin D deficiency.

 

Treatment for Vitamin D Deficiency

Treatment for vitamin D deficiency involves getting more vitamin D — through diet and supplements. Although there is no consensus on vitamin D levels required for optimal health — and it likely differs depending on age and health conditions — a concentration of less than 20 nanograms per milliliter is generally considered inadequate, requiring treatment.

Guidelines from the Institute of Medicine increased the recommended dietary allowance (RDA) of vitamin D to 600 international units (IU) for everyone aged 1-70, and raised it to 800 IU for adults older than 70 to optimize bone health. The safe upper limit was also raised to 4,000 IUs.

If you don’t spend much time in the sun or always are careful to cover your skin (sunscreen inhibits vitamin D production), you should speak to your doctor about taking a vitamin D supplement, particularly if you have risk factors for vitamin D deficiency.

Further Reading:

http://www.webmd.com/diet/vitamin-d-deficiency

—-

Doenças autoimunitárias e neurodegenerativas: sobre a gravidade da deficiência de hormônio imunoregulador e o tratamento com reposição da Vitamina D – Dr. Cícero Galli Coimbra PhD, MD, Presidente do Instituto de Investigação e Tratamento de Autoimunidade

Doenças autoimunitárias e neurodegenerativas: sobre a gravidade da deficiência de hormônio imunoregulador e o tratamento com reposição da Vitamina D – Dr. Cícero Galli Coimbra PhD, MD, Presidente do Instituto de Investigação e Tratamento de Autoimunidade

 155336_345790338850350_1260490623_n

Jornalista portador de esclerose múltipla dirigiu vídeo retratando a experiência de pacientes beneficiados: Vitamina D – por uma outra terapia

http://www.youtube.com/watch?feature=player_detailpage&v=erAgu1XcY-U

http://biodireitomedicina.wordpress.com/2012/04/12/vitamina-d-por-uma-outra-terapia/

 

Mais de 10 anos de tratamento com vitamina D para esclerose múltipla

http://www.youtube.com/watch?feature=player_detailpage&v=fQN32qR_M2Y

Entrevistas com Junia, Márcia e Nayra sobre a experiência da família com o tratamento da vitamina D. Nayra descobriu a EM há mais de 10 anos e é provavelmente uma das pacientes mais antigas tratando a EM com o Dr. Cícero Coimbra

 

Dr Cicero Galli Coimbra e Daniel Cunha – Vitamina D Sem Censura

http://www.youtube.com/watch?feature=player_detailpage&v=v4E4_vCdo-0

Dr. Cícero Galli Coimbra e Marcelo Palma – Esclerose múltipla e o tratamento  com a  vitamina D – 28.01.13 – TV Mundi

http://www.youtube.com/watch?v=hv6tD3B0Nlo&list=PLeqEGmvbpULNrc8biL5LF9Mp3-WbJT2Ao

http://www.youtube.com/watch?list=PLeqEGmvbpULNrc8biL5LF9Mp3-WbJT2Ao&feature=player_detailpage&v=hv6tD3B0Nlo

Entrevistas com Dr. Cícero Galli Coimbra sobre o hormônio-vitamina D http://www.youtube.com/playlist?list=PLeqEGmvbpULN2NfNfnLU6bYse4fp9alQS

Vitamina D é um hormônio vital para preservação e recuperação da saúde

http://www.facebook.com/VitaminaD.HormonioVital

 

Vitamina D – 10.000 UI diárias podem preservar sua saúde e sua vida – legendado

http://www.youtube.com/watch?v=PhRJelmTUpQ

 

“Infelizmente este conhecimento não tem sido levado aos livros textos de medicina e isso gera esse desconhecimento, não só do público em geral mas até da classe médica em relação á gravidade dessa situação sobre a deficiência desse hormonio esteroide, conhecido como vitamina D. Esta deficiencia torna as 229 funções do sistema imunológico do próprio organismo das pessoas desregulado, permitindo desenvolver qualquer doença, o que levou á pandemia do mundo atual.” – Dr. Cicero Galli Coimbra, medico neurologista, Phd., MD, neurocirurgião, neurocientista, professor na UNIFESP

 https://objetodignidade.wordpress.com/2012/12/09/por-um-novo-paradigma-de-conduta-e-tratamento-estamos-vivendo-uma-defasagem-entre-o-conhecimento-cientifico-e-a-pratica-medica-dr-cicero-galli-coimbra/

DANOS À SAÚDE PODEM SER EVITADOS

A vital importância do hormônio conhecido por Vitamina D3 para a preservação ou recuperação de sua saúde de doenças autoimunes: exijam que seus médicos se atualizem

23/12/2012 — Celso Galli Coimbra

http://biodireitomedicina.wordpress.com/2012/12/23/vitamina-d3-e-sua-saude/

 

Vitamina D revoluciona tratamento da esclerose múltipla e todas autoimunes

http://biodireitomedicina.wordpress.com/2010/08/03/vitamina-d-pode-revolucionar-o-tratamento-da-esclerose-multipla/

• a ingestão de 10.000 UI / dia de vitamina D3 não tem qualquer toxicidade.

Doses elevadas de vitamina D precisam de acompanhamento medico permanente, exames clínicos, dieta e hidratação diárias orientadas por medico atualizado para não danificar a saúde do paciente.

 

Colecalciferol, a vitamina D3,10.000 UI ou 10 ng/ml tem valor preventivo e não tem efeitos colaterais . Esta dose é vendida nas farmácias nos Estados Unidos sem receita medica, porque não faz mal algum. Previne doenças.

 

OS PERIGOSOS LAÇOS DA MEDICINA COM A INDÚSTRIA FARMACÊUTICA – VITAMINA D COMENTADO

25/12/2012 — Celso Galli Coimbra

http://biodireitomedicina.wordpress.com/2012/12/25/os-perigosos-lacos-da-medicina-com-a-industria-farmaceutica-vitmina-d-comentado/

 

A responsabilidade Civil e Criminal Médica na Desinformação às pessoas – Revista VEJA, 2.304: “O que você não sabe sobre a Vitamina do Sol. Ela continua a surpreender a medicina com novos efeitos benéficos.”

20/01/2013 — Celso Galli Coimbra

http://biodireitomedicina.wordpress.com/2013/01/20/a-responsabilidade-civil-e-criminal-medica-na-desinformacao-as-pessoas-revista-veja-2-304-o-que-voce-nao-sabe-sobre-a-vitamina-do-sol-ela-continua-a-surpreender-a-medicina-com-novos-efe/

”ATENÇÃO: o uso preventivo do Vitamina D3 é DIFERENTE do uso terapêutico deste hormônio-vitamina, que exige sempre a orientação e acompanhamento de médico com treinamento adequado para ser responsável pela avaliação caso a caso e a específica determinação de dosagem, em contrário haverá sérios danos à saúde.”

—————

 

“NAS CÉLULAS DO SISTEMA IMUNOLÓGICO, a vitamina D tem a função de produzir o que se chama de tolerância imunológica, ou seja, de impedir que essas células agridam o próprio organismo, que é o que acontece nas doenças autoimunitárias”, explica Coimbra. Nas pessoas com pré-disposição genética para doenças autoimunitárias, a transformação da vitamina D inativa em ativa (hidroxilase) dentro das células do sistema imunológico é lenta, o que favorece o surgimento desse tipo de doença. “Hoje, já se sabe que o risco de esclerose múltipla aumenta quando se têm níveis baixos de vitamina D. O que propomos é a elevação dos níveis de vitamina D ao ponto máximo que não provoque efeitos tóxicos ao organismo. O sucesso do tratamento com vitamina D vem sendo demonstrado.”

 

“A esclerose múltipla, bem como as outras doenças do sistema imunológico, é um mal dos tempos modernos – e isso também tem a ver com o sol. Nossos antepassados sofriam muito menos com isso. “Nossos avós tinham uma vida na lavoura, iam à feira livre fazer compras. Hoje, nós pegamos o metrô, descemos num shopping center, entramos num carro com Insulfim, descemos na garagem de um prédio e subimos de elevador. Como toda doença autoimunitária, a esclerose múltipla aumentou muito nos dias atuais. Nosso nível de exposição solar é hoje quase o mesmo que o dos ratos de laboratório”, adverte Cícero Coimbra.”

Fonte: www.biodireitomedicina.worpdpress.com

 

Desenvolvido no Brasil em 2003, pelo neurologista Dr. Cícero Galli Coimbra, PhD, MD, da Universidade Federal de São Paulo (Unifesp), o tratamento com vitamina D consiste na reposição deste natural hormonio imunoregulador presente no organismo de todos os seres vivos há 750 milhões de anos. Segundo o médico, cerca de 70% das pessoas que sofrem de esclerose múltipla ou outras doenças autoimunes apresentam níveis muito baixos desse nutriente, o que se correlaciona com uma frequência maior de manifestações – surtos no caso da esclerose múltipla e com sequelas neurológicas mais acentuadas após cada ocorrência.

 

Desde então, a experiência clínica de Dr. Cícero Galli Coimbra com pacientes de esclerose múltipla no país tem apresentado quadros de estabilidade da doença, regressão de sequelas — com o retorno às atividades físicas — e até mesmo melhoras em lesões no cérebro e na medula, reveladas por ressonâncias magnéticas. O mesmo protocolo é eficaz no tratamento de outras doenças autoimunitárias, como artrite reumatoide, lúpus, psoríase, vitiligo, diabetes e hipotireoidismo.

 

Dr. Cicero Galli Coimbra fala sobre prevenção e tratamento de doenças neurodegenerativas e autoimunes com Vitamina D

Baixos índices de vitamina D3 no sangue estão diretamente associados ao estresse emocional ou sofrimento. Em casos de doenças autoimunitárias, tais como a esclerose múltipla, artrite reumatoide, psoriase, hipertireoidismo, hipotireoidismo, lupus, vitiligo, por exemplo, existe deficiência de vitamina D confirmada em exames de sangue. Esta deficiência de vitamina D torna as pessoas mais suscetíveis à depressão e aos estados de sofrimento emocional, que são as condições adequadas à perda de massa neural, o envelhecimento do sistema nervoso. Por outro lado, a solução simples, para estas pessoas, é o consumo de altas doses de vitamina D3. A vitamina D é capaz de produzir um estado de bem-estar indescritível, e unida ao estado de tranquilização, permite a obtenção de uma condição de estabilização e recuperação do sistema nervoso. É importante que se saiba, em condições de equilíbrio — vitaminas deficientes complementadas e o aspecto emocional tranquilo –, voltam a nascer células-tronco, e novos neuronios, todos os dias

Estas informações foram expostas na entrevista com Dr. Cícero Galli Coimbra sobre o sistema nervoso, o estresse emocional, depressão, doenças e o envelhecimento dos neurônios. A importancia da Vitamina D foi comentada e explicada por Dr. Cícero Galli Coimbra em:

http://www.youtube.com/watch?feature=player_detailpage&v=yRQkITHjZ5k

“a situação fundamental é a mesma: a existência de um distúrbio metabólico evidente e corrigível, capaz de explicar os eventos fisiopatológicos conhecidos, e cuja correção pode deter a progressão da doença (interrompendo a continuidade da morte neuronal crônica, recuperando células neuronais já afetadas pelo processo neurodegenerativo — mas que não atingiram ainda o ponto de irreversibilidade), promover a recuperação total em casos de início recente, ou ao menos parcial das deficiências neurológicas nos casos mais avançados  (minimizando seqüelas permanentes) e impedir a morte.” [1]

[1]Dr. Cícero Galli Coimbra

PhD, MD, Médico Neurologista e Professor Livre-Docente

Departamento de Neurologia e Neurocirurgia — Universidade Federal de São Paulo — Unifesp/EPM — Sofrimento emocional. — Em defesa da administração de doses elevadas de riboflavina associada à eliminação dos fatores desencadeantes no tratamento (…).

Disponivel em http://www.unifesp.br/dneuro/nexp/riboflavina/c.htm

 

 ‘‘O benefício da vitamina D fica ainda mais nítido, diz Coimbra, se observarmos que os casos de esclerose múltipla são muito mais frequentes nos países nórdicos, como as nações escandinavas e o Canadá, onde a exposição da população aos raios solares é muito baixa. O sol, como se sabe, é a principal fonte de vitamina D com a qual contamos. “A radiação solar da manhã e do final da tarde faz com que o nosso organismo produza vitamina D. Uma pessoa que fique na beira da piscina de sunga, com 90% do corpo exposto ao sol por apenas 10 minutos, produz mais vitamina D do que a contida na dose diária normalmente recomendada pelo médicos. Mas atenção: o mesmo não acontece com o sol do meio-dia, que provoca câncer de pele”, orienta o médico.’’-

Sobre a vitamina D, Dr. Cícero Galli Coimbra explica que “essa substancia é na realidade um hormônio esteroide e que, por infelicidade, entre 1918 e 1922, foi chamada de vitamina D antes que se conhecesse qual era a verdadeira estrutura química dessa substancia. É importante que se saiba que este hormonio é o principal determinante do estado de saúde do mundo moderno, hoje vivendo uma pandemia de doenças autoimunitárias, neurodegenerativas e todos os tipos de infecções.”

“O hormonio esteroide conhecido como vitamina D, é uma substancia química que controla 229 funções em cada uma de todas as nossas células do sistema imune. A deficiencia deste hormônio esteroide é praticamente um pré-requisito para desenvolver qualquer doença autoimunitária, para desenvolver câncer, doenças cardiovasculares, diabetes, infecções. Em termos de deficiência de vitamina D, durante a gestação, leva ao nascimento de crianças autistas.”

Fonte: http://biodireitomedicina.wordpress.com/

Sua depressão pode ser devido à deficiência de D de vitamina

04/04/2013 — Celso Galli Coimbra

http://biodireitomedicina.wordpress.com/2013/04/04/sua-depressao-pode-ser-devido-a-deficiencia-de-d-de-vitamina/

Falta de luz solar piora estado de pessoas com depressão. Falta do hormônio-vitamina D

04/04/2013 — Celso Galli Coimbra

http://biodireitomedicina.wordpress.com/2013/04/04/falta-de-luz-solar-piora-estado-de-pessoas-com-depressao-falta-do-hormonio-vitamina-d/

O Hormônio-Vitamina D interfere com cerca de 3.000 genes associados a muitas doenças, diminuindo os seus riscos

04/04/2013 — Celso Galli Coimbra http://biodireitomedicina.wordpress.com/2013/04/04/vitamina-d-interfere-com-cerca-de-3-000-genes-associados-a-muitas-doencas-diminuindo-os-seus-riscos/

__

A verdade sobre a vitamina D é uma enorme ameaça para o sistema estabelecido do lucro médico-indústrias farmaceuticas

 

• a ingestão de 10.000 UI / dia de vitamina D3 não tem qualquer toxicidade.

Doses elevadas de vitamina D precisam de acompanhamento medico permanente, exames clínicos, dieta e hidratação diárias orientadas por medico atualizado para não danificar a saúde do paciente.

 

Colecalciferol, a vitamina D3,10.000 UI ou 10 ng/ml tem valor preventivo e não tem efeitos colaterais . Esta dose é vendida nas farmácias nos Estados Unidos sem receita medica, porque não faz mal algum. Previne doenças.

 

OS PERIGOSOS LAÇOS DA MEDICINA COM A INDÚSTRIA FARMACÊUTICA – VITAMINA D COMENTADO

25/12/2012 — Celso Galli Coimbra

http://biodireitomedicina.wordpress.com/2012/12/25/os-perigosos-lacos-da-medicina-com-a-industria-farmaceutica-vitmina-d-comentado/

 

“As doses diárias de 10.000 unidades de colecalciferol devem ser tomadas por todas pessoas. Essa quantidade previne todas as doenças inclusive à autoimunidade. Com 10.000 unidades a pessoa sai da deficiencia de vitamina D. A dose de 1.000 unidades não tira as pessoas da deficiencia de vitamina D.’’ – Dr. Cicero Galli Coimbra, medico neurologista, Phd., professor na Universidade Federal de São Paulo, Presidente do Instituto de Investigação e Tratamento de Autoimunidade

https://objetodignidade.wordpress.com/2013/01/21/as-doses-diarias-de-10-000-unidades-de-colecalciferol-devem-ser-tomadas-por-todas-pessoas-essa-quantidade-previne-todas-as-doencas-inclusive-a-autoimunidade-com-10-000-unidades-a-pessoa-sai/

 

A prescrição diária de 10.000 UIs de Vitamina D representaria para a indústria farmacêutica uma perda de 40% de uma receita de trilhões de dólares

https://objetodignidade.wordpress.com/2013/01/16/a-prescricao-diaria-de-10-000-uis-de-vitamina-d-representaria-para-a-industria-farmaceutica-uma-perda-de-40-de-uma-receita-de-trilhoes-de-dolares/

 

“10.000 unidades todos os dias NÃO CAUSAM INTOXICAÇAO. Nos Estados Unidos, doses de 10.000 unidades de vitamina D são vendidas nas farmácias, sem receita médica. É assim porque a dose de 10.000 unidades não tem efeitos colaterais e não causa intoxicação” – Dr. Cícero Galli Coimbra

https://objetodignidade.wordpress.com/2012/11/10/10-000-unidades-todos-os-dias-nao-causam-intoxicacao-nos-estados-unidos-doses-de-10-000-unidades-de-vitamina-d-sao-vendidas-nas-farmacias-sem-receita-medica-e-assim-porque-a-dose-de-10-00/

 

Dr. Cícero Galli Coimbra – Doenças Autoimunes e Vitamina D – “Se a natureza não precisasse de 10.000 unidades todo o dia, não formava uma quantidade tão grande em tão poucos minutos.”

https://objetodignidade.wordpress.com/2012/11/04/dr-cicero-galli-coimbra-doencas-autoimunes-e-vitamina-d-se-a-natureza-nao-precisasse-de-10-000-unidades-todo-o-dia-nao-formava-uma-quantidade-tao-grande-em-tao-poucos-minutos/

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PORQUE A VERDADE SOBRE A VITAMINA D É UMA ENORME AMEAÇA PARA O SISTEMA ESTABELECIDO DO LUCRO MÉDICO-INDÚSTRIAS

 

1) A vitamina D é livre (você pode obtê-lo, o hormonio esteroide, a partir do sol, sem receita médica).

2) Vitamina D previne mais de uma dúzia de doenças cronicas e condições de saúde (osteoporose, câncer, diabetes, MS, e outros) que fornecem alto lucro.

3) A vitamina D é extremamente segura, mesmo quando tomada em forma de suplemento, porque é uma vitamina natural / hormônio que o corpo reconhece por ser produzido a partir da exposição ao UVB solar.

http://www.naturalnews.com/031577_vitamin_D_scientific_research.html#ixzz2PDWvdCFO

 

 

Vitamina D em medicina preventiva: estamos ignorando as provas?

“Os dados epidemiológicos indicam também um baixo status da vitamina D na tuberculose, artrite reumatóide, esclerose múltipla, doenças inflamatórias intestinais, hipertensão e certos tipos de câncer.”

https://objetodignidade.wordpress.com/2009/08/28/vitamina-d-em-medicina-preventiva-estamos-ignorando-as-provas/

.A Zittermann.

 

Erros declaratórios da morte encefálica

por Celso Galli Coimbra

http://www.youtube.com/watch?v=egD3g9K1qY8&list=PLeqEGmvbpULMSipZ__vfDS5LKkkaShFE5

Dr. Cícero Galli Coimbra explica o Mal de Alzheimer: causas multifatoriais com destaque à depressão e a deficiência do hormonio imunoregulador vitamina D — um distúrbio metabólico http://www.youtube.com/watch?feature=player_detailpage&v=93sSODBmNJM

Suplementos de vitamina D podem reduzir risco de Alzheimer — 10.000 UI, não menos

http://biodireitomedicina.wordpress.com/2012/12/04/suplementos-de-vitamina-d-podem-reduzir-risco-de-alzheimer-10-000-ui-nao-menos/

Alzheimer: Vitamina D diminui riscos das mulheres padecerem da doença

http://biodireitomedicina.wordpress.com/2012/12/04/alzheimer-vitamina-d-diminui-riscos-das-mulheres-padecerem-da-doenca/

Vitamina D e Alzheimer — Vitamin D may reduce the risk of dominantly inherited Alzheimer’s disease

http://biodireitomedicina.wordpress.com/2012/07/26/vitamina-d-e-alzheimer-vitamin-d-may-reduce-the-risk-of-dominantly-inherited-alzheimers-disease/

Nutrientes contra Parkinson e Alzheimer

http://biodireitomedicina.wordpress.com/2009/09/17/nutrientes-contra-parkinson-e-alzheimer/

 

POR UM NOVO PARADIGMA DE CONDUTA E TRATAMENTO Por Dr. Cícero Galli Coimbra

Médico Internista e Neurologista

Professor Associado Livre-Docente da Universidade Federal de São Paulo

Presidente do Instituto de Investigação e Tratamento de Autoimunidade

http://www.institutodeautoimunidade.org.br/novo-paradigma.html

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Tratamento com a Vitamina D, Cura e Prevenção — Low vitamin D levels ‘linked to Parkinson’s disease’

30-year study

https://objetodignidade.wordpress.com/2013/02/19/tratamento-com-a-vitamina-d-cura-e-prevencao-low-vitamin-d-levels-linked-to-parkinsons-disease/

Tratamento com a Vitamina D, Cura e Prevenção — Role of Vitamin D in Parkinson’s Disease

https://objetodignidade.wordpress.com/2013/02/19/tratamento-com-a-vitamina-d-cura-e-prevencao-role-of-vitamin-d-in-parkinsons-disease/

 

POR 30 ANOS, EXTENSA REVISÃO DE TODA A PESQUISA ANTERIOR CONFIRMA QUE BAIXO NÍVEL DE VITAMINA D É UMA SENTENÇA DE MORTE

http://biodireitomedicina.wordpress.com/2013/02/14/vitamina-d-reportagem-com-dr-cicero-galli-coimbra-e-daniel-cunha-na-rede-record/

Tradução de Celso Galli Coimbra

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Veja links sobre o mesmo assunto:

1. http://biodireitomedicina.wordpress.com/2010/08/03/vitamina-d-pode-revolucionar-o-tratamento-da-esclerose-multipla/

2. http://biodireitomedicina.wordpress.com/2012/04/12/vitamina-d-por-uma-outra-terapia/

3. http://biodireitomedicina.wordpress.com/2011/03/23/informacoes-medicas-sobre-a-prevencao-e-tratamento-de-doencas-neurodegenerativas-e-auto-imunes-como-parkinson-alzheimer-lupus-psoriase-vitiligo-depressao/

4. http://biodireitomedicina.wordpress.com/2010/03/20/vitamina-d-pode-combater-males-que-mais-matam-pessoas-no-mundo/

5. http://biodireitomedicina.wordpress.com/2012/05/28/folha-de-sao-paulo-terapia-polemica-usa-vitamina-d-em-doses-altas-contra-esclerose-multipla/

6. http://biodireitomedicina.wordpress.com/2012/06/18/taxas-baixas-de-vitamina-d-na-maioria-da-populacao-preocupam-especialistas/

“(…) cerca de 70% da população mundial apresenta taxas inadequadas de vitamina D, substância que, dentro do corpo, trabalha como um hormônio. O fenômeno da insuficiência não poupa nem países tropicais, como o Brasil, e a defasagem tende a ser maior nas grandes cidades, já que, dentro de casa, no carro ou no escritório, as pessoas acabam fugindo do sol. De acordo com o endocrinologista Geraldo Santana, do Instituto Mineiro de Endocrinologia, “a deficiência de vitamina D é um achado frequente e também preocupante devido à importante ação da substância no organismo.”

http://biodireitomedicina.wordpress.com/2012/06/29/vitamina-d-sem-censura-dr-cicero-galli-coimbra-e-daniel-cunha/

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VitaminDCouncil

Vitamin D and Risk of Ischemic Heart Disease

http://www.youtube.com/watch?feature=player_detailpage&v=jTLwD7hpjCs

Apelo do Dr. Rath às pessoas da Alemanha, da Europa e de todo mundo, Berlim 13.03.2012

https://www.youtube.com/watch?feature=player_detailpage&list=HL1352566764&v=VFJsicKGho0

Vitamina D

visualizar lista de reprodução completa ( 5 vídeos)

http://www.youtube.com/watch?v=erAgu1XcY-U&list=PL301EAE2D5602A758&feature=g-all-a

Vitamina D – Por uma outra terapia (Vitamin D – For an alternative therapy)

http://www.youtube.com/watch?feature=player_detailpage&v=erAgu1XcY-U

◊ Dr. Cícero Galli Coimbra PhD., MD., é médico graduado pela Universidade Federal do Rio Grande do Sul (1979), possui título de especialista em medicina interna (1981) e neurologia (1983) pela mesma instituição, e em neurologia pediátrica (1985) pelo Jackson Memorial Hospital da Universidade de Miami, EUA. Obteve o título de mestre (1988) e doutor (1991) em Neurologia pela Universidade Federal de São Paulo e pós-doutorado (1993) pela Universidade de Lund, Suécia. Atualmente é Professor Livre Docente do Departamento de Neurologia e Neurocirurgia da Universidade Federal de São Paulo, onde dirige o Laboratório de Fisiopatologia Clínica e Experimental. Atua na área de Medicina (Neurologia e Clínica Médica), com ênfase em doenças neurodegenerativas e autoimunitárias.

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·       A Autoimunidade tem cura com a Vitamina D – Instituto de Investigação e Tratamento da Autoimunidade

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