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1.
Hum Antibodies ; 24(3-4): 71-76, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27792005

RESUMO

BACKGROUND: Multiple sclerosis (MS) as a complex neurological disease can be due to vitamin D deficiency. CYP27B1 is referred to as a vitamin D metabolizing enzyme. MATERIALS AND METHODS: This study compared the expression level of CYP27B1 in Relapsing-Remitting MS (RRMS) patients with normal individuals in Iran. The RNA was extracted from 50 RRMS patients and 50 normal controls. Quantitative RT-PCR was adopted to measure the expression level of CYP27B1 gene. RESULTS: The expression level of CYP27B1gene was significantly lower in the RRMS patients than their normal counterparts (P value = 0.04). Also, the RRMS females participating had a significant reduction in CYP27B1 gene expression compared to normal females (P-Value = 0.01). In addition, the correlation between CYP27B1 expression level, and the risk of Expanded Disability Status Scale of Kurtzke (EDSS) was not linear. Additionally, there was no significant correlation between expression status of CYP27B1gene and duration of the disease. CONCLUSION: A significant decrease in the expression level of CYP27A1 in female patients could indicate their greater vulnerability to MS than the male patients.


Assuntos
25-Hidroxivitamina D3 1-alfa-Hidroxilase/genética , Esclerose Múltipla Recidivante-Remitente/genética , Vitamina D/sangue , 25-Hidroxivitamina D3 1-alfa-Hidroxilase/imunologia , Adulto , Estudos de Casos e Controles , Regulação para Baixo , Feminino , Expressão Gênica , Cadeias HLA-DRB1/genética , Cadeias HLA-DRB1/imunologia , Humanos , Fatores Imunológicos/uso terapêutico , Interferon beta/uso terapêutico , Masculino , Pessoa de Meia-Idade , Esclerose Múltipla Recidivante-Remitente/tratamento farmacológico , Esclerose Múltipla Recidivante-Remitente/imunologia , Esclerose Múltipla Recidivante-Remitente/patologia , Fatores Sexuais
2.
Biomed Res Int ; 2016: 1375858, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27403416

RESUMO

The primary function of vitamin D is to regulate calcium homeostasis, which is essential for bone formation and resorption. Although diet is a source of vitamin D, most foods are naturally lacking vitamin D. Vitamin D is also manufactured in the skin through a photolysis process, leading to a process called the "sunshine vitamin." The active form of vitamin D, 1,25-dihydroxyvitamin D (calcitriol), is biosynthesised in the kidney through the hydroxylation of 25-hydroxycholecalciferol by the CYP27B1 enzyme. It has been found that several immune cells express the vitamin D receptor (VDR) and CYP27B1; of the latter, synthesis is determined by several immune-specific signals. The realisation that vitamin D employs several molecular mechanisms to regulate innate immune responses is more recent. Furthermore, evidence collected from intervention studies indicates that vitamin D supplements may boost clinical responses to infections. This review considers the current knowledge of how immune signals regulate vitamin D metabolism and how innate immune system function is modulated by ligand-bound VDR.


Assuntos
Imunidade Inata/imunologia , Receptores de Calcitriol/imunologia , Transdução de Sinais/imunologia , Vitamina D/imunologia , 25-Hidroxivitamina D3 1-alfa-Hidroxilase/imunologia , Animais , Humanos , Lignanas
3.
J Steroid Biochem Mol Biol ; 163: 173-82, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27210415

RESUMO

Male fetus and neonates show increased immune vulnerability compared to females, which results in a higher risk of perinatal infections. These differences could partially be due to sex steroids differential modulation of vitamin D metabolism; since calcitriol, the most active vitamin D metabolite, regulates immune responses and transcriptionally induces the antimicrobial peptide cathelicidin in the human placenta. Calcitriol availability depends on CYP27B1 and CYP24A1 expression, the cytochromes involved in its synthesis and degradation, respectively. However, the effects of testosterone upon these enzymes and the final biological outcome upon the calcitriol-dependent immune-target cathelicidin in the placenta have not been studied. In this study we show that testosterone significantly inhibited CYP27B1 while stimulated CYP24A1 gene expression in cultured trophoblasts. These effects were accompanied by CREB activation through cAMP-independent and androgen receptor-dependent mechanisms. Male placental cotyledons showed reduced basal CYP27B1 and cathelicidin gene expression compared to females (P<0.05). Testosterone concentration was higher in the cord blood of male neonates (P=0.007), whereas cathelicidin levels were lesser compared to females (P=0.002). Altogether our results suggest that male placentas produce less cathelicidin due to decreased calcitriol bioavailability. We propose that the observed sex-dependent differences in placental vitamin D metabolism contribute in fetal responses to infections and could partially explain why the increased male fetuses immune vulnerability. Moreover, gestational hyperandrogenemia could adversely affect placental vitamin D metabolism independently of fetal sex.


Assuntos
25-Hidroxivitamina D3 1-alfa-Hidroxilase/genética , Catelicidinas/genética , Testosterona/farmacologia , Trofoblastos/efeitos dos fármacos , Vitamina D3 24-Hidroxilase/genética , Vitamina D/metabolismo , 25-Hidroxivitamina D3 1-alfa-Hidroxilase/imunologia , Peptídeos Catiônicos Antimicrobianos , Catelicidinas/imunologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/imunologia , Feminino , Feto , Regulação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Imunidade Inata , Masculino , Gravidez , Cultura Primária de Células , Receptores Androgênicos/genética , Receptores Androgênicos/imunologia , Caracteres Sexuais , Transdução de Sinais , Testosterona/metabolismo , Trofoblastos/citologia , Trofoblastos/imunologia , Vitamina D/farmacologia , Vitamina D3 24-Hidroxilase/imunologia
4.
Eur J Immunol ; 44(6): 1781-90, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24643654

RESUMO

The active vitamin D metabolite 1α,25-dihydroxyvitamin D (1,25[OH]2 D) potently inhibits DC priming of T-cell activation, suggesting that it mediates a homeostatic role in this context. Therefore, careful regulation of 1,25[OH]2 D levels is necessary to avoid inappropriate inhibition of T-cell activation. Cell-autonomous control of vitamin D activity can be modulated by the action of the vitamin D-activating and -inactivating hydroxylases, CYP27B1, and CYP24A1, respectively. We show that in comparison to macrophages, human monocyte-derived DCs exhibit significantly less activation of 25-dihydroxyvitamin D to 1,25[OH]2 D, and that DCs predominantly express a truncated CYP27B1 transcript that may contribute to the deficiency in activation of vitamin D. Furthermore, in response to stimulation with 1,25[OH]2 D, upregulation of the inactivating enzyme CYP24A1 curtailed the functional effects of vitamin D in DCs, but not macrophages. Production of 1,25[OH]2 D by macrophages was adequate to induce expression of vitamin D-responsive genes by DCs, inhibit DC maturation in response to innate immune stimulation and DC-dependent T-cell responses. Our data suggest that in comparison to macrophages, differential regulation of hydroxylases limits autocrine vitamin D activity in DCs, and that paracrine activation of vitamin D exerts a more potent mechanism for homeostatic control of DC function.


Assuntos
25-Hidroxivitamina D3 1-alfa-Hidroxilase/imunologia , Calcitriol/imunologia , Células Dendríticas/imunologia , Ativação Linfocitária/fisiologia , Esteroide Hidroxilases/imunologia , Linfócitos T/imunologia , Células Dendríticas/citologia , Feminino , Homeostase/fisiologia , Humanos , Macrófagos/citologia , Macrófagos/imunologia , Masculino , Monócitos/citologia , Monócitos/imunologia , Linfócitos T/citologia , Vitamina D3 24-Hidroxilase
5.
Immunology ; 139(1): 121-8, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23289765

RESUMO

The ability of T cells to activate antimicrobial pathways in infected macrophages is essential to host defence against many intracellular pathogens. Here, we compared the ability of two T-cell-mediated mechanisms to trigger antimicrobial responses against Mycobacterium tuberculosis in humans, CD40 activation and the release of interferon-γ (IFN-γ). Given that IFN-γ activates a vitamin D-dependent antimicrobial response, we focused on induction of the key components of this pathway. We show that activation of human monocytes via CD40 ligand (CD40L) and IFN-γ, alone, and in combination, induces the CYP27b1-hydroxylase, responsible for the conversion of 25-hydroxyvitamin D (25D) to the bioactive 1,25-dihydroxyvitamin D (1,25D), and the vitamin D receptor (VDR). The activation of the vitamin D pathway by CD40L and IFN-γ results in up-regulated expression of the antimicrobial peptides, cathelicidin and DEFB4, as well as induction of autophagy. Finally, activation of monocytes via CD40L and IFN-γ results in an antimicrobial activity against intracellular M. tuberculosis. Our data suggest that at least two parallel T-cell-mediated mechanisms, CD40L and IFN-γ, activate the vitamin D-dependent antimicrobial pathway and trigger antimicrobial activity against intracellular M. tuberculosis, thereby contributing to human host defence against intracellular infection.


Assuntos
Ligante de CD40/imunologia , Interferon gama/imunologia , Monócitos/imunologia , Mycobacterium tuberculosis/imunologia , Receptores de Calcitriol/imunologia , Tuberculose/imunologia , 25-Hidroxivitamina D3 1-alfa-Hidroxilase/imunologia , Peptídeos Catiônicos Antimicrobianos/imunologia , Ligante de CD40/agonistas , Ligante de CD40/metabolismo , Calcitriol/imunologia , Feminino , Humanos , Interferon gama/agonistas , Interferon gama/metabolismo , Masculino , Monócitos/microbiologia , Linfócitos T/imunologia , beta-Defensinas/imunologia
6.
PLoS Pathog ; 8(11): e1003017, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23166493

RESUMO

Toll-like receptors (TLR) are important in recognizing microbial pathogens and triggering host innate immune responses, including autophagy, and in the mediation of immune activation during human immunodeficiency virus type-1 (HIV) infection. We report here that TLR8 activation in human macrophages induces the expression of the human cathelicidin microbial peptide (CAMP), the vitamin D receptor (VDR) and cytochrome P450, family 27, subfamily B, polypeptide 1 (CYP27B1), which 1α-hydroxylates the inactive form of vitamin D, 25-hydroxycholecalciferol, into its biologically active metabolite. Moreover, we demonstrate using RNA interference, chemical inhibitors and vitamin D deficient media that TLR8 agonists inhibit HIV through a vitamin D and CAMP dependent autophagic mechanism. These data support an important role for vitamin D in the control of HIV infection, and provide a biological explanation for the benefits of vitamin D. These findings also provide new insights into potential novel targets to prevent and treat HIV infection.


Assuntos
25-Hidroxivitamina D 2/farmacologia , Autofagia/efeitos dos fármacos , HIV-1/metabolismo , Receptores de Calcitriol/imunologia , Receptor 8 Toll-Like/agonistas , Vitaminas/farmacologia , 25-Hidroxivitamina D 2/imunologia , 25-Hidroxivitamina D3 1-alfa-Hidroxilase/imunologia , Peptídeos Catiônicos Antimicrobianos/imunologia , Autofagia/imunologia , Feminino , Infecções por HIV/tratamento farmacológico , Infecções por HIV/imunologia , Humanos , Ligantes , Masculino , Vitaminas/imunologia
7.
J Immunol ; 189(11): 5155-64, 2012 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-23087405

RESUMO

1,25-Dihydroxyvitamin D(3) [1,25(OH)(2)D(3)], the active form of vitamin D, exerts potent effects on several tissues including cells of the immune system, where it affects T cell activation, differentiation and migration. The circulating, inactive form of vitamin D, 25(OH)D(3), is generally used as an indication of vitamin D status. However, use of this precursor depends on its uptake by cells and subsequent conversion by the enzyme 25(OH)D(3)-1α-hydroxylase (CYP27B1) into active 1,25(OH)(2)D(3). Using human T cells, we show in this study that addition of inactive 25(OH)D(3) is sufficient to alter T cell responses only when dendritic cells (DCs) are present. Mechanistically, CYP27B1 is induced in DCs upon maturation with LPS or upon T cell contact, resulting in the generation and release of 1,25(OH)(2)D(3), which subsequently affects T cell responses. In most tissues, vitamin D binding protein acts as a carrier to enhance the use of vitamin D. However, we show that vitamin D binding protein modulates T cell responses by restricting the availability of inactive 25(OH)D(3) to DC. These data indicate that the level of free 25(OH)D(3) available to DCs determines the inflammatory/regulatory balance of ensuing T cell responses.


Assuntos
25-Hidroxivitamina D3 1-alfa-Hidroxilase/metabolismo , Calcifediol/imunologia , Calcitriol/imunologia , Células Dendríticas/imunologia , Linfócitos T/imunologia , 25-Hidroxivitamina D3 1-alfa-Hidroxilase/imunologia , Calcifediol/metabolismo , Calcitriol/metabolismo , Comunicação Celular/efeitos dos fármacos , Comunicação Celular/imunologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/imunologia , Técnicas de Cocultura , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/enzimologia , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/imunologia , Humanos , Lipopolissacarídeos/imunologia , Lipopolissacarídeos/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Cultura Primária de Células , Transdução de Sinais/efeitos dos fármacos , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Fator de Crescimento Transformador beta/imunologia , Fator de Crescimento Transformador beta/farmacologia
8.
Autoimmun Rev ; 11(2): 84-7, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21864722

RESUMO

Vitamin D is synthesized from cholesterol in the skin (80-90%) under the sunlight and then metabolized into an active D hormone in liver, kidney and peripheral immune/inflammatory cells. These endocrine-immune effects include also the coordinated activities of the vitamin D-activating enzyme, 1alpha-hydroxylase (CYP27B1), and the vitamin D receptor (VDR) on cells of the immune system in mediating intracrine and paracrine actions. Vitamin D is implicated in prevention and protection from chronic infections (i.e. tubercolosis), cancer (i.e. breast cancer) and autoimmune rheumatic diseases since regulates both innate and adaptive immunity potentiating the innate response (monocytes/macrophages with antimicrobial activity and antigen presentation), but suppressing the adaptive immunity (T and B lymphocyte functions). Vitamin D has modulatory effects on B lymphocytes and Ig production and recent reports have demonstrated that 1,25(OH)2D3 does indeed exert direct effects on B cell homeostasis. A circannual rhythm of trough vitamin D levels in winter and peaks in summer time showed negative correlation with clinical status at least in rheumatoid arthritis and systemic lupus erythematosus. Recently, the onset of symptoms of early arthritis during winter or spring have been associated with greater radiographic evidence of disease progression at 12 months possibly are also related to seasonal lower vitamin D serum levels.


Assuntos
Imunidade Adaptativa , Linfócitos B/efeitos dos fármacos , Imunidade Inata , Receptores de Calcitriol/imunologia , Vitamina D/imunologia , 25-Hidroxivitamina D3 1-alfa-Hidroxilase/imunologia , 25-Hidroxivitamina D3 1-alfa-Hidroxilase/metabolismo , Imunidade Adaptativa/efeitos dos fármacos , Aromatase/imunologia , Aromatase/metabolismo , Inibidores da Aromatase/imunologia , Inibidores da Aromatase/metabolismo , Inibidores da Aromatase/farmacologia , Artrite Reumatoide/tratamento farmacológico , Artrite Reumatoide/imunologia , Artrite Reumatoide/metabolismo , Artrite Reumatoide/patologia , Linfócitos B/citologia , Linfócitos B/imunologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/imunologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Sistema Endócrino/efeitos dos fármacos , Sistema Endócrino/imunologia , Feminino , Homeostase/efeitos dos fármacos , Homeostase/imunologia , Humanos , Imunidade Inata/efeitos dos fármacos , Lúpus Eritematoso Sistêmico/tratamento farmacológico , Lúpus Eritematoso Sistêmico/imunologia , Lúpus Eritematoso Sistêmico/metabolismo , Lúpus Eritematoso Sistêmico/patologia , Receptores de Calcitriol/metabolismo , Estações do Ano , Pele/imunologia , Pele/metabolismo , Tuberculose/tratamento farmacológico , Tuberculose/imunologia , Tuberculose/metabolismo , Tuberculose/patologia , Vitamina D/metabolismo , Vitamina D/farmacologia
9.
J Hepatol ; 54(5): 887-93, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21145801

RESUMO

BACKGROUND & AIMS: Vitamin D is an important immune modulator and preliminary data indicated an association between vitamin D deficiency and sustained virologic response (SVR) rates in hepatitis C virus (HCV) genotype 1 patients. We, therefore, performed a comprehensive analysis on the impact of vitamin D serum levels and of genetic polymorphisms with functional relevance within the vitamin D cascade on chronic hepatitis C and its treatment. METHODS: Vitamin D serum levels, genetic polymorphisms within the vitamin D receptor and 1α-hydroxylase were determined in a cohort of 468 HCV genotype 1, 2, and 3 infected patients who were treated with interferon-alfa based regimens. RESULTS: Chronic hepatitis C was associated with a high incidence of severe vitamin D deficiency compared to controls (25(OH)D(3)<10 ng/ml in 25% versus 12%, p<0.00001). 25(OH)D(3) deficiency correlated with SVR in HCV genotype 2 and 3 patients (50% and 81% SVR for patients with and without severe vitamin D deficiency, respectively, p<0.0001). In addition, the CYP27B1-1260 promoter polymorphism rs10877012 had substantial impact on 1,25-dihydroxyvitamin D serum levels (72, 61, and 60 pmol/ml for rs10877012 AA, AC, and CC, respectively, p=0.04) and on SVR rates in HCV genotype 1, 2, and 3 infected patients (77% and 65% versus 42% for rs10877012 AA, AC, and CC, respectively, p=0.02). CONCLUSIONS: Chronic hepatitis C virus infection is associated with vitamin D deficiency. Reduced 25-hydroxyvitamin D levels and CYP27B1-1260 promoter polymorphism leading to reduced 1,25-dihydroxyvitamin D levels are associated with failure to achieve SVR in HCV genotype 1, 2, and 3 infected patients.


Assuntos
25-Hidroxivitamina D3 1-alfa-Hidroxilase/genética , Antivirais/uso terapêutico , Hepacivirus/efeitos dos fármacos , Hepatite C Crônica , Interferon-alfa/uso terapêutico , Deficiência de Vitamina D/genética , 25-Hidroxivitamina D3 1-alfa-Hidroxilase/imunologia , Adulto , Idoso , Calcifediol/sangue , Farmacorresistência Viral/imunologia , Feminino , Genótipo , Hepacivirus/genética , Hepacivirus/imunologia , Hepatite C Crônica/tratamento farmacológico , Hepatite C Crônica/genética , Hepatite C Crônica/imunologia , Humanos , Masculino , Pessoa de Meia-Idade , Polimorfismo Genético/imunologia , Regiões Promotoras Genéticas/genética , Regiões Promotoras Genéticas/imunologia , Receptores de Calcitriol/genética , Estudos Retrospectivos , Deficiência de Vitamina D/imunologia , Adulto Jovem
10.
Endocrinol Metab Clin North Am ; 39(2): 365-79, table of contents, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20511058

RESUMO

Interaction with the immune system is one of the most well-established nonclassic effects of vitamin D. For many years this was considered to be a manifestation of granulomatous diseases such sarcoidosis, in which synthesis of active 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) is known to be dysregulated. However, recent reports have supported a role for 1,25(OH)(2)D(3) in mediating normal function of the innate and adaptive immune systems. Crucially, these effects seem to be mediated via localized autocrine or paracrine synthesis of 1,25(OH)(2)D(3) from precursor 25-hydroxyvitamin D(3), the main circulating metabolite of vitamin D. The ability of vitamin D to influence normal human immunity is highly dependent on the vitamin D status of individuals, and may lead to aberrant response to infection or autoimmunity in those who are lacking vitamin D. The potential health significance of this has been underlined by increasing awareness of impaired vitamin D status in populations across the globe. This article describes some of the recent developments with respect to vitamin D and the immune system, and possible clinical implications.


Assuntos
Imunidade Adaptativa , Doenças Autoimunes/imunologia , Sistema Imunitário , Imunidade Inata , Vitamina D/imunologia , 25-Hidroxivitamina D3 1-alfa-Hidroxilase/genética , 25-Hidroxivitamina D3 1-alfa-Hidroxilase/imunologia , Animais , Apresentação do Antígeno/imunologia , Peptídeos Catiônicos Antimicrobianos/imunologia , Linfócitos B/imunologia , Doença de Crohn/imunologia , Células Dendríticas/imunologia , Diabetes Mellitus Tipo 1/imunologia , Humanos , Macrófagos/imunologia , Camundongos , Esclerose Múltipla/imunologia , Linfócitos T/imunologia , Tuberculose/imunologia , Vitamina D/metabolismo , Vitamina D/uso terapêutico
11.
J Immunol ; 182(7): 4296-305, 2009 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-19299729

RESUMO

The addition of monophosphoryl lipid A, a minimally toxic derivative of LPS, to nonmucosally administered vaccines induced both systemic and mucosal immune responses to coadministered Ags. This was dependent on an up-regulated expression of 1alpha-hydroxylase (CYP27B1, 1alphaOHase), the enzyme that converts 25-hydroxycholecalciferol, a circulating inactive metabolite of vitamin D(3), into 1,25(OH)2D(3) (calcitriol). In response to locally produced calcitriol, myeloid dendritic cells (DCs) migrated from cutaneous vaccination sites into multiple secondary lymphoid organs, including classical inductive sites of mucosal immunity, where they effectively stimulated B and T cell immune responses. The endogenous production of calcitriol by monophosphoryl lipid A-stimulated DCs appeared to be Toll-IL-1R domain-containing adapter-inducing IFN-beta-dependent, mediated through a type 1 IFN-induced expression of 1alphaOHase. Responsiveness to calcitriol was essential to promote the trafficking of mobilized DCs to nondraining lymphoid organs. Collectively, these studies help to expand our understanding of the physiologically important roles played by locally metabolized vitamin D(3) in the initiation and diversification of adaptive immune responses. The influences of locally produced calcitriol on the migration of activated DCs from sites of vaccination/infection into both draining and nondraining lymphoid organs create a condition whereby Ag-responsive B and T cells residing in multiple lymphoid organs are able to simultaneously engage in the induction of adaptive immune responses to peripherally administered Ags as if they were responding to an infection of peripheral or mucosal tissues they were designed to protect.


Assuntos
Adjuvantes Imunológicos/farmacologia , Colecalciferol/metabolismo , Células Dendríticas/imunologia , Receptores Toll-Like/imunologia , Vacinas/imunologia , 25-Hidroxivitamina D3 1-alfa-Hidroxilase/imunologia , 25-Hidroxivitamina D3 1-alfa-Hidroxilase/metabolismo , Animais , Linfócitos B/imunologia , Western Blotting , Linfócitos T CD4-Positivos/imunologia , Calcitriol/imunologia , Calcitriol/metabolismo , Movimento Celular/imunologia , Colecalciferol/imunologia , Células Dendríticas/metabolismo , Imunidade nas Mucosas/imunologia , Interferon Tipo I/imunologia , Interferon Tipo I/metabolismo , Lipídeo A/análogos & derivados , Lipídeo A/imunologia , Lipídeo A/farmacologia , Ativação Linfocitária/imunologia , Tecido Linfoide/citologia , Tecido Linfoide/imunologia , Camundongos , Camundongos Transgênicos , Receptores Toll-Like/metabolismo
12.
J Immunol ; 182(7): 4289-95, 2009 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-19299728

RESUMO

The active form of vitamin D, 1,25-dihydroxyvitamin D (1,25(OH)(2)D) enhances innate immunity by inducing the cathelicidin antimicrobial peptide (hCAP). In monocytes/macrophages, this occurs primarily in response to activation of TLR, that induce expression of the vitamin D receptor and localized synthesis of 1,25(OH)(2)D from precursor 25-hydroxyvitamin D(3) (25OHD). To clarify the relationship between vitamin D and innate immunity, we assessed changes in hCAP expression in vivo and ex vivo in human subjects attending a bone clinic (n = 50). Of these, 38% were vitamin D-insufficient (<75 nM 25OHD) and received supplementation with vitamin D (50,000 IU vitamin D(2) twice weekly for 5 wk). Baseline 25OHD status or vitamin D supplementation had no effect on circulating levels of hCAP. Therefore, ex vivo changes in hCAP for each subject were assessed using peripheral blood monocytes cultured with 10% autologous serum (n = 28). Under these vitamin D "insufficient" conditions the TLR2/1 ligand 19 kDa lipopeptide or the TLR4 ligand LPS, monocytes showed increased expression of the vitamin D-activating enzyme CYP27b1 (5- and 5.5-fold, respectively, both p < 0.01) but decreased expression of hCAP mRNA (10-fold and 30-fold, both p < 0.001). Following treatment with 19 kDa, expression of hCAP: 1) correlated with 25OHD levels in serum culture supplements (R = 0.649, p < 0.001); 2) was significantly enhanced by exogenous 25OHD (5 nM); and 3) was significantly enhanced with serum from vivo vitamin D-supplemented patients. These data suggest that a key role of vitamin D in innate immunity is to maintain localized production of antibacterial hCAP following TLR activation of monocytes.


Assuntos
Peptídeos Catiônicos Antimicrobianos/imunologia , Imunidade Inata , Monócitos/imunologia , Deficiência de Vitamina D/imunologia , Vitamina D/análogos & derivados , 25-Hidroxivitamina D3 1-alfa-Hidroxilase/biossíntese , 25-Hidroxivitamina D3 1-alfa-Hidroxilase/imunologia , Peptídeos Catiônicos Antimicrobianos/metabolismo , Catelicidinas , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Feminino , Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Monócitos/metabolismo , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Receptor 1 Toll-Like/imunologia , Receptor 1 Toll-Like/metabolismo , Receptor 2 Toll-Like/imunologia , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/imunologia , Receptor 4 Toll-Like/metabolismo , Vitamina D/imunologia , Vitamina D/metabolismo , Vitamina D/uso terapêutico , Deficiência de Vitamina D/metabolismo
13.
J Immunol ; 181(10): 7115-20, 2008 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-18981132

RESUMO

An essential function of the innate immune system is to directly trigger antimicrobial mechanisms to defend against invading pathogens. In humans, one such pathway involves activation by TLR2/1L leading to the vitamin D-dependent induction of antimicrobial peptides. In this study, we found that TLR2/1-induced IL-15 was required for induction of CYP27b1, the VDR and the downstream antimicrobial peptide cathelicidin. Although both IL-15 and IL-4 triggered macrophage differentiation, only IL-15 was sufficient by itself to induce CYP27b1 and subsequent bioconversion of 25-hydroxyvitamin D3 (25D3) into bioactive 1,25D3, leading to VDR activation and induction of cathelicidin. Finally, IL-15-differentiated macrophages could be triggered by 25D3 to induce an antimicrobial activity against intracellular Mycobacterium tuberculosis. Therefore, IL-15 links TLR2/1-induced macrophage differentiation to the vitamin D-dependent antimicrobial pathway.


Assuntos
Diferenciação Celular/imunologia , Interleucina-15/metabolismo , Macrófagos/citologia , Receptor 1 Toll-Like/metabolismo , Receptor 2 Toll-Like/metabolismo , Vitamina D/metabolismo , 25-Hidroxivitamina D3 1-alfa-Hidroxilase/imunologia , 25-Hidroxivitamina D3 1-alfa-Hidroxilase/metabolismo , Peptídeos Catiônicos Antimicrobianos/imunologia , Peptídeos Catiônicos Antimicrobianos/metabolismo , Expressão Gênica , Humanos , Interleucina-15/imunologia , Macrófagos/microbiologia , Macrófagos/fisiologia , Receptores de Calcitriol/imunologia , Receptores de Calcitriol/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Receptor 1 Toll-Like/imunologia , Receptor 2 Toll-Like/imunologia , Vitamina D/imunologia
14.
J Clin Endocrinol Metab ; 91(9): 3566-74, 2006 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-16787983

RESUMO

CONTEXT: 25-Hydroxyvitamin D can be activated to 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] by the rate-limiting enzyme 1alpha-hydroxylase in cells of the immune system under control of immune stimuli, such as interferon-gamma (IFNgamma). In pathological situations, such as sarcoidosis, this can lead to systemic excess of 1,25(OH)(2)D(3) and hypercalcemia. OBJECTIVE: The aim of this study was to elucidate the intracellular pathways used by the immune system to tightly regulate 1,25(OH)(2)D(3) production in monocytes and macrophages. DESIGN: Human monocytic THP1-cells were differentiated and activated by IFNgamma and a secondary stimulus, such as lipopolysaccharide or phorbol myristate acetate. 1alpha-Hydroxylase mRNA levels were quantified by real-time RT-PCR. The involvement of different signaling pathways in the regulation of this enzyme was investigated using specific pharmacological inhibitors, whereas phosphorylation of signal transducer and activator of transcription 1alpha and CCAAT/enhancer binding protein beta was investigated by Western blotting. RESULTS: In undifferentiated monocytic THP1 cells, IFNgamma needs to be combined with a second stimulus, such as lipopolysaccharide, to induce 1alpha-hydroxylase. In contrast, in phorbol myristate acetate-differentiated THP1 macrophages, IFNgamma alone induces 1alpha-hydroxylase and to much higher levels. Many different signaling pathways need to be activated concurrently to allow immune-mediated 1alpha-hydroxylase up-regulation. We show involvement of the Janus kinase-signal transducer and activator of transcription, MAPK, and nuclear factor-kappaB pathways, with a crucial role for the transcription factor CCAAT/enhancer binding protein beta. Furthermore, histone remodeling involving histone deacetylases and histone acetylase p300 is required. CONCLUSION: The present findings indicate that IFNgamma-mediated 1,25(OH)(2)D(3) production, as observed in granulomatous diseases such as sarcoidosis, will take place only under conditions where the necessary other signaling pathways are also activated.


Assuntos
25-Hidroxivitamina D3 1-alfa-Hidroxilase/imunologia , Interferon gama/imunologia , Monócitos/imunologia , Fator de Transcrição STAT1/imunologia , 25-Hidroxivitamina D3 1-alfa-Hidroxilase/genética , Antracenos/farmacologia , Western Blotting , Proteína beta Intensificadora de Ligação a CCAAT/genética , Proteína beta Intensificadora de Ligação a CCAAT/imunologia , Diferenciação Celular/imunologia , Linhagem Celular , Indução Enzimática , Flavonoides/farmacologia , Humanos , Imidazóis/farmacologia , Proteínas Quinases Ativadas por Mitógeno/imunologia , Monócitos/citologia , Monócitos/enzimologia , Inibidores de Proteínas Quinases/farmacologia , Piridinas/farmacologia , RNA/química , RNA/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição STAT1/genética , Transdução de Sinais , Acetato de Tetradecanoilforbol/imunologia
15.
J Bone Miner Res ; 21(1): 37-47, 2006 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-16355272

RESUMO

UNLABELLED: Monocytes express 1alpha-hydroxylase, the enzyme responsible for final hydroxylation of vitamin D3, in response to IFNgamma and CD14/TLR4 activation. Cross-talk between the JAK-STAT, the NF-kappaB, and the p38 MAPK pathways is necessary, and direct binding of C/EBPbeta to its recognition sites in the promoter of the 1alpha-hydroxylase gene is a prerequisite. INTRODUCTION: The activated form of vitamin D3, 1,25(OH)2D3, known for its action in bone and mineral homeostasis, has important immunomodulatory effects. 1,25(OH)2D3 modulates the immune system through specific nuclear receptors, whereas macrophages produce 1,25(OH)2D3. In monocytes, the expression of 1alpha-hydroxylase, the enzyme responsible for final hydroxylation of vitamin D3, is regulated by immune stimuli. The aim of this study was to elucidate the intracellular pathways through which interferon (IFN)gamma and Toll-like receptor (TLR) modulation regulate expression of 1alpha-hydroxylase in monocytes/macrophages. MATERIALS AND METHODS: Monocytes were isolated from peripheral blood mononuclear cells (PBMCs) and stimulated with IFNgamma (12.5 U/ml) and/or lipopolysaccharide (LPS; 100 ng/ml) for 48 h. The following inhibitors were used: janus kinase (JAK) inhibitor AG490 (50 microM), NF-kappaB inhibitor sulfasalazine (0.25 mM), p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 (5 microM). 1alpha-hydroxylase mRNA expression was monitored by qRT-PCR. Phosphorylation of transcription factors was studied by Western blotting. Transfection of mutated or deletion promoter constructs, cloned in the pGL3-luciferase reporter plasmid, were performed in the RAW264.7 cell line. Cells were stimulated with IFNgamma (100 U/ml) and LPS (100 microg/ml), and promoter activity was studied. Binding of signal transducer and activator of transcription (STAT)1alpha, NF-kappaB, and C/EBPbeta to their respective binding sites in the promoter was analyzed by gel shift assays. RESULTS: 1alpha-hydroxylase mRNA expression in monocytes is synergistically induced by IFNgamma and CD14/TLR4 ligation and paralleled by 1,25(OH)2D3 production. This induction requires the JAK-STAT, NF-kappaB, and p38 MAPK pathways. Each of them is essential, because blocking individual pathways is sufficient to block 1alpha-hydroxylase expression (JAK inhibitor, 60% inhibition, p < 0.01; NF-kappaB inhibitor, 70% inhibition, p < 0.05; p38 MAPK inhibitor, 95% inhibition, p < 0.005). In addition, we show the involvement of the p38 MAPK pathway in phosphorylation of C/EBPbeta. Direct binding of C/EBPbeta to its recognition sites in the 1alpha-hydroxylase promoter is necessary to enable its immune-stimulated upregulation. CONCLUSION: IFNgamma and CD14/TLR4 binding regulate expression of 1alpha-hydroxylase in monocytes in a synergistic way. Combined activation of the JAK-STAT, p38 MAPK, and NF-kappaB pathways is necessary, with C/EBPbeta most probably being the essential transcription factor controlling immune-mediated transcription.


Assuntos
25-Hidroxivitamina D3 1-alfa-Hidroxilase/biossíntese , Conservadores da Densidade Óssea/farmacologia , Calcifediol/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Monócitos/enzimologia , Transdução de Sinais/efeitos dos fármacos , 25-Hidroxivitamina D3 1-alfa-Hidroxilase/imunologia , Conservadores da Densidade Óssea/imunologia , Proteína beta Intensificadora de Ligação a CCAAT/imunologia , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Calcifediol/imunologia , Células Cultivadas , Regulação Enzimológica da Expressão Gênica/imunologia , Humanos , Interferon gama/imunologia , Interferon gama/farmacologia , Receptores de Lipopolissacarídeos/imunologia , Receptores de Lipopolissacarídeos/metabolismo , Monócitos/citologia , Monócitos/imunologia , NF-kappa B/imunologia , NF-kappa B/metabolismo , Proteínas Tirosina Quinases/imunologia , Proteínas Tirosina Quinases/metabolismo , Transdução de Sinais/imunologia , Receptor 4 Toll-Like/imunologia , Receptor 4 Toll-Like/metabolismo
16.
FASEB J ; 18(2): 332-4, 2004 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-14657005

RESUMO

According to the present paradigm, 1alpha,25-dihydroxyvitamin D3 [1alpha,25-(OH)2D3] is a biologically active hormone; whereas 25-hydroxyvitamin D3 (25OHD3) is regarded as a prohormone activated through the action of 25-hydroxyvitamin D3 1alpha-hydroxylase (1alpha-hydroxylase). Although the role of vitamin D3 in the regulation of growth and differentiation of prostatic epithelial cells has been well studied, its action and metabolism in prostatic stroma are still largely unknown. We investigated the effects of 25OHD3 and 1alpha,25-(OH)2D3 on two human stromal primary cultures termed P29SN and P32S. In a cell proliferation assay, 25OHD3 was found at physiological concentrations of 100-250 nM to inhibit the growth of both primary cultures, whereas 1alpha,25-(OH)2D3 at a pharmacological concentration of 10 nM exhibited the growth-inhibitory effects on P29SN cells but not on P32S cells. Quantitative real-time RT-PCR analysis revealed that both 25OHD3 and 1alpha,25-(OH)2D3 induced 25-hydroxyvitamin D3 24-hydroxylase (24-hydroxylase) mRNA in a dose- and time-dependent manner. By inhibiting 1alpha-hydroxylase and/or 24-hydroxylase enzyme activities, the induction of 24-hydroxylase mRNA by 250 nM 25OHD3 was clearly enhanced, suggesting that 1alpha-hydroxylation is not a prerequisite for the hormonal activity of 25OHD3. Altogether our results suggest that 25OHD3 at a high but physiological concentration acts as an active hormone with respect to vitamin D3 responsive gene regulation and suppression of cell proliferation.


Assuntos
Calcifediol/farmacologia , Próstata/citologia , Células Estromais/efeitos dos fármacos , 25-Hidroxivitamina D3 1-alfa-Hidroxilase/antagonistas & inibidores , 25-Hidroxivitamina D3 1-alfa-Hidroxilase/imunologia , 25-Hidroxivitamina D3 1-alfa-Hidroxilase/metabolismo , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Inibidores das Enzimas do Citocromo P-450 , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Relação Dose-Resposta a Droga , Indução Enzimática/efeitos dos fármacos , Hormônios/farmacologia , Humanos , Hidroxilação/efeitos dos fármacos , Masculino , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Esteroide Hidroxilases/antagonistas & inibidores , Esteroide Hidroxilases/genética , Esteroide Hidroxilases/metabolismo , Células Estromais/citologia , Células Estromais/enzimologia , Células Estromais/metabolismo , Fatores de Tempo , Vitamina D3 24-Hidroxilase
17.
FEBS Lett ; 390(1): 10-4, 1996 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-8706816

RESUMO

A mitochondrial cytochrome P450 fraction catalyzing 1 alpha- and 27-hydroxylation but not 24-hydroxylation of 25-hydroxyvitamin D3 was purified from pig kidney. The ratio between the 1 alpha- and 27-hydroxylase activities was the same in all purification steps including a side fraction. Attempts to separate the 1 alpha- and 27-hydroxylase activities were unsuccessful. A monoclonal antibody directed against purified pig liver CYP27 recognized a protein of the same apparent M(r) and immunoprecipitated both the 1 alpha- and 27-hydroxylase activities towards 25-hydroxyvitamin D3 in the purified kidney enzyme fraction as well as in a solubilized, crude cytochrome P450 extract considered to represent the major part of the 25-hydroxyvitamin D3 hydroxylases in kidney mitochondria. Taken together, the results from the purification and the experiments with CYP27 antibody, substrate inhibition, and recombinant expressed human liver CYP27 strongly indicate that CYP27 is able to catalyze 1 alpha-hydroxylation but not 24-hydroxylation of 25-hydroxyvitamin D3 in kidney. In conclusion, the results provide evidence for a role for CYP27 as a major renal mitochondrial 25-hydroxyvitamin D3 1 alpha-hydroxylase.


Assuntos
25-Hidroxivitamina D3 1-alfa-Hidroxilase/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Córtex Renal/enzimologia , Mitocôndrias Hepáticas/enzimologia , Mitocôndrias/enzimologia , Esteroide Hidroxilases/metabolismo , 25-Hidroxivitamina D3 1-alfa-Hidroxilase/imunologia , 25-Hidroxivitamina D3 1-alfa-Hidroxilase/isolamento & purificação , Animais , Anticorpos Monoclonais , Western Blotting , Colestanotriol 26-Mono-Oxigenase , Cromatografia por Troca Iônica , Clonagem Molecular , Sistema Enzimático do Citocromo P-450/imunologia , Sistema Enzimático do Citocromo P-450/isolamento & purificação , Eletroforese em Gel de Poliacrilamida , Escherichia coli , Humanos , Cinética , Peso Molecular , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Esteroide Hidroxilases/imunologia , Esteroide Hidroxilases/isolamento & purificação , Suínos
18.
J Bone Miner Res ; 6(2): 199-204, 1991 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-2028838

RESUMO

We demonstrate the unique capability of monoclonal antibodies for the specific immunodetection and characterization of two antigenic proteins occurring in normal chick kidney mitochondrial extracts. The antigens were adsorbed to cyanogen bromide-activated Sepharose gel coupled to monoclonal antibodies (MAbs) of the IgM class raised against cytochrome P450(1) alpha, which inhibit equally the 25-hydroxyvitamin D3 1 alpha- and 24-hydroxylase catalytic activities (Mandel et al. 1990 J Clin Lab Immunol, in press). The two identified antigenic proteins are polypeptides with apparent molecular weights of 57,000 and 55,000 daltons. The 1 alpha-hydroxylase cytochrome P450 has been shown to have a molecular weight of 57,000 daltons (Mandel et al. 1990 Biochim Biophys Acta 1034:239-246). The optimal antigen:gel ratio for maximal antigen binding as cytochrome P450 heme, which was determined spectrally, was found to be 1.3 nmol cytochrome P450 per g MAb-coupled Sepharose. At this ratio the total binding capacity of the gel was 1 nmol cytochrome P450 per g Sepharose. The two polypeptides were desorbed with 0.1% Emulgen 911 in 25% glycerol at pH 3.0 and separated by SDS-gel electrophoresis. The amino-terminal sequences of the two antigens were determined by automated Edman degradation with an on-line analyzer of PTH derivatives. The sequences in both antigens were 100% homologous. Complete amino acid composition analysis also revealed that their amino acid compositions were highly similar. These findings suggest that the smaller protein may be a proteolytic cleavage product of the 1 alpha-hydroxylase P450 cytochrome and may represent a putative 24-hydroxylase antigen.


Assuntos
25-Hidroxivitamina D3 1-alfa-Hidroxilase/imunologia , Anticorpos Monoclonais , Sistema Enzimático do Citocromo P-450/imunologia , Rim/química , Mitocôndrias/química , Proteínas/química , Sequência de Aminoácidos , Aminoácidos/análise , Animais , Galinhas , Cromatografia de Afinidade , Eletroforese em Gel de Poliacrilamida , Immunoblotting , Dados de Sequência Molecular , Peso Molecular , Proteínas/imunologia , Homologia de Sequência do Ácido Nucleico
19.
Histochemistry ; 95(1): 37-42, 1990.
Artigo em Inglês | MEDLINE | ID: mdl-2286531

RESUMO

Chick 25-hydroxyvitamin D3-1 alpha-hydroxylase, a cytochrome P-450 monooxygenase with a molecular weight of 57 kDa, can be isolated as described by Mandel et al. (1990 b). Under normal physiological circumstances, it occurs exclusively in kidney mitochondria. An isozyme of the 1 alpha-hydroxylase, known as the 24-hydroxylase, which uses the same substrate to yield an isomeric product, is also a cytochrome P-450 monooxygenase, has a molecular weight of 55 kDa, and like-wise occurs in kidney mitochondria. The amino-terminal sequences of the first 10 residues of the two isozymes are 100% homologous. Monoclonal antibodies of the IgM class raised against the 1 alpha-hydroxylase, which quantitatively discriminate against other P-450 cytochromes of mitochondrial or microsomal origin, recognize and interact with the 24-hydroxylase as an antigen. In the present study we show that the intestine, which is the only non-renal tissue with demonstrable 24-hydroxylase activity, gives a positive peroxidase-antiperoxidase immunohistochemical reaction using the monoclonal antibodies against the 1 alpha-hydroxylase. The reactions revealed that the antigen in the kidney is restricted to the cortical proximal tubular cells while in the intestine, the antigen is localized in the enterocytes of the villi. In kidney medullary or intestinal crypt cells, or in liver, heart and lung tissues where 1 alpha-hydroxylase or 24-hydroxylase activity could not be detected using cell or tissue homogenates, the immunohistochemical reactions were also negative.(ABSTRACT TRUNCATED AT 250 WORDS)


Assuntos
25-Hidroxivitamina D3 1-alfa-Hidroxilase/análise , Sistema Enzimático do Citocromo P-450/análise , Mucosa Intestinal/enzimologia , Rim/enzimologia , Esteroide Hidroxilases/análise , 25-Hidroxivitamina D3 1-alfa-Hidroxilase/imunologia , 25-Hidroxivitamina D3 1-alfa-Hidroxilase/metabolismo , Animais , Calcifediol/metabolismo , Cartilagem Articular/patologia , Galinhas , Sistema Enzimático do Citocromo P-450/imunologia , Sistema Enzimático do Citocromo P-450/metabolismo , Dieta , Técnicas Imunoenzimáticas , Fígado/enzimologia , Pulmão/enzimologia , Masculino , Miocárdio/enzimologia , Esteroide Hidroxilases/imunologia , Esteroide Hidroxilases/metabolismo , Deficiência de Vitamina D/enzimologia , Deficiência de Vitamina D/patologia , Vitamina D3 24-Hidroxilase
20.
Endocrinology ; 123(5): 2491-8, 1988 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-3049051

RESUMO

Monoclonal antibodies (MAbs) have been produced which recognize specific epitopes on bovine renal mitochondrial vitamin D3 1 alpha- and 24-hydroxylases. Renal mitochondria cytochrome P-450s were partially purified to 0.5-2 nmol/mg by Emulgen 911 and cholate solubilization, followed by chromatography on a 2-(4,6-dichloro-O-biphenyloxy)ethylamine HBR affinity column. Reduced carbon monoxide difference spectra determined that this preparation contained 0.5-2 nmol P-450/mg protein. This preparation contained both 1 alpha- and 24-hydroxylase activities, and Eadie-Hofstee plots of product formation as a function of substrate concentrations have maximum velocities of 1.4 and 4 pmol product/30 min.mg protein and Km values of 690 and 1300 nM, respectively. Bovine renal hydroxylases were isolated by immunoprecipitation from this partially purified P-450 preparation with a polyclonal antibody specific for rat liver microsomal cytochrome P-450 RLM5. This polyclonal antibody immunoprecipitated both 1 alpha- and 24-hydroxylase activities as well as renal mitochondrial cytochrome P-450, as determined by reduced CO spectra. Bovine renal mitochondrial components were immunoisolated and used to immunize BALB/c mouse spleen cells in vitro. MAbs then produced were screened for 1) immunoisolation of renal mitochondrial hydroxylase activity from a partially purified preparation, 2) immunohistochemical detection of antigen in renal proximal tubule cells, and 3) immunoquantitation of renal hydroxylases in a solid phase sandwich (enzyme-linked immunosorbent assay) and by 4) Western blot analysis. MAbs were isolated with specifically immunoprecipitated 1 alpha-hydroxylase activity, 24-hydroxylase activity, or both. In 10 micron sections of bovine kidney, antibodies detected antigen only in proximal tubule cells on the basal surface, which is rich in mitochondria. No antigen was detected in sections of pancreas or liver. In the solid phase sandwich enzyme-linked immunosorbent assay, MAbs detected 1 alpha and 24-hydroxylases only in renal mitochondria and not in liver microsomes or adrenal gland mitochondria. In a Western blot, MAbs specific for epitopes expressed on both hydroxylases detected a single band(s) at 52,000-53,000 daltons. Apparently it is not possible to discriminate between hydroxylases by sodium dodecyl sulfate-polyacrylamide gel electrophoresis Western blots. By these criteria, MAbs have been generated which are specific to epitopes expressed on bovine renal mitochondrial 1 alpha- and 24-hydroxylases.


Assuntos
25-Hidroxivitamina D3 1-alfa-Hidroxilase/imunologia , Anticorpos Monoclonais/imunologia , Rim/enzimologia , Esteroide Hidroxilases/imunologia , 25-Hidroxivitamina D3 1-alfa-Hidroxilase/análise , Animais , Especificidade de Anticorpos , Western Blotting , Bovinos , Sistema Enzimático do Citocromo P-450/imunologia , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Técnicas de Imunoadsorção , Cinética , Camundongos , Camundongos Endogâmicos BALB C , Mitocôndrias/enzimologia , Esteroide Hidroxilases/análise , Distribuição Tecidual , Vitamina D3 24-Hidroxilase
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