Low Vitamin-D Levels Combined with PKP3-SIGIRR-TMEM16J Host Variants Is Associated with Tuberculosis and Death in HIV-Infected and -Exposed Infants.

BACKGROUND: This study examined the associations of 25-hydroxyvitamin D and specific host genetic variants that affect vitamin D levels or its effects on immune function, with the risk of TB or mortality in children. METHODS: A case-cohort sample of 466 South African infants enrolled in P1041 trial (NCT00080119) underwent 25-hydroxyvitamin D testing by chemiluminescent immunoassay. Single nucleotide polymorphisms (SNPs) that alter the effect of vitamin D [e.g. vitamin D receptor (VDR)], vitamin D levels [e.g. vitamin D binding protein (VDBP)], or toll like receptor (TLR) expression (SIGIRR including adjacent genes PKP3 and TMEM16J) were identified by real-time PCR. Outcomes were time to TB, and to the composite of TB or death by 192 weeks of follow-up. Effect modification between vitamin D status and SNPs for outcomes was assessed. FINDINGS: Median age at 25-hydroxyvitamin D determination was 8 months; 11% were breastfed, 51% were HIV-infected and 26% had low 25-hydroxyvitamin D (<32ng/mL). By 192 weeks, 138 incident TB cases (43 definite/probable, and 95 possible) and 26 deaths occurred. Adjusting for HIV status and potential confounders, low 25-hydroxyvitamin D was associated with any TB (adjusted hazard ratio [aHR] 1.76, 95% CI 1.01-3.05; p = 0.046) and any TB or death (aHR 1.76, 95% CI 1.03-3.00; p = 0.038). Children with low 25-hydroxyvitamin D and TMEM 16J rs7111432-AA or PKP3 rs10902158-GG were at increased risk for probable/definite TB or death (aHR 8.12 and 4.83, p<0.05) and any TB or death (aHR 4.78 and 3.26, p<0.005) respectively; SNPs in VDBP, VDR, and vitamin D precursor or hydroxylation genes were not. There was significant interaction between low 25-hydroxyvitamin D and, TMEM 16J rs7111432-AA (p = 0.04) and PKP3 rs10902158-GG (p = 0.02) SNPs. CONCLUSIONS: Two novel SNPs, thought to be associated with innate immunity, in combination with low vitamin D levels were identified as increasing a young child's risk of developing TB disease or death. Identifying high-risk children and providing targeted interventions such as vitamin D supplementation may be beneficial. TRIAL REGISTRATION: ClinicalTrials.gov NCT00080119.

Behind the scenes of vitamin D binding protein: more than vitamin D binding.

Although being discovered in 1959, the number of published papers in recent years reveals that vitamin D binding protein (DBP), a member of the albuminoid superfamily, is a hot research topic. Besides the three major phenotypes (DBP1F, DBP1S and DBP2), more than 120 unique variants have been described of this polymorphic protein. The presence of DBP has been demonstrated in different body fluids (serum, urine, breast milk, ascitic fluid, cerebrospinal fluid, saliva and seminal fluid) and organs (brain, heart, lungs, kidneys, placenta, spleen, testes and uterus). Although the major function is binding, solubilization and transport of vitamin D and its metabolites, the name of this glycoprotein hides numerous other important biological functions. In this review, we will focus on the analytical aspects of the determination of DBP and discuss in detail the multifunctional capacity [actin scavenging, binding of fatty acids, chemotaxis, binding of endotoxins, influence on T cell response and influence of vitamin D binding protein-macrophage activating factor (DBP-MAF) on bone metabolism and cancer] of this abundant plasma protein.

Degalactosylated/Desialylated Bovine Colostrum Induces Macrophage Phagocytic Activity Independently of Inflammatory Cytokine Production.

BACKGROUND/AIM: Colostrum contains antibodies, such as immunoglobulin G (IgG), immunoglobulin A (IgA) and immunoglobulin M (IgM), and, therefore, has potent immunomodulating activity. In particular, IgA has an O-linked sugar chain similar to that in the group-specific component (Gc) protein, a precursor of the Gc protein-derived macrophage-activating factor (GcMAF). In the present study, we investigated the macrophage-activating effects of degalactosylated/desialylated bovine colostrum. RESULTS: We detected the positive band in degalactosylated/ desialylated bovine colostrum by western blotting using Helix pomatia agglutinin lectin. We also found that degalactosylated/ desialylated bovine colostrum could significantly enhance the phagocytic activity of mouse peritoneal macrophages in vitro and of intestinal macrophages in vivo. Besides, degalactosylated/desialylated bovine colostrum did not mediate the production of inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß). CONCLUSION: Similar to the use of GcMAF, degalactosylated/desialylated bovine colostrum can be used as a potential macrophage activator for various immunotherapies.

Presence of auto-antibody against two placental proteins, annexin A1 and vitamin D binding protein, in sera of women with pre-eclampsia.

Pre-eclampsia (PE) is one of the most complex and life-threatening pregnancy disorders. PE is characterized by maternal hypertension and proteinuria. There is much evidence to support an immunological etiology for PE and auto-immunity is considered a predisposing factor for PE. The aim of the present study was the investigation of placental proteins as targets for auto-antibodies in PE patients. 2D-PAGE technique was used for separation of the total human placental proteins. After separation, protein spots were transferred to the PVDF membranes and blotted with sera from 20 PE patients and compared with membranes blotted with 20 sera from normal women. MALDI TOF/TOF mass spectrometry technique was used for identification of differentially blotted spots. Moreover, the results of mass analysis were confirmed using western blot with commercial mAbs and RT-PCR technique. The results indicated that two placental proteins, annexin A1 and vitamin D binding protein (DBP), might be targeted by PE sera. The expression of annexin A1 and DBP was also confirmed at RNA level using the RT-PCR technique. Furthermore, the mass results were confirmed by western blotting with commercial mAbs against two targeted proteins. The data of the present study suggest two new placental proteins, annexin A1 and DBP, as placental immune targets. Considering the relation among vitamin D deficiency, increased risk of PE, and the role of annexin A1 in the resolution of inflammation, production of antibody against annexin A1 and DBP may be considered a new auto-immune hypothesis in pre-eclampsia that calls for further investigation in future work.

New perspectives on the vitamin D binding protein.

The serum vitamin D binding protein (DBP), also known as GC-globulin, is a multifunctional protein known for its role in the transport of vitamin D metabolites. DBP also binds fatty acids and actin monomers, preventing their polymerization that could be detrimental in the circulatory system. DBP may have immune functions independent of its role as a transporter of vitamin D. Because of the abundance of DBP, many aspects of its basic biochemistry were quickly established. Other features of vitamin D action, particularly transcriptional mechanisms of regulation, received greater focus and early interest in DBP centred on its value as a tool for population genetics because of its intriguing genetic variations. Nonetheless, knowledge of DBP mechanisms in physiology was obtained, and functions beyond vitamin D ligand binding were identified. With the recent increased attention regarding the benefits of vitamin D (bone health and immunological regulation), there has been a resurgence of interest in DBP. Because DBP is the primary transporter of vitamin D, it has a role in maintaining the total levels of vitamin D for the organism and in regulating the amounts of free (unbound) vitamin D available for specific tissues and cell types to utilize. This review will describe the findings on the basic biochemistry and molecular biology of DBP, the studies that elucidated its biological functions and highlight these results in light of the current renewed interest in vitamin D and human health, as well as the debate over what constitutes sufficient levels of vitamin D.

Phenotype of Gc-globulin influences the macrophage activating factor (MAF) levels in serum.

BACKGROUND: Gc-globulin is a polymorphic protein with three phenotypes: Gc 1-1, Gc 2-1 and Gc 2-2. Deglycosylation of Gc-globulin results in a Gc-macrophage activating factor (Gc-MAF). This study investigated the potential of MAF as a tumour marker and the influence of Gc-phenotypes on serum MAF-concentrations. METHODS: Gc-phenotype of 98 healthy individuals and 60 cancer patients was determined. MAF-levels of healthy individuals and cancer patients were analysed according to their Gc-phenotype using a Helix pomatia agglutinin-based ELISA. ROC curves analysed the efficiency of MAF as a tumour marker. RESULTS: MAF-levels between controls and patients were significantly different (p<0.001). No phenotypic differences were found in the patients. In comparison with the controls, MAF-values were significantly lower in cancer patients carrying Gc 1-1 (p<0.01) and Gc 2-1 (p<0.001). No difference was observed in Gc 2-2 phenotype. Diagnostic accuracy of MAF as a tumour marker also demonstrated pronounced differences between Gc-phenotypes. CONCLUSIONS: Gc-phenotype is a confounding factor when interpreting MAF-data. The value of MAF as a tumour marker varies according to phenotype. Future studies on MAF will have to consider the effect of Gc-phenotype.

Gc protein (vitamin D-binding protein): Gc genotyping and GcMAF precursor activity.

The Gc protein (human group-specific component (Gc), a vitamin D-binding protein or Gc globulin), has important physiological functions that include involvement in vitamin D transport and storage, scavenging of extracellular G-actin, enhancement of the chemotactic activity of C5a for neutrophils in inflammation and macrophage activation (mediated by a GalNAc-modified Gc protein (GcMAF)). In this review, the structure and function of the Gc protein is focused on especially with regard to Gc genotyping and GcMAF precursor activity. A discussion of the research strategy "GcMAF as a target for drug discovery" is included, based on our own research.

Therapeutic potential of vitamin D-binding protein.

Vitamin D-binding protein (DBP) is a multi-functional plasma protein with many important functions. These include transport of vitamin D metabolites, control of bone development, binding of fatty acids, sequestration of actin and a range of less-defined roles in modulating immune and inflammatory responses. Exploitation of the unique properties of DBP could enable the development of important therapeutic agents for the treatment of a variety of diseases.

Measurement of vitamin D-binding protein in pleural fluids and sera by means of a turbidimetric immunoassay measuring system.

BACKGROUND: Vitamin D-binding protein (DBP) has been recognized as a multifunctional plasma protein that can modulate certain immune and inflammatory responses. There may be differences between the DBP concentrations in pleural fluids from various diseases involving a variety of possible responses in the pleural cavity. METHODS: An anti-DBP polyclonal antibody was prepared using commercially available DBP to establish a quantitative measuring system for DBP. With a rabbit antibody, a turbidimetric immunoassay (TIA) was developed for DBP with an automatic analyzer. Using this measuring system, the concentrations of DBP were compared with the protein concentration in pleural fluid and serum specimens from patients with various diseases. RESULTS: The fluid DBP concentrations in transudative (n=11) and exudative (n=41) effusions were 71.9+/-21.2 and 180.7+/-43.7 mg/l, respectively. Among the exudative effusions, the fluid DBP concentrations in the bacterial (n=10), tuberculous (n=13), and malignant (n=18) effusions were 218.8+/-37.3, 186.7+/-26.2, and 155.1+/-41.3 mg/l, respectively. The DBP fluid/serum ratio and the fluid DBP/protein ratio in bacterial effusions were significantly higher than those in tuberculous (p<0.005, p<0.05, respectively) and malignant effusions (p<0.0005, p<0.005, respectively), although no statistically significant differences in the serum DBP/protein ratio between those effusions were found. CONCLUSIONS: Using the TIA assay, the DBP concentrations in bacterial pleural effusions were significantly higher than in tuberculous and malignant effusions.

Characterization of 25-hydroxyvitamin D binding protein from intestinal cells.

We have previously purified a cytosolic vitamin D metabolite binding protein (cDBP) from rat enterocytes, which has characteristics distinct from other vitamin D binding proteins. In these studies, we demonstrate that cDBP in a semi-purified fraction from human intestinal cells (Caco-2 cells) binds 25-hydroxyvitamin D (25OHD) with at least a 1000-fold greater affinity than 1, 25-dihydroxyvitamin D (1,25(OH)(2)D) or 24,25-dihydroxyvitamin D. Treatment of cells with 1,25(OH)(2)D reduced 25OHD binding to approximately one third that of the untreated cells (0.42 CPM/mg total protein vs 1.34 CPM/mg total protein, respectively). Finally, the cDBP is not immunoreactive to antibodies prepared against the C-terminus of the nuclear vitamin D receptor (VDR). In summary, cDBP bound 25OHD with greater affinity than either 1,25(OH)(2)D or 24,25 dihydroxyvitamin D, the cytosolic binding activity was down-regulated by 1,25(OH)(2)D and cBDP is distinct from the nuclear VDR.

Structurally well-defined macrophage activating factor derived from vitamin D3-binding protein has a potent adjuvant activity for immunization.

Freund's adjuvant produced severe inflammation that augments development of antibodies. Thus, mixed administration of antigens with adjuvant was not required as long as inflammation was induced in the hosts. Since macrophage activation for phagocytosis and antigen processing is the first step of antibody development, inflammation-primed macrophage activation plays a major role in immune development. Therefore, macrophage activating factor should act as an adjuvant for immunization. The inflammation-primed macrophage activation process is the major macrophage activating cascade that requires participation of serum vitamin D3-binding protein (DBP; human DBP is known as Gc protein) and glycosidases of B and T lymphocytes. Stepwise incubation of Gc protein with immobilized beta-galactosidase and sialidase efficiently generated the most potent macrophage activating factor (designated GcMAF) we have ever encountered. Administration of GcMAF (20 or 100 pg/mouse) resulted in stimulation of the progenitor cells for extensive mitogenesis and activation of macrophages. Administration of GcMAF (100 pg/mouse) along with immunization of mice with sheep red blood cells (SRBC) produced a large number of anti-SRBC antibody secreting splenic cells in 2-4 days. Thus, GcMAF has a potent adjuvant activity for immunization. Although malignant tumours are poorly immunogenic, 4 days after GcMAF-primed immunization of mice with heat-killed Ehrlich ascites tumour cells, the ascites tumour was no longer transplantable in these mice.

Epitope analysis of monoclonal antibodies to human Gc globulin (vitamin D-binding protein).

Gc globulin, also called vitamin D-binding protein, is a human 56 kDa plasma protein. Antigenic epitopes of the protein recognized by 12 different monoclonal antibodies (MoAbs), including the previously prepared MoAb E12 of which binding to Gc is inhibited by actin, were analyzed in relation to the functional domains of the protein. To map the epitopes recognized by the respective antibody, the reactivities of the MoAbs were tested by immunoblotting against the recombinant Gc fragments expressed in E. coli cells, and then a competitive ELISA was performed. The results showed that the antibodies were classified into at least six groups. Furthermore, in addition to E12, actin inhibited the reactivity of MoAb 21, of which the epitope was located within residues Asp320 to Glu379, in an ELISA in the presence of the ligand. This result must indicate that this antibody-binding site is near the site that interacts with actin. In contrast, no inhibitory effect by 25-hydroxyvitamin D to any antibodies was observed. Furthermore, all antibodies, including E12 and 21, reacted with membrane-bound Gc of lymphocytes by an immunofluorescence study, suggesting that Gc is unlikely to bind to the plasma membrane in its interaction with actin.

Bacterial expression of human vitamin D-binding protein (Gc2) in functional form.

In this report, we report the first expression of human vitamin D-binding protein (hDBP), a serum protein with several functions and a multidomained structure, in Escherichia coli. The recombinant protein (reDBP) was expressed as a fusion partner of glutathione S-transferase in order to facilitate proper folding of the reDBP; E. coli-expressed DBP was found to be fully functional with respect to vitamin D sterol binding, interaction with actin, and cross-reactivity with anti-DBP antibody. Furthermore, both natural DBP and reDBP were affinity-labeled with 25-hydroxyvitamin D3-3-bromo[1-14C]acetate in a similar fashion. Availability of an expression system for hDBP in functional form provides opportunity to develop mutants and truncated DBPs to study multiple ligand-binding properties of this protein in relationship with its structure.

Deglycosylation of serum vitamin D3-binding protein by alpha-N-acetylgalactosaminidase detected in the plasma of patients with systemic lupus erythematosus.

A serum glycoprotein, Gc protein (vitamin D3-binding protein), can be converted by beta-galactosidase of B cells and sialidase of T cells to a potent macrophage-activating factor (MAF), a protein with N-acetylgalactosamine as the remaining sugar moiety. Thus, Gc protein is the precursor for MAF. Treatment of Gc protein with immobilized beta-galactosidase and sialidase generates a remarkably high titered macrophage-activating factor (GcMAF). When peripheral blood monocytes/ macrophages (designated macrophages) of 33 systemic lupus erythematosus patients were incubated with GcMAF (100 pg/ml), the macrophages of all patients were activated as determined by superoxide generation. However, the precursor activity of patient plasma Gc protein was lost or reduced in these patients. Loss of the precursor activity was the result of deglycosylation of plasma Gc protein by alpha-N-acetylgalactosaminidase activity found in the patient plasma. Levels of plasma alpha-N-acetylgalactosaminidase activity in individual patients had an inverse correlation with the MAF precursor activity of their plasma Gc protein. Deglycosylated Gc protein cannot be converted to macro-phage-activating factor. The resulting defect in macro-phage activation may lead to an inability to clear pathogenic immune complexes. Thus, elevated plasma alpha-N-acetylgalactosaminidase activity resulting in the loss of MAF precursor activity and reduced macro-phage activity may play a role in the pathogenesis of systemic lupus erythematosus.

Vitamin D3-binding protein as a precursor for macrophage activating factor in the inflammation-primed macrophage activation cascade in rats.

When rat peritoneal nonadherent cells were treated with inflammatory lipid metabolites and cultured with adherent cells in 1% fetal calf serum (FCS) supplemented medium RPMI 1640 (FCS medium) for 3 hr, markedly enhanced phagocytic and superoxide generating capacities of macrophages were observed. Stepwise preparation of conditioned medium of lysophosphatidylcholine (lyso-Pc)-treated B cells and untreated T cells in FCS medium generated a potent macrophage activating factor whereas cultivation of lyso-Pc-treated B cells alone in a 1% adult rat serum supplemented medium efficiently generated the macrophage activating factor. Generation of macrophage activating factor requires a precursor protein, serum vitamin D3-binding protein (DBP), as well as participation of lymphocyte glycosidases. The lyso-Pc-inducible beta-galactosidase of B lymphocytes and the Neu-1 sialidase of T lymphocytes modified bovine DBP (bDBP) to yield the macrophage activating factor, a protein with N-acetylgalactosamine as the remaining sugar. In contrast, lyso-Pc-inducible beta-galactosidase of B cells alone modified rat DBP (rDBP) to yield the macrophage activating factor, a protein with N-acetylgalactosamine as the remaining sugar. Thus, we conclude that bDBP carries a trisaccharide composed of N-acetylgalactosamine, galactose, and sialic acid while rDBP carries a disaccharide composed of N-acetylgalactosamine and galactose.

Roles of beta-galactosidase of B lymphocytes and sialidase of T lymphocytes in inflammation-primed activation of macrophages.

The outer surface of mouse B lymphocytes carries constitutive and inducible beta-galactosidase isozymes. A brief (30 min) treatment of B lymphocytes with lysophosphatidylcholine (lyso-Pc) immediately induced an approximate 3-fold higher beta-galactosidase activity than the constitutive isozyme of untreated B lymphocytes. Thus, the lyso-Pc-inducible isozyme is not a de novo enzyme. Outer surface of mouse T lymphocytes carries constitutive (non-Neu-1) and inducible (Neu-1) sialidase isozymes. The lyso-Pc-inducible beta-galactosidase of B lymphocytes and the Neu-1 sialidase of T lymphocytes were required for conversion of vitamin D3-binding protein (Gc protein) to a potent macrophage activating factor. This enzymatic generation of the macrophage activating factor was mediated via enzyme-associated receptors.

Human testis vitamin D binding protein involved in infertility.

Immunoglobulin G (IgG) fraction was prepared from a serum obtained from an infertile woman containing antisperm antibodies that induced head-to-head agglutination of human sperm. The antibodies in the IgG fraction interacted with a 60-kD protein found in human testes determined by Western blot. The 60-kD protein was purified from human testis by isoelectric focusing (IEF), affinity chromatography on blue sepharose column, and preparative electrophoresis with electroelution. The purified 60-kD protein migrated as a single homogeneous band when analyzed by SDS-PAGE. The amino acid sequence of the N-terminus was determined. The initial 10 amino acid residues were identical to the human serum vitamin D binding protein (VDBP). Polyclonal antibodies were raised against the 60-kD protein. The polyclonal anti-60-kD antibodies and the anti-VDBP antibodies obtained from a commercial source immobilized human sperm in vitro. The interacting antigens were located on the postacrosomal region and midpiece of human sperm, as determined by an immunofluorescence method. The IgG fraction prepared from the serum of an infertile woman interacted with the human testis 60-kD protein but failed to stain serum VDBP. The results suggest that the 60-kD and VDBP are related proteins but not identical entities and that the 60-kD protein contains a unique structural group lacking in serum VDBP. Production of antibodies against the unique structure of the 60-kD protein may be the cause of the infertility.