Vitamin D-binding protein deficiency: an underrecognized Mendelian disorder of vitamin D metabolism.

Vitamin D-binding protein (VDBP) deficiency is a recently discovered apparently benign biochemical disorder that can masquerade as treatment-resistant vitamin D deficiency and is likely underrecognized. We present the case of a child with persistently low 25OH vitamin D levels despite replacement therapy. Exome sequencing revealed a novel homozygous nonsense variant in the GC gene, leading to undetectable levels of VDBP. Interestingly, exome sequencing also revealed a homozygous loss-of-function variant in ZNF142, which likely explains the additional clinical features of recurrent febrile convulsions and global developmental delay. Our findings corroborate the two previously reported patients with autosomal recessive VDBP deficiency caused by biallelic GC variants and emphasize the importance of measuring VDBP levels in cases of apparent vitamin D deficiency that is treatment-resistant. We also urge caution in concluding "atypical" presentations without careful investigation of a potential dual molecular diagnosis.

Vitamin D-binding protein-loaded PLGA nanoparticles suppress Alzheimer's disease-related pathology in 5XFAD mice.

The aggregation and accumulation of amyloid beta (Aß) peptide is believed to be the primary cause of Alzheimer's disease (AD) pathogenesis. Vitamin D-binding protein (DBP) can attenuate Aß aggregation and accumulation. A biocompatible polymer poly (D,L-lactic acid-co-glycolic acid) (PLGA) can be loaded with therapeutic agents and control the rate of their release. In the present study, a PLGA-based drug delivery system was used to examine the therapeutic effects of DBP-PLGA nanoparticles in Aß-overexpressing (5XFAD) mice. DBP was loaded into PLGA nanoparticles and the characteristics of the DBP-PLGA nanoparticles were analyzed. Using a thioflavin-T assay, we observed that DBP-PLGA nanoparticles significantly inhibited Aß aggregation in vitro. In addition, we found that intravenous injection of DBP-PLGA nanoparticles significantly attenuated the Aß accumulation, neuroinflammation, neuronal loss and cognitive dysfunction in the 5XFAD mice. Collectively, our results suggest that DBP-PLGA nanoparticles could be a promising therapeutic candidate for the treatment of AD.

Is α-N-acetylgalactosaminidase the key to curing cancer? A mini-review and hypothesis.

In the constant battle against cancer cells, macrophages are of great importance. Their activation is achieved through various mechanisms such as Vitamin D binding protein (VDBP or Gc). After undergoing modifications via enzymes secreted by stimulated lymphocytes, VDBP is modified into Macrophages Activator Form/Factor (Gc-MAF). Some studies (particularly those focusing on cancer) have reported that an enzyme known as α-N-acetylgalactosaminidase (nagalase) facilitates the deglycosylation of Gc-MAF, which in turn inhibits the activation of macrophages. The aim of this review was to evaluate studies associated with nagalase and its escalation in various diseases and to propose hypothetical solutions in order to neutralize the effects of nagalase in cancer patients.

Vitamin D binding protein (VDBP) hijacks twist1 to inhibit vasculogenic mimicry in hepatocellular carcinoma.

Rationale: Vitamin D (VD) has been suggested to have antitumor effects, however, research on the role of its transporter vitamin D-binding protein (VDBP, gene name as GC) in tumors is limited. In this study, we demonstrated the mechanism underlying the inhibition of vasculogenic mimicry (VM) by VDBP in hepatocellular carcinoma (HCC) and proposed an anti-tumor strategy of combining anti-PD-1 therapy with VD. Methods: Three-dimensional cell culture models and mice with hepatocyte-specific GC deletion were utilized to study the correlation between VDBP expression and VM. A patient-derived tumor xenograft (PDX) model was further applied to validate the therapeutic efficacy of VD in combination with an anti-PD-1 drug. Results: The study revealed that VDBP expression is negatively correlated with VM in HCC patients and elevated VDBP expression is associated with a favorable prognosis. The mechanism studies suggested VDBP hindered the binding of Twist1 on the promoter of VE-cadherin by interacting with its helix-loop-helix DNA binding domain, ultimately leading to the inhibition of VM. Furthermore, VD facilitated the translocation of the vitamin D receptor (VDR) into the nucleus where VDR interacts with Yin Yang 1 (YY1), leading to the transcriptional activation of VDBP. We further demonstrated that the combination of VD and anti-PD-1 led to an improvement in the anti-tumor efficacy of an anti-PD-1 drug. Conclusion: Collectively, we identified VDBP as an important prognostic biomarker in HCC patients and uncovered it as a therapeutic target for enhancing the efficacy of immune therapy.

Vitamin D binding protein-macrophage activating factor inhibits HCC in SCID mice.

BACKGROUND: A high incidence of recurrence after treatment is the most serious problem in hepatocellular carcinoma (HCC). Therefore, a new strategy for the treatment of the disease is needed. The aim of the present study was to investigate whether vitamin D binding protein-macrophage activating factor (DBP-maf) is able to inhibit the growth of HCC. METHODS: The effects of DBP-maf on endothelial cells and macrophage were evaluated by WST-1 assay and phagocytosis assay, respectively. Human HCC cells (HepG2) were implanted into the dorsum of severe combined immunodeficiency (SCID) mice. These mice were divided into control and DBP-maf treatment groups (n = 10/group). The mice in the treatment group received 40 ng/kg/d of DBP-maf for 21 d. RESULTS: DBP-maf showed anti-proliferative activity against endothelial cells and also activated phagocytosis by macrophages. DBP-maf inhibited the growth of HCC cells (treatment group: 126 ± 18mm(3), untreated group: 1691.5 ± 546.9mm(3), P = 0.0077). Histologic examinations of the tumors revealed the microvessel density was reduced and more macrophage infiltration was demonstrated in the tumor of mice in the treatment group. CONCLUSION: DBP-maf has at least two novel functions, namely, an anti-angiogenic activity and tumor killing activity through the activation of macrophages. DBP-maf may therefore represent a new strategy for the treatment of HCC.

Immunotherapy with GcMAF revisited - A critical overview of the research of Nobuto Yamamoto.

This overview describes the research of Nobutu Yamamoto (Philadelphia) concerning immunotherapy with GcMAF for patients with cancer and for patients infected with pathogenic envelope viruses. GcMAF (Group-specific component Macrophage-Activating Factor) is a mammalian protein with an incredible potency to directly activate macrophages. Since the late 1980s Yamamoto's investigations were published in numerous journals but in order to understand the details of his research, a minute survey of many of his patents was required. But even then, regrettably, a precise description of his experiments was sometimes lacking. This overview tries to summarize all of Yamamoto's research on GcMAF, as well as some selected more recent papers from other investigators, who tried to verify and/or reproduce Yamamoto's reports. In my opinion the most important result of the GcMAF research deserves widespread renewed attention: human GcMAF injections (100 ng per week, intramuscular or intravenous) can help to cure patients with a great variety of cancers as well as patients infected with pathogenic envelope viruses like the human immunodeficiency virus 1 (HIV-1), influenza, measles and rubella (and maybe also SARS-CoV-2). From Yamamoto's data it can be calculated that GcMAF is a near-stoichiometric activator of macrophages. Yamamoto monitored the progress of his immunotherapy via the serum level of an enzyme called nagalase (α-N-acetylgalactosaminidase activity at pH 6). I have extensively discussed the properties and potential catalytic site of this enzyme activity in an Appendix entitled: "Search for the potential active site of the latent α-N-acetylgalactosaminidase activity in the glycoproteins of some envelope viruses".

Immunotherapy of HIV-infected patients with Gc protein-derived macrophage activating factor (GcMAF).

Serum Gc protein (known as vitamin D3-binding protein) is the precursor for the principal macrophage activating factor (MAF). The MAF precursor activity of serum Gc protein of HIV-infected patients was lost or reduced because Gc protein is deglycosylated by alpha-N-acetylgalactosaminidase (Nagalase) secreted from HIV-infected cells. Therefore, macrophages of HIV-infected patients having deglycosylated Gc protein cannot be activated, leading to immunosuppression. Since Nagalase is the intrinsic component of the envelope protein gp120, serum Nagalase activity is the sum of enzyme activities carried by both HIV virions and envelope proteins. These Nagalase carriers were already complexed with anti-HIV immunoglobulin G (IgG) but retained Nagalase activity that is required for infectivity. Stepwise treatment of purified Gc protein with immobilized beta-galactosidase and sialidase generated the most potent macrophage activating factor (termed GcMAF), which produces no side effects in humans. Macrophages activated by administration of 100 ng GcMAF develop a large amount of Fc-receptors as well as an enormous variation of receptors that recognize IgG-bound and unbound HIV virions. Since latently HIV-infected cells are unstable and constantly release HIV virions, the activated macrophages rapidly intercept the released HIV virions to prevent reinfection resulting in exhaustion of infected cells. After less than 18 weekly administrations of 100 ng GcMAF for nonanemic patients, they exhibited low serum Nagalase activities equivalent to healthy controls, indicating eradication of HIV-infection, which was also confirmed by no infectious center formation by provirus inducing agent-treated patient PBMCs. No recurrence occurred and their healthy CD + cell counts were maintained for 7 years.

Vitamin D Binding Protein and Vitamin D Levels in Multi-Ethnic Population.

INTRODUCTION: Low levels of 25-hydroxyvitamin D (25(OH)D) has been associated with many negative health outcomes including falls and fractures. 25(OH)D is largely bound to vitamin D binding protein (VDBP). There is increasing evidence that free or bioavailable 25(OH)D may be a better measure of vitamin D deficiency. OBJECTIVE: To determine the prevalence of 25(OH)D deficiency and VDBP levels in multi-ethnic population, and its impact on muscle strength. DESIGN AND METHODS: Cross-sectional study of older adults in Western region of Singapore. 295 participants from three ethnic groups were selected from the Healthy Older People Everyday (HOPE) cohort for measurements of total 25(OH)D and VDBP levels. Total 25(OH)D, VDBP, frailty status, Timed-Up-and-Go (TUG) and grip strength (GS) were assessed. Albumin, free and bioavailable 25(OH)D were only available for 256 participants. RESULTS: 53% of Malay and 55% of Indians were deficient in 25(OH)D compared with 18.2% of ethnic Chinese participants. Chinese also had higher total 25(OH)D concentrations with a mean of 29.1 ug/l, (p = <0.001). Chinese had the lowest level of VDBP (169.6ug/ml) followed by Malay (188.8 ug/ml) and Indian having the highest (220.1 ug/ml). Calculated bioavailable and free 25(OH)D levels were significantly higher in Chinese, followed by Malays and Indians, which also correlated with better grip strength measures amongst the Chinese. CONCLUSION: The Malays and Indians had overall lower free, bioavailable and total 25(OH)D compared with ethnic Chinese. Chinese ethnic group also had the lowest VDBP and better overall grip strength.

Total and Free Circulating Vitamin D and Vitamin D-Binding Protein in Relation to Colorectal Cancer Risk in a Prospective Study of African Americans.

Background: Previous studies rarely evaluated the associations between vitamin D-binding protein and free vitamin D with colorectal cancer risk. We assessed these biomarkers and total 25-hydroxyvitamin D in relation to colorectal cancer risk in a sample of African Americans.Methods: Cases comprised 224 African American participants of the Southern Community Cohort Study diagnosed with incident colorectal cancer. Controls (N = 440) were selected through incidence density sampling and matched to cases on age, sex, and race. Conditional logistic regression was used to calculate odds ratios (OR) and 95% confidence intervals (CI) for associations between biomarker levels and colorectal cancer risk.Results: Vitamin D was inversely associated with colorectal cancer risk where the OR per-SD increase in total and free 25-hydroxyvitamin D were 0.82 (95% CI, 0.66-1.02) and 0.82 (95% CI, 0.66-1.01), respectively. Associations were most apparent among cases diagnosed >3 years after blood draw: ORs for the highest tertile versus the lowest were 0.69 (95% CI, 0.21-0.93) for total 25-hydroxyvitamin D and 0.71 (95% CI, 0.53-0.97) for free 25-hydroxyvitamin D. Inverse associations were seen in strata defined by sex, BMI, and anatomic site, although not all findings were statistically significant. Vitamin D-binding protein was not associated with colorectal cancer risk.Conclusions: Our findings suggest that total and free 25-hydroxyvitamin D may be inversely associated with colorectal cancer risk among African Americans.Impact: These findings highlight a potential role for vitamin D in colorectal cancer prevention in African Americans. Cancer Epidemiol Biomarkers Prev; 26(8); 1242-7. ©2017 AACR.

Effects of vitamin D(3)-binding protein-derived macrophage activating factor (GcMAF) on angiogenesis.

BACKGROUND: The vitamin D(3)-binding protein (Gc protein)-derived macrophage activating factor (GcMAF) activates tumoricidal macrophages against a variety of cancers indiscriminately. We investigated whether GcMAF also acts as an antiangiogenic factor on endothelial cells. METHODS: The effects of GcMAF on angiogenic growth factor-induced cell proliferation, chemotaxis, and tube formation were examined in vitro by using cultured endothelial cells (murine IBE cells, porcine PAE cells, and human umbilical vein endothelial cells [HUVECs]) and in vivo by using a mouse cornea micropocket assay. Blocking monoclonal antibodies to CD36, a receptor for the antiangiogenic factor thrombospondin-1, which is also a possible receptor for GcMAF, were used to investigate the mechanism of GcMAF action. RESULTS: GcMAF inhibited the endothelial cell proliferation, chemotaxis, and tube formation that were all stimulated by fibroblast growth factor-2 (FGF-2), vascular endothelial growth factor-A, or angiopoietin 2. FGF-2-induced neovascularization in murine cornea was also inhibited by GcMAF. Monoclonal antibodies against murine and human CD36 receptor blocked the antiangiogenic action of GcMAF on the angiogenic factor stimulation of endothelial cell chemotaxis. CONCLUSIONS: In addition to its ability to activate tumoricidal macrophages, GcMAF has direct antiangiogenic effects on endothelial cells independent of tissue origin. The antiangiogenic effects of GcMAF may be mediated through the CD36 receptor.

Immunotherapy of BALB/c mice bearing Ehrlich ascites tumor with vitamin D-binding protein-derived macrophage activating factor.

Vitamin D3-binding protein (DBP; human DBP is known as Gc protein) is the precursor of macrophage activating factor (MAF). Treatment of mouse DBP with immobilized beta-galactosidase or treatment of human Gc protein with immobilized beta-galactosidase and sialidase generated a remarkably potent MAF, termed DBPMAF or GcMAF, respectively. The domain of Gc protein responsible for macrophage activation was cloned and enzymatically converted to the cloned MAF, designated CdMAF. In Ehrlich ascites tumor-bearing mice, tumor-specific serum alpha-N-acetylgalactosaminidase (NaGalase) activity increased linearly with time as the transplanted tumor cells grew in the peritoneal cavity. Therapeutic effects of DBPMAF, GcMAF, and CdMAF on mice bearing Ehrlich ascites tumor were assessed by survival time, the total tumor cell count in the peritoneal cavity, and serum NaGalase activity. Mice that received a single administration of DBPMAF or GcMAF (100 pg/mouse) on the same day after transplantation of tumor (1 x 10(5) cells) showed a mean survival time of 35 +/- 4 days, whereas tumor-bearing controls had a mean survival time of 16 +/- 2 days. When mice received the second DBPMAF or GcMAF administration at day 4, they survived more than 50 days. Mice that received two DBPMAF administrations, at days 4 and 8 after transplantation of 1 x 10(5) tumor cells, survived up to 32 +/- 4 days. At day 4 posttransplantation, the total tumor cell count in the peritoneal cavity was approximately 5 x 10(5) cells. Mice that received two DBPMAF administrations, at days 0 and 4 after transplantation of 5 x 10(5) tumor cells, also survived up to 32 +/- 4 days, while control mice that received the 5 x 10(5) ascites tumor cells only survived for 14 +/- 2 days. Four DBPMAF, GcMAF, or CdMAF administrations to mice transplanted with 5 x 10(5) Ehrlich ascites tumor cells with 4-day intervals showed an extended survival of at least 90 days and an insignificantly low serum NaGalase level between days 30 and 90.

Case Report: GcMAF Treatment in a Patient with Multiple Sclerosis.

BACKGROUND/AIM: Gc protein-derived macrophage-activating factor (GcMAF) has various functions as an immune modulator, such as macrophage activation, anti-angiogenic activity and anti-tumor activity. Clinical trials of second-generation GcMAF demonstrated remarkable clinical effects in several types of cancers. Thus, GcMAF-based immunotherapy has a wide application for use in the treatment of many diseases via macrophage activation that can be used as a supportive therapy. Multiple sclerosis (MS) is considered to be an autoimmune disorder that affects the myelinated axons in the central nervous system (CNS). This study was undertaken to examine the effects of second-generation GcMAF in a patient with MS. RESULTS: This case study demonstrated that treatments of GcMAF in a patient with MS have potent therapeutic actions with early beneficial responses, especially improvement of motor dysfunction. CONCLUSION: GcMAF shows therapeutic potency in the treatment of MS.

Oral Colostrum Macrophage-activating Factor for Serious Infection and Chronic Fatigue Syndrome: Three Case Reports.

BACKGROUND: Gc protein-derived macrophage-activating factor (GcMAF) immunotherapy has been steadily advancing over the last two decades. Oral colostrum macrophage-activating factor (MAF) produced from bovine colostrum has shown high macrophage phagocytic activity. GcMAF-based immunotherapy has a wide application for use in treating many diseases via macrophage activation or for use as supportive therapy. RESULTS: Three case studies demonstrate that oral colostrum MAF can be used for serious infection and chronic fatigue syndrome (CFS) without adverse effects. CONCLUSION: We demonstrate that colostrum MAF shows promising clinical results in patients with infectious diseases and for symptoms of fatigue, which is common in many chronic diseases.

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.

Effects of vitamin D binding protein-macrophage activating factor (DBP-MAF) infusion on bone resorption in two osteopetrotic mutations.

Osteopetrosis is a heterogeneous group of bone diseases characterized by an excess accumulation of bone and a variety of immune defects. Osteopetrosis (op) and incisors absent (ia) are two nonallelic mutations in the rat which demonstrated these skeletal defects as a result of reduced bone resorption. Osteopetrotic (op) rats have severe sclerosis as a result of reduced numbers of osteoclasts which are structurally abnormal. The sclerosis in ia rats is not as severe as in op mutants; they have elevated numbers of osteoclasts, but they are also morphologically abnormal, lacking a ruffled border. Both of these mutations have defects in the inflammation-primed activation of macrophages. They demonstrate independent defects in the cascade involved in the conversion of vitamin D binding protein (DBP) to a potent macrophage activating factor (DBP-MAF). Because this factor may also play a role in the pathogenesis of osteoclastic dysfunction, the effects of ex vivo-generated DBP-MAF were evaluated on the skeletal system of these two mutations. Newborn ia and op rats and normal littermate controls were injected with DBP-MAF or vehicle once every 4 days from birth until 2 weeks of age, at which time bone samples were collected to evaluate a number of skeletal parameters. DBP-MAF treated op rats had an increased number of osteoclasts and the majority of them exhibited normal structure. There was also reduced bone volume in the treated op animals and an associated increased cellularity of the marrow spaces. The skeletal sclerosis was also corrected in the ia rats; the bone marrow cavity size was significantly enlarged and the majority of the osteoclasts appeared normal with extensive ruffled borders.

The toothless osteopetrotic rat has a normal vitamin D-binding protein-macrophage activating factor (DBP-MAF) cascade and chondrodysplasia resistant to treatments with colony stimulating factor-1 (CSF-1) and/or DBP-MAF.

The osteopetrotic rat mutation toothless (tl) is characterized by little or no bone resorption, few osteoclasts and macrophages, and chondrodysplasia at the growth plates. Short-term treatment of tl rats with colony-stimulating factor-1 (CSF-1) has been shown to increase the number of osteoclasts and macrophages, producing dramatic resolution of skeletal sclerosis at some, but not all, sites. Defects in production of vitamin D-binding protein-macrophage activating factor (DBP-MAF) have been identified in two other independent osteopetrotic mutations of the rat (op and ia), and two in the mouse (op and mi), in which macrophages and osteoclasts can be activated by the administration of exogenous DBP-MAF. The present studies were undertaken to examine the histology and residual growth defects in tl rats following longer CSF-1 treatments, to investigate the possibility that exogenous DBP-MAF might act synergistically with CSF-1 to improve the tl phenotype, and to assess the integrity of the endogenous DBP-MAF pathway in this mutation. CSF-1 treatment-with or without DBP-MAF-induced resorption of metaphyseal bone to the growth plate on the marrow side, improved slightly but did not normalize long bone growth, and caused no improvement in the abnormal histology of the growth plate. Injections of lysophosphatidylcholine (lyso-Pc) to prime macrophage activation via the DBP-MAF pathway raised superoxide production to similar levels in peritoneal macrophages from both normal and mutant animals, indicating no defect in the DBP-MAF pathway in tl rats. Interestingly, pretreatments with CSF-1 alone also increased superoxide production, although the mechanism for this remains unknown. In summary, we find that, unlike other osteopetrotic mutations investigated to date, the DBP-MAF pathway does not appear to be defective in the tl rat; that additional DBP-MAF does not augment the beneficial skeletal effects seen with CSF-1 alone; and that the growth plate chondrodystrophy seen in this mutation is unaffected by either molecule. Thus, the tl mutation intercepts the function of a gene required for both normal endochondral ossification and bone resorption, thereby uncoupling the coordination of skeletal metabolism required for normal long bone growth.

Case report: A breast cancer patient treated with GcMAF, sonodynamic therapy and hormone therapy.

BACKGROUND: Gc protein-derived macrophage-activating factor (GcMAF) occurs naturally in the human body. It has various functions, such as macrophage activation and antitumor activities. Recently, immunotherapy has become an attractive new strategy in the treatment of cancer. GcMAF-based immunotherapy can be combined with many other therapies. Sonodynamic therapy (SDT) using low-intensity ultrasound is a novel therapeutic modality. Ultrasound has been demonstrated to activate a number of sonosensitive agents allowing for the possibility of non-invasive targeted treatment for both superficial and deep-seated tumors. The current case study demonstrates that GcMAF and SDT can be used in combination with conventional therapies in patients with metastatic cancer, especially where treatment options are limited due to factors such as toxicity. This case study also suggests a new concept of cancer treatment using local destruction of cancer tissue, in this case conducted with SDT, to be used in combination with GcMAF immunotherapy as a systemic treatment.

Vitamin D binding protein: a multifunctional protein of clinical importance.

Since the discovery of group-specific component and its polymorphism by Hirschfeld in 1959, research has put spotlight on this multifunctional transport protein (vitamin D binding protein, DBP). Besides the transport of vitamin D metabolites, DBP is a plasma glycoprotein with many important functions, including sequestration of actin, modulation of immune and inflammatory responses, binding of fatty acids, and control of bone development. A considerable DBP polymorphism has been described with a specific allele distribution in different geographic area. Multiple studies have shed light on the interesting relationship between polymorphisms of the DBP gene and the susceptibility to diseases. In this review, we give an overview of the multifunctional character of DBP and describe the clinical importance of DBP and its polymorphisms. Finally, we discuss the possibilities to use DBP as a novel therapeutic agent.