Molecular activity of vitamin D and the Vitamin D Receptor in chronic disease

A number of studies have suggested that patients with chronic inflammatory diseases are deficient in 25-hydroxyvitamin-DThe vitamin D metabolite widely (and erroneously) considered best indicator of vitamin D "deficiency." Inactivates the Vitamin D Nuclear Receptor. Produced by hydroxylation of vitamin D3 in the liver. (25-D) and that consuming greater quantities of vitamin D, which further elevates 25-D levels, alleviates symptoms of disease. Some years ago, molecular biology identified 25-D as a secosteroid. Secosteroids would typically be expected to depress inflammation, which is in line with the reports of symptomatic improvement. The simplistic first-order mass-action model used to guide the early vitamin studies has given way to a more complex description of action. When active, the Vitamin D nuclear receptor (VDR) affects transcription of at least 913 genes and impacts processes ranging from calcium metabolism to expression of key antimicrobial peptides.

Located in the nucleus of a variety of cells including immune cells, the VDR is a control system of sorts. When exposed to infection and damage, especially that which is caused by pathogens, the body begins to convert the inactive form 25-D into the active form, 1,25-D. As cellular concentrations of 1,25-D increase, 1,25-D activates the VDR, turning on any number of tens of thousands of genes the receptor transcribes. The activation of certain genes leads to the synthesis of antimicrobial peptides. The antimicrobial peptides are the body's “natural antibiotics” and have a potent anti-bacterial effect.

However, bacteria create ligands, which like 25-D, inactivate the VDR and, in turn, the innate immune responseThe body's first line of defense against intracellular and other pathogens. According to the Marshall Pathogenesis the innate immune system becomes disabled as patients develop chronic disease.. This allows the microbes to proliferate. In response, the body increases production of 1,25-D from 25-D, leading to one of the hallmarks of chronic inflammatory disease: a low 25-D and a high 1,25-D.

This pattern is a result of the disease process rather than a cause. For a variety of reasons, neither increased consumption of vitamin D nor the body's synthesis of additional 1,25-D is ultimately effective at combatting infection.

1,25-D is different than 25-D in that it possesses a single 1-alpha hydroxylation. It is this additional hydroxylation, which stabilizes helix 12 in the Vitamin D ReceptorA nuclear receptor located throughout the body that plays a key role in the innate immune response. binding the promoter which allow activation of the VDR and leads to the transcription of thousands of genes.

A general appreciation for how 25-D and 1,25-D compete for nuclear receptors gets to the heart of their opposing roles in the body. The VDR is foremost a control system. Under most circumstances, the active form, 1,25-D, acts as the “on” switch and the inactive form, 25-D, is the “off” switch.¹ 25-D is not completely inactive, but it does not and cannot activate the VDR.

Further underscoring this role for these two D metabolites is that 25-D and 1,25-D “happen” to share similar binding affinities for the VDR. According to molecular modeling by Trevor Marshall, PhD, 1,25-D has an affinity of 8.48 (as measured by nanomolar Kd) and 25-D has an affinity of 8.36.² It would seem that activation of the Vitamin D nuclear receptor is achieved by a delicate balance between the concentrations of a number of endogenous hormones. Indeed, at the risk of overgeneralization, the body increases and decreases the production of 1,25-D to control the innate immune response.

As mentioned before, exposure to injury and infection enhances production of 1,25-D, which in turn leads to the creation of antimicrobial peptides and activation of TLR2a receptor which is expressed on the surface of certain cells and recognizes native or foreign substances and passes on appropriate signals to the cell and/or the nervous system..

However, certain feedback mechanisms are also in place, which allow the body to limit the production of 1,25-D to just that amount needed for proper transcriptional activation of the VDR.

• When the VDR is activated, it transcribes the gene for the enzyme CYP24A1, which inactivates conversion of 25-D into 1,25-D.
• An activated VDR also controls 1,25-D concentration by limiting transcription of the gene CYP27B1, which converts 25-D into 1,25-D.³

The Antimicrobial Peptide Database lists hundreds of antimicrobial peptides known to kill or inhibit the reproduction of bacteria,⁴ 793 AmPs found in animals as of January 5, 2009. The sheer diversity of these proteins coupled with the fact that they have been conserved over millenia suggests that enough pathogenic bacteria exist in sufficient quantities to warrant the evolution of these defense mechanisms. It would seem that is in the strong interest of the human body to destroy or disrupt these bacteria.

Pathogenic bacteria are likewise driven by evolutionary impetus: it's in their interest to disrupt the proteins, which interfere with their growth. In what way or ways could bacteria interrupt production of the AmPs?

According to one researcher, it is nearly impossible for bacteria to develop resistance to the AmPs:

Acquisition of resistance by a sensitive microbial strain against antimicrobial peptides is surprisingly improbable.

Michael Zasloff ⁵

However, what if it were possible to disrupt the expression of the Vitamin D Receptor by secreting ligands, which bind to and inactivate the receptor? Such bacteria would have an undeniable reproductive advantage.

Think about this for a minute – if you were a persistent pathogen, wouldn’t it seem a good idea to disable your host’s ability to produce antimicrobial peptides? And if you discovered that disabling just one receptor, the VDR, would get rid of both cathelicidin Family of antimicrobial peptides found primarily in immune cells and transcribed by the Vitamin D Receptor. and defB2, wouldn’t you try to evolve a mechanism for doing that?

Trevor Marshall, PhD

Capnine is a protein created by bacteria, a protein which binds to and antagonizes or inactivates the VDR. The secretion of capnine and substances like it fulfills an important evolutionary need for bacteria: to disrupt the innate immune response.

Since the VDR at heart at innate immune system, bacteria can survive by discovering how to disable it through a variety of different actions. Actions accumulate and are more powerful that individual actions.

In keeping with evolutionary theory, a growing number of substances and species have been shown to downregulate the activity of the VDR:

• “Gliding” biofilmA structured community of microorganisms encapsulated within a self-developed protective matrix and living together. bacteria have been shown to created Capnine – Capnine is a 2-amino-3-hydroxy-15-methylhexadecane-1-sulfonic acid, and is created by the genera Cytophaga, Capnocytophaga, Sporocytophaga, and Flexibacter.⁶⁷ The secretion of capnine meets an important evolutionary need for bacteria. Capnine possesses a high affinity for the VDR as evidenced by the fact that molecular modeling shows that its stable in the ligand binding pocket. Molecular modeling further shows that when capnine is docked in the VDR, it inactivates the receptor.
• Borrelia burgdorferi – Live Borrelia burgdorferi reduced VDR expression in monocytes (phagocytes) by 50 times, and lysates (“dead” Borrelia) reduced it by 8 times⁸
• Mycobacterium tuberculosis – shown to downregulate the VDR 3.3-fold ⁹ which makes sense given that an active VDR helps phagocytes to suppress the intracellular growth of M. tuberculosis ^{10 11}

The following substances reduce the number of VDR, without which immune function is limited:

• Caspase-3 – A protein which cleaves (breaks apart) the VDR structure and thus limits the ability of VDR to perform gene transcription.¹²

According to the Marshall Pathogenesis, pathogens' production of ligands, which bind to and antagonize or inactivate the Vitamin D Receptor, is the fundamental process by which chronic inflammatory disease occurs. The consumption of other immunosuppressive substances also has an effect.

One promising area for future research is to fully characterize the breadth and diversity of proteins created by bacteria in infected cells.

One can see the effects of a dysfunctional VDR in knockout mice, mice genetically engineered to be born without the receptor. These mice demonstrate what it is like to have a VDR completely blocked by bacterial ligands.

Mice without a VDR have been shown in separate studies to be born with alopecia, an inflammatory condition in which organisms have no hair¹³ and age prematurely.¹⁴

Further validating the Marshall Pathogenesis model is this: other research in VDR knockout mice has shown a marked increase, by a factor of ten, in serum 1,25-D and a clear reduction - to almost undetectable levels - in serum 25-D. Such levels persisted at seven weeks until the mice eventually died.¹⁵

• Epstein-Barr virus – shown to downregulate expression of the VDR by a factor of about five ¹⁶
• HIV – inhibits conversion of 25-D into 1,25-D (see below) ¹⁷

There is strong evidence that patients with inflammatory disease have elevated levels of 1,25-D.

It is sometimes thought that the liver and kidney are the only sites for conversion of 25-D into 1,25-D, but there is evidence that this process happens outside those organs – not coincidentally, at the very sites where patients report symptoms of chronic disease. High levels of 1,25-D and the enzyme which leads to the production of 1,25-D, 1 α-hydroxylase, have been found at various locations where the human body needs a strong host defense.¹⁸

• skin cells of sarcoidosis patients – Sarcoidosis patients have a variety of skin symptoms including bumps, ulcers, or discolored skin. Zehnder et al found increased expression of the enzyme 1 α-hydroxylase – the enzyme which converts 25-D into 1,25-D – in the skin cells of sarcoidosis patients.¹⁹ They write:

In particular, the expression of 1α-OHase [1 α-hydroxylase] by activated macrophages and epidermal keratinocytes [skin cells] suggests a role for 1,25(OH)2D3 [1,25-D] as an immunomodulatory and/or antiproliferative hormone.

• synovial fluid surrounding the joints of patients with rheumatoid arthritis – Mawer et al found that 1,25-D levels were particulary elevated in the synovial fluid surrounding the joints of patients with rheumatoid arthritis (RA).²⁰ In this study, median serum levels of 1,25-D at baseline was not elevated in the RA patients — only 24 pg/ml. Thus, the extrarenal synthesis of 1,25-D was not obvious from the routine blood test for 1,25-D. There is no reason to think that the metabolism of other diseases is any different.
• immune cells including macrophages – Research has also shown that 1,25-D is synthesized in cells of the immune system, including the T cells and antigen-presenting cells²¹ as well as the macrophages.^{22 23} The fact that the immune cells are a site for 1,25-D synthesis is notable, because it is these cell types, especially macrophages, which are often infected by the Th1 pathogens.

There is strong evidence that the extrarenal enzyme located in macrophages plays a major role in certain granulomatous conditions (e.g., sarcoidosis), causing uncontrolled elevations of blood 1,25-(OH)2D3 levels….

Glenville et al.²⁴

• respiratory epithelial cells – The primary lung epithelial cells, which play an important role in the defense against airborne pathogens, have been shown to express high baseline levels of activating 1 α-hydroxylase and low levels of inactivating 24-hydroxylase. This activity leads to increased expression of genes by the Vitamin D Receptor.²⁵

These disease site-specific peaks in 1,25-D somewhat undercuts the validity of the 1,25-D serum blood test as the gold standard for measuring the presence of chronic disease. There is currently no clinically available whole body test for elevated 1,25-D. For this reason, the ultimate measure of diseases treatable by the Marshall Protocol is the therapeutic probeA brief trial of the Marshall Protocol to see if it will generate an immunopathological response. The "gold standard" for testing whether a patient is a good candidate for the MP..

A cohort of 100 patients was shown to have significantly (p<0.0001) higher serum levels of 1,25-D compared to the standard range. This data was described in Dr. Greg's Blaney's 2008 presentation at the International Congress on Autoimmunity in Portugal

As 1,25-D increases, it sometimes leaks into the bloodstream where it can be detected by measures of the metabolite in blood serum, but certainly not always.

A number of studies have demonstrated that the level of the hormone 1,25-D rises in patients with certain chronic diseases. One study found that in a cohort of 88 Crohn's disease patients, 35 patients or 40% had elevated levels of 1,25-D, which the authors defined as above 60 pg/mL.²⁶

Bell listed the following diseases and conditions, which manifest with high levels of 1,25-D: tuberculosis, AIDS with Pneumocystis carinii pneumonia, AIDS with cytomegalovirus infection, disseminated candidiasis, leprosy, rheumatoid arthritis, silicone-induced granulomas, Wegerner’s granulomatosis, Hodgkin’s disease, lymphoma, histocytic lymphoma, T-cell leukemia, plasma cell granuloma, leiomyoblastoma, seminoma, and subcutaneous fat necrosis.²⁷

Dr. Greg Blaney, a physician who practices in Vancouver, British Columbia (a setting with relatively infrequent sunlight), found that of a group of 100 patients with a variety of chronic inflammatory diseases, 85 had elevated measures of 1,25-D, which was defined as greater than or equal to 110 pmol/L.

Kavathia et al found that in patients with sarcoidosis, those with high serum levels of 1,25-D have more pronounced chronic treatment needs.²⁸

The active vitamin D metabolite, 1,25-D, serves an important role in fighting infection, but high levels of the hormone contribute to symptoms of disease by virtue of its interactions with the body's other nuclear receptors.

Molecular modeling data show that at high levels, 1,25-D not only binds the VDR but also has a strong affinity for other key receptors that control the body's major hormonal systems including those that regulate the body's sex, thyroid, and adrenal hormones. As 1,25-D rises, it pushes out the molecules that are meant to control these receptors. Compromising these receptors can disrupt the body's ability to regulate temperature, libido, and any number of other functions.[table of affinities needed]

Molecular research also shows that, like the VDR, several of these nuclear receptors (including the alpha/beta thyroid receptors, glucocorticoid receptor, and androgen receptor) also express many families of antimicrobial peptides. A recent analysis of AmP expression by Brahmachary showed that the glucocorticoid receptor, the androgen receptor, and the Vitamin D Receptor, seem to be in control of 20, 17 and 16 families respectively, out of 22 analyzed.²⁹ This means that when elevated 1,25-D displaces their endogenous ligands, the body's overall AmP expression is thwarted to an even greater degree. This further impairs the innate immune system's ability to combat chronic pathogens.

1,25-D has a very high affinity for the alpha thyroid nuclear receptorIntracellular receptor proteins that bind to hydrophobic signal molecules (such as steroid and thyroid hormones) or intracellular metabolites and are thus activated to bind to specific DNA sequences which affects transcription. (ThRa) having a Kd value of 8.41. Normally levels of the endogenous ligand for ThRa known as T3 (which has a Kd 7.20 for ThRa) keep 1,25-D out of the binding pocket, but as 1,25-D rises due to VDR dysregulation it starts to proportionately displace T3 and block transcription by ThRa. The same thing should happen with thyroid beta – 1,25-D has a Kd of 8.44 for that receptor.

When 1,25-D displaces T3, the genes with ThRa promoters are no longer transcribed, resulting in the phenomenon known as thyroid hormone resistance. Since related nuclear receptors work as a group, when transcription by ThRa is dysregulated, system wide gene transcription is also affected.

1,25-D has a kD of 8.05 for the androgen receptor, and a Kd of 8.12 for the glucocorticoid receptor. Elevated 1,25-D can displace cortisol and testosterone from their target receptors as well, leading to an array of other hormonal imbalances.

A primary action of 1,25-D is that a high level in susceptible individuals (e.g. during pregnancy and sun holidays) causes the cellular membrane protein TACO to allow tiny bacteria to freely enter and exit the immune cells, without causing the cells to die in the process.

Dr. Andy Wright has taken photos of [bacteria freely entering and exiting cells], and it obviously would allow the pathogen(s) to spread without restraint.

Trevor Marshall, PhD

One of the hallmarks of late-stage inflammatory diseases is a very low 1,25-D with HIV/AIDS being the most commonly cited example.

HIV is a viral infection, and AIDS is the syndrome, which results – according to the Marshall Pathogenesis, at least – by a dysregulated vitamin D metabolism. As evidenced by the subset of people who survive for decades with HIV, the virus itself is not necessarily deadly. Instead, it is the co-infections which are the proximate cause of the disease. One can define the breadth of AIDS-related complications by the extent and number of co-infections such as pneumonia, herpes, Candida, etc.

Supporting this hypothesis, a number of terminal AIDS patients have neglible levels of 1,25-D. 18 of 29 patients in a study of AIDS patients had undetectable levels of the metabolite.³⁰ The patients with depressed levels of 1,25-D were characterized by advanced clinical HIV infection, low CD4+ lymphocyte counts, and high serum levels of tumor TNF-alpha – all indication of more severe forms of the disease.

The exact mechanism by which 1,25-D is downregulated is not entirely known, but it is highly likely that it is caused by pathogens. Haug et al theorized that TNF-alpha and possibly other cytokinesAny of various protein molecules secreted by cells of the immune system that serve to regulate the immune system. – which pathogens are known to create³¹ – inhibit conversion of 25-D into 1,25-D in late-stage cases of HIV/AIDS.³²