Pivotal Nexus Points from Health to Disease to Disaster

Loss of health starts with chronic inflammation. In most cases diet is the predominant source of inflammation, but infections (typically opportunistic cryptic bacterial, viral and fungal biofilms often ‘farmed’ by and supporting protist parasites) also increase the chemical ante, heightening the cytokine crescendo.

Inflammation sets the stage for damaging ‘friendly fire’ by faulty processing of communicating proteins at the focal location where lymphocytes and antigen-presenting cells congregate.

Inflammatory cytokines and the inflammatory response cascade ratchet up genetically from activation of nuclear factor-kappa beta (NF-kB), and vice versa. Multiple cytokine mRNAs (at least IL-6, GM-CSF and TNF-a) share the same transcription factor, NF-kB.

Cytokines strongly modulate drive and fatigue. For example, interferon-alpha (IFN-a), interleukin-2 (IL-2), or tumor necrosis factor (TNF) have been used to treat cancer or hepatitis, and they typically cause a "cytokine syndrome" of fatigue, fever, brain fog, myalgia, apathy and depression.

Inflammation causes a disruption of the integrity of the endothelial extracellular matrix at sites of local inflammation. NF-kB activation shuts down the expression of genes involved in heparin sulfate proteoglycan (HSPG) synthesis and this makes the tissue/blood barrier leaky.

Locally this facilitates the recruitment of lymphocytes and neutrophils for defense, but systemically it leads to leaky endothelial gut/kidney/brain barriers that permit bacterial invasion.

MODS (multiple organ dysfunction syndrome) results from exaggerated and uncontrolled inflammatory immune, metabolic, vascular, neural and endocrine responses to a causative agent. These responses are dynamic, variable, interconnected and confounding to most current thinking, parabolic or non-linear in nature.

Responses vary not only in different hosts, but change at different time intervals in the same host. The reactions form an ever-changing, interconnecting network or non-linear system, so that a straight line analytical approach would fail to evaluate emergent properties of this network or system.

MODS is a further evolution of the systemic inflammatory response syndrome (SIRS). SIRS due to infection is called sepsis. SIRS is present when any two or more of the symptoms listed below are seen in a patient: 1. Temperature (too high or too low) > 38.0C or < 36.0C; 2. Respiratory rate > 20/min - respiratory alkalosis, PCO2 < 32 mm Hg (local or systemic metabolic inflammatory oxidative alkalosis too);3. Tachycardia > 90/min;4. White blood cells too high or too low > 12,000/mm3 or < 4,000/mm3 or > 10% band forms.

Here's a snapshot of an immune system interaction:

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Dendritic cells are stellate or tree-like cells (Greek, dendron, tree) that are found in lymphoid or immune organs, and at the interfaces between our bodies and the environment. The epidermal layer of the skin has a rich network of dendritic cells. In addition, dendritic cells line the surfaces of the airway and intestine, where they function as sentinels that sample proteins and particulates from the environment.

Dendritic cells arise from proliferating progenitors (stem cells), primarily in the bone marrow, a process driven by chemical messengers, to become precursors such as the monocytes in blood, and these in turn give rise to immature dendritic cells or macrophages. Endothelial cells, fibroblasts, chondroblasts, osteoblasts, chondroclasts and osteoclasts are also derived from stem cells that have responded to their individual cytokine commands.

As sentinels, dendritic cells patrol the body seeking out foreign invaders, whether these are bacteria, viruses, or dangerous toxins. After capturing the invaders, called antigens, dendritic cells digest them into smaller pieces and display the antigenic fragments on their cell surfaces. A changed cell surface of a barrier cell attracts the attention of opportunistic planktonic microorganisms for possible attachment and invasion or subsequent development and organization of pathogenic biofilm.

Mobile dendritic cells then travel to lymph nodes or spleen where they stimulate other cells of the immune system to make vigorous responses, in particular, the B cells that make antibodies to neutralize the invaders and killer T cells that launch specific attacks to destroy them.

The dendritic cell (which has a DNGR-1 receptor) also mobilizes an inflammatory immune response after sensing necrosis (abnormal cell death). Tumors trigger this type of immune ‘danger’ reaction as well since they often contain clusters of cells undergoing necrosis due to limited oxygen supply.

Dendritic cells are equally responsible for immune tolerance, which silences dangerous immune cells and prevents them from attacking innocuous materials in the body or the body's own tissues. Dendritic cells carry on their surface high levels of major histocompatibility complex (MHC) products, which are critically recognized by T-lymphocytes.

Two mechanisms allow dendritic cells to induce tolerance. The antigen-loaded immature dendritic cells can silence T cells by either deleting them (via inducing apoptosis) or by inducing regulatory T cells that suppress the reactions of other immune cells. When the dendritic cells subsequently mature in response to infection, the preexisting tolerance nullifies any reaction to innocuous antigens and allows the dendritic cells to focus immune response on the pathogen.

Failure to silence heightened immunity can lead to autoimmune diseases like systemic lupus erythematosus, rheumatoid arthritis and multiple sclerosis. If dendritic cells are too tolerant, a permissive environment for chronic infectious agents, such as virus is created. Biofilm infections and tumors use similar strategies to exploit the ‘excessively tolerant’ function of dendritic cells, shut down normal immune defenses and perpetuate disease.

Dendritic cells are major immune stimulators and are quite potent.

In fact, a dendritic cell to T cell ratio of 1 to 100 initiates vigorous and optimal responses. Moreover, the dendritic cells directly activate both helper T cells as well as killer T cells. Once activated by dendritic

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cells, the T cells can also interact vigorously with other antigen presenting B cells and macrophages to further magnify immune responses from these cells.

For coordinating these various processes efficiently and precisely, the dendritic cells are considered to be ’conductors of the immune orchestra.’ An incompatible food, infectious agent like a virus or bacterium is introduced to the body, and the dendritic cells stimulate and coordinate an appropriate response by the complement system, killer T- cell and macrophage attack.

Threatening agents have surface antigenic molecules that the body uses for identification. Macrophages actually engulf and digest invaders. They remove the antigen molecules from the invader’s surface and act as APC (Antigen Presenting Cells) presenting examples of the antigen on its surface to recruited T (thymus) and or B (bone) lymphocytes (which produce receptors that fit the challenging antigen’s shape as a means of identifying it).

Next, two types of cells are produced, effectors and helpers. The helpers are those that "remember" the invading antigen by multiplying and generating more cells with surface receptors that fit it. These cells circulate throughout the body watching for the invaders return appearance. If it reappears, these cells stimulate the clonal production of both helper cells like themselves and effector cells which actually attack the challenging antigen.

Cytokines

Various stages of that process are controlled by local hormones, messenger proteins called cytokines. There are over 100 different often structurally unrelated cytokines which fall into several families such as Interleukins (IL), Interferons (IFN) and Tumor Necrosis Factors (TNF). Cytokines often work by modulating genetic transcription, controlling and coordinating immune cell production.

Most cytokines are very short lived, only working over short distances. Generally this short existence means that a cytokine acts only on the cell that produced it, having an "autocrine" effect. Those cytokines acting on other cells exhibit a "paracrine" effect. Usually that means other cells in the immediate area or micro-environment of the cell producing the cytokine.

Immediately after a T helper cell becomes activated (upon encountering an antigen), the cell secretes interleukin-2 (IL-2), which acts on the T helper cell to make it (and its progeny) divide rapidly. Such a positive feedback loop has the potential to start a runaway chain reaction.

The initial critical stimulators that favor Th1 vs. Th2 responses are largely unknown. Intracellular viral and bacterial antigens favor Th1 and extracellular bacterial antigens, protozoa and parasites favor Th2, whereas low concentration of antigen favors Th1 and high concentration favors Th2. Both responses produce TNF-b and GM-CSF (granulocyte–macrophage colony-stimulating factor).

Chemotactic chemokines

One of the first things that happen with a threat is that a generic immune cell called a macrophage, like a cop on the lung beat, sounds an alarm by making a protein, MCP-1 (monocyte chemotactic protein-1). MCP-1 combines with CCR=2 (chemotactic chemokine receptor-2) which calls monocytes to the area.

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CCR-2 also induces monocytes to perform a more special function: produce two more proteins, TNF-a and inducible nitric oxide synthase (iNOS).

Monocytes morph next into macrophages called dendritic cells (DCs), or more precisely a special subset of dendritic cells called Tip-DCs (TNF-iNOS-producing DCs). Tip-CDs are necessary for killer T cells to function properly in clearing virus. Virus-infected cells secrete interferon, a chemical that rouses natural killer cells.

A potent killer CD8+ T-cell recall response can result in complete protection from an otherwise lethal respiratory virus infection. CD8+ T lymphocytes have several important antiviral effector activities at their disposal, including the perforin/granzyme system, interferon (IFN)- , TNF- and TNF- .

Activated human NK cells and immature dendritic cells provide an immune ‘control switch.’

Contact-dependent interactions between activated human NK cells and immature dendritic cells (iDCs) provide a ‘control switch’ for the immune system. At low NK/DC ratios, this interaction dramatically amplifies DC responses, whereas at high ratios it completely turns off their responses. Macrophages can produce IL-12 (NK activator) or TGF-b (Th inhibitor).

Specifically, cultured activated human NK cells with iDCs, at low NK/DC ratios (1:5), led to exponential increases in DC cytokine production, which were completely dependent on cell-to-cell contact. DC maturation was also driven by cognate interactions with NK cells and maturation was dependent on endogenously produced TNF-a in culture.

At slightly higher NK/DC ratios (5:1), inhibition of DC functions was the dominant feature due to potent killing by the autologous NK cells. Resting NK cells also stimulate autologous DC maturation in a TNF-a/ contact-dependent manner, however, increasing the NK/DC ratio only enhances this effect.

NK cells have increased turnover rates during viral infection, that this increase in turnover is accompanied by NK cell division, and that this results in increases in the numbers of NK cells. NK cells can produce INF-g (Th2 inhibitor). Th2 cytokines increase antibody inflammatory immunity while depressing macrophage activation and cellular mediated immunity.

Epithelial chemokine expression leads to the massive recruitment of host inflammatory cells, which is associated with observed morbidity and mortality. Neutralization of one important chemokine, monocyte chemoattractant protein (MCP)-1 (or CCL2), significantly abrogates the inflammation and self injury associated with CD8+ T-cell recognition of alveolar antigen.

A pharmaceutical that modulates CCR-2 but does not completely knock out its benefits is a peroxisome proliferator-activated receptor-gamma (PPAR-g) compound called pioglitazone. Another compound of this class is rosiglitazone, marketed under the trade name Avandia, as a treatment for type II diabetes. Pre-treating mice with pioglitazone significantly reduced the lethality and weight loss related to infection with virulent strains of flu virus (mortality went from 90% to 50%).

PPARs are members of the nuclear hormone receptor superfamily of ligand-activated transcription factors that heterodimerize with the retinoid X receptor (RXR) to regulate gene expression. Peroxisomal proliferator-activated receptor (PPAR) has been shown to decrease the inflammatory response via

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transrepression of proinflammatory transcription factors. PPAR may also increase catalase expression genetically, thus down-regulating the inflammatory response via scavenging of reactive oxygen species.

Inflammatory cytokines and activated macrophages

IFN- g, (mostly made by T-cells and NK cells) has antiviral, immune regulatory and anti-tumor properties. This interferon was originally called macrophage-activating factor, and is especially important in the maintenance of chronic inflammation. The persistence of TNF and IL-6 in serum, rather than peak levels correlate with multiorgan dysfunction syndrome development and predict a poor outcome.

TNF is hub of the cytokine network.

TNF- α and IL-1 (mostly made by macrophages) both affect a wide variety of cells to induce many similar inflammatory reactions: fever, production of cytokines, endothelial gene regulation, chemotaxis, leukocyte adherence and activation of fibroblasts. These cytokines are responsible for the systemic effects of inflammation, such as loss of appetite and increased heart rate.

The cytokine TNF-α is another example of an endogenous signaling molecule that exerts hormetic beneficial effects at low doses but toxic effects at high doses. TNF- α plays a key role in the killing and removal of infectious agents and damaged cells within the affected tissue, and TNF- α can prevent the death of cells that are not severely damaged.

TNF- α exerts its beneficial effects by activating an adaptive stress response pathway involving the transcription factor NF-κB. NF-κB induces the expression of genes that help cells resist stress including anti-apoptotic Bcl-2 family members and antioxidant enzymes.

However, excessive long-term production of TNF- α and NF-κB can damage and kill normal cells including neurons; such toxic actions are mediated by stimulation of the production of toxic chemicals by inflammatory immune cells such as macrophages/microglia.

Microglia are the resident macrophages of the brain and exist in the adult brain in ramified "resting" states. However, they rapidly respond to damage in the brain and are transformed into "activated" microglia. Microglia have very diverse effector functions, in line with macrophage populations in other organs. The term activated microglia needs to be qualified to reflect the distinct and very different states of activation-associated effector functions in different disease states.

Interferon- (IFN- ) and lipopolysaccharide (LPS) classically activate macrophages. Activated macrophages are a more mixed group of cells than originally thought, with different physiologies and

performing distinct immunological functions.

Classically activated microglia have migratory and phagocytic properties, a high proliferative capacity, express MHC Ags and release cytokines (IL-1 or TNF- ), chemokines, reactive oxygen as well as reactive nitrogen species. Microglia proliferation is a key component of the response to infection or damage in the brain.

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Exposure of macrophages to interleukin (IL)-4 or glucocorticoids induces a population of cells that up-regulates certain phagocytic receptors but does not produce nitrogen radicals and is therefore relatively poor at killing intracellular pathogens. Instead, these alternatively activated cells produce several components involved in the synthesis of extracellular matrix, suggesting their primary role is tissue repair rather than microbial killing.

Macrophages exposed to classical activating signals in the presence of immunoglobulin G (IgG) immune complexes induce production of a cell type fundamentally different from the classically activated

macrophage. These cells generate large amounts of IL-10 and thus are potent inhibitors of acute cellular inflammatory responses to bacterial endotoxin. These activated macrophages are called type 2-activated macrophages because of their ability to induce T helper cell type 2 (Th2) responses, which are predominated by IL-4 signaling, leading to IgG class-switching by B cells.

There are basically three different populations of activated macrophages with three distinct biological roles. The first is the classically activated macrophage whose duty is as an effector cell in Th1 cellular immune responses. The second type of cell, the alternatively activated macrophage, is involved in immune suppression and tissue repair. The third group is the type 2-activated macrophage, which is anti-inflammatory and preferentially induces Th2-type humoral-immune responses to antigen. Together, these three populations of cells form a regulatory network to provide a balanced immune response of appropriate magnitude.

Overactive cell-mediated immune response

If naïve T cells are chronically Th1 helper polarized, an overactive cell-mediated immune response can result. Persistently high secretion of IL-12 will cause Th1 cells to produce large amounts of pro-inflammatory cytokines like IFN-γ and TNF-α. These cytokines further activate macrophages to produce additional pro-inflammatory mediators (i.e. IL- 12 and IL-18) in a positive feedback loop that has potential pathological consequences.

Persistent Th1-mediated inflammation of the gastrointestinal tract is associated with pathologies like Crohn’s disease, H. pylori gastritis, cellular autoimmunity, chronic recurrent inflammation and likely atherosclerosis, rheumatoid arthritis, multiple sclerosis and systemic lupus erythematosus.

The Th-1 cytokines, IFN- and TNF- , also inhibit erythropoiesis, while the Th-2 cytokines, IL-4 and IL-10, down-regulate the production of Th-1 cytokines and facilitate red blood cell formation in certain circumstances.

An oversupply of proinflammatory cytokines like TNF- α, IL-6, IL-1, IL-8 plus virus-releasing neuraminidase can produce potent oxidative chemistry that overwhelms antioxidant systems (if the alarm messaging is not modulated), leading to shock. Absence of modulatory residential T cells with Toll-like receptors allows an unleashed innate immune response which can be a direct cause of death.

Septic shock is the nation’s 10th most frequent cause of death and the leading cause of hospital-related mortality. The incidence of sepsis and septic shock continues to increase with 400,000 cases of sepsis and 200,000 episodes of septic shock estimated to occur annually, resulting in more than 100,000 deaths.

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Bacterial infections, signaled by the lipopolysaccharides (LPS) that are part of the bacterial cell wall, mimic our endotoxin (messenger molecule of alarm) and aggressively stimulate the inflammatory response which may overwhelm its stop signals and sometimes spins out of control. Bacterial cell wall parts, like lipopolysaccharide or lipid A, are intensely pyrogenic (inflammatory and fever-inducing).

The primary symptoms of a cytokine storm are high fever, swelling and redness, extreme fatigue and nausea. Scarily, sepsis can progress swiftly from chills and fever with shallow breathing, to suffocate in lungs flooded with mucus, with dilated and leaky blood vessels, fibrin deposition with intravascular coagulation as well as a drop in blood pressure with lack of blood supply to the body’s organs, multiple organ failure and eventually death.

Hypercoagulability

Platelets are granule-containing cellular fragments critical for blood clotting and sealing off wounds. Platelets also contain granules like mast cells, monocytes and macrophages. They also release substances that activate components of the immune system.

Circulating platelets and chemo-attractant proteins, such as the CC chemokine RANTES, contribute to the activation and interaction of monocytes and endothelium and may thereby play a pivotal role in the pathogenesis of inflammatory and atherosclerotic disease. High levels of plasma IL-8 correlate with poor outcome. While low levels of RANTES correlate with poor outcome.

RANTES is a member of a large family of cytokines, called chemokines, which play a regulatory role in inflammatory processes. RANTES expression dramatically increases in inflammatory sites. In addition, megakaryocytes, some tumors and some fetal tissues express high levels of RANTES. Measles virus, a member of the Paramyxoviridae family, induces RANTES expression by astrocytes.

Induction of VEGF levels occurs at a later time point than TNF- , IL-1 and IL-6. VEGF is a late marker of sepsis and appears downstream of early response cytokines. The cytokine storm associated with sepsis contributes to the increase in VEGF levels. Since VEGF sensitizes endothelial cells to the effects of low TNF- concentrations, high VEGF levels observed in sepsis may accentuate cytokine storm activation. VEGF induces endothelial permeability, an effect that may contribute to the morbidity and mortality in sepsis.

Sepsis is associated with a time-dependent increase in circulating levels of vascular endothelial growth factor (VEGF) in animal and human models of sepsis. In addition to its role in promoting endothelial permeability and proliferation, VEGF may contribute to inflammation and coagulation. Adenovirus-mediated over expression of VEGF exacerbated lipopolysaccharide-mediated toxic effects.

Anti-angiogenesis products are Artemisia annua (Chinese wormwood), Viscum album (European mistletoe), Curcuma longa (turmeric), Scutellaria baicalensis (Chinese skullcap), resveratrol and proanthocyanidin (grape seed extract), Magnolia officinalis (Chinese magnolia tree), Camellia sinensis (green tea), Ginkgo biloba (ginkgo), quercetin, Poria cocos, Zingiber officinalis (ginger), Panax ginseng, Rabdosia rubescens hora (Rabdosia) and Chinese destagnation herbs. These herbs act as biologic modifiers, as adaptogens and as enhancers of conventional therapy.

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Chinese wormwood (Artemisia annua) is an anti-malaria herb whose active chemical is artemisinin. It causes apoptosis (natural cell death) and lowers VEGF in cancer cells.

European mistletoe (Viscum album) is used against cancer in anthroposophic and homeopathic medicine. It lowers VEGF and induces apoptosis.

Turmeric (Curcuma longa) contains the active ingredient, curcumin, which improves treatments with chemotherapy and radiotherapy and lowers VEGF. Nitric oxide (NO) levels correlate with tumor growth. Curcumin reduces with cellular production of NO.

Chinese skullcap (Scutellaria baicalensis) contains baicalin and baicalcin. Skullcap is anti-angiogenic and even works against advanced prostate cancer.

Resveratrol and proanthocyanidin (grape seed extracts) from grapes and wine. They inhibit angiogenesis. One study showed that proanthocyanidin can increase angiogenesis in wound healing.

Chinese magnolia tree (Magnolia officinalis) seed cones contain honokiol blocks angiogenesis.

Milk thistle (Silybum marianum) fruit and seeds contain silibinin and silymarin. They are polyphenolic flavonoids which suppress VEGF. This has been seen in human ovarian cancer.

Green tea (Camellia sinensis) contains polyphenols and catechins which reduce breast cancer and VEGF. Epigallocatechin-3 gallate (EGCG) is a catechin comes from powdered green tea. The suggested dose is 7-8 Japanese cups (120 mL) per day. The caffeine of green tea improves anti-angiogenesis. The gastrointestinal effects and nervousness from caffeine limit the dosage. Polyphenon E, a standardized extract containing EGCG was tested with a maximum effective tolerated dose up to 2000mg twice per day and benefits were still increasing.

Ginkgo (Ginkgo biloba) contains the flavonoid ginkgolide B which lowers VEGF. Quercetin is a flavone contained in apples, raspberries, red grapes, onions, citrus fruit, cherries, broccoli and leafy green vegetables. Quercitin blocks angiogenesis and improves the anticancer effects of tamoxifen.

Panax ginseng contains ginsenosides which are anticancer and anti-angiogenic.

The catastrophic antiphospholipid syndrome (CAPS) affects mainly small vessels predominantly supplying organs. It is characterized by elevations of antibodies directed toward negatively charged phospholipids, as measured by anticardiolipin antibody assays and/or positive lupus anticoagulant tests. Thrombocytopenia is usually marked, and a Coombs positive microangiopathic-type anemia may accompany the condition.

Features of disseminated intravascular coagulation may be evident in some patients. It is fatal in approximately 50% of cases reported. Treatment should include not only adequate anticoagulation with

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intravenous heparin but also full doses of intravenous corticosteroids, to offset the systemic inflammatory response syndrome that occurs as a result of the extensive tissue damage. Parenteral antibiotics should be administered early if infection is suspected.

By molecular mimicry, a process common to many immunologic conditions (e.g., the role of Chlamydia and certain strains of Shigella or Salmonella in the genesis of arthritis in HLA-B27 positive individuals), it has been demonstrated that some infectious agents can induce not only antiphospholipid antibodies (aPLs) but also anti b2-GPI antibodies. There are seven proteins with sequence homology to GDKU2 proteins (present in the b2-GPI binding site for aPL in viruses).

Enterotoxins produced by Enterococci or Staphylococci may activate T-cells, resulting in B-cell activation. Those B-cells already programmed to produce aPL will then “overproduce” with consequent effects on clotting. Newly formed clots themselves, in patients with pre-existing hypercoagulability, continue to provide coagulation activation products such as prothrombin activation products F1 and 2, thrombin-antithrombin (TAT) complexes and protein C activation peptide, producing a thrombotic storm.

Increased production of cytokines, such as TNF-a and IL-1 and 6, result in the “cytokine storm” responsible for some of the major clinical manifestations of the condition. These include cerebral edema (causing the initial confusion and deterioration of consciousness), myocardial dysfunction and manifestations of adult respiratory distress syndrome (ARDS). These symptoms are superimposed on the microangiopathic pathology.

Effective intravenous heparin administration is essential to inhibit ongoing clotting and to lyse existing clot. Because of the extreme hypercoagulability existing in patients with CAPS, more than the usual doses of heparin are often administered (e.g., 15,000-20,000 units daily).

Although oral fibrinolytics (serripeptidase, nattokinase, lumbrokinase) work well, in severe cases of intractable CAPS in which patients do not respond to heparin, the use of intravenous fibrinolytics, such as tissue plasminogen activator (tPA) or streptokinase may be justified. Hemorrhage induced by these fibrinolytics can be treated with blood transfusions.

Intravenous steroids in supra-physiologic doses (methyl prednisolone, 1000 mg daily) may be administered short-term for 3-5 days to treat the excessive cytokine response in patients with CAPS. Adrenal hypofunction, which may be clinically undetectable, can be safely shored up long-term with appropriate low-dose oral replacement therapy (5-20 mg hydrocortisone early in the day).

Virus uses subterfuge to survive, use and maintain cytokine dysregulation.

For a virus to survive and reproduce in an organism, it uses strategies to escape and misdirect host immune response. A primary evasive tactic utilized by the SARS virus, like influenza, is to inhibit host corticosteroid stress response.

The virus expresses amino acid sequences that are molecular mimics of adrenocorticotropin hormone (ACTH). When antibodies are produced against these viral antigens, they also bind to the host's own

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ACTH, which limits adrenal stress response by compromising ACTH stimulation of secretion of corticosteroids.

Monocytes and macrophages are important mediators of innate-immune responses because of their phagocytic potential and ability to produce various cytokines. They also induce specific immune

responses by presenting antigens to effector T cells.

Cytokines influence monocytes/macrophages in two different but partly overlapping ways. They regulate the proliferation and differentiation of these cells, and they are responsible for the attraction and functional activation of mature monocytes or macrophages. Cytokines exert their actions via specific receptors and signaling pathways.

Cytokines can regulate immune response via positive- and negative-feedback circuits. When cytokines amplify a specific immune response, they tend to stabilize mRNAs that participate in such a process.

Examples of those cytokines are the proinflammatory cytokines, IL-1 and TNF- , which have been found to stabilize a significant number of cytokines and chemokines, including IFN- -inducible protein 10 (IP-10), growth-related oncogene proteins and fractalkine (SCYD1), as well as several genes involved in regulation of inflammatory responses such as TNF- -induced protein 3, NF- B I and mannose-binding

lectin 2 (MBL2).

Lipopolysaccharide (LPS) initiates a signaling cascade in macrophages, which engages transcriptional induction such as NF- B-mediated gene expression and post-transcriptional mechanisms, involving activation of the p38 MAPK, central in mRNA stabilization of many of the proinflammatory cytokines.

This pathway affects the activity and subcellular localization of several RNA-binding proteins such as TTP, which promote mRNA decay. Cytokines use p38 MAPK signaling and notably, its target MK2 to trigger mRNA stabilization. For example, using this pathway, IFN- amplifies immune responses such as increasing NK activity and up-regulating MHC expression.

Antigenic, endotoxin and other stressor molecules cause the body’s immune cells to produce inflammatory cytokines. The acute inflammatory response requires constant stimulation to be sustained. Normally, inflammatory mediators have short half lives and are quickly degraded in the tissue.

Molecular mimicry

Self proteins, proteins of infectious agents such as viruses and bacteria as well as food proteins are made up of strings of amino acids. In molecular mimicry, a short series of amino acids, perhaps 5-15 in one protein is very similar to a sequence of amino acids in another protein.

T cells have receptors which bind to short segments (less than10 amino acids) of a foreign protein. A macrophage engulfs a foreign protein and breaks it down into fragments and then "presents" it to millions of circulating T cells.

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A T cell with a matching receptor binds onto the presented protein fragment, becomes activated and stimulates other portions of the immune system to begin an active response against all proteins which contain a similar looking amino acid sequence. A variety of similar, yet somewhat different amino acid chains can be recognized by the same T cell.

If the protein fragment from a foreign invader which is presented to the T cell closely resembles part of a self protein then the activated immune system will not only attack all foreign invaders which have the same string of amino acids but will also attack the similar string in a self protein. Parts of proteins in various foods and infectious agents resemble parts of various self proteins.

Sometimes three way mimicry occurs with a protein fragment from a food closely resembling that of an infectious agent which in turn closely resembles part of a self protein. In Celiac disease, part of the gliadin molecule (found in various grains such as wheat and rye), part of adenovirus 12 and part of a gut protein all closely resemble each other and the result of such mimicry is an immune attack on the gut when food containing gliadin protein is eaten.

A similar three way mimicry occurs between a cell wall protein in grains and legumes, part of the Epstein Barr virus and part of the collagen in joints. This leads to rheumatoid arthritis in genetically susceptible people. For type 1 diabetes, parts of milk proteins and viral proteins mimic proteins in the insulin-producing beta cells of the pancreas.

The tyrosine phosphatase IA-2 is a molecular target of pancreatic islet autoimmunity in type 1 diabetes. This dominant IA-2 epitope peptide also has 75-45% identity and 88-64% similarity to over 8-14 amino acid sequences in Dengue, cytomegalovirus, measles, hepatitis C and canine distemper viruses as well as the bacterium Haemophilus influenzae. Three other IA-2 epitope peptides are 71-100% similar over 7-12 amino acids to herpes, rhino-, hanta- and flaviviruses.

Two others are 80-82% similar over 10-11 amino acids to sequences in milk, wheat and bean proteins. T-cell activation by rotavirus and possibly other viruses and dietary proteins, could trigger or exacerbate beta-cell autoimmunity through molecular mimicry with IA-2 and for rotavirus (glutamic acid decarboxylase).

For multiple sclerosis (MS), numerous viruses and bacteria have amino acid strings which mimic parts of proteins in the myelin proteins of the central nervous system. Undoubtedly food proteins (like gliadin) also contain such mimicking protein fragments and thus two and three way mimicry is a viable theory for why the immune system attacks myelin and causes MS.

An important part of molecular mimicry is what exact string of amino acids is presented to the immune system, since that will determine if part of a self protein is mimicked or not. That is why MS and other autoimmune diseases are strongly dependent on one’s genetic makeup as well as individual digestive idiosyncrasies and why only a small percentage of the population contracts these diseases.

Lipopolysaccharides of bacterial cell walls (and toasted glycoproteins in foods) which imitate alarming endotoxin can be mimicked as well. Crohn’s disease is a chronic granulomatous inflammation of the GI tract, first described in the beginning of the 20th century. The histological similarity with intestinal tuberculosis led to exploration of the involvement of mycobacteria or mycobacterial antigens, in the etiology.

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A major defense mechanism against mycobacterial lipid antigens is the CD1 system which includes complement molecules for antigen presentation and natural killer T cells for recognition and subsequent production of cytokines like If-g and TNF-a. These cytokines upregulate the inflammatory process and promote granulomatous transformation.

Various food additives (emulsifiers, thickeners, surface-finishing agents and contaminants like plasticizers) share structural domains with mycobacterial lipids. These compounds can stimulate the CD1 system in the GI mucosa by molecular mimicry and thus trigger the pro-inflammatory cytokine cascade. The ‘fertile field’ concept involves three mechanisms: molecular mimicry, bystander activation and viral persistence. Any given individual may be repeatedly exposed to a potential immunogen without any untoward consequences; but that under certain circumstances, for example, if the person has a viral infection at the time of exposure, infection alters the immunological environment in which the antigen was encountered, leading to a more profound immune response.

Viruses express themselves when we are stressed and cellular immunity is depressed.

As cytokines and leukotrienes increase so does cortisol. Bursts of cortisol responsively activate steroid receptors, blocking NF-kB activation and inflammation. However, if one is out of circadian rhythm (due to weak breakfast or inadequate sleep) or one has an established biofilm (which engenders chronic inflammation), cortisol is constantly produced. This diminishes cellular immunity and cytokine response, until adrenal exhaustion sets in and this buffer is lost

The upper respiratory tract is of easy access to pathogens, and although it has evolved a number of defensive barriers to avoid invasion, acute and chronic infections of the ears, nose and throat are common and present a huge challenge to the health-care system. The same can be said for the gastrointestinal tube, from mouth to anus.

Most infections are viral, mild and self-limiting. However, viruses change or ‘condition’ the surface ecology of the barrier tissues, inviting opportunistic bacterial attachment and established biofilm formation. Once formed, biofilm becomes a sophisticated multicellular organism with many stealth survival strategies.

Subsequent pathogenic biofilm development protects and supports an established resistant pleomorphic infection that engenders inflammation as a food source. When inflammatory response regulators become exhausted or overwhelmed, an exaggerated oxidative immune reaction becomes responsible for considerable morbidity and has substantial potential for life-threatening sequela.

Rats with tumors show higher levels of three pro-inflammatory cytokines (IL-1& b, IL-6 and TNF-a) in their tumors, their blood and their brains. All of these have been linked to changes in behavior following infection or brain injury. Raised levels of these cytokines, at even moderate levels, have been linked to depression, learning difficulties and emotional problems in humans.

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When these agents are released by the immune cells there is an inevitable "friendly fire" effect that can result in the death of nearby healthy cells. Problems arise when the body is unable to turn off cytokine production and the body is overwhelmed with a cascading chemical crescendo that creates a ‘cytokine storm’, causing a destructive oxidative inflammatory response that can bubble out of control causing varying degrees of autoimmune breakdown from tooth decay and periodontal diseases to arthritis, diabetes, atherosclerosis and cancer, or explode untethered, even leading to sudden death.

Typically, the body's immune system sends out killer T cells to puncture and dismantle invading harmful cells. The ‘Spanish flu’ virus had a coating which prevented this action from killing them and instead just divided them into two parts which independently lived and just replicated more rapidly, continuing to stimulate the chemistry that upregulates immune response. This viral survival strategy creates an overwhelming cytokine-release syndrome (cytokine storm), making the body fill up with mucus; especially the lungs, and one literally suffocates and then drowns in mucus.

Steroids

Relatively short-term high-dose steroid injections (thus not inactivated by the liver), whether intramuscular or intravenous cause many activated macrophages to commit apoptosis (cell suicide) and thus modulate the immune response rather than suppress it. The goal is to buy enough time to allow inherent systems to successfully counteract the infection.

With any kind of infection (parasitic, bacterial or viral), steroid treatment after five to seven days becomes immunosuppressive. Therefore, the way to handle a cytokine storm is to give large declining doses early in series along with a non-steroidal anti-inflammatory with the goal to be weaned off the steroids by day five and certainly by day seven. Often by modulating the response successfully, the steroids can be removed by day two or three.

Stop the storm

Histamine causes itching, pain, mucus production and constriction of the bronchioles as well as hypotension through dilatation and leakage of blood vessels. Antihistamines help by preventing histamine from attaching to a cellular receptor, H1. Attachment to this receptor is necessary for histamine activity to occur.

The first generation antihistamines that cause drowsiness and dry mouth also centrally block the activity of the parasympathetic system, which stimulates mucus secretion. The newer (non-sedating) antihistamines do not have the same degree of effectiveness for treating the sneezing and mucus nasal discharge (of colds), but can create additive systemic effect with less drowsiness.

Younger people or those with a robust immune system surprisingly tend to have a stronger exacerbation of cytokine storm.

In large cities, ‘rapid transit’ refers to the efficient movement of people allowing rapid entry and exit from a transportation system. A rapid transit mechanism also occurs in the immune system to allow immediate activation of the immune cells toward microbes, transient mode of action and swift recovery.

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Post-transcriptional regulation of genetically manufactured cytokines contributes significantly to this rapid transit by several mechanisms, including mRNA stability modulation and translational control; collectively, they modulate the expression of key gene products involved in the immune response.

Septic shock, a dangerous, potentially fatal runaway immune response, is ultimately controlled by one’s own variable function of a genetic on/off switch called the AUF1 gene. The AUF1 gene is released and begins action once the immune response is initiated and after cytokine production gets underway.

AUF1 forms a complex with cap-dependent translation initiation factors and heat shock proteins (Hsp27) to attract mRNA degradation machinery. The terminating action is pronounced. Messenger RNAs (mRNAs) which are blueprints for genetic production of very specific proinflammatory cytokines, namely IL-1 b, TNF-a and COX-2 are quickly dismantled. Degrading their mRNAs shuts off production of these incendiary cytokines.

The AUF1 gene produces a built-in protector that can stop an infection from progressing to septic shock. It does so initially when silenced by allowing cytokine production and then when activated, quickly dismantling the production of these proteins. AUF1 acts like a cytokine on/off switch.

Initially the RNA-binding protein, AUF1 is isomerized, interfering with its mRNA decay-promoting function, contributing to early and rapid response of T cell activation and cytokine production. In later phases of the inflammatory process, cytokine shut-off can be facilitated by AUF1 via post-transcriptional mechanisms including mRNA destabilization and translational arrest.

The p40 AUF1 isoform selectively plays a critical, positive role in interleukin-10 (IL-10) expression upon exposure to lipopolysaccharides (LPS). IL-10 is an immune modulatory cytokine that regulates inflammatory responses of mononuclear phagocytes (monocytes and macrophages). Although most cytokines have a role in stimulating immunity (IL-10 can go both ways), IL-27, actually suppresses CD4+ T cells, the 'helper' T cells that orchestrate the immune system's response to infections.

Post-transcriptional control plays a significant part in specific pathways that regulate T cell activation and involves several types of modulators, including suppressive cytokines, miRNA and adaptors. When cytokines such as IFN- /ß, IL-4 and IL-10 dampen the immune response, they tend to destabilize mRNAs involved. IFNs such as IFN-ß or - , when in combination with LPS, induces zinc-dependent TTP transcription and thereby reduce proinflammatory cytokines.

Mononuclear cells exposed to microbes or microbial products secrete many proinflammatory cytokines followed by delayed onset of anti-inflammatory IL-10. IL-10 suppresses immune responses by inhibiting cytokine production by mononuclear phagocytes.

IL-10 is a potent, suppressive cytokine, which can inhibit macrophage action and generation of Th2 lymphocytes. It is capable of inhibiting proinflammatory cytokines and Th1 cytokines, such as TNF- , IFN- , IL-2, IL-3 and GM-CSF. IL-10 destabilizes TNF- mRNA and probably others (by inhibiting the binding of HuR to the AREs).

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Mechanisms of IL-10-suppressive action, though incompletely understood, are diverse and depend upon the gene of interest, the nature of the stimulus and the cell type. IL-10 can reduce the level of gene transcription, decrease the stability of specific mRNAs and reduce their translation.

One of the most important protein complexes involved in maintaining correct RNA levels in eukaryotic cells is the primitive exosome (from the ever-present ‘RNA world’), a complex consisting almost exclusively of exoribonucleolytic proteins. The exosome also has a prominent role in gene silencing as well as in regulating the expression of a wide variety of noncoding RNAs.

RNA-binding proteins HuR and AUF1 bind to many common AU-rich target mRNAs and exert opposing influence on target mRNA stability. In the nucleus, both proteins were found together within stable ribonucleoprotein complexes; in the cytoplasm, HuR and AUF1 bind to target mRNAs individually, HuR co-localizing with the translational apparatus and AUF1 with the exosome.

HuR translocates from the nucleus to the cytoplasm and stabilizes ARE-mRNAs, and pin1 isomerizes another RNA-binding protein, AUF1, leading to loss of its mRNA decay-promoting function. These events contribute to early and rapid response of T cell activation and cytokine production. (In later phase, shut-off is facilitated by post-transcriptional mechanisms including mRNA destabilization and translational arrest.)

HuR stabilizes many AU-rich mRNAs including those that participate in immunity and extending inflammation such as TNF- , IL-3, IL-8 and the urokinase activator, urokinase-type plasminogen activator. The composition and fate (stability, translation) of HuR- and/or AUF1-containing ribonucleoprotein complexes depend on the target mRNA of interest, RNA-binding protein abundance, stress condition and subcellular compartment.

Down-regulation of AUF1 induces DNA methyltransferase 1 (DNMT1) expression by stabilizing its mRNA. Depletion of AUF1 protein (besides allowing cytokine storm) in nontransformed human fibroblasts also leads to increased levels of DNMT1 protein and global genomic methylation.

The enzyme DNA methyltransferase 1 (DNMT1) is responsible for maintenance and propagation of DNA methylation patterns. DNMT1 protein over-expression and tumor suppressor gene hyper-methylation characterizes a number of different tumors.

MicroRNA silencing machinery (drosha and dicer) is not limited to negative control of T helper cell

activation but itself, has a direct innate immunity function. Drosha and dicer can act as intracellular, antiviral enzymes against HIV infected human monocytic cells. Another important role of dicer occurs in the development of regulatory T cells.

Virus infection can initiate or accelerate autoimmune disease via epitope (antigenic determinant, that part recognized by antibodies, B cells or T cells) spreading as well as molecular mimicry. This can lead to the development of an inflammatory region with activated APCs and possible presentation of self antigens. Or, viral infective strategy could lead to immune suppression and chemokine gradients of anti-

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inflammatory cytokines such as IL-10 or TGF-ß with activation-induced apoptotic cell death of auto-reactive cells.

Different viruses code for different viral proteins, which can control the cell’s machinery for their own growth or to evade immune cells. Viruses can stabilize mRNAs of gene products that are essential to the virus life cycle. For successful takeover of the cell, viruses have the ability to shut-off cellular mRNA biogenesis and induce rapid degradation of selected cellular mRNAs, particularly those required for host defense.

Can the RNA decay-promoting activity of the RNA-binding proteins be harnessed as an antiviral therapeutic strategy? The zinc-dependant TTP binds directly to the AU-rich HIV-1 RNA and enhances multiple splicing, which leads to a reduction of HIV-1 virons.

Metabolic syndrome X and inflammation

The spectrum of diseases which involve insulin insensitivity (e.g. obesity, periodontal diseases, gall bladder disease, endometriosis, osteoporosis, type 2 diabetes, atherosclerosis and cancer) also show increased cytokine production and elevated markers of inflammation. Evidence actually favors chronic inflammation as a trigger for chronic insulin insensitivity, rather than the reverse situation.

Inflammation causes diabetes, and the diabetic state promotes inflammation. HbA1c (glycosylated hemoglobin) representing AGEs (glycated dietary glycoproteins) messaging and NF-kB (upstream inflammatory genetic trigger) were correlated with each other, and also with hyperglycemia and lipid peroxidation. NF-kB promotes asthma; neurodegeneration; ischemia/reperfusion injury; hepatitis; glomerulonephritis; inflammatory bowel disease; rheumatoid arthritis; and probably most other diseases driven by inflammation including atherosclerosis and cancer.

In mice, increase in serum leptin correlates with disease susceptibility, reduction in food intake due to inflammatory anorexia and decrease in body weight. Acute starvation, which prevents the increase in serum leptin, delays autoimmune disease onset and attenuates clinical symptoms by inducing a T helper 2 cytokine switch. Leptin secretion by activated T cells sustains their proliferation in an autocrine loop, since anti-leptin receptor antibodies are able to inhibit the proliferative response of auto-reactive T cells (in vitro).

NF-kB is a protein which activates the genes responsible for the production of pro-inflammatory cytokines and inflammatory enzymes (such as COX-2 and others). NF-kB has also been linked to cancer and autoimmune diseases. Aging increases levels of NF-kB (which can be decreased by many herbs).

Regulation and inhibition of NF-kB is a key focus for the suppression of chronic inflammation. While NF-kB is an important part of the immune response, chronic states of inflammation and illness result when it becomes upregulated. Whereas low levels enhance survival , high levels of NF-kB can be very destructive to the tissues of the body and even play a role in cancer development.

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NF-kB regulates inflammatory factors which affect the initiation and progression of autoimmune diseases: (1) pro-inflammatory cytokines (TNF-a and IL-1b) and (2) pro-inflammatory enzymes (COX-2, 5-LOX and iNOS).

While cytokine production is initiated by activation of NF-kB, cytokines also act as a stimulus for the production of more NF-kB. This can produce an upward cycling of inflammation which can become chronic and subtly destructive to the tissues in the body or acute, leading to shock and sudden death.

Efficient recognition of the foreign antigens in microbes, such as bacterial surface molecules and viral dsRNA, is accomplished by cells of the innate immunity, mostly macrophages derived from dendritic cells (DC). Cell membrane members of the toll-receptor (TLR) family are responsible for creating balanced

response of the host’s border barrier cells to microbes, reducing inflammation in the presence of friendly commensal microorganisms and up-regulating the immune chemical cascade in response to pathogens.

TLR4 activation encompasses several post-transcriptional, positive regulatory events, which lead to amplification of LPS-induced signaling and ultimately, production of proinflammatory cytokines. These cytokines and other mediators, including vascular endothelial growth factor, the solute carrier family 11 member 1 and NO, can be up-regulated at the level of mRNA stability in LPS-stimulated, monocytes.

Upon activation of TLR4 by LPS, the adaptor protein MyD88 is recruited to the receptor and couples with IL-1 receptor–associated kinase (IRAK)1 and -4, causing IRAK activation and recruitment into a complex with TNF receptor-associated factor 6 (TRAF6). This pathway finally culminates in a cascade of signaling events including NF-kB activation, MAPK activation and transcriptional activation of proinflammatory cytokines.

AUF1, known to destabilize target mRNA, is increased fourfold in JNK-activated cells. A variety of other stress-related stimuli, such as p38 MAPK activation and phorbol ester, upregulates AUF1 expression in

cultured cardiac cells as well.

JNK activation leads to a marked loss in B56 protein expression and transcript (which binds to and inactivates AUF1) that is associated with a striking increase in mRNA instability. Signaling-induced mRNA instability and perhaps its counterpart mRNA stability are associated with a range of cardiovascular pathologies, including pressure overload, sepsis and ischemia.

The life cycle of dendritic cells (DCs) must be precisely regulated for proper functioning of adaptive immunity. However, signaling pathways actively mediating DC death remain enigmatic. Here is one mechanism of hierarchical transcriptional control of DC life and death.

Binding of TNF receptor superfamily (TNFR-SF) members to DCs and cognate contact with T cells results in quantitatively balanced NF-kB and c-Jun N-terminal kinase (JNK)-mediated activator protein-1 (AP-1) induction and strongly enhances DC longevity.

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Specific blockade of NF-kB in DCs induces strongly augmented JNK/AP-1 activity because of elevated levels of reactive oxygen species. Here, DC activation by TNFR-SF members or T cells induces DC death by apoptosis.

Specific inhibition of JNK/AP-1 rescues DCs from apoptosis and restores TNFR-SF member- and T-cell-mediated survival. JNK/AP-1 activity is under negative feedback control of NF-kB and can trigger apoptosis in DCs. Thus, feedback-controlled signaling amplitudes of two transcriptional pathways decide life or death of a DC.

This pathway ultimately affects the activity and subcellular localization of several RNA-binding proteins such as TTP, which promote mRNA decay. The zinc-dependant finger-protein tristetraprolin (TTP) promotes destabilization of TNF and GM-CSF (granulocyte-macrophage colony-stimulating factor) mRNAs. Like TTP, AUF1 acts as another negative-feedback inhibitor of the innate inflammatory response to endotoxin.

The direct interaction of AUF1 with cytokines (and subsequent reduction in inflammation) is impaired by binding with heat shock proteins (HSPs). HSPs are mimicked systemically by glycosylation of high levels of serum glucose and fructose, or eating browned or crisped foods, or smoking sugar-cured tobacco, which both contain caramelized sugars, AGEs (advanced glycation end-products).

Secondary or nutritional hyperparathyroidism (HPT)

The elevated parathyroid hormone (PTH) and disordered mineral metabolism associated with secondary hyperparathyroidism (HPT) complicate the clinical course of most patients with late-stage chronic kidney disease (CKD) and, when advanced, are associated with markedly increased morbidity and mortality.

The hallmark of secondary HPT is the high levels of circulating PTH, which result from increased PTH secretion, increased PTH gene expression and synthesis, and increased parathyroid gland (PT) cell proliferation.

Lack of adequate dietary calcium or magnesium from vegetables or excessive phosphorous from processed foods commonly seen in the standard American diet (SAD) results in a secondary or nutritional hyperparathyroidism.

The effect of calcimimetics on PTH gene expression is post transcriptional and correlated with differences in protein–RNA binding and posttranslational modifications of the trans acting factor AUF1 in the parathyroid gland. Calcimimetic agents (calcium and magnesium) are effective tools in the management of secondary hyperparathyroidism, acting through allosteric modification of the calcium-sensing receptor (CaR) on the parathyroid gland (PT) to decrease parathyroid hormone (PTH) secretion and PT cell proliferation.

Parathyroid hormone mRNA binding proteins include AUF1 which stabilizes and KSRP which destabilizes the parathyroid hormone mRNA. Uremia and activation of the CaR mediated by calcimimetics modify

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AUF1 posttranslationally. These modifications in AUF1 correlate with changes in protein–PTH mRNA binding and PTH mRNA levels.

The calcimimetic-mediated decrease in PTH mRNA levels is of particular clinical relevance because the parathyroid gland possesses only a limited amount of preformed PTH secretory granules. The gland contains enough preformed PTH to maintain secretion for only about 90 minutes after the initiation of hypocalcemia. Accordingly, more prolonged secretion, as would be seen in patients with secondary HPT, is dependent on increased PTH synthesis as well as decreased PTH breakdown in the gland.

The hypocalcemia-induced increase in PTH gene expression by stabilization of the PTH transcript may have an important role in this effect. Calcimimetics decrease PTH gene expression posttranscriptionally. 1,25(OH)2 vitamin D3 potently decreases PTH gene transcription. Combined use of 1,25(OH)2 vitamin D3 and a calcimimetic is a synergistic choice to decrease PTH gene expression and PTH secretion.

The expression of many genes involved in growth regulation, including proto-oncogenes (such as c-fos, c-myc, and c-jun), growth factors and their receptors (GM-CSF and VEGF), cytokines (TNF ), and cell cycle regulatory genes (cyclin A, B1, and D1, p21), is mainly controlled by modulation of their mRNA stability. Depending on the class of ARE, the level of p37AUF1 over-expression relative to the other isoforms and the abundance and/or activity of the auxiliary factors with which it interacts, AUF1 may act either as a stabilizing or a destabilizing factor.

Cell cycle regulation of AUF1 can posttranscriptionally control DNA methyltransferase 1 (DNMT1) mRNA and is critical for maintaining the integrity of genomic methylation levels. AUF1 maintains a dynamic balance of growth control by coordinating the expression of genes with opposite functions.

Depletion of AUF1 protein in nontransformed human fibroblasts leads to increased levels of DNMT1 protein and global genomic methylation. DNMT1 protein over expression and tumor suppressor gene hypermethylation characterize a number of different tumors, and these elevated levels are believed to contribute directly to tumor growth.

NF-kB resides in the cytoplasm of the cell and is bound to its inhibitor. Injurious and inflammatory stimuli, such as free radicals, release NF-kB from its inhibitor. NF-kB moves into the nucleus and activates genes responsible for expressing inflammatory cytokines, collagen-digesting metalloproteinases (MMPs) and COX-2, which triggers massive production of inflammatory prostaglandins.

Extracellular matrix (ECM) and matrix metalloproteinases (MMPs)

Matrix metalloproteinases (which are zinc-dependant) and their natural inhibitors are ingredients of a fundamental cellular toolbox for effecting environmental change. These enzymes enable cells to alter their relationship to the environment by directly cleaving structural macromolecules of the extracellular matrix (ECM). They also play an important regulatory role in matrix remodeling by catalyzing the processing of inactive matrix metalloproteinase and cytokine precursors.

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‘Matrikines’, small peptides derived from the degradation of extracellular matrix macromolecules, are able to modulate connective tissue cell activity and matrix metalloproteinase expression and/or activation. IL-1 is one of the main cytokines involved in matrix remodeling. IL-1 is also involved in fibrosis. TNF-a is also strongly involved in matrix remodeling. The enzyme which liberates TNF-a from the plasma membrane is TNF-a convertase (TACE).

An imbalance between activated MMPs and their endogenous inhibitors leads to pathologic breakdown of the extracellular matrix during periodontitis. MMPs in inflamed gingival tissue of adult periodontitis patients, like those in gingival crevicular fluid, originate primarily from infiltrating polymorphonucleocytes rather than resident gingival cells (fibroblasts and epithelial cells) or monocyte/macrophages. MMP’s pathologically-elevated tissue-degrading activities can be directly inhibited by pharmacologic levels of doxycycline.

Increase in TACE expression contributes, at least in part, to the rise in TNF-a after oxygen-glucose deprivation (OGD) of rat forebrain slices mimicking cerebral ischemia and subsequent reperfusion, and iNOS expression in OGD-subjected brain slices results from TACE activity and subsequent increase in TNF-a levels. Tetracyclines are potent inhibitors of 2 major matrix metalloproteinases which have been implicated in connective tissue degradation: collagenase and Type IV collagenase/gelatinase, even at doses low enough to not alter systemic balance of bacteria.

When stimulated by cytokines, smooth muscle cells produce activated forms of MMPs. Constitutive and cytokine-induced enzymes can digest all the major components of the vascular ECM. Since these mediators augment the production of MMPs without appreciably affecting the synthesis of tissue inhibitors of MMPs, locally secreted cytokines may tip the regional balance of MMP activity in favor of vascular matrix degradation.

MMPs are implicated in various steps of development of metastasis, through their ability to degrade the extracellular matrix. Increased MMP activity of tumor cells has been associated with a higher metastatic potential. Inhibitors of MMPs have been shown to effectively reduce or prevent the formation of metastases. The family of tetracyclines is able to inhibit matrix metalloproteinase activity through chelation of the zinc ion at the active site of the enzyme.

Hormesis and NF-kB

Free radicals, heavy metals, toxins, AGEs and organophosphate pesticides are examples of substances that activate NF-kB. Cytokines such as interleukin-1 (IL-1) can also stimulate NF-kB.

Antioxidants can reduce the activation of NF-kB, including green tea polyphenols; resveratrol from red wine; vitamins C and E; curcumin; silymarin in milk thistle; and glutathione. The entire family of flavonoids and carotenoids found in fruits and vegetables that have antioxidant functions is capable of reducing free-radical activation of NF-kB.

The surprising paradox is that these polyphenol plant pigments are active toxic molecules made to combat stressors and are interpreted by our genes as instructions to hormetically up-regulate

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manufacture of antioxidant and detoxification systems, thus improving survival fitness. For example, curcurmin can kill a wide variety of tumor cell types through diverse mechanisms.

Another key to glutathione function is substances that maintain manufactured genetically-regulated glutathione in its active reduced state, which includes supplements such as NADH, lipoic acid, coenzyme Q10 and vitamins C, Ds and Es. The anti-inflammatory omega-3 oils reduce NF-kB activity as well as reduce IL-1 production, which in turn, helps activate NF-kB.

Macrophage migration inhibitory factor (MIF)

Macrophage migration inhibitory factor (MIF) is a pituitary hormone. It is also a versatile pro-inflammatory cytokine (lymphokine), released from T-cells, monocytes and macrophages, after one’s exposure to bacterial endotoxins and exotoxins (LPS, toxic shock syndrome toxin-1 and streptococcal pyogenic exotoxin), malaria pigment (hemozoin) or cytokines (TNF-α and INF-γ).

MIF normally circulates in the blood at 2-4ng/mL. Macrophage cells and T-cells contain preformed pools of MIF (2-4 fg) so it can be rapidly secreted after exposure to endotoxins or cytokines such as TNF-α or some interferons. In fact, secretion and further translation is induced by levels of LPS 10-100 times lower than those needed to induce TNF-α production.

As regulators of the inflammatory response, MIF and TNF-α have similar actions, both ratcheting up inflammation and macrophage response to the point that their actions can be lethal, resulting in toxic shock syndrome. In addition, MIF and TNF-α induce each other; working together to stimulate an effective inflammatory response.

Several clinical trials involving a chimeric anti-TNF- α antibody have shown marked clinical benefit in most patients with Crohn's disease, verifying the importance of TNF- α in the pathogenesis of Crohn's.

Virus primes the pump.

TNF-α seems the principal mediator of mortality in response to genital HSV-2 infection. TNF-α has a lead role in neuropathology, as opposed to a direct destructive effect of virus within the CNS. In the immune compromised patient as well as newborns, the course of the infection can be quite severe, even ultimately resulting in death.

In response to CNS virus infection, TNF-α is produced by astrocytes, microglia, neurons and infiltrating hematopoietic cells. TNF-α regulates leukocyte trafficking by inducing a number of factors, including cell adhesion molecules (ICAM-1 and VCAM-1), selectins (E and P selectins) as well as chemokine expression. At the cellular level, TNF-α synergizes with gamma interferon to induce nitric oxide.

The majority of trigeminal ganglia (TGs) are latently infected with alpha-herpes viruses [herpes simplex virus type-1 (HSV-1) and varicella-zoster virus (VZV)]. Whereas HSV-1 periodically reactivates in the TGs, VZV reactivates very rarely.

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CD8, IFN-g, TNF-a, IP-10 and RANTES transcripts were significantly induced in TGs latently infected with HSV-1 but not in uninfected TGs. The persisting lymphocytic cell infiltration and the elevated CD8 and cytokine/chemokine expression in the TGs demonstrate for the first time that latent herpes viral infection in humans is accompanied by a chronic inflammatory process at an immune privileged site but without any neuronal destruction. The chronic immune response seems to maintain viral latency and influence viral reactivation.

Viral infection significantly increases the risk of cutaneous drug reaction (CDR). Proinflammatory cytokines TNF- α and IL-1b induce a variety of biochemical responses in cells, including decreases in glutathione. Interestingly, TNF- α is substantially increased in the skin of patients with severe bullous blisters and large areas of skin sloughing seen with CDR (seen most often in response to NSAIDS or antibiotics).

Antibiotics would be a typical choice for killing infecting bacteria. In the case of cryptic, chronic infections, however, application of therapeutic antibiotics might be problematic. The established infections likely have produced privileged local ecologies, rich in nutritious inflammatory exudate, isolated from the vascular system and protected by their biofilm.

Alternatively, the quick death of bacteria and release of pyrogenic factors may produce life-threatening inflammation that requires careful support. Attack a cryptic biofilm infection aggressively with a combination of herbs, enzymes, chelators and antibiotics and anticipate need for suppression of cytokine storms.

By their multiple immunological and biochemical effects, viral infections “prime” the cutaneous environment for an exaggerated response to agents that results in an infectious biofilm formation or a cutaneous drug reaction. Epstein-Barr virus influences the Th1/Th2 balance toward the production of Th1 cytokines and chemokines. Viral and bacterial infections can trigger the release of a variety of cytokines that up-regulate the expression of key immune-mediating molecules in keratinocytes and Langerhans’ cells (dendritic cells in epidermis).

Herpes simplex virus (HSV)-associated erythema multiforme (HAEM) falls into a clinical spectrum that ranges from mild erythema multiforme (EM) through the more severe disorders of Stevens-Johnson

syndrome (SJS) and toxic epidermal necrolysis (TEN).

The molecular hallmark of HAEM is the presence and expression of the RNA that is the HSV DNA polymerase gene (Pol) in lesional skin and circulating CD34+ stem cells. The CD34+ cells are increased in number in peripheral blood collected during acute HAEM episodes, suggesting that infected endogenous stem cells contribute to HAEM pathogenesis.

Skin clues

Psoriasis (eczema, atopic dermatitis or herpetiform dermatitis) is a chronic inflammatory cutaneous disorder with itchy blisters which can turn life-threatening with large sheets of broken and wet interconnecting hives, filled with purulence in the extreme. T cells and cytokines are of major

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importance in the development of this frequent autoimmune disease. The cutaneous and systemic over expression of several proinflammatory cytokines occurs, particularly type-1 cytokines such as IL-2, IL-6, IL-8, IL-12, IFN-g and TNF- α.

The over expression of these proinflammatory cytokines is responsible for initiation, maintenance and recurrence of skin lesions. The cellular composition of the inflammatory infiltrate within the psoriatic plaques as well as the keratinocyte hyper proliferation appears to be directed by cytokines as well.

Over expression of the chemo-attractant IL-8 contributes to the accumulation of granulocytes, typically found in psoriatic lesions. In contrast to the over expression of proinflammatory cytokines, a relatively low level of expression of type-2 anti-inflammatory cytokines IL-1RA and IL-10 is found, suggesting an insufficient counter regulatory capacity in psoriasis (likely depending on individual genetic polymorphisms).

Psoriasis is a T cell-mediated autoimmune chronic skin disease that is strongly associated with streptococcal throat infections. M protein has been described as the major virulence factor of Streptococcus pyogenes, although other streptococcal antigens may be involved. An aggressive Th1 cytokine response to M protein in psoriatic patients causes the cellular responses involved in psoriasis, while healthy subjects typically respond without proinflammatory Th2 increase, but enhanced suppressive IL-10 producing regulatory T cells.

And on to the cytokine storm of cancer

A variety of malignancies present first as auto-immune rheumatic syndromes like psoriasis. The clinical and pathologic features of classical Hodgkin lymphoma reflect an abnormal immune response due to the elaboration of a variety of cytokines by the malignant Reed-Sternberg (RS) cells or cytokine triggers from biofilm in surrounding tissues.

The involvement of Epstein-Barr virus, cytokines and/or oncogenes in promotion is suspected, although the precise mechanisms leading to transformation or tumor progression are still elusive. Activation of NF-kB as well as autocrine secretion of interleukin-13 are both implicated.

Molecular mimicry plays a part in human cytokine and cytokine response pathway genes created by the Kaposi's sarcoma-associated herpes virus (KSHV). Four virus proteins similar to two human macrophage inflammatory protein (MIP) chemokines, interleukin-6 (IL-6) and interferon regulatory factor (IRF) are encoded by the KSHV genome.

Interleukin 10 (IL-10) acts as human cytokine synthesis inhibitory factor (CSIF). cDNA clones encoding human IL-10 (hIL-10) have been isolated from a tetanus toxin-specific human T-cell clone. Like mouse IL-10, hIL-10 exhibits strong DNA and amino acid sequence homology to an open reading frame in the Epstein-Barr virus.

A successful immune response requires a delicate balance among antigen drive, lymphocyte proliferation and lymphocyte apoptosis. In MALT (mucosa-associated lymphoid tissue) lymphomas,

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unchecked proliferation of auto-reactive B-cells can lead to autoimmune disease, e.g., Sjogren’s syndrome or Hashimoto’s disease as well as lymphoma.

In a parallel fashion, defective immune regulation of responding T cells following chronic EBV infection may result in a cytokine storm, leading to a hemophagocytic syndrome and, rarely, to T-cell lymphomas. Hemophagocytosis is phagocytosis by macrophages of erythrocytes, leukocytes, platelets, and their precursors in bone marrow and other tissues. The syndrome is an uncommon blood disorder that, typically, clinically manifests as fever, enlarged spleen and jaundice with laboratory findings of increased lymphocytes and macrophages.

Chlamydia psittaci , Borrelia burgdorferi and Campylobacter jejuni share many traits with Helicobacter pylori, because they are also associated with chronic persistent infection; induce via NF-kB a polyclonal

lymphoid infiltrate in extranodal mucosa-associated sites; and have been implicated as cofactors in the development of both carcinoma and lymphoma.

Interaction of bacterial or viral antigens with host T-cells and antigen-presenting cells leads to a complex cascade resulting in clonal B-cell or plasma cell expansion. Although all immune responses are

in effect clonal, mucosa-associated lymphoid tissue (MALT) lymphomas are the result of immune responses gone awry, in which persistence of the antigen leads eventually to an autonomous clonal proliferation. Surprisingly, cell proliferation in gastric MALT lymphoma is directed at auto-antigens, and not at H. pylori.

In Epstein-Barr virus (EBV) infection, the virus immortalizes B lymphocytes and cytotoxic T lymphocytes (CTLs) which are directed toward both latent and lytic viral antigens expressed on EBV-infected B-cells. Various EBV-associated diseases occur as a result of this disruption of immune surveillance. In the majority of EBV-associated hemophagocytic syndrome cases, the major cell types containing EBV DNA are not B-cells, but clonally proliferating T-cells or NK-cells.

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Latent membrane protein 1 (LMP1) of Epstein–Barr virus (EBV) is an integral membrane protein which has transforming potential and is necessary but not sufficient for B-cell immortalization by EBV. LMP1 molecules aggregate in the plasma membrane and recruit tumor necrosis factor receptor (TNF-R) -associated factors (TRAFs) which are involved in the signaling cascade leading to NF- B activation by LMP1. Thus, LMP1 acts like a constitutively activated receptor, mimicking and usurping our own cytokine action.

A virus-encoded mimic of cellular receptors of cytokines is commonly found in a number of viruses (a viroceptor). Viroreceptors are expressed in members of the pox virus, herpes virus and retrovirus families and were probably derived from captured and modified cellular genes. Some cytokine imitators are also likely to be proteins unrelated to known cellular proteins.

Four virus proteins similar to two human macrophage inflammatory protein chemokines, IL-6, and interferon regulatory factor, are encoded by the Kaposi's sarcoma-associated herpes virus (KSHV) genome. These viral genes may form part of the response to host defenses contributing to virus-induced neoplasia and may be relevant to KSHV and HIV-I interactions.

Most of these receptor-like proteins are either secreted glycoproteins or are located at the infected cell surface. It is believed that the function of these viral-receptor mimics is to intercept the activity of the similar cytokine, thus effectively short-circuiting host immune responses to the viral infection.

Proliferation of these cells then produces severe immune reactions in the host, and the clinical features related to massive cytokine production at the onset of disease are unique and distinct from other EBV-associated diseases.

In the treatment of EBV- hemophagocytic syndrome cases, therapeutic infusion of EBV-specific cytotoxic T cells appears to be ineffective. Eradication of EBV-containing cells is useful but not sufficient to save lives, because of high incidence of acute mortality due to cytokine-induced multiple organ failure and low white blood cell-associated opportunistic infections.

The optimal treatment strategy for this disease consists of three steps: (1) control of cytokine storm including hyper-coagulatable state with multiple organ failure, (2) control of opportunistic infections, and (3) eradication of clonally proliferating EBV-containing T- or NK- cells by immune chemotherapy and, if necessary, stem cell/bone marrow transplantation (SCT/BMT).

Malignant Hodgkin's-Reed-Sternberg cells secrete interleukin-1 (IL-1), IL-5, IL-6, IL-9, TNF- α, macrophage colony-stimulating factor, transforming growth factor-beta, and, less frequently, IL-4 and granulocyte colony-stimulating factor. These cytokines are responsible for the increased cellular reaction and fibrosis observed in involved lymph nodes, as well as the accompanying immune suppression.

In atopic dermatitis, a common precursor of lymphoma, epidermal IL-4 exerts its effect on mast cells for their proliferation and for their Th2 cytokine production and secretion along with the cytokines released by infiltrating Th2 cells. The activities of these Th2 cytokines (IL-4, IL-5, IL-13) result in hives. IL-4, with its known functional role in ɛ chain switching, activates B cells to produce large quantities of IgE. By

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binding to the surface IgE receptor of mast cells, these abundant IgE activate them to degranulate and secrete even more Th2 cytokines and other proinflammatory molecules.

If this inflammatory chemistry is not quickly dismantled, cytokine storm may gather force. Anaplastic large cell lymphomas may also result in an apparent cytokine storm, with fevers, vascular leakage and altered blood counts that indicate anemia, thrombocytopenia, leukopenia and leukocytosis.

Most Hodgkin’s cases are characterized by expression of tumor necrosis factor receptor (TNFR) family members and their ligands, as well as an unbalanced production of Th2 cytokines and chemokines (cytokines with chemo-attractant properties). Large amounts of growth factor transcripts are present in a variety of malignant lymphomas.

The significance of growth factors is still unclear, since they are also used for repair. There is certainly increased alarm signaling, but it is likely loss of physiologic down-regulation within the cytokine network which encourages neoplastic cell growth.

US Patent 5833996 describes how injected immune regulatory material derived from Mycobacterium vaccae, especially dead cells of M. vaccae, is useful for the treatment of pathological conditions (other than mycobacterial disease and arthritic disease) in a patient who’s IgG shows an abnormally high proportion of agalactosyl IgG (significantly increased in patients with celiac disease, Crohn's disease, RA, ankylosing spondylitis and psoriatic arthritis). Binding and uptake of agalactosyl IgG by the mannose receptor on macrophages and dendritic cells generates epitopes recognized by T cells.

Reduce signaling crescendo with blocking mannose docking

Many bacteria also use the mannose receptor to initially adhere to our barrier tissues. When the receptor s filled with any of the alcohol sugars (mannitol, xylitol, erythritol or sorbitol), bacteria can’t stick, allowing them to be washed away, reducing infections substantially.

When incorporated into biofilm, these alcohol sugars weaken the pathogens’ primary protective proteoglycan structure. These dietary five-sided sugars are also bound by this receptor, making it more difficult for transient monocytes to become attached and morph into long-lived cytokine-producing macrophages as well as for agalactosyl IgGs to upregulate the immune inflammatory response that changes barrier ecology, encouraging and feeding pathogenic biofilm formation.

All allergens and auto-antigens have a triplet of basic amino acids that are likely involved in the initial aberrant presentation of these antigens as a result of the internalization by the carbohydrate-binding domain of mannose receptors on the surface of inflammation-stimulated immune cells. IL-1 b, the inflammatory cytokine, also has a basic triplet.

The special circumstances that lead to allergy and autoimmunity result in the binding of self-proteins or allergens to the mannose receptor and result in antibody production. The mannose receptor has an exposed tryptophan that may also bind numerous plant products (cranberry for example).

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In fact, the ‘antioxidant’ phytochemicals (alkaloids, flavonoids and terpenoids), are an abundant and varied group of chemicals that would bind to the exposed tryptophan of the mannose receptor and also compete for binding with antigenic basic amino acid triplets.

Mannitol receptor adhesion of the monocyte to the intimal wall is the initiating factor in atherosclerosis, allowing its conversion to a macrophage and development eventually to a cholesterol-accumulating foam cell. This engorged macrophage’s uptake of lipoproteins with continual release of reactive oxygen species and cytokines synergistically contribute to atherosclerotic progression.

M. vaccae extracts also treat chronic inflammatory disorders (other than arthritic disease) caused or accompanied by an abnormally high release from macrophages of interleukin-6 and/or tumor necrosis factor (primary biliary cirrhosis, sarcoidosis, ulcerative colitis, systemic lupus erythematosus (especially when accompanied by Sjogren's syndrome), multiple sclerosis, Guillain-Barre syndrome, primary diabetes mellitus and perhaps some aspects of graft rejection).

Sugar chains of adhesion molecules such as integrins and CD44 also influence the metastasis of cancer cells. Serum IgG oligosaccharide chains without galactose (agalactosyl IgG oligosaccharide) significantly increased with tumor progression of lung and gastric cancers. A marked increase of agalactosyl IgG oligosaccharide in cancer patients is associated with carcinogenesis and metastasis.

Herbs are adaptogenic and amphoteric

Adaptogens modulate our response to stress (physical, environmental, or emotional) and help regulate the interconnected endocrine, immune, and nervous systems. This re-regulation of a disordered or highly stressed system is achieved by metabolic regulators such as cytokines, catecholamines, glucocorticoids, cortisol, serotonin, nitric oxide (NO), cholecystokinin, corticotrophin-releasing factor (CRF), and sex hormones.

This broad array of biochemical activators helps explain why adaptogens also have anti-inflammatory, antioxidant, anxiolytic, antidepressant, nervine and amphoteric effects as well.

Amphoteric herbs normalize function, so if it is overreacting, they calm; if it is under reacting they stimulate. This classification is not found in conventional western medicine, only in the herbal realm. The medicinal mushrooms in use today are Chaga, Maitake, Shitake and Reishi (Gano derma). Additionally, the Traditional Chinese Medicine herb Astragalus is also amphoteric to the immune system.

Reduce the crescendo with turmeric 95% with 5% curcurmin @ 900mg. Take 4 900mg tabs a day to stop a cytokine storm if you become infected. Make curry a regular part of your diet.

Green tea (a possible Tamiflu/Relenza alternative) is a very effective antiviral. Tea catechins also decrease the production of TNF- α and inhibit release of virus via neuraminidase.

Boswellia serrata is an Indian herb also known as Shallaki. Its resin contains boswellic acids, which inhibit 5-lipoxygenase, leukotriene synthesis and leukocyte elastase. There are significant anti-inflammatory effects of Boswellia extract in rheumatoid arthritis, ulcerative colitis and Crohn's disease.

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Cat’s claw (ARG’s or Enzymatic Therapy’s Samento) with no toxic TOAs, more potent than Cat's claw products derived from the bark, benefits both the natural and acquired immune systems. It decreases the production of the cytokine TNF- α, but the number of white blood cells increases during treatment.

Artemisinin is anti-malarial and anticancer. The anticancer effect is in the range of concentration between nanomolar and micromolar blood levels. Artemisinin influences genes that control regulation of proliferation, angiogenesis and apoptosis (cell suicide of defective or infected cells), slowing tumor development.

Iron glycine sulfate and transferrin are used to increase the effectiveness (via oxidative stress) of artemisunate and artemisinin against leukemia and astrocytoma (brain tumor) cancer cells. Milk thistle, a liver protective herb, is used as part of the protocol and liver enzymes are monitored. Lactoferrin facilitates the entry of iron into the cells and also has immune modulating effects.

Artemesinin treats Schistomsoma (flukes), Pneumocystis carnii (a cause of pneumonia in AIDS), Toxoplasma gondii (parasite), human cytomegalovirus, Herpes simplex virus as well as hepatitis C and B. Artemisinin inhibits nitric oxide synthesis and is effective in brain malaria. There are few problems with side effects and it is generally safe for children. The active chemical of Artemisia annua (qinhao, sweet wormwood) is artemisinin, which is a sesquiterpene lactone. (Such compounds are anti-inflammatory.)

Vitamin D suppresses proinflammatory cytokines and increases anti-inflammatory cytokines. It also increases production of two hundred known antimicrobial peptides which directly and rapidly destroy the cell walls of bacteria, fungi and viruses, including the influenza virus, and play a key role in keeping the lungs free of infection, minimizing risk to deadly cytokine storm. Vitamin D is a steroid hormone that shows remarkable antibiotic properties.

VD3 genetically orchestrates cytokine production leading to differentiation (lineage maturation) and mitosis (multiplication) of these immune cells in subtle, sometimes complex ways. For example; 1,25dihydroxyD3 blocks animal models of (autoimmune) diabetes without generalized immune suppression. On the other hand, VD3 actually increases the immune response by macrophages versus bacteria. Serum levels of 1,25dihydroxyD3 typically coincide (as does selenium) with survival rates among those infected by HIV.

The autoimmune damage generated by multiple sclerosis (MS) is typically associated with an elevated ratio of Th1 to Th2 cells. VD3 at least partly corrects that imbalance. VD3 influences ratios between these T-helpers reducing the over-proliferation of T and B leukocytes. This is partially caused at the hematopoietic level, influencing blood cell creation in the bone marrow as well as later during leukocyte maturation in the thymus. Vitamin D receptors have been found in both T and B lymphocytes maturing in the thymic medulla.

While glucocorticoid steroids limit maturation of many of these lymphocytes by stimulating apoptosis, some of those resistant to glucocorticoids may be eliminated by VD3’s ability to signal their differentiation along a pathway also leading to apoptosis (rather than maturity as cytotoxic cells). Vitamin D receptors are also found in cells of the lymph nodes and tonsils where VD3 down-regulates effector cell expression.

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Thus VD3 actually has a biphasic effect on the immune system. Rather than a simple down regulation of immune response it blunts generalized over-proliferation of both lymphocytes and monocytes while sharpening immune system attack on specific targets.

Vitamin E supplemented as its four tocopherols and four tocotrienols is an immune booster and also decreases the production of the cytokine TNF- α. It is very suitable for immune compromised people, especially the elderly. Its effects are enhanced when taken with Vitamin C.

St John’s Wort (Hypericum) is a very effective antiviral and decreases production of the cytokine IL-6.

Osha (Ligusticum porteri), a Native American herb was used by the Native American population with noticeable benefit seen during the 1917-1918 Spanish flu pandemic that killed tens of millions of people. Those who took this native herb only got a relatively mild case of the flu without the deadly cytokine storm.

Viral neuraminidase is an enzyme on the surface of influenza viruses that enables the virus to be released from the host cell and proliferate. Scuttellaria (Skullcap) herb is used as a tea. It has no side effects, is a mild tranquilizer and likely inhibits neuraminidase, which is needed by the H5N1 virus to reproduce. Resveratrol inhibits neuraminidase while also sending a message to infected cells to stop manufacturing viruses and commit cell suicide (apoptosis) to avoid transmitting them.

Perhaps the cytokine storm is not a mistake, but in fact an ancient mammalian group survival strategy for dealing with virulent pandemic flu. Viral epidemics have been around for longer than mammals, and have infected animal and bird populations regularly for millions of years.

Possibly when faced with this sort of viral challenge, the response is a sort of programmed societal "apoptosis", a planned person (as opposed to cell) death? By dying quickly and horribly, the rest of the population is protected by similar strategy: (1) being warned that a plague has arrived, and (2) the person dies quickly and does not circulate amongst the group further spreading the infection.

Zinc (try L-OptiZinc with 30-60 mg of zinc, which is a zinc-methionine complex). Zinc has direct activity against a range of viruses. Zinc is a mild to moderate inhibitor of neuraminidase. Zinc ions have a slight inhibitory action on all strains of neuraminidase tested. Zinc reduces TNF, IL-1 and IL-8 production. Zinc can also turn down TNF, ICAM and other cytokines involved in over reaction to H5N1 and other viruses.

The hydroxyl radical wreaks havoc by reacting with lipids in the cellular membranes, nucleotides in DNA, and sulfhydryl groups on proteins. Iodide neutralizes hydrogen peroxide by converting it first to hypiodious acid and then water, thereby blocking its conversion into the hydroxyl radical. Iodide defends brain cells from lipid peroxidation, rendering them less susceptible to free radicals; and it increases the antioxidant status of human serum similar to that of vitamin C.

Iodine induces apoptosis, programmed cell death, destroying cells that represent a threat to the integrity of the organism, like cancer cells and cells infected with viruses.

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Proteolytic enzymes are potent anti-inflammatory agents (papain and bromelain). They increase antioxidant enzymes (SOD, catalase and glutathione peroxidase) while reducing inflammatory cytokines TNF-a and IL-1b.

Allopurinol might slow or inhibit cytokine storming. Allopurinol inhibits the enzyme xanthine oxidase (also present in pasteurized milk) and can lead to reduced levels of uric acid. It is typically used to prevent gout (won’t help to end a gout attack, but can help prevent one). It also reduces the release TNF-, and might, therefore, interrupt the chain of events enough to prevent cytokine storm. It is an inexpensive and relatively safe pharmaceutical.

Paradoxically, allopurinol is one of the drugs thought to occasionally trigger Stevens-Johnson Syndrome (SJS). This is a potentially life-threatening cytokine storm where the epidermis separates from the dermis creating many large bullous blisters. Symptoms sometimes start with painful rashes in the mouth. That said, allopurinol might be a potentially useful pharmaceutical in the arsenal to tone down systemic inflammatory response.

Patients with severe granulocytopenia (low white blood cell counts) are more susceptible to severe infections and sepsis. Because circulating concentrations of uric acid are increased in neutropenic mice, the effect of lowering uric acid with allopurinol and sodium bicarbonate was tested in neutropenic mice challenged with LPS. Treated mice had an improved survival and a reduced proinflammatory cytokine production versus controls.

When macrophages from normal and neutropenic mice were stimulated ex vivo with LPS, the macrophages from neutropenic animals synthesized more cytokines in response to LPS. Neutropenia also activates other hematopoietic regulatory cytokines such as granulocyte-macrophage colony-stimulating factor, which further stimulates production of proinflammatory cytokines. A product of leukocyte damage (eg, uric acid) may be responsible for the increased cytokine production during neutropenia.

Hyperuricemia induced by repeated administrations of uric acid in normal mice led to increased TNF production after LPS. Neutropenic mice respond with enhanced cytokine production and increased susceptibility to an LPS challenge, and hyperuricemia likely plays an important role in this phenomenon.

Cannabinoids also inhibit IL-2 & TNF-α. Heavy pot users are more prone to viral infections due to the inhibition of immune and inflammatory pathways. In all likelihood, smoking marijuana while in the throes of influenza is not too good an idea (same applies to smoking anything, since that really stresses the lungs!). Luckily, California pharmacies supply cannabis-containing teas, cookies, cakes and brownies (watch out for the sugar!). Depending on availability, cannabinoids likely fall into the category of agents that can temporarily impair the pro-inflammatory response.

Herbs known to reduce inflammation include ginger, turmeric, pokeroot, cleavers, devil’s claw, licorice, autumn saffron, boswelia, curcurmin, arnica, bromelain, German chamomile, licorice, white willow, witch hazel and capsaicin.

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Spice components, such as curcurmin from turmeric and capsaicin from red pepper reduce inflammation by influencing arachidonic acid metabolism and also the secretion of lysosomal enzymes by macrophages. Curcurmin and capsaicin also inhibit secretion of collagenase, elastase and hyaluronidase demonstrating that they reduce release of pro-inflammatory mediators like eicosanoids.

Many plant chemicals have effects on at least one of the following proinflammatory cytokines: interleukin-1 (IL-1), interleukin-6 (IL-6), tumor necrosis factor (TNF) and interferon (IFN).

Herbs that reduce or modulate the secretion of one or more of these cytokines include Acalypha wilkesiana (Copperleaf), Acanthopanax gracilistylus root, Allium sativum (garlic), curcurmin, Echinacea purpurea, Grifola frondosa (maitaiki mushroom), Panax ginseng, Polygala tenuifolia root, Silybum marianum (milk thistle), Tinospora cordifolia, Uncaria tomentosa (cat’s claw) and Withania somnifera (Ashwagandha).

Propionibacterium acnes, an anaerobic pathogen, participates in the pathogenesis of acne by inducing certain inflammatory mediators. These intermediaries include reactive oxygen species (ROS) and pro-inflammatory cytokines. ROS, interleukin-8 (IL-8) and TNF-α are often used as the major criteria for the evaluation of anti-inflammatory activity.

To explore the anti-inflammatory effects of herbs, polymorphonuclear leukocytes (PMNL) and monocytes were treated with culture supernatant of P. acnes in the presence or absence of herbs. Rubia cordifolia (Indian madder), Curcuma longa (turmeric), Hemidesmus indicus (Indian sarsaparilla) and Azadirachta indica (neem) caused significant suppression of ROS from PMNL. Sphaeranthus indicus (East Indian globe thistle) caused a smaller, but still important suppression of ROS. Aloe vera had no effect on ROS production.

When monocytes were induced to produce proinflammatory cytokines, maximum suppression was shown by Azadirachta indica and Sphaeranthus indicus, followed by Hemidesmus indicus, Rubia cordifolia and Curcuma longa. Aloe vera also showed insignificant inhibitory activity. These herbs effectively reduce the cellular production of P. acnes-induced ROS and pro-inflammatory cytokines.

When a patient has a Th1-cell dominant disorder, such as Crohn’s disease, then administration of specific probiotic strains can be given to promote secretion of IL-10 and TGF-β cytokines and, therefore, down-regulate chronic Th1 cell-associated inflammation and promote a return to balanced immunity.

Inflamed Crohn’s disease mucosa shows significant reduction in the production of TNF-α by when cultured with L. casei or L. bulgaricus, but not with L. crispatus or E. coli. Bifidobacterium genomic DNA induced secretion of the anti-inflammatory IL-10 by peripheral blood mononuclear cells. Total bacterial DNA from feces collected after probiotic administration modulated the immune response by a decrease of IL-1b and an increase of IL-10.

Saccharomyces boulardii is probiotic yeast successfully used to prevent antibiotic induced diarrhea, preclude relapse of Clostridium difficile and Crohn's disease. The secretion of proinflammatory cytokines such as IL-1b is decreased in the infected cells incubated with Saccharomyces boulardii, but

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anti-inflammatory cytokine levels such as IL-4 and IL-10, however, are found to be higher. S. boulardii also induces a decrease in TNF- α and related apoptosis in enterohemorrhagic Escherichia coli-infected T84 cells.

Alternately, if a person has a Th2 cell dominant disorder, like allergies, specific probiotic strains can be given to help release TGF-β, reduce IL-4 and IL-5 or increase IL-12 and, therefore, shift the immune system back to a balanced Th1:Th2 response. L. rhamnosus GG can reduce allergic disease symptoms in humans. Different Lactobacillus strains reduce IL-4 levels (a Th2 cytokine) and enhance production of Th2 cell-related cytokines, supporting a more balanced Th1/ Th2 response.

Other subsets of T cells, formerly called suppressor T cells or type 1 T regulatory cells (Treg) or Type 3 T regulatory cells (Th3) help regulate T-helper cell functions and maintain intestinal immune homeostasis. For example, Treg cells predominantly secret IL-10, a cytokine that down-regulates Th1 activity and, therefore, reduces Th1-associated inflammation.

Adequately primed Th3 cells primarily secrete TGF-β, which helps modulate both Th1 and Th2 activity. Th3 cells are important in the maintenance of mucosal immunity and, therefore, the prevention of pathology, with certain probiotic strains can moderate these regulatory responses. For example, L. paracasei (NCC2461) stimulates in vitro regulatory T cells to produce TGF-b and IL-10. Artificial induction of the oral tolerance response, via the administration of strain-specific probiotics helps modulate hypersensitivity reactions.

Avoid fanning the flames

The following immune-upregulating substances may be best to avoid during a viral pandemic, since they may increase capacity to trigger a life-threatening cytokine storm: Elderberry juice (Sambucal) – Avoid, elderberry increases production of cytokines TNF- α and IL-6. This substance is very effective against the common flu but may not be desirable for the H5N1 virus.

Micro algae (Chlorella and Spirulina) – Avoid, sea greens increases production of cytokine TNF- α.

Honey – Avoid, bee nectar increases production of cytokines TNF- α and IL-6.

Chocolate – Avoid, it increases production of cytokines TNF- α and IL-6.

Echinacea – Avoid, it increases production of cytokines TNF- α and IL-6. Although it is often effective for normal flu, it may increase the chance of cytokine storms for H5N1.

Kimchi – Avoid, this fermented food increases production of cytokines TNF- α and IL-6.

Dairy products and bananas add to inflammation-increased massive mucus production.

Calcium and perhaps magnesium compete with zinc, selenium and manganese (tend to increase the activity of virus-releasing neuraminidases). Cancer is associated with high copper levels (low zinc levels).

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The cancer-promoting activity of angiogenesis is copper dependent. Copper chelation to lower copper levels is one way to treat cancer.

Iron or copper can exacerbate inflammation, enhancing hydroxyl radical production significantly with presence of reduced glutathione, cysteine, ascorbic acid and selected catechols, but not by mannitol, melatonin or tyramine. Use caution with therapeutic levels of thiols and ascorbate for attempting to quench oxy-radical-induced tissue damage in environments where free redox-active metal ions may be present.

Copper and iron function both as foci for site-specific peroxidative activity and as catalysts to promote the pro-oxidant properties of certain endogenous reducing systems, thereby elevating rather than diminishing aggressively destructive ·OH levels.

TNF/iNOS-producing dendritic cells are the necessary evil of lethal influenza virus infection

One of the first things that happens when we breathe an immune system irritant is that a dendritic cell or macrophage, like a cop patrolling on the lung beat, sounds an alarm by making a protein, MCP-1 (monocyte chemotactic protein-1).

MCP-1 combines with CCR-2 (chemotactic chemokine receptor-2) which calls to the area another cell called a monocyte. CCR-2 also induces the monocyte to perform a more special function: produce two more proteins, tumor necrosis factor-alpha (TNF-a) and inducible nitric oxide synthase (iNOS).

Monocytes now morph into a dendritic cell (DC), or more precisely a special subset of dendritic cells called Tip-DCs (TNF-iNOS-producing DCs). There are many other kinds of immune cells in the mix: natural killer (NK) cells, neutrophils, other kinds of DCs and macrophages. However, with highly virulent strains, mostly Tip-DC accumulates in the lungs.

Dendritic cells also function in the later phase of the immune response. They are antigen presenting cells (APCs) that take the pathogen and broadcast its description to other cells in the immune system, cells like the killer T cells.

The number of Tip-DCs is significantly elevated in mice infected with highly pathogenic influenza A viruses. These Tip-DCs function locally in the respiratory tract as APCs, and they are required for the full realization of protective CD8+ T cell-mediated immunity via functioning killer T cells. In addition, their complete absence from the lungs of CCR2−/− mice is associated with severe disease, indicating that Tip-DC elimination is not a viable therapeutic option.

In other words, when it comes to Tip-DC, you can't live with them and you can't live without them (in terms of infection with virulent flu virus). A drug that affects CCR-2 but doesn't completely knock it out belongs to an interesting class of compounds called peroxisome proliferator-activated receptors (PPARs), of which there are three (alpha, beta and gamma).

The gamma subtype provides a drug called pioglitazone. Another compound of this class is rosiglitazone, marketed under the trade name Avandia, as a treatment for type II diabetes.

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By pre-treating mice with pioglitazone, the lethality and weight loss related to infection with the virulent strains of flu virus was reduced significantly (mortality went from 90% to 50%). There was also a reduction in inflammatory response, associated with less buildup of Tip-DCs in the mouse lungs.

Avandia has increased risk of heart attack for those taking it long term for diabetes. On the other hand, balancing that risk against death from cytokine storm due to a pandemic flu virus suggests it might be worthwhile to consider as part of the pharmaceutical arsenal should a pandemic develop.

Migration inhibitory factor (MIF) is a pivotal proinflammatory cytokine

MIF is a pivotal molecule in inflammation, playing important roles in the "bio-vicious-gate" of circulation collapse. MIF-related pathology underlies sepsis, fibrosis and the massive mucus production of asthma or acute respiratory distress syndrome, hives of atopic dermatitis, rheumatoid arthritis, nephropathy and tumors.

In patients in the throes of sepsis, MIF concentrations are 10-20 times higher than normal. If MIF goes down, the chance that patients will survive sepsis is increased dramatically. MIF levels are doubled in autoimmune diseases like rheumatoid arthritis and diabetes.

MIF exerts its function through interaction with a body of signal molecules that are involved in functional regulation of several end-point molecules such as, carbon monoxide, insulin, complement C5a, nitric oxide, inducible nitric oxide synthase and glucocorticoids.

Nitric oxide, a short-lived free radical–generating gas, is an important signal-transduction molecule in many cell types, regulating such diverse functions as vasomotor tone, neurotransmission, mediation of immune responses and inflammatory cell adhesion to the vessel wall.

In the cardiac muscle cell, the major physiological role of NO involves depression of contractility and electrophysiological stabilization through elevation of intracellular cGMP. A rise in myocardial NO levels likely accounts for the contractile depression seen during sepsis.

These downstream mediators are linked to vascular tone differentially, and function with cardiac muscle cell, vessel endothelial and smooth muscle cell and then lead to constitutive myocardial dysfunction and vascular dilation, ultimately unbalancing systemic circulation.

Thus, MIF functions as a critical upstream molecular switch to change the downstream action of signals in sepsis-associated circulatory collapse.

The macrophage is a pivotal mediator of innate immunity and a front line of host response to tissue invasion. Mycobacterium tuberculosis is one of the few pathogens able to reside and replicate within the toxic environment of the macrophage. Hormetic strategies likely help this pathogen adapt, since low concentrations of reactive nitrogen intermediates (RNI) may inhibit replication but do not eradicate M. tuberculosis. In culture, clinical M. tuberculosis isolates survive and reproduce in the presence of lower concentrations of RNI as well as or better than they survive and reproduce in medium alone.

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Once activated, macrophages produce an assortment of microbicidal effectors and immune regulatory cytokines that act to eliminate the invasive agent and influence the course of the ensuing, cognate immune response. An overly robust macrophage response, however, can lead to pathological results that contribute significantly to septic shock, autoimmune diseases and various granulomatous disorders.

Apoptosis limits the productive life of activated macrophages and restricts the expression of their inflammatory products to the site of tissue invasion, eventually terminating the immune response. The LPS-induced apoptotic response in macrophages (a bacterial ploy) requires the production of nitric oxide (NO), the intracellular accumulation of the tumor suppressor gene product p53 and activation of a caspase-dependent cell-suicide pathway.

MIF suppresses the activation pathway leading to LPS-stimulated macrophage apoptosis by functionally inactivating p53 activity via inducing COX-2. The COX-2 isozyme is of critical importance in the tissue pathology associated with septic shock. Curbing of p53 function via MIF induction of COX-2 in macrophages is likely an important mechanism for the overall proinflammatory actions of MIF.

Inhibition of MIF-induced COX-2 activity by NSAIDs may underlie the cancer protective effect of these drugs and supports a potentially fundamental link between MIF expression and p53 function in a variety of cell systems. In the cell, tumor suppressor p53 protein binds DNA, which in turn stimulates another gene to produce a protein called p21 that interacts with a cell division-stimulating protein (cdk2). When p21 is complexed with cdk2 the cell cannot pass through to the next stage of cell division.

Blocked or mutant p53 can no longer bind DNA in an effective way, and as a consequence the p21 protein is not made available to act as the 'stop signal' for cell division. Thus cells divide uncontrollably, and form tumors. Recovery from infections sometimes triggers shrinkage of tumors and cancer cure.

A recombinant form of migration inhibitory factor (MIF) is able to activate, in a dose-dependent manner, murine macrophages to express nitric oxide (NO) synthase and to produce high levels of NO. The time course of the induction of NO synthase is similar to that produced by IFN-g.

MIF can synergize with IFN-g in the induction of NO synthesis, and the induction of NO synthase by both MIF and IFN-g is sensitive to inhibition by steroids (dexamethasone). However, unlike IFN-g-induced NO generation, MIF is sufficient for the induction of the enzyme, does not synergize with LPS, and is highly sensitive to inhibition by transforming growth factor.

MIF is released by the anterior pituitary gland as a consequence of the systemic stress response and is also expressed by macrophages, T cells and eosinophils after immune/inflammatory activation. MIF has the capacity to override the immunosuppressive effects of glucocorticoids, partly accounting for the proinflammatory role of MIF in conditions like septic shock, arthritis and glomerulonephritis.

Additional biological activities for MIF include the regulation of macrophage and T cell activation, IgE synthesis, insulin release and carbohydrate metabolism, cell growth and apoptosis as well as tumor angiogenesis.

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Insulin-induced change in vascular tone correlates with protein MIF. MIF can promote excessive release of insulin. Thus the link of “MIF-insulin-vasodilation” formed in septic context finally results in consistent circulation dysfunction.

Nitric oxide (NO) is a highly reactive gas that can easily pass the lipid bilayer through zones of tissue, by which it creates areas of vasodilation effect and contributes to host immunity. The production of NO and its inducible nitric oxide synthase (iNOS) are subsequently increased if MIF is inhibited or blocked during infection.

Apoptotic cell death is involved in myocardial dysfunction and vascular paralysis in people or rodents with sepsis. Gene p53 is a fundamental tumor suppressor and apoptosis regulator. Gene p53 functions in inflammation by correlating with MIF, and thus results in pathological effects that contribute substantially to septic shock. In sepsis, the complement system plays a crucial part in endotoxin-induced vasodilation, increased vascular permeability and induction of heart muscle cell apoptosis.

The liver and kidney toxic iminoquinone metabolite of acetaminophen, N-acetyl-p-benzoquinone imine (NAPQI), binds to and inhibits both the isomerase and the biological activities of MIF. These acetaminophen metabolites and several related chemotypes irreversibly inhibit both the enzymatic and biological activities of MIF, perhaps the mode of action of this risky pain reliever, which becomes especially liver and kidney toxic when glutathione is exhausted.

Acidity frees up minerals and alkalinity binds

The underlying basis of cellular reactivity in internal environment and microenvironment relies on a variety of positive ionic elements, known as cations. These major mineral ions (calcium, magnesium, zinc) play many roles as critical regulators through a complex network intertwined with MIF, finally resulting in decrease in responsiveness to circulation active agents.

Acute respiratory distress syndrome (ARDS)

Imbibing one or two alcoholic beverages per day is associated with a longer healthier life due to a compensatory stress response called hormesis. One of the paradoxes of biological systems is that little stresses increase capacity to handle larger stresses that would be overwhelming without gentler previous priming.

Mild stresses from alcohol, calorie restriction, radiation, poisonous plant phytochemicals, exercise, large doses of antioxidants as well as discussing ‘passionate pet peeves’ are most often mediated through generated reactive oxygen species. These oxidative irritants make us stronger and live longer by encouraging production of more mitochondria as well as enhancing increased genetic production of catalytic antioxidant and detoxification systems.

A family of histone deacetylases called sirtuins has played pivotal roles in stress resistance through a vast expanse of evolutionary history. The sirtuins are activated by various types of stress, and can protect cells against energy deprivation and oxidative stress. Two other types of stress resistance

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proteins are chaperones such as heat-shock protein 70 and glucose-regulated protein 78, and antioxidant enzymes such as Mn-superoxide dismutase and glutathione peroxidase.

Nerve cell networks are the ‘first responders’ to environmental challenges. They perceive the challenge and orchestrate coordinated adaptive responses that typically involve autonomic, neuroendocrine and behavioral changes. In addition to direct adaptive responses of neurons to environmental stressors, cells subjected to a stressor produce and release molecules such as growth factors, cytokines and hormones that alert adjacent and even distant cells to impending danger.

The discoveries that some molecules (e.g., carbon monoxide and nitric oxide) and elements (e.g., selenium and iron) that are toxic at high doses also play primary roles in cellular signaling or metabolism suggest that during evolution, organisms (and their nervous systems) co-opted environmental toxins and used them to their advantage.

Oxygen, carbon monoxide, iron and selenium are all toxic when present in high amounts, but are commonly used by cells. Oxygen is used for the production of cellular energy (ATP) and carbon monoxide is an intercellular signaling molecule in blood vessels and nerve cells. Iron and selenium are important for the proper functions of proteins such as hemoglobin and antioxidant enzymes

Low doses of radiation or other perceived stresses cause conformational changes in cell membrane structure, which turn on intracellular signal transduction, inducing gene expression. If DNA is damaged, genes for DNA repair are also expressed. The expression of these genes causes the creation of proteins used for repair, triggers apoptosis of defective or infected cells and produces cytokines for activating immune cells as well as regulating cell functions.

The hormetic biphasic nature of responses to neurotransmitters, neurotrophic factors, cytokines and drugs of abuse as well as treatments for a range of neurological disorders is underappreciated and often ignored in basic and applied neuroscience research. With unrelenting or overwhelming stress, suppressive feed-back loops become inadequate, creating catastrophic disorder, the cytokine storm.

Alcohol abuse, even in otherwise healthy individuals, causes significant mineral loss as well as oxidant stress within the alveolar space and impairs both alveolar epithelial and alveolar macrophage function via common pathologic physiological mechanisms. Alcohol abuse independently increases the risk of developing the acute respiratory distress syndrome (ARDS) by as much as three to four fold.

Acute respiratory distress syndrome (ARDS), the most severe form of acute lung injury, is a devastating clinical syndrome with a high mortality rate of 30–60%. Predisposing factors for ARDS are many and include sepsis, aspiration, pneumonia and infections with SARS or avian flu.

In the case of Swine flu, sometimes instead of typical transitioning into recovery, inflammatory signaling becomes acute and life threatening. Mucus builds up in the lungs and creates acute respiratory distress syndrome which is triggered by a cytokine storm.

Anytime there is mucus or fluid build-up, the kidneys and liver are begging to be flushed. Drink at least 2 quarts (8 cups) of spring water and at least 2-4 cups of Essiac tea or Amazon Herb Company’s Shipibo

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Treasure tea. The medicinal herbs and bark in these teas are anti-viral and reduce inflammation while flushing toxins from the body.

At present, there are no effective drugs for improving the clinical outcome of ARDS. ACE2 was identified as the critical SARS receptor in vivo and ACE2 is expressed in lungs. The effect of ACE2 gene deficiency was examined in mice that mimicked common lung failure pathology in humans, where ACE2 protects lungs from acute injury induced by acid aspiration and sepsis.

Angiotensin II

ARDS disease pathogenesis was mapped to the ACE-angiotensin II-angiotensin receptor 1a (AT1a) pathway, while ACE2 and the angiotensin 2 receptor (AT2) negatively regulate lung function and lung edemas. Importantly, recombinant human ACE2 protects mice from severe acute lung failure. ACE2 has a critical function in acute lung injury, making it a possible therapy for an acute syndrome affecting millions of people worldwide every year.

In humans, some strains of bird flu kill by causing lung failure. At autopsy, the lungs of people who die of bird flu resemble those of people who have died from SARS or influenza A infection, two other lethal viruses. The lung becomes invaded by macrophages and the lung's alveoli get filled up with mucus. Relatively little virus is present. What kills people is a cytokine-driven overly-exuberant immune response to a continually proliferating new virus, not the virus itself.

Using readily available pharmaceuticals might provide a universal preventive against death from most viral diseases by blocking angiotensin II. ACE inhibitors and ARBs block the action of angiotensin II, which causes blood vessels to narrow. As a result, blood vessels tend to relax and open up, making it easier for blood to flow through the vessels, reducing blood pressure. They also increase the release of sodium and water into the urine, additionally lowering blood pressure.

Angiotensin II gets the immune response started and keeps it going. Angiotensin I-converting enzyme (ACE), the enzyme which produces angiotensin II, is expressed on macrophages when they become activated. All immune cells, including macrophages, contain receptors for angiotensin II.

Blocking angiotensin II production with a suitable ACE inhibitor, or the action of angiotensin II at its receptors with an angiotensin II receptor blocker (ARB) tends to tone down host immune response. GenoMed has seen this approach work for several autoimmune diseases over the past year and a half. The patient's blood pressure guides whether an ACE inhibitor or an ARB is used. ARBs at low doses can tone down immune system response without lowering blood pressure at all.

ARBs are prescribed for controlling high blood pressure, treating heart failure and preventing kidney failure in people with diabetes or high blood pressure. Since these medications have effects that are similar to those of ACE inhibitors, they are often used when an ACE inhibitor cannot be tolerated by patients.

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According to big Pharma, ARBs are well-tolerated by most individuals. However, the most common side effects are: annoying cough, elevated potassium levels, low blood pressure, dizziness, headache, drowsiness, diarrhea, abnormal taste sensation (metallic or salty taste) and rash. Compared to ACE inhibitors, cough occurs less often with ARBs. The most serious, but less usual side effects are kidney failure, liver failure, allergic reactions, a decrease in white blood cells and swelling of tissues (angioedema). ARBs may cause birth defects if taken during pregnancy.

Natural substances with ACE inhibition function are: Garlic, Seaweed (Wakame, etc), Tuna protein / muscle(?mercury), Sardines, Hawthorne berry, Bonito fish (dried), Resveratrol, Pycnogenol, Casein / Hydrolyzed whey and Hydrolyzed wheat germ isolate, Sour milk, Gelatin, Sake, EFAs (omega-3s), Chicken egg yolks, Zein, Dried salted fish, Fish sauce and Zinc.

Natural substances with ARB function are: Potassium (K+), Fiber, Garlic, Vitamin C, Vitamin B6 (Pyridoxine), Co-enzyme Q10, Celery (4 stalks), Gamma linolenic acid (GLA) and DGLA.

Extracellular Matrix and Cytokines: A Functional Unit

The extracellular matrix (ECM) and soluble mediators like cytokines can influence the behavior of cells in very distinct as well as cooperative ways. One group of ECM molecules which shows an especially broad cooperation with cytokines and growth factors are the proteoglycans. Proteoglycans can interact with their core proteins as well as their glycosaminoglycan chains with cytokines.

These interactions can modify the binding of cytokines to their cell surface receptors or they can lead to the storage of the soluble factors in the matrix. Human polymorphonuclear leukocytes (PMN) release large quantities of hydrogen peroxide in response to TNF, but only when the cells are adherent to surfaces coated with extracellular matrix proteins. Proteoglycans themselves likely have cytokine activity.

Interaction of heparin sulfate chains with fibroblast growth factor-2 (FGF-2, basic FGF) is a prototype example for the interaction of heparin-binding cytokines with heparin sulfate proteoglycans that illustrates different levels of mutual dependence of the cytokine network and the ECM.

Heparin binding mediates the interaction between most growth factors or cytokines and their cell surface receptors. Heparin inhibits NF- B activation in angiotensin II-stimulated kidney mesangial cells that express either normal or chemically altered glycosaminoglycan. Alterations in heparin sulphate glycosaminoglycan chemistry or metabolism under pathological conditions, such as diabetic nephropathy, have direct functional consequences for the local effect of angiotensin II.

Many viruses and bacteria bind to cell surfaces via heparin sulfate. Acidic polysaccharides form the matrix of biofilms. Heparin and nucleic acids can also serve this function. Heparin binds to basic amino acids in proteins via hydrophobic interactions. Heparin binding domains are groups of basic amino acids (K for lysine and R for arginine) of proteins that bind the common acidic extracellular polysaccharide heparin.

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Aromatic dyes, such as berberine (which confounds the multidrug-resistant pumps that enable survival of pathogenic inhabitants of the biofilm), bind to heparin through similar hydrophobic interactions.

Antimicrobial peptides, like defensins, have groups of basic amino acids. Heparin binding domains excised from proteins as peptides are antimicrobial. Stomach proteases cleave around heparin-binding domains to produce antimicrobial peptides. Intestinal proteases cleave within heparin-binding domains and inactivate bacterial and viral agglutinins.

Angiotensin II also plays an important role in skin wound healing, accelerating keratinocyte and fibroblast migration in a process mediated by heparin-binding epidermal growth factor (EGF)-like shedding.

Apoptosis

Apoptosis is the process of controlled cellular death where activation of specific death-signaling pathways leads to deletion of cells from tissue without triggering inflammation. Unplanned cell death by necrosis provokes inflammatory response. Planned death-signaling pathways can be activated in response to receptor–ligand interactions, environmental factors such as ultraviolet light and redox potential as well as internal factors that are encoded in the genes for development, growth or repair.

The activation of interleukin-1 -converting enzyme (ICE)-like and cysteine protease protein (CPP)-32-like cysteine proteases is required to mediate TNF- -induced apoptosis of human umbilical venous cells. Endothelial-derived nitric oxide (NO) as well as exogenous NO donors inhibit TNF- -induced cysteine protease activation. Shear stress-mediated NO formation interferes with cell death signal transduction and likely contributes to endothelial cell integrity by inhibition of apoptosis.

Ultimately, apoptosis results in fragmentation of the DNA, a decrease in cell volume and envelopment of the apoptotic cell’s prepackaged organelles by nearby phagocytes. The useful organelles are utilized and damaged structures or toxins are oxidized in peroxisomes. Inhibition or activation of apoptosis can lead to human disease either because infected or defective 'undesired' cells have apoptosis cancelled and develop prolonged survival or because nerve, barrier or structural 'desired' cells die prematurely.

During acute inflammation the cytokines granulocyte colony-stimulating factor and granulocyte/ macrophage colony-stimulating factor prolong the survival of neutrophils, and thus enhance neutrophil orchestrated inflammation. One theory that links apoptosis with acute respiratory distress syndrome (ARDS) is that neutrophil apoptosis resolves inflammation, and predicts that stress-driven inhibition of neutrophil apoptosis or inhibition of clearance of apoptotic neutrophils fans the flames of ARDS.

On the other hand, the epithelial hypothesis suggests that the lung injury seen during ARDS is caused by apoptotic death of alveolar epithelial cells in response to soluble mediators such as soluble Fas ligand and predicts that blockade of such inhibitors would be helpful in preventing or treating ARDS. These two theories are not mutually exclusive, and both are likely congruent, with the same mitochondrial signals being received by two different tissue types, but each with their own programmed response.

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Several factors modulate Fas-mediated apoptosis of alveolar epithelial cells that creates part of the pathogenesis of acute lung injury. Surfactant protein A (SP-A), the primary protein present in pulmonary surfactant, is an inhibitor of type II apoptosis. This is important because, in patients with early ARDS, the concentration of SP-A is decreased in BAL fluid.

The lower concentration of SP-A would favor apoptosis of type II cells in these patients. Another important modulator of Fas ligand in the lungs is angiotensin II. Epithelial cells interact with angiotensin II via the angiotensin receptor subtype AT1, and this interaction is required for Fas-mediated apoptosis of alveolar epithelial cells in vitro.

In ARDS the concentration of angiotensin-converting enzyme, which catabolizes the conversion of angiotensin I to angiotensin II, is increased in BAL fluid. Therefore, in early ARDS a combination of three factors favors alveolar epithelial apoptosis: increased concentrations of soluble Fas ligand; decreased concentrations of SP-A; plus increased concentrations of angiotensin-converting enzyme and angiotensin II.

There is a molecular link between SARS pathogenesis and the role of the renin-angiotensin system (RAS) in lung failure. Recombinant ACE2 protein could therefore not only be a treatment to block the spreading of SARS but modulation of the RAS could be also used to protect SARS patients, and possibly people infected with other viruses such as avian influenza strains, from developing acute lung failure and ARDS.

Glutathione

Chronic alcohol ingestion decreases the levels of the antioxidant glutathione within the alveolar space by as much as 80–90%, and, as a consequence, impairs alveolar epithelial surfactant production and barrier integrity, decreases alveolar macrophage function and renders the lung susceptible to oxidant-mediated injury. These changes are often subclinical and may not manifest as detectable lung impairment until challenged by an acute insult such as sepsis or trauma.

Reduced glutathione (GSH) is a linear tripeptide of L-glutamine, L-cysteine and glycine. Technically N-L-gamma-glutamyl-cysteinyl glycine or L-glutathione, the molecule has a sulfhydryl (SH) group on the cysteinyl portion, which accounts for its strong electron-donating character.

As electrons are lost, the molecule becomes oxidized, and two such molecules become linked (dimerized) by a disulfide bridge to form glutathione disulfide or oxidized glutathione (GSSG). This linkage is reversible upon re-reduction.

Glutathione is under tight homeostatic control both intracellularly and extracellularly. A dynamic balance is maintained between GSH synthesis, recycling it from oxidized GSSG and its utilization. Reduced glutathione is essential for maintaining normal red-cell structure and for keeping hemoglobin in the ferrous state. Red cells with a lowered level of reduced glutathione are more susceptible to hemolysis.

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Intracellular reduction-oxidation status is increasingly recognized as a primary regulator of cellular growth and development. The relative reduction-oxidation state of the cell depends primarily on the precise balance between concentrations of reactive oxygen species and the cysteine-dependent antioxidant thiol buffers glutathione and thioredoxin, which by preferentially reacting with reactive oxygen species, protect other intracellular molecules from oxidative damage.

The transsulfuration pathway constitutes the major route of cysteine biosynthesis, and is central to controlling the intracellular reduction-oxidation state and the balance between self-renewal and differentiation programs.

Glutathione (GSH), the smallest intracellular thiol molecule, has tremendous reducing capacity and serves at a potent universal antioxidant in biological systems. Its levels are tightly regulated and a fine balance exists between GSH synthesis and oxidation. Oxidized glutathione (GSSG) is converted back to GSH by glutathione reductase that utilizes the reducing equivalents from NADPH to convert GSSG to 2GSH.

Mammalian cells exposed to increased oxidative stress exhibit lower GSH/GSSG ratios. GSH deficiencies have been reported in various pulmonary diseases, liver cirrhosis, hepatitis C infection, diabetes and chronic fatigue syndrome. In addition, commonly used drugs, such as acetaminophen can also reduce GSH levels in the liver.

GSH depletion leads to cell death, and has been documented in many degenerative conditions. Mitochondrial GSH depletion may be the ultimate factor determining vulnerability to oxidant attack. The GSH/GSSG ratio is varied in different cell microenvironments, to customize the redox milieu of each area for its specialized functions. The glutathione status of a cell (that is, the excess of reduced over oxidized glutathione) is likely the most accurate single indicator of its health.

For health maintenance, use selenium in a good bioavailable form, in a low dose (such as 200mcg). This tends to provide good anti-inflammatory and anti-cancer benefits due to enhancing cellular pathways of GSSH-GSH metabolism. Avoid long-term higher doses of selenium. High dose selenium induces ROS (the superoxide anion) and incites problems.

Glutathione, cysteine and certain sulfur-containing proteins (methionine, taurine and cystine) form an important "pool" of compounds that are responsible for maintaining the proper oxidation state in the body. These particular sulfur-containing compounds return (reduce) certain oxidized compounds back to their normal forms.

Glutathione itself is an important water-soluble antioxidant and free-radical scavenger. Additionally, glutathione, via enzymic reactions in cells, can reduce oxidized vitamin C back to vitamin C, and vitamin C can reduce oxidized vitamin E back to the stabilized form of vitamin E.

Glutathione synthesis involves two closely linked, enzymatically-controlled reactions that utilize ATP. First, cysteine and glutamate are combined by gamma-glutamyl cysteinyl synthetase. Second, GSH synthetase combines gamma-glutamylcysteine with glycine to generate glutathione.

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As glutathione levels rise, they self-limit further GSH synthesis; otherwise, cysteine availability is usually rate-limiting. Fasting, protein-energy malnutrition, or other dietary amino acid deficiencies, especially of glutamine or glycine, limit glutathione synthesis.

Glutathione recycling is catalyzed by glutathione disulfide reductase, which uses reducing equivalents from NADPH (reduced niacin) to reconvert GSSG to 2GSH. The reducing power (ability to donate hydrogen ions) of ascorbate or vitamin E helps conserve systemic glutathione.

Donated hydrogen ions from ascorbate or reduced glutathione decolorize a blue 2,6 indolchlorophenol dye and thus make the ‘lingual ascorbic acid test’ a useful clinical tool to evaluate (in real time) the staged potential depletion of initially ascorbate, then followed by exhaustion of reduced glutathione.

Glutathione is used as a cofactor by (1) multiple peroxidase enzymes, to detoxify peroxides generated from oxygen radical attack on biological molecules; (2) transhydrogenases, to reduce oxidized centers on DNA, proteins, and other biomolecules; and (3) glutathione S-transferases (GST) to conjugate gluathione with endogenous substances (e.g., estrogens), exogenous electrophiles (e.g., arene oxides, unsaturated carbonyls, organic halides, and diverse xenobiotics. Low GST activity may increase risk for disease. But paradoxically, some glutathione conjugates can also be toxic.

Toxic overload, direct attack by free radicals and other oxidative agents can also deplete glutathione. The homeostatic glutathione redox cycle attempts to keep glutathione repleted as it is being consumed. Unless the diet is totally fresh and raw, amounts available from foods are limited (less that 150 mg/day), and oxidative depletion can easily outpace synthesis.

The liver is the largest glutathione reservoir. Liver parenchymal cells synthesize GSH for conjugation of oxidative cytochrome P450 enzymes as well as numerous other metabolic requirements and then export GSH as a systemic source of SH-reducing power. Glutathione is carried in the bile to the intestinal luminal compartment. Epithelial tissues of the kidney tubules, intestinal lining and lung have substantial oxidative P450 activity along with modest capacity to export glutathione.

Glutathione equivalents circulate in the blood predominantly as cystine, the oxidized and more stable form of cysteine. Cells import cystine from the blood, reconvert it to cysteine (likely using ascorbate as cofactor), and from it synthesize GSH. Conversely, inside the cell, glutathione helps re-reduce oxidized forms of other antioxidants, such as coenzyme Q10, ascorbate and tocopherols.

Glutathione is an extremely important cell protectant. It directly quenches reactive hydroxyl free radicals, other oxygen-centered free radicals and radical centers on DNA and other biomolecules. GSH is an integral oxidant scavenger, reacting as either a one-electron donor to radicals or a two-electron donor to electrophiles. Glutathione is a primary protectant of skin, lens, cornea and retina against radiation damage and other biochemical foundations of P450 detoxification in the liver, kidneys, lungs, intestinal, epithelia and other organs.

Glutathione is the essential cofactor for many enzymes that require thiol-reducing equivalents, and helps keep redox-sensitive active sites on enzymes in the necessary reduced state. Higher-order thiol

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cell systems, the metallothioneins, thioredoxins and other redox regulator proteins are ultimately regulated by glutathione levels (and the GSH/GSSG redox ratio). GSH/GSSG balance is crucial to homeostasis, stabilizing the cellular biomolecular spectrum as well as facilitating cellular performance and survival.

Glutathione and its metabolites also interface with energy production and neurotransmitter syntheses through several important metabolic pathways. Glutathione availability down-regulates the pro-inflammatory potential of leukotrienes and other eicosanoids. Recently discovered S-nitroso metabolites, generated from glutathione and NO (nitric oxide), further diversify glutathione's impact on metabolism.

Almost of half of people living today have limited capacity to get rid of their accumulations from the avalanche of modern environmental toxins due to missing parts of the genetic machinery necessary to produce or recycle glutathione.

Pneumonia is the source of sepsis in 60% of alcoholic subjects and is the most common source of sepsis

in nonalcoholic subjects (35%). The risks of pneumonia and ARDS are tightly linked for most and this connection is even greater in the context of alcohol abuse.

Chronic alcohol insults impair salivary secretion, promote gingivitis and increase colonization of the mouth and pharynx with gram-negative bacteria. Alcoholics with pneumonia are more likely to be infected with either serious gram-negative pathogens such as Klebsiella pneumoniae or to develop bacteremia and shock from even less-aggressive pathogens, like Streptococcus pneumoniae.

Depletion of glutathione is critical in the pathogenesis of alcohol-mediated disease, particularly in the liver and in the lung, but in other target tissues as well. Glutathione is utilized in multiple important pathways including detoxification of peroxides, conjugation to xenobiotics and other toxic molecules to facilitate their excretion and control of oxidant-mediated induction of inflammatory cytokines.

Glutathione depletion precedes the development of the typical histological changes of alcohol-mediated hepatic toxicity. Liver tissue glutathione levels are decreased in chronic alcoholics whether or not there is evidence of cirrhosis.

In animal tests, excessive alcohol administration decreases glutathione synthesis and increases glutathione turnover independently of glutathione oxidation. In addition, alcohol ingestion decreases glutathione transferase and glutathione peroxidase activity.

Acetaldehyde may decrease glutathione levels by binding to cysteine or glutathione itself, although

alcohol-induced suppression of hepatic glutathione levels cannot simply be accounted for by acetaldehyde or alcohol levels alone. The precise mechanisms by which alcohol decreases glutathione levels in the liver are unknown, but, glutathione depletion is the primary underlying causative factor in alcohol-mediated liver disease.

A fundamental aspect of the alcoholic lung is evidence of chronic oxidative stress and depletion of glutathione within the alveolar air space, as well. Dietary supplementation with glutathione precursors

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decreases cytokine- and/or oxidant-induced apoptosis, preserves surfactant synthesis and secretion, restores barrier function, prevents activation of matrix metalloproteinases during endotoxemia, and maintains surfactant composition as well as limits acute lung injury during sepsis.

It is not known why procysteine, but not N-acetylcysteine (which is such an effective treatment for excessive mucus or acetaminophen toxicity created by glutathione exhaustion), restores and/or

maintains both the cytosolic and mitochondrial glutathione pools during chronic alcohol ingestion.

Many studies implicate mitochondrial glutathione depletion as a fundamental feature of alcohol-induced lung dysfunction as well as in alcohol-induced liver injury, providing strong evidence that mitochondrial dysfunction is a common mechanism by which alcohol abuse leads to tissue injury.

Methyl donors (SAMe, folic acid, B6, B12, betaine or trimethylglycine, MSM)

SAM supplementation (S-adenosylmethionine, betaine, B6, B12 and folate) may attenuate acute liver disease (ALD) by decreasing oxidative stress through the up-regulation of glutathione synthesis, reducing inflammation via the down-regulation of TNF- and the up-regulation of IL-10 synthesis, increasing the ratio of SAM to S-adenosylhomocysteine (SAH) and inhibiting the apoptosis of normal hepatocytes while stimulating apoptosis of abnormal liver cancer cells.

Folate deficiency may accelerate or promote ALD by increasing hepatic homocysteine and SAH concentrations; decreasing hepatic SAM and glutathione concentrations and the SAM-SAH ratio; increasing cytochrome P4502E1 activation and lipid peroxidation; up-regulating endoplasmic reticulum stress markers, including sterol regulatory element–binding protein-1, and proapoptotic gene caspase-12 as well as decreasing global DNA methylation.

Betaine may attenuate ALD by increasing the synthesis of SAM and, eventually, glutathione, decreasing the hepatic concentrations of homocysteine and SAH. This increases the SAM-SAH ratio, which can trigger a cascade of events that lead to the activation of phosphatidylethanolamine methyltransferase, increased phosphatidylcholine synthesis and formation of VLDL for the export of triacylglycerol from the liver to the circulation. Additionally, decreased concentrations of homocysteine can down-regulate endoplasmic reticulum stress, which leads to less apoptosis and fatty acid synthesis.

Glutathione is a tripeptide found in all cells in the body, including the bile, the epithelial lining fluid of the lungs, and (at much smaller concentrations) in the blood. Glutathione is the smallest intracellular protein thiol (molecule containing an SH or sulfhydryl group) in the cells. Its water-solubility and minuteness allows its pluripotent antioxidant action and supports a multifaceted thiol exchange system, which regulates cell activity.

Glutathione transferases catalyze the conjugation of glutathione to various electrophilic compounds. Soluble glutathione transferases make up a superfamily of evolutionarily related enzymes. Primarily, glutathione transferases catalyze nucleophilic substitution or addition reactions, but they can also act as peroxidase, isomerase, or just as a binding protein sequestering hydrophobic molecules.

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The ‘natural’ substrates of glutathione transferases range from molecules of foreign origin (e.g., diol epoxides of benzo[a]pyrene), to by-products of cellular metabolism (e.g., phospholipid hydroperoxides). In addition to the electrophilic center, most substrates have hydrophobicity in common.

The two cytosolic enzymes involved in producing glutathione are both ATP-dependent. Therefore, a reduction in ATP energy could have a direct negative effect on the production of glutathione. On the other hand, it takes three glutathiones to make each ATP, the chemical currency of energy (and we make half our body weight in ATP each day).

The mitochondria are exposed to oxygen free radicals produced by the energy making processes, yet cannot make their own GSH for protection; they must expend energy to import it from the surrounding cellular cytosol. Mitochondria do genetically contain their own antioxidant protective enzymes that are inducible (including superoxide dismutase and catalase).

In regenerating rat livers after partial (70%) hepatectomy, after 24 hours both the rate of adenosine triphosphate (ATP) synthesis and the amount of mitochondrial glutathione were depressed by 50% compared to sham-operated animals.

The kinetics of recovery were different, in that the total amount of mitochondrial glutathione was completely restored 48 hours after partial hepatectomy, whereas 72 hours were needed for the recovery of ATP production by oxidative phosphorylation. The decrease in the rate of energy production from oxidative phosphorylation was due to decreased catalytic subunit beta-F1 of the ATP synthase complex (which paralleled diminished imported intramitochondrial glutathione).

The central cysteine is the key to the protection afforded by glutathione. Its sulfur atom scavenges destructive molecules like peroxides and free radicals, converting them to harmless compounds. In the liver, the enzyme glutathione S-transferase takes the sulfur from glutathione and attaches it to toxic molecules, making them more soluble and easier to eliminate. Cysteine by itself would probably serve as well, but free cysteine is quite reactive and would be toxic at this high level.

Sepsis in rats was produced by the method of cecal ligation and needling perforation (CLP). After 15 hours the liver adenosine triphosphate (ATP) level and ketone body ratio decreased significantly. But in rats pretreated with antioxidant methyl palmitate 24 hours prior to CLP, the ATP level returned to normal and ketone body ratio remained at significantly higher values. After CLP, the liver lipoperoxide (LPO) concentration increased and glutathione (GSH) contents decreased significantly.

Both LPO and GSH returned to normal when rats were treated with methyl palmitate before injury. Septic hepatic dysfunction is primarily orchestrated via many toxic mediators (ROS and cytokines) released by activated Kupffer cells (liver macrophages).

Under pathological conditions, primarily when glutathione is depleted, this reversible and physiological role may switch to an irreversible and pathological effect in causing nitrosylation of complex I, inhibition of respiration and cell death.

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This intertwined apoptotic process is reminiscent of our ancient evolutionary history that led to the collaboration between smaller bacterial prokaryocytes and larger eukaryotes, ultimately evolving into this intriguing synergistic obligatory symbiotic relationship that we mammals have with our mitochondria.

Although apoptosis involves altruistic individual cellular death for the development or survival of an organism (or a cooperative community of cells in biofilm), much of the process of cell suicide seems to be a residual expression of some conflict between the needs and demands of the eukaryotic cell on the one hand and of the prokaryotic mitochondria on the other.

Nitric oxide (NO)

NO is regarded as an extremely potent vasodilator, has a role a primary signaling molecule in the early differentiation events of embryonic stem cells, acts to signal adaptive responses of the cardiopulmonary vasculature and enhances endothelial barrier functions. NO has potential application as a topical antimicrobial agent, regulates vascular tone and neurotransmission as well as the regulation of cell death.

NO signaling is deceptively simple. In nearly immeasurable quantities, NO stimulates soluble guanylate cyclase to produce cGMP, which in turn affects intracellular calcium levels as a basic switch to modulate many cellular activities, including the eccentric muscle fiber contraction that creates vasodilation.

What is often lost in describing the chemistry of NO signaling is its extremely diffusive nature compared with other signaling molecules. NO’s moderate half-life also allows it to coordinate and integrate physiological responses within small clusters of cells within tissues, over time.

A crucial aspect of NO signaling is to act as a shock absorber to dampen physiological responses to prevent potentially parasitic oscillations from overwhelming complex homeodynamic control systems. Its diffusive nature also allows NO to act as a retrograde neuromessenger that can transiently affect thousands of synapses within specific areas (of the brain).

Excessive stimulation of this system, such as working in a nitroglycerin factory or by combining a male potency pill with a blood pressure reduction pill may in the short term may cause fainting or priapus, but in the long term can create excessive oxidative stress, especially when an established cryptic biofilm infection continuously triggers the unbridled production of peroxynitrites.

The formation of reactive nitrogen species is not the usual consequence of synthesizing NO. NO is efficiently removed by reacting with circulating oxyhemoglobin to form nitrate, which prevents even the highest rates of NO synthesis from directly reacting with oxygen to form significant amounts of nitrogen dioxide.

However, the simultaneous activation of free-radical superoxide synthesis along with NO will completely transform the biological actions of NO by forming peroxynitrite. Several enzyme complexes, such as NADPH oxidases (NADPHox) and xanthine oxidase (from pasteurized cow’s milk protein), can be activated in many cellular systems to actively produce large amounts of superoxide.

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What happens when superoxide and NO are produced simultaneously in close proximity? Modestly increasing superoxide and NO each at a 10-fold greater rate will increase peroxynitrite formation by 100-fold. Under pro-inflammatory conditions, simultaneous production of superoxide and NO can each be strongly activated to increase production 1,000-fold, which increases peroxynitrite formation by a 1,000,000-fold.

TNF-a inhibits respiratory electron flow and thus increases mitochondrial oxidant generation.

In skeletal muscle of both rodents and humans, a diet high in fat increases the H2O2-emitting potential of mitochondria, shifts the cellular redox environment to a more oxidized state, and decreases the redox-buffering capacity in the absence of any change in mitochondrial respiratory function. Increased mitochondrial H2O2 emission could occur as a result of an increased rate of superoxide production, a decreased rate of H2O2 scavenging in the matrix, or a combination of both.

Transitioning to a high-fat diet initially induces an increase, not a decrease, in mitochondrial biogenesis and fatty acid oxidative capacity in skeletal muscle, presumably as an adaptive response to the elevated lipid load. Deteriorations in mitochondrial structure and function in skeletal muscle of mice appear only after several months of high-fat feeding, well after insulin resistance has developed.

It seems that mitochondrial dysfunction, similar to insulin resistance, is a consequence rather than a primary cause of the altered cellular metabolism that develops with nutritional overload.

Under resting conditions, the rate of electron leak from complex I is extremely sensitive to redox state/membrane potential, such that even a small surplus of reducing equivalents would be predicted to elicit an exponential increase in the rate of superoxide production and H2O2 emission from mitochondria. Attenuating mitochondrial H2O2 emission, either by treating rats with a mitochondrial-targeted antioxidant or by genetically engineering the over expression of catalase in mitochondria of muscle in mice, completely preserves insulin sensitivity despite a high-fat diet.

Hyperglycemia and diabetes mellitus are associated with an exponential increase in superoxide anion (O2

–·) production. Without superoxide, the formation of nitrogen dioxide by the reaction of NO with oxygen is miniscule by comparison. However, NO and superoxide do not even have to be produced within the same cell to form peroxynitrite, because NO can so readily move through membranes and between cells.

Insulin stimulates glucose uptake and flux through metabolism, generating an increase in mitochondrial H2O2 emission that is in turn buffered by GSH. The response to carbohydrate ingestion, however, is likely transient, owing to the rapid clearance of glucose. By contrast, a diet high in fat generates a persistent reduction in GSH/GSSG (evident even after 12-hour fast), suggesting that the clearance and metabolism of dietary lipids elicit a more sustained elevation in H2O2 emission, shifting the cellular redox environment to a more persistent oxidized state.

Superoxide is converted to hydrogen peroxide (H2O2) by superoxide dismutase enzymes (MnSOD, CuZnSOD, and ECSOD). Both synthesis of GSH and efflux of GSSG from cells are acutely influenced by several factors in addition to H2O2, including nitric oxide and fatty acids.

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Several interrelated cellular signaling molecules are involved in the adaptive process of hormesis. Examples include the gases oxygen, carbon monoxide and nitric oxide, the neurotransmitter glutamate,

the calcium ion and TNF. In each case low levels of these signaling molecules are beneficial and protect

against disease, whereas high levels can cause the dysfunction and/or death of cells.

In the much broader context of redox systems biology, redox signaling mechanisms function well below the threshold of “oxidative stress” and are critical to maintaining cellular homeostasis.

The muscle redox environment, as reflected by both the GSH/GSSG ratio and GSH t, is remarkably sensitive to nutritional intake, shifting to a more oxidized state in response to both acute (e.g., glucose ingestion) and chronic (e.g., high-fat diet) food ingestion. Cytosolic GSH is the primary redox buffer for H2O2 emitted from mitochondria.

Unsaturated (n-3) PUFAs increase oxidative stress. In combination with the rapid colonocyte oxidation of butyrate, these protective fatty acids increase cellular reactive oxygen species (ROS) in a manner sufficient to induce apoptosis in a rat model of experimentally induced colon cancer.

Yet ROS can also damage and potentially mutate DNA; therefore, cells employ several defenses against

ROS including upregulating antioxidant enzymes such as superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT).

Although these enzymes are key players in preventing cellular damage caused by endogenous ROS, over expression or addition of these enzymes to tissue systems might enhance tumor formation and block the action of several chemotherapeutic drugs by suppressing ROS-induced apoptosis.

Blocking the inflammatory NF-κB pathway (either pharmacologically or genetically) protects against induced insulin resistance created by the high-fat diet. One of the things that regulates NF-kB (the pro-inflammatory transcription factor that causes the expression of iNOS and other downstream mediators) is NO. NO inhibits NF-kB and reduces the expression of iNOS and by feedback reduces the level of NO that is produced by that iNOS. PPPR-g agonists also have effects mediated through NO and NO has similar effects all by its self.

N-acetyl-L-cysteine (NAC), a sulfhydryl containing thiol antioxidant, is used in the treatment of acetaminophen-induced liver toxicity, immune modulation in HIV patients and cardiovascular diseases, for cancer prevention and as chelating agent in acute heavy metal poisoning. NAC inhibits activation of redox-sensitive transcription factors such as NF- B.

Nitric oxide is synthesized in three ways. Neuronal nitric oxide synthesis occurs, as the name implies, by isoforms in the nervous system. Inducible NO or Type II is released by macrophage activity and type III endothelial NO synthesis takes place in specialized endothelial cells.

iNOS is not post transcriptionally regulated the way that eNOS and nNOS are regulated. It is pretty much open-loop control, the cells make a certain amount of iNOS and they then make NO levels commensurate with that iNOS level until the iNOS is degraded. If you make too much iNOS, you are upregulated until it is degraded.

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Low basal NO turns up the “gain” on the immune system by disinhibiting NF-kB so more iNOS and more cytokines are produced. Normally, this is for local control, where specific tissue compartments have a lower NO level.

A local infection lowers NO level, likely due to proteolytic cleavage of xanthine oxidoreductase, from increased metabolic activity and more superoxide, due to superoxide and inflammation from other immune cells. The respiratory burst triggered by immune cell stimulation causes low NO and ratchets the immune system into the “on” state. Low NO also potentiates mast cell degranulation, unleashing allergic response.

Normal systemic NO from moderate continuous immune system stimulation, as from parasitic worms, may be one of the things that normally modulates the immune system and reduces systemic inflammation and also things like inflammatory bowel disease. Therapeutic parasitic worms are useful immune modulators for IBD and Crohn’s.

The ‘hygiene hypothesis’ theorizes that immune system imbalance from being ‘too clean’ and from not having occasional parasitic worm infestations is important in development of allergies and asthma.

Allergies can be thought of simplistically as coming from too high a “gain” on the immune system. All important systems in physiology have automatic gain control. When it isn’t doing anything, it turns up the “gain” until it is doing something. In the absence of an infection, that may be the source of the chronic inflammation that is observed in metabolic syndrome. Basal NO level is an important part of automatic gain control.

Turning up the “gain” on the immune system might be expected to help resolve minor infections, but would tend to exacerbate chronic inflammatory disorders, maybe create more cancer and cause more (and more severe) cytokine storm-type reactions.

Time constants of the various immune system sub-routines are very important. Getting them slightly out of sync or out of phase could cause big problems. Time constants are necessarily coupled to fluid flow and turnover in the extravascular space, which is not as consistent over the whole body as is blood flow.

Perhaps by increasing NO/NOx levels before contracting the flu, it will then turn down the gain and turn any cytokine storm into a mild but effective drizzle.

Arginine is the substrate for nitric oxide synthase, but NO production from NOS is controlled independently of the arginine supply. Within broad limits, arginine does not work to raise the basal NO level long term. Basal NO level is too important for physiology to allow it to be regulated by dietary levels of arginine.

The production of nitric oxide is elevated in populations living at high-altitudes, which helps these people avoid hypoxia by aiding in dilation of the vasculature.

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Nitric oxide is absorbed systemically via the pulmonary capillary bed. When the pulmonary capillaries are adequately oxygenated, NO combines with saturated hemoglobin to form methemoglobin (a hematogenous pigment formed from hemoglobin by oxidation of the iron atom from the ferrous to the ferric state) and nitrite. NO and nitrite are the predominant byproducts that enter the systemic circulation. Excess nitrate is eliminated through normal urine output.

When applied externally, urine or urea has a very beneficial effect on wound healing, including infected wounds, burns and ulcerating tumors. Urea crystals may be directly packed into a wound or a strong or saturated solution may be applied. In this way, urea removes the foul odor often associated with an ulcerating tumor or other dead or putrefying tissue.

One can shower less and encourage a biofilm of ammonia oxidizing bacteria on the skin. Perhaps this is the natural way to regulate basal NO levels, the way that our bodies evolved to do so. Sweating during a fever is a way to supplement NO/NOx levels by turning the ammonia in sweat into NO and nitrite which is rapidly absorbed.

The most important time to have a high NO level is before the infection happens. That increases basal mitochondria number, which increases the capacity to tolerate mitochondria destruction that occurs during the cytokine storm.

Nitric oxide (NO), a widespread physiological messenger that acts as endothelial relaxation factor may also act as an endogenous mitochondrial inhibitor, which may be crucial in septic shock, which is likely to be partly a mitochondrial disease.

Mitochondrial Ca2+ uptake serves to modulate the rate of [Ca2+] wave propagation in astrocytes. Glutamate neurotoxicity may trigger the collapse of mitochondrial potential and cell death through the coincidence of mitochondrial calcium accumulation and NO production. One major result of the mitochondrial Ca2+ uptake pathway is the up-regulation of the TCA cycle activity by a high intramitochondrial [Ca2+].

Mitochondrial metabolism is central to the process involved in transduction of a rise in [glucose] to initiate insulin secretion. The potentiation of insulin secretion follows the up-regulation of the TCA cycle, which generates an increase in intracellular glutamate following a rise in intramitochondrial Ca2+. The glutamate appears then to act as an intracellular signal that primes the exocytotic secretory machinery, further increasing insulin secretion.

In early biochemical change in the Parkinsonian substantia nigra (SN) is reduction in total glutathione (GSH + GSSG) levels in affected dopaminergic neurons prior to depletion in mitochondrial complex I activity, dopamine loss and cell death. Total glutathione depletion in these cells results in selective complex I inhibition via a reversible thiol oxidation event. Selenium (cofactor for most active form of glutathione) deficiency impairs the ability of mouse neutrophils to kill C. albicans.

Inhibition of complex I may occur either by direct nitric oxide (NO) but not peroxinitrite-mediated inhibition of complex I or through H2O2-mediated inhibition of the tricarboxylic acid (TCA) cycle enzyme

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alpha-ketoglutarate dehydrogenase (KGDH) which supplies NADH as substrate to the complex. Activity of both enzymes is reduced in Parkinson’s disease.

While glutathione depletion causes a reduction in spare KGDH enzymatic capacity, it produces a complete collapse of complex I reserves and significant effects on mitochondrial function. NO is likely the primary agent involved in preferential complex I inhibition following acute glutathione depletion in dopaminergic cells.

Brain ischemia/reperfusion (I/R) injury is a complex, multistage process. It is initiated by the immediate damage caused by hypoxia as well as by the return of oxygenated blood, which then injures the glutathione-depleted arterioles. So-called secondary damage involves neuronal injury from excitatory amino acids glutamate and aspartate, intracellular Ca2+ accumulation, free radical generation and apoptosis as well as microvascular processes.

Nitric oxide can contribute to reperfusion injury when an excessive amount produced during reperfusion (following a period of ischemia) reacts with superoxide to prolifically produce the damaging oxidant peroxynitrite.

There is accumulating evidence that inflammatory-immunologic reactions are also involved in the pathogenesis of cerebral ischemia. Inflammatory cells such as neutrophils and macrophages infiltrate the ischemic brain in various models of ischemic stroke. In addition, dendritic cells resident in brain, such as astrocytes, microglia and endothelia, have been found to be activated by cerebral I/R.

These cells then become immunologically reactive and interact with each other by producing inflammatory mediators, including eicosanoids, reactive oxygen species (ROS), cytokines and adhesion molecules. These molecules appear responsible for the accumulation of inflammatory cells in the injured brain, and the resulting oxidative cascade may negatively affect the survival of neurons subjected to ischemic injury.

ROS produced by the immune system are well-recognized toxic metabolites that can directly cause damage to brain cells. It has become apparent that ROS have a much broader role in the regulation of the immune response itself.

For example, lower levels of ROS genetically up-regulate production of antioxidant and detoxification systems, reducing inflammation, promoting fitness and survival. High levels of ROS serve as endogenous signals of alarm, whether environmentally introduced or released by injured tissue, that trigger

aggressive activation of the inflammatory response.

ROS are important regulators of many intracellular signaling pathways, leading to the release of inflammatory mediators as well as those that represent reaction of different cell types to the mediators. The signaling pathways involve G proteins, protein tyrosine kinases, protein tyrosine phosphatases, Janus kinases, mitogen-activated kinases, caspases and a variety of transcription factors.

Increased level of glutathione peroxidase activity in transgenic mice modulates inflammatory response in focal brain I/R model of stroke and decreases the sensitivity of brain cells to induced cell death.

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Intracellular reduction-oxidation status is increasingly recognized as a primary regulator of cellular growth and development. The relative reduction-oxidation state of the cell depends primarily on the precise balance between concentrations of reactive oxygen species and the cysteine-dependent antioxidant thiol buffers glutathione and thioredoxin, which by preferentially reacting with reactive oxygen species, protect other intracellular molecules from oxidative damage.

The transsulfuration pathway constitutes the major route of cysteine biosynthesis, and may thus be central in controlling the intracellular reduction-oxidation state and the balance between self-renewal and differentiation programs.

S-adenosylmethionine (SAMe)

All organisms synthesize S-adenosylmethionine (SAMe). A large fraction of all genes is SAMe dependent methyltransferases. SAMe-dependent methylation has been shown to be central to many biological processes, including recycling glutathione. Methionine adenosyltransferase (MAT, the enzyme that makes SAMe), MAT1A is specifically expressed in adult liver.

It now appears that growth factors, cytokines and hormones regulate liver MAT mRNA levels and enzyme activity. SAMe should not be seen only as an intermediate metabolite in methionine catabolism, but also as an intracellular control switch that regulates essential hepatic functions such as regeneration, differentiation and the sensitivity to injury.

Recommended doses of SAMe vary depending on the health challenge. Depression: 800 - 1,600 mg of SAMe per day, in two divided doses (morning and afternoon). Osteoarthritis: 600 - 1,200 mg per day in two to three divided doses. Fibromyalgia: A dosage of 400 mg two times per day for 6 weeks. Alcoholic liver disease: 600 - 1,200 mg per day by mouth in divided doses for 6 months enhances liver function.

Side effects may include dry mouth, nausea, gas, diarrhea, headache, anxiety, a feeling of elation, restlessness and insomnia. For this reason, do not take SAMe at night. Large doses of SAMe may cause mania (abnormally elevated mood). Start at a low dose and gradually increase it; do not exceed recommended doses.

It’s the liver and pancreas.

The body’s coagulation system is part of its defense against pathogens and trauma. When an injury occurs, the acute phase proteins go into warp drive by increasing their production to help the body heal.

A partial list of acute phase proteins includes: Fibrinogen (which can increase up to four times normal), Factors V & VIII (provide for Thrombin generation), von Willebrand Factor (prevents blood loss), C-Reactive Protein (responds to infections: TNF-alpha, IL6, IL8, IL1, others), Complement C4(b) Binding Protein (Protein S carrier protein which decreases Protein S availability resulting in hypercoagulability), Lp(a) and PAI-1 (decreases fibrinolysis), alpha-2-AntiPlasmin (protects fibrin from being degraded), Complement C3 and C4, and adhesion molecules ICAM-1 VCAM-1.

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These proteins are part of the “fight or flight” process. If we choose to fight, the body is prepared for the potential wounds of warfare. These preparations are to limit blood loss as much as to wall off a pathogen invasion.

In chronic illness, fibrin formation and deposition can coat the endothelial cells (EC) of the microvasculature (capillaries). This inhibits the natural fibrinolytic activation of plasminogen to plasmin, due to the ‘Teflon®-like’ deposits on the EC surfaces, encouraging biofilm formation. Microorganisms can continually upregulate immune response leading to cytokine storm, but are protected from it by survival strategies of the biofilm. Native fibrinolytics are produced by the pancreas.

When the pancreas is exhausted or inflamed, fibrinolytic activity is compromised. The only way to clean the linings of the tubes and ducts of the body is to use external fibrinolytic activators and sometimes even metal chelators.

Effective fibrinolytic activators include: serapeptidases, endozyme, nattokinase and lumbrokinase. Digestive enzymes like bromelain, Wobenzym®or pancreatic enzymes provide lesser amounts of fibrinolytic activity, but not on a scale which can remove a lot of fibrin and digest biofilm unless taken by the handful.

Early acinar glutathione depletion of the pancreas, associated with diminished ATP, likely plays a role in the premature alkaline activation of digestive enzymes by impairment of the integrity of the cytoskeleton and cell organelles or lowered defense capabilities against oxidant stress, leading to chronic or acute pancreatitis. Acute pancreatitis is often complicated by multi-organ dysfunction, which is thought to occur in part by macrophage infiltration into the pancreas.

Mononuclear cells throughout the body are induced to produce large amounts of cytokines during acute pancreatitis. Macrophages can be induced by specific activated pancreatic enzymes (elastase, carboxypeptidase A and lipase) to produce TNF-α. This process is dependent on IKB-13 degradation and NF-kB activation, suggesting that these enzymes trigger this second messenger system through specific membrane-bound receptors.

Activation of protease-activated receptor PAR-2 with trypsin, which can be released after pancreatitis induction, positively regulates the transcript level of MIF and increased MIF results in exaggerated pulmonary expression of toll-like receptor TLR-4, leading to the development of acute lung injury with a subsequent infectious attack.

Resultant diminished pancreatic production of amylases then disables our primary enzymatic defense against the proteoglycan structure of opportunistic biofilm organisms, encouraging them to proliferate. Also lacking glutathione and energy, phagocytic immune cells become sluggish, refuse to ingest invaders and stop patrolling barrier tissues, again heartening biofilm growth.

Glutathione sources

Sulfur-rich foods support formation of glutathione. The main ones in the diet are runny egg yolks, garlic, onions and cruciferous vegetables (broccoli, kale, collards, cabbage, cauliflower, watercress).

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Bioactive whey protein is great source of cysteine and the amino acid building blocks for glutathione synthesis. Most dairy is factory-farmed and harmfully altered by pasteurization and homogenization as well as fractionation. Look for raw or fermented sustainably farmed milk products that contain whey.

For whey protein to most effectively raise glutathione, it is made and individually packaged from cold-processed non-denatured proteins (denatured means deformed protein structure). Choose raw non-pasteurized and sustainably produced milk sources that contain no pesticides, hormones or antibiotics. Immunocal is a prescription version of bioactive non-denatured whey protein that is even listed in the Physician's Desk Reference.

Exercise boosts glutathione levels by creating mild oxidative stress, which triggers a change in gene response, that upregulates production of glutathione and other antioxidant systems. Mild stress in many forms hormetically helps boost immunity, improve detoxification and enhance inherent antioxidant defense systems.

Start slowly and build up to 30 minutes a day of vigorous aerobic exercise like walking or jogging, or play various sports. One should be breathing heavily, but still be able to talk. Strength training for 20 minutes 3 times a week is also helpful. A good measure of optimal hormetic efficacy is an exercise experience that could be repeated again without undue strain. Sunshine provides everything positive for the body that exercise does, just to a smaller degree.

It would seem to be easy just to take glutathione as a pill. But the body mostly digests this simple tripeptide protein into its amino acid precursors, and you receive primarily topical anti-inflammatory benefits in the gut, with only mild systemic effect, since the production and recycling of glutathione in the body is under tight homeostatic enzymatic control and requires many different cofactors.

N-acetyl-cysteine (NAC) has been used for years to help treat asthma and lung disease and to treat people with life-threatening liver failure from Tylenol overdose. It is also given to prevent kidney damage from dyes used during x-ray studies.

Alpha lipoic acid independently recycles glutathione and is a close second to glutathione in importance in our cells and is involved in energy production, blood sugar control, brain health and detoxification. The body usually makes it, but given all the stresses we are under, we often become depleted and it becomes conditionally essential.

NADPH (reduced niacin) is pivotal for defense against ROS and maintenance of cellular redox homeostasis. NADPH is required for maintenance of the thioredoxin and glutathione redox systems.

Methylation nutrients (folate, vitamins B6, B12 and betaine) are perhaps the most critical to keep the body producing glutathione. Methylation (creating the most basic building block in the body) and the production and recycling of glutathione are the two most important biochemical functions in the body.

Supplement with folate (especially in the active methyl form), B6 (in active form of P5P) and B12 (in the active form of methylcobalamin). Adding trimethyl glycine (betaine hydrochloride) enhances stomach

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acidity, thus increasing absorption of proteins, minerals and the other methylating B vitamins necessary for recycling glutathione as well as boosting glycine, one of the three amino acids that comprise it.

Selenium and zinc contain viral expression and boost glutathione activity

Cells have many enzymatic and nonenzymatic mechanisms to remove or detoxify ROS to prevent oxidative stress. Nonenzymatic defenses include small molecules such as vitamins C and E, the tripeptide glutathione and the dithiol-containing thioredoxins.

Enzymatic mechanisms include zinc, copper and manganese-dependant superoxide dismutases (SODs) that convert superoxide anion to hydrogen peroxide and oxygen, whereas the selenium-dependant glutathione dependent peroxidases and catalase convert hydrogen peroxide to water and oxygen.

Selenium is an essential component or cofactor of enzymes throughout metabolism, such as glutathione peroxidase (GPx), thioredoxine reductase and iodine deiodinase. GPx acts against hydrogen peroxide and lipid peroxidation and is an important line of defense against free radicals; thioredoxine reductase is involved in nucleus redox status; and iodine deiodinase is involved in thyroid hormone metabolism, which is frequently impaired in critically ill patients.

Selenium also has an anticarcinogenic effect likely induced by the production of methyselenol, a selenium metabolite that affects gene expression and modifies cell cycling and immune function.

Selenium deficiency enhances the virulence or progression of some viral infections. The increased oxidative stress resulting from selenium deficiency induces mutations or changes in expression of some viral genes.

When selenium-deficient mice are inoculated with a relatively harmless strain of coxsackievirus, mutations occur in the viral genome that results in a more virulent form of the virus, which causes an inflammation of the heart muscle (myocarditis). Once mutated, this form of the virus also causes myocarditis in mice that are not selenium deficient, demonstrating that the increased virulence is due to a change in the virus rather than the effects of selenium deficiency on the host immune system.

Selenium deficiency results in decreased activity of glutathione peroxidase, increasing oxidative damage as well as the likelihood of mutations in the viral genome. Coxsackievirus has been isolated from the blood of some sufferers of Keshan disease, suggesting that it may be a cofactor in the development of the cardiomyopathy associated with this selenium deficiency seen in humans.

Common coxsackievirus mutates into the deadly, rapidly reproducing strain when an infected person or animal is deficient in selenium or vitamin E. The coxsackievirus in animals eating a selenium-rich diet does not mutate. However, the mutated virus might infect and be deadly to a person or animal eating adequate selenium.

Genes in HIV control the formation of selenocysteines, proteins with a voracious appetite for selenium. The virus makes glutathione like us, to enhance its survival. When the virus depletes all of the selenium in an HIV-infected cell, it reproduces and begins attacking other cells in search of more selenium. The more selenium that is stolen by the virus, the less remains available for the body's immune system.

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Eventually, immunity becomes so compromised that AIDS patients become vulnerable to life-threatening "opportunistic" infections.

Folks with a functional selenium deficit have suboptimal immune status and a deficit in viral handling. Selenium supplementation (100 µg Se per day) increases plasma selenium concentrations, the body exchangeable selenium pool (measured by using 74Se) and lymphocyte phospholipid as well as cytosolic glutathione peroxidase activities.

Selenium supplements augmented the cellular immune response through an increased production of interferon and other cytokines, an earlier peak T cell proliferation and an increase in T helper cells. Humoral immune responses are unaffected.

Selenium-supplemented subjects show more rapid clearance of poliovirus. Poliovirus reverse

transcriptase–polymerase chain reaction products recovered from the feces of supplemented subjects also contained fewer mutations.

Sulfur dioxide, a byproduct of the burning of fossil fuels, reacts with selenium compounds in the soil, making the mineral more difficult to absorb by plants. Fossil fuel burning that creates acid rain likely contributes to a gradual decrease of selenium in the food chain.

Supplemental selenium would do two favorable things. First, it would provide what the HIV virus needs so it wouldn't spread throughout the body, creating a biochemical stalemate of sorts. Second, it would help keep the person's overall immune system functioning, so it could resist the secondary infections that usually kill HIV patients.

In human studies, zinc deficiency results in decreased production of interleukin 2 (IL-2) and gamma interferon (cytokines associated with Th1 cells), whereas the production of IL-4, -6, and -10 (cytokines associated with Th2 cells) is not affected.

Topical application of zinc sulfate has also been found effective in the treatment of HSV infection in both HSV types 1 and 2. Zinc acts in several ways to inhibit viruses. Zinc may block the binding of rhinovirus virions to the cell surface. Zinc may block the protease activity in rhinovirus, thereby preventing the breakdown of the virus polypeptide necessary to generate individual functional proteins.

Zinc compounds owe their anti-HIV-1 effects to inhibition of HIV-1 DNA-to-RNA transcription, rather than inhibition of the adsorption or penetration, the way zinc salts inhibit respiratory syncytial virus (RSV) plaque formation, major cause of pediatric lower respiratory tract disease.

Manganese superoxide dismutase (MnSOD) is the principal antioxidant in mitochondria. A deficiency in manganese causes skeletal deformation in animals and inhibits the production of collagen in wound healing (an expression of scurvy with blood and fluid leaking from the capillaries due to a cytokine storm).

Manganese is a popular remedy for strains, sprains and inflammation due to its ability to increase the level or activity of SOD thus increasing antioxidant activity. Those with rheumatoid arthritis or other inflammatory conditions have an increased need for manganese.

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MnSOD plays a role in the alteration of immune function seen upon infection of B-cells with measles virus. Intracellular MnSOD inhibits proliferation of B-cells and also decreases the titer of virus produced from infected cells.

Vaccinia virus is the most studied member of the poxvirus family, a group of large DNA viruses that replicate in the cell cytoplasm. Within the Poxviridae, proteins related to SOD are present in the leporipoxviruses myxoma virus and Shope fibroma virus, the molluscipoxvirus molluscum contagiosum virus and the entomopoxvirus B Amsacta moorei virus. It is likely that Escherichia coli or poxviruses use their nonfunctional SOD-like protein to inhibit or regulate cellular SOD function or for some other purpose.

Molecular mimicry may lead to antibodies against our own SOD, disabling defenses. Another example is the Epstein-Barr virus, which induces autoantibodies against cellular MnSOD, thus contributing to viral pathogenesis. Or, the regulation of cellular SOD in poxvirus-infected cells might disrupt the balance of oxidants and antioxidants. Further, since oxidative stress can induce necrosis or apoptosis, this may aid virus dissemination.

Japanese encephalitis virus (JEV) infection induces the generation of superoxide anion and nitric oxide in rat cortical glial cells. Manganese superoxide dismutase, but not copper/zinc superoxide dismutase is activated by JEV infection.

Additionally, an increase in the cellular oxidant status results in activation of transcriptional factors, such as NF- B, that may be necessary for replication of some viruses. Biofilm generates inflammatory exudates as a favorite food. In addition, the Tat protein from human immunodeficiency virus type 1 induces the activation of NF- B, which is important for virus replication, by down-regulation of cellular MnSOD.

Because mitochondria are the main source for free-radical production, the MnSOD is the most important antioxidant defense enzyme. The expression of mitochondrial manganese superoxide dismutase (MnSOD) in human glial cells is significantly increased in acute infection. With persistent viral and nutritional stress, the expression of MnSOD eventually becomes drastically down regulated.

This down regulation of MnSOD expression in the chronic stress model is likely due to repression of antioxidant defense. The down regulation of MnSOD expression leads to an increase of free-radical

production and explains why cells in the chronic stress model are more vulnerable to other oxidative stress influences.

Reactive oxygen and nitrogen species such as superoxide and nitric oxide are released into the extracellular spaces by inflammatory and airway epithelial cells. These molecules likely exacerbate lung

injury after influenza virus pneumonia. Enhancing extracellular superoxide dismutase in the conducting and distal airways of the mouse lung minimizes influenza-induced lung injury by both ameliorating inflammation and attenuating oxidative stress.

Glisodin® increases the body’s production of SOD, Glutathione peroxidase and Catalase; it has the capability of stimulating the body’s defense against free radicals and positively influences the immune system. A proprietary wheat protein matrix protects the SOD activity from destruction by gastric acidity allowing it to be assimilated by the intestinal cells.

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Antioxidants protect the body from the damaging effects of free radicals. There are two categories of antioxidants: antioxidant enzyme systems and nonenzymatic dietary antioxidants.

Superoxide dismutase (SOD) is a zinc-dependent enzyme system able to convert superoxide radicals into the less toxic hydrogen peroxide. SOD requires mineral cofactors to function: zinc and either manganese or copper. Lack of any of these minerals allows an excess of super oxide, exponentially increasing production of peroxynitrites from NO, unleashing the cytokine storm. Lack of zinc to produce cytokine quenching finger proteins further extends the storm’s fury.

Other enzymes (catalase and glutathione peroxidase) then convert the hydrogen peroxide into oxygen and water. Catalase is a heme-requiring enzyme; glutathione peroxidase is the pivotal selenium-containing enzyme that requires glutamine, cysteine and glycine.

The ability of these enzymes to work depends on the availability of these potentially toxic minerals (if in excess) needed as cofactors: manganese, selenium, zinc, copper and iron.

The body will bind and sequester these metals to protein carriers, liketransferrin and ceruloplasmin, to make them unavailable for conversion to ROS. (Iron and copper have a dual role in the production and destruction of ROS. While both are necessary for protective enzymes, they can, when unbound and in the presence of hydrogen peroxide, catalyze the production of the toxic hydroxyl radical.)

Nonenzymatic antioxidants are the other line of defense against ROS and include vitamins E and C, carotenes, glutathione, uric acid, taurine and phytochemicals. All these antioxidants are found in food; they work by intercepting and stabilizing the ROS. This is known as free-radical scavenging.

The vitamin E group is the most important antioxidant within lipid membranes, preventing cell membrane damage by scavenging the peroxyl radical.

Vitamin C (ascorbate) is the most abundant water-soluble antioxidant with a vast array of ROS it can protect against. In addition, it prevents the conversion of nitrites to the carcinogenic nitrosamines and can regenerate vitamin E moieties once it gets used up scavenging free radicals.

Carotenes, especially beta-carotene, are potent scavengers of peroxyl and hydroxyl radicals. They can also protect lipid membranes.

Glutathione is a pivotal antioxidant, scavenging free radicals, removing hydrogen and lipid peroxides, and preventing the oxidation of various substances in the body. Many other functions of glutathione involve nutrient metabolism, the regulation of gene expression, DNA and protein synthesis, cell proliferation, cytokine production and immune response.

A protein deficiency will cause a glutathione deficiency, as it is synthesized from the amino acids cysteine, glutamate and glycine, with cysteine usually the rate-limiting amino acid. Taurine, cystine and methionine can be used as a precursor to cysteine, while glutamine is effective as a precursor for glutamate and tryptophan for glycine.

When the heart is stressed, the protective effect of glutamine is related to a strengthening of the myocardial membrane by its membrane stabilizing action. It also counters free radicals by its

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antioxidant property as well as its ability to maintain the activities of free radical scavenging enzymes and the level of GSH (which protects myocardial membranes against oxidative damage by decreasing lipid peroxidation).

In people with HIV/AIDS, decreased levels of antioxidants and increased oxidative stress have been documented. As oxidative stress increases, so does viral replication, which increases the destruction of CD4+ T cells and encourages the disease’s progression.

A group of antioxidants including lipoic acid, vitamins C and E (balanced in its eight natural forms) work together to recycle reduced glutathione. Conventional antioxidant supplements (such as vitamins A, C and E) are known as “consumable” antioxidants because they are used up as they neutralize free radicals on a one-to-one basis.

Dietary plant polyphenols (flavonoids) increase expression of an important enzyme in both cellular antioxidant defenses and detoxification of poisons, gamma-glutamylcysteine synthetase, rate limiting in the synthesis of the most important onboard antioxidant in cells, glutathione.

The blend of herbal phytonutrients in Protandim signal the genes to produce 300% more glutathione as well as increasing other special antioxidant enzyme systems, superoxide dismutase (SOD) and catalase (CAT) that work synergistically together with glutathione as the body’s first line of defense against free radicals. Protandim is a mix of:

Milk thistle extract (Silybum marianum) (seed) Bacopa extract (Bacopa monnieri) (aerial part) Ashwagandha (Withania somnifera) (root) Green tea extract (Camellia sinensis) (leaf) Turmeric extract (Curcuma longa) (rhizome)

These antioxidant enzyme systems are “catalytic”, which means that SOD and CAT are not used up when they neutralize free radicals, they inherently recycle. A single daily caplet of Protandim creates a cascade of the body’s natural catalytic antioxidants that are able to trap millions of free radicals per second, on a continuous basis.

The small, free radical molecule nitric oxide (NO; N=O) is a major signal transduction molecule in vertebrates (animals). There are three forms of nitric oxide synthase - a neuronal type called nNOS, an epithelial type called eNOS and an inducible form called iNOS. In endothelial or epithelial cells, NO causes vascular dilation by controlling smooth muscle contractility. Nitric oxide is converted into cGMP, which becomes the secondary messenger that causes smooth muscle relaxation, resulting in blood vessel dilation.

In the central nervous system it affects synaptic transmission stimulating learning and memory capacity. In blood plasma, NO induces platelet aggregation, an important factor in wound healing and blood coagulation. Hemoglobin is a major transport vehicle for NO in blood.

Inducible NOS is only expressed under certain conditions like immune system regulation by cytokines or pathological induction in the presence of endotoxins (bacterial lipopolysaccharide) and cytotoxins (which affect cytokine secretion). NO production is a stress response and can lead to either tissue injury

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because of its radical chemistry, or be cytoprotective, protecting cells from damage by destroying pathogenic microorganisms first.

Nitric oxide and particularly its superoxide derivative peroxynitrite cause DNA damage in the bacteria. Peroxynitite (ONOO) induces DNA damage through chemical modifications (mutations) while NO inhibits ribonucleotide reductase. Both DNA damage and reductase inhibition keep the microorganism in a state of energy costly nucleotide synthesis and repair mode. This leads eventually to cell death by energy depletion of bacterial cells.

Inhibition of catalase activity by cytokines is nitric oxide dependent. This inhibition may confer increased susceptibility to cytokine- or nitric oxide-induced cell killing. Such defense mechanisms, however, have their draw backs. Inducible NOS, which is expressed as an emergency mechanism to suppress tumor growth in gastric epithelia, breast tissue and the brain, is linked to septic shock.

Bacterial endotoxins (e.g. from H.pylori or E.coli infections) induce the iNOS gene, which in turn produces high levels of NO damaging pathogenic DNA and inhibiting respiration (inhibits metabolic energy production needed for cell division). Produced in response to bacterial infections, free radicals do not discriminate between pathogenic DNA from host DNA.

Host DNA is designed to have more antioxidant shields (like glutathione), but they may be exhausted or overwhelmed. Overstimulation of iNOS induces cell and tissue damage, contributing to autoimmune diseases like atherosclerosis, osteoarthritis, rheumatoid arthritis and asthma as well as when unbuffered, even the development of fatal septic shock.

Peroxynitrite, hydrogen peroxide and dinitrotrioxide all have been linked to cell suicide or programmed cell death (apoptosis) through protein nitration and increased mutagenesis. Mutations are a consequence of DNA stand breakage and guanine nitration.

For example, acute neural toxicity is linked to the overproduction of peroxynitrite, which inhibits respiratory enzymes and also damages DNA by covalent bond formation to DNA and removal of bases. Inhibitors of nitric oxide synthase and antioxidants are known to have neuroprotective properties because the limit the formation of highly reactive nitrogen containing radicals.

The free radical chemistry in cells can be prevented or at least diminished by adding antioxidants or free radical scavengers, molecules which have a high affinity and strongly react with these free radicals. Antioxidants tend to be either hydrophilic or hydrophobic.

Hydrophilic antioxidants include glutathione peroxidase, Fe(II) chelators like the proteins ceruloplasmin and transferrin, and hydroxylated aromatic molecules like uric acid or ascorbate (vitamin C). Hydrophobic (fat-soluble) antioxidants include flavin-nucleotide or carotene containing proteins and vitamin Es. Lipoic acid is ampiphillic, being soluble in both water and fat.

Melatonin too is a major physiological antioxidant (and hormone) by directly reacting with hydroxyl and peroxyl radicals, or by stimulating the expression of superoxide dismutase, glutathione peroxidase or glutathione reductase. Melatonin also inhibits nitric oxide synthetase. Melatonin inhibits the production of adhesion molecules that promote the sticking of leukocytes to endothelial cells, attenuating transendothelial cell migration and edema.

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Glutathione is a cofactor for a number of enzymes, and its presence is essential for maximal enzyme activity by the inducible macrophage nitric oxide synthase (iNOS), which produces the reactive nitric oxide radical. Hepatocytes contain substantial quantities of glutathione, and this important tripeptide is decreased in liver cells stressed by ischemia/reperfusion or endotoxemia.

Endotoxemia also induces the synthesis of inflammatory cytokines that result in the production of nitric oxide from hepatocytes by iNOS, suggesting that liver cells may be attempting to synthesize nitric oxide at times when intracellular glutathione is reduced.

Nitric oxide reacts with intracellular glutathione and activates the hexose monophosphate shunt in human neutrophils, giving evidence for S-nitrosoglutathione as a bioactive intermediary. At levels sufficient to inhibit chemoattractant-induced superoxide anion production, nitric oxide causes a depletion of measurable intracellular glutathione.

Depletion of intracellular glutathione is accompanied by a rapid and concurrent activation of the hexose monophosphate shunt (HMPS) following exposure to nitric oxide. Kinetic studies showed that nitric oxide-dependent activation of the HMPS was reversible and paralleled nitric oxide-induced glutathione depletion.

The increase in eNOS in response to red wine involves several polyphenolic compounds with a major contribution from trans-resveratrol and lesser contributions from cinnamic and hydroxycinnamic acids, cyanidin, and some phenolic acids. NO and NO donors increase cellular GSH in cells. The mechanism of this effect is unclear but likely involves genetic upregulation of the normal GSH synthetic pathways as a hormetic response to mild stress.

Arginine is a conditionally essential amino acid. Arginine is key in nitric oxide (NO) production, which dilates bloods vessels and improves blood flow. This may benefit heart health and circulation in general. High doses of arginine may also stimulate immunity as well as the secretion of growth hormone.

Arginine, along with glycine and methionine, is also used to produce creatine. In addition, arginine may promote wound repair, may help decrease the severity or incidence of migraine headaches and may help improve some types of erectile dysfunction.

However, NOS can be converted to a major ROS generator, as seen in vascular endothelium exposed to increased oxidant or hemodynamic stress. Active NOS3 is a homodimer that generates NO and L-citrulline from L-arginine. When exposed to oxidant stress, including peroxynitrite (ONOO–), or deprived of its reducing cofactor tetrahydrobiopterin (BH4) or substrate L-arginine, NOS3 uncouples to the monomeric form that generates O2

– rather than NO. Oral supplementation with BH4 prevents NOS3 uncoupling and markedly blunts ROS generation.

Extracellular glutathione peroxidase (eGPx) is produced by airway epithelium and alveolar macrophages, secreted into the surface epithelial lining fluid and functions as a first-line defense against inhaled ROS. NO, produced by NO synthase 2 (NOS2) combines rapidly with ROS to form highly reactive peroxynitrites. Hyperoxia increases NOS2 mRNA in airway epithelial cells by 2.5-fold but does not increase eGPx mRNA.

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In contrast, cigarette smoke upregulates eGPx mRNA over 2-fold in airway epithelial cells and alveolar macrophages but does not affect NOS2 expression. Laboratory exposure of respiratory epithelial cells to ROS or RNS also increases eGPx expression. So, susceptibility of the airway to oxidative injury is influenced by distinctly dissimilar molecular responses in the airway to different inhaled ROS.

Glutathione (GSH), a reduced thiol that modulates redox state and forms adducts of nitric oxide (NO),

improves endothelium-dependent vasomotion and NO activity in atherosclerosis. Thiol supplementation with GSH improves human endothelial dysfunction by enhancing NO activity.

Steven N. Green, DDS, 10261 SW 72 St., #106, Miami, FL 33173, 305-273-7779

Antiagingdentist.com or sngreen.com, ddsgreen@bellsouth.net, October 5, 2009

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