This story began 12 years ago, when I received a phone call from a prospective Canadian patient asking my advice about treating hepatitis C virus (HCV). He had acquired both hepatitis B and C infections from blood products used to treat hemophilia approximately 34 years earlier. Together we evaluated many claims of cure; tried promising remedies; and checked serum viral load, liver enzymes, and level of liver fibrosis. (Initially his biopsy revealed a level 3 cirrhosis, and FibroScan score >20.) Follow-up fibrosis studies were measured with the FibroScan. Years later his liver is now in the range of normal fibrosis (FibroScan 4.7). Remarkably, his serum HCV level was unchanged, still at 1 to 10 million copies/ml.
A Failure Leading to Success
One notable therapeutic failure in our search was using a gliadin remedy, which promised improved liver enzymes levels in hepatitis. But the molecule apparently suppressed or depleted his antiviral immune response, allowing his hepatitis B viral load to skyrocket to 2.1 billion copies/ml. (This was one of the highest viral loads every recorded in Canada.) When gliadin therapy was terminated, the rebound immune response produced a life-threatening liver flare, with liver enzymes (ALT) over 600.
Ironically, this failure was the doorway to ultimate success. In an effort to save his life, and avoid a liver transplant, we tried a bioflavonoid immune modulating therapy that ultimately became a vital component of the antifibrotic protocol.
Genesis of the Theory and Therapy
Peter Schmid, MD, was the central figure in (1) proposing the mechanism of HCV cellular damage and its subsequent development of fibrosis/cirrhosis, (2) theorizing which biomolecular countermeasures would effectively interrupt and reverse that damage, (3) assembling the treatment protocol (38 nutrients at doses of proven experimental efficacy) to counter those pathological processes, and (4) personally acquiring one of the first FibroScan units, at great expense, which opened the potential to test the antifibrotic protocol's efficacy. This project was the natural outcome of his passion and 20 years of work in hepatology.
Schmid theorized that HCV damages hepatocytes by the intracellular effects associated with (1) chronic NF-kB stimulation, (2) the cascade of events (cytokine release, apoptosis, and molecular damage) surrounding oxidative stress, and (3) the ultimate conversion of stellate cells into collagen secreting myofibroblasts. (Note: Fibrous tissue generation is designed to repair acute damage, but under chronic stimulation it results in fibrosis/cirrhosis.)
Schmid researched the literature for natural remedies that could normalize these intracellular cytokines. Likewise, he sought remedies to interrupt the fire of oxidative stress as a possible point of intervention. He reasoned that ROS (reactive oxygen species) attract macrophages, and macrophages signal the activation of stellate cells to secrete collagen. In the end, the theory guided him to a successful treatment protocol. Over a period of years, my patient's liver enzymes returned to normal, his fibrosis reversed, and his health returned. Other patients were subsequently given the protocol, with similar results.
Proof of Theory
My patient's medical doctor was Lorne Tyrrell, MD, a world-class researcher in virology and past chair of the Department of Medicine at University of Alberta. Tyrrell was astonished at the reversal of cirrhosis and wanted to confirm the efficacy of the antifibrotic protocol using chimeric mice (i.e., mice with human livers, and deficient adaptive immune systems – a mouse system and technology that he had developed).
After success with my patient, Schmid began an informal clinical trial on a cohort with HCV, as well as other liver conditions. The antifibrotic protocol proved effective in reducing liver enzymes, reversing fibrosis, and increasing quality of life for a number of patients.
The protocol was then formulated and marketed as a nutritional product. After a year of successful therapy with patients, it was tested at the University of Alberta. Based on the positive results of the university study, the antifibrotic protocol is now available as a "medical food" only by prescription from a licensed medical practitioner.
Liver fibrosis is a major consequence of HCV infection. An estimated 170 to 200 million people worldwide are chronic HCV carriers. After acute infection, HCV becomes chronic in 65% to 80% of its victims. Chronic infection can lead to: (1) chronic active hepatitis, (2) cirrhosis, and (3) hepatocellular carcinoma. These sequella manifest 10, 20, and 25 years respectively after the initial infection. End-stage liver disease from HCV is the leading indication for liver transplantation in North America and Western Europe. After transplant, the liver is universally reinfected, and approximately 15% develop HCV-related cirrhosis within 2 years after transplantation.
Chronic HCV infection takes a highly variable disease course, and the factors involved in hepatocyte injury and the progression of liver disease are complex and not fully understood. The antifibrotic protocol was effective in clinical pilot trials, so it seemed worthy of spending the money and time to formally prove efficacy under conditions of strictly controlled trials. Hepatitis Technologies contracted with Tyrrell to conduct an experiment modeling HCV infection in chimeric mice.
Testing Chimeric Mice
Human livers were transplanted into a special strain of mice by injecting human hepatocytes into their spleens, after killing the mouse hepatocytes with carbon tetrachloride. The transplanted hepatocytes then migrated to and colonized the liver. The mice were then infected with HCV and divided into two groups: an infected but untreated control group, and a group receiving the antifibrotic protocol.
Response of Chimeric Mice to HCV
Examination of the chimeric mice's response to infection revealed hepatic effects similar to those seen in human HCV patients. There was: (1) stimulation of NF-kB, (2) induction of interferon response genes (to produce interferon, which inactivates viruses), (3) changes in expression of genes involved in lipid metabolism (this reflects the increased metabolic demand on the cell due to the virus. Lipid metabolism is also an indirect indication of increased oxidative stress, since lipid metabolism inherently produces ROS), (4) cytokines and chemokines induced in the human hepatocytes, and (5) HCV-induced apoptosis in infected chimeric mouse livers, similar to that seen in liver biopsies from HCV-infected patients.
Mouse Immune Response
The fact that there is no adaptive immune system (antibody-producing B cells or T cell-mediated immunity) in this variety of mice gives evidence to the theory that hepatocyte damage is triggered by the innate immune system's response to the HCV.
In fact, there is very little adaptive immune response against HCV, since the virus hides in cholesterol particles. The fatty viral envelope has a natural affinity to lipid particles, essentially bathing itself in fat. This makes the virus almost invisible to the adaptive immune system, which explains why all attempts to develop an anti-HCV vaccine or therapy have failed. HCV antibodies do reduce the rate of infection, by inactivating viruses after release from an infected hepatocyte. But antibodies and T cells cannot detect or deactivate viruses hidden in particles of cholesterol.
In the mouse experiment, similar damage to infected human hepatocytes was seen, which validates the theory that the adaptive immune response (T and B cells) was insignificant in the disease process. Thus, the majority of HCV-induced hepatocyte damage appears to come from the response to the innate immune system (which includes: NF-kB stimulation of the protective response, apoptosis, ROS, and inflammation). The response to the innate and adaptive immune response is the generation of a chronic signal sent to stellate cells to convert to myofibroblasts, which in turn secrete fibrous tissue in an inappropriate attempt to repair an ongoing, low-intensity injury.
Examination of HCV Cytological Effects
We shall now review a few ultrastructural cell phenomena and molecular pathways triggered by the HCV infection.
1. After penetration into the cell, the HCV RNA begins to utilize the cell's metabolic machinery to produce viral proteins. This process overwhelms the capacity of the cell to fold the proteins produced and creates a condition known as endoplasmic reticulum (ER) stress. This process increases apoptosis, macrophage chemotaxis, ROS release, and tissue inflammation.
2. The stellate cells activate into myofibroblasts under the influence of the inflammatory signals of ROS, TNF, and TGF-B. The subsequent secretion of collagen explains the development of cirrhosis in HCV.
3. HCV stimulates NF-kB activation, which in turn activates 300 genes, in an effort to keep the cell alive. But NF-kB was designed to respond to acute viral, bacterial, radiation, and chemical threats. Since HCV is usually chronic, NF-kB remains continuously activated for years. NF-kB deploys intracellular ROS defenses to damage the genome of invaders. But the result is occasional collateral damage to the cell's genome, which explains the prevalence of hepatocellular cancer in HCV.
Endoplasmic Reticulum Stress
ER stress is a sequence of cellular events triggered when the proteins produced by ribosomes on the endoplasmic reticulum (ER) surface are not fully folded by the ER. This backlog of unfolded proteins triggers a response in the cell called the unfolded protein response (UPR). The UPR is a complex sequence of molecular triggers: (1) which stop the production of more protein, and (2) upregulate the production of chaperone molecules to help in folding the recently assembled protein strands. (3) If the UPR is unsuccessful in clearing the ER of unfolded protein, an alternate molecular pathway is triggered that leads to cell apoptosis.
Sequence of Events in HCV-Infected Hepatocytes
1. HCV is an RNA virus, which utilizes (hijacks) the cellular machinery to synthesize the proteins needed to assemble more HCV particles. The very large quantity of HCV protein transcribed by the endoplasmic reticulum requires more chaperone molecules to accomplish this increased folding load. This added folding burden overwhelms the available chaperone molecule-folding capacity, producing a backlog of unfolded proteins.
2. This backup of unfolded HCV protein triggers the UPR (unfolded protein response), which attempts to clear the backlog of unfolded HCV protein, by stimulating the production of proteins (Bax and BiP), which triggers the production of more chaperone molecules. If the UPR is insufficient, cellular messengers are produced that trigger apoptosis.
3. Likewise, the intracellular presence of the HCV virus and its proteins reduce activation of NF-kB, which places the cell at greater risk for apoptosis.
4. In response to apoptosis of other liver cells, the innate immune system (macrophages, dendritic cells, and NK cells) responds with ROS secretion and phagocytosis of the intracellular debris. Likewise, the NK cells release TNF, FAS, or TRAIL messenger molecules, which all facilitate apoptosis.
5. The apoptotic sequence completes when the intracellular machinery produces sufficient concentration of death ligands, which activate the death receptors and ultimately trigger apoptosis.
Role of NF-KB
Like all viruses, HCV stimulates NF-kB activation, which triggers interferon gene transcription and an antiviral response. This may result in a net pro- or antiapoptotic signal, depending on the balance of the intracellular signals. While HCV tries to inhibit NF-kB activation to enhance its own survival (e.g., inhibiting antiviral interferon production), the actual levels of NF-kB internal to an infected cell are raised compared with the uninfected cell. And while NF-kB helps the cell survive an acute attack, ROS secretion and other defensive biochemicals can be toxic to the cell and damage the genome (ultimately putting the patient at risk for liver cancer). Thus, chronic activation of NF-kB results in ongoing damage to the cell. NF-kB is vital in responding to acute threats and damage, but damaging in the case of chronic stressors – hence the therapeutic goal of supplying nutrients that lower NF-kB.
NF-kB is an important intracellular messenger molecule that activates the cell's innate immune response. NF-kB is stored in an inactive form in the cytosol until activated by cell stressors (such as bacteria, viruses, and pro-inflammatory cytokines such as TNF and IL1B). Viruses triggers NF-kB activation upon recognition by the pattern recognition receptors: Toll-like receptors on the cell's surface and RIG receptors intracellularly. NF-kB, once activated, stimulates a rapid nuclear response, to transcribe many genes and produce cell-protective proteins. (This response includes antiviral interferons, and a vast array of cell survival, protective, and inflammatory responses.) NF-kB helps protect the cell from apoptosis by stimulating mitochondrial transmembrane molecules of the BCL-2 family, which inhibit apoptosis. But HCV inhibits the production of NF-kB, resulting in the blunting of signals that would protect against apoptosis. Thus HCV interferes with NF-kB, which adds to the apoptotic pressure.
Tumor Necrosis Factor (TNF)
TNF is an immune cell cytokine (released by macrophages, NK cells, and lymphocytes), whose secretion is integral to fighting infections. Oxidative stress and inflammation stimulate its secretion, which in turn promote inflammation. TNF secretion contributes to cell death in the form of both apoptosis and necrosis of hepatocytes, both of which positively reinforce the pro-inflammatory cascade. Hepatic damage results from the body's immune response and ER stress.
TNF secretion and NF-kB activation are part of the body's natural response to HCV infection. The central place of TNF in promoting the inflammatory cascade suggests that intervention to reduce this cytokine is a potentially effective strategy in attenuating fibrogenesis.
HCV and Fibrosis
Chronic HCV infection adds a bias toward apoptosis through ER stress and NF-kB interference. Apoptosis attracts macrophages, which secrete oxidative species (a molecular offense meant to damage the genome of the replicating virus). The cellular debris created by apoptosis attracts macrophages, which secrete more ROS, which attracts more macrophages. The oxidative environment stimulates macrophages to release the cytokine TGF-B (transforming growth factor beta, a multieffect cell-signaling molecule), which can activate stellate cells to convert into myofibroblasts, which secrete collagen in an effort to repair the tissue integrity after damage. And while an important survival response for acute injury, chronic secretion causes fibrosis and shrinkage of the liver. Thus, we see the importance of quickly quenching ROS after clearance of apoptotic cellular debris.
The mouse experiment results were consistent with: (1) HCV induction of hepatocyte stress and damage, (2) provoking a innate immune response leading to apoptosis, (3) elevated ROS tissue levels, (4) macrophage invasion, (5) conversion of stellate cells to myofibroblasts, (6) unregulated secretion of extracellular fibrin, and (7) development of fibrosis.
HCV-Induced Tissue Changes
Histologic exam of the mouse-human livers revealed signs of hepatocyte damage and increased inflammation in the parenchyma. This experiment was the first report of HCV infection producing oxidative and ER stress in the absence of an adaptive immune response. The study confirmed that the HCV itself greatly stimulates intracellular ROS production. Until this experiment, the prevailing opinion was that the body's own adaptive immune system produced the oxidative stress that resulted in cellular damage and apoptosis.
The researchers stained the chimeric mouse livers for collagen and quantified the results. They found that significant fibrosis developed in HCV-infected chimeric mice within 2 months postinfection.
HepTech's Test Protocol
The HCV-infected mice were fed an antifibrotic/antioxidant protocol and their livers were examined after 2 months of infection. The protocol consisted of antioxidants, as well as vitamins and cofactors designed to feed into the glutathione homeostasis cycle. During the course of infection and treatment with the antifibrotic protocol, viral titers were measured, and there were no significant changes in levels of HCV viral titers. There was no change in mortality during the course of the study, which gives some indication of the safety of the antifibrotic protocol.
The livers of infected mice on the antifibrotic protocol were compared with those of infected mice not receiving the protocol. The results showed a decrease in lobular inflammation and an increase in portal inflammation. Notably, there was a decrease in the amount of apoptosis in liver sections. There was a highly significant decrease in the number of activated stellate cells when liver sections were stained (using antibodies specific for smooth muscle actin – an indicator of conversion into myofibroblasts). Importantly, there was a highly significant decrease in the amount of collagen I deposited in infected livers, as measured by staining. Thus, it appears that the fibrosis induced by HCV in chimeric mice with mouse/human livers can be suppressed by an antioxidant-rich antifibrotic protocol.
A New Possibility in Cirrhosis
Conventional medical-science wisdom holds that liver cirrhosis is irreversible. But this paradigm was upended with the observation that when the fibrosis-generating stimuli are removed, a reduction in fibrosis staging is frequently achieved. To a remarkable degree, all but the most advanced stages of cirrhosis are reversible after elimination of toxic, infectious, immune, or metabolic causes of inflammation.
It appears that oxidative stress may be a key factor determining the severity of the damage caused by the infection, which in turn opens the possibility of another effective therapy to deal with the effects of HCV infection.
Hepatitis B and C viruses affect people in different ways, and with varying degrees of severity. The level of damage caused by increased ROS production may simply correspond to the degree of the individual's antioxidant capacity overwhelm.
Summary of Mouse Test Results
Tyrrell directed the study on the efficacy of the Hepatitis Technologies Antifibrotic Protocol in preventing or reversing liver cell damage and fibrosis in HCV-infected transgenic mice engrafted with human hepatocytes. These mice can be infected with HCV, and their human livers respond much as do livers in humans. The results of these studies show that the antifibrotic protocol resulted in decreases in HCV-induced liver cell apoptosis, the stimulation of stellate cells, and the extent of HCV-induced fibrosis. These results have not yet been published, but the trend is clear in the treated animals.
Future Phase 3 Human Trial
So far, the evidence (both anecdotal and research-based) indicates that this multipronged approach is effective in fighting fibrogenesis. The University of Alberta animal study was completed and sufficiently convincing to merit a human phase 3 trial. Enrollment will soon begin to test the effectiveness of this antifibrotic protocol against other types of cirrhosis, including NASH, autoimmune, alcohol-induced, and viral hepatitis.
The human phase 3 trial will include only proven cirrhotic participants who have failed standard medical therapy. This cohort will use only the antifibrotic therapy. Hepatic panels, FibroScan readings, and quality of life assessments will measure the success of treatment.
The Antifibrotic Protocol
1. Intervention against ROS: Glutathione repletion was chosen as a primary intervention, since glutathione depletion is a hallmark of hepatitis C infection. People with strong antioxidant systems and antioxidant-rich diets may be less affected than people whose repletion is low and whose intracellular antioxidant systems are depleted. Dietary antioxidants cannot eradicate the virus; however, they can significantly strengthen and normalize the body's natural protective interdependent chain of antioxidants.
ROS production is a necessary part of the immune response, but it must be modulated properly to prevent damage to the cell's genetic material, enzymes, cellular structures, metabolites, and cofactors. A replete antioxidant system helps provide the needed protection against the ROS produced in HCV infection.
2. Botanical Support: The second category of therapeutic intervention was curcumin, resveratrol, and 8 other GRAS (generally recognized as safe) botanical extracts, with all nutrients included at therapeutic dosage. Each botanical is well known as an effective anti-inflammatory agent. Interruption of the self-sustaining oxidative cycle of inflammation and apoptosis is crucial, since the unregulated inflammatory cycle stimulates the macrophage secretion of TGF-B and the conversion of stellate cells into collagen-secreting myofibroblasts. Therefore, these botanical anti-inflammatory agents can act in direct ways to modulate the body's inappropriately intense and destructive response to HCV infection.
3. Polyenylphosphatidyl Choline (Ethanol-Free): A third strategy for controlling the damage caused by hepatitis C was based on the diligent work of Harvard researcher, Dr. C. S. Lieber. His career involved conducting over 80 studies demonstrating significant fibrosis regression in patients with alcoholic cirrhosis, using only the oral ethanol-free form of polyenylphosphatidylcholine. PPC is a key membrane phospholipid with a polyunsaturated tail, which increases membrane fluidity. (Note: PPC is not to be confused with phosphatidylcholine). While Lieber's studies did not involve PPC use in viral hepatitis patients, it has never been shown that the process of fibrogenesis differs significantly due to different stimuli, whether alcohol, viral, steatosis, or autoimmune. Treatment of the chimeric human liver mouse system included a combination of antioxidants, GRAS botanicals, and PPC cocktail. The theory was that synergistic effects could be achieved by simultaneous administration of multiple effective therapies, all at therapeutic dosage.
The HepTech Antifibrotic Protocol proved efficacious first with clinical anecdotes (FibroScans, hepatic panels, and patient reports), which supported the theory that reversal of HCV-induced fibrotic damage is possible. The mouse study gave scientific validation to the hypothesis. The antifibrotic protocol is based upon (1) reducing chronic inflammation by increasing glutathione and antioxidant repletion, (2) reducing chronic NF-kB activation using herbal phytochemicals, and (3) increasing membrane fluidity with PPC. Fibrosis reversal proceeds when resorption of fiber exceeds production. With regression of the fibrotic infiltrate, the natural regeneration of liver tissue can proceed, thereby allowing gradual restoration of liver function.
Article based upon the research and writing of Peter Schmid, MD; Lorne Tyrrell, MD; Michael Joyce, PhD; and Helen Watt, MD. Studies funded by an Innovation Alberta Research Grant and Nutraceutical Research Group Inc./Hepatitis Technologies.
Thomas Lee Abshier, ND, practices as a naturopathic physician in Portland, Oregon, where he has maintained a general family practice including internal medicine and counseling for the past 20 years. Dr. Abshier graduated from National College of Naturopathic Medicine in 1987 after pursuing a wide range of life and educational experiences, including a bachelor of science from the UCLA School of Engineering, US Naval Nuclear Power School, submarine service as a US Naval officer, and various business enterprises before pursuing his career in naturopathic medicine.
The FibroScan Technology
The FibroScan unit is a European technology (not yet available in the US) that uses reflected ultrasonic waves, processed by an algorithm, to give a "fibrosis score" between 0 and 40. The FibroScan rating scale: 0–4 is normal, 4–8 is mild fibrosis, 8–15 is moderate fibrosis, 15–20 severe fibrosis, and >20 cirrhosis. The sonic scores correlate well with biopsy, are harmless, and allow for frequent repeat analysis and disease staging as the pathology progresses or recedes. This technology is used almost exclusively throughout all of Europe and many parts of Asia. It may someday replace most needlepoint biopsies.