Archive for March, 2014

Report From San Francisco I: The “Father of Autoimmunity” Speaks

March 31, 2014

When the "Father of Autoimmunity' went to medical school 'autoimmunity' was not believed to exist

When the “Father of Autoimmunity’ went to medical school ‘autoimmunity’ was not believed to exist

Dr. Noel Rose M.D., PhD is  the Director of the Center for Autoimmune Disorders at Johns Hopkins University. Dr. Rose has been studying autoimmunity for over sixty years  but he was a pretty darn spry 80-something at the conference.

When I got close enough to  ask him a  quick question I would have sworn he was a well-preserved sixty something. The bow tie,though, still apparently in  fashion in some medical circles, gave it away as did his statement that the medical schools of his day scoffed, yes, scoffed at the idea that the body could attack itself.

In fact, in 1956 Dr. Rose was the first researcher to introduce autoimmunity as a cause of disease. In an interview he stated

“They were skeptical. People weren’t really prepared to believe it because it seemed so bizarre that you would develop an immune response to your own body. We had all been taught in medical school that the immune response is only directed to what’s foreign. But now we know that isn’t true.”

He’s one of the few people who could can say they wrote the book on it because he did. He wrote the first book on autoimmunity and it’s still being revised today. (Get the latest 1300 page edition for a cool $215)

Dr. Rose probably didn’t appear to know a lot about Chronic Fatigue Syndrome, but he does know  a lot about autoimmunity and with the Rituximab trials putting that subject on everyone’s mind it was good to get a primer on it.

Dr. Rose started off explaining the basics of the autoimmune response by stating that it’s a completely  natural response. We’re all doing autoimmune responses all  the time.  The reason for that is that ‘we all'; that is all the organisms on earth,  share proteins and enzymes and that means that the immune system is going to, in its fervor to rid the body of a pathogen, will inevitably accidentally attack the body instead at times. That means we  come loaded from the get-go with auto -antibodies and  self reactive T and B-cells and also that  we have a built-in way of suppressing those cells.

It’s when that suppression system goes down that you have trouble.

Males and females generally have equal rates of autoimmunity until puberty when the hormones kick in. Rates begin to even out  more in seniors

Males and females generally have equal rates of autoimmunity until puberty when the hormones kick in. Rates begin to even out more in seniors

An autoimmune response can affect essentially any organ disease in the body, and while  autoimmune disorders can show up very differently, they also share many features. They’re common – they’ve been diagnosed in about 20 million people in the U.S., but probably closer to 50 million Americans have one. They’re ‘chronic’  (usually life-long). They mainly affect women and they’re heavily hereditary.

There are no known cures, they’re expensive – costing the U.S. about a hundred billion (gulp) dollars yearly, and, in contrast to cancer and heart disease, they are on the rise.  About 80 autoimmune diseases have been identified and more are coming.

To the question why there are so many autoimmune  diseases, he stated

 “We think it’s the fact that the immune system is always looking for new pathogenic organisms and being very broad in its abilities to recognize organisms that it also responds to things within our own bodies that are of the same or a very similar substance.”

 

The Sex Bias

Hormones clearly play a major factor in the female predilection for autoimmune disorders.  Rates of autoimmune disorders in children  prior to puberty are equal  in males and females and pregnancy can have a huge effect on symptoms, with some women feeling much better.

They’re believed to  mostly result when  a genetic weakness plus an endocrine effect (female bias) bumps up against an environmental trigger such as an infection. Bad genes account for thirty percent of  the risk which means that 70% of it is probably some chance environmental factor you would have surely avoided if only you’d known.

Known environmental triggers include viruses (in particular EBV), bacteria (streptoccocus), drugs, foods (excess salt), pollutants such as mercury, hormones, stress (important but hard to quantitate). Because the exposure usually comes months or even years before you come down with an autoimmune disorder finding, the triggering factor is  often very  difficult.

gene

Genes account for 30% of the risk for getting an autoimmune disorder. Environmental triggers (infection, toxin, stress, drug) are far more important.

Celiac disease is one of the few autoimmune disorders in which the triggering factor is clear: gluten. Gluten-free diets may be important for more than Celiac patients, however. Dr. Rose believes they  can help people with other kinds of autoimmune disorders.

“Interestingly,” he added, “the gluten free diet may also be helpful for people who don’t have celiac disease but who have other forms of autoimmune disease. It’s just speculative, but as gluten free diets are more available, other people are trying them and often feel better.”

If you have a family member with an autoimmune disorder your risk of developing one jumps over 10-fold (from  .4% to 7%). If your twin has an autoimmune disorder you’ve got a 30% chance that you will come down with one  as well.

Once autoimmunity gets going it usually causes more damage and inflammation and generates more autoimmunity. One of the common signatures is  multiple autoantibodies to multiple antigens.

Treatment

Like ME/CFS and many other chronic illnesses, treatment is focused on symptom alleviation not curing the disorder. Progress is being made, however.

 “We still don’t have a cure, but new treatments have been introduced in the past 15, 20 years for autoimmune diseases like lupus and MS that are remarkable and very much improved. As we understand more about the autoimmune response we find more ways of developing drugs that will intervene, that will benefit the patient by at least alleviating the symptoms even if they won’t cure the disease.”

Autoimmune or Autoinflammatory?

Some disorders that have a strong inflammatory component and look like autoimmune disorders  are not auto-immune disorders; they’re auto-inflammatory disorders.  People with the autoinflammatory disorders carry  loads of auto-antibodies, but lack the self-reactive T and B cells found in true autoimmune disorders.  With regards to treatment  it’s often  a distinction without a difference because both involve the  innate immune system and they’re often  treated the same way.

innate immune diagram

Problems with the innate immune system (NK cell, neutrophils, complement, histamine, etc. ) are behind autoinflammatory disorders

The term  autoinflammatory only showed up in the scientific literature for the first time in 1990’s. Autoinflammatory disorders involve dysregulation of the innate immune system (NK cells, dendritic cells, macrophages,etc.) that result in high rates of inflammation.

Autoinflammatory disorders originally included ‘recurrent fever syndromes’, then branched out to include some genetic disorders, hard to understand disorders like Crohn’s disease, and now may include such common disorders as type II diabetes, gout and atherosclerosis. The  decreased adaptive immune response and increased innate immune response found in autoinflammatory disorders suggest aging could be included in this category. (In the Conference we’ll see some preliminary evidence of increased aging in ME/CFS)

We don’t know if some  individuals with ME/CFS have an autoimmune or an autoinflammatory disorder. Rose didn’t appear to know much about ME/CFS but after his  talk I asked Rose what I thought was THE question; the question that was not given to him  during the Q&A session even though I wrote it out twice,  the second time at least semi-legibly, and  that was:

“What does a positive response to Rituximab in a gender  specific disorder tell you about autoimmunity or  an auto-inflammatory response?” The MAN said it meant it could be either.

 ME/CFS Autoimmune Studies Underway

 “We hope this study will identify a pathogen as a likely causative agent of the disease in order to focus future study. Dr. Elledge

dorsal root ganglia

One CFIDS Association of America study will target autoantibodies to the dorsal root ganglia that the Light’s believe play a role in ME/CFS

Besides the two Rituximab trials two studies, both using the CFIDS Association of America’s Biobank, will shed light on the role autoimmunity plays in  ME/CFS. One promising study lead by Dr. Elledge of Harvard University will determine what the autoantibodies present in ME/CFS patients blood are targeting and what viruses the antibodies present are associated with.

“…autoantibodies to brain and dorsal root ganglia have been demonstrated in chronic post-Lyme disease syndrome, an entity essentially identical with chronic fatigue syndrome/fibromyalgia” Dr. Cooperstock

Another lead by Dr. Cooperstock will look for autoantibodies to nervous system targets including the dorsal root ganglia that feature in some theories of ME/CFS.

EBV I: A Deficient Immune Response, Increased Levels of Epstein-Barr Virus Opens Up EBV Question in Chronic Fatigue Syndrome Again

A Long History

Although altered EBV-specific antibody titers have been repeatedly demonstrated in CFS, no clear evidence for chronic EBV replication has been obtained so far. Authors

Perhaps the most common viral trigger for chronic fatigue syndrome (infectious mononucleosis, aka glandular fever) or Epstein Barr Virus (EBV) is a herpesvirus almost all adults have been exposed to and carry, usually in latent form in their cells.

conflicting signs

Conflicting results have made it difficult to determine the role EBV plays in ME/CFS. Will this German study signal a change?

EBV infection was proposed as the cause of chronic fatigue syndrome not long after the disorder became prominent in the 1980s, but inconsistent study results in the 1980s and 1990s followed by Straus’s 2000 paper (which suggested the search for herpesvirus infections in ME/CFS was over) put a damper on EBV research efforts.

From 2000 to the present only Dr. Lerner with his stream of positive studies (but sometimes challenging study designs) and Dr. Glaser published fairly consistently on EBV in ME/CFS. Recently Dr. Lipkin stated (unpublished) he found no evidence of active EBV infection using high throughput sequencing in the plasma of hundreds of ME/CFS patients.

Despite study inconsistencies, EBV has remained a pathogen of interest in ME/CFS. Both Lerner and Glaser have produced evidence suggesting that a defective form of the EBV virus may be causing the symptoms in some people with ME/CFS. Recent studies suggesting that EBV triggers autoimmune disorders are intriguing given the successful ME/CFS Rituximab treatment trials.

EBV’s ability to reactivate during stress and in hypoxic conditions may have implications for its possible role in ME/CFS as well. A recent laboratory study suggesting that high rates of oxidative stress can reactivate EBV and that antioxidants (including NAC, catalase, and L-glutathione) might be helpful in reducing EBV reactivation is intriguing given the high rates of oxidative stress found in ME/CFS.

Now, in a surprising turn, German researchers have not only put the spotlight back on EBV, but have dug deeper into EBV, ME/CFS, and the associated immune response than any group has before.

The Study

“Taken together, our study provides clear evidence that deficiency of EBV-specific immune response is present in CFS” The authors

The adaptive part of the immune system, the one that takes time to kick in, comes in the form of B-cell produced antibodies that lock onto proteins the virus produces and cytotoxic T-cells that attempt to the kill the virus. (B-cell’s attack the virus in the blood and cytotoxic T-cell attack virally infected cells.)

broken

Ohio State researchers believe a defective form of EBV that is spewing out proteins may be causing ME/CFS

Noting some unusual findings in their lab, these researchers looked at these antibodies and T-cells to see if people with chronic fatigue syndrome were mounting an effective immune response against EBV. They also looked for direct evidence of an active EBV infection.

EBV replication occurs when the virus produces proteins in a sequence that allows it to build another virus. One theory, developed by Dr. Lerner and a group at The Ohio State University (that includes Drs. Ariza, Glaser, and Williams), proposes that EBV undergoes ‘abortive replication’ in some people with ME/CFS. In abortive replication, a defective form of EBV produces early proteins, but is unable to produce later ones. The Ohio State group believes continual production of these proteins is causing a chronic inflammatory state in some people with ME/CFS.

Results

First, the German researchers found evidence of primary EBV infection or reactivation (increased IgM antibodies to a late EBV protein in @15% of patients vs 3% of controls) in significantly more ME/CFS patients than controls. The fact that this could be a ‘primary infection’; i.e. it represents the first time these patients are exposed to the virus is intriguing. A primary infection of EBV early in life usually leads to nothing more than a cold; a primary infection later in life can have serious consequences including infectious mononucleosis.

Having found evidence that an active EBV infection was more common in people with ME/CFS than controls, they looked to see if a reduced immune response was responsible for that.

The first hint of a reduced immune response to EBV in ME/CFS came in the form of a lack of antibodies to EBV-produced proteins VCA and EBNA1.

But first, a short antibody primer:

antibody

Antibodies attack pathogens in the blood; cytotoxic T-cells attack them in the cell

Antibody Types

  • IgG antibodies are ‘memory antibodies’ that travel through our system looking for evidence that a pathogen is present. Once your B-cells have mounted an attack against a pathogen, they are always present in our system. Therefore, IgG antibodies are not evidence of an ongoing infection.
  • IgM antibodies are attack proteins associated with a pathogen. High IgM titers to a viral protein generally reflect a primary infection.

With two types of antibodies being manufactured against a range of viral proteins the situation becomes complicated, but a healthy immune system should produce an array of both IgG and IgM antibodies that can detect (IgG) and inhibit (IgM) pathogens (found outside cells) at different stages of their lifecycles.

As they dug deeper, the German researchers found holes in the immune response to EBV in ME/CFS patients.

Immune Holes to Epstein-Barr Virus Found

Immune Hole #1 – reduced antibody response

Evidence of a impaired B-cell response to EBV first came in the form of missing IgG antibodies to VCA and EBNA in 13% of ME/CFS patients compared to 4% of controls. This indicated that 13% of their ME/CFS study population did not have some of the memory B-cells needed to detect an EBV infection.

Increased IgM antibody responses in ME/CFS (17.5% in ME/CFS vs 4% in controls), on the other hand, suggested active and perhaps primary EBV infections were more commonly found in ME/CFS patients.

All told, 30% of the ME/CFS patients either had reduced IgG (EBNA-IgG) or increased IgM (VCA) responses to EBV.

That finding prompted a deeper look, and a much larger study that found no IgG response to a protein expressed during latency by EBV (called EBNA-1 protein) in 10% of IgG positive ME/CS patients. This indicated that the immune systems of approximately 10% of the ME/CFS group were unable to detect a very early stage of EBV latency.

EBV

The EBNA-1 protein featured in many of the tests helps EBV maintain its latency in B-cells

Latency – For EBV to maintain itself in the body over time, it needs to be able to maintain itself in B-cells in a process called latency. EBNA-1 is a protein that helps maintain EBV’s viral genome in the earliest stages of latency.

The authors noted that people with severe infectious mononucleosis and chronic active Epstein-Barr virus disease have similar findings (although it’s not clear why, given that EBNA-1 is not involved, so far as we know, in replication).

That brings up the question of how many people with ME/CFS would have fit into the category of severe mononucleosis at the time they got ill. The Dubbo studies found that more severe infections greatly increase the risk of coming down with ME/CFS.

Immune Hole #2 – reduced frequencies of two B-cell antibody producing cells

Intrigued by the findings, the German researchers dug deeper into the immune response to EBV. They took blood (PBMCs) from ME/CFS patients and then stimulated it with CpG, SAC, and PWM for seven days, and found reduced frequencies of B-cells producing antibodies against VCA and EBNA-1, and for the first time they found evidence of immune deficiencies in most people with ME/CFS.

No less than 59% of ME/CFS samples had a diminished response to a later stage EBV protein (VCA) produced during the late stage of lytic replication, and a whopping 76% of ME/CFS samples had a diminished response to the EBNA-1 protein. With the VCA finding we have evidence suggesting many people with ME/CFS may have trouble controlling EBV replication.

Calling the findings ‘remarkable’, the authors suggested that either the memory B-cells associated with these EBV antigens had been lost or had failed to develop into antibody-secreting cells.

Immune Hole #3 – Reduced T-cell response to EBNA-1

A similar deficiency in the T-cell response to EBNA-1 indicated that both arms of the adaptive immune response to Epstein-Barr Virus, the B-cells and the T-cells, had difficult recognizing and responding to this protein.

Citing other disorders such as HIV, they suggested that persistent EBV reactivation in ME/CFS had driven the T-cell response in ME/CFS into ‘exhaustion’. (A similar suggestion has been made with regard to natural killer cells that attack pathogens early in an infection, which use killing methods similar to those employed by T-cells.)

Further analysis suggested that T-cell suppressor cells which decrease B-cell responses were not responsible for the B-cell suppression found. Normal B-cell responses to herpes simplex and cytomegalovirus suggested that the deficient B-cell responses were associated with EBV and not other herpesviruses.

cytotoxic T-cell

Lower cytotoxic T-cell responses to EBNA-1 could be associated with an increased risk of autoimmune disorders

Immune Hole #4 – reduced T-cell response to EBNA-1, reduced T-cell responses to EBV

Next they explored T-cell induced cytokine production. The T-cells should produce an array of cytokines against EBV. About 20% fewer ME/CFS patients (70% of controls vs 50% of ME/CFS patients) were able to mount an IFN-y response against EBV.

Looking specifically at the latency associated EBNA-I protein, they found the startling result that no ME/CFS patients mounted an IFN-y response against it.

They also found that ME/CFS patients produced significantly lower amounts of the pro-inflammatory cytokine TNF-a in response to EBV. Finally, a lower percentage of patients produced IL-2 as well. The reduced cytokine production suggested cytotoxic T-cells, one of the big guns of the adaptive immune response, were not being strongly activated in response to EBV.

Immune Hole #5 – Reduced frequencies of EBNA-1 specific T-cells

The researchers dug deeper still. Next they stimulated the blood (PBMCs) of ME/CFS patients and healthy controls (n=40) with the EBNA-1 protein, expanded the cells in the presence of IL-2 and Il-7, and then checked the T-cell response to them. Specific types of T-cells should be produced to attack EBV, but reduced frequencies of EBV-specific T-cells occurred in about 50% of the ME/CFS samples. That again suggested the cytotoxic T-cell response to the EBNA-1 protein was substantially reduced in ME/CFS.

Direct Evidence of Active EBV Infection

“Remarkably, in line with this finding we could provide evidence of enhanced viral load of EBV by detection of EBV DNA in a significantly higher proportion of patients compared to healthy controls.” The authors

Using a real-time PCR test in the whole blood that looked for ‘low-copy’ numbers (<1,000-2930 copies/ml) they found evidence of increased EBV viral load in 7.2% of 290 ME/CFS patients. When they dug deeper and did the same test in PBMCs in a subset of patients, they found that a whopping 55% of patients (vs 13% of controls) tested positive for EBV.

The viral loads were far below those found in other EBV associated illnesses such as infectious mononucleosis or post-transplant EBV infections, and there was no evidence of lytic replication (i.e., full replication of the virus), but something the authors called ‘latency-associated replication’ was common in people with ME/CFS, yet not in healthy controls.

The Lipkin Study

Using PCR the German researchers found much higher rates of EBV infection in PBMCs vs whole blood and no evidence of EBV infection in plasma.

plasma-blood

Was looking for EBV in plasma somehow a mistake?

Neither the Lipkin CFI ME/CFS pathogen study nor the CFIDS Association of America BSR study found evidence of EBV infection in ME/CFS. According to Russell Fleming’s transcript of the Lipkin talk, the CFI study looked in the plasma of both the Montoya and the ME/CFS experts’ samples.

Whole blood contains plasma, red blood cells, white blood cells, and platelets. Plasma makes up 55% of blood volume and contains water (90%), proteins, nutrients, waste products, clotting factors, hormones and carbon dioxide. It does not contain red or white blood cells or thrombocytes.

EBV DNA has certainly been found successfully in plasma before and plasma has been used to track EBV activation. Serum/plasma EBV PCR kits are available for purchase. Researchers search and find EBV in plasma frequently.

Dr. Chia, however, reportedly stated he believes the use of plasma rather than blood was a serious mistake, and the Germans were able to find evidence of EBV activation in blood but not in plasma.

EBV (and CMV and HHV-6) are ‘cell-associated viruses. The only times their DNA escapes the cells is when the cell dies (and the DNA goes into the plasma) or when the virus is replicating. Otherwise the virus sits in a latent or semi-latent state in the cells
Medical dogma states if you can’t find EBV in the plasma, the infection is not active. EBV DNA can be found in the plasma when EBV replication rates are high, as sometimes occurs in transplant patients, but it’s not likely to be found in the smoldering infection believed present in ME/CFS. Many researchers do not accept the idea of a smoldering infection that pumps out proteins which trigger an inflammatory response.

The German researchers are deepening their study of EBV and ME/CFS and currently evaluating antibody responses against a broader variety of EBV peptides derived from 8 different proteins. They are also quantifying the levels of memory B-cells targeting EBNA-1 and VCA.

Conclusion

“We think the altered pattern of the specific immune response to EBV may be suitable as a diagnostic marker for CFS.” Authors

ball of string

The harder they looked, the more they found …

It was if these researchers kept pulling a string that got longer and longer. First their interest was piqued by some paradoxical antibody findings, then they found widespread deficiencies in some antibody responses and T-cell responses, and finally they saw evidence of an active EBV infection in the blood of 55% people with ME/CFS (vs 7% of controls).

Much is still unclear. The EBNA-1 protein that the immune systems of ME/CFS patients had trouble responding to is associated with ‘early latency’, not EBV replication. The authors’ reference to ‘latency associated replication’ is unclear given that latency is not usually associated with replication. When asked what importance their findings have for EBV reactivation or EBV survival or  more severe casesof infectious mononucleosis in ME/CFS, the authors stated they can’t answer those questions yet.

Some researchers believe, however, that reduced cytotoxic T-cell responses to EBV increase the risk for autoimmune disorders. (We’ll be covering that possibility in the next blog.) These findings also suggest that the proposal by Lerner and the OSU group of Drs. Ariza, Glazer, and Williams that an abortive lytic process (smoldering EBV infection) is present in many people with ME/CFS may be correct.

While it will take more work to determine what these findings mean for ME/CFS,  the broad range of dysfunction  found and the high rate of active EBV infection (in plasma) would appear to put this pathogen back into play in a meaningful way in ME/CFS.