Abstract
Aristo Vojdani obtained his MSc and PhD in the fields of microbiology and clinical immunology from Bar-Ilan University in Israel. He then went on to do postdoctoral studies in comparative immunology at the University of California, Los Angeles (UCLA), and research in tumor immunology at Charles Drew/UCLA School of Medicine and Science. He is an Adjunct Associate Professor in the Department of Preventive Medicine at Loma Linda University in California, and an Adjunct Professor at the Lincoln College of Professional, Graduate, and Continuing Education at the National University of Health Sciences. Dr. Vojdani's research has resulted in the development of more than 300 antibody assays for the detection of autoimmune disorders and other diseases. He is the CEO and Technical Director of Immunosciences Lab in Los Angeles, California, and is also the Chief Scientific Advisor for Cyrex Labs in Phoenix, Arizona.
When I started in Israel, I did not know much Hebrew, so I was struggling to get to the same level as Israeli students. By my third year, my Hebrew had improved significantly, and I received a 100% in my second immunology course, clinical immunology. It was the first time in my life I scored a 100%. I went from the Ds to Cs and Bs, until finally I got an A+. That score was followed by an invitation by the professor to work in his laboratory during the summer doing research.
At this time, we did not know much about the different white blood cells. We knew about T cells and B cells but not T-cell subpopulations such as Th1, Th2, Th3, and Th17. And cytokines were unknown.
When I started at Bar-Ilan, it was a small university with about 5,000 students. It has since grown to become one of the largest universities in Israel. Some of my classmates at university went on to the Weizmann Institute, but the majority of them came to the United States for their postdoctoral studies. For example, today, one of my classmates is a senior investigator in the Immunoregulation Section of the National Eye Institute at the National Institutes of Health (NIH). She originally worked on immune tolerance in fish and then in mice. This led to her research in the induction of tolerance against a very important autoimmune disease of the eye called uveitis.
In 1976, I finished my PhD at Bar-Ilan University, and I did one year of research at the Weizmann Institute and at one of the hospitals associated with Tel-Aviv University. I then went back to Iran for two years to teach immunology. However, when the revolution happened, I came to UCLA for my postdoctoral studies. This is when I started to research invertebrate immunology, specifically the earthworm immune system. I discovered that earthworms produce significant amounts of lectins and agglutinins, which I was able to use to separate T cells and B cells. After that, I moved to Charles Drew/UCLA School of Medicine and Science where I became an assistant professor and then an associate professor. During my four to five years at Charles Drew, I worked on the role of environmental toxins and cell-mediated immunity in mice, for which I was awarded several NIH grants. I left to work in a biotechnology company, eventually starting my own clinical immunology testing lab called Immunosciences Lab in 1988.
After I had established my own laboratory, I started researching the role of environmental toxins on the human immune system. I published an article related to the environmental toxin formaldehyde. 1 Around 1989, my colleagues and I studied a group of individuals living in mobile homes who had become seriously sick. After drawing blood from these subjects, we measured antibodies against formaldehyde, isocyanate, trimellitic anhydride, and dinitrophenol. I used the knowledge and experience I got from my professor in Israel who was using penicillin to bind to red blood cells. I used the same methodology to bind small molecules, such as formaldehyde or isocyanate, to human serum albumin, and then measured antibodies against these neoantigens. We found very high levels of antibodies against formaldehyde, which was released from materials used in making the mobile homes. We didn't find high levels of formaldehyde in the controls who were not exposed to this chemical.
My co-authors and I had great difficulties publishing our articles in scientific journals about 30 years ago. It was unbelievable. It was rejection after rejection. Finally, we found a journal that was more sympathetic to the subject. That journal was Archives of Environmental Health.
Even today, after publishing many articles over the years, I have still had problems publishing articles about the production of antibodies against very small molecules called haptens. Haptens are tiny molecules that by themselves are not antigenic but can bind to human tissue, after which they can be recognized by the immune system, which then produces antibodies against them. For example, one article was about bisphenol A bound to human serum albumin. We found antibodies in patients with thyroiditis. Journal editors to whom we submit our manuscripts do not understand how it is possible. They say that we are detecting antibodies against chemicals that are too small to make antibodies against. We try to tell them that when the haptens bind to human tissue, they become immunologically detectable, but they continue to push back against the evidence.
Another issue that we got pushback on was the idea of mold or chemical sensitivities. We would test for mycotoxin or silicone antibodies. We used to get lab samples from several clinics all over the country. We found that for some people with fibromyalgia or chronic fatigue symptoms, not only were their immune systems making antibodies against mycotoxins and silicone, but the ratio of helper/suppressor cells was 5.8 or higher. For reference, the normal range at that time was 2.0. So these individuals had immune activation and possible autoimmunities. On the other hand, a subgroup of patients had a helper/suppressor ratio of 0.5. Everybody thought these individuals had acquired immune deficiency syndrome. Nobody knew what was wrong. Then, I spoke to the patients' practitioners about their medical histories and found the answer. These patients had silicone breast implants. 2,3 Or they were exposed to mold and mycotoxins. That is where I became more interested in this field of exposure to molds and mycotoxins and other environmental toxins.
At Immunosciences, I performed antibody testing against mold and mycotoxins in samples from >200,000 individuals. My team and I published some findings in different journals, including the Archives of Environmental Health. 4,5 We found that individuals exposed to mold not only had antibodies to mycotoxin, they also produced other tissue antibodies. Rheumatoid factor was elevated, and we also detected smooth-muscle antibodies, myelin basic protein antibodies, and myelin oligodendrocyte glycoprotein antibodies. In fact, we found more brain antibodies in individuals exposed to molds and mycotoxins than any other groups. We think that mycotoxins bind to human tissue and form a neoantigen, and then the immune system makes antibody against the complex. Then, through epitope spreading, the body starts making antibodies to everything around it.
So, in my opinion, measuring mycotoxins and other toxins in urine and blood is not reliable. It is more important to measure the adductomes in the blood. From a pathological point of view, if the toxin or its metabolites bind to human tissue, then it is a completely different story.
Lyme disease is another example. 6 Several years ago, I cultured lymphocytes with the Borrelia antigen from patients with Lyme disease. I looked for the rate of DNA synthesis or cytokine production in the samples from these patients, and found it to be significantly elevated. However, it was also elevated in people who did not have Lyme disease. Perhaps they had Epstein–Barr virus (EBV) infection or other abnormalities, which cross-reacted with the Borrelia antigen. So, I offered this test for only a couple of years until I found out that there was a lot of cross-reactivity between a variety of pathogens and even food antigens with Borrelia burgdorferi. Memory lymphocytes react not only to specific antigens, but also to cross-reactive antigens. Imagine if a patient was infected with Yersinia enterocolitica. That patient's immune system will produce antibodies against Yersinia, and as a result memory lymphocytes against Y. enterocolitica will be present in that person's body for many years. Now, Y. enterocolitica cross-reacts with EBV, certain food antigens, and B. burgdorferi. These memory lymphocytes will recognize Y. enterocolitica toxin, as well as EBV toxin and certain food antigens. So, these memory lymphocytes of patients with food immune reactivity, EBV, or Y. enterocolitica infection will react nonspecifically to Borrelia antigens in culture, resulting in the production of cytokines, which can be measured. Any antigen that cross-reacts with Borrelia antigen will do the same thing.
That is why I stopped doing this test. But there are other labs that are still doing this test. How do we know if the cytokines produced are due to exposure to Borrelia antigen, to EBV, or to Y. enterocolitica, or even to food antigens? So, if a laboratory is offering a test for Lyme disease based on this test, in my opinion, it is misleading the practitioners. And this false determination of Lyme disease may result in many months or years of unjustified antibiotic treatment, which can have deleterious effects on the microbiome, which we now know has such an important role in the function and regulation of the immune system, and can affect a person's health in different parts of the body, including the brain.
Let us take mycoplasma as an example. About 18 years ago, based on false claims that the U.S. government weaponized Mycoplasma fermentans, many people started blaming every single disease on mycoplasma. 8,9 It became so important that the Department of Defense (DOD) got involved and funded a major research project to investigate the matter. In this project, the DOD sent blood samples from patients who had Gulf War syndrome and from controls to six different laboratories for the detection of mycoplasma by polymerase chain reaction (PCR). Of the six laboratories, four of them were associated with different universities, and the other two were clinical laboratories, of which Immunosciences Lab was one. After two years of investigation, our tests showed that there was no significant difference between results from the Gulf War syndrome patients and controls; the rate of mycoplasma detection was very low, about 15%.
So, what is the value of this research if you find the same rate of positivity by PCR in healthy subjects as well as in Gulf War syndrome patients? So, after two years and spending >$1 million, the conclusion was that there was no conclusion.
However, it is possible that if you are exposed to mycoplasma, and you make antibody against mycoplasma, because of the similarity of mycoplasma to human tissue, that antibody might induce autoimmunity in the future. Looking for the antibody is more important than looking at the level of mycoplasma.
The same thinking applies to Lyme disease. I used to do PCR testing for Lyme. Only 3–5% of PCRs that I performed were positive. I had to stop doing this type of testing because it was a waste of money to do PCR on blood samples because Borrelia, when it becomes chronic, might be in the joints or even in the brain—so why should we look for it in the blood? I honestly believe the antibody is the best test. I have actually seen several cases of patients who tested strongly positive by DNA detection and lymphocyte culture in other laboratories, who then tested absolutely negative with antibody testing by both ELISA and Western blot in Immunosciences Lab and others.
So, as a practitioner, what do you do? Are you going to put this patient on months or years of antibiotics based on a false-positive test, thus completely destroying the patient's microbiome? However, when it comes to antibodies, there are accepted criteria by the Centers for Disease Control and Prevention for determination of Lyme disease by antibody testing. At Immunosciences and other reputable labs, we follow strict proficiency testing protocols established by the College of American Pathology (CAP), the established regulatory body for lab testing. In this fashion, reputable labs achieve almost 100% correlation regarding specificity and sensitivity with the desired results established by CAP. So, if I were a practitioner, I would rely on the testing methodology based on the rigidly tested government-approved criteria. I still believe that Western blot and ELISA are the best tests for Lyme disease.
About 12 years ago, I developed my own patented version of ELISA for Lyme disease, which was based on in vivo–induced antigen technology. 6 This method is based on measuring antibodies, not only against Borrelia grown in culture, but also Borrelia in vivo in the human system because Borrelia expresses different new antigens when it enters the human body. Borrelia changes its antigenic structure in human tissue in order to hide from the immune system, and our body makes antibodies against Borrelia grown in culture, as well as those expressed in the human system. In our version of ELISA, we do determinations for antibodies against 12 different antigens, six of them from Borrelia burgdorferi, three of them against subspecies, and three against the co-infections Babesia, Ehrlichia, and Bartonella, which are sometimes mistakenly misdiagnosed as Borrelia. This is the most comprehensive way of testing for Lyme disease and associated disorders.
Practitioners should know that the prevalence of Lyme disease varies from state to state. 10 We have two extremes: one saying that everybody has Lyme disease, and the other, here in Los Angeles, where they say there is no such thing as Lyme disease in Los Angeles or in California.
We do have Lyme disease in Los Angeles because we have deer in Los Angeles. In my neighborhood where I live, I see many deer. So, there is Lyme disease in Los Angeles. But not everybody has Lyme disease. Testing has to be done but only based on evidence. That is why functional medicine is evidence-based medicine. Whether it is in Los Angeles or Connecticut, we can only say that an individual has Lyme disease if the clinical symptomatology correlates with reliable laboratory antibody testing such as ELISA or Western blot. We do not want to expose an individual to months or years of antibiotics unnecessarily. We all know that giving antibiotics irresponsibly can do so much damage to the gut.
Just this month, Scientific American has this topic on the cover. 11 Our seventh sense is the interaction of the gut, brain, and immune system. So, when we destroy the gut, we destroy the immune system. When we destroy the immune system, we destroy brain function. We have to be very careful. We must treat based only on evidence.
So, in this case, these antibodies are predictive. But we also determined that certain foods can cross-react with ZnT8 or GAD65. 13 Certain foods can cross-react with thyroid hormone. So, how can we differentiate where these predictive antibodies are coming from? Are they coming from the food? Are they coming from the infection?
For example, the membranes of chlamydia or mycoplasma can cross-react with phospholipids. If the patient has phospholipid antibodies, how do we know if it's from mycoplasma or a real reaction against human phospholipids? Those are the kinds of questions that we need to have answers for. The more research that I have done over the past five years, the more we find cross-reactivity between food, pathogens, and human tissue. Therefore, we have to do testing that will find these triggers that are cross-reacting with each other, and once we identify them, we can remove them, whether it is food from the patient's diet, toxic chemicals from the patient's environment, or an infection that has to be cured.
We are learning that if someone has an autoimmune disease, for example, and they produce antibodies that cross-react to an antigen in certain foods, for example in egg yolk, then I would remove egg yolk from the diet. It just makes sense.
However, today, we can say that if certain foods cross-react with ZnT8, then remove those foods from the diet of your child. Do not wait another 10 years until that child develops full-blown type 1 diabetes. Dealing with general inflammation and leaky gut might be helpful, but testing for food antibodies is important. Some people call these food allergies, but that is completely misleading. I call them food-associated autoimmunities, which is the title of my to-be-published book, which will be available around January or February next year. In this situation, food turns our immune system against us. If we have proof that a food antigen cross-reacts with ZnT8, GAD65, or insulin, that food should definitely be removed from the diet of the child. Once the triggers are removed, it is hoped that the level of antibodies in the blood will decline.
Take the name ALCAT. The name originally stood for Antigen Leukocyte Cellular Antibody Test, but it would appear that the second “A” in the acronym is completely misleading, since, as described, this method does not actually measure antibodies. 14
Cytotoxic testing involves putting a drop of blood under the microscope, mixing in food, and then looking for a change in white blood cell sizing. The problem with this is that the exposure of white blood cells to impure food antigens, toxic chemicals, or medication can induce nonspecific cell swelling, apoptosis, or necrosis. And nowhere in this process is testing for antibodies conducted, as the A in ALCAT implies.
The MRT also bears a deceptive name. The name seems to indicate that the test measures cytokines and other proinflammatory mediators that are released when the immune system destroys invading pathogens or antigens. Instead, the test as described measures volumetric changes in white blood cells. 14
The third test has changed its name from ELISA/ACT to LRA. In the field of immunology, actual lymphocyte response testing pertains to separating lymphocytes from all other components of the blood, including plasma and red blood cells, then culturing these white blood cells in the presence or absence of mitogens, antigens such as food chemicals bound to protein, and any other required factors in a very special medium in a very special environment that is required for tissue culture. LRA instead relies on unproven claims that it uses enzyme-enhanced ELISA technology to measure all three delayed-allergy pathways and the reaction of white blood cells to 400 items, including sensitivity to chemicals.
Both the new versions of cytotoxic testing as well as crude testing for IgG by ELISA and microarray (a mini-version of ELISA) that are being promoted for food allergy testing are misleading clinicians and the public. For example, there are labs that are marketing the test that obviously don't understand the principle of ELISA; you can tell this is so because they include sugar as one of the food items they test for. I only used sugar as a negative control when I originally developed my test because the human body does not produce antibody against pure sugar. Only when the sugar molecule binds to human tissue or transforms into an advanced glycation end product can it be used for testing.
Tests such as this and the labs that market them do not adhere to the four core principles of reliable and accurate lab testing: purity of the antigen, optimization of the antigen, validation of the assay, and testing in duplicate. These are the principles that Immunosciences Lab and Cyrex Lab follow. These are the core principles that all reputable labs should follow to have credibility in their test results. If these principles are not followed, then a patient's results are subject to false positives and false negatives with potentially life-threatening consequences.
Even if and when the core principles of lab testing are followed, I think there is another mistake being made in that the term “food allergy” is being applied too broadly. Laboratories should stop marketing these tests as food allergy panels. Food allergy, strictly speaking, refers only to IgE-mediated reactions to food, and not IgG, IgA, IgM, or cytotoxic testing. Cyrex Lab correctly promotes their testing as being about food cross-reactivity with human tissue and its possible contribution to autoimmune diseases. That is the focus of my book.
Examples of food cross-reacting with human tissue are gliadin and the cerebellum; casein and myelin oligodendrocyte glycoprotein; tomato, corn, spinach, and soy with human aquaporin; seaweed with islet cells and ZnT8. These are all discussed in some of my previous articles. 13,16
There is also a problem with the measurement of zonulin levels. I have seen results from four different laboratories. All laboratories had the same reference ranges. However, every laboratory, before reporting the results of any test, is supposed to establish the performance characteristics of the test. This entails very extensive work, establishing reference intervals or reference ranges. If these laboratories went through all of that, why are their reference ranges exactly the same? In my opinion, it seems that these laboratories are using the reference ranges recommended by the manufacturer's kit, which are indicated as being for research purposes only. The law says that because this kit is only for research, the labs must establish their own reference ranges by running 120 or more samples from healthy subjects. This whole field is unfortunately suffering from many such problems. Laboratories are reporting results before going through the extensive validation steps required by law.
So, to answer your question, in my opinion, zonulin levels or LPS levels in the blood versus antibodies are two different stories. At least antibodies are stable in the blood for 15–30 days or even longer. In my own recent study published in the World Journal of Gastroenterology, I found that due to fluctuation, a single measurement of zonulin levels is not recommended for the assessment of intestinal barrier integrity. Instead, the measurement of IgG and IgA antibodies against zonulin is proposed for an accurate evaluation of intestinal barrier integrity. Therefore, I recommend the antibodies, and hopefully we will have more research in this field.
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