Our Immune System: The Sixth Sense?

I came across an article implying another link between our immune system, behavior, and psychology, and could not resist digging a little further and sharing what I read.
Although it is often understood, accepted, and sometimes overlooked that someone suffering from either chronic illness, an acute life-changing medical event, or a terminal disease would experience feelings of depression or even, at times, to end their own life - what if, instead, the immune-boosting medications they were taking or the immunological processes at work in their system was supplying the fuel for their depression?
According to McElroy, 12-30 percent of patients of various diseases with an inflammatory component, also suffer from depressive disorders (News Bureau, 7/27/04). Glassman and Miller, similarly, found that "45% of malignant melanoma patients treated with high-dose alpha interferon developed major depression" (Biological Psychiatry, August, 2007). The statistics abound with depression and suicide suspiciously following administration of two immune-boosters - alpha interferon and interleukin-2 (New Scientist, 16 June 2001).
Dantzer and Kelley believe that the cytokines, boosted by drugs, such as those listed above, activate the enzyme indoleamine-2,3-dioxygenase, which catabolizes tryptophan, thereby preventing it from being turned into serotonin - a known brain regulatory chemical and focus of much of the anti-depressant industry today (News Bureau, 7/24/04). They also invoked fever and sickness-related behavior in mice whose brains were directly injected with cytokines and then found that if they blocked pathways from brain to body, they could inhibit these same sickness behaviors (News Bureau, 7/27/04).
Maes found that people suffering from depression had higher than expected immune system markers: natural-killers cells, monocytes, and macrophages, as opposed to an expected supressed immune system (New Scientist, 16 June 2001). There was even evidence from Germany of a possible viral link to depression, though results were unable to be replicated (New Scientist, 16 June 2001).
The vote is still out it seems - Glassman and Miller site inconsistencies and obviously a complex relationship between cytokines and depression, with much left to be identified and understood (Biological Psychiatry, August 2007).
What if depression were somehow linked to viral infection? What if we could prevent depression associated with illness or major medical procedures with the simple administration of SSRIs? Is the immune system really our sixth sense, telling our bodies to display symptoms of illness and depression? The questions seem neverending.

References:

"Scientists Build on Case Connecting Inflammatory Disease and Depression." McElroy, Molly; News Bureau, 7/27/2004, University of Illinois at Urbana-Champaign.
"A Mind Under Seige." Brown, Phyllida; New Scientist, 16 June 2001.
"Where there is Depression, there is Inflammation, Sometimes!" Glassman, Alexander H., and Miller, Gregory E.; Biological Psychiatry, Volume 62, Issue 4, 15 August 2007, Pages 280-281.

our venerable leaders...

Here is a quote from presidential candidate Mike Huckabee (1992):

"If the federal government is truly serious about doing something with the AIDS virus, we need to take steps that would isolate the carriers of this plague," Huckabee wrote.
"It is difficult to understand the public policy towards AIDS. It is the first time in the history of civilization in which the carriers of a genuine plague have not been isolated from the general population, and in which this deadly disease for which there is no cure is being treated as a civil rights issue instead of the true health crisis it represents."

Camel Antibodies are Strange

I mentioned in class that camel antibodies are heat-resistant, and someone has developed a camel antibody-based dip-stick test for caffeine in hot drinks (see Nature, 11 May 2006, p.169). Becky Buckley told me afterwards that camel Abs are structurally odd, so I did some reading, and find that a majority of them have no L chains, but are H chain dimers! So the MW is more like 100,000 than 150,000. In human Abs, it's hard to get 2 H chains to dimerize, because their association surfaces (part of the V region framework) are "looking" for L chains. So some clever types have "camelized" human H chains, mutating the amino acids that repel other H chains, and successfully made H chain dimers, some of which are biologically active. Nature and biotechnology, aren't they both lovely?

Pig Brain as Antigen?

I realize this post is too late for further class credit, but its too interesting to not post. Therefore I'll keep it short.

I found this story in the press from Minnesota. See the link:

http://news.postbulletin.com/newsmanager/templates/localnews_story.asp?z=2&a=317960

It appears workers in a meat processing plant may have introduced pig brain as antigen while using pressurized air to clean the word environment. There are MS like symptoms (disease unknown at this time), and all affected employees worked near a machine desposing of brain. This sounds like the example JJ gave in class about the lab tech exploding a glass blender, thereby "inoculating" himself with novel antigen.

Very interesting..Do you guys think the symptoms are immunological? What are the hypothetical mechanism? Or alternatives?

Maternal-Fetal Immunology and Preeclampsia

Preeclampsia is a disease unique to pregnant humans characterized by elevated blood pressures (BPs) and proteinuria. The mild form of preeclampsia is defined as BPs >140/>90 on two occasions, 6 hours apart, as well as >300mg of protein in the urine when collected over 24 hours. When a patient meets the criteria for severe preeclampsia her BPs are >160/>110 with >5 grams of protein in a 24 hour urine collection. These findings must occur at > 20 weeks of pregnancy.
While preeclampsia only affects 5-8% of pregnancies, it is responsible for major maternal and fetal morbidity and mortality worldwide due to: seizures (eclampsia), stroke, renal failure, pulmonary edema, placental abruption, and hemorrhage. Currently, the only “cure” for preeclampsia is delivery. Since preeclampsia also can occur in pregnancies that do not contain a fetus (i.e. molar pregnancies) and is more common in multiple gestations (i.e. twins, triplets, etc.), it is believed that the placenta is the main perpetrator in the pathogenesis of the disease. (1,2)
The placenta is therefore under intense study in attempt to understand the etiology of preeclampsia. The Maternal-Fetal immune interface involves several areas of placental growth and development that are likely pivotal in the development of preeclampsia. The human placenta is the organ that connects the mother’s blood supply, via the uterus, to the developing fetus, via the umbilical vein.
One cell type pertinent to placental immunology is the syncytiotrophoblast. These cells line the fetal “capillaries,” aka the chorionic villi. Syncytiotrophoblasts are unique in that they express no HLA. Therefore, they are “immunologically privileged” in that they cannot be recognized by the maternal immune system and do not present foreign antigen (i.e. fetal antigen). Syncytiotrophoblasts are the barrier between the fetal circulation and the intervillous space, which is where the maternal blood supply empties, creating the main communication between the mother and growing fetus.(3)
As the human placenta develops, invasive cytotrophoblasts (cells generated from the developing embryo) penetrate into the muscular layer of the uterus (myometrium) that contains a copious blood supply. It is thought that uterine Natural Killer (uNK) cells aid in this invasion. The uNK cells are specialized to produce cytokines and growth factors that promote cytotrophoblast growth and invasion, but they are only weakly cytotoxic.(4)
Ultimately, the cytotrophoblasts alter the architecture of the maternal “spiral” arteries (those arteries that penetrate the myometrium and “empty” into the intervillous space) and become essentially the endothelial lining of these arteries. This converts uterine blood flow to the spiral arteries from a high velocity, high resistance area in the non-pregnant state into a low-velocity, low resistance area in pregnancy that aids in the perfusion of the placenta and, thus, the fetus.(5)
Tremendous research is going into the investigation of preeclampsia and how cytotrophoblastic invasion of the myometrium is involved in the development of this disease. Cytotrophoblasts (which are fetal) can express HLA-C, HLA-E, or HLA-G. It is the combination of these with the highly polymorphic uNK cell Ig-like receptors (KIRs) that have been implicated heavily in the facilitation or inhibition of the cytotrophoblastic invasion. Proper or improper invasion either protects against or stimulates the pathogenesis of preeclampsia. This depends on how well the fetal HLA type “interacts” with the maternal uNK KIRs.
Uterine NK KIRs recognize HLA-C. However, as the KIRs genes are polymorphic, they can be variable in their ability to express activating receptors. For example, individuals with the KIRs “AA” phenotype usually have no activating receptors while those individuals who express the “B” haplotype have copious activating receptor expression. So, depending on the maternal KIRs phenotype and fetal HLA-C epitopes expressed, invasion of the maternal vasculature by cytotrophoblasts is either hindered or facilitated.(6)
If invasion into the maternal vasculature by cytotrophoblasts is hindered, development of the placenta is impaired. Early in pregnancy, this triggers oxidative stress in the placenta in its attempt to improve communication with the maternal blood supply. This oxidative stress leads to the release of many of the factors identified previously in the serum of women who will ultimately develop the clinical syndrome of preeclampsia. These factors include sFlt-I (soluble VEGF-receptor I), syncytiotrophoblastic debris, complement, endothelial microparticles, as well as other yet unidentified players. Release of these factors also contributes to the clinical characteristics of the disease.
The release of these factors by a “sick” placenta leads to endothelial dysfunction in the maternal circulation and an amplified maternal systemic inflammatory response that is manifested as labile elevated blood pressures and proteinuria resulting from intermittent vasospasm.(7,8) In preeclampsia, therefore, the maternal-fetal immune response is deranged. Initiated by poor placentation, preeclampsia begins as a local pathology early in pregnancy that becomes amplified and systemic in the third trimester. The resultant cascade of events, including endothelial damage and metabolic derangement, represent the clinical illness that we ultimately diagnose as preeclampsia. (9)


1 Roberts JM, Taylor RN, Musci TJ, Rodgers GM, Hubel CA, McLaughlin MK. Preeclampsia: an endothelial cell disorder. Am J Obstet Gynecol 1989; 161(5): 1200-4.
2 Redman CWG, Sargent IL. Latest advances in understanding preeclampsia. Science. 2005;308:1592-1594.
3 Sibai B, Dekker G, Kupferminc M. Pre-eclampsia. Lancet 2005;365(9461):785-99.
4 Redman CWG. Immunology of preeclampsia. Society of Gynecologic Investigation annual meeting. Feb 2006:19-20
5 Redman CWG, Sacks GP, Sargent IL. Preeclampsia: an excessive maternal inflammatory response to pregnancy. Am J Obstet Gynecol. 1999;180:499-506.
6 Hiby, SE, Walker JJ, O’Shaughnessy KM, et al. Combinations of maternal KIR and fetal HLA-C genes influence the risk of preeclampsia and reproductive success. J Exp Med. 2004;200:957-965.
7 Levine RJ, Maynard SE, Qian C, Lim KH, England LJ, Yu KF, et al. Circulating angiogenic factors and the risk of preeclampsia. N Engl J Med 2004; 350(7):672-83.
8 Maynard SE, Min JY, Merchan J, Lim KH, Li J, Mondal S, et al. Excess placental soluble fms-like tyrosine kinase 1 (sFlt1) may contribute to endothelial dysfunction, hypertension, and proteinuria in preeclampsia. J Clin Invest 2003;111(5):649-58.
9 McMaster MT, Zhou Y, Fisher SJ. Abnormal placentation and the syndrome of preeclampsia. Semin Nephrol 2004; 24(6):540-7.

Good news, anyone?

Congrats to JennaL7630, whose paper "Environmental Predictors of Human West Nile Virus Infections, Colorado" was published in the last issue of Emerging Infectious Diseases; most people should be able to read it by clicking here.

And also to StephenB7630, who took a week off recently to get married!

Any other good news?

I also want to add that:

AlisaR495
JessicaAr495
DanielO495

...are all graduating this December from the U of A!!

Congrats to all!! : )

Jenean07630 has been awarded a pre-doctoral fellowship from the Breast Cancer Research Program sponsored by the Department of Defense. Good work!

T cells are involved with ischemia reperfusion injury to the kidney

This article examined the role of T cells in acute renal failure (ARF). In vitro, ARF can be studied by performing bilateral ischemia (BI) on both kidneys in mice. This is done by clamping both the right and left kidney peduncles for around 22 minutes, restricting blood flow to both kidneys. After 22 minutes, the clamps are removed, and blood flow returns to the kidney.

Of course, ischemia-reperfusion injury results from doing BI on the kidneys. This causes pro inflammatory cytokines to be released throughout the blood, thus damaging other parts of the body. Recently, it was found that T cells are directly involved with ischemia reperfusion injury (Savransky et. Al, 2006).

Researchers in this article tested their hypothesis by looking at mice with deficient T cell receptors (TCR). They found that mice lacking the TCR were essentially protected against ischemia reperfusion injury; specifically, deficient TCR mice had a lower level of TNF-α and IL-6 (Savransky et. Al, 2006). The researchers used techniques like flow cytometry to confirm their TCR deficient mice were truly deficient, as well as an myeloperoxidase (MPO) assay to determine if neutrophils and macrophages had infiltrated the kidney tissue. However, they found no significant difference in neutrophil and macrophage infiltration at 24 hr between WT and TCR deficient mice (Savransky et. Al, 2006).

I personally found this article interesting because of the nature of my work. I do research on kidney failure and I thought it was great to find an article that correlates between my research and the topics of this class. I’m curious to know if anyone has further tested the data these researchers found by depleting macrophages in WT mice. Specifically, if you inject WT mice with LEC (liposome encapsulating clodronate, a macrophage ‘killer’) and then perform BI in the same fashion as these researchers, would you also get decreased TNF-α and IL-6 results?

Reference

“Role of the T-cell receptor in kidney ischemia-reperfusion injury.”

V Savransky, R R Molls, M Burne-Taney, C-C Chien, L Racusen and H Rabb.

Kidney International (2006) 69, 233-238.

Toxicity of NSAID's in Rat Models

As we read a couple weeks ago in the review articles about the ‘Use of Non Steroidal Anti-inflammatory Drugs’ NSAID’s are very controversial in clinical use as they have shown increase use risk of cardiovascular and cerebrovascular events. This article was very good review article to warn people of the clinical cases that have been reported but there was no lab research done on these drugs. The article “Comparison of the Intestinal Toxicity of Celecoxib, a Selective COX-2 Inhibitor, and Indomethacin in the Experimental Rat” gives a new perspective on NSAID’s and compares two NSAID’s, Celecoxib a selective COX-2 inhibitor and Indomethacin a non-selective COX inhibitor. The claim is that all the problems with NSAID’s are that they cause a ‘topical’ effect and inhibit mucosal constitutive COX-1 enzyme. They used both in vivo and in vitro methods of Celecoxib and Indomethacin to test intestinal permeability and inflammation along with pro inflammatory proteins and production of ulcers. They found that Indomethacin, a non-selective COX inhibitor had a significant damaging effect to the intestinal mucosa, where Celecoxib, a selective COX inhibitor showed less intestinal damage, lower PGF levels and did not show the ‘topical’ effect on the mitochondria.
This article in essence showed a in vivo and in vitro with rat models and found evidence that shows that different NSAID’s may have different effects. Granted that this study was more focused on the digestive system and most of the problems reported have been with the cardiovascular and cerebrovascular systems. It does shed a new light on NSAID’s and maybe the idea to look at research of NSAID’s effect on the heart and brain.

Comparison of the Intestinal Toxicity of Celecoxib, a Selective COX-2 Inhibitor, and Indomethacin in the Experimental Rat
J.A. Tibble, G. Sigthorsson, R. Foster, I Bjarnason

Turmeric Supplements Shown To Prevent Rheumatoid Arthritis Flares

As I last blogged, Turmeric has been used for centuries for treatment of inflammatory disorders. It seems as if it is a too good to be true cure all for numerous ailments though, as there is not much information regarding its true efficiency. The research article entitled "Turmeric Extracts Containing Curcumoids Prevent Experimental Rheumatoid Arthritis" sheds new light on turmeric stating that it does have an antiarthritic effect in Rheumatoid Arthritis (RA). However, do not bother reading the article for yourself because it is actually quite difficult to decipher from which turmeric extract they are speaking of through out the article's entirety. So I will just give you the condensed version!
The curcuminoids extracted from turmeric are believed to inhibit the production of the cytokines TNF-alpha, and IL-1B along with PGE2. They are thought to be inhibitors of transcription factor activation such as the activation of NF-kB. If this is activated during a bout of inflammation it will inevitably lead to increased expression of cytokines, chemokines, and COX-2 (inflammatory protein), which are the mediators of joint inflammation in RA. Random over the counter supplements of turmeric were chosen and then tested to find that the majority of them were actually composed of less than 50% curcuminoids. The researchers prepared and chemically characterized a complex turmeric extract depleted in essential oils and composed of less than 50% curcuminoids, isolating it from a commercial source of turmeric rhizome, and then tested it for the in vitro antiinflammatory efficiency along with its in vivo antiarthritic efficiency. The efficiency of this oil-depleted turmeric fraction was then compared with commercial curcumin product (which have greater than 90% curcuminoids). The animal model used in the experiment was a streptococcal cell wall (SCW) induced arthritis, which is the animal model for rheumatoid arthritis. Over a 28 day time course, female Lewis rats having SCW developed an acute phase of joint swelling to be followed by a chronic phase of inflammation that was characteristic of actual joint destruction.
The oil-depleted turmeric extract (40.6% curcuminoid by weight) was administered intraperitoneally daily starting 4 days before the SCW administration. This was shown to significantly inhibit joint inflammation in the acute inflammatory phase by 64% and the chronic destructive phase of arthritis was inhibited 72%. Alternatively, if treatment was administered after the acute phase but before the start of the chronic phase, the same dose did not have any effect on joint swelling. Also, as would be expected, four days before administering SCW treatment with a lower dose of the turmeric fraction showed no effect on joint swelling.
In contrast to how ineffective a low dose of turmeric fraction was on joint swelling, administration with the same lose does of purified curcuminoids (93.6% curcuminoid by weight) starting four days prior to SCW administration significantly inhibited joint inflammation in both acute and chronic phases of arthritis (75% and 68% inhibition). The turmeric fraction dose had to be increased by six-fold in order to have these same effects. The remaining components of the turmeric fraction , besides the three major curcuminoids, may actually be preventing the antiarthritic effect of the curcuminoids.
A granulomatous inflammatory response occurs in the liver and spleen of the rats at the sites of SCW deposition. A high does of turmeric fraction prevented hepatic granulomas. A low dose of purified curcuminoids had no effect on the formation of granuloma formation. The fact that a high dose of the turmeric fraction inhibited granuloma formation most like indicates that a higher dosage of curcuminoids is necessary to induce an antiinflammatory effect.
Essentially this study provides evidence that turmeric may actually be a legitimate antiinflammatory agent. Turmeric products seem to be useful only for the prevention of arthritis but not for treatment during active inflammation. I will be interested to see in the next couple of years what further research states about turmeric. But what do you think...do you guys believe that this is enough evidence for further clinical testing of curcuminoids from turmeric supplements for inhibiting RA flares?



Turmeric Extracts Containing Curcuminoids Prevent Experimental Rheumatoid Arthritis
Janet L. Funk, Janice N. Oyarzo, Jennifer B. Frye, et al.

Ebola Virus Can Recombine

Ebola virus was discovered in 1976 and has since, swept across Africa causing fever, hemorrhage and death in up to 90% of people who are infected. Just this last August, 76 people were affected in the Democratic Republic of Congo. This disease is equally fatal to ape populations. The lowland gorilla is now classified as an endangered species due to multiple outbreaks over the last few years.

Research comparing recent samples from six gorillas and a chimpanzee revealed that the virus from all animals have similar genes. The genes are also homologous to Ebola virus obtained from human samples in the region. However, when compared to a broader range of Ebola virus samples, the virus sequences map to two separate strains. Gene sequences of Ebola virus in human samples taken before 1996 are all more similar to each other than to viral sequences from human and ape samples since then.

This in itself suggests mutation of the virus, but what is truly frightening is that some human patients carry a copy of one gene specific to each strain suggesting that the virus has recombined. This is rarely seen in RNA viruses, and has never been characterized in Ebola’s broader family of filoviruses to date.

The obvious consequence of recombination capable viruses is that vaccines developed to target specific protein on the Ebola may become ineffective once the protein profile changes.

Mackenzie, Debora. “Ebola evolves deadly new trick”. New Scientist October 13-19, 2007, 12.

Proceedings of the National Academy of Sciences, DOI: 10.1073/pnas.0704076104.

IL-1 and TNF-a: Fascinating past and present

IL-1 and TNF-a: Looking to the past for answers

I recently wrote a review of the discovery of IL-1 and TNF-a for the Rheumatology Report Volume 2, Number 1 (Fall 2007). I took excerpts from this review for this blog and expanded on those I felt were most interesting to share.

For hundred of years, physicians and scientists have endeavored to discover why fever occurs and regresses in human disease. The discovery of cytokines and their inhibitors has led to a more complex understanding of the regulation and dysregulation of the immune system. The history of the discovery TNF-a and the biologic TNF-a inhibitors will be addressed as well as IL-1b and its inhibitor IL-1Ra. A review of the role of the IL-1b and TNF-a in synovial inflammation and potential targets for treatment of Rheumatoid Arthritis including gene based therapy, vaccine therapy and mesenchymal stem cell therapy will be outlined.

Coley’s toxin and the discovery of TNF-a

IN 1891, Dr. William Coley frustrated by the inability of aggressive surgery to result in a cure of sarcoma searched through the medical records of his hospital. Dr. Coley found the records of a 7 year old child who recovered from sarcoma following an infection with erysipelas. Dr Coley then proceeded to treat a patient with lymphoma with multiple injections of streptococcal cultures at 3-4 day intervals. The tumor underwent necrosis after the patient developed an attack of erysipelas and the patient remained disease free for 8 years. Throughout the remainder of his career, Dr. Coley used injection of streptococci and heat killed streptococci plus Serratia marcescens, (“Coley’s toxin”) to treat patients with malignant tumors. The dose of “Coley’s toxin” needed to produce a temperature of 40-40.5 degrees Celsius in order to induce necrosis of the tumor. Coley observed that infection resulted in a systemic response that resulted in tumor destruction.1 Many years later in 1975, an endotoxin-induced serum factor was found to be responsible for the necrosis of tumors and named Tumor Necrosis Factor. 2

Inhibition of TNF

In 1988, an inhibitory protein of TNF was isolated from the urine of febrile patients. 3 The protein inhibited with the function of TNF by blocking the binding of TNF to its’ receptor. The protein was found to bind both TNF-a and TNF-b. 4 In subsequent years, many members of the TNF-family members and their receptors were discovered as well their roles in rheumatic disease and novel ways to block their activity were developed. 5 In 1993, chimeric monoclonal antibodies to TNF-a were used to block its activity and to successfully treat rheumatoid arthritis. 6 Modification of a TNF receptor for the successful treatment of rheumatoid arthritis was accomplished in 1997 with the design of a recombinant soluble TNF receptor (p75) linked to the Fc portion of human IgG1. 7 The soluble TNF receptor (p75) binds and blocks the activity of both TNF-a and TNF-b.

The History of IL-1 and its Inhibition

In 1926, Zinsser and Tamiya discovered that contact with tissues of animals infected with tuberculosis resulted in the production of a toxic factor in uninfected cells. They suspected that a protein constituent of mycobacterial growth or of the mycobacterium itself stimulated cells to liberate a toxic substance on other cells or within the cell itself. 8 These observations may have been the first described effects of IL-1.

In the 1970s, numerous investigators simultaneously identified factors that modulate lymphocyte function including lymphocyte-activating factor, mitogenic protein, helper peak-1, t cell-replacing factor III, T cell-replacing factorMF, B cell-activating factor and B cell differentiation factor. It was determined that these factors were identical and in 1979 all were renamed IL-1. 9 The pleiotropic actions of IL-1 explain its simultaneously description and discovery by multiple investigators. In subsequent years, different receptors and inhibitors of IL-1 were discovered, importantly IL-1 Receptor Antagonist (IL-1Ra) at the University of Colorado. The first clinical observation of variations of IL-1Ra was in children with systemic juvenile inflammatory arthritis and reported in 1987. 10 In 1996, the use of recombinant human IL-1Ra, anakinra, was used to treat patients with rheumatoid arthritis. 11 More recently, IL-1Ra has been successfully used in treating patients with juvenile onset and adult onset Still’s disease. 12-14

How the synovium may be subject to inflammation in rheumatoid arthritis

The initial site of inflammation in rheumatoid arthritis has remained elusive. Ochi et al reported on the ability of fibroblastic stromal cells from the bone marrow to migrate to the joint space and form synovial tissue in the mouse model of collagen-induced arthritis. This specific population of fibroblast stromal cells can act as nurse-like cells and have the ability to interact with lymphocytes and monocytes and induce cellular differentiation and promote biological activities that mimic features of rheumatoid inflammation. 15 The findings of this study provide evidence of the bone marrow’s ability to maintain a disease state and to direct localization to the joints. It has yet to be established how the nurse-like cells of fibroblast stromal origin migrate from the bone marrow to the joint space.

A mechanism by which antibodies may gain access to joint compartment was reported by Binstadt et al, 2006. Using intravital imaging, they identified that arthritogenic antibodies from the K/BxN arthritis model caused vasopermeability localized to sites leading to the development of arthritis in normal mice. This vasopermeability was dependent upon mast cells, neutrophils and FcgRIII but not complement, TNF or IL-1. 16 Neither of these studies identified the initial site of inflammation or trigger of inflammation in rheumatoid arthritis, but provided insight into the mechanisms of continued disease activity and identify potential targets for future treatment modalities.

Cadherin- 11, an adhesion molecule, was shown to play a critical role in establishing synovial cell- to cell- contact necessary for synovial lining formation. Caherin-11 is necessary for the establishment of the K/BxN serum mediated transfer of arthritis as cadherin-11-null mice failed to develop inflammatory arthritis and had a poorly organized synovium. Similarly, use of anti-cadherin 11 in established arthritis helped to ameliorate disease in the K/BxN serum transfer mouse model of arthritis.17

IL-1b and TNF-a induce the overgrowth of synovial cells

In collagen-induced arthritis, IL-1b and to a lesser extent TNF-a were found to induce the expression of synoviolin in mouse synovial fibroblasts. IL-1b induced synoviolin transcription, which in turn enhanced IL-1b induced synovial fibroblast proliferation. The synovial fibroblasts produced more IL-1b for the induction of synoviolin leading to a positive feedback loop that may be critical in maintenance of rheumatoid inflammation. 18

Targeting inflammatory cytokines through gene based therapy

In a phase-I trial, fibroblast-like synoviocytes were transduced with a retroviral vector containing the gene for IL-1Ra and injected into the metacarpophalangeal joints. The transfer and expression of IL-1Ra was safely and successfully accomplished but no follow-up clinical studies are planned using this method. 19

A phase I dose escalation trial using delivery of a recombinant adeno-associated virus containing the TNF-receptor-Fc immunoglobulin fusion gene (tgAAC94) has been conducted and a phase I/II trial was being conducted when a study participant died.19 The woman had a low-grade fever and fatigue for several days before she received a second dose of active drug, dosed at10 trillion tgAAC94 particles per milliliter. Within a few days after the second injection, her condition worsened and she died of histoplasmosis. Last month, at the American College of Rheumatology meeting in Boston, results of an investigation into the woman’s death were released. Genetic analyses of tissue samples from the woman showed 500,000 copies/microgram of tgAAC94 in the injected knee, but fewer than 30 copies/microgram in other tissues including the other knee, liver, spleen, tonsil, and bowel. Wild-type AAV copies were found at low or undetectable levels in the various tissues. The investigation found no evidence that the agent had been contaminated with Histoplasma. 20 The FDA announced this week that the trial has been re-opened for enrollment.

Before gene therapy is successful, it needs to be determined if neutralizing antibodies to the viral vectors will render the therapy ineffective. Additionally, methods for producing vectors needs to be optimized and how therapeutic gene expression will be regulated in the human host needs to be established. 19 Methods to measure the suppression of systemic TNF activity attributable to gene therapy need to be designed to determine the activity of the gene therapy. Moreover, there are numerous safety concerns to be addressed.

TNF-a kinoid vaccination as a potential therapy for rheumatoid arthritis

Le Buanec et al, 2006 have developed a vaccine using a keyhole limpet hemocyanin-hTNF-a immunogen. When injected into hTNF-a transgenic mice in incomplete Freund’s adjuvant, a high-titer of neutralizing antibodies to hTNF-a were produced that eliminated the bioactivity of hTNF-a and resulted in reversal of arthritis. 21 Further studies are needed in other mouse models and caution should be used in the interpretation of these results, as completely limiting the function of TNF-a in humans would likely result in deleterious effects including increased risk of infection and malignancy.

Immunomodulation through the use of mesenchymal stromal cells

The report of an interdisciplinary meeting addressing the potential role for multipotent mesenchymal stromal cells in the pathogenesis and management of autoimmune diseases was published in January 2007. Mesenchymal stromal cells, derived from the bone marrow, in vitro exhibit antiproliferative effects on T and B lymphocytes, dendritic cells, natural killer cells and B cell tumor cell lines. 22,23 In the collagen-induced arthritis mouse model, injecting a murine mesenchymal cell line systemically or intra-articularly was not effective in modulating disease and labeled mesenchymal stromal cells were not found in the articular injection sites. The lack of therapeutic effect was attributed to increased levels of TNF-a and its reversal of the immunosuppressive properties of the mesenchymal stromal cells. 24 More data is needed on the optimal source of mesenchymal stromal cells as those from autoimmune diseased patients may not be equivalent to those from healthy individuals.25 Additionally, optimal timing, location and number of cells to be given have yet to be determined. 23

Final Thoughts

The extensive scientific and clinical work over the last century has led to exciting discoveries in immune system biology and the pathogenesis of autoimmune diseases. The experiences of the past with infectious agents and the responses they elicit led to the discovery of cytokines and there potential use in altering immune response to benefit individuals. Although blocking of one cytokine with current biologic therapies has led to improved management of rheumatoid arthritis, for many patients blocking only one cytokine may not be sufficient. 26 New ways of targeting cytokines and their regulation may be possible in the future with gene therapy, immunization therapies and tissue specific synovial based therapies.
We shall not cease from exploration
And the end of all our exploring
Will be to arrive where we started
And know the place for the first time.

Little Gidding V, Four Quartets, T.S. Eliot (1943)

For an interesting discussion of Dr. Coley’s work and progress made after his death see: http://www.coleytoxins.com/1893htm.

References

1. Bickels J, Kollender Y, Merinsky O, Meller I. Coley's toxin: historical perspective. Isr Med Assoc J. Jun 2002;4(6):471-472.
2. Carswell EA, Old LJ, Kassel RL, Green S, Fiore N, Williamson B. An endotoxin-induced serum factor that causes necrosis of tumors. Proc Natl Acad Sci U S A. Sep 1975;72(9):3666-3670.
3. Seckinger P, Isaaz S, Dayer JM. A human inhibitor of tumor necrosis factor alpha. J Exp Med. Apr 1 1988;167(4):1511-1516.
4. Engelmann H, Aderka D, Rubinstein M, Rotman D, Wallach D. A tumor necrosis factor-binding protein purified to homogeneity from human urine protects cells from tumor necrosis factor toxicity. J Biol Chem. Jul 15 1989;264(20):11974-11980.
5. Ashkenazi A. Targeting death and decoy receptors of the tumour-necrosis factor superfamily. Nat Rev Cancer. Jun 2002;2(6):420-430.
6. Elliott MJ, Maini RN, Feldmann M, et al. Treatment of rheumatoid arthritis with chimeric monoclonal antibodies to tumor necrosis factor alpha. Arthritis Rheum. Dec 1993;36(12):1681-1690.
7. Moreland LW, Baumgartner SW, Schiff MH, et al. Treatment of rheumatoid arthritis with a recombinant human tumor necrosis factor receptor (p75)-Fc fusion protein. N Engl J Med. Jul 17 1997;337(3):141-147.
8. Zinsser H, Tamiya T. An Experimental Analysis of Bacterial Allergy. J Ex Med. 1926;44:753-776.
9. Revised nomenclature for antigen-nonspecific T cell proliferation and helper factors. J Immunol. Dec 1979;123(6):2928-2929.
10. Prieur AM, Kaufmann MT, Griscelli C, Dayer JM. Specific interleukin-1 inhibitor in serum and urine of children with systemic juvenile chronic arthritis. Lancet. Nov 28 1987;2(8570):1240-1242.
11. Campion GV, Lebsack ME, Lookabaugh J, Gordon G, Catalano M. Dose-range and dose-frequency study of recombinant human interleukin-1 receptor antagonist in patients with rheumatoid arthritis. The IL-1Ra Arthritis Study Group. Arthritis Rheum. Jul 1996;39(7):1092-1101.
12. Fitzgerald AA, Leclercq SA, Yan A, Homik JE, Dinarello CA. Rapid responses to anakinra in patients with refractory adult-onset Still's disease. Arthritis Rheum. Jun 2005;52(6):1794-1803.
13. Vasques Godinho FM, Parreira Santos MJ, Canas da Silva J. Refractory adult onset Still's disease successfully treated with anakinra. Ann Rheum Dis. Apr 2005;64(4):647-648.
14. Aarntzen EH, van Riel PL, Barrera P. Refractory adult onset Still's disease and hypersensitivity to non-steroidal anti-inflammatory drugs and cyclo-oxygenase-2 inhibitors: are biological agents the solution? Ann Rheum Dis. Oct 2005;64(10):1523-1524.
15. Ochi T, Yoshikawa H, Toyosaki-Maeda T, Lipsky PE. Mesenchymal stromal cells. Nurse-like cells reside in the synovial tissue and bone marrow in rheumatoid arthritis. Arthritis Res Ther. 2007;9(1):201.
16. Binstadt BA, Patel PR, Alencar H, et al. Particularities of the vasculature can promote the organ specificity of autoimmune attack. Nat Immunol. Mar 2006;7(3):284-292.
17. Lee DM, Kiener HP, Agarwal SK, et al. Cadherin-11 in synovial lining formation and pathology in arthritis. Science. Feb 16 2007;315(5814):1006-1010.
18. Gao B, Calhoun K, Fang D. The proinflammatory cytokines IL-1beta and TNF-alpha induce the expression of Synoviolin, an E3 ubiquitin ligase, in mouse synovial fibroblasts via the Erk1/2-ETS1 pathway. Arthritis Res Ther. 2006;8(6):R172.
19. Adriaansen J, Vervoordeldonk MJ, Tak PP. Gene therapy as a therapeutic approach for the treatment of rheumatoid arthritis: innovative vectors and therapeutic genes. Rheumatology (Oxford). Jun 2006;45(6):656-668.
20. Gever J. www.medpagetoday.com/MeetingCoverage/ACRMeeting/tb/7390.
21. Le Buanec H, Delavallee L, Bessis N, et al. TNFalpha kinoid vaccination-induced neutralizing antibodies to TNFalpha protect mice from autologous TNFalpha-driven chronic and acute inflammation. Proc Natl Acad Sci U S A. Dec 19 2006;103(51):19442-19447.
22. Di Nicola M, Carlo-Stella C, Magni M, et al. Human bone marrow stromal cells suppress T-lymphocyte proliferation induced by cellular or nonspecific mitogenic stimuli. Blood. May 15 2002;99(10):3838-3843.
23. Tyndall A, Walker UA, Cope A, et al. Immunomodulatory properties of mesenchymal stem cells: a review based on an interdisciplinary meeting held at the Kennedy Institute of Rheumatology Division, London, UK, 31 October 2005. Arthritis Res Ther. 2007;9(1):301.
24. Djouad F, Fritz V, Apparailly F, et al. Reversal of the immunosuppressive properties of mesenchymal stem cells by tumor necrosis factor alpha in collagen-induced arthritis. Arthritis Rheum. May 2005;52(5):1595-1603.
25. Del Papa N, Quirici N, Soligo D, et al. Bone marrow endothelial progenitors are defective in systemic sclerosis. Arthritis Rheum. Aug 2006;54(8):2605-2615.
26. Dayer J. How Far Are We From Understanding Rheumatoid Arthritis? Ann Rheum Dis. 2007;66(Suppl II)(9).

HIV is a superantigen for B-cells

I have worked on a variety of human immunodeficiency virus (HIV) projects, but only recently became aware of not-so-new research suggesting the HIV envelope glycoprotein, gp 120, is an Ig superantigen for a subpopulation of B cells. A superantigen has been defined as an antigen binding 5-25% of the T or B cell population, dramatically more than the <0.01% of a conventional antigen (Goodglick, 1995).

It is well known that infection with HIV depletes CD4+ T cells. B cell repertoires, which lack CD4, are also impacted by the virus (Berberian, 1993). Antibody specificity is determined by the variable regions of their heavy (VH) and light chains. The VH genes are divided into 7 families (VH1-7) that are distinguished by at least 80% nucleotide homology in certain VH framework regions. In adult peripheral B cells, VH3 comprises about half of the expressed VH repertoire (Scamurra, 2000). HIV gp120 stimulates endogenous B cells from the VH3 family by binding to membrane Ig, outside the conventional, (hypervariable) Ag-binding site. HIV-1 gp120 has thus been labeled a superantigen, binding an estimated 4-6% of the B cell population (Goodglick, 1995). This binding results in an increase in VH3 B cells in early disease. This overactive humoral state is followed by a decline in B cell response which is prior to the significant depletion of CD4+ T cells (Karray, 1997). The resulting B cell loss and/or shift in distribution may result in impaired humoral responses to infections and vaccines (Scamura, 2000).

After writing the above, I received some pre-blog feedback from Elisabeth Bowers, a pre-doctoral fellow in the Microbiology department working in this area. She noted that a direct association between the increase in VH3 B cells in early disease and the B cell dysfunction seen during infection has not been shown. (B cell dysfunction includes hypergammaglobulinemia –a high amount of antibody in the blood, lymphadenopathy, and increased risk of B cell lymphomas.) The B cell dysfunction may be, in part, a result of increased VH3 B cells, but much of the response is directed against HIV. “So the current hypothesis for some (but not all) of the B cell dysfunction is humoral immune reaction to the virus itself, leading to B cells producing virus-specific antibodies, and ‘bystander’ B cell responses – B cells that are not specific to virus, but still become activated anyway (these may not all be VH3 B cells) (Shirai, 1992; Amadori, 1990; Schnittman, 1986). B cell abnormalities have been directly associated with high viral loads (Viau, 2007).”

Berberian, et al., “Immunolglobulin VH3 gene products: natural ligands for HIV gp120”. Science. 1993. 261: 1588-91.

Goodglick, et al,. “Mapping the Ig superantigen-binding sit of HIV gp120”. The Journal of Immunology. 1995. 155: 5151-59.

Karray, et. al., “Identification of the B cell superantigen-binding site of HIV-1 gp120”. PNAS. 94: 1356-60.

Scamurra et al., “Impact of HIV-1 infection of Vh3 gene repertoire of naïve human B cells”. The Journal of Immunology. 2000. 164: 5482-91.

Shirai et al., “Human Immunodeficiency Virus Infection Induces Both Polyclonal and Virus-specific B Cell Activation”. The Journal of Clinical Investigation. 1992. 89: 561-566.

Amadori et al., “B-cell activation and HIV-1 infection: deeds and misdeeds”. Immunology Today. 1990. 11(10): 374-379.

Schnittman et al., “Direct Polyclonal Activation of Human B Lymphocytes by the Acquired Immune Deficiency Syndrome Virus”. Science. 1986. 233: 1084-1086.

Viau et al., “Direct impact of inactivated HIV-1 virions on B lymphocyte subsets”. Molecular Immunology. 2007. 44: 2124-2134.

Possible reasons why the Merck HIV vaccine failed

Going along with the theme of the previous few blog posts, I was going to blog about the failed Merck HIV vaccine trials.  Instead, I decided to delve a little bit deeper into the hypotheses attempting to explain WHY this vaccine, even the promising results of increased CD8+ killer T cell numbers, may have ultimately failed.

The MRKAd5 vaccine was based on the premise of inserting copies of 3 artificially generated HIV genes into a genetically engineering viral vector (2).  The vector used in the HIV vaccine, and many other viral vaccines, is adenovirus5, an inactivated form of a "common cold" virus.  The hopes for this vaccine were that the immune system would recognize the viral components enough that if a vaccinated individual were exposed to HIV, there would be enough of a response to prevent, or at least delay, the onset of AIDS (1).  However, after a conference in Seattle to discuss the new evidence that the MRKAd5 vaccine is "not efficacious" and may result in INCREASED risk for HIV infection (3), several ideas have been brought forth in an attempt to explain why the vaccine failed.  One theory is based on the idea that if a vaccinated volunteer was previously exposed to adenovirus5 (the viral backbone of the vaccine), the subsequent response would be biased towards CD4+ cells rather than the Cd8+ cells the vaccine was intended to amplify.  An increase in CD4+ cells only results in the production of more cells that the HIV virus can go on to infect (Dr. Keith Gottesdiener, unpublished data). Some preliminary results suggest that some people who have prior immunity to adenovirus5 may indeed be the ones who are at higher risk for HIV infection.  The second theory depends on the idea that perhaps this system generates too much antigen for a prolonged period of time.  The excess antigen causes "exhaustion" of the immune cells, which then fail to proliferate and die (6).

Regardless of the actual unforeseen mechanisms that caused the MRKAd5 vaccine to fail,  the outcome of this trial raises questions concerning the use of similar adenovirus vectors in vaccine creation.  Several other vaccine trials, including an Ebola vaccine and another HIV vaccine, have been slowed, postponed or altered so that the test population pool are individuals with low risk of exposure to colds (4).  It also reminds us that while data generated in model organisms is invaluable, care must be taken when extrapolating the putative animal model into human applications, as we've seen several times during this course.  In the case of the MRKAd5 vaccine, the data generated in primates suggested that the vaccine upregulated the number of CD8+ cells (7), although later on, it was shown in mouse that these additional cells may not be completely functional (2).

1.  Ho, DD.  (2007).  A Shot in the Arm for AIDS Vaccine Research.  PLoS Med 2(2) :e36.

2.  Lin, SW et al.  (2007).  Recombinant adeno-associated virus vectors induce functionally impaired transgene product-specific CD8 T cells in mice.  J Clin Invest.  2007 Nov. 15 [Epub ahead of print].

3.  Cohen, J.  (2007).  Did Merck's vaccine cause harm?  Science 312(5853):1048-9.

4.  Stark, K.  (2007).  HIV trial's halt reverberates.  www.philly.com/philly/business/20071116_HIV_trials_halt_reverberates.html

5.  Cohen, J.  (2007).  Promising AIDS vaccine's failure leaves field reeling.  Science 318(5847):28.

6.  Kaiser Daily AIDS/HIV report from November 16, 2007.  www.kaisernetwork.org/daily_reports/rep_index.cfm?DR_ID=48904

7.  Shiver, JW and Emini EA.  (2004).  Recent advances in the development of HIV-1 vaccines using replication-incompetent adenovirus vectors.  Annu Rev Med 55:355-72.

Believe them or not, interesting associations among “old friends”, gut dysfunction and autism

Ever since our first Immunology class, the “old friends”, commensal flora, had caught my attention since they seemed to be able to survive from human immune system’s attack and I was very interested in finding out how they do that. It turned out that this is still a largely unknown question. People agree that the establishment of commensal flora after birth represents a major developmental and functional stimulus to the immune system. The fighting between the immune system and the commensal flora finally creates a mucosal immune homeostasis (1). In addition to stimulate immune system to mature, the “old friends” also support digestion, absorption and metabolism; they compete with pathogens in our intestine for space and resources too (2).

The most interesting thing I found during my web search about commensal flora is a hypothesis on the association between autism and gut dysfunction which could be caused by the altered “old friends”. Autism is a developmental disorder for which there is no drug or even a known cause. One of the hypotheses of its cause is the alteration of gut flora leads to increases in intestinal permeability, also known as “leaky gut” which allows the passage of neuroactive peptides digested from food origin into the blood and then into the cerebrospinal fluid to interfere directly with the function of the CNS (3). In the presence of an alteration of the gut flora, or immune dysregulation, or agenetic predisposition, there is a sustained chronic inflammation and release of calprotectin from neutrophils. The inflammatory response of the intestinal epithelial cells to pathogeneic and altered gut flora can lead to increases in intestinal permeability thus allow the passage of toxins, microbes, undigested food, waste or larger than normal macromolecules (2). Digestion products of natural foods such as cow’s milk and bread are able to enter the blood through the leaky mucosa and induce antigenic responses, as well as interfere directly with the central nervous system. Casomorphins and gliadomorphins are good examples as the short chain neuroactive peptides digested from dietary gluten and casein in the lumen of the small intestine by the action of pancreatic and intestinal peptidases. These peptides, which are structurally similar to endorphins, are called exorphins to reflect their dietary origin (3). Interestingly, a substantial group of autistic children have been diagnosed to have increased intestinal permeability (4, 5).

It seems that the true, unaltered “old friends” are really versatile good friends.


Reference:
1. http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=eurekah.section.
2. http://littlecanaries.org/gastro.htm
3. White, JF. Intestinal Pathophysiology in Autism Exp Biol Med (Maywood) 2003, 228, 639–649.
4. Horvath K, Zielke RH, Collins J, et al. Secretin improves intestinal permeability in autistic children. J Pediatr Gastroenterol Nutr 2000;31: S30-31.
5. D'Eufemia P, Celli M, Finocchiaro R, et al. Abnormal intestinal permeability in children with autism. Acta Paediatr 1996;85: 1076-1079.

HIV vaccines

The pursuit to develop a vaccine against HIV has been a subject of some discussion in our class. Unfortunately, it does not seem likely that a vaccine would be able to prevent HIV infection. However, the development of a T-cell vaccine against HIV would be a powerful tool in the toolbox of HIV treatment. A T-cell vaccine aimed at the period of susceptibility directly following infection would lower the initial “burst of viremia” and lead to better patient outcomes. Mellors et al. (1996) found that the viremia measurements at initial infection were powerful predictors of prognosis for progression from HIV to AIDS and for AIDS-related death. Likelihood of transmission to secondary hosts is also predicted by viremia. A vaccine that could lessen the impact of HIV infections in individuals and dampen the spread of the disease would represent an amazing stride forward.

The infection and slow depletion of CD4+ T-cells that characterizes HIV leads to the eventual loss of immune function and overt AIDS. The idea behind Merck’s recent STEP vaccine trial was to immunize recently infected HIV patients with a recombinant adenovirus (serotype 5, hence the name: Ad5) vector carrying three genes contained within the HIV genome: gag, pol, and nef. There are only 9 identified genes within the viral genome of HIV. This immunization would prime the T-cells to recognize the virion and preempt systemic invasion. The results of the trial were abysmal. Of 3000 high-risk patients that were initially enrolled, randomized, blinded and treated, 82 people were infected with HIV over the course of 90 weeks. Efficacy of the treatment was based on viral load setpoint and HIV infection rates. 4.6% of treatment cases went on to develop HIV, while 3.1% of placebo controls developed HIV. Perhaps the vaccine was worse than futile, but actually increased the incidence of infection. As a result, Merck halted the trial.

Vaccines of this vector variety have been found to be effective in eliciting immune responses in Rhesus monkeys. However, many human populations have been found to have pre-existing immunity to Ad5. This immunity limits the immunogenicity of recombinant Ad5 vaccines and might explain the failure of the STEP trial. However, the creation of chimeric vectors may restore the immunogenicity of the Ad5 viral coat.

On a related note, resistance to progression of HIV-1 infection has been found to be associated with the allele HLA-B*5701. This emphasizes the important role that class I-restricted CD8+ T-cells can play in resistance to disease progression.

References

STEP Study Summary; Press briefing, November 7, 2007. http://www.hvtn.org/media/pr/STEPPressbriefingNov2007.pdf

M.I. Johnson and A.S. Fauci. 2007. An HIV Vaccine – Evolving Concepts. New England Journal of Medicine 356:2073-2081.

J.W. Mellors, C.R. Rinaldo Jr., P. Gupta, R.M. White, J.A. Todd, L.A. Kingsley. 1996. Prognosis in HIV-1 Infection Predicted by the Quantity of Virus in Plasma. Science 272(5265): 1167-1170.

HIV virus sequence compendium 2005. http://www.hiv.lanl.gov/content/hiv-db/COMPENDIUM/2005/0.pdf. Accessed: 11/21/2007.

Conrad.org. Novel Adenovirus Vector-Based Vaccines for HIV-1. D. Barouch presenting, November 17, 2006. http://www.conrad.org/pdf/BiomarkersMtg.Barouch.pdf

Migueles, S.A. et al. 2000. HLA B*5701 is highly associated with restriction of virus replication in a subgroup of HIV-infected long term nonprogressors. PNAS Immunology 97(6): 2709-2714.

Good news from the virology frontlines

To date, HIV treatment regimens have primarily targeted virus enzymes or the process of virus-cell fusion, but not the integrated proviral DNA. Current highly antiretroviral therapy (HAART) targets the viral reverse transcriptase, protease and fusion proteins and this regime has transformed HIV-1 infection into a chronic disease and curtailed the mobidity of infected individuals⁽¹⁾…that is, if you have access to antiretroviral drugs.

Since traditional vaccine aproaches have, up to date, failed miserably⁽²⁾, new therapeutic interventions must be brought to the clinic.

A new attractive alternative has just been published⁽³⁾ which is the specific erradication of the integrated HIV-1 provirus⁽⁴⁾ in the host’s DNA.

In brief, the authors evolved a CRE recombinase⁽⁵⁾⁽⁶⁾ which specifically recognizes sequences present in HIV-1 Long Terminal Repeats (LTRs)⁽⁷⁾ and “splices out” the proviral DNA in cells infected with HIV-1.

This piece of work is quite interesting as proof-of-principle but it’s unlikely to be of immediate therapeutic use, although other authors have been able to engineer Cre recombinases which are cell membrane permeable and can even cross the blood-brain barrier⁽⁸⁾.

Stay tuned…

References:

1) Lalezari JP et al. “Enfuvirtide, an HIV-1 fusion inhibitor, for drug-resistant HIV infection in North and South America.” N Engl J Med. 2003 May 29;348(22):2175-85. Epub 2003 Mar 13

2) http://www.nature.com/news/2007/071114/pdf/450325a.pdf

3) Sarkar, Indrani et al. “HIV-1 proviral DNA excision using an evolved recombinase” Science. 2007 Jun 29;316(5833):1912-5

4) For a small review on the life cycle of a retrovirus: http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=mcb.figgrp.1437

5) For more on CRE recombinase:

http://en.wikipedia.org/wiki/Cre_recombinase

(6) Glaser S, et al “Current issues in mouse genome engineering.” Nat Genet. 2005 Nov;37(11):1187-93.

(7) For more on LTR’s please visit:

http://www.stanford.edu/group/nolan/tutorials/retcl_3_ltrs.html

(8) Jo, Dawoong et al. “Epigenetic regulation of gene structure and function with a cell-permeable Cre reombinase” Nat Biotechnol. 2001 Oct;19(10):929-33

Something to chew on (with your turkey)

When you are sitting down to your Thanksgiving dinner, and already dreaming about the soup that will be made out of the leftovers, chew on this (slightly silly) tid-bit...chicken soup has anti-inflammatory properties! In a special report (CHEST 2000, 118:1150-1157), Rennard et al. studied the effects of chicken soup on neutrophil chemotaxis in vitro. Sure, in vivo would be better, but that's for another study!
The study prepared a tasty soup (with matzoh balls) from scratch and tested it at different stages of preparation, and compared this to commercially available soups.
Overall, chicken soup was found to inhibit neutrophil chemotaxis compared to media in a concentration-dependent manner. (and luckily, chicken soup was not toxic to neutrophils)
When comparing the commercially available soups (13 different brands) to the homemade soup, the authors found that most inhibited chemotaxis, but some (such as Campbell's Ramon noodles, chicken flavor) enhanced chemotaxis.
Overall, a nice warm bowl of chicken (or turkey) soup may decrease inflammation as well as loosening nasal secretions (due to the vapor), and perhaps improve cilia function (CHEST 1978;10:408-410)

Happy Thanksgiving!!

Where is the peanut allergy vaccine?

In our last class, Dr. Cohen mentioned that some anti-IgE antibodies have been tested in people with food allergies, most interestingly in people with allergies to peanuts. Peanut allergies seem to be on the rise in children in recent years, and can substantially decrease the quality of life for a person (and the families of people) with such allergies. A study published by Leung et al., in 2003 examined the effects of a humanized IgG monoclonal antibody against IgE called TNX-901. This antibody prevents IgE from binding to the Fc receptor on mast cells.

A placebo-controlled, double blind food challenge established that four subcutaneous injections (450 mg) of TNX-901 every four weeks (for a total study time of 16 weeks) raised the allergen tolerance threshold from one-half peanut to nine peanuts in subjects. While this is not enough to allow a person with peanut allergies to begin eating peanuts regularly, it is probably sufficient to prevent a deadly reaction in case of accidental exposure – and provide some peace of mind for those that must vigilantly avoid peanut exposure.

Although the results of the initial study were very promising, a legal battle over infringement rights between the drug’s maker Tanox, Inc., and Novartis and Genentech has shelved the drug indefinitely. A similar anti-IgE drug, omalizumab, is approved to treat asthma and is sometimes used off-label for those with extreme food allergies (for more on the mechanisms of this drug see Holgate et al., 2005). However, drug trials approving its use in food allergies, and thus insurance coverage for those who need it for such reasons, remain a few years off. In the end, it seems a drug that was fast-tracked because of its promising initial results has been delayed by years because of legal issues.

Holgate ST et al. (2005). Anti-immunoglobulin E treatment with omalizumab in allergic diseases: an update on anti-inflammatory activity and clinical efficacy. Clin Exp Allergy. 35(4):408-16.

Leung DY et al. (2003). Effect of anti-IgE therapy in patients with peanut allergy. N Engl J Med. 348(11):986-93.

The Great Diet Debate

Cardiovascular disease is one of the major killers in today’s society. There are many risk factors that contribute to cardiovascular disease, some that are modifiable and others that are not. One of the major factors that are modifiable is obesity and hypercholesterolemia. Prevention and treatment for heart disease are very important and sometimes go hand in hand. The South Beach Heart Program is a diet that claims to ‘detect, prevent and even reverse heart disease.’ Studies of this program however did show that LDL levels did not go down and with that in mind high LDL levels is a major factor for cardiovascular disease. So what do the people on this diet do if the diet is not lowering there LDL levels, they take statins, cholesterol lowering drugs. Here is were I believe the problem lies, and that is with the idea that we as a society want a drug or pill to take to help us when there is a cheaper and just as effective way to obtain our optimal health and that is diet and exercise. Diet and exercise can help an individual so much to achieve a healthy life but they need to be done together and be a part of a lifestyle. People who have made intensive changes to their lifestyle with diet and exercise for a year have dropped their LDL levels 40 percent and that is equal to the statin Lipitor. I have no problem with statins and I know that the results that statins get are desired ones, yet I think this falls in line with us as a society being lazy and wanting pill to help us and us not wanting to do physical labor as exercise. Diet and exercise can do so much more than just a pill can do and just help with LDL levels; it can help with the other risk factors for cardiovascular disease as obesity, which can lead to many other problems like diabetes and hypertension. The underlying factor that I believe is and should be is that diet and exercise are vital for us to live and maintain a healthy life as individuals.

How does codeine release histamine?

Simple answer: not known. There are 2 commonly used histamine releasers, codeine and compound 48/80, a synthetic polyamine. Codeine apparently is idiosyncratic: histamine release induced by codeine is not blocked by the opiate antagonist naloxone, and other opiates like fentanyl do not release histamine. Both codeine and 48/80 probably engage the downstream signaling pathway of the IgE receptor, mimicking crosslinking. This can be a side effect of codeine-related medications, as the other Dr. Cohen can attest, after being covered in hives when given some oxycodone.

Type 1 Diabetes and Viral Infections

Type 1 Diabetes has a long standing history as a largely genetic autoimmune disease passed through families. This is of course supported by looking at the genes of an individual and specifically looking at his or her HLA region to give a percent of risk of diabetes. This was only a potential risk and even with continuing markers for diabetes, antibodies being one, we have yet to be able say for sure whether or not a child will get diabetes until they do. A new component has emerged to identify both a genetic and environmental component to disease progression or, as far as immunology is concerned, a link between adaptive and innate immunity in type 1 Diabetes.

The role of the innate immune system in the development of type 1 diabetes in at risk individuals is currently under investigation and while the complete mechanism is still unclear what is clear is that enterovirus infections are involved. A 2005 paper by Merja Roivainen outlines the potential pathways that EV may take in the destruction of insulin producing β-cells and potential clinical applications. At approximately 80% loss of pancreatic insulin producing β-cells diabetes becomes symptomatic and treatment is required. In previous studies by the same author they were able isolate enterovirus RNA via PCR from the postmortem islet cells of T1D (1). Other studies have also demonstrated a link between enterovirus infection and type 1 diabetes, atherosclerosis disease, myocardial infarction, bronchiolitis and asthma (1). While this may be well established it is unclear as to a mechanism for the virus to ultimately induce or influence disease.

There are three major theories on possible mechanisms for the virus to damage β-cells. First, upon reaching β-cells the virus destroys islet cells and exposes the immune system to “previously hidden self-components” (1). This is similar the case we discussed in class where a researcher accidentally exposed his immune system to brain tissue and his immune attacked his brain, which it otherwise never saw, causing encephalitis. Second, there may be a bystander component to the viral infection where the pancreatic islet cells are damaged by being too close to the viral infection. Lastly there also may be a molecular mimicry component in which the immune system reacts to the virus and, because of similarity between autoantigens such as GAD-65 and HSP-60 and viral proteins 2c and VP0, to the islet cells. An interesting genetic component that is also related is an new SNP that has been associated with diabetes on a protein that is involved in the innate immune response. A single nucleotide polymorphism on the IFIH1 (Inerferon-Induced Helicase 1), a protein that dices incoming viral dsRNA and also is involved in intracellular signaling for the release of cytokines and chemokines in, has been associated with diabetes (2). A viral trigger to diabetes does however provide new hope to potential for clinical applications for prevention.

Clinical immunizations may be the future of diabetes prevention but there are some hurdles to overcome. We know little about what specific strains are causing the progression of diabetes and given that there are currently one hundred known enteroviruses that can infect humans. Also there could be only single amino acid differences between one virus that causes diabetes and one that doesn’t so given that viral RNA replication in a host is error-prone it is likely that an immunization wouldn’t be able to keep-up with the continually mutating virus. While these may be true continued research in viruses my one day both wipe them of the face of the earth and cure disease.

1. Roivainen, Merja. Enertoviruses: New findings on the role of enteroviruses in type 1 diabetes. The Int. J. of Biochem and Cell Bio., 2006. 38:721-725.

2. Smyth, Deborah, Jason Cooper, Rebecca Bailey, Sarah Field, Oliver Burren, Luc Smink, Cristina Guja, Constantin Ionescu-Tirgoviste, Barry Widmer, David Dunger, David Savage, Neil Walker, David Clayton and John A Todd. A genome-wide association study of nonsynonymous SNPs identifies a type 1 diabetes locus in the interferon-induced helicase (IFIH1) region. Nature Genetics, 2006. 38:6, 617-619.

"Spicing Up" of the Immune System by Curcumin

It is quite possible that scarfing down some Indian food could actually strengthen your immune system! Turmeric is a Indian curry spice in which its orange-yellow coloring comes from curcumin. As it is most often known to be an antiinflammatory agent, curcumin has recently proven to be an agent that varies activation of T cells, B cells, macrophages, neutrophils, natural killer cells, and dendritic cells. Curcumin downregulates the expression of several proinflammatory cytokines such as TNF, IL-1, IL-2, IL-6, IL-8, IL-12, along with chemokines, through the inactivation of the transcription factor NF-kB. In low doses curcumin can also increase antibody responses. Reportedly curcumin has been beneficial in treating patients with arthritis, allergies, asthma, atherosclerosis, heart disease, Alzheimer's disease, diabetes, and cancer maybe from its regulatory effect on the immune system.
Turmeric is actually the rhizome powder from a certain herb in the ginger family. For those of you who do not know, rhizome is the stem of a plant found underground. Turmeric is a major cash crop in Asia, India, and China and is largely used as a spice in curries, food additive, and pigment. It also has medicinal purposes as it has been used to treat rheumatism, body aches, skin diseases, diarrhea, inflammations and several other conditions. Ladies forget the Midol, cause Turmeric is here! It is considered to be an emmenagogue which stimulates blood flow to the pelvis/uterus but also is considered to be a carminative or drug used against cramps!
Several studies have shown that curcumin regulates both the activation and the proliferation of T cells. In one particular study curcumin inhibited the proliferation of lymphocytes induced by concanavalin A, phytohemagglutinin, and phorbol-12-myristate-13-acetate of lymphocytes in human spleen. Curcumin, in the same study, inhibited IL-2 synthesis and IL-2 induced proliferation of lymphocytes. From these results curcumin appears to be immunosuppressive by way of regulating IL-2.
Curcumin also has the ability to affect autoimmune diseases due to its ability to modulate immune cells and immune cell cytokines. Alzheimer's disease is known for amyloid plaques forming on the brain due to inflammation. Curcumin downregulates cytokines, such as TNF-alpha, IL-1beta, activities in blood monocytes as well as reducing the amyloid-beta plaque formation. In asthma, curcumin reduces lymphocyte production of IL-2, IL-5, GM-CSF, and IL-4. For all you inflammatory bowel diseases lovers out there, cucumin has got something for you too! In this disease it reduces inflammatory cytokine levels, impeding NO and O2 production along with suppressing NF-kB activation in colon epithelium.


Ganesh Chandra Jagetia, Bharat B. Aggarwal
Journal of Clinical Immunology, Vol 27, No. 1 January 2007

Celiac Disease

Celiac Disease is an autoimmune disorder that is becoming more recognized among the medical profession and is a disease that is being diagnosed on a more frequent basis based on the growing knowledge of the disease. Celiac disease is linked to interactions between gluten and immune, genetic, and environmental factors (1). One known cause of celiac disease is the ingestion of gluten in genetically predisposed people (1). Gluten is the protein storage for wheat, barley, and rye, a major food component ingested by most individuals. Celiac disease was once known as celiac sprue, a rare malabsorption syndrome of childhood and is now known as a common condition affecting many organ systems (1).

According to Green et. al., gluten is digested by luminal enzymes into amino acids and peptides. The main peptide used in the innate immune system is the gliadin peptides which cause a change in the epithelium. Gliadin peptides cause damage to the epithelial cells causing an over expression of Interleukin-15. When Interleukin 15 is expressed, intraepithelial lymphocytes are activated which causes the activated cells to become cytotoxic and kill the enterocytes marked with MIC-A (major-histocompatibility-complex class I chain-related A) (1). In the adaptive immune system gliadin causes changes in the lamina propria. Gliadin enters the lamina propria during infection, where gliadin is deamidated by tissue transglutaminase. The adaptive immune response is mediated by the glidian-reactive CD4+ T cells which are bound to HLA class II molecules on APCs (1). Interferon-γ cytokines are released by the T cells causing an inflammatory response.

CD8+ T lymphocytes represent one of the diagnostic hallmarks of Celiac disease (3). These CD8+ T lymphocytes recognize peptides in HLA Class I molecules which are found in patients with celiac disease. According to Gianfrani et. al., the peptide A-gliadin is selectively recognized by CD8+ T lymphocytes from HLA-DQ2 or HLA-DQ8 celiac patients. Studies have shown that gliadin-specific CD8+ T cells infiltrating celiac mucosa, along with CD4+ T cells may play an important role in CD pathogensis (3).

Genetic factors of the disease seem to be strongly linked in determining individuals who have a high risk of developing the disease. Because the disease is linked to the adaptive immune response, specific class II HLA genes play a specific role on the development of celiac disease. Persons with alleles that encode for HLA-DQ2 or HLA-DQ8 have a high risk of developing (2). Green et al. say that the presence of these HLA genes is necessary but not sufficient in the development of Celiac disease because there are many people who do not have Celiac Disease but carry these alleles. It is known that HLA-DQ2 is found in 90-95% of patients with celiac disease, while the HLA-DQ8 is identified in the rest of the patients (1). The presence of absence in these HLA genes is important in determining who should be tested for the disease if symptoms appear.

Diagnosing celiac disease has become increasingly easier with HLA assessment, biopsy, and serological testing. Antibody testing is used to diagnose celiac disease. The main antibodies used are the antigliadin antibodies, connective tissue antibodies, and antibodies that are directed against tissue transglutaminase (2). Patients who have celiac disease often have low IgA endomysial antibodies and IgA antitissue antibodies against tissue transglutaminase. If levels from serological testing indicate a possibility of disease, then a biopsy of the small intestine is performed.

It is interesting to note that both the innate and adaptive immune response is involved in Celiac disease. Many studies have shown that the activation of the innate immune response sets the stage for activation of the adaptive immune response, by the activating of dendritic cells (3). There are still many unanswered questions as to the true mechanisms behind this disease, but with the knowledge of the immune response pathways, there is a better understanding of the processes and roles that are actually occurring in Celiac’s Disease.

(1) Peter H.R. Green, M.D., and Christophe Cellier, M.D., Ph.D. Celiac Disease. N Engl. J Med. 2007 Oct 25;357(17):1731-43.

(2) Armin Alaedini, Haruka Okamoto, Chiara Briani, Kurt Wollenberg, Holly A. Shill, Khalafalla O. Bushara, Howard W. Sander, Peter H. R. Green, Mark Hallett, and Norman Latov. Immune Cross-Reactivity in Celiac Disease: Anti-Gliadin Antibodies Bind to Neuronal Synapsin I. Journal of Immunology. May 2007; 178: 6590 - 6595.

(3) Gianfrani C, Auricchio S, Troncone R. Adaptive and innate immune responses in celiac disease. Immunol Lett. 2005 Jul 15;99(2):141-5.

Using Immunity in Cancer Therapy

One in two Americans will be diagnosed with cancer at some point in their lives. Traditional options for treating these patients usually involve surgery, chemotherapy, and/or radiation therapy. The goal of these modalities is to destroy or remove the cancer cells. However, the limitation of these therapies is that they are highly-toxic or invasive and as a result the side effects can be debilitating. A relatively new approach to improve cancer treatment involves engaging the body’s immune system to recognize and destroy the cancer cells; this biological approach is termed immunotherapy.

To date, a common and somewhat successful approach to immunotherapy has involved the use of monoclonal antibodies (mAb). In this type of therapy, donor antibodies are injected into a vein and go on to attach to specific antigens, some attach to cancer cells and others to cells that help tumors grow. However, one limitation of this approach is that tumor specific antigens are often intracellular and therefore out of reach of the humoral immune response. To overcome this issue an alternative or complimentary therapy is required.

The concept of lymphocyte immunotherapy was assisted by the evolution of organ transplantation. In transplant recipients, graft-versus-host disease (GVHD), where the transplant T-cells attack host tissue, is an undesirable complication. However, some leukemia and lymphoma patients who experience mild GVHD have a demonstrated a decreased risk of recurrent cancer. In these cases it appears that the transplant cells reject the cancer cells of the host. This concept helped to generate the idea of utilizing immuno-competent donor T-cells to treat cancer patients.

The use of monoclonal T-cells or T-cell receptors (mTCRs) is being investigated as viable approach. This is based on the ability of lymphocyte receptors to recognize peptide or major histamine complex (MHC) molecules in tumor cells. As with mAb therapy, a key challenge is to generate a therapeutic response that has high specificity for the tumor cells so that normal tissue is not destroyed. One significant study found effective results when CD4+ T cells, together with CD8+ T cells, and interleukin-2 (IL-2) were transferred to melanoma patients. T-cells proliferated and attacked tumor cells and there was significant regression of the metastatic melanoma in 6 of the 13 patients.

Immunotherapy does not come without side effects. Thankfully, they are often considered mild compared to chemotherapy. Five patients in the aforementioned study exhibited onset of antimelanocyte autoimmunity. Four of these patients developed vitiligo and one developed uveitis. Vitiligo is a condition where the melanocytes are destroyed and skin pigmentation is lost on patches of the skin. Uveitis is inflammation of the interior eye that can result in blindness. Presence of these side effects indicates that although there are benefits, the treatment needs to be refined. Although immunotherapy is still in the early stages of development, great advancements have been made in the last decade. It will be interesting to see what the future holds.

References:
1. Davis ID, Jefford M, Parente P, Cebon J. Rational approaches to human cancer immunotherapy. J Leukoc Biol 2003;73:3-29.
2. Dudley ME, Wunderlick JR, Robbins PF, et al. Cancer regression and autoimmunity in patients after clonal repopulation with antitumor lymphocytes. Science 2002;298:850-4.

New Markers For Airway Inflammation In Asthma?

Biopsies, sputum eosinophil levels and exhaled nitric oxide levels are the tests used currently when monitoring airway inflammation in asthma patients. The 2006 study by Piacentini suggests an easier method for testing inflammation. Piacentini used subjects with mild asthma and control subjects without asthma to test different markers of inflammation. The subjects rested for one hour before the initial test and rested for ten minutes between tests. Room temperature, humidity and axillary body temperature were strictly controlled.
This study found a positive correlation between end-expiratory manoeuvre plateau temperature (PLET), eosinophil percentage in sputum and exhaled nitric oxide. The use of salbutamol, a bronchodilator, caused an increase in PLET for both the asthmatics and the controls. The study also found no correlation between the above numbers and the rate on increase in exhaled breath temperature, which was a previously suggested method for measuring inflammation. This study also found that using a microbial filter during exhalation tests skewed the test curves and should not be used.
The conclusion is that PLET (exhaled air temperature) is a good marker of airway inflammation in asthma patients and could be used in adjusting treatment plans for asthma patients. More studies need to be done to determine a standardized procedure for testing PLET.

Piacentini GL, Peroni D, Crestani E, Zardini F, Bodini A, Costella S, Boner.
Exhaled air temperature in asthma: methods and relationship with markers of disease. Clinical and Experimental Allergy, 37, 415-419

Paredi P, Kharitonov SA, Barnes PJ. Faster rise of exhaled breath temperature in asthma: a novel marker of airway inflammation? Am J Respir Crit Care Med 2002; 165:181–4.

Paredi P, Kharitonov SA, Barnes PJ. Correlation of exhaled breath temperature with bronchial blood flow in asthma. Respir Res 2005; 6:15.

Piacentini GL, Bodini A, Zerman L et al. Relationship between exhaled air temperature and exhaled nitric oxide in childhood asthma. Eur Respir J 2002; 20:108–11.

Tonsils - in or out?

So, this post is a more personal one.

I have my tonsils. I've often wondered why others have theirs removed, since I've always considered them a part of my immune system. I came across this article: "The rise and decline of tonsillectomy in twentieth-century America" by Gerald N. Grob, which as its title describes, explains the history of tonsillectomy in America.

I'm amazed that so many physicians in the early 20th century really believed in the "focal infection theory," or the idea that the tonsils, as diseased organs contributed to systemic disease / infection, such as rheumatism or endocarditis...especially with no evidence! Wow. Fortunately, through numerous epidemiologic studies, and continued scrutiny, tonsillectomy has been on the decline...especially now with the success of antibiotics for various bacterial infections.

I had fun with this article, as it satisfied my curiosity, and wanted to share it with you.


J Hist Med Allied Sci. 2007 Oct;62(4):383-421. Epub 2007 Apr 10.

Tight Skin, Crusty Organs, and Novel Immunoglobulin Function

Tight Skin, Crusty Organs, and Novel Immunoglobulin Function: A Peculiar Tale of Stimulatory IgG Antibodies and Systemic Sclerosis

Considering its fund-hindering prevalence, it is unlikely that few reading this have ever heard of Systemic Sclerosis, or Scleroderma (SSc) so here’s a brief description…I stress “brief”: Scleroderma is a chronic connective tissue autoimmune disease characterized by systemic tissue fibrosis (i.e. too much collagen in places it shouldn’t be) with symptoms that can range from mild to life threatening (1).

“So where do the antibodies come into play, and what’s all this stimulatory jazz,” you might be asking? Rightly so. Stimulatory antibodies, until recently a curiosity associated mainly with Graves’ disease and hyperthyroidism, are immunoglobulins with the unusual ability to bind to a cell’s receptor and stimulate some form of physiological function or process not originally thought to be associated with the immune system. In the case of Graves’ disease, stimulatory antibodies bind (in excess) to thyrotropin receptors on the thyroid and stimulate the release of thyroid hormone (2). Since the body can regulate Thyroid Stimulating Hormone production but not, apparently, autoantibody production the result is chronic thyroid over-stimulation with hyperthyroidism ensuing. It is important to remember that until recently Graves’ disease was the only condition in which this stimulatory autoantibody phenomenon was known to occur. Enter Scleroderma. In a recent study by Baroni et al., 46 SSc patients (all that were in the study) were shown to have elevated serum stimulatory IgG levels (3). These IgG autoantibodies bound to, and stimulated, Platelet-Derived Growth Factor Receptors (PDGFR) resulting in excess collagen production. More importantly, none of the 75 controls, who displayed other autoimmune diseases such as Raynauds’s Phenomenon, Systemic Lupus Erythematosus, Rheumatoid Arthritis, and Idopathic Pulmonary Fibrosis, displayed elevated PDGFR stimulatory IgG levels. With additional experiments Baroni et al. was able to tease out the pathways resulting in increased fibroblast (cells that make collagen) activity: 1) SSc autoantibody IgGs bind to PDGFR, 2) This binding increases Reactive Oxygen Species (ROS) production, 3) Long term accumulation of ROS stimulates collagen-gene expression, 4) Too much collagen equals thick skin and crunchy-stiff blood vessels. …Okay, so step 4 was not in their original paper, but you can see how it is a logical corollary.

Hopefully by now you are thinking, “So what?” Well, Gleicher et al. realized the importance immediately and discusses it in his recent paper published last June (4). Basically, the finding by Baroni et al. (stimulatory IgGs in SSc patients) changes the way we must view the role of autoantibodies in human physiology and autoimmune disease. It has been known for some time that even healthy people occasionally harbor low levels of autoantibodies in their system. It is only when these concentrations rise excessively that they become problematic. That there is a stimulatory (Gleicher uses the term “functional”) role for autoantibodies in Graves’ disease and Scleroderma, and that there might be similar undiscovered autoantibodies with similar functions in other diseases, brings with it a new paradigm in which to view autoimmune function and future research.

1. LeRoy EC, Black C, Fleischmajer R, Jablonska S, Krieg T, Medsger TA, Jr., Rowell N, Wollheim F. Scleroderma (systemic sclerosis): classification, sub-sets and pathogenesis. J. Rheumatol 1988; 15:202-5.

2. Morgenthaler NG, Hodak K, Seissler J, Steinbrenner H, Pampel I, Gupta M, McGregor AM, Scherbaum WA, Banga JP. Direct binding of thyrotropin receptor autoantibody to in vitro translated thyrotropin receptor: a comparison to radioreceptor assay and thyroid stimulating bioassay. Thyroid 1999 May; 9(5):466-75.

3. Baroni SS, Olivieri A, Campelli N, Luchetti M, Poloni A, Trappolini S, Moroncini G, Bacigalupo A, Leoni P, Avvedimento EV, Gabrielli A. Stimulatory autoantibodies to PDGF receptor in patients with extensive chronic graft-versus-host disease. Blood 2007 Jul 1;110(1):237-41.

4. Gleicher N, Barad D, Weghofer A. Functional autoantibodies, a new paradigm in autoimmunity? Autoimmun Rev 2007 Nov;7(1):42-5.