The Heterogeneity of B-cell Memory and Vaccine Design

The establishment of long-term humoral memory to a pathogen is an important goal of vaccination. One of the important component of it is belong to memory B cells. This cells will help us from the future re-attack of pathogens by surveying their specific antigen in the periphery for affinity maturation. Classically the marker for B cell memory was expressed by CD27. However recent finding suggest the heterogeneity of the memory compartment that involving CD27- memory B cells. If a vaccine has been designed to non-specific memory B cell, it potentially will result in loss of maintenance and/or establishment of an important subset of memory populations. Consequently, this will lead to improper localization and ineffective response that result in loss of vaccine efficacy. Therefore it would be challenging to study the environment that create this subset memory cells and to understand their property in vivo for the better vaccine design.

Reference:
http://www.frontiersin.org/b_cell_biology/10.3389/fimmu.2011.00077/full

Behavioral Conditioning and the Immune System

This is in follow-up to our class discussion of how immunosuppression can be classically conditioned. We discussed a study in which cyclosporin (an immunosuppressive drug) was given to rats along with a sweet-tasting drink. After multiple pairings, just the sweet-tasting drink was given and it was observed that the rats had the same immunosuppressive response WITHOUT the cyclosporin! This is fascinating because it means that the immune system can actually learn to associate environmental cues with a response.

Another study (Goebel, et al, 2002) found this same effect in humans. After pairing cyclosporin with a novel-tasting drink four times, just the novel-tasting drink produced the same immunosuppressive effects. These effects included reduced IL-2 and IFN-y mRNA expression in peripheral blood lymphocytes, and less secretion of IL-2 and IFN-y by CD3+CD4+ (Th) lymphocytes.

An antihistamine effect has also been conditioned in humans (Goebel, et al, 2008). Patients with allergic rhinitis were given the anti-histamine desloratadine along with a novel-tasting drink once daily for 5 consecutive days. Nine days later, some were re-exposed to the novel-tasting drink along with a placebo pill, some were given water with a placebo pill, and some were given water and the desloratadine. The water-plus-placebo group showed reduction in subjective total symptom scores and histamine skin prick test results, but no basophil inhibition (did the placebo pill act as a weaker-acting conditioned stimulus?). The group who received the novel-tasting drink with a placebo pill showed reduction in subjective total symptom scores, reduction in skin prick reactions to histamine, and basophil inactivation in degrees that were similar to the group who received the desloratadine and water.

What about immune system activation? Studies show that it can also be classically conditioned! In one such study (Pacheco-Lopez, G. et al, 2004), researchers injected the superantigen staphylococcal enterotoxin B (SEB) into rats while giving them a saccharine-flavored drink. One week later, presenting just the drink induced a strong taste aversion response and increased plasma levels of corticosteroid, INF-y, and IL-2.

What does this mean? It means that strong signals associated with immune-acting drugs are noticed by the immune system. After multiple exposures, the effect of a drug on the immune system is not only due to specific drug mechanisms anymore, but includes the effects of learned responses. So, there must be some link between the CNS gustatory system and the immune response. There are so many questions about how this actually occurs, making it a totally exciting field for more research!

Goebel, M., et al (2008). Behavioral conditioning of antihistamine effects in patients with allergic rhinitis. Psychotherapy and Psychosomatics,77(4), 227-34. http://proquest.umi.com/pqdweb?index=0&did=1479529181&SrchMode=1&sid=1&Fmt=6&VInst=PROD&VType=PQD&RQT=309&VName=PQD&TS=1324060097&clientId=18952

http://www.ncbi.nlm.nih.gov/pubmed/18418029

Goebel, M., et al (2002). Behavioral conditioning of immunosuppression is possible in humans. The FASEB Journal, 16, 1869-1873. http://www.fasebj.org/content/16/14/1869.full

Pacheco-Lopez, G. et al (2004). Behavioural endocrine immune-conditioned response is induced by taste and superantigen pairing. Neuroscience, 129, 555–562. http://www.sciencedirect.com/science?_ob=MiamiImageURL&_cid=271071&_user=418620&_pii=S0306452204007742&_check=y&_coverDate=2004-01-01&view=c&wchp=dGLbVBA-zSkzS&md5=206c181608005e8a34afe66bf9cb7a8c/1-s2.0-S0306452204007742-main.pdf

Diabetes, a rising epidemic

Diabetes mellitus is a lifelong disease of high levels of sugar in the blood. Diabetes affects over 20 million Americans. Over 40 million Americans have prediabetes and the incidence of this disease is increasing every year. The disease is characterized by any combination of the pancreas dysfunction (not making enough insulin), cells’ inability to respond to insulin normally, and the body’s inability to move sugar into the fat, liver, and muscle cells for energy storage. There are three types of diabetes:

Type I: can occur at any age, but most often diagnosed in young children, teens or young adults. This is an autoimmune disease characterized by the immune system T lymphocytes destroying the beta cells (insulin secretors) of the pancreatic islet.

Type II: is generally adult-onset and makes up the majority of diabetics. There is an increasing incidence of the onset of Type II in young children, teens, and young adults because of an increasingly high rate of obesity.

Gestational diabetes: high blood sugar of the mother developing during pregnancy

Diabetes can be tested by urine analysis for high blood content, fasting blood glucose test, hemoglobin A1c, and an oral glucose tolerance test.

The autoimmune response is detectable in type I diabetes (T1D) by screening for autoantibodies. Autoantibodies are detectable pre-diagnosis in the months or years preceding clinical disease. Studies by various labs around the world indicate that the disease can be diagnosed by the presence of two or more of the autoantibodies (some are beta cell specific) to glutamate decarboxylase (GAD65), insulin, islet antigen (IA-2) and zinc transporter 8 (ZnT8). These are biomarkers or indicators of beta cell destruction. T-lymphocytes mediate the beta cell destruction. They receive signals from antigen presenting cells, such as dendritic cells and macrohpages and receive help from B-lmphocytes to initiate a complete immunological response. This results in impairment of beta cell function, progressive destruction of beta cells, and consequently the development of T1D. Throughout progression of disease these autoantigens undergo epitope spreading, leading to an increase in antigenic determinants.

Thus the goal of many studies is to understand the etiology of T1D and design methods to intervene the immunological response before disease onset. Autoantigens thus far identified include GAD65, insulin, IA-2, ZnT8, Chromagranin A, and Insulin amyloid peptide (IAPP).

ZnT8 was recently discovered in the lab of John Hutton of the Barbara Davis Diabetes Center and is a major target of humoral and cell-mediated autoimmunity. ZnT8, like insulin and islet-specific glucose 6 phosphatase related protein (IGRP) are found in the pancreatic beta cell and is more restricted in its tissue distribution than other autoantigens. Goals in the lab include ZnT8 autoantibody prevalence (wildtype and polymorphisms have been identified), antibody titer, epitope specificity pre and post-diabetes onset, relationship to C-peptide responses, and effects on insulin secretion and beta cell function. From an immunological point of view, we are interested in specificity of T cell and B cell responses and identifying specific T cell and B cell clones.

http://www.ncbi.nlm.nih.gov/pubmedhealth/PMH0002194/

Skyler JS. Immune intervention for type 1 diabetes mellitus. Int J Clin Pract Suppl. 2011, (170):61-70.

Wenzlau JM, Walter M, Gardner TJ, Frisch LM, Yu L, Eisenbarth GS, Ziegler AG, Davidson HW, Hutton JC. Kinetics of the post-onset decline in zinc transporter 8 autoantibodies in type 1 diabetic human subjects. J Clin Endocrinol Metab. 2010, 95(10):4712-9.

What the Cat Dragged In

This time, it’s Toxoplasma gondii, one of the strangest parasites I have ever heard of, and one that is estimated to infect up to a staggering 1/3 of the world’s human population. (Infection is much higher in developing countries; recent testing in the US determined that infection rates are around 10% of the population).3

Tox. gondii begins its life cycle in warm-blooded mammal intermediate hosts, preferably rodents that will be eaten by cats, thus allowing the parasite to complete its life cycle and sexually reproduce. While most rats avoid cats and cat odors, rats infected with toxoplasmosis suddenly display unique behavioral changes. These rats develop what has been called a “fatal attraction” to cats, increasing their chances of being eaten and thereby increasing the parasite’s chances of sexual reproduction. The rats become more active, less fearful, slower to react, slower to learn, less averse to cat odors, and less averse to cat-populated areas. It is interesting that this effect is so selective; in other matters such as competition for mates and social status, the rats behave normally.1 6

Humans can become infected with Tox. gondii if they are exposed to oocytes found in animal meat and cat feces. Transmission can also occur through the placenta, but only in immunosuppressed mothers who are initially infected while pregnant. Once oocytes are injested by the intermediate host (rat/human/other mammal), the parasite undergoes asexual reproduction, with rapidly dividing tachyzoites and more slowly dividing bradyzoites. These go on to form cysts in the host’s brain, heart, and other tissues. An innate and adaptive response in the host is activated, and antibody is made. Acute toxoplasmosis is a relatively mild and short-lived disease in most immunocompetent people, and it transitions quickly into the latent phase: the cysts remain, potentially for the rest of the host’s life.5

The most interesting thing about these cysts is that they may actually cause a change in a human host’s personality. Multiple studies have concluded that infected humans have slower reaction times, have been in more traffic accidents, and are less novelty-seeking. Infected woman may be more warm and prone to guilty feelings, and infected men may be more suspicious and have higher testosterone levels.2 (However, it should be noted that many of these human studies are correlational. In fact, one reported study has shown that infection with CMV is also correlated with less novelty-seeking.4 So, there is some evidence that the mechanism may be general brain infection and not a specific parasitic action.) Tox. gondii is associated with incidences of schizophrenia and mood disorders, and may affect the host’s levels of dopamine. One recent study actually found that infected men were less averse to the smell of cat urine, while infected women were more averse (aversions to other animals’ urine were normal).2

Tox. gondii is only one example out of many parasites that have learned to control their host’s behavior in order to increase their reproductive potential. It is fascinating to me that this is even possible, and especially that infection with Tox. gondii is so rampant throughout the world.

Take-home message? Keep your cat away from raw meat!

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1 Berdoy, M., Webster, J.P., & Macdonald, D.W. (2000). Fatal attraction in rats infected with Toxoplasma gondii. Proceedings. Biological Sciences/The Royal Society, 267(1452), 1591-4. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1690701/pdf/11007336.pdf

2 Flegr, J., et al. (2011). Fatal attraction phenomenon in humans - cat odour attractiveness increased for toxoplasma-infected men while decreased for infected women. PLoS Neglected Tropical Diseases, 5(11), e1389. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3210761/?tool=pubmed

3 Jones, J. L., et al. (2007). Toxoplasma gondii infection in the United States, 1999–2004, decline from the prior decade. American Journal of Tropical Medicine and Hygiene, 77(3), 405-10. http://www.ajtmh.org/content/77/3/405.long

4 Novotná , et al. (2005). Probable neuroimmunological link between Toxoplasma and cytomegalovirus infections and personality changes in the human host. BMC Infectious Diseases, 5, 54. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1187888/?tool=pubmed

5 Tenter, A.M., Heckeroth, A.R., & Weiss, L.M. (2000). Toxoplasma gondii: from animals to humans. International Journal for Parisitology, 30(12-13), 1217-1258. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3109627/?tool=pubmed

6 Webster, J.P. (2007). The effect of Toxoplasma gondii on animal behavior: playing cat and mouse. Schizophrenica Bulletin, 33(3), 752-756. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2526137/?tool=pubmed

Drinking Parasites or Fecal Transplant?

Parasite drinking

http://www.npr.org/2010/12/02/131753267/eat-your-worms-the-upside-of-parasites

Fecal Transplants

http://www.sciencedaily.com/releases/2011/10/111031114945.htm

Inflammatory bowel disease (IBD) is a group gut inflammatory conditions that affect the colon and small intestine. Two types exist, Crohn’s disease and ulcerative colitis. The main difference is the location and nature of the inflammation. Chron’s disease is usually classified as an autoimmune disease and can affect any part of the gastrointestinal tract from mouth to anus. Ulcerative colitis is a bowel disease in which the immune system appears to attack the lining of the colon, causing devastating ulcers. I will discuss the pros and cons of each treatment method.

Fecal microbiotoa transplants (FMT): The studies cited that treatment was effective in >80% (sometimes even 98%) of patients that suffered from diarrhea (caused by C. difficile infections) and did not have to remain on therapies post FMT. The cons to FMT were that some patients didn’t respond at all and for the case studies of IBD, multiple treatments (ie: daily rectal FMT for 1^st month, followed by weekly FMTs until 1FMT/6wks). However, it seems that the IBD patients are cured and even remained off therapy for the next four years.

Therapies for C. Difficile infection include antibiotics, probiotics, toxin-binding medications, and colonectomies (surgery to removed affected parts). Other studies by Furrie et al, and Kruis et al, have show that the use of probiotics have been effective in treating inflammation in patients with ulcerative colitis and even as successful as using antibiotics.

Parasite drinks – a drink with Trichuris suis ova taken 2x monthly decreased symptoms of IBD in many patients (Summers, et al 2003). Croese et al, 2006 also show that use of hookworms (Necator americanus) was effective in the treatment of IBD. There is increasing evidence, by Loke in the parasite drinking article and others, that the administration of worm eggs improves IBD symptoms. The possible mechanism is that worm ingestion causes the production of IL-22, which promotes epithelial growth and healing, and perhaps other ways to benefit the immune system. The con of this particular example is that once the worms begin to die, the disease comes back. Also, if the worms get too large, they can cause blockage of the GI tract and patients would then have to take medications to help expel them.

A possible mechanism of action is that the immune response is pushing more towards a TH2 response. Typically bacteria and viruses elicit a Th1 response, down regulating the Th2 response. The Hygiene hypothesis proposes that insufficient stimulation of Th1 can lead to an overactive Th2 response to self, thus infection with fecal matter or worms may positively select for genes, that historically and evolutionarily were beneficial wherein the contemporary, more sterile environment of today have deleteriously selected against that pressure. Fumagalli, et al (2009) studied autoimmune diseases and influences by interleukin gene polymorphisms and pathogens. They concluded that there were positive selective pressures induced by the presence of worms that had protective effects and identified potential IL-related SNPs for further study.

Croese J, O’neil J, Masson J, Cooke S, Melrose W, Pritchard D, Speare R. A proof of concept study establishing Necator americanus in Chrohn’s patients and reservoir donors. Gut. 2006, 55(1):136-7.

Fumagalli M, Pozzoli U, Cagliani R, Comi GP, Riva S, Clerici M, Bresolin N, Sironi M. Parasites represent a major selective force for interleukin genes and shape the genetic predisposition to autoimmune conditions. J Exp Med. 2009, 206(6):1395-408.

Furrie E, Macfarlane S, Kennedy A, Cummings JH, Walsh SV, O’neil DA, Macfarlane GT. Synbiotic therapy (Bifidobacterium longum/Synergy 1) initiates resolution of inflammation in patietns with active ulcerative colitis: a randomized controlled pilot trial. Gut. 2005, 54(2):242-9.

Kruis W, Fric P, Pokrotneiks J, Lukas M, Fixa B, Kascak M, Kamm MA, Weismueller J, Beglinger C, Stolte M, Wolff C, Schulze J. Maintaining remission of ulcerative colitis with the probiotic Escherichia coli Nissle 1917 is as effective as with standard mesalaine. Gut. 2004, 53(11):1617-23.

Summers RW, Elliott DE, Qadir K, Urban JF Jr, Thompson R, Weinstock JV. Trichuris suis seems to be safe and possibly effective in the treatment of inflammatory bowel disease. Am J Gastroenterol. 2003, 98(9):2034-41.

Immunity and Alcoholism

The effects of alcohol on the immune system have been debated for many years. On one hand a small amount probably has no effect or possibly even a beneficial effect, but excessive amounts lead to increased infections. It may be an obvious connection that too much of anything will lead to health problems, but what is the mechanism involved? Will knowing this mechanism teach us anything about how the immune system works?

I was in a lab that wanted to find this out, and my work led to a paper in 2007. In summary we found that our mouse model for chronic alcohol abuse compromises the Th1 driven delayed type hypersensitivity reaction. The cell population we found to be responsible were a subpopulation of dendritic cells that were CD11c+CD8α+ which were impaired in their ability to activate Th1 cells. This is predicted to be due to glutathione depletion and resulting oxidative stress.

Since I have shifted my interest to cancer, I was curious as to what has been done since 2007 regarding the effects alcohol has on adaptive immunity. I was surprised to find a recent paper that cited my work. What Porretta et al did was look for the impact of alcohol on BCG-Induced Immunity Against M. Tuberculosis.

Tuberculosis is one of the many known infections that alcoholics have a greater change of succumbing to. The infection can either remain in a latent state (held at bay by the Th1 response) or through several mechanisms become active. Alcoholism can be a factor in driving the infection to the active state. Using the same mouse model as we used for studying the alcoholic’s immune system, they vaccinated mice with BCG, a live vaccine of the attenuated strain and followed up with pulmonary challenge. They found that alcohol consumption prior to vaccination impaired the ability of the vaccine to protect the mice from infection, but that alcohol consumption after vaccination produced had no effect on the effectiveness of the vaccine.

Since BCC is not effective in producing the Th1 and CTL immunity essential to recover from tuberculosis, and therefore not a popular vaccine in America. New vaccines are being developed and tested in the developing world where the cases of TB are higher. Since alcoholism is also high in developing countries, the current trials need to conducted without the added variable of alcohol.

Porretta et al (2011) The Impact of Alcohol on BG-Induced Immunity Against Mycobacterium tuberculosis. Alcohol Clin Exp Res 35:1530-1538.

Heinz R, Waltenbaugh C (2007) Ethanol consumption modifies dendritic cell antigen presentation in mice. Alcohol Clin Exp Res 31:1759–1771.

Virus Hunter

An article in the November 7, 2011 issue of TIME magazine entitled "Virus Hunter" caught my eye as we discussed HIV tranmission in class.  The article highlights an organization, Global Viral Forecasting (GVF), and its founder, Nathan Wolfe, who aim to prevent new pathogens from spreading from animals to humans, stopping deadly pandemics before they start.  Wolfe has set up projects in Africa, Southeast Asia and southern China, which are all hot spots where humans and wild animals intermingle and new viruses can leap from one species to another.

In 1998, Wolfe published a paper that raised the possible links between the hunting of wild animals, a popular source of protein in Central Africa, and the spread of emerging infectious diseases.  Viruses make the leap between species when bodily fluids are shared, which tends to happen when one animal hunts, kills and eats another.  Wolfe's goal is to collect blood samples from bush meat hunters and their prey to find out what microbes are out there.  GVF distributes filter paper to villages throughout the country and when hunters make a kill, they squeeze a few drops of blood from the animal onto the paper, noting what they butchered, when and where. 

In 2004, Wolfe and his colleagues found evidence that simian foamy virus (SFV) had spread to Cameroonian hunters, a novel primate retrovirus from the same genetic tree that produced HIV - so called because of the way it makes infected cells appear under a microscope.  SFV hasn't been connected to any known symptoms.  However, this discovery was proof for Wolfe that his organization, GVF, was effective in finding new viruses that could possibly trigger pandemics in the future.  He hopes that identifying these new viruses before they spread will inspire a switch from chasing pandemics to predicting and preventing them. 

I was intrigued by this story and thought it was worth sharing.  Unfortunately, to access this story online you have to be a TIME subscriber, but I'm including the link to the story, just in case you are a subscriber:
http://www.time.com/time/magazine/article/0,9171,2097962,00.html
 

Microparticles, chemokines, DNA and siRNA- Mix well and inject… what do you get? More effective immunotherapy?!

This is totally cool and also a good review of many of the mechanisms we learned this semester. This group in Texas is making an effort to more effectively eliminate tumor cells in patients with malignant non-Hodgkin's lymphomas (NHL) of B-cell lineages by priming (ie adding an adjuvant) to a DNA vaccination. The working model being- that relapse following an initial anti-tumor treatment is due to inadequate removal of tumor cells. They’ve engineered microparticles loaded with chemokines, tumor specific DNA antigen, and an IL-10 silencing mechanism (siRNA).

Immature DCs are recruited via the chemokines, take in the microparticles by endocytosis, and travel to lymph nodes presenting the tumor specific DNA antigen in MHCII on their surface. This elicits a T cell response and, because of the siRNA, gene expression of IL-10 in the DCs is silenced at the post-transcriptional level. This shifts the immune response towards a Th1 response. Remember, IL-10 from DCs would normally act to inhibit a Th1 response and encourage a Th2 response.

An increased Th1 response means more CD8+ CTLs are activated in the lymph nodes following DC contact and with help from CD4+ Th1s releasing IL-2. There is clonal expansion and the activated, tumor cell-specific CTLs then travel to the site of antigen recognition where they do a couple of things. 1. They administer the “kiss of death” to the lymphoma cells exhibiting the antigen for which they are specific and, 2. they release IFNgamma, upon engagement of the TCR with the lymphoma cell. IFNgamma is pro-inflammatory and leads to activation of macrophages (M0) into classically activated “angry” M1s.

This study found a significantly increased CD8+ cytotoxic T cell (CTL) response and stronger CD4+ CTL activity when compared to the DNA vaccine alone.

Source:

An injectable synthetic immune-priming center mediates efficient T-cell class switching and T-helper 1 response against B cell lymphoma.

Journal of Controlled Release, Volume 155, Issue 2, 30 October 2011, Pages 184-192

Ankur Singh, Hong Qin, Irina Fernandez, Jinsong Wei, Jian Lin, Larry W. Kwak, Krishnendu Roy