Measurement of airway inflammation in asthmatic children

A group in Italy has been searching for a way to screen children for asthma, and get a reliable indicator of the level of inflammation in the airway. Initially the group looked into measurements of the temperature of exhaled breath to provide a marker of airway remodeling (1). The increase in the exhaled breath temperature (EBT) has been hypothesized to result from increased vascularity of the airway wall (1). Other studies have noted the correlation between inflammation and temperature and given mechanistic hypothesis (4). Previous studies also showed preliminary results that linked metalloprotineinase-9 (MMP-9) to the remodeling process. The preliminary data showed a correlation when children were exposed to allergens between the exhaled temperature and MMP-9, measured through enzyme-linked immunoabsorbant assays on sputum.

The same group abandoned the tempurature measurement to persue measurements of exhaled nitric oxide (eNO) and fractional exhaled nitric oxide (FENO). The measurements of FENO have been shown to correlate with eosinophilic inflammation in asthma patients. The studies indicated that allergen avoidance modifies inflammatory parameters and that the initial extracellular bioelectrical conductivity measurements provided a reliable non-invasive method to assess airway inflammation (2). The method was also tested for correlation against the Childhood Asthma Control Test (C-ACT), when used together produce a reliable diagnostic and predicting tool (3).

(1) Piacentini, G.L. , et. Al. Exhaled breath temperature as a marker of airway remodeling in asthma, a preliminary study, Allergy 2008:63; 484-485
(2) Peroni, D.G., et. Al . Bioimpedance monitoring of airway inflammation in asthmatic allergic children. Allergologia et Immunopathologia 2008
(3) Piacentini, G.L. , et. Al. Childhood Asthma Control Test and airway inflammation evaluation in asthmatic children. Allergy 2009
(4) Piacentini, G.L. , et. Al. Exhaled air temperature in asthma: methods and relationship with markers of disease. Clin Exp Allergy, 2007; Mar;37(3): 415-419

operational tolerance post liver transplants

One of the interesting groups of patients that I have come across in the last 1 1/2 years are the patients who receive liver transplant and over time develop "operational tolerance" of their graft, meaning that they are able to wean off immunosuppressive agents over time.  Operational tolerance is defined clinically as the "lack of acute or chronic rejectionn and graft survival with normal function and histology in an immunosuppressant-free, immunocompetent host." 

Anectodally, we see this most commonly with teenagers who have become non-compliant with their immunosuppressive regiments.  And of the solid organ transplants, liver transplants are most likely to become tolerated by the host's immune system.  In general, children are more likely than adults to develop operational tolerance.  In the literature, 3-20% of patients may be able to wean successfully off of immunosuppressants.

This brings the questions--why do some patients reject the graft and others become tolerant?  How can we predict which patients will develop operational tolerance?

A study in Brussels observed that pediatric patients who developed operational tolerance had higher levels of IL-10 and decreased TNF-alpha and IFN-gamma levels soon after transplant (2 hours, 1 day and 4 days).  This could potentially identify a cytokine profile that could possibly predict patients who would become tolerant.  Other studies have suggested that there is an elevated number of T regs in patients who develop tolerance (this would correlate with higher amounts of IL-10, which is produced by T regs as we are aware). 

T regs express CD4/CD25 and the transcription factor FoxP3.  There are studies that suggest that FoxP3 can also be induced by peripheral T cells that are CD4+/CD25-.

This study (Pons et al) set out to monitor T cells in patients s/p liver transplant as immune suppression was weaned.  They selected 12 adults who had:  received a liver 2+ years prior to the start of the study, were 1+ years past a rejection episode, and had no evidence of autoimmune liver disease or viral infections.  They were monitored prospectively as their immunosuppression (cyclosporine and prednisone) was gradually weaned.  Flow cytometry was used to identify lymphocytes, and FoxP3 mRNA levels were detected by PCR.  The researchers looked at populations of T lymphocytes (CD3+CD4+, CD3+CD8+, CD3+Vdelta+), B lymphocytes (CD19+), and NK cells (CD3_CD16+CD56+) prior to withdrawal of immunosuppression and then at various periods during withdrawal of immunosuppression.  They compared these findings to another group s/p liver transplant who were immunosuppressant-dependant (ID) and another group who was healthy (H).  There was a similar profile of T regs (CD4+CD25+) in all immunosuppressed patients. 

Over the course of withdrawal of immunosuppression, 5/12 patients developed tolerance.  The percentage of CD3+, CD4+, CD8+, CD19+, CD3-CD16+CD56+, CD8+CD28- populations did not change during withdrawal (in the patients who ended up becoming tolerant and those who ended up rejecting).  However, in the group that became tolerant of their graft, T regs increased significantly during the withdrawal (beginning at the time of assessment pre-withdrawal and ending at the tolerance point).  The group who rejected did not exhibit this increase in T regs.  They noticed a similar pattern with FoxP3 mRNA expression.

This study was unable to predict (prior to immunosuppression withdrawal) which patients will become tolerant.  However, they were able to determine that T regs and FoxP3 expression dramatically changes in patients who become tolerant.

Continued research in this area will greatly influence care of patients post transplant.  In the past, it has been gold standard to continue immunosuppression, tolerating the side effect profiles of the medications.  Identifying patients who will successful become tolerant will help to titrate medications, improve quality of life, and decrease harmful side effects of the immunosuppression medications.


Pons JA et al, FoxP3 in peripheral blood is associated with operational tolerance in liver transplant patients during immunosuppression withdrawal.  Transplantation. 2008 Nov 27;86(10):1370-8.

Orlando G et al, Operational Tolerance after Liver Transplantation.  J Hepatology 2009;50:1247-57.

Allogeneic Hematopoetic Stem Cell Transplantation for Sickle Cell Disease

Background
Myeloablative allogeneic hematopoietic stem-cell transplantation is curative in children with sickle cell disease, but in adults the procedure is unduly toxic. Graft rejection and graft-versus-host disease (GVHD) are additional barriers to its success. We performed nonmyeloablative stem-cell transplantation in adults with sickle cell disease.
Methods
Ten adults (age range, 16 to 45 years) with severe sickle cell disease underwent nonmyeloablative transplantation with CD34+ peripheral-blood stem cells, mobilized by granulocyte colony-stimulating factor (G-CSF), which were obtained from HLA matched siblings. The patients received 300 cGy of total-body irradiation plus alemtuzumab before transplantation, and sirolimus was administered afterward.
Results
All 10 patients were alive at a median follow-up of 30 months after transplantation (range, 15 to 54). Nine patients had long-term, stable donor lymphohematopoietic engraftment at levels that sufficed to reverse the sickle cell disease phenotype. Mean ± SE) donor–recipient chimerism for T cells (CD3+) and myeloid cells (CD14+15+) was 53.3 ± 8.6% and 83.3 ± 10.3%, respectively, in the nine patients whose grafts were successful. Hemoglobin values before transplantation and at the last follow-up assessment were 9.0± 0.3 and 12.6 ± 0.5 g per deciliter, respectively. Serious adverse events included the narcotic-withdrawal syndrome and sirolimus-associated pneumonitis and arthralgia. Neither acute nor chronic GVHD developed in any patient.
Conclusions
A protocol for nonmyeloablative allogeneic hematopoietic stem-cell transplantation that includes total-body irradiation and treatment with alemtuzumab and sirolimus can achieve stable, mixed donor–recipient chimerism and reverse the sickle cell phenotype. (ClinicalTrials.gov number, NCT00061568.)




As the authors note in their abstract, the myeloablative transplantation of allogeneic stem cells has been extremely successful (90-95%) in curing children with sickle cell disease. However, the myeloablation process has shown unacceptable toxicity in human adult patients, preventing clinicians from successfully treating these patients. The authors thus tested a nonmyeloablative procedure on a murine model to determine safety and efficacy, then progressed to this study on 10 adult human patients. Instead of attempting to block T cell activation as in normal myeloablative procedures, the drug combination used by this team inhibits T cell proliferation. The inhibition of T cell proliferation causes anergy in the host’s Tcells, limiting their reactivity to antigens, promoting T cell tolerance to the new graft tissue. With this treatment, the authors were successful at reversing the sickle cell disease phonotype in 9 out of 10 patients, and managed to prevent GVHD in all patients. This is promising research into being able to reverse the disease state in adults.

1. Hsieh, M. M.; Kang, E. M.; Fitzhugh, C. D.; Link, M. B.; Bolan, C. D.; Kurlander, R.; Childs, R. W.; Rodgers, G. P.; Powell, J. D.; Tisdale, J. F., Allogeneic Hematopoietic Stem-Cell Transplantation for Sickle Cell Disease. N Engl J Med 2009, 361 (24), 2309-2317.

Cord Blood IgE as a Predictor of Atopy

This longitudinal study was conducted with a group of mothers living in Vancouver and Winnipeg, Canada. Mothers of high risk children were recruited in their third trimester and ‘high risk’ was defined as having at least one parent with asthma or two parents with other IgE allergic diseases. Then house dust samples were collected and a questionnaire regarding health and exposures were completed before birth and at ages 1, 2 and 7. Additionally at years 2 and 7 allergy skin tests were performed on the following allergens: house dust mite, cat and dog dander, peanuts, eggs, cow’s milk, wheat and soy. Atopy was defined as one or more positive skin tests for any allergen. Also, cord blood IgE (CD-IgE) levels were collected at birth.
CD-IgE was detectable (≥0.5 KU/l) in 19.3% of the 285 children. A high percentage of children with detectable CD-IgE had a maternal history of asthma (p-value of 0.001), maternal atopy [adjusted OR 2.21 (1.01-4.84)], and were born in the winter months [adjusted OR 4.07 (1.68-9.83)]. Additionally at 7 years, detectable CD-IgE was associated with increased risks for atopy [OR 2.22 (1.11-4.41)], positive skin test reactions against any aeroallergen [OR 2.25 (1.13-4.47)] and recurrent wheeze [OR 2.51 (1.09-5.76)].
The data that they collected looks really interesting so make sure to look at the two tables they provided. Although this was a small study, the authors noted that this information can lead to targeted prevention efforts for infants who may be at higher risk for developing asthma. I think it’s most interesting that being born in the winter months was found to have a significant association with detectable CD-IgE. This is probably due to the increased exposure to inhalant allergens after birth but who knew the season you were born in may have that effect!
You can find the article at:
http://0-www3.interscience.wiley.com.impulse.ucdenver.edu/journal/123193720/issue
Elevated cord blood IgE is associated with recurrent wheeze and atopy at 7 yrs in a high risk cohort
Ferguson, Alexander A (12/2009). "Elevated cord blood IgE is associated with recurrent wheeze and atopy at 7 yrs in a high risk cohort". Pediatric allergy and immunology (0905-6157), 20 (8), p. 710.

RXR Activation: Hope for New Parkinson's Disease Treatment

Parkinson’s disease is a neurodegenerative disease, which effects the dopamine-releasing cells in the brain. There has been a few experiments run attempting to rescue the dopamine neurons. This is done using chemicals that interact with the retinoid X receptor (RXR). The chemicals that will be investigated to interact with these receptors are known as RXR ligands. The article, which was published in journal BMC Neuroscience, describes the use of two cellular events which lead to the neuronal damage due to Parkinson’s disease. The two ligands examined in the experiment were the two RXR ligands LG268 and XCT. Specifically, they were studied to determine the neuroprotective function of the two.

Susanna Kjellander worked with a team of researchers from the Ludwig Institute for Cancer Research, Sweden, to test both the ligands and the two destructive Parkinson’s pathways. As a result she was able to make the following conclusion; "Nuclear hormone receptors like RXR and the Nurr1-RXR receptor heterodimer are emerging as interesting factors in Parkinson's research. It is unclear exactly how neurons are damaged in Parkinson's disease, but it is suggested that oxidative damage and energy depletion in the brain are involved. By activating RXR, neurons can be rescued from this degeneration."

In the study, there were two different dopaminergic cells (DA cells) to resemble the conditions present in persons with Parkinson’s. After studying these models, the researchers discovered the two RXR-activating ligands studied were able to selectively protect dopaminergic neurons from the stress induced in the model itself.

The results of this experiement were then confirmed to be accurate in a novel system in which dopaminergic neurons generated from mouse embryonic stem cells were treated with the neurotoxin. From this information they were able to theorize that "The regulation of RXR activity holds promise to contribute to a novel, alternative strategy to treat Parkinson's disease."

To reference the original article, it can be reached at http://www.sciencedaily.com/releases/2009/12/091210193158.htm. The article may also be searched by "RXR Activation: Hope for New Parkinson's Disease Treatment" ScienceDaily (Dec. 11, 2009).

Thiamine Found Important for Diabetics

What was once termed “adult-onset diabetes” due to the fact that the vast majority of individuals with the disease were not in their youth, type 2 diabetes now effects people of all ages, and is one of the fastest growing health problems in the United States today. The most common procedure for treating an individual with type 2 diabetes is to simply limit the amount of sugars and refined carbohydrates in their diet. However, I came across an article, which proposed that a simple B vitamin referred to as Thiamine (B1) may play a key role in the treatment of the disease.

The complications that often accompany the disease are devastating to the effected individual, as they might include nerve damage, eye problems, cardiovascular disease, and kidney damage. Also, kidney failure usually occurs 15 to 20 years following the onset of the disease.

A major contributing factor to this grim prognosis lies in the fact that most people are under the influence that by tightly regulating blood glucose levels with diet, oral medication, or insulin, one would likely avoid any complications. However, recent studies have shown that while these things may in fact delay complications, it often does not prevent their onset. This was found to be especially true with diabetic nephropathy, which occurs when the capillaries inside the glomerulus are destroyed. This leads to thickening and scarring of the glomerulus, resulting in the drastic decrease in normal renal function. The first detectable symptoms of diabetic nephropathy are an increased level of albumin in the urine, known as microalbuminuria. Research led by Dr. Naila Rabbani and Professor Paul J Thornalley at Warwick Medical School, University of Warwick, in collaboration with researchers at the University of Punjab and Sheik Zaid Hospital, Lahore, Pakistan, discovered that high doses of Thiamine administered orally can significantly decrease the secretion of albumin, and reverse the progression of early kidney disease in type 2 diabetics.

The subjects (40) for the study were type 2 diabetics between the ages of 35 and 65, and administered 100mg of thiamine three times a day for three months. The control group was administered a placebo. Of those given thiamine, there was a 41% decrease in albumin excretion, and 35% of the subjects given thiamine returned to their normal levels after the thiamine treatment.

Another study led by Professor Paul Thornalley was able to prove that thiamine deficiency plays a significant role in a vast array of vascular complications, and even diabetic neuropathy, as a result of diabetes. They also discovered that thiamine levels in type 1 and type 2 diabetes were 76% and 75% lower than the control, respectively. This drastic decrease in thiamine levels is not due to a low dietary intake of thiamine, but a significant increase in urinary output, therefore supplemental thiamine for diabetics may prove essential.

Possibly one of the most beneficial thiamine treatments, although synthetic, is Benfotiamine, which is chemically similar to the allithiamines one would find in garlic. One of the reasons Benfotiamine is so effective is its ability to readily cross lipid bilayers. This allows the compound to reach relatively high levels in the tissues, while avoiding being excreted rapidly. Benfotiamine has been shown to block three of the four metabolic pathways responsible for the progression of vascular disease in diabetics, and also demonstrated an ability to reduce Advanced Glycation Endproducts (AGEs).

For more information, the article can be found at http://www.NaturalNews.com/025136_thiamine_diabetic_diabetics.html or search “Thiamine Found Important for Diabetics”; by: Patty Donovan, citizen journalist.

Transmissible cancers

Came across a paper in Cell published in 2006 about the clonal origin of a transmissible cancer (CTVT) found in dogs. This was the first time I have heard about a tumor cell itself acting as the transmissible agent. The tumor cell itself was shown to be transferred as an allograft came from three different experiments: 1. CTVT can only be experimentally induced by transplanting living tumor cells, and not by lysates or killed cells. 2. The karyotype of the of the tumor cells is aneuploid but striking similar in tumors collected from various regions of unrelated dogs. And 3. A LINE-1 element upstream of c-myc was found in every tumor line examined. This tumor is passed from dog to dog usually by coitus but also can occur from licking and biting. This tumor is not fatal and usually is treatable. This cell has evolved into a parasite which represents the oldest somatic mammalian cell in continuous propagation.

For obvious reasons, Immunologists had a very hard time believing that the transmissible agent was the cell itself. The immune system is built to kill anything that is not recognized as self. So how did this cell evolve in such a way as to evade the hosts immune system? This was one of the questions that this paper sought to answer. As it turns out, MHC class I are downregulated in each of these tumors and class II are absent. Vertebrates has evolved NK cells to detect cells (typically tumors) that do not express MHC I and II and kill them because the normal acquired immune system would not be able to pick up these cells. These CTVT cells seemed to have evolved an expression level of MHC I that is high enough to keep the hosts NK cells at bay but low enough as to not illicit an immune response.

I found this paper really interesting and the authors and myself wonder why this isnt something that has occurred more often. At any rate, its amazing to see evolution in action, even at the cellular level. And we as researchers should look closely at this because it seems not a matter of 'if' but 'when' something like this will be seen in humans.

Murgia, C., Pritchard, JK., Kim, SY., Fassati, A., and Weiss, RA. Clonal Origin and Evolution of Transmissible Cancer. (2006) Cell 126, 477-487

Inflammation and the Host Response to Injury

As we all learnt in this class inflammation is one of fundamental protection mechanisms in human biology. This is how we protect ourselves from foreign invaders as well as abnormal cell inside of our body. But gone awry inflammation that spreads beyond the primary locus or fails to subside poses serious chronic and acute health risks for millions of people.

Each year about 60 million Americans sustain injuries. Trauma accounts for more than 12% of all medical spending in America. After the first 24 hours following the injury the primary cause of death is Multiple Organ Failure (MOF). It presents as an excessive inflammatory response directed towards patient’s own tissues. It is closely related to Systemic Inflammatory Response Syndrome (SIRS) and Sepsis. Sepsis alone accounts for up to 215,000 American’s lifes every year, according to the National Institute of General Medical Sciences. We referred to SIRS in Immu 7630 course as cytokine storm.

Growing evidence suggests that genetic factors drive key aspects of an individual’s inflammatory outcome. Scientists studying inflammation are trying to identify the genes that drive inflammation as well as biomarkers from throughout the course of inflammation. Genomic studies, in addition to their proteomic and metabolomic cousins, aim to resolve an age-old mystery: namely, why some patients recover readily from inflammation while others suffer and die from it. Ideally, new gene based discoveries will provide diagnostic biomarkers to predict who among these patients will react poorly to inflammation and why. If doctors could reliably predict this outcome in advance, they might tailor antibiotics and other treatment options to a patient’s own inflammatory system, potentially saving lives.

Among the numerous programs moving inflammation research forward is an effort funded by a National Institute of General Medical Sciences “glue grant,” so named because it “glues together” multidisciplinary efforts to tackle biomedical questions beyond the means of any one research group. This program, called Inflammation and the Host Response to Injury, strives to determine why patients can have dramatically different outcomes after traumatic injuries and burns.

UCD and Denver Health Medical Center are part of this multicenter research study.

In order to find immuno-inflammatory biomarkers for developing MOF Glue Grant is using genomic and proteomic analyses:


mRNA Isolation for Protein Coding

The Glue Grant investigators nationwide are isolating messenger RNA (mRNA) from blood and other available tissues. Isolation of the mRNA molecule is key to finding expressed genes in the vast expanse of the human genome. Since mRNA is very unstable outside of a cell, we are using a special enzyme called reverse transcriptase to convert it to complementary DNA (cDNA) which is a much more stable compound than mRNA and is presumed to represent the sequences of the genes being expressed. Sequences contained in the cDNA are used to generate expressed sequence tags (ESTs) that code for the protein in question or represent non-translated regions. ESTs are powerful tools for gene mapping and gene discovery. In the search for known genes, they greatly reduce the time needed to locate a gene. ESTs provide sequences that can be generated rapidly and inexpensively, in that only one sequencing experiment is needed per each cDNA generated and ESTs do not have to be confirmed for sequencing errors.

Only a fraction of genes are turned on, and it is the subset that is "expressed" that confers the unique properties to each cell type. Gene expression is the term used to describe the transcription of the information contained within the DNA into mRNA molecules that are then translated into the proteins that perform most of the critical functions of cells. Gene expression is a highly complex and tightly regulated process that allows a cell to respond dynamically both to environmental stimuli and its own changing needs. This mechanism acts as both an "on/off" switch to control which genes are expressed in a cell as well as a "volume control" that increases or decreases the level of expression of particular genes as necessary.

Microarray technology is applied to the samples to collect gene expression data. This technology works through the ability of a given mRNA molecule to bind specifically to, or hybridize to, the DNA template from which it originated. By using an array that contains many DNA samples, we can determine - in a single experiment - the expression levels of thousands of genes within a cell by measuring the amount of mRNA bound to each site on the array. The amount of mRNA bound to the spots on the microarray is precisely measured, thus generating a profile of gene expression in the cell. Microarray expression analysis are performed on the samples to determine the level, or volume, at which a certain gene is expressed. The arrays used in this analysis are called expression chips or "Gene Chips". We use the expression chips to detect expression patterns, that is, whether or not a particular gene is being expressed more or less under certain circumstances, and to examine changes in gene expression over a given period of time.

Detection and Analyses of SNPs

A single nucleotide polymorphism (SNP) is a small genetic alteration that can occur in a patient's DNA sequence. An SNP variation occurs when a single nucleotide (adenine, cytosine, thymine, or guanine) is replaced by one of the other three nucleotides. Each patient's genetic material contains a unique genetic pattern that is composed of several different genetic variations, including SNPs. It is believed that SNPs can serve both as a biological marker for pinpointing a disease or condition and may be directly associated with or causative of a certain disease or condition.

Proteomic Analyses

Cytokine mediators, leukocyte phenotypes, and cell signaling intermediates (e.g., protein kinases, NF-ê B family) will be analyzed from blood and plasma of trauma patients. Of particular interest are investigations of changes in pro-inflammatory and anti-inflammatory cytokines in isolated populations of cells from these patients. There are several cytokines of interest that include TNF, IL-1, IL-6, IL-8, IL-10, IL-12, IL-18, IL-1ra, p55, and p75.

Glue Grant study is still underway and the final results are yet to be analyzed…

For more information please visit www.gluegrant.org or search PubMed for “Inflammation and the host response to injury: a Large-Scale Collaborative Project”.

More news on my new favorite spice, tumeric! In a recent report researchers at the Univ. of Michigan found that the active ingredient in tumeric, curcumin, when applied to breast cancer cells in culture, was able to repress the growth of cancer stem cells, while not affecting the growth on non-stem cells. The same activity was seen with the active ingredient from black pepper, piperinem and was greatest when the two compounds were used together. Is this anti-stem cell activity a component in the anti-inflammatory mechanism used by curcumin, or perhaps an entirely separate effect?

This stem-cell specific growth inhibition may provide a better approach to limiting the growth of tumor cells in situ. Current treatments, such as standard chemotherapy or radiation, are thought to indiscriminately inhibit the growth of all cancer cells. But research has shown that many cancers are the result of cancerous stem cells driving the growth of tumors. And treatments that can specifically target these stem cells may provide better eradication than current treatments which are thought to often miss total irradication of the cancer stem cells.

Of note, the concentration of curcumin applied in culture is far greater (some 20 fold greater) than what could be obtained in the diet - supplementation would be required. Also, the study was an in vitro cell culture study, with all of the limitations inherent to cell culture models. But, the authors report that they hope to conduct Phase I safety trials as soon as next year.

1. Madhuri Kakarala, Dean E. Brenner, Hasan Korkaya, Connie Cheng, Karim Tazi, Christophe Ginestier, Suling Liu, Gabriela Dontu and Max S. Wicha. (2009) Targeting breast stem cells with the cancer preventive compounds curcumin and piperine. Breast Cancer Research and Treatment, Vol 66(3), p123-133

Hygiene Hypothesis goes in utero!

On the blog and in class we have discussed the Hygiene Hypothesis. I was very interested to read today in a review article in JEM that there seems to be a growing body of evidence indicating that bacterial exposure in pregnant mothers can reduce the incidence of asthma and allergy in their children.

Of note, newborns of mothers exposed to a barn environment during pregnancy displayed enhanced Treg activity. A parallel study looking at PBMCs from 5-13 year-old children of farm-exposed mothers showed decreased sensitization. The really COOL thing is that the decreased sensitization was unrelated to the children's farm exposure at the time the PBMCs were taken. Instead the effects were exclusively related to the mothers' farm exposures during pregnancy.

This (and other data you can read in the article) suggests that during a child's development in the womb its immune system can be programmed to some degree. Why do you think maternal programming might exist? Is this exposure more relevant than the child's own exposure?

Soothing signals: transplacental transmission of resistance to asthma and allergy

Patrick G. Holt and Deborah H. Strickland

J. Exp. Med. 2009;0 200924691-20092469, Published online Dec 7 2009, 10.1084/jem.20092469.

Placebo Effect

I thought the conversation about the saccharine water and the placebo effect in mice was very interesting. I was wondering if any studies have been done that can show that behavior can also negatively impact the immune system.

It seems that when pregnant women get sick from a certain type of food during pregnancy, they tend to feel nauseous or get ill from that food later on after the pregnancy. I've heard similar stories with people that get food poisoning. I wonder if this has to do with the body thinking that it will get sick from that same food because it has in the past. I would think that this would be a "reverse" placebo-effect in the sense that something that causes problems once in the body will be associated in the mind as always being a problem causer.

Aging, DNA damage, and chronic inflammation?

Hi all,

Recently I found a research article that describes experiments that show for the first time a possible cause of the chronic inflammation that seems to be implicated in the etiology of so many chronic medical conditions. This chronic inflammation becomes more common and widespread as people age, or when they somehow sustain considerable damage to a particular tissue over time. One underlying cause of this age-associated inflammation turns out to be unrepairable DNA damage which accumulates in non-cycling cells of our bodies (either quiescent or senescent) as we age. This accumulation of DNA damage in cells as we age has been known for some time - but what this paper, by Campisi et. al. [1] at the Lawrence Berkeley National Laboratory, unexpectedly showed is that these damaged cells secrete a unique and powerful battery of inflammatory cytokines. As we age we constantly secrete more and more of these inflammatory cytokines in our tissues, which can lead to the development or exacerbation of all of the chronic inflammatory conditions which we have studied this semester

As an aside, I found this paper on the Faculty of 1000 Biology web-site (http://f1000biology.com/guardpages/evaluation/1145049//article/article.asp%253Fid%253D1145049%2526view%253D%2526style%253D). This web-site is a very cool service which provides a synopsis and critique of the most interesting current papers in biology, as judged by the "Faculty of 1000" - generaly recognized respected researchers in their fields. So, if you can afford the subcription fee, you can have the hottest papers in your research area identified and critiqued by experts and sent straight to your e-mailbox! You can also get a copy at the cool free-access Public Library of Science (PLoS) website, http://www.plosbiology.org/article/info:doi%2F10.1371%2Fjournal.pbio.0060301.

Anyways, back to the paper. In their experiments, the authors modified commercial antibody arrays to substantially improve their range and sensitivity, and then measured the proteins secreted by senescent cells in culture, as well as from tissue samples that were collected from prostate cancer patients both before and after completing a DNA-damaging form of chemotherapy. They found that different types of cells from different tissue all secrete a very similar set of proteins when they senesce in response to DNA-damaging radiation or chemotherapy. The authors propose to call this common response to DNA damage and senescence "senescence-associated secretory phenotype", or SASP. Importantly, they then showed that it occurs not only in cultured cells, but also to cells in vivo in response to exogenously introduced DNA damage (and by extension to normally produced endogenous oxidative DNA damage). The SASP develops slowly over several days and only after DNA damage of sufficient magnitude to induce senescence. The SASP featured high levels of secreted inflammatory cytokines, immune modulators, and growth factors, all of which are associated with inflammation and malignancy (IL-6, IL-8, GRO-α, MCP-1, GM-CSF...).

Note: For those interested, the primary authors followed up this seminal line of experiments with further work clarify some of the molecular interactions involved in this inflammatory phenotype of aging and/or damaged cells, finding connections with the well characterized dna repair/maintenance genes ATM, NBS1 and CHK2, but not the cell-cycle arrest enforcers p53 and pRb... [2]


- Brian P


1. Jean-Philippe Coppé, Christopher K. Patil, Francis Rodier, Yu Sun, Denise P. Muñoz, Joshua Goldstein, Peter S. Nelson, Pierre-Yves Desprez, Judith Campisi (2007) Senescence-Associated Secretory Phenotypes Reveal Cell-Nonautonomous Functions of Oncogenic RAS and the p53 Tumor Suppressor, PloS Biology, www.plosbiology.org




2. Francis Rodier1,2, Jean-Philippe Coppé1, Christopher K. Patil1, Wieteke A. M. Hoeijmakers1,4, Denise P. Muñoz2, Saba R. Raza1,5, Adam Freund1,3, Eric Campeau1,6, Albert R. Davalos1 & Judith Campisi1,2 (2009) Persistent DNA damage signalling triggers senescence-associated inflammatory cytokine secretion, Nature Cell Biology 11, 973 - 979.

Pharmacist's Letter Continued!

For those of you in the UA course, I presented info from the pharmacist's letter database on turmeric. The database is designed to show any info that is related to medications including natural supplements that allow pharmacists to convey information including interactions, safety, and effectiveness. Recall that turmeric is only possibly safe when used orally or topically in medicinal amounts and likely safe when used in amounts commonly found in food. I found similar information using the same database regarding the scientific article from last Monday where they tested several active spice-derived components and how effective they were at being anti-inflammatories. This may be a long blog so sorry in advance. I just found it really interesting. I tried to bold anything related to inflammation to bring attention to them.

1. Black Mustard (allyl isothiocyanate)
-Uses: the oil is used for the common cold, rheumatism and osteoarthritis; orally - used to induce vomiting, a diuretic, and an appetite stimulant; topically - pneumonia, osteoarthritis, aching feet, and as a counterirritant (something that causes inflammation in one area to lessen it in another)
-Safety: likely safe when used orally in the amounts commonly found in foods; there is insufficient reliable information available about the safety of black mustard when used orally or topically for medicinal purposes
-Effectiveness: There is insufficient reliable information about the effectiveness of black mustard
-Mechanism of action: applicable part are the seed, oil, leaf, and powder; the powder contains glucosinolate sinigrin, which produces allyl isothiocyanate when mixed with warm water that has strong antimicrobial (bacterial and fungal) properties and can act as a counterirritant when diluted; seed, oil, and powder can have powerful irritant properties that can cause pain and increase inflammation of the skin
-Interactions with Herbs & Supplements, Drug, Foods, Lab Test, and Disease or Conditions - None known
-Adverse Reactions: Large amounts orally - vomiting, stomach pain, diarrhea, cardiac failure, breathing difficulties, coma, and possibly death
-The interesting thing I found was that this drug is such a potent so when applied topically for too long (longer than 10 minutes), it can lead to severe skin irritation and even burns and the concentrations as a counterirritant is 0.5% to 5% and used 3-4 times a day

2. Garlic (diallyl disulfide)
-Uses: hypertension, hyperlipidemia, coronary heart disease, earaches, chronic fatigue syndrome, prevention of colon cancer and other cancers of the breast, lung, prostrate, and bladder, osteoarthritis, allergic rhinitis, colds, flu, TB, whooping cough, and a lot of other random things; topically - oil is used for scalp diseases and vaginitis
Safety: Likely safe when used orally and appropriately; has been used safely in clinical studies lasting up to 7 years without significant toxicity; possibly unsafe when used topically - can cause severe skin irritation; possible safe for children in appropriate oral amounts and possibly unsafe in large amounts; pregnancy - likely safe when used orally in amounts commonly found in foods; possibly unsafe - orally in medicinal amounts
-Effectiveness: possibly effective to treat atherosclerosis at 300 mg per day, some evidence associated with decreasing the risk of developing stomach cancer, and come clinical research shows that taken orally, garlic can modestly reduce blood pressure
-Mechanism of action: bulb mostly used as an antihyperlipidemic, antihypertension, antifungal, and has also been reported to have antibacterial, antihelmintic, antiviral, antispasmodic, antithrombotic, and an immunostimulant
-Based on this info and other info that I found off the database, I didn't find any clinical evidence that suggests that garlic has no evidence of having anti-inflammatory properties. If it did, this database would discuss it because it talks about everything related in clinical terms regarding supplements.

3. Ginger (zingerone)
-Uses: Orally - motion sickness, morning sickness, dyspepsia, rheumatoid arthritis, loss of appetite, osteoarthritis, migraines, headache, anorexia, cough, bronchitis, stomachache, and others; Fresh ginger - orally for malaria, rheumatism, and toothaches
-Safety: likely safe - when used orally and appropriately; has been used in clinical trials (includes trial info in the database)
-Effectiveness: can reduce severity of nausea and vomiting in pregnant patients and postoperative patients and insufficient reliable evidence to treat chemo-induced nausea and vomiting; preliminary evidence shows modest benefits in OA - extract at 170 mg a day three times daily for 3 weeks is effective in reducing pain; some preliminary evidence shows that ginger might help in decreasing joint pain in patients with RA
-Mechanism of action: pharmacological properties - antipyretic, analgesic, anti-inflammatory, sedative, antibiotic, and other properties

4. Black Pepper (piperine)
-Uses: stomach upset, bronchitis, and cancer
-Safety: Likely safe when used orally in amounts commonly found in foods; possibly safe when used orally and appropriately in medicinal amounts; black pepper oil is used topically and is nonirritating and typically well tolerated; likely safe in children, pregnancy, and lactation
-Effectiveness: Insufficient reliable information about the effectiveness of black pepper
-Mechanism of Action: a typical diet provides 360 mg of black pepper provides about 60-110 micromolar of piperine that seems to increase oral absorption of drugs and other substances
-There doesn't seem to be much clinical evidence that black pepper works as an anti-inflammatory

In conclusion, only two out of the four anti-inflammatory agents mentioned in the article seem to have any clinical evidence. It will be interesting to see if new research sheds any more light on black pepper and garlic.

Does air pollution exacerbate asthma?

A 2007 article in the Annals of Allergy, Asthma & Immunology asked the very salient question: does air pollution exacerbate asthma?

The objective of the article was to investigate associations between traffic and outdoor air pollution levels near residences and poorly controlled asthma among adults diagnosed as having asthma in Los Angeles and San Diego counties.

The authors estimated traffic density (TD) within 500 feet of 2001 California Health Interview Survey (CHIS) respondents' reported residential cross-street intersections. They then assigned annual average concentrations at government monitoring stations within a 5 mile radius of reported residential cross-street intersections of:

1. ozone

2. nitrogen dioxide

3. particulate matter (PM) 2.5 and 10 micrometers or less in diameter

4. carbon monoxide

In order to obtain the study population, 19,664 individuals were interviewed; 2,237 (11.4%) were diagnosed by a physician as having asthma. These individuals’ residence locations were entered into a database. The final study population was 1,609 adults (others were excluded because of missing data).

Exposure evaluation was evaluated by examining traffic density, which was based on a 500 foot buffer around person's probable home street segment. From the nearest measuring station within 5 miles of the respondent, the authors determined annual average concentrations of: ozone, nitrogen dioxide, particulate matter 2.5 micrometers or less or 10 micrometers or less, carbon monoxide.

For the statistical analysis, traffic density was divided into three tiers:

1. Low TD (20th percentile or less)

2. Medium TD (21-80 percentil)

3. High TD (> 80th percentile)

The results can be summarized in the following 4 main points:

1. There was a 2-fold increase in poorly controlled asthma among asthmatic adults (OR 2.11, 95% CI 1.38-3.23) in the highest quintile of traffic density after adjusting for age, sex, race, and poverty.

2. Similar increases were seen for nonelderly adults, men, and women. The strongest associations were seen in elderly adults (OR 3, 95% CI 1.13-7.91).

3. Ozone exposures were associated with poorly controlled asthma among two subgroups:

- elderly adults (OR 1.7, CI 0.91-3.18 per 1 pphm)

- men (OR 1.76, CI 1.05-2.94 per 1 pphm)

4. Particulate matter 10 micrometers or less affected primarily women (OR 2.06, CI 1.17-3.61), even at levels below national air quality standard.

The authors’ conclusion? Heavy traffic and high air pollution levels near residences are associated with poorly controlled asthma.

While this may seem like a relatively straightforward conclusion based on the data, this study had some very significant caveats to keep in mind:

1. Misclassification of exposures from stations:

- Traffic pollutants can have a strong spatial gradient, meaning levels detected at measuring stations might not reflect actual exposure levels of individuals.

2. Resident variability:

- The study did not take into account variables such as personal mobility, occupation-related exposures, or indoor exposures.

3. Temporal ambiguity

- This study was based on cross-sectional survey data, so the length of time any individual lived in a neighborhood was unknown.

4. Medication use

- Individuals’ use of medications was unknown; use could reduce symptoms and lead to underestimated prevalence of poorly controlled asthma.

Y-Y Meng, et al. Traffic and outdoor air pollution levels near residences and poorly controlled asthma in adults. Annals of Allergy, Asthma, & Immunology 2007;98:455-463.

Novel NF-Kb links inflammation and cancer promotion

A recent paper by researchers at the University of California [1] reveals a novel mechanism by which chronic inflammation may play a decisive role in the promotion of cancer. This mechanism is a sort of molecular “cross-talk” between two processes in cells which were thought to be distinct – cell development and inflammation. This cross-talk comes through the actions of a protein, p100, which has been found to be crucial in a cell’s normal developmental metabolic pathways as well as in the cell’s response to inflammation. Novel cancer therapeutic treatments which target developmental pathways linked to chronic inflammation via p100 may prove effective in conjunction with the use of current anti-inflammatories such as NSAIDS.

NF-κB activity is inducible by a diverse range of stimuli including pathogen derived substances and intercellular mediators of inflammation. It is known that NF-κB is normally repressed by three different intracellular inhibitors (IκB-a,b,e proteins),and that during classical inflammatory signaling these inhibitors are released from binding NF-κB, resulting in its activation. A second, independent NF-κB activation pathway is thought to be activated in response to cell developmental signals rather than inflammatory signals. The molecular mechanism of this particular pathway has remained uncertain, but the results of this study show that a fourth IκB inhibitor (p100) is involved in the activation of NF-κB in developmental pathway signaling, such as during lymphoid organogenesis (see included figure).

This diagram reveals the cross-talk between NF-κB activation via inflammatory or developmental signaling – inflammatory signaling can activate developmental (cell reproduction and differentiation) pathways to some extent and vice-verse. And further work by the researchers showed that the “cross-activation” of developmental pathways by classical inflammatory signaling was much stronger, and of longer duration, than the normal developmental activation signal strength. For example, a 1 hr pulse of TNF-a stimulation resulted in an elevation of p100 protein of about 4-fold, that persisted for more than 20 hr. Remarkably, the researchers also showed that the strength of this cross-talk increased with repeated signaling – in other words, the longer cells were exposed to inflammatory signals via TNF-a, the more they were stimulated to reproduce! This is in marked contrast to the normally accepted role of TNF-a in signaling cell death.

The researchers speculate that in epithelial cells that are exposed to prolonged periods of inflammation, such as they might experience during chronic inflammatory diseases, the amount of cross-talk between the classic TNF-a mediated inflammatory cell death pathway and the p100 mediated developmental cell growth pathway may profoundly shift, with the inflammatory signals generating a stronger and stronger cell growth signal in place of the expected cell death signal. This could be the mechanism responsible for chronic inflammations being able to promote the growth of many types of cancers – after prolonged exposure, inflammatory TNF-a signals that were originally responsible for inducing cell death (and resolving inflammation and tissue repair processes) become instead growth stimulating signals that drive epithelial cells toward unrestrained cancerous growth.


1. Basak S, Kim H, Kearns JD, Tergaonkar V, O'Dea E, Werner SL, Benedict CA, Ware CF, Ghosh G, Verma IM, Hoffmann A.(2007) A fourth IkappaB protein within the NF-kappaB signaling module. Cell. Vol 128(2) p369-81.

NSAIDS and Cancer Prevention

Over the last four decades dozens of epidemiological, clinical and experimental studies have established nonsteroidal anti-inflammatory drugs (NSAIDs) as promising epithelial cancer chemopreventive agents. The long-term use of aspirin and other NSAIDs has been shown to reduce the risk of cancer of the colon and other gastrointestinal organs, as well as cancer of the breast, prostate, lung, and skin. In a recent review, Harris[1] et. al. comprehensively reviewed the published research on NSAIDs and cancer incident. Data from 91 epidemiologic studies were analyzed for the dose response of relative cancer risk and level of NSAID intake for ten prevalent human cancers. The results showed a significant exponential decline in the risk with increasing intake of NSAIDs (primarily aspirin or ibuprofen) for 7 out of the 10 cancers, including the four major types: colon, breast, lung, and prostate cancer. Consistent daily intake of NSAIDs (primarily aspirin), produced risk reductions of 73% for stomach, 69% for esophageal, 63% for colon, 47% for ovarian, 39% for breast and prostate, and 36% for lung! NSAID effects became apparent after five or more years of use and were stronger with longer duration.

Despite general consensus as to the effectiveness of NSAIDs for cancer prevention, unresolved questions with regard to safety, efficacy, optimal treatment regimen, and mechanism of action currently limit the clinical application of NSAIDs to the prevention of cancer. Also, the development of safe and effective NSAIDs for chemoprevention is complicated by the potential that rare, serious toxicity may offset the benefit of treatment with these drugs given to healthy individuals who have a low risk of developing cancer. However, I believe there is growing support for the view that a full understanding of the role of NSAIDs in the prevention and treatment of epithelial cancers will be an integral part of the development of effective future treatments for reducing mortality and morbidity from most cancers.


1. Harris RE, Beebe-Donk J, Doss H, Burr Doss D. Aspirin, ibuprofen, and other non-steroidal anti-inflammatory drugs in cancer prevention: a critical review of non-selective COX-2 blockade (review). Oncol Rep. 2005 Apr;13(4):559-83.

Chronic Beryllium Disease --going beyond HLA-DPB1?

I have been working for several years with a doctor who does research with chronic beryllium disease (CBD). If you recall from our notes, this is a disease which is caused by inhalation of beryllium dust and an immune response which leads to granuloma formation in the lung. There is a known association between certain HLA-DPB1 alleles and CBD and we are always looking for ways to better understand what this means. The article “Electrostatic Potential on Human Leukocyte Antigen: Implications for Putative Mechanism and Chronic Beryllium Disease” describes how the authors constructed structural models of HLA-DP representing several distinct allotypes and how they calculated the electrostatic potential on the surface of these models. They determine that the higher the negative charge for the HLA-DPB1 alleles the more likely that the person would have chronic beryllium disease. These charges are then related back to certain alleles. I guess I have a question for anyone who may be reading this: Does the electrostatic charge give you any new meaningful information?

The article's link in PubMed.

H1N1 Influenza strains contain H5N1 genetic material...Are the young and healthy at risk of a massive cytokine storm induced by virus infection?

Human infection with highly pathogenic avian influenza A viruses (HPAIV) such as H5N1 subtypes are characterized by inner bleedings and a massive overproduction of cytokines known as cytokine storm (Schmolke et al., 2009). Recent reports have linked the genomes of the last three pandemic influenza viruses as having emerged from gene reassortment events and possessing HA genes of avian influenza origin. Thus, these findings provoke the following question: If existing influenza strains (i.e. 2009 H1N1 influenza) undergo gene reassortment similar to H5N1 strains, is it possible that the resulting human adapted form could manifest itself as a deadly pandemic targeting healthy individuals by the onset of a massive cytokine storm?

Review of what the “cytokine storm” is: when the body’s normal immune system overacts to an intruder such as a virus, by overproduction of signaling chemicals (known as cytokines and chemokines) that help mobilize immune cells capable of removing infectious agents from the body. The extremely high levels of cytokines that are produced as a consequence of a healthy immune system are over-exaggerated and fatally damaging to tissues and organs. Death will usually result from multisystem organ failure.

I came across two articles I’d like to reference in regard to this blog. Below is a brief summary of each paper, highlighting the author’s findings which are relevant to this blog topic.

The first paper published in July 2009 in Science (Rebecca J.Garten et al., 2009) involved a large collaborative effort of many authors representing the WHO, CDC, NIH and various health organizations. The objective of this study was to characterize the antigenic and genetic variations of 2009 H1N1 circulating in humans. In their attempts to do this, they analyzed genome sequences isolated from 2009 H1N1 patients with previous strains of influenza including classical swine, human seasonal, American avian, North American and Eurasian swine strains. This paper examines the antigenic capabilities of influenza and how it has evolved overtime resulting in the emergence of pandemic flu strains. The paper provides the lineages and reassortment events that have taken place overtime and which gave rise to three pandemic influenza viruses (1918 H1N1, 1957 H2N2, and 1968 H3N2). The lineage suggests that all three strains originated from nonhuman reservoirs and the HA genes of all, originated from avian influenza virus. Now the most recent 2009 A/H1N1 strain contains a never before seen combination of gene segments which appear to be derived from Eurasian swine and classical swine lineages (Garten et al., 2009).

The second paper, by Schmolke M. et al., 2009 in the Journal of immunology by a group in Germany presents in accordance with numerous reports, the observation that the high fatality rate of avian influenza is a consequence of lethal cytokine storm induced by A/H5N1 virus both in humans and animals (Deng RM. et al., 2008; Claas EC. et al., 1998). The significance of the study is based on the concern of highly pathogenic avian influenza viruses (HPAIV) being directly transmitted to humans as a possible source of a new influenza pandemic. They performed mRNA profiling on human umbilical vein endothelial cells (HUVEC) infected with H5N1 based on the idea that endothelial cells are a major source of cytokines and chemokines, and relevant for systemic viral dissemination. Their results confirm clustering and upregulation of inflammatory/immune response which are accompanied by a massive systemic production of cytokines and chemokines, characteristic of a cytokine storm.

It’s frightening to acknowledge the ongoing threats of the 2009 H1N1 swine flu and the still present Asian avian flu virus (AAV H5N1) and its pandemic potential. In regards to the influenza strain reassortment and antigenic evolution, sequence variation in the gene segments of the H5N1 strain have been shown to contribute to viral virulence. It will be interesting to compare the updated sequencing of gene segment among the 2009 H1N1 strains with previously pandemic strains which could suggest whether it may or may not be likely to elicit such a strong cytokine response associated with highly virulent strains such as H5N1.

Based on the recent publications mentioned above I think it is obvious that 1) the new swine flu influenza A strain appears to be a combination of previous bird, swine and human genetic make-up. In addition, all three show lineage to have originated from avian flu. 2) H5N1 the highly virulent avian flu strain causes lethal cytokine storm in young and healthy individuals. These ideas together implicate that H1N1 viruses have pandemic potential and historically support the possibility that healthy young adults may be more susceptible to severe infection due to a strong immune system capable of eliciting a lethal cytokine storm. If uncontrolled, such a cytokine storm occurring in vital organs can lead to organ failure and death.

What would be the best approach in preventing an influenza pandemic or in addressing the potential damage it could elicit by inducing a cytokine storm in infected individuals? Should health officials stringently monitor strains of the 2009 A(H1N1) virus for any antigenic and genetic changes? In the case of infected patients, should the use of prophylactics such as immune suppressors or antivirals be used to minimize the possibility of the cytokine storm?

References:

Garten RJ et al., 2009. Antigenic and genetic characteristics of swine-origin 2009 A(H1N1) influenza viruses circulating in humans. Science. 325:197-201.

Schmolke M, Viemann D, Roth J, Ludwig S. 2009. Essential impact of NF-κB signaling on the H5N1 influenza A virus-induced transcriptome. J Immunol. 183:5180-5189.

Deng RM, Korteweg LC, Gao Z, McNutt MA, Ye J, Zhang I, Gu J. 2008. Distinctly different expression of cytokines and chemokines in the lungs of two H5N1 avian influenza patients. J Pathol. 216:328-336.

Claas EC, Osterhaus AD, van Beek R, De Jong JC, Rimmelzwaan GF. 1998. Human influenza A H5N1 virus related to a highly pathogenic avian influenza virus. Lancet. 351:472-477.

Genetic Variation In The Maintenance of HIV Seronegative Status Among High-Risk Populations

Chemokine receptors have proved to be important in maintaining seronegative status among individuals who have a high risk for contracting the HIV virus. Cultures of lymphocytes and macrophages have shown that those who are relatively resistant to HIV infection secrete high levels of co-receptors CCL3, CCL4, and CCL5 when they are inoculated with this virus. It has been discovered that HIV resistant people are homozygous for an allelic, nonfunctional version of the CCR5 co-receptor, D32. In the Caucasian population, the prevalence of this frameshift mutation (coupled with a protein truncation) in the homozygous form is fairly high at about 1%. The heterozygous allelic form of this mutation may provide some modest protection against the sexual transmission of the HIV virus, and could even slow the progression of existing infections. In addition to this, variation in the promoter region of the CCR5 gene has been identified in both Caucasians and African Americans. Differences in promoter regions have also been associated with different rates of progression of the disease. This evidence shows that CCR5 is the major macrophage and T-lymphocyte co-receptor used by HIV to establish initial infection. Interestingly, this also offers hope that primary HIV infection can be blocked by anti-CCR5 receptors.

Janeway, Charles. Janeway's Immunology - 7th Edition / Kenneth Murphy, Paul Travers, Mark Walport. Garland Science. 2008.