Upregulation of TNF-a leads to working memory deficits

Evidence from humans suggests that most chronic pain patients (without brain involvement) experience working memory deficits. However, the mechanism responsible for this effect has remained elusive. This effect does not seem dependent on pain level though, as acute pain patients do not have memory deficits, nor do the deficits improve in chronic pain patients on opioid therapy.

To look at the mechanisms responsible, researchers performed spared nerve injury (SNI) on a group of rats. They found that SNI rats performed worse on a radial arm maze, specifically a test designed to test short-term memory (whether they re-entered an arm they had already entered). This memory effect was present even in a small number of animals who received an SNI, but did not develop pain-related behaviors.

TNF-a has been shown to inhibit LTP in the hippocampus, and the researchers looked at the relationship between TNF-a and working memory deficits. TNF-a levels were increased in the CSF and hippocampus of SNI rats, and administration of TNF-a antibody or synthesis inhibitor reduced the amount of working memory errors. Conversely, administration of recombinant TNF-a into the ventricles or hippocampus of non-SNI animals resulted in working memory deficits similar to those in the SNI animals.

This shows that elevated levels on TNF-a in chronic disease states can cause impairment of working memory, and that reductions of TNF-a improves these deficits. It highlights how uncontrolled inflammation or cytokine secretion can have detrimental effects on cognition, even when there is no brain involvement in the original problem.

http://www.ncbi.nlm.nih.gov/pubmed/21289602

SBrookshire495K

Metal as an Antigen?

Who’s scared of a little metal? YOU SHOULD BE. With an atomic number of 4 and an atomic weight of 9, the lightest metal on the periodic table is beryllium (Be). Beryllium’s properties make it very valuable. It is used in many industries including aerospace, defense and ceramics. It is also used in everyday objects such as light fixtures, golf clubs and electronics. Even though beryllium has properties such as conductance and weight that make it advantageous, there is a flip side to the coin. Beryllium, in the form of particulate dust, can and does get inhaled by those who mine, machine and form the metal into objects used everyday. This inhalation causes a disease in the susceptible known as berylliosis or Chronic Beryllium Disease.

Chronic Beryllium Disease (CBD) is a CD4 (helper) T cell mediated disease. In this affliction, inhaled particles of beryllium are taken up by alveolar macrophages (those in the lung). Up to 14% of this endocytized beryllium may be presented (unpublished results) in the contest of specific MHC class II on the macrophage to beryllium responsive helper T cells. This presentation causes a signaling cascade through the T cell receptor (TCR) and leads to events including macrophage cell death by apoptosis, T cell expansion, T cell pro-inflammatory cytokine release and granuloma formation in the lungs. These granulomas cause a slew of problems that can never be fully resolved.

Presentation of beryllium is performed mostly by a subset of MHC class II known as HLA-DP. Remember that HLA-DP, HLA-DR and HLA-DQ are the three HLA in the human genome. Within the HLA-DP there are subsets and the one most responsible for CBD is HLA-DP0201. In a paper by Richeldi et al. they found that 83% of subjects suffering form CBD were HLA-DP0201, compared to 30% of the unaffected population.

However, there are also other HLA including some DR and DQ subtypes that are found in CBD patients. After several years of work researchers have found that all of the HLA types (DR, DQ or DP) that confer susceptibility to beryllium have a glutamic acid corresponding to position 69 (Glu69) of the HLA-DP beta-pleated sheet. Recently, researchers at University of Colorado Denver have shown that substitution of amino acids in the HLA-DP0201 abrogates the T cell response to beryllium.

Performing a series of experiments involving amino acid substitutions, researchers were able to observe that replacement of certain amino acids affected the T cells beryllium specific response. The substitution of the glutamic acid at position 69 of the beta chain with the uncharged amino acid alanine either interferes with T cell recognition or HLA-DP0201 presentation. Either way, T cell response was totally abolished. This abolishment of beryllium specific T cell response was also observed during substitution of several other beta chain amino acids that have been modeled to have interaction with the TCR of beryllium responsive T cells. Since the T cell can not respond to the beryllium (either because the TCR can not interact with the MHC or the MHC has been changed so that it cannot present beryllium to the TCR) it will not make pro-inflammatory cytokines or direct the alveolar macrophages to wall off the insoluble beryllium in a granuloma.

This study has highlighted the importance of the delicate balance between the MHC and the TCR in interactions to develop disease. If a particular MHC cannot present a particular antigen there can be no disease. Also, if the responsive TCR cannot bind the MHC with a particular antigen, there can be no disease.

Mutagenesis of beryllium-specific TCRs suggests an unusual binding topology for antigen recognition.

Bowerman NA, Falta MT, Mack DG, Kappler JW, Fontenot AP.

J Immunol. 2011 Oct 1;187(7):3694-703. Epub 2011 Aug 26.

You've received antivenom following a North American crotalid envenomation, but you're still not out of the woods yet!

There are approximately 100,000 deaths worldwide from snakebites each year. In North America, venomous snakebites are often the result of envenomation from the genus Crotalidae, which includes The Western Diamondback (Crotalus atrox), the Eastern Diamondback (Crotalus adamanteus), and the Mojave rattlesnake (Crotalus scutulatus). Bites from these snakes are usually treated with Crotalidae Polyvalent Immune Fab (ovine)[FabAV]. For many years Antivenin (Crotalidae) Polvalent [Equine] (ACP) was the sole treatment for crotalid envenomation in North America. It is/was composed of purified horse sera containing IgG from horses immunized with crotalid snake venom. ACP was known for causing possible hypersensitivity reactions, including anaphylaxis and serum sickness. FabAV is made from sheep (ovine) and is composed of Fab fragments and is less immunogenic. FabAV seems to work well, but patients need to be closely monitored to determine how many repeated doses are required. A case report from 2008 published in Clinical Toxicology describes a 24-year old man who was intoxicated and fell into his western diamondback rattlesnake enclosure and was bitten on both hands and the right side torso by two diamondbacks. When he arrived at the hospital he was hypotensive, tachycardic, and thrombocytopenic with a platelet count of 17/nl. Several hours after administration of current FabAV protocol the patient's hematocrit continued to drop. The patients blood pressure upon arrival was 83/48mmHg and heart rate was 104 bpm. The patient received 5 vials over a 1 hour period. 5 additional vials were given over the next hour. Blood pressure improved to 114/86 mmHg and platelet count improved to 206/nl. He was then given 2 vials every six hours for the next 18 hours. The next morning the patient had a platelet count of 15/nl and hematocrit decreased from 37.8% to 21.6%. The patient was given packed red blood cells and platelets over the next 12 hours and his hematocrit and platelet counts stabilized. The patient remained in the hospital for eleven days while doctors worked hard at maintaining adequate blood stats. It took the patient 14 days for his own body to keep his hematocrit and platelet counts within physiological range. So, FabAV may work well at minimizing tissue damage and possible death from crotalid envenomation, but extensive patient monitoring and repeated FabAV doses are very important.

Clinical Toxicology (2008) 46, 823-826

mPGES-1 knockout mice display reduced pain and tumor progression in a bone cancer model

Anti-inflammatory drugs such as NSAIDS inhibit the formation of prostaglandins, particularly PGE2. There are several isoforms of the enzyme directly responsible for the formation of PGE2, and one of these, mPGES-1 (microsomal prostaglandin E synthase-1) is an inducible enzyme. Thus, the expression of mPGES-1 is increased in inflamed tissues, and this increased expression correlates with an increase in COX-2. To see if this enzyme represents a possible target of drugs, bone cancer pain was evaluated in mPGES-1 knockout mice.
mPGES-1 KO mice exhibited a delay in exhibiting pain related behaviors as well as a decrease in the size of the tumor, relative to wild type mice. Levels of PGE2 were markedly reduced in mPGES-1 KO mice as well as the level of another pro-nociceptive molecule, CGRP. Wild type mice given celecoxib had similar levels of pain related behaviors and bone loss as mPGES-1 KO mice.
This suggests that inhibition of inflammatory molecules, particularly PGE2, may help reduce bone cancer related pain and the progression of the tumor. mPGES-1 may be a suitable target for drug development because it may reduce pain and inflammation, while avoiding the side effects associated with both selective and non-selective COX inhibitors.

http://www.ncbi.nlm.nih.gov/pubmed/21324324

SBrookshire495K

The Adzuki Bean...A Potential Natural Immuno-modulator

Just as the first major anti-inflammatory drug to become ubiquitous was based on a folk remedy of chewing or making tea from willow bark, it is likely that others exist naturally that have yet to be identified. Perhaps there even exist others that more specific or potent. When taken out of context, this is the basic assumption of most naturopathic arguments. Where naturopathy becomes most dubious for me is when its proponents insist that natural remedies are more efficacious simply due to the state of being in their “natural” form. Indeed, willow bark is thousands of times less potent volume-for-volume than is isolated salicylic acid.

That aside, I had to seek out any natural-based anti-inflammatory therapies that seemed to be promising in an attempt to appear unbiased about the subject. The Adzuki bean (Vigna angularis; alternatively ‘Azuki’ or ‘Aduki’) is a commonly cultivated bean throughout Asia, especially northeastern Asia. It forms the basis for many Asian dishes and its extract is prescribed to treat inflammation and arthritis in Asian countries [Yu 1197.] Yu, et al, investigated the immune-modulatory activity of an ethanol extract of the bean and found it to down-regulate several critical immune-signaling and transcription factors [Yu 1202.]

Mukai and Sato narrowed the focus further to the active properties of the bean’s coat as the most promising target for further research, as this portion of the bean contains the many polyphenols that they described as responsible for attenuating vascular oxidative stress and inflammation in hypertensive rats [Mukai 17.] Of the most interest to this, our section on anti-inflammatories, is that Mukai described a “significant” decrease in both iNOS and COX-2 expression upon treatment with the bean coat preparation [Mukai Figs. 4 & 5.] Importantly, Yu, et al, were also able to show that the immune-modulatory components of the bean were orally-available [Yu 1204.]

Is it likely that western doctors will begin prescribing beans to chronic inflammation patients? Probably not; however, it may just be a matter of time before we see polyphenols isolated from the Adzuki bean serve as a basis for synthesis, or a direct source of anti-inflammatory drugs themselves.

[For ease of reference, each paper is linked to PubMed, but full-text versions are available as of this post through the UA library online after login.]

Mukai > Pubmed #20185287: http://www.ncbi.nlm.nih.gov/pubmed/20185287

Yu > Pubmed #21821108: http://www.ncbi.nlm.nih.gov/pubmed/21821108

Inflammation vs Obesity in Insulin Resistance

Since the value of the research discussed in the lay article “Enzyme boosts metabolism, prevents weight gain in mice” is hidden by what seems to be the author’s lack of knowledge in this subject area, here is a summary from the paper itself:

Information surrounding obesity, inflammation, and insulin resistance is usually presented in a linear fashion—obesity causing widespread inflammation leading to insulin resistance and therefore type II diabetes. To get a better grasp on the relationships between these three, Ping Jiao et. al. (2012) engineered a mouse that was inflamed though not obese. More importantly they had over expressed a pathway in these mice specifically involved in obesity-related inflammation, achieved by expressing a constituently active form of IKKβ that is found in human adipocytes. This essentially just skipped the first part of this cause and effect relationship, looking at inflammation independent of obesity.

Four groups of mice were studied—those that expressed IKKβ were compared to those without after given either a high fat or a regular chow diet. The researchers concluded that these transgenic mice not only had a lower body weight, but also had improved tolerance to glucose and insulin despite increased inflammation. The presence of inflammation was determined by looking at the levels of mRNA coding for TNFα, IL-6, and MCP-1 where the transgenic mice had statistically increased amounts of both IL-6 and MCP-1, though no increase in TNFα. Separate tests determined possible mechanisms behind the increase in energy expenditure, where mRNA levels in brown adipose tissue and muscle were quantified. Increased expression of genes involved in thermogenesis and beta-oxidation in these tissues seem to play a key role in the increased metabolism.

These findings suggest that it is the direct consequences of obesity rather than inflammation that attribute to decreased insulin sensitivity. As Xu mentions in the lay article “Lower body weight is always a beneficial thing for influencing insulin sensitivity…Reduced adiposity wins over increased inflammation.” Possible explanations may be the decrease in free fatty acids or increased energy expenditure of the thinner, inflamed mice. This was an interesting twist after a semester discussing the detriments of unregulated inflammation. Thoughts?

Sources:

Enzyme boosts metabolism, prevents weight gain in mice.

http://medicalxpress.com/news/2011-11-enzyme-boosts-metabolism-weight-gain.html

Constitutive Activation of IKKβ in Adipose Tissue Prevents Diet-Induced Obesity in Mice.

Jiao, Ping, Feng B, Ma J, Nie Y, Paul E, Li Y, Xu H.

Endocrinology. January 2012; 153(1)

Note: I was unable to find the research article for free the second time I tried, but have it on my computer if anyone has trouble finding it.

CCR5 and Type 1 Diabetes

Earlier in our class, Dr. Cohen discussed the Chemokine receptor 5 (CCR5) and it’s effect on resistence and progression to AIDS for individuals with a mutant version of this gene. One project that I participated on was looking at CCR5 and any observable effects on patients with Type I diabetes.

As briefly mentioned in class, there is a mutant form of the CCR5 gene in which a 32bp deletion results in a truncated non-functional protein that is not expressed on the cell surface of mononuclear cells. A number of studies have looked at the association of CCR5D32 and it’s effects on disease. Individuals with CCR5D32 have either strong resistance to HIV infection (homozygous) or delayed progression to AIDS (heterozygous). In rheumatoid arthritis there is a negative association of the polymorphism suggesting that it influences the development of RA, as well as the clinical course and severity. For the study I participated on, we wanted to see what (if any) effects that this gene polymorphism has on Type I diabetes. Specifically, if it influences the development of persistent islet autoimmunity (IA) and/or the progression to T1D. Other studies looking at this with limited/specific populations have mixed results. A small report from Hungary shows no association while to others with larger cohorts report partial protection from progression to T1D.

In our study, we genotyped children with the high risk HLA DR3/4-DQ8 genotype as well as the offspring of T1D parents. Included in the cohort were high-risk T1D individuals, siblings and offspring of relatives of T1D individuals, as well as general population newborns. This information was coupled with the IA information for all. (Type 1 diabetes is usually preceded by the presence of autoantibodies directed toward pancreatic islet cell antigens. These antibodies can be present months to years before the actual clinical diagnosis of diabetes and can serve as predictive markers of T1D).

The data for persistent IA and progression to T1D was grouped by genotypes and analyzed by survival analysis. The comparisons for the development of persistent IA showed significant levels of protection from having at least one D32 allele for both high-risk T1D individuals as well as the normal population group. The data also suggest that there is protection from the progression to T1D, however the sample size was too small for this result to have statistical significance.

Although not yet published, this study showed an association with the CCR5 polymorphism and the clinical course of T1D and also agreed with two other published studies. Combined with other genetic markers, perhaps the CCR5 gene could prove to be of benefit in disease prediction.

Sources:

1. Steck, AK. Chemokine Receptor CCR5D32 is associated with protection from persistent Islet Autoimmunity. Not published.

2. Int J Mol Med. 2008 Nov;22(5):669-75. Frequency of CCR5-Delta32 deletion in human immunodeficiency virus type 1 (HIV-1) in healthy blood donors, HIV-1-exposed seronegative and HIV-1-seropositive individuals of southern Brazilian population.

3. J Diabetes Complications. 2003 Nov-Dec;17(6):387-91. CC-chemokine receptor CCR5-del32 mutation as a modifying pathogenetic factor in type I diabetes.

4. Pediatr Res. 1999 Jul;46(1):82-4. Chemokine receptor CCR2 and CCR5 polymorphisms in children with insulin-dependent diabetes mellitus.

5. Diabetes Care. 2004 Feb;27(2):497-502. Is presence of islet autoantibodies at birth associated with development of persistent islet autoimmunity? The Diabetes Autoimmunity Study in the Young (DAISY).

Spinal Cord Injury, Inflammation, and Antioxidants

A recent 2011 article published in the Journal of Biomedical Science shows promise for reducing the extent of spinal cord injury due to inflammation by using antioxidants. Following spinal cord injury (SCI), Proteins related to oxireduction and signal transduction can contribute to extensive inflammation. Events like apoptosis, necrosis, metabolic disturbances, destruction of microvasculature, inflammation, demyelination, glial scar formation as well as many other changes occur immediately following an acute or chronic spinal cord injury and can persist for weeks. Researchers measured a large list of factors at day 1 and day 14. Factors like galectin-3 involved in cell differentiation, heat shock beta-1 protein involved in oxireduction, were all up regulated. Factors like Fascin-1 involved in actin filament binding, Dihydropyrimidinase-protein 2 involved in neurogenesis, were down regulated at day 14 compared to day 1. This research gives a lot of information as to the extensive list of biomolecules responsible for cellular repair, inflammation, and regeneration following acute to chronic spinal
cord injury and many possible targets for inflammation modulation and cellular repair.

Wang et. al Journal of Biomedical Science 2011, 18:13
http//www.jbiomedsci.com/content/18/1/13

Increase Aorta Elasticity with Fish Oil

In a recent study published In the European Journal of Clinical nutrition researchers found the consumption of fish oil showed increased elasticity of the aorta in hypertensive overweight individuals. 52 overweight and hypertensive individuals from a community in Northwest China were divided in half. 26 were given 3 gram fish oil capsules containing 540mg EPA and 360mg DHA, and 26 were given a placebo. Radial arterial pressure waves were measured non-invasively by arterial tonometer sensor array every 2 weeks for 8 weeks. Vascular structural changes in hypertensive patients show increased structural alterations in intima-media, Plaque formation, stenosis, Endothelial dysfunction, and changes in the mechanical ability of arteries. Patients were also checked for a fasting blood sample Measuring serum lipids, sVCAM-1, Leptin, insulin, glucose, and free fatty acid composition. Results show a marked increase in n3 polyunsaturated fatty acid levels and large artery elasticity. No changes were observed in the other blood measurements including blood pressure. It appears that long term use of fish oil may prove to be cardio protective even in individuals who are already overweight and hypertensive.

Go to Sleep or Have Your Heart Dissected!

This seems to be the message according to a 2003 article in the American Journal of Respiratory and Critical Care Medicine. In the article titled, Obstructed Sleep Apnea and Thoracic Aorta Dissection, researchers investigated a possible link between 19 patients presenting aortic dissection and the occurrence of obstructive sleep apnea syndrome (OSAS). The study was conducted because of a look into how OSAS can cause negative intrathoracic pressures that may put mechanical stress on the aorta wall. The study shows a link between OSAS and hypertension, a major factor in aortic dissection, as well as, impaired left ventricular function, gastroesophageal reflex, and an increase in sysltolic and diastolic aortic diameters during apnea and hypopnea. Patients having to breathe against an occluded airway showed a mean intrathoracic pressure of -60cmH2o. The study included 19 patients who had aortic dissection and 19 patients with hypertension. 13 of 19 in each group (68%) had sleep apneas with a prevalence of apnea-hypopnea index of more than 5 per hour with some as high as 30 or more per hour. The real danger seems to be that the aortic transmural pressure is dangerously out of range. Being that blood pressure is generally higher in patients with OSAS and the intrathoracic pressures are out of range in addition to the frequent occurrence of the insult presented by the occlusion and movement of the pharynx, the aortic wall is stressed. The study suggests that sleep therapy is important in OSAS patients to help prevent a vascular insult that could possibly lead to aortic dissection.

Michrochimerism

This week we discussed the concept of fetal cells in maternal circulation, in particular the implication of the Rh factor. I found an interesting article that reviews the concept of Michrochemerism (MC).

A normal pregnancy is accompanied by major physiological changes. Adaptation by the mother to the foreign fetus requires major immunolical changes that even to this date are not completely understood. It is clear however, that the fetus is not protected from the maternal immune system by physical barriers such as the placenta. Extant research supports that the two circulations are in close approximation and there is bidirectional flow of maternal and fetal cells throughout the pregnancy. The maternal system does recognize the fetal cells' paternal antigens and produces antigen specific cytotoxic T cells and antibodies. These are thought to be prevented from attacking fetal cells by the stimulation of paternal antigen specific Treg cells.

MC refers to the DNA or low levels of viable cells present in the circulation of and/or organs of an individual that clearly originate from another genetically distinct individual. MC was first identified in mice. In 1996 the first identification of MC in humans was found. The prescence of male progenator cells was found in the blood of women. MC appears to be able to live in the maternal blood for years.

MC is thought to engraft in the bone marrow of the mother and differentiate into leukocytic cells. Data supports the theory that MC cells are progenator stems cells capable of multiple differentiation depending on their location. It is currently thought that the maternal immune response toward fetal cells is suppressed when maternal and fetal circulations intersect in the syncitiotrophoblast. Here the unique pregnancy associated human leukocyte antigen HLA-G is expressed in the cells. It is thought HLA-G induces the production of treg cells and suppress NK cells. T regs suppress the maternal immune response to fetal cells.

The exact role of MC in women's health is as yet unclear. Study is hampered by difficulty in measurement. Some theories implicate MC in the role of autoimmune disease in women whereas other theories propose beneficial health effects. Implications for future research are huge.

Groer, M. W., Manion, M., Szkeres, C., & El-Badri, N. (2011). Fetal michrochimerism and women's health: a new paradigm. Biological Research for Nursing, 13(4), 346-350.

ImSAIDS: A New Approach to Inflammation Modulation

Immune Selective Anti-Inflammatory Derivatives (ImSAIDS) are a new class of immune-therapeutic agents under active research by several laboratories and which are already produced for veterinary use by the IMULAN corporation right here in Arizona. Currently, the most interest is in the tripeptide phenylalanine-glutamate-glycine in its D-isomeric form (feG.) feG has been shown to modulate states of activation and migration capability on neutrophils circulating within an experimental system (Mathison, et al. 2003.) The specific mechanism of action appears to be by disruption of leukocyte migration into inflamed tissues, resulting in a lower rate of free radical production and oxidative damage therein.

The primary therapeutic advantage of agents like feG over traditional steroids and NSAIDS is that ImSAIDS do not cause a systemic reduction in immune activity; instead, they tend to down-regulate acute immune responses (Mathison, et al. 2003.) Two clinically-pertinent examples of destructive acute immune responses have been studied and found to be potential targets for feG therapy, anaphylaxis and spinal cord inflammation after injury. When rats were challenged with a pro-anaphylatic antigen, Turesin, et al, showed that feG was effective at preventing a destructive, systemic inflammatory response (2002.) Bao, et al, studied a rat model of acute physical spinal damage and showed that feG was capable of preventing neutrophil activity in damages tissue by as much as 48% resulting in a marked reduction in secondary, inflammation-related damage to spinal cord tissue (2006.) A secondary advantage observed in all known studies on feG to date is that therapy with the agent appears to present no potential toxicity.

IMSAIDS are currently being investigated for the treatment of sepsis, anaphylaxis, acute trauma, pancreatitis, cystitis, allergies and asthma. In theory, they could be applied to any situation where an inappropriate or exaggerated immune response is present, with the added benefit of not completely attenuating the immune system. They will, no doubt, be a welcome addition to the arsenal of rheumatologists and other clinicians when they are studied in humans to a greater extent.

A comprehensive review article can be found at: http://www.journal-inflammation.com/content/7/1/49

Mathison -> PubMed 12659660: http://www.ncbi.nlm.nih.gov/pubmed/12659660

Turesin -> PubMed 12199907: http://www.ncbi.nlm.nih.gov/pubmed/12199907

Bao -> PubMed 16581192: http://www.ncbi.nlm.nih.gov/pubmed/16581192

Oxidative Stress and Alzheimer's Disease

A recent study has linked the inability to remove reactive oxygen species, specifically the superoxide ion, and acceleration of Alzheimer's Disease symptoms in a mouse model.

Oxidative Stress

The production of reactive oxygen species (ROS) drives oxidative stress and the damage associated. A major anti-oxidant enzyme, superoxide dismutase (SOD), is one of the many ways cells are able to neutralize ROS. There are three isoforms of SOD: SOD1 (CuZn-SOD) which is located in the cytosol,nucleus, and the intermembrane space of the mitochondrias, SOD2 (Mn-SOD) which is located in the mitochondrial matrix, and SOD3(CuZn-SOD/EC-SOD) which is located in the extracellular matrix. A previous study had shown that SOD1 knockout caused increased drusen formation, which correlates with age-related macular degeneration . Drusen deposits are thought to possibly contain amyloid B oligomers, the same oligomers that are associated with Alzheimer's Disease.
SOD1 Knockout

By removing SOD1(SOD1 +/-, SOD1-/-) from the amyloid precursor protein-overexpressing mouse model of Alzheimer's Disease, several effects of oxidative damage were studied. Deficiency of SOD1 caused accelerated amyloid B oligomerization, increased tau phosphorylation, and decreased synaptophysin (a protein expressed on presynaptic vesicles). All of these correspond with the worsening of cognitive function associated with Alzheimer's. An increase in advance glycation end-products (AGEs) were seen and have been shown in other studies to possibly effect histamine release from mast cells present in the brain. Furthermore, activated microglia and astrocytes were seen to increase, associated with increase plaque formation and subsequent neuroinflammation. To further establish the significance of SOD activity in human Alzheimer's patients, levels of the three SOD isoforms were determined from human Alzheimer's patients and age-matched controls. It was found that SOD1 was significantly decreased, while SOD2/3 were unchanged.
Implications for Alzheimer's Disease

Alzheimer's is a complex neurodegenerative disease with many possible factors effecting the onset and progression of symptoms. Oxidative stress clearly plays a role, but it is still undetermined the exact role and the extent to which it participates in the pathogenesis of Alzheimer's. Identification of possible pathways to counteract the role of oxidative stress in Alzheimer's could lead to therapeutic treatments. In the case of SOD1, possible targets could be SOD1 replacement (mimetics) or dietary intake of Cu (thought to stabilize activity), although currently mouse models of Alzheimer's are the only evidence for the benefits of any treatment.

References

Murakami et al. 2011. SOD1 DEFICIENCY DRIVES AMYLOID B OLIGOMERIZATION AND MEMORY LOSS IN A MOUSE MODEL OF ALZHEIMER’S DISEASE. JBC. M111.279208.

Sick et al. 2010. Advanced glycation end products (AGEs) activate mast cells. BJP. 161:442-455.

Venom, just what the doctor could order some day...

Following our class discussion on bee string therapy, I found a review article detailing the current findings on venom and possible therapeutic features across several species. Here's a brief overview:

Bee Venom

Key players: Apamin, adolapin, mast cell degranulating peptide

Pros: The proteins mentioned above have been found to have an inhibitory role in leukocyte migration as well as downregulation of TNF-a, tumor growth, and an upregulation in IL10. Overall, the components display anti-inflammatory effects that should be beneficial against Experimental Autoimmune Encephalomyelitis (EAE) and Multiple Sclerosis (MS).

Cons: Melittin and Phospholipase A2, proteins also found in bee venom, are believed to contribute to the irritation and hypersensitivity association with bee stings. Also, in Wesselius' study, there were no significant decrease in new-gadolinium enhancing lesions or other symptoms for MS patients. The therapy itself involved patients receiving up to 20 stings (ouch!) to the leg in one setting, its hard to imagine the pain and discomfort of the treatment outweighs the slight benefits.

Sea Anemone Venom

Key Player: ShK Toxin. The toxin gets its name from the species it is isolated from, stichodactyla helianthus, which is found off the coast of Cuba. The K comes from the fact that this toxin is a potassium channel inhibitor.

Pros: ShK blocks potassium channel Kv1.3, a voltage-gated K channel expressed in T cells. In blocking this channel, ShK reduces T cell activation, proliferation, and cytokine production and has been found to protect against T cell-mediated autoimmunity in animal models.

Cons: The author seemed rather positive about this form of treatment, looking for more research to understand side effects and long term consequences.

Scorpion Venom

Key players: Kaliotoxin, charybdotoxin, iberiotoxin.

Pros: Kaliotoxin has similar Kv1.3 channel blocking activity and was found in inhibit the delayed-type hypersensitivity respone towards myelin basic protein. Furthermore, charybdotoxin was found to share several immunosuppressive effects.

Cons: Kaliotoxin is also selective against the Kv1.1 channel expressed in the nodal regions of neurons, more research will need to conducted to identify potential neuroprotective or degenerative effects on this channel. Furthermore, the author mentioned more research is needed to understand the effect of altered potassium signalling and specificity on the microglial cells and astrocytes.

Snake Venom

Key players: Cobra venom factor, dendrotoxin-1, ancrod

Pros: Cobra venom factor has shown to mimic complement component C3b, which competes for endogenous C3 and reduces serum complement levels, greatly enhancing the symptoms of EAE in rat models. Dendrotoxin-1 is also a Kv1.3 channel as seen in sea anemones and scorpions and has similar effects. Finally, ancrod is a fibrinogen degrading protein that reduces the amount of ectopic fibrin deposits in brain lesions. Fibrin was also found to have a down-regulatory role on myelin regeneration, and thus the removal of fibrin from lesions is protective.

Cons: Trials have been very complex, utilizing different venomous proteins from different species of snakes and thus require more work before advancing into later trials of testing.

Bottom line: Evidence does support that several of these venomous proteins expressed by different species can play important roles in the treatment of neurodegenerative disorders. However, at this point in research most are being used in an animal EAE model and need more understanding before advancing to human MS trials. Furthermore, the author of this review does not mention administration methods during the discussion of therapeutic effects. The potency and dose-dependence in artificial administration will need to be considered.

Source:

Mirshafiey, Abbas. 2007. Venom therapy in multiple sclerosis. Neuropharmacology 53: 353-361

Alzheimer’s Disease: Antibodies to the Rescue?

Effective drug therapies have evaded researchers in hopes of overcoming Alzheimer’s disease (AD). The disease exhibits a complexity in its pathogenesis and progression, making it difficult to tackle the disease. With several hallmarks establishing AD, it offers researchers a variety of promising targets to understand and combat AD. Examples of promising targets would be inflammation, insulin resistance, neurofibrillary tangles, and beta-amyloid peptide fragments.

In our class discussion, we focused on how inflammation plays a role in AD. By the presence of inflammation, the immune system will recruit cytokines/chemokines that have shown to play roles in the neurodegeneration of AD. Discovering the role inflammation played in AD showed promising targets for drug therapies; however, the utilization of anti-inflammatories has not delivered the anticipated results in the arrest of AD progression, the restoration in cognitive function, or prevention.

The search in another area…

The beta-amyloid peptide that develops into plaques and oligomers is of great interest to researchers. The reasoning behind their interest is that when the parent amyloid protein is cleaved into the beta amyloid peptide fragments they become sticky allowing them to accumulate and cause plaques, which disrupts the neuronal network. The peptides also have the ability to stick to each other causing oligomers, which float into the synaptic space disrupting the ability for neurons to pass along information via signals. Both the plaques and synaptic disruption leads to necrosis of the neuron, which leads to brain shrinkage due to the lost of the neuronal network. The lost of the neuronal network produces the diminishing effects of brain function; as more of the network is lost, the severity of cognitive decline is exhibited.

Researchers hypothesize that beta-amyloid peptide is a major player in the development of AD. Targeting the beta-amyloid sticky fragments is plausible approach in developing effective drug therapies for AD. There are several strategies in approaching the problematic beta-amyloid peptide: blocking enzymes that produced the beta amyloid fragment, blocking aggregation of beta-amyloid into plaques, and the removal of the beta-amyloid fragments. Each of the strategies all target to result in the reduction of levels to inhibit plaque and oligomer formations.

What role can antibodies play?

Researchers believe they have found that by proper clearance of excess beta-amyloid peptide can halt the progression of AD and restore cognitive function. A way they attempt to accomplish this clearance is by employing the individual’s immune system. There is a nasal vaccine that uses active immunization by delivering a synthetic version of the beta-amyloid peptide with a carrier protein to induce an immune response. By building this response, the hope is for the individual’s immune system to successfully clear the beta-amyloid peptides before they become problematic by using anti beta-amyloid protein antibodies. The antibodies are suspected to be effective in clearing plaques after formation or preventing formation, giving the use of the vaccine a broad spectrum to be utilized in different stages of AD. There has been attempt (name of vaccine is AN1792) in 2002 to use this immunotherapy vaccine, but was terminated in phase 2 trials due to the development of meningoencephalitis and leukoencephalopathy in a small percentage of patients. However, the results from the clinical trials did indicate the vaccine was successful in the attempt to clear amyloid beta plaques. Due to those results, there is currently a second attempt (name of vaccine is ACC-001) with the same approach; however, to avoid the adverse effects from the first attempt; a modification was made by reducing the size of the synthetic beta-amyloid peptide that accompanies the carrier protein to induce an immune response; this vaccine is currently in phase 2 trials. Another way to induce antibody activity is instead of stimulating the individual’s immune system to develop antibody defense is to utilize passive immunization; the patient is delivered a vaccine that already contains the antibody (vaccine named is AAB-001), which this method may be more preferable due to the ability to regulate dosing of antibodies. It is currently in trials and showing great promise of reducing beta-amyloid plaques and restoring cognitive function. One last approach to mention in immunotherapies for AD is the use of intravenous immunoglobulin (IVIg) to reduce beta-amyloid levels, which has shown to be effective in the reduction of plaques and restoration of cognitive function; however, I question the practicality of this method. The IVIg is a collection of Ig antibodies from a pool of human donors- the pool consists of plasma from a large population of human blood donors. The availability and cost of IVIg are going to create obstacles in utilizing this immunotherapy in AD. Considering the growing rate of people being diagnoses with AD, makes it unlikely to be a source of future therapies also since it is a limiting resource, which not to mention this type of immunotherapy should first be available to individuals who lack or have impaired immune systems. In my opinion, this approach won’t be as reliable as the previous mention immunotherapies.

Are antibodies enough?

Though we have witness through mice models the reduction in beta-amyloid levels leads to reversing the cognitive decline, these results most likely are obtained in mice models that exhibit solely beta-amyloid pathology and may not have the other hallmarks present. With that in mind, we can easily expect the arrest of AD progression and reverse cognitive decline if there are no other components of AD to consider in the progression. My concern is that hallmarks of AD are not subsequent of one another, meaning that just by eliminating beta-amyloid plaques will eliminate inflammation, neurofibrillary tangles, increased microgliosis, etc. For example, in a paper earlier discussed in class, we examined how inflammation could recruit cytokines/chemokines that will initiate signaling cascades that will induce hyper phosphorylation of the tau protein in axons of neurons, which leads to tangles. These tangles lead to the disruption of signals being prorogated down the axon. When neurons are defective in their signaling it can lead to cell death, which means we will still witness the neurodegeneration of an individual without the beta-amyloid factor.

I think in our future we will find that AD will be combated with a combo-therapy treatment. I believe a reduction approach should be taken when discovering therapies and understanding AD. As researchers focus on individual hallmarks, it will allow them to work on individual areas of the puzzle. By success of clinical trials and understanding of the individual hallmarks, will enable researchers the ability to put together the whole puzzle on how to effectively treat AD.

Resources:

http://www.alzforum.org/drg/drc/detail.asp?id=102

http://www.alz.org/

Rojo, et. al Neuroinflammation: Implications for the Pathogenesis and Molecular
Diagnosis of Alzheimer’s Disease. Archives of Medical Research 39 (2008) 1e16

B Cells Involved in Regulation....REALLY??!!

Dr. Cohen spoke briefly in class about recent studies showing that B cells had other roles besides their main role in antibody secretion. I wanted to shed some light on this and did a little researching and found a great article that shows some of the other roles B cells play in immune responses. We are all aware that T regulatory cells are one of the key players in immunosuppression; however, in the last 10 years more has been found on B cells and their role in immunosuppression.

It appears the B cells secrete IL-10, which is known as an anti-inflammatory. IL-10 has many different effects, but it's main role is regulating antigen presenting cells and T lymphocytes. In antigen presenting cells, IL-10 inhibits the secretion of pro-inflammatory cytokines, TNF alpha and IL-1(beta). IL-10 affects differentiated CD4+ T cells; more specifically, it showed that it reduced proliferation of Th1 and Th2. Th17 and cytotoxic T cells are unaffected by IL-10 so many believe these T cells are regulated by T regulatory and other cytokines.

Multiple Mechanisms of Immune Suppression by B Lymphocytes.

Klinker MW, Lundy SK.

Mol Med. 2011 Oct 24. doi: 10.2119/molmed.2011.00333.

Breast Cancer and Macrophages

I am completing my first rotation lab next week and wanted to share with everyone what my research has been on. The lab I joined is interested in studying how involution can lead to pregnancy associated breast cancer.

All women have an increased risk of developing breast cancer for the first 5-10 years after giving birth. One possible reason for this link is involution. Involution occurs after lactation (or after childbirth if the mother doesn't breast feed). It is the body's way of restructuring the mammary tissue to return back to its pre-pregnancy state. This is done in part by apoptosis of epithelial cells in the mammary gland. Other mammary epithelial cells become phagocytic and eat the dead cells, but macrophages will also arrive at the scene.

It has been shown that these macrophages get stimutated to the M2 variety of macrophage. Because M2 macrophages are anti-inflammatory, which is tumor promotional, these M2 macrophages may be what links breast cancer with pregnancy.

My project was to test an in vitro model of this mechanism with mouse cells. Before I started my project, mammary epithelial cells were grown, stimulated to become phagocytic, and then fed apoptotic cells. This was all done in order to mimic what occurs in an involuting mammary gland. The supernatant (conditioned media) was collected off these cell cultures and used for my experiments.

I first wanted to verify that I could get M2 macrophages from activation with the supernatant. I did this by taking bone marrow derived macrophages activated with the conditioned media. There are several controls for the conditioned media as well as controls of known M1 and known M2 macrophages. The phenotypes of the conditioned media activated macrophages were confirmed to match the M2 phenotype by western blots of the cytokines produced.

To test whether the macrophages activated by conditioned media would respond to antigen in an inflammatory way, I stimulated the cells with LPS and measured pro-inflammatory cytokines. This data is still being analyzed.

I noticed that one of the cytokines, CD86, was lowered in both the M2 macrophages and my conditioned media activated macrophages. CD86 is required to activate T cells, so having decreased amounts would indicate that they may not be able to activate T cells. A future direction would be to do a T cell activation assay to test this out.

So far my data supports the hypothesis. Unfortunately, there is only so much that can be done in one rotation.