Pediatric Brain Tumors Exhibit Distinct Phenotypes: Should Therapies Be Individualized?

Tumors are never something to joke around about; they are dangerous, unpredictable, and they can even be fatal. They can appear nearly anywhere in the human body, at basically any point in time. In fact, one of the most dangerous habitats for a tumor is the brain. A complicated and mysterious part of the human body already, the brain controls almost everything, and a tumor appearing in this region can be devastating. They are difficult to remove, and they can have disastrous mental and physical consequences. Even worse, they can present themselves at any age. This makes children susceptible, and what could be worse than that? With around 1,500 children diagnosed a year, pediatric brain tumors are not the most common cause of death in children (1). However, they are the most common type of pediatric tumor, and they have the highest mortality rate over all other childhood cancers. Despite its reputation, this deadly disease has had no improvements regarding treatment methods in the past. Until recently even, the standard treatment was radiation exposure and chemotherapy, which were often coupled with horrible, debilitating side effects. Now, a new form of treatment has been devised due to its tumor specificity – immunotherapy.

"Types of Brain and Spinal Cord Tumors in Children." Johns Hopkins Medicine.
Johns Hopkins University, Hospital, and Health System, 2013. Web. 10 Dec. 2013.

Studies have shown that there have been positive correlations between host immunity and survival rate in children diagnosed with brain tumors. Still, immunotherapy has demonstrated to be largely ineffective due to the immunosuppression induced by brain tumors because it tampers with the immune system-supressing qualities of the exogenous therapy. Several scientists researched pediatric brain tumor types further, however, in order to better understand immunophenotypes – the immunological characteristics – of these tumors. They hoped to shed some light on the subject in order to be able to better treat these afflicted children and give them more of a fighting chance.

               

In Andrea Griesinger and her colleagues’ study, they measured the phenotype and frequency of tumor-infiltrating leukocytes in the four most common types of child brain tumors*(4). They began by surgically taking tumor samples from forty-two patients at the Children’s Hospital in Colorado; they also took five non-tumor samples for a control group. Then, the tissue samples were disaggregated and frozen, before they were eventually suspended, stained, and analyzed via a FACS analysis and a gene expression analysis (2, 3). The FACS analysis then sorted the variety of cells into two or more containers based on their fluorescence while the gene expression analysis quantified the expression levels of certain genes.

             

Can Antibiotic Exposure Influence Development of Allergic Diseases?

A recent study provides certain implications for an association between exposure to antibiotics at a young age and the development of allergic diseases, primarily asthma, in early childhood.¹

            At some point, most people have gone to the doctor’s office and left with a prescription for an antibiotic. In today’s world, antibiotics have developed a connotation as a medicine that can ward off all sorts of sicknesses, which is only partially true. What many people don’t realize is that antibiotics are strictly useful for fighting bacterial infections and will have no effect on viral illnesses.* Although antibiotics have saved countless lives since the discovery of penicillin, there are some concerns about their use.

There two main negative consequences of using antibiotics more liberally than in the past: some unhealthy bacteria have increased resistance to treatment and administration of antibiotics can lead to decreased levels of healthy bacteria. The first consequence relates to overprescribing antibiotics for patients that may not be suffering from a bacterial infection. Every time a person takes antibiotics, he or she increases the likelihood that bacteria in the body will become resistant, which makes it difficult to treat later infections.^#

Commensal Bacteria

The second consequence, which directly relates to the study in question, has to do with the healthy bacteria that reside in the human gastrointestinal tract. When antibiotics are introduced to the body during infancy, a critical period for the development of the immune system, disruption to gut microflora can occur.¹ This could possibly predispose patients to the development of an allergic phenotype. Research shows that disruptions in the normal growth of gastrointestinal bacteria can prevent regulatory T cells from properly dampening the immune system’s response to respiratory allergens.^$  For more information, click here. Reduced diversity of microbes in infant excrement has also been connected to an increased risk of allergic diseases late in childhood.²

Allergic diseases develop when a person's immune system becomes sensitized to a normally harmless antigen. Type I hypersensitivity is a category of allergic reaction in which CD4+ Th2 cells that interact with these antigens stimulate B-cells to produce Immunoglobulin E (IgE) antibodies. These antibodies will then mark the specific antigen for destruction by other immune cells.⁺ Once the individual has been initially exposed and developed the specific antibodies, a subsequent exposure to the allergen will result in an allergic reaction. For more information, click here.

Mechanisms of Allergic Response

Autoimmunity, Rheumatoid Arthritis, and Abatacept

                        Figure 1: Example of rheumatoid arthritis effects.

Retrieved from Cedars-Sinai (2013) (http://www.cedars-sinai.edu/Patients/Health-Conditions/Arthritis---Rheumatoid-Arthritis-Osteoarthritis-and-Spinal-Arthritis.aspx)                      

You’ve likely heard of rheumatoid arthritis (RA), an autoimmune disease that results in a chronic, systemic inflammatory disorder. More than 2 million adults in the United States suffer from this disease, with women being two to three times more likely to develop it than men (UCSF Medical Center 2013; http://www.ucsfhealth.org/conditions/rheumatoid_arthritis/). The disease can occur at any age, although it typically affects those over 40 years old (Mayo Clinic 2013; http://www.mayoclinic.com/health/rheumatoid-arthritis/DS00020). RA is caused by an autoimmune attack on antigens expressed in the synovial tissue and cartilage of joints. Wrists, fingers, knees, feet, and ankles are most commonly affected. Early phase symptoms are characterized by morning stiffness in the affected joints. Over time, inflammation, cartilage destruction, and bone erosion may lead to deformations and crippling. The general explanation for these effects is as follows. The immune system attacks the synovium, which is the lining of the membrane that surrounds joints. Inflammation ensues which results in the thickening of the synovium and can eventually lead to the destruction of the cartilage and bone within the joint. The tendons and ligaments that hold the joint together also weaken and stretch, and can suffer degradation as a result of proteases (enzymes that break down proteins) secreted by activated macrophages (phagocyte meaning that they engulf solid particles). In this fashion, the joint gradually loses its shape and alignment, leading to deformities and crippling.

                            Figure 2: RA commonly affected joints and impact on joints.

Retrieved from Atlantic Apothecary (2013) (http://www.atlanticapothecary.com/disease-state-management/arthritis/rheumatoid-arthritis/)

So, more specifically, how does this all happen? Activated macrophages and DCs extravasate (essentially escape from a blood vessel into tissues) into the joint and produce large amounts of pro-inflammatory cytokines (intercellular mediators), especially TNF. The blood vessels in the inflamed joint take on the characteristics of high endothelial venules (HEVs), which specializes them for lymphocyte (small white blood cell) extravasation (Villani 2012; http://www.ipbs.fr/?High-endothelial-venules-HEVs). The inflammation is perpetuated by the eventual infiltration of the joint by CD4⁺ Th effectors (white blood cells that assist other immune cells) and CD8⁺ CTLs (white blood cells responsible for causing cell death of infected/damaged cells), which produce cytokines like TNF and IL-17. RA synovial tissues also contain “ectopic” germinal centers (sites where mature B lymphocytes proliferate, differentiate, and mutate and switch the class of their antibodies), meaning that these structures have developed in the wrong tissue. Plasma cells in these abnormal germinal centers produce autoantibodies (an antibody formed in response to and reacting against an antigenic constituent of its own tissues) directed against antigens (substance that induces an immune antibody response) in the synovial membrane and cartilage. Common markers of RA include the presence of rheumatoid factor and anti-citrullinated protein antibodies (ACPA) in the serum. These markers represent autoantibodies that have significant diagnostic values (da Mota et al. 2012; http://www.ncbi.nlm.nih.gov/pubmed/22187055).

 Figure 3: General immune overview of RA joint. Retrieved from Nutrition Remarks (2013) (http://www.nutritionremarks.com/2013/03/09/fish-oil-can-reduce-rheumatoid-arthritis-flu/)

ACPAs are autoantibodies present in the majority of patients with RA. They have proven to be useful biomarkers and allow for the diagnoses of RA at an early stage (da Mota et al. 2012; http://www.ncbi.nlm.nih.gov/pubmed/22187055). During inflammation, in a process known as citrullination, arginine residues in proteins can be converted to citrulline ones (Suurmond et al. 2011; http://www.ncbi.nlm.nih.gov/pubmed/21339220). If this change significantly alters the shape of the proteins, they may be seen as antigens and an immune response will be generated. Autoantibodies are generated against these citrullinated proteins (including fibrinogen or vimentin for example), forming the basis of an autoimmune disease. It is important to note that rheumatoid arthritis patients can be either ACPA-positive or ACPA-negative, and this status can have a significant influence on the intensity and therapy of RA. In fact, ACPA-positive and ACPA-negative RA have been recognized as distinct disease sub-entities, which demonstrate significant differences with regards to HLA-association, genetic and environmental risk factors, disease phenotype, and treatment response. 

See See How CCL19 Effects Eosinophilic Pneumonia

  

Eosinophils are granulocytes that contain basic granules (i.e. secretory vesicles) that kill large parasites and are linked to various forms of allergies.  Eosinophilic pneumonia (EP) is a broadly defined disease that is characterized by an infiltration of eosinophils in lung alveolar tissue.  EP includes Churg-Strauss Syndrome (a rare autoimmune disease), chronic EP and acute EP (the difference between the two is the presence of eosinophils in the blood/tissues and only the tissue, respectfully).  Individuals with EP are often found to have an increased concentration of macrophages and dendritic cells, important innate immune response mediators.  Symptoms include shortness of breath, weight loss, fever, and even respiratory failure, while causes range from parasitic infection, immune system dysfunction, medication, and environmental stimuli like tobacco smoke and dust.  Although symptoms can be serious, few is known about the cellular mechanisms behind EP, specifically macrophage and DC recruitment into the lungs.  Therefore in response, Nureki et al. investigated EP further by seeing if either, both, or neither CCL19 and CCL21 (molecules that attract motile cells with a specific receptor to a specific location) bound to CCR7 expressed on DCs and macrophages, homing them into the lungs.

In order to extract cells present in alveoli of patients with EP and control individuals as noninvasively as possible, the researchers performed Bronchoalveolar lavage (BAL).  BAL is a procedure in which fluid (BALF) in released into the lungs and recollected (via bronchoscope.  Once BALF was collected, cytokines/chemokines were measured by enzyme-linked immunosorbent assay (ELISA), which is used to measure the concentration of antigens via antibody (complementary binding molecules) detection.  Finally, levels of proteins on cell surfaces were detected by immunocytochemistry, a technique that uses antibody binding and further bound-antibody detection.

Implications of Chronic Alcoholism for HIV Infection

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Alcohol dependence is the most common form of drug abuse in the US, with around 7% of the population meeting criteria for alcoholism (Grant 1994). We all know that the effects of alcohol are wide-ranging, impacting both behavior and physiology; it impairs judgement, motor skill, etc. It has been known to weaken the immune system, and research in the past few years has linked the effects of alcohol to HIV infection. Common sense tells us that alcohol promotes risky behaviors, including those that increase the possibility of HIV infection (sex, drugs, etc.). Recent interest in binge drinking and HIV has produced data showing alcohol in a binge pattern changed the proportion of immune cells after SIV infection in rhesus macaques, a common animal model for HIV, and may even increase the disease course (Molina et al 2006, Poonia et al 2006). But researchers at the Scripps Research Institute have also showed that chronic alcoholism may generate microenvironments in the body that are more vulnerable to HIV infection.

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To accomplish this study, the researchers developed a protocol in which rhesus macaques self-administered alcohol orally, twice a day for 30 days before infection and during infection. To do this, they mixed alcohol and an orange flavored drink in different ratios, and conditioned the animals to drink the solution (replacing water with the alcohol solution for a short duration). The final concentration given to the animals was 6% alcohol, similar to the alcohol content of most beers, and animals drank enough alcohol per day to cause blood alcohol levels that in humans are greater than the legal driving limit and would decrease motor skill. When the animals were conditioned, they were infected with SIVmac251, a strain of SIV that induces high peak and steady viral loads and is also known to infect the CNS (Burdo et al 2005). SIVs, simian immunodeficiency viruses, are retroviruses that infect non-human primates and produces symptoms and changes in physiology that mirror those induced by HIV infection (it is generally believed that HIV originated from SIV crossing the species barrier to humans).

A New Mechanism of Cell Death by HIV

            It’s a disease that is well known all over the world: human immunodeficiency virus, better known as HIV.  It is often talked about in tandem with acquired immunodeficiency syndrome, or AIDS, which develops in HIV patients over time and is the end stage of the disease.  HIV originated in chimpanzees as simian immunodeficiency virus (SIV) and transferred over into humans in the 1800s.  The first cases in the United States were reported in 1981.  Throughout the 1980s, cases increased dramatically, peaking in the early 1990s.  However, a breakthrough in drug treatment for people living with HIV and HIV prevention campaigns helped to bring the number of cases back down.  The drug therapy known as antiretroviral therapy (ART) is still used today as the main way to help people with HIV live normal lives, hopefully preventing/delaying the progression to AIDS.  They target different points in the HIV virus replication cycle to try and slow down its progression through the body.

Figure 1: HIV replication in a cell.  It is able to dump its
contents into the cell, reverse transcribe its RNA,
integrate it into host DNA, and use the host to create new viral copies.

HIV is a retrovirus, a form of RNA virus that can be inserted into the host DNA, and then uses host cells to replicate.  The virus comes with all sorts of proteins that let it do this.  For example, reverse transcriptase allows the viral RNA to be turned into viral DNA.  Integrase allows it to be inserted into the host DNA to then use the host’s own protein making mechanisms to make new viruses and viral proteins.  Figure 1 shows an outline of HIV replication.  It is possible to trace the viral RNA to viral DNA to host DNA and then back out to spread to other cells. 

HIV also specifically infects a certain class of immune cells known as CD4+ T cells.  CD4+ T cells are called this due to the presence of a cell surface receptor called CD4.  These cells can differentiate into all different subtypes of CD4+ T cells called T helper cells, or Th cells, whose name describes their function: they “help” other immune cells mount responses to pathogens.  CD4+ T cells and all of their progeny are crucial for providing immunity to all sorts of infections, pathogens, and the like.  HIV comes into play and infects the CD4+ T cells.  It creates a chain reaction, infecting, spreading, and slowly killing all the CD4+ T cells in the body.  In simple terms: HIV is slowly knocking out an entire branch of the immune system.  Any further immune function that would need a CD4+ cell to work won’t be able to work once the CD4+ T cells are gone.  Figure 2 lays this out in a graph showing CD4+ T cells in blue, viral RNA copies in read, and time in weeks on the x-axis.  It is possible to see that as the RNA copies go up, the CD4+ T cells go way down.  Many of the ART drugs target these proteins that prevent the HIV from infecting cells as easily or spreading once it has infected a cell.  However, it is still unknown how HIV actually kills CD4+ T cells.

Figure 2: Timeline of HIV infection.  As viral RNA increases,
CD4+ cells decrease.  A latent period exists where the
person may not know they are infected
until their cell count reaches a certain point.

Cooper et al demonstrate one potential way HIV could kill CD4+ T cells.  They first infected cells with HIV and stained for a particular viral protein called p24.  They noticed that the CD4+ T cells that were killed didn’t express this viral protein, while cells that weren’t killed did.  Next, they looked at whether these cells that were lacking expression of this viral protein had been infected with the virus before they died.  More T cells were infected with HIV that also encoded for green fluorescent protein (GFP), which fluoresces green.  GFP is often used as an indicator for protein production. The gene for GFP is placed within the HIV genome, so if HIV proteins are being produced, GFP will also be produced.  If the cells are dead, no GFP will be detectable.  This is a commonly used method to visualize and also quantify protein production.  They analyzed the cells for GFP expression and cell viability, as well as viral cDNA.  Non-viable GFP- cells were found to have copies of viral cDNA.  When viable GFP+ cells were watched over time, the researchers saw that many of these cells eventually died (therefore losing their GFP expression) but retained viral cDNA.  These data together suggest that the cells that were killed died after successful HIV gene expression.