The human immune system
Immunology is the most difficult issue in my physiology. Although I had graduated from a great extended test "Immunology" with Professor. Superior of Krč, an expert in immunology feel. Of the course the best is still remember the feeling of amazement that I seized after they learned chapters on the maturation of T lymphocytes and the development of antibodies. In a magazine, which name I have forgotten a long time ago I read that the total number of human genes is about a third involved the construction of the nervous system, the third building on the immune system and the remaining third for all other tasks. I do not know how this statement still applies today, but I really can say that the immune system is about as complex as the brain, with the difference that the immune system does not anatomical clues to his understanding.
How it works in the immune system?
Experts Immunology perhaps forgive me when I say that the immune system most of all reminds totalitarian police apparatus:
All cells and molecules of the human body before birth is registered and established as "self." For this purpose the thymus ( thymus ), in which the police cells lymphocytes ) are educated so as to be able to react against all possible and impossible molecules, except their own.
If later in life gets into the body of a foreign molecule, there will be a certain number of immune cells that have the ability to detect it. Foreign molecule transported to the lymph nodes, which works as a police station. There is the case first considered. If the immune system decides against that foreign molecule to react, will be lead by dendritic cells rapid process of mutation and selection, which in the course of 3-4 days leads to an amazing optimize receptor rozeznávajícího the foreign molecule and cloning the original cell that carried it. Clones with an optimized receptor can then go on traveling through the body at which seek and destroy cells containing the foreign molecule (this is called type 1 response, whether cellular or cytotoxic response). A second possibility is that the optimized receptor clones start to be discharged freely into the extracellular space and finds himself diffusion of target molecules on the surface of microbes should be (this is called type 2 response, whether humoral or humoral response, lat. Humor = juice).
Intracellular parasites (e.g. viruses) and tumors of the body resists that each cell (except red blood cells) on the surface of MHC molecules bears, functioning as an identity card. MHC molecules are presented as if the photo of the internal environment nositelské cells - each individual MHC molecule is wearing glued randomly selected fragment of intracellular proteins. Cells which correspond to fragments of foreign molecules are destroyed. Those cells that would seek to avoid detection by reduced expression of MHC, are killed by a special group of supervisory cells - NK cells.
Basic concepts of immunology
Let me mention in a brief list of basic immunological terms, which in their texts often mention - please keep in mind that the immune system is still much more complicated:
- Passive immunity - Mechanical protection represented by the skin, mucosa, mucus, working ciliated epithelia sweeping dirt from our bronchi, chemical protection acidity of skin, stomach, cervix, bacteriolytic lysozyme in saliva and tears, peroxidase system (lactoperoxidase DUOX), Placenta or the blood-brain barrier etc.
- Active immunity - reaction of the immune system caused by the presence of the pathogen in the body.
- Innate immunity - innate immune system's ability to recognize common pathogens. As for the robber is typical that is poorly shaved and costs with a club around the corner, and pathogens have some common molecular features, according to which can recognize without delay. E.g. leukocytes have on their surface several types of so-called. TLR receptors (TLR1-13) recognizing polysaccharides of bacteria or fungi inside the cells is an enzyme with the ability to discern DICER1 chop and double-stranded RNA viruses, etc.
- Acquired immunity - Our immune system has the ability to the infection in record time to develop and produce large amounts of specific antibodies tailored against specific antigens.
- Antigen - Any foreign molecule that elicits an immune response. Typically the antigens speak style "anti-specifically recognizes the antigen something something".
- Antigen receptor - a sort of negative impression of criminals. They are molecules that fit its shape exactly to foreign antigens. These include the BCR (B cell antigen receptor), TCR (T cell antigen receptor) and immunoglobulins, or antibodies which are essentially free-floating antigen receptors secreted in a large amount of B cells. In the process of maturation of the immune system before birth, our body creates the myriad of antigenic receptors which have the ability to recognize all possible and impossible foreign molecules. Receptors that react with self molecules are destroyed in the process of maturation.
- Antibodies (immunoglobulins) - An antibody, or immunoglobulin is a freely floating protein very similar to antigen receptors of B and T lymphocytes. Source of antibodies by B lymphocytes. The shape reminds immunoglobulin clothespin with oddly shaped heads, fitting a negative impression on foreign molecules. Immunoglobulin is composed of two parts: the larger (ie. The heavy chain) and smaller (ie. The light chain). Both parts undergoing intense gene manipulation for the first time in the maturation of the immune system before birth, and once the infection, even when they are deliberately mutated and selected to be as tightly bound to the antigen. Immunoglobulins are further divided into subtypes (IgA, IgD, IgE, IgG, IgM), which differ in small details.
- MHC (histocompatibility complex main, major histocompatibility complex) - All body cells have a duty to prove its surface sufficiently high number of molecules MHC I with bound randomly selected strings of their internal proteins. It can be likened to a civil ID card with a photo glued internal peptides owner. If the cell presenting foreign peptides, T C cells directs cell apoptosis and impaired even helps make perforations of the wall using a special poison perforin. The same fate meets a cell, does not show if the sufficient amount of MHC I, which oversees on NK cells. MHC class II molecule on the other hand is not an identity card, but the police file of photographs of criminals. MHC class II molecules on its surface carrying professional immune cells. If the foreign MHC II peptide attached, it means that the appropriate immune cell just solve the case.
- White blood cells (leukocytes) - Police Cell. They are divided into ordinary guards (granulocytes) and specialists (lymphocytes). A special type of leukocytes are monocytes, cellular garbage collectors, who have the immune response is always busy.
- Granulocytes - Guard serving with determination samurai. They have the shortest lifespan of all cells. Although immune responses participate, or it will survive, commit to each case after several days of removal services program (apoptosis). Durability in combat tank is about 15 minutes and can therefore be at a sacrifice durability of components for other benefits (e.g. turbine engine in Abrams or Wankel at British tanks bud2002cwh ). As well as on granulocytes, which the selection of destructive microbes against pozřeným not unlike normal cells to ensure their health. Granulocytes have a wide range of molecular weaponry that are in their cytoplasm seen as grains - granules. According weapons are divided into three types:
- neutrophil granulocytes - The most abundant type.
- eosinophilic granulocytes - Moderately abundant.
- basophils - rarer, except that they were very similar to the so-called. mast cells (mast cells), sedentary living life as stationary defense in the tissues.
The methods granulocyte counts eating microbes, burning acids, superoxide, peroxide, hypochlorite (drugstore known as Savo) and other corrosive liquids, destruction by digestive enzymes, and among other things, large amounts of nitric oxide which is otherwise appropriate concentrations necessary and beneficial component of cellular communication . The immune system uses a truly all possible methods, including the now loudly roztrubované DNA nanotechnology - that seems granulocytes using DNA as a building material for the construction of cages for tiny microbes ( wartha2007net ).
- Lymphocytes - Police specialists. When the lineman to bring the District lymph nodes of MHC II case with a foreign peptide glued, not just the immune response. The case is solved soon, says, consider the mitigating and aggravating circumstances and the overall situation in the organism. Research shows that the lymphocytes in lymphoid tissue still somewhere in a hurry and still argue with other cells. When a decision is made on the immune response of B and T lymphocytes are grouped around dendritic cells and begin the process of mutation and clonal selection (directed evolution), whereby during 3-4 days of high optimizes antigen receptor, first foreign molecule rozeznavší. Lymphocytes are divided into:
- B lymphocytes - They are responsible for production of antibodies (immunoglobulins). Both B cells and T cells have antigen receptors - molecules capable of recognizing foreign framework is. While the T-lymphocytes but their antigen receptors (TCR), leaving on the surface of B lymphocytes are famous by being discharged into the environment as antibodies. B cells also have their surface antigen receptor (BCR), which are basically antibody bound to the cell surface. In the aftermath of the immune response of B lymphocytes transformed into memory cells that provide long-lasting immunity.
- T lymphocytes - Lustration cells. Divided into:
- T C (cytotoxic) lymphocytes - Check civilians. They have the ability to destroy suspicious cells so they are called 'cytotoxic'. They bear on the surface "controller card civilians," CD8 molecule that binds to the "identity card" MHC I . With TCR receptor, which is equivalent to the antibodies of B cells, T lymphocytes C watching a picture of the internal environment of cells MHC I glued card and decide whether it is necessary to destroy the cell. Ways in which B and T lymphocytes through MHC controlling BCR and TCR receptors also relates Research prof. Superior .
- H T (helper, "helper") lymphocytes - Investigation. They bear on the surface "card interrogator" CD4, which binds to the 'police file' of MHC II and unfortunately also the HIV virus. It is divided into several other subtypes, the most important are T H 1, to authorize the operation C for the T cells and T H 2, which in turn allow the activity of B cells.
- T S (suppressor, "buffer") lymphocytes - (more recently, they reportedly say to them T reg, regulatory) - State defenders. They have access to various files and block the immune response.
- T M (memory, "memory") lymphocytes - Police archive. After a successful immune response is part of veterans of different classes of T and B cell changes to memory cells that have long retain the ability to re-establish the appropriate antibodies. Creating the appropriate memory cells is the principle of vaccination.
- further special class of cells - have different special skills, e.g. by bacteria able to ferret out the specific bacterial metabolites or absent in the body etc.
- NK cells - disciplinary supervision to wear identity cards. Diseased cells could theoretically can avoid destruction by T C lymofocytem that MHC I were not carrying card at all. For such a case there are NK cells that destroy cells with too little expression of MHC I. NK acronym derived from "natural killer" which is but a phrase that I do not like. NK cells also have many other functions.
- Monocytes - Cell garbage collectors, but who also hold police and combat functions. All this division at the guards, specialists and garbage is just an example - the division of labor between cells not from the perspective of human occupation no logic. Theoretically, all cells can do everything (they have a complete set of genes). When monocytes operate in tissues, they are called macrophages (literally great devourers). When you settle down somewhere, spread around a network of numerous projections and they are called dendritic (tree-like) cells. Part of the monocytes penetrate the brain, where they must behave quietly and discipline as a hotel maid, to disturb the neurons in the thinking - so they say monocytes microglia. Microglia, however, must have the ability in the critical situation in an instant change in the hotel bouncer and cope with the invaders without the aid of lymphocytes, which usually do not go to the brain.
- Maturation of the immune system - With the T and B lymphocytes, our immune system's ability to respond to pathogens with which we have never met neither we nor our ancestors. This is so because shortly before the birth of our immune system performs registration of all the body's own molecules. They are trained billions of T and B lymphocytes capable of recognizing all possible and impossible molecules, except those that are our body's own.
University of T lymphocytes is thymus (thymus, hence the T) in which the first leads to their multiplication, the development of individuality targeted gene manipulations in antigen receptor TCR and subsequent selection. When selecting the first destroyed by those T cells that do not have sufficient distinctive talent. In the second round they are destroyed those that react against self-derived structures (hence e.g. congenital syphilis no immune response). B lymphocytes in mammals through the same training in the bone marrow or lymph nodes, but the birds have to train special body of B lymphocytes, a sort of appendix cloaca (bursa of Fabricius, hence B). Training survive about 2% účastnivších the cells undergo apoptosis 98% and is eliminated by macrophages.
Trained lymphocytes then enters a waiting phase. If during the life encounter with a foreign peptide, which thou-so corresponds to their antigenic receptors are activated, and in agreement with other cells can become major players in the process of affinity maturation, which makes for ultrafast directed evolution (somatic hypermutation and clonal selection ). Ultrafast because it does not wait until it has finished cutting - the eukaryotic cell cycle would last at least 24 hours and it would be beset for four days only 4 iterations. Instead, the mutated genes selected by immediate testing of their protein product. The result is a highly optimized antigen receptor. If you Golem XIV Stanisława Lema complains that evolution is moving from the initial molecular genius for getting worse technical solution ( work here , unfortunately on the net only in English.), It can add to that that the immune system of the genius remains under pressure parasites partly preserved.
- Communication molecules - All components of the immune system cooperate. For example, only B cells have the ability to produce specific immunoglobulins, they are then used by all but immune cells and complement. Immune globulin can be considered as information molecule - it is negative or rather negative impression of the face criminals. In addition, the immune system uses a number of other communication molecules:
- cytokines, chemokines - Common name for communication molecules bíkovinného character. These include interleukins, interferons, and dozens of other peptides with ugly names.
- interleukins - Protein molecules generally managing communication strategies and tactics of the immune response: They invoke fever, leukocyte proliferation of controls, controls inflammatory response, proclaim martial law, etc.
- interferons - Three types of antiviral peptides alarm.
- TNF (tumor necrosis factors) - Multifunctional immune communication peptides in human pundits earned fame as an anticancer molecule alarm.
- eicosanoids (prostaglandins, leukotrienes, thromboxanes and other so called. nonclassical eicosanoids) - Communications molecules derived from dvacetiuhlíkové (εικοσι = 20) fatty acids, arachidonic acid. It belongs to the world of essential fatty acids well known omega-3 and omega-6 fatty acids, formerly known as vitamin F. Our first organism to ω-3 and ω-6 unsaturated bonds allocates more ω-9 and ω-12, thereby forming arachidonic acid. From that further enzymes are produced using specialized communication eicosanoids. The key enzyme in their production is cyclooxygenase, which is the goal of the action of the most common analgesics . Acetylsalicylic acid (aspirin) , paracetamol (aspirin) and ibuprofen (Brufen) share the same main effect - blocking cyclooxygenase.
- Complement - part interacting protein molecules (designated C1 to C9, possibly more than the nine) that are capable of individually recognizing and destroying, or at least a tag pathogens. C1 molecule recognizes the pathogen either directly or by immunoglobulin molecules C2-C8 then consider the situation to avoid accidentally destroying the good cells and then creates C9 attack complex, which perforates the membrane targets. Own cells additionally protects against complement antidote Protektin that inhibits the activity of C9 molecules. Like roller-delayed conclusion šturmkvéru 45 or mechanical ladybird, about which so likes to tell our cybernetic theorist and Slovak immigrant Jozef Kelemen , complement is an example of genius in simplicity. Magazines us now slowly starting to bomb the news about how in America there are 'smart' antibiotics, so we did not feel inferior, it's good to know that up to 5% of our blood serum protein molecules are the smartest antibiotics - complement.
- Defensins - Effective antibiotics produced by our organism. It can say that in terms of common microbes, we rank among poisonous animals.
- α-defensins - Weapon of granulocytes and other immune cells.
- β-defensins - Anti-bacterial toxins that exclude the surface of our skin, mucous membranes and body cavities.
- Apoptosis - Controlled cell removal. Cells undergoing her elderly if they are damaged DNA, if they are parasitized, or if they have in themselves a suspected tumor growth. The term "cell suicide" I consider it misleading. (I and others consider the "death" as entirely unscientific term, but more on that later.)
- Autoimmunity - Like any system, even in the immune errors occur. There are quite a few cases when certain proteins in our cells appear in the course of life and lymphocytes must understand that these proteins are not hostile. Lymphocytes alone can bring even without the erroneous judgment and accidentally start the autoimmune response. Immune errors are very dangerous because they can easily mistake their own cells to destroy the population, as is necessary with type 1 diabetes mellitus . The immune system would also not overreact to dozens of species of beneficial bacteria, fungi and (maybe) useful viruses occurring on the skin and in the digestive tract.
Immune system problems
It appears that most of lifestyle diseases, or diseases of longevity is somehow associated with a malfunctioning immune system. Mechanisms of aging already know a few. We oxidative theory of aging and antioxidant theory of aging DNA, telomere shortening theory, the mitochondrial theory of aging ... I think that this list should add a theory deterioration of the immune system. It's not just allergies and asthma, as well as arthritis, osteoarthritis , diabetes , or seemingly unrelated to atherosclerosis . For all these diseases should pay attention immunomodulatory drugs.
Many natural adaptogens (ai neadaptogenů) affect the immune system. For adaptogens sensu stricto interaction could be expected on the axis of glucocorticoids, the immunomodulatory meaning is well known ( Sapolsky2000hdg ). A large amount of scientific effort has been devoted to a model adaptogen ginseng and medicinal fungi Polyporales groups , which are just immunomodulatory effect deems most important. Another known immunomodulatory adaptogens is purple coneflower (Echinacea purpurea) , membranous tragacanth (Astragalus membranaceus). Active substances are triterpenoids ( christensen2009gcb , Paterson2006gtf ) and other secondary metabolites ( Percival2000uem , Block2003ise ), but all these plants / fungi are very important also specific proteoglycans / polysaccharides.
SUMMARY immunomodulatory effects
The immune response can be roughly divided into two types, which compete with each other:
- cytotoxic (anti-virus, tumors): -> maturation of T H 1 -> activation of T lymphocytes C
- antibody (against most bacteria): -> maturation of T H 2 -> B cell activation
When the disease, the immune system must properly assess the type of risk and consider what type of pathogen is concentrated. Antigens from the affected tissue are transported to the lymph nodes where there is a production of interleukin IL-2 and start the immune response. Type of response is the result of discussions on antigen leads T H lymphocytes and dendritic cells (DC). Opinion cells express through communication cytokines:
- DB type 1 T H 1 - Production of IFN-γ (gamma interferon), TNF (tumor necrosis factor alpha), IL-12
- DB type 2 T H 2 - production of interleukins IL-4, IL-10
The final decision is in the hands of DB - at your discretion controlled maturation of T H cells to either T H 1 type (cytotoxic response) or Type T H 2 (antibody response).
It is not possible in a single article to cover all natural medicaments with effect on immunity. There is more detail devote only a model adaptogen ginseng . Immunomodulatory effects of natural medicines are in common and are referred to on the site (see, for example, medicinal fungi Polyporales groups which are polysaccharides and proteoglycans particularly important).
The fact that ginseng affects the immune system, is now proven beyond doubt ( christensen2009gcb , Choi2008bcp , Xiang2008cau ). Ginseng strengthens the immune system especially against viruses and tumors. Cancer can among other things look as immune to failure . In cancer ginseng welcome support means, with or without certain direct anticancer effect .
Content ginseng substances influence the choice of the immune response. Christensen2009gcb (ch. "Immunomodulatory effects") referencuje thesis that ginseng promotes a cytotoxic response (against tumors and viruses). In contrast, lee2004gre shows that the majority ginseng panaxosid Rg 1 has the effect of just the opposite. Thirdly, ginseng has proven anti-inflammatory and antiallergic effect. A comprehensive scientific view of the immunomodulatory effects of ginseng is not available, but the current data confirm that its content components have contradictory effects on immunity, which is typical for the effect of adaptogens .
The stimulatory effect of ginseng on the immune system
In general adaptogens and ginseng specifically occurs in several aspects to the contradictory action of drugs . Specifically, ginseng reduces inflammation , act against autoimmunity and are often applied where the doctors usually prescribe immunosuppressive corticosteroids. It is therefore the ginseng immunosuppressive?
The answer to this question is not clear. Acute fever (ie. Infections) are one of the few traditional contraindications ginseng. I therefore believe that at least some infections calming effect of ginseng on white blood cells are not welcome. Ginseng but by far not merely immunosuppressive agents (which, moreover, are not even mentioned corticosteroids Sapolsky2000hdg ). For many bacterial and viral infections, most Ginseng helps - immunity improves.
For specific ginseng polysaccharides was found able to inhibit Staphylococcus aureus septicemia in an optimum dose of 25 micrograms / kg ( lim2002aep , lim2004iap ). The effect was associated with a multiple increase in NO production and cytokines (TNF-α, IL-1, IL-6 and IFN-γ), macrophages.
The effect of ginseng on the ability of macrophages to respond to infection during prolonged stress ( Pannacci2006pgm ). It is believed that the long term stress reduces immunity ( Viswanathan2005saa ), but short-term stress activates macrophages ( Berczi1998scn ). Ginseng polysaccharides ginsan (25mg / kg / day p.o.) in mice, increased the expression of the TLR receptor of macrophages (these receptors non-specifically recognize pathogens) and thus the ability of macrophages to respond to infection ( Ahn2006igi ). Ginseng also positively affects anti-cancer immunity .
Inhibitory and anti-inflammatory effect of ginseng
Effect against autoimmunity
- Ginseng polysaccharides in a mouse model of autoimmune counteract brain damage. ( Hwang2011app )
Specific immunomodulatory effects:
- On monocytes / macrophages: Panaxosidy Rb 1 and Rb 2 suppressed production of TNF-α in mouse and human macrophages stimulated with lipopolysaccharides with IC 50 ~ 50 or ~ 25μM ( Cho2001vie ). According Lee2002fma red ginseng increased production of TNF-α by macrophages. In contrast, according Cho2001vie , ginsenosides Rb 1, Rb 2, Rg 1 are potent inhibitors of TNF-α production by macrophages stimulated with bacterial lipopolysaccharide. Said panaxosidy additionally inhibit the production of other inflammatory cytokines such as IL-6 and IL-1β ( Rhule2006pna ).
- For lymphocytes of mouse spleen was found that pxsd. Rb 1 and Re at a concentration of about 100 mA significantly increased the proliferation of mitogen-stimulated T H (CD4 +) and B lymphocytes, Rg 1 on it had no effect, while Rb 2 it decreased the IC 50 of about 25μM. Rb 2 also suppressed production of IL-2 the lymphocytes after stimulation with Concanavalin IC50 ~ 13.3μM. UT C (CD8 +) lymphocytes results were different - Rb 2 and Rb 1 do not mask their proliferation after stimulation with IL-2, while Re and Rg 1 their propagation limiting IC 50 57.5, respectively 64.7μM ( Cho2002gfp ). According lee2004gre , gssd. Rg 1 promotes maturation of T H 2 cells and the production of IL-4. In contrast, Lee2006grh argue that gssd. Rg1 promotes maturation of T H 1 cells.
- Pxsd. F 1 and Rg 1 in culture of mouse splenocytes to selectively enhance the production of type 2 cytokines (IL-4 by splenocytes, IL-12 by macrophages) and their transcription factor GATA-3, while pxsd. Rh 1 and 20 (R) -Rh 1 pordukci selectively increase the cytokine type 1 (IFN-γ in splenocytes) and their transcription factor T-bet. All of these pxsd. (F 1, Rg 1, Rh 1 and 20 (R) -Rh 1) increases the binding of the transcription factor NF-kB to the DNA. It is interesting that the maximum increase in cytokines have been observed at concentrations of 5μM and 10 mm packing, higher concentrations of cytokines increase again less at 50μM more than twice less than 10 mm packing ( Yu2005pgd ).
- On dendritic cells: According Takei2004dcm and Takei2008dcp , compound K and gssd. 20 (S) -PPT affect monocytic dendritic cells towards a type 1 immune responses, these studies discuss the possibility of an anti-cancer effect of the ginseng and the opportunity for cancer immunotherapy affected by dendritic cells.
- NK cells: Choi2008bcp discussion ability of ginseng extract and specifically Panaxosidy Rh 2 to restore NK cell activity and immune cell damage after experimental mitomycin.
- Granulocyte: gssd. Re activates neutrophilic granulocytes against tumor cells ( Plohmann1997iae ).
- Flu: ginseng effect against influenza is elaborated in the article flu and viral diseases .
- HIV: ginseng according Choi2008bcp suppress reproduction of HIV and alleviates the course of AIDS.
- On lymphocyte adhesion to the endothelium and diapedesi Some panaxosidy, e.g. notoginsenosid R 1 ( Chen2008enr ) or ginsenoside Rb 1 ( He2007peg ) reduce lymphocyte adhesion to the endothelium, which is explained by the observed effect against atherosclerosis and inflammation .
Immunomodulatory polysaccharides and proteoglycans
The immunomodulatory effects of natural medicines play an important role polysaccharides and proteoglycans. This is so, even ginseng ( Sun2011sba , Yun1993iat ), American ginseng ( Assinewe2002epp , Wilson2013uai Azike2015ssi ) and other herbs. Again, if we take the example of P. ginseng, for example acidic polysaccharides ginsenan PA and PB ginsenan increases the serum IgG and activate phagocytosis ( Tomoda1993cta , Tomoda1994csg ). Information in this field is still evolving, relevant publications of the immunomodulatory effects of ginseng polysaccharides include lim2004iap , Choi2008rga , Yoo2012peg , Wang2013mmb and more.
Another classic adaptogens with immunomodulatory effects
- Japanese ginseng (Panax japonicus) mouse model improves the recovery of the damaged immune system. ( Zhang2011epi )
Proteoglycan fraction of Reishi activating immunity in human monocytes (22364151) and improve immunity in a mouse model of immunosuppression (22,403,542). Otherwise, work on the effect of Reishi on the immune system is a large amount, and most of them I comment section of their antitumor effects .
Other immunomodulatory adaptogens
Immunomodulatory effect is always one of the first things to be examined at adaptogens. Just briefly, relevant reference point for hailing ( Rasool2006irw , Minhas2012tew , Ku2014wii , Ichikawa2006wpa ), ginger ( Lakhan2015zep ), turmeric ( Lakhan2015zep ), prickly pear ( Zhao2012ieo ) Cordyceps ( Lee2014aec ).