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Before every war, plans are hatched out so a positive outcome can be achieved. Movements of the enemy, amount of military forces, abilities in dry land, air and sea. All these are required in order to maximize the chances to win a war. The same background is used in the Immunology sector of Biological/Biochemical Sciences but in the microcosm. Studies of the immune system, factors from which the system is consisted, specific movements of receptors, antigens, antibodies, intracellular abilities, surface abilities etc. Understanding the pathway in immunology is half of the success to win the "war" because different approaches can be tested in order to cease an overexpression, or improve a downregulated protein.
An ideal example of an immunological strategic plan, is the study of the interaction between CD14 (and receptors that accompany this molecule) and Gram Negative Bacteria. The response of CD14 to these Bacteria during infection but also the whole mechanism from this reaction gave birth for many ideas that led to various experiments. So what is the background of these two opponents?
The CD14 receptor is a 356 amino acid membrane glycoprotein12 where the C-terminal leader sequence of 28-30 amino acids is replaced by a glycosyl phosphatidylinositol (GPI) anchor after translation.13. CD14 was characterized as a receptor (co-receptor along with the Toll-like receptor TLR 4 and MD-2) for bacterial endotoxin (LPS) in 1990, almost a decade before the discovery and characterization of TLR, and can be regarded as the first described pattern-recognition receptor .
Because of this fact CD14 is not a transmembrane protein but is anchored to the cellular membrane through GPI linkage. The membrane expressed CD14 (mCD14) is founded on the surface of mature myeloid cells14.GPI anchored proteins, (like mCD14), are gathered in membrane microdomains which are involved in cellular processes like transcytosis,19 and transmembrane signalling.21 CD14 also exists in a soluble form (sCD14), that can be found with two different molecular weights in serum.22 The sCD14s have an important role in LPS mediated activation of CD14 negative cells (epithelial and muscle cells)16 24 but the biological differences between these two forms are unknown.
CD14 is a molecule with a wide range of functions. In addition to functioning as a pattern recognition receptor for a variety of microbial ligands, CD14 also acts as a receptor for endogenous molecules like intercellular adhesion molecule (ICAM)-3 on the surface of apoptotic cells, amyloid peptid, ceramide, and urate crystals.
As mentioned above CD14 is contributing in the recognition of bacterial LPS. The contribution of CD14 is directly related to Toll-Like Receptor 4 of the TLRs family. TLRs are type I transmembrane proteins with parts outside of the cell containing leucine-rich repeats that determine the recognition of Pathogen-associated molecular patterns (PAMPs). In order for a downstream signal transaction to occur, domains like Toll-interleukin 1 receptors in the cell (TIR) and Transmembrane domains are required. From recent studies, 10 and 12 TLRs (operational) have been identified both in humans and mice, with the other TLRs (from TLR1-TLR9) being preserved in human and mice respectively. TLR11, TLR12 and TLR13 have been lost from the human genome. Studies from experiments with mice deficient in every type of TLR showed that TLRs have an apparent activity regarding PAMP recognition and immune responses3.
LPS is not founded in every bacterium but specifically to Gram Negative bacteria. Their name was adopted by the Bacteria's failure in giving a violet color in a Gram Staining Protocol3233. Bacteria that retain the violet color are called Gram Positive Bacteria but they have a crucial difference from the negative ones. They do not carry LPS in their cell wall. Thus CD14 would not have a role regarding signal transaction from a Gram Positive Bacteria infection, and that clarified that LPS is the endotoxin that triggers an immune signal cascade. Bacteria in the category of Gram-Negative ones are Escherichia, Bartonella, Enterobacter, Hemophilus, Pseudomonas, Salmonella, Shigella, Vibrio, etc.
Identifying the reasons for the initiation of this immune response, made it only natural to investigate the order of the signal transaction and point out specifically the role of each protein in this signal repertoire.
Lipopolysaccharide (LPS) monomers are extracted from bacterial membranes by the serum protein LPS-binding protein (LBP) which transfers the LPS monomer to a lipid-binding site on CD14 in the membrane of phagocytes. CD14 promotes the binding of LPS to the TLR4-MD-2 complex, which signals to the cell interior. In the absence of CD14, TLR4-MD-2 can still function with some forms of LPS or with much higher LPS concentrations. Besides CD14 and TLR4-MD-2, there are also more cell surface molecules which could detect LPS such as the ion channels, the macrophage scavenger receptor (or MSR), and CD11b/CD18. Signaling in the interior of the cell depends on many different factors like the binding of the TLR domain that lies in the cell, to IRAK (IL-1 receptor-associated kinase, TIR (Toll/IL-1 receptor homology domain), MyD88 (myeloid differentiation protein 88), and TIRAP (TIR domain containing adapter protein also called MyD88- adapter-like protein or Mal), and prevented by another protein called Tollip (Toll-interacting protein). It is important to mention that a MyD88 independent pathway exists, and from this pathway TIRAP/Mal gives a signal with the assistance of the interferon regulatory factor (IRF)-3and an RNA-dependent protein kinase (PKR).
Through this mechanism CD14/Gram Negative Bacteria cascade was scattered in many different domains in order to find ways of preventing various diseases.
If we were in the position to make an experiment based on this information, the first thing that could come in our mind would be just the elimination of CD14 from this signal transaction pattern.
Thus a study in CD14-deficient mice was carried out revealing reduce distribution of Gram-Negative Bacteria in the host and resistance to endotoxin shock3232.
This study is supposed to evaluate the role of CD14, in shock caused from endotoxin/LPS or from Gram-negative bacteria. They came to the conclusion that when CD14 was not present LPS's effects were significantly reduced in the hosts. In this study the hosts were CD14-deficient mice and proved not to be sensitive against endotoxin/LPS-induced shock. Through the study different amounts of endotoxin/LPS were injected and in a portion that kills totally normal mice, CD14-deficient mice produce small amounts of TNFÎ± and IL-6, which are known to assist in causing an endotoxin shock.
Through these different reactions in the mutated mice, cells managed to survive from these high doses of endotoxin/LPS or Gram-negative bacteria with no prove for an appearance of shock in any case. This revealed that CD14 has a crucial part in the shock expression that is caused from LPS. When incidents of shock did not occur, reduced Gram-Negative bacterial infections showed also up on table. From this observation one could assume that the same factors that deactivate or eliminate CD14 could be the reason of the reduced infections from Gram-Negative Bacteria.
Since there are two types of CD14 (in solid state on the cell surface but also in a soluble state), it was quit expected to try and find differences between the two states. Thus a study on the soluble CD14 (sCD14) gave rise to some interesting results. It showed that even though both CD14s can distinguish Lipid A from LPS's region, sCD14 could identify Lipid A structure in a more sensitive way than membrane CD14 (mCD14)22. This showed the importance of sCD14 when defenses of the infected host are activated. The results of this study could provide an understanding of the mechanism that lies behind the recognition of Lipid A. Further on, other study indicates that gram-negative organisms activate endothelium from a pathway that depends on sCD14. A hypothesis was made that complexes of LPS-sCD14 derived from Gram-Negative Bacteria's membrane and bind to an endothelial receptor which is not yet clarified.
This interaction activates NF-kB, new gene transcription, but also the induction of an endothelial cell activation phenotype. The studies are setting a general assumption on determining whether therapies which target sCD14 for elimination, or try to extinguish this endothelial cell receptor for the bacterium-sCD14complex could be in patients' advance regarding the risk for gram-negative infections1.
More studies were carried out regarding CD14 for its ability to make use of Hydrophilic Regions so it can detect LPS232, or as a complex CD14/LBP of this signal pattern21 or how CD14 responds using different types of Gram-Negative Bacteria11.
Everything that consists of the reaction cascade of the CD14/Gram-Negative bacteria was studied in depth. Thus studies on the rest participating molecules were carried out.
Toll-like receptors were thoroughly investigated alone1 or in combination. For example from TLR4-MD-2 complex it was found that MD-2 has a vital role for triggering a signal for activation through TLR4, the correct allocation of TLR4 and interacting with LBS22. Studies for different type of receptors were initiated (TLR2) with also different cluster differentiation molecules (CD36) in order to make comparisons with the existing knowledge but also the differences of the mechanism when receptor changes221.
Throughout these long year studies many "secrets" were revealed regarding the interaction between CD14 and Gram-Negative Bacteria specifically. As it was firstly said the purpose of knowing the "opponents" moves is to try and acquire a positive outcome in order to cease or induce specific functions that could lead to diseases or cures respectively. Understanding how the signal transaction of this interaction works, the scientific society focused on uncovering the results of these immune responses in different type of cells located in different places in the body and with immunotherapy suppress or eliminate any unwanted results.
Common shock reactions come from conditions like Sepsis. Sepsis labels a quite complicated clinical syndrome that has its outcome from a harmful or damaging host response to infection. Because of the tremendous effort to understand the pathogenetic mechanisms, important progress has been made that gave an understanding not only for the process of sepsis, but also basic principles foremost bacterial-host interactions. It is important to mention that Gram-negative bacteria are reported in half of the cases of sepsis.
There was a study that showed significant benefits by using low-dose corticosteroids. As a first try there were experiments of very high doses of steroids, since it is known that sepsis means an uncontrolled inflammatory response. Results were not encouraging since there were no positive survival rates. On the same subject another research group found out, that patients in the advanced stages of septic shock had low amounts in adrenaline and because of that, low-dose replacement steroids was thought to be beneficial. The better ways to comprehend immunopathology of sepsis has revealed many other approaches, and more strategies are at different stages of development. Examples could be therapies focused at bacterial targets, like new anti-endotoxin molecules (bactericidal/permeability-increasing protein), or modified lipoproteins. It is of great importance to mention that both absorb and neutralize LPS. In recent studies results showed that oxidized phospholipids can interfere with binding of LPS to LBP74.
Studies that are of great importance make use of Toll-Like Receptors for treating Cancer via Immunotherapy.
The best understood family of pattern recognition receptors is the Toll-like receptors (TLRs). From all the TLRs, 10 are known in humans. TLR9 has the ability to detect unmethylated CpG dinucleotides, ("CpG motifs"), which are quite common in the genomes of most bacteria and DNA viruses, suppressed and methylated in vertebrate genomes. For immune therapy of cancer, infectious disease, and other conditions, synthetic oligodeoxynucleotides containing CpG motifs ("CpG ODN") have been developed. Most types of immune cells do not express TLR9, and so are not activated directly by CpG ODN. Among resting human immune cells TLR9 is expressed primarily or exclusively in B cells and in plasmacytoid dendritic cells (pDC), a specialized type of DC which produces most of the type I interferons (IFN) that are made in response to infection by viral and other intracellular pathogens. Therefore, the immune effects of CpG ODN in humans result directly and indirectly from activating TLR9-expressing pDC and B cells.
CpG ODN have anti-tumor activity in many mouse models. In relatively small tumors CpG monotherapy can be sufficient to induce a T cell mediated regression, but to induce rejection of larger tumors the CpG ODN often needs to be combined with other effective anti-tumor strategies, such as vaccines, monoclonal Ab, radiation therapy, surgery, and chemotherapy. Chemotherapy usually has been considered to be immune suppressive, but studies of humans receiving taxane chemotherapy actually show increased T cell and NK cell function, possibly due to induction of proinflammatory cytokine production, homeostatic leukocyte proliferation, and reversal of the immune suppressive effects of regulatory T cells (Treg).
Many other projects are currently running, focusing in completely different areas that could be affected by immunological responses. Instead of preventing the immunological response through bacteria, some scientists are trying to promote it like probiotic which are known of promoting health1. Some other studies focus on more daily issues like obesity.
Toll-like receptors recognize the flagella of bacteria and activate the immune system. Obesity is associated with an increase in immune system activity and this may contribute to a range of other symptoms associated with obesity, including elevated risk for cardiovascular disease and type 2 diabetes ( 3).
Understanding the Mechanisms that consists the immune system is the gate to comprehend the reasons of infection and ways to prevent them. In this essay many results from different studies were presented. The clue that exists in every study is the general knowledge of the interaction between CD14 and Gram-Negative Bacteria. That is the foundation of every study and building upon it led to more sophisticated studies of great depth and detail. By increasing the information of every study it gets us closer to a well shaped and spherical image of the incidents that are occurring due to immune response. In a battle many fights are lost. But learning from the mistakes that resulted in losing a fight could reinforce future methods so they can be more consistent and accurate, and the meaning of this kind of war could mean the discovery of drugs and medicines leading eventually to the cure.