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\\n\\nLaunching 2021
\\n\\nArtificial Intelligence, ISSN 2633-1403
\\n\\nVeterinary Medicine and Science, ISSN 2632-0517
\\n\\nBiochemistry, ISSN 2632-0983
\\n\\nBiomedical Engineering, ISSN 2631-5343
\\n\\nInfectious Diseases, ISSN 2631-6188
\\n\\nPhysiology (Coming Soon)
\\n\\nDentistry (Coming Soon)
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\\n\\nNote: Edited in October 2021
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\n\nDesigned to cover fast-moving research fields in rapidly expanding areas, our Book Series feature a Topic structure allowing us to present the most relevant sub-disciplines. Book Series are headed by Series Editors, and a team of Topic Editors supported by international Editorial Board members. Topics are always open for submissions, with an Annual Volume published each calendar year.
\n\nAfter a robust peer-review process, accepted works are published quickly, thanks to Online First, ensuring research is made available to the scientific community without delay.
\n\nOur innovative Book Series format brings you:
\n\nIntechOpen Book Series will also publish a program of research-driven Thematic Edited Volumes that focus on specific areas and allow for a more in-depth overview of a particular subject.
\n\nIntechOpen Book Series will be launching regularly to offer our authors and editors exciting opportunities to publish their research Open Access. We will begin by relaunching some of our existing Book Series in this innovative book format, and will expand in 2022 into rapidly growing research fields that are driving and advancing society.
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\n\nArtificial Intelligence, ISSN 2633-1403
\n\nVeterinary Medicine and Science, ISSN 2632-0517
\n\nBiochemistry, ISSN 2632-0983
\n\nBiomedical Engineering, ISSN 2631-5343
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\n\nDentistry (Coming Soon)
\n\nWe invite you to explore our IntechOpen Book Series, find the right publishing program for you and reach your desired audience in record time.
\n\nNote: Edited in October 2021
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These invaders include viruses, bacteria, protozoa or even larger parasites. Any Human body is continuously exposed to pathogenic microorganisms. The immune system is composed of two major subdivisions of immune system, the innate or nonspecific immune system and the adaptive or specific immune system [1].
The innate immune system is our first line of defense against invading organisms while the adaptive immune system acts as a second line of defense and gives protection against re-exposure to the same pathogen. Each of the major subdivisions of the immune system has both cellular and humoral components by which they carry out their protective function and help each other to do these functions. Since pathogens may replicate intracellularly (viruses and some bacteria and parasites) or extracellularly (most bacteria, fungi and parasites), different components of the immune system have evolved to protect against these different types of pathogens [2].
Include first line of defense which acts before invasion of pathogenic microbes. And the second line of defense which acts after invasion. The anatomical barriers that works mainly against infections with microbial invaders. This first line of defense represented by the epithelial surfaces and skin form the physical barriers that are very impermeable to most infectious agents [1].
The shedding of skin epithelium also helps remove bacteria and other infectious agents that have adhered to the epithelial surfaces. Movement due to cilia or peristalsis helps to keep air passages and the gastrointestinal tract free from microorganisms. The trapping effect of mucus that lines the respiratory and gastrointestinal tract helps protect the lungs and digestive systems from infection [2].
Chemical barriers like Lysozyme and phospholipase found in saliva and other secretions can breakdown the cell wall of bacteria and destabilize bacterial membranes. The low pH of gastric secretions prevents the growth of bacteria [3].
The microbiota of the skin and in the gastrointestinal tract can prevent the colonization of pathogenic bacteria by secreting toxic substances or by competing with pathogenic bacteria for nutrients or attachment to cell surfaces. They represent the biological barriers of the innate immunity [2].
The anatomical barriers are very effective in preventing colonization of tissues by microorganisms. However, when there is damage to tissues the anatomical barriers are breeched and infection happens. Once infectious agents have penetrated tissues, another innate defense mechanisms comes into play, namely acute inflammation as the second line of innate immune defense. Many Humoral and cellular factors play an important role in inflammation against microbial invasion, which is characterized by edema and the activation of phagocytic cells [4].
These humoral factors are found in serum or they are formed at the site of infection. They contain Complement system, Interferons and Lysozymes. The most important humoral barrier is the Complement system, since it acts as with the phagocytic cells as a bridge between specific and non-specific immune response. Complement system represents a set of glycoproteins in blood. Once they are activated after rapid cascade events that can lead to increase vascular permeability, activation of phagocytic cells, opsonization of bacteria and lysis [5].
Complement glycoproteins are synthesized by liver cells (hepatocytes) and macrophages and many other cell (e.g. gut epithelial cells). All normal individuals have complement components in their blood. This system can be activated by [1, 2]:
Antigen-antibody complexes containing IgG or IgM activate complement by the classical pathway that starts with C1 (complement 1).
Membranes and cell walls of microbial organisms (e.g. Lipopolyccharides layer [LPS] of gram –ve bacteria) and many other substances can activate complement by the alternative pathway.
Proteolytic enzymes released either from microbes or from host cells during immune defence mechanisims, can also activate the complement system by breaking down critical components.
The complement system takes part in both specific and non-specific resistance and generates a number of products of biological and immunological importance. The functions of the complement system are summarized in Table 1 [3, 5]:
No. | Function |
---|---|
1. | Binding and neutralizing foreign substances that activate it. |
2. | Induce the ingestion of complement-coated substances by phagocytic cells (help fn the opsonization process when C3b and C4b linked with the surface of microorganisms and attach to Complement receptor on phagocytic cells then induce phagocytosis). |
3. | Activation of many cells including polymorphonuclear cells (PMNs) and macrophages. |
4. | Have roles in regulation of antibody responses. |
5. | Clearance of immune complexes and apoptotic cells. |
6. | Have roles in inflammation and tissue damage. |
7. | Some components (C3a, C4a and C5a), have role in Anaphylaxis (a dangerous case of type 1 hypersensitivity), hence they are called anaphylotoxins. |
8. | Some complement components acts as chemotactic facters e.g. C5a and MAC. |
The functions of the complement system.
On the other side; the cellular factors are the main line of defense in the nonspecific immune system, they are listed in the Table 2 [2, 5].
No. | Cell | Function |
---|---|---|
1. | Neutrophils | Polymorphonuclear cells (PMNs) migrate to the site of infection where they phagocytose invading organisms and kill them intracellularly. In addition, PMNs contribute to tissue damage that occurs during inflammation. |
2. | Macrophages | Tissue macrophages and activated monocytes, which differentiate into macrophages, also function in phagocytosis and intracellular killing of microorganisms. In addition, macrophages are capable of extracellular killing of infected or transformed cells (self-target). Furthermore, macrophages have role in tissue repair and act as antigen presenting cells APC, which are required for the induction of specific immune responses. |
3. | Natural killer | NK cells can nonspecifically kill virus infected and tumor cells. These cells are not part of the inflammatory response but they are important in nonspecific immunity to viral infections and tumor surveillance. |
4. | Eosinophils | Eosinophils have proteins in granules that are effective in killing certain parasites. |
Cellular factors of the nonspecific immune system and their function.
Phagocytosis is a very important process during non-specific immune response when specialized cells engulf foreign body like bacteria or molecule like toxin or virus. The phagocytosis has four steps, Figure 1 [2]:
Chemotaxis. Phagocytic cells response and migrate to the site of infection or injury by the effect of complement products and cytokines released from tissue macrophages that have encountered bacteria or any foreign body in tissue.
Endocytosis. Starts with pseudopodia formation then phagocytic cells bind to the foreign body by: Fc receptors–Bacteria with IgG antibody on their surface have the Fc region exposed and this part of the Ig molecule can bind to the receptor on phagocytes. Complement receptors–Phagocytic cells have a receptor for the complement C3b. Scavenger receptors mainly for invading bacteria.
Phagolysosome formation and degradation of foreign substances. After attachment of the bacteria the phagocyte begins to extend pseudopods around the bacteria and surround and engulf them forming the phagosome. During phagocytosis the granules or lysosomes of the phagocytes bind or fuse with the phagosome and empty their contents. The result is the foreign bodies or bacteria engulfed in the phagolysosome which have the contents of the granules or lysosomes.
Phagocytosis process steps.
Intracellular killing and Digestion (Lysis and excretion): There are three means of killing the microorganisms inside phagocytic cells; either Oxygen dependent killing by formation of NADPH using Oxygen, then production of the toxic oxygen compounds like H2O2 and hydroxyl radical (OH•). These compounds are toxic to microbes and kill them, Oxygen independent killing by production of toxic hypochlorite (OCl-) and singlet oxygen (1O2) from H2O2 using the enzyme Myeloperoxidase that released into the phagolysosome or Nitric oxide dependent killing by Toxic nitric oxide synthesis and production (NO) when microorganism binds to the macrophage because of cytokines release (TNF-α and IFN-γ) [3].
Oxygen -dependent killing and Oxygen -independent killing both are called the Respiratory burst. After killing, the enzymatic system of the cell will digest all the phagosome components then absorb the useful materials and excrete the residues to the environment (blood) by fusing the phagolysosome with the cell membrane.
The cells that able to do phagocytosis are (monocytes, macrophage, PMNs and dendric cells). The results of phagocytosis are either a complete destruction of foreign body and excretion (PMNs). Or a complete destruction of foreign body and some parts (polypeptides) of it will be processed and presented on the surface of the phagocytic cells (monocytes, macrophage and dendric cells) then the phagocytic cell will be antigen presenting cell (APC) [2, 3].
These cells are the messengers between innate (non-specific) immunity and the adaptive (specific). Specialized APC are macrophage (MØ), B-cells and Dendric cells (DC) [3].
Roles of Antigen Presenting Cell (APC) can be summarized as [5]:
Engulfment of foreign Ag, processing it and presenting it (or a olypeptide from it) on the surface near the Major Histocompatibility Complex MHC class I or II.
Communication during the immune response between immune cells especially T- cells to induce the proper immune response cellular or humoral.
Secretion of cytokines which are substances (glycoproteins) that regulate the immune response.
B-cells or B-lymphocytes, T-cells or T-lymphocytes (T-helper cells including Th1 and Th2, T-Cytotoxic Tc and T-suppressor Ts) [3].
Natural killer cells (NK) have no CD markers on the surface so they are usually called null cells [3].
It is important to know that B-cells are able to be APC by internalization of Ag inside the cell and do the processing and presenting, which will be discussed later. Also Dendric cells (DC) are cells found only in the mammalian immune system; their function is to engulf and process Ag then present it on the surface to other immune cells. Found in tissues that in contact with external environment such as skin, lung, stomach and intestine [2].
In the mucosal surfaces and sites, the mucosal immune response come to play role in resistance against infection establishment. Many lymphoid tissues are associated with mucosa which are usually called mucosa-associated tissues play major role in protection since they are rich with both T-cells and B-cells, produce many types of Lymphokines that acts as signals of the immune system actions, produce IgA (sIgA Secretory IgA); the main effective immunoglobulin type in the surfaces of the body and the most important part is that mucosal surfaces have the receptors of microbiota that play as a biological barrier and support innate immunity. Many secretions are also produced by the mucosa to protect surfaces like gastric acid and continuous mucous secretion and shedding helps in renewing normal flora population and shed colonized pathogens.
Mucosa-Associated Lymphoid Tissues (MALT) Include the lymphoid tissues of the intestinal tract, genitourinary tract, tracheobronchial tree, and mammary glands. All of the mucosa-associated lymphoid tissues are unencapsulated and contain both T and B lymphocytes [2, 5].
It is found along the digestive tract. Three major areas of GALT that can be identified are the tonsils, the Peyer’s patches, located on the submucosa of the small intestine, and the appendix. In addition, scanty lymphoid tissue is present in the lamina propria of the gastrointestinal tract [3, 5].
Tonsils, located in the oropharynx, are predominantly populated by B-lymphocytes and are the site of antigenic stimulation.
Peyer’s patches (PPs), they are lymphoid structures disseminated through the submucosal space of the small intestine
Physiological roles of secondary lymphoid organs:
The follicles of the intestinal Peyer’s patches are extremely rich in B-cells, which differentiate into IgA-producing plasma cells.
T-lymphocytes are also present in the intestinal mucosa, the most abundant of them expressing membrane markers that are considered typical of memory helper T-cells. This population is involved in the induction of humoral immune responses (HMI) [1, 2].
B-cells have normal Ag receptors on the surface they are natural Igs, these Igs are able to form Ag-Ab complex on the surface of B-cell. This complex will be internalized inside B-cell, then the foreign Ag will be processed within B-cell and presented (or polypeptides from it) on the surface of B-cell near MHC class II and now B-cell is APC.
T-helper (Th) cells come near the APC B-cell and by the help of TCR and CD4; Th will interact and communicate with APC B-cell and Th cell will be activated and release cytokines or lymphokines (IL-2, IL-4, IL-5 and IFN-γ), these products will induce other B-cells for dividing, proliferation and differentiation. IgM will be the first Ig produced then B- cell will switch to make IgG. This response is called T-dependent Ag immune response. The other type of response is T-independent Ag immune response, this type of Ag stimulates B-cells without need for T-helper lymphocytes interfere [1, 2].
After B-cells activation, series of events happen (proliferation, clonal expansion, division and maturation), ending with Ab and memory B-cells production. These series of events called B-cell Maturation. During the second exposure to the same Ag that started the first immune response (perhaps after year from first exposure), the B-memory cells will remember the Ag and will be activated and divide into a clone of plasma cells to start the Secondary immune response (Memory response) [3].
Antibodies or Immunoglobulins (Ig) that are produced after specific humoral response are in five types; IgG; IgM, IgA, IgD and IgE based on differences in the amino acid sequences in the constant region of the heavy chains. In addition, the classes of immunoglobulins can be divided into subclasses based on small differences in the amino acid sequences in the constant region of the heavy chains [1].
This response occurs against cells, which are called Target cells. During both HMI and CMI, T-helper cells recognize foreign Ag processed on the surface of APC. If this Ag was processed and presented near MHC class II, then Th cells will activate HMI by B- cells activation, but if the presented Ag on APC was near MHC class I, then Th cells will activate CMI by activation of Tc, NK and MØ. Th cells able to activate and regulate CMI and HMI by many cytokines production.
In addition, in both CMI and HMI, when Th cells recognize the foreign Ag, Th cells will start T-cells activation by series of events (expanding, clonal proliferation and differentiation), then become mature to give specific activated T-helper cells in HMI and give specific activated T-helper cells and memory T-cells in CMI [2, 5].
Role of CMI response: is the defense against Tumor cells or cancer cells, Grafts Rejection, against Intracellular parasite infected cells with foreign Ag presented near MHC class I. Target cell is the infected cell with parasite and Types 4 hypersensitivity (Delayed type of hypersensitivity) [5].
When T-helper cells recognize foreign Ag on the surface of target cell in association with (or near) MHC class I. The TCR and CD4+ play role in recognition. Then Th cell will be activated and produce cytokines (especially IL-2 and IFN- γ). These cytokines will activate Tc CD8+ cells, MØ and NK cells. This activation will increase these cells ability for killing and became more effecter.
After T-cytotoxic cells and NK cells activation by Th cells, T-cytotoxic cells come into close contact with target cell; they will bind to the Ag by their specific Ag receptors. While NK cells will attach to Ag (on Target cell surface) by their non-specific receptors for Ag.
T-cytotoxic cells and NK cells will kill target cells by the following mechanisms [1, 2, 3, 4, 5]:
Direct contact killing: Production of perforin, which is a protein able to form pores in target cell membrane at the point of contact between Tc cell and target cell, lead to osmotic lysis of target cell.
Indirect killing: By secretion of a toxin protein in the space between the two cells, which causes fragmentation of target cell nuclear DNA, then the death of target cell by Apoptosis: the programmed cell death.
Antibody-dependent cellular cytotoxicity (ADCC) killing: it is specific mode of killing occurs when the parasites Ags have ability to induce both HMI and CMI, target cells will be coated with specific Abs formed after HMI against some parts of intracellular parasite like virus. These Abs will bring Tc and NK cells very close to the target cell by acting like a bridge because Tc and NK have receptors to the constant region of Ab. Then Tc and NK cells will be activated and kill the target cell by extracellular products (toxins and enzymes).
This type of CMI occurs when the foreign Ag persist for long time (e.g.
It is the first exposure to the Ag resulting of forming specific Abs and memory B-cells for HMI or T-cells and memory T-cells for CMI, the phases are, Figure 2 [1, 2, 3, 4, 5]:
Latent Phase: start after first time exposure to an Immunogen or after induction, include the followings
No Ab level increase (Steady titer).
Recognizing Ag as foreign after processing the Ag inside APC.
Cellular proliferation and differentiation.
Duration of this phase (period) is variable depending on many factors (Ag immunogenicity, Ag dose, Ag solubility, Ag route of immunization or exposure).
Logarithmic phase: starts when Ab titer begin to increase (active biosynthesis of Ab), last for 10-14 days till reach peak.
Steady phase: starts when the rates of both formation (synthesis) and catabolism are equal, then serum concentration of Ab is constant.
Decline phase: starts when the Ab titer starts to fall down due to increase Ab catabolism rate than synthesis.
Primary and secondary immune response.
Note: during early primary response, IgM class antibodies is predominant and first rise than IgG appears later [2, 5].
It is the second exposure to the same immunogen that induced the first immune response (after booster dose of vaccination) may be after weeks, months, or even years later, includes [1, 2, 3, 4, 5]:
Accelerated or fast appearance of Abs.
Shorter latent period.
Rapid rate of Ab synthesis.
Higher peak titer of Ab.
More presence of memory cells.
Dose of immunogen needed is lower than primary.
Predominant Ab Class is IgG.
Long standing steady phase, whereas Ab titer will stay high longer time.
Negative phase: occur between primary and secondary Immune response when immunogen second dose is small and/or there is pre-existing antibodies from the first immune response (primary), then immunogen will be all consumed in Ag-Ab complex formation and phagocytosed then removed with no induction to secondary immune response [5].
The bacterial antigens are the components or products of pathogens that are able to induce the immune defenses of the host to defend against, and to eliminate, the pathogen or disease. As with all
Somatic antigens are the O side chain of LPS; they are heat stable and alcohol resistant. Cross-absorption studies individualize a large number of antigenic factors, 67 of which are used for serological identification. O factors labeled with the same number are closely related.
Surface antigens, commonly in enteric bacteria (e.g.,
Flagellar antigens are heat-labile proteins. Mixing
Innate immunity barriers play a good role in defense against
Primary infections with
In the
The organisms usually multiply in intestinal lymphoid tissue and are excreted in stools. However, in the case of
The presence of increasingly large numbers of bacteria in the bloodstream (called bacteremia) is responsible for an increasingly high fever, rising in stages throughout the first week to 39/40/41°C and may last throughout the four to eight weeks of the disease, in untreated individuals. Other symptoms include constipation (initially), extreme fatigue, headache and joint pain. Further symptoms: leukopenia, bradycardia, splenic swelling, abdominal roseola, beginning in the third-week diarrhea, sometimes with intestinal bleeding due to ulceration of the Peyer’s patches and inflammation of the gallbladder, severe irritation and inflammation of the lining of the abdominal cavity, called peritonitis, which is frequently a fatal outcome of typhoid fever [5, 12].
From the mesenteric lymph nodes, viable bacteria and LPS (endotoxin) will be released into the bloodstream resulting in septicemia. Moreover the effect of LPS as pyrogenic toxin, it causes activation of the complement alternative pathway which ends with membrane attack complex MAC, and that will increase LPS levels in bloodstream due to breakage of more bacterial cells leading to more harmful pyrogenic effects. The fever rises to a high plateau, and the spleen and liver become enlarged. Rose spots or rash usually on the skin of the abdomen or chest may be seen in some cases. Another scientific fact, LPS can induce both T-Dependent and T-Independent specific immune response. Specific antibodies against
The complications of typhoid fever include liver and spleen enlargement (sometimes so extreme that the spleen ruptures), anemia (low red blood cell count due to blood loss from the intestinal bleeding), joint infections (especially frequent in patients with sickle cell anemia and immune system disorders), pneumonia (due to a superimposed infection, usually by
Due to that
Antigenic variation can occur due to that
Cellular mediated immunity is induced after APC formation, since
Cytokines of both Th1 and Th2 levels increase during Salmonellosis, Interlukins (IL-1, IL-2, IL-4, IL-6, IL-8, IL-9, IL-10, IL-13, IL-15, IL-17). Also Interferon-gamma (IFN-γ) play a great role during cellular immune response and its levels elevates in patients’ blood even after cure. Another important cytokine is Tumor necrosis factor-alpha (TNF-α), its levels raise upon infection start and stay elevated along the disease time [5, 12].
After manifest or subclinical infection, some individuals continue to harbor
In the case of carriers with gall stones, surgery may need to be performed to remove the gall bladder, because the
Despite of that some patients with
However, relapses may occur in 2–3 weeks after recovery despite specific antibodies titer rise. Secretory IgA antibodies may prevent attachment of
Some genetic factors can make person susceptible host for re-infection easier like persons with S/S hemoglobin (sickle cell disease) are susceptible to
The incidence of human disease decreases when the level of development of a country increases (like controlled water sewage systems, improve hygiene, pasteurization of milk and dairy products). Bad ways in having food like eating raw or undercooked egg can cause illness due to these bacteria called
Plasmid-borne antibiotic resistance is very frequent among and can be considered as a virulence factor upon ongoing infections.
Vaccination is very good health measure in eradication of
Early research produced two vaccines made from the entire (whole-cell) bacterium. The first one became available in the 1890s, the second in 1952. Both protected about 65% of recipients. However, the frequency and severity of the adverse effects they caused dissuaded many countries from using them. These shortcomings, combined with drug treatment failures, as a result of increasingly widespread resistance to antibiotic therapy [12, 13, 14].
Before the end of the 20th century, two new-generation typhoid vaccines had entered the scene. One, named (Ty21) and first licensed in 1983, is given in three to four oral doses and consists of a live but genetically modified
Meanwhile, third-generation typhoid vaccines are under trial. One is a Vi conjugate vaccine that protects about 85% of recipients, according to late-stage clinical trials, and appears to be effective in children under two years of age. A second candidate vaccine, is, like Ty21a, a live attenuated vaccine but, unlike Ty21a, can be given in a single oral dose [15].
Three types of typhoid vaccines are currently available for use nowadays:
Oral live-attenuated vaccine.
Heat-phenol-inactivated vaccine; killed bacterial vaccine.
The Vi capsular polysaccharide vaccine for intramuscular use.
A fourth vaccine, an acetone-inactivated parenteral vaccine, is currently available only to the armed forces. While Typhoid fever vaccinations for tourists and travelers to the endemic areas is best be done with the oral attenuated vaccine Virotif Ty 21a.
Despite of that; No typhoid vaccine is 100% effective and provide only short-term protection (sometimes for a few months), it is not a substitute for being careful and elevate hygiene [15].
Blindness due to AMD is of great concern to the ageing elderly population since the prevalence of the disease in Europe, in those aged 60 years and over has been estimated to be 27.7% with a projected increase in numbers from 67 to 77 million by 2050 [1]. Clinically, AMD is broadly divided into early, intermediate and late (or advanced) forms. Early AMD is characterised by the presence of large drusen and pigmentary abnormalities such as hypo- or hyperpigmentation of the fundus. Progression to the late form results in geographic atrophy of the RPE followed by photoreceptor degeneration, known as ‘dry’ AMD. The late phase is also associated with secondary complications of neovascular episodes (comprising 10-20% patients), these being designated as ‘wet’ AMD.
The wet form of the disease can lead to rapid visual loss and considerable efforts at intervention have resulted in anti-vascular endothelial growth factor (anti-VEGF) intra-vitreal injections with a considerable degree of success in managing the neovascularisation, but the underlying progression of the disease is not altered. Thus, the vast majority of AMD patients (falling in the ‘dry’ AMD category) still await the development of a suitable treatment modality that can either slow or arrest the progression of the disease [2, 3].
The pathophysiology of AMD is highly complex due to the diverse genetic associations and considerable gene-environmental interactions exacerbated by the additional association of dietary and cardiovascular risk factors [4, 5, 6]. Furthermore, all these factors are superimposed on the normal ageing changes in the visual unit making it very difficult to nominate specific targets for intervention. Since age is the highest risk factor for the development of AMD, an understanding of the inherent stresses in the visual system would allow us to predict the likely effect of additional risk factors providing a more targeted approach towards therapy.
Briefly, in the visual unit, the photoreceptor is the primary site of sustained damage producing highly toxic compounds that can trigger an inflammatory response. However, this damage is rapidly transferred to the RPE by the daily shedding of outer segment discs and phagocytosis. Since the RPE also operates in the same oxidative environment as the photoreceptor cell, the engulfed discs undergo further damage resulting in compromised lysosomal degradation. Non-degradable material, comprising mainly lipofuscin-related products is either packaged and stored in the RPE or extruded as membraneous debris onto Bruch’s membrane. With age, this debris accumulates in Bruch’s compromising its ability to transport nutrients, anti-oxidants, and vitamins essential for RPE and photoreceptor function. The toxic metabolites in Bruch’s are pro-inflammatory mediators and in the normal elderly, lead to a low-grade inflammatory response [7]. In advanced ageing associated with AMD, a chronic inflammatory response is precipitated leading to the death of RPE and photoreceptors.
For therapeutic intervention to be effective, the functional aspects of the RPE and Bruch’s membrane need to be restored. We will examine the compositional and functional alterations of ageing RPE and Bruch’s membrane, nominate suitable targets for intervention, and assess the potential for amphipathic saponin molecules to reverse these ageing changes as a potential therapy for dry AMD.
Photoreceptor physiology is dependent on the supportive roles provided by the RPE and Bruch’s membrane. Inherent stresses in these compartments lead to morphological and functional deterioration manifesting as ‘normal’ ageing changes but in the advanced ageing scenario of AMD culminate in the transition to pathology. The stresses within each compartment of the visual unit will be identified, providing therapeutic targets for intervention.
The photoreceptor is a highly specialised neuronal cell capable of detecting a single photon of light. Absorption of light by rhodopsin (R) present in the outer segment disc membranes leads to isomerization of the 11-cis retinal chromophore to its all-trans form (AT-RL), producing activated rhodopsin (R*). Amplification of the light signal begins by rapid lateral diffusion of R* over the disc membrane and interaction with many transducin molecules. Such high mobility of R* requires a very fluid membrane conferred by the high level of unsaturated docosahexanoic acid (DHA) in its membrane phospholipids. Further enzymatic amplification of the light signal by the guanalate cyclase-phosphodiesterase system leads to closure of sodium channels in the outer segment membrane resulting in hyperpolarisation of the cell and concomitant modulation of transmitter release. To meet the energy demands of these processes, the photoreceptor maintains the highest rates of oxidative metabolism of any cell in the body. Associated with this activity is the release of damaging oxygen radicals by the mitochondrial electron transport chain.
The presence of toxic retinoids, highly unsaturated fatty acids, high oxygen tension, high oxidative metabolism, and light is an explosive mixture for the generation of free-radical mediated damage. Since released AT-RL is highly toxic, it is rapidly reduced to all-trans retinol. However, AT-RL can react with phosphatidylethanolamine to form retinylidene-phosphatidylethanolamine (NRPE) [8]. NRPE can react with a second molecule of AT-RL to form a bis-retinoid. Further modifications produce a variety of all-trans retinal dimers including A2E, the auto-fluorescent fluorophore of lipofuscin [9]. These bis-retinoids can undergo photo-oxidation to form oxo-aldehydes which then react with proteins to form advanced glycation end-products (AGEs) that are triggers of inflammatory processes [10].
Peroxidation of polyunsaturated fatty acids such as DHA results in fragmentation of the molecule leading to a mixture of compounds that bind to proteins [11, 12]. Oxidation of DHA produces carboxy-ethyl-pyrrole (CEP)-protein adducts. Thus, oxidation of PUFAs results in lipid aggregates, lipid-protein complexes, protein cross-link formation and CEP-adducts. These CEP-adducts have been localised to the RPE and drusen and being strongly immunogenic, activate the immune system [13].
Some protection from oxidative damage is afforded by the impressive anti-oxidant machinery (vitamins C&E, macular pigments, and enzymes such as catalase, peroxidase, and superoxide dismutase) [14, 15]. However, this protection in photoreceptors is dependent on an adequate supply of anti-oxidants and essential metals for the enzymic system by the RPE and Bruch’s membrane. Despite these protective mechanisms, considerable damage is sustained by photoreceptors. Fortunately for the photoreceptor, this damage is confined to the outer segment discs and transferred to the RPE.
Therapeutic intervention to combat this damage has been considered resulting in the Age-Related Eye Disease Study (AREDS) vitamin and anti-oxidant supplements and their effectiveness will be discussed later.
The RPE operates in the same oxidative environment as the photoreceptor cell and therefore, the toxic reactions initiated in the outer segments will continue in the phagolysosome. Lysosomal enzymes hydrolyse the normal, undamaged protein and lipid components. recycling the base metabolites back to the photoreceptor cell. Damaged proteins, lipid-derived adducts, protein cross-links due to lipid-carbonyl attack, and aggregated lipid complexes that are no longer susceptible to lysosomal enzymes remain in the phago-lysosomal sac [16]. The lysosomal hydrolysis of bis-retinoids results in the formation of the primary age pigment, A2E. A2E and other bis-retinoids undergo further oxidation to produce a variety of toxic products that not only damage lysosomal enzymes but also damage the lysosomal membrane inhibiting the proton pumps with the subsequent increase in pH that will further diminish lysosomal enzyme activity [17].
Un-hydrolysed lipoprotein and aggregated protein complexes together with bis-retinoids are packaged and stored as the auto-fluorescent pigment lipofuscin in membrane enclosed sacs. Lipofuscin content of the RPE increases with age and can amount to nearly 20% of cytoplasmic volume in the elderly [18]. Increased oxidative stress is inferred from the accumulation of AGEs in both ageing RPE and Bruch’s membrane [19]. The RPE has a battery of anti-oxidants and a robust enzymic machinery to neutralise the oxidative stress and again, the components of the protective machinery are supplied by transport across Bruch’s membrane. However, the age-related accumulation of bis-retinoids and damaged proteins suggests that the anti-oxidant system is not effective in tackling this threat.
The primary functions of the RPE are (a) phagocytosis of shed outer segment discs and their degradation, (b) vectorial transport of nutrients, lipids, metals, vitamins and anti-oxidants, and the removal of waste products generated in the photoreceptor cell, and (c) fluid transport from the sub-retinal space to the choroid. The effect of the age on the various functional parameters of the RPE are poorly understood. One report has suggested that phagocytic activity is halved between the ages of 30 and 80 years [20]. Another important function of the RPE is the delivery of nutrients, anti-oxidants, vitamins, etc supplied by the choroidal circulation to the photoreceptor cell. Since the RPE is the site of the outer blood-retinal barrier, all metabolites must cross the interior of the cell to gain access to photoreceptors. Therefore, transport across the RPE is mediated by passive diffusion or facilitated by active and passive carriers in the membrane. Most active carriers utilise the sodium electro-chemical gradient generated primarily by mitochondrial respiration [21]. However, A2E generated in the RPE binds to cytochrome C of the electron transport chain impairing mitochondrial respiration and this is expected to impact on the effectiveness of active carrier transport [22, 23].
There is little information of the effect of age on the activity of ligand carriers of the RPE due largely to interference from the adjacent Bruch’s membrane. This is best illustrated with the transport of retinol (vitamin A). In elderly subjects and patients with early AMD, the recovery in dark-adaptation following a strong bleach is delayed [24, 25]. This delay is thought to be due to low levels of retinoids in the RPE and therefore slower transfer of 11-cis retinal to photoreceptors for regeneration of rhodopsin. Lowered levels of retinoids in the RPE could be due to lowered uptake by the RPE itself or diminished transport of retinol across Bruch’s membrane. The fact that there is improvement in dark-adaptation following vitamin A supplementation would suggest inefficient delivery across Bruch’s, rather than reduced uptake by the RPE as the contributary factor [26].
Fluid transport is another important function carried out by the RPE. Retinal fluids (originating from retinal capillary beds and retinal metabolism) are transported out by the RPE predominantly by an active process [27, 28]. The daily output of fluid from the RPE has been determined to be about 0.13 ± 0.11 μl/hour/mm2 and metabolic insufficiency in the RPE would lead to fluids accumulating on top of the RPE resulting in macular oedema and/or retinal detachment [29, 30].
Therapeutic intervention in support of the RPE would require effective delivery of anti-oxidants and strengthening of its metabolic capability so as to reduce the generation of toxic products and assist in their rapid removal.
Bruch’s membrane mediates the exchange of nutrients and waste products between the choroidal blood supply and the RPE. An age-related compromise in these functions will reduce the capacity to supply essential nutrients to the RPE and photoreceptor cells increasing the risk of damage in these compartments.
The most obvious morphological change in Bruch’s with age is increased thickness from about 1.5 μm in the young to 5.5 μm in the elderly [31]. This is due primarily to the deposition of normal and abnormal extracellular matrix (ECM) material. In the elderly, cross-linked and denatured (damaged) collagen accounts for nearly 50% of total collagen in Bruch’s membrane [32]. There is also an increase in oxidative and non-enzymic glycosylation of proteins and lipids leading to the accumulation of toxic advanced glycation end-products, AGEs [33]. The membrane also shows an exponential increase in the level of lipid-rich debris [34]. Most of this debris arises from inefficient phagocytic processing of damaged outer segment discs in the RPE that is then extruded onto Bruch’s membrane. This material then undergoes further oxidative modification with both the inherent matrix proteins and with passer-by constituents leading to further damage and deposition. Finally, the lipid components undergo free-energy driven aggregation leading to the accumulation of 100 nm diameter lipid-rich particles observed in the inner collagenous layer of Bruch’s membrane [35].
Thus, in addition to the toxic metabolites mentioned above, deposits in Bruch’s contain phospholipids, triglycerides, cholesterol, cholesterol esters, peroxidised lipids and apolipoproteins, immunoglobulins, amyloid, complement, and proteins specific to RPE function [36]. Heavy metal deposition has also been demonstrated that stabilises the debris in Bruch’s [37].
The above changes result in gross morphological alteration of ageing Bruch’s membrane that are expected to be detrimental to its transport functions (Figure 1).
Morphology of ageing Bruch’s membrane. With age, Bruch’s becomes thicker and contains a lot of debris rich in lipids, and abnormal matrix and non-matrix material. The increase in thickness alone will reduce the diffusional gradients for the transport of nutrients and waste products. Vertical bar denotes the thickness of Bruch’s membrane. ICL, inner collagenous layer, EL, elastin layer; OC, outer collagenous layer. Bar marker: 1 μm.
Mechanisms exist to counteract the deleterious changes described above for Bruch’s membrane. This involves the continuous synthesis and degradation of the extracellular matrix, the latter process being mediated by the matrix metalloproteinase (MMP) system [38]. Although this system performs well in the young, it deteriorates rapidly with age and more so in AMD [39].
Since Bruch’s membrane is crucial for the exchange of nutrients and waste products, a deficiency in its transport functions will increase the risk of damage in the RPE and photoreceptor compartments for the reasons outlined earlier. The extent to which the compositional alterations of ageing Bruch’s impact on its ability to remove fluids into the choroidal circulation, to supply adequate levels of essential nutrients, antioxidants, and vitamins to the RPE and photoreceptors, to maintain the rejuvenation potential of its membrane, and to modulate the occurrence of inflammatory responses will now be examined in both normal ageing and in the advanced ageing scenario of AMD.
The capacity for fluid transport across a membrane is designated by its hydraulic conductivity. As previously indicated, the daily output of fluid from the RPE and onto Bruch’s membrane is about 0.13 ± 0.11 μl/hour/mm2. To effectively transport this amount of fluid, Bruch’s needs to have a minimum hydraulic conductivity of 0.65 x10−10 m/s/Pa, and this level is referred to as the failure threshold [40, 41]. If hydraulic conductivity falls below this level, then fluid will accumulate on top of the membrane leading to a RPE detachment. Hydraulic conductivity of human Bruch’s has been determined in 56 donors spanning the age range 1-91 years (Figure 2, modified from reference [40]). Conductivity was shown to decline exponentially with age and in the semi-log plot, the transformation is shown as a straight line. The half-life of the decay process was 16 years, i.e., conductivity was halved for every 16 years of life. Excess capacity is present in the younger population but with age, there is a drift towards the failure threshold. Extrapolating the straight line shows that the shelf-life of human Bruch’s is about 123 years, but in the data of Figure 2, two of the normal donors have already reached the failure threshold. Bruch’s from AMD donors showed a faster rate of decline in hydraulic conductivity [40] and as such, complications of RPE detachment are observed in about 12-20% of AMD patients [42].
Semi-logarithmic plot to show the exponential decay in the hydraulic conductivity of human Bruch’s with age. (Modified from reference [
For an effective therapeutic intervention in AMD, the exponential decay line in Figure 2 needs to be elevated so as to avoid the failure threshold within the life-time of an individual.
Metabolites ranging from the simple sugars and amino acids to the much larger lipo-protein complexes are released from the fenestrated endothelium of the choriocapillaris vessels and traverse Bruch’s by passive diffusion. Most of the essential metabolites such as heavy metals, vitamins (including vitamin A), and lipids are transported bound to carrier proteins that generally have a hydrodynamic radius of about 3-12 nm.
To assess the effect of age on the diffusional status of human Bruch’s membrane, a FITC-albumin test probe was utilised that has a hydrodynamic radius of about 3.5 nm, similar to most carrier proteins. Diffusional experiments were conducted in standard Using chambers utilising isolated Bruch’s membrane preparations from 33 donors, age range 12-92 years. The diffusional status of Bruch’s membrane was observed to decrease exponentially with age, with a half-life of 18 years (Figure 3) [43]. Thus, over a human life-span, diffusional status was reduced by about 10-fold. We do not know the value of the failure threshold for diffusion across Bruch’s membrane. However, since most elderly subjects show delayed dark-adaptation due to inefficient transport of vitamin A, the albumin diffusion values of subjects aged 77-87 years (0.024 nmol/6 mm/hour) were taken as the failure threshold.
The effect of age on the diffusion of albumin across human Bruch’s. Semi-logarithmic plot showing the decay half-life to be 18 years.
Other in-vitro studies utilising serum proteins (MW 40-200 kDa) or FITC-dextran molecules (MW 21 kDA, radius 3.3 nm) have also shown a >10-fold reduction in diffusion capability over a human life-span [44, 45].
In AMD, the reduction in diffusional transport across Bruch’s membrane was much more severe compared to age-matched controls [45]. This reduction in transport is expected to impact on the nutritional and anti-oxidant support of both RPE and photoreceptor cells, increasing oxidative stress. Similarly, transport in the opposite direction i.e., removal of toxic waste products from Bruch’s membrane will also be diminished leading to greater oxidative modifications and generation of further toxic products.
As with hydraulic conductivity, for effective therapeutic intervention, the diffusional decay curves should be elevated away from the failure threshold.
Reduced diffusion within Bruch’s membrane will also affect the protective mechanisms that depend on rapid mobility such as the interactions of complement factor H (CFH) with its many ligands and activation of pro-MMP2 in the regeneration of Bruch’s. These aspects are described below.
The ECM of Bruch’s is continuously regenerated by coupled processes of synthesis and degradation. This ensures that damaged material is removed and replaced by new ECM components synthesised by the RPE, thereby maintaining the transport integrity of Bruch’s membrane. Since abnormal collagen accumulates in ageing Bruch’s (amounting to 50% of total collagen in the elderly), the regeneration process appears to be dysfunctional [32]. Little is known about the synthetic rate of ECM by the ageing RPE but the accumulation of damaged ECM components suggests problems with the degradation machinery.
Matrix degradation is mediated by a family of proteolytic enzymes called the matrix metalloproteinases (MMPs). These are synthesised in the RPE and released into Bruch’s membrane as latent pro-enzymes (pro-MMPs) that on activation, following the removal of a small inhibitory peptide, can degrade almost all components of the ECM [38, 46]. In Bruch’s, the major MMP species are pro-MMP2 and pro-MMP9, the former being the homeostatic enzyme in the system and the latter being the inducible form. Activation of pro-MMP2 occurs on the basolateral surface of the RPE by the initial formation of a binary complex between the membrane bound MMP-14 and the tissue inhibitor of MMPs, TIMP2. This then binds pro-MMP2 to form a tertiary complex that then results in the hydrolysis of the inhibitory peptide on pro-MMP2 by a second molecule of MMP-14, to release activated MMP-2 [47].
Thus, optimal pro-MMP2 activation requires adequate levels of pro-MMP2 and TIMP2 and good mobility of these two components within the matrix of Bruch’s to interact with the MMP-14 enzyme on the RPE basal membrane. The age-related reduction in diffusion within Bruch’s is expected to compromise this activation potential (Figure 3). Furthermore, pro-MMP2 covalently binds pro-MMP9 to form the high molecular weight complex termed HMW2, reducing its level for the activation process [48]. A polymorphism in the microsatellite region of the MMP9 gene (present in most AMD patients) results in elevated levels of pro-MMP9 in both plasma and Bruch’s membrane, increasing the potential for further sequestration of pro-MMP2 from the activation step [39, 49, 50, 51].
The gross alterations of ageing Bruch’s membrane together with the reduction in diffusional competence are expected to hamper the mobility of pro-MMP2 and TIMP2, diminishing the activation of this MMP. Thus, levels of activated MMP2 decrease with age, and in AMD, the level was reduced by 50% compared to age-matched controls [39]. It should also be noted that activated MMP2 may not be able to diffuse adequately to interact with its substrate and in the gross morphology of Figure 1, may be trapped within the membrane. The decreased turnover of Bruch’s leads to the deleterious morphological and functional changes described earlier culminating in diminished support of RPE and photoreceptors.
Many of the toxic products produced in the RPE and present in Bruch’s (as outlined earlier), including A2E, bis-retinoids, malonaldehyde, carbonyl lipids, C-reactive protein, etc., are capable of activating the complement system [52]. Thus, in elderly subjects, the exponential increase in A2E in Bruch’s may be associated with a low-grade complement activation [53, 54]. A low-grade inflammatory response may be beneficial for eliminating toxic metabolites present in Bruch’s or in drusen and may serve to prevent the transition from normal ageing to pathology. Thus, the presence of the membrane attack complex and other complement factors in drusen and inter-capillary columns may allow their removal by macrophages [55].
However, indiscriminate activation of the complement system can lead to a chronic inflammatory response damaging RPE and photoreceptor cells. CFH (a 155 kDa glycoprotein) plays important roles in modulating the activation of the complement cascade. Firstly, it can bind to the toxic entities to prevent complement activation and secondly, by binding to the C3b complement component, block the progression of the cascade [56, 57, 58].
Levels of CFH in Bruch’s are maintained by synthesis in the RPE and binding to glycosaminoglycans in the membrane, and delivery of plasma-derived CFH. With age, and under oxidative stress, the production of CFH by the RPE is reduced [59, 60]. Similarly, the nearly 10-fold decrease in diffusion across elderly Bruch’s is expected to compromise delivery from the blood. Furthermore, in the presence of inflammatory activity in Bruch’s, CFH is nitrated [61]. This nitrated CFH does not bind lipid peroxidation products nor C3b, diminishing its protective ability. Also, plasma levels of nitrated CFH are elevated in AMD patients and this may contribute to AMD progression [61].
A polymorphism in the CFH gene (Tyr402His) has been detected in about 50% of AMD patients [62, 63]. This mutated CFH shows diminished binding to toxic ligands such as malondialdehyde and C-reactive protein, and thus becomes ineffective in modulating the inflammatory response [64, 65, 66]. Mutated CFH also shows poor binding to heparin sulphate in Bruch’s and hence its enhanced presence in Bruch’s is compromised [67].
Ageing changes in Bruch’s and the RPE therefore compromise the protective effects of CFH and in the aged AMD patient may exacerbate the inflammatory response leading to the death of RPE and photoreceptors.
Oxidative damage in the RPE and Bruch’s membrane is the primary driver of ageing changes in the normal elderly and more so in patients with AMD. These ageing changes diminish the supply of key anti-oxidants and vitamins required to combat oxidative stress and therefore a vicious cycle is set-up that leads to the degenerative changes in AMD.
Anti-oxidant and vitamin supplement regimes have been devised as a possible interventionist measure to reduce oxidative stress and hopefully slow the progression of the disease. Thus, the AREDS dietary supplementation cocktail was devised (vitamin C (500 mg), vitamin E (400 IU), beta-carotene (15 mg), zinc oxide (80 mg), and cupric oxide (2 mg)) and initial results showed it to be effective in reducing the risk of visual loss [68, 69]. The supplement was further modified (as the AREDS 2 formulation) by removing beta-carotene and adding lutein (10 mg) and zeaxanthine (2 mg) but did not confer any additional benefits [70].
Despite the wide use of AREDS supplements for over 10 years, controversy remains as to its usefulness since it does not prevent legal blindness in advanced AMD [71]. It has been pointed out that the earlier reported decrease in progression was related to the occurrence of neovascularisation rather than slowing the progression of dry AMD [72].
Given the fact that the diffusion of metabolites across Bruch’s membrane is reduced by nearly 10-fold in the elderly, and perhaps more so in AMD, one must question the likely effectiveness of such dietary supplementation. The major problem with supplementation therapies is that they do not address transport in the opposite direction across Bruch’s membrane i.e., the removal of toxic metabolites that are the likely triggers of neovascular and inflammatory episodes.
For effective therapeutic intervention, it would be ideal to improve the bi-directional transport pathways across Bruch’s membrane, to improve nutritional and anti-oxidant delivery and to remove toxic waste products. This would require the destabilisation and dispersal of the lipid-rich debris and the removal of normal and damaged proteinaceous deposits. Such a strategy would also release trapped activated MMP enzymes that could participate in hydrolysing the altered collagenous components. The expected improvement in intra-membrane mobility would favour greater activation of pro-MMP2, kick-starting the normal rejuvenation machinery in Bruch’s membrane. Therapeutic success would be realised if the transport decay curves shown in Figures 2 and 3 could be elevated so that they no longer crossed the failure threshold within the lifetime of an individual. The potential implementation of such a strategy using saponin molecules is discussed next.
Saponins are amphipathic molecules that have hydrophobic and hydrophilic domains that can partition into lipoidal deposits and assist dispersal [73, 74, 75, 76]. Saponins extracted from the ginseng plant (Panax ginseng CA Meyer) have a 4-membered triterpenoid ring and are often referred to as ginsenosides or steroidal glycosides because of the structural similarity to the cholesterol molecule (Figure 4A). The type and number of sugar units attached at sites R1, R2, and R3 gives rise to a myriad of species and over 30 have been structurally characterised. Saponins extracted from the roots of the ginseng plant were separated on Silica Gel thin-layer-chromatography (TLC) plates using a solvent mixture comprising chloroform: methanol: acetic acid: water (50:30:8:3 v/v) and colour developed by spraying with 20% sulphuric acid in methanol and heating to 100 oC for 5 minutes (Figure 4B). Since the separation was dependent on the degree of hydrophilic/hydrophobic properties, each spot on the chromatogram represents a collection of several species.
Saponins extracted from the ginseng plant. (A) Structural similarity of saponins to the cholesterol molecule. Sugar attachment sites R1, R2, and R3 lead to the diversity of saponin species. (B) TLC of extracted saponins. G1 to G11 denote the major spots, each spot comprising several species.
These saponin molecules not only bind to various lipid classes, they also display transition metal chelating properties [77, 78]. Thus, saponins can chelate the heavy metal deposits in Bruch’s membrane and therefore assist in destabilising the lipid aggregates.
The potential for saponin mediated dispersal of lipids was assessed in both isolated deposits and intact human Bruch’s membrane. Bruch’s from four donor eyes (ages 50 and 82 years) was homogenised in Tris-buffer and spun to obtain pellets containing the deposits. Pellets were resuspended in Tris buffer containing ginseng-derived saponins in the range 0-1.2 mg/ml and incubated for 12 hours at 37oC. Samples were then spun and any lipids released into the supernatants extracted with chloroform: methanol (2:1 v/v). Lipids were then separated on Silica Gel thin-layer chromatography (TLC) plates using solvent system #1, chloroform: methanol: acetic acid: water (50:30:8:3 v/v) and solvent system #2, heptane: diethyl ether: acetic acid (70:30:2 v/v). Lipid spots were visualised by staining with amido-black 10B stain and following densitometry, levels quantified with reference to standard curves. Saponins were observed to rapidly release various lipid classes from the deposits in a dose dependent manner (Figure 5).
Saponin-mediated release of lipids from deposits extracted from Bruch’s membrane. Extracted deposits were incubated with saponins in the range 0-1.2 mg/ml for 12 hours, spun, and lipids present in the supernatant quantified. Saponins released cholesterol, cholesterol esters, phospholipids, and triglycerides in a dose-dependent manner. Data is given as Mean ± SD.
In addition, 14 Bruch’s preparations were obtained from 4 human donors (age range 64-75 years) and mounted in Ussing chambers. All chambers were perfused with Tris buffer to remove loosely attached debris and then half the chambers were incubated with Tris buffer and the other half with Tris buffer containing 4.6 mg/ml saponins for 12 hours at 37oC. Chambers were rinsed in Tris buffer and the content of the various lipid classes present in Bruch’s membrane quantified as detailed above. The content of lipids in the samples incubated with saponins was significantly reduced compared to controls (Figure 6). Thus, saponin molecules are able to solubilise and disperse the lipid deposits in Bruch’s membrane.
Saponin-mediated release of lipids from intact human Bruch’s membrane. The level of the major lipid classes was reduced following saponin treatment. Data is given as Mean ± SD.
Soluble proteins that are either damaged due to chemical modification or denatured tend to unfold exposing the hydrophobic regions to the aqueous environment. This leads to aggregation and deposition within Bruch’s membrane. The possibility that saponins could interact with these ‘amphipathic’ structures to de-segregate and solubilise them was also assessed. Bruch’s membrane from four human donors (age range 49-77 years) was mounted in 8 Ussing chambers and perfused with Tris buffer. The perfusate was collected every 5 hours and the protein content determined. As indicated in Figure 7, perfusion with Tris buffer resulted in slow release of loosely adherent proteins up to the fourth perfusion period (20 hours). In four chambers, the perfusion fluid was then switched to one containing 167 μg/ml of saponin Rb1. This resulted in the rapid and copious release of further, presumably trapped proteins from the membrane (Figure 7). The possibility that trapped MMP enzymes may also have been released is assessed below.
Solubilisation and release of trapped proteins from Bruch’s membrane. On perfusion with saline, loosely adherent proteins are released slowly. When there was no further release (after period 4), the perfusion medium was switched to one containing Rb1, resulting in the copious release of trapped proteins. TBS-Tris buffered saline; Rb1-Ginsenoside Rb1. Data as Mean ± SD.
MMPs are detected by the technique of gelatine zymography. This is standard electrophoresis but with the gel containing 1% gelatine, a substrate for MMPs. Samples are electrophoresed with the MMPs migrating according to molecular weight, the smaller ones running fastest. Following electrophoresis, the gel is incubated in Tris-buffer (containing calcium) for a period of 18-24 hours to allow the MMP enzymes to digest the gelatine in their locality. The gel is then stained with Coomassie Blue and after de-staining, regions containing gelatine stain blue but where the gelatine has been hydrolysed (by MMP enzymes), the region is colourless. Reversing the grey-scale of the gel image shows the MMP regions as dark bands and the identity of the MMP is confirmed from its molecular weight.
To assess the likely effect of saponins on release of trapped MMP enzymes, Bruch’s membrane from donors aged 49-71 years was mounted in Ussing chambers and perfused for a period of 12 hours to remove plasma-derived and loosely bound MMP species. Half the chambers were then perfused with Tris buffer for three periods of 3 hours each and the remaining half with saponins at a level of 4.6 mg/ml. After each period, the perfusate was collected and examined by zymography. Incubation with Tris did not show the release of any MMP species (Figure 8A). However, perfusion with saponins showed a trace release of MMPs in the first period followed by greater release in the two subsequent periods (Figure 8B). The release profiles showed the presence of activated MMP9 and MMP2. If this release occurred in vivo, it would kick start the MMP degradation machinery, rejuvenating the membrane.
Saponin-mediated release of trapped MMP enzymes. (A) Perfusion with Tris buffer did not release any MMP species from Bruch’s membrane. (B) Saponin perfusion released trapped MMP species and in particular activated forms of MMPs 2&9. FCS-foetal calf serum standard. P- pro-MMPs; A-activated MMPs. From reference [
Saponins have been shown to disperse and release lipid deposits, protein aggregates and trapped active MMP enzymes as described earlier. This is akin to reversing the ageing process of Bruch’s membrane. The likely impact of these changes on the functional properties of Bruch’s has also been assessed by monitoring hydraulic conductivity and diffusional status.
Hydraulic conductivity measurements were undertaken in 23 preparations of Bruch’s membrane from donors over the age range 13-90 years. After determining basal hydraulic conductivity, samples were incubated with saponins (4.6 mg/ml) for 24 hours at 37oC. After a thorough rinse in Tris buffer, the hydraulic conductivity was re-assessed. Treatment with saponins improved the hydraulic conductivity about 2-fold (p < 0.0001) elevating the exponential decay curves upwards, away from the failure threshold (Figure 9).
Effect of saponins on the hydraulic conductivity of human Bruch’s membrane. Saponins improved the hydraulic conductivity by 2-fold across the age range examined (p<0.0001). Decay curves were elevated so that failure threshold was met 19 years later. Modified from reference [
The curves were shifted by 19 years, reversing the ageing decline in fluid transport. Such a shift means that the curves will meet the failure threshold outside the normal human life span. In AMD, this improvement will reduce the risk of pigment epithelial detachments that normally affects 12-20% of these patients. These results were obtained after a single exposure to the saponins. A second subsequent exposure to the saponins (1.15 mg/ml) further increased the improvement in hydraulic conductivity indicative of potential to further elevate the decay curves (Figure 10). This is the likely result of continued removal of lipid and protein debris and release of MMP enzymes.
Effect of repeated saponin exposure on the hydraulic conductivity of Bruch’s membrane. Initial exposure to saponins improved hydraulic conductivity by over 2-fold. This was further increased by a second exposure.
Diffusional status of Bruch’s was assessed by following the transport of an FITC-labelled albumin test molecule (MW 65 kDa, hydrodynamic radius 3.5 nm) across the membrane, at a concentration gradient of 0.1 mM. Bruch’s was obtained from 21 human donors (age range 12-92 years) and the basal diffusion rate was first determined. After a thorough wash in Tris buffer for 3 hours to remove all traces of albumin, the samples were incubated with saponin solution (4.6 mg/ml) for 24 hours at 37oC. Following a rinse with Tris buffer, the diffusional status was re-assessed. Exposure to saponins improved the diffusional status of Bruch’s membrane by 2-fold (p<0.0001) shifting the ageing decay lines upwards, effectively reversing the ageing process by ~15 years (Figure 11).
Effect of saponins on the diffusional status of Bruch’s membrane. Saponin incubation improved diffusion (p<0.0001) elevating the decay curves upwards so that they reached failure thresholds 15 years later. Modified from reference [
Saponin mediated improvement in diffusion within Bruch’s membrane would allow greater delivery of nutrients, vitamins and anti-oxidants to both the RPE and photoreceptor cells. Reduction in oxidative stress would result in reduced production of A2E and other bis-retinoids minimising the generation of pro-inflammatory mediators. Similarly, the removal of toxic waste products from Bruch’s would be facilitated. This would also improve the mobility of TIMP2 and proMMP2 allowing greater activation of the MMP species and thus improved turnover of Bruch’s membrane. Furthermore, improved mobility of CFH would minimise the risk of inflammatory involvement. In AMD, these changes are expected to slow the degenerative phase of the disease.
In-vitro work, using donor human Bruch’s preparations has demonstrated the potential for saponin molecules to disperse and remove lipoidal and proteinaceous debris, releasing trapped activated MMP enzymes that can then mediate the normal degradation processes essential for rejuvenation of the membrane. Associated with these changes was a significant improvement in the bi-directional transport properties of Bruch’s membrane. In vivo, improved transport would considerably augment the delivery of protective anti-oxidants and related nutrients to the RPE and photoreceptor cells and more importantly stimulate the removal of toxic products that underlie the progression of the disease.
Saponins constitute a large mixture of amphipathic molecules, the diversity being due to the varied nature of sugar residues at the several attachment sites on the aglycone ring structure. Individual species, such as ginsenoside Rb1 or Compound K have been shown to improve the functional status of Bruch’s membrane preparations [43]. These compounds could be administered by intra-ocular injection as a potential therapy. But being very difficult to synthesise, reliance would have to be on isolation and purification from natural sources and therefore not likely to be cost-effective.
The alternative would be to utilise a varied mixture of saponins and this has many advantages. Firstly, they can be administered orally and in the Far East have been used as nutritional supplements for centuries. Secondly, and most importantly, a mixture can target a broad spectrum of substrates that are normally encountered in the deposits in Bruch’s membrane. From the pharmacokinetic data currently available for saponins, and our in-vitro dose response curves, calculations suggest that a 200 mg dose of an appropriate saponin mixture, taken twice daily, should significantly improve the transport characteristics of Bruch’s membrane over a period of 4-6 months. Work is in progress to develop a clinical protocol for assessing the usefulness of saponin-mediated therapy for dry AMD.
Unlike other therapeutic interventions in AMD where outcome has been judged by following the progression of the disease over a period of 2-5 years, the saponin intervention can be assessed at 4-6 months using dark-adaptation kinetics. Since the saponin intervention aims at improving the transport systems in Bruch’s membrane, the increased delivery of vitamin A would supplement the retinoid stores in the RPE allowing faster delivery of 11-cis retinal to the photoreceptor, and hence faster dark adaptation would be expected as proof of principle of saponin intervention.
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All published Book Chapters are licensed under a Creative Commons Attribution 3.0 Unported License. Monographs are licensed under the Creative Commons Attribution-NonCommercial 4.0 International (CC BY-NC 4.0) license granted to all others. Our Copyright Policy aims to guarantee that original material is published while at the same time giving significant freedom to our Authors. IntechOpen upholds a flexible Copyright Policy meaning that there is no copyright transfer to the publisher and Authors hold exclusive copyright to their work.
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He was elected a Yangtze River Scholars Distinguished Professor in 2013, a member of the International Statistical Institute (ISI) in 2016, a member of the board of the International Chinese Statistical Association (ICSA) in 2018, and a fellow of the Institute of Mathematical Statistics (IMS) in 2021. He received the ICSA Outstanding Service Award in 2018 and the National Science Foundation for Distinguished Young Scholars of China in 2012. He serves as a member of the editorial board of Statistics and Its Interface and Journal of Systems Science and Complexity. He is also a field editor for Communications in Mathematics and Statistics. His research interests include biostatistics, empirical likelihood, missing data analysis, variable selection, high-dimensional data analysis, Bayesian statistics, and data science. He has published more than 190 research papers and authored five books.",institutionString:"Yunnan University",institution:{name:"Yunnan University",country:{name:"China"}}},{id:"1177",title:"Prof.",name:"António",middleName:"J. R.",surname:"José Ribeiro Neves",slug:"antonio-jose-ribeiro-neves",fullName:"António José Ribeiro Neves",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/1177/images/system/1177.jpg",biography:"Prof. António J. R. Neves received a Ph.D. in Electrical Engineering from the University of Aveiro, Portugal, in 2007. Since 2002, he has been a researcher at the Institute of Electronics and Informatics Engineering of Aveiro. Since 2007, he has been an assistant professor in the Department of Electronics, Telecommunications, and Informatics, University of Aveiro. He is the director of the undergraduate course on Electrical and Computers Engineering and the vice-director of the master’s degree in Electronics and Telecommunications Engineering. He is an IEEE Senior Member and a member of several other research organizations worldwide. His main research interests are computer vision, intelligent systems, robotics, and image and video processing. He has participated in or coordinated several research projects and received more than thirty-five awards. He has 161 publications to his credit, including books, book chapters, journal articles, and conference papers. He has vast experience as a reviewer of several journals and conferences. As a professor, Dr. Neves has supervised several Ph.D. and master’s students and was involved in more than twenty-five different courses.",institutionString:null,institution:{name:"University of Aveiro",country:{name:"Portugal"}}},{id:"11317",title:"Dr.",name:"Francisco",middleName:null,surname:"Javier Gallegos-Funes",slug:"francisco-javier-gallegos-funes",fullName:"Francisco Javier Gallegos-Funes",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/11317/images/system/11317.png",biography:"Francisco J. Gallegos-Funes received his Ph.D. in Communications and Electronics from the Instituto Politécnico Nacional de México (National Polytechnic Institute of Mexico) in 2003. He is currently an associate professor in the Escuela Superior de Ingeniería Mecánica y Eléctrica (Mechanical and Electrical Engineering Higher School) at the same institute. His areas of scientific interest are signal and image processing, filtering, steganography, segmentation, pattern recognition, biomedical signal processing, sensors, and real-time applications.",institutionString:"Instituto Politécnico Nacional",institution:{name:"Instituto Politécnico Nacional",country:{name:"Mexico"}}},{id:"428449",title:"Dr.",name:"Ronaldo",middleName:null,surname:"Ferreira",slug:"ronaldo-ferreira",fullName:"Ronaldo Ferreira",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/428449/images/21449_n.png",biography:null,institutionString:null,institution:{name:"University of Aveiro",country:{name:"Portugal"}}},{id:"165328",title:"Dr.",name:"Vahid",middleName:null,surname:"Asadpour",slug:"vahid-asadpour",fullName:"Vahid Asadpour",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/165328/images/system/165328.jpg",biography:"Vahid Asadpour, MS, Ph.D., is currently with the Department of Research and Evaluation, Kaiser Permanente Southern California. He has both an MS and Ph.D. in Biomedical Engineering. He was previously a research scientist at the University of California Los Angeles (UCLA) and visiting professor and researcher at the University of North Dakota. He is currently working in artificial intelligence and its applications in medical signal processing. In addition, he is using digital signal processing in medical imaging and speech processing. Dr. Asadpour has developed brain-computer interfacing algorithms and has published books, book chapters, and several journal and conference papers in this field and other areas of intelligent signal processing. He has also designed medical devices, including a laser Doppler monitoring system.",institutionString:"Kaiser Permanente Southern California",institution:null},{id:"169608",title:"Prof.",name:"Marian",middleName:null,surname:"Găiceanu",slug:"marian-gaiceanu",fullName:"Marian Găiceanu",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/169608/images/system/169608.png",biography:"Prof. Dr. Marian Gaiceanu graduated from the Naval and Electrical Engineering Faculty, Dunarea de Jos University of Galati, Romania, in 1997. He received a Ph.D. (Magna Cum Laude) in Electrical Engineering in 2002. Since 2017, Dr. Gaiceanu has been a Ph.D. supervisor for students in Electrical Engineering. He has been employed at Dunarea de Jos University of Galati since 1996, where he is currently a professor. Dr. Gaiceanu is a member of the National Council for Attesting Titles, Diplomas and Certificates, an expert of the Executive Agency for Higher Education, Research Funding, and a member of the Senate of the Dunarea de Jos University of Galati. He has been the head of the Integrated Energy Conversion Systems and Advanced Control of Complex Processes Research Center, Romania, since 2016. He has conducted several projects in power converter systems for electrical drives, power quality, PEM and SOFC fuel cell power converters for utilities, electric vehicles, and marine applications with the Department of Regulation and Control, SIEI S.pA. (2002–2004) and the Polytechnic University of Turin, Italy (2002–2004, 2006–2007). He is a member of the Institute of Electrical and Electronics Engineers (IEEE) and cofounder-member of the IEEE Power Electronics Romanian Chapter. He is a guest editor at Energies and an academic book editor for IntechOpen. He is also a member of the editorial boards of the Journal of Electrical Engineering, Electronics, Control and Computer Science and Sustainability. Dr. Gaiceanu has been General Chairman of the IEEE International Symposium on Electrical and Electronics Engineering in the last six editions.",institutionString:'"Dunarea de Jos" University of Galati',institution:{name:'"Dunarea de Jos" University of Galati',country:{name:"Romania"}}},{id:"4519",title:"Prof.",name:"Jaydip",middleName:null,surname:"Sen",slug:"jaydip-sen",fullName:"Jaydip Sen",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/4519/images/system/4519.jpeg",biography:"Jaydip Sen is associated with Praxis Business School, Kolkata, India, as a professor in the Department of Data Science. His research areas include security and privacy issues in computing and communication, intrusion detection systems, machine learning, deep learning, and artificial intelligence in the financial domain. He has more than 200 publications in reputed international journals, refereed conference proceedings, and 20 book chapters in books published by internationally renowned publishing houses, such as Springer, CRC press, IGI Global, etc. Currently, he is serving on the editorial board of the prestigious journal Frontiers in Communications and Networks and in the technical program committees of a number of high-ranked international conferences organized by the IEEE, USA, and the ACM, USA. He has been listed among the top 2% of scientists in the world for the last three consecutive years, 2019 to 2021 as per studies conducted by the Stanford University, USA.",institutionString:"Praxis Business School",institution:null},{id:"320071",title:"Dr.",name:"Sidra",middleName:null,surname:"Mehtab",slug:"sidra-mehtab",fullName:"Sidra Mehtab",position:null,profilePictureURL:"https://s3.us-east-1.amazonaws.com/intech-files/0033Y00002v6KHoQAM/Profile_Picture_1584512086360",biography:"Sidra Mehtab has completed her BS with honors in Physics from Calcutta University, India in 2018. She has done MS in Data Science and Analytics from Maulana Abul Kalam Azad University of Technology (MAKAUT), Kolkata, India in 2020. Her research areas include Econometrics, Time Series Analysis, Machine Learning, Deep Learning, Artificial Intelligence, and Computer and Network Security with a particular focus on Cyber Security Analytics. Ms. Mehtab has published seven papers in international conferences and one of her papers has been accepted for publication in a reputable international journal. She has won the best paper awards in two prestigious international conferences – BAICONF 2019, and ICADCML 2021, organized in the Indian Institute of Management, Bangalore, India in December 2019, and SOA University, Bhubaneswar, India in January 2021. Besides, Ms. Mehtab has also published two book chapters in two books. Seven of her book chapters will be published in a volume shortly in 2021 by Cambridge Scholars’ Press, UK. Currently, she is working as the joint editor of two edited volumes on Time Series Analysis and Forecasting to be published in the first half of 2021 by an international house. Currently, she is working as a Data Scientist with an MNC in Delhi, India.",institutionString:"NSHM College of Management and Technology",institution:{name:"Association for Computing Machinery",country:{name:"United States of America"}}},{id:"226240",title:"Dr.",name:"Andri Irfan",middleName:null,surname:"Rifai",slug:"andri-irfan-rifai",fullName:"Andri Irfan Rifai",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/226240/images/7412_n.jpg",biography:"Andri IRFAN is a Senior Lecturer of Civil Engineering and Planning. He completed the PhD at the Universitas Indonesia & Universidade do Minho with Sandwich Program Scholarship from the Directorate General of Higher Education and LPDP scholarship. He has been teaching for more than 19 years and much active to applied his knowledge in the project construction in Indonesia. His research interest ranges from pavement management system to advanced data mining techniques for transportation engineering. He has published more than 50 papers in journals and 2 books.",institutionString:null,institution:{name:"Universitas Internasional Batam",country:{name:"Indonesia"}}},{id:"314576",title:"Dr.",name:"Ibai",middleName:null,surname:"Laña",slug:"ibai-lana",fullName:"Ibai Laña",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/314576/images/system/314576.jpg",biography:"Dr. Ibai Laña works at TECNALIA as a data analyst. He received his Ph.D. in Artificial Intelligence from the University of the Basque Country (UPV/EHU), Spain, in 2018. He is currently a senior researcher at TECNALIA. His research interests fall within the intersection of intelligent transportation systems, machine learning, traffic data analysis, and data science. He has dealt with urban traffic forecasting problems, applying machine learning models and evolutionary algorithms. He has experience in origin-destination matrix estimation or point of interest and trajectory detection. Working with large volumes of data has given him a good command of big data processing tools and NoSQL databases. He has also been a visiting scholar at the Knowledge Engineering and Discovery Research Institute, Auckland University of Technology.",institutionString:"TECNALIA Research & Innovation",institution:{name:"Tecnalia",country:{name:"Spain"}}},{id:"314575",title:"Dr.",name:"Jesus",middleName:null,surname:"L. Lobo",slug:"jesus-l.-lobo",fullName:"Jesus L. Lobo",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/314575/images/system/314575.png",biography:"Dr. Jesús López is currently based in Bilbao (Spain) working at TECNALIA as Artificial Intelligence Research Scientist. In most cases, a project idea or a new research line needs to be investigated to see if it is good enough to take into production or to focus on it. That is exactly what he does, diving into Machine Learning algorithms and technologies to help TECNALIA to decide whether something is great in theory or will actually impact on the product or processes of its projects. So, he is expert at framing experiments, developing hypotheses, and proving whether they’re true or not, in order to investigate fundamental problems with a longer time horizon. He is also able to design and develop PoCs and system prototypes in simulation. He has participated in several national and internacional R&D projects.\n\nAs another relevant part of his everyday research work, he usually publishes his findings in reputed scientific refereed journals and international conferences, occasionally acting as reviewer and Programme Commitee member. Concretely, since 2018 he has published 9 JCR (8 Q1) journal papers, 9 conference papers (e.g. ECML PKDD 2021), and he has co-edited a book. He is also active in popular science writing data science stories for reputed blogs (KDNuggets, TowardsDataScience, Naukas). Besides, he has recently embarked on mentoring programmes as mentor, and has also worked as data science trainer.",institutionString:"TECNALIA Research & Innovation",institution:{name:"Tecnalia",country:{name:"Spain"}}},{id:"103779",title:"Prof.",name:"Yalcin",middleName:null,surname:"Isler",slug:"yalcin-isler",fullName:"Yalcin Isler",position:null,profilePictureURL:"https://s3.us-east-1.amazonaws.com/intech-files/0030O00002bRyQ8QAK/Profile_Picture_1628834958734",biography:"Yalcin Isler (1971 - Burdur / Turkey) received the B.Sc. degree in the Department of Electrical and Electronics Engineering from Anadolu University, Eskisehir, Turkey, in 1993, the M.Sc. degree from the Department of Electronics and Communication Engineering, Suleyman Demirel University, Isparta, Turkey, in 1996, the Ph.D. degree from the Department of Electrical and Electronics Engineering, Dokuz Eylul University, Izmir, Turkey, in 2009, and the Competence of Associate Professorship from the Turkish Interuniversity Council in 2019.\n\nHe was Lecturer at Burdur Vocational School in Suleyman Demirel University (1993-2000, Burdur / Turkey), Software Engineer (2000-2002, Izmir / Turkey), Research Assistant in Bulent Ecevit University (2002-2003, Zonguldak / Turkey), Research Assistant in Dokuz Eylul University (2003-2010, Izmir / Turkey), Assistant Professor at the Department of Electrical and Electronics Engineering in Bulent Ecevit University (2010-2012, Zonguldak / Turkey), Assistant Professor at the Department of Biomedical Engineering in Izmir Katip Celebi University (2012-2019, Izmir / Turkey). He is an Associate Professor at the Department of Biomedical Engineering at Izmir Katip Celebi University, Izmir / Turkey, since 2019. In addition to academics, he has also founded Islerya Medical and Information Technologies Company, Izmir / Turkey, since 2017.\n\nHis main research interests cover biomedical signal processing, pattern recognition, medical device design, programming, and embedded systems. He has many scientific papers and participated in several projects in these study fields. He was an IEEE Student Member (2009-2011) and IEEE Member (2011-2014) and has been IEEE Senior Member since 2014.",institutionString:null,institution:{name:"Izmir Kâtip Çelebi University",country:{name:"Turkey"}}},{id:"339677",title:"Dr.",name:"Mrinmoy",middleName:null,surname:"Roy",slug:"mrinmoy-roy",fullName:"Mrinmoy Roy",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/339677/images/16768_n.jpg",biography:"An accomplished Sales & Marketing professional with 12 years of cross-functional experience in well-known organisations such as CIPLA, LUPIN, GLENMARK, ASTRAZENECA across different segment of Sales & Marketing, International Business, Institutional Business, Product Management, Strategic Marketing of HIV, Oncology, Derma, Respiratory, Anti-Diabetic, Nutraceutical & Stomatological Product Portfolio and Generic as well as Chronic Critical Care Portfolio. A First Class MBA in International Business & Strategic Marketing, B.Pharm, D.Pharm, Google Certified Digital Marketing Professional. Qualified PhD Candidate in Operations and Management with special focus on Artificial Intelligence and Machine Learning adoption, analysis and use in Healthcare, Hospital & Pharma Domain. Seasoned with diverse therapy area of Pharmaceutical Sales & Marketing ranging from generating revenue through generating prescriptions, launching new products, and making them big brands with continuous strategy execution at the Physician and Patients level. Moved from Sales to Marketing and Business Development for 3.5 years in South East Asian Market operating from Manila, Philippines. Came back to India and handled and developed Brands such as Gluconorm, Lupisulin, Supracal, Absolut Woman, Hemozink, Fabiflu (For COVID 19), and many more. In my previous assignment I used to develop and execute strategies on Sales & Marketing, Commercialization & Business Development for Institution and Corporate Hospital Business portfolio of Oncology Therapy Area for AstraZeneca Pharma India Ltd. Being a Research Scholar and Student of ‘Operations Research & Management: Artificial Intelligence’ I published several pioneer research papers and book chapters on the same in Internationally reputed journals and Books indexed in Scopus, Springer and Ei Compendex, Google Scholar etc. Currently, I am launching PGDM Pharmaceutical Management Program in IIHMR Bangalore and spearheading the course curriculum and structure of the same. I am interested in Collaboration for Healthcare Innovation, Pharma AI Innovation, Future trend in Marketing and Management with incubation on Healthcare, Healthcare IT startups, AI-ML Modelling and Healthcare Algorithm based training module development. I am also an affiliated member of the Institute of Management Consultant of India, looking forward to Healthcare, Healthcare IT and Innovation, Pharma and Hospital Management Consulting works.",institutionString:null,institution:{name:"Lovely Professional University",country:{name:"India"}}},{id:"1063",title:"Prof.",name:"Constantin",middleName:null,surname:"Volosencu",slug:"constantin-volosencu",fullName:"Constantin Volosencu",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/1063/images/system/1063.png",biography:"Prof. Dr. Constantin Voloşencu graduated as an engineer from\nPolitehnica University of Timișoara, Romania, where he also\nobtained a doctorate degree. He is currently a full professor in\nthe Department of Automation and Applied Informatics at the\nsame university. Dr. Voloşencu is the author of ten books, seven\nbook chapters, and more than 160 papers published in journals\nand conference proceedings. He has also edited twelve books and\nhas twenty-seven patents to his name. He is a manager of research grants, editor in\nchief and member of international journal editorial boards, a former plenary speaker, a member of scientific committees, and chair at international conferences. His\nresearch is in the fields of control systems, control of electric drives, fuzzy control\nsystems, neural network applications, fault detection and diagnosis, sensor network\napplications, monitoring of distributed parameter systems, and power ultrasound\napplications. He has developed automation equipment for machine tools, spooling\nmachines, high-power ultrasound processes, and more.",institutionString:'"Politechnica" University Timişoara',institution:null},{id:"221364",title:"Dr.",name:"Eneko",middleName:null,surname:"Osaba",slug:"eneko-osaba",fullName:"Eneko Osaba",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/221364/images/system/221364.jpg",biography:"Dr. Eneko Osaba works at TECNALIA as a senior researcher. He obtained his Ph.D. in Artificial Intelligence in 2015. He has participated in more than twenty-five local and European research projects, and in the publication of more than 130 papers. He has performed several stays at universities in the United Kingdom, Italy, and Malta. Dr. Osaba has served as a program committee member in more than forty international conferences and participated in organizing activities in more than ten international conferences. He is a member of the editorial board of the International Journal of Artificial Intelligence, Data in Brief, and Journal of Advanced Transportation. He is also a guest editor for the Journal of Computational Science, Neurocomputing, Swarm, and Evolutionary Computation and IEEE ITS Magazine.",institutionString:"TECNALIA Research & Innovation",institution:{name:"Tecnalia",country:{name:"Spain"}}},{id:"275829",title:"Dr.",name:"Esther",middleName:null,surname:"Villar-Rodriguez",slug:"esther-villar-rodriguez",fullName:"Esther Villar-Rodriguez",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/275829/images/system/275829.jpg",biography:"Dr. Esther Villar obtained a Ph.D. in Information and Communication Technologies from the University of Alcalá, Spain, in 2015. She obtained a degree in Computer Science from the University of Deusto, Spain, in 2010, and an MSc in Computer Languages and Systems from the National University of Distance Education, Spain, in 2012. Her areas of interest and knowledge include natural language processing (NLP), detection of impersonation in social networks, semantic web, and machine learning. Dr. Esther Villar made several contributions at conferences and publishing in various journals in those fields. Currently, she is working within the OPTIMA (Optimization Modeling & Analytics) business of TECNALIA’s ICT Division as a data scientist in projects related to the prediction and optimization of management and industrial processes (resource planning, energy efficiency, etc).",institutionString:"TECNALIA Research & Innovation",institution:{name:"Tecnalia",country:{name:"Spain"}}},{id:"49813",title:"Dr.",name:"Javier",middleName:null,surname:"Del Ser",slug:"javier-del-ser",fullName:"Javier Del Ser",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/49813/images/system/49813.png",biography:"Prof. Dr. Javier Del Ser received his first PhD in Telecommunication Engineering (Cum Laude) from the University of Navarra, Spain, in 2006, and a second PhD in Computational Intelligence (Summa Cum Laude) from the University of Alcala, Spain, in 2013. He is currently a principal researcher in data analytics and optimisation at TECNALIA (Spain), a visiting fellow at the Basque Center for Applied Mathematics (BCAM) and a part-time lecturer at the University of the Basque Country (UPV/EHU). His research interests gravitate on the use of descriptive, prescriptive and predictive algorithms for data mining and optimization in a diverse range of application fields such as Energy, Transport, Telecommunications, Health and Industry, among others. In these fields he has published more than 240 articles, co-supervised 8 Ph.D. theses, edited 6 books, coauthored 7 patents and participated/led more than 40 research projects. He is a Senior Member of the IEEE, and a recipient of the Biscay Talent prize for his academic career.",institutionString:"Tecnalia Research & Innovation",institution:{name:"Tecnalia",country:{name:"Spain"}}},{id:"278948",title:"Dr.",name:"Carlos Pedro",middleName:null,surname:"Gonçalves",slug:"carlos-pedro-goncalves",fullName:"Carlos Pedro Gonçalves",position:null,profilePictureURL:"https://s3.us-east-1.amazonaws.com/intech-files/0030O00002bRcmyQAC/Profile_Picture_1564224512145",biography:'Carlos Pedro Gonçalves (PhD) is an Associate Professor at Lusophone University of Humanities and Technologies and a researcher on Complexity Sciences, Quantum Technologies, Artificial Intelligence, Strategic Studies, Studies in Intelligence and Security, FinTech and Financial Risk Modeling. He is also a progammer with programming experience in:\n\nA) Quantum Computing using Qiskit Python module and IBM Quantum Experience Platform, with software developed on the simulation of Quantum Artificial Neural Networks and Quantum Cybersecurity;\n\nB) Artificial Intelligence and Machine learning programming in Python;\n\nC) Artificial Intelligence, Multiagent Systems Modeling and System Dynamics Modeling in Netlogo, with models developed in the areas of Chaos Theory, Econophysics, Artificial Intelligence, Classical and Quantum Complex Systems Science, with the Econophysics models having been cited worldwide and incorporated in PhD programs by different Universities.\n\nReceived an Arctic Code Vault Contributor status by GitHub, due to having developed open source software preserved in the \\"Arctic Code Vault\\" for future generations (https://archiveprogram.github.com/arctic-vault/), with the Strategy Analyzer A.I. module for decision making support (based on his PhD thesis, used in his Classes on Decision Making and in Strategic Intelligence Consulting Activities) and QNeural Python Quantum Neural Network simulator also preserved in the \\"Arctic Code Vault\\", for access to these software modules see: https://github.com/cpgoncalves. He is also a peer reviewer with outsanding review status from Elsevier journals, including Physica A, Neurocomputing and Engineering Applications of Artificial Intelligence. Science CV available at: https://www.cienciavitae.pt//pt/8E1C-A8B3-78C5 and ORCID: https://orcid.org/0000-0002-0298-3974',institutionString:"University of Lisbon",institution:{name:"Universidade Lusófona",country:{name:"Portugal"}}},{id:"310576",title:"Prof.",name:"Erick Giovani",middleName:null,surname:"Sperandio Nascimento",slug:"erick-giovani-sperandio-nascimento",fullName:"Erick Giovani Sperandio Nascimento",position:null,profilePictureURL:"https://intech-files.s3.amazonaws.com/0033Y00002pDKxDQAW/ProfilePicture%202022-06-20%2019%3A57%3A24.788",biography:"Prof. Erick Sperandio is the Lead Researcher and professor of Artificial Intelligence (AI) at SENAI CIMATEC, Bahia, Brazil, also working with Computational Modeling (CM) and HPC. He holds a PhD in Environmental Engineering in the area of Atmospheric Computational Modeling, a Master in Informatics in the field of Computational Intelligence and Graduated in Computer Science from UFES. He currently coordinates, leads and participates in R&D projects in the areas of AI, computational modeling and supercomputing applied to different areas such as Oil and Gas, Health, Advanced Manufacturing, Renewable Energies and Atmospheric Sciences, advising undergraduate, master's and doctoral students. He is the Lead Researcher at SENAI CIMATEC's Reference Center on Artificial Intelligence. In addition, he is a Certified Instructor and University Ambassador of the NVIDIA Deep Learning Institute (DLI) in the areas of Deep Learning, Computer Vision, Natural Language Processing and Recommender Systems, and Principal Investigator of the NVIDIA/CIMATEC AI Joint Lab, the first in Latin America within the NVIDIA AI Technology Center (NVAITC) worldwide program. He also works as a researcher at the Supercomputing Center for Industrial Innovation (CS2i) and at the SENAI Institute of Innovation for Automation (ISI Automação), both from SENAI CIMATEC. He is a member and vice-coordinator of the Basic Board of Scientific-Technological Advice and Evaluation, in the area of Innovation, of the Foundation for Research Support of the State of Bahia (FAPESB). He serves as Technology Transfer Coordinator and one of the Principal Investigators at the National Applied Research Center in Artificial Intelligence (CPA-IA) of SENAI CIMATEC, focusing on Industry, being one of the six CPA-IA in Brazil approved by MCTI / FAPESP / CGI.br. He also participates as one of the representatives of Brazil in the BRICS Innovation Collaboration Working Group on HPC, ICT and AI. He is the coordinator of the Work Group of the Axis 5 - Workforce and Training - of the Brazilian Strategy for Artificial Intelligence (EBIA), and member of the MCTI/EMBRAPII AI Innovation Network Training Committee. He is the coordinator, by SENAI CIMATEC, of the Artificial Intelligence Reference Network of the State of Bahia (REDE BAH.IA). He leads the working group of experts representing Brazil in the Global Partnership on Artificial Intelligence (GPAI), on the theme \"AI and the Pandemic Response\".",institutionString:null,institution:null},{id:"241400",title:"Prof.",name:"Mohammed",middleName:null,surname:"Bsiss",slug:"mohammed-bsiss",fullName:"Mohammed Bsiss",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/241400/images/8062_n.jpg",biography:null,institutionString:null,institution:null},{id:"276128",title:"Dr.",name:"Hira",middleName:null,surname:"Fatima",slug:"hira-fatima",fullName:"Hira Fatima",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/276128/images/14420_n.jpg",biography:"Dr. Hira Fatima\nAssistant Professor\nDepartment of Mathematics\nInstitute of Applied Science\nMangalayatan University, Aligarh\nMobile: no : 8532041179\nhirafatima2014@gmal.com\n\nDr. Hira Fatima has received his Ph.D. degree in pure Mathematics from Aligarh Muslim University, Aligarh India. Currently working as an Assistant Professor in the Department of Mathematics, Institute of Applied Science, Mangalayatan University, Aligarh. She taught so many courses of Mathematics of UG and PG level. Her research Area of Expertise is Functional Analysis & Sequence Spaces. She has been working on Ideal Convergence of double sequence. She has published 17 research papers in National and International Journals including Cogent Mathematics, Filomat, Journal of Intelligent and Fuzzy Systems, Advances in Difference Equations, Journal of Mathematical Analysis, Journal of Mathematical & Computer Science etc. She has also reviewed few research papers for the and international journals. 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The motor of the society is the industry and the research of this topic has to be empowered in order to increase and improve the quality of our lives.",coverUrl:"https://cdn.intechopen.com/series_topics/covers/22.jpg",hasOnlineFirst:!0,hasPublishedBooks:!0,annualVolume:11418,editor:{id:"27170",title:"Prof.",name:"Carlos",middleName:"M.",surname:"Travieso-Gonzalez",slug:"carlos-travieso-gonzalez",fullName:"Carlos Travieso-Gonzalez",profilePictureURL:"https://mts.intechopen.com/storage/users/27170/images/system/27170.jpeg",biography:"Carlos M. Travieso-González received his MSc degree in Telecommunication Engineering at Polytechnic University of Catalonia (UPC), Spain in 1997, and his Ph.D. degree in 2002 at the University of Las Palmas de Gran Canaria (ULPGC-Spain). He is a full professor of signal processing and pattern recognition and is head of the Signals and Communications Department at ULPGC, teaching from 2001 on subjects on signal processing and learning theory. His research lines are biometrics, biomedical signals and images, data mining, classification system, signal and image processing, machine learning, and environmental intelligence. He has researched in 52 international and Spanish research projects, some of them as head researcher. He is co-author of 4 books, co-editor of 27 proceedings books, guest editor for 8 JCR-ISI international journals, and up to 24 book chapters. He has over 450 papers published in international journals and conferences (81 of them indexed on JCR – ISI - Web of Science). He has published seven patents in the Spanish Patent and Trademark Office. He has been a supervisor on 8 Ph.D. theses (11 more are under supervision), and 130 master theses. He is the founder of The IEEE IWOBI conference series and the president of its Steering Committee, as well as the founder of both the InnoEducaTIC and APPIS conference series. He is an evaluator of project proposals for the European Union (H2020), Medical Research Council (MRC, UK), Spanish Government (ANECA, Spain), Research National Agency (ANR, France), DAAD (Germany), Argentinian Government, and the Colombian Institutions. He has been a reviewer in different indexed international journals (<70) and conferences (<250) since 2001. He has been a member of the IASTED Technical Committee on Image Processing from 2007 and a member of the IASTED Technical Committee on Artificial Intelligence and Expert Systems from 2011. \n\nHe has held the general chair position for the following: ACM-APPIS (2020, 2021), IEEE-IWOBI (2019, 2020 and 2020), A PPIS (2018, 2019), IEEE-IWOBI (2014, 2015, 2017, 2018), InnoEducaTIC (2014, 2017), IEEE-INES (2013), NoLISP (2011), JRBP (2012), and IEEE-ICCST (2005)\n\nHe is an associate editor of the Computational Intelligence and Neuroscience Journal (Hindawi – Q2 JCR-ISI). He was vice dean from 2004 to 2010 in the Higher Technical School of Telecommunication Engineers at ULPGC and the vice dean of Graduate and Postgraduate Studies from March 2013 to November 2017. 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Because of the close relationship between structure and function, studies in human physiology and anatomy seek to understand the mechanisms that help the human body function. The series on human physiology deals with the various mechanisms of interaction between the various organs, nerves, and cells in the human body.",coverUrl:"https://cdn.intechopen.com/series_topics/covers/12.jpg",keywords:"Anatomy, Cells, Organs, Systems, Homeostasis, Functions"},{id:"13",title:"Plant Physiology",scope:"Plant Physiology explores fundamental processes in plants, and it includes subtopics such as plant nutrition, plant hormone, photosynthesis, respiration, and plant stress. In recent years, emerging technologies such as multi-omics, high-throughput technologies, and genome editing tools could assist plant physiologists in unraveling molecular mechanisms in specific critical pathways. The global picture of physiological processes in plants needs to be investigated continually to increase our knowledge, and the resulting technologies will benefit sustainable agriculture.",coverUrl:"https://cdn.intechopen.com/series_topics/covers/13.jpg",keywords:"Plant Nutrition, Plant Hormone, Photosynthesis, Respiration, Plant Stress, Multi-omics, High-throughput Technology, Genome Editing"}],annualVolumeBook:{},thematicCollection:[],selectedSeries:null,selectedSubseries:null},seriesLanding:{item:{id:"6",title:"Infectious Diseases",doi:"10.5772/intechopen.71852",issn:"2631-6188",scope:"This series will provide a comprehensive overview of recent research trends in various Infectious Diseases (as per the most recent Baltimore classification). Topics will include general overviews of infections, immunopathology, diagnosis, treatment, epidemiology, etiology, and current clinical recommendations for managing infectious diseases. Ongoing issues, recent advances, and future diagnostic approaches and therapeutic strategies will also be discussed. This book series will focus on various aspects and properties of infectious diseases whose deep understanding is essential for safeguarding the human race from losing resources and economies due to pathogens.",coverUrl:"https://cdn.intechopen.com/series/covers/6.jpg",latestPublicationDate:"August 16th, 2022",hasOnlineFirst:!0,numberOfOpenTopics:4,numberOfPublishedChapters:124,numberOfPublishedBooks:13,editor:{id:"131400",title:"Prof.",name:"Alfonso J.",middleName:null,surname:"Rodriguez-Morales",fullName:"Alfonso J. Rodriguez-Morales",profilePictureURL:"https://mts.intechopen.com/storage/users/131400/images/system/131400.png",biography:"Dr. Rodriguez-Morales is an expert in tropical and emerging diseases, particularly zoonotic and vector-borne diseases (especially arboviral diseases). He is the president of the Travel Medicine Committee of the Pan-American Infectious Diseases Association (API), as well as the president of the Colombian Association of Infectious Diseases (ACIN). He is a member of the Committee on Tropical Medicine, Zoonoses, and Travel Medicine of ACIN. He is a vice-president of the Latin American Society for Travel Medicine (SLAMVI) and a Member of the Council of the International Society for Infectious Diseases (ISID). Since 2014, he has been recognized as a Senior Researcher, at the Ministry of Science of Colombia. He is a professor at the Faculty of Medicine of the Fundacion Universitaria Autonoma de las Americas, in Pereira, Risaralda, Colombia. He is an External Professor, Master in Research on Tropical Medicine and International Health, Universitat de Barcelona, Spain. He is also a professor at the Master in Clinical Epidemiology and Biostatistics, Universidad Científica del Sur, Lima, Peru. In 2021 he has been awarded the “Raul Isturiz Award” Medal of the API. Also, in 2021, he was awarded with the “Jose Felix Patiño” Asclepius Staff Medal of the Colombian Medical College, due to his scientific contributions to COVID-19 during the pandemic. He is currently the Editor in Chief of the journal Travel Medicine and Infectious Diseases. His Scopus H index is 47 (Google Scholar H index, 68).",institutionString:"Institución Universitaria Visión de las Américas, Colombia",institution:null},subseries:[{id:"3",title:"Bacterial Infectious Diseases",keywords:"Antibiotics, Biofilm, Antibiotic Resistance, Host-microbiota Relationship, Treatment, Diagnostic Tools",scope:"