\\n\\n
Released this past November, the list is based on data collected from the Web of Science and highlights some of the world’s most influential scientific minds by naming the researchers whose publications over the previous decade have included a high number of Highly Cited Papers placing them among the top 1% most-cited.
\\n\\nWe wish to congratulate all of the researchers named and especially our authors on this amazing accomplishment! We are happy and proud to share in their success!
Note: Edited in March 2021
\\n"}]',published:!0,mainMedia:null},components:[{type:"htmlEditorComponent",content:'IntechOpen is proud to announce that 191 of our authors have made the Clarivate™ Highly Cited Researchers List for 2020, ranking them among the top 1% most-cited.
\n\nThroughout the years, the list has named a total of 261 IntechOpen authors as Highly Cited. Of those researchers, 69 have been featured on the list multiple times.
\n\n\n\nReleased this past November, the list is based on data collected from the Web of Science and highlights some of the world’s most influential scientific minds by naming the researchers whose publications over the previous decade have included a high number of Highly Cited Papers placing them among the top 1% most-cited.
\n\nWe wish to congratulate all of the researchers named and especially our authors on this amazing accomplishment! We are happy and proud to share in their success!
Note: Edited in March 2021
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He has received many awards and honors in India and abroad including various Young Scientist Awards, BBSRC India Partnering Award (UK), Dr. JC Bose National Award of Department of Biotechnology, Min. of Science and Technology, Govt. of India, and Fellow of various internationally prestigious societies/academies including Royal College of Pathologists (UK), Royal Societies of Biology and Chemistry, London, United Kingdom, Academy of Translational Medicine Professionals, Austria, and is named as the Global Leader in Science by The Scientist magazine (USA) and International Opinion Leader/Expert involved in the vaccination for JE by IPIC (UK).",institutionString:"King George's Medical University",position:null,outsideEditionCount:0,totalCites:0,totalAuthoredChapters:"10",totalChapterViews:"0",totalEditedBooks:"6",institution:{name:"King George's Medical University",institutionURL:null,country:{name:"India"}}}],coeditorOne:null,coeditorTwo:null,coeditorThree:null,coeditorFour:null,coeditorFive:null,topics:[{id:"912",title:"Virology",slug:"pure-microbiology-virology"}],chapters:[{id:"61641",title:"Introductory Chapter: Human Influenza A Virus Infection - Global Prevalence, Prevention, Therapeutics, and Challenges",slug:"introductory-chapter-human-influenza-a-virus-infection-global-prevalence-prevention-therapeutics-and",totalDownloads:631,totalCrossrefCites:1,authors:[{id:"158026",title:"Prof.",name:"Shailendra K.",surname:"Saxena",slug:"shailendra-k.-saxena",fullName:"Shailendra K. 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From chapter submission and review, to approval and revision, copyediting and design, until final publication, I work closely with authors and editors to ensure a simple and easy publishing process. I maintain constant and effective communication with authors, editors and reviewers, which allows for a level of personal support that enables contributors to fully commit and concentrate on the chapters they are writing, editing, or reviewing. I assist authors in the preparation of their full chapter submissions and track important deadlines and ensure they are met. I help to coordinate internal processes such as linguistic review, and monitor the technical aspects of the process. As an ASM I am also involved in the acquisition of editors. Whether that be identifying an exceptional author and proposing an editorship collaboration, or contacting researchers who would like the opportunity to work with IntechOpen, I establish and help manage author and editor acquisition and contact."}},relatedBooks:[{type:"book",id:"3311",title:"Current Perspectives in HIV Infection",subtitle:null,isOpenForSubmission:!1,hash:"1bcacf84d50370cac414fea1616244c6",slug:"current-perspectives-in-hiv-infection",bookSignature:"Shailendra K. Saxena",coverURL:"https://cdn.intechopen.com/books/images_new/3311.jpg",editedByType:"Edited by",editors:[{id:"158026",title:"Prof.",name:"Shailendra K.",surname:"Saxena",slug:"shailendra-k.-saxena",fullName:"Shailendra K. 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Sometimes, these tests are the only methods exploring the functional deficits of the patient in otherwise normal structure. In contrast, ocular electrophysiological tests may explore normal function in cases with functional vision loss. In this chapter, we will focus on the clinical use of ocular electrophysiological tests after a short explanation about recording parameters. A basic clinical use of ocular electrophysiological tests will be discussed rather than detailed and theoretical explanations.
Full-field electroretinogram (ERG) represents a mass-response of the retina to a full-field flash of light. The resultant single waveform is the total response of the retina. The details of full-field ERG recording techniques may be obtained from ISCEV (International Society for Clinical Electrophysiology of Vision) standards [1]. The functions of rod, cones and inner retinal layers may be recorded separately by changing stimulus parameters and the adaptive state of the eye to the light.
In a typical full-field ERG (Figure 1), five recordings are performed. At first, the patient is dark adapted for at least 20 minutes. A dim white or blue flash light [2.0 log unit below the standard flash intensity (Standard flash: 3.0 cds/m2)] is used to stimulate the retina. This response is shown as ‘DA 0.01 response’ in the latest ISCEV guideline for full-field ERG recording. At that flash intensity, only rod photoreceptors are stimulated and the resultant waveform belongs to rod functions. In that response, only a positive b wave originated from rod ON-bipolar cells is recorded. This means that DA 0.01 response is an indirect indicator for rod photoreceptor function. Secondly, DA 3.0 response is recorded. A standard flash light is used to stimulate both rod and cone photoreceptors and combined response of rod and cone photoreceptors is recorded. The first negative peak, a-wave is caused by the hyperpolarization of photoreceptors. However, in DA 3.0 response, a-wave has a bifid configuration which makes the evaluation of photoreceptor function problematic. For this reason, ISCEV recommended the use of bright flash ERG recordings (DA 10.0 ERG or DA 30.0 ERG) for photoreceptor function evaluation. In these brighter light levels, a–wave has a clear single peak. Oscillatory potentials, which reflects amacrine cell function is recorded using standard flash light intensity under dark-or light-adapted (LA; at least 10-min of light adaptation using a background luminance of 30cd/m2) conditions. LA 3.0 response is generated within cone system. LA 3.0 30 Hz flicker response is the most sensitive indicator of cone system however, it arises in the inner retina and cannot be used to localize the level of abnormality within the cone system [2].
Each retina consists of approximately 4-5 million of cone and 100-120 million of rod photoreceptors. The rods contain light-sensitive pigment rhodopsin with a spectral absorption peak at 496 nm. Each cone contains one of three types of color sensitive pigments. L, M and S-cones (L: long wavelength cones, M: middle wavelength cones, S: small wavelength cones) have peak absorption spectra at 558nm, 531nm and 419nm, respectively. L,M and S cones is also named as red cones, green cones and blue cones with respect to colors of peak-sensitized light [3].
It is apparent from the stimulation technique that small areas of retinal dysfunction cannot be explored by full-field ERG, such as cases with Stargardt macular dystrophy or age-related macular degeneration, macular edema, etc. The cone photoreceptors are the most heavily packed in the macula, however 85-90 percent of cone photoreceptors reside in extra-macular retina. For this reason, full-field ERG is not a good way to investigate functional status or follow-up of retinal diseases known to be restricted to the macular area. Full-field ERG should be used for generalized retinal dysfunction.
A representative full-field ERG response from a healthy subject.
Full-field ERG responses belonging to a healthy subject and a 21-year old male with retinitis pigmentosa. DA 0.01 response is non-recordable. DA 3.0 response has a very low b wave amplitude. LA 3.0 and LA 3.0 30 Hz responses are reduced.
The original report of the ERG in primary retinitis pigmentosa revealed nondetectable or very small responses but these patients usually had advanced disease with attenuation of retinal vessels and extensive pigmentary changes in the retina. However, later studies showed that in the early stages of the disease, the ERG amplitudes are generally subnormal when the patient is asymptomatic. In that stage, however, delays in the implicit times helps in the establishing of widespread progressive forms of retinitis pigmentosa [4].
Retinitis pigmentosa has mainly three types of genetic transmission, autosomal dominant, autosomal recessive and X-linked recessive forms. Almost 50% of patients are sporadic retinitis pigmentosa patients which means that the most common form is this form of the disease. The worst prognosis is seen in X-linked recessive inheritance. These patients generally have non-recordable rod and cone ERG until the end of first decade. However, autosomal dominant type has the best prognosis, and patients with autosomal dominant inheritance may have good rod and cone functions until the fourth and fifth decades [4].
In cases with non-recordable full-field ERG, the follow-up of the macular function may be performed with multifocal ERG, focal ERG or pattern ERG. This will be discussed in the next parts of this chapter.
Negative ERG is seen in stationary night blidness but is not limited to this condition. A normal a wave and reduced b wave means that there is problem in the transmission of electrical biopotential from the photoreceptors to the inner retinal layers. The retina has a dual circulation. Photoreceptors are nourished by choroidal circulation while inner retinal layers are nourished by retinal circulation. The biopotential cannot be transferred to inner retinal layers if a problem exist in the retinal circulation. For this reason, central retinal artery obstruction, central retinal vein obstruction may cause negative ERG. Similarly, juvenile retinoschisis is one of the causes of negative ERG.
Full-field ERG responses belonging to a healthy subject and a 23-year old male with cone-dystrophy. DA 0.01 and DA 3.0 responses are normal. LA 3.0 response is almost non-recordable. LA 3.0 30 Hz responses are very much reduced.
Full-field ERG responses belonging to a healthy subject and a 20-year old male with congenital stationary night blindness. DA 0.01 is non-recordable, DA 3.0 response has a negative configuration that is b-wave amplitude is lower than a-wave amplitude. LA 3.0 response and LA 3.0 30 Hz responses are mildly reduced.
Full-field electroretinogram with very delayed rod response, a rhomboid a-wave and supernormal b-wave in bright-flash rod-cone response and very reduced cone responses in a patient with KCNV2 mutation (Used with permission of Journal of Retina-Vitreus, 2011).
Chloroquine/hydroxychloroquine is used in the treatment of rheumatoid arthritis, systemic lupus erithematosis, and malarial fever. Both drugs have an affinity to melanin and tends to accumulate in the choroid, ciliary body, and retinal pigment epithelium. When the degenerative changes are limited to the macular area, normal or subnormal full-field ERG responses are obtained. In the late stages of the toxic effect, peripheral pigmentary changes become apparent. In this stage, minimal or non-recordable responses are obtained. Because full-field ERG responses are minimally affected in the early stages, this test is not recommended to detect early functional deficits. Instead, central 10/2 visual field testing and multifocal ERG is more appropriate for this purpose [19].
Multifocal ERG, first developed by Sutter and Tran in 1991 [28], provides a topographic map of the retinal function. As discussed above, full-field ERG is a mass response of the retina and small areas of retinal dysfunction cannot be explored with full-field ERG. At that point, multifocal ERG has its own advantages. By using a single electrode, multifocal ERG technique allows the recording of the functions of 61, 103 or even more retinal areas in less than 7-8 minutes. The recordings belong to central 30 to 50 degrees of the retina. For this reason, it is an excellent tool in detecting macular function. Multifocal ERG is a reproducible technique although very small responses are produced in each hexagonal area [29].
Multifocal ERG responses may be presented as single waveforms for each hexagonal area, ring analysis beginning from the most central to the periphery of the stimulated area and 3-D presentation (Figure 6).
A normal multifocal ERG response output. Upper left: Plots diagram showing single responses from each retinal area. Upper right: Ring analyses. The upper rings show central retinal functions, the lower rings show peripheral functions. Lower right: 2-D amplitudes with color-coded diagram. Lower left: 3-D amplitudes.
The multifocal ERG stimulus is displayed on a video monitor. The stimulus consists of a pattern of hexagonal areas which are scaled to produce equal ERG responses from the retina (Figure 7). During stimulation, the display appears to flicker because each hexagon goes through a pseudo-random sequence (the m-sequence) of black and white presentations. Each hexagon has a probability of 0.5 of being white or black on each frame change [30]. Complex mathematical analyses between each retinal response and pseudo-random m sequence provide local retinal responses belonging to each hexagonal area.
Multifocal ERG stimulus
Central areolar choroidal dystrophy, first described by Nettleship in 1884, is a macular dystrophy characterized by the development of fine, mottled, depigmented retinal pigment epithelium in the macula. After several decades the pathognomonic zone of circumscript atrophy, affecting retina, retinal pigment epithelium and choriocapillaris, develops in the macular region of the eye [31, 32]. Although, most cases are sporadic, autosomal dominant and recessive inheritance patterns have been reported [33]. In a recent study, we have showed that mfERG responses were reduced corresponding to the areas of ophthalmoscopically visible lesion and there were significant correlations between foveal retinal sensitivity in the Humprey visual field and mfERG P1/N1 amplitudes (Figure 8) [34].
Color fundus photographs, pattern deviation of Humphrey visual fields and multifocal ERG results of four patients with central areolar choroidal dystrophy. Central responses are markedly reduced and delayed in multifocal ERG. (Used with permission of Wichtig Editore. From. ‘Multifocal electroretinogram and central visual field testing in central areolar choroidal dystrophy‘‘, Gundogan et al, European Journal of Ophthalmology, Volume 20, Number 5, 2010).
Multifocal ERG was used to evaluate macular function and the response of macular edema to different types of treatment in different types of macular edema. In one of them we showed that multifocal ERG is not a good way to monitor the macular function in
One of the most important studies on the use of multifocal ERG for glaucoma detection was performed by Sutter and Bearse [43]. The authors used a mathematical algorithm to extract a component with a latency, which increased in proportion to the estimated length of the ganglion cell axons from the site of stimulation to the optic nerve head. The authors found that glaucomatous damage may reduce the magnitude of this component (optic nerve head component) [39, 43].
Focal ERG is used to record local ERG response. In contrast to multifocal ERG stimulus, a direct focal light is used over the retinal area being tested, mostly the macular or foveal region. The response to such a stimulus is about a few microvolts, for this reason, signal-to-noise ratio is low in focal ERG. To overcome this issue, hundreds of stimulus should be used to have a reliable average response. Second problem in focal ERG is the scattered light. The original ring-shaped light is scattered in the eye and may easily stimulate the area outside the intended retinal area. For this reason, the stimulating light is encircled by an annulus ring of steady background light that is typically brighter than the test stimulus. However, this is not an unproblematic solution, because it use of a brighter background light prevents the recording of rod functions [44]. Focal ERG is generally not used in routine clinical practice in most electrophysiological units because of these difficulties and the emergence of multifocal ERG in 1992.
Pattern ERG is a retinal response to a checkerboard pattern stimulus with alternating black and white squares. In low temporal frequencies (<6 reversals per second), a positive component, P50 (positive peak around 50th milliseconds), and a negative component, N95 (negative component around 95th milliseconds), are observed. Sometimes, a negative component around 35 milliseconds may be recorded (N35).This response to low-frequency stimulus is called ‘transient PERG. (Figure 9)
A typical pattern ERG recording.
In high temporal frequencies (>7 reversals per second), P50 and N95 peaks are merged into a sinusoidal waveform, dominated by the N95 component. This response is called ‘steady-state pattern ERG’. In steady-state pattern ERG, it is impossible to distinguish the original P50 and N95 peaks [45].
Initially it was thought that PERG is almost totally originated from ganglion cell functions. However, later studies showed that P50 peak has an earlier component originated from cells distal to the ganglion cells and reflecting mostly the macular function [46-49]. In two reports [50, 51], it has been detected that some pattern ERG response still may be recorded after post-traumatic and surgical optic nerve section despite no light perception. In one of them, P50 amplitude reduction with P50 latency shortening was observed. These findings too imply that pattern ERG is not completely originated from ganglion cells. In addition, shortening of the P50 latency caused the theory that a later part of the P50 response is related with ganglion cell function and P50 latency shortens if ganglion cell function extinguishes.
It is apparent from the Figure 9 that P50 amplitude reduction is accompanied by a secondary N95 reduction, as N95 amplitude is measured from P50 peak to N95 trough. However, this is not the same for N95 amplitude reduction. N95 amplitude reduction may be selective. For this reason, the ratio of N95 amplitude to P50 amplitude has an importance in detecting whether the visual loss is related to macular disease or ganglion cell disease. If N95/P50 ratio is normal, then it may be thought that the visual loss may be attributed to macular disease. If the ratio is lower than normal (which is called as ‘selective N95 reduction’), visual loss may be attributed to ganglion cell disease. N95/P50 ratio is about 1.5 in the author’s electrophysiology laboratory.
The first reports about N95 in optic nerve demyelination were presented by Holder. Holder reported that pattern ERG abnormalities could be limited to N95 component. The author also reported that there was a 40% pattern ERG abnormality among 200 patients with optic nerve demyelination, however 85% of the abnormalities were detected in N95 [52, 53].
Figure 10 and figure 11 show P50 and N95 results of 382 patients with optic nerve demyelination. As shown in Figure 10, most of the patients have normal P50 amplitudes despite prolonged P100 latency in pattern VEP. However Figure 11 shows that N95/P50 ratio decreases as P100 latency increases [52].
Pattern ERG P50 amplitudes in patients with optic nerve demyelination. (Used with permission of Pergamon. From. ‘Pattern Electroretinography (PERG) and an Integrated Approach to Visual Pathway Diagnosis ‘‘, Holder GE, Progress in Retinal and Eye Research, Volume 20, Number 4, 2001).
Pattern ERG N95/P50 ratio in patients with optic nerve demyelination. (Used with permission of Pergamon. From. ‘Pattern Electroretinography (PERG) and an Integrated Approach to Visual Pathway Diagnosis ‘‘, Holder GE, Progress in Retinal and Eye Research, Volume 20, Number 4, 2001).
Ventura et al.[56] investigated the steady-state pattern ERG responses with PERGLA paradigm in 200 glaucoma suspects with increased optic disc cupping and normal visual field and in 42 patients with early manifest glaucoma. The PERG was abnormal in amplitude, phase, or inter-ocular asymmetry in amplitude and phase in 52% of glaucoma suspect patients and 69% of EMG patients. The pattern ERG amplitude was correlated weakly with both mean deviation and vertical C/D (p=0.05). The correlation between pattern ERG amplitude and MD and C/D was stronger for inter-ocular differences rather than absolute measures. Inter-ocular pattern ERG amplitude asymmetry was positively correlated with the severity of the disease. Compared to white glaucoma suspects, a lower pattern ERG amplitude was found in black glaucoma suspects and early manifest glaucoma patients, but not in black glaucoma controls.
Unlike full-field ERG, electro-oculogram (EOG) is not a stimulated response. EOG records the continuous resting potential across the retinal pigment epithelium which is named as ‘transepithelial potential’. This potential is only about a few millivolts. Transepithelial potential is mainly generated by retinal pigment epithelium. However, as well as the integrity of the retinal pigment epithelium, photoreceptor and interphotoreceptor matrix integrity and function should be intact. For this reason, EOG is decreased in photoreceptor diseases, retinal detachment and other generalized outer retinal damages in addition to primary retinal pigment diseases such as Best disease.
The resting potential across the retinal pigment epithelium is not a steady potential. In the dark-adaptation, transepithelial potential is decreased to a minimum value (dark trough) after about 12 minutes. In the light-adaptation, the transepithelial potential increases to a peak value (light-peak) after about 7-12 minutes [57]. The ratio of light-peak to dark-trough is called Arden ratio or ‘EOG ratio’. This value should be 1.8 or greater in normal subjects and considered abnormal under 1.6. In the author’s institution, Arden ratio is 2.35±0.44 (mean±SD) [58]. Figure 12 shows EOG recordings of a patient with Best disease. There is almost no light peak with light stimulation in EOG. Arden ratios are 1.18 in the right eye and 1.17 in the left eye.
Fundus photo of a patient with Best disease and EOG recordings. Arden ratio is 1.18 in OD and 1.17 in OS. This implies very small change in transepithelial potential with light stimulation.
Visual evoked potential (VEP) represents the cortical response to a checkerboard-pattern stimulus (pattern VEP) or a flash stimulus (flash VEP). Pattern VEP components that are commonly measured are N75, P100 and N135 peaks (Figure 13).
Representative pattern VEP waveforms to five consecutive check sizes.
The amplitudes of the peaks are measured from the peak of the one component to the trough of the preceding component. P and N refer to positive and negative voltages recorded at the occipital electrode with respect to the voltage at the reference electrode.
Flash VEP components are defined as N1, P1, N2, P2, etc (Figure 14).
Flash VEP responses in a patient with intravitreal hemorrhage in the left eye. Left-eye flash VEP responses are very much reduced.
VEP response primarily reflects the central retinal function although the stimulated retinal area in pattern VEP and flash VEP is about 50 degrees and full retinal areas, respectively. There are three main reasons for this contribution of the central retina [59]. (1).The central visual field is represented at the outer surface of the visual cortex while peripheral retina is represented at the deep surfaces of the calcarine sulcus. Active electrode in VEP recordings is placed approximately 2 cm above the protuberentia occipitalis externa which is the nearest point to the surface of the visual cortex. (2) Cortical magnification phenomenon. In the central retina each photoreceptor transmits its signal almost to one ganglion cell, while many photoreceptors converges on a single ganglion cell in the peripheral retina. Thus, more than 50% of the cells in the visual cortex represent approximately central 10 degrees of the retina. (3) In PVEP testing, small checkerboard stimuli may be used. These small sized stimuli may only be resolved by the central retina which has the highest concentration of photoreceptors.
Because of the reflection of the central retinal function, pattern VEP is used to estimate visual acuity in many clinical situations besides optic nerve function [60]. An impaired VEP is anatomically non-specific. However, a through ocular examination including the retina, optic nerve and brain frequently explores the localization of the problem [61]. Pattern VEP is more valuable than flash VEP in the clinical evaluations of the visual pathway. However, flash VEP is valuable in the situations of fixation problem, mature cataract, intravitreal hemorrhage, ocular trauma or any other circumstance that prevents patient cooperation. In these situations, flash VEP gives important knowledge about visual status.
Pattern VEP recording requires fixation to a point in the screen. Impaired VEP responses may be produced by deliberate poor fixation, defocusing to the fixation point, or conscious suppression.[61-64] This is an important issue in the evaluation of patients with functional visual loss. Voluntary flash VEP suppression is more difficult, because it does not require fixation.
Pattern VEP traces belonging to a –normal and a multiple sclerosis patient. P100 latency to 2 degree check size is about 150 ms in the left eye while it is about 100 ms in the right eye.
Figure 16 shows PVEP recordings to 5-consecutive check sizes of an African woman with no light perception in the left eye for 2 years. Biomicroscopic and fundoscopic examinations were unremarkable. No relative afferent pupillary defect was detected. Pattern VEP responses in both eyes were totally in the normal limits in terms of P100 amplitude and latency values. In this patient, we were able to show that the patient was capable of reading at least 0.3 in Snellen chart from 6 meters with the use of polaroid glasses.
PVEP response to five consecutive check sizes in a malingerer who claimed no light perception in the left eye.
Full-field ERG is invaluable in generalized retinal diseases. Pattern ERG is complimentary test for full-field ERG, because it may localize the problem to macula or ganglion cells. Multifocal ERG is used to evaluate central retinal function. EOG is the recording of transepithelial potential. VEP is a cortical potential that is the end of visual pathway, for this reason it gives important knowledge about the ‘vision’ itself. The ophthalmologist can localize the visual problem with a thorough understanding of the origins of these tests.
In the last decade, we observed a massive upsurge of studies in the field of extracellular vesicles (EVs) [1]. As it is known now, EVs can be loaded with different therapeutic molecules and transport them to recipient cells with little interrogation by the immune system. This property of EVs prompts new possibilities for treatment in various clinical settings [2, 3, 4]. In this chapter, we review the biology of EVs as a universal cellular component from a broader perspective, and afterward provide an updated view on red blood cell extracellular vesicles (RBCEVs), their merits and potential applications in therapeutics [5].
\nWolf was the first to discover small procoagulant structures derived from activated platelets in human blood and named them “platelet dust” in 1967. He separated the small structures by ultracentrifugation and further characterized them using an electron microscopy [6]. In 1987, Johnstone further studied the formation of such vesicles in the duration of sheep reticulocytes maturation
EVs are a heterogeneous class of cell-derived structures with a lipid bilayer membrane, which comprise exosomes, microvesicles, and apoptotic bodies. They are either of the endosomal origins or are shed from the plasma membrane under physiological and pathological conditions. Additionally, they are present in almost all biological fluids, such as blood, urine, breast milk, cerebrospinal fluid, saliva, semen, etc. [11, 12, 13, 14, 15, 16, 17]. Further characterizations are based on the different sizes and biogenesis of EVs. Exosomes generally range from 50 to 150 nm in diameter and are secreted from endosomal multivesicular bodies, whereas microvesicles are larger vesicles ranging from 100 to 500 nm in diameter and are formed through a budding or exocytosis process of the plasma membrane [11, 18, 19, 20, 21, 22, 23]. Apoptotic bodies are much larger, ranging from 800 to 5000 nm in diameter, and are generated by blebbing of plasma membrane from cells undergoing apoptosis. Hence, apoptotic bodies represent the fragments of dying cells and differ from exosomes and microvesicles in property (Figure 1) [17, 18, 19, 20, 21, 22]. In this chapter, we will collectively term both exosomes and microvesicles as EVs with apoptotic bodies excluded.
\nBiogenesis and composition of extracellular vesicles. Extracellular vesicles (EVs) are composed of exosomes, microvesicles, and apoptotic bodies. Exosomes are typically of endosomal origins and are the smallest among them with 50 to 150 nm in diameter. Microvesicles are larger in size from 100 to 500 nm in diameter and are generated through an outward budding or exocytosis of the plasma membrane. Apoptotic bodies are usually the largest ranging from 800 to 5,000 nm in diameter and are generated by blebbing of plasma membrane from cells undergoing apoptosis. Major components of EVs are lipids, proteins, and nucleic acids. Due to different biogenesis mechanisms, the compositions of exosomes and microvesicles do vary.
The components of EVs are mainly proteins, lipids, and nucleic acids. However, due to different biogenesis mechanisms, the compositions of exosomes and microvesicles do vary slightly [11, 24, 25, 26]. Proteins that are associated with endocytic pathways can be usually found in EVs, such as flotillin and annexin. Some of the biogenesis-associated proteins, such as Tsg101 and Alix, and common tetraspanins, such as CD9 and CD81, are commonly used as EVs markers with CD63 which is mostly regarded as a marker of exosomes. However, currently, there lack well-defined protein markers to distinguish exosomes and microvesicles [11, 24, 25, 26]. Lipid components of EVs include phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingomyelin, cholesterol, and so on, which can be found in plasma membrane as well. As microvesicles are formed by budding from plasma membrame, the lipid composition of microvesicles resembles that of plasma membrane of the cells more while exosomes are of higher levels in sphingomyelin, cholesterol, and phosphatidylserine [27, 28, 29]. It is noteworthy that many nucleic acid species are highly enriched in EVs. The lipid bilayer structure of EVs acts as a natural shelter against degrading nucleases in the extracellular environment and protects the nucleic acid cargo under adverse conditions such as long-term storage and multiple freeze-thaw cycles. In the recent decade, reports have it that many mRNAs, microRNAs, and other non-coding RNAs are discovered in EVs (Figure 1) [30, 31, 32].
\nAs EVs are abundant and widely distributed in biological fluids and carry bioactive cargo, they influence various biological processes of the donor and recipient cells [33]. The intercellular communication can occur between cells by transferring EVs that act as an exchange mediator of proteins, lipids, and RNAs. Thus, EVs have a fundamental role to play in important biological processes such as the exchange of surface membrane and horizontal RNA transport between neighboring and remote cells [18]. This aspect is being extensively investigated in cancers [34], neurodegenerative diseases [35], autoimmune disorders [36], aging [37], and so on. The bioactive cargo encapsulated by EVs contain valuable information from the source of diseases, which can serve as robust biomarkers in diagnostics and status snapshots in treatment monitoring [38, 39]. The endogenous property of transporting molecules by EVs inspires researchers to utilize them as a superb delivery platform of therapeutic agents as well [40].
\nSimilar to EVs released from other cells, EVs in the circulation carry biomarkers originated from the donor cells [41]. Usually EVs contain various markers which indicate their origins, e.g., CD235a (also called GPA) for RBCs, CD41 for platelets, and CD11c for dendritic cells [42, 43, 44]. RBCs express classes of CD59 and DAF, known as complement inhibitors, and signaling of CD47 and SHPS-1 molecules on the cell surface to protect themselves against endogenous elimination [45, 46]. For instance, the RBCs membrane protein CD47 inhibits RBCs phagocytosis via macrophages by binding to the inhibitory receptor signal regulatory protein alpha (SIRPα). The presence of such proteins on the surfaces of RBCEVs may help RBCEVs to escape from the clearance by macrophages if they carry CD47 on their surfaces [47, 48, 49]. Mature RBCs lack nuclei and most of the intracellular membrane structures; hence, EVs released from mature RBCs are microvesicles derived from the plasma membrane (Figure 2). Even with the same cell origins, the protein or lipid compositions of EVs may differ on account of the lateral cell membrane variation. Further proteomic assays have illustrated that to some extent, the proteomic spectrum difference of EVs and the releasing cells can be attributed to the stimulating conditions during EVs biogenesis [50]. Microvesicles derived from RBCs are reported to be different in protein contents when produced naturally
Using red blood cell extracellular vesicles (RBCEVs) for therapeutic delivery. Calcium ionophore is added to RBCs which simulates the release of microvesicles, the only type of RBCEVs. Naturally, RBCEVs contain hemoglobin, Alix, TSG101, and some microRNAs in their lumen. They also display stomatin (STOM) and glycophorin A (GPA) on their membrane. RBCEVs can be loaded with therapeutic molecules including RNAs, proteins, and chemical drugs for delivery of these molecules to other cell types.
There are many studies of RBCEVs under diseased conditions with malaria being frequently reported. Mantel and colleagues illustrated that EVs from human RBCs infected with
Chang and colleagues demonstrated the ability of RBCEVs to efficiently deliver ultra-small superparamagnetic iron oxide particles into human bone marrow mesenchymal stem cells for cellular magnetic resonance imaging
Up to now, standardized protocols for EVs isolation for either scientific research or clinical application are lacking [56]. One of the commonly used methods to obtain EVs is ultrafiltration with subsequent differential ultracentrifugation. Ultrafiltration followed by liquid size exclusion chromatography suits the large-scale demand of isolating EV for therapeutics as the method results in, on the one hand, a significantly higher EV yield and, on the other hand, the well-preserved biophysical properties of the purified EVs [57]. Usman and colleagues provided a lab-based approach to purify RBCEVs using ultracentrifugation with sucrose cushion. To begin with, RBCs in whole blood were separated from white blood cells and plasma by low centrifugation and using leukodepletion filters. Then, the isolated RBCs were diluted in PBS and treated with 10 mM calcium ionophore overnight which can stimulate the release of RBCEVs and significantly increase the yield. In order to purify RBCEVs, RBCs and cell debris were removed by several rounds of low-speed centrifugation. Later, the resulting supernatants were passed through 0.45 μm syringe filters. Afterward, the RBCEVs were concentrated using ultracentrifugation at 100,000 ×
For therapeutic agents to be loaded into EVs, two major strategies currently have been applied. The first option is to load the therapeutic molecules, such as RNAs, into the EVs after EVs isolation, while the second one is conducted during EV biogenesis. These methods are also known as post-loading and pre-loading, respectively. The pre-loading encapsulation approach is also referred to as the endogenous method as it uses the cellular machinery in order to load small RNA into EVs. The pre-loading approach has been shown to work for the packaging of both siRNA and miRNA into EVs. The post-loading method artificially introduces RNAs into EVs, whereas pre-loading is performed in the EVs biogenesis. Post-loading can be subdivided into passive loading, such as by physical incubation, and active loading with instances of electroporation or sonication. Furthermore, the functional small RNAs delivery using electroporated EVs has been shown to be a success in several reports but it depends on the small RNA species [58, 59, 60, 61, 62]. Usman and colleagues used the electroporation method for post-loading of RNAs into RBCEVs [55]. Ideally, various therapeutic molecules including ASOs, siRNAs, gRNAs, mRNAs, plasmid DNA, proteins, peptides, and chemical drug compounds can be loaded into RBCEVs using electroporation (Figure 2). Other post-loading methods such as mild sonication and physical incubation may be applicable to RBCEVs as tested for other types of EVs. Labeling of EVs is then required to examine the efficiency of delivery to target cells. Various methods and techniques have been applied to label EVs, with most common methods being incubation with biotinylated radioisotope, substrate of luciferase, fluorescence lipophilic dye, streptavidin-conjugated fluorescence dyes, or the use of other modified proteins [55, 64, 65, 66].
\nDue to their innate function on cell-cell communication, EVs can be used effectively for drug delivery [12, 67, 68, 69]. The biggest advantage of EVs drug delivery is probably that EVs can be taken from an organism and returned to the same organism
It has been reported that diversified types of cells, including RBCs, endothelial cells, monocytes, granulocytes, and platelets, release EVs. Additionally, EVs can be isolated by various methods from cell culture media, plasma, and other biofluids [23, 41, 70, 71]. Although several research groups have demonstrated the advantages of using EVs for RNA delivery, there are still issues with EVs generated from fibroblasts and dendritic cells being not permanently available from all subjects [69, 72]. RBCs are readily obtainable from any human subject and easy to store, and blood transfusion has been a relatively safe, well-established, and routine medical procedure for decades which makes RBCEVs easy to obtain and safe to use. Thus, EVs from whole plasma are easily accessible and substantially present, but these EVs are derived from various cell types, e.g., nucleated cells which represent a risk for horizontal gene transfer [63]. Therefore, obtaining ultrapure RBCEVs solely derived from RBCs is highly preferred as RBCs lack both nuclear and mitochondrial DNA, which means that RBCEVs for pharmaceutical purpose avoid the risk of horizontal gene transfer. RBCEVs formation has been extensively investigated and described in the recent years. Therefore, with such knowledge, RBCEVs are safer and less complicated to use [73, 74, 75, 76].
\nConsequently, RBCEVs possess several features which make them better suitable for clinical applications than EVs from other cell types. First of all, blood units are easily accessible from existing blood banks. A large scale of RBCEVs can be produced at low cost as RBCs are the most abundant cell type in the body (84% of all cells) and, during their 120-day lifespan, RBCs continue to release RBCEVs, leading to an approximate 20% loss in RBCs volume and an increase of around 14% in hemoglobin concentration [23, 77, 78, 79]. Additionally, RBCEVs are obtainable for allogeneic and autologous transfusion from the patients’ own blood. A large number of RBCs (~1012 cells/L) are obtainable from each blood unit. Thus, there exists no need to expand cells in culture and no risk of the emergence of mutations
EVs are shed from the plasma membrane or released by endosomal pathways under both physiological and diseased conditions. Intercellular communication is one of the best known functions of EVs by far, which provides the possibility to utilize the EVs natural vehicle property of transporting nucleic acids, proteins, and lipids for drug delivery. Recent studies demonstrate that human RBCEVs can be developed as robust delivery platform for multiple therapeutic RNAs in cancer treatment. RBCEVs feature multiple benefits as compared to EVs from other cell types. They are easily obtainable in large amounts, can be frozen and thawed multiple times without significant compromise, are nontoxic and nonimmunogenic, can reach remote tissues in the body with minimal hindrance by physiological barriers, and do not contain DNA or other unpredictable contents which could result in horizontal gene transfer. By obtaining RBCEVs directly from the patient, they are safe to use allogeneic treatments and possess no risk of emerging mutations during expansion by cell culture. Thus, RBCEVs show promising advantages in overcoming various limitations of cell-based therapeutics. All in all, RBCEVs need further research in order to establish them as a new source and promising approach for practical therapeutics in clinical use.
\nWe would like to thank our colleagues at City University of Hong Kong including Chin Siew Mei, Waqas Muhammad Usman, Tin Chanh Pham, Luyen Tien Vu, Boya Peng, Thach Tuan Pham, Abdullah Faqeer, Yeokyeong Kim, Seongkyeol Kim, Likun Wei, Ching Yee Moo, Ru Zhen, and Migara Kavishka Jayasinghe for their support. We are grateful to the generous funding from the Hong Kong Health, and Medical Research Fund (grant 03141186) and the Hong Kong Research Grants Council (grant 21106616).
\nThe authors declare no conflict of interest.
General requirements for Open Access to Horizon 2020 research project outputs are found within Guidelines on Open Access to Scientific Publication and Research Data in Horizon 2020. The guidelines, in their simplest form, state that if you are a Horizon 2020 recipient, you must ensure open access to your scientific publications by enabling them to be downloaded, printed and read online. Additionally, said publications must be peer reviewed.
',metaTitle:"Horizon 2020 Compliance",metaDescription:"General requirements for Open Access to Horizon 2020 research project outputs are found within Guidelines on Open Access to Scientific Publication and Research Data in Horizon 2020. The guidelines, in their simplest form, state that if you are a Horizon 2020 recipient, you must ensure open access to your scientific publications by enabling them to be downloaded, printed and read online. Additionally, said publications must be peer reviewed. ",metaKeywords:null,canonicalURL:null,contentRaw:'[{"type":"htmlEditorComponent","content":"Publishing with IntechOpen means that your scientific publications already meet these basic requirements. It also means that through our utilization of open licensing, our publications are also able to be copied, shared, searched, linked, crawled, and mined for text and data, optimizing our authors' compliance as suggested by the European Commission.
\\n\\nMetadata for all publications is also automatically deposited in IntechOpen's OAI repository, making them available through the Open Access Infrastructure for Research in Europe's (OpenAIRE) search interface further establishing our compliance.
\\n\\nIn other words, publishing with IntechOpen guarantees compliance.
\\n\\nRead more about Open Access in Horizon 2020 here.
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\n\nMetadata for all publications is also automatically deposited in IntechOpen's OAI repository, making them available through the Open Access Infrastructure for Research in Europe's (OpenAIRE) search interface further establishing our compliance.
\n\nIn other words, publishing with IntechOpen guarantees compliance.
\n\nRead more about Open Access in Horizon 2020 here.
\n\nWhich scientific publication to choose?
\n\nWhen choosing a publication, Horizon 2020 grant recipients are encouraged to provide open access to various types of scientific publications including monographs, edited books and conference proceedings.
\n\nIntechOpen publishes all of the aforementioned formats in compliance with the requirements and criteria established by the European Commission for the Horizon 2020 Program.
\n\nAuthors requiring additional information are welcome to send their inquiries to funders@intechopen.com
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I am also a member of the team in charge for the supervision of Ph.D. students in the fields of development of silicon based planar waveguide sensor devices, study of inelastic electron tunnelling in planar tunnelling nanostructures for sensing applications and development of organotellurium(IV) compounds for semiconductor applications. I am a specialist in data analysis techniques and nanosurface structure. I have served as the editor for many books, been a member of the editorial board in science journals, have published many papers and hold many patents.",institutionString:null,institution:{name:"Sheffield Hallam University",country:{name:"United Kingdom"}}},{id:"54525",title:"Prof.",name:"Abdul Latif",middleName:null,surname:"Ahmad",slug:"abdul-latif-ahmad",fullName:"Abdul Latif Ahmad",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:null},{id:"20567",title:"Prof.",name:"Ado",middleName:null,surname:"Jorio",slug:"ado-jorio",fullName:"Ado Jorio",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:{name:"Universidade Federal de Minas Gerais",country:{name:"Brazil"}}},{id:"47940",title:"Dr.",name:"Alberto",middleName:null,surname:"Mantovani",slug:"alberto-mantovani",fullName:"Alberto Mantovani",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:null},{id:"12392",title:"Mr.",name:"Alex",middleName:null,surname:"Lazinica",slug:"alex-lazinica",fullName:"Alex Lazinica",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/12392/images/7282_n.png",biography:"Alex Lazinica is the founder and CEO of IntechOpen. After obtaining a Master's degree in Mechanical Engineering, he continued his PhD studies in Robotics at the Vienna University of Technology. Here he worked as a robotic researcher with the university's Intelligent Manufacturing Systems Group as well as a guest researcher at various European universities, including the Swiss Federal Institute of Technology Lausanne (EPFL). During this time he published more than 20 scientific papers, gave presentations, served as a reviewer for major robotic journals and conferences and most importantly he co-founded and built the International Journal of Advanced Robotic Systems- world's first Open Access journal in the field of robotics. Starting this journal was a pivotal point in his career, since it was a pathway to founding IntechOpen - Open Access publisher focused on addressing academic researchers needs. Alex is a personification of IntechOpen key values being trusted, open and entrepreneurial. Today his focus is on defining the growth and development strategy for the company.",institutionString:null,institution:{name:"TU Wien",country:{name:"Austria"}}},{id:"19816",title:"Prof.",name:"Alexander",middleName:null,surname:"Kokorin",slug:"alexander-kokorin",fullName:"Alexander Kokorin",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/19816/images/1607_n.jpg",biography:"Alexander I. Kokorin: born: 1947, Moscow; DSc., PhD; Principal Research Fellow (Research Professor) of Department of Kinetics and Catalysis, N. Semenov Institute of Chemical Physics, Russian Academy of Sciences, Moscow.\r\nArea of research interests: physical chemistry of complex-organized molecular and nanosized systems, including polymer-metal complexes; the surface of doped oxide semiconductors. He is an expert in structural, absorptive, catalytic and photocatalytic properties, in structural organization and dynamic features of ionic liquids, in magnetic interactions between paramagnetic centers. The author or co-author of 3 books, over 200 articles and reviews in scientific journals and books. He is an actual member of the International EPR/ESR Society, European Society on Quantum Solar Energy Conversion, Moscow House of Scientists, of the Board of Moscow Physical Society.",institutionString:null,institution:{name:"Semenov Institute of Chemical Physics",country:{name:"Russia"}}},{id:"62389",title:"PhD.",name:"Ali Demir",middleName:null,surname:"Sezer",slug:"ali-demir-sezer",fullName:"Ali Demir Sezer",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/62389/images/3413_n.jpg",biography:"Dr. Ali Demir Sezer has a Ph.D. from Pharmaceutical Biotechnology at the Faculty of Pharmacy, University of Marmara (Turkey). He is the member of many Pharmaceutical Associations and acts as a reviewer of scientific journals and European projects under different research areas such as: drug delivery systems, nanotechnology and pharmaceutical biotechnology. Dr. Sezer is the author of many scientific publications in peer-reviewed journals and poster communications. Focus of his research activity is drug delivery, physico-chemical characterization and biological evaluation of biopolymers micro and nanoparticles as modified drug delivery system, and colloidal drug carriers (liposomes, nanoparticles etc.).",institutionString:null,institution:{name:"Marmara University",country:{name:"Turkey"}}},{id:"61051",title:"Prof.",name:"Andrea",middleName:null,surname:"Natale",slug:"andrea-natale",fullName:"Andrea Natale",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:null},{id:"100762",title:"Prof.",name:"Andrea",middleName:null,surname:"Natale",slug:"andrea-natale",fullName:"Andrea Natale",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:{name:"St David's Medical Center",country:{name:"United States of America"}}},{id:"107416",title:"Dr.",name:"Andrea",middleName:null,surname:"Natale",slug:"andrea-natale",fullName:"Andrea Natale",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:{name:"Texas Cardiac Arrhythmia",country:{name:"United States of America"}}},{id:"64434",title:"Dr.",name:"Angkoon",middleName:null,surname:"Phinyomark",slug:"angkoon-phinyomark",fullName:"Angkoon Phinyomark",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/64434/images/2619_n.jpg",biography:"My name is Angkoon Phinyomark. I received a B.Eng. degree in Computer Engineering with First Class Honors in 2008 from Prince of Songkla University, Songkhla, Thailand, where I received a Ph.D. degree in Electrical Engineering. My research interests are primarily in the area of biomedical signal processing and classification notably EMG (electromyography signal), EOG (electrooculography signal), and EEG (electroencephalography signal), image analysis notably breast cancer analysis and optical coherence tomography, and rehabilitation engineering. I became a student member of IEEE in 2008. During October 2011-March 2012, I had worked at School of Computer Science and Electronic Engineering, University of Essex, Colchester, Essex, United Kingdom. In addition, during a B.Eng. I had been a visiting research student at Faculty of Computer Science, University of Murcia, Murcia, Spain for three months.\n\nI have published over 40 papers during 5 years in refereed journals, books, and conference proceedings in the areas of electro-physiological signals processing and classification, notably EMG and EOG signals, fractal analysis, wavelet analysis, texture analysis, feature extraction and machine learning algorithms, and assistive and rehabilitative devices. I have several computer programming language certificates, i.e. Sun Certified Programmer for the Java 2 Platform 1.4 (SCJP), Microsoft Certified Professional Developer, Web Developer (MCPD), Microsoft Certified Technology Specialist, .NET Framework 2.0 Web (MCTS). 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