\\n\\n
IntechOpen was founded by scientists, for scientists, in order to make book publishing accessible around the globe. Over the last two decades, this has driven Open Access (OA) book publishing whilst levelling the playing field for global academics. Through our innovative publishing model and the support of the research community, we have now published over 5,700 Open Access books and are visited online by over three million academics every month. These researchers are increasingly working in broad technology-based subjects, driving multidisciplinary academic endeavours into human health, environment, and technology.
\\n\\nBy listening to our community, and in order to serve these rapidly growing areas which lie at the core of IntechOpen's expertise, we are launching a portfolio of Open Science journals:
\\n\\nAll three journals will publish under an Open Access model and embrace Open Science policies to help support the changing needs of academics in these fast-moving research areas. There will be direct links to preprint servers and data repositories, allowing full reproducibility and rapid dissemination of published papers to help accelerate the pace of research. Each journal has renowned Editors in Chief who will work alongside a global Editorial Board, delivering robust single-blind peer review. Supported by our internal editorial teams, this will ensure our authors will receive a quick, user-friendly, and personalised publishing experience.
\\n\\n"By launching our journals portfolio we are introducing new, dedicated homes for interdisciplinary technology-focused researchers to publish their work, whilst embracing Open Science and creating a unique global home for academics to disseminate their work. We are taking a leap toward Open Science continuing and expanding our fundamental commitment to openly sharing scientific research across the world, making it available for the benefit of all." Dr. Sara Uhac, IntechOpen CEO
\\n\\n"Our aim is to promote and create better science for a better world by increasing access to information and the latest scientific developments to all scientists, innovators, entrepreneurs and students and give them the opportunity to learn, observe and contribute to knowledge creation. Open Science promotes a swifter path from research to innovation to produce new products and services." Alex Lazinica, IntechOpen founder
\\n\\nIn conclusion, Natalia Reinic Babic, Head of Journal Publishing and Open Science at IntechOpen adds:
\\n\\n“On behalf of the journal team I’d like to thank all our Editors in Chief, Editorial Boards, internal supporting teams, and our scientific community for their continuous support in making this portfolio a reality - we couldn’t have done it without you! With your support in place, we are confident these journals will become as impactful and successful as our book publishing program and bring us closer to a more open (science) future.”
\\n\\nWe invite you to visit the journals homepage and learn more about the journal’s Editorial Boards, scope and vision as all three journals are now open for submissions.
\\n\\nFeel free to share this news on social media and help us mark this memorable moment!
\\n\\n\\n"}]',published:!0,mainMedia:{caption:"",originalUrl:"/media/original/237"}},components:[{type:"htmlEditorComponent",content:'
After years of being acknowledged as the world's leading publisher of Open Access books, today, we are proud to announce we’ve successfully launched a portfolio of Open Science journals covering rapidly expanding areas of interdisciplinary research.
\n\n\n\nIntechOpen was founded by scientists, for scientists, in order to make book publishing accessible around the globe. Over the last two decades, this has driven Open Access (OA) book publishing whilst levelling the playing field for global academics. Through our innovative publishing model and the support of the research community, we have now published over 5,700 Open Access books and are visited online by over three million academics every month. These researchers are increasingly working in broad technology-based subjects, driving multidisciplinary academic endeavours into human health, environment, and technology.
\n\nBy listening to our community, and in order to serve these rapidly growing areas which lie at the core of IntechOpen's expertise, we are launching a portfolio of Open Science journals:
\n\nAll three journals will publish under an Open Access model and embrace Open Science policies to help support the changing needs of academics in these fast-moving research areas. There will be direct links to preprint servers and data repositories, allowing full reproducibility and rapid dissemination of published papers to help accelerate the pace of research. Each journal has renowned Editors in Chief who will work alongside a global Editorial Board, delivering robust single-blind peer review. Supported by our internal editorial teams, this will ensure our authors will receive a quick, user-friendly, and personalised publishing experience.
\n\n"By launching our journals portfolio we are introducing new, dedicated homes for interdisciplinary technology-focused researchers to publish their work, whilst embracing Open Science and creating a unique global home for academics to disseminate their work. We are taking a leap toward Open Science continuing and expanding our fundamental commitment to openly sharing scientific research across the world, making it available for the benefit of all." Dr. Sara Uhac, IntechOpen CEO
\n\n"Our aim is to promote and create better science for a better world by increasing access to information and the latest scientific developments to all scientists, innovators, entrepreneurs and students and give them the opportunity to learn, observe and contribute to knowledge creation. Open Science promotes a swifter path from research to innovation to produce new products and services." Alex Lazinica, IntechOpen founder
\n\nIn conclusion, Natalia Reinic Babic, Head of Journal Publishing and Open Science at IntechOpen adds:
\n\n“On behalf of the journal team I’d like to thank all our Editors in Chief, Editorial Boards, internal supporting teams, and our scientific community for their continuous support in making this portfolio a reality - we couldn’t have done it without you! With your support in place, we are confident these journals will become as impactful and successful as our book publishing program and bring us closer to a more open (science) future.”
\n\nWe invite you to visit the journals homepage and learn more about the journal’s Editorial Boards, scope and vision as all three journals are now open for submissions.
\n\nFeel free to share this news on social media and help us mark this memorable moment!
\n\n\n'}],latestNews:[{slug:"webinar-introduction-to-open-science-wednesday-18-may-1-pm-cest-20220518",title:"Webinar: Introduction to Open Science | Wednesday 18 May, 1 PM CEST"},{slug:"step-in-the-right-direction-intechopen-launches-a-portfolio-of-open-science-journals-20220414",title:"Step in the Right Direction: IntechOpen Launches a Portfolio of Open Science Journals"},{slug:"let-s-meet-at-london-book-fair-5-7-april-2022-olympia-london-20220321",title:"Let’s meet at London Book Fair, 5-7 April 2022, Olympia London"},{slug:"50-books-published-as-part-of-intechopen-and-knowledge-unlatched-ku-collaboration-20220316",title:"50 Books published as part of IntechOpen and Knowledge Unlatched (KU) Collaboration"},{slug:"intechopen-joins-the-united-nations-sustainable-development-goals-publishers-compact-20221702",title:"IntechOpen joins the United Nations Sustainable Development Goals Publishers Compact"},{slug:"intechopen-signs-exclusive-representation-agreement-with-lsr-libros-servicios-y-representaciones-s-a-de-c-v-20211123",title:"IntechOpen Signs Exclusive Representation Agreement with LSR Libros Servicios y Representaciones S.A. de C.V"},{slug:"intechopen-expands-partnership-with-research4life-20211110",title:"IntechOpen Expands Partnership with Research4Life"},{slug:"introducing-intechopen-book-series-a-new-publishing-format-for-oa-books-20210915",title:"Introducing IntechOpen Book Series - A New Publishing Format for OA Books"}]},book:{item:{type:"book",id:"3341",leadTitle:null,fullTitle:"Sustainable Radio Frequency Identification Solutions",title:"Sustainable Radio Frequency Identification Solutions",subtitle:null,reviewType:"peer-reviewed",abstract:"Radio frequency identification (RFID) is a fascinating, fast developing and\r\nmultidisciplinary domain with emerging technologies and applications. It is characterized\r\nby a variety of research topics, analytical methods, models, protocols, design principles and\r\nprocessing software. With a relatively large range of applications, RFID enjoys extensive\r\ninvestor confidence and is poised for growth. \r\nA number of RFID applications proposed or already used in technical and scientific\r\nfields are described in this book. Sustainable Radio Frequency Identification Solutions comprises\r\n19 chapters written by RFID experts from all over the world. In investigating RFID solutions\r\nexperts reveal some of the real-life issues and challenges in implementing RFID.",isbn:null,printIsbn:"978-953-7619-74-9",pdfIsbn:"978-953-51-6411-1",doi:"10.5772/174",price:139,priceEur:155,priceUsd:179,slug:"sustainable-radio-frequency-identification-solutions",numberOfPages:370,isOpenForSubmission:!1,isInWos:null,isInBkci:!1,hash:"2adaeecfea5c7ebc03b4bb7764c3bdd3",bookSignature:"Cristina Turcu",publishedDate:"February 1st 2010",coverURL:"https://cdn.intechopen.com/books/images_new/3341.jpg",numberOfDownloads:85307,numberOfWosCitations:53,numberOfCrossrefCitations:37,numberOfCrossrefCitationsByBook:3,numberOfDimensionsCitations:76,numberOfDimensionsCitationsByBook:3,hasAltmetrics:1,numberOfTotalCitations:166,isAvailableForWebshopOrdering:!0,dateEndFirstStepPublish:"April 24th 2012",dateEndSecondStepPublish:"May 15th 2012",dateEndThirdStepPublish:"August 19th 2012",dateEndFourthStepPublish:"November 17th 2012",dateEndFifthStepPublish:"December 17th 2012",currentStepOfPublishingProcess:5,indexedIn:"1,2,3,4,5,6,7",editedByType:"Edited by",kuFlag:!1,featuredMarkup:null,editors:[{id:"9302",title:"Dr.",name:"Cristina",middleName:null,surname:"Turcu",slug:"cristina-turcu",fullName:"Cristina Turcu",profilePictureURL:"https://mts.intechopen.com/storage/users/9302/images/system/9302.jpg",biography:"Cristina Turcu is an Associate Professor of Software Engineering and Artificial Intelligence at Stefan cel Mare University of Suceava, Romania. She received her Diploma (M.Sc.) in Automatics and Computers and her Doctorate (Ph.D.) in Automatics, in 1991 and 2000, respectively, both from the Gheorghe Asachi Technical University of Iasi, Romania where she has been Head of Computer Department since 2004. Her research interests include software engineering, RFID applications for the end-consumer, and intelligent systems. Dr. Turcu is an Editor of four books and has served on various program committees of conferences in computing and RFID systems. She also has served as a reviewer for numerous referred journals and conferences. She is the Editor in Chief of the International Journal of Radio Frequency Identification & Wireless Sensor Networks. 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Rajesh Banu",coverURL:"https://cdn.intechopen.com/books/images_new/6839.jpg",licenceType:"CC BY 3.0",editedByType:"Edited by",editors:[{id:"218539",title:"Dr.",name:"Rajesh Banu",middleName:null,surname:"Jeyakumar",slug:"rajesh-banu-jeyakumar",fullName:"Rajesh Banu Jeyakumar"}],productType:{id:"1",title:"Edited Volume",chapterContentType:"chapter",authoredCaption:"Edited by"}}},ofsBook:{item:{type:"book",id:"11433",leadTitle:null,title:"Human Migration in the Last Three Centuries",subtitle:null,reviewType:"peer-reviewed",abstract:"
\r\n\tIn March 2022, another book on human migration seems important when the events or tragedies unfolding in Eastern Europe are considered. People have always migrated and have moved, but, specifically looking at the last three hundred years, involuntary migration is on the rise. Involuntary migration does not only affect Europe; Asia, Africa, and North as well as South America, have had their fair share of natural catastrophes, invasions, and wars.
\r\n\tThis book will intend to look at different migrant patterns, voluntary and involuntary migration, over the last three centuries. What influenced people to leave their home countries, family, and friends and settle somewhere else? The book may include histories of the 19th century, consider tragedies and movements activated by political events in the 20th century, and/or look at recent events of the 21st century. Push and pull factors are important points. While most of us may be influenced in a negative way by the current happenings in Eastern Europe, the Russian invasion and resulting tragedies also demonstrate some very positive human traits – the preparedness of Ukraine’s surrounding countries to help those in need and to provide a safe place for the present.
\r\n\tWhether one looks at voluntary or involuntary migration into any country, after a period of adjustment, migrants do play a positive role. The research found that migrants contribute to the economy (food, shelter, employment, tax) and enrich a country’s cultural norms. Prerequisites for successful settlements are that the host society adopts a tolerant approach and that the migrants recognize the law and the language of the host country. Nothing is ever easy or without controversy, but I am a migrant (German Australian), and life in Australia has been relatively harmonious. Issues that could be considered in the book are multicultural societies (do monocultural societies still exist?) and theories of acculturation versus integration (settlement processes).
\r\n\tTwo further issues are very important in relation to human migration. There is climate change, global warming, and the environment, which clearly affect people’s movement. Small island populations are very concerned about rising sea levels. 2021 has also seen floods costing human lives: Turkey (August 2021), Brazil (December 2021), Chile (January 2021), and South India (November 2021), to name but a few. In Australia (March 2022), farms and whole townships in New South Wales and Queensland have been flooded for the second time in five years, and plans to resettle these towns are considered. Official and social media provide ample coverage of the events, which leads me to the next issue. There is today’s very important role of the media, of the official and social media. We are constantly bombarded with images of human war tragedies and flood victims. People in industrialized, western countries must be the best-informed populace. How far do the images and up-to-date TV news influence us, make us change our behavior, and perhaps even consider us more generous than we have been?
\r\n\tClimate change and the media are relatively new to the human migration debate, but both issues play important parts, and some interesting discussions are appreciated.
\r\n\t
Skin melanoma is a devastating disease, frequently diagnosed in human and dogs, accounting for 0,8% - 2% of all skin tumors in latter species [1].
Melanocytic tumours are common neoplasms in dogs, accounting for 4 to 7% of neoplastic lesions in general, and up to 7% of all malignant tumours [2,3]. They generally arise on the oral cavity (Figure 1), lip, skin (Figure 2) and digit, amongst other locations (Figure 3) [4].
Canine oral melanoma (courtesy of Dr. Abel Fernandes).
Canine cutaneous melanoma.
Canine ocular melanoma.
Biologic behaviour of these tumours is often related to its location. Over 85% of melanocytic lesions located on haired skin are described as of benign behaviour. The majority of oral and mucocutaneous junction melanomas, with the exception of the eyelid, and 50% of digital melanomas originated from the nail bed are reported as malignant [5,6].
Cutaneous canine melanocytic lesions are usually benign [7,8]. They are generally detected at a late stage, when excision is rarely curative and metastasis is often detectable in regional lymph nodes [7]. Malignant tumors are found most frequently on the head, ventral abdomen, and scrotum [7]; in the last one, they represent 3,1% of all cutaneous malignant melanomas and 4,7% of scrotal tumours [9]. Amelanotic lesions can occur as cutaneous neoplasms, but are more frequent in the oral cavity, and tend to be behaviorally malignant [10].
Metastasis are often found on regional lymph nodes and lungs, but organs as brain, heart and spleen are also commonly affected [11].
Veterinary nomenclature of canine melanocytic tumours has been subjected to many controversies and changes over time. Through this article, in accordance with the revised World Health Organization classification system, and in order to simplify and avoid confusion, the authors describe benign lesions as melanocytoma, whereas malignant lesions are referred to as melanoma [12].
Etiology of this neoplasms is still uncertain, and several factors may be related, such as consanguinity, trauma, chemical exposure, hormones and genetic susceptibility [10]. Unlike humans, ionizing solar radiation exposure doesn’t seem to be related to canine melanoma initiation [4].
Canine melanocytic tumour diagnosis often represents a challenge for the pathologist, since a high number of neoplastic lesions are quite similar in terms of its clinical and histologic appearance, including carcinoma, sarcoma, lymphoma and plasmocitoma, amongst others [10]. Indeed, cutaneous neoplasms with malignant behavior are more difficult to distinguish histologically from benign neoplasms than oral or lip neoplasms [13].
Diagnosis is usually based on fine-needle aspiration citology, but biopsy for histopathological examination is essential to determine its malignant potential [4,14]. The most reliable histologic criteria is the mitotic index, defined as the total number of mitotic figures observed per ten high-power light microscopic fields, which is known to be 90% accurate [5,6,15].
Immunochemistry has arised as an extremely useful tool, for both diagnostic and prognostic purposes. A positive diagnostic for melanocytic neoplasms is obtained with a positive labelling of S100 protein, vimentine, Neuron Specific Enolase (
The treatment of choice for local cutaneous melanomas is surgical excision; tumours with benign histopathology criteria have an excellent prognostic after this surgery. However, for malignant tumors, the prognostic is guarded, since metastatic rates of 30-75% have been reported [5,6].
Alternative therapy methods described in the literature include systemic chemotherapy, radiotherapy [17], photodynamic therapy [18], local hyperthermia [19,20] and intralesional injection of cisplatin or carboplatin.
The poor responses to the conventional therapy are leading to a development of new immunotherapy procedures – including intralesional adenoviral vector-mediated transfer of CD40L, a tumor necrosis factor gene [21], therapy with a plasmid DNA encoding staphylococcal enterotoxin B [22], and systemic tratment with liposome-encapsulated muramyl tripeptide [23].
At last, the most promising therapy appears to be a xenogenetic human tyrosinase DNA vaccine, with minimal local reaction and no systemic toxicity signs, and a great clinical responde with significant increasing of the survival time [24].
In this paper, the authors aim to contribute to the understanding of melanocytic tumours in the dog, making a critical review of the literature and discussing the parameters currently considered valid for diagnosis use in canine melanocytic neoplams.
Cutaneous melanocytic tumours are most common in older dogs (mean age of 9 years old) [25], with a higher mean age for dogs with malignant melanocytic tumours (12 years). However, age has not been related with the patient clinical outcome and survival time [26].
Although all breeds of dog (and crossbred animals) may be affected, some breeds are reported as predisposed, including Schnauzer, Doberman, Scottish Terrier, Irish Setter, Golden Retriever, Chow Chow, Cocker Spaniel, German Shepherd and Rottweiler [4,27]. Breed predisposition is thought to be related to an underlying genetic risk and/or increased pigmentation in the described above breeds [4].
One study [6] has also established a relationship between patient breed and tumour behavior, likely due to genetic susceptibility, as prior described. In the referred work, melanocytic lesions tended to be behaviorally benign in Doberman pinschers and miniature schnauzers, while miniature poodles were the mostly affected breed with malignant melanoma. However, it must be noted that oral neoplasms were also included in that study.
An early report described a higher frequency of these lesions in male dogs [28], but recent literature denies gender predisposition [6,27,29-31].
Melanin is a dark-brown pigment synthetized by melanocytes, dendritic cells found within the basal layer of the epidermis. These cells are dispersed from each other, located between basal keratinocytes, forming adherent and regulatory junctions mediated by epithelial cadherin (E-cadherin) molecules. After its synthesis, melanin is retained in melanosomas and transferred to the adjacent keratinocytes [32].
Conversion of normal melanocytes to clusters of neoplastic melanocytes is a process composed by a series of events: initiation, promotion, transformation and metastasis [4].
Little is known about initiation on most animal melanomas, but ionizing solar radiation exposure – the main initiator factor in Human melanomas [33] - doesn’t seem to be related to canine melanomas [4]. A higher incidence of spontaneously mutated cells due to familiar clustering through inbreeding may be a critical initiation factor in domestic animals.
Malignant transformation of canine cutaneous melanocytomas is very uncommon. Regarding cutaneous melanomas, there are a few published case reports, including one by Valentine and team [34], which have described a single case of malignant transformation of a congenital melanocytic nevus is a Golden retriever. Conroy [35] described two cases of melanoma originated from junctional or dermal hamartomas and a single case of a primary melanomas originated from a subcutaneous melanocytoma have also been reported [36]. In summary, canine cutaneous melanomas are thought to arise
Promotion phase is related to mutated cells proliferation, with subsequent amplification of cell population and origin of additional mutations [4]. Melanoma promoters include chronic trauma, chemical exposure, drugs and hormones [10], and its action results in reactive hyperplasia of the epithelium, with disruption of regular keratinocyte-melanocyte interactions and proliferation of initiated cells.
The next step in carcinogenesis involves a series of transformation events. Recent developments in genetic and molecular study techniques have identified the role of a few tumour suppressors in melanoma cell lines, giving new insights on the importance of these molecules in canine melanoma development. A reduction or loss of
After local proliferation phase, neoplastic cells may acquire a malignant behavior, and disseminate through hematic or lymphatic vessels to various other organs, originating secondary neoplasms known as metastasis. This complex process has it start with loss of adhesion and detachment of neoplastic cells from the primary mass, hematic and lymphatic vessels intravasion and attachment and proliferation within a secondary location [4].
Metastasis process is dependent of various adhesion molecules regulation by neoplastic cells. Several studies have shown an association between decreased and altered expression of E-cadherin, a calcium-dependent adhesion molecule responsible for melanocyte-keratinocyte interaction, and canine cutaneous melanoma progression [39,40]. CD44, a second transmembrane glycoprotein which facilitates metastasis, is required for several processes, including hyaluronate degradation, cell aggregation and migration, angiogenesis and hematopoiesis [4]. Down-regulation of regular CD44 plus up-regulation of CD44v5 has also been associated with melanoma metastasis, particularly with lymph node metastasis [41].
Autonomous growth is a key requirement for both primary and secondary neoplastic development. The most important autocrine growth factors in animal melanoma include basic fibroblast growth factor (
Macroscopically, canine malignant melanoma cannot be differentiated from melanocytoma [42]. Melanomas in dogs tend to be dermal in location, unlike Human melanomas – which are intraepidermal with some degree of dermal invasion. Prognostic schemes, such as Clark’s level or Breslow thickness, built nased on depht of dermal invasion, are not applicable on canine lesions [43].
Canine melanocytomas share some aspects with Human benign melanocytic lesions, in terms of clinical evolution most common metastatic locations [42], and genetic alterations [44].
Cutaneous melanocytomas are usually symmetrical, circumscribed, but encapsulated [43], solitary, black, brown, or gray cutaneous alopecic nodules [43,45] with a variable size with range of 1-4 cm in diameter (Figure 4 and Figure 5) [43]. Epidermis is usually intact, and alopecia is frequent. Epidermal cells may be hyperpigmented, and the majority of dermal cells are replaced by the tumoral ones, which in larger masses might also extends into the subcutaneous tissue. The tumors may have a varieated appearance, with areas of pigmentation intermingled with no pigmented regions [42].
Canine cutaneous malignant melanomas can vary considerably in appearance, regardless of the location. Melanomas tend to be asymmetrical. The asymmetry may be most readily recognizable in the epidermal component of junctional tumors [43]. Melanomas size vary from some milimiteres to as large as 10 centimeters in diameter (mean range being 1 to 3 cm in diameter) [43], but this is not a reliable indicator of malignancy [13,42]. The color is variable, ranging from gray or brown to black, red, or even dark blue [7]. Cutaneous melanoma presentation includes smooth domes, sessile nodules, polypoid, plaquelike [7,43], or even lobulated masses [7]. The larger ones are often ulcerated [7,43]. The tumors may invade deeply into the subcutaneous tissue and along fascial planes [42].
Canine cutaneous melanoma.
Canine cutaneous melanoma.
Microscopic examination of a cytological specimen obtained through fine needle aspiration has become a valuable technique to obtain a preliminary, and often definitive, diagnosis [46]. Being a quick, non-evasive and inexpensive procedure, it can also provide information on the stage, prognosis and metastasis evidence. Its main limitation reside on the fact that non-pigmented melanomas may strongly resemble other neoplastic lesions, and the amount of cytological sample might be very small and not fully representative of the lesion [4,46].
Several studies in Human cancer have described a strong accuracy in cytological examination in comparison with histopathological findings [47,48], but there are only few studies on the subject in Veterinary Medicine [49]. For instance, Ghisleni and others [50] evaluated a series of cutaneous and subcutaneous masses from dogs and cats through histopathology and cytology, describing an agreement between both techniques in 90.9% of the samples.
Melanocytic tumours are characterized by the presence of cells with abundant cytoplasmatic melanin granules. Neoplastic cells may appear with an epithelial (cohesive cells), mesenchymal (single oval or spindle-shaped cells) or round cell morphology. Nuclei may present a central or eccentric location, and is often solitary, though multinucleated forms are occasionally found. Nucleoli tends to be very prominent, with variable shapes such as round, oval or angular. These cells have a light basophilic cytoplasm, with a moderate to high nuclear-cytoplasmatic ratio. Varying degrees of pigmentation might be found within the same tumour smear [4,46], (Figure 6 and Figure 7).
Malignant criteria consist, most importantly, of marked anisokaryosis and nuclear pleomorphism, but also of the presence of large and atypical nucleoli [46]. Mitotic index, the most reliable criteria in histopathological evaluation, has no use in cytology. Regional lymph nodes are the most commonly evaluated site while monitoring for metastasis [4].
Canine cutaneous melanoma (Wright, 100x).
Canine cutaneous melanoma (Wright, 400x).
Histological characteristics of canine melanocytic neoplasms were defined by World Health Organization, in
Canine cutaneous melanocytoma (H&E, 200x).
Canine cutaneous melanoma (H&E, 200x).
Histological appearance does not always correlate well with biological behavior [8]. More recent studies have provided a numerical ‘‘tumor score’’ taking into consideration mitotic index, nuclear atypia, inflammation, necrosis and volume which appears to have improved correlation between histology and behavior [8].
The term nevus, commonly used in describing pigmented melanocytic lesions of the epidermis and dermis in humans, is not used in veterinary dermatopathology [7].
In this chapter we review several histological parameters on their ability to diagnose and predict prognosis (i.e., prediction of mortality) of canine melanocytic neoplasms. Malignancy celular features include a characteristic large nucleous, nuclear atypia, hyperchromasia, abnormal chromatin clumping and anomalous mitotic figures [51].
Melanocytic neoplasms are generally composed of one of the following cell types: epithelioid (Figure 10), spindle, mixed (Figure 11), dendritic [51,52], and round cells [43]. Other less commonly described cell types include signet ring and ballon cells [51]. All these cell types may occur either alone or in combination [43]. In melanomas, ganglion cell and multinucleated giant cell forms also may be observed [43]. The epithelioid cell type is the most common type in all locations, whereas a mixture of cell types is seen with less frequency [51].
The epithelioid cells are round, with discrete cell borders, abundant glassy cytoplasm, appearing arranged in sheets and larger nests [43]. Similar to their spindle-shaped counterparts melanomas may exhibit large ovoid nuclei and prominent nucleoli [7,43], marked anisokaryosis and variable chromatin patterns [43].
The spindle cell tumors are arranged in streams and interweaving bundles, resembling fibrosarcoma or neurofibrosarcoma presentation. In malignant lesions, the nuclei are large and fusiform with prominent nucleoli [7,43], and moderate to marked nuclear pleomorphism is seen [43]. Spindle cell predominant morphology was statistically associated with benignity in one study (in 71% melanocytomas in contrast to 29% melanomas) [43]. The mixed type consists of both cell morphologies and patterns [7].
Dendritic melanocytes have a highly angular shape, sometimes with long cytoplasmic processes, and are usually arranged in small nests [43] or organized in tightly swirling streams, often with a fingerprint pattern [7]. The dendritic or whorled forms occurs only in the skin [53].
Round cells tumors have round to polygonal cells arranged in sheets as dense packets of cells, the packets being separated by a scant stroma. The nuclei is large and round in melanomas [43].
The signet-ring cell tumors consist of compact neoplastic clusters, with round to ovoid cells presenting a faintly-eosinophilic cytoplasm and an intensely-stained periphery. A vesicular nuclei, crescent-shaped, and with a peripheral location, gives the cell a signet-ring appearance [52].
Epithelioid cell canine cutaneous melanoma (H&E, 400x).
Mixed cell canine cutaneous melanoma (H&E, 400x).
Balloon cells are found organized in groups, separated by collagenous septa [52]. The cells are round to polyhedral and have clear or faintly eosinophilic cytoplasm [43,52]. In melanomas, the nuclei are round situated mainly at the periphery of the tumor cells [52]. Usually contains one central prominent nucleolus [7], which sometimes is difficult to detect. Heterochromatin, which is sparse, is dispersed throughout the nuclei [52].
Benign melanocytic cells present an enlarged vesicular nuclei with small nucleoli. Nuclear shape vary on according to the predominant tumour cell type. Mitotic figures are rare, and mitotic atypia is rarely observed. [43].
Although cell type appears to play some role in the prognosis of ocular melanocytic neoplasms, this feature lacked significance in prognosis of melanocytic tumors occurring in the mouth, feet, buccal mucosa and skin of dogs in several studies [8]. On the contrary, the epithelioid shape was associated with an unfavorable course, for 8/15 cases. The difference seems to be significant (p = 0,03) [12].
One of the major criteria of malignancy in melanocytic neoplasms arising at any pigmented anatomic site is nuclear atypia. This feature is more valuable in epithelioid tumours than in spindle, whorled type or signet-ring cells, due to the insufficient nuclear detail associated with the later neoplasms [8,44,54]. However, not all studies are consensual [12].
Well-differentiated tumoral melanocytes have a small nucleus with one central nucleolus [8]. In contrary, undifferentiated tumours generally present cells with multiple, large, irregular and eccentrically nucleoli [8].
Several criteria are used to estimate nuclear atypia, including the percentage of nuclei involved [8]. Figures 12 and 13 (below) present moderate and severe nuclear atypia, respectively.
In animal cutaneous and eye melanocytic neoplasms, mitotic index (MI) is the most reliable histological feature for distinguishing malignant from benign tumors [44,51], and also in predicting the clinical course of the disease [8]. In cutaneous melanoma, an MI of ≥3/10 hpf is significantly correlated with decreased survival [55].
The number of mitoses is usually lower in melanocytomas [<3 mitotic figures per 10 high power fields (hpf)] than melanomas [42,44,53].
In one study, the mitotic index was strongly correlated with the clinical outcome of tumors. For tumors with a favorable outcome, the mean value of the number of mitosis (on 10 randomly selected high power fields) was 1,98 (from 0 to 27). For tumors with a malignant behavior, it was 18,53 (from 0 to 75) [12].
The evaluation of the MI in conjunction with nuclear atypia classification (Figure 13) offers a more precise value the histological diagnosis [8].
Canine cutaneous melanoma with moderate diferenciation (H&E, 400x).
Canine cutaneous melanoma with evident nuclear atypia and numerous mitosis (arrow), (H&E, 400x).
Cellular pleomorphism criteria include several features, such as cell size and shape, pigmentation degree and nuclear features (including prominence of nucleoli and chromatin pattern). The usefulness of these parameters as individual prognostic factors is doubtful; however, it increases when these are used together [8,27].
Degree of pigmentation is highly variable, even within a single smear (Figures 14, 15 and 16). Even on histological evaluation of amelanotic-classified tumours is common to detect a few very fine pigment granules in some cells. These are generally punctuate, spherical or elongated, as observed in pigmented keratinocytes. Cells with a very fine pigment may present a dusty gray appearance, instead of the typical granulation image [7]. In summary, it can be difficult to accurately diagnose an amelanotic melanocytic neoplasm and to define the degree of pigmentation [13].
Individual tumoral cells possess a different amount of melanin, which granules are generally small and uniform in size within the same cell. Splindle, round to polygonal, and balloon cells have sparsely-distributed melanin granules, while large epithelioid and dendritic cells are known to have a higher degree of pigmentation [43].
The majority of canine melanocytomas (except for ballon cell ones) have marked to moderate melanin pigmentation overall, especially in the superficial aspect of the tumors [43]. Similarly to cellular morphology, the degree of pigmentation of neoplastic cells was not an indicator of prognosis [12].
Canine cutaneous melanoma with marked pigmentation (H&E, 400x).
Canine cutaneous melanoma with moderate pigmentation (H&E, 400x).
Amelanotic melanoma (H&E, 400x).
Junctional activity refers to the proliferation of neoplastic melanocytes at the interface between the epidermis and dermis or epithelium and submucosa [53].
The presence or absence of junctional activity is not specific to melanoma and often occurs in melanocytomas [7], however, malignant melanomas arising in the skin often show marked junctional activity, (Figure 17 and Figure 18) [42].
Junctional activity was not statistically associated with survival for skin neoplasms in one study [8]. In contrast, another work considered junctional activity as an independent prognostic factor (p =0,0239) for cutaneous melanocytic neoplasms, and found that its occurrence was associated with a longer survival time (p = 0,0046) [55].
Canine cutaneous melanoma without junctional activity (H&E, 200x).
Canine cutaneous melanoma with junctional activity (H&E, 400x).
The presence of intraepidermal tumoural cells can be graded as absent, slight (25% of neoplastic melanocytes in epidermis), moderate or prominent (more than 50% of tumour cells are present in the epidermis) [44]. However, this feature appears not to be of prognostic significance [8].
In a recent study was found that the samples of canine melanomas presented medium to prominent scatter of intraepidermal melanocytes, lower pigmentation, and higher nesting of intraepidermal melanocytes, in comparison with melanocytomas [44].
In most melanocytomas, the overlying epidermis is hyperpigmented, regardless of the presence or absence of intraepidermal clusters of neoplastic melanocytes [43].
Canine melanomas are most frequently ulcerated [44] and particularly larger masses [43] than melanocytomas.
According to Laprie and team [55], ulceration might be taken as a prognostic marker for this neoplasms. In the referred work, the presence of an ulcerated epidermis was associated with a shorter survival time (p = 0,0023) and shown to be an independent prognostic factor (p = 0,0065) [55]. However, two other studies found no correlation between ulceration and clinical evolution of lip, nail bed [27] or cutaneous melanocytic neoplasms [12,27].
Melanocytic tumours strictly limited to the dermis, with a shallow depth, are associated with a greater survival time (p < 0,0001), and deep level of infiltration has been shown to be a significant prognostic factor (p = 0,0012) [55]. One other study that evaluated the level of invasion (in cutaneous and subcutaneous tissues) concluded that tumors confined to the superficial dermis were associated with a benign course in 94% of cases. On the other hand, tumors reaching the deep dermis and the subcutis showed a malignant behavior; however, the sample number were too low to allow for a conclusion [12].
Necrosis is a common feature, particularly in larger masses [43]. The presence of necrosis was correlated with malignancy and with a short survival time in a study set of 389 melanocytic neoplasms containing both benign and malignant lesions from various locations (mouth, feet and lip, skin) [8]. In other report with a set of 38 malignant melanomas from various locations, no correlation was found with survival time [54]. In summary, necrosis is considered of limited prognostic value in animals [7].
Melanocytomas include dermal, compound, and balloon cell tumors, as well as multiple dysplastic melanocytoma syndrome in dogs [43].
Dermal melanocytoma are strictly intradermal in location and larger tumours may extend into the subcutis [43]. Melanocytomas are generally composed by spindle cells disposed in bundles, nests and whorles, with a moderate cellular concentration and a lack of stromal collagen [43,45]. Melanophages might be dispersed throughout the tumoral nodule or in aggregates [43]. Mitotic figures are ocasionally seen (inferior to 1 per 10 high power fields), and mitotic atypia is not observed [43].
Some dermal melanocytomas are composed of epithelioid or dendritic cells that are heavily pigmented [43]. Nuclear morphology may be obscured by the large amount of pigment [43]. Although nuclei may be large, there is minimal nuclear pleomorphism [43].
A compound melanocytoma has a wedge-shaped configuration, and its description refers to the fact of including both junctional and dermal components. A numerous and densely packed tumor cell population is present in the dermis, while a variable amount of tumor cells accumulate in clusters and nests within the epidermis, along the dermal–epidermal junction and in the outer follicular wall – this pattern is referred to as ‘junctional activity’ [43].g
Balloon cell melanocytoma have a dermal location, and are predominantly composed of large round cells [43,45], although some fusiform or polygonal melanocytes might be present [42], with an abundant, pale eosinophilic and finely granular cytoplasm [42,43,45]. These lesions often lack readily visible pigmentation; however, dust-like melanin granules may be detected in small numbers [42,43]. Nuclei are small, uniform, and ovoid [43,45] and mitotic figures are rarely observed [42,43].
Multiple dysplastic melanocytoma syndrome mostly resemble compound melanocytomas on low magnification. However, their incidence increases in larger lesions. Also, cytologic atypia and mitotic figures are present and some of the proliferating melanocytes have irregularly shaped and enlarged hyperchromatic nuclei [43].
Canine melanocytoma–acanthomas are mixed tumors that are composed of a benign melanocytic proliferation, resembling compound melanocytoma, and a benign epithelial proliferation [43,45]. The epithelial component usually appears follicular and resembles an isthmus-type tricholemmoma or infundibular keratinizing acanthoma [43]. The epithelial population forms a mass in the dermis composed of cords and nests with occasional small cystic structures containing keratin [45]. Melanocytic cells form nests in the epidermis and sometimes in the cords of epithelial cells within the dermal mass; melanocytic spindle cells can form whorls and bundles between the epithelial cords and nests [45]. A dermal melanocytoma– acanthoma has been immunophenotyped in a German Shepherd dog identifying the presence of keratinocytes and melanocytes [56].
Dermal melanomas have no junctional activity, but in some cases the tumour might extend deeply into the subcutaneous tissue. There might be a predominance of a certain cell type, but most tumors reveal a mixture of spindle cells, round to polygonal cells, and/or epithelioid cells. While spindle cells are poorly pigmented, more round and epithelioid cells tend to have a moderate to abundant amont of melanin granule [43]. Other important features in this kind of neoplastic lesions include a marked nuclear pleomorphism, nucleolar prominence, a moderate mitotic rate (3 or greater per 10 high power fields), atypical mitotic figures, asymmetry of the tumor nodule and a lymphoplasmacytic cell population [43].
Melanomas have an obvious ‘junctional activity’ pattern, with tumor cells distributed through the dermal–epidermal junction, as well as at higher levels of the epidermis, particularly in those of the nail bed and lip [43]. The intraepidermal element is mainly composed of epithelioid melanocytes, disposed individually or arranged in nests and clusters [43]. Tumours with numerous melanocytes distributed through all levels of the epidermis are referred to as lesions with a ‘pagetoid’ pattern [43]. Other melanomas present numerous individual neoplastic melanocytes present within the basal cell layer only, which is also referred to as an ‘atypical lentiginous infiltrate’ [43].
Spindle cell and desmoplastic melanomas, a subgroup of dermal melanomas, is composed predominantly of spindled melanocytes densely packed, or arranged loosely within abundant pale stroma [43]. Occasionally, there is a prominent fibroblastic component associated with the spindle shaped melanocytes, and collagen may become more abundant than the tumor cells [43]. The vast majority are amelanotic, thus a Fontana–Masson stain usually is necessary to detect the presence of melanin granules; cells are usually arranged in bundles or palisades, mimicking tumors of neural origin [43].
Balloon cell melanoma (clear cell melanoma), possess large cells with a clear eosinophilic cytoplasm [43]. The majority of balloon cell melanomas are amelanotic [43]. Some dust-like melanin granules may be present in a few tumor cells, and Fontana–Masson staining may be required for their demonstration [43,45]. These cells have a large vesicular nuclei with a prominent nucleoli [7,43]. Mitotic activity is generally low [43,45] and these dermal masses exhibit no junctional activity [45].
Signet-ring melanomas are composed of round to polygonal cells [45,52], with a pale eosinophilic cytoplasm and a darker periphery [52]. The nuclei are vesicular, crescent shaped and located at the periphery, giving the cells the appearance of signet-rings [52]. Nucleoli are prominent and occasional multinucleated cells may be present [45].
The histological diagnosis of melanoma can be a challenge for the pathologist, especially in amelanotic tumours. On the other hand, in tumours heavily pigmented the observation of cellular features could be very difficult, requiring the use of bleaching, a histochemical method where melanin is extracted [7].
In cases where the diagnosis of a melanocytic tumor is not evident, histochemical methods specific for the cells producing melanin, such as DOPA (dihydroxyphenylalanine) reaction can be used [7,43].
A diagnosis of melanocytic tumors in dogs is not always easy to obtain only by conventional histological methods. Melanoma is often similar to other tumours types and has a highly variable histologic pattern which implies an accurate differential diagnosis. Furthermore, the distinction between benign and malignant tumors is not always easy [7]. Thus, it is essential the research of additional tools that can be used in melanocytic tumours diagnosis and to achieve a more accurate prognosis [57].
Several markers are used to evaluate the presence of proteins normally found in melanocytes or in cells of neuroectoderm origin. The most common antibodies used are S-100 protein,
In the absence of an ideal marker that excludes definitively other tumour types, confirms a diagnosis of canine melanoma and positively reacts with tumour cells in all melanomas, a diagnostic panel must be performed, including different antibodies [58-61].
The proliferative activity has provided valuable information on cell growth kinetics and consequently tumour behavior in melanocytic tumours [63]. There are some markers that estimate tumor proliferation, by identifying steps associated with cell cycle [64].
MIB-1 is the monoclonal antibody which is reactive against Ki-67 nuclear antigen, a protein present in all active phases of the cell cycle (G1, S, G2 and M) while being absent in resting phase (G0) [65,66].
In canine melanocytic tumours, Ki-67 may be useful in distinction between benign and malignant tumors: melanocytomas seem to have a significant lower growth fraction than malignant melanomas [67]. Furthermore, KI-67 could be an important prognosis factor in canine cutaneous melanocytic tumours [64,68].
Changes in DNA content may reflect chromosomal alterations and represent tumour genetic instability [69-72]. Changes in the DNA ploidy constitute an early event in carcinogenesis [73,74] and detection of aneuploid cell population of pre-neoplastic lesions can be considered a factor risk of the emergence of malignant tumours [75]. Although usually benign tumors were diploid [76] and aneuploidy were malignant [77], diploidy is not synonymous of a benign behavior [78]. Moreover, not all malignant tumors are aneuploid [79,80].
There are few studies about the ploidy assessment in canine melanocytic tumors [81,82] and its usefulness is still discussed. DNA index and ploidy balance seem to provide an additional tool to evaluate melanocytic tumors, being useful in the distinction of benign and malignant melanocytic tumours, mainly in amelanotic lesions [82]. Flow cytometry apparently has a limited utility for predicting the biological behavior of pigmented canine melanomas. DNA content and nuclear morphometric variables have little value in predicting survival time [83].
The c-Kit protein (CD117), a transmembrane receptor that belongs to RTK III family, is a growth factor for melanocyte migration and proliferation. A loss-of-function KIT mutation are usually related with human melanocytic tumors [84,85].
In canine cutaneous melanocytic tumours, c-kit immunolabeling (both extension and intensity) were generally higher in melanocytomas than in malignant melanomas. The lack of c-kit expression in canine cutaneous malignant melanomas might be used as a criteria of tumor aggressiveness, helping to achieve a proper diagnosis [86].
MMPs are zinc- and calcium-dependent proteases that promove not only the disruption and remodeling of structural barriers [87-89] but also a response to signaling molecules, acting as ligand for cellular adhesion receptors [90-92].
MMP-2 is widely distributed and constitutively expressed by most cells [93,94]. This protease has major roles reducing cell adhesion, stimulating cell migration and differentiation, and acting as an anti-inflammatory factor [95]. MMP-9 expression is normally induced, while almost MMPs are constitutively secreted after their translation [93,96], and may act as anti- or pro-inflammatory factors [95].
MMPs play a pivotal role in cancer development and progression [92,94,96-98] contributing to tumour proliferation, invasion, intravasation into circulation, extravasation, migration to metastatic sites and angiogenesis [90,94,99-101], deregulate the balance between growth and antigrowth signals in the tumour microenvironment [97,102,103], orchestrate inflammation [94,102,104], and evade apoptosis [89,91,102].
In canine cutaneous melanocytic tumours, MMP-2 and MMP-9 may be taken as a complement to histology in tumour diagnosis, especially in borderline lesions. Both MMP-2 and MMP-9 were expressed in the majority of canine cutaneous melanocytic tumours. MMP-2 is most commonly expressed in melanocytomas than in melanomas [105]. MMP-9 was overexpressed in malignant melanomas, compared with its expression in melanocytomas [106].
Additionally, in canine malignant melanomas a switch may occur in the MMP expression profile during tumour progression; meaning that the aggressiveness, evaluated by nuclear grade, seems to be associated with a decrease of MMP-9 and an increase of MMP-2 expression [105].
The tumour associated inflammatory infiltrate may be modulate and determine tumor behavior [107]. The most studied cells in CCMT are macrophages and T-lymphocyte, however, studies on the matter are scarce.
Macrophages constitute the most abundant leukocytes in the tumor environment, recruited by a number of chemoattractants that are produced by the tumor cells and tumor-associated stroma [108,109]. TAMs play a critical role in tumour progression and invasion by inducing neovascularization, suppressing immunocompetent cells and supporting cancer stem cells [110-113].
One study published by our group found that canine cutaneous melanocytomas present a lower number of TAMs than malignant melanomas. TAMs could constitute an important marker of canine melanocytic aggressiveness, being implicated in the progression of melanocytic precursor lesions to malignant melanoma [114].
In spite of the fact that the role of the T-lymphocytic infiltrate in cancer tumourigenesis remains controversial, several studies showed that the presence of TILs are related to tumoural behavior in different tumour types [115,116].
Preliminary studies of our group showed that there is a difference between TILs in benign and malignant melanocytic neoplasms, whereas all melanocytomas presented little or even absence of TILs and melanomas had a more intense TILs, (Figure 19), [117].
Abundant CD3+TILs in canine cutaneous melanoma (IHC, 100x), courtesy of Dr. Patricia Monteiro.
Epithelial cadherin (E-cadherin) is a transmembranar glycoprotein which belongs to a family of cell to cell adhesion molecules dependent of the present of calcium molecules to bind cytoskeleton proteins through catenins. A decreased or altered expression of E-cadherin molecules often represents an increased invasiveness of tumour cells and ultimately malignancy in animal and human epithelial tumours [118,119].
In canine melanocytic tumours, the benign lesions present a membranous labelling (Figure 20) and the malignant ones an erroneous labelling, with a cytoplasmatic predominant immunostaining. Additionally, a loss of E-cadherin expression is noted in melanoma [120]. A loss of membrane E-cadherin/β-catenin complex is also detected in canine melanoma showing that a disruption of E-cadherin/β-catenin complexes and a increase of β-catenin may be associated with canine melanocytic tumours progression and aggressiveness [121].
Strong membranar E-cadherin expression (IHC, 200x), courtesy of Dr. Mariana Santos.
Cyclooxygenase (COX), also known as the prostaglandin H-synthase, is an enzyme envolved on prostanoids biosynthesis. Cox-1 and Cox-2 are the two cyclooxigenase isoforms identified to date, similar in its structure but produced by different genes. The biological functions are also different: Cox-1, constitutively expressed in many tissues, plays an important role in the regulation of normal physiological, while Cox-2 is usually absent from normal cells but induced by growth factors, inflammatory reactions, tumour promoters and oncogenes [122-125].
Cox-1 and Cox-2 expression was recently described in canine melanocytic tumours [126-128]. Cox-1 is expressed in almost every tumours, both benign and malignant melanocytic skin lesions. Regarding Cox-2, melanocytomas did not present a positive immunolabelling, but in melanomas Cox-2 expression was present in more than 50% of the tumours [114,127]. The differences observed suggest that Cox-2 expression could be a useful tool in canine melanoma diagnosis, particularly in borderline lesions.
COX-2 expression was also observed in tumours with epithelium ulceration, necrosis, high mitotic index and nuclear grade and in less pigmented neoplasms, which could represent the higher aggressiveness of Cox-2 positive melanocytic tumours. In canine malignant melanomas, Cox-2 is associated with a higher cellular proliferation [114]. Besides the relation with tumour behavior [127], Cox-2 over-expression relates with a poor overall survival [129].
Vascular system is essential in oxygen and nutrients supply, elimination of metabolism products and promoting efficient access of leukocytes [130].
Angiogenesis is a complex process by which new blood vessels develop from pre-existing vasculature [131-133]. It is a fundamental requirement for organs development. Angiogenesis is also implicated in the pathogenesis of different pathological alterations, such as cancer and inflammation [134-136].
The tumor-associated neovascularization, by establishing continuity with the systemic circulation, allows tumor cells expressing their critical advantage in growth and facilitates metastization [137-139].
Angiogenesis is a delicate process, tightly regulated by the balance of pro- and anti-angiogenic factors [140]. Among the angiogenic factors, VEGF is the more powerful and ubiquitous vascular endothelial growth factor, capable of inducing proliferation, migration, specialization and survival of endothelial cells [141,142]. Functions of VEGF family members related to neoplastic pathogenesis are linked not only to its angiogenic capacity [143], but also with the lymphangiogenesis [144,145], immunosuppression [146-148], stimulation and recruitment of endothelial and hematopoietic precursors of the bone marrow [149,150] and anti-apoptotic activity [150].
In canine melanomas, the only study published on VEGF expression is in oral melanomas. High blood concentrations of VEGF were correlated to a shorter survival time in dogs receiving definitive therapy and were associated with tumour stage [151].
This is a promising area, since VEGF may be a good indicator of preneoplastic change in melanocytic lesions [137,152]. VEGF plays a role in human melanoma progression [153,154] with a strong involvment in the switch from the radial to the vertical growth phase and the metastatic phase. So, anti-angiogenic agents might even interfere with or block melanoma progression [155].
Preliminary studies performed by Gomes J and Pires I (data unpublished) show that VEGF may be useful as a discriminating factor between malignant melanoma and benign, since it is more intensely expressed in melanomas (Figure 21) than in melanocytomas.
Canine cutaneous melanoma with a strong and diffuse VEGF expression (IHC, 200x), courtesy of Dr. Joana Gomes.
Tumor angiogenesis can be estimated through a quantification of microvessel density (MVD). The most widely used method is the immunohistochemical methods, in which specific markers for endothelial cells are employed, as von Willebrand factor (Figure 22), CD34 and CD31 [156-158].
MVD seems to be important for diagnostic purposes in canine MT’s. MVD is significantly higher in melanoma than in melanocytomas [159] and its expression has been associated with a high mitotic index, necrosis and ulceration (study performed by Gomes J and Pires I, data unpublished). However, its prognostic significance is still discussable [159,160].
Tumoural neovessels positive to von Willebrand factor (IHC, 400x), courtesy of Dr. Joana Gomes.
The incidence of melanoma is increasing annually both in man and in dog. Given that dogs and humans share the same environment and similarities between human and canine melanoma it is urgent to discuss common mechanisms in melanoma development in both species.
Melanoma diagnosis in dogs can be challenging due to the variety of its histological appearances, especially when pathologists are facing amelanotic or metastatic lesions. Although the definitive diagnosis of a melanoma is often difficult by the lack of specific markers that can distinguish these lesions, immunohistochemistry plays a key role in the differential diagnosis with other neoplasms.
Additionally, the distinction between benign and malignant melanocytic tumours is not always easy, especially in borderline lesions, thus the importance of a strong knowledge of new markers of malignancy for the establishment of a definitive diagnosis and a correct therapy managment and prognosis establishment.
Skeletal muscle is the most abundant tissue in human body. Skeletal muscle accounts for approximately 20% of our resting energy expenditure [1], and composes 30–40% of one’s body mass [2] depending on their fitness level [3]. As a part of the musculoskeletal system, skeletal muscle is connected to the skeleton to form part of the mechanical system that moves the limbs and other parts of the body. While skeletal muscle refers to multiple bundles of cells called muscle fibers, the composition of the individual fibers is different between muscle types. In this review, we describe how muscle fiber types are specified during embryonic myogenesis, what potential factors would be involved in the changes of fiber type composition, and how fiber type variations are influenced by specific disease conditions. Knowing the functional role of how muscle fibers contribute to and are affected by skeletal muscle diseases aids in our understanding of the disease and provides insight to mechanisms of prevention, treatment, or cure of these conditions.
\nSkeletal muscle plays important roles in the body that are concerned with movement, posture, and balance under voluntary control. Skeletal muscles are one of three major muscle types, the others being cardiac muscle and smooth muscle, and it is the most common of the three types of muscle in the body. As one component of the musculoskeletal system, skeletal muscle is attached to bones by tendons, and they produce all the movements of body parts in relation to each other. Unlike smooth muscle and cardiac muscle, skeletal muscle is under voluntary control. Similar to cardiac muscle, however, skeletal muscle is striated; it has long, thin, multinucleated fibers (known as myofibers).
\nSkeletal muscle function seems to be maintained across mammals, but the composition of the individual fibers is different between muscle types [4]. Fiber type composition is initially defined in each muscle during embryonic myogenesis. In this section, we will go through the basic fundamentals of skeletal muscle development and fiber type specification (Figure 1).
\nSkeletal muscle differentiation and fiber type specification. The terminal differentiation starts when Pax3+ and/or Pax7+ progenitors begin to express Myf5 or MyoD as committed myoblasts. Theses myoblasts gradually express myogenin (MyoG) and form single-nucleated nascent myotubes and multi-nucleated myotubes with myosin heavy chain (MyHC+). Actin, myosin, and elastic myofilaments are arranged to form organized sarcomeres within the myotubes. Primary myofibers express four isoforms of MyHC: MyHC I/β, MyHC-α, MyHC-emb and MyHC-peri. Development of fiber type continues as satellite cells differentiate and the fibers become innervated, forming mature fiber types. Different isoforms of myosin, MyHC I/β. MyHC-α, MyHC 2A, and MyHC 2x, are expressed. This figure is modified from Jiwlawat et al. [
Studies investigating embryonic myogenesis have been extensively conducted in the embryos of zebrafish, chicken, and mice. After an embryo is generated, three germ layers (ectoderm, endoderm, and mesoderm) are formed. The mesoderm is characterized as paraxial, intermediate, and lateral mesoderm. The formation of skeletal muscle initiates from the paraxial mesoderm in early embryogenesis. In response to the signals from the notochord, neural tube, and surface ectoderm, the paraxial mesoderm forms segmented spheres termed somites. The somites are located as a pair on either side of the neural tube and the notochord and develop in a rostral-caudal direction. The somite is further specified as the dermomyotome, myotome, and sclerotome. The cells in the dermomyotome express the paired box transcription factors Pax3 and Pax7 [5, 6]. The cells in the dorsomedial and ventrolateral portions of the dermomyotome will give rise to the epaxial (primaxial) and hypaxial (abaxial) myotomes, respectively. Myf5-positive cells in the epaxial myotomes differentiate and form the trunk and back muscles. In contrast, MyoD-positive progenitors de-laminate and migrate from the hypaxial myotome into the developing limb as the source of limb muscles. MyoD and Myf5 are expressed in committed muscle cells, and are located in the myotome, which is form the maturation of dermomyotome lips [7, 8, 9]. The ventrolateral lip of the dermomyotome contributes to the hypaxial myotome, which is a source of precursor cells that form the trunk and thoracic vertebral column muscles. The dorsomedial lip of the dermomyotome contributes to the epaxial myotome, which is a source of muscles of the back. The process of myotome maturation originally initiates at the rostral part of the embryo and then extends to the tail [7].
\nThe terminal differentiation of progenitors and myoblasts initiates when myogenic progenitors in the dermomyotome stop dividing and exit the undifferentiated stage [10]. The progenitors differentiate into committed myoblasts, and form nascent myotubes following the maturation of the myotome [11]. More specifically, Pax3 and/or Pax7-positive proliferating progenitors are withdrawn from the cell cycle once the differentiation step is initiated (Figure 1). These progenitors become committed myoblasts expressing Myf5 and/or MyoD and then form the nascent myosin heavy chain-positive myotubes with myogenin-positive nuclei.
\nTwo waves of myotube formation occur during skeletal muscle development, and sequentially give rise to primary and secondary myotubes [12]. Primary myotubes are generated from fusion of early myoblasts, and then align between muscle tendons. Late-stage myoblasts proliferate on the surface of primary myotubes and fuse to form secondary myotubes, and motor axons initiate innervation to the myotubes [12]. At this point, primary and secondary myotubes express specific isoforms of myosin heavy chain (MyHC), which can be used to broadly define two distinct fiber types, slow-twitch Type I and fast-twitch Type II myofibers. Primary myotubes preferentially express Type I fibers [13, 14], while Type II fibers appear later during myogenesis [15, 16]. Single-nucleated myotubes then fuse with the nearby myotubes to form multi-nucleated myotubes. Thick-myosin and thin-actin filaments within the myotube begin organization and form a sarcomere structure, which is the functional unit of muscle contraction. Well-organized sarcomeric structure gives rise to a striation pattern in myotubes, representing many chains of myofibrils.
\nPrimary myogenesis starts during the embryonic stage, when somatic stem cells express the genes Pax3 and Pax7 (Figure 1). This transforms the cells into myogenic progenitors, which migrate from the dermomyotome to form myocytes and primary myofibers. At this point of embryonic myogenesis, three isoforms of myosin heavy chain are expressed; slow MyHC (MYH7), MyHC-emb (MYH3), and MyHC-peri (MYH8) [17]. These primary myofibers serve as a template for the skeletal muscle to mature and differentiate. Secondary myogenesis progresses as satellite cells differentiate, become innervated, and mature myofibers are formed. In whole, genetic influences and motor neuron innervation during developmental differentiation determines the fiber types that one is born with [17]. Fiber type ratios determined at birth are not concrete throughout one’s life however, as skeletal muscle chemical properties can change over time to meet physiological or pathological demands.
\nSkeletal muscle tissue in humans is heterogeneous, composed of a variety of molecules [4]. The main functional proteins and structures within the muscle are maintained, such as mitochondria network, myosin, actin and titin. Yet, the specific isoforms of the molecules and the concentration of each monomer differ between skeletal muscles all throughout the body. These heterogeneous tissues are a resultant factor of evolution which allows each muscle to have a specialized function. The size of each whole muscle is determined by both the number and the diameter of muscle fibers that compose it. Individual muscle fibers are multi-nucleated, with each nucleus controlling the protein type, myosin that is translated in its surrounding. This is known as a nuclear domain [18].
\nMyosin is the main protein within skeletal muscle, and the certain isoform that is expressed determines the rate at which the muscle contracts, as well as its physiological properties. Within a single sarcomere of a skeletal muscle fiber, myosin heads and actin interact to form cross bridges. ATP hydrolyzation via ATPase is responsible for the energy to cause cycling of the myosin head and actin connections, which ultimately causes the muscle contraction. The type of myosin expressed is one factor that ultimately determines the fiber type. There are 11 total isoforms of myosin known to mammals [4, 19], which when expressed in different ratios compose a fiber type with distinct physiological properties. As discussed above, there are two categories of adult muscle fiber types in humans; Type I and Type II fibers (Figure 1).
\nType I and Type II fibers are classified based on their myosin isoform, velocity of contraction and presence of physiological enzymes [3]. Type I fibers are also known as slow oxidative. Compared to Type II, they contain a higher number of oxidative enzymes and a lower number of glycolytic enzymes. They are rich in mitochondria and have a great capillary network to perfuse the fibers [20]. This contributes to their oxidative capacity. Type I muscle fibers predominantly contain myosin isoforms MyHC I/β or MyHC-α, encoded by the gene MYH7 [17] and they contract slower and are more resistant to fatigue than Type II fibers. Because of their endurance properties, Type I fibers are commonly found in muscles mainly involved in posture, such as erector spinae, hamstrings, and gastrocnemius muscles.
\nType II fibers on the other hand are fast to fatigue, as they have low oxidative capacity. These fiber types are recruited in short bursts of movement or power [3]. This is due to their greater maximal velocity of shortening, and abundance of glycolytic enzymes [3]. This in turn allows for quick energy utilization due to increased ATPase activity. There are two subcategories in human Type II fibers; Type IIa and Type IIx. Type IIa are classified as fast-oxidative glycolytic, a sort of combination between fast and slow contraction rates. Type IIx are fast glycolytic, having the fastest rate of contraction of all the human fiber types, yet the shortest time to fatigue. MyHC isoform genes MYH2 and MYH1 are expressed respectively in Type IIa and Type IIx fibers. The myosin protein isoforms present in each subtype are termed MyHC-2A and MyHC-2X [17].
\nSkeletal muscles are innervated by motor neurons which are responsible for the initiation of muscle contraction. Motor units are formed, consisting of a single alpha motor neuron that originates in the spinal cord that innervates a group of skeletal muscle fibers, all of the same fiber type. Changes in motor unit innervation of the skeletal muscle has shown to change the properties of fiber types innervated, therefore motor units too are contributors to the determinants of fiber type [21].
\nSkeletal muscles have the property of plasticity. This means the composition of fiber types within a given skeletal muscle can change when under the influence of physiological changes such as mechanical stress and unloading. Further, abnormal health conditions caused by diseases and injuries also triggers significant changes of muscle fiber types [22]. The size and functional capacity of the muscle can be decreased upon injury, disease, or excess weight. As a result, scar tissue, connective tissue, or fat can take up mass that was once occupied by functional muscle [18]. When muscles become denervated, there is a tendency for slow to fast fiber transition [3]. This carries heavy implications for training status and disease state in humans [3, 23, 24].
\nThe physiological and pathological changes influence the levels of trophic factors, hormones, and nerve signaling associated with the muscle, which result in adaptive changes in muscle fibers. The relative amounts of these factors and the extent of the changes that they can make are ultimately determined by genomic background and epigenetic control in individuals. The genes that one inherits controls and determines 40–50% of the ratio of Type I fibers within a muscle [3]. This means that physiological stressors can impact the plasticity of the muscles to a point, but in the end one’s genetic make-up determines the extent to which the fiber types within the muscle can switch [3]. Like all cells in the body, the different fiber types contain the same genomic DNA sequence. MYH genes have been hypothesized to be clustered in a manner to facilitate temporal and spatial expression of these related genes [23]. Slow MyHC isoforms are located on chromosome 14, while chromosome 17 contains the fast and embryonic MYH genes in a cluster. The difference in gene expression, and resultant protein levels in a specific cell, are controlled by epigenetic mechanisms. As fiber types shift within a lifetime, the epigenetic profile within the cell is also affected, specifically in the amount of acetylation or deacetylation within the genome. This change is mostly seen within differentiating satellite cells, which are not fully mature [23]. Further, variations in expression levels of genes controlling systems such as mitochondrial biogenesis, glucose/lipid metabolism, cytoskeletal function, hypoxia, angiogenesis, and circulatory homeostasis would influence muscle fiber type. The frequency of alleles within a genome also impact the fiber type development [3]. Overall, there are many genetic factors at play such as single gene effects, gene–gene interactions and gene–environment interactions [3].
\nNeuromuscular diseases are caused by functional defects of skeletal muscles, directly via muscle pathology or indirectly via disruption of the nervous system. Most of these diseases are multi-facetted, and terminally result in wasting and atrophy of skeletal muscles. These abnormal conditions often lead to disabilities and complete loss of muscle function, with little to no cure. Pathology is best understood at the cellular level, and here we explore how the progression of the disease is involved in the changes of muscle fiber types, and how changes in fiber type may serve as a protective mechanism. Diseases covered in this chapter are mainly genetic in nature, having an uncontrollable disruption in cellular function that results in disease. This can either be inherited from previous ancestors or be sporadic in nature. This section will introduce several names of muscle and motor neuron diseases; however, this is not an exhaustive list.
\nSarcopenia is a term that refers to the loss of lean body mass, particularly skeletal muscle, with an increase in aging [25]. This can be diagnosed through weakness within the body, difficulties walking, or dual-energy absorptiometry, which is a machine that tells the exact body composition of fat, bone mass and tissue. Sarcopenia at the individual fiber level is characterized by a loss of satellite cells associated with Type II fibers [18]. Organelles affected in the myofibers include a decreased amount of mitochondria, an alteration in the sarcoplasmic reticulum, and hindered excitation-contraction coupling. Both Type I and II fibers have shown to be affected by losing their maximal force in both men and women. This is attributed to a loss of myosin expression within the cell, or oxidation of the myosin protein which inhibits the formation of crosslinks [18]. Surprisingly, the expression levels of myosin isoform MYH7, that of slow muscle fibers, are not affected [26].
\nMuscular dystrophies are a group of muscle diseases that result in the wasting of skeletal muscles, caused by muscle fiber necrosis [27]. The dystrophies involve mutations in genes that encode functional proteins involved in dystrophin or enzymes that modify the dystrophin proteins [18]. These mutations affect velocity of cross bridge cycling of actin filaments on myosin and of particular interest, they change the quality and force production of Type I and Type II fibers [18]. Apoptosis and necrosis in fiber types are a trademark of the disease, with caspase 3 being a known apoptotic gene that is upregulated in muscular dystrophies compared to unaffected individuals [27].
\nIn Duchenne Muscular Dystrophy, Type II muscle fibers are the first to be affected with Type I muscle fibers following late in the disease progression [26]. Remaining Type I fibers are not similar to those found in healthy muscle. Degeneration and regeneration of diseased fibers is hypothesized to take place, due to coexpression of fetal MYH and slow MYH genes in adult muscle fibers [28]. Since Type II fibers are the most commonly affected in Duchenne Muscular Dystrophy, it is thought that inducing the expression of Type I fibers will alleviate both the symptoms and progression of Duchenne Muscular Dystrophy. A similar trend was found in another type of muscular dystrophy, Facioscapulohumeral Muscular Dystrophy, as there is an early decrease in Type II fibers and an overall increase in the number of Type I fibers [29]. On the contrary, in myotonic dystrophy, Type I fibers are affected, as they atrophy more frequently and they lose a greater amount of force generation compared to Type II fibers [30, 31, 32]. One hypothesis for this fiber type susceptibility to disease states is variation to transcriptional control of muscle fiber type. Genetic manipulations and pharmacological interventions have shown the effect of fiber type switching on disease sates in mice [26]. For example, over expression of the transcriptional coactivator PPARGC1A rescues the cellular defects cause by the Dmdmdx mutation via increased expression of Type I fiber contractile machinery and oxidative enzymes [26].
\nMotor neuron diseases are characterized by the progressive degeneration of motor neurons with subsequent functional loss. In the motor system, motor neuron axons carry the motor impulses from the spinal cord to the voluntary muscles. Innervation of alpha motor neurons from the central nervous system has a large part in determining the fiber type that is expressed within muscles. Motor units innervate muscle fibers in an “all or none” fashion, meaning a single motor unit innervates Type I and each subcategory of Type II fibers individually, and all the fibers that the motor unit innervate are of the same fiber type.
\nCo-expression of fiber types within a single muscle fiber has been seen in motor neuron diseases such as Amyotrophic Lateral Sclerosis (ALS) and Spinal Muscular Atrophy [33]. In these diseases, specifically ALS, studying disease influence on skeletal muscles would provide valuable insight to the mechanisms of disease progression. Changes in muscle fiber types may occur at the early stage of diseases by reduced inputs from motor neurons, as disconnections between muscle and axon terminals have been observed in animal models of motor neuron diseases before symptom onset. Studying the disease onset in skeletal muscles has the potential to reveal the catastrophic pathology influence and the body’s compensatory mechanisms to counteract disease progression [34].
\nSwitches in muscle fiber type has been observed in patients in motor neuron diseases, however the switches cannot prevent the ultimate outcome: apoptosis and necrosis of individual muscle fibers [27]. Often, motor neuron diseases are diagnosed clinically via histochemical staining of muscle biopsies. Necrosis can be easily seen as fat or scar tissue under the microscope, but apoptosis is harder to identify due to the lack of inflammatory response from the body [27]. Denervation of the muscle results in upregulation of pro-apoptotic genes, such as bax and anti bcl-2, which are upregulated due to intrinsic cell stress. Muscle fiber atrophy is hypothesized to be caused by apoptosis induced degradation of a fiber’s nuclei. This includes destruction of the nuclear lamina, the nuclear envelope, and DNA destruction [27].
\nAmyotrophic Lateral Sclerosis (ALS), also known as Lou Gehrig’s disease, is a fatal neurodegenerative disease caused by the selective loss of motor neurons in the spinal cord and brain stem. Motor neuron degeneration and neuromuscular junction denervation rapidly result in decreased motor function. Death typically results 3–5 years after diagnosis due to respiratory failure after loss of diaphragm control. About 90% of ALS cases occur sporadically; the remaining 10% are familial components. Approximately 70–80% of familial ALS have mutations of the Cn2+/Zn2+ superoxide dismutase 1 (
Although a disease cause of sporadic ALS has not been specified, this disease is generally regarded as resulting from factors involving environment, lifestyle, aging, and genetic predisposition [36]. Several proposed pathological mechanisms of disease include protein misfolding and aggregation, glutamate excitotoxicity, oxidative stress, mitochondrial dysfunction, glial cell activation and related inflammatory processes, and axonal transport defects [37].
\nALS causes motor neuron death and gradual denervation of skeletal muscles over time. This denervation causes loss of muscle function and muscular atrophy within affected cells, ultimately resulting in cellular death due to apoptosis. While many of existing ALS therapies are expected to promote motor neuron survival in the spinal cord or motor cortex [38], looking at the pathologies within skeletal muscle gives support for the “dying-back” hypothesis. This hypothesis states that irregularities within the skeletal muscle are the primary cause of ALS, denervating muscle and motor neuron [39]. Based on this hypothesis, possible contributions of skeletal muscles and neuromuscular junctions in ALS pathology have been proposed in recent studies. Specifically, our research group also reported that using stem cells to deliver growth factors directly into the skeletal muscle could restore motor function in a rat model of familial ALS [40, 41, 42]. Our approach can sufficiently protect motor neurons by preventing the “dying back” of these cells from the skeletal muscle in ALS.
\nWhile the majority of ALS patients have limb onset, about 25% of cases eventually diagnosed with ALS have bulbar onset which strikes the corticobulbar area in the brain stem. This section controls muscles in the face, neck and head. Bulbar onset usually affects voice and swallowing first. Patients with bulbar onset have a correlation between the age of death and the loss of slow tonic fibers, although there is neither correlation seen in spinal onset ALS nor controls [34].
\nALS pathology affects skeletal muscle in many ways, which seems to influence muscle fiber type changes. Autopsies show fiber atrophy, fiber grouping, fiber splitting, with increased fatty tissue and connective tissue [43]. Interestingly, unlike atrophy from exercise, ALS shows a fast to slow fiber type switch [39, 44]. In the hindlimb muscle (tibialis anterior muscle) of pre-symptomatic ALS model mice, there is denervation of the most forceful and fast to fatigue fibers Type IIB (only found in mice). This results in transitions to fast motor units with intermediate fatigue and fatigue resistant fibers. Although this transition is present, it is not a sudden change nor a complete loss of Type IIB fibers [44]. Biopsies taken from atrophied skeletal muscles in patients with ALS have shown that individual muscle fibers contain myosin isoforms corresponding to both fiber Types I and II, termed a mixed fiber type. An early pattern of denervation can be detected and has the potential to be used for diagnostic purposes. This pattern is individual fibers with a mixed fiber type and little fiber type grouping, all within an atrophying muscle [45].
\nIt has been reported that specific muscle groups such as extraocular muscles are relatively spared from the disease phenotype in ALS [46]. Motility of the eye is often maintained in ALS patients [47] and autopsies have shown the extraocular muscles do have some muscle fiber pathology compared to control, but in relation to other ALS affected skeletal muscles in the body, the extraocular muscles were well preserved [43]. The pathology that was seen include change in fiber type composition, the cellular architecture, and decreased overall MyHC content. Embryonic MyHC was almost nonexistent in the extraocular muscles in those affected by ALS [43].
\nThis preservation of extraocular skeletal muscle is accredited to the distinct fiber type composition within the extraocular muscles. Extraocular muscles have a unique myosin expression that is not found in skeletal muscles located other places of the body. Along with Type I and Type II fibers, a special myosin isoform, MyHC extraocular, is present and Type I fibers seem to express two separate forms of MyHC, of specific interest MyHC α cardiac. [43]. Embryonic MyHC has notable expression in the extraocular muscles, as healthy human controls show co-expression of embryonic MyHC in Type II fibers, while ALS patients had no embryonic MyHC expression [43, 48].
\nAlthough there is great speculation, the exact mechanism of why extraocular muscles are spared in ALS is unknown. However, one interesting hypothesis is the multiple innervations of slow tonic fibers serve as a protective mechanism against the neurodegenerative disease [48]. It has also been found that the motor neurons of the extraocular muscles have different surface markers than motor neurons found elsewhere in the body, suggesting they have properties that make the neurons less susceptible to disease [49]. As an additional note, similar specific insusceptibility in the extraocular muscles has also been observed in Duchenne Muscular Dystrophy [50].
\nAnother question is how sex influences fiber type specification in the muscle during ALS pathology. The exact etiology of ALS is still uncertain, but most epidemiological studies have shown a higher incidence of ALS in men than women. Interestingly, sexual dimorphism in disease onset and progression is also observed in rodent models of familial ALS [51, 52]. Although it is still uncertain whether such sexual differences are originated from the intrinsic difference in individual cells [53], further studies would be required to answer this question.
\nSpinal Muscular Atrophy (SMA) is a group of motor neuron diseases, which are autosomal recessive in nature. Each SMA type has a different clinical outcome, however all SMA types commonly demonstrate motor neuron degeneration caused by insufficient expression of a specific protein named Survival of motor neuron (SMN) [54]. The clinical severity of SMA ranges from I–IV, with IV being the least severe. I is infantile SMA that causes death early in childhood and IV involves some motor neuron loss, but allows for a normal life expectancy [55].
\nAll cases of SMA result from reductions in levels of the SMN protein. Specifically, SMA is caused by deletion or mutation of the survival motor neuron gene (SMN1). The SMA disease is present in a spectrum of disease severities ranging from infant mortality, in the most severe cases, to minor motor impairment, in the mildest cases. The variability of disease severity inversely correlates with the copy number, and thus expression of a second, partially functional survival motor neuron gene, SMN2.
\nIn type III SMA-induced mice, muscle atrophy resulted in a transition to slower, oxidative phenotype. This meaning that there were more Type I fibers in the soleus muscle and Type II fibers in fast twitch muscles transitioned to a more oxidative fiber type [54]. These same mice also had smaller motor neurons units than controls and the Type I motor neurons decreased in size as the disease progressed. Other studies that have used type III SMA-induced mice have shown to have increased fiber type grouping compared to wild type [56].
\nThere has been evidence that these pathological changes in muscle fiber types can be reversed. Swimming aided the mice to regain more glycolytic fast twitch fibers, and restore Type I motor unit size close to wild type levels [54]. Running produced more Type I fibers compared to sedentary SMA mouse control [54] and was able to restore SMA fast fiber types. Upon completion of exercise intervention by type III SMA-induced mice, their structure and number of the Type I fibers were comparable to controls [54].
\nIn humans, it has been shown that innervation of fibers in children with SMA (specifically Werdnig-Hoffmann disease) is incomplete. This results in atrophy of fibers and the inability of fetal MyHC to switch to adult Type I and Type II myosin. When this observation was tracked through childhood it showed that in infancy, there is a large increase in the number of Type I fibers, and no detectable Type II fibers by 20 months. This further emphasizes the need for motor neuron innervation for Type II fibers to prevail [4, 57].
\nMuscle fiber type composition is primarily determined during development but will be altered by physiological and pathological conditions. Significant changes of fiber type composition have been identified in the muscles with a background of major neuromuscular diseases. To further understand the roles of muscle fiber composition in skeletal muscle development and diseases, additional studies using new research approaches may help us understand how muscle fiber type specification occurs during development and disease conditions. For instance, skeletal muscle cell culture derived from human pluripotent cell resources can provide a new tool to study how human skeletal myocytes differentiate into myotubes with specific fiber types in culture [58, 59]. These studies could highlight what specific mechanisms are involved in the significant changes of fiber type composition and ratio in the skeletal muscle during embryonic myogenesis and under disease conditions, and how these changes of muscle fiber types impact on muscle physiology and pathology.
\nThis work was supported by grants from the ALS Association (15-IIP-201, Masatoshi Suzuki), NIH/NINDS (R01NS091540, Masatoshi Suzuki), and the University of Wisconsin Foundation (Masatoshi Suzuki).
\nThe authors declare that there is no conflict of interest regarding the publication of this paper.
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Currently, he is a professor of Orthodontics. He holds a Certificate of Advanced Study type A in Technology of Biomaterials used in Dentistry (1995); Certificate of Advanced Study type B in Dento-Facial Orthopaedics (1997) from the Faculty of Dental Surgery, University Denis Diderot-Paris VII, France; Diploma of Advanced Study (DESA) in Biocompatibility of Biomaterials from the Faculty of Medicine and Pharmacy of Casablanca (2002); Certificate of Clinical Occlusodontics from the Faculty of Dentistry of Casablanca (2004); University Diploma of Biostatistics and Perceptual Health Measurement from the Faculty of Medicine and Pharmacy of Casablanca (2011); and a University Diploma of Pedagogy of Odontological Sciences from the Faculty of Dentistry of Casablanca (2013). 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He also obtained an MSc in Molecular and Genetic Medicine, and a Ph.D. in Clinical Immunology and Human Genetics from the University of Sheffield, UK. He also completed a short-term fellowship in Pediatric Clinical Immunology and Bone Marrow Transplantation at Newcastle General Hospital, England. Dr. Rezaei is a Full Professor of Immunology and Vice Dean of International Affairs and Research, at the School of Medicine, Tehran University of Medical Sciences, and the co-founder and head of the Research Center for Immunodeficiencies. He is also the founding president of the Universal Scientific Education and Research Network (USERN). Dr. Rezaei has directed more than 100 research projects and has designed and participated in several international collaborative projects. He is an editor, editorial assistant, or editorial board member of more than forty international journals. He has edited more than 50 international books, presented more than 500 lectures/posters in congresses/meetings, and published more than 1,100 scientific papers in international journals.",institutionString:"Tehran University of Medical Sciences",institution:{name:"Tehran University of Medical Sciences",country:{name:"Iran"}}},{id:"180733",title:"Dr.",name:"Jean",middleName:null,surname:"Engohang-Ndong",slug:"jean-engohang-ndong",fullName:"Jean Engohang-Ndong",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/180733/images/system/180733.png",biography:"Dr. Jean Engohang-Ndong was born and raised in Gabon. After obtaining his Associate Degree of Science at the University of Science and Technology of Masuku, Gabon, he continued his education in France where he obtained his BS, MS, and Ph.D. in Medical Microbiology. He worked as a post-doctoral fellow at the Public Health Research Institute (PHRI), Newark, NJ for four years before accepting a three-year faculty position at Brigham Young University-Hawaii. Dr. Engohang-Ndong is a tenured faculty member with the academic rank of Full Professor at Kent State University, Ohio, where he teaches a wide range of biological science courses and pursues his research in medical and environmental microbiology. Recently, he expanded his research interest to epidemiology and biostatistics of chronic diseases in Gabon.",institutionString:"Kent State University",institution:{name:"Kent State University",country:{name:"United States of America"}}},{id:"188773",title:"Prof.",name:"Emmanuel",middleName:null,surname:"Drouet",slug:"emmanuel-drouet",fullName:"Emmanuel Drouet",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/188773/images/system/188773.png",biography:"Emmanuel Drouet, PharmD, is a Professor of Virology at the Faculty of Pharmacy, the University Grenoble-Alpes, France. As a head scientist at the Institute of Structural Biology in Grenoble, Dr. Drouet’s research investigates persisting viruses in humans (RNA and DNA viruses) and the balance with our host immune system. He focuses on these viruses’ effects on humans (both their impact on pathology and their symbiotic relationships in humans). He has an excellent track record in the herpesvirus field, and his group is engaged in clinical research in the field of Epstein-Barr virus diseases. He is the editor of the online Encyclopedia of Environment and he coordinates the Universal Health Coverage education program for the BioHealth Computing Schools of the European Institute of Science.",institutionString:null,institution:{name:"Grenoble Alpes University",country:{name:"France"}}},{id:"131400",title:"Prof.",name:"Alfonso J.",middleName:null,surname:"Rodriguez-Morales",slug:"alfonso-j.-rodriguez-morales",fullName:"Alfonso J. Rodriguez-Morales",position:null,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},{id:"332819",title:"Dr.",name:"Chukwudi Michael",middleName:"Michael",surname:"Egbuche",slug:"chukwudi-michael-egbuche",fullName:"Chukwudi Michael Egbuche",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/332819/images/14624_n.jpg",biography:"I an Dr. Chukwudi Michael Egbuche. I am a Senior Lecturer in the Department of Parasitology and Entomology, Nnamdi Azikiwe University, Awka.",institutionString:null,institution:{name:"Nnamdi Azikiwe University",country:{name:"Nigeria"}}},{id:"284232",title:"Mr.",name:"Nikunj",middleName:"U",surname:"Tandel",slug:"nikunj-tandel",fullName:"Nikunj Tandel",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/284232/images/8275_n.jpg",biography:'Mr. Nikunj Tandel has completed his Master\'s degree in Biotechnology from VIT University, India in the year of 2012. He is having 8 years of research experience especially in the field of malaria epidemiology, immunology, and nanoparticle-based drug delivery system against the infectious diseases, autoimmune disorders and cancer. He has worked for the NIH funded-International Center of Excellence in Malaria Research project "Center for the study of complex malaria in India (CSCMi)" in collaboration with New York University. The preliminary objectives of the study are to understand and develop the evidence-based tools and interventions for the control and prevention of malaria in different sites of the INDIA. Alongside, with the help of next-generation genomics study, the team has studied the antimalarial drug resistance in India. Further, he has extended his research in the development of Humanized mice for the study of liver-stage malaria and identification of molecular marker(s) for the Artemisinin resistance. At present, his research focuses on understanding the role of B cells in the activation of CD8+ T cells in malaria. Received the CSIR-SRF (Senior Research Fellow) award-2018, FIMSA (Federation of Immunological Societies of Asia-Oceania) Travel Bursary award to attend the IUIS-IIS-FIMSA Immunology course-2019',institutionString:"Nirma University",institution:{name:"Nirma University",country:{name:"India"}}},{id:"334383",title:"Ph.D.",name:"Simone",middleName:"Ulrich",surname:"Ulrich Picoli",slug:"simone-ulrich-picoli",fullName:"Simone Ulrich Picoli",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/334383/images/15919_n.jpg",biography:"Graduated in Pharmacy from Universidade Luterana do Brasil (1999), Master in Agricultural and Environmental Microbiology from Federal University of Rio Grande do Sul (2002), Specialization in Clinical Microbiology from Universidade de São Paulo, USP (2007) and PhD in Sciences in Gastroenterology and Hepatology (2012). She is currently an Adjunct Professor at Feevale University in Medicine and Biomedicine courses and a permanent professor of the Academic Master\\'s Degree in Virology. She has experience in the field of Microbiology, with an emphasis on Bacteriology, working mainly on the following topics: bacteriophages, bacterial resistance, clinical microbiology and food microbiology.",institutionString:null,institution:{name:"Universidade Feevale",country:{name:"Brazil"}}},{id:"229220",title:"Dr.",name:"Amjad",middleName:"Islam",surname:"Aqib",slug:"amjad-aqib",fullName:"Amjad Aqib",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/229220/images/system/229220.png",biography:"Dr. Amjad Islam Aqib obtained a DVM and MSc (Hons) from University of Agriculture Faisalabad (UAF), Pakistan, and a PhD from the University of Veterinary and Animal Sciences Lahore, Pakistan. Dr. Aqib joined the Department of Clinical Medicine and Surgery at UAF for one year as an assistant professor where he developed a research laboratory designated for pathogenic bacteria. Since 2018, he has been Assistant Professor/Officer in-charge, Department of Medicine, Manager Research Operations and Development-ORIC, and President One Health Club at Cholistan University of Veterinary and Animal Sciences, Bahawalpur, Pakistan. He has nearly 100 publications to his credit. His research interests include epidemiological patterns and molecular analysis of antimicrobial resistance and modulation and vaccine development against animal pathogens of public health concern.",institutionString:"Cholistan University of Veterinary and Animal Sciences",institution:null},{id:"62900",title:"Prof.",name:"Fethi",middleName:null,surname:"Derbel",slug:"fethi-derbel",fullName:"Fethi Derbel",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/62900/images/system/62900.jpeg",biography:"Professor Fethi Derbel was born in 1960 in Tunisia. He received his medical degree from the Sousse Faculty of Medicine at Sousse, University of Sousse, Tunisia. He completed his surgical residency in General Surgery at the University Hospital Farhat Hached of Sousse and was a member of the Unit of Liver Transplantation in the University of Rennes, France. He then worked in the Department of Surgery at the Sahloul University Hospital in Sousse. Professor Derbel is presently working at the Clinique les Oliviers, Sousse, Tunisia. His hospital activities are mostly concerned with laparoscopic, colorectal, pancreatic, hepatobiliary, and gastric surgery. He is also very interested in hernia surgery and performs ventral hernia repairs and inguinal hernia repairs. He has been a member of the GREPA and Tunisian Hernia Society (THS). During his residency, he managed patients suffering from diabetic foot, and he was very interested in this pathology. For this reason, he decided to coordinate a book project dealing with the diabetic foot. Professor Derbel has published many articles in journals and collaborates intensively with IntechOpen Access Publisher as an editor.",institutionString:"Clinique les Oliviers",institution:null},{id:"300144",title:"Dr.",name:"Meriem",middleName:null,surname:"Braiki",slug:"meriem-braiki",fullName:"Meriem Braiki",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/300144/images/system/300144.jpg",biography:"Dr. Meriem Braiki is a specialist in pediatric surgeon from Tunisia. She was born in 1985. She received her medical degree from the University of Medicine at Sousse, Tunisia. She achieved her surgical residency training periods in Pediatric Surgery departments at University Hospitals in Monastir, Tunis and France.\r\nShe is currently working at the Pediatric surgery department, Sidi Bouzid Hospital, Tunisia. Her hospital activities are mostly concerned with laparoscopic, parietal, urological and digestive surgery. She has published several articles in diffrent journals.",institutionString:"Sidi Bouzid Regional Hospital",institution:null},{id:"229481",title:"Dr.",name:"Erika M.",middleName:"Martins",surname:"de Carvalho",slug:"erika-m.-de-carvalho",fullName:"Erika M. de Carvalho",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/229481/images/6397_n.jpg",biography:null,institutionString:null,institution:{name:"Oswaldo Cruz Foundation",country:{name:"Brazil"}}},{id:"186537",title:"Prof.",name:"Tonay",middleName:null,surname:"Inceboz",slug:"tonay-inceboz",fullName:"Tonay Inceboz",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/186537/images/system/186537.jfif",biography:"I was graduated from Ege University of Medical Faculty (Turkey) in 1988 and completed his Med. PhD degree in Medical Parasitology at the same university. I became an Associate Professor in 2008 and Professor in 2014. I am currently working as a Professor at the Department of Medical Parasitology at Dokuz Eylul University, Izmir, Turkey.\n\nI have given many lectures, presentations in different academic meetings. I have more than 60 articles in peer-reviewed journals, 18 book chapters, 1 book editorship.\n\nMy research interests are Echinococcus granulosus, Echinococcus multilocularis (diagnosis, life cycle, in vitro and in vivo cultivation), and Trichomonas vaginalis (diagnosis, PCR, and in vitro cultivation).",institutionString:"Dokuz Eylül University",institution:{name:"Dokuz Eylül University",country:{name:"Turkey"}}},{id:"71812",title:"Prof.",name:"Hanem Fathy",middleName:"Fathy",surname:"Khater",slug:"hanem-fathy-khater",fullName:"Hanem Fathy Khater",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/71812/images/1167_n.jpg",biography:"Prof. Khater is a Professor of Parasitology at Benha University, Egypt. She studied for her doctoral degree, at the Department of Entomology, College of Agriculture, Food and Natural Resources, University of Missouri, Columbia, USA. She has completed her Ph.D. degrees in Parasitology in Egypt, from where she got the award for “the best scientific Ph.D. dissertation”. She worked at the School of Biological Sciences, Bristol, England, the UK in controlling insects of medical and veterinary importance as a grant from Newton Mosharafa, the British Council. Her research is focused on searching of pesticides against mosquitoes, house flies, lice, green bottle fly, camel nasal botfly, soft and hard ticks, mites, and the diamondback moth as well as control of several parasites using safe and natural materials to avoid drug resistances and environmental contamination.",institutionString:null,institution:{name:"Banha University",country:{name:"Egypt"}}},{id:"99780",title:"Prof.",name:"Omolade",middleName:"Olayinka",surname:"Okwa",slug:"omolade-okwa",fullName:"Omolade Okwa",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/99780/images/system/99780.jpg",biography:"Omolade Olayinka Okwa is presently a Professor of Parasitology at Lagos State University, Nigeria. She has a PhD in Parasitology (1997), an MSc in Cellular Parasitology (1992), and a BSc (Hons) Zoology (1990) all from the University of Ibadan, Nigeria. She teaches parasitology at the undergraduate and postgraduate levels. She was a recipient of a Commonwealth fellowship supported by British Council tenable at the Centre for Entomology and Parasitology (CAEP), Keele University, United Kingdom between 2004 and 2005. She was awarded an Honorary Visiting Research Fellow at the same university from 2005 to 2007. \nShe has been an external examiner to the Department of Veterinary Microbiology and Parasitology, University of Ibadan, MSc programme between 2010 and 2012. She is a member of the Nigerian Society of Experimental Biology (NISEB), Parasitology and Public Health Society of Nigeria (PPSN), Science Association of Nigeria (SAN), Zoological Society of Nigeria (ZSN), and is Vice Chairperson of the Organisation of Women in Science (OWSG), LASU chapter. She served as Head of Department of Zoology and Environmental Biology, Lagos State University from 2007 to 2010 and 2014 to 2016. She is a reviewer for several local and international journals such as Unilag Journal of Science, Libyan Journal of Medicine, Journal of Medicine and Medical Sciences, and Annual Research and Review in Science. \nShe has authored 45 scientific research publications in local and international journals, 8 scientific reviews, 4 books, and 3 book chapters, which includes the books “Malaria Parasites” and “Malaria” which are IntechOpen access publications.",institutionString:"Lagos State University",institution:{name:"Lagos State University",country:{name:"Nigeria"}}},{id:"273100",title:"Dr.",name:"Vijay",middleName:null,surname:"Gayam",slug:"vijay-gayam",fullName:"Vijay Gayam",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/273100/images/system/273100.jpeg",biography:"Dr. Vijay Bhaskar Reddy Gayam is currently practicing as an internist at Interfaith Medical Center in Brooklyn, New York, USA. 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Through the application of epidemiological skill, classical and molecular virological skills, he investigates viruses of economic and public health importance for the mitigation of the negative impact on people, animal and the environment in the context of Onehealth. \r\nDr. Meseko’s field experience on animal and zoonotic diseases and pathogen dynamics at the human-animal interface over the years shaped his carrier in research and scientific inquiries. He has been part of the investigation of Highly Pathogenic Avian Influenza incursions in sub Saharan Africa and monitors swine Influenza (Pandemic influenza Virus) agro-ecology and potential for interspecies transmission. 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His research work has been published in various high-impact factor journals (Science, PNAS, Nature Medicine) with a high number of citations. He has received many awards and honors in India and abroad including various Young Scientist Awards, BBSRC India Partnering Award, and Dr. JC Bose National Award of Department of Biotechnology, Min. of Science and Technology, Govt. of India. Dr. Saxena is a fellow of various international societies/academies including the Royal College of Pathologists, United Kingdom; Royal Society of Medicine, London; Royal Society of Biology, United Kingdom; Royal Society of Chemistry, London; and Academy of Translational Medicine Professionals, Austria. He was named a Global Leader in Science by The Scientist. He is also an international opinion leader/expert in vaccination for Japanese encephalitis by IPIC (UK).",institutionString:"King George's Medical University",institution:{name:"King George's Medical University",country:{name:"India"}}},{id:"94928",title:"Dr.",name:"Takuo",middleName:null,surname:"Mizukami",slug:"takuo-mizukami",fullName:"Takuo Mizukami",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/94928/images/6402_n.jpg",biography:null,institutionString:null,institution:{name:"National Institute of Infectious Diseases",country:{name:"Japan"}}},{id:"233433",title:"Dr.",name:"Yulia",middleName:null,surname:"Desheva",slug:"yulia-desheva",fullName:"Yulia Desheva",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/233433/images/system/233433.png",biography:"Dr. Yulia Desheva is a leading researcher at the Institute of Experimental Medicine, St. Petersburg, Russia. 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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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