\\n\\n
More than half of the publishers listed alongside IntechOpen (18 out of 30) are Social Science and Humanities publishers. IntechOpen is an exception to this as a leader in not only Open Access content but Open Access content across all scientific disciplines, including Physical Sciences, Engineering and Technology, Health Sciences, Life Science, and Social Sciences and Humanities.
\\n\\nOur breakdown of titles published demonstrates this with 47% PET, 31% HS, 18% LS, and 4% SSH books published.
\\n\\n“Even though ItechOpen has shown the potential of sci-tech books using an OA approach,” other publishers “have shown little interest in OA books.”
\\n\\nAdditionally, each book published by IntechOpen contains original content and research findings.
\\n\\nWe are honored to be among such prestigious publishers and we hope to continue to spearhead that growth in our quest to promote Open Access as a true pioneer in OA book publishing.
\\n\\n\\n\\n
\\n"}]',published:!0,mainMedia:null},components:[{type:"htmlEditorComponent",content:'
Simba Information has released its Open Access Book Publishing 2020 - 2024 report and has again identified IntechOpen as the world’s largest Open Access book publisher by title count.
\n\nSimba Information is a leading provider for market intelligence and forecasts in the media and publishing industry. The report, published every year, provides an overview and financial outlook for the global professional e-book publishing market.
\n\nIntechOpen, De Gruyter, and Frontiers are the largest OA book publishers by title count, with IntechOpen coming in at first place with 5,101 OA books published, a good 1,782 titles ahead of the nearest competitor.
\n\nSince the first Open Access Book Publishing report published in 2016, IntechOpen has held the top stop each year.
\n\n\n\nMore than half of the publishers listed alongside IntechOpen (18 out of 30) are Social Science and Humanities publishers. IntechOpen is an exception to this as a leader in not only Open Access content but Open Access content across all scientific disciplines, including Physical Sciences, Engineering and Technology, Health Sciences, Life Science, and Social Sciences and Humanities.
\n\nOur breakdown of titles published demonstrates this with 47% PET, 31% HS, 18% LS, and 4% SSH books published.
\n\n“Even though ItechOpen has shown the potential of sci-tech books using an OA approach,” other publishers “have shown little interest in OA books.”
\n\nAdditionally, each book published by IntechOpen contains original content and research findings.
\n\nWe are honored to be among such prestigious publishers and we hope to continue to spearhead that growth in our quest to promote Open Access as a true pioneer in OA book publishing.
\n\n\n\n
\n'}],latestNews:[{slug:"stanford-university-identifies-top-2-scientists-over-1-000-are-intechopen-authors-and-editors-20210122",title:"Stanford University Identifies Top 2% Scientists, Over 1,000 are IntechOpen Authors and Editors"},{slug:"intechopen-authors-included-in-the-highly-cited-researchers-list-for-2020-20210121",title:"IntechOpen Authors Included in the Highly Cited Researchers List for 2020"},{slug:"intechopen-maintains-position-as-the-world-s-largest-oa-book-publisher-20201218",title:"IntechOpen Maintains Position as the World’s Largest OA Book Publisher"},{slug:"all-intechopen-books-available-on-perlego-20201215",title:"All IntechOpen Books Available on Perlego"},{slug:"oiv-awards-recognizes-intechopen-s-editors-20201127",title:"OIV Awards Recognizes IntechOpen's Editors"},{slug:"intechopen-joins-crossref-s-initiative-for-open-abstracts-i4oa-to-boost-the-discovery-of-research-20201005",title:"IntechOpen joins Crossref's Initiative for Open Abstracts (I4OA) to Boost the Discovery of Research"},{slug:"intechopen-hits-milestone-5-000-open-access-books-published-20200908",title:"IntechOpen hits milestone: 5,000 Open Access books published!"},{slug:"intechopen-books-hosted-on-the-mathworks-book-program-20200819",title:"IntechOpen Books Hosted on the MathWorks Book Program"}]},book:{item:{type:"book",id:"5410",leadTitle:null,fullTitle:"Metamaterials - Devices and Applications",title:"Metamaterials",subtitle:"Devices and Applications",reviewType:"peer-reviewed",abstract:"Metamaterials have become one of the most important emerging technologies in the scientific community due to its unusual electromagnetic properties. 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Borja received his MSc degree in telecommunication engineering and PhD degree from the Universidad Politécnica de Valencia, Valencia, Spain, in 2005 and 2009, respectively. From 2005 to 2006, he was with the Communication Group, University of Birmingham, where he was involved with the research and development of metamaterial-based antennas. He then joined, from 2007 to 2008, the Institut d’Électronique de Microélectronique et de Nanotechnologies (IEMN), Université des Sciences et Technologies de Lille 1; his research activity included the design of metamaterial-based structures with frequency-selective properties. Since 2009, he has been with the Departamento de Ingeniería Eléctrica, Electrónica, Automática y Comunicaciones, Universidad de Castilla-La Mancha, where he is an assistant lecturer. He has published more than 70 papers in peer-reviewed international journals and conference proceedings and frequently acts as a reviewer for several technical publications. 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Milk is a biological fluid designed to contain all nutritional requirements of a specific mammalian newborn; therefore, the composition of milk differs by the needs of the neonate of different species. Although much research has been devoted to milk composition in the domestic horse, donkey’s milk has recently aroused scientific interest, above all among paediatric allergologists and nutritionists. Clinical studies have demonstrated that donkey milk may be considered a good replacer for dairy cow’s milk in feeding children with severe Ig-E mediated cow’s milk protein allergy, when human milk can not be given [1]. For these patients, donkey milk is not only useful [2], but also safer compared with milk obtained by other mammalian species [3], due to the high similarity with human milk, especially considering protein fractions content [4, 5].
In 1838, a Swedish scientist called Jacob Berzelius suggested the term “protein” after the greek word “proteios”, which means “primary” or “of the first rank”; since then, many scientific discoveries have been made about these large molecules indispensable for the good functioning of the body’s cells, tissues and organs. In the past, it was believed that the function of proteins was restricted to tissue-formation, while carbohydrates and lipids provided energy to the body. Another out-of-date concept was the belief that dietary proteins were completely hydrolyzed in the gastro-intestinal tract and only free amino acids could be adsorbed from the gut. The new concept is that macro- and microelements (such as vitamins and minerals) may interact to perform different functions in the body [6]; amino acids and peptides formed in the digestion of natural proteins are adsorbed and incorporated (anabolism) into various tissues and organs as body proteins.
Milk protein is a very heterogeneous group of molecules and, for ease description, could be classified into five main categories: caseins, whey proteins, milk fat globule proteins, enzymes and other miscellaneous minor proteins [7]. Milk proteins appear to be an exciting link between nutrition, dietetics and therapy; today, consumers can expect more than just nutrition from intake of milk proteins. In fact, milk contains a variety of bioactive compounds with special properties associated with the development, growth and survival of infants beyond those provided by nutrition alone [8]. The major antimicrobial proteins in milk are immunoglobulins, lactoferrin, lactoperoxidase and lysozyme [9]. Immunoglobulins (IgG, IgM and secretory IgA) act by a specific mode of action involving antigen-antibody reactions. The other three proteins are non-specific protective factors, and their antimicrobial mechanisms of action differ from each other.
Lactoferrin, also called lactotransferrin, is an iron binding protein present in milk, saliva, tears and mucus secretions, its bacteriostatic effects are usually attributed to lactoferrin’s ability to bind environmental iron ions. Lactoferrin inhibits the growth of many kinds of Gram-negative and Gram-positive bacteria as well as some species of fungi and yeast [9]. Lactoperoxidase, a heme-containing protein, catalyzes an oxidation reaction involving hydrogen peroxide (H2O2) and functions as a component of host defense system. The lactoperoxidase system is known to be effective to preserve raw milk without refrigeration and it is used in the production of many dairy products [10].
Lysozyme exerts its antimicrobial activity by the hydrolysis of glycosidic bonds of mucopolisaccarides in bacterial cell walls [11]. Lysozyme, together with other peptides including immunoglobulins, lactoferrin and lactoperoxidase, is active in the infant’s digestive tract in order to reduce the incidence of gastro-intestinal infections [12].
Food allergy is the clinical syndrome resulting from sensitization of an individual to dietary proteins or other food allergens present in the intestinal lumen [13]. Food allergy is much more common among children than adults, and is more common among younger children than older children [14]. Cow Milk Allergy (CMA) is a frequent disease in infants, but its etiologic mechanisms are not clear [15]. Clinical symptomology for patients allergic to bovine milk proteins include: rhinitis, diarrhea, vomiting, asthma, anaphylaxis, urticaria, eczema, chronic catarrh, migraine, colitis and epigastric distress. Cow milk allergy is clinically an abnormal immunological reaction to cow milk proteins, which may be due to the interaction between one or more milk proteins and one or more immune mechanisms, and resulting in immediate IgE-mediated reactions [16]. On the other side, reactions not involving the immune system are defined as cow milk protein intolerance.
Cow milk is one of the most common food allergies in children, occurring in between 0.3 and 7.5% of the infant population [17]. The clinical diagnosis of milk allergy differs widely due to the multiplicity of symptoms. Cow milk contains more than 20 proteins (allergens) that can cause allergic reactions [18]. The main proteins are casein and whey protein; casein is fractionated into α-, β- and k-casein, whey proteins include α-lactalbumin, β-lactoglobulin, bovine serum albumin (BSA) and immunoglobulin (Igs). In addition to those, several minor proteins are also present in cow milk. Most studies revealed that casein and β-lactoglobulin are the main allergens in cow milk. The type of immune response after intrusion of foreign proteins is extremely variable, depending on the animal species, the age of the host, the quality and the quantity of antigens absorbed, the location of the absorption, the pathophysiological state and the genetic background [19].
For human beings cow’s milk represents the most common feeding during the infant weaning, but also the first allergen in life. The European Academy of Allergy and Clinical Immunology distinguishes allergy from intolerance [20]. Allergy is an adverse reaction to food with an involvement of the immune system; intolerance is an adverse reaction to food that does not involve the immune system, does not reply to a precise and single fault and shows different symptoms. In many countries cow’s milk is the most important food allergen in babies and children [21]. Adverse reactions to cow’s milk were found in 2% of babies during the first year of life: 30% of cases at the first month, 60% before the third and 96% within the twelfth [22, 23]. Symptoms can even appear during the breast-feeding because newborn reacts against a small amount of cow milk proteins present in maternal milk [24]. Children followed for the first 3 years of life, 56% of cases had recovered from cow’s milk allergy at 1-year age, 77% at 2 years and 87% at 3 years age [25] (Host and Halken, 1990). However allergy can persist for all life.
Considering the possible use of alternative milk sources for human in cases of cow’s milk allergy, the use of goat’s milk should be avoided because of the high risk of cross-reactivity, while mare’s and donkey’s milks, used in popular practice for allergic children, are valid alternative protein sources when appropriately evaluated from the hygienic point of view [26].
The donkey (Equus asinus) is a member of the horse family and its progenitor was the small gray donkey of northern Africa (Equus africanus) domesticated around 4000 BC on the shores of the Mediterranean Sea. It worked together with humans for centuries; the most common role was for transport. It still remains an important work animal in the poorer Regions.
Compared with ruminant’s milk, donkey milk has been studied less in the past, but in the last years research interest and capital investment in donkey milk have increased because its composition is similar to that of human milk (see Table 1).
The protein composition is significantly different from cow’s milk: the total content is lower (13-28 mg/ml) and quite similar to that of human and mare milk: this condition avoids an excessive renal load of solute [28]. The main difference is the proportion of whey proteins: they are 35-50% of the nitrogen fraction while they represent only 20% in cow’s milk [29]. Comparing donkey’s and mares milk, the casein to whey protein ratio in mares milk is 0.2:1 immediately post-partum, and changes to 1.2:1 during the first week of lactation [30].
Donkey | Human | |
pH | 7.0-7.2 | 7.0-7.5 |
Protein (g/100g) | 1.5-1.8 | 0.9-1.7 |
Fat (g/100 g) | 0.3-1.8 | 3.5-4.0 |
Lactose (g/100 g) | 5.8-7.4 | 6.3-7.0 |
Ash (g/100 g) | 0.3-0.5 | 0.2-0.3 |
Total Solids (g/100 g) | 8.8-11.7 | 11.7-12.9 |
Caseins (g/100 g) | 0.64-1.03 | 0.32-0.42 |
Whey Proteins (g/100 g) | 0.49-0.80 | 0.68-0.83 |
The donkey’s three major whey proteins are α-lactalbumin, β-lactoglobulin and lysozyme. Donkey’s milk α-lactalbumin has two isoforms with different isoeletric point [31]. Recently, it has been shown that α-lactalbumin presents antiviral, antitumor, and anti-stress properties. In particular in human breast milk it was shown that the α-lactalbumin forms a complex with oleic acid called HAMLET (Human Alpha-lactalbumin Made Lethal to Tumor cells) that proved to be able to induce tumour-selective apoptosis. This complex may be considered as a potential therapeutic agent against various tumour cells [32]. Furthermore it was shown that α-lactalbumin possesses anti-inflammatory activity exerted by the inhibition of cyclooxygenase-2 (COX-2) and phospholipase A2 [33].
One of the main allergens in children is β-lactoglobulin that is the major whey protein in cow milk [17] while it is absent in human milk [34]. In donkey milk the content of β-lactoglobulin is approximately 40% of the whey proteins equal to the level in mare milk and lower than that in cow milk [34]. This condition may be related to the hypoallergenic characteristic of donkey milk [1, 12, 35]. The mechanism for tolerance may be related to the specific levels of the major allergenic components in the milk. Donkey’s milk has three genetic variants for β-lactoglobulin: one presents three amino acid substitutions while the others have two amino acid exchanges [29]. Donkey milk β-lactoglobulin is a monomer whereas this protein is a dimer in ruminant’s milk. β-lactoglobulin is a protein of the lipocalin family and has high affinity for a wide range of compounds opening the way to various suppositions about its function. In fact it has been shown that this protein is involved in hydrophobic ligand transport and uptake, enzyme regulation, and the neonatal acquisition of passive immunity, other authors demonstrated that β-lactoglobulin forms complexes with folic acid suggesting that these complexes could be used as an effective carrier of folic acid in functional foods [36].
The high content of lysozyme may be responsible for the low bacterial concentration in donkey milk [4, 5]. Donkey milk lysozyme presents two isoforms that differ in three amino acid substitutions at position 48, 52 and 61 [29]. The concentration of lysozyme in human milk increases strongly after the second month of lactation, suggesting that this enzyme plays an important role in fighting infections in breast-fed infants during the late lactation [30].
The lactose content (7%) of donkey milk is similar to that of human milk and is much higher if compared with cows milk. The high content is responsible for the good palatability and facilitates the intestinal absorption of calcium that is essential for infant’s bone mineralization. Donkey milk shows a lower fat content compared to human milk, presenting for this reason a reduced energetic value [37]. The large number of fatty acids present in the lipid fraction of milk makes it one of the most complex naturally occurring fats. Saturated fatty acids are the most represented class in donkey’s milk compared to monounsaturated and polyunsaturated fatty acids, even though a wide variability can be observed in the data available in literature, most likely related to dietary and/or body condition differences [4]. Within a well balanced and integrated diet, donkey’s milk is a good source of essential fatty acids; this category of fatty acids are very important in the diet of patients with Cow Milk Allergy (CMA), especially if affected by multiple food allergy. These subjects are in fact at risk of developing a deficiency in essential fatty acids and particularly in PUFA n – 3, which are absolutely necessary for adequate growth, neurological development and cardiovascular health [38]. Donkey’s milk shows an high content of both linoleic (C18:2) and linolenic (C18:3) acids, respectively 9.0 g/100 g and 5.1 g/100 g of total fatty acids, when compared with ruminant species milks, in which the contents of the above mentioned Polyunsaturated Fatty Acids are always lower. To increase the total fat content in donkey milk, clinical studies [17, 35] suggested to enrich donkey milk with medium-chain triglycerides, in order to obtain a final fat content similar to human milk.
The mineral composition is very close to that of human milk except for the highest level of calcium and phosphorus but the Ca-P ratio is similar. The milk produced in the first month of lactation, when it is the only nutritional source for the foal, contained the highest levels of mineral elements that may be related to the fast growth stage of the foal. Afterwards, the mineral supply in milk decreases considerably.
Basically, donkey milk has nutritional properties that make it more similar to human milk than another mammalian one. Therefore it could be used not only as a breast milk substitute for allergic children but also as a new dietetic food for human consumption. This chapter would be a further contribution to increase the characterization of donkey milk, evaluating the nutritional qualities of donkey milk using different proteomic approaches.
The term proteome was introduced to describe “all proteins expressed by a genome or tissue [39]. Another definition of proteome, but similar, is: “a set of all expressed proteins in a cell, tissue or organism at a certain point in time” [40]. After genomics and transcriptomics, proteomics is considered the next and more articulate step in the study of biological systems. In fact, while the genome is more or less constant, any proteins may exist in multiple forms that vary from cell to cell and from time to time. In fact the proteome analysis reveals translational, post-translational modification, regulatory and degradation processes that affect protein structure, localization, function, and turnover. Proteomics is the study of multi-protein systems and their roles as part of a larger system or network. Therefore the context of proteomics is system biology, rather than structural biology, since it is a tool used to characterize the behaviour of the system rather than the behaviour of any single component.
Milk proteins have been studied in depth for well over 50 years and lot of studies were performed in order to analyse the various milk protein components, in various milk from different mammals. However, many questions concerning milk protein expression, structure and protein modifications remain still not completely covered such as some details of protein modifications due to disease and processing. The milk proteome is extremely complex because of the presence of post-translational modifications, alternative splicing and different genetic variants. The post-translational modifications are: glycosylation, phosphorylation, disulphide bonds formation and proteolysis, they create a large number of different protein variants from a single gene product. In milk the molecular composition of proteins is very important since it influences the functional properties of milk proteins such as solubility, clotting aptitude, thermal denaturation and the nutritional properties of the milk. Usually, the analytical procedures used for structural analysis of milk proteins are based on chromatographic techniques (ionic exchange, reversed phase chromatography, size-exclusion chromatography) followed by one-dimensional electrophoretic techniques (PAGE, Urea-PAGE and SDS-PAGE), or more efficiently on bi-dimensional electrophoresis technique (2-DE).
In our work we approached the study of donkey milk protein profile by different techniques for protein separation: initially they were based on chromatographic techniques followed by sodium dodecyl sulphate polyacrylamide gel electrophoresis SDS-PAGE [5]. Successively, the milk was analyzed through two-dimensional electrophoresis (2-DE) followed by N-terminal sequencing, in order to give a more detailed panoramic view of the proteins that are present in donkey milk [41].
Donkey’s milk casein fraction was characterized by different chromatographic approaches using an Äkta Purifier HPLC system: ion-exchange chromatography, and reversed-phase. After chromatography, each protein was subjected to SDS-PAGE. The purified caseins were identified by N-terminal sequencing [5]. By cation-exchange chromatography (Mono S HR 5/5 column, GE Healthcare,1.0 ml bed volume), performed at pH 5 and 7, followed by 15% SDS-PAGE (Mini Protean III apparatus, Bio-Rad) it was possible to separate 9 peaks that were identified as β-caseins (sequence: REKEELNVSS) and αS1-caseins (sequence: RPKLPHRQPE), having different molecular weight, as shown in figure 1 and table 2.
Cationic-exchange chromatography on HPLC (Mono S HR 5/5) analysis on whole casein performed at pH 5.5 (A) and at pH 7.0 (B) [5] (copyright permission obtained).
Peak/Chromatography | Protein | kDa | N-terminal sequence |
A: cationic exchange, pH 5.5 | Β-casein | 35.40 | REKEELNVS |
C, D, E: cationic exchange, pH 5.5 | αs1-casein αs1-casein | 33.00 30.70 | RPKLPHRQPE RPKLPHRQPE |
F: cationic exchange, pH 7.0 | Β-casein | 33.30 | REKEELNVS |
G, H, I, J: cationic exchange, pH 7.0 | αs1-casein αs1-casein | 31.30 29.40 | RPKLPHRQPE RPKLPHRQPE |
Reversed-phase chromatography on HPLC (RP-HPLC) followed by 15% SDS-PAGE and N-terminal analysis was performed on the skimmed donkey’s milk giving as a result three main peaks (K, L, M) identified as lysozyme (sequence, KVFSKXELA), α-lactalbumin, (sequence, KQFTKXELSQVLXSM), and β-lactoglobulin (sequence TNIPQTMQ), respectively (Figure 2A and\n\t\t\t\t\ttable 3). RP-HPLC was also performed on the donkey’s milk casein fraction after their precipitation from skimmed milk at pH 4.6. Five peaks were recovered (N-R) each of them submitted to 13% SDS-PAGE and N-terminal analysis and the results, showed in figure 2B and in table 3, indicated mainly the presence of αS1-caseins and β-caseins. Furthermore, the β-casein sequence of peak R (REKEALNV) showed an E→A substitution in the fifth aminoacid [5].
This study revealed the presence of β-caseins (sequence: REKEELNVSS) and αs1-caseins (sequence: RPKLPHRQPE), which presented marked homology with αs1- and β-caseins from mare’s milk [42], while the presence of other types of caseins, such as αs2-, γ- and k- were not determined in donkey milk. This result show another high similarity between donkey and human milk: in fact, the presence of αs2-caseins in human milk has not been demonstrated [34].
Peak/RP-HPLC | Protein | kDa | N-terminal sequence |
K | Lysozyme | 14.60 | KVFSKXELA |
L | α-lactalbumin | 14.12 | KQFTKXELSQVLXSM |
M | β-lactoglobulin | 22.40 | TNIPQTMQ |
P | αs1-casein | 33.30 | RPKLPHQPE |
Q | β-casein | 37.50 | REKEELNVS |
R | β-casein | 37.50 | REKEALNVS |
A) Reversed-phase HPLC of: (A) skimmed donkey’s milk and (B) casein fraction [5] (copyright permission obtained).
Thanks to RP-HPLC analysis, it was possible also to calculate the lysozyme, β-lactoglubulin and α-lactalbumin concentrations (in mg/ml) at different stages of lactation (60, 90, 120, 160 and 190 days after parturition), the results are shown in Table 4.
Days after parturition | Lysozyme (mg/ml) | β-lactoglobulin | α-lactalbumin |
60 | 1.34 | Not determined | 0.81 |
90 | 0.94 | 4.13 | 1.97 |
120 | 1.03 | 3.60 | 1.87 |
160 | 0.82 | 3.69 | 1.74 |
190 | 0.76 | 3.60 | 1.63 |
The amount of lysozyme in donkey’s milk varied considerably during the different stages of lactation, with a mean value of 1.0 mg/ml, and proved to be higher with respect tothat in bovine (traces), human (0.12 mg/ml) and goat’smilk (traces), whereas, it was very close to mare’s milk (0.79 mg/ml) [43]. The mean β-lactoglobulin content in donkey’s milk (3.75 mg/ml) was very close to that of bovine milk (3.3 mg/ml) and pony mare’s milk (3.0 mg/ml), whereas in human milk the β-lactoglobulin is absent [34]. The α-lactalbumin content increased in the three months after parturition till the value of 1.8 mg/ml, close to the α-lactalbumin content in human milk (1.6 mg/ml) but lowest compared to the pony mare’s α-lactalbumin content (3.3 mg/ml) [5].
Lactoferrin was purified by a cationic exchange chromatography (Mono S HR5/5 column) and its identity was confirmed by N-terminal sequencing and by western blot analysis using anti-lactoferrin antibodies (see figure 3A) [41]. The quantitative determination of donkey’s milk lactoferrin (Figure 3B) gave a result of 0.080 ±0.0035 g/L, similar to that found in mare (0.1 g/L), cow (0.02-0.2 g/L), goat (0.06-0.40 g/L), and sheep milk (0.135 g/L), but lower when compared with the lactoferrin content in human milk, in which values are usually in the range 1.0-6.0 g/L [44, 45].
Lactoferrin is an iron-binding protein that displays many biological functions: regulation of iron homeostasis, cellular growth, anti-microbial and anti-viral functions, and protection against cancer development and metastasis [46]. Lactoferrin exerts its antibacterial effect by two different mechanisms involving two separate domains of the protein. In the first one, the antimicrobial effect is due to the high iron binding affinity of the protein that deprives some iron-requiring bacteria of iron and consequently inhibits their growth [46, 47]. The second antimicrobial property is due to the cationic domain at N-terminus directly responsible for the bactericidal effect [48].
A) 12% SDS-PAGE (lane 1) and immunoblotting using anti-lactoferrin antibodies (lane 2) of lactoferrin purified by Mono S column. St: Bio-Rad low-molecular-weight standard (phosphorylase b, 97.4 kDa; bovine serum albumin, 66.2 kDa; ovalbumin 45.0 kDa; carbonic anhydrase, 31 kDa; soybean trypsin inhibitor, 21.5 kDa; lysozyme, 14.4 kDa. B) Calibration line of standard solutions of lactoferrin (20-100 final μg).
Lactoperoxidase is a glycoprotein consisting of a single peptide chain with a molecular weight of 78.0 kDa. This enzyme exerts its antimicrobial action through the oxidation of thiocyanate ions (SCN-) by hydrogen peroxide, both present in biological fluids and also in milk. Lactoperoxidase activity in skimmed donkey milk was evaluated by a continuous spectrophotometric rate determination using as substrate 2,2’-Azinobis (3-Ethylbenzthiazoline-6-Sulfonic Acid) [49]. In donkey milk the activity of lactoperoxidase is very low, 4.83±0.35 mU/mL. The enzyme quantification was achieved by a calibration line obtained by plotting the nanograms of peroxidase standard solutions against the enzymatic activity The mean (± SD) concentration of donkey milk lactoperoxidase was calculated to be 0.11±0.027 mg/L, close to the value obtained with human milk (0.77±0.38 mg/L) [50]. In table 5 the concentration of three proteins with antimicrobial effect are compared from donkey, human and cow milk. From these data is evinced that human and donkey milk contain considerable amounts of lysozyme and lactoferrin but lactoperoxidase is present only in small amounts.
Milk | Lactoperoxidase (mg/L) | Lysozyme (g/L) | Lactoferrin (g/L) |
Human | 0.77 | 0.12 | 0.3-4.2 |
Donkey | 0.11 | 1.0 | 0.080 |
Bovine | 30-100 | trace | 0.10 |
Whole casein was obtained from skimmed milk by adjusting the pH to 4.6 with 10% (v/v) acetic acid and centrifuging at 3000xg for 10 min in order to obtain a supernatant of whey proteins and the isoelectrically precipitated caseins. In the 2-DE analysis [41], the first dimension was an isoelectric focusing (IEF) performed using a pre-cast immobilized pH gradient gel strip Immobiline DryStrip, (IPG-strip, length 18 cm) in the pH range of 4-7 for the casein fraction and in the pH range of 3-11 for the whey proteins. The second dimension, performed using a Protean II apparatus (Bio-Rad, 180 x 200 x 1.5 mm), consisted of a 13% SDS-PAGE for the casein fraction and a 15% or a 7.5% SDS-PAGE for the whey proteins. After staining, the gel were analyzed using PD-Quest software (Version 7.1.1; Bio-Rad) in order to define spot-intensity calibration, spot detection, background abstraction, calibration, and calculation of molecular mass and pI. The spots considered interesting and quantitatively significant were subjected to N-terminal analysis for their identification. The results of donkey milk 2-DE analysis is shown in figure 4.
Two-dimensional electrophoresis analysis of donkey milk casein fraction. The first dimension was performed in the pH range of 4-7, the second dimension consisted of a 13%SDS-PAGE. St: Bio-Rad low molecular weight standard (phosphorylase b, 97.4 kDa; bovine serum albumin, 66.2 kDa; ovalbumin 45.0 kDa; carbonic anhydrase, 31 kDa; soybean trypsin inhibitor, 21.5 kDa; lysozyme, 14.4 kDa). [41] (copyright permission obtained).
The casein fraction (pH range 4-7, figure 4) showed the presence of 13 major protein spots: eight of them were identified as β-caseins (N-terminal sequence: RKEELNVSS) with a pI values ranging from 4.63 to 4.95. Furthermore four β-caseins spots (from spot A to D) showed molecular weights ranging from 33.10 to 33.74 kDa whereas the other four β-caseins spots (from spot E to H) displayed molecular weights ranging from 31.15 to 32.15 kDa, with a difference of about 1000 aminoacids. This results are in good agreement with [52] who demonstrated the presence of a full-length β-casein variant carrying 7, 6, 5 phosphate groups, with a pI of 4.74, 4.82, 4.91 respectively and a spliced variant (-923 aminoacids), carrying 7, 6, 5 phosphate groups with a pI of 4.64, 4.72, 4.80 respectively. On the basis of these observations, and looking at figure 4, it may be evinced that the spots B, C and D may correspond to the full-length forms of β-caseins (pI: 4.72, 4.82, 4.92 respectively) whereas the spots E, F, and G may correspond to the spliced variants of β-caseins (pI: 4.68, 4.80, 4.88). The other remaining five spots (from I to N) were identified as αs1-caseins (N-terminal sequence: RPKLPHRPE) with a pI values ranging from 4.92 to 5.36 (see figure 4). In donkey milk an heterogeneity for the αs1-casein was found [52], assigned to either discrete phosphorylation (5, 6 and 7 phosphate/mole) or non-allelic spliced forms. In our work we found in donkey milk five αs1-caseins: three of them showed a high molecular weight (about 31. 3 kDa) and probably correspond to the full-length phosphorylated forms, whereas two αs1- caseins showed a lowest molecular weight (about 28 kDa) therefore they may correspond to the spliced variants. In our study the presence of αs2-casein and κ-casein were not demonstrated probably because of their low amount in donkey milk. Another group of authors [53] identified in donkey milk the presence of a weak spot identified as αs2-casein and three very weak spots identified as κ-casein. Therefore, the heterogeneity shown in the whole casein analysis by 2-DE may be due to a variable degree of phosphorylation and to spliced forms of αs1- and β-caseins [54-56].
The whey fraction was analyzed by 2-DE in a pH range of 3-11 for the first dimension but with two different polyacrylamide gel percentages in the second dimension in order to have a better differentiation and identification of the low- and high-molecular weight whey proteins as shown in figure 5 A and B.
Two-dimensional electrophoresis analysis of donkey milk whey protein fraction. The first dimension was performed in the pH range of 3-11. The second dimension was carried out by: A) 15% SDS-PAGE for the identification of the low molecular weight whey proteins, B) 7.5 % SDS-Page for the identification of high molecular weight whey proteins. The standard (st) was the same as figure 4. [41] (copyright permission obtained).
The separation of low-molecular-weight whey proteins achieved by 2-DE (first dimension: IPG-strip, pH 3-11, second dimension: 15% SDSPAGE) revealed the presence of two isoforms of α-lactalbumin (Figure 5A) corresponding to the spots R and S. This result is in agreement with [57] who observed oxidized methionine forms for α-lactoalbumin (Met 90), due to in vivo oxidative stress that give rise to two α-lactalbumin isoforms. Furthermore from 2-DE, three isoforms of donkey milk β-lactoglobulin (Figure 5A), corresponding to the spots O, P, and Q, were observed. In this case from literature it is known that in donkey milk, this protein exists under two different forms, named β-lactoglobulin I, that constitutes the major form (80%), and β-lactoglobulin II [58, 59] that constitutes the minor form (20%). Successively, a genetic variant for β-lactoglobulin I (named β-lactoglobulin I B) with three amino acid substitutions explained by the degeneracy of the genetic code was found [29], and two genetic variants for β-lactoglobulin II (named β-lactoglobulin II B and C). Successively another β-lactoglobulin II variant (named D) was detected as minor component in the whey fraction of donkey milk [57]. Finally, after 2-DE experiments, only one spot corresponding to donkey milk lysozyme was detected (Figure 5A, spot T) even if from literature the presence of two donkey milk lysozyme genetic variants that arise from an oxidized methionine residue at position 79 [29, 57]. Finally, Figure 5B shows the electropherogram for the donkey milk high molecular-weight whey proteins separated by 2-DE (first dimension: IPG-strip pH 3-11, second dimension: 7.5% SDS-PAGE). By N-terminal sequencing it was possible to assign the spot U to serum albumin (kDa/ pI: 62.7/7.1) and the spot V to lactoferrin (kDa/pI: 77.0/9.8), already discussed in the section 5.2 [41].
Recent clinical evidence has renewed the interest in donkey milk because of high tolerability in infants with cows’ milk protein allergy. To be successful as a substitute for human milk in infant nutrition, donkey milk must be capable of performing many biological functions associated with human milk. The specific protein fraction in donkey milk can be a good indicators of its potential role. In this study, donkey milk whey proteins were analyzed by 2-DE and were also quantified. From the proteomic map was revealed the presence of two isoforms of α-lactalbumin, three isoforms of β-lactoglobulin, lysozyme, albumin and lactoferrin.
The high lysozyme and α-lactalbumin content found in donkey milk may be responsible for the low bacterial count reported in literature. Lysozyme, lactoperoxidase and lactoferrin have been recognized as antimicrobial and bacteriostatic agents and could be useful to prevent intestine infections in infants. Their action may extend the conservation of fresh donkey milk and the relative potential commercial supply.
On the basis of results obtained donkey milk may be considered suitable for feeding young children affected by severe cow’s milk allergy. In the past it has been widely used to replace human milk because its chemical composition and particularly protein content are close to that of human. Great attention must be obviously given to the hygienic characteristics of donkey milk production, in order to consider this milk a valid substitute of hydrolysed proteins or soy-bean derived formulae in the treatment of infants with cow’s milk protein allergy.
Food is called functional if it contains one or more components that can provide a benefit to human health, beyond their traditional nutritional role. Donkey milk may be configured as functional food in early childhood and not only.
Pain is intrinsically private, and the concept of pain is difficult to describe and assess due to its subjective nature and individuals’ unique experiences of pain [1, 2]. Up until the mid-1980s, clinicians believed that infants, toddlers and persons with disabilities, specifically those with significant communication difficulties, either do not have pain or may have very high pain thresholds [3, 4, 5]. These myths and beliefs were reinforced by McCaffery’s widely accepted definition of pain at that time that stated that “pain is what the person says it is and exists whenever he or she says it does” [6, p. 95]. By default, McCaffery’s definition therefore suggested that all persons with the inability to communicate their pain verbally (including the aforementioned) may not have pain.
\nIn addition to their limited verbal ability to express pain, communication vulnerable children’s neurology may also impact on their ability to show other tell-tale signs of pain that transform the parts of the brain responsible for the expression of pain [5]. For this reason, clinicians repeatedly overlooked other signs of pain [4], such as changes in the children’s behaviour (withdrawal, acting clownish, having mood changes, displaying aggressive behaviour or exhibiting extreme tantrums) or changes in positioning (refusing to use the body part where pain is). This is because children with communication challenges may not display pain in the typical ways such as by crying or through facial changes [7, 8, 9, 10]. Clinicians often mistakenly regard these kinds of “different reactions to pain” as challenging behaviour and not as children’s alternative attempts of trying to express their pain [11].
\nLately, clinicians have started to acknowledge that the inability to communicate pain verbally does not negate the likelihood that a person is in pain or that they require applicable pain-relieving treatment [3, 10]. The International Association for the Study of Pain (IASP) updated the definition of pain in July 2020 [2, p. 2] to: “An unpleasant sensory and emotional experience associated with, or resembling that associated with, actual or potential tissue damage”. According to Raja et al. [2] the IASP also added six key notes as an expansion to the definition and to provide further context to the definition and the etymology of the word “pain.” Additional notes to the latest pain definition for example highlight that a person’s report of their pain should be acknowledged and respected and that verbal expressions of pain is only one of many behaviours to express pain [2]. Nevertheless, irrespective of patients’ ability or inability to verbally self-report their pain, it remains the ethical obligation of all clinicians to acknowledge and relieve the most vulnerable patients’ pain [12].
\nChildren with severe physical, sensory and/or cognitive disabilities affecting their receptive and expressive communication may not be able to verbally communicate their pain and other pain-related experiences [10, 13]. Children with languages or cultures different to those of the treating clinicians or with limited proficiency in the latter’s language often do not have the vocabulary to express their pain [14]. Furthermore, children who are receiving treatment in intensive care units – where medical intervention such as sedation, intubation or tracheotomy can influence their ability to verbally communicate – as well as children receiving palliative end-of-life support may also not be able to communicate verbally [13]. Authors refer to these groups of children as communication vulnerable [13, 14, 15]. Communication vulnerability is defined as a reduced ability in respect of expressive and/or receptive communication and can involve permanent vulnerability (such as children with severe communication disabilities) or temporary vulnerability (such as patients in critical care units receiving medical interventions that may influence their ability to speak) [16, 17].
\nThe inability to express pain verbally may result in communication breakdowns between the child and the clinician, which could result in risks such as non-treatment, adverse medical outcomes and increased anxiety for both patients and clinicians [18]. Clinicians often find it demanding to assess pain in communication vulnerable children [7, 19], as they have to attempt to interpret the children’s bodily movements, facial expressions and physiological signs [7]. As mentioned earlier, children with communication disabilities may express their pain in atypical ways that could influence clinicians’ interpretation of the children’s pain [10, 11, 19]. In the latest recommendations for clinicians to follow during pain assessment of those unable to self-report, Herr et al. proposed that as a first step, clinicians should become aware of potential causes of pain [20]. The second step in pain assessment is to try to obtain self-report from all patients [20]. Therefore, it is vital that alternative means of communication should be investigated to enable children with severe communication difficulties to self-report their pain.
\nHay et al. [21] promoted the use of self-reporting as the primary method for measuring the intensity and other features of pain. Thus, it was recommended that parents’ proxy reports of their children’s pain should only be used once the children’s reports were in doubt [21, 22]. Research has confirmed that speaking children themselves can give a clear self-report of their pain experience by verbally expressing their pain or using various pain assessment tools such as the Coloured Analogue Scale or the Faces Pain Scale-Revised [23]. However, Schiavenato and Craig [24] are of the opinion that pain assessment tools do not do justice to a patient’s pain experience as they oversimplify the demands for rating pain intensity without taking the type of pain into consideration. For this reason, a possible solution should be found for how communication vulnerable children can self-report their pain in ways other than by verbal accounts.
\nClinicians’ expertise to support communication vulnerable children in pain depends on the availability of reliable and valid information about the existence and precise nature of the child’s distress [25]. Self-report and observational measures of pain can be reviewed from the perspective of a model of human communication [26]. Therefore, to gain a better understanding of this complex pain communication process, clinicians and researchers need to grasp the challenges that children with disabilities – and particularly those who are communication vulnerable – may encounter when trying to express their pain. The social communication model of pain [26, 27] offers an inclusive theoretical framework to be used in this chapter, because it explains the dynamic interaction between the biological, psychological and social determinants of pain [28]. An adapted social communication model of pain for communication vulnerable children based on the model proposed by Craig [27, 28] warrants further discussion in this chapter.
\nCommunication plays an important part in any action that aims to improve health [29]. Communication is a social, dynamic and interchanging reciprocal process that involves persons (acting as a sender or receiver) [30]. Communication comprises verbal (speech) as well as non-verbal modes (gestures, a shared glance, facial expression) [31]. Symbols (abstract or concrete) are used to convey information from the sender to the receiver in order to achieve a shared meaning in a specific context or environment [30]. In other words, communication involves sender(s) and receiver(s) conveying information through a communication channel. Effective communication occurs when the intent and meaning of one person (e.g. the sender) is understood by another person (e.g. the receiver) [31]. For communication vulnerable children, this communication process poses a serious challenge, due to their inability to communicate verbally (i.e. the communication intent is lost if the receiver does not understand the communication channel used by the sender). Although these children may have the desire to communicate their pain, research indicates that communication vulnerable children often opt not to communicate their pain because their previous communication attempts were ignored, or simply because it takes too much physical effort trying to communicate their pain [32].
\nThe social communication model of pain was developed as a framework to explain how pain is experienced and to describe the multifaceted communication process required to adequately express and interpret pain and to have pain understood by others [26, 27]. The social communication model of pain underlines both the role of the sender who is the person in pain (e.g. the communication vulnerable child) and the ability of the receiver as the observer of the pain (e.g. clinicians) in understanding the experience of pain. Biomedical models, in contrast, focus on the sensory characteristics of pain, with no emphasis on the social factors of pain [26, 27, 33]. Since this chapter will proceed to focus on pain communication of communication vulnerable children, Figure 1 depicts the suggested adaptations to the social communication model of pain (based on Craig [27, 28]) as it relates to communication vulnerable children’s expression of pain.
\nAdapted social communication model of pain for communication vulnerable children (as based on Craig [27, 28]).
A proposed three-step pain communication process altered for communication vulnerable children highlights the different factors that may intervene in the children’s pain expression, the pain assessment and the accompanying treatment [10, 27, 28]:
\nPain experience – the inward personal painful pain experience that happens over time and is stimulated by both interpersonal and intrapersonal (biological and psychological) factors involves the status of the child before the event;
\nMessage – the encoding of the pain experience (e.g. the child’s understanding or making sense of the pain) and the expression of pain through expressive behaviours such as crying, exclamations or (verbal) self-report to make the pain known to observers (e.g. clinicians or parents);
\nObserver (receiver of message) – the process whereby observers decipher or decode pain behaviours to react and respond by providing appropriate pain management or pain-relieving treatment [34].
This model also highlights the possibility that observers’ own perceptions and responses to pain as based on their own pain experiences may influence their understanding of pain as well as how they will respond to the child’s pain experience. Although researchers and clinicians should be aware of their own bias towards pain, it will not be dealt with in further detail in this chapter.
\nIn short, the adapted social communication model of pain proposes that the communication of pain begins with the communication vulnerable child who experiences pain (A); it continues to describe how this experience influences the child to make sense of the pain (B) and to express it in atypical ways or by means of augmentative and alternative communication (AAC) modes. These pain expressions are made known to the observers (C), who decode the child’s pain to take appropriate pain management actions. The adapted social communication model of pain can thus be used to help researchers and clinicians to understand the pain in communication vulnerable children. Examples are children with a variety of disabilities such as Down syndrome, intellectual disabilities, autism spectrum disorder (ASD) or cerebral palsy (CP), or children who experience temporary communication vulnerability due to medical interventions such as intubation.
\nThe model considers that there are many ways that a child can encode (B) their pain experience (A). Thus, when decoding the child’s pain, observers (C) need to be open to other modes that children may use to communicate their pain. A child’s self-report of pain is influenced by the pain context as well as their emotional, sensory, cognitive, developmental and cultural composition [2, 35, 36]. The social factors and reciprocal, repeating and dynamic effects of pain communication are acknowledged during this pain account within human beings [28]. In the social communication model of pain, a clear distinction is made between historical and current biological and social factors. For example, intrapersonal factors refer to a person’s temperament to react based on their biological, psychological and social histories. Craig [28] highlights that, during the pain event (A), the immediate social and physical environment has a powerful effect on both the person in pain (e.g. the communication vulnerable child) and on the observers (C). The internal subjective pain experiences of communication vulnerable children will now be discussed based on the adapted social communication model of pain for communication vulnerable children (Figure 1).
\nThe way persons express pain can give insight into their pain experiences. Pain expressions involve the person’s observable response (such as their pain behaviours) to a noxious stimulus, whereas a person’s pain experience is private and internal and involves severity of discomfort [27]. Based on their own experiences with pain, each individual displays different potential behavioural reactions to pain [27]. For example, children who have had negative pain experiences during needle procedures may exhibit more severe responses to pain because of their previous negative experiences. Additionally, their individual biological capabilities trigger their complicated expressions of pain [2]. Children with significant communication difficulties have different disability diagnoses with unique pain-related experiences related to these disabilities (e.g. children with CP or ASD).
\nAlong with biological capabilities, the constructs behind pain expression are the impact of language and cognitive development as well as social interaction and experiences. The expansion of pain-related vocabulary progresses along a similar sequence as does natural language development [37, 38]. The theoretical constructs that underlie pain expression within communication vulnerable children with various aetiologies will now be discussed in more detail.
\nAll children experience pain on a regular basis. Young children with typical development may respond to everyday pain such as bumps and bruises by crying, verbalisations or spoken words to express their pain experiences. They usually start to use the word “pain” by the age of 6 years [37]. On the contrary, children with disabilities might have more pain incidents more often than their peers without disabilities. For example, children with disabilities may experience more acute pain incidents due to needle procedures (such as blood drawing or receiving blood transfusions) and recurring medical procedures and treatments (such as range-of-motion manipulation during physiotherapy for children with CP) to maintain their health [3, 39, 40].
\nYoung children with CP experience high occurrences of chronic and acute pain [19, 41]. In an Australian study conducted by Ostojic and colleagues [19] to determine the prevalence of pain in children with CP, they found that two in three children with CP experienced acute pain and one in three children had chronic pain. Furthermore, the study revealed that children with CP, functioning on levels IV and V of the Gross Motor Function Scale (GMFCS), have a bigger risk of suffering from chronic pain [19]. This group also has communication challenges and may need alternative means to communicate their pain [19, 41]. Multi-factorial reasons for pain in children with CP could include spasticity, contractures and the incapacity to walk [19, 41, 42]. Spasticity and the inability to change their positioning to decrease pressure on certain body parts may also lead to contractures, musculoskeletal and gastrointestinal pain [43]. In a study among children with CP in South African schools, Adolfsson and colleagues [44] found that South African children with CP often experience hip dislocations– resulting from spasticity that caused hip displacements and ultimately lead to hip dislocations. As such, persons with CP have to undergo constant surgical procedures and medical interventions throughout their life span in an attempt to correct or rehabilitate orthopaedic problems associated with their condition [41, 43, 45]. All these procedures, including range-of-motion manipulation and assisted stretching, are painful experiences [44]. Communication through the use of AAC communication strategies is therefore crucial for children with CP to ensure that they can express their pain and receive appropriate pain treatment [41].
\nChildren with intellectual disabilities are at risk of experiencing a variety of painful somatic conditions due to comorbidities such as contractures, gastro-oesophageal refluxes, and epilepsy [11, 46]. These children with intellectual disabilities often experience socio-communicative deficits typical of children with ASD, for example they may not use facial expressions or make eye contact to display pain or other emotions [11, 46, 47]. Children with intellectual disabilities also express their pain consistent with their level of cognitive and physical development and not necessarily consistent with their chronological age [46]. Some atypical expressions, such as hand flapping or hand rubbing, smiling or freezing has been observed when children with intellectual disabilities were not able to verbalise their pain [5]. Yet, according to Doody and Bailey [9], children with intellectual disability who are unable to communicate their pain in a typical manner seem to have less opportunity to receive pain treatment.
\nChildren with Down syndrome also fall in the group of children with intellectual disability who can be expected to experience pain as a result of their disability. They are at high risk of secondary pain-related experiences such as the development of hip abnormalities and oral health issues [3, 48]. Children with Down syndrome have higher occurrences than their peers with typical development of dental problems due to frequent incidence of periodontal disease and chronic facial pain disorders [3]. They may also experience chronic pain due to congenital heart anomalies, bone fractures due to osteoporosis, or eczema – to name a few conditions [5]. Davies [48] reported that, compared to their siblings with typical development, children with Down syndrome have a decreased tendency to react to pain – but that does not mean that they are unresponsive to pain. Due to lower cognitive functioning, children with Down syndrome may not have the ability to localise the painful stimulus, because their pain-related vocabulary only tends to develop at a later stage. Their limited pain-related vocabulary may thus influence their ability to communicate pain [38].
\nAs with children with CP, children with intellectual disabilities such as Down syndrome or ASD also experience a large number of pain incidents and they are sometimes two to three times more at risk of an injury than their peers with typical development [10, 49]. Children with ASD often display challenging and self-injurious behaviour, as well as extreme tantrums that could lead to injury and pain [10]. Some children with ASD may also have trouble expressing their pain, due to their typical delay in language development and possible cognitive impairment [10, 50]. If children with ASD do use speech, they struggle to convey their emotions and the intensity of their pain experiences due to their monotone intonation. In addition, they do not usually use the same facial expressions and gestures that their peers with typical development would do to express their feelings. The pain expressions of children with ASD are distinctively individual and may differ from those of the larger population, considering the fact that children with ASD experience socio-communicative impairments and therefore may not understand social closeness as their peers with typical development would do [3].
\nBesides the communication difficulties of children with disabilities, this chapter also focuses on children who experience a temporary communication vulnerability due to medical procedures (such as intubation) or life-threatening conditions (such as cancer). For example, critically ill children who have been admitted to paediatric intensive care units suffer a temporary loss of their expressive or receptive communication [13]. These communication vulnerable children show stress, frustration and anxiety, and are at a greater risk of being treated incorrectly by clinicians who wrongly decode the children’s pain message [15, 51]. Even clinicians such as nurses often mention their feelings of frustration when they find it difficult to grasp what their paediatric patients are trying to communicate [7]. The vast significance of efficient alternative means of communication to ensure safe treatment of paediatric patients is therefore emphasised [15].
\nSpoken language is seen as the ultimate means of communicating pain [52]. Language and cognitive development influence children’s use of words to describe their pain experiences in such a way that observers (clinicians) can decode the message correctly and respond appropriately with pain-relieving treatment [53]. Language learning occurs within a physical and social context determined by actual people, objects, activities and events in the child’s environment [54]. Children learn about new concepts in the world while interacting with their physical environment, which forms the foundation for their lexical development [54]. For example, the words parents use to communicate with their children during painful experiences enable children to acquire new pain-related vocabulary [38]. Parents tend to talk to their children about pain on an age-appropriate level, thus enlarging their children’s pain-related vocabulary. For example, when a child cries when injured, the parent might respond with exclamations or words such as “Ouch! You got hurt!” thereby enabling the child to add meaning to the painful experience and to expand their repertoire of pain-related vocabulary [55].
\nHowever, since children with severe communication difficulties do not have the same contact with their social environment as their peers with typical development, they may find the language-learning process challenging [54]. Whereas children with typical development gain new knowledge about the world they live in through their encounters with their environment, children with disabilities have reduced access to their environment. This makes it more challenging for them to acquire new concepts without having the relevant previous knowledge to build on [54]. It is consequently the adults’ responsibility to guarantee that children with severe communication difficulties are exposed to a social environment that includes people, objects and possible pain experiences. This exposure to facilitate children’s language development can be achieved for instance through play activities like doctor-doctor play with peers [54].
\nLanguage development corresponds with cognitive development and as children mature cognitively, they can describe their pain more successfully [52]. Younger children tend to explain the bodily sensations they experience during pain in a more concrete manner (such as ‘my stomach hurts’) due to their limited cognitive and language skills [56]. As children’s thinking develops on a symbolic level, they start to use more graphic descriptors such as “terrible” or “beating”, while older children start to add intensifiers, such as “really bad” when describing their pain [53]. Since children with severe communication difficulties may not be able to verbally express their pain, Johnson et al. [57] proposed that clinicians such as speech-language therapists provide these children with preselected pain vocabulary that can be added to their AAC system to enable them to express their pain appropriately.
\nApart from disability aetiology, language or cognitive development, gender is another intrapersonal factor that might have an impact on the development of children’s pain-related vocabulary [37, 38, 58, 59].
\nGender differences in pain expression and pain-related vocabulary – despite similar pain experiences – are often highlighted in literature [38, 60, 61, 62]. As girls typically develop expressive vocabulary sooner than boys, Frank et al. [38] found a slight advantage in girls’ pain-related vocabulary, which may imply that pain-related language acquisition could be related to other factors. For example, girls tend to be more emotive and more expected to complain and also report their pain experiences more frequently than boys [52]. Contrary to girls, boys tend to be more passive or have more anger-related vocabulary in response to pain due to an injury [38]. In the event of communication vulnerable children, the differences between the reactions to pain by boys and girls are not clear [8]. However, it was found in literature that adult observers’ responses to children’s pain experiences tend to be influenced by gender-stereotyped attitudes, and that girls were treated in a different way than boys [62, 63, 64]. Clinicians are often biased and expect girls to experience more pain than boys [63, 64, 65].
\nAccording to the adapted social communication model of pain, interpersonal factors such as family settings, children’s social and cultural environment, as well as previous hospitalisations may further influence children’s experience and expression of pain [27].
\nThough some characteristics of pain-related language seem to be universal, substantial influences of family and ethnic contexts are also repeated in the specificity of pain-related language due to the nature of the social setting in which children are growing up [55, 66]. The entire family is affected by children’s chronic pain experiences and these experiences are often stressful for other family members as well. The treatment prescribed to manage the child’s pain can result in interferences in planned family events, thus upsetting or disrupting the overall family system [66].
\nFrom the perspective of the family systems theory, family dynamics influence the way children understand and talk about their pain [22, 62]. Parents are the role models for their children to learn words to express pain [55]. As children’s cognitive and social skills develop, they learn to talk about pain by observing how their parents respond to and talk about their children’s pain experiences [66]. Parents’ socio-economic background, education and age may influence the way in which they respond to their children’s pain. For example, in an American study by Rowe [67] – who investigated why parents from different socio-economic statuses communicate in different ways with their children – it was found that more educated parents and parents from advantaged backgrounds tended to talk more often to their children and use a bigger variety of words and longer utterances thereby expanding their children’s language ability and pain-related vocabulary. Younger parents also tended to use different pain words in comparison with older parents [68].
\nBirth order also impacts on children’s development of pain-related vocabulary [38]. Younger children observe their older siblings’ use of pain words, which stimulates their own development of pain-related vocabulary [38]. It was reported that the presence of one or more older siblings has an impact on children’s use of pain words compared to those children without older siblings [38]. Moreover, children with siblings who had previously been hospitalised had a larger vocabulary than those with siblings who had never been hospitalised before. This suggests that experience plays a role in the learning of pain language because these children had to deal with the illness or hospitalisations of their sibling(s) [38].
\nApart from family practices, children develop an understanding of pain-related language within their sociolinguistic environment [66, 69]. Children’s language is influenced by their cultural beliefs, social groups and communities [69]. There are differences between the beliefs of diverse cultures and their views on parents’ roles in their children’s language development. In some cultures, parents may not react to their children’s utterances: they are of the opinion that adults must not teach children to talk, as they will eventually learn to talk on their own [67]. Some family and cultural beliefs can also result in disparities in the way children learn about pain and react to pain [36]. In some Nguni and Sotho cultures in South Africa, for example, boys are taught that they may not express their pain, because showing or expressing pain is a sign of weakness or lack of courage [70].
\nClinicians should therefore acknowledge cultural differences and try to understand the culture of the communication vulnerable child. They should ask detailed questions to help understand the child’s pain condition and to prevent any misunderstanding [52]. Clinicians should for instance be aware of the fact that in some cultures it is considered disgraceful to ask for pain relief, while people in other cultures believe that a godly intervention will relieve pain when necessary [62, 70].
\nChildren’s understanding (and the significance) of their first painful experience due to tissue injury will intensify with experience – either through positive or negative contextual associations [2]. Children learn the use of the word “pain” through their experiences related to injury [58]. Hospitalisations help children to develop pain vocabulary based on their personal experiences with pain. Therefore, children with previous hospitalisations who experience pain events more often and who have learnt and processed the concept of pain (and pain management) tend to have a larger pain-related vocabulary than those who have never been admitted to hospital before [38].
\nFrom the discussion above, it is clear that communication vulnerable children experience challenges to express their pain and need alternative means – such as AAC strategies and systems – to communicate their pain. AAC involves a variety of communication strategies that can be used to aid communication attempts of persons with communication challenges to either augment their speech or to be used as an alternative means to speech [31]. Regarding the adapted social communication model of pain, one can agree that when the communication vulnerable child is offered the use of AAC to express their pain (A), the form (or communication mode used) is less important than ensuring that the message (B) is understood by the observer (C). AAC systems are classified as either unaided or aided. Unaided AAC systems are defined as the use of only body parts to convey messages such as by pointing, making gestures, body language movements, facial expressions, and manual signing [31]. Aided AAC systems include low-technology aids that need no electronic programming (e.g. pen and paper, and symbol-based communication boards), as well as high-technology aids such as speech-generating devices [71]. Clinicians should be encouraged to incorporate AAC strategies and tools to enable communication vulnerable child patients to communicate their pain.
\nAAC strategies and systems have been successfully used with communication vulnerable children in various settings, including hospital settings [13, 16, 17, 18, 71, 72, 73]. Next, some potential AAC strategies are proposed to support communication vulnerable children to express their pain in order for observers (C) to understand the messages (B). The suggested AAC strategies will focus mainly on low-technology systems although all these strategies could also be incorporated in apps on digital mobile devices (smart phones or tablets) to enable communication vulnerable children as well as clinicians and researchers to gain a history of pain communication and subsequent pain treatment [74].
\nCommunication boards are low-technology AAC systems used to display pictures (photographs, line drawings or graphic symbols) to enable communication vulnerable persons to communicate [31]. When designing a communication board, aspects such as the type of symbol (photograph, type of line drawing, graphic symbols or written words), the symbol size (to best accommodate the child’s visual and motor skills), symbol colour (to ensure contrast and increase the ease of finding a word within a particular word class), board layout and display (e.g. using the left-to-right Fitzgerald-key outlay as a precursor to reading), as well as the child’s vocabulary need should be taken into consideration [71]. For example, children with physical disabilities or limited range of movement may not be able to access symbols that are too far apart.
\nJohnson et al. [53] conducted a scoping review to compile a list of children’s pain-related vocabulary in an attempt to provide clinicians and parents with possible pain words that children would typically use to express their pain. In this scoping review, 17 studies from diverse cultures in countries such as the United States of America, Canada, Finland, Kuwait, South Africa, Spain and Sweden were included. It was interesting to note that the meaning of children’s pain-related words in the native language translated to the same English word or words [53]. The study also showed that clinicians from different countries could use this list of pain-related words to compile basic pain-related communication boards that could be further individualised for their communication vulnerable paediatric clients [53].
\nIn a follow-up pilot study by Gerber [75], 6- to 9-year-old children were asked to choose which symbols from two symbol sets, namely Picture Communication Symbols [PCS™] and Bildstöd symbols (
The discussion earlier in this chapter makes it clear that children’s previous negative pain-related encounters influence how they perceive new pain experiences. Furthermore, since children with ASD and intellectual disabilities need routine to function optimally, visual schedules can be used to great benefit to prepare them for specific medical procedures. This preparation could reduce their anxiousness due to unfamiliarity with the procedure or previous negative experiences [15, 76]. A visual schedule should include a step-by-step and easily understandable format with pictures accompanied by written words (see Figure 2 for example). These will provide children with the necessary information to help them feel that they are in control of the imminent frightening procedures [77]. Visual schedules can be offered either in paper format (low-technology) or, where applicable, in a video story-based format.
\nVisual schedule of a needle procedure.
Children with severe physical disabilities and limited movements may not be able to use their fingers to point to choices on a low-technology communication board. Therefore, the use of eye gaze displays is proposed. With eye gaze, the child is instructed to use their eyes to look at a picture or word on the display and then glance at the communication partner (observer), who will then verbally confirm the child’s selection [15]. Figure 3 is an example of an eye-gaze flipchart display.
\nEye gaze flip chart.
During pain assessments of communication vulnerable children, clinicians or researchers can also ask the child pain-related questions providing them three options: “Yes”, “No”, “Not sure”. Communication vulnerable children often have clear yes/no responses (e.g. head nodding to indicate “Yes”). Should communication vulnerable children have no typical yes/no responses, the clinician can ask the child to blink their eyes (“Yes”), close their eyes (“No”), or to look away to indicate that they are not sure what to answer. In this case, the clinician should refrain from asking more than one close-ended question at a time (e.g. “Does it hurt?” and “Do you hurt in your [body part]?”). The clinician should rather ask only one question (e.g. “Does it hurt?”) to ensure that the child can give an appropriate response.
\nAppropriate pain management relies on the ability to accurately assess pain. For children, a common method to communicate pain is the use of pain scales [13]. Pain scales that are often used in clinical and research practice typically depict faces, colours or numeric grading [13]. An example of faces pain scales that are built on how children communicate their feeling(s) in a facial expression is the Faces Pain Scale-Revised (FPS-R) [78]. Colours and numeric grading are typically used in analogue scales that are based on increments to indicate pain severity, and these allow children to show that a somewhat larger or smaller pain is experienced (examples are the Colour Analogue Scale (CAS) [79]; and the Numeric Rating Scale (NRS) [80]). In a systematic review by Birnie et al. [23] on recommendations for the selection of children’s self-report rating scales for pain intensity, the FPS-R, CAS and NRS were recommended for self-report of acute pain. However, though these self-report scales are freely available, clinicians and researchers should keep in mind that they may not be effective for everyone [13]. For example, while some of these scales may not need expressive language, receptive language skills are crucial, as children are expected to comprehend and know the meaning of words such as “hurt” or “pain” when using these scales [26].
\nThis chapter aimed to address communication vulnerable children’s experiences of pain and their need for alternative ways to express their pain so as to receive appropriate pain treatment. The concept of communication vulnerability was explained framed in the context of the adapted social communication model of pain for communication vulnerable children. According to this model, there are many ways in which communication vulnerable children can encode (B) their pain experience (A). The model also emphasises the need for observers (C) to be open to other communication modes that children may use to communicate their pain. The discussion centred on the pain experiences of communication vulnerable children such as children with Down syndrome, with intellectual disabilities, autism or cerebral palsy, as well as of children in intensive care settings who experience temporary communication vulnerability. The chapter concludes with suggestions on how AAC strategies can be used to support communication vulnerable children in communicating their pain.
\nThe author would like to thank and acknowledge Prof Stefan Nilsson from Gothenburg University and Prof Juan Bornman from the Centre for AAC, University of Pretoria for their valuable comments to improve the content of this manuscript. The author would further like to thank Ms Olivia Loots for the drawings as portrayed in the three figures in this chapter.
\nThe funding from the National Research Foundation in South Africa to this project is also acknowledged.
\nThe author declares no conflict of interest.
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