Breast Feeding: Ocular and Hematopoietic Effects

**51**

**Chapter 4**

**Abstract**

retinal disease

that cannot be fed with breast milk.

conditions, and complications.

Relationship between Ocular

*Erdinc Bozkurt and Hayrunisa Bekis Bozkurt*

Morbidity and Infant Nutrition

The nutrition of the constantly growing and developing infant even after birth has an undeniable contribution to the development of eyes, which can be considered as the extension of the brain. Therefore, the elucidation of these physiological developments is valuable in terms of preventing pathological conditions. During the first six months of an infant's life, nutrition is provided through breast milk or infant formula, and after the sixth month, there is a transition to additional food. Breast milk is, thus, considered as 'miracle food', with a growing body of research being undertaken to investigate its relationship with orbital diseases and reporting that breast milk reduces ocular morbidity. Breast milk is an accessible, economical and important nutrition source for eye development and infant health. The developments in recent years have resulted in the content of formula being closer to that of breast milk, which can positively affect the neurovisional development of babies

**Keywords:** breastfeeding, formula, infant, visual development, refractive disorders,

Orbital development begins in fourth to sixth weeks of the intrauterine period. The eye develops from the surface ectoderm, neural ectoderm, and mesoderm. The optic nerve is also seen as an extension of the brain [1]. There is a significant relationship between the mother's diet during pregnancy and the orbital development of the infant. In recent years, the concept of the "first 1000" days emerged to refer to the process during pregnancy and the first two years after birth. The first 1000 days play a key role in many stages of life [2], and this period is also important in terms of healthy eye development and the prevention of orbital diseases, comorbid

The mother's diet during pregnancy is very valuable in terms of providing essential fatty acids and amino acids, which are necessary for the development of the orbital tissues of the infant. Studies have shown this developmental process is positively affected by a diet rich in phospholipids (PL), phosphatidylcholine (PC), phosphatidylethanolamine (PE), N-acylphosphatidylethanolamine (NAPE), phosphatidylinositol (PI), and phosphatidylserine (PS) [3]. Similarly, the mother's malnutrition during breastfeeding in the first year after birth negatively affects the orbital and brain development of the infant. In the complex process of orbital and visual development, tissues and cells need many minerals, vitamins and nutrients to continue their functions [4]. For example, in this process, vitamin A plays a vital

**1. Nutrition in the first year of life and orbital development**

#### **Chapter 4**

## Relationship between Ocular Morbidity and Infant Nutrition

*Erdinc Bozkurt and Hayrunisa Bekis Bozkurt*

#### **Abstract**

The nutrition of the constantly growing and developing infant even after birth has an undeniable contribution to the development of eyes, which can be considered as the extension of the brain. Therefore, the elucidation of these physiological developments is valuable in terms of preventing pathological conditions. During the first six months of an infant's life, nutrition is provided through breast milk or infant formula, and after the sixth month, there is a transition to additional food. Breast milk is, thus, considered as 'miracle food', with a growing body of research being undertaken to investigate its relationship with orbital diseases and reporting that breast milk reduces ocular morbidity. Breast milk is an accessible, economical and important nutrition source for eye development and infant health. The developments in recent years have resulted in the content of formula being closer to that of breast milk, which can positively affect the neurovisional development of babies that cannot be fed with breast milk.

**Keywords:** breastfeeding, formula, infant, visual development, refractive disorders, retinal disease

#### **1. Nutrition in the first year of life and orbital development**

Orbital development begins in fourth to sixth weeks of the intrauterine period. The eye develops from the surface ectoderm, neural ectoderm, and mesoderm. The optic nerve is also seen as an extension of the brain [1]. There is a significant relationship between the mother's diet during pregnancy and the orbital development of the infant. In recent years, the concept of the "first 1000" days emerged to refer to the process during pregnancy and the first two years after birth. The first 1000 days play a key role in many stages of life [2], and this period is also important in terms of healthy eye development and the prevention of orbital diseases, comorbid conditions, and complications.

The mother's diet during pregnancy is very valuable in terms of providing essential fatty acids and amino acids, which are necessary for the development of the orbital tissues of the infant. Studies have shown this developmental process is positively affected by a diet rich in phospholipids (PL), phosphatidylcholine (PC), phosphatidylethanolamine (PE), N-acylphosphatidylethanolamine (NAPE), phosphatidylinositol (PI), and phosphatidylserine (PS) [3]. Similarly, the mother's malnutrition during breastfeeding in the first year after birth negatively affects the orbital and brain development of the infant. In the complex process of orbital and visual development, tissues and cells need many minerals, vitamins and nutrients to continue their functions [4]. For example, in this process, vitamin A plays a vital role for photoreceptors, vitamin C is important for the development of the aqueous humor, and fatty acids are essential for the development of the optic nerve and myelin sheath [5]. In recent years, with the development of technology, premature infants now live longer, and the nutrition and supportive treatments for premature newborns have become even more important. In these infants, retinopathy of prematurity (ROP), which can cause blindness when not treated early, is seen very often due to hyperoxia, long stay in mechanical ventilation, infection, prematurity, low birth weight, and low-calorie maternal diet [6]. A diet rich in vitamins A and E, and longer duration of breastfeeding decreases the requirement of surgery to treat ROP [7, 8].

The nutrition of the constantly growing and developing infant even after birth has an undeniable contribution to the development of eyes, which can be considered as the extension of the brain. Therefore, the elucidation of these physiological developments is valuable in terms of preventing pathological conditions [9]. During the first six months of an infant's life, nutrition is provided through breast milk or infant formula, and after the sixth month, there is a transition to additional food. In this chapter, we discuss breast milk, infant formula, and their relationship with ocular morbidity.

#### **2. Breast milk: the best nutrition for orbital development**

After the baby is born, breastfeeding especially in the first six months is considered as a fundamental right by WHO and necessary actions are taken to ensure this for every child [10]. Breast milk is the most suitable food for a newborn in terms of the balance of nutrients it contains [11]. Studies have shown that breast milk protects the infant against many diseases, malignancies, obesity, and malnutrition [12]. Degeneration occurs in retinal ganglion cells and photoreceptors, especially when the infant's diet is deficient in taurine found at high levels in breast milk [13]. Many bioactive compounds, such as α-lactalbumin, lactoferrin and immunoglobulins, which have antioxidant properties, are known to be important for brain and orbital development, as well as immunomodulatory functions [14]. Being rich in glial cell-line derived neurotrophic factor (GDNF) and oligosaccharides, breast milk acts as an important stimulator for healthy development [15]. The presence of many growth factors and the importance of the baby being in proper osmolarity and balance for the full development of the intestines and kidneys further increase the value of breast milk [16]. Breast milk is, thus, considered as 'miracle food', with a growing body of research being undertaken to investigate its relationship with orbital diseases and reporting that breast milk reduces ocular morbidity [17].

#### **3. New formulas enriched with vitamin and minerals**

In circumstances where breast milk is insufficient or contraindicated, the use of formula approaching breast milk in terms of content has increased in recent years. Infant formula can be divided into three groups as soy-based, cow milk-based and hypoallergic or amino acid-based special foods produced for special conditions, such as metabolic diseases [18]. However, there have been discussions of safety for formula in terms of infant health since its first use [19]. Due to the high levels of renal solute load of the foods used in the first year of life, infant formula still lags behind breast milk, and there are only limited scientific studies on the relationship between infant formula and highly critical molecules for brain and eye development, . Considering the positive impact of breast milk on the immune system, the

**53**

*Relationship between Ocular Morbidity and Infant Nutrition*

food given to the infant in case of breast milk deficiency or contraindication should be "closest" to breast milk [20]. With the latest technologies, the enrichment of formula foods with prebiotics, probiotics, oligosaccharides, and various vitamins and minerals has made significant contributions to infant nutrition [21]. In particular, molecules which take part in the development of visual functions, such as docosahexaenoic acid (DHA), vitamin A, and vitamin E have started to be added to infant formula. In a study on baby rhesus monkeys, it was observed that formula enriched with carotenoid increased the amount of lutein in the brain and tissues and positively affected their development, but not to the extent of the positive

A healthy and balanced nutrition in childhood is very important for not only growth and development and but also prevention of diseases. In terms of eye health, it is important to ensure that the infant receives age-appropriate nutrition with breast milk and foods close to breast milk in content and quality, which will protect her/him against malnutrition and obesity and an adequate intake of vita-

Breast milk contains an average of 1.1% protein, 4.2% fat, and 7.0% carbohydrates, and is a miraculous nutrient in which every 100 g contains 72 kcal energy [23]. Breast milk also has vitamins E and C, as well as enzymes, such as superoxide dismutase, catalase, and glutathione peroxidase with strong antioxidant properties. In this way, it protects the infant from eye damage that may be caused by oxidative stress [24]. Generally, breast milk has been found to be inadequate in certain vitamins and minerals, such as vitamin D, iodine, iron, and vitamin K. Since the deficiency of these vitamins and minerals can affect the infant systematically, going beyond eye diseases, they are usually added to the infant's diet as supple-

Breast milk supports the infant's growth and development through its content of long-chain polyunsaturated essential fatty acids, such as arachidonic acid, long chain polyunsaturated fatty acids (LC-PUFAs) (20: 4n-6) and DHA (22: 6n-3), linoleic (LA; 18: 2n-6) and alpha-linolenic (18: 3n-3) acid [26]. DHA and arachidonic acid (AA) in the membrane lipids of the brain and retina are critical for visual and neuronal functions. Taking these substances with diet in the first year of life is

The retina (and the crystalline lens to a lesser extent) has reduced light-sourced oxidative damage by vitamin E and C, and carotenoids (lutein and zeaxanthin), which are intensely present in the macular region. Vitamins E and C and lutein cannot be synthesized by the infant, and therefore should be taken in through the diet [28]. Dietary carotenoid, lutein and zeoxanthin are known to be protective against some eye diseases, such as macular degenerations [29]. It was observed that the serum lutein level of infants fed with breast milk was higher than that of infants fed with formula [30]. This indicates that infants fed with breast milk may be resistant to many eye diseases caused by oxidative damage due to its higher antioxidant levels than formula. Vitamin A is an important vitamin used by photoreceptors in visual physiology. Adequate intake of vitamin A, which is found in breast milk and formula foods, has been shown to reduce the severity of ROP through vascular endothelial growth factor (VEGF-A). In addition, preterm infants fed with breast milk have higher serum insulin-like growth factor-1 (IGF-1) levels than those who

*DOI: http://dx.doi.org/10.5772/intechopen.92162*

**4. Breastfeeding and ocular disease**

**4.1 Breastfeeding and visual development**

important for the visual development of infants [27].

effect of breast milk [22].

mins and minerals.

ments [25].

*Relationship between Ocular Morbidity and Infant Nutrition DOI: http://dx.doi.org/10.5772/intechopen.92162*

*Infant Feeding - Breast versus Formula*

ROP [7, 8].

ocular morbidity.

role for photoreceptors, vitamin C is important for the development of the aqueous humor, and fatty acids are essential for the development of the optic nerve and myelin sheath [5]. In recent years, with the development of technology, premature infants now live longer, and the nutrition and supportive treatments for premature newborns have become even more important. In these infants, retinopathy of prematurity (ROP), which can cause blindness when not treated early, is seen very often due to hyperoxia, long stay in mechanical ventilation, infection, prematurity, low birth weight, and low-calorie maternal diet [6]. A diet rich in vitamins A and E, and longer duration of breastfeeding decreases the requirement of surgery to treat

The nutrition of the constantly growing and developing infant even after birth has an undeniable contribution to the development of eyes, which can be considered as the extension of the brain. Therefore, the elucidation of these physiological developments is valuable in terms of preventing pathological conditions [9]. During the first six months of an infant's life, nutrition is provided through breast milk or infant formula, and after the sixth month, there is a transition to additional food. In this chapter, we discuss breast milk, infant formula, and their relationship with

After the baby is born, breastfeeding especially in the first six months is considered as a fundamental right by WHO and necessary actions are taken to ensure this for every child [10]. Breast milk is the most suitable food for a newborn in terms of the balance of nutrients it contains [11]. Studies have shown that breast milk protects the infant against many diseases, malignancies, obesity, and malnutrition [12]. Degeneration occurs in retinal ganglion cells and photoreceptors, especially when the infant's diet is deficient in taurine found at high levels in breast milk [13]. Many bioactive compounds, such as α-lactalbumin, lactoferrin and immunoglobulins, which have antioxidant properties, are known to be important for brain and orbital development, as well as immunomodulatory functions [14]. Being rich in glial cell-line derived neurotrophic factor (GDNF) and oligosaccharides, breast milk acts as an important stimulator for healthy development [15]. The presence of many growth factors and the importance of the baby being in proper osmolarity and balance for the full development of the intestines and kidneys further increase the value of breast milk [16]. Breast milk is, thus, considered as 'miracle food', with a growing body of research being undertaken to investigate its relationship with orbital diseases and reporting that breast milk reduces ocular morbidity [17].

In circumstances where breast milk is insufficient or contraindicated, the use of formula approaching breast milk in terms of content has increased in recent years. Infant formula can be divided into three groups as soy-based, cow milk-based and hypoallergic or amino acid-based special foods produced for special conditions, such as metabolic diseases [18]. However, there have been discussions of safety for formula in terms of infant health since its first use [19]. Due to the high levels of renal solute load of the foods used in the first year of life, infant formula still lags behind breast milk, and there are only limited scientific studies on the relationship between infant formula and highly critical molecules for brain and eye development, . Considering the positive impact of breast milk on the immune system, the

**2. Breast milk: the best nutrition for orbital development**

**3. New formulas enriched with vitamin and minerals**

**52**

food given to the infant in case of breast milk deficiency or contraindication should be "closest" to breast milk [20]. With the latest technologies, the enrichment of formula foods with prebiotics, probiotics, oligosaccharides, and various vitamins and minerals has made significant contributions to infant nutrition [21]. In particular, molecules which take part in the development of visual functions, such as docosahexaenoic acid (DHA), vitamin A, and vitamin E have started to be added to infant formula. In a study on baby rhesus monkeys, it was observed that formula enriched with carotenoid increased the amount of lutein in the brain and tissues and positively affected their development, but not to the extent of the positive effect of breast milk [22].

A healthy and balanced nutrition in childhood is very important for not only growth and development and but also prevention of diseases. In terms of eye health, it is important to ensure that the infant receives age-appropriate nutrition with breast milk and foods close to breast milk in content and quality, which will protect her/him against malnutrition and obesity and an adequate intake of vitamins and minerals.

#### **4. Breastfeeding and ocular disease**

#### **4.1 Breastfeeding and visual development**

Breast milk contains an average of 1.1% protein, 4.2% fat, and 7.0% carbohydrates, and is a miraculous nutrient in which every 100 g contains 72 kcal energy [23]. Breast milk also has vitamins E and C, as well as enzymes, such as superoxide dismutase, catalase, and glutathione peroxidase with strong antioxidant properties. In this way, it protects the infant from eye damage that may be caused by oxidative stress [24]. Generally, breast milk has been found to be inadequate in certain vitamins and minerals, such as vitamin D, iodine, iron, and vitamin K. Since the deficiency of these vitamins and minerals can affect the infant systematically, going beyond eye diseases, they are usually added to the infant's diet as supplements [25].

Breast milk supports the infant's growth and development through its content of long-chain polyunsaturated essential fatty acids, such as arachidonic acid, long chain polyunsaturated fatty acids (LC-PUFAs) (20: 4n-6) and DHA (22: 6n-3), linoleic (LA; 18: 2n-6) and alpha-linolenic (18: 3n-3) acid [26]. DHA and arachidonic acid (AA) in the membrane lipids of the brain and retina are critical for visual and neuronal functions. Taking these substances with diet in the first year of life is important for the visual development of infants [27].

The retina (and the crystalline lens to a lesser extent) has reduced light-sourced oxidative damage by vitamin E and C, and carotenoids (lutein and zeaxanthin), which are intensely present in the macular region. Vitamins E and C and lutein cannot be synthesized by the infant, and therefore should be taken in through the diet [28]. Dietary carotenoid, lutein and zeoxanthin are known to be protective against some eye diseases, such as macular degenerations [29]. It was observed that the serum lutein level of infants fed with breast milk was higher than that of infants fed with formula [30]. This indicates that infants fed with breast milk may be resistant to many eye diseases caused by oxidative damage due to its higher antioxidant levels than formula. Vitamin A is an important vitamin used by photoreceptors in visual physiology. Adequate intake of vitamin A, which is found in breast milk and formula foods, has been shown to reduce the severity of ROP through vascular endothelial growth factor (VEGF-A). In addition, preterm infants fed with breast milk have higher serum insulin-like growth factor-1 (IGF-1) levels than those who

feed with formula [31]. A high IGF-1 level decreases the severity of ROP by ensuring the normal development of retinal vascularization [32].

#### **4.2 Refractive disorders and breastfeeding**

Refractive disorders (error) represent a mismatch between the focal and axial lengths of the eye. The 10th revision of the International Classification of Diseases, defines this disorder as blurring in vision as a result of a defect in focusing light on the retina [33]. At birth, the average cycloplegic refractive error for the infant usually ranges from ±1.50 dioptre (D) to ±2.50 D, with standard deviations from +1.00 to +2.50 D [34]. At the age of six to 72 months, the eyes undergo a process of emmetropization in which the average refractive error decreases in both myopic and hypermetropic infants [35].

Refractive disorders are the most common cause of visual impairment and the second most common cause of treatable visual deficiency [36]. The frequency of refractive disorders may vary according to ethnicity, age, and the development level of the country [37]. According to a study carried out in the USA in 2015, the frequency of visual impairment in children between the ages of three and five is estimated to be around 1.5%, which is expected to increase in the near future [38]. Among the most important etiological causes of refractive disorders are genetics and gene-environment interactions [39–41]. In addition, nutrition is considered to have an impact on refractive disorder and orbital health. It is stated that the prevalence of refractive disorders, such as ametropia, anisometropia and astigmatism is high, especially in African societies exposed to malnutrition [37].

Recently, the effect of breast milk intake, which is the most important source of infant nutrition, on refractive disorders has been investigated. It is thought that breastfeeding is important for visual development and orbital growth during the infantile period [42]. In one of these studies, it was shown that breast milk-fed children of mothers with a DHA-rich diet had better stereoscopic vision than the formula-fed children [43].

Refractive disorders can be basically classified as myopia, hyperopia, and astigmatism. Myopia is one of the most common visual impairments and has become an important public health problem due to its increasing prevalence in recent years. While the prevalence of ametropia, anisometropia, and astigmatism is high in Africa, the frequency of myopia is low in developing countries while it is higher in developed societies, such as the United States of America (USA) and the United Kingdom (UK) [44]. This is not a surprising finding considering that myopia is associated with reading and using technological devices, which require looking at objects closely.

In a study carried out in Singapore, high levels of unsaturated fatty acids and cholesterol intake were associated with longer AL, although they could not be directly associated with myopia [45]. In another study undertaken by Liu et al. in China, it was found that feeding the infant with breast milk for the first six months was associated with hyperopic spherical equivalent refraction (SER) and less myopia and was not related to AL [46]. However, in the study of Growing Up in Singapore Towards Healthy Outcomes (GUSTO), infant feeding at the sixth, ninth and 12th months did not affect the myopia levels at the age of 3.5 years [47].

There are also many studies suggesting that breastfeeding has no effect on myopia. In a study on strabismus, amblyopia and refractive disorder (STARS) conducted with 797 children in Singapore, 65.4% of the sample were myopic, of which 8.5% were breastfed. In that study, it was stated that there was no relationship between breast milk and myopia [48]. In another study supporting the STARS study, it was reported that breast milk had no effect on myopia [49]. In a study

**55**

breast milk [62].

*Relationship between Ocular Morbidity and Infant Nutrition*

data to support the presence of such relationship.

**4.3 Breastfeeding and retinal disease**

vitamins and minerals [58, 59].

which included 311 children in Iran (grades 1–5), the rate of breastfeeding for more than six months was 85% while the frequency of myopia (SE of at least −0.50 D) was 5.2%. It was stated that the breastfeeding of the infant for the first six months had no significant effect on vision level or refractive error [50]. A study undertaken in the UK suggested that other factors, such as parental education status, gender, maternal age, and order of birth were more important for visual development and myopia in early life than the type of infant feeding [49]. The existence of different opinions and findings on this subject indicates that further detailed studies

Another refractive defect is hypermetropia, which occurs due to either the AL of the eye being shorter than normal or refractive structures such as cornea and lens having less refractive power than normal [51]. In a study investigating the relationship between hyperopia and breastfeeding, it was stated that breastfeeding resulted in a significantly high rate of hyperopia. This increased incidence of hyperopia was associated with various reasons, such as ethnicity, presence of refractive disorder, ethnic and sociocultural structure, and content of the mother's milk [52]. Bozkurt et al. stated that although breastfeeding leads to a hyperopic shift, it has no effect on

Another refractive disorder, astigmatism, occurs as a result of the refractive parts of the eye (cornea and lens) not producing an equal amount of refraction on each meridian, leading to images not being focused on a point on the fovea. Generally, the breaking force of the vertical axis of the anterior aspect of the cornea is 0.5 D more than its horizontal axis. This condition, known as physiological astigmatism, is reduced to zero by the cornea posterior face and lens. Lenticular (lens-dependent) astigmatism is rarer. The image of an object being in the form of two separate lines, 90° perpendicular to each other in two planes, is called regular astigmatism, which is the most common type of astigmatism in the clinic [54]. The prevalence of astigmatism can be seen in different countries at different frequencies. While this rate is 2.2% in Nepal, it reaches 82.2% in Singapore [55, 56]. Since there is only limited information in the literature investigating the relationship between breastfeeding and astigmatism, we consider that there is not yet enough

The retina, which is considered an extension of the brain, has two major layers as the outer retinal pigment epithelium and the inner neurosensory layer. The neurosensory layer is formed by a photoreceptor layer, bipolar ganglion, amacrine and horizontal cells, and support cells similar to neuroglia [57]. When the pathologies that may occur with inflammation, trauma, autoimmune or epigenetic mechanisms in each layer are examined, it is seen that they are associated with the deficiency of

In addition to except vitamins K and D, breast milk is very rich in other vitamins and minerals that are indispensable elements of a healthy and balanced nutrition. Furthermore, polyunsaturated fatty acids and antioxidants in breast milk play an important role in the development of the eye and neuronal structure in the first months of life [48]. The leading chemical structures that contribute to the development of the brain and retina in breast milk are LA, α-LA, AA, and DHA [60], which are commonly called LC-PUFAs. In animal experiments, DHA deficiency has been shown to cause the impairment of neuronal and retinal functions [61]. It has also been reported that the photoreceptor external segments of monkeys fed with a diet devoid of taurine amino acid are degenerated, and taurine is abundantly found in

*DOI: http://dx.doi.org/10.5772/intechopen.92162*

are needed.

SER [53].

#### *Relationship between Ocular Morbidity and Infant Nutrition DOI: http://dx.doi.org/10.5772/intechopen.92162*

*Infant Feeding - Breast versus Formula*

hypermetropic infants [35].

formula-fed children [43].

objects closely.

feed with formula [31]. A high IGF-1 level decreases the severity of ROP by ensur-

Refractive disorders (error) represent a mismatch between the focal and axial lengths of the eye. The 10th revision of the International Classification of Diseases, defines this disorder as blurring in vision as a result of a defect in focusing light on the retina [33]. At birth, the average cycloplegic refractive error for the infant usually ranges from ±1.50 dioptre (D) to ±2.50 D, with standard deviations from +1.00 to +2.50 D [34]. At the age of six to 72 months, the eyes undergo a process of emmetropization in which the average refractive error decreases in both myopic and

Refractive disorders are the most common cause of visual impairment and the second most common cause of treatable visual deficiency [36]. The frequency of refractive disorders may vary according to ethnicity, age, and the development level of the country [37]. According to a study carried out in the USA in 2015, the frequency of visual impairment in children between the ages of three and five is estimated to be around 1.5%, which is expected to increase in the near future [38]. Among the most important etiological causes of refractive disorders are genetics and gene-environment interactions [39–41]. In addition, nutrition is considered to have an impact on refractive disorder and orbital health. It is stated that the prevalence of refractive disorders, such as ametropia, anisometropia and astigmatism is

Recently, the effect of breast milk intake, which is the most important source of infant nutrition, on refractive disorders has been investigated. It is thought that breastfeeding is important for visual development and orbital growth during the infantile period [42]. In one of these studies, it was shown that breast milk-fed children of mothers with a DHA-rich diet had better stereoscopic vision than the

Refractive disorders can be basically classified as myopia, hyperopia, and astigmatism. Myopia is one of the most common visual impairments and has become an important public health problem due to its increasing prevalence in recent years. While the prevalence of ametropia, anisometropia, and astigmatism is high in Africa, the frequency of myopia is low in developing countries while it is higher in developed societies, such as the United States of America (USA) and the United Kingdom (UK) [44]. This is not a surprising finding considering that myopia is associated with reading and using technological devices, which require looking at

In a study carried out in Singapore, high levels of unsaturated fatty acids and cholesterol intake were associated with longer AL, although they could not be directly associated with myopia [45]. In another study undertaken by Liu et al. in China, it was found that feeding the infant with breast milk for the first six months was associated with hyperopic spherical equivalent refraction (SER) and less myopia and was not related to AL [46]. However, in the study of Growing Up in Singapore Towards Healthy Outcomes (GUSTO), infant feeding at the sixth, ninth

and 12th months did not affect the myopia levels at the age of 3.5 years [47]. There are also many studies suggesting that breastfeeding has no effect on myopia. In a study on strabismus, amblyopia and refractive disorder (STARS) conducted with 797 children in Singapore, 65.4% of the sample were myopic, of which 8.5% were breastfed. In that study, it was stated that there was no relationship between breast milk and myopia [48]. In another study supporting the STARS study, it was reported that breast milk had no effect on myopia [49]. In a study

ing the normal development of retinal vascularization [32].

high, especially in African societies exposed to malnutrition [37].

**4.2 Refractive disorders and breastfeeding**

**54**

which included 311 children in Iran (grades 1–5), the rate of breastfeeding for more than six months was 85% while the frequency of myopia (SE of at least −0.50 D) was 5.2%. It was stated that the breastfeeding of the infant for the first six months had no significant effect on vision level or refractive error [50]. A study undertaken in the UK suggested that other factors, such as parental education status, gender, maternal age, and order of birth were more important for visual development and myopia in early life than the type of infant feeding [49]. The existence of different opinions and findings on this subject indicates that further detailed studies are needed.

Another refractive defect is hypermetropia, which occurs due to either the AL of the eye being shorter than normal or refractive structures such as cornea and lens having less refractive power than normal [51]. In a study investigating the relationship between hyperopia and breastfeeding, it was stated that breastfeeding resulted in a significantly high rate of hyperopia. This increased incidence of hyperopia was associated with various reasons, such as ethnicity, presence of refractive disorder, ethnic and sociocultural structure, and content of the mother's milk [52]. Bozkurt et al. stated that although breastfeeding leads to a hyperopic shift, it has no effect on SER [53].

Another refractive disorder, astigmatism, occurs as a result of the refractive parts of the eye (cornea and lens) not producing an equal amount of refraction on each meridian, leading to images not being focused on a point on the fovea. Generally, the breaking force of the vertical axis of the anterior aspect of the cornea is 0.5 D more than its horizontal axis. This condition, known as physiological astigmatism, is reduced to zero by the cornea posterior face and lens. Lenticular (lens-dependent) astigmatism is rarer. The image of an object being in the form of two separate lines, 90° perpendicular to each other in two planes, is called regular astigmatism, which is the most common type of astigmatism in the clinic [54]. The prevalence of astigmatism can be seen in different countries at different frequencies. While this rate is 2.2% in Nepal, it reaches 82.2% in Singapore [55, 56]. Since there is only limited information in the literature investigating the relationship between breastfeeding and astigmatism, we consider that there is not yet enough data to support the presence of such relationship.

#### **4.3 Breastfeeding and retinal disease**

The retina, which is considered an extension of the brain, has two major layers as the outer retinal pigment epithelium and the inner neurosensory layer. The neurosensory layer is formed by a photoreceptor layer, bipolar ganglion, amacrine and horizontal cells, and support cells similar to neuroglia [57]. When the pathologies that may occur with inflammation, trauma, autoimmune or epigenetic mechanisms in each layer are examined, it is seen that they are associated with the deficiency of vitamins and minerals [58, 59].

In addition to except vitamins K and D, breast milk is very rich in other vitamins and minerals that are indispensable elements of a healthy and balanced nutrition. Furthermore, polyunsaturated fatty acids and antioxidants in breast milk play an important role in the development of the eye and neuronal structure in the first months of life [48]. The leading chemical structures that contribute to the development of the brain and retina in breast milk are LA, α-LA, AA, and DHA [60], which are commonly called LC-PUFAs. In animal experiments, DHA deficiency has been shown to cause the impairment of neuronal and retinal functions [61]. It has also been reported that the photoreceptor external segments of monkeys fed with a diet devoid of taurine amino acid are degenerated, and taurine is abundantly found in breast milk [62].

Another important molecule, phosphatidylcholine, is essential for the synthesis of phospholipids, DNA methylation, and the neurodevelopmental process of infants. In an animal experiment study conducted by Surzenko et al., it was shown that a low choline diet during pregnancy affected the retinal development and function in the fetus, and choline provided differentiation and proliferation in retinal progenitor cells [63]. Infants received enough choline and LC-PUFAs from breast milk, which are very important for the brain, retinal and neurovisional development. In another study, it was emphasized that formula foods containing choline and LC-PUFAs were required for growth and development in non-breastfed infants [64].

The thickness of the retinal nerve fiber layer (RNFL) can offer an idea about retinal structures in the early period. Conditions occurring in the retinal layers, such as edema and atrophy can be objectively evaluated using optical coherence tomography. In a study undertaken by Bozkurt et al., stated that the retinal nerve fiber layer was thicker in formula-fed children, than breast-fed infant, which might be related to the content of formula. However, a definitive conclusion could not be reached as to whether the thickening of RNFL in formula-fed infants was a healthy neurotrophy or an adaptive change [53].

Another important eye disease associated with infant nutrition is ROP, which occurs in preterm babies (birth week <37) with an immature retinal structure and not fully developed retinal vasculature. Today, ROP is considered as the most important cause of preventable blindness in childhood in developed and developing countries all over the world [65]. There are many studies showing the relationship between ROP and breast milk intake. For example, Okamoto et al. reported that the content of breast milk could reduce ROP severity and the ingredients in breast milk could protect premature infants from blindness [66]. In another study, Hylander et al. found that antioxidant substances, such as inositol, vitamin E, and beta carotene in breast milk prevented ROP development, and formula-fed infants had a higher ROP frequency than breast-fed infants, especially due to the absence of inositol in standard formula [67]. In another study supporting this study, the supplementation of infant diet with inositol was shown to reduce the incidence of ROP [68]. ROP is the most serious retinal disease that causes the deficiency of vision in the neonatal period, secondary to retinal detachment. According to the literature, breast milk intake should be encouraged to reduce the harm caused by ROP [66, 67].

In conclusion, breast milk is an accessible, economical and important nutrition source for eye development and infant health. The developments in recent years have resulted in the content of formula being closer to that of breast milk, which can positively affect the neurovisional development of babies that cannot be fed with breast milk.

**57**

**Author details**

Erdinc Bozkurt1

Turkey

\* and Hayrunisa Bekis Bozkurt<sup>2</sup>

\*Address all correspondence to: drerdincbozkurt@hotmail.com

provided the original work is properly cited.

1 Department of Ophthalmology, Kafkas University, Faculty of Medicine, Kars,

2 Department of Pediatrics, Kafkas University, Faculty of Medicine, Kars, Turkey

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

*Relationship between Ocular Morbidity and Infant Nutrition*

*DOI: http://dx.doi.org/10.5772/intechopen.92162*

*Relationship between Ocular Morbidity and Infant Nutrition DOI: http://dx.doi.org/10.5772/intechopen.92162*

*Infant Feeding - Breast versus Formula*

neurotrophy or an adaptive change [53].

infants [64].

ROP [66, 67].

with breast milk.

Another important molecule, phosphatidylcholine, is essential for the synthesis of phospholipids, DNA methylation, and the neurodevelopmental process of infants. In an animal experiment study conducted by Surzenko et al., it was shown that a low choline diet during pregnancy affected the retinal development and function in the fetus, and choline provided differentiation and proliferation in retinal progenitor cells [63]. Infants received enough choline and LC-PUFAs from breast milk, which are very important for the brain, retinal and neurovisional development. In another study, it was emphasized that formula foods containing choline and LC-PUFAs were required for growth and development in non-breastfed

The thickness of the retinal nerve fiber layer (RNFL) can offer an idea about retinal structures in the early period. Conditions occurring in the retinal layers, such as edema and atrophy can be objectively evaluated using optical coherence tomography. In a study undertaken by Bozkurt et al., stated that the retinal nerve fiber layer was thicker in formula-fed children, than breast-fed infant, which might be related to the content of formula. However, a definitive conclusion could not be reached as to whether the thickening of RNFL in formula-fed infants was a healthy

Another important eye disease associated with infant nutrition is ROP, which occurs in preterm babies (birth week <37) with an immature retinal structure and not fully developed retinal vasculature. Today, ROP is considered as the most important cause of preventable blindness in childhood in developed and developing countries all over the world [65]. There are many studies showing the relationship between ROP and breast milk intake. For example, Okamoto et al. reported that the content of breast milk could reduce ROP severity and the ingredients in breast milk could protect premature infants from blindness [66]. In another study, Hylander et al. found that antioxidant substances, such as inositol, vitamin E, and beta carotene in breast milk prevented ROP development, and formula-fed infants had a higher ROP frequency than breast-fed infants, especially due to the absence of inositol in standard formula [67]. In another study supporting this study, the supplementation of infant diet with inositol was shown to reduce the incidence of ROP [68]. ROP is the most serious retinal disease that causes the deficiency of vision in the neonatal period, secondary to retinal detachment. According to the literature, breast milk intake should be encouraged to reduce the harm caused by

In conclusion, breast milk is an accessible, economical and important nutrition source for eye development and infant health. The developments in recent years have resulted in the content of formula being closer to that of breast milk, which can positively affect the neurovisional development of babies that cannot be fed

**56**

### **Author details**

Erdinc Bozkurt1 \* and Hayrunisa Bekis Bozkurt<sup>2</sup>

1 Department of Ophthalmology, Kafkas University, Faculty of Medicine, Kars, Turkey

2 Department of Pediatrics, Kafkas University, Faculty of Medicine, Kars, Turkey

\*Address all correspondence to: drerdincbozkurt@hotmail.com

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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[13] Gaucher D, Arnault E, Husson Z, Froger N, Dubus E, Gondouin P, et al. Taurine deficiency damages retinal neurones: Cone photoreceptors and retinal ganglion cells. Amino Acids.

[14] Ballard O, Morrow AL. Human milk composition: Nutrients and bioactive factors. Pediatric Clinics.

[15] Rodrigues D, Li A, Nair D, Blennerhassett MJ. Glial cell linederived neurotrophic factor is a key neurotrophin in the postnatal enteric nervous system. Neurogastroenterology

and Motility. 2011;**23**(2):e44-e56

[17] Aksoy A, Ozdemir M, Aslan L, Aslankurt M, Gul O. Effect of breast feeding on ocular morbidity. Medical

[18] Martin CR, Ling P-R, Blackburn GL. Review of infant feeding: Key features

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*Infant Feeding - Breast versus Formula*

[35] Saunders KJ, Woodhouse JM, Westall CA. Emmetropisation in human infancy: Rate of change is related to initial refractive error. Vision Research.

associated with prenatal and postnatal dietary factors: A report from a population-based cohort study. The American Journal of Clinical Nutrition.

[44] Morgan IG, Ohno-Matsui K, Saw SM. Myopia. Lancet. 2012;**379**(9827):1739-1748

[45] Lim LS, Gazzard G, Low Y-L, Choo R, Tan DT, Tong L, et al. Dietary factors, myopia, and axial dimensions in children. Ophthalmology. 2010;**117,** 

[46] Liu S, Ye S, Wang Q, Cao Y, Zhang X. Breastfeeding and myopia: A cross-sectional study of children aged 6-12 years in Tianjin, China. Scientific

[47] Chua SYL, Sabanayagam C, Tan CS, Lim LS, Toh JY, Chong YS, et al. Diet and risk of myopia in three-year-old Singapore children: The GUSTO cohort. Clinical and Experimental Optometry.

Reports. 2018;**8**(1):10025

2018;**101**(5):692-699

[48] Chong Y-S, Liang Y, Tan D, Gazzard G, Stone RA, Saw S-M. Association between breastfeeding and likelihood of myopia in children. JAMA. 2005;**293**(24):2999-3002

[49] Rudnicka AR, Owen CG, Richards M, Wadsworth ME, Strachan DP. Effect of breastfeeding and sociodemographic factors on visual outcome in childhood and adolescence. The American Journal of Clinical Nutrition. 2008;**87**(5):1392-1399

[50] Shirzadeh E, Kooshki A, Mohammadi M. The relationship between breastfeeding and

measurements of refraction and visual acuity in primary school children. Breastfeeding Medicine. 2016;**11**:235-238

[51] Flitcroft DI. Emmetropisation and the aetiology of refractive errors. Eye (London, England). 2014;**28**(2):169-179

2001;**73**(2):316-322

**5**:993-997

[36] Murthy GV, Gupta SK, Ellwein LB, Munoz SR, Pokharel GP, Sanga L, et al. Refractive error in children in an urban population in New Delhi. Investigative Ophthalmology & Visual Science.

[37] Miller D, Atebara N, Fellenz M, Rosenthal P, Schechter R, West CJB, et al. Section 3: Optics, refraction, and contact lenses. In: Basic and Clinical Science Course. San Francisco: American Academy of Ophthalmology;

[38] Varma R, Tarczy-Hornoch K, Jiang X. Visual impairment in preschool children in the United States: Demographic and geographic variations from 2015 to 2060. JAMA Ophthalmology. 2017;**135**(6):610-616

[39] Saw SM, Chua WH, Hong CY, Wu HM, Chan WY, Chia KS, et al. Nearwork in early-onset myopia. Investigative Ophthalmology & Visual

[40] Seet B, Wong TY, Tan DT, Saw SM, Balakrishnan V, Lee LK, et al. Myopia in Singapore: Taking a public health approach. The British Journal of Ophthalmology. 2001;**85**(5):521-526

[41] Morgan I, Rose K. How genetic is school myopia? Progress in Retinal and

Eye Research. 2005;**24**(1):1-38

[42] Heller CD, O'Shea M, Yao Q, Langer J, Ehrenkranz RA, Phelps DL, et al. Human milk intake and retinopathy

of prematurity in extremely low birth weight infants. Pediatrics.

[43] Williams C, Birch EE, Emmett PM, Northstone K. Stereoacuity at age 3.5 y in children born full-term is

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[53] Bozkurt E, Bozkurt HB, Ucer MB. Comparative effect of feeding human milk as opposed to formula on visual function and ocular anatomy. Breastfeeding Medicine. 2019;**14**(7):493-498

[54] Grzybowski A, Kanclerz P. Beginnings of astigmatism understanding and management in the 19th century. Eye & Contact Lens. 2018;**44**(Suppl 1):S22-Ss9

[55] Pokharel GP, Negrel AD, Munoz SR, Ellwein LB. Refractive error study in children: Results from Mechi zone. Nepalese Journal of Ophthalmology. 2000;**129**(4):436-444

[56] Woo WW, Lim KA, Yang H, Lim XY, Liew F, Lee YS, et al. Refractive errors in medical students in Singapore. Singapore Medical Journal. 2004;**45**(10):470-474

[57] Burns SA, Elsner AE, Sapoznik KA, Warner RL, Gast TJ. Adaptive optics imaging of the human retina. Progress in Retinal and Eye Research. 2019;**68**:1-30

[58] Evans JR, Lawrenson JG. Antioxidant vitamin and mineral supplements for slowing the progression of age-related macular degeneration. The Cochrane Database of Systematic Reviews. 2017;**7**:CD000254

[59] Zhao Y, Feng K, Liu R, Pan J, Zhang L, Lu X. Vitamins and mineral supplements for retinitis pigmentosa. Journal of Ophthalmology. 2019;**2019**

[60] Innis SM. Impact of maternal diet on human milk composition and neurological development of infants. The American Journal of Clinical Nutrition. 2014;**99**(3):734S-741S

[61] Jeffrey BG, Mitchell DC, Hibbeln JR, Gibson RA, Chedester AL, Salem N Jr. Visual acuity and retinal function in infant monkeys fed long-chain PUFA. Lipids. 2002;**37**(9):839-848

[62] Liu A, Terry R, Lin Y, Nelson K, Bernstein PS. Comprehensive and sensitive quantification of long-chain and very long-chain polyunsaturated fatty acids in small samples of human and mouse retina. Journal of Chromatography. A. 2013;**1307**:191-200

[63] Surzenko N, Trujillo-González I, Zeisel SH. Low intake of choline during pregnancy leads to aberrant retinal architecture and poor visual function in the offspring. The FASEB Journal. 2016;**30**(1\_supplement):679.9

[64] Mun JG, Legette LL, Ikonte CJ, Mitmesser SH. Choline and DHA in maternal and infant nutrition: Synergistic implications in brain and eye health. Nutrients. 2019;**11**(5)

[65] Quimson SK. Retinopathy of prematurity: Pathogenesis and current treatment options. Neonatal Network. 2015;**34**(5):284-287

[66] Okamoto T, Shirai M, Kokubo M, Takahashi S, Kajino M, Takase M, et al. Human milk reduces the risk of retinal detachment in extremely low-birthweight infants. Pediatrics International. 2007;**49**(6):894-897

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**63**

**Chapter 5**

**Abstract**

sodium caseinate

**1. Introduction**

leukemia, and lymphoma [2].

Caseins as Regulators of

*Edgar Ledesma-Martinez, Vanihamin Domínguez-Meléndez,* 

The main physiological role of casein, the main protein component in the milk, is to be a source of amino acids that are required for the growth of the neonate; therefore, casein is considered a highly nutritious protein. Over time, it has been revealed that casein is a protein whose physiological importance reaches levels far superior to the food field, having a wide array of biological activities including antimicrobial activities, facilitating absorption of nutrients, as well as acting as a growth factor and an immune stimulant. Here we analyze how caseins can exert numerous hematopoietic and immunomodulatory actions, their role in granulopoiesis, monocytopoiesis, and lymphopoiesis from the early stages of postnatal development seemingly throughout life, and we wonder if casein could be useful to fight pathogens resistant to antibiotics, inducing a strong immune response in immunosuppressed patients, or even be a prophylactic strategy to prevent infections.

*Itzen Aguiñiga-Sánchez and Edelmiro Santiago-Osorio*

**Keywords:** granulopoiesis, monocytopoiesis, lymphopoiesis, milk proteins,

(HPCs), which give rise to all hematopoietic cell lineages [1].

Hematopoiesis is a process that includes the formation, maturation, and differentiation of blood cells. These cells have a relatively short life in circulation, so blood is a tissue with a high rate of renewal. The production of hematopoietic cells depends on a highly specialized bone marrow microenvironment, which regulates the quiescence, differentiation, and self-renewal of a rare population of multipotent cells known as hematopoietic stem cells (HSCs) and hematopoietic progenitor cells

The hematopoietic system plays numerous essential roles in human health and

Studies in the past have consistently demonstrated that diet and nutritional status can significantly alter organismal physiology [3]. Thus, Kornberg et al. demonstrated over 60 years ago that amino acids were required for granulocyte and erythrocyte production; now, it is evident that disruption of dietary and metabolic factors [4], such as inadequate or imbalanced intake of macronutrients (carbohydrates, proteins, and fats) and micronutrients (vitamins and minerals), also known as "malnutrition," alters hematopoiesis and is generally associated with health risk

disease. Failure to maintain homeostasis in the blood system results in a range of human diseases, including anemia, hemophilia, immunodeficiency, allergies,

Hematopoiesis

#### **Chapter 5**

## Caseins as Regulators of Hematopoiesis

*Edgar Ledesma-Martinez, Vanihamin Domínguez-Meléndez, Itzen Aguiñiga-Sánchez and Edelmiro Santiago-Osorio*

#### **Abstract**

The main physiological role of casein, the main protein component in the milk, is to be a source of amino acids that are required for the growth of the neonate; therefore, casein is considered a highly nutritious protein. Over time, it has been revealed that casein is a protein whose physiological importance reaches levels far superior to the food field, having a wide array of biological activities including antimicrobial activities, facilitating absorption of nutrients, as well as acting as a growth factor and an immune stimulant. Here we analyze how caseins can exert numerous hematopoietic and immunomodulatory actions, their role in granulopoiesis, monocytopoiesis, and lymphopoiesis from the early stages of postnatal development seemingly throughout life, and we wonder if casein could be useful to fight pathogens resistant to antibiotics, inducing a strong immune response in immunosuppressed patients, or even be a prophylactic strategy to prevent infections.

**Keywords:** granulopoiesis, monocytopoiesis, lymphopoiesis, milk proteins, sodium caseinate

#### **1. Introduction**

Hematopoiesis is a process that includes the formation, maturation, and differentiation of blood cells. These cells have a relatively short life in circulation, so blood is a tissue with a high rate of renewal. The production of hematopoietic cells depends on a highly specialized bone marrow microenvironment, which regulates the quiescence, differentiation, and self-renewal of a rare population of multipotent cells known as hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs), which give rise to all hematopoietic cell lineages [1].

The hematopoietic system plays numerous essential roles in human health and disease. Failure to maintain homeostasis in the blood system results in a range of human diseases, including anemia, hemophilia, immunodeficiency, allergies, leukemia, and lymphoma [2].

Studies in the past have consistently demonstrated that diet and nutritional status can significantly alter organismal physiology [3]. Thus, Kornberg et al. demonstrated over 60 years ago that amino acids were required for granulocyte and erythrocyte production; now, it is evident that disruption of dietary and metabolic factors [4], such as inadequate or imbalanced intake of macronutrients (carbohydrates, proteins, and fats) and micronutrients (vitamins and minerals), also known as "malnutrition," alters hematopoiesis and is generally associated with health risk

markers [3]. In this sense, the organismal diet has emerged as an important regulator of adult HSC function [5].

Of the different types of malnutrition, protein restriction results from insufficient protein ingestion that can modify physiological responses and induce cellular disturbances, especially in tissues with a high rate of cellular renewal and proliferation, such as hematopoietic tissue; such process requires a large supply of nutrients as well as an organized structure for proliferation [6]. Protein malnutrition causes modifications to the blood tissue, hampering the development and maturation of hematopoietic cells, and these changes could be the cause of anemia, leukopenia, and bone marrow hypoplasia [1].

Protein malnutrition can disrupt numerous processes in hematopoiesis, causing damage to the hematopoietic niche, stromal cells, and the extracellular matrix, and they can result in cell death in bone marrow [1]. However, these issues are not only a consequence of inadequate nutrient supply. Here, we analyze how protein intake, in particular, caseins, the main proteinaceous component of milk, can exert numerous hematopoietic and immunomodulatory actions in addition to performing their nutritional properties [7] from the early stages of postnatal development seemingly throughout life.

#### **2. Casein**

Milk proteins can be broadly classified into three categories: caseins, whey proteins, and mucins [8], as proteins present in the milk fat globule membrane. In milk, caseins interact with calcium phosphate, forming large stable colloidal particles termed micelles. The micelles consist of casein molecules, calcium, inorganic phosphate, and citrate ions [9]. These micelles make it possible to maintain a supersaturated calcium phosphate concentration in milk, providing the newborn with sufficient calcium phosphate for the mineralization of calcified tissues [10].

Casein (from the Latin word *caseus* for cheese) comprises the major protein component of milk of most mammals [11], but relative proportions of caseins differ widely between species. In this sense, caseins comprise approximately 80% of the total protein in ruminant milk [12], but only about 55% of the total protein in horse milk [13].

Casein, which is a phosphoprotein, contains 0.7–0.9% phosphorus that is covalently bound to the protein by serine ester linkages [9], composed of many hundreds of individual amino acids, each of which may have a positive or a negative charge depending on the pH of the milk system. All amino acids that are essential to humans are present in casein in high proportions, with the possible exception of cysteine [9]. It is widely accepted that the main physiological role of casein in the milk system is to be a source of amino acids that are required for the growth of the neonate; therefore, casein is considered a highly nutritious protein. However, the dominant physiological role of the casein micelle system is to prevent pathological calcification of the mammary gland [14]. Over time, it has been revealed that casein is a protein whose physiological importance reaches levels far superior to the food field, having a wide array of biological activities including antimicrobial activities, facilitating absorption of nutrients, as well as acting as a growth factor and an immune stimulant [15].

Caseins are consist of at least three and normally four gene products and further divided into αS1-casein, αS2-casein, β-casein, and κ-casein in farm animals [11, 16] and human, and each has slightly different properties that are caused by small variations in their amino acid content. The four different types are known to occur in at least 10 genetic variants (A1–A3 and B–H) from which the A2, A1, and B forms are the most prevalent [9].

**65**

*Caseins as Regulators of Hematopoiesis DOI: http://dx.doi.org/10.5772/intechopen.91881*

the first year of lactation [8].

Casein is the major component of bovine milk, whereas whey is predominant in human milk. The human milk whey/casein ratio changes over the course of lactation, declining from 90/10 in colostrum (days [d] 0–5) to 65/35 in transitional milk (d6–15), then 60/40 beginning at 1 month postpartum, and continuing throughout

Caseins are synthesized in the mammary gland and are under multihormonal control, and in the bovine genome, they are linked within a 200-kb region on

Bovine milk caseins are composed mainly of equal amounts of β-casein and αS1 casein [11] also contains κ-caseins [18], whereas human milk contains β- and κ-casein and a low concentration of α-casein. The whey/casein ratio in the formula is similar to that of mature human milk (60/40), but the formula contains all bovine milk caseins. The concentrations of total caseins and β- and κ-casein increase slightly between early and transitional milk before declining and remaining relatively stable in mature milk. In contrast, the concentration of α-casein is constant throughout lactation [19]. αS2-casein is the most calcium-sensitive member of the casein family; the sensitivity is potentially due to its high ester phosphate content, which ranges from 10 to 13 phosphate groups per peptide chain [20]. αS2-casein comprises up to 10% of the casein fraction in bovine milk; it consists of two major and several minor components that exhibit varying levels of posttranslational phosphorylation [21] as well as minor degrees of intermolecular disulfide bonding [22]. αS1-casein is only found in trace amounts in human milk (between 3 and 540 μg/mL postpartum) [23] and is thus unlikely to function as a significant amino acid source for breastfed infants [24]. β-Casein has 209 amino acids. The presence of proline or histidine at the 67th position of β-casein allows the distinction between two types of milk, A1 and A2; otherwise, there are no other differences between the two caseins. A1 β-casein is a major variant of β-casein in the milk of the common dairy cows of north European origin: Friesian, Ayrshire, British Shorthorn, and Holstein. A2 β-casein is predominantly found in the milk of Channel Island cows, Guernsey and Jersey cows, Southern French breeds, Charolais and Limousin cows [25], and Zebu original cattle of Africa. The presence of proline or histidine at the 67th position of β-casein is associated with a major effect in terms of bioactive peptide release by different gastrointestinal enzymes [26]; thus, a bioactive seven-amino-acid peptide, β-casomorphin-7 (BCM-7), can be more easily released in the small intestine by digestion of A1 β-casein with pepsin, leucine aminopeptidase, and elastase, but the

chromosome 6, in the order αS1-, β-, αS2- and κ-casein [17].

alternative proline at position 67 prevents a cleavage at this site [27].

**3. Casein and hematopoietic tissue**

κ-Casein contains only one cysteine residue [28, 29], which implies that it is unable to form homomultimers, but it is capable of making one intermolecular disulfide bond [10]. In bovine milk, κ-casein exists as homomultimers cross-linked by random disulfide bonds [22], and it plays a key role in maintaining the stability and solubility of the micelle. Thus, the other caseins do not seem to have a role that requires well-defined structures, and κ-casein may well be more structured to fulfill its function as the interface between the calcium-sensitive caseins and milk serum [30]. In that role, κ-casein naturally resides at the surface of the casein micelle [31].

Low protein intake can affect all systems and organs, but it primarily affects tissues with a high rate of cell turnover, such as hematopoietic tissue [6]. Recently, Hastreiter et al. [32] compared a low-protein diet based on 20 g/kg casein with a control diet based on 120 g/kg casein, and they showed that male C57BL/6 mice after the period of malnutrition presented with peripheral leukopenia and a

#### *Caseins as Regulators of Hematopoiesis DOI: http://dx.doi.org/10.5772/intechopen.91881*

*Infant Feeding - Breast versus Formula*

tor of adult HSC function [5].

and bone marrow hypoplasia [1].

throughout life.

**2. Casein**

milk [13].

markers [3]. In this sense, the organismal diet has emerged as an important regula-

Of the different types of malnutrition, protein restriction results from insufficient protein ingestion that can modify physiological responses and induce cellular disturbances, especially in tissues with a high rate of cellular renewal and proliferation, such as hematopoietic tissue; such process requires a large supply of nutrients as well as an organized structure for proliferation [6]. Protein malnutrition causes modifications to the blood tissue, hampering the development and maturation of hematopoietic cells, and these changes could be the cause of anemia, leukopenia,

Protein malnutrition can disrupt numerous processes in hematopoiesis, causing damage to the hematopoietic niche, stromal cells, and the extracellular matrix, and they can result in cell death in bone marrow [1]. However, these issues are not only a consequence of inadequate nutrient supply. Here, we analyze how protein intake, in particular, caseins, the main proteinaceous component of milk, can exert numerous hematopoietic and immunomodulatory actions in addition to performing their nutritional properties [7] from the early stages of postnatal development seemingly

Milk proteins can be broadly classified into three categories: caseins, whey proteins, and mucins [8], as proteins present in the milk fat globule membrane. In milk, caseins interact with calcium phosphate, forming large stable colloidal particles termed micelles. The micelles consist of casein molecules, calcium, inorganic phosphate, and citrate ions [9]. These micelles make it possible to maintain a supersaturated calcium phosphate concentration in milk, providing the newborn with sufficient calcium phosphate for the mineralization of calcified tissues [10]. Casein (from the Latin word *caseus* for cheese) comprises the major protein component of milk of most mammals [11], but relative proportions of caseins differ widely between species. In this sense, caseins comprise approximately 80% of the total protein in ruminant milk [12], but only about 55% of the total protein in horse

Casein, which is a phosphoprotein, contains 0.7–0.9% phosphorus that is covalently bound to the protein by serine ester linkages [9], composed of many hundreds of individual amino acids, each of which may have a positive or a negative charge depending on the pH of the milk system. All amino acids that are essential to humans are present in casein in high proportions, with the possible exception of cysteine [9]. It is widely accepted that the main physiological role of casein in the milk system is to be a source of amino acids that are required for the growth of the neonate; therefore, casein is considered a highly nutritious protein. However, the dominant physiological role of the casein micelle system is to prevent pathological calcification of the mammary gland [14]. Over time, it has been revealed that casein is a protein whose physiological importance reaches levels far superior to the food field, having a wide array of biological activities including antimicrobial activities, facilitating absorption of nutrients, as well as acting as a growth factor and an immune stimulant [15]. Caseins are consist of at least three and normally four gene products and further divided into αS1-casein, αS2-casein, β-casein, and κ-casein in farm animals [11, 16] and human, and each has slightly different properties that are caused by small variations in their amino acid content. The four different types are known to occur in at least 10 genetic variants (A1–A3 and B–H) from which the A2, A1, and B forms are the

**64**

most prevalent [9].

Casein is the major component of bovine milk, whereas whey is predominant in human milk. The human milk whey/casein ratio changes over the course of lactation, declining from 90/10 in colostrum (days [d] 0–5) to 65/35 in transitional milk (d6–15), then 60/40 beginning at 1 month postpartum, and continuing throughout the first year of lactation [8].

Caseins are synthesized in the mammary gland and are under multihormonal control, and in the bovine genome, they are linked within a 200-kb region on chromosome 6, in the order αS1-, β-, αS2- and κ-casein [17].

Bovine milk caseins are composed mainly of equal amounts of β-casein and αS1 casein [11] also contains κ-caseins [18], whereas human milk contains β- and κ-casein and a low concentration of α-casein. The whey/casein ratio in the formula is similar to that of mature human milk (60/40), but the formula contains all bovine milk caseins. The concentrations of total caseins and β- and κ-casein increase slightly between early and transitional milk before declining and remaining relatively stable in mature milk. In contrast, the concentration of α-casein is constant throughout lactation [19].

αS2-casein is the most calcium-sensitive member of the casein family; the sensitivity is potentially due to its high ester phosphate content, which ranges from 10 to 13 phosphate groups per peptide chain [20]. αS2-casein comprises up to 10% of the casein fraction in bovine milk; it consists of two major and several minor components that exhibit varying levels of posttranslational phosphorylation [21] as well as minor degrees of intermolecular disulfide bonding [22]. αS1-casein is only found in trace amounts in human milk (between 3 and 540 μg/mL postpartum) [23] and is thus unlikely to function as a significant amino acid source for breastfed infants [24].

β-Casein has 209 amino acids. The presence of proline or histidine at the 67th position of β-casein allows the distinction between two types of milk, A1 and A2; otherwise, there are no other differences between the two caseins. A1 β-casein is a major variant of β-casein in the milk of the common dairy cows of north European origin: Friesian, Ayrshire, British Shorthorn, and Holstein. A2 β-casein is predominantly found in the milk of Channel Island cows, Guernsey and Jersey cows, Southern French breeds, Charolais and Limousin cows [25], and Zebu original cattle of Africa. The presence of proline or histidine at the 67th position of β-casein is associated with a major effect in terms of bioactive peptide release by different gastrointestinal enzymes [26]; thus, a bioactive seven-amino-acid peptide, β-casomorphin-7 (BCM-7), can be more easily released in the small intestine by digestion of A1 β-casein with pepsin, leucine aminopeptidase, and elastase, but the alternative proline at position 67 prevents a cleavage at this site [27].

κ-Casein contains only one cysteine residue [28, 29], which implies that it is unable to form homomultimers, but it is capable of making one intermolecular disulfide bond [10]. In bovine milk, κ-casein exists as homomultimers cross-linked by random disulfide bonds [22], and it plays a key role in maintaining the stability and solubility of the micelle. Thus, the other caseins do not seem to have a role that requires well-defined structures, and κ-casein may well be more structured to fulfill its function as the interface between the calcium-sensitive caseins and milk serum [30]. In that role, κ-casein naturally resides at the surface of the casein micelle [31].

#### **3. Casein and hematopoietic tissue**

Low protein intake can affect all systems and organs, but it primarily affects tissues with a high rate of cell turnover, such as hematopoietic tissue [6]. Recently, Hastreiter et al. [32] compared a low-protein diet based on 20 g/kg casein with a control diet based on 120 g/kg casein, and they showed that male C57BL/6 mice after the period of malnutrition presented with peripheral leukopenia and a

reduction in lymphocytes and monocytes, especially in granulocytic cells associated with bone marrow hypoplasia. Therefore, hematopoietic stem cell (Lin-Sca-1+c-Kit+-LSK) and progenitor cell (CD45+CD34+) populations were decreased in malnourished animals [33], but also low protein intake induced a specific reduction in granulocyte-monocyte progenitors (Lin-IL7r-c-Kit+Sca-1-CD16/32high), which explains, in part, why there was a reduction in mature granulocytes [32].

It is well known that in protein malnutrition states, the number of granulocytic cells, especially neutrophils, is reduced, which predisposes patients to higher susceptibility to infection [34, 35]. However, this involvement in hematopoiesis cannot be explained only because there are not enough amino acids to support the requirements of an expanding tissue; other cellular mechanisms are involved. In this sense, Hastreiter et al. showed that there is an impaired ability of c-Kit+ cells from the bone marrow of malnourished animals to produce CFU-GEMM and CFU-GM cells, which are myeloid progenitors and, consequently, are the cells responsible for granulopoiesis [36]; this malfunction is related to Kit+ cells exhibiting reduced expression of the receptor of granulocyte colony-stimulating factor (G-CSFr), which is a granulopoietic cytokine [32].

Interestingly, Domínguez-Melendez et al. showed that administration of casein as sodium caseinate in BALB/c mice increased the percentage of myeloid precursors from bone marrow and increased the total number of bone marrow leukocytes resulting from cell proliferation. They also found that casein induced proliferation and activation of granulocytes, and it increased the serum concentration of cytokines such as G-CSF, M-CSF, and GM-CSF [37]. These cytokines are key to the proliferation, differentiation, and activation of granulomonocytic cells, which in turn induce multiple functions within the immune response; since these cytokines regulate inflammation, as is the case for monocyte-macrophages, they are responsible for phagocytosis, which is a crucial event in fungal and bacterial infections; once activated, macrophages are the bridge between activated CD4+ lymphocytes and the adaptive immune response [38]. For the lymphoid lineage in vivo, sodium caseinate also influences the induction of IL-7, a key cytokine involved in lymphopoiesis of B cells, which are key cells for the adaptive immune response, since once activated, they are responsible for producing antibodies that they will opsonize foreign antigens to facilitate their elimination [39].

This suggests that casein may be linked to the development of the immune system in the early stages of life, and it may be relevant throughout life as a way of activating the immune system; this notion has been demonstrated experimentally by mice that, when injected with lethal doses of bacteria, can survive only after inducing protection by granulocytes with administration of casein [40].

Studies of the effect of casein or sodium caseinate on hematopoietic tissue in vivo and in vitro are limited, and most of them do not include non-casein protein experimental controls. In some cases, there could be controversy in its effects observed due to the presence of a general source of protein or if they were specific to casein. In this sense, it would be more than advisable for future casein work to consider the inclusion of non-casein protein controls.

#### **4. The role of caseins in granulopoiesis and monocytopoiesis**

Caseins and sodium caseinate have been studied for almost four decades, where, from the beginning, it was clear that there was a proinflammatory effect of casein on myeloid lineage cells; this activity was demonstrated by Lotem and Sachs working group, which showed that sodium caseinate had the ability to differentiate a leukemic cell line of myeloid origin toward granulocytes and macrophages

**67**

*Caseins as Regulators of Hematopoiesis DOI: http://dx.doi.org/10.5772/intechopen.91881*

in mice via inflammation and via the activity of T lymphocytes in the peritoneal cavity [41]. Later, this same group showed that the inflammation caused by sodium caseinate had the ability to induce the production of G-CSF and GM-CSF in vitro and in vivo [42]. Another study showed that protein deprivation, such as dietary casein restriction, in rats directly resulted in a decrease in erythropoietin, which is a hormone that is directly related to the proliferation of the erythroid lineage [43]. In a similar study, the role of casein on this lineage was reconfirmed, since the restriction of protein in standard diets in mice once again demonstrated the involvement of proteins in the proliferation of erythroid progenitors in mice [44]. Subsequently, it was shown that after intraperitoneal inoculation of casein, both the production of G-CSF and GM-CSF were rapidly induced, and the high concentration of both cytokines caused a high migration of neutrophils only at the site of inoculation, but they revealed no increase in their percentage in the bone marrow [45]. Interestingly, in a study carried out by the Noursadeghi group, it was shown that after previous inoculation of casein, protection could be given to mice treated with lethal doses of bacteria, and this was due to defensive ability of activated neutrophils recruited by G-CSF induced by that intraperitoneal (IP) injection of casein [40]. Regarding the casein and sodium caseinate fractions on myeloid cell lines, one study showed that in vitro sodium caseinate had the ability to inhibit the proliferation of a myeloid cell line 32D without inhibiting its viability. On the other hand, α-casein exhibited the ability to inhibit the proliferation of a myeloid tumor line, which is the case for WEHI-3 and sodium caseinate of tumor lines J-774 and P388 at different concentrations. In this same study, it was shown that casein fractions α-, β- and κ-casein could induce differentiation of the 32D cell line but not the WEHI-3 tumor cell line, but the study interestingly demonstrated that sodium caseinate and a-casein have the ability to induce M-CSF secretion in the 32D cell line [46]. The same group of researchers subsequently demonstrated that sodium caseinate has the ability to induce the differentiation of the granulocytic lineage in vitro in the same way that G-CSF does, and G-CSF is the specific cytokine required for the differentiation of this lineage; these results demonstrated that these cells have the ability to induce the production of functional M-CSF, a key cytokine in the activation and differentiation of the monocytic lineage [47]. The Vordenbaumen group demonstrated that macrophages of human origin could be stimulated by human αS1-casein to produce GM-CSF and that αS1-casein activated the p38 MAPK pathway, which is an important signal in cells of hematopoietic origin. Interestingly, this group found that αS1-casein was linked to specific receptors for the protein in most of the macrophages analyzed and not only that but also that it could induce the production of IL-1 and IL-6, which positions it as an excellent immunomodulatory protein [48]. This same group subsequently demonstrated that αS1-casein in human milk has the ability to differentiate human monocytes from macrophages, and they also increase the phagocytic capacity of the monocytes in vitro once stimulated with the protein [49]. On the other hand, the Santiago-Osorio group showed that the IP inoculation of BALB/c mice with sodium caseinate induces in vivo proliferation of the myeloid lineage in the bone marrow. Interestingly, it was observed that the granulocytes had the ability to incorporate BrdU, a thymidine analyte that is incorporated into proliferating cells; further, the cells exhibited a greater ability to phagocytose when

compared to cells from mice that had not received treatment [37].

Regarding the influence of caseins or sodium caseinate on the lymphoid lineage,

the role of these proteins is not prominent, but they have interesting functions

**5. The role of caseins in lymphopoiesis**

#### *Caseins as Regulators of Hematopoiesis DOI: http://dx.doi.org/10.5772/intechopen.91881*

*Infant Feeding - Breast versus Formula*

which is a granulopoietic cytokine [32].

foreign antigens to facilitate their elimination [39].

consider the inclusion of non-casein protein controls.

reduction in lymphocytes and monocytes, especially in granulocytic cells associated with bone marrow hypoplasia. Therefore, hematopoietic stem cell (Lin-Sca-1+c-Kit+-LSK) and progenitor cell (CD45+CD34+) populations were decreased in malnourished animals [33], but also low protein intake induced a specific reduction in granulocyte-monocyte progenitors (Lin-IL7r-c-Kit+Sca-1-CD16/32high), which

It is well known that in protein malnutrition states, the number of granulocytic cells, especially neutrophils, is reduced, which predisposes patients to higher susceptibility to infection [34, 35]. However, this involvement in hematopoiesis cannot be explained only because there are not enough amino acids to support the requirements of an expanding tissue; other cellular mechanisms are involved. In this sense, Hastreiter et al. showed that there is an impaired ability of c-Kit+ cells from the bone marrow of malnourished animals to produce CFU-GEMM and CFU-GM cells, which are myeloid progenitors and, consequently, are the cells responsible for granulopoiesis [36]; this malfunction is related to Kit+ cells exhibiting reduced expression of the receptor of granulocyte colony-stimulating factor (G-CSFr),

Interestingly, Domínguez-Melendez et al. showed that administration of casein as sodium caseinate in BALB/c mice increased the percentage of myeloid precursors from bone marrow and increased the total number of bone marrow leukocytes resulting from cell proliferation. They also found that casein induced proliferation and activation of granulocytes, and it increased the serum concentration of cytokines such as G-CSF, M-CSF, and GM-CSF [37]. These cytokines are key to the proliferation, differentiation, and activation of granulomonocytic cells, which in turn induce multiple functions within the immune response; since these cytokines regulate inflammation, as is the case for monocyte-macrophages, they are responsible for phagocytosis, which is a crucial event in fungal and bacterial infections; once activated, macrophages are the bridge between activated CD4+ lymphocytes and the adaptive immune response [38]. For the lymphoid lineage in vivo, sodium caseinate also influences the induction of IL-7, a key cytokine involved in lymphopoiesis of B cells, which are key cells for the adaptive immune response, since once activated, they are responsible for producing antibodies that they will opsonize

This suggests that casein may be linked to the development of the immune system in the early stages of life, and it may be relevant throughout life as a way of activating the immune system; this notion has been demonstrated experimentally by mice that, when injected with lethal doses of bacteria, can survive only after

Studies of the effect of casein or sodium caseinate on hematopoietic tissue in vivo and in vitro are limited, and most of them do not include non-casein protein experimental controls. In some cases, there could be controversy in its effects observed due to the presence of a general source of protein or if they were specific to casein. In this sense, it would be more than advisable for future casein work to

inducing protection by granulocytes with administration of casein [40].

**4. The role of caseins in granulopoiesis and monocytopoiesis**

Caseins and sodium caseinate have been studied for almost four decades, where, from the beginning, it was clear that there was a proinflammatory effect of casein on myeloid lineage cells; this activity was demonstrated by Lotem and Sachs working group, which showed that sodium caseinate had the ability to differentiate a leukemic cell line of myeloid origin toward granulocytes and macrophages

explains, in part, why there was a reduction in mature granulocytes [32].

**66**

in mice via inflammation and via the activity of T lymphocytes in the peritoneal cavity [41]. Later, this same group showed that the inflammation caused by sodium caseinate had the ability to induce the production of G-CSF and GM-CSF in vitro and in vivo [42]. Another study showed that protein deprivation, such as dietary casein restriction, in rats directly resulted in a decrease in erythropoietin, which is a hormone that is directly related to the proliferation of the erythroid lineage [43]. In a similar study, the role of casein on this lineage was reconfirmed, since the restriction of protein in standard diets in mice once again demonstrated the involvement of proteins in the proliferation of erythroid progenitors in mice [44]. Subsequently, it was shown that after intraperitoneal inoculation of casein, both the production of G-CSF and GM-CSF were rapidly induced, and the high concentration of both cytokines caused a high migration of neutrophils only at the site of inoculation, but they revealed no increase in their percentage in the bone marrow [45]. Interestingly, in a study carried out by the Noursadeghi group, it was shown that after previous inoculation of casein, protection could be given to mice treated with lethal doses of bacteria, and this was due to defensive ability of activated neutrophils recruited by G-CSF induced by that intraperitoneal (IP) injection of casein [40]. Regarding the casein and sodium caseinate fractions on myeloid cell lines, one study showed that in vitro sodium caseinate had the ability to inhibit the proliferation of a myeloid cell line 32D without inhibiting its viability. On the other hand, α-casein exhibited the ability to inhibit the proliferation of a myeloid tumor line, which is the case for WEHI-3 and sodium caseinate of tumor lines J-774 and P388 at different concentrations. In this same study, it was shown that casein fractions α-, β- and κ-casein could induce differentiation of the 32D cell line but not the WEHI-3 tumor cell line, but the study interestingly demonstrated that sodium caseinate and a-casein have the ability to induce M-CSF secretion in the 32D cell line [46]. The same group of researchers subsequently demonstrated that sodium caseinate has the ability to induce the differentiation of the granulocytic lineage in vitro in the same way that G-CSF does, and G-CSF is the specific cytokine required for the differentiation of this lineage; these results demonstrated that these cells have the ability to induce the production of functional M-CSF, a key cytokine in the activation and differentiation of the monocytic lineage [47]. The Vordenbaumen group demonstrated that macrophages of human origin could be stimulated by human αS1-casein to produce GM-CSF and that αS1-casein activated the p38 MAPK pathway, which is an important signal in cells of hematopoietic origin. Interestingly, this group found that αS1-casein was linked to specific receptors for the protein in most of the macrophages analyzed and not only that but also that it could induce the production of IL-1 and IL-6, which positions it as an excellent immunomodulatory protein [48]. This same group subsequently demonstrated that αS1-casein in human milk has the ability to differentiate human monocytes from macrophages, and they also increase the phagocytic capacity of the monocytes in vitro once stimulated with the protein [49]. On the other hand, the Santiago-Osorio group showed that the IP inoculation of BALB/c mice with sodium caseinate induces in vivo proliferation of the myeloid lineage in the bone marrow. Interestingly, it was observed that the granulocytes had the ability to incorporate BrdU, a thymidine analyte that is incorporated into proliferating cells; further, the cells exhibited a greater ability to phagocytose when compared to cells from mice that had not received treatment [37].

#### **5. The role of caseins in lymphopoiesis**

Regarding the influence of caseins or sodium caseinate on the lymphoid lineage, the role of these proteins is not prominent, but they have interesting functions

in vivo and in vitro; for instance, the proteolytic activity of leukocytes can be induced by β-casein in vitro [50]. In another work, it was demonstrated that peptides derived from αS1-casein increased the concentration of IFN-γ by stimulating CD8+ T cells, and IFN-γ is a potent inhibitor of Th2 lymphocyte-dependent events as well as an inhibitor of the production of IgE [51]. Another working group demonstrated that β-casein rather than κ-casein is mainly responsible for inhibiting spleen CD3+ T lymphocytes [52]. In a more detailed work, the group of Santiago Osorio demonstrated that the IP inoculation of BALB/c mice with sodium caseinate decreases the proliferation of B/B220+ lymphocytes in bone marrow, but this lineage increases proliferation in the spleen; this observation suggests that the IP injection induces extramedullary lymphopoiesis, while the treatment does not increase the proliferation of lineage lymphocytes specifically in the thymus and has only very subtle effects in the spleen without affecting the viability of both lineages. It should be noted that the production of IL-2, IL-7, and IL-15 is increased, and they are key interleukins involved in lymphopoiesis of both T cells and B cells in mice [53]. Although there is currently clear evidence for the role of caseins or sodium caseinate on the lymphoid lineage, there is more evidence of their effect on the myeloid lineage, which may be due to the characteristics of the protein and its particular influence on these cells. However, the field is still open to further exploration of the role of sodium caseinate or caseins on the lymphoid lineage, since its role is not yet clear.

#### **6. Inflammation and immune system enhancement by caseins**

Milk is a complex physiological liquid that simultaneously provides nutrients and bioactive components, including prebiotics, immune proteins, and the microbiome of human milk itself; the establishment of symbiotic microflora and the development of gut-associated lymphoid tissues facilitate the successful postnatal adaptation of the newborn infant by stimulating cellular growth and digestive maturation [9]. Breastfeeding is associated with a decreased incidence of gastrointestinal (GI) tract infections [54, 55], which is corroborated by several studies that have correlated breastfeeding with a lower incidence of necrotizing enterocolitis in humans and animal models [56, 57].

The antimicrobial activity in milk is greater than the sum individual immunoglobulin and of whey proteins such as lactoferrin, lactoferricins, lactoperoxidase, lysozyme, lactenin, casecudubs, etc. [58]; this activity could be also associated with gut-colonizing bacteria that prevent adhesion and colonization of pathogenic bacteria while stimulating mucosal cell proliferation and enhancing immune development [59]; a portion of these antimicrobial activities are performed by caseins, most likely κ-casein fucose carbohydrate residues.

Purified human κ-casein inhibits specific adhesion of *Helicobacter pylori* to mucous cells at the human gastric surface. The inhibitory activity is abolished by the oxidation of metaperiodate and is considerably reduced by preincubation with alpha-l-fucosidase but not with α-N-acetylneuraminidase or endo-β-galactosidase. Thus, glycosylated κ-casein is likely important for the inhibition of *Helicobacter pylori* adhesion and, therefore, infection. This could explain why breastfeeding may protect against *Helicobacter pylori* infection during early life and how the speciesspecific glycosylation patterns in human bovine κ-casein partly determine both the narrow host spectrum of this human gastric pathogen and the capacity to resist infection [60].

Unphosphorylated αS1-casein in breast milk may contribute to the development of the immune system before major colonization of the gut by microbes occurs by

**69**

*Caseins as Regulators of Hematopoiesis DOI: http://dx.doi.org/10.5772/intechopen.91881*

TLR4 [64].

vided by casein injection [40].

development of the neonate?

**7. Toll-like receptors (TLRs) and caseins**

triggering immune responses to potential pathogens, including pathogen-associated molecules such as LPS. Moreover, αS1-casein by itself gives rise to sustained specific IgG antibody production in individuals who nursed [61]. Early infantile autoantibody production in turn is speculated to confer protection from pathogens [62]. αS1-casein activates the secretion of the proinflammatory cytokines GM-CSF (granulocyte-macrophage colony-stimulating factor), IL1-β (interleukin 1β), IL-6 (interleukin 6), and chemokine IL-8 (interleukin 8) in human monocytes via the mitogen-activated protein kinase p38 (MAPK-p38) signaling pathway [24, 49]. Human unphosphorylated αS1-casein induces Toll-like receptor 4 (TLRs) mediated expression of the proinflammatory cytokines IL-1β, GM-CSF, and IL-6 in monocytic cells [63] and induces the differentiation of monocytes toward macrophages [49, 64], but this process is not dependent upon LPS. Interestingly, a posttranslational modification in αS1-casein (a phosphorylation event) inhibits binding to TLR4, which acts as an off switch for proinflammatory effects [48]. Ectopic expression (outside the mammary gland) of αS1-casein has been detected in inflamed tissues such as synovial cells and cartilage of rheumatoid arthritis, osteoarthritis, and multiple sclerosis patients [63, 65–67], prostate hyperplasia [68], and lymph nodes of encephalomyelitic mice [67]. Hence, αS1-casein may constitute an autogenous stimulus that upholds chronic autoimmune inflammation via

On the other hand, it is known that casein and sodium caseinate are agents that can induce inflammation when inoculated intraperitoneally, and it has been shown that sodium caseinate and casein can stimulate neutrophils to produce proinflammatory cytokines, such as M-CSF, in vitro [47]. Not only that, but it is known that they can induce in this same lineage signaling pathways involved in inflammation, such as p38 MAPK, which in turn stimulates the production of key cytokines both in inflammation and in hematopoietic cell differentiation processes [69]. However, casein inoculation can induce a rapid accumulation of neutrophils within 3 h due to selective release of mature cells from the bone marrow; then, a significant increase in the concentrations of granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) occurs in the peritoneal cavity [45], but the accumulation did not affect the serum values of TNFα, IL-1β, or IL-6. As demonstrated by Noursadeghi, inflammation induced by casein was associated with higher serum G-CSF concentrations, and administration of an Ab that neutralized this cytokine completely nullified protection against *Escherichia coli* infection after casein pretreatment. Injection of recombinant murine G-CSF between 3 and 24 h before infection conferred the same protection that was pro-

Caseins serve as a source of amino acids but also perform a range of functions, including improving micronutrient bioavailability, stimulating intestinal growth and maturation, supporting immunologic defense, shaping the microbiome, and enhancing learning and memory [19]. Some bioactive peptides in milk act in variable ways as antihypertensives, antithrombotic agents, opioids, antimicrobials, cytomodulators, and immunomodulators [70]. How can this be possible, if proteins are degraded in the gastrointestinal tract to yield the essential amino acids for the

Bioactive milk peptides were first described in 1950, when Mellander (1950) reported that ingestion of casein-derived phosphorylated peptides led to enhanced vitamin D-independent calcification in rachitic infants. While bioactive peptides

#### *Caseins as Regulators of Hematopoiesis DOI: http://dx.doi.org/10.5772/intechopen.91881*

*Infant Feeding - Breast versus Formula*

role is not yet clear.

humans and animal models [56, 57].

most likely κ-casein fucose carbohydrate residues.

in vivo and in vitro; for instance, the proteolytic activity of leukocytes can be induced by β-casein in vitro [50]. In another work, it was demonstrated that peptides derived from αS1-casein increased the concentration of IFN-γ by stimulating CD8+ T cells, and IFN-γ is a potent inhibitor of Th2 lymphocyte-dependent events as well as an inhibitor of the production of IgE [51]. Another working group demonstrated that β-casein rather than κ-casein is mainly responsible for inhibiting spleen CD3+ T lymphocytes [52]. In a more detailed work, the group of Santiago Osorio demonstrated that the IP inoculation of BALB/c mice with sodium caseinate decreases the proliferation of B/B220+ lymphocytes in bone marrow, but this lineage increases proliferation in the spleen; this observation suggests that the IP injection induces extramedullary lymphopoiesis, while the treatment does not increase the proliferation of lineage lymphocytes specifically in the thymus and has only very subtle effects in the spleen without affecting the viability of both lineages. It should be noted that the production of IL-2, IL-7, and IL-15 is increased, and they are key interleukins involved in lymphopoiesis of both T cells and B cells in mice [53]. Although there is currently clear evidence for the role of caseins or sodium caseinate on the lymphoid lineage, there is more evidence of their effect on the myeloid lineage, which may be due to the characteristics of the protein and its particular influence on these cells. However, the field is still open to further exploration of the role of sodium caseinate or caseins on the lymphoid lineage, since its

**6. Inflammation and immune system enhancement by caseins**

Milk is a complex physiological liquid that simultaneously provides nutrients and bioactive components, including prebiotics, immune proteins, and the microbiome of human milk itself; the establishment of symbiotic microflora and the development of gut-associated lymphoid tissues facilitate the successful postnatal adaptation of the newborn infant by stimulating cellular growth and digestive maturation [9]. Breastfeeding is associated with a decreased incidence of gastrointestinal (GI) tract infections [54, 55], which is corroborated by several studies that have correlated breastfeeding with a lower incidence of necrotizing enterocolitis in

The antimicrobial activity in milk is greater than the sum individual immunoglobulin and of whey proteins such as lactoferrin, lactoferricins, lactoperoxidase, lysozyme, lactenin, casecudubs, etc. [58]; this activity could be also associated with gut-colonizing bacteria that prevent adhesion and colonization of pathogenic bacteria while stimulating mucosal cell proliferation and enhancing immune development [59]; a portion of these antimicrobial activities are performed by caseins,

Purified human κ-casein inhibits specific adhesion of *Helicobacter pylori* to mucous cells at the human gastric surface. The inhibitory activity is abolished by the oxidation of metaperiodate and is considerably reduced by preincubation with alpha-l-fucosidase but not with α-N-acetylneuraminidase or endo-β-galactosidase. Thus, glycosylated κ-casein is likely important for the inhibition of *Helicobacter pylori* adhesion and, therefore, infection. This could explain why breastfeeding may protect against *Helicobacter pylori* infection during early life and how the speciesspecific glycosylation patterns in human bovine κ-casein partly determine both the narrow host spectrum of this human gastric pathogen and the capacity to resist

Unphosphorylated αS1-casein in breast milk may contribute to the development of the immune system before major colonization of the gut by microbes occurs by

**68**

infection [60].

triggering immune responses to potential pathogens, including pathogen-associated molecules such as LPS. Moreover, αS1-casein by itself gives rise to sustained specific IgG antibody production in individuals who nursed [61]. Early infantile autoantibody production in turn is speculated to confer protection from pathogens [62].

αS1-casein activates the secretion of the proinflammatory cytokines GM-CSF (granulocyte-macrophage colony-stimulating factor), IL1-β (interleukin 1β), IL-6 (interleukin 6), and chemokine IL-8 (interleukin 8) in human monocytes via the mitogen-activated protein kinase p38 (MAPK-p38) signaling pathway [24, 49].

Human unphosphorylated αS1-casein induces Toll-like receptor 4 (TLRs) mediated expression of the proinflammatory cytokines IL-1β, GM-CSF, and IL-6 in monocytic cells [63] and induces the differentiation of monocytes toward macrophages [49, 64], but this process is not dependent upon LPS. Interestingly, a posttranslational modification in αS1-casein (a phosphorylation event) inhibits binding to TLR4, which acts as an off switch for proinflammatory effects [48]. Ectopic expression (outside the mammary gland) of αS1-casein has been detected in inflamed tissues such as synovial cells and cartilage of rheumatoid arthritis, osteoarthritis, and multiple sclerosis patients [63, 65–67], prostate hyperplasia [68], and lymph nodes of encephalomyelitic mice [67]. Hence, αS1-casein may constitute an autogenous stimulus that upholds chronic autoimmune inflammation via TLR4 [64].

On the other hand, it is known that casein and sodium caseinate are agents that can induce inflammation when inoculated intraperitoneally, and it has been shown that sodium caseinate and casein can stimulate neutrophils to produce proinflammatory cytokines, such as M-CSF, in vitro [47]. Not only that, but it is known that they can induce in this same lineage signaling pathways involved in inflammation, such as p38 MAPK, which in turn stimulates the production of key cytokines both in inflammation and in hematopoietic cell differentiation processes [69]. However, casein inoculation can induce a rapid accumulation of neutrophils within 3 h due to selective release of mature cells from the bone marrow; then, a significant increase in the concentrations of granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) occurs in the peritoneal cavity [45], but the accumulation did not affect the serum values of TNFα, IL-1β, or IL-6. As demonstrated by Noursadeghi, inflammation induced by casein was associated with higher serum G-CSF concentrations, and administration of an Ab that neutralized this cytokine completely nullified protection against *Escherichia coli* infection after casein pretreatment. Injection of recombinant murine G-CSF between 3 and 24 h before infection conferred the same protection that was provided by casein injection [40].

#### **7. Toll-like receptors (TLRs) and caseins**

Caseins serve as a source of amino acids but also perform a range of functions, including improving micronutrient bioavailability, stimulating intestinal growth and maturation, supporting immunologic defense, shaping the microbiome, and enhancing learning and memory [19]. Some bioactive peptides in milk act in variable ways as antihypertensives, antithrombotic agents, opioids, antimicrobials, cytomodulators, and immunomodulators [70]. How can this be possible, if proteins are degraded in the gastrointestinal tract to yield the essential amino acids for the development of the neonate?

Bioactive milk peptides were first described in 1950, when Mellander (1950) reported that ingestion of casein-derived phosphorylated peptides led to enhanced vitamin D-independent calcification in rachitic infants. While bioactive peptides

can be generated from a variety of foods, milk proteins are generally regarded as a very rich source; as a result, they have become fundamental constituents of several commercially available functional food products and ingredients [19].

What is the bridge that connects casein, the genesis of myeloid and lymphoid hematopoietic cells, and the activation of the immune system? This linking role may be the direct responsibility of TLRs, which are receptors that recognize at least α-casein and β-casein [71] and are expressed in granulocytes, macrophages, and B and T lymphocytes; TLRs are capable of activating these cells to produce key cytokines for both proliferation and activation of the innate and adaptive immune response, such as TNF-α [72], G-CSF, IL-2 [73, 74], and IL-7 [75].

In this sense, there is evidence that at least β-casein can influence B lymphocytes via TLR4 [76, 77], and a recent study showed that casein binds directly to TLR4 of CD8+ T lymphocytes [64], although it has also been shown that β-casein can influence the activation and production of histamine through a kinase-dependent mechanism PI3 [78].

Regarding the role of TLR in the production of key cytokines for the activation and proliferation of both T and B lymphocytes, TLR4 of T lymphocytes is involved in the production of IL-2 [73, 74].

For myeloid cells, neutrophils have been shown to use both TLR4 and TLR2 for survival and activation [79]. Thus, TRL4 is essential for the production of G-CSF in neutrophils stimulated with *Clostridium* [80], and another study showed that in addition to G-CSF, TLR4 is capable of inducing the expression of GM-CSF, which plays a fundamental role in the activation and differentiation of both neutrophils and monocytes [81].

TLRs have been shown to be essential for the activation of monocytic cells, and it plays a role in the production of GM-CSF by activating the transcription factor PU.I, which plays a fundamental role in this lineage [82]. In dendritic cells derived from macrophages, TLRs are involved in the synthesis of IL-7, which is an essential interleukin for the maintenance of LT CD8+ [75]. Therefore, casein or sodium

#### **Figure 1.**

*Action mechanism of caseins to induce hematopoiesis and immunomodulation. Casein is highly likely to induce TLR4 activation, after which the expression of GM-CSF, G-CSF, M-CSF, and IL-7 is induced in both myeloid and lymphoid cells. This massive cellular activation of hematopoietic cells and immune responses explain the antimicrobial and immunomodulatory effects of casein.*

**71**

**Author details**

molecule [83].

**Acknowledgements**

Edgar Ledesma-Martinez1

Itzen Aguiñiga-Sánchez1

Mexico City, Mexico

Veracruz City, Mexico

, Vanihamin Domínguez-Meléndez2

and Edelmiro Santiago-Osorio1

caseinate could stimulate TLR4 via the production of at least IL-7 in dendritic cells

It is clear that there is a close relationship between TLR4 and cells of myeloid and lymphoid origin, as these cells use TLR4 both in their proliferation and in their activation. This relationship then entails the production of cytokines of myeloid and lymphoid origin, and these cytokines in turn are key pieces for the proliferation and activation of these same cells. Thus, TLR4-bound caseins or sodium caseinate are highly likely to induce TLR4 activation, after which the expression of GM-CSF, G-CSF, M-CSF, and IL-7 is induced in both myeloid and lymphoid cells (**Figure 1**). It is even possible that indirectly, by stimulating other cell types, TLR4 could induce the activation of cells that can influence the erythroid and megakaryocyte lineage. Then, this massive cellular activation of hematopoietic cells and immune responses could explain the antimicrobial and immunomodulatory effects of casein as well as the activity of casein as an antihypertensive, antithrombotic, and antioxidant

Here, we can reveal that activation of the innate immune system by casein could be useful to fight pathogens resistant to antibiotics, as has been suggested [40], so casein could be used to induce a strong immune response in immunosuppressed patients [84, 85]; it could be used as a prophylactic strategy to prevent infections.

This work was financially supported by DGAPA PAPIIT IN221017 & IN229820.

1 Hematopoiesis and Leukemia Laboratory, Research Unit on Cell Differentiation

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

and Cancer, FES Zaragoza, National Autonomous University of Mexico,

2 Center for Health Studies and Services (CESS), University of Veracruz,

\*Address all correspondence to: edelmiro@unam.mx

provided the original work is properly cited.

,

\*

*Caseins as Regulators of Hematopoiesis DOI: http://dx.doi.org/10.5772/intechopen.91881*

derived from macrophages.

#### *Caseins as Regulators of Hematopoiesis DOI: http://dx.doi.org/10.5772/intechopen.91881*

*Infant Feeding - Breast versus Formula*

mechanism PI3 [78].

and monocytes [81].

in the production of IL-2 [73, 74].

can be generated from a variety of foods, milk proteins are generally regarded as a very rich source; as a result, they have become fundamental constituents of several

What is the bridge that connects casein, the genesis of myeloid and lymphoid hematopoietic cells, and the activation of the immune system? This linking role may be the direct responsibility of TLRs, which are receptors that recognize at least α-casein and β-casein [71] and are expressed in granulocytes, macrophages, and B and T lymphocytes; TLRs are capable of activating these cells to produce key cytokines for both proliferation and activation of the innate and adaptive immune

In this sense, there is evidence that at least β-casein can influence B lymphocytes

Regarding the role of TLR in the production of key cytokines for the activation and proliferation of both T and B lymphocytes, TLR4 of T lymphocytes is involved

For myeloid cells, neutrophils have been shown to use both TLR4 and TLR2 for survival and activation [79]. Thus, TRL4 is essential for the production of G-CSF in neutrophils stimulated with *Clostridium* [80], and another study showed that in addition to G-CSF, TLR4 is capable of inducing the expression of GM-CSF, which plays a fundamental role in the activation and differentiation of both neutrophils

TLRs have been shown to be essential for the activation of monocytic cells, and it plays a role in the production of GM-CSF by activating the transcription factor PU.I, which plays a fundamental role in this lineage [82]. In dendritic cells derived from macrophages, TLRs are involved in the synthesis of IL-7, which is an essential interleukin for the maintenance of LT CD8+ [75]. Therefore, casein or sodium

*Action mechanism of caseins to induce hematopoiesis and immunomodulation. Casein is highly likely to induce TLR4 activation, after which the expression of GM-CSF, G-CSF, M-CSF, and IL-7 is induced in both myeloid and lymphoid cells. This massive cellular activation of hematopoietic cells and immune responses explain the* 

via TLR4 [76, 77], and a recent study showed that casein binds directly to TLR4 of CD8+ T lymphocytes [64], although it has also been shown that β-casein can influence the activation and production of histamine through a kinase-dependent

commercially available functional food products and ingredients [19].

response, such as TNF-α [72], G-CSF, IL-2 [73, 74], and IL-7 [75].

**70**

**Figure 1.**

*antimicrobial and immunomodulatory effects of casein.*

caseinate could stimulate TLR4 via the production of at least IL-7 in dendritic cells derived from macrophages.

It is clear that there is a close relationship between TLR4 and cells of myeloid and lymphoid origin, as these cells use TLR4 both in their proliferation and in their activation. This relationship then entails the production of cytokines of myeloid and lymphoid origin, and these cytokines in turn are key pieces for the proliferation and activation of these same cells. Thus, TLR4-bound caseins or sodium caseinate are highly likely to induce TLR4 activation, after which the expression of GM-CSF, G-CSF, M-CSF, and IL-7 is induced in both myeloid and lymphoid cells (**Figure 1**). It is even possible that indirectly, by stimulating other cell types, TLR4 could induce the activation of cells that can influence the erythroid and megakaryocyte lineage. Then, this massive cellular activation of hematopoietic cells and immune responses could explain the antimicrobial and immunomodulatory effects of casein as well as the activity of casein as an antihypertensive, antithrombotic, and antioxidant molecule [83].

Here, we can reveal that activation of the innate immune system by casein could be useful to fight pathogens resistant to antibiotics, as has been suggested [40], so casein could be used to induce a strong immune response in immunosuppressed patients [84, 85]; it could be used as a prophylactic strategy to prevent infections.

#### **Acknowledgements**

This work was financially supported by DGAPA PAPIIT IN221017 & IN229820.

### **Author details**

Edgar Ledesma-Martinez1 , Vanihamin Domínguez-Meléndez2 , Itzen Aguiñiga-Sánchez1 and Edelmiro Santiago-Osorio1 \*

1 Hematopoiesis and Leukemia Laboratory, Research Unit on Cell Differentiation and Cancer, FES Zaragoza, National Autonomous University of Mexico, Mexico City, Mexico

2 Center for Health Studies and Services (CESS), University of Veracruz, Veracruz City, Mexico

\*Address all correspondence to: edelmiro@unam.mx

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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[26] Ul Haq MR, Kapila R, Shandilya UK, Kapila S. Impact of milk derived β-casomorphins on physiological functions and trends in research: A review. International Journal of Food Properties. 2014;**17**:1726-1741. DOI: 10.1080/10942912.2012.712077

[27] Truswell AS. The A2 milk case: A critical review. European Journal of Clinical Nutrition. 2005;**59**:623-631. DOI: 10.1038/sj.ejcn.1602104

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[31] Holt C. Structure and stability of bovine casein micelles. Advances in Protein Chemistry. 1992;**43**:63-151. DOI: 10.1016/S0065-3233(08)60554-9

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antigen-specific IFN-γ production from CD8+ T cells by a single amino acidsubstituted peptide derived from bovine α(s1)-casein. Clinical Immunology and

Altendorfer I, Bleck E, Jose J,

10.1186/1471-2172-14-46

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[53] Domínguez-Meléndez V, Aguiñiga-Sánchez I, Moreno-Fierros L, Torres B, Osorio ES. El caseinato de sodio incrementa número de linfocitos B en ratones. Biomédica. 2017;**37**:571-576. DOI: 10.7705/biomedica.v34i2.3604

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10.1242/dmm.000315

31 January 2020]

DOI: 10.1006/clin.1998.4585

*Caseins as Regulators of Hematopoiesis DOI: http://dx.doi.org/10.5772/intechopen.91881*

*Infant Feeding - Breast versus Formula*

G-CSF receptor on granulocytic progenitor cells causes neutropenia in protein malnutrition. Nutrition. 2020;**69**:1-8. DOI: 10.1016/j.

more mechanism of action. Antibodies. 2019;**8**:52. DOI: 10.3390/antib8040052

[40] Noursadeghi M, Bickerstaff MCM,

Pepys MB. Production of granulocyte colony-stimulating factor in the nonspecific acute phase response enhances host resistance to bacterial infection. Journal of Immunology. 2002;**169**:913-919. DOI: 10.4049/

[41] Lotem J, Sachs L. Control of in vivo differentiation of myeloid leukemic cells. III. Regulation by T lymphocytes and inflammation. International Journal

of Cancer. 1983;**32**:781-791. DOI:

[42] Lotem J, Sachs L. Independent regulation of myeloid cell growth and differentiation inducing proteins: In vivo regulation by compounds that induce inflammation. International Journal of Cancer. 1985;**35**:93-100. DOI:

[43] Okano M, Ohnota H, Sasaki R.

erythropoiesis in rats by reducing serum erythropoietin concentration and the population size of erythroid precursor

10.1002/ijc.2910320620

10.1002/ijc.2910350115

jn/122.7.1376

stem.5530110407

Protein deficiency impairs

cells. The Journal of Nutrition. 1992;**122**:1376-1383. DOI: 10.1093/

[44] Barceló AC, Alippi RM, Boyer P, Olivera MI, Mide SM, Caro J, et al. Impaired response of polycythemic mice to erythropoietin induced by protein starvation imposed after hormone administration. Stem Cells. 1993;**11**:296-302. DOI: 10.1002/

[45] Metcalf D, Robb L, Dunn AR, Mifsud S, Di Rago L. Role of granulocyte-macrophage colonystimulating factor and granulocyte colony-stimulating factor in the development of an acute neutrophil inflammatory response in mice. Blood.

Herbert J, Moyes D, Cohen J,

jimmunol.169.2.913

[33] Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method. Methods. 2001;**25**:402-408. DOI: 10.1006/

[34] Scrimshaw NS, Viteri FE. INCAP

nutrition: Malnutrition, infection and immunity. The Journal of Nutrition. 2003;**133**:336S-340S. DOI: 10.1093/

[36] Chee LCY, Hendy J, Purton LE, McArthur GA. The granulocyte-colony stimulating factor receptor (G-CSFR) interacts with retinoic acid receptors (RARs) in the regulation of myeloid differentiation. Journal of Leukocyte Biology. 2013;**93**:235-243. DOI: 10.1189/

[37] Domínguez-Melendez V,

DOI: 10.1128/jvi.01270-18

[39] Sawa T, Kinoshita M, Inoue K, Ohara J, Moriyama K. Immunoglobulin for treating bacterial infections: One

Silvestre-Santana O, Moreno-Fierros L, Aguiñiga-Sánchez I, Martínez L, Marroquin-Segura R, et al. Sodium caseinate induces mouse granulopoiesis. Inflammation Research. 2012;**61**:367- 373. DOI: 10.1007/s00011-011-0421-7

[38] Harmon JR, Spengler JR, Coleman-McCray JD, Nichol ST, Spiropoulou CF, McElroy AK. CD4 T cells, CD8 T cells, and monocytes coordinate to prevent rift valley fever virus encephalitis. Journal of Virology. 2018;**92**:e01270-18.

studies of kwashiorkor and marasmus. Food and Nutrition Bulletin. 2010;**31**:34-41. DOI: 10.1177/156482651003100105

[35] Keusch GT. The history of

nut.2019.06.021

meth.2001.1262

jn/133.1.336s

jlb.1211609

**74**

1996;**88**:3755-3764. DOI: 10.1182/blood. v88.10.3755.bloodjournal88103755

[46] Ramos-Mandujano G, Weiss-Steider B, Melo B, Córdova Y, Ledesma-Martínez E, Bustos S, et al. Alpha-, beta- and kappa-caseins inhibit the proliferation of the myeloid cell lines 32D cl3 and WEHI-3 and exhibit different differentiation properties. Immunobiology. 2008;**213**:133-141. DOI: 10.1016/j.imbio.2007.07.004

[47] Santiago-Osorio E, Mora L, Bautista M, Montesinos JJ, Martínez I, Ramos-Mandujano G, et al. Sodium caseinate induces secretion of macrophage colony-stimulating factor from neutrophils. Immunobiology. 2010;**215**:332-339. DOI: 10.1016/j. imbio.2009.03.003

[48] Vordenbäumen S, Saenger T, Braukmann A, Tahan T, Bleck E, Jose J, et al. Human casein alpha s1 induces proinflammatory cytokine expression in monocytic cells by TLR4 signaling. Molecular Nutrition & Food Research. 2016;**60**:1079-1089. DOI: 10.1002/ mnfr.201500792

[49] Vordenbäumen S, Braukmann A, Altendorfer I, Bleck E, Jose J, Schneider M. Human casein alpha s1 (CSN1S1) skews in vitro differentiation of monocytes towards macrophages. BMC Immunology. 2013;**14**:46. DOI: 10.1186/1471-2172-14-46

[50] Verdi RJ, Barbano DM. Properties of proteases from milk somatic cells and blood leukocytes. Journal of Dairy Science. 1991;**74**:2077-2081. DOI: 10.3168/jds.S0022-0302(91)78379-3

[51] Totsuka M, Kakehi M, Kohyama M, Hachimura S, Hisatsune T, Kaminogawa S.Enhancement of antigen-specific IFN-γ production from CD8+ T cells by a single amino acidsubstituted peptide derived from bovine α(s1)-casein. Clinical Immunology and

Immunopathology. 1998;**88**:277-286. DOI: 10.1006/clin.1998.4585

[52] Bonomi F, Brandt R, Favalli S, Ferranti P, Fierro O, Frøkiær H, et al. Structural determinants of the immunomodulatory properties of the C-terminal region of bovine β-casein. International Dairy Journal. 2011;**21**:770- 776. DOI: 10.1016/j.idairyj.2011.04.012

[53] Domínguez-Meléndez V, Aguiñiga-Sánchez I, Moreno-Fierros L, Torres B, Osorio ES. El caseinato de sodio incrementa número de linfocitos B en ratones. Biomédica. 2017;**37**:571-576. DOI: 10.7705/biomedica.v34i2.3604

[54] Gill HS, Doull F, Rutherfurd KJ, Cross ML. Immunoregulatory peptides in bovine milk. British Journal of Nutrition. 2018;**84**:111-117. DOI: 10.1017/S0007114500002336

[55] M. Knopfler, How Compatible is Cow' s Milk with the Human Immune System?, 2016. Available from: https:// touroscholar.touro.edu/sjlcas [Accessed: 31 January 2020]

[56] Meinzen-Derr J, Poindexter B, Wrage L, Morrow AL, Stoll B, Donovan EF. Role of human milk in extremely low birth weight infants' risk of necrotizing enterocolitis or death. Journal of Perinatology. 2009;**29**:57-62. DOI: 10.1038/jp.2008.117

[57] Sodhi C, Richardson W, Gribar S, Hackam DJ. The development of animal models for the study of necrotizing enterocolitis. Disease Models & Mechanisms. 2008;**1**:94-98. DOI: 10.1242/dmm.000315

[58] Park YW. Bioactive peptides in milk and dairy products: A review. Korean Journal for Food Science of Animal Resources. 2015;**35**:831-840. DOI: 10.5851/kosfa.2015.35.6.831

[59] Ward TL, Hosid S, Ioshikhes I, Altosaar I. Human milk metagenome: A functional capacity analysis.

BMC Microbiology. 2013;**13**:1. DOI: 10.1186/1471-2180-13-116

[60] Hernell O, Hansson L. Human milk k-casein and inhibition of helicobacter pylori adhesion to human gastric mucosa. Journal of Pediatric Gastroenterology and Nutrition. 1995;**21**:288-296. DOI: 10.1097/00005176-199510000-00006

[61] Petermann K, Vordenbäumen S, Maas R, Braukmann A, Bleck E, Saenger T, et al. Autoantibodies to αs1 casein are induced by breast-feeding. PLoS One. 2012;**7**:e32716. DOI: 10.1371/ journal.pone.0032716

[62] Barbouche R, Forveille M, Fischer A, Avrameas S, Durandy A. Spontaneous IgM autoantibody production in vitro by B lymphocytes of normal human neonates. Scandinavian Journal of Immunology. 1992;**35**:659-667. DOI: 10.1111/j.1365-3083.1992.tb02972.x

[63] Galligan CL, Baig E, Bykerk V, Keystone EC, Fish EN. Distinctive gene expression signatures in rheumatoid arthritis synovial tissue fibroblast cells: Correlates with disease activity. Genes and Immunity. 2007;**8**:480-491. DOI: 10.1038/sj.gene.6364400

[64] Saenger T, Vordenbäumen S, Genich S, Haidar S, Schulte M, Nienberg C, et al. Human αS1-casein induces IL-8 secretion by binding to the ecto-domain of the TLR4/MD2 receptor complex. Biochimica et Biophysica Acta, General Subjects. 2019;**1863**:632-643. DOI: 10.1016/j.bbagen.2018.12.007

[65] Ungethuem U, Haeupl T, Witt H, Koczan D, Krenn V, Huber H, et al. Molecular signatures and new candidates to target the pathogenesis of rheumatoid arthritis. Physiological Genomics. 2010;**42A**:267-282. DOI: 10.1152/physiolgenomics.00004.2010

[66] Karlsson C, Dehne T, Lindahl A, Brittberg M, Pruss A, Sittinger M, et al. Genome-wide expression profiling

reveals new candidate genes associated with osteoarthritis. Osteoarthritis and Cartilage. 2010;**18**:581-592. DOI: 10.1016/j.joca.2009.12.002

[67] Otaegui D, Mostafavi S, Bernard CCA, de Munain AL, Mousavi P, Oksenberg JR, et al. Increased transcriptional activity of milk-related genes following the active phase of experimental autoimmune encephalomyelitis and multiple sclerosis. Journal of Immunology. 2007;**179**:4074-4082. DOI: 10.4049/jimmunol.179.6.4074

[68] Xu K, Ling MT, Wang X, Wong YC. Evidence of a novel biomarker, αs1- Casein, a milk protein, in benign prostate hyperplasia. Prostate Cancer and Prostatic Diseases. 2006;**9**:293-297. DOI: 10.1038/sj.pcan.4500872

[69] Van Overmeire E, Stijlemans B, Heymann F, Keirsse J, Morias Y, Elkrim Y, et al. M-CSF and GM-CSF receptor signaling differentially regulate monocyte maturation and macrophage polarization in the tumor microenvironment. Cancer Research. 2016;**76**:35-42. DOI: 10.1158/0008-5472. CAN-15-0869

[70] Hayes M, Stanton C, Fitzgerald GF, Ross RP. Putting microbes to work: Dairy fermentation, cell factories and bioactive peptides. Part II: Bioactive peptide functions. Biotechnology Journal. 2007;**2**:435-449. DOI: 10.1002/ biot.200700045

[71] Lewis SL, Van Epps DE. Demonstration of specific receptors for fluoresceinated casein on human neutrophils and monocytes using flow cytometry. Inflammation. 1983;**7**:363- 375. DOI: 10.1007/BF00916301

[72] Arruda-Silva F, Bianchetto-Aguilera F, Gasperini S, Polletti S, Cosentino E, Tamassia N, et al. Human neutrophils produce CCL23 in response to various TLR-agonists and TNFα. Frontiers in Cellular and Infection

**77**

*Caseins as Regulators of Hematopoiesis DOI: http://dx.doi.org/10.5772/intechopen.91881*

fcimb.2017.00176

10.1093/mmy/myy048

10.1038/icb.2008.80

jf0610864

[74] Seydoux E, Liang H, Dubois Cauwelaert N, Archer M, Rintala ND, Kramer R, et al. Effective combination adjuvants engage both TLR and inflammasome pathways to promote potent adaptive immune responses. Journal of Immunology. 2018;**201**:98- 112. DOI: 10.4049/jimmunol.1701604

[75] Carreno BM, Becker-Hapak M, Linette GP. CD40 regulates human dendritic cell-derived IL-7 production that, in turn, contributes to CD8+

[76] Tobita K, Kawahara T, Otani H. Bovine β-casein (1-28), a casein

phosphopeptide, enhances proliferation and IL-6 expression of mouse CD19+ cells via toll-like receptor 4. Journal of Agricultural and Food Chemistry. 2006;**54**:8013-8017. DOI: 10.1021/

[77] Kawahara T, Katayama D, Otani H. Effect of-casein (1-28) on proliferative responses and secretory functions of human immunocompetent cell lines. Bioscience, Biotechnology, and Biochemistry. 2004;**68**:2091-2095

[78] Smuda C, Wechsler JB, Bryce PJ. TLR-induced activation of neutrophils promotes histamine production via a PI3 kinase dependent mechanism. Immunology Letters. 2011;**141**:102-108. DOI: 10.1016/j.imlet.2011.08.002

[79] Sabroe I, Prince LR, Jones EC, Horsburgh MJ, Foster SJ, Vogel SN, et al. Selective roles for Toll-like receptor (TLR)2 and TLR4 in the regulation

antigen-specific expansion. Immunology and Cell Biology. 2009;**87**:167-177. DOI:

T-cell

of neutrophil activation and life span. Journal of Immunology. 2003;**170**:5268-5275. DOI: 10.4049/

[80] Takehara M, Seike S, Sonobe Y, Bandou H, Yokoyama S, Takagishi T, et al. Clostridium perfringens α-toxin impairs granulocyte colony-stimulating factor receptor-mediated granulocyte

[81] Hayashi F, Means TK, Luster AD. Toll-like receptors stimulate human

[82] Sadeghi K, Wisgrill L, Wessely I, Diesner SC, Schuller S, Dürr C, et al. GM-CSF down-regulates TLR expression via the transcription factor PU.1 in human monocytes. PLoS One. 2016;**11**:e0162667. DOI: 10.1371/journal.pone.0162667

[83] Meisel H, Bockelmann W. Bioactive peptides encrypted in milk proteins: Proteolytic activation and throphofunctional properties. In: van Leeuwenhoek A editor. Int. J. Gen. Mol. Microbiol, 1999. pp. 207-215. DOI:

[84] Millon L, Manteaux A, Reboux G, Drobacheff C, Monod M, Barale T, et al. Fluconazole-resistant recurrent oral candidiasis in human immunodeficiency virus-positive patients: Persistence of Candida albicans strains with the same genotype. Journal of Clinical Microbiology. 1994;**32**:1115-1118. Available from: http://www.ncbi.nlm. nih.gov/pubmed/8027327 [Accessed: 31

[85] Popa LG, Popa MI, Mihai IR. Casecontrol study to evaluate the link between immunosuppression and Candida spp. infection. Roumanian Archives of Microbiology and Immunology. 2005;**64**:72-76

10.1023/A:1002063805780

January 2020]

production while triggering septic shock. Communications Biology. 2019;**2**:1-12. DOI: 10.1038/

neutrophil function. Blood. 2003;**102**:2660-2669. DOI: 10.1182/

s42003-019-0280-2

blood-2003-04-1078

jimmunol.170.10.5268

Microbiology. 2017;**7**:176. DOI: 10.3389/

[73] Rossato L, dos Santos SS, Ferreira LG, de Almeida SR. The importance of Toll-like receptor 4 during experimental *Sporothrix brasiliensis* infection. Medical Mycology. 2019;**57**:489-495. DOI:

*Caseins as Regulators of Hematopoiesis DOI: http://dx.doi.org/10.5772/intechopen.91881*

*Infant Feeding - Breast versus Formula*

10.1186/1471-2180-13-116

BMC Microbiology. 2013;**13**:1. DOI:

reveals new candidate genes associated with osteoarthritis. Osteoarthritis and Cartilage. 2010;**18**:581-592. DOI:

[67] Otaegui D, Mostafavi S, Bernard CCA, de Munain AL, Mousavi P, Oksenberg JR, et al. Increased transcriptional activity of milk-related genes following the active phase of experimental autoimmune encephalomyelitis and multiple sclerosis. Journal of

Immunology. 2007;**179**:4074-4082. DOI:

[68] Xu K, Ling MT, Wang X, Wong YC. Evidence of a novel biomarker, αs1- Casein, a milk protein, in benign prostate hyperplasia. Prostate Cancer and Prostatic Diseases. 2006;**9**:293-297.

10.4049/jimmunol.179.6.4074

DOI: 10.1038/sj.pcan.4500872

CAN-15-0869

biot.200700045

[71] Lewis SL, Van Epps DE.

[72] Arruda-Silva F, Bianchetto-Aguilera F, Gasperini S, Polletti S, Cosentino E, Tamassia N, et al. Human neutrophils produce CCL23 in response to various TLR-agonists and TNFα. Frontiers in Cellular and Infection

Demonstration of specific receptors for fluoresceinated casein on human neutrophils and monocytes using flow cytometry. Inflammation. 1983;**7**:363- 375. DOI: 10.1007/BF00916301

[69] Van Overmeire E, Stijlemans B, Heymann F, Keirsse J, Morias Y, Elkrim Y, et al. M-CSF and GM-CSF receptor signaling differentially regulate monocyte maturation and macrophage polarization in the tumor microenvironment. Cancer Research. 2016;**76**:35-42. DOI: 10.1158/0008-5472.

[70] Hayes M, Stanton C, Fitzgerald GF, Ross RP. Putting microbes to work: Dairy fermentation, cell factories and bioactive peptides. Part II: Bioactive peptide functions. Biotechnology Journal. 2007;**2**:435-449. DOI: 10.1002/

10.1016/j.joca.2009.12.002

[60] Hernell O, Hansson L. Human milk k-casein and inhibition of helicobacter pylori adhesion to human gastric mucosa. Journal of Pediatric Gastroenterology and Nutrition. 1995;**21**:288-296. DOI: 10.1097/00005176-199510000-00006

[61] Petermann K, Vordenbäumen S, Maas R, Braukmann A, Bleck E, Saenger T, et al. Autoantibodies to αs1 casein are induced by breast-feeding. PLoS One. 2012;**7**:e32716. DOI: 10.1371/

[62] Barbouche R, Forveille M, Fischer A, Avrameas S, Durandy A. Spontaneous IgM autoantibody production in vitro by B lymphocytes of normal human neonates. Scandinavian Journal of Immunology. 1992;**35**:659-667. DOI: 10.1111/j.1365-3083.1992.tb02972.x

[63] Galligan CL, Baig E, Bykerk V, Keystone EC, Fish EN. Distinctive gene expression signatures in rheumatoid arthritis synovial tissue fibroblast cells: Correlates with disease activity. Genes and Immunity. 2007;**8**:480-491. DOI:

[64] Saenger T, Vordenbäumen S, Genich S, Haidar S, Schulte M, Nienberg C, et al. Human αS1-casein induces IL-8 secretion by binding to the ecto-domain of the TLR4/MD2 receptor complex. Biochimica et Biophysica Acta, General Subjects. 2019;**1863**:632-643. DOI: 10.1016/j.bbagen.2018.12.007

[65] Ungethuem U, Haeupl T, Witt H, Koczan D, Krenn V, Huber H, et al. Molecular signatures and new candidates to target the pathogenesis of rheumatoid arthritis. Physiological Genomics. 2010;**42A**:267-282. DOI: 10.1152/physiolgenomics.00004.2010

[66] Karlsson C, Dehne T, Lindahl A, Brittberg M, Pruss A, Sittinger M, et al. Genome-wide expression profiling

10.1038/sj.gene.6364400

journal.pone.0032716

**76**

Microbiology. 2017;**7**:176. DOI: 10.3389/ fcimb.2017.00176

[73] Rossato L, dos Santos SS, Ferreira LG, de Almeida SR. The importance of Toll-like receptor 4 during experimental *Sporothrix brasiliensis* infection. Medical Mycology. 2019;**57**:489-495. DOI: 10.1093/mmy/myy048

[74] Seydoux E, Liang H, Dubois Cauwelaert N, Archer M, Rintala ND, Kramer R, et al. Effective combination adjuvants engage both TLR and inflammasome pathways to promote potent adaptive immune responses. Journal of Immunology. 2018;**201**:98- 112. DOI: 10.4049/jimmunol.1701604

[75] Carreno BM, Becker-Hapak M, Linette GP. CD40 regulates human dendritic cell-derived IL-7 production that, in turn, contributes to CD8+ T-cell antigen-specific expansion. Immunology and Cell Biology. 2009;**87**:167-177. DOI: 10.1038/icb.2008.80

[76] Tobita K, Kawahara T, Otani H. Bovine β-casein (1-28), a casein phosphopeptide, enhances proliferation and IL-6 expression of mouse CD19+ cells via toll-like receptor 4. Journal of Agricultural and Food Chemistry. 2006;**54**:8013-8017. DOI: 10.1021/ jf0610864

[77] Kawahara T, Katayama D, Otani H. Effect of-casein (1-28) on proliferative responses and secretory functions of human immunocompetent cell lines. Bioscience, Biotechnology, and Biochemistry. 2004;**68**:2091-2095

[78] Smuda C, Wechsler JB, Bryce PJ. TLR-induced activation of neutrophils promotes histamine production via a PI3 kinase dependent mechanism. Immunology Letters. 2011;**141**:102-108. DOI: 10.1016/j.imlet.2011.08.002

[79] Sabroe I, Prince LR, Jones EC, Horsburgh MJ, Foster SJ, Vogel SN, et al. Selective roles for Toll-like receptor (TLR)2 and TLR4 in the regulation

of neutrophil activation and life span. Journal of Immunology. 2003;**170**:5268-5275. DOI: 10.4049/ jimmunol.170.10.5268

[80] Takehara M, Seike S, Sonobe Y, Bandou H, Yokoyama S, Takagishi T, et al. Clostridium perfringens α-toxin impairs granulocyte colony-stimulating factor receptor-mediated granulocyte production while triggering septic shock. Communications Biology. 2019;**2**:1-12. DOI: 10.1038/ s42003-019-0280-2

[81] Hayashi F, Means TK, Luster AD. Toll-like receptors stimulate human neutrophil function. Blood. 2003;**102**:2660-2669. DOI: 10.1182/ blood-2003-04-1078

[82] Sadeghi K, Wisgrill L, Wessely I, Diesner SC, Schuller S, Dürr C, et al. GM-CSF down-regulates TLR expression via the transcription factor PU.1 in human monocytes. PLoS One. 2016;**11**:e0162667. DOI: 10.1371/journal.pone.0162667

[83] Meisel H, Bockelmann W. Bioactive peptides encrypted in milk proteins: Proteolytic activation and throphofunctional properties. In: van Leeuwenhoek A editor. Int. J. Gen. Mol. Microbiol, 1999. pp. 207-215. DOI: 10.1023/A:1002063805780

[84] Millon L, Manteaux A, Reboux G, Drobacheff C, Monod M, Barale T, et al. Fluconazole-resistant recurrent oral candidiasis in human immunodeficiency virus-positive patients: Persistence of Candida albicans strains with the same genotype. Journal of Clinical Microbiology. 1994;**32**:1115-1118. Available from: http://www.ncbi.nlm. nih.gov/pubmed/8027327 [Accessed: 31 January 2020]

[85] Popa LG, Popa MI, Mihai IR. Casecontrol study to evaluate the link between immunosuppression and Candida spp. infection. Roumanian Archives of Microbiology and Immunology. 2005;**64**:72-76

Section 4

Breast Feeding:

Microbiological Aspects

**79**

Section 4
