f78 nBos d 8 Casein, Milk

Allergen

Cow's milk is popularly consumed throughout the world. Allergy to cow's milk is prevalent among infants and young children during the first year of life, affecting 0.5-3% at the age of 1 year. Bos d 8 (casein) has been reported as a major allergenic protein in cow's milk. According to a number of studies, sensitization towards Bos d 8 ranges between 0.9-65% of patients with cow's milk allergy. Bos d 8 has been found to be a highly heat-resistant protein, and its allergenicity is found to remain despite heating at 120oC (for 15 min). Therefore, specific immunoglobulin E (sIgE) levels of Bos d 8 may help assess the likelihood of tolerance to cooked or baked milk. Further, low sIgE levels to Bos d 8 have been reported to indicate tolerance towards cow's milk. Clinical manifestations of Bos d 8-mediated allergy majorly include atopic dermatitis. Moreover, Bos d 8 sIgE in serum has been regarded as a beneficial tool for predicting resolution and management with respect to introducing baked milk products into the diet. Cross-reactivity has been observed between Bos d 8 (casein) from cow's milk and casein from goat, sheep, and human milk.

Epidemiology

Worldwide distribution

Cow's milk is a common food consumed worldwide by infants, children, and adults (Muthukumar, Selvasekaran et al. 2020). It is considered one of the major food allergens prevalent among infants and young children, affecting 0.5-3% with cow's milk allergy (CMA) at the age of 1 year (Skripak, Matsui et al. 2007, Flom and Sicherer 2019), with a high prevalence observed during the first year of life (Kaczmarski, Wasilewska et al. 2013). However, the majority of the children outgrow their CMA by the age of 5 years and develop tolerance (Ito, Futamura et al. 2012).

Bos d 8 (casein) has been reported as a major allergenic protein in cow's milk (Wal 2002). According to the studies reported in the literature, the prevalence of sensitization toward casein is varied (range 0.9%-65%) among patients with CMA, which might be due to the diverse study population (Wal 2002, Kaczmarski, Wasilewska et al. 2013, Chen, Lee et al. 2014, Tedner, Söderhäll et al. 2021).

A US-based study was conducted on 78 children (7.9-14.4 years) with moderate to severe atopic dermatitis (AD). The study reported Bos d 8 to be responsible for generating the highest specific immunoglobulin E (sIgE) level among milk-allergic children (Frischmeyer-Guerrerio, Rasooly et al. 2019).

A study was conducted in Japan on 83 children (0.8-15.8 years) with suspected CMA. The CMA children (n=61; clinically diagnosed) were claimed to possess a considerably higher level of Bos d 8 sIgE compared to the non-CMA children (Ito, Futamura et al. 2012).

A study in the Netherlands included 28 adults (≥16 years) CMA patients [18 assessed via skin prick test (SPT) and 10 via double-blind placebo-controlled food challenge] in the study group and 25 individuals in the control group (cow milk sensitized but tolerant individuals). The study detected an elevated level (p=0.016) of Bos d 8 sIgE in individuals present in the study group compared to the control group (Lam, van Hoffen et al. 2008).

Environmental Characteristics

Source and tissue

Cow's milk contains mainly two types of proteins, including casein/Bos d 8 (constituting about 80% of the total milk protein) and whey (comprising about 20% of the total milk protein) (Flom and Sicherer 2019). Bos d 8 (casein) is further made up of four different fractions, αS1, αS2, β, and κ-caseins. Hydrolysis of β-casein has been found to yield three γ–caseins (γ1, γ2, and γ3) (Restani, Ballabio et al. 2009). The most important allergen identified in the casein fraction is αS1 casein (Hochwallner, Schulmeister et al. 2014).

Bos d 8 is found to be a member of the secretory calcium-binding phosphoprotein family that contains phosphate groups with a strong binding affinity for polyvalent cations (like calcium). They help in the secretion and transportation of phosphate and calcium as well as retention-mediated nutrient digestion and absorption in the stomach (Villa, Costa et al. 2018).

The International Union of Immunological Societies (IUIS) has listed Bos d 8, a casein protein, as a food allergen from cow's milk (WHO/IUIS 2019).

Clinical Relevance

Disease severity

CMA is considered one of the most common pediatric food allergies (Agyemang, Saf et al. 2019). Milk allergy can be either IgE-mediated or non-IgE-mediated (Walsh, Meyer et al. 2016). The clinical manifestations of cow milk protein allergy are reported to be of two types, such as immediate (shortly after consumption) and delayed (after hours or days of consumption) responses (De Greef, Hauser et al. 2012). The immediate reactions are generally IgE-mediated and can lead to angioedema, urticaria, aggravation of AD, vomiting, and in some cases, anaphylaxis, as well as respiratory symptoms (breathing, wheezing, coughing) (De Greef, Hauser et al. 2012, Ito, Futamura et al. 2012, Flom and Sicherer 2019). On the other hand, the majority of the delayed reactions are non-IgE mediated (Walsh, Meyer et al. 2016) and can lead to gastrointestinal symptoms (like enteropathy or proctocolitis) or skin reactions (such as AD) (De Greef, Hauser et al. 2012, Ito, Futamura et al. 2012).

Bos d 8 has been found to be a highly heat-resistant protein among all other milk proteins. The allergenicity of Bos d 8 has been found to remain despite heating at 120oC (for 15 min) (Tsabouri, Douros et al. 2014).

Therefore, sIgE level to Bos d 8 may help assess the likelihood of tolerance to cooked or baked milk, and 75% of children with CMA may actually tolerate baked milk products such as muffins, waffles, cakes and breads (Nowak-Wegrzyn, Bloom et al. 2008, Caubet, Nowak-Wegrzyn et al. 2013, Ford, Bloom et al. 2013). High sIgE levels of Bos d 8, however, indicate an allergy to all forms of milk (Caubet, Nowak-Wegrzyn et al. 2013, Ford, Bloom et al. 2013), and low sIgE levels to Bos d 8 indicate the patient may develop tolerance (Sicherer and Sampson 1999).

This heat-resistant property of Bos d 8 has been validated by a study conducted on 134 CMA children (subjected to oral food challenges to various baked milk products). The study demonstrated significantly lower Bos d 8 sIgE among the baked-milk tolerant group compared to the baked-milk reactive group (Agyemang, Saf et al. 2019).

A prospective, observational, real-life study was conducted on 100 clinically diagnosed CMA children (79 with AD and 29 without AD). Children with AD showed cutaneous reactions as the symptom of CMA. The study reported that the children affected with AD exhibited an elevated level of sIgE towards Bos d 8, particularly at 6 months (age) (Giannetti, Cipriani et al. 2019).

Cross-reactive molecules

Milk of different ruminant species (cow, goat, buffalo, and sheep) has been reported to possess Bos d 8 proteins bearing high sequence homology (80 to >90%), which could probably be the reason for cross-reactivity between these mammalian milk (Villa, Costa et al. 2018). A prospective cohort study on 38 children (<14 years) with CMA-associated symptoms detected the rate of cross-sensitization (SPT-based) between goat milk and cow milk to be 63.2% (24/38) (Ehlayel, Bener et al. 2011).

A broad dissimilarity (less Bos d 8 and more whey protein) in protein distribution has been observed in donkey and horse (mare) compared to cow's milk, which has been believed to be responsible for the low cross-reactivity of these milk with cow's milk (Tsabouri, Douros et al. 2014). Similarly, a lower molecular similarity between cow and camel milk has been identified as responsible for a weak cross-reactivity between these two types of milk. A prospective cohort study identified cross-sensitization between camel and cow milk in 20% (7/38) of children with CMA (Ehlayel, Bener et al. 2011).

Moreover, in vitro cross-reactivity between cow milk Bos d 8 and soy protein A5-B3 glycinin molecule (11 S globulin of soybean) has been reported (Rozenfeld, Docena et al. 2002, Tsabouri, Douros et al. 2014).

Molecular Aspects

Biochemistry

Bos d 8 (casein) consists of four key proteins, αS1, αS2, β, and κ- caseins, constituting 40%, 12.5%, 35%, and 12.5%, respectively of the casein fraction in milk (Villa, Costa et al. 2018). The aggregation of these four principal casein proteins in an ordered fashion is known as micelles, spherical with a diameter of 100 to 300 nm. The micelle's inner part is composed of α and β-caseins, whereas the κ- casein is found on the outer surface (Hochwallner, Schulmeister et al. 2014).

Bos d 8 is reported to have a highly amphipathic structure due to the presence of hydrophobic and hydrophilic patches. The distribution of charges on the Bos d 8 molecule is non-uniform because of the variations in the nature of the residues (hydrophobic, polar, and charged). The surface of this protein has been found to be water-insoluble and extremely active (Villa, Costa et al. 2018).

Bos d 8 possesses a coiled structural conformation, further stabilized by hydrophobic interactions (Wal 2002). The presence of multiple proline residues and the absence of disulfide bonds facilitate the bending of the Bos d 8 protein chain, thereby preventing the formation of an ordered/compact (rigid) secondary structure (Fiocchi, Brozek et al. 2010).  

Further, Bos d 8 has been claimed to possess weak immunogenic potential due to its structural flexibility. This protein has been found prone to enzymatic degradation by proteolytic enzymes (such as proteinases and exopeptidases) during gut digestion. However, this protein is found to be resistant to other denaturing agents, including heat treatment or urea (Villa, Costa et al. 2018).

Isoforms, epitopes, antibodies

Food allergens (like milk; Bos d 8) have been reported to possess linear epitopes. Various sequential epitopes have been found to be present on the αS1-casein, which could be responsible for the initiation of allergic reactions. Moreover, the epitope distribution sites of αS2, β, and κ- caseins have been identified; although, a lack of sufficient data on their allergenic activity has been reported (Hochwallner, Schulmeister et al. 2014).

Five IgE-binding epitopes of Bos d 8 (2 on κ- casein, 2 on αS1 casein, and 1 on αS2 casein) were identified by a study majorly in patients with a persistent milk allergy. Furthermore, the binding of IgE antibodies to at least one of the three epitopes (αS1 casein, αS2 casein, and κ- casein) was found in all persistent CMA patients (Jarvinen, Beyer et al. 2002).

Cross-reactivity due to structural similarity

According to a study, the milk of cows, goats, and sheep has been found to possess identical amino acid sequences (>85%) (El-Agamy 2007). As a result of structural homology (primary structure of α-casein) between cow, goat, and sheep milk, studies have claimed a high level of clinical cross-reactivity between these milk (Spuergin, Walter et al. 1997, Kattan, Cocco et al. 2011, Tsabouri, Douros et al. 2014).

Additionally, β-caseins from both human and bovine milk have been found to exhibit around 50% of sequence homology (Villa, Costa et al. 2018). Moreover, the common regions (specifically the C-terminus moiety) shared by the β-caseins of human and bovine milk are considered the primary phosphorylation site, which is involved in the cross-reactivity between both the milk (Tsabouri, Douros et al. 2014).

Diagnostic Relevance

Disease Severity

Milk-sIgE testing is used as an aid in diagnosing milk allergy and monitoring disease development (Panel, Boyce et al. 2010). A direct correlation has been observed between elevated levels of Bos d 8 sIgE and persistent CMA. In contrast, lower levels of Bos d 8 sIgE are found to be associated with higher chances of tolerance towards baked milk products. Hence, Bos d 8 sIgE in serum is regarded as a beneficial tool for predicting resolution and management with respect to introducing baked milk products into the diet (Flom and Sicherer 2019). The cut-off decision points (combined with clinical history and physician expertise) for sIgE to Bos d 8 can be used by physicians to identify the ideal candidates for baked milk OFCs and improve the management of children with suspected CM allergy. As per a US-based study, the positive and negative decision points for reactivity to baked milk were 20.2 kUA/L and approximately < 0.94 kUA/L, respectively, for Bos d 8 sIgE (Caubet, Nowak-Wegrzyn et al. 2013).

The presence of an increased level of sIgE to Bos d 8 has been reported to be correlated with milk allergy among children and may be linked with persistent CMA (Ito, Futamura et al. 2012). According to a study, children with persistent CMA have been found to possess significantly higher ratios of sIgEs to linear vs conformational epitopes to α- and β-casein (p<0.005 and p<0.002, respectively) compared to the individuals outgrowing milk allergy (Vila, Beyer et al. 2001). A high concentration of sIgE towards Bos d 8 might be a prognostic factor of a reaction to baked milk in children with CMA. Measurement of κ-casein has been suggested as the most beneficial marker to predict reactivity to baked milk and allergies (Popielarz and Krogulska 2021).

Exposure

The route of exposure for the Bos d 8 component found in milk is through ingestion (Kaczmarski, Wasilewska et al. 2013).

However, direct skin contact (cutaneous exposure) mediated anaphylaxis in a 12-month-old child post-exposure to Bos d 8 milk allergen-containing ointment in diaper rash has been reported in a case study (Jarmoc and Primack 1987).

Also, inhalation-mediated allergic reactions (asthma and rhinitis) toward milk protein Bos d 8 (present in a dermatological powder) were observed in a 44-year-old nurse with a positive SPT towards Bos d 8 (Bonadonna, Senna et al. 2003).

Explained Results

Allergen information

Bos d 8 (casein) is a major allergenic protein in cow's milk (Wal 2002). It is a heat-resistant protein sensitive to enzymatic digestion in the gut (Verhoeckx, Vissers et al. 2015, Villa, Costa et al. 2018).

Clinical relevance

Typical symptoms of CMA include skin reactions (angioedema, urticaria, and atopic dermatitis), GI symptoms, respiratory systems, and in some(De Greef, Hauser et al. 2012, Ito, Futamura et al. 2012, Flom and Sicherer 2019) cases, anaphylaxis.

Cross-reactivity

Bos d 8 (casein) from cow's milk has shown extensive cross-reactivity with casein from goat, sheep, and human milk (Spuergin, Walter et al. 1997, Kattan, Cocco et al. 2011, Tsabouri, Douros et al. 2014). Low cross-reactivity has been reported between bovine milk and soy, donkey, horse (mare), and camel milk (Ehlayel, Bener et al. 2011, Tsabouri, Douros et al. 2014).