Infant food allergies are a serious and often life-threatening health problem, affecting approximately 4% of children and their families worldwide . These allergies may cause anaphylactic shock or even death, but there is currently no drug therapy for a milk protein allergy. The only way to avoid this type of allergy is to eliminate the harmful milk protein from the diet [23, 24]. Management strategies for a milk protein allergy include removing dairy products from exclusively breastfed babies and eliminating milk protein from the mother’s diet. Breast milk is a source of "standard nutrition" for infants with milk protein allergies and should be promoted as much as possible . Data on the prevalence of reproducible clinical responses to milk protein in breastfed children are very limited, but the prevalence has been reported to be approximately 0.5% . Although b-lactoglobulin originating from cow’s milk can be detected in the breast milk of 95% of lactating women, the amount is insignificant to many infants with a mild to moderate milk protein allergy . For babies who are allergic to milk protein from breastfeeding, mothers should be encouraged to continue breastfeeding while avoiding milk protein in their diet . However, if breastfeeding or maintenance of breastfeeding is not possible in patients with a milk protein allergy, a hydrolyzed milk formula or amino acid formula should be provided.
If the symptoms of infants who are breastfed or fed only formula milk cannot be relieved, it is recommended that extensively hydrolyzed formula milk is used. Hypoallergenic products are either extensively hydrolyzed milk powder (ehMF) composed of small peptides < 1.5 kDa or amino acid formulas composed of essential and nonessential amino acids. The latter is recommended for intolerant infants affected by ehMF [25, 28]. Amino acid-based formulas (AAFs) are usually used to address complex milk protein allergies, including various food allergies or formulas that do not tolerate large amounts of hydrolysis. In our study, infants with a milk protein allergy were treated with a dietary intervention (involving amino acid formulas or deeply hydrolyzed formulas or mothers avoiding the consumption of milk), and the allergic symptoms gradually decreased.
Calprotectin reflects the migration of neutrophils to the intestinal lumen, so it can be used as a sensitive marker of intestinal inflammation . Under normal circumstances, its concentration in feces is six times the concentration in plasma , emphasizing the potential of fecal calprotectin as an accurate biomarker of intestinal inflammation. The evidence supporting the use of fecal calprotectin as a marker of intestinal inflammation is sufficient and continues to accumulate . In addition, fecal calprotectin remains stable in the feces for more than a week, so it is a useful marker of intestinal inflammation  and can be measured within a few hours using a simple ELISA test; therefore, the test results are quickly available for effective clinical decision making. Calprotectin has attracted increasing attention in studies of food allergies [10, 14, 32, 33]. Fecal calprotectin may play an important role in food allergies, and it is speculated that in addition to being an inflammatory factor, calprotectin has a role in the process of food allergies, possibly as a trigger that amplifies the cascade reaction of allergic-related and inflammatory factors in allergic responses . In response to food allergens, eosinophils and neutrophils are activated, while neutrophils and epithelial cells in the intestinal mucosa activate calprotectin, resulting in increased levels of calprotectin . Most studies have indicated that the activation of Th2 cells is an important step in the immune mechanism of food allergies [15, 34, 35]. In the process of allergies, there are inflammatory features of the Th2 cytokine environment (such as increased eosinophils and mast cells) and local eosinophilia during allergies. The activation of granulocytes and neutrophils is recruited and activated. It can be speculated that this may lead to an increase in the expression and secretion of calprotectin, which indicates that there may be a process similar to inflammation in an allergic state . Our previous study indicated that calprotectin may activate DCs and related signal transduction through the action of TLR4 to promote the differentiation of initial CD4 + T cells into Th2-type cells and then cause allergy-related immune responses, leading to the occurrence of allergies. The increase in S100A8/A9 amplifies the cascade of allergic and inflammatory factors in food allergies . Baldassarre et al  compared the fecal calprotectin levels of 30 milk protein allergic infants with rectal bleeding and healthy infants of the same age. The fecal calprotectin level of infants with a milk protein allergy was significantly higher than that of the control group (325.89 vs. 131.97 µg/g). Four weeks after removing milk from the diet, the fecal calprotectin level dropped by 50%; however, it was still higher than that of the control group (157.5 vs. 93.72, p = 0.03). Beşer et al  found that infants who were allergic to milk protein had significantly lower fecal calprotectin levels after eliminating milk protein from the diet than before eliminating milk protein from the diet. In addition, the level of fecal calprotectin before the milk protein elimination diet was significantly higher than that of healthy infants (p = 0.011). In a preliminary study of 6 patients with a milk allergy , fecal calprotectin levels were measured before dietary intervention and at 3 and 6 weeks after the initiation of protein hydrolysate formula feeding. The fecal calprotectin level before dietary intervention was 135–1537 mg/L (average: 557 mg/L) and decreased to 42–219 mg/L (average: 163 mg/L) after six weeks. The fecal calprotectin level of infants with a milk protein allergy was compared with that before treatment; after treatment, the fecal calprotectin level decreased, and the clinical symptoms were alleviated , which is consistent with our results. In this study, the median fecal calprotectin values of 90 infants with a milk protein allergy before intervention and at the first and second follow-up visits were 410 µg/g, 253 µg/g and 160 µg/g, respectively. With the extension of dietary intervention and treatment time, allergic symptoms improved, and the level of fecal calprotectin in infants with a milk protein allergy gradually decreased. This suggested that fecal calprotectin may be useful for determining relapses and follow-ups after diagnosis of a milk protein allergy, particularly an allergy with GI involvement. Tracking fecal calprotectin levels might reveal increases or reductions in disease activity, and it may be useful as an inexpensive, simple, and noninvasive test to demonstrate and assess disease activity in infants with a milk protein allergy. The level of fecal calprotectin may be used to monitor the improvement in intestinal allergies in infants with a milk allergy, which may be used as a possible biological indicator for follow-up and monitoring of intestinal allergies.
In this study, the number of eosinophils in the infants with a milk protein allergy was much greater than that in the infants with a non-milk protein allergy, and with dietary intervention and treatment, the number of eosinophils in the allergic infants gradually decreased and tended to be normal. We found that the eosinophil count (495/mm3) of the infants with a milk protein allergy was significantly lower at the first follow-up visit than before dietary intervention (655/mm3) and that the number of eosinophils at the second follow-up visit (330/mm3) was significantly lower than that at the first follow-up visit (495/mm3). In a similar study by Dogan, E et al.  found significantly higher eosinophil cationic protein levels (51.45 ng/mL) and blood eosinophil counts (475/mm3) in infants with a milk protein allergy than in controls (17.55 ng/mL, 300/mm3). Recently, Li, J et al.  found that in 6-month-old infants with a milk protein allergy, eosinophil counts were higher than those of the nonallergic group (0.89 ± 0.45/mm3 and 0.26 ± 0.12/mm3, respectively, p < 0.01). All of the above studies have found that infants with a milk protein allergy have an increased number of eosinophils in their blood. The eosinophils formed in the bone marrow have large cytoplasmic granules, which contain eosinophil cationic protein, eosinophil protein X, and eosinophil-derived neurotoxin . The eosinophil cationic protein encoded by the RNASE3 gene is a cytotoxic protein that enters the surrounding tissues when activated eosinophils degranulate and manifests as an increase in the level of eosinophils in the surrounding tissues . Therefore, the response to Th2-induced allergic diseases (such as milk protein allergy, asthma and inflammatory diseases) may increase the number of circulating eosinophils and eosinophil cationic protein levels. Eosinophilic cationic protein is one of the four main basic proteins in specific granules in the cytoplasm of eosinophils. It can reflect the activity of eosinophils and elevated levels in body fluids such as saliva, serum and feces in the course of inflammatory processes and allergic diseases . When inflammation and allergic reactions occur in the body, the level of fecal calprotectin and the number of eosinophils are increased [10, 38, 41], so the level of fecal calprotectin and the number of eosinophils are positively correlated.
In this study, we also recorded the changes in the baby's weight and length before and after the intervention. From the results of our data analysis, after dietary intervention, the growth indicators of allergic children increased significantly during the first and second follow-ups of the intervention. A study showed that  infants allergic to milk protein who received amino acid-based infant formula gained weight and exhibited a decreased allergic performance. Children have poor growth related to low-grade inflammation affecting GI barrier function, leading to suboptimal nutrient absorption . A diet that does not contain milk protein, especially a large amount of hydrolyzed formula, can reduce GI symptoms due to changes in immune mechanisms and exercise capacity (for example, reducing gastric emptying time) . There is evidence that hydrolyzed infant formula may have a long-term preventive effect on the development of allergic symptoms . Our research results are similar to the above published studies, and we found that through a dietary intervention, the height and weight of the child gradually increased, which was better than the levels before the intervention.
This study shows that the fecal calprotectin level of infants with a milk protein allergy is significantly higher than that of healthy infants without an allergy. The level of fecal calprotectin was negatively correlated with the growth and development of the infants with a milk allergy. These infants were treated with a dietary intervention, and the symptoms of intestinal allergies were improved. The level of fecal calprotectin also decreased with the remission of allergic symptoms. The lower the level of fecal calprotectin was, the better the growth and development of the infants with a milk allergy. Fecal calprotectin may be used as a possible marker for monitoring intestinal hypersensitivity in infants. The level of fecal calprotectin may be used to monitor the improvement in intestinal allergy in infants with a milk allergy and may be used as a biological indicator for follow-up and monitoring of intestinal allergy. However, in a recent review by Xiong et al  including thirteen studies with IgE-mediated and non-IgE-mediated milk protein allergies, the authors concluded that the available evidence was not sufficient to confirm the utilization of fecal calprotectin, neither for diagnosis nor for the monitoring of a milk protein allergy. This may be due to minor infections or even non-GI infections that may affect the level of fecal calprotectin. This fact could result in uncertain clinical interpretations if fecal calprotectin is used as a biomarker for milk protein allergy diagnosis in infants, as mild infections are frequent at this age . More studies are needed in the future to determine the value of fecal calprotectin levels in allergic diseases.
Our study has several limitations. First, stool samples were collected from the children’s diapers. Olafsdottir et al  reported that this method of collection increases the fecal calprotectin concentration by up to 30% because water is absorbed into the diaper. This could yield measured fecal calprotectin levels that are higher than those actually present; therefore, direct stool collection during excretion may be more practical . Second, we did not draw blood to detect IgE in the milk protein allergy group, and we could not determine whether the children had IgE-mediated or non-IgE-mediated allergic reactions. Third, this study had a small sample size and short follow-up time, and a large-sample study with a longer follow-up time is needed to investigate the role of calprotectin in the intestinal tract of children with food allergies. Finally, we did not follow up with healthy children in this study.