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Clin Exp Immunol. Jan 1999; 115(1): 91–94.
PMCID: PMC1905198

Human milk 90K (Mac-2 BP): possible protective effects against acute respiratory infections

Abstract

Eighty-six children fed human milk were followed prospectively from birth to 12 months of age to assess the effect of milk 90K, a secreted glycoprotein with immune-stimulatory properties, on development of acute respiratory infections (ARI). The level of human milk 90K was inversely related to episodes of ARI (r = − 0.34; P = 0.001). The average 90K level in human milk fed to children who did not develop ARI was significantly higher than in milk fed to children in whom infection occurred on multiple occasions (156.6 ± 144.8 μg/ml versus 70.9 ± 92.3 μg/ml; P = 0.001). These data suggest that the protective effects of human milk against ARI may be due in part to immune maturation effects by secreted 90K.

Keywords: human milk, immune stimulation, 90K (Mac-2 BB), cytokines, acute respiratory infections

INTRODUCTION

Acute respiratory infections (ARI) are a major cause of morbidity and mortality among infants and children living in the industrialized world [1,2].The frequency is higher than in adults because of children's immunological immaturity and naivety. Four to six ARI per year are expected until about 6 years of age, when adequate immune mechanisms develop and sufficient immunity to the most common viruses and bacteria is acquired [3]. Recently, the prophylactic effects of human breast milk in preventing ARI have been described [4,5]. At least in part, this is due to bioactive components present in milk that modulate the immune response of the infant [6,7]. The milk substances involved in these processes have not been characterized. However, a glycoprotein of molecular weight > 400 kD has been described that is able to neutralize respiratory syncytial virus [8]. Similarly, high molecular weight material has been identified in human serum that interferes with the attachment and infectivity of hepatitis A virus to various cell lines [9].

Previously, we have identified a human tumour-derived antigen, designated 90K, in the culture supernatant of human breast cancer cells [10]. A series of analyses has established that 90K is a widely expressed, secreted, approx. 90-kD serum glycoprotein found both in normal individuals and at elevated levels in the serum of cancer patients as well as sufferers from other non-malignant diseases such as HIV or hepatitis B virus (HBV)/hepatitis C virus (HCV) [1113]. Cloning and sequencing studies have shown that 90K is identical to the human Mac-2 binding protein (Mac-2 BP), a ligand for the lactose/galactose-specific S-type lectin Mac-2 [14]. The 32-kD lectin Mac-2, originally identified as a cell-associated macrophage antigen. [15], is a bifunctional secreted protein with a lectin domain capable of binding laminin [16,17] and which, in association with its ligand, 90K/Mac-2 BP, may serve as a bridge between the macrophages and the extracellular matrix, microorganisms, or other cells bearing galactosylated proteins. 90K itself has been shown to be a potent immune stimulator with positive effects on cell-mediated immune responses [14]. We previously found that 90K is present in large amounts in human milk and speculated that the glycoprotein may be one of the milk substances affording protection to the breast-fed infant against infections [18].

In the present study we measured 90K levels in human milk fed to infants followed prospectively from birth to 12 months of age to determine the effect of the glycoprotein on development of ARI.

MATERIALS AND METHODS

Case definition

Inclusion criteria were: (i) absent congenital disease (including cystic fibrosis and primitive immune deficiencies) and perinatal HIV-1 infection; (ii) gestational age between 38 and 42 weeks; (iii) birth weight within 25 and 75 centile for gestational age; (iv) available sample of maternal early milk; (v) exclusive breast feeding for at least 15 days.

ARI was defined as a febrile (fever > 38°C) disease involving the upper and/or lower respiratory airways [19,20]. Temperature was measured by electronic devices. Signs should fulfil criteria for diagnosis of acute pharyngitis and nasopharyngitis, acute laryngitis, otitis media, bronchitis, bronchiolitis and pneumonia. Febrile rhinitis was not included in the ARI definition.

To be assured that infants met inclusion criteria, familial history was carefully collected, serum IgM and IgA levels and lymphocyte counts were evaluated, absence of HIV-1 antibodies was demonstrated by immunoenzymatic and Western blot methods, and determination of immunoreactive trypsinogen in blood spots.

Data set

Infants (n = 112) born between 2 May and 14 July 1996 and consecutively referred to the Neonatology Unit of the University of Chieti Medical School were enrolled. Of them, 86 children (44 males and 42 females) met the inclusion criteria and entered the present study. Data set included one twin pair.

Study design

Infants were followed up to the age of 12 months. Experienced paediatricians carefully collected history of respiratory infections during monthly telephone interviews and visits carried out at Neonatology Unit every 3 months. Parents received instructions to take their children to the family paediatrician or our Neonatology Unit whenever they manifested fever. In addition, parents and family paediatricians received instructions to fill in a form with children's clinical history and occurrence of respiratory infections, according to the above definition. Completed forms were returned at the next visit, during which parents were also questioned about any other information that might have been relevant. Data were entered in a specific database which also included children's gestational age, mode of delivery, birth weight, gender, and information concerning parental tobacco smoking, day care attendance, and family crowding. Smoking was evaluated as a dichotomic variable (yes or no) and crowding was assessed by family member (including siblings) numbers.

Informed consent was obtained and the study was approved by the Hospital's Ethic Committee.

Human milk

Milk was collected by means of manual expression into polypropylene containers. The samples were transferred to the laboratory, centrifuged at 3000 g and stored at −20°C until assayed. Milk was collected once within 2 days of delivery.

Determination of 90K in human milk

The solid-phase ELISA was used and performed in triplicate [18]. Pooled human milk was used as a reference for each assay. The data obtained from the reference were used to adjust the day-to-day and the plate-to-plate variations of results with subject human milk. The lower limit of detection in this assay was 31 ng/ml.

Statistical analysis

Two-group comparison was tested with the use of the Mann–Whitney U-test. The Spearman regression coefficient was used to examine the magnitude of selected associations.

RESULTS

Population (Table 1)

Table 1
Characteristics of infants enrolled

Eighty-six infants were examined. Male and female infants were equally distributed. Duration of breast feeding ranged from 0.5 to 12 months.

90K in human milk

90K was detectable in all milk samples. The level of 90K ranged from 8.5 to 643.8 μg/ml (mean ± s.d., 115.5 ± 125.8 μg/ml).

Occurrence of ARI

At 1 year old, 45 children (52%) had a history of ARI: 16 (19%) had one episode and 29 (34%) had two or more episodes. The mean number of episodes of ARI was 2.2 ± 1.2 with a maximum of five episodes (Table 1). Forty-one children (48%) never had ARI.

Human milk 90K and episodes of ARI

The level of human milk 90K was inversely related to episodes of ARI (r = − 0.34, P = 0.001; Fig. 1). There was no difference in average duration of breast feeding between infants who did and infants who did not develop ARI (4.7 ± 3.3 months and 5.1 ± 3.1 months, respectively). However, the average 90K levels in milk fed to infants without ARI were 156.6 ± 144.8 compared with 70.9 ± 92.3 (P = 0.001) in milk fed to infants who developed ARI. No significant difference in the timing of milk collection was observed between the ARI and non-ARI group (30.1 ± 19.1 h versus 26.3 ± 20.2 h post-birth, P = NS).

Fig. 1
Relationship between number of episodes of acute respiratory infection (ARI) and level of human milk 90K. Spearman's regression coefficient was used to determine the P value.

Day care attendance, parental tobacco smoking, or family crowding were not different among the two groups (Table 2).

Table 2
Characteristics of infants with and without acute respiratory infection (ARI)

DISCUSSION

In the present study we found that children fed human milk containing high levels of 90K suffered from ARI less frequently than children consuming milk with low 90K levels. These data suggest that 90K in human milk may be protective against ARI. The protective effect seems to be related to the amount of 90K ingested during the first few days, as subsequently milk 90K concentrations rapidly decline in all mothers [18]. Thus, 90K may act by priming some defence mechanism in the infant early in life.

Although the protective value of human milk against infant ARI has been well recognized, the exact nature of such a protection is not well understood. Two general mechanisms have been proposed to explain the manner by which human milk may protect infants from infections. One is the interaction between specific constituents in milk and epithelial surfaces or specific substances in the gastrointestinal lumen during digestion and absorption of the milk [2123]. The other mechanism is the possible modulation of the infant immune system by bioactive substances in the milk, which results in selective production of immune factors by the infant [6,24]. Our hypothesis is that 90K belongs to this last class of substances. In support of this hypothesis are the findings of previous studies showing that 90K enhances the generation of cytotoxic effector cells (natural killer (NK) and lymphokine-activated killer (LAK)) [14] as well as the secretion of IL-1, IL-6 and tumour necrosis factor-alpha (TNF-α) from peripheral blood mononuclear cells [25]. In addition, it was found in a previous study that 90K at low concentrations also has a stimulatory effect on monocytes [26]. 90K in milk may therefore play a defensive role by stimulating the production of cytokines from mononuclear cells present in the milk [27,28] which in turn may act on the oropharyngeal and mucosal associated lymphoid tissues. From these sites, activated lymphocytes and monocytes can influence other distant lymphoid and non-lymphoid tissues, thus inducing immunostimulation.

In conclusion, we suggest that milk 90K may possess a beneficial effect on the neonate by affording protection against ARI. Once this role is firmly established, it will be possible to evaluate the clinical usefulness of 90K supplements as a therapeutic agent to prevent ARI, at least in high-risk infants.

Acknowledgments

This work was supported in part from a grant from Associazione Italiana per la Ricerca sul Cancro (A.I.R.C.).

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