Survival of Immunoglobulins from Human Milk to Preterm Infant Gastric Samples at 1, 2, and 3 h Postprandial.

BACKGROUND: Human milk immunoglobulins (Ig) are an important support for the naïve infant immune system; yet the extent to which these proteins survive within the infant digestive tract, particularly for preterm infants, is poorly studied. OBJECTIVES: Our objective was to evaluate the survival of human milk Igs in the preterm stomach across postprandial time. METHODS: Human milk and infant gastric samples were collected from 11 preterm (23-32 weeks gestational age) mother-infant pairs within 7-98 days postnatal age. Preterm gastric samples were collected 1, 2, and 3 h after the beginning of the feeding. Samples were analyzed for concentration of total IgA (secretory IgA [SIgA]/IgA), total secretory component (SC/SIgA/SIgM), total IgM (SIgM/IgM), and IgG via enzyme-linked immunosorbent assay. Ig-chain fragment peptides were determined using peptidomic analysis. One-way analysis of variance with repeated measures followed by Tukey's multiple comparison tests was applied. RESULTS: Concentrations of total IgA were lower in the gastric contents at 3 h postprandial compared with human milk and gastric contents at 1 and 2 h. Human milk SC/SIgA/SIgM, IgG, and total IgM concentrations remained stable in the preterm stomach across postprandial time. Peptide counts from the Ig alpha-chain and the Ig gamma-chain increased in gastric contents from 1 to 2 h postprandial. Peptide counts from the human milk Ig-chain, Ig-chain, and SC were stable across postprandial time. These peptides from Ig-chains were not present in human milk but were released in the stomach due to their partial degradation. CONCLUSIONS: Human milk total SC (SIgA/SC/SIgM), total IgM, and IgG survived mostly intact through the preterm infant stomach, while total IgA was -partially digested.

Comparison of Human Milk Immunoglobulin Survival during Gastric Digestion between Preterm and Term Infants.

Human milk provides immunoglobulins (Igs) that supplement the passive immune system of neonates; however, the extent of survival of these Igs during gastric digestion and whether this differs between preterm and term infants remains unknown. Human milk, and infant gastric samples at 2 h post-ingestion were collected from 15 preterm (23⁻32 week gestational age (GA)) mother-infant pairs and from 8 term (38⁻40 week of GA) mother-infant pairs within 7⁻98 days postnatal age. Samples were analyzed via ELISA for concentration of total IgA (secretory IgA (SIgA)/IgA), total secretory component (SC/SIgA/SIgM), total IgM (SIgM/IgM), and IgG as well as peptidomics. Total IgA concentration decreased by 60% from human milk to the preterm infant stomach and decreased by 48% in the term infant stomach. Total IgM and IgG concentrations decreased by 33% and 77%, respectively, from human milk to the term infant stomach but were stable in the preterm infant stomach. Release of peptides from all Ig isotypes in the term infant stomach was higher than in the preterm stomach. Overall, the stability of human milk Igs during gastric digestion is higher in preterm infant than in term infants, which could be beneficial for assisting the preterm infants' immature immune system.

Saliva electrophoretic protein profiles in infants: changes with age and impact of teeth eruption and diet transition.

OBJECTIVE: The objective of this study was to describe the changes in salivary protein profiles in infants between the ages of 3 and 6 months, and to evaluate the impact of teeth eruption and introduction of solid foods on such profiles. DESIGN: 73 infants were followed longitudinally at 3 and 6 months of age. Their whole saliva proteins were separated by SDS-PAGE electrophoresis and semi-quantified by image analysis. Amylase activity was also measured on a sub-sample of the population (n=42 infants). Bands which abundance was significantly different between the two ages according to paired comparisons were identified by mass spectrometry techniques. RESULTS: Out of 21 bands, 13 were significantly different between 3 and 6 months of age. Two short variants of amylase increased in abundance with age, as did amylase activity. Other changes possibly translated developmental physiological events, for example maturation of the adaptive immune system. The balance between S-type cystatins and cystatins A and B was modified, in favour of S-type cystatins at 6 months of age. Teeth eruption resulted in an increase in albumin abundance, whilst introduction of solid foods was associated with higher levels of ß-2 microglobulin and S-type cystatins. CONCLUSIONS: Salivary profiles were modified substantially between the ages of 3 and 6 months. Both teeth eruption and diet had an impact on abundance changes for some proteins, revealing dynamic interactions between saliva proteome, oral physiology and diet.

Isolation of tracheal aspirate mesenchymal stromal cells predicts bronchopulmonary dysplasia.

BACKGROUND: We have isolated mesenchymal stem cells (MSCs) from tracheal aspirates of premature infants with respiratory distress. Under the influence of transforming growth factor ß, MSCs differentiate into α-smooth-muscle actin-expressing myofibroblasts. Myofibroblasts are increased in the lungs of patients with bronchopulmonary dysplasia (BPD), a chronic lung disease of prematurely born infants. OBJECTIVE: We tested whether isolation of MSCs from tracheal aspirates of premature infants with respiratory distress during the first week of life correlates with BPD. METHODS: Eighty-four infants born at a gestational age of <33 weeks and requiring mechanical ventilation were studied. Aspirates were collected during suctioning and centrifuged. Cell pellets were resuspended in culture medium and plated. Adherent cells were grown to confluence. RESULTS: MSCs were isolated from the tracheal aspirates of 56 infants; 28 aspirate samples showed no MSCs. There was no statistical difference in gestational age or birth weight between the MSC and no-MSC groups. In the MSC group, 12 infants died and 25 developed BPD, as defined by a requirement for supplemental oxygen at 36 weeks' postmenstrual age. In the no-MSC group, 6 infants died and 1 developed BPD. Accounting for potential influences of gender, birth weight, gestational age, number of tracheal aspirate samples taken, and the duration of endotracheal intubation (up to 7 days), isolation of MSCs increased the adjusted odds ratio of BPD more than 21-fold (95% confidence interval: 1.82-265.85). CONCLUSIONS: Isolation of tracheal aspirate MSCs predicts the development of BPD, which suggests that MSCs play an important role in the pathogenesis of this disease.

The pulpal origin of immunoglobulins in dentin beneath caries: an immunohistochemical study.

Immunoglobulins localized in uninfected dentin beneath caries are thought to be protective, but their origin remains controversial. We reasoned that the localization and dominance of serum IgG1 would support the pulpal origin of the immunoglobulins while a predominance of secretory component (SC) bearing IgA1 and IgA2 would support their salivary origin. The prevalence and staining intensity of IgG1, IgA1, IgA2, IgM, and SC in uninfected dentinal tubules beneath shallow, deep caries, and noncaries teeth were examined immunohistologically. SC was only localized in caries, and IgG1 was the predominant subclass in uninfected dentinal tubules beneath shallow and deep caries, followed by IgA1. In noncaries teeth, IgG1 was localized on the pulpal end. The intensity of IgG1 was significantly higher than either IgA1 or IgA2 in both shallow and deep caries. Our data support the serum origin of immunoglobulins in uninfected dentin beneath caries.

Secretory immune system in human embryonic and fetal development: joining chain and immunoglobulin transport (Review).

The role of joining (J) chain, one of the protein components of the secretory immune system (SIS), in the immune reactions of the human embryo and fetus was analyzed on the basis of data from the literature and our previous studies. All organs and structures, including extra-corporeal ones, of 18 embryos (4-8 weeks of development) and 45 fetuses (9-38 weeks) were studied using methods of pathomorphology, immunohistochemistry and morphometry. This approach enabled us to analyze the problem in the whole organism throughout its embryonic and fetal development. J chain, as well as polymeric immunoglobulin (Ig) receptor-secretory component (pIgR/SC) and Igs, are already widely distributed in 4-week-old embryos before the appearance of the common immune system. The whole complex of protein components of the SIS was seen in mucous layers, and in blood-tissue and tissue-tissue barrier structures. Therefore, we can consider two parts of the SIS: mucosal and barrier. Already in embryos, an increase in the functional activity of the SIS following massive antigenic attack in cases of acute chorioamnionitis reflects the increased exocrine secretion of Igs. The J chain appears to participate in the endocytosis but not exocytosis of Igs. J chain and Igs, but not pIgR/SC, were present in cells of the heart, endocrine glands, gonads and some other organs. The exocrine secretion of Igs, the main function of the SIS, is absent in these organs, and, they are therefore, not considered part of the SIS.

Free secretory component from cystic fibrosis sputa displays the cystic fibrosis glycosylation phenotype.

Secretory IgA contributes to humoral defense mechanisms against pathogens targeting mucosal surfaces, and secretory component (SC) fulfills multiple roles in this defense. The aims of this study were to quantify total SC and to analyze the form of free SC in sputa from normal subjects, subjects with asthma, and subjects with cystic fibrosis (CF). Significantly higher levels of SC were detected in CF compared with both other groups. Gel filtration chromatography revealed that SC in CF was relatively degraded. Free SC normally binds interleukin (IL)-8 and inhibits its function. However, in CF sputa, IL-8 binding to intact SC was reduced. Analysis of the total carbohydrate content of free SC signified overglycosylation in CF compared with normal subjects and subjects with asthma. Monosaccharide composition analysis of free SC from CF subjects revealed overfucosylation and undersialylation, in agreement with the reported CF glycosylation phenotype. SC binding to IL-8 did not interfere with the binding of IL-8 to heparin, indicating distinct binding sites on IL-8 for negative regulation of function by SC and heparin. We suggest that defective structure and function of SC contribute to the characteristic sustained inflammatory response in the CF airways.

Decreased excretion of antimicrobial proteins and peptides in saliva of patients with oral candidiasis.

BACKGROUND: Antimicrobial peptides in saliva appear to play a crucial role in the regulation of oral Candida growth, and study on antimicrobial excretion in saliva and oral candidiasis appears useful for the analysis of pathophysiology of oral candidiasis. METHODS: To clarify the role of saliva in the regulation of oral Candida growth, the levels of antimicrobial proteins and peptides and their excretion rates were examined in saliva obtained from 50 patients with oral candidiasis and 35 healthy individuals. RESULTS: The inhibitory activities of patients' saliva against Candida adhesion with HeLa cells and against Candida growth (radiolabeled glucose incorporation) were lower than those of saliva from the healthy controls. The salivary levels of lactoferrin (Lf; 11 +/- 9 microg/ml), secretory immunoglobulin A (sIgA; 160 +/- 37 microg/ml), beta-defensin 1 (375 +/- 37 ng/ml), and beta-defensin 2 (412 +/- 51 ng/ml) in the patients were largely lower than those in the control group (33 +/- 14 microg/ml, 204 +/- 51 microg/ml, 452 +/- 89 ng/ml, and 530 +/- 142 ng/ml, respectively), although the transferrin (Tf) and secretory component (SC) levels were almost same in both groups, and alpha-defensin 1 was slightly increased in the patient group (660 +/- 115 ng/ml vs. 467 +/- 168 ng/ml). In addition, the excretion rates of the proteins and peptides were largely decreased in the patients (Tf: 14 +/- 2 microg/10 min vs. 34 +/- 7 microg/10 min; Lf: 18 +/- 11 microg/10 min vs. 139 +/- 43 microg/10 min; sIgA: 300 +/- 132 microg/10 min vs. 900 +/- 207 microg/10 min; SC: 112 +/- 46 microg/10 min vs. 292 +/- 64 microg/10 min; alpha-defensin 1: 1223 +/- 431 ng/10 min vs. 2044 +/- 612 ng/10 min; beta-defensin 1: 687 +/- 243 ng/10 min vs. 1985 +/- 295 ng/10 min; and beta-defensin 2: 784 +/- 299 ng/10 min vs. 2288 +/- 278 ng/10 min). CONCLUSION: These results conclusively suggest that oral candidiasis is associated with salivary gland hypofunction and that decreases of salivary antibacterial proteins induce Candida overgrowth.

An immunohistochemical study of the secretory immune system in human fetal membranes and decidua of the first trimester of pregnancy.

PROBLEM: We analyzed the presence and distribution of components of the secretory immune system (SIS) in human fetal membranes (amnion, yolk sac, chorion) and decidua from the first trimester of pregnancy. MATERIALS AND METHODS: Specimens from 17 embryos (4-8 weeks of pregnancy) and nine fetuses (9-12 weeks) were divided into those that had not been exposed to massive foreign antigenic effects (Group I, n = 18) and those that had suffered acute chorioamnionitis (Group II, n = 8). RESULTS: Positive immunostaining for secretory component (SC), joining (J) chain, IgG, IgA, and macrophages was seen from 4 to 5 weeks of development and then during the whole first trimester of pregnancy in the syncytio- and cytotrophoblasts, the amniotic epithelium, the yolk sac endoderm and decidual cells. Macrophages with J chain, IgG and IgA were found in embryonic tissues on week 4, whereas lymphocytes, including those synthesizing IgA and IgM, appeared only at the end of the first trimester of pregnancy. In the decidua, lymphocytes and macrophages were recognized during the whole period of study. In cases with chorioamnionitis (Group II), reactivity of IgG and IgA in the mentioned cells of fetuses decreased sharply while the rate of immunoreactivity of SC and J chain as well as the number of T and B lymphocytes did not change. In the decidua, the number of immune reactive cells sharply increased with the appearance of plasma cells. CONCLUSIONS: In the fetal membranes and decidua all the SIS components are present. We suggest that two SIS, maternal and fetal, participate in the formation of the barrier between a mother and the fetus. Both these systems have different origin and cellular content as well as different immune reactions.

Secretory component, J chain, and immunoglobulins in human embryos and fetuses of the first trimester of pregnancy: immunohistochemical study.

In our previous studies, we described the development of the secretory (mucosal) immune system (SIS) in human fetuses in the second trimester of pregnancy. In the present study, we examined the presence and distribution of components of this system in human embryos and early fetuses in the first trimester. An immunohistochemical study was performed on 17 embryos and 9 fetuses (4 to 12 wk of development) using antibodies against secretory component (SC), joining (J) chain, immunoglobulins (IgA, IgM, IgG), subsets of T and B lymphocytes, and macrophages. Cells positive for SC, J chain, and IgG were found in epithelial tissues from wk 4 of pregnancy. In the internal organs, such as the myocardium and endocardium, capillary endothelium, epithelium of the kidney tubules and some others, only J chain and immunoglobulins were seen. IgA was weakly reactive in tissues where SC and/or J chain were presented. IgM was very weak or absent. Among the cellular components of the SIS, only macrophages were seen in 4-wk-old embryos. CD3+ and CD20+ lymphocytes were found at wk 7 to 8. IgA- and IgM-positive lymphocytes appeared at the end of wk 9. The SIS is widespread in embryonic and early fetal periods and begins to function before the appearance of the common immune system in the developing organism. The first functional components of the SIS, such as IgG and IgA observed in this study, are most probably of maternal origin.

Immunohistochemical expression of vimentin and secretory component antigens in endometrial hyperplasia and neoplasia.

Vimentin is an intermediate filament protein normally expressed in mesenchymal cells, but evidence is accumulating in the literature which suggests that the aberrant expression of vimentin in epithelial cancer cells might be related to local invasiveness and metastatic potential. Previous studies strongly support the implication of vimentin in the metastatic progression of breast and cervical lesions. The secretory component is isolated from human colostrum and is of help in more precise grading of endometrial carcinoma. In this study we examined vimentin and secretory component (SC) expression in adenomatous hyperplasia, atypical adenomatous hyperplasia and well-differentiated adenocarcinoma (cribriform pattern). The results showed decreased expression of vimentin and increased expression of the secretory component as the lesion progressed to malignancy.

The distribution of secretory immunoglobulin A in the liver of patients with hepatolithiasis.

Patients with hepatolithiasis are known to be complicated with bile stasis and bacterial infections in the stone-containing ducts. It has been suspected that a decrease in portal venous flow is important in the progression of hepatolithiasis. This study was done to find if the affected liver in hepatolithiasis is inflamed or has lowered local immunity by an examination of the distribution of secretory immunoglobulin A and proliferating cell nuclear antigen in the intrahepatic biliary tracts in operative specimens of 36 patients with hepatolithiasis. The number of biliary epithelia stained for the immunoglobulin was greater when the cholangitis was more severe up to a point; in advanced cholangitis, with severe parenchymal atrophy or proliferating epithelia, there were fewer cells stained for the immunoglobulin than in mild cholangitis. In hepatolithiasis, secretory immunoglobulin A decreases when inflammatory changes become severe and there is parenchymal atrophy caused by stenosis or obstruction of portal branch.

Lymphoid-epithelial secretory immune system in human fetuses in the second trimester of gestation.

The development of the secretory immune system (SIS) in the respiratory, digestive, and urogenital tracts and other organs of fetuses in the second trimester of gestation is described. Tissues of all internal organs of human fetuses (n = 36) that had died between 13 and 25 weeks of gestation were studied immunohistochemically for the presence of secretory component (SC), J chain, IgA, IgM, IgG, macrophages, and different subsets of lymphocytes. We found protein elements of the SIS in fetuses during the entire second trimester in the epithelium of the digestive, respiratory, and urinary tracts; in hepatocytes; in the epithelium of the bile duct, renal tubules, and all the urinary tract; in the salivary glands, pancreas, and thyroid; in the epithelium of the Fallopian tubes and uterus; in the epididymis and the rete testes; in the skin; and in other organs. Immunocompetent cells, including IgA- and IgM-secreting cells, were located in these organs under the epithelium and sometimes between epithelial cells. In fetuses with acute infection, the number of immunocompetent cells was higher, reflecting a whole-immune system reaction, including the SIS. We conclude that the fetal SIS is a ramified, defensive immune system that is distributed throughout most organs of epithelial origin in second-trimester fetuses, and that it reacts against intrafetal infiltration by foreign antigens.

The secretory immune system as part of the placental barrier in the second trimester of pregnancy in humans.

The objective of this study was to describe the development of the secretory immune system (SIS) in the placenta of 32 human fetuses who had died from different causes during the second trimester of pregnancy. The distribution of SIS protein elements, including the secretory component (SC), J-chain, IgA, IgM, IgG, as well subsets of lymphocytes and macrophages, were studied by immunohistochemical methods. Both the fetal and maternal parts of the placenta were found to contain these elements. In the fetal part, the immunoglobulins, SC and J chain were located in the syncytio- and cytotrophoblast of the chorion and in the epithelium of the amnion. The villous stroma contained a small amount of different subsets of lymphocytes. Macrophages accounted for up to 45% of the stromal cells of the villi and contained IgG and J-chain. In the maternal part of the placenta, the SIS proteins were in the decidual cells. Relatively few lymphocytes and macrophages were observed in the decidual stroma. Our data suggest that the placenta has two different SIS, one in the fetal part and the other in the maternal part. They differ in their structure and orientation: the maternal SIS protects the mother against paternal antigens from the fetus, while the fetal SIS protects the fetus against macromolecules and infectious agents penetrating from the mother. The placenta represents the largest extracorporal immune system that is functionally active during the whole second trimester of gestation. We suggest that the concept of placental barrier should be expanded to include both fetal and maternal parts of the SIS, fetal membranes and the decidual tissue.

Lymphocyte homing and Ig secretion in the murine mammary gland.

In mice the majority of the immunoglobulins (Ig) in milk belongs to the IgA class. Prior to its transepithelial transportation into the milk, dimeric IgA (dIgA) is bound to the transmembrane form of the secretory component or polymeric Ig receptor (SC/pIgR). The latter is synthesized in the epithelial cells lining the ducts and alveoli of the mammary gland. A candidate for playing the role of adhesion molecule to primed lymphocytes present in the murine mammary gland might be the mucosal addressin cell adhesion molecule-1 (MAdCAM-1). We studied the correlation between the levels of IgA in colostrum and milk, the number of IgA producing plasma cells in the mammary gland and the expression of MAdCAM-1 in mammary gland endothelial cells during pregnancy and lactation. The relation between the IgA levels in the milk and the expression levels of pIgR in mammary gland epithelial cells was also investigated. We found that the expression of MAdCAM-1 and pIgR starts in early-mid pregnancy; the number of IgA-producing plasma cells and the IgA concentration in milk increase from early lactation onwards. The MAdCAM-1 expression declines during lactation whereas the pIgR levels and IgA-producing plasma cell numbers rise until the end of lactation. Because the MAdCAM-1 level starts to rise several days before the rise of the IgA-producing plasma cell level, MAdCAM-1 cannot be the rate determining factor governing extravasation of primed B cells to the mammary gland. We also conclude that the pIgR is present in sufficient amounts to enable increasing S-IgA secretion into the milk during lactation.

Chewing stimulates secretion of human salivary secretory immunoglobulin A.

Immunoglobulin A (IgA) is the most abundant immunoglobulin in saliva and other mucosal secretions and plays an important role in mucosal immunity. The present study examined whether secretion of IgA, like other salivary proteins, is increased by reflex stimulation. Parotid saliva was collected from subjects into separate vials under resting conditions and during chewing-stimulated secretion over 45 min. Enzyme-linked immunosorbent assay (ELISA) indicated that chewing increased IgA secretion. The extent and pattern of the increase were similar to those of total protein and acinar cell amylase. SDS gel electrophoresis and Western blotting showed that high-molecular-weight forms of IgA-containing secretory component predominated in all saliva samples. Secretory component, the cleaved epithelial receptor for polymeric IgA, was secreted in a pattern very similar to that of IgA. It is concluded that chewing stimulates epithelial cell transcytosis of IgA and increases secretion of secretory IgA into saliva.

Secretory IgA, free secretory component and IgD in saliva of newborn infants.

AIM: To determine levels of secretory IgA (sIgA), free secretory component (FSC) and IgD in saliva of newborn infants at the age of 1 day and to evaluate the detection patterns, the influence of saliva flow and the relation to serum derived proteins. METHODS: Seventy-three healthy newborn infants were studied. Saliva was obtained from the bottom of the mouth and buccal sulci using a sterile polyethylene tube connected to a syringe. SIgA, FSC, IgD and albumin were measured by radial immunodiffusion. RESULTS: SIgA was detected in 74.0% of all saliva samples, whereas detection rates for FSC and IgD were 94.5% and 75.3%, respectively. Investigation of detection patterns and their relation to saliva flow indicated that secretion of sIgA and FSC into the oral cavity is under similar regulation. Levels of IgD were found to be independent from saliva flow, as well as from concentrations of serum-derived proteins suggesting different regulative mechanisms compared to sIgA and FSC. The flow rate of unstimulated whole saliva in newborn infants was found to be 15 times lower compared to adolescents, emphasizing the role of saliva flow as a limiting factor for secretion of sIgA and FSC. CONCLUSION: SIgA, FSC and IgD can be determined in saliva of newborn infants even in the first day of life. The saliva flow rate has to be considered when evaluating the function and biological relevance of the oral mucosal immune system of newborn infants shortly after birth.

Cigarette smoke decreases the expression of secretory component in human bronchial epithelial cells, in vitro.

Epithelial secretory component (SC) is thought to be essential for immunologic protection of the respiratory tract from viral and bacterial infection, since it transports polymeric IgA from the basolateral to the luminal surface of epithelial cells. We have hypothesized that recurrent infection in airways of cigarette smokers is at least partly a consequence of cigarette smoke-induced downregulation of the expression and/or release of SC from airway epithelial cells, subsequently resulting in decreased transcytosis of secretory IgA to the airway lumen. To test this hypothesis, we have cultured human bronchial epithelial cells (HBEC) from surgical tissues and exposed these for 20 minutes to either air or cigarette smoke. Following exposure to cigarette smoke the HBEC cultures were incubated for a further period of up to 24 h, during which time separate cultures were processed by immunocytochemistry for the presence of SC, in a time-dependent manner. The stained HBEC cultures were evaluated by colour image analysis for the percentage of total cells staining for SC. Exposure to cigarette smoke significantly decreased the percentage of total HBEC staining for secretory component from a baseline value (median and interquartile[IQ]1, IQ3) of 35.9% (26.5, 41.6) to 15.7% (8.2, 25.4; p < 0.05) 1 h after exposure, compared with exposure to air. The percentage of cells staining for secretory component were further reduced to 5.3% (3.3, 6.4; p < 0.01), 6 h after exposure, compared to exposure to air. After incubation for 24 h following exposure to cigarette smoke, there was gross cell damage and the cells were not suitable for immunocytochemical analysis. These results suggest that short-term exposure to cigarette smoke may compromise the immune barrier function of the airway mucosa by decreasing the expression and/or release of epithelial SC, thereby decreasing the transcytosis of IgA necessary for inactivating the microbial pathogens in the airway lumen.