Immune profiling of breast milk from mothers with treated celiac disease.

BACKGROUND: The protective effect of breastfeeding on celiac disease (CD) onset is controversial. We studied a wide range of milk components in milk produced by celiac mothers following long-term gluten-free diet (GFD) in comparison to milk produced by healthy mothers. METHODS: Breast-milk samples from celiac (n = 33) and healthy (n = 41) mothers were obtained during the first year of lactation. A panel of bioactive components was analyzed by enzyme-linked immunosorbent assay in the aqueous fraction. We studied molecules involved in defenses, immunoregulation, and strengthening of the gut-epithelial barrier. RESULTS: During late lactation (from 6 to 12 months after delivery), the content of total immunoglobulin A (IgA) and IgM was significantly lower in the milk produced by celiac patients. Nevertheless, gliadin (GFD)-specific IgA relative contribution was higher in this group, in contrast to tetanus toxoid-specific antibodies. The balance between pro-inflammatory and anti-inflammatory molecules was different. While interleukin-6, tumor necrosis factor-α, and monocyte chemoattractant protein-1 were most frequently found in samples from celiac mothers, soluble Toll-like receptor-2 prevalence was lower. CONCLUSIONS: We describe differences between the innate and adaptive immune profile of milk produced by celiac and healthy mothers. These results might explain previous controversial reports about breastfeeding and CD protection. IMPACT: In spite of a long-term adherence to GFD, the milk produced by mothers with CD exhibit a different immune profile, in relation with some immunoregulatory factors and antibody content. This work shows a more comprehensive characterization of milk from celiac mothers, including macronutrients, lysozymes, growth factors, and immunoregulatory components that had not been studied before. The present study widens the available data regarding the characteristics of human milk of celiac mothers following GFD. Further follow-up studies of the health of children who were breastfed by celiac mothers will be necessary in order to also estimate the impact of the present results therein.

Impact of Holder pasteurization on immunological properties of human breast milk over the first year of lactation.

BACKGROUND: The timing of milk donations to human milk banks ranges from a few days to more than 1 year after delivery, and the Holder method is used for pasteurization. We evaluated the effect of temporal variation and thermal treatment on the immunological properties of milk. METHODS: We analyzed 73 milk samples, raw and after pasteurization, donated at different lactation stages. We studied antibodies, lysozyme, cytokines, soluble receptors, and factors with impact on barrier function. We also evaluated in vitro the capacity of milk to modulate nuclear factor-κB (NF-κB) signaling in an HT-29 epithelial cell line stimulated with tumor necrosis factor-α (TNF-α). RESULTS: With few exceptions, immune components exhibited their highest levels in colostrum, and were stable in the various stages of mature milk. Pasteurization altered the immunological composition of milk, and very drastically for some components. Raw milk of the first year reduced NF-κB activation in HT-29 cells treated with TNF-α to approximately the same extent, and Holder pasteurization significantly affected this capacity. CONCLUSIONS: Overall, the present work reports that mature donated milk is equally valuable over the first year of lactation, but warns about drastic losses of anti-inflammatory properties during Holder pasteurization that could be critical for the health of preterm infants.

Enamel organ proteins as targets for antibodies in celiac disease: implications for oral health.

Enamel defects in permanent and deciduous teeth may be oral manifestations of celiac disease. Sometimes they are the only sign that points to this underdiagnosed autoimmune pathology. However, the etiology of these specific enamel defects remains unknown. Based on previously reported cross-reactivity of antibodies to gliadin with the enamel proteins, amelogenin and ameloblastin, we analyzed (using immunohistochemistry) the ability of anti-gliadin IgG, produced during untreated disease, to recognize enamel organ structures. We used swine germ teeth as a tissue model because they are highly homologous to human teeth in terms of proteins and development biology. Strong staining of the enamel matrix and of the layer of ameloblasts was observed with serum samples from women with celiac disease; high IgG reactivity was found against both gliadin peptides and enamel matrix protein extract, but there was no IgG reactivity against tissue antigens. In line with these findings, the gamma globulin fraction from gliadin-immunized BALB/c mice showed a similar staining pattern to that of amelogenin-specific staining. These results strongly suggest a pathological role for antibodies to gliadin in enamel defect dentition for both deciduous and permanent teeth, considering that IgG can be transported through the placenta during fetal tooth development.

Acute and chronic sleep restriction differentially modify maternal behavior and milk macronutrient composition in the postpartum rat.

BACKGROUNDS: Sleep restriction is considered a stressful condition itself, causing a wide variety of physiological alterations, from cognitive and hormonal to immunological status. In addition, it is established that stress in mother rats can modify milk ejection, milk composition, and maternal care of the pups. Also, sleep disturbances during the early stages of motherhood are a common feature of all studied species. In this context, while the impacts of sleep disruption in non-lactating animals were extensively investigated, its repercussions during the initial phases of motherhood have been poorly explored. Therefore, we wonder if maternal behavior, milk ejection and its macronutrient composition would be disrupted when mother rats are subjected to an additional acute or chronic sleep restriction to the already existing sleep disturbances. METHODS: Lactating rats were implanted with unilateral electrodes for polysomnographic recordings and for deep brain electrical stimulation into mesopontine waking-promoting area (for sleep deprivation). During the early postpartum period (postpartum day 5-9), mother rats were randomly assigned into one of three groups: chronic sleep restriction group (CSR; 6 h of sleep deprivation/day for five consecutive days), acute sleep restriction group (ASR; 6 h of sleep deprivation only for one day), or undisturbed group (control group). Active maternal behaviors (retrievals of the pups into the nest, mouthing, lickings [corporal and anogenital] and sniffing the pups) and passive maternal behaviors (kyphotic and supine nursing postures) were evaluated during a 30 min period without sleep restriction immediately after the sleep restriction or control period. The litter weight gain was assessed every day, and on the last experimental session mothers were milked for posterior macronutrients analysis (protein, carbohydrates and fat). RESULTS: When compared to control group, CSR decreased the amount of milk ejected in the middle days of the sleep restriction period, while ASR did not affect this parameter. Moreover, ASR reduced milk protein content compared to control and CSR groups. Finally, compared to the control group, CSR reduced active maternal behaviors towards the end of the treatment days. CONCLUSIONS: We demonstrated that not only acute but also chronic sleep restriction impacts on the postpartum period, each one affecting different aspects of maternal behavior and lactation. Our results suggest the existence of a homeostatic recovery mechanism in breastfeeding during CSR, possibly ensuring the survival of the litter, while the decline in active maternal behaviors appears to be cumulative.

Hyperthyroidism keeps immunoglobulin levels but reduces milk fat and CD11b/c+ cells on early lactation.

Thyroid hormones influence mammary gland differentiation and lactation by binding to thyroid hormone receptors. Hyperthyroidism disrupts pregnancy and lactation, affecting offspring growth and milk production. Despite maternal milk is a vital source of bioactive compounds and nutrients for newborns, it is unclear whether hyperthyroidism alters its composition, mainly immune factors. Therefore, our work aimed to evaluate the influence of hyperthyroidism on milk quality and immunological parameters during early lactation. Twelve-week-old female Wistar rats received daily injections of 0,25 mg/kg T4 (HyperT, n = 20) or vehicle (control, n = 19) starting 8 days before mating and continuing throughout pregnancy. Rats were euthanized on day 2 of lactation for analyzing the impact of hyperthyroidism on mammary gland, serum and milk samples. HyperT pups exhibited reduced weight, length and head circumference with altered serum hormones, glucose and albumin levels. HyperT mammary gland analysis revealed structural changes, including decreased alveolar area, adipose tissue, increased connective tissue and reduced epithelial elongation, accompanied by decreased TRß1 RNA expression. HyperT milk displayed lower caloric value and fat concentration. HyperT animals exhibited altered milk immune cell counts, displaying increased numbers of CD45+ and CD3+ cells and decreased CD11b/c+ cells without changes on milk and serum IgA, IgG and IgG2a levels. In summary, we have demonstrated that hyperthyroidism affects mammary gland morphology, disrupts pup development and alters biochemical and immunological parameters. Our findings highlight the impact of maternal hyperthyroidism on offspring early development and milk immune composition, underscoring the importance of thyroid function in maternal and neonatal immune health.

Study of tissue transglutaminase spliced variants expressed in THP-1 derived macrophages exhibiting distinct functional phenotypes.

Tissue transglutaminase (TG2) expressed in monocytes and macrophage is known to participate in processes during either early and resolution stages of inflammation. The alternative splicing of tissue transglutaminase gene is a mechanism that increases its functional diversity. Four spliced variants are known with truncated C-terminal domains (TGM2_v2, TGM2_v3, TGM2_v4a, TGM2_v4b) but scarce information is available about its expression in human monocyte and macrophages. We studied the expression of canonical TG2 (TGM2_v1) and its short spliced variants by RT-PCR during differentiation of TPH-1 derived macrophages (dTHP-1) using two protocols (condition I and II) that differ in Phorbol-12-myristate-13-acetate dose and time schedule. The production of TNF-α and IL-1ß in supernatant of dTHP-1, measured by ELISA in supernatants showed higher proinflammatory milieu in condition I. We found that the expression of all mRNA TG2 spliced variants were up-regulated during macrophage differentiation and after IFN-γ treatment of dTHP-1 cells in both conditions. Nevertheless, the relative fold increase or TGM2_v3 in relation with TGM2_v1 was higher only with the condition I. M1/M2-like THP-1 macrophages obtained with IFN-γ/IL-4 treatments showed that the up-regulation of TGM2_v1 induced by IL-4 was higher in relation with any short spliced variants. The qualitative profile of relative contribution of spliced variants in M1/M2-like THP-1 cells showed a trend to higher expression of TGM2_v3 in the inflammatory functional phenotype. Our results contribute to the knowledge about TG2 spliced variants in the biology of monocyte/macrophage cells and show how the differentiation conditions can alter their expression and cell function.

Effects of human donor milk on gut barrier function and inflammation: in vitro study of the beneficial properties to the newborn.

Introduction: The gastrointestinal and immune systems of premature infants are not fully developed, rendering them more vulnerable to severe complications like necrotizing enterocolitis. Human milk offers a rich array of bioactive factors that collectively contribute to reducing the incidence of gut infections and inflammatory conditions. When a mother's milk is unavailable, preterm infants are often provided with donor human milk processed in Human Milk Banks. However, it remains uncertain whether pasteurized milk confers the same level of risk reduction as unprocessed milk. This uncertainty may stem from the well-documented adverse effects of heat treatment on milk composition. Yet, our understanding of the comprehensive impact on protective mechanisms is limited. Methods: In this study, we conducted a comparative analysis of the effects of raw versus pasteurized milk and colostrum versus mature milk on cellular functions associated with the gut epithelial barrier and responses to inflammatory stimuli. We utilized THP-1 and HT-29 cell lines, representing monocyte/macrophages and gut epithelial cells, respectively. Results: Our observations revealed that all milk types stimulated epithelial cell proliferation. However, only raw colostrum increased cell migration and interfered with the interaction between E. coli and epithelial cells. Furthermore, the response of epithelial and macrophage cells to lipopolysaccharide (LPS) was enhanced solely by raw colostrum, with a milder effect observed with mature milk. In contrast, both raw and pasteurized milk diminished the LPS induced response in monocytes. Lastly, we examined how milk affected the differentiation of monocytes into macrophages, finding that milk reduced the subsequent inflammatory response of macrophages to LPS. Discussion: Our study sheds light on the impact of human milk on certain mechanisms that potentially account for its protective effects against necrotizing enterocolitis, highlighting the detrimental influence of pasteurization on some of these mechanisms. Our findings emphasize the urgency of developing alternative pasteurization methods to better preserve milk properties. Moreover, identifying the key components critically affected by these protective mechanisms could enable their inclusion in donor milk or formula, thereby enhancing immunological benefits for vulnerable newborns.

Hypoxia and inflammation conditions differentially affect the expression of tissue transglutaminase spliced variants and functional properties of extravillous trophoblast cells.

PROBLEM: Persistent hypoxia and inflammation beyond early pregnancy are involved in a bad outcome because of defective trophoblast invasiveness. Tissue transglutaminase (TG2) coregulates several cell functions. An aberrant expression and/or transamidation activity could contribute to placental dysfunction. METHOD OF STUDY: The first-trimester trophoblast cell line (Swan-71) was used to study TG2 expression and cell functions in the absence or presence of inflammatory cytokines (TNF-α, IL-1ß) or chemical hypoxia (CoCl2 ). We analyzed The concentration of cytokines in the supernatant by ELISA; Cell migration by scratch assay; NF-κB activation by detection of nuclear p65 by immunofluorescence or flow cytometry using a Swan-71 NF-κB-hrGFP reporter cell line. Tissue transglutaminase expression was analyzed by immunoblot and confocal microscopy. Expression of spliced mRNA variants of tissue transglutaminase was analyzed by RT-PCR. Transamidation activity was assessed by flow cytometry using 5-(biotinamido)-pentylamine substrate. RESULTS: Chemical hypoxia and TGase inhibition, but not inflammatory stimuli, decreased Swan-71 migration. IL-6 production was also decreased by chemical hypoxia, but increased by inflammation. Intracellular TGase activity was increased by all stimuli, but NF-κB activation was observed only in the presence of proinflammatory cytokines. TG2 expression was decreased by CoCl2 and TNF-α. Translocation of TG2 and p65 to nuclei was observed only with TNF-α, without colocalization. Differential relative expression of spliced variants of mRNA was observed between CoCl2 and inflammatory stimuli. CONCLUSION: The observed decrease in total TG2 expression and relative increase in short variants under hypoxia conditions could contribute to impaired trophoblast invasion and impact on pregnancy outcome.

Predictive value of blood measurement of Complement System proteins and metabolic components for early detection of obstetric complications linked to poor placental function.

Preeclampsia-eclampsia syndrome (PES) is associated with severe obstetric complications and there are no efficient methods available for an early detection. We studied blood concentration of some immunological and metabolic markers in association with obstetric outcome in healthy pregnant women and patients with obstetric risk factors, by ELISA and biochemical tests. Patients with complications showed higher levels of CRP and C4 positively correlated with Triglycerides and Cholesterol concentrations. Our results provide evidence that Immunological and metabolic alterations contribute to obstetric complications and that biomarkers linked to these alterations could be useful for an early detection of these problems.

Antibody Profile of Colostrum and the Effect of Processing in Human Milk Banks: Implications in Immunoregulatory Properties.

BACKGROUND: When feeding preterm infants, donor milk is preferred if the mother's own milk is unavailable. Pasteurization may have detrimental effects on bioactivity, but more information is needed about its effects on the immunological compounds. Research aim: This work has two main aims: evaluate the antibody profile of colostrum and study the quantitative variations in the antibodies' level and specific reactivity after undergoing Holder pasteurization. The authors focused on immunoregulatory components of colostrum (antidietary antibodies and TGF-ß2) in the neonatal gut. METHODS: This is a descriptive cross-sectional study of a convenience sample of 67 donated colostrum samples at different days after delivery, both raw and pasteurized. Antibody profiles were analyzed at different times during breastfeeding, and total and specific antibodies (IgM, IgA, and IgG subclasses) were compared with tetanus toxoid and ovalbumin using enzyme-linked immunosorbent assay. The processing effect on total and specific antibodies, as well as TGF-ß2, was evaluated by paired analyses. RESULTS: No variations in immunological compounds were observed throughout the colostrum stage. The TGF-ß2, antibodies' concentrations, and antibodies' specific reactivity after pasteurization did not vary significantly as days of lactation varied. Changes in antibody levels were dependent on isotype and IgG subclass, and IgG4 showed remarkable resistance to heating. Moreover, the effect of the pasteurization on specific reactivity was antigen dependent. CONCLUSION: The supply of relevant immunological components is stable throughout the colostrum stage. The effects of pasteurization on antibodies depend on isotype, subclass, and specificity. This information is relevant to improving the immunological quality of colostrum, especially for preterm newborns.