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.

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.

Development of novel fluorescent particles applicable for phagocytosis assays with human macrophages.

Phagocytosis is a fundamental process for removal of pathogens and for clearance of apoptotic cells. The objective of this work was the preparation of fluorescent microspheres by a simple method and the evaluation of its applicability in phagocytosis assays by using different human derived cells, differentiated THP-1 cell line and blood monocytes, with flow cytometry measurements for functionality assays. Our results show that microparticles are efficiently internalised in a non-opsonised form and in dose-dependent manner by both cellular types. Concerning mechanism we determined that tTG-ß3 integrin signaling could be involved in the uptake of these particles.

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.

Tissue transglutaminase on trophoblast cells as a possible target of autoantibodies contributing to pregnancy complications in celiac patients.

PROBLEM: Women with celiac disease (CD) are often affected by atypical presentations of the disease associated with reproductive disorders as a main extra-digestive complaint. Here, we analyzed if autoantibodies against tissue transglutaminase (tTG) in sera from CD patients with reproductive disorders could display direct effects through their interaction with tTG expressed on trophoblast cells and phagocytes inducing tissue damage and interfering in the clearance of trophoblast apoptotic bodies. METHOD OF STUDY: Sera from CD women with reproductive disorders were obtained, and their ability to induce apoptosis of Swan-71 (cytotrophoblast cell line) and to modulate the wound-healing and phagocytes process was tested. RESULTS: Swan-71 cells expressed tTG and CD sera displayed a significant decrease in trophoblast cell migration and a delay in injury healing on trophoblast cells, compared with those observed with control sera. Moreover, CD sera significantly reduced trophoblast cell proliferation and increased apoptosis levels in comparison with those observed in the control sera. Finally, autoantibodies against tTG interfere in the clearance of trophoblast apoptotic bodies through a mechanism involving MFG-E8 (milk fat globulin-EGF factor 8)-tTG binding. CONCLUSION: The anti-tTG antibodies might contribute to trophoblast damage and disrupt the phagocytosis process of apoptotic bodies that could promote a pro-inflammatory microenvironment.

Enamel defects associated with coeliac disease: putative role of antibodies against gliadin in pathogenesis.

Enamel defects in the permanent teeth of patients with coeliac disease (CD) are often reported as an atypical manifestation, sometimes being suggestive of an undiagnosed atypical disease. We proposed to explore the pathogenesis of these oral defects, which are poorly studied. Sequence analyses of proteins from gluten (gliadins) and of proline-rich enamel proteins (amelogenin and ameloblastin) suggested the presence of common antigenic motifs. Therefore, we analyzed, by ELISA and western blotting, the reactivity of sera from patients with CD against gliadin and enamel-derived peptides. Correlation analyses between the levels of specific antibodies against gliadin and enamel derived peptides and inhibition experiments confirmed the presence of cross-reactive antibodies. Immunoblot analysis revealed that the most prominent component in enamel matrix derivative (of approximately 18.6 kDa), identified by an amelogenin-specific antibody, is recognized by sera from patients with CD; in addition, several fractions of pure gliadin were recognized by amelogenin-specific antibody. In agreement, sera from mice immunized with enamel matrix-derived proteins generated antibodies that recognized a peptide (of approximately 21.2 kDa) derived from gliadin. In conclusion, antibodies against gliadin generated in patients with CD can react in vitro with a major enamel protein. The involvement of anti-gliadin serum in the pathogenesis of enamel defects in children with untreated CD can be hypothesized on the basis of these novel results.

Celiac disease and gyneco-obstetrics complications: can serum antibodies modulate tissue transglutaminase functions and contribute to clinical pattern?

PROBLEM: Untreated celiac disease (CD) is often associated with early miscarriages, infertility, and alterations in menstrual cycle. Tissue transglutaminase (tTG) antibodies could be involved by interfering with tTG transamidating activity and/or biological functions mediated by its interaction with fibronectin (FN). METHOD OF STUDY: The correlation between the presence of extra-digestive disorders and the reactivity of sera against tTG-FN and its effects on tTG transamidating activity was analyzed in a group or 50 women with recently diagnosed CD. RESULTS: Heterogeneous behavior was observed among serum samples derived from patients with different complaints, suggesting that differences in fine specificity patterns could condition clinical outcome. Sera from women with gynecological and/or obstetric problems induced significant inhibition of in vitro enzymatic activity in comparison with those without these kinds of disorders. CONCLUSIONS: The significant correlation observed between serum effects and clinical profile suggests a putative involvement of tTG-specific antibodies in gynecological and/or obstetric disorders during active CD.

UDP-N-acetyl-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 6 (ppGalNAc-T6) mRNA as a potential new marker for detection of bone marrow-disseminated breast cancer cells.

The evaluation of disseminated epithelial tumor cells in patients with early stages of breast cancer has generated considerable interest because of its potential association with poor clinical outcome. Considering that O-glycosylation pathways are frequently altered in breast cancer, we performed this work to evaluate the potential usefulness of UDP-N-acetyl-D-galactosamine:polypeptide N-acetylgalactosaminyltransferases (ppGalNAc-Ts) (a family of glycosyltransferases which catalyze the first key step of mucin-type O-glycosylation) to detect disseminated cells in bone marrow samples from patients with operable breast cancer. Using RT-PCR assays, we studied the gene expression of 9 enzymes (ppGalNAc-T1-T9). Among the ppGalNAc-Ts expressed by breast tumors (-T1, -T2, -T3, -T6 and -T7), the best specificity (negative results on all PBMN cell samples from healthy donors) was shown for ppGalNAc-T6. Thus, we selected this enzyme as a target gene for further evaluation. ppGalNAc-T6 mRNA was found in 22/25 (88%) breast cancer samples, in all 3 human breast cancer cell lines evaluated (MCF-7, ZR75-1 and T47D), in 1/30 (3%) PBMN cells and 0/19 bone marrow samples obtained from patients without cancer. Using this method, 22/61 (36%) patients with breast cancer, who underwent curative surgery, showed positive ppGalNAc-T6 mRNA in bone marrow aspirates obtained prior to surgery, including 11/34 patients with stage-I or -II, without histopathological lymph node involvement. In a preliminary follow-up evaluation, 19/61 patients experienced recurrence of the disease. ppGalNAc-T6 was positive in 11/19 (57.9%) of these patients. Interestingly, in the group of patients without lymph node involvement, disease recurrence was observed in 54.5% of patients who showed ppGalNAc-T6 mRNA-positive bone marrow aspirates and only in 4.3% of patients when ppGalNAc-T6 was negative (p = 0.014). These results indicate that ppGalNAc-T6 mRNA could be a specific marker applicable to the molecular diagnosis of breast cancer cells dissemination.

Immunohistochemical analysis of MUC5B apomucin expression in breast cancer and non-malignant breast tissues.

A deregulation of several MUC genes (MUC1, MUC2, MUC3, MUC5AC, and MUC6) was previously demonstrated in breast carcinomas. Considering that recently we found the "non-mammary" MUC5B mRNA in primary breast tumors (Berois et al. 2003), we undertook the present study to evaluate the expression profile of MUC5B protein product in breast tissues, using LUM5B-2 antisera raised against sequences within the non-glycosylated regions of this apomucin. Expression of MUC5B by breast cancer cells was confirmed by immunocytochemistry, in situ hybridization, and Western blot on MCF-7 cancer cells. Using an immunohistochemical procedure, MUC5B apomucin was detected in 34/42 (81%) primary breast tumors, in 13/14 (92.8%) samples of non-malignant breast diseases, in 8/19 (42.1%) samples of normal-appearing breast epithelia adjacent to cancer, and in 0/5 normal control breast samples. The staining pattern of MUC5B was very different when comparing breast cancer cells (cytoplasmic) and non-malignant breast cells (predominantly apical and in the secretory material). We analyzed MUC5B mRNA expression using RT-PCR in bone marrow aspirates from 22/42 patients with breast cancer to compare with MUC5B protein expression in the primary tumors. Good correlation was observed because the six MUC5B-positive bone marrow samples also displayed MUC5B expression in the tumor. Our results show, for the first time at the protein level, that MUC5B apomucin is upregulated in breast cancer. Its characterization could provide new insights about the glycobiology of breast cancer cells.

Clinical evaluation of a panel of mRNA markers in the detection of disseminated tumor cells in patients with operable breast cancer.

Persistent high mortality rates in breast cancer patients, in spite of latest advances in diagnosis and therapy, affirm the necessity of new developments in tumor biology prognostic factors. Immunocytochemical detection of disseminated breast cancer cells in bone marrow has been frequently associated with a decrease in disease-free survival as an independent prognostic factor, but methods based on molecular biology procedures must still be validated. Considering tumor heterogeneity, the multimarker approach has been suggested as a better strategy than individual marker assays. The aim of this work was evaluation of the prognostic value of a multimarker reverse-transcriptase polymerase chain reaction (RT-PCR) assay, associating four mRNA markers for the detection of disseminated breast cancer cells. We compared the prognostic significance of cytokeratin 19 (CK19), carcinoembryonic antigen (CEA), mammaglobin (MG) and the mucin MUC5B mRNA in bone marrow aspirates in the follow-up of 80 operable breast cancer patients. The best prognostic value for clinical outcome was seen for CEA mRNA, not improved for any association with other markers. Unexpectedly, some tumor mRNA in bone marrow correlates with a favorable clinical outcome, especially MUC5B. Therefore, our results suggest that not all epithelial or tumor markers have the same significance in predicting the metastatic potential of disseminated cancer cells. New parameters are needed for the identification of individual patients at high risk of tumor recurrence. Multimarker RT-PCR assays could be a good approach, but they should be performed associating mRNA markers that are able to predict tumor aggressiveness associated with poor outcome and not just epithelial markers, which only indicate the mere presence of tumor cells.

Detection of bone marrow-disseminated breast cancer cells using an RT-PCR assay of MUC5B mRNA.

The evaluation of disseminated epithelial tumor cells in breast cancer patients has generated considerable interest due to its potential association with disease recurrence. Our work was performed to analyze the usefulness of 5 mucin genes expression (MUC2, MUC3, MUC5B, MUC6 and MUC7), using RT-PCR assays, to detect disseminated cancer cells in patients with operable breast cancer. The highest frequencies of positive RT-PCR tests in breast tumor extracts were observed for MUC5B (7/15) and MUC7 (5/12). The best specificity, negative results on all peripheral blood mononuclear (PBMN) cell samples from healthy donors, were shown for MUC2, MUC5B and MUC6 RT-PCR assays. Thus, we selected MUC5B as a target gene for further evaluation. Using a nested RT-PCR, MUC5B mRNA transcripts were detected in 16/31 primary breast tumors (but not in 36 samples of normal PBMN cells) and in the human MCF-7 breast cancer cell line but not in BT20, MDA, T47D and ZR-75 breast cancer cell lines, indicating that MUC5B mRNA is expressed in a population of breast cancer cells. Using this method, 9/46 patients (19.5%) who underwent curative surgery showed positive MUC5B mRNA in bone marrow aspirates obtained prior to surgery, including 5/24 patients (20.8%) with stage I or II breast cancer, without histopathologic lymph node involvement. These results indicate that MUC5B mRNA could be a specific marker applicable to the molecular diagnosis of breast cancer cell dissemination. A comparative evaluation between MUC5B mRNA, cytokeratin 19 (CK19) mRNA and carcinoembryonic antigen (CEA) mRNA in all bone marrow aspirates suggests a putative complementation for molecular detection of disseminated carcinoma cells. Considering that breast cancer is characterized by a great phenotypic heterogeneity, the use of multimarker approach could contribute to tumor cell detection in bone marrow and blood.