Diet Modulates the High Sensitivity to Systemic Infection in Newborn Preterm Pigs.

Background: Preterm infants are born with an immature immune system, limited passive immunity, and are at risk of developing bacteremia and sepsis in the postnatal period. We hypothesized that enteral feeding, with or without added immunoglobulins, improves the clinical response to systemic infection by coagulase negative staphylococci. Methods: Using preterm cesarean delivered pigs as models for preterm infants, we infused live Staphylococcus epidermidis (SE, 5 × 109 colony forming units per kg) systemically 0-3 days after birth across five different experiments. SE infection responses were assessed following different gestational age at birth (preterm vs. term), enteral milk diets (bovine colostrum, infant formula with or without added porcine plasma) and with/without systemic immunoglobulins. Pigs infected with SE were assessed 12-48 h for clinical variables, blood bacteriology, chemistry, hematology, and gut dysfunction (intestinal permeability, necrotizing enterocolitis lesions). Results: Adverse clinical responses and increased mortality were observed in preterm vs. term pigs, when infected with SE just after birth. Feeding bovine colostrum just after birth improved blood SE clearance and clinical status (improved physical activity and intestinal structure, fewer bone marrow bacteria), relative to pigs fed infant formula. A few days later, clinical responses to SE bacteremia (hematology, neutrophil phagocytic capacity, T cell subsets) were less severe, and less affected by different milk diets, with or without added immunoglobulins. Conclusion: Prematurity increases the sensitivity of newborn pigs to SE bacteremia, potentially causing sepsis. Sensitivity to systemic SE infection decreases rapidly in the days after preterm birth. Both age and diet (parenteral nutrition, colostrum, milk, formula) may influence gut inflammation, bacterial translocation and systemic immune development in the days after birth in preterm newborns.

Tendril extract of Cucurbita moschata suppresses NLRP3 inflammasome activation in murine macrophages and human trophoblast cells.

Inflammation is the root cause of many diseases that pose a serious threat to human health. Excessive inflammation can also result in preterm birth or miscarriage in pregnant women. Pumpkin (Cucurbita moschata Duchesne, CMD) is a well-known traditional health food and medicinal herb used in many countries to treat diabetes, obesity, osteoporosis, cancer and other diseases. In this study, we investigated the effects of hot water extract derived from the tendrils of C. moschata Duchesne (TCMD) on NLRP3 inflammasome activation in murine macrophages and human trophoblast cells. The TCMD treatment of LPS-primed bone marrow-derived macrophages (BMDMs) and human trophoblast cells attenuated NLRP3 inflammasome activation induced by inflammasome activators such as ATP, nigericin, and monosodium urate (MSU). TCMD treatment suppressed IL-1ß secretion in a dose-dependent manner, without affecting IL-6 secretion. In addition, TCMD inhibited NLRP3-dependent pyroptosis in BMDMs. TCMD also suppressed the release of mature IL-1ß and activation of cleaved-caspase-1 via limited ASC oligomerization. Furthermore, TCMD significantly inhibited IL-1ß secretion and pyroptotic cell death in human trophoblast cells. These results suggest that TCMD exhibits anti-inflammatory effects mediated via inhibition of NLRP3 inflammasome activation suggesting therapeutic potential against inflammatory diseases, preterm birth, and miscarriage.

Does Caesarean Section or Preterm Delivery Influence TGF-ß2 Concentrations in Human Colostrum?

Human colostrum (HC) is a rich source of immune mediators that play a role in immune defences of a newly born infant. The mediators include transforming growth factor ß (TGF-ß) which exists in three isoforms that regulate cellular homeostasis and inflammation, can induce or suppress immune responses, limit T helper 1 cells (Th1) reactions and stimulate secretory immunoglobulin A (IgA) production. Human milk TGF-ß also decreases apoptosis of intestinal cells and suppresses macrophage cytokine expression. The aim of the study was to determine the concentration of TGF-ß2 in HC obtained from the mothers who delivered vaginally (VD) or by caesarean section (CS), and to compare the concentrations in HC from mothers who delivered at term (TB) or preterm (PB). In this study, 56% of preterm pregnancies were delivered via CS. The concentrations of TGF-ß2 were measured in HC from 299 women who delivered in the 1st Department of Obstetrics and Gynaecology, Medical University of Warsaw: 192 (VD), 107 (CS), 251 (TB), and 48 (PB). The colostrum samples were collected within 5 days post-partum. TGF-ß2 levels in HC were measured by the enzyme-linked immunosorbent assay (ELISA) test with the Quantikine ELISA Kit-Human TGF-ß2 (cat.no. SB250). Statistical significance between groups was calculated by the Student t-test using StatSoft Statistica 13 software. The mean TGF-ß2 concentration in patients who delivered at term or preterm were comparable. The levels of TGF-ß2 in HC were higher after preterm than term being 4648 vs. 3899 ng/mL (p = 0.1244). The delivery via CS was associated with higher HC concentrations of TGF-ß2. The levels of TGF-ß2 were significantly higher in HC after CS than VD (7429 vs. 5240 ng/mL; p = 0.0017). The data from this study suggest: caesarean section was associated with increased levels of TGF-ß2 in HC. The increased levels of TGF-ß2 in HC of women who delivered prematurely require further research. Early and exclusive breast-feeding by mothers after caesarean section and premature births with colostrum containing high TGF-ß2 levels may prevent the negative impact of pathogens which often colonize the gastrointestinal tract and may reduce the risk of chronic diseases in this group of patients.

Preterm Birth, Inflammation and Infection: New Alternative Strategies for their Prevention.

BACKGROUND: Worldwide, the progress in reducing neonatal mortality has been very slow. The rate of preterm birth has increased over the last 20 years in low-income and middle-income countries. Its association with increased mortality and morbidity is based on experimental studies and neonatal outcomes from countries with socioeconomic differences, which have considered implementing alternative healthcare strategies to prevent and reduce preterm births. METHODS: Currently, there is no widely effective strategy to prevent preterm birth. Pharmacological therapies are directed at inhibiting myometrial contractions to prolong parturition. Some drugs, medicinal plants and microorganisms possess myorelaxant, anti-inflammatory and immunomodulatory properties that have proved useful in preventing preterm birth associated with inflammation and infection. RESULTS: This review focuses on the existing literature regarding the use of different drugs, medicinal plants, and microorganisms that show promising benefits for the prevention of preterm birth associated with inflammation and infection. New alternative strategies involving the use of PDE-4 inhibitors, medicinal plants and probiotics could have a great impact on improving prenatal and neonatal outcomes and give babies the best start in life, ensuring lifelong health benefits. CONCLUSION: Despite promising results from well-documented cases, only a small number of these alternative strategies have been studied in clinical trials. The development of new drugs and the use of medicinal plants and probiotics for the treatment and/or prevention of preterm birth is an area of growing interest due to their potential therapeutic benefits in the field of gynecology and obstetrics.

Preterm Life in Sterile Conditions: A Study on Preterm, Germ-Free Piglets.

Preterm infants born with immature organ systems, which can impede normal development, can also be highly sensitive to different biological and/or environmental factors. Animal models could aid in investigating and understanding the effects of different conditions on the health of these immunocompromised infants. The epitheliochorial placentation of the pig prevents the prenatal transfer of protective colostral immunoglobulins. Surgical colostrum-deprived piglets are free of maternal immunoglobulins, and the cells that are normally provided via colostrum. We bred preterm germ-free piglets in sterile conditions and compared them with their term counterparts. Enterocyte development and intestinal morphology, tight junction proteins claudin-1 and occludin, pattern-recognizing receptors, adaptor molecules and coreceptors (RAGE, TLR2, TLR4, TLR9, MyD88, TRIF, MD2, and CD14), and inflammasome NLRP3 transcription were all evaluated. The production of inflammatory mediators IFN-α, IL-4, IL-6, IL-8, IL-10, IL-12/23 p40, TNF-α, IFN-γ, and high mobility group box 1 (HMGB1) in the intestine of germ-free piglets was also assessed. In the preterm germ-free piglets, the ileum showed decreased lamina propria cellularity, reduced villous height, and thinner and less distinct stratification - especially muscle layer, in comparison with their term counterparts. Claudin-1 transcription increased in the intestine of the preterm piglets. The transcription levels of pattern-recognizing receptors and adaptor molecules showed ambiguous trends between the groups. The levels of IL-6, IL-8, IL-10, and TNF-α were increased in the preterm ileum numerically (though not significantly), with statistically significant increases in the colon. Additionally, IL-12/23 p40 and IFN-γ were statistically significantly higher in the preterm colon. Both blood plasma and intestinal HMGB1 levels were nonsignificantly higher in the preterm group. We propose that the intestine of the preterm germ-free piglets showed "mild inflammation in sterile conditions." This model, which establishes preterm, hysterectomy-derived germ-free piglets, without protective maternal immunoglobulins, can be used to study influences of microbiota, nutrition, and therapeutic interventions on the development and health of vulnerable immunocompromised preterm infants.

Levels of innate immune factors in preterm and term mothers' breast milk during the 1st month postpartum.

There is a paucity of data on the effect of preterm birth on the immunological composition of breast milk throughout the different stages of lactation. We aimed to characterise the effects of preterm birth on the levels of immune factors in milk during the 1st month postpartum, to determine whether preterm milk is deficient in antimicrobial factors. Colostrum (days 2-5 postpartum), transitional milk (days 8-12) and mature milk (days 26-30) were collected from mothers of extremely preterm (<28 weeks of gestation, n 15), very preterm (28-<32 weeks of gestation, n 15), moderately preterm (32-<37 weeks of gestation, n 15) and term infants (37-41 weeks of gestation, n 15). Total protein, lactoferrin, secretory IgA, soluble CD14 receptor (sCD14), transforming growth factor-ß2 (TGF-ß2), α defensin 5 (HD5), ß defensins 1 (HBD1) and 2, IL-6, IL-10, IL-13, interferon-γ, TNF-α and lysozyme (LZ) were quantified in milk. We examined the effects of lactation stage, gestational age, volume of milk expressed, mode of delivery, parity and maternal infection on milk immune factor concentrations using repeated-measures regression analysis. The concentrations of all factors except LZ and HD5 decreased over the 1st month postpartum. Extremely preterm mothers had significantly higher concentrations of HBD1 and TGF-ß2 in colostrum than term mothers did. After controlling for other variables in regression analyses, preterm birth was associated with higher concentrations of HBD1, LZ and sCD14 in milk samples. In conclusion, preterm breast milk contains significantly higher concentrations of some immune proteins than term breast milk.

The effects of vitamin D plus calcium supplementation on metabolic profiles, biomarkers of inflammation, oxidative stress and pregnancy outcomes in pregnant women at risk for pre-eclampsia.

BACKGROUND: The present study was designed to examine the effects of vitamin D plus calcium administration on metabolic profiles and pregnancy outcomes among women at risk for pre-eclampsia. METHODS: In a prospective, double-blind, placebo-controlled trial, 60 women at risk for pre-eclampsia were randomised to take either 50 000 IU vitamin D3 every 2 weeks plus 1000 mg day(-1) calcium supplements (as calcium carbonate) (n = 30) or to receive placebos at the same times (n = 30) from 20 to 32 weeks of gestation. Fasting blood samples were taken at baseline and 12 weeks after intervention to determine related variables. Newborn anthropometric measurements were determined. RESULTS: Taking combined cholecalciferol and calcium supplements, compared to placebo, led to significant reductions in fasting plasma glucose (FPG) [mean (SD)] [-5.7 (5.5) versus -0.6 (12.6) mg dL(-1) , P = 0.04], serum insulin concentrations [-2.8 (6.0) versus +7.7 (9.8) µIU mL(-1) , P < 0.001], homeostasis model of assessment-insulin resistance [-0.8 (1.3) versus +1.6 (2.2), P < 0.001], homeostatic model assessment-beta cell function [-8.2 (25.8) versus +32.6 (41.3, P < 0.001] and a significant rise in quantitative insulin sensitivity check index score [+0.02 (0.02) versus -0.02 (0.02, P < 0.001]. Additionally, pregnant women who received cholecalciferol plus calcium supplements had increased serum high-density lipoprotein (HDL)-cholesterol [+4.6 (8.3) versus -2.9 (7.7) mg dL(-1) , P = 0.001] and plasma total glutathione (GSH) concentrations [+23.4 (124.0) versus -94.8 (130.2) µm, P = 0.001] compared to placebo. However, after adjustment for the baseline levels, maternal age and baseline body mass index, the effects on FPG levels (P = 0.13) and systolic blood pressure (P = 0.13) disappeared. CONCLUSIONS: Vitamin D plus calcium administration for 12 weeks had beneficial effects on glycaemic status, HDL-cholesterol, GSH and blood pressure among women at risk for pre-eclampsia.

Bacteriological, biochemical, and immunological properties of colostrum and mature milk from mothers of extremely preterm infants.

OBJECTIVES: The objective of this work was to elucidate the influence of extremely premature birth (gestational age 24-27 weeks) on the microbiological, biochemical, and immunological composition of colostrum and mature milk. METHODS: A total of 17 colostrum and 34 mature milk samples were provided by the 22 mothers of extremely preterms who participated in this study. Bacterial diversity was assessed by culture-based methods, whereas the concentration of lactose, glucose, and myo-inositol was determined by a gas chromatography procedure. Finally, the concentrations of a wide spectrum of cytokines, chemokines, growth factors, and immunoglobulins were measured using a multiplex system. RESULTS: Bacteria were present in a small percentage of the colostrum and milk samples. Staphylococci, streptococci, and lactobacilli were the main bacterial groups isolated from colostrum, and they could be also isolated, together with enterococci and enterobacteria, from some mature milk samples. The colostrum concentrations of lactose and glucose were significantly lower than those found in mature milk, whereas the contrary was observed in relation to myo-inositol. The concentrations of most cytokines and immunoglobulins in colostrum were higher than in mature milk, and the differences were significant for immunoglobulin G3, immunoglobulin G4, interleukin (IL)-6, interferon-γ, interleukin-4 (IL-4), IL-13, IL-17, macrophage-monocyte chemoattractant protein-1 and macrophage inflammatory protein-1ß. CONCLUSIONS: The bacteriological, biochemical, and immunological content of colostrum and mature milk from mothers of extremely preterm infants is particularly valuable for such infants. Efforts have to be made to try that preterm neonates receive milk from their own mothers or from donors matching, as much as possible, the gestational age of the preterm.

Change in mononuclear leukocyte responsiveness in midpregnancy and subsequent preterm birth.

OBJECTIVE: To estimate the associations of change in immune response with preterm delivery, omega-3 supplementation, and fish diet. METHODS: This was an ancillary study to a randomized trial of omega-3 fatty acid supplementation for the prevention of recurrent preterm birth. In vitro maternal peripheral blood mononuclear leukocyte production of the anti-inflammatory cytokine, interleukin-10, and the proinflammatory cytokine, tumor necrosis factor-α, in response to stimulation with lipopolysaccharide, was measured at 16-22 weeks of gestation (baseline) and again at 25-28 weeks of gestation (follow-up) among women with prior spontaneous preterm birth. Changes in concentrations from baseline to follow-up ([INCREMENT]) were compared separately among groups defined by gestational age category at delivery, fish diet history, and omega-3 compared with placebo treatment assignment with Kruskal-Wallis tests. RESULTS: Interleukin-10 [INCREMENT] differed by gestational age category among 292 women with paired assays. Concentrations increased less in women delivering between 35 and 36 6/7 weeks of gestation (48.9 pg/mL) compared with women delivering at term (159.3 pg/mL) and decreased by 65.2 pg/mL in women delivering before 35 weeks of gestation (P=.01). Tumor necrosis factor-α Δ also differed by gestational age category among 319 women, but the pattern was inconsistent. Those delivering between 35 and 36 6/7 weeks of gestation exhibited decreased concentrations of tumor necrosis factor-α at follow-up compared with baseline (-356.0 pg/mL); concentrations increased among women delivering before 35 weeks of gestation and those delivering at term, 132.1 and 86.9 pg/mL (P=.03). Interleukin-10 Δ and tumor necrosis factor-α Δ were unaffected by either omega-3 supplementation or fish diet. CONCLUSION: Recurrent preterm birth was associated with decreased peripheral blood mononuclear leukocyte production of interleukin-10 in response to a stimulus during the second trimester. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov, www.clinicaltrials.gov, NCT00135902. LEVEL OF EVIDENCE: II.

Surfactant protein (SP)-A suppresses preterm delivery and inflammation via TLR2.

Toll like receptors (TLRs) are pattern-recognition molecules that initiate the innate immune response to pathogens. Pulmonary surfactant protein (SP)-A is an endogenously produced ligand for TLR2 and TLR4. SP-A has been proposed as a fetally produced signal for the onset of parturition in the mouse. We examined the effect of interactions between SP-A and the pathogenic TLR agonists lipopolysaccharide (LPS), peptidoglycan (PGN) and polyinosinic:cytidylic acid (poly(I:C)) (ligands for TLR4, TLR2 and TLR3, respectively) on the expression of inflammatory mediators and preterm delivery. Three types of mouse macrophages (the cell line RAW 264.7, and fresh amniotic fluid and peritoneal macrophages, including macrophages from TLR4 and TLR2 knockout mice) were treated for up to 7 hours with pathogenic TLR agonists with or without SP-A. SP-A alone had no effect upon inflammatory mediators in mouse macrophages and did not independently induce preterm labor. SP-A significantly suppressed TLR ligand-induced expression of inflammatory mediators (interleukin (IL)-1ß, tumor necrosis factor (TNF)-α and the chemokine CCL5) via a TLR2 dependent mechanism. In a mouse inflammation-induced preterm delivery model, intrauterine administration of SP-A significantly inhibited preterm delivery, suppressed the expression of proinflammatory mediators and enhanced the expression of the CXCL1 and anti-inflammatory mediator IL-10. We conclude that SP-A acts via TLR2 to suppress TLR ligand-induced preterm delivery and inflammatory responses.

Dydrogesterone supplementation in women with threatened preterm delivery--the impact on cytokine profile, hormone profile, and progesterone-induced blocking factor.

Progesterone is indispensable in creating a suitable endometrial environment for implantation, and also for the maintenance of pregnancy. Successful pregnancy depends on an appropriate maternal immune response to the fetus. A protein called progesterone-induced blocking factor (PIBF) acts by inducing Th2-dominant cytokine production to mediate the immunological effects of progesterone. The aim of this prospective study was to compare serum concentrations of progesterone (P), estradiol (E2), anti-inflammatory (IL-10) and pro-inflammatory (IL-6, TNFα, IFNγ) cytokines, and serum PIBF concentrations in women with threatened preterm delivery who were given progesterone supplementation (study group) with those of women with threatened preterm delivery who were not given progesterone supplementation (control group). After dydrogesterone treatment of patients in the study group, serum PIBF as well as progesterone concentrations significantly increased. Women in this group had significantly higher serum levels of IL-10 than controls. The length of gestation was significantly higher in the group of women who were given progesterone supplementation. Our data suggest that dydrogesterone treatment of women at risk of preterm delivery results in increased PIBF production and IL-10 concentrations, and lower concentrations of IFNγ.

Attenuated interleukin-8/leukocyte immunoresponse in preterm infants compared with term infants hospitalized with respiratory syncytial virus bronchiolitis: a pilot study.

Decreased transplacental transfer of antibodies and altered immunoresponsiveness may place preterm (PT) infants at higher risk for serious consequences from respiratory syncytial virus (RSV) bronchiolitis. We hypothesize that among infants hospitalized with RSV bronchiolitis, immune response in PT infants may be different when compared with that of term infants. Nasal-wash samples were collected from 11 PT (<37 weeks of gestation) and 13 term infants (≥37 weeks of gestation) hospitalized with RSV bronchiolitis. Severity of illness (clinical score [CS]), admission peripheral oxygen saturation, and days subjects required supplemental oxygen were compared. Nasal-wash leukocyte count as well as cytokines for interleukin (IL)-8, IL-4, and interferon-γ (IFN-γ) were assayed. No significant differences in CS, admission SaO(2), and O(2) days were seen between PT and term infants. Nasal-wash leukocyte counts and IL-8 levels were higher in term infants compared with PT and correlated with severity (higher CS) in term (p < 0.05) but not in PT (p > 0.05) infants. IL-4 and IFN-γ levels did not differ between the 2 groups (p > 0.05). PT infants hospitalized with RSV bronchiolitis have lower nasal-wash leukocyte counts and a less robust IL-8 response than term infants, and only in term infants did IL-8 levels correlate with clinical disease severity.

Preconception omega-3 fatty acid supplementation of adult male mice with a history of developmental 2,3,7,8-tetrachlorodibenzo-p-dioxin exposure prevents preterm birth in unexposed female partners.

We have recently reported that adult male C57BL/6 mice exposed in utero to the environmental toxicant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) confer an increased risk of preterm birth (PTB) to unexposed females. Risk of PTB was coincident with decreased placental progesterone receptor (Pgr) mRNA expression and increased toll-like receptor 4 (Tlr4) mRNA expression, suggesting that toxicant exposure induced a heightened inflammatory response at the maternal-fetal interface. Since omega-3 fatty acids exhibit anti-inflammatory activity, in this study, we provided TCDD-exposed males a fish oil-enriched diet prior to mating. Although PTB was common in control females mated to TCDD-exposed males on the standard diet, fish oil supplementation of TCDD-exposed males eliminated PTB in unexposed partners. We also determined the influence of preconception, paternal fish oil supplementation on the placental inflammatory response in late pregnancy (E18.5) by examining the expression of Pgr and Tlr4 mRNA as well as the expression of 15-hydroxyprostaglandin dehydrogenase (PGDH). PGDH catabolizes the inflammatory PGE2 to an inactive form; thus, reduced expression of this enzyme would promote tissue inflammation. Compared with control pregnancies, examination of E18.5 placentas arising from TCDD-exposed males on the standard diet revealed a significant increase in Tlr4 mRNA expression corresponding to a reduction in Pgr mRNA and PGDH protein expression. In contrast, fish oil supplementation of toxicant-exposed males led to normalization of placental expression of both Pgr and Tlr4 mRNA and a marked increase in PGDH expression. These studies suggest that a paternal preconception diet that includes omega-3 fatty acids prevents the toxicant-associated increase in the placental inflammatory response at late gestation, preventing PTB.