Electrospun Scaffold for Biomimic Culture of Caco-2 Cell Monolayer as an In Vitro Intestinal Model.

The Caco-2 cell monolayer has been extensively used for the high-throughput assessing of nutrient absorption, screening of drug permeability, and studying the intestinal physiological process in vitro. The most used Caco-2 cell model is the Transwell model with polycarbonate microporous membranes. However, Caco-2 cells in the classical Transwell model need 21 days to gain an intact and mature monolayer. Electrospun nanofiber scaffolds mimicking the natural extracellular matrix could improve cell adhesion, proliferation, and expression, whereas there are no reports that intestinal cells were cultured on the electrospun nanofiber scaffolds. Here, electrospun polylactic acid (PLA) nanofiber scaffolds were chosen as the ideal scaffolds for Caco-2 cell monolayers to construct a modified Transwell. Cell morphology and polarity were studied. Monolayer barrier properties were assessed by measuring transepithelial electrical resistance (TEER) and the leakage of phenol red. As found, intact Caco-2 cell monolayers were formed on the PLA nanofiber scaffolds after 4 days of culture. After 4 days, the TEER increased to 422 Ω·cm2 and the apparent permeability coefficients of phenol red decreased to 1.0 ± 0.1 × 10-6 cm/s, suggesting that Caco-2 cell monolayers developed a formidable barrier to small molecules on the surface of PLA nanofiber scaffolds. Microvilli and tight junctions were clearly visible after day 3. Besides, Caco-2 cell monolayers on the surface of PLA nanofiber scaffolds presented higher differentiation properties than on the surface of the polycarbonate microporous membrane in traditional Transwell including higher alkaline phosphatase activity and higher P-gp activity. Results of quercetin absorption and probiotics adhesion demonstrated that Caco-2 cell monolayers formed on the surface of PLA nanofiber scaffolds also had better physiological function and prediction function in vitro. Overall, the present study indicated that the Transwell with the structurally and functionally biomimetic electrospun PLA nanofiber scaffold could be potentially developed as a promising in vitro intestinal model.

Bifidobacterium longum Subspecies infantis (B. infantis) in Pediatric Nutrition: Current State of Knowledge.

Abstract: Since originally isolated in 1899, the genus Bifidobacterium has been demonstrated to predominate in the gut microbiota of breastfed infants and to benefit the host by accelerating maturation of the immune response, balancing the immune system to suppress inflammation, improving intestinal barrier function, and increasing acetate production. In particular, Bifidobacterium longum subspecies infantis (B. infantis) is well adapted to the infant gut and has co-evolved with the mother-infant dyad and gut microbiome, in part due to its ability to consume complex carbohydrates found in human milk. B. infantis and its human host have a symbiotic relationship that protects the preterm or term neonate and nourishes a healthy gut microbiota prior to weaning. To provide benefits associated with B. infantis to all infants, a number of commercialized strains have been developed over the past decades. As new ingredients become available, safety and suitability must be assessed in preclinical and clinical studies. Consideration of the full clinical evidence for B. infantis use in pediatric nutrition is critical to better understand its potential impacts on infant health and development. Herein we summarize the recent clinical studies utilizing select strains of commercialized B. infantis.

Maternal administration of probiotics promotes brain development and protects offspring's brain from postnatal inflammatory insults in C57/BL6J mice.

Neonatal morbidities are associated with long term neurological deficits in life and have also been associated with dysbiosis. We tested whether optimizing the neonate's microbiome through maternal probiotic supplementation can improve offspring's neurodevelopmental outcomes. Maternal LB supplementation, carried out by giving Lactobacillus acidophilus and Bifidobacterium infantis (LB) to pregnant C57/BL6J mice daily from E16 to weaning, significantly suppressed postnatal peripheral proinflammatory insult-induced systemic inflammation and normalized compromised blood-brain barrier permeability and tight junction protein expression in the offspring at pre-weaned age. Maternal LB exposure also regulated markers associated with leukocyte transendothelial migration, extracellular matrix injury and neuroinflammation. The suppressed neuroinflammation by maternal LB supplementation was associated with reduced astrocyte/microglia activation and downregulation of the transcriptional regulators CEBPD and IκBα. Furthermore, maternal LB supplementation promoted neuronal and oligodendrocyte progenitor cell development. Our study demonstrates the efficacy of maternal LB supplementation in modulating systemic and central nervous system inflammation as well as promoting neural/oligodendrocyte progenitor development in the offspring. This evidence suggests that maternal probiotic supplementation may be a safe and effective strategy to improve neurological outcomes in the offspring.

Short chain fatty acids produced by colonizing intestinal commensal bacterial interaction with expressed breast milk are anti-inflammatory in human immature enterocytes.

Necrotizing enterocolitis (NEC) is a devastating intestinal emergency that affects ten percent of very low birth weight premature babies and costs society in both expense and heartache. It is probably caused by an inappropriate interaction of colonizing bacteria with an immature intestine. A possible preventative measure is to feed prematures their mother's expressed breast milk in conjunction with a probiotic. This synbiotic prevention reduces the severity and incidence of this condition. This study was designed to determine the mechanism of the synbiotic effect in human and mouse fetal intestine. Breast milk interacting with a NEC preventative probiotic such as Bifidobacterium infantis can produce increased levels of short chain fatty acids (acetate, propionate and butyrate) (SCFAs). SCFAs are known to be anti-inflammatory in mature enterocytes and immunocytes. Very little is known about their role in immature intestine. When exposed to a human fetal cell line, fetal intestinal organoids and fetal mouse intestine, these SCFAs were anti-inflammatory. Their mechanism of anti-inflammation differed from those reported for mature cells by involving the G-protein coupled receptor (GPR 109A) and inhibiting histone deacetylase 4 and 5. These bacterial metabolites may help explain the synbiotic anti-inflammatory effect of breast milk and probiotics given to premature infants at risk for NEC.

Reintroducing B. infantis to the cesarean-born neonate: an ecologically sound alternative to "vaginal seeding".

There is a burgeoning literature highlighting differences in health outcomes between babies born vaginally and by caesarean section (c-section) This has led to the suggestion that infants born by c-section may benefit from vaginal swabbing/seeding. Here, we discuss from an ecological perspective that it is gut-adapted, not vagina-adapted microbes that are likely to take up residence in the gut and have the most beneficial impact on the developing neonate. Further, we caution the practice of 'vaginal seeding' may be potentially unsafe and also give parents and health professionals a false sense of action in restoring the infant gut microbiome following c-section. Instead, we argue that restoring B. longum subsp. infantis, which has evolved to colonize the infant gut, is a safe and ecologically-sound approach to restoring the gut microbiome of infants born by c-section.

Ingestion of Bifidobacterium longum subspecies infantis strain CCFM687 regulated emotional behavior and the central BDNF pathway in chronic stress-induced depressive mice through reshaping the gut microbiota.

Increasing evidence points to the effect of the gut microbiota on central nervous system functions. Supplementation of certain microbial strains has been demonstrated to alleviate depressive behaviors and neurological abnormalities. This study took the approach to screen for an anti-depressive Bifidobacterium longum strain from fourteen candidates and systematically verified its effect in a chronic stress-induced depression mice model. B. longum subsp. infantis strain CCFM687 could significantly enhance the biosynthesis of 5-hydroxytryptamine (5-HTP) in vitro in RIN14B cells through up-regulation of the Tph1 gene expression. Administration of CCFM687 in mice significantly improved the scores in behavioral tests and increased the level of 5-HTP and serotonin (5-HT) in the prefrontal cortex (PFC) of the brain. The brain-derived neurotrophic factor (BDNF) in the PFC was also increased, possibly through the 5-HT1A-CREB-BDNF pathway. In addition, CCFM687 alleviated the hyperactivity of the hypothalamic-pituitary-adrenal (HPA) axis response and accordingly reversed the peripheral inflammation status. Moreover, the stress-induced structural and functional dysbiosis of the gut microbiome was improved by CCFM687, through increased alpha diversity and abundance of butyrate-producing bacteria, in conjunction with inhibition of pathogenic gene expression. In summary, these results indicate that supplementation of B. longum subsp. infantis strain CCFM687 may prevent the onset of depression from chronic stress, and RIN14B could serve as an efficient cell model for rapid screening of anti-depressive probiotics.

Early-life gut microbiome modulation reduces the abundance of antibiotic-resistant bacteria.

Background: Antibiotic-resistant (AR) bacteria are a global threat. AR bacteria can be acquired in early life and have long-term sequelae. Limiting the spread of antibiotic resistance without triggering the development of additional resistance mechanisms is of immense clinical value. Here, we show how the infant gut microbiome can be modified, resulting in a significant reduction of AR genes (ARGs) and the potentially pathogenic bacteria that harbor them. Methods: The gut microbiome was characterized using shotgun metagenomics of fecal samples from two groups of healthy, term breastfed infants. One group was fed B. infantis EVC001 in addition to receiving lactation support (n = 29, EVC001-fed), while the other received lactation support alone (n = 31, controls). Coliforms were isolated from fecal samples and genome sequenced, as well as tested for minimal inhibitory concentrations against clinically relevant antibiotics. Results: Infants fed B. infantis EVC001 exhibited a change to the gut microbiome, resulting in a 90% lower level of ARGs compared to controls. ARGs that differed significantly between groups were predicted to confer resistance to beta lactams, fluoroquinolones, or multiple drug classes, the majority of which belonged to Escherichia, Clostridium, and Staphylococcus. Minimal inhibitory concentration assays confirmed the resistance phenotypes among isolates with these genes. Notably, we found extended-spectrum beta lactamases among healthy, vaginally delivered breastfed infants who had never been exposed to antibiotics. Conclusions: Colonization of the gut of breastfed infants by a single strain of B. longum subsp. infantis had a profound impact on the fecal metagenome, including a reduction in ARGs. This highlights the importance of developing novel approaches to limit the spread of these genes among clinically relevant bacteria. Future studies are needed to determine whether colonization with B. infantis EVC001 decreases the incidence of AR infections in breastfed infants. Trial registration: This clinical trial was registered at ClinicalTrials.gov, NCT02457338.

Probiotics SOD inhibited food allergy via downregulation of STAT6-TIM4 signaling on DCs.

Bacterial probiotics are of increasing use against intestinal disorders such as food allergy. However, the detailed molecular mechanism underlying probiotics-mediated anti-allergic effect remains unknown. In the present study, we orally treated OVA-sensitized mice with Bifidobacterium infantis (BB) for two weeks. It was found that OVA specific-IgE and-IgG levels in serum were significantly decreased after BB administration. BB treatment also significantly reduced the release of IL-4, -5, -13 in splenocytes. Besides, after challenge with OVA, the occurrence of temperature drop and diarrhea was dramatically reduced in BB group. Moreover, the protective effect of BB on allergic mice was correlated with its antioxidative enzyme, superoxide dismutase (SOD). The antioxidative effect of BB on Dendritic cells (DCs0 was further demonstrated to be mediated by cAMP/PKA signaling. We also found that the mRNA and protein expression levels of TIM4 were attenuated in BB group. Finally, ChIP-qPCR assay studies indicate that BB reduced the binding of STAT6 to its response elements in the TIM4 promoter. In conclusion, orally administration of BB protected allergic mice via attenuation of oxidative stress, which further reduced TIM4 expression by inhibiting its transcription factor STAT6.

Rapid assessment of viable but non-culturable Bacillus coagulans MTCC 5856 in commercial formulations using Flow cytometry.

Accurate enumeration of bacterial count in probiotic formulation is imperative to ensure that the product adheres to regulatory standards and citation in consumer product label. Standard methods like plate count, can enumerate only replicating bacterial population under selected culture conditions. Viable but non culturable bacteria (VBNC) retain characteristics of living cells and can regain cultivability by a process known as resuscitation. This is a protective mechanism adapted by bacteria to evade stressful environmental conditions. B. coagulans MTCC 5856(LactoSpore®) is a probiotic endospore which can survive for decades in hostile environments without dividing. In the present study, we explored the use of flow cytometry to enumerate the viable count of B. coagulans MTCC 5856 under acidic and alkaline conditions, high temperature and in commercial formulations like compressed tablets and capsules. Flow cytometry (FCM) was comparable to plate count method when the spores were counted at physiological conditions. We show that VBNC state is induced in B. coagulans MTCC 5856by high temperature and acidic pH. The cells get resuscitated under physiological conditions and FCM was sensitive to detect the VBNC spores. Flow cytometry showed excellent ability to assess the viable spore count in commercial probiotic formulations of B. coagulans MTCC 5856. The results establish Flow cytometry as a reliable method to count viable bacteria in commercial probiotic preparations. Sporulation as well as existence as VBNC could contribute to the extreme stability of B. coagulans MTCC 5856.

Bifidobacterium Infantis Ameliorates Chemotherapy-Induced Intestinal Mucositis Via Regulating T Cell Immunity in Colorectal Cancer Rats.

BACKGROUND/AIMS: Intestinal mucositis (IM) is a commonly encountered side effect in cancer patients receiving chemotherapy. This study aimed to investigate the effect of Bifidobacterium infantis (B. infantis) in attenuating the severity of chemotherapy-induced intestinal mucositis by regulating the T cell subsets in rats with colorectal cancer (CRC). METHODS: Thirty male Sprague-Dawley (SD) rats were injected dimethyl hydrazine (DMH) subcutaneously for 10 weeks, and then injected SW480 cells in rectal mucosa to create a CRC model, and the rats were randomly divided into three groups: Control group (saline + saline), Chemotherapy group (saline + 5-FU+Oxaliplatin), B. infantis group (B. infantis + 5-FU+Oxaliplatin). IM was evaluated based on diarrhea severity, intestinal villus height, crypt depth, pro-inflammatory cytokines (IL-6, IL-1ß, TNF-α), T cell subsets (CD4+ IL17A+ cells and CD4+ CD25+ Foxp3+ Tregs) and related cytokine profiles. RESULTS: The results showed that the B. infantis group demonstrated a higher body weight (BW) and intestinal villus height and a deeper crypt depth compared to the Chemotherapy group. The level of IL-6, IL-1ß and TNF-α which increased by chemotherapy, was lowered by B. infantis administration. Real time reverse transcription- polymerase chain reaction (RT-PCR) showed B. infantis reduced relative expression of Th17 and Th1 cells related cytokines, and increased relative expression of CD4+ CD25+ Foxp3+ Tregs related cytokines. Furthermore, Flow cytometry analysis showed B. infantis reduced CD4+ IL17A+ cells and increased CD4+ CD25+ Foxp3+ Tregs in mesenteric lymph nodes (MLNs) compared to the Chemotherapy group. CONCLUSION: B. infantis effectively attenuates chemotherapy-induced intestinal mucositis by decreasing Th1 and Th17 response and increasing CD4+ CD25+ Foxp3+ Tregs response.

Lactobacillus acidophilus/Bifidobacterium infantis probiotics are associated with increased growth of VLBWI among those exposed to antibiotics.

We performed an observational study with very-low-birth weight infants (VLBWI) ≤33 weeks of gestation born in centers of the German Neonatal Network (GNN; (total n = 8534, n = 6229 received probiotics). The primary objectives of our study were (a) to assess the effect of Lactobacillus acidophilus/Bifidobacterium infantis probiotics on growth in VLBWI during primary stay in hospital and (b) to determine whether this effect is modified by antibiotic exposure. In linear regression models the administration of probiotics was independently associated with improved weight gain [g/d; effect size B = 0.62 (95% CI: 0.37-0.87), p < 0.001], and higher growth rates for body length [(mm/d; B = 0.06 (95% CI: 0.04-0.08), p < 0.001] and head circumference [mm/d; B = 0.03, 95% CI: 0.02-0.04, p < 0.001]. This effect was pronounced in infants with postnatal exposure to antibiotics; i.e. weight gain [g/d; B = 0.66 (95% CI: 0.32-1), p < 0.001], growth rate body length [(mm/d; B = 0.09 (95% CI: 0.06-0.12), p < 0.001] and head circumference [mm/d; B = 0.04, 95% CI: 0.02-0.06, p < 0.001]. In the small subgroup that was available for analysis at 5-year-follow-up (with probiotics: n = 120 vs. without probiotics: n = 54) we noted a sustained effect of probiotics in infants who received postnatal antibiotics. Probiotics may improve growth in antibiotic-treated infants which needs to be confirmed in randomized-controlled trials.