Live Lactobacillus Rhamnosus GG Research Articles

Lactobacillus rhamnosus GG colonization in early life regulates gut-brain axis and relieves anxiety-like behavior in adulthood

Evidence reveals that gut dysbiosis is involved in bidirectional interactions in gut-brain axis and participates in the progress of multiple disorders like anxiety. Gut microbes in early life are crucial for establishment of host health. We aimed to investigate whether early life probiotics Lactobacillus rhamnosus GG (LGG) colonization could relieve anxiety in adulthood through regulation of gut-brain axis. Live or fixed LGG was gavaged to C57BL/6 female mice from day 18 of pregnancy until natural birth, and newborn mice from day 1 to day 5 respectively. In this study, we found that live LGG could be effectively colonized in the intestine of offspring. LGG colonization increased intestinal villus length and colonic crypt depth, accompanied with barrier function protection before weaning. Microbiota composition by 16S rRNA sequencing showed that some beneficial bacteria, such as Akkermansia and Bifidobacteria, were abundant in LGG colonization group. The protective effect of LGG on gut microbiota persisted from weaning to adulthood. Intriguingly, behavioral results assessed by elevated plus mazed test and open field test demonstrated relief of anxiety-like behavior in adult LGG-colonized offspring. Mechanically, LGG colonization activated epithelial growth factor receptor (EGFR) and enhanced serotonin transporter (SERT) expression and modulated serotonergic system in the intestine, and increased brain-derived neurotrophic factor and γ-aminobutyric acid receptor levels in the hippocampus and amygdala. Blocking EGFR blunted LGG-induced the increased SERT and zonula occludens-1 expression. Collectively, early life LGG colonization could protect intestinal barrier of offspring and modulate gut-brain axis in association with relief of anxiety-like behavior in adulthood.

Lactobacillus rhamnosus GG induces cGAS/STING- dependent type I interferon and improves response to immune checkpoint blockade

ObjectiveOur goals were to evaluate the antitumour efficacy of Lactobacillus rhamnosus GG (LGG) in combination with immune checkpoint blockade (ICB) immunotherapies on tumour growth and to investigate the underlying mechanisms.DesignWe...

Low‐fat fermented milk with a combination of fructo‐oligosaccharides and live Lactobacillus rhamnosus GG (ATCC 53103), Streptococcus thermophilus (Z57) and Lactobacillus bulgaricus (LB2), and defence against reactivation of Herpes simplex virus in the orolabial epithelia: evaluation of a health claim pursuant to Article 13(5) of Regulation (EC) No 1924/2006

Following an application from Granarolo S.p.A., submitted for authorisation of a health claim pursuant to Article 13(5) of Regulation (EC) No 1924/2006 via the Competent Authority of Italy, the EFSA Panel on Dietetic Products, Nutrition and Allergies (NDA) was asked to deliver an opinion on the scientific substantiation of a health claim related to a low-fat fermented milk and defence against reactivation of Herpes simplex virus (HSV) in the orolabial epithelia. The scope of the application was proposed to fall under a health claim based on newly developed scientific evidence. The food which is the subject of the health claim, a low-fat fermented milk with a combination of fructo-oligosaccharides and live Lactobacillus rhamnosus GG (ATCC 53103), Streptococcus thermophilus (Z57) and Lactobacillus delbrueckii subsp. Bulgaricus (LB2), is sufficiently characterised. Defence against reactivation of HSV in the orolabial epithelia is a beneficial physiological effect. The Panel considers that no conclusions can be drawn from three out of the four human intervention studies, which were provided by the applicant, for the scientific substantiation of the claim. In weighing the evidence, the Panel took into account that one human intervention study from which scientific conclusions can be drawn does not show a consistent effect of daily consumption of the low-fat fermented milk on the reduction in incidence of herpes labialis (HL) lesions after ultraviolet B (UVB) exposure and that no convincing evidence was provided for a mechanism by which the low-fat fermented milk could contribute to the defence against reactivation of HSV in the orolabial epithelia. The Panel concludes that a cause and effect relationship has not been established between the consumption of the low-fat fermented milk, which is the subject of the health claim, and defence against reactivation of HSV in the orolabial epithelia.

Administration of probiotics Lactobacillus rhamnosus GG and Lactobacillus gasseri K7 during pregnancy and lactation changes mouse mesenteric lymph nodes and mammary gland microbiota

The milk and mammary gland (MG) microbiome can be influenced by several factors, such as mode of delivery, breastfeeding, maternal lifestyle, health status, and diet. An increasing number of studies show a variety of positive effects of consumption of probiotics during pregnancy and breastfeeding on the mother and the newborn. The aim of this study was to investigate the effect of oral administration of probiotics Lactobacillus gasseri K7 (LK7) and Lactobacillus rhamnosus GG (LGG) during pregnancy and lactation on microbiota of the mouse mesenteric lymph nodes (MLN), MG, and milk. Pregnant FVB/N mice were fed skim milk or probiotics LGG or LK7 resuspended in skim milk during gestation and lactation. On d 3 and 8 postpartum, blood, feces, MLN, MG, and milk were analyzed for the presence of LGG or LK7. The effects of probiotics on MLN, MG, and milk microbiota was evaluated by real-time PCR and by 16S ribosomal DNA 454-pyrosequencing. In 5 of 8 fecal samples from the LGG group and in 5 of 8 fecal samples from the LK7 group, more than 1×103 of live LGG or LK7 bacterial cells were detected, respectively, whereas no viable LGG or LK7 cells were detected in the control group. Live lactic acid bacteria but no LGG or LK7 were detected in blood, MLN, and MG. Both probiotics significantly increased the total bacterial load as assessed by copies of 16S ribosomal DNA in MLN, and a similar trend was observed in MG. Metagenomic sequencing revealed that both probiotics increased the abundance of Firmicutes in MG, especially the abundance of lactic acid bacteria. The Lactobacillus genus appeared exclusively in MG from probiotic groups. Both probiotics influenced MLN microbiota by decreasing diversity (Chao1) and increasing the distribution of species (Shannon index). The LGG probiotic also affected the MG microbiota as it increased diversity and distribution of species and proportions of the genera Lactobacillus and Bifidobacterium. These results provide evidence that probiotics can modulate the bacterial composition of MLN and MG microbiota in ways that could improve the health of the MG and, ultimately, the health of the newborn.

Lactobacillus rhamnosus GG and its SpaC pilus adhesin modulate inflammatory responsiveness and TLR-related gene expression in the fetal human gut.

Bacterial contact in utero modulates fetal and neonatal immune responses. Maternal probiotic supplementation reduces the risk of immune-mediated disease in the infant. We investigated the immunomodulatory properties of live Lactobacillus rhamnosus GG and its SpaC pilus adhesin in human fetal intestinal models. Tumor necrosis factor (TNF)-α mRNA expression was measured by qPCR in a human fetal intestinal organ culture model exposed to live L. rhamnosus GG and proinflammatory stimuli. Binding of recombinant SpaC pilus protein to intestinal epithelial cells (IECs) was assessed in human fetal intestinal organ culture and the human fetal intestinal epithelial cell line H4 by immunohistochemistry and immunofluorescence, respectively. TLR-related gene expression in fetal ileal organ culture after exposure to recombinant SpaC was assessed by qPCR. Live L. rhamnosus GG significantly attenuates pathogen-induced TNF-α mRNA expression in the human fetal gut. Recombinant SpaC protein was found to adhere to the fetal gut and to modulate varying levels of TLR-related gene expression. The human fetal gut is responsive to luminal microbes. L. rhamnosus GG significantly attenuates fetal intestinal inflammatory responses to pathogenic bacteria. The L. rhamnosus GG pilus adhesin SpaC binds to immature human IECs and directly modulates IEC innate immune gene expression.

Probiotic Bacteria Induce Maturation of Intestinal Claudin 3 Expression and Barrier Function

An immature intestinal epithelial barrier may predispose infants and children to many intestinal inflammatory diseases, such as infectious enteritis, inflammatory bowel disease, and necrotizing enterocolitis. Understanding the factors that regulate gut barrier maturation may yield insight into strategies to prevent these intestinal diseases. The claudin family of tight junction proteins plays an important role in regulating epithelial paracellular permeability. Previous reports demonstrate that rodent intestinal barrier function matures during the first 3 weeks of life. We show that murine paracellular permeability markedly decreases during postnatal maturation, with the most significant change occurring between 2 and 3 weeks. Here we report for the first time that commensal bacterial colonization induces intestinal barrier function maturation by promoting claudin 3 expression. Neonatal mice raised on antibiotics or lacking the toll-like receptor adaptor protein MyD88 exhibit impaired barrier function and decreased claudin 3 expression. Furthermore, enteral administration of either live or heat-killed preparations of the probiotic Lactobacillus rhamnosus GG accelerates intestinal barrier maturation and induces claudin 3 expression. However, live Lactobacillus rhamnosus GG increases mortality. Taken together, these results support a vital role for intestinal flora in the maturation of intestinal barrier function. Probiotics may prevent intestinal inflammatory diseases by regulating intestinal tight junction protein expression and barrier function. The use of heat-killed probiotics may provide therapeutic benefit while minimizing adverse effects.

Modelling food allergy with mice models

Food allergies are adverse immune reactions to food proteins that can range from immediate, potentially life-threatening reactions to chronic disorders such as atopic dermatitis and allergic gastrointestinal disorders. These adverse reactions can be IgE-mediated; cells mediated or result from a combination of both. No effective preventive strategy or curative protocol is currently established. Various mouse models have been developed which mirror some of the key elements of food allergies to a high degree. These models have extended our understanding on the immunological and patho-physiological mechanisms of the allergic immune response and have been used for the initial testing of preventive and therapeutic agents. In particular the prenatal and early postnatal period seem to be a critical window for the establishment and maintenance of a normal immune response towards food allergens. This presentation will focus on the role of the maternal adaptive immune response and the nature of the diaplacental antigen transfer during the prenatal period in preventing the onset of allergies in the offspring. In addition, during the early postnatal period several host factors can influence the acquisition of oral tolerance. The interaction of the developing immune system with microbial structures seems to play a decisive role for the induction of local and systemic tolerance. Several studies demonstrated that continuous administration of live Lactobacillus rhamnosus GG (LGG) during gestation and the breastfeeding period inhibited the onset of allergen-induced sensitization and airway disease in the offspring which is associated with the induction of T-regulatory cells. Recent findings suggest that heat treated and soluble factors may also have the ability to suppress the allergic immune response. These data may help to interpret previous data from successful clinical trials and provide an outlook on future intervention strategies.

Live and Heat-Killed Lactobacillus rhamnosus GG: Effects on Proinflammatory and Anti-Inflammatory Cytokines/Chemokines in Gastrostomy-Fed Infant Rats

Lactobacillus rhamnosus GG (LGG), a probiotics, ameliorates intestinal and other organ inflammation in infant rats. The hypothesis is that live and heat-killed LGG have similar effects on decreasing the inflammatory response induced by E. coli lipopolysaccharide (LPS) in the infant rat. Using a gastrostomy-fed rat model, 7-d-old rat pups were gastrostomy fed with or without live LGG (10(8) or 10(12) cfu x L(-1) x kg(-1) x d(-1)) for 6 d. In a separate experiment, LPS was administered to rat pups with or without live or heat-killed LGG (10(8) cfu x L(-1) x kg(-1) x d(-1)). Cytokine/chemokine proteins were determined by ELISA or multiplex assay. Both live and heat-killed LGG decreased LPS-induced cytokine-induced neutrophil chemoattractant-1 (CINC-1) production in liver and plasma (p < 0.05) and also showed a trend (p = 0.09) in lungs. Live and heat-killed LGG ameliorated LPS-suppressed IL-10 level in lungs (p < 0.05). Both forms of LGG decreased IL-1b production in liver. There was no difference between low and high doses of live LGG in the production of CINC-1, TNF-alpha, and myeloperoxidase (MPO). There was a trend of increase of claudin-1 in both live and heat-killed groups (p = 0.08). In conclusion, both live and heat-killed LGG provided by the enteral route decrease LPS-induced proinflammatory mediators and increase anti-inflammatory mediators.

Live and Ultraviolet-Inactivated Lactobacillus Rhamnosus GG Decrease Flagellin-Induced Interleukin-8 Production in Caco-2 Cells

Probiotics are widely used in the treatment and prevention of gastrointestinal problems. However, in some immune-compromised populations, the administration of live microorganisms may not be appropriate. A potential alternative to live microorganisms is to inactivate them as long as the beneficial function is retained. We hypothesized that UV-inactivated Lactobacillus rhamnosus GG (LGG) could downregulate interleukin-8 (IL-8) production in intestinal epithelial cells stimulated by the pathogenic ligand, flagellin, using similar mechanisms as live LGG. Caco-2 cells were pretreated with live or UV-inactivated LGG at 10 11 colony-forming units/L and stimulated by flagellin at a dose of 500 μg/L. IL-8 production was measured by ELISA, inhibitor of κB (I κB) and ubiquitinated-I κB (Ub-I κB) expression by immunoblotting and nuclear factor (NF) κB localization by immunofluorescence staining. Flagellin induced a 17-fold increase in IL-8 production compared with control (P < 0.05), whereas pretreatment with either live LGG or UV-inactivated LGG resulted in 66 and 59% decreases, respectively, compared with the flagellin group (P < 0.05). Flagellin-induced NF κB nuclear translocation was prevented by both live and UV-inactivated LGG. Flagellin decreased I κB, which was reversed by either live or UV-inactivated LGG (P < 0.05). UV-inactivated LGG decreased Ub-I κB expression (P < 0.05), although live LGG had no effect. This study supports the concept that UV-inactivated and live LGG are equally effective in decreasing IL-8 production in the intestinal epithelium. Although the mechanism involves different pathways, both alter cytoplasmic I κB, thereby inhibiting NF κB nuclear translocation.

Ingestion of heat‐treated Lactobacillus rhamnosus GG prevents development of atopic dermatitis in NC/Nga mice

Continuous oral administration of live Lactobacillus rhamnosus GG (L. GG) to pregnant subjects with atopic dermatitis and their children, suppressed the frequency of atopic dermatitis. The details of mechanisms and immune systems involved in this suppressive effect, however, remain speculative. We sought to clarify suppressive mechanisms of L. GG on atopic dermatitis by using NC/Nga mice, a model of human atopic dermatitis. Maternal mice and infant mice were fed with food containing or not containing heat-treated L. GG during pregnancy and breastfeeding, and after weaning. Control NC/Nga mice raised under an air-uncontrolled condition spontaneously manifested typical skin lesions very similar to those in patients with atopic dermatitis. On the other hand, administration of food containing heat-treated L. GG inhibited the onset and development of atopic skin lesions, accompanied by smaller numbers of mast cells and eosinophils in the affected skin sites. Mice fed with L. GG showed a significant increase in plasma IL-10 levels compared with control mice, while there was no significant difference in the proportion of splenic CD4(+)CD25(+) regulatory T cells between mice fed with L. GG and control mice. The IL-10 mRNA expression was enhanced in both Peyer's patches and mesenteric lymph nodes in mice fed with L. GG. These findings suggest that some components of heat-treated L. GG may have an ability to delay the onset and suppress the development of atopic dermatitis, probably through a strong induction of IL-10 in intestinal lymphoid organs and systemic levels.

Alive and Dead Lactobacillus rhamnosus GG Decrease Tumor Necrosis Factor-α–Induced Interleukin-8 Production in Caco-2 Cells

Certain probiotic bacteria show anti-inflammatory activity after being heat killed, whereas others do not, suggesting that the gastrointestinal environment may alter the activity of probiotic bacteria. Occasionally, probiotics are provided when a patient is also being treated with oral antibiotics; this may have an effect on the probiotic activity. We hypothesized that Lactobacillus rhamnosus GG (LGG) are capable of downregulating tumor necrosis factor-alpha (TNFalpha)-induced interleukin (IL)-8 production under all 3 of these conditions, and that LGG act through the nuclear factor kappaB (NFkappaB)/inhibitor of kappaB (IkappaB) pathway. Caco-2 cells were treated with live or heat-killed LGG in doses ranging from 10(4) to 10(10) cfu/L, in the presence or absence of antibiotics and TNFalpha in the media. TNFalpha-induced production of IL-8 by Caco-2 cells was modulated by LGG under all 3 conditions. However, higher doses of live LGG without TNFalpha in the presence or absence of antibiotics in vitro induced the production of IL-8 (P = 0.001). Heat-killed LGG also blunted the TNFalpha-induced IL-8 production (P < 0.01), but by itself did not increase IL-8 production at higher doses as markedly as live LGG (P < 0.05). LGG reduced the TNFalpha-induced NFkappaB translocation to the nucleus and lessened the decrease in IkappaB in the cytoplasm (P < 0.05). LGG reduced TNFalpha-induced IL-8 production by affecting the NFkappaB/IkappaB pathway in Caco-2 cells. High doses of live LGG markedly increased IL-8 production, but heat-killed LGG caused only a slight increase in IL-8. Thus, heat-killed LGG may effectively ameliorate inflammation with a lower potential than live LGG at high doses to cause inflammation.

Stability and activity of specific antibodies against Streptococcus mutans and Streptococcus sobrinus in bovine milk fermented with Lactobacillus rhamnosus strain GG or treated at ultra-high temperature.

Passive local immunization against dental caries is a promising approach to its prevention, as clinical evidence of active oral or nasal immunization is still limited and controversial. By means of systemic immunization of pregnant cows with a multivalent vaccine, high titres of IgG antibodies against human cariogenic bacteria, Streptococcus mutans and Streptococcus sobrinus, were produced in bovine colostrum. The purified immune product (IP) of this preparation has a number of anticariogenic properties, such as inhibition of streptococcal adherence to saliva-coated hydroxyapatite and inhibition of glucosyltransferase enzymes. This study investigated whether IP antibodies remained active and functional when added to ultra-high temperature (UHT)-treated milk or to Lactobacillus rhamnosus GG (LGG)-fermented milk stored for an extended time. LGG was chosen because of its widely known health benefits in humans and animals. A commercial UHT toddler's milk was supplemented with IP and stored for 2 months at 5, 21 and 30 degrees C. The antistreptococcal titres in UHT milk did not decline at any temperature during storage, and UHT-IP inhibited the adherence of S. mutans for up to 2 months. This was not the case with UHT toddler's milk without IgG antibodies. Milk was fermented with live LGG cells in the presence or absence of 5% IP. The antistreptococcal titres declined to about 30% of the original titres after storage. Fresh milk alone slightly enhanced streptococcal adhesion but fresh milk with IP inhibited the adherence of S. mutans by over 50%. LGG-positive fermented milk without antibodies also inhibited (P < 0.05) the adhesion by about 40%. In both LGG-fermented and UHT immune milk, the activity of antibodies against cariogenic streptococci was maintained during the expected shelf-life of these products. From the anticariogenic point of view it may be beneficial to add bovine-specific antibodies against mutans streptococci to probiotic LGG-containing milk products.