Characterization of the pathophysiological determinants of diarrheagenic Escherichia coli infection using a challenge model in healthy adults.

An experimental human challenge model with an attenuated diarrheagenic Escherichia coli (E. coli) strain has been used in food intervention studies aimed to increase resistance to E. coli infection. This study was designed to refine and expand this challenge model. In a double-blind study, healthy male subjects were orally challenged with 1E10 or 5E10 colony-forming units (CFU) of E. coli strain E1392/75-2A. Three weeks later, subjects were rechallenged with 1E10 CFU of E. coli. Before and after both challenges, clinical symptoms and infection- and immune-related biomarkers were analyzed. Subset analysis was performed on clinically high- and low-responders. Regardless of inoculation dose, the first challenge induced clinical symptoms for 2-3 days. In blood, neutrophils, CRP, CXCL10, and CFA/II-specific IgG were induced, and in feces calprotectin and CFA/II-specific IgA. Despite clinical differences between high- and low-responders, infection and immune biomarkers did not differ. The first inoculation induced protection at the second challenge, with a minor clinical response, and no change in biomarkers. The refined study design resulted in a larger dynamic range of symptoms, and identification of biomarkers induced by a challenge with the attenuated E. coli strain E1392/75-2A, which is of value for future intervention studies. Addition of a second inoculation allows to study the protective response induced by a primary infection.Clinicaltrials.gov registration: NCT02541695 (04/09/2015).

Temporal Changes of Protein Composition in Breast Milk of Chinese Urban Mothers and Impact of Caesarean Section Delivery.

Human breast milk (BM) protein composition may be impacted by lactation stage or factors related to geographical location. The present study aimed at assessing the temporal changes of BM major proteins over lactation stages and the impact of mode of delivery on immune factors, in a large cohort of urban mothers in China. 450 BM samples, collected in three Chinese cities, covering 8 months of lactation were analyzed for α-lactalbumin, lactoferrin, serum albumin, total caseins, immunoglobulins (IgA, IgM and IgG) and transforming growth factor (TGF) ß1 and ß2 content by microfluidic chip- or ELISA-based quantitative methods. Concentrations and changes over lactation were aligned with previous reports. α-lactalbumin, lactoferrin, IgA, IgM and TGF-ß1 contents followed similar variations characterized by highest concentrations in early lactation that rapidly decreased before remaining stable up to end of lactation. TGF-ß2 content displayed same early dynamics before increasing again. Total caseins followed a different pattern, showing initial increase before decreasing back to starting values. Serum albumin and IgG levels appeared stable throughout lactation. In conclusion, BM content in major proteins of urban mothers in China was comparable with previous studies carried out in other parts of the world and C-section delivery had only very limited impact on BM immune factors.

Lactobacillus paracasei reduces intestinal inflammation in adoptive transfer mouse model of experimental colitis.

Studies showed that specific probiotics provide therapeutic benefits in inflammatory bowel disease. In vitro evidence suggested that Lactobacillus paracasei also called ST11 (CNCM I-2116) is a potent strain with immune modulation properties. However, little is known about its capacity to alleviate inflammatory symptoms in vivo In this context, the main objective of this study was to investigate the role of ST11 on intestinal inflammation using the adoptive transfer mouse model of experimental colitis. Rag2(-/-) recipient mice were fed with ST11 (10(9) CFU/day)a month prior toinduce colitis by adoptive transfer of naive T cells. One month later, in clear contrast to nonfed mice, weight loss was significantly reduced by 50% in ST11-fed mice. Further analysis of colon specimens revealed a significant reduction neutrophil infiltration and mucosal expression of IL1ß, IL-6, and IL12 proinflammatory cytokines, whereas no consistent differences in expression of antibacterial peptides or tight junction proteins were observed between PBS and ST11-fed mice. All together, our results demonstrate that oral administration of ST11 was safe and had a significant preventive effect on colitis. We conclude that probiotics such as Lactobacillus paracasei harbor worthwhile in vivo immunomodulatory properties to prevent intestinal inflammation by nutritional approaches.

Potential role of the intestinal microbiota of the mother in neonatal immune education.

Mucosal dendritic cells are at the heart of decision-making processes that dictate immune reactivity to intestinal microbes. They ensure tolerance to commensal bacteria and a vigorous immune response to pathogens. It has recently been demonstrated that the former involves a limited migration of bacterially loaded dendritic cells from the Peyer's patches to the mesenteric lymph nodes. During lactation, cells from gut-associated lymphoid tissue travel to the breast via the lymphatics and peripheral blood. Here, we show that human peripheral blood mononuclear cells and breast milk cells contain bacteria and their genetic material during lactation. Furthermore, we show an increased bacterial translocation from the mouse gut during pregnancy and lactation and the presence of bacterially loaded dendritic cells in lactating breast tissue. Our observations show bacterial translocation as a unique physiological event, which is increased during pregnancy and lactation. They suggest endogenous transport of intestinally derived bacterial components within dendritic cells destined for the lactating mammary gland. They also suggest neonatal immune imprinting by milk cells containing commensal-associated molecular patterns.

Intake of a milk-based wolfberry formulation enhances the immune response of young-adult and aged mice.

Aging is associated with alterations of immune responses. Wolfberry, a popular Chinese functional ingredient, is prized for its anti-aging properties; however, little is known about the immunological effect of wolfberry intake. The purpose of this study was to examine the effect of dietary intake of a milk-based formulation of wolfberry, named Lacto-Wolfberry, on in vivo and ex vivo parameters of adaptive immunity in young-adult and aged mice. Over 44 days, young-adult (2 months) and aged (21 months) C57BL/6J mice were fed ad libitum with a controlled diet and received drinking water supplemented or not with 0.5% (wt/vol) Lacto-Wolfberry. All mice were immunized on day 15 and challenged on day 22 with a T cell- dependent antigen, keyhole limpet hemocyanin (KLH). Lacto-Wolfberry supplementation significantly increased in vivo systemic immune markers that are known to decline with aging. Indeed, both antigen-(KLH) specific humoral response and cell-mediated immune responses in young-adult and aged mice were enhanced when compared to their respective controls. No significant effect of Lacto-Wolfberry supplementation was observed on ex vivo spleen cells proliferative response to mitogens and on splenocyte T cell subsets. In conclusion, dietary intake of Lacto-Wolfberry may favorably modulate the poor responsiveness to antigenic challenge observed with aging.

Effect of Lactobacillus paracasei NCC2461 on antigen-specific T-cell mediated immune responses in aged mice.

Aging is associated with a reduced capacity to mount proper immune responses, in particular to vaccines. Probiotic lactic acid bacteria may improve the immune status of the elderly; however, there is little evidence showing an effect of these bacteria on humoral and cellular immune responses. In the present study, the immunomodulatory capacity of the probiotic Lactobacillus paracasei NCC2461 combined or not with a prebiotic composition, FOS/inulin, was examined in aged mice. Male C57BL/6J mice (21-months-old) were allocated to one of three groups fed ad libitum for 44 days with different diets: a normal diet (control), a normal diet plus NCC2461 given in the drinking water, or a diet containing FOS/inulin plus NCC2461 in the drinking water. All mice were immunized on day 15 and challenged on day 22 with keyhole limpet hemocyanin (KLH). T helper (Th)1 cell-dependent immune responses (anti-KLH immunoglobulin G(2a) [IgG(2a)] levels and delayed type hypersensitivity response) were increased significantly in NCC2461-supplemented mice when compared to controls. Supplementation with FOS/inulin did not further improve the immune-enhancing effect mediated by the probiotic. Splenocyte proliferation, T cell subsets, systemic total IgG levels, and mucosal total IgA responses were not affected. Interestingly, supplementation with NCC2461 modulated the intestinal microbiota composition by increasing the numbers of bifidobacteria and lactobacilli. In conclusion, oral intake of L. paracasei NCC2461 by aged mice enhanced the specific adaptive immune response to in vivo antigenic challenge without altering other cellular and humoral immune responses. The poor responsiveness to antigenic challenge, frequently observed in elderly people, may be improved by supplementation with L. paracasei NCC2461.

Bacterial imprinting of the neonatal immune system: lessons from maternal cells?

OBJECTIVE: We examined the presence of a natural bacterial inoculum in breast milk and its intracellular transport from the maternal intestine to the breast through the circulation. METHODS: Breast milk and peripheral blood were collected aseptically from healthy donors at various times after delivery, and the presence of viable bacteria was determined through plating. Temporal temperature gradient gel electrophoresis was used to examine the bacterial ribosomal DNA content in milk cells, maternal peripheral blood mononuclear cells, and feces and in corresponding infant feces. Blood from nongravid nonlactating women served as control samples. Bacterial translocation to extraintestinal tissues was also evaluated in virgin, pregnant, and lactating mice. RESULTS: Breast milk contained a low total concentration of microbes of <10(3) colony-forming units per mL. Temporal temperature gradient gel electrophoresis revealed that maternal blood and milk cells contained the genetic material of a greater biodiversity of enteric bacteria. Some bacterial signatures were common to infant feces and to samples of maternal origin. Bacterial translocation from the gut to mesenteric lymph nodes and mammary gland occurred during late pregnancy and lactation in mice. CONCLUSIONS: Bacterial translocation is a unique physiologic event, which is increased during pregnancy and lactation in rodents. Human breast milk cells contain a limited number of viable bacteria but a range of bacterial DNA signatures, as also found in maternal peripheral blood mononuclear cells. Those peripheral blood mononuclear cells showed greater biodiversity than did peripheral blood mononuclear cells from control women. Taken together, our results suggest that intestinally derived bacterial components are transported to the lactating breast within mononuclear cells. We speculate that this programs the neonatal immune system to recognize specific bacterial molecular patterns and to respond appropriately to pathogens and commensal organisms.