Karine Vidal

Modulation of Neonatal Microbial Recognition: TLR-Mediated Innate Immune Responses Are Specifically and Differentially Modulated by Human Milk

The mechanisms controlling innate microbial recognition in the neonatal gut are still to be fully understood. We have sought specific regulatory mechanisms operating in human breast milk relating to TLR-mediated microbial recognition. In this study, we report a specific and differential modulatory effect of early samples (days 1–5) of breast milk on ligand-induced cell stimulation via TLRs. Although a negative modulation was exerted on TLR2 and TLR3-mediated responses, those via TLR4 and TLR5 were enhanced. This effect was observed in human adult and fetal intestinal epithelial cell lines, monocytes, dendritic cells, and PBMC as well as neonatal blood. In the latter case, milk compensated for the low capacity of neonatal plasma to support responses to LPS. Cell stimulation via the IL-1R or TNFR was not modulated by milk. This, together with the differential effect on TLR activation, suggested that the primary effect of milk is exerted upstream of signaling proximal to TLR ligand recognition. The analysis of TLR4-mediated gene expression, used as a model system, showed that milk modulated TLR-related genes differently, including those coding for signal intermediates and regulators. A proteinaceous milk component of ≥80 kDa was found to be responsible for the effect on TLR4. Notably, infant milk formulations did not reproduce the modulatory activity of breast milk. Together, these findings reveal an unrecognized function of human milk, namely, its capacity to influence neonatal microbial recognition by modulating TLR-mediated responses specifically and differentially. This in turn suggests the existence of novel mechanisms regulating TLR activation.

Lipoteichoic Acids from Lactobacillus johnsonii Strain La1 and Lactobacillus acidophilus Strain La10 Antagonize the Responsiveness of Human Intestinal Epithelial HT29 Cells to Lipopolysaccharide and Gram-Negative Bacteria

ABSTRACT Intestinal epithelial cells (IECs) respond to lipopolysaccharide (LPS) from gram-negative bacteria in the presence of the soluble form of CD14 (sCD14), a major endotoxin receptor. Since sCD14 is also known to interact with gram-positive bacteria and their components, we looked at whether sCD14 could mediate their effects on human IECs. To this end, we examined the production of proinflammatory cytokines following exposure of the IECs to specific gram-positive bacteria or their lipoteichoic acids (LTAs) in the absence and presence of human milk as a source of sCD14. In contrast to LPS from Escherichia coli or Salmonella enteritidis, neither the gram-positive bacteria Lactobacillus johnsonii strain La1 and Lactobacillus acidophilus strain La10 nor their LTAs stimulated IECs, even in the presence of sCD14. However, both LTAs inhibited the sCD14-mediated LPS responsiveness of IECs. We have previously hypothesized that sCD14 in human milk is a means by which the neonate gauges the bacterial load in the intestinal lumen and liberates protective proinflammatory cytokines from IECs. The present observations suggest that gram-positive organisms, via their LTAs, temper this response and prevent an exaggerated inflammatory response.

Goji berry effects on macular characteristics and plasma antioxidant levels.

Purpose. Goji berry (Lycium barbarum L.) is purported to benefit vision because of its high antioxidant (especially zeaxanthin) content, although this effect has not been demonstrated in high-quality human studies. The purpose of this study was to evaluate the effects of daily supplementation with a proprietary milk-based formulation of goji berry, Lacto-Wolfberry (LWB), on macular characteristics and plasma zeaxanthin and antioxidant capacity levels in elderly subjects. Methods. This was a double-masked, randomized, placebo-controlled trial in healthy elderly subjects (range, 65 to 70 years) receiving 13.7 g/d of LWB (n = 75) or placebo (n = 75) for 90 days. Subjects underwent direct ophthalmic examination to assess pigmentation and soft drusen count in the macula and a blood draw to measure plasma zeaxanthin level and total antioxidant capacity. Results. The placebo group demonstrated hypopigmentation and soft drusen accumulation in the macula, whereas the LWB group remained stable. Both plasma zeaxanthin level and antioxidant capacity increased significantly in the LWB group, by 26% and 57%, respectively, but did not change in the placebo group. No product-related adverse events were reported in either group. Conclusions. Overall, daily dietary supplementation with goji berry for 90 days increases plasma zeaxanthin and antioxidant levels as well as protects from hypopigmentation and soft drusen accumulation in the macula of elderly subjects. However, the mechanism of action is unclear, given the lack of relationship between change in plasma zeaxanthin and change in macular characteristics.

Osteoprotegerin in Human Milk: A Potential Role in the Regulation of Bone Metabolism and Immune Development

Osteoprotegerin (OPG) is a member of the tumor necrosis factor superfamily. It is a soluble “decoy” receptor for tumor necrosis factor–related apoptosis-inducing ligand and ligand of the receptor activator of NF-κB. As such, OPG inhibits osteoclast activity and regulates the immune system. Human milk is a complex biologic fluid that supplies nutritional and protective factors to the breast-fed infant. In the present study, human milk samples at various times postpartum were assessed for the presence of OPG. Using biochemical as well as immunologic and biologic techniques we showed that human milk contains OPG at a level that is 1000-fold higher than that found in normal human serum. We observed that human breast milk cells and the human mammary epithelial cell line MCF-7 express OPG, indicating that both cell types are possible sources of milk OPG in vivo. In vitro studies demonstrated that milk OPG is biologically active and suggested that it may contribute to the antiresorptive activity of milk on bone, as well as tumor necrosis factor–related apoptosis-inducing ligand–induced inhibition of T cell proliferation. OPG-like activity was also observed in bovine colostrum and milk. Furthermore, we were able to detect human OPG in the sera of rats gavaged with human milk. We discuss the relevance of our findings for the breast-fed infant and for the prevention of immune and bone disorders.

Immunomodulatory Effects of Dietary Supplementation with a Milk-Based Wolfberry Formulation in Healthy Elderly: A Randomized, Double-Blind, Placebo-Controlled Trial

Wolfberry (fruit of Lycium barbarum) has been prized for many years in China for its immunomodulatory property and its high specific antioxidant content. However, clear clinical evidence demonstrating the effect of wolfberry dietary supplementation is still lacking. After our earlier report showing that a proprietary milk-based wolfberry formulation (Lacto-Wolfberry) enhances in vivo antigen-specific adaptive immune responses in aged mice, the present study aimed at demonstrating the effect of dietary Lacto-Wolfberry supplementation on immune functions in the elderly, especially vaccine response known to decline with aging. A 3-month randomized, double-blinded, placebo-controlled study was conducted on 150 healthy community-dwelling Chinese elderly (65-70 years old) supplemented with Lacto-Wolfberry or placebo (13.7 grams/day). Immune response to influenza vaccine was assessed in the study, along with inflammatory and physical status. No serious adverse reactions were reported during the trial, neither symptoms of influenza-like infection. No changes in body weight and blood pressure, blood chemistry or cells composition, as well as autoantibodies levels were observed. The subjects receiving Lacto-Wolfberry had significantly higher postvaccination serum influenza-specific immunoglobulin G levels and seroconversion rate, between days 30 and 90, compared with the placebo group. The postvaccination positive rate was greater in the Lacto-Wolfberry group compared to the placebo group, but did not reach statistical significance. Lacto-Wolfberry supplementation had no significant effect on delayed-type hypersensitivity response and inflammatory markers. In conclusion, long-term dietary supplementation with Lacto-Wolfberry in elderly subjects enhances their capacity to respond to antigenic challenge without overaffecting their immune system, supporting a contribution to reinforcing immune defense in this population.

Bifidobacterium lactis attenuates onset of inflammation in a murine model of colitis

AIM To assess the anti-inflammatory effect of the probiotic Bifidobacterium lactis (B. lactis) in an adoptive transfer model of colitis. METHODS Donor and recipient mice received either B. lactis or bacterial culture medium as control (deMan Rogosa Sharpe) in drinking water for one week prior to transfer of a mix of naive and regulatory T cells until sacrifice. RESULTS All recipient mice developed signs of colonic inflammation, but a significant reduction of weight loss was observed in B. lactis-fed recipient mice compared to control mice. Moreover, a trend toward a diminution of mucosal thickness and attenuated epithelial damage was revealed. Colonic expression of pro-inflammatory and T cell markers was significantly reduced in B. lactis-fed recipient mice compared to controls. Concomitantly, forkhead box protein 3, a marker of regulatory T cells, was significantly up-regulated by B. lactis. CONCLUSION Daily oral administration of B. lactis was able to reduce inflammatory and T cells mediators and to promote regulatory T cells specific markers in a mouse model of colitis.

Prognostic values of α2-macroglobulin, fibrinogen and albumin in regards to mortality and frailty in old rats

The study aimed to determine if acute phase proteins (APP) are markers of frailty in old rats. We evaluated in male Wistar rats at 96 weeks of age (n=72) whether single measurements of alpha(2)-macroglobulin, fibrinogen and albumin are predictive of mortality, body weight loss and inflammatory status during a 10-week follow-up period. Rats were clustered depending on levels of these APP at baseline. Rats with extremely high levels of alpha(2)-macroglobulin or fibrinogen (upper quartiles), or extremely low level of albumin (lower quartile), had an 11.6, 8.1 and 5.3-fold higher risk of mortality, respectively, than other rats. Body weight loss was negatively correlated with alpha(2)-macroglobulin, a trend was observed with fibrinogen (P=0.08) but not with albumin. Rats with fibrinogen levels >4.0 g/L or alpha(2)-macroglobulin levels >91 mg/L (respective top halves) at 96 weeks of age had higher levels of alpha(2)-macroglobulin and fibrinogen and lower levels of albumin throughout the follow-up period and higher levels of sTNFR-1 and lipopolysaccharide-binding protein at 106 weeks of age. Highest levels of alpha(2)-macroglobulin, fibrinogen and lowest albumin were predictive of mortality, whereas moderate levels of alpha(2)-macroglobulin and fibrinogen were, according to body weight loss and inflammatory status, markers of frailty in old rats.

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.

Dietary Supplementation with Lacto-Wolfberry Enhances the Immune Response and Reduces Pathogenesis to Influenza Infection in Mice

Despite the availability of vaccines, influenza is a considerable public health problem, which emphasizes the need for development of additional strategies to enhance host defense against influenza. Wolfberry, or goji berry, long used as a medicinal food in China, has recently been shown to improve immune response in mice. Because immune response plays a key role in the bodys defense against pathogens, we hypothesized that wolfberry may increase host resistance to influenza infection by enhancing immune response. To test this hypothesis, we fed adult mice (4 mo old) a milk-based preparation of wolfberry called Lacto-Wolfberry (LWB) for 4 wk and then infected them with influenza A/Puerto Rico/8/34 (H1N1) while continuing the same experimental diets. Viral titer, lung pathology, and immune response were determined at different time points postinfection. LWB supplementation prevented infection-induced weight loss and reduced lung pathology on days 6 and 9 postinfection (P < 0.05). LWB-fed mice showed overall, significantly higher concanavalin A-induced IL-2 production (P < 0.05). Furthermore, we found positive correlations between weight loss and lung viral titer, pathology score, TNFα, and IL-6 production as well as negative correlations with T cell proliferation and IL-2 production (all P ≤ 0.05). These results indicate that LWB supplementation can attenuate symptoms and pathology of influenza infection by decreasing inflammatory cytokines in lungs while enhancing systemic T cell-mediated function as measured by their ability to produce IL-2.

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.