Incorporation of cis-9,trans-11 or trans-10,cis-12 conjugated linoleic acid into plasma and cellular lipids in healthy men

This study investigated the incorporation of cis-9,trans-11 conjugated linoleic acid (c9,t11 CLA) and trans-10,cis-12-CLA (t10,c12 CLA) into plasma and peripheral blood mononuclear cell (PBMC) lipids when consumed as supplements highly enriched in these isomers. Healthy men (n = 49, age 31 +/- 8 years) consumed one, two, and four capsules containing similar to600 mg of either c9,t11 CIA or t10,c12 CLA per capsule for sequential 8 week periods followed by a 6 week washout before consuming the alternative isomer. Both isomers were incorporated in a dosedependent manner into plasma phosphatidylcholine (PC) (c9,t11 CLA r = 0.779, t10,c12 CLA r = 0.738; P < 0.0001) and cholesteryl ester (CE) (c9,t11 CLA r = 0.706, t10,c12 CLA r = 0.788; P < 0.0001). Only t10,c12 CLA was enriched in plasma nonesterified fatty acids. Both c9,t11 CIA and t10,c12 CLA were incorporated linearly into PBMC total lipids (r = 0.285 and r = 0.273, respectively; P < 0.0005). The highest concentrations of c9,t11 CLA and t10,c12 CLA in PBMC lipids were 3- to 4-fold lower than those in plasma PC and CE. These data suggest that the level of intake is a major determinant of plasma and PBMC CLA content, although PBMCs appear to incorporate both CLA isomers less readily.

Measles vaccination and antibody response in autism spectrum disorders

Objective: To test the hypothesis that measles vaccination was involved in the pathogenesis of autism spectrum disorders (ASD) as evidenced by signs of a persistent measles infection or abnormally persistent immune response shown by circulating measles virus or raised antibody titres in children with ASD who had been vaccinated against measles, mumps and rubella (MMR) compared with controls. Design: Case-control study, community based. Methods: A community sample of vaccinated children aged 10-12 years in the UK with ASD (n = 98) and two control groups of similar age, one with special educational needs but no ASD (n = 52) and one typically developing group (n = 90), were tested for measles virus and antibody response to measles in the serum. Results: No difference was found between cases and controls for measles antibody response. There was no dose-response relationship between autism symptoms and antibody concentrations. Measles virus nucleic acid was amplified by reverse transcriptase-PCR in peripheral blood mononuclear cells from one patient with autism and two typically developing children. There was no evidence of a differential response to measles virus or the measles component of the MMR in children with ASD, with or without regression, and controls who had either one or two doses of MMR. Only one child from the control group had clinical symptoms of possible enterocolitis. Conclusion: No association between measles vaccination and ASD was shown.

In vitro immunomodulatory activity of Lactobacillus fermentum CECT5716 and Lactobacillus salivarius CECT5713: two probiotic strains isolated from human breast milk

Commensal bacteria, including some species of lactobacilli commonly present in human breast milk, appear to colonize the neonatal gut and contribute to protection against infant infections, suggesting that lactobacilli could potentially modulate immunity. In this study, we evaluated the potential of two Lactobacillus strains isolated from human milk to modulate the activation and cytokine profile of peripheral blood mononuclear cell (PBMC) subsets in vitro. Moreover, these effects were compared to the same probiotic species of non-milk origin. Lactobacillus salivarius CECT5713 and Lactobacillus fermentum CECT5716 at 105, 106 and 107 bacteria/mL were co-cultured with PBMC (106/mL) from 8 healthy donors for 24 h. Activation status (CD69 and CD25 expressions) of natural killer (NK) cells (CD56+), total T cells (CD3+), cytotoxic T cells (CD8+) and CD4+ T cells was determined by flow cytometry. Regulatory T cells (Treg) were also quantified by intracellular Foxp3 evaluation. Regarding innate immunity, NK cells were activated by addition of both Lactobacillus strains, and in particular, the CD8+ NK subset was preferentially induced to highly express CD69 (90%, p<0.05). With respect to acquired immunity, approximately 9% of CD8+ T cells became activated after co-cultivation with L. fermentum or L salivarius. Although CD4+ T cells demonstrated a weaker response, there was a preferential activation of Treg cells (CD4+CD25+Foxp3+) after exposure to both milk probiotic bacteria (p<0.05). Both strains significantly induced the production of a number of cytokines and chemokines, including TNFα, IL-1β, IL-8, MIP-1α, MIP-1β, and GM-CSF, but some strain-specific effects were apparent. This work demonstrates that L salivarius CECT5713 and L. fermentum CECT5716 enhanced both natural and acquired immune responses, as evidenced by the activation of NK and T cell subsets and the expansion of Treg cells, as well as the induction of a broad array of cytokines.

Effect of olive oil on immune function in middle-aged men

Consumption of diets rich in monounsaturated fatty acids (MUFAs) has been linked with a low prevalence of atherosclerosis and there has been great interest in the effects of MUFAs on lipoprotein metabolism. Less attention has been paid to the effects of MUFAs on the immune system, yet cells of the immune system are an inherent part of the inflammatory events involved in atherosclerosis and several animal studies showed that olive oil has some potent immunomodulatory actions. We therefore considered it important to investigate the effects of chronic consumption of MUFAs on several immune cell functions in healthy humans. Healthy middle-aged males entered a doubleblind, randomized, controlled trial in which they consumed either a MUFA diet or a control diet for 2 mo. There was a significant decrease in the expression of intercellular adhesion molecule 1 by peripheral blood mononuclear cells from subjects consuming the MUFA diet. Consumption of the MUFA diet did not affect natural killer cell activity or proliferation of mitogen-stimulated leukocytes. The effects of a MUFA-rich diet on adhesion molecule expression may have implications for the influence of dietary fat on inflammatory diseases, including atherosclerosis.

Comparative effects of six probiotic strains on immune function in vitro

There is considerable interest in the strain specificity of immune modulation by probiotics. The present study compared the immunomodulatory properties of six probiotic strains of different species and two genera in a human peripheral blood mononuclear cell (PBMC) model in vitro. Live cells of lactobacilli (Lactobacillus casei Shirota, L. rhamnosus GG, L. plantarum NCIMB 8826 and L. reuteri NCIMB 11951) and bifidobacteria (Bifidobacterium longum SP 07/3 and B. bifidum MF 20/5) were individually incubated with PBMC from seven healthy subjects for 24 h. Probiotic strains increased the proportion of CD69+ on lymphocytes, T cells, T cell subsets and natural killer (NK) cells, and increased the proportion of CD25+, mainly on lymphocytes and NK cells. The effects on activation marker expression did not appear to be strain specific. NK cell activity was significantly increased by all six strains, without any significant difference between strains. Probiotic strains increased production of IL-1β, IL-6, IL-10, TNF-α, granulocyte-macrophage colony-stimulating factor and macrophage inflammatory protein 1α to different extents, but had no effect on the production of IL-2, IL-4, IL-5 or TNF-β. The cytokines that showed strain-specific modulation included IL-10, interferon-γ, TNF-α, IL-12p70, IL-6 and monocyte chemotactic protein-1. The Lactobacillus strains tended to promote T helper 1 cytokines, whereas bifidobacterial strains tended to produce a more anti-inflammatory profile. The results suggest that there was limited evidence of strain-specific effects of probiotics with respect to T cell and NK cell activation or NK cell activity, whereas production of some cytokines was differentially influenced by probiotic strains.

Effects of oxidised low density lipoprotein on dendritic cells: a possible immunoregulatory component of the atherogenic micro-environment?

Objective: The objective of this study was to explore the relationship between low density lipoprotein (LDL) and dendritic cell (DC) activation, based upon the hypothesis that reactive oxygen species (ROS)-mediated modification of proteins that may be present in local DC microenvironments could be important as mediators of this activation. Although LDL are known to be oxidised in vivo, and taken up by macrophages during atherogenesis; their effect on DC has not been explored previously. Methods: Human DCs were prepared from peripheral blood monocytes using GM-CSF and IL-4. Plasma LDLs were isolated by sequential gradient centrifugation, oxidised in CuSO4, and oxidation arrested to yield mild, moderate and highly oxidised LDL forms. DCs exposed to these LDLs were investigated using combined phenotypic, functional (autologous T cell activation), morphological and viability assays. Results: Highly-oxidised LDL increased DC HLA-DR, CD40 and CD86 expression, corroborated by increased DC-induced T cell proliferation. Both native and oxidised LDL induced prominent DC clustering. However, high concentrations of highly-oxidised LDL inhibited DC function, due to increased DC apoptosis. Conclusions: This study supports the hypothesis that oxidised LDL are capable of triggering the transition from sentinel to messenger DC. Furthermore, the DC clustering–activation–apoptosis sequence in the presence of different LDL forms is consistent with a regulatory DC role in immunopathogenesis of atheroma. A sequence of initial accumulation of DC, increasing LDL oxidation, and DC-induced T cell activation, may explain why local breach of tolerance can occur. Above a threshold level, however, supervening DC apoptosis limits this, contributing instead to the central plaque core.

The effect of cruciferous and leguminous sprouts on genotoxicity, in vitro and in vivo

Vegetable consumption is associated with a reduced risk of colorectal cancer, which is the second most common cancer after lung/breast cancer within Europe. Some putative protective phytochemicals are found in higher amounts in young sprouts than in mature plants. The effect of an extract of mixed cruciferous and legume sprouts on DNA damage induced by H(2)O(2) was measured in HT29 cells using single cell microgelelectrophoresis (comet). Significant antigenotoxic effect (P < or = 0.05) was observed when HT29 cells were pre-incubated with the extract (100 and 200 microL/mL) for 24 hours and then challenged with H(2)O(2). A parallel design intervention study was carried out on 10 male and 10 female healthy adult volunteers (mean age = 25.5 years) fed 113 g of cruciferous and legume sprouts daily for 14 days. The effect of the supplementation was measured on a range of parameters, including DNA damage in lymphocytes (comet), the activity of various detoxifying enzymes (glutathione S-transferase, glutathione peroxidase, and superoxide dismutase), antioxidant status using the ferric reducing ability of plasma assay, plasma antioxidants (uric acid, ascorbic acid, and alpha-tocopherol), blood lipids, plasma levels of lutein, and lycopene. A significant antigenotoxic effect against H(2)O(2)-induced DNA damage was shown in peripheral blood lymphocytes of volunteers who consumed the supplemented diet when compared with the control diet (P = 0.04). No significant induction of detoxifying enzymes was observed during the study, neither were plasma antioxidant levels or activity altered. The results support the theory that consumption of cruciferous vegetables is linked to a reduced risk of cancer via decreased damage to DNA.

Evidence of immunomodulatory effects of a novel probiotic, Bifidobacterium longum bv. infantis CCUG 52486

Bifidobacterium longum bv. infantis CCUG 52486 was originally isolated from healthy elderly subjects and demonstrated to have particular ecological fitness and anti-pathogenic effects. Bifidobacteria are commonly associated with immunomodulatory properties, especially in older people, but this strain has not been investigated for effects on immune function. This study aimed to explore the immunomodulatory effects of this novel probiotic, compared with three commercial strains, B. longum SP 07/3, L. rhamnosus GG (L.GG) and L. casei Shirota (LcS). Human peripheral blood mononuclear cells (PBMCs) were isolated from fasting blood of young or older volunteers and exposed to probiotic strains or Con A. NK activity and activation, and cytokine release were enhanced by all probiotics with strain-specificities. The effect of B. infantis on NK activity was influenced by ageing. Except for L.GG, probiotics increased IFN-γ production to a much greater degree in young subjects, and increased IL-6 production to a much greater degree in older subjects. Based on IL-10/IL-12 ratios, B. infantis resulted in the most anti-inflammatory profile of all of the probiotics. These results suggest that B. infantis CCUG 52486 has strong immunomodulatory potential compared with well-known commercial strains, and that the immune response to probiotics may be influenced by ageing.

Immunomodulatory effects of a probiotic drink containing Lactobacillus casei Shirota in healthy older volunteers

PURPOSE: There is growing evidence that probiotics confer health benefits to the host by modulating immune function, especially in older people, where immunosenescence is a feature even of healthy ageing. The aim of this study was to investigate the effect of a probiotic drink containing Lactobacillus casei Shirota (LcS) on immune function in a healthy non-immunocompromised older population. METHODS: Thirty healthy old volunteers were recruited into a randomized placebo-controlled, single-blind crossover study. The volunteers were supplemented with the probiotic drink containing 1.3 × 10(10) CFU LcS or skimmed milk per day for 4 weeks, followed by 4 weeks of washout and were crossed over to the other treatment. Peripheral blood and saliva samples were collected at baseline and end of each treatment. RESULTS: Probiotic consumption was associated with a significant increase in natural killer (NK) cell activity relative to baseline and a significant decrease in the mean fluorescence intensity of CD25 expression in the resting T cells compared with placebo. Additionally, there was a trend towards an increased ratio of IL-10 to IL-12 relative to baseline after LcS intake. CONCLUSIONS: Consumption of a probiotic drink containing LcS improved NK cell activity and tended to produce a more anti-inflammatory cytokine profile in an older population.

Ovarian expression of insulin-like peptide 3 (INSL3) and its receptor (RXFP2) during development of bovine antral follicles and corpora lutea and measurement of circulating INSL3 levels during synchronized estrous cycles

Insulin-like peptide 3 (INSL3), a major product of testicular Leydig cells, is also expressed by the ovary but its functional role remains poorly understood. Here, we quantified expression of INSL3 and its receptor RXFP2 in theca interna (TIC) and granulosa (GC) compartments of developing bovine antral follicles and in corpora lutea (CL). INSL3 and RXFP2 mRNA levels were much higher in TIC than GC and increased progressively during follicle maturation with INSL3 peaking in large (11-18mm) estrogen-active follicles and RXFP2 peaking in 9-10mm follicles before declining in larger (11-18mm) follicles. Expression of both INSL3 and RXFP2 in CL was much lower than in TIC. In situ hybridization and immunohistochemistry confirmed abundant expression of INSL3 mRNA and protein in TIC. These observations indicate follicular TIC rather than CL as the primary site of both INSL3 production and action, implying a predominantly auto-/paracrine role in TIC. To corroborate the above findings, we showed that in vitro exposure of TIC to a luteinizing concentration of LH greatly attenuated expression of both INSL3 and its receptor while increasing progesterone secretion and expression of STAR and CYP11A1. Moreover, in vivo, a significant cyclic variation in plasma INSL3 was observed during synchronized estrous cycles. INSL3 and estradiol-17β followed a similar pattern, both increasing after luteolysis, before falling sharply after the LH surge. Thus, theca-derived INSL3, likely from the dominant pre-ovulatory follicle, is detectable in peripheral blood of cattle and expression is down-regulated during luteinisation induced by the pre-ovulatory LH surge. Collectively, these findings underscore the likely role of INSL3 as an important intrafollicular modulator of TIC function/steroidogenesis, whilst raising doubts about its potential contribution to CL function.

Genetic heterogeneity in erythropoietic protoporphyria: a study of the enzymatic defect in nine affected families

Erythropoietic protoporphyria (EPP) is associated with a deficiency of protohaem ferrolyase. We have used a novel assay for this enzyme based on its ability to utilize zinc as a substrate to investigate the inheritance of EPP in nine affected families. Zinc chelatase activity was markedly reduced in peripheral blood mononuclear cells from 14 EPP patients (mean, 3.3 nmol Zn protohaem/h/mg protein; range, 0.3-8.0) when compared with 41 controls (16.8 +/- 3.6) p less than 0.01. In three families with parent-to-child transmission of disease, the asymptomatic parent had an enzymatic activity within the normal range. In three pedigrees where the parents were asymptomatic, enzymatic activities were below the 95% confidence limits in both. Zinc chelatase activity was below the mean control value in 17 of the 18 parents in nine affected pedigrees, and six of seven asymptomatic offspring of patients with protoporphyria. The findings suggest that EPP is not transmitted as a simple dominant trait and that inheritance of more than one gene may be required for disease expression.

Impact of polysialylated CD56 on natural killer cell cytotoxicity

Background Siglec-7, a sialic acid binding inhibitory receptor expressed by NK cells is masked in vivo by a so far unknown ligand. It shows a strong binding prevalence for α-2,8-linked disialic acids in vitro. Results Here we describe the expression of PSA-NCAM (α-2,8-linked polysialic acid modified NCAM) on functional adult peripheral blood natural killer cells and examine its possible role in masking Siglec-7. Unmasking of Siglec-7 using Clostridium perfringens neuraminidase massively reduces NK cell cytotoxicity. By contrast a specific removal of PSA using Endo-NF does not lead to a reduction of NK cell cytotoxicity. Conclusion The results presented here therefore indicate that PSA-NCAM is not involved in masking Siglec-7.

1,8-Cineol inhibits nuclear translocation of NF-κB p65 and NF-κB-dependent transcriptional activity

Natural plant-derived products are commonly applied to treat a broad range of human diseases, including cancer as well as chronic and acute airway inflammation. In this regard, the monoterpene oxide 1,8-cineol, the active ingredient of the clinically approved drug Soledum®, is well-established for the therapy of airway diseases, such as chronic sinusitis and bronchitis, chronic obstructive pulmonary disease and bronchial asthma. Although clinical trials underline the beneficial effects of 1,8-cineol in treating inflammatory diseases, the molecular mode of action still remains unclear. Here, we demonstrate for the first time a 1,8-cineol-depending reduction of NF-κB-activity in human cell lines U373 and HeLa upon stimulation using lipopolysaccharides (LPS). Immunocytochemistry further revealed a reduced nuclear translocation of NF-κB p65, while qPCR and western blot analyses showed strongly attenuated expression of NF-κB target genes. Treatment with 1,8-cineol further led to increased protein levels of IκBα in an IKK-independent matter, while FRET-analyses showed restoring of LPS-associated loss of interaction between NF-κB p65 and IκBα. We likewise observed reduced amounts of phosphorylated c-Jun N-terminal kinase 1/2 protein in U373 cells after exposure to 1,8-cineol. In addition, 1,8-cineol led to decreased amount of nuclear NF-κB p65 and reduction of its target gene IκBα at protein level in human peripheral blood mononuclear cells. Our findings suggest a novel mode of action of 1,8-cineol through inhibition of nuclear NF-κB p65 translocation via IκBα resulting in decreased levels of proinflammatory NF-κB target genes and may therefore broaden the field of clinical application of this natural drug for treating inflammatory diseases.

Bacillus coagulans GBI-30, 6086 modulates Faecalibacterium prausnitzii in older men and women

Abstract Background: Advancing age is linked to a decrease in beneficial bacteria such as Bifidobacterium spp. and reduced aspects of innate immune function. Objectives: We investigated whether daily consumption of a probiotic [Bacillus coagulans GBI-30, 6086 (BC30); GanedenBC30] could improve immune function and gut function in men and women aged 65–80 y, using a double-blind, placebo-controlled crossover design. Method: Thirty-six volunteers were recruited and randomly assigned to receive either a placebo (microcrystalline cellulose) or the probiotic BC30 (1 3 109 colony-forming units/capsule). Volunteers consumed 1 treatment capsule per day for 28 d, followed by a 21-d washout period before switching to the other treatment. Blood and fecal samples were collected at the beginning and end of each treatment period. Fecal samples were used to enumerate bacterial groups and concentrations of calprotectin. Peripheral blood mononuclear cells (PBMCs) were extracted from whole blood to assess natural killer cell activity and lipopolysaccharide (LPS)-stimulated cytokine production. C-reactive protein concentrations were measured in plasma. Results: Consumption of BC30 significantly increased populations of Faecalibacterium prausnitzii by 0.1 log10 cells/mL more than during consumption of the placebo (P = 0.03), whereas populations of Bacillus spp. increased significantly by 0.5 log10 cells/mL from baseline in volunteers who consumed BC30 (P = 0.007). LPS-stimulated PBMCs showed a 0.2 ng/mL increase in the anti-inflammatory cytokine IL-10 28 d after consumption of BC30 (P < 0.05), whereas the placebo did not affect IL-10, and no overall difference was found in the effect of the treatments. Conclusions: Daily consumption of BC30 by adults aged 65–80 y can increase beneficial groups of bacteria in the human gut and potentially increase production of anti-inflammatory cytokines. This study shows the potential benefits of a probiotic to improve dysbiosis via modulation of the microbiota in older persons. J Nutr doi: 10.3945/jn.114.199802.

Targeted activation of toll-like receptors: conjugation of a toll-like receptor 7 agonist to a monoclonal antibody maintains antigen binding and specificity

Therapeutic activation of Toll-like receptors (TLR) has potential for cancer immunotherapy, for augmenting the activity of anti-tumor monoclonal antibodies (mAbs), and for improved vaccine adjuvants. A previous attempt to specifically target TLR agonists to dendritic cells (DC) using mAbs failed because conjugation led to non-specific binding and mAbs lost specificity. We demonstrate here for the first time the successful conjugation of a small molecule TLR7 agonist to an anti-tumour mAb (the anti-hCD 20 rituximab) without compromising antigen specificity. The TLR7 agonist UC-1V150 was conjugated to rituximab using two conjugation methods and yield, molecular substitution ratio, retention of TLR7 activity and specificity of antigen binding were compared. Both conjugation methods produced rituximab-UC-1V150 conjugates with UC-1V150 : rituximab ratio ranging from 1:1 to 3:1 with drug loading quantified by UV spectroscopy and drug substitution ratio verified by MALDI TOF mass spectroscopy. The yield of purified conjugates varied with conjugation method, and dropped as low as 31% using a method previously described for conjugating UC-1V150 to proteins, where a bifunctional crosslinker was firstly reacted with rituximab, and secondly to the TLR7 agonist. We therefore developed a direct conjugation method by producing an amine-reactive UV active version of UC-1V150, termed NHS:UC-1V150. Direct conjugation with NHS:UC-1V150 was quick and simple and gave improved conjugate yields of 65-78%. Rituximab-UC-1V150 conjugates had the expected pro-inflammatory activity in vitro (EC50 28-53 nM) with a significantly increased activity over unconjugated UC-1V150 (EC50 547 nM). Antigen binding and specificity of the rituxuimab-UC-1V150 conjugates was retained, and after incubation with human peripheral blood leukocytes, all conjugates bound strongly only to CD20-expressing B cells whilst no non-specific binding to CD20-negative cells was observed. Selective targeting of Toll-like receptor activation directly within tumors or to DC is now feasible.