Improving selection of markers in nutrition research: evaluation of the criteria proposed by the ILSI Europe Marker Validation Initiative.

The conduct of high-quality nutrition research requires the selection of appropriate markers as outcomes, for example as indicators of food or nutrient intake, nutritional status, health status or disease risk. Such selection requires detailed knowledge of the markers, and consideration of the factors that may influence their measurement, other than the effects of nutritional change. A framework to guide selection of markers within nutrition research studies would be a valuable tool for researchers. A multidisciplinary Expert Group set out to test criteria designed to aid the evaluation of candidate markers for their usefulness in nutrition research and subsequently to develop a scoring system for markers. The proposed criteria were tested using thirteen markers selected from a broad range of nutrition research fields. The result of this testing was a modified list of criteria and a template for evaluating a potential marker against the criteria. Subsequently, a semi-quantitative system for scoring a marker and an associated template were developed. This system will enable the evaluation and comparison of different candidate markers within the same field of nutrition research in order to identify their relative usefulness. The ranking criteria of proven, strong, medium or low are likely to vary according to research setting, research field and the type of tool used to assess the marker and therefore the considerations for scoring need to be determined in a setting-, field- and tool-specific manner. A database of such markers, their interpretation and range of possible values would be valuable to nutrition researchers.

Type I interferons protect neonates from acute inflammation through interleukin 10-producing B cells.

Newborns and infants are highly susceptible to viral and bacterial infections, but the underlying mechanism remains poorly understood. We show that neonatal B cells effectively control the production of proinflammatory cytokines by both neonatal plasmacytoid and conventional dendritic cells, in an interleukin (IL) 10-dependent manner, after Toll-like receptor (TLR) 9 triggering. This antiinflammatory property of neonatal B cells may extend to other TLR agonists (Pam3CSK4, lipopolysaccharide, and R848) and viruses. In the absence of B cells or of CD5(+) B cell subsets, neonatal mice developed stronger inflammatory responses and became lethally susceptible to CpG challenge after galactosamine sensitization, whereas wild-type (WT) mice were resistant. Paradoxically, interferon (IFN)-alpha/beta enhanced the inflammatory response to CpG challenge in adult mice, whereas they helped to control neonatal acute inflammation by stimulating the secretion of IL-10 by neonatal B cells. Finally, WT neonatal B cells rescued IL-10(-/-) neonates from a lethal CpG challenge, whereas IFN-alpha/beta receptor-deficient B cells did not. Our results show that type I IFNs support a negative regulatory role of neonatal B cells on TLR-mediated inflammation, with important implications for neonatal inflammation and infection.

Monitoring immune modulation by nutrition in the general population: identifying and substantiating effects on human health.

Optimal functioning of the immune system is crucial to human health, and nutrition is one of the major exogenous factors modulating different aspects of immune function. Currently, no single marker is available to predict the effect of a dietary intervention on different aspects of immune function. To provide further guidance on the assessment and interpretation of the modulation of immune functions due to nutrition in the general population, International Life Sciences Institute Europe commissioned a group of experts from academia, government and the food industry to prepare a guidance document. A draft of this paper was refined at a workshop involving additional experts. First, the expert group defined criteria to evaluate the usefulness of immune function markers. Over seventy-five markers were scored within the context of three distinct immune system functions: defence against pathogens; avoidance or mitigation of allergy; control of low-grade (metabolic) inflammation. The most useful markers were subsequently classified depending on whether they by themselves signify clinical relevance and/or involvement of immune function. Next, five theoretical scenarios were drafted describing potential changes in the values of markers compared with a relevant reference range. Finally, all elements were combined, providing a framework to aid the design and interpretation of studies assessing the effects of nutrition on immune function. This stepwise approach offers a clear rationale for selecting markers for future trials and provides a framework for the interpretation of outcomes. A similar stepwise approach may also be useful to rationalise the selection and interpretation of markers for other physiological processes critical to the maintenance of health and well-being.

Thrombospondin 1 is an autocrine negative regulator of human dendritic cell activation.

Thrombospondin 1 (TSP) elicits potent antiinflammatory activities in vivo, as evidenced by persistent, multiorgan inflammation in TSP null mice. Herein, we report that DCs represent an abundant source of TSP at steady state and during activation. Human monocyte-derived immature dendritic cells (iDCs) spontaneously produce TSP, which is strongly enhanced by PGE2 and to a lesser extent by transforming growth factor (TGF) beta, two soluble mediators secreted by macrophages after engulfment of damaged tissues. Shortly after activation via danger signals, DCs transiently produce interleukin (IL) 12 and tumor necrosis factor (TNF) alpha, thereby eliciting protective and inflammatory immune responses. Microbial stimuli increase TSP production, which is further enhanced by IL-10 or TGF-beta. The endogenous TSP produced during early DC activation negatively regulates IL-12, TNF-alpha, and IL-10 release through its interactions with CD47 and CD36. After prolonged activation, DCs extinguish their cytokine synthesis and become refractory to subsequent stimulation, thereby favoring the return to steady state. Such "exhausted" DCs continue to release TSP but not IL-10. Disrupting TSP-CD47 interactions during their restimulation restores their cytokine production. We conclude that DC-derived TSP serves as a previously unappreciated negative regulator contributing to arrest of cytokine production, further supporting its fundamental role in vivo in the active resolution of inflammation and maintenance of steady state.

Antigen persistence is required for dendritic cell licensing and CD8+ T cell cross-priming.

It has been demonstrated that CD4(+) T cells require Ag persistence to achieve effective priming, whereas CD8(+) T cells are on "autopilot" after only a brief exposure. This finding presents a disturbing conundrum as it does not account for situations in which CD8(+) T cells require CD4(+) T cell help. We used a physiologic in vivo model to study the requirement of Ag persistence for the cross-priming of minor histocompatibility Ag-specific CD8(+) T cells. We report inefficient cross-priming in situations in which male cells are rapidly cleared. Strikingly, the failure to achieve robust CD8(+) T cell activation is not due to a problem with cross-presentation. In fact, by providing "extra help" in the form of dendritic cells (DCs) loaded with MHC class II peptide, it was possible to achieve robust activation of CD8(+) T cells. Our data suggest that the "licensing" of cross-presenting DCs does not occur during their initial encounter with CD4(+) T cells, thus accounting for the requirement for Ag persistence and suggesting that DCs make multiple interactions with CD8(+) T cells during the priming phase. These findings imply that long-lived Ag is critical for efficient vaccination protocols in which the CD8(+) T cell response is helper-dependent.

Does amygdalar perfusion correlate with antidepressant response to partial sleep deprivation in major depression?

This study used functional MRI (fMRI) to clarify the sites of brain activity associated with the antidepressant effects of sleep deprivation (SD). We hypothesized: (1) baseline perfusion in right and left amygdalae will be greater in responders than in nonresponders; (2) following partial sleep deprivation (PSD), perfusion in responders' right and left amygdalae would decrease. Seventeen unmedicated outpatients with current major depression and eight controls received perfusion-weighted fMRI and structural MRI at baseline and following 1 night of late-night PSD. Baseline bilateral amygdalar perfusion was greater in responders than nonresponders. Clusters involving both amygdalae decreased from baseline to PSD specifically in responders. Right amygdalar perfusion diverged with PSD, increasing in nonresponders and decreasing in responders. These novel amygdalar findings are consistent with the overarousal hypothesis of SD as well as other functional imaging studies showing increased baseline amygdalar activity in depression and decreased amygdalar activity with remission or antidepressant medications.

Dendritic-cell maturation alters intracellular signaling networks, enabling differential effects of IFN-alpha/beta on antigen cross-presentation.

The broad and often contrasting effects of type I interferons (IFNs) in innate and adaptive immunity are belied by the signaling via a single receptor, IFN-alpha receptor (IFNAR). Here, we show that IFN-alpha/beta induces opposing effects on the immunologic outcome of antigen cross-presentation depending on dendritic cell (DC) maturation status. Despite equivalent IFNAR expression, immature conventional DCs (cDCs) activate STAT1 in response to IFN-alpha/beta, whereas exposure of mature DCs to IFN-alpha/beta results in signaling via STAT4. Microarray analysis revealed numerous transcriptional changes resulting from the altered signaling. Importantly, STAT1 signaling resulted in significant inhibition of CD40L-induced IL-12 production, accounting for the inhibition of CD8+ T-cell activation. These data provide evidence for a molecular switch in signaling pathways concomitant with DC maturation that offers a novel mechanism by which DCs modulate the integration of signals from the surrounding environment.

Decreased perfusion in young alcohol-dependent women as compared with age-matched controls.

AIM: To use the superior spatial resolution of magnetic resonance imaging (MRI) to examine differences in cerebral perfusion between young alcohol dependent and normal women. METHODS: Eight alcohol dependent women and 8 controls (all ages 18-25) received single-slice resting perfusion-weighted MRI (directly proportional to brain blood flow), with slices located above the corpus callosum. RESULTS: Alcohol-dependent women had decreased perfusion in prefrontal and left parietal regions. CONCLUSIONS: Reduced perfusion has not previously been reported in young, physically healthy alcohol dependent females, yet is consistent with previously reported decreased cerebral activity in alcohol dependence.

Semimature stage: a checkpoint in a dendritic cell maturation program that allows for functional reversion after signal-regulatory protein-alpha ligation and maturation signals.

CD47 on live cells actively engages signal-regulatory protein-alpha (SIRP-alpha) on phagocytes and delivers a negative signal that prevents their elimination. We evaluated the biological consequences of SIRP-alpha ligation on the dendritic cell (DC) response to maturation signals and the potential interplay with the IL-10/IL-10R inhibitory pathway. At first, CD47/SIRP-alpha allowed the generation of mature migratory DCs not producing IL-12, IFN-gamma-inducible protein-10, and CCL19. Rather, they secreted neutrophils attracting chemokine CXCL5 and IL-1beta, reflecting a partial block in functional DC maturation. Afterward, semimature DCs functionally regressed in an IL-10-independent fashion toward cells that retrieved the cardinal features of immature DCs: re-expression of CCR5, loss of DC-lysosome-associated membrane protein, high endocytosis, and impaired allostimulatory functions. The global gene expression profile of IL-10 and SIRP-alpha-ligated DC demonstrated two distinct molecular pathways. IL-10R and SIRP-alpha expression were reciprocally down-regulated by CD47 and IL-10, respectively. These results emphasize that the SIRP-alpha pathway might be part of the molecular machinery used by the DC to dampen or resolve an inflammatory response in an IL-10-independent manner.

IL-2 is required for the activation of memory CD8+ T cells via antigen cross-presentation.

Dendritic cells (DCs) are capable of capturing exogenous Ag for the generation of MHC class I/peptide complexes. For efficient activation of memory CD8(+) T cells to occur via a cross-presentation pathway, DCs must receive helper signals from CD4(+) T cells. Using an in vitro system that reflects physiologic recall memory responses, we have evaluated signals that influence helper-dependent cross-priming, while focusing on the source and cellular target of such effector molecules. Concerning the interaction between CD4(+) T cells and DCs, we tested the hypothesis that CD40 engagement on DCs is critical for IL-12p70 (IL-12) production and subsequent stimulation of IFN-gamma release by CD8(+) T cells. Although CD40 engagement on DCs, or addition of exogenous IL-12 are both sufficient to overcome the lack of help, neither is essential. We next evaluated cytokines and chemokines produced during CD4(+) T cell/DC cross talk and observed high levels of IL-2 produced within the first 18-24 h of Ag-specific T cell engagement. Functional studies using blocking Abs to CD25 completely abrogated IFN-gamma production by the CD8(+) T cells. Although required, addition of exogenous IL-2 did not itself confer signals sufficient to overcome the lack of CD4(+) T cell help. Thus, these data support a combined role for Ag-specific, cognate interactions at the CD4(+) T cell/DC as well as the DC/CD8(+) T cell interface, with the helper effect mediated by soluble noncognate signals.

Thrombospondin/CD47 interaction: a pathway to generate regulatory T cells from human CD4+ CD25- T cells in response to inflammation.

Thymus-derived CD4+ CD25+ T regulatory cells (Tregs) are essential for the maintenance of self-tolerance. What critical factors and conditions are required for the extra-thymic development of Tregs remains an important question. In this study, we show that the anti-inflammatory extracellular matrix protein, thrombospondin-1, promoted the generation of human peripheral regulatory T cells through the ligation of one of its receptor, CD47. CD47 stimulation by mAb or a thrombospondin-1 peptide induced naive or memory CD4+ CD25- T cells to become suppressive. The latter expressed increased amounts of CTLA-4, OX40, GITR, and Foxp3 and inhibited autologous Th0, Th1, and Th2 cells. Their regulatory activity was contact dependent, TGF-beta independent, and partially circumvented by IL-2. This previously unknown mechanism to induce human peripheral Tregs in response to inflammation may participate to the limitation of collateral damage induced by exacerbated responses to self or foreign Ags and thus be relevant for therapeutic intervention in autoimmune diseases and transplantation.

A two-step induction of indoleamine 2,3 dioxygenase (IDO) activity during dendritic-cell maturation.

Prostaglandins, a family of lipidic molecules released during inflammation, display immunomodulatory properties in several models. One use includes exposure of monocyte-derived dendritic cells (DCs) to a cocktail of cytokines that contains prostaglandin E2 (PGE2) for purposes of maturation; such cells are currently being used for cancer immunotherapy trials. Our analysis of the transcription profile of DCs matured in the presence of tumor necrosis factor alpha (TNFalpha) and PGE2 revealed a strong up-regulation of indoleamine 2-3 dioxygenase (IDO), an enzyme involved in tryptophan catabolism and implicated in both maternal and T-cell tolerance. Using quantitative assays to monitor levels of IDO mRNA, protein expression, and enzyme activity, we report that PGE2 induces mRNA expression of IDO; however, a second signal through TNF receptor (TNF-R) or a Toll-like receptor (TLR) is necessary to activate the enzyme. Interestingly, use of TNFalpha, lipopolysaccharide, or Staphylococcus aureus Cowan I strain (SAC) alone does not induce IDO. The effect of PGE2 is mediated by activation of adenylate cyclase via the Gs-protein-coupled receptor E prostanoid-2 (EP2). A better understanding of these regulatory mechanisms and the crosstalk between TNF-R/TLR and EP2 signaling pathways will provide insight into the regulation of T-cell activation by DCs and may help to improve existing immunotherapy protocols.

IFN-alpha/beta enhances BCR-dependent B cell responses.

Type I interferon (IFN-I) is constitutively produced in the bone marrow (BM), and induced at sites of inflammation and following infection by viruses or microorganisms. We have previously shown that IFN-I regulates the generation and selection of normal B cell populations in the BM. In the present work, we assess the effects of IFN-I on mature B cell function by monitoring the responses of IFN-alpha/beta-treated murine splenic B cells to apoptotic, mitogenic and activating stimuli. A similar analysis is performed on BM mature B cells obtained from wild-type or IFN-I receptor-deficient mice. IFN-alpha/beta is shown to induce B cells to a state of partial activation characterized by the up-regulation of CD69, CD86 and CD25 molecules in the absence of either proliferation or terminal differentiation. B cells treated with IFN-alpha/beta show an increased survival and resistance to Fas-mediated apoptosis. IFN-alpha/beta also enhances B cell responses to BCR ligation such as calcium fluxes, IgM internalization, induction of activation markers and proliferation. These results indicate that in addition to its inhibitory effect on viral replication and T cell apoptosis, IFN-alpha/beta plays an essential role during an inflammatory response by lowering the threshold for B cell induction, thereby promoting fast and polyclonal antibody responses.

Type I Interferon controls the onset and severity of autoimmune manifestations in lpr mice.

Type I Interferons (IFN-I) are immunoregulatory cytokines that enhance activation and survival of many cellular components of the immune system. In the present work, we evaluated the effect of IFN-I on the development of the lymphoproliferative disorder in Fas-defective lpr mice. We report that sustained injection of polyinosinic:polycytidylic acid, a potent inducer of IFN-I, in B6 lpr mice resulted in a dramatic aggravation of the renal disease, higher titers of autoantibodies, a 10-fold increase in serum Ig and accumulation of activated lymphocytes. Moreover, introducing a null mutation for the IFN-I-Receptor gene into the lpr background resulted in dramatic decrease of immune complexes deposition in the kidney and reduced lymphadenopathy. While several recent reports correlated serum levels of IFN-alpha with disease activity in systemic Lupus erythematosus patients, our findings establish a causal link from IFN-I production to the onset and severity of another related autoimmune syndrome.