The Human Microbiome Project (HMP), funded as an initiative of the NIH Roadmap for Biomedical Research (http://nihroadmap.nih.gov), is a multi-component community resource. The goals of the HMP are: (1) to take advantage of new, high-throughput technologies to characterize the human microbiome more fully by studying samples from multiple body sites from each of at least 250 “normal” volunteers; (2) to determine whether there are associations between changes in the microbiome and health/disease by studying several different medical conditions; and (3) to provide both a standardized data resource and new technological approaches to enable such studies to be undertaken broadly in the scientific community. The ethical, legal, and social implications of such research are being systematically studied as well. The ultimate objective of the HMP is to demonstrate that there are opportunities to improve human health through monitoring or manipulation of the human microbiome. The history and implementation of this new program are described here.

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Interferon T (IFNT), originally identified as a pregnancy recognition hormone, is a type I interferon that is related to the various IFNat species (IFNais). Ovine IFNT has antiviral activity similar to that of human IFNaA on the Madin-Darby bovine kidney (MDBK) cell line and is equally effective in inhibiting cell proliferation. In this study, IFNT was found to differ from IFNaLA in that it was >30-fold less toxic to MDBK cells at high concentrations. Excess IFNT did not block the cytotoxicity ofIFNaA on MDBK cells, suggesting that these two type I IFNs recognize the type I IFN receptor differently on these cells. In direct binding studies, 251I-lFNT had a Kd of 3.90 x 10-10 M for receptor on MDBK cells, whereas that of 1251-IFNaA was 4.45 x 10-11 M. Consistent with the higher binding affinity, IFNaA was severalfold more effective than IFNT in competitive binding against 1251-IFNT to receptor on MDBK cells. Paradoxically, the two IFNs had similar specific antiviral activities on MDBK cells. However, maximal IFN antiviral activity required only fractional occupancy of receptors, whereas toxicity was associated with maximal receptor occupancy. Hence, IFNaA, with the higher binding affinity, was more toxic than IFN-. The IFNs were similar in inducing the specific phosphorylation of the type I receptor-associated tyrosine kinase Tyk2, and the transcription factors Statla and Stat2, suggesting that phosphorylation of these signal transduction proteins is not involved in the cellular toxicity associated with type I IFNs. Experiments using synthetic peptides suggest that differences in the interaction at the N termini of IFN-and IFNa with the type I receptor complex contribute significantly to differences in high-affinity equilibrium binding of these molecules. It is postulated that such a differential recognition of the receptor is responsible for the similar antiviral but different cytotoxic effects of these IFNs. Moreover, these data imply that receptors are "spare" with respect to certain biological properties, and we speculate that IFNs may induce a concentrationdependent selective association of receptor subunits.

Staphylococcal enterotoxins are a family of structurally related proteins that are produced by Staphylococcus aureus. In addition to their role in the pathogenicity of food poisoning, these microbial superantigens have profound effects on the immune system, which makes them useful tools for understanding its mechanism of action. These molecules (24-30 kDa) are highly hydrophilic and exhibit low alpha helix and high beta pleated sheet content, suggesting a flexible, accessible structure. Staphylococcal enterotoxins are among the most potent activators of T lymphocytes known. The receptors for staphylococcal enterotoxins on antigen-presenting cells are major histocompatibility complex (MHC) class II molecules. Further, the alpha-helical regions of the class II molecule are essential for function and appear to interact directly with the NH2-terminal region of staphylococcal enterotoxins such as SEA. Recent studies have shown that a complex of staphylococcal enterotoxin and MHC class II molecules is required for binding to the V beta region of the T cell antigen receptor. Staphylococcal enterotoxin mitogenic activity is dependent on induction of interleukin 2, which may be intimately involved in the mechanism of toxicity. The mouse minor lymphocyte stimulating (M1s) "endogenous" self-superantigen has been shown to be a retroviral gene product, so this too is apparently a microbial superantigen. An understanding of the mechanisms of action of these microbial superantigens has implications for normal and pathological immune functions.

Staphylococcal enterotoxins (SE) are exoproteins produced by Staphylococcus aureus that act as superantigens and have been implicated as a leading cause of food-borne disease and toxic shock. Little is known about how these molecules penetrate the gut lining and gain access to both local and systemic immune tissues. To model movement in vitro of staphylococcal enterotoxins, we have employed a monolayer system composed of crypt-like human colonic T-84 cells. SEB and SEA showed comparable dose-dependent transcytosis in vitro, while toxic shock syndrome toxin (TSST-1) exhibited increased movement at lower doses. Synthetic peptides corresponding to specific regions of the SEB molecule were tested in vitro to identify the domain of the protein involved in the transcytosis of SE. A toxin peptide of particular interest contains the amino acid sequence KKKVTAQELD, which is highly conserved across all SE. At a toxin-to-peptide ratio of 1:10, movement of SEB across the monolayers was reduced by 85%. Antisera made against the SEB peptide recognized native SEB and also inhibited SEB transcytosis. Finally, the conserved 10-amino-acid peptide inhibited transcytosis of multiple staphylococcal enterotoxins, SEA, SEE, and TSST-1. These data demonstrate that this region of the staphylococcal enterotoxins plays a distinct role in toxin movement across epithelial cells. It has implications for the prevention of staphylococcal enterotoxin-mediated disease by design of a peptide vaccine that could reduce systemic exposure to oral or inhaled superantigens. Since the sequence identified is highly conserved, it allows for a single epitope blocking the transcytosis of multiple SE.

Prostaglandin E2 (PGE2) regulates production of a wide array of cytokines. We have found that PGE2 can upregulate the levels of both interleukin-10 (IL-10) and IL-6 produced by activated murine macrophages, but the molecular pathways leading to their augmentation differ. Synthesis of IL-10 in response to PGE2 is dependent on p38 MAP kinase activity, whereas synthesis of IL-6 is not. Evidence to support this derives from two experimental approaches. First, we established that PGE2 is effective in elevating IL-10 levels only when it is added to cells in which p38 kinase has been activated. In contrast, PGE2 can augment IL-6 levels regardless of whether or not p38 kinase is active. Second, we showed that inhibitors that are selective for p38 kinase prevent the IL-10 response to PGE2 but not the IL-6 response. We found that p38 kinase inhibitors are able to inhibit IL-6 production in activated macrophages, but this occurs primarily as a result of their concurrent inhibition of cyclooxygenase-2 and endogenous PGE2 synthesis. These results indicate that macrophage IL-10 and IL-6 expression is differentially regulated by PGE2 and p38 MAP kinase in murine inflammatory macrophages.

The NIH sponsored a scientific workshop, “Soy Protein/Isoflavone Research: Challenges in Designing and Evaluating Intervention Studies,” July 28–29, 2009. The workshop goal was to provide guidance for the next generation of soy protein/isoflavone human research. Session topics included population exposure to soy; the variability of the human response to soy; product composition; methods, tools, and resources available to estimate exposure and protocol adherence; and analytical methods to assess soy in foods and supplements and analytes in biologic fluids and other tissues. The intent of the workshop was to address the quality of soy studies, not the efficacy or safety of soy. Prior NIH workshops and an evidence-based review questioned the quality of data from human soy studies. If clinical studies are pursued, investigators need to ensure that the experimental designs are optimal and the studies properly executed. The workshop participants identified methodological issues that may confound study results and interpretation. Scientifically sound and useful options for dealing with these issues were discussed. The resulting guidance is presented in this document with a brief rationale. The guidance is specific to soy clinical research and does not address nonsoy-related factors that should also be considered in designing and reporting clinical studies. This guidance may be used by investigators, journal editors, study sponsors, and protocol reviewers for a variety of purposes, including designing and implementing trials, reporting results, and interpreting published epidemiological and clinical studies.

The related staphylococcal toxins staphylococcal enterotoxin A (SEA) and toxic shock syndrome toxin 1 (TSST-1) are microbial superantigens. They require interaction with class II major histocompatibility complex (MHC) molecules to activate T cells. We have previously identified a binding site on SEA, the N-terminal 45 amino acids, as well as its corresponding receptor on the MHC antigen, residues 65-85 of the fi chain. To further characterize the structural basis for SEA binding to class II MHC molecules we have examined its relationship to TSST-1 binding. Both toxins bound similarly to murine A20 cells, but blockage of binding was observed only with the homologous toxin, which suggests that the binding sites for the two toxins on A20 cells are distinct. In contrast, specific binding of SEA was greater than that of TSST-1 on human Raji Staphylococcal enterotoxin A (SEA) is the most potent T-cell mitogen known, stimulating DNA synthesis, interferon-y production, and interleukin 2 production at concentrations as low as 10-16 M (1-3). It has been described as a microbial superantigen because of its ability to stimulate all T cells bearing particular T-cell antigen receptor VB regions (4, 5). Antigen-presenting cells are required for SEA activity (6, 7), and recently it has been shown that class II major histocompatibility complex (MHC) molecules are the specific receptors on the antigen-presenting cell for SEA (8-10). Unlike classically presented antigens SEA is not processed prior to binding (6,7,11), nor is its presentation restricted by polymorphic portions of class II molecules (7).The N-terminal region of SEA has been identified previously as a site on SEA that is responsible for its interaction with MHC class II antigens (12). Moreover, the same region of SEA is capable ofbinding to class II antigens from different species-i.e., human leukocyte antigens (HLA) and murine immune-associated antigens (Ia) (12). SEA and staphylococcal enterotoxin B (SEB) are reported to compete for binding to HLA-DR, implying that they bind to the same region on the MHC molecule (10). In contrast, SEB and toxic shock syndrome toxin 1 (TSST-1) have been shown to interact at different sites (13). We have further examined the complex binding ofmicrobial superantigens to class II MHC molecules by using synthetic peptides, and we have detected multiple binding sites for SEA on human Raji cells, one of which overlaps with that for TSST-1. MATERIALS AND METHODSToxins. SEA and TSST-1 were obtained from Toxin Technology (Madison, WI). SEA was homogenous by SDS gel electrophoresis (1). For biotinylation, 1 mg ofSEA or TSST-1 was dissolved in 50 mM sodium bicarbonate buffer (pH = 9.6) with 0.02 mg of sulfosuccinimidyl 6-(biotinamide) hexanoate (NHS-LC-biotin; Pierce) and incubated on ice for 2 hr. 125The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.