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Phagosomes were purified from human neutrophils ingesting Salmonella typhimurium opsonized with adsorbed normal human serum or with rabbit IgG. Constituents within the phagosome were endogenously labeled by supplying the cells with 125INa during phagocytosis. Lactoferrin and vitamin B12 binding protein (TC1 and TC3), markers for specific granules, were present in the phagosomes from neutrophils ingesting S. typhimurium opsonized with IgG but were 3.5- to 5-fold less prominent in phagosomes from cells phagocytosing Salmonella bearing C3 fragments only. In contrast, iodinated azurophilic granule components, most prominently defensins, were the major constituents in phagosomes prepared under both opsonization conditions. Furthermore, labeled complement (CR1 and CR3) and immunoglobulin (Fc gamma RIII) receptors were incorporated in the phagosome regardless of the ligand mediating phagocytosis. These results suggest that the ligand-receptor interactions mediating phagocytosis influence incorporation of neutrophil-specific granule contents into phagosomes.

Research shows that chronic granulomatous diseases of childhood may be inherited through defective neutrophil cytosol factors.

Competing Interests: POTENTIAL CONFLICT OF INTEREST: Dr Sicherer served as a member of the National Institute of Allergy and Infectious Diseases (NIAID) expert panel and as the American Academy of Pediatrics representative of the coordinating committee of the updated guidelines discussed in this article; Dr Eichenfield served as a member of the expert panel and as the American Academy of Dermatology representative of the coordinating committee of the updated guidelines discussed in this article; Dr Sampson served as a member of the NIAID expert panel of the updated guidelines discussed in this article; is a consultant to Allertein Therapeutics, developing nanoparticle-based therapy for treating peanut allergy; and is employed by DBV Technologies, developing a patch for epicutaneous treatment of food allergies; and Dr Rotrosen has indicated he has no potential conflicts of interest to disclose.

Background: Food allergy is an important public health problem because it affects children and adults, can be severe and even life-threatening, and may be increasing in prevalence. Beginning in 2008, the National Institute of Allergy and Infectious Diseases, working with other organizations and advocacy groups, led the development of the first clinical guidelines for the diagnosis and management of food allergy. A recent landmark clinical trial and other emerging data suggest that peanut allergy can be prevented through introduction of peanut-containing foods beginning in infancy., Objectives: Prompted by these findings, along with 25 professional organizations, federal agencies, and patient advocacy groups, the National Institute of Allergy and Infectious Diseases facilitated development of addendum guidelines to specifically address the prevention of peanut allergy., Results: The addendum provides 3 separate guidelines for infants at various risk levels for the development of peanut allergy and is intended for use by a wide variety of health care providers. Topics addressed include the definition of risk categories, appropriate use of testing (specific IgE measurement, skin prick tests, and oral food challenges), and the timing and approaches for introduction of peanut-containing foods in the health care provider's office or at home. The addendum guidelines provide the background, rationale, and strength of evidence for each recommendation., Conclusions: Guidelines have been developed for early introduction of peanut-containing foods into the diets of infants at various risk levels for peanut allergy., (Published by Elsevier Inc.)

Immune tolerance-inducing therapies reprogramme immune cells to eliminate pathogenic immune responses while preserving protective immunity. The Immune Tolerance Network (ITN), sponsored by the US National Institutes of Health, was established in 1999 to evaluate new tolerance-inducing therapies and carry out mechanistic studies using a unique interactive approach in partnership with industry, academia and foundations. Ten years later, the ITN has carried out approximately 36 clinical trials and tolerance studies examining innovative tolerogenic approaches in the settings of allergy, autoimmune diseases and organ transplantation. ITN investigators have published more than 80 original research papers based on this work. This Timeline article summarizes the progress and challenges of clinical research in the ITN.

Complexities in sample handling, instrument setup and data analysis are barriers to the effective use of flow cytometry to monitor immunological parameters in clinical trials. The novel use of a central laboratory may help mitigate these issues.

Since 1991, the National Institute of Allergy and Infectious Diseases (NIAID) has funded four consecutive research initiatives to investigate the problem of high asthma prevalence, morbidity and mortality in poor urban communities. The multi-site studies conducted under these initiatives have identified key risk factors for asthma morbidity and novel interventions to improve asthma control. NIAID focuses its asthma and allergy programs on understanding the interaction of the immune system with allergens and infectious agents and identifying genetic and epigenetic elements that influence the immune system. A key goal in this field is to define mechanisms of immune system deviation and immune tolerance and apply this knowledge to generate improvements in asthma care and allergen immunotherapy. A related goal is to further understand the environmental, social, and immunological elements that impact on the development of inner-city asthma through in-depth characterization and longitudinal follow-up of inner-city children from the time of birth. In the past 5 years, NIH budgetary constraints have imposed many challenges for the academic research community. Despite these constraints, NIAID has maintained its support of a highly productive asthma and allergy research program.

The U.S. National Institutes of Health Roadmap and the U.S. Food and Drug Administration's Critical Path Initiative have endorsed the establishment of large academic clinical research networks as part of the solution to the growing divide between increased R&D spending and the lagging number of new drugs making it to market. Clearly, the role of these networks as translational science incubators that complement industry-sponsored programs is laudable and much-needed. However, the path to success for such organizations is less clear. Here, drawing on the experiences of the Immune Tolerance Network, a multidisciplinary clinical research network founded in 1999, we discuss some of the barriers inherent in developing such consortia and offer firsthand insight into the planning, resources, and organizational infrastructure required for a successful research program.

There have been enormous advances in the field of immunology over the past 3 decades, and those advances have had a positive effect on many subspecialties of medicine. Opportunities for even more notable advances remain. However, present and projected budget constraints for the National Institutes of Health have created formidable challenges. This commentary addresses the opportunities and challenges for the field of immunology during a period of restricted budgets.

Immune tolerance therapies are designed to reprogram immune cells in a highly specific fashion to eliminate pathogenic responses while preserving protective immunity. A concept that has tantalized immunologists for decades, the development of tolerance-inducing therapies, would revolutionize the management of a wide range of chronic and often debilitating diseases by obviating the need for lifelong immunosuppressive regimens. The advances of the past decade have provided a more detailed understanding of the molecular events associated with T-cell recognition and activation. Building on these advances, immunologists have demonstrated the feasibility of various tolerance-inducing approaches in small- and large-animal models of autoimmunity, allergy, and transplant graft rejection. Unprecedented opportunities to test these approaches in a variety of human diseases have now emerged. To capitalize on these advances, the National Institutes of Health recently established the Immune Tolerance Network (ITN), an international consortium of more than 70 basic and clinical immunologists dedicated to the evaluation of novel tolerance-inducing therapies and associated studies of immunologic mechanisms. By using a unique interactive approach to accelerate the development of clinical tolerance therapies, the ITN is partnering with the biotechnology and pharmaceutical industries to examine innovative tolerogenic approaches in a range of allergic and autoimmune diseases and to prevent graft rejection after transplantation. Two years since its inception, the ITN now has approximately 2 dozen clinical trials or tolerance assays studies ongoing or in later stages of protocol development. This report summarizes the rationale for emphasizing clinical research on immune tolerance and highlights the progress of the ITN.

Superoxide production by phagocytic white blood cells requires the assembly of an NADPH oxidase from membrane and cytosolic proteins. Recombinant cytosolic proteins p47phox and p67phox and neutrophil membranes were used to purify a third cytosolic component that is necessary and sufficient for cell-free reconstitution of NADPH oxidase. The component was isolated as a complex of rho-GDP dissociation inhibitor (rho-GDI) and two members of the rho subfamily of ras-related guanine nucleotide binding proteins, rac2 and CDC42Hs. Oxidase reconstitution with these pure cytosolic proteins was unaffected by GTP gamma S but was inhibited by GDP beta S, suggesting that the active complex contained endogenous bound GTP. Direct binding of rho-GDI to the GTP gamma S-bound forms of these G-proteins was demonstrated by gel filtration following exchange with radiolabeled guanine nucleotide. rho-GDI was shown to be nonessential for cell-free oxidase reconstitution in experiments that compared the activities of pure recombinant forms of these G-proteins. Recombinant rac augmented superoxide production, while recombinant CDC42Hs, which shares 70% amino acid sequence identity with rac, did not. Three highly conserved regions of rac1 and rac2 were noted as markedly divergent in CDC42Hs. It is proposed that one or more of these regions of rac may be involved in the specific interaction of rac with the other NADPH oxidase protein(s).

The phagocyte respiratory burst oxidase is a flavin-adenine dinucleotide (FAD)-dependent dehydrogenase and an electron transferase that reduces molecular oxygen to superoxide anion, a precursor of microbicidal oxidants. Several proteins required for assembly of the oxidase have been characterized, but the identity of its flavin-binding component has been unclear. Oxidase activity was reconstituted in vitro with only the purified oxidase proteins p47phox, p67phox, Rac-related guanine nucleotide (GTP)-binding proteins, and membrane-bound cytochrome b558. The reconstituted oxidase required added FAD, and FAD binding was localized to cytochrome b558. Alignment of the amino acid sequence of the beta subunit of cytochrome b558 (gp91phox) with other flavoproteins revealed similarities to the nicotinamide adenine dinucleotide phosphate (reduced) (NADPH)-binding domains. Thus flavocytochrome b558 is the only obligate electron transporting component of the NADPH oxidase.

The phagocyte NADPH oxidase is a multicomponent membrane-bound electron transport chain that catalyzes the reduction of O2 to superoxide. Cytochrome b558, the terminal electron donor to O2, is an integral membrane heterodimer containing 91- and 22-kDa subunits (gp91-phox and p22-phox, respectively). Synthetic peptides, whose amino acid sequences correspond to a gp91-phox carboxyl-terminal domain, inhibit superoxide production by blocking assembly of the oxidase from membrane and cytosol components. In this study, we examined the amino acid sequence requirements of a series of synthetic truncated gp91-phox peptides for inhibition of human neutrophil NADPH oxidase activation. RGVHFIF, corresponding to gp91-phox residues 559-565, was the minimum sequence capable of inhibiting superoxide generation. Contributions of individual amino acids to overall RGVHFIF inhibitory activity were determined by comparing the abilities of alanine-substituted RGVHFIF peptides to inhibit superoxide production. Substitution of alanine for arginine, valine, isoleucine, or either of the phenylalanines (but not glycine or histidine) within RGVHFIF resulted in loss of inhibitory activity. Synthetic gp91-phox carboxyl-terminal peptides are likely to be competitive inhibitors of the corresponding carboxyl-terminal domain of native gp91-phox by virtue of amino acid identity. We conclude that properties of arginine valine, isoleucine, and phenylalanine side chains within an RGVHFIF-containing domain of gp91-phox contribute significantly to cytochrome b558-mediated activation of the oxidase.

The phagocyte NADPH oxidase is a complex system consisting of membrane and cytosolic components that must assemble at the membrane for proper activation. Studies of patients with chronic granulomatous diseases of childhood have enabled the molecular characterization of these components, which has led to studies defining their interaction during NADPH complex assembly. Understanding NADPH oxidase assembly provides an opportunity to develop therapeutics for the regulation of this important reaction of inflammation.

Professional phagocytes contain a unique NADPH oxidase responsible for the production of microbicidal oxidants. Activation of this oxidase requires participation of cytosolic and membrane proteins, but the interactions of these components are incompletely understood. Patients with autosomal recessive Chronic Granulomatous Diseases (CGD) are characterized by functional defects in phagocyte oxidase activity resulting from a deficiency of either a 47 kDa (p47) or a 67 kDa (p67) cytosolic oxidase component. Cytosols from such patients are valuable for biochemical studies of the oxidase, but are not generally available because CGD is a rare disorder. The present study illustrates means of producing cytosols functionally and immunochemically deficient in either p47 or p67. Cytosol from monocytes cultured for 6 days is immunochemically deficient in p47 but not p67, while cytosol from HL-60 cells induced with retinoic acid for 3 days is deficient in p67 but not p47. Each of these cytosols fail to generate superoxide when added to neutrophil membranes in a cell-free assay but complement each other when combined. Complementation studies in which these cytosols were mixed in the cell-free assay with p47- or p67- deficient CGD cytosol established the functional characteristics of the experimentally produced cytosols.

Ascorbic acid uptake was investigated in isolated, plated human neutrophils using high-performance liquid chromatography with coulometric electrochemical detection. Freshly isolated neutrophils contained 1.3 mM ascorbic acid and accumulated significantly greater amounts when physiologic concentrations of the vitamin were present in the extracellular buffer. In several different buffers uptake was dependent on the presence of calcium and magnesium. Under these conditions, scintillation spectrometry of [14C]ascorbic acid in conjunction with high-performance liquid chromatography was suited for measuring ascorbic acid transport.

Adherence of Candida species to host tissues and nonbiologic materials has been studied in vivo and in vitro. Attachment of Candida albicans to mucosal cells, fibrin-platelet matrices, vascular endothelial cells, and plastic materials has been examined to elucidate early events in the pathogenesis of mucosal colonization and infection, candidal endocarditis, tissue invasion from the intravascular space, and infection of prosthetic devices. Adherence of C. albicans and Candida tropicalis exceeds that of less virulent Candida species, and germinated C. albicans cells adhere to host tissues more readily than do yeast-phase organisms. The adhesin of Candida that mediates attachment has yet to be characterized at the molecular level; however, on the basis of competitive inhibition by crude and purified cell wall products, blocking by antibody and lectin, and controlled degradation of the cell surface of Candida, it appears that mannans and mannoproteins are important constituents of the adhesin. The methods currently used to assay adherence of Candida all have limitations, and an approach to resolving these limitations is one of several areas that warrant further investigation.