Do the contemporary dietary patterns of children align with national food and nutrient recommendations?

BACKGROUND: Childhood nutrition is important in optimising growth, development and future health. The present study compared dietary intakes of Australian children aged 4-8 years with (i) Australian Guide to Healthy Eating (AGHE) food group recommendations and (ii) age-specific Nutrient Reference Values (NRVs), in addition to (iii) describing food group intakes of children meeting key NRVs. METHODS: Data were obtained from a representative sample of children (n = 789) from the National Nutrition and Physical Activity Survey between May 2011 and June 2012. Parent-reported 24-h recall dietary data were disaggregated into five core food groups, along with energy-dense, nutrient-poor (EDNP) foods, with intakes being compared with AGHE recommendations. Food group intakes were compared for children meeting the NRVs for 10 nutrients used for the development of AGHE food groups. Chi-squared and t-tests were performed to determine differences in food group intakes with P < 0.05 considered statistically significant. RESULTS: Only one child met the recommended daily servings for all AGHE core food groups and none met both core and energy-dense, nutrient-poor (EDNP) food group recommendations. The lowest level of alignment (percentage meeting recommendations) was for vegetables (4.6%) and the highest was for fruit (47.7%). Mean (SD) daily intake of EDNP foods [4.7 (3.2) serves day-1 ] accounted for 38.4% of total energy intakes. Children meeting key NRVs (n = 395) consumed greater daily servings of fruit [2.2 (1.7)], dairy [2.2 (1.2)] and EDNP foods [5.0 (3.4)] compared to the total sample (n = 789). CONCLUSIONS: Significant discrepancies exist between contemporary dietary patterns of Australian children and national recommendations. Future AGHE revisions should incorporate greater diversity of consumption patterns, including sub-categories of EDNP foods.

Fluorescence resonance energy transfer analysis of cell surface receptor interactions and signaling using spectral variants of the green fluorescent protein.

BACKGROUND: Fluorescence resonance energy transfer (FRET) is a powerful technique for measuring molecular interactions at Angstrom distances. We present a new method for FRET that utilizes the unique spectral properties of variants of the green fluorescent protein (GFP) for large-scale analysis by flow cytometry. METHODS: The proteins of interest are fused in frame separately to the cyan fluorescent protein (CFP) or the yellow fluorescent protein (YFP). FRET between these differentially tagged fusion proteins is analyzed using a dual-laser FACSVantage cytometer. RESULTS: We show that homotypic interactions between individual receptor chains of tumor necrosis factor receptor (TNFR) family members can be detected as FRET from CFP-tagged receptor chains to YFP-tagged receptor chains. Noncovalent molecular complexation can be detected as FRET between fusions of CFP and YFP to either the intracellular or extracellular regions of the receptor chains. The specificity of the assay is demonstrated by the absence of FRET between heterologous receptor pairs that do not biochemically associate with each other. Interaction between a TNFR-like receptor (Fas/CD95/Apo-1) and a downstream cytoplasmic signaling component (FADD) can also be demonstrated by flow cytometric FRET analysis. CONCLUSIONS: The utility of spectral variants of GFP in flow cytometric FRET analysis of membrane receptors is demonstrated. This method of analyzing FRET allows probing of noncovalent molecular interactions that involve both the intracellular and extracellular regions of membrane proteins as well as proteins within the cells. Unlike biochemical methods, FRET allows the quantitative determination of noncovalent molecular associations at Angstrom level in living cells. Moreover, flow cytometry allows quantitative analyses to be carried out on a cell-by-cell basis on large number of cells. Published 2001 Wiley-Liss, Inc.

Conjugation of fluorochromes to monoclonal antibodies.

UNLABELLED: Detection of cell surface molecules labeled by monoclonal or polyclonal antibodies conjugated to a fluorochrome is probably the most widely used application of flow cytometry. This unit contains protocols for tagging monoclonal antibodies with fluorescein, biotin, Texas Red, and phycobiliproteins. In addition, it provides a procedure for preparing a PE-Texas Red tandem conjugate dye that can then be used for antibody conjugation. These protocols enable investigators to label antibodies of their choice with multiple fluorochromes and permit more combinations of antibodies for multicolor flow applications. KEYWORDS: flow cytometry; monoclonal antibodies; fluorochromes; antibody labeling.

A regulatory role for ADP-ribosylation factor 6 (ARF6) in activation of the phagocyte NADPH oxidase.

In activated neutrophils NADPH oxidase is regulated through various signaling intermediates, including heterotrimeric G proteins, kinases, GTPases, and phospholipases. ADP-ribosylation factor (ARF) describes a family of GTPases associated with phospholipase D (PLD) activation. PLD is implicated in NADPH oxidase activation, although it is unclear whether activation of PLD by ARF is linked to receptor-mediated oxidase activation. We explored whether ARF participates in NADPH oxidase activation by formyl-methionine-leucine-phenylalanine (fMLP) and whether this involves PLD. Using multicolor forward angle light scattering analyses to measure superoxide production in differentiated neutrophil-like PLB-985 cells, we tested enhanced green fluorescent fusion proteins of wild-type ARF1 or ARF6, or their mutant counterparts. The ARF6(Q67L) mutant defective in GTP hydrolysis caused increased superoxide production, whereas the ARF6(T27N) mutant defective in GTP binding caused diminished responses to fMLP. The ARF1 mutants had no effect on fMLP responses, and none of the ARF proteins affected phorbol 12-myristate 13-acetate-elicited oxidase activity. PLD inhibitors 1-butanol and 2, 3-diphosphoglycerate, or the ARF6(N48R) mutant assumed to be defective in PLD activation, blocked fMLP-elicited oxidase activity in transfected cells. The data suggest that ARF6 but not ARF1 modulates receptor-mediated NADPH oxidase activation in a PLD-dependent mechanism. Because PMA-elicited NADPH oxidase activation also appears to be PLD-dependent, but ARF-independent, ARF6 and protein kinase C may act through distinct pathways, both involving PLD.

Vitamin A deficiency in mice causes a systemic expansion of myeloid cells.

To examine the role of retinoids in hematopoietic cell growth in vivo, we studied female SENCAR mice made vitamin A deficient by dietary restriction. Deficient mice exhibited a dramatic increase in myeloid cells in bone marrow, spleen, and peripheral blood. The abnormal expansion of myeloid cells was detected from an early stage of vitamin A deficiency and contrasted with essentially normal profiles of T and B lymphocytes. This abnormality was reversed on addition of retinoic acid to the vitamin A-deficient diet, indicating that the myeloid cell expansion is a direct result of retinoic acid deficiency. TUNEL analysis indicated that spontaneous apoptosis, a normal process in the life cycle of myeloid cells, was impaired in vitamin A-deficient mice, which may play a role in the increased myeloid cell population. Quantitative reverse transcriptase-polymerase chain reaction analysis of purified granulocytes showed that expression of not only RAR, but RXRs, 2 nuclear receptors that mediate biologic activities of retinoids, was significantly reduced in cells of deficient mice. This work shows that retinoids critically control the homeostasis of myeloid cell population in vivo and suggests that deficiency in this signaling pathway may contribute to various myeloproliferative disorders.

Measurement of molecular interactions in living cells by fluorescence resonance energy transfer between variants of the green fluorescent protein.

Many signal transduction pathways operate through oligomerization of proteins into multi-subunit complexes. Although biochemical assays can identify potential protein-protein interactions, studying these interactions in living cells is more challenging. Fluorescence resonance energy transfer (FRET) has been used as a "spectroscopic ruler" to measure molecular proximity, but these methods have been limited by the need for chemical labeling of target proteins or labeled antibodies. We present methods for examining interactions between target proteins molecularly fused to cyan and yellow variants of the green fluorescent protein (GFP) by FRET in living cells. Flow cytometric and microscope-based methods are described that have been applied to a variety of interacting proteins.

Peripheral blood-derived CD34+ progenitor cells: CXC chemokine receptor 4 and CC chemokine receptor 5 expression and infection by HIV.

The present study demonstrates cell surface expression of both CXC chemokine receptor 4 (CXCR4) and CC chemokine receptor 5 (CCR5), major coreceptors for T cell-tropic and macrophage-tropic strains of HIV, respectively, on CD34+ progenitor cells derived from the peripheral blood. CD34+ progenitor cells were susceptible to infection by diverse strains of HIV, and infection could be sustained for prolonged periods in vitro. HIV entry into CD34+ progenitor cells could be modulated by soluble CD4, HIV gp120 third variable loop neutralizing mAb and the cognate ligands for the CXCR4 and CCR5 HIV coreceptors. This study suggests that a significant proportion of the circulating progenitor cell pool may serve as a reservoir for HIV that is capable of trafficking the virus to diverse anatomic compartments. Furthermore, the infection and ultimate destruction of these progenitor cells may explain in part the defective lymphopoiesis in certain HIV-infected individuals despite effective control of virus replication during highly active antiretroviral therapy.

Impaired generation of bone marrow B lymphocytes in mice deficient in C/EBPbeta.

CAAT/enhancer binding proteins (C/EBP) are a family of transcription factors that mediates adipocyte differentiation and the regulation of genes expressed in immune responses and inflammation, such as interleukin-6 (IL-6), IL-8, and granulocyte colony-stimulating factor (G-CSF). We investigated the role of C/EBPbeta (NF-IL6) in the generation of bone marrow B lymphocytes by taking advantage of C/EBPbeta-/- mice. We found that the expansion of bone marrow (BM) B lymphocytes was impaired in long-term lymphoid cultures from C/EBPbeta-/- mice. Consistent with this finding, the number of BM B cells was decreased in C/EBPbeta-/- mice. Both the levels of IL-7 gene expression and bioactive IL-7 from BM stromal cells were decreased in C/EBPbeta-/- mice. Furthermore, the proliferative responsiveness of BM B-cell precursors to IL-7 was also reduced as compared to wild-type mice, indicating that C/EBPbeta is required for the generation of BM B cells induced by IL-7. Accordingly, IL-7 stimulates the C/EBPbeta DNA-binding activity of normal BM pre-B lymphocytes as well as of 70Z/3 pre-B cells. These results point to C/EBPbeta as a critical signaling molecule in BM B lymphopoiesis.

Characteristics of a murine gammaherpesvirus infection immunocompromised mice.

BACKGROUND-MATERIALS: Mice with normal or impaired immune function were studied for responses to intranasal infection with MHV68, a gammaherpesvirus that acutely infects lung epithelial cells and establishes latency in B cells. Infection of normal mice induced a vigorous pulmonary inflammatory response composed of T, B, and NK cells and macrophages and stimulated activation and proliferation of T and B cells in spleen. METHODS-RESULTS-CONCLUSIONS: Resolution of the infection was associated with induction of MHV68-specific antibodies, but virus-specific cytotoxic T cells were not detected. Mice inoculated with retroviruses that induce severe immunodeficiency unexpectedly cleared MHV68 from lung in the same time-frame as controls and failed to develop latency as determined by infectious center tests of spleen cells. In contrast, control of MHV68 infection in spleen and/or lung was impaired in mice deficient in CD4+ or CD8+ T cells or both T cell subsets, B cells, IFN-gamma, or inducible nitric oxide synthase (iNOS). Infection was uniformly lethal in nude and iNOS-deficient mice and killed one-third of IFN-gamma-deficient mice. These results indicate that resistance to MHV68 is markedly influenced by expression of IFN-gamma from T cells leading to induction of iNOS and generation of nitric oxide.

Perceived self-care information needs and information--seeking behaviors before and after elective spinal procedures.

Patients undergoing elective lumbar spinal surgical procedures pose a challenge to nurses who provide discharge instruction, because the decreased length of stay (LOS) severely limits time for comprehensive discharge instruction. The perspectives of 15 adult patients on their perceived self-care information needs and information seeking behaviors following elective spinal surgical procedures were examined. Content analytic techniques were used to categorize responses. Preoperatively, a majority of the subjects (93.3%) indicated that the neurosurgeon rather than the nurse, was anticipated to be the sole source of information related to self-care needs. Postdischarge, more than half of the subjects reported that they had difficulty describing the teaching session because they were either too sedated due to the analgesia, or were experiencing extreme pain at the time the discharge instruction was being delivered. Results substantiate the importance of supplementing oral discharge instruction with comprehensive written discharge instruction and of increasing public awareness of the teaching expertise of nurses.

Characterization of the expression and gene promoter of CD22 in murine B cells.

CD22 is a B cell-restricted surface molecule which may play an important role in interactions between B cells and other cells and in regulating signals through the B cell receptor (BCR) complex. Here we have examined whether the mouse is a suitable in vivo model for studying CD22 functions. In primary and secondary lymphoid organs of adult mice CD22 is on all mature B cells, including resting IgM+IgD+ B cells, IgG+ HSA(lo) memory B cells, syndecan+ plasma cells and CD5+ B cells, but it is not on immature IgM+IgD- B cells. Biochemical analysis revealed that murine CD22 is associated with the IgM receptor in some, but not all, CD22+ B leukemic and lymphoma cell lines; as with human CD22, murine CD22 is rapidly phosphorylated on tyrosine after ligation of the BCR. In the CD22- murine pro-B cell line, FEMCL, CD22 expression was inducible by treatment with phorbol 12-myristate 13-acetate. A genomic fragment of the cd22b allele containing 1.3 kb 5' of exon 1 was sequenced in order to identify potential DNA regulatory elements in the CD22 promoter region. Consensus sequences for transcription factor binding sites including PU.1, AP-1, AP-2, C/EBP and SP-1 were present, but no classical TATA elements or initiator motifs were evident at relevant positions. The 1.3-kb promoter fragment 5' of exon 1 was sufficient for directing basal promoter activity in B and T cells. There was no significant sequence similarity between the murine and human cd22 gene promoters, although both contain repetitive elements and Sp-1 and AP1 binding sites. Thus, murine CD22 shares a number of features with human CD22 and the mouse provides a suitable model system for elucidating the function of CD22 in vivo.

Characterization of a new antigen expressed by B and myeloid lineage cells identified by the monoclonal antibody LIP-6.

The LIP-6 MAb was produced against the undifferentiated cell line bh2-1 and recognizes an antigen expressed on all pre-B and B cell lines tested and some myeloid lineage lines. FACS analysis of normal tissues showed that LIP-6 is expressed on B lineage cells at all stages of differentiation, from bone marrow pre-B to plasma cells. T cells and thymocytes are LIP-6-, and splenic CD11b+ cells are heterogeneous for LIP-6 expression. The LIP-6 MAb was shown to precipitate a major 75-kDa and a minor 85-kDa protein under reducing conditions and a large protein of > 240 kDa under nonreducing conditions. Removal of N-linked sugars from the reduced lysates resulted in a single 65-kDa protein, suggesting that there is differential glycosylation of a single 65-kDa protein that forms disulfide-linked multimers. Finally, the LIP-6 antigen was shown not to be linked to the cell surface via a GPI linkage.

Contribution of B cell subsets to delayed development of MAIDS in xid mice.

C57BL/6 (B6) mice develop a syndrome of progressive lymphoproliferation and immunodeficiency, murine AIDS (MAIDS), when infected with an etiologic replication-defective virus termed BM5def. Induction of MAIDS requires the presence of CD4+ T cells and B cells. B6 mice with altered conventional B cell function and a deficit in CD5+ B cells due to the xid mutation develop disease with a greatly prolonged latency. The association of this mutation with resistance to MAIDS was confirmed in studies of P.xid mice. To test the hypothesis that conventional B cells are required for rapid induction of disease, B6.xid mice were injected with spleen cells from nude mice or were given bone marrow from aged donors. Both sets of recipients developed advanced disease by 10 weeks post infection, suggesting that resistance to MAIDS in xid mutants may be due to effects of B cells other than the CD5+ subset.

Effects of exogenous, nonleukemogenic, ecotropic murine leukemia virus infections on the immune systems of adult C57BL/6 mice.

Mouse AIDS (MAIDS) develops in mice infected with a mixture of replication-competent ecotropic and mink lung cell focus-inducing murine leukemia viruses and an etiologic replication-defective virus. Helper viruses are not required for induction of MAIDS, but the time course of disease is accelerated in their presence. To understand the possible contributions of ectropic murine leukemia viruses to MAIDS pathogenesis, we biologically cloned a series of viruses from the MAIDS-inducing LP-BM5 virus mixture. These viruses were examined for replication in tissues of infected mice and for effects on the immune system. All virus stocks replicated efficiently in mice. Infected animals showed slight lymphadenopathy and splenomegaly due primarily to B-cell proliferation associated with differentiation to immunoglobulin secretion resulting in twofold increases in serum immunoglobulin M levels; however, B-cell responses to helper T-cell-independent antigens were increased rather than decreased as in MAIDS. Analyses of CD8+ T-cell function showed that cytotoxic T-lymphocyte responses to alloantigens were comparable in control and infected mice. Finally, we showed that infection resulted in enhanced expression of transcripts for interleukin-10, interleukin-4, and gamma interferon. These cytokines can all contribute to B-cell activation and may promote the expansion of a target cell population for the MAIDS defective virus.

Outcomes analysis in cranial base surgery - preliminary results.

A system of analysis addressing predictors of management outcomes in Cranial Base Surgery has yet to be published. We therefore report data on seventy-nine consecutive patients undergoing surgery for tumors involving the cranial base, excluding patients with the diagnosis of pituitary microadenoma. Outcomes were defined prospectively in terms of completeness of tumor resection, complications of treatment with emphasis on neurological morbidity, and return to work or independent living. Also, preoperative features are analyzed as influencing cost of treatment, estimated in terms of the number of surgical procedures required, duration of hospital and Intensive Care Unit stay, and time taken to return to work. Preliminary analysis of data reveals that severe brainstem compression, large tumor size (average diameter > 3 cm), high cavernous sinus grade, and tumor encasement of major cerebral arteries are associated with incomplete tumor resection (p < 0.05). Patient age greater than 65, preoperative Karnofsky Performance Score (KPS) less than 80, and severe brainstem compression are associated with increased risk of stroke (p < 0.05). Age greater than 65 and preoperative KPS less than 80 are associated with an increased length of stay (p < 0.05). Other untoward events did not occur with sufficient frequency to reach statistical significance. A model of outcomes analysis in Cranial Base Surgery is proposed utilizing a database to incorporate a group of non-operated patients and include quality of life measurements in long-term patient follow-up.

Long-term lymphoid reconstitution of SCID mice suggests self-renewing B and T cell populations in peripheral and mucosal tissues.

Peyer's patch, peripheral lymph node, and mesenteric lymph node cells were transferred to immunodeficient SCID mice to assess the long-term (150-300 days) potential of these cells to repopulate the host's immune system. Results demonstrate that, irrespective of donor population, total serum Ig and isotype distribution appear normal within 4 weeks of reconstitution and remain at normal levels for up to one year following cell transfer. At the cellular level, each donor population reconstitutes splenic T and B cell compartments in a progressive and quantitatively indistinguishable manner. Immunohistological analyses of reconstituted mice indicate that, although some qualitative differences are evident, normal splenic composition and architecture are observed. In contrast, gut reconstitution varies significantly with donor population. Peyer's patch cells yield normal-appearing gut tissue with extensive infiltration of the lamina propria and intraepithelial compartments by T cells and IgA-secreting plasma cells. Peripheral lymph node cells give rise to T cells found almost exclusively in the lamina propria, while IgA secreting plasma cells are rarely detected. The course and extent of reconstitution further suggest that all donor populations contain long-lived T and B cells as well as self-renewing lymphocytes capable of extensive expansion. This latter observation has potentially important implications for both transplantation biology and gene therapy applications.

Changes in the subsets of CD4+ T cells in Trypanosoma musculi infection: delay of immunological cure in young mice and the weak ability of aged mice to control the infection.

After a 3 week course (approximately), during which there is marked lymphoid hyperplasia, Trypanosoma musculi infections in young-adult mice are cured by an immune mechanism involving antibodies of the IgG2a isotype. Both the lymphoid hyperplasia and IgG2a antibody response are T-cell-dependent events and both processes appear to be defective in aged mice. The purpose of the studies reported here was to elucidate the effects of T. musculi infection on subsets of T cells for two reasons: (i) to gain insight into the probable roles of selected cytokines (IL-2, IL-4 and IFN-gamma) in facilitating the production of curative, IgG2a antibodies, and (ii) to examine the hypothesis that aging affects the competence of CD4+ T cells to participate in immunological control of infections. The major conclusions from these studies are that: (i) T. musculi infection of mice induces rapid change in the CD4+ T cell population toward predominance of the activated or memory (CD45RBloCD44hi) phenotype, cells which produce IFN-gamma, II-3, IL-4 and IL-5, accompanied by profound inhibition of IL-2 production, and (ii) in the old mice these changes are superimposed on the natural age-associated changes in the same direction (i.e. toward predominance of CD45RBloCD44hi T cells). Thus, in the old animals, the combined changes of aging and infection, moving in the same direction, are devastating, resulting in the aged animals being unable, or barely able, to control infection.