IRF4 Deficiency Leads to Altered BCR Signalling Revealed by Enhanced PI3K Pathway, Decreased SHIP Expression and Defected Cytoskeletal Responses.

The graded expression of transcription factor interferon regulatory factor 4 (IRF4) regulates B cell development and is critical for plasma cell differentiation. However, the mechanisms, by which IRF4 elicits its crucial tasks, are largely unknown. To characterize the molecular targets of IRF4 in B cells, we established an IRF4-deficient DT40 B cell line. We found that in the absence of IRF4, the expression of several molecules involved in BCR signalling was altered. For example, the expression of B cell adaptor for PI3K (BCAP) was upregulated, whereas the SHIP (SH2-containing Inositol 5?-Phosphatase) expression was downregulated. These molecular unbalances were accompanied by increased BCR-induced calcium signalling, attenuated B cell linker protein (BLNK) and ERK activity and enhanced activity of PI3K/protein kinase B (Akt) pathway. Further, the IRF4-deficient cells showed dramatically diminished cytoskeletal responses to anti-IgM cross-linking. Our results show that IRF4 has an important role in the regulation of BCR signalling and help to shed light on the molecular mechanisms of B cell development and germinal centre response.

Negative regulation of interleukin 2 transcription by the glucocorticoid receptor.

Glucocorticoid-dependent transcriptional enhancement is known to occur through the interaction of the glucocorticoid receptor (GR) with specific DNA response elements. In contrast, negative regulation of gene expression by this class of hormone is less well understood. Glucocorticoids are potent immunosuppressive agents acting primarily by inhibiting T lymphocyte activation and lymphokine production. Interleukin 2 (IL-2) gene expression, a critical early event during T lymphocyte activation, is inhibited in glucocorticoid-sensitive cells by hormone treatment. We have studied the mechanism of this inhibition. In transgenic mice carrying c-myc linked to the IL-2 enhancer, mitogen-induced expression of the transgene is inhibited by concurrent glucocorticoid treatment, while a similar transgene construct driven by three copies of the binding site for nuclear factor of activated T cells is not inhibited. Cotransfection experiments into glucocorticoid-insensitive jurkat cells show that the NH2 terminus of the glucocorticoid receptor is dispensable for inhibition of the IL-2 enhancer but that an intact DNA binding domain, although not necessarily binding to DNA, is required. Hybrid GRs containing the DNA binding domains of either the estrogen receptor (ER) or thyroid receptor, as well as the entire wild-type ER, all function as repressors of the IL-2 enhancer. We have localized the site of inhibition to two sequences located in the proximal half of the enhancer. These sequences bind a similar, if not identical, inducible nuclear factor that has biologic characteristics that distinguish it from AP-1. The mechanism of IL-2 inhibition likely involves direct interactions between the GR and this factor.

A network analysis of the single nucleotide polymorphisms in acute allergic diseases.

BACKGROUND: Genetics of acute allergies has focused on identifying single nucleotide polymorphisms (SNPs) within genes relevant in the pathogenesis. In this study, we begin a systems biology analysis of the interconnectivity and biological functions of these genes, their transcripts and their corresponding proteins. METHODS: The literature (Pubmed) was searched for SNPs within genes relevant in acute allergic diseases. The SNP-modified genes were converted to corresponding proteins and their protein-protein interactions were searched from six different databases. This interaction network was analysed with annotated vocabularies (ontologies), such as Gene Ontology, Reactome and Nature pathway interaction database. Time-series transcriptomics was performed with nasal epithelial cells obtained from allergic patients and their healthy control subjects. RESULTS: A total of 39 genes with SNPs related to acute allergic diseases were found from a literature search. The corresponding proteins were then hooked into a large protein-protein interaction network with the help of various databases. Twenty-five SNP-related proteins had more than one interacting protein and a network contained 95 proteins, and 182 connections could be generated. This network was 10-fold enriched with protein kinases and proteins involved in the host-virus interaction compared with background human proteome. Finally, eight of the 95 nodes on our network displayed nasal epithelial transcriptomal regulation in a time-series analysis collected from birch allergic patients during the spring pollen season. CONCLUSIONS: Signal transduction with special reference to host-virus interactions dominated in the allergy-related protein interaction network. Systems level analysis of allergy-related mutation can provide new insights into pathogenetic mechanisms of the diseases.

Time-series nasal epithelial transcriptomics during natural pollen exposure in healthy subjects and allergic patients.

BACKGROUND: The role of epithelium has recently awakened interest in the studies of type I hypersensitivity. OBJECTIVE: We analysed the nasal transcriptomics epithelial response to natural birch pollen exposure in a time series manner. METHODS: Human nasal epithelial cell swabs were collected from birch pollen allergic patients and healthy controls in winter season. In addition, four specimens at weekly intervals were collected from the same subjects during natural birch pollen exposure in spring and transcriptomic analyses were performed. RESULTS: The nasal epithelium of healthy subjects responded vigorously to allergen exposure. The immune response was a dominating category of this response. Notably, the healthy subjects did not display any clinical symptoms regardless of this response detected by transcriptomic analysis. Concomitantly, the epithelium of allergic subjects responded also, but with a different set of responders. In allergic patients the regulation of dyneins, the molecular motors of intracellular transport dominated. This further supports our previous hypothesis that the birch pollen exposure results in an active uptake of allergen into the epithelium only in allergic subjects but not in healthy controls. CONCLUSION: We showed that birch pollen allergen causes a defence response in healthy subjects, but not in allergic subjects. Instead, allergic patients actively transport pollen allergen through the epithelium to tissue mast cells. Our study showed that new hypotheses can arise from the application of discovery driven methodologies. To understand complex multifactorial diseases, such as type I hypersensitivity, this kind of hypotheses might be worth further analyses.

Expression of LIF and LIF receptor beta in Alzheimer's and Parkinson's diseases.

BACKGROUND: Signaling through the leukemia inhibitory factor (LIF) receptor (LIFR) is crucial for nervous system development. There are few studies concerning the expression of LIF and LIFR in normal and degenerating adult human brain. OBJECTIVES: To study the expression of LIF and LIFR in Alzheimer's disease (AD), Parkinson's disease (PD), and control brains. PATIENTS AND METHODS: LIF and LIFR mRNA copy numbers were determined by quantitative real-time RT-PCR from four brain regions of 34 patients with AD, 40 patients with PD, and 40 controls. Immunohistochemistry was performed in seven PD and in four AD patients and in seven normal controls. RESULTS: In general, the LIF copy numbers were 1 log higher than the LIFR copy numbers. In the AD brains, LIF expression was higher than in the controls in the hippocampus and in the temporal cortex, and in the PD brains in the hippocampus and in the anterior cingulated cortex. Expressions of LIF and LIFR in different brain regions were opposite except for the AD hippocampus and PD anterior cingulated cortex, where the expression patterns were parallel. CONCLUSIONS: Co-operative expression of LIF and LIFR in AD hippocampus and PD anterior cingulated cortex may indicate a role for LIF in neuronal damage or repair in these sites.

Birch pollen allergen Bet v 1 binds to and is transported through conjunctival epithelium in allergic patients.

BACKGROUND: Previous work in type-I pollen allergies has mainly focused on lymphocytes and immune responses. Here, we begin to analyse with a systems biology view the differences in conjunctival epithelium obtained from healthy and allergic subjects. METHODS: Transcriptomics analysis combined with light and electron microscopic analysis of birch pollen allergen Bet v 1 located within conjunctival epithelial cells and tissues from birch allergic subjects and healthy controls was carried out. RESULTS: Bet v 1 pollen allergen bound to conjunctival epithelial cells within minutes after the exposure even during the nonsymptomatic winter season only in allergic, but not in healthy individuals. Light- and electron microscopy showed that Bet v 1 was transported through the epithelium within lipid rafts/caveolae and reached mast cells only in allergic patients, but not in healthy individuals. Transcriptomics yielded 22 putative receptors expressed at higher levels in allergic epithelium compared with healthy specimens. A literature search indicated that out of these receptors, eight (i.e. 37%) were associated with lipid rafts/caveolae, which suggested again that Bet v 1 transport is lipid raft/caveola-dependent. CONCLUSIONS: We show a clear difference in the binding and uptake of Bet v 1 allergen by conjunctival epithelial cells in allergic vs healthy subjects and several putative lipid raft/caveolar receptors were identified, which could mediate or be co-transported with this entry. The application of discovery driven methodologies on human conjunctival epithelial cells and tissues can provide new hypotheses worth a further analysis to the molecular mechanisms of a complex multifactorial disease such as type-I birch pollen allergy.

Twinfilin is required for actin-dependent developmental processes in Drosophila.

The actin cytoskeleton is essential for cellular remodeling and many developmental and morphological processes. Twinfilin is a ubiquitous actin monomer-binding protein whose biological function has remained unclear. We discovered and cloned the Drosophila twinfilin homologue, and show that this protein is ubiquitously expressed in different tissues and developmental stages. A mutation in the twf gene leads to a number of developmental defects, including aberrant bristle morphology. This results from uncontrolled polymerization of actin filaments and misorientation of actin bundles in developing bristles. In wild-type bristles, twinfilin localizes diffusively to cytoplasm and to the ends of actin bundles, and may therefore be involved in localization of actin monomers in cells. We also show that twinfilin and the ADF/cofilin encoding gene twinstar interact genetically in bristle morphogenesis. These results demonstrate that the accurate regulation of size and dynamics of the actin monomer pool by twinfilin is essential for a number of actin-dependent developmental processes in multicellular eukaryotes.

A randomized, controlled trial of tonsillectomy in periodic fever, aphthous stomatitis, pharyngitis, and adenitis syndrome.

OBJECTIVE: We carried out a prospective, randomized, controlled trial to clarify the effect of tonsillectomy on the clinical course of periodic fever, aphthous stomatitis, pharyngitis, and adenitis (PFAPA) syndrome. STUDY DESIGN: Twenty-six consecutive children (mean age 4.1 years) with at least 5 PFAPA attacks were recruited from 3 tertiary care pediatric hospitals during 1999-2003 and randomly allocated to tonsillectomy or follow-up alone. They were all followed up with symptom diaries for 12 months. Tonsillectomy was allowed after 6 months in the control group if the attacks recurred. RESULTS: Six months after randomization all 14 children in the tonsillectomy group and 6/12 children in the control group (50%) were free of symptoms (difference 50%, 95% confidence interval 23% to 75%, P < .001). Tonsillectomy was performed on 5/6 of the patients in the control group who still had symptoms after 6 months. The remaining unoperated child in the control group had recurrences of the fever episodes throughout the follow-up, but the symptoms became less severe, and the parents did not choose tonsillectomy. CONCLUSION: Tonsillectomy appeared to be effective for treating PFAPA syndrome. The fever episodes ceased without any intervention in half of the control subjects. We conclude that although the mechanisms behind this syndrome are unknown, tonsillectomy can be offered as an effective intervention for children with PFAPA.

Bioactivities of alternative protein sources and their potential health benefits.

Increasing the utilisation of plant proteins is needed to support the production of protein-rich foods that could replace animal proteins in the human diet so as to reduce the strain that intensive animal husbandry poses to the environment. Lupins, quinoa and hempseed are significant sources of energy, high quality proteins, fibre, vitamins and minerals. In addition, they contain compounds such as polyphenols and bioactive peptides that can increase the nutritional value of these plants. From the nutritional standpoint, the right combination of plant proteins can supply sufficient amounts of essential amino acids for human requirements. This review aims at providing an overview of the current knowledge of the nutritional properties, beneficial and non-nutritive compounds, storage proteins, and potential health benefits of lupins, quinoa and hempseed.

Analysis of risk factors for childhood persistent middle ear effusion.

Although exposure to infectious agents and parental smoking are known to influence the overall risk of otitis media, these risk factors do not appear to be linked with the tendency to develop chronic otitis media with effusion (COME) instead of recurrent acute otitis media (RAOM). The genetic inflammatory response type of the child appears to influence the risk of persistent middle ear effusion in COME. Two different clinical presentations of childhood otitis media are encountered: RAOM; and COME, which is associated with persistent effusion in the middle ear. The objective of this study was to assess putative factors that may regulate the development of persistent middle ear effusion in COME. In total, 159 children with RAOM and their parents (n=304), and 55 children with COME and their parents (n=110) were evaluated. All the children with COME or RAOM were aged <4 years. There was no difference in the frequency of attendance at day care outside the home, number of siblings or parental smoking between children with RAOM and those with COME. The frequency of parental allergy and asthma was lower among children with COME than those with RAOM.

Protein kinase C subtypes in endothelial cells.

Activation of protein kinase C (PKC) has been linked to the regulation of class II expression on endothelial cells by interferon-gamma (IFN-gamma). PKC subtypes in endothelial cells were analyzed using three different approaches, the immunoperoxidase staining of native and IFN-gamma stimulated cells cultured on chamber slides as well as immuno- and Northern blotting. All approaches revealed that of the conventional subtypes, alpha is the predominant form of PKC in endothelial cells. Even though IFN-gamma is able to induce PKC translocation to particulate fractions, no translocation was detected in histological stainings. Western blot studies as well as mRNA studies revealed that IFN-gamma is unable to increase the total amount of PKC in endothelial cells.

Protein kinase C is crucial in signal transduction during IFN-gamma induction in endothelial cells.

We have demonstrated that IFN-gamma, a potent peptide mediator in inflammatory responses, operates via the protein kinase C dependent transduction pathway in the induction of class II MHC antigens on rat microvascular endothelial cells. Stimulators of protein kinase C, like PMA, replaced IFN-gamma in the induction of MHC class II on endothelial cells in a dose-dependent manner. Selective enzyme inhibitors of protein kinase C, H-7 as well as sphingosine down-regulated the IFN-gamma induced class II expression in a dose-dependent manner. Addition of cAMP or cGMP in the culture, had no effect on the class II expression on the endothelial cells. Transient rise of cytosolic Ca2+ by calcium ionophore A23187, or a calmodulin antagonist W-7, had no effect on the IFN-gamma induced class II expression.

Down-regulation of monocytic VLA-4 leads to a decreased adhesion to VCAM-1.

The alpha 4 beta 1 integrin VLA-4 is expressed on practically all leukocytes, except on mature granulocytes. Here we show that in vitro treatment of monocytic cells with phorbol-12-myristate-13-acetate (PMA) leads to a selective decrease in the VLA-4 alpha-chain expression, both at the RNA and protein level. Meanwhile the expression of beta 1 and that of alpha 5, another alpha-chain associating with beta 1, was seen to increase. The decrease of alpha 4 expression was restricted to monocytic cells, and was not observed on other VLA-4-positive cells tested (MOLT-4 T cells and HOS sarcoma cells). The down-regulation of the VLA-4 alpha-chain was followed by a decreased binding capacity of the cells to recombinant VCAM-1. This data indicates that while previous findings show that the integrin-dependent adhesion may rapidly be regulated by altering the avidity of the interacting molecules, their quantitative modulation also has a clear impact on adhesion.

Leukotriene B4 increases the lymphocyte binding to endothelial cells.

Leukotrienes are potent mediators of local microvascular environment. Leukotriene B4 treatment of cultured endothelium increases the binding of lymphocytes to endothelial cell monolayers within minutes. This effect is dose-dependent and reversible upon removal of the leukotriene. Pretreatment of lymphocytes slightly decreases the binding and pretreatment of both lymphocytes and endothelium with leukotriene B4 prior to the adherence assay did not alter the binding. These results suggest that leukotriene B4 regulates exclusively the vascular side, but not the white cell side of this interaction.

High endothelial cells synthesize and degrade sLex. Putative implications for L-selectin-dependent recognition.

L-selectin guides lymphocytes into peripheral lymphoid tissues by recognizing glycoprotein ligands decorated with 6-sulfated sialyl Lewis x (sulfo sLex). Here we have used a rat peripheral lymph node high endothelial cell line (Ax) to study in detail the synthesis, expression and degradation of sLex epitope. We show here that Ax cells possess active alpha(1,3)fucosyltransferase Fuc-TVII, the enzyme responsible for the final fucosylation of sialyl-N-acetyllactosamine during sLex synthesis, and express sLex on the cell surface. Furthermore, these cells degrade sLex, primarily by desialylating it to neutral Lex epitopes by alpha(2,3)sialidase(s).

Bioavailability of vitamin D from wild edible mushrooms (Cantharellus tubaeformis) as measured with a human bioassay.

BACKGROUND: The bioavailability of vitamin D from mushrooms in humans is unknown. OBJECTIVE: We investigated the bioavailability of vitamin D from wild edible mushrooms (Cantharellus tubaeformis) using the increase in serum 25-hydroxyvitamin D concentrations as a measure of vitamin D bioavailability. DESIGN: Twenty-seven volunteers with serum 25-hydroxyvitamin D concentrations <60 nmol/L (mean : 38.5 nmol/L; range: 15-60 nmol/L) were randomly divided into 3 groups of 9 persons each. For 3 wk, excluding Saturdays and Sundays, group 1 received mushrooms (C. tubaeformis) providing 14 microg ergocalciferol/d with their lunch, group 2 (control) received an ergocalciferol supplement providing 14 microg/d, and group 3 (also a control) received no supplementation. RESULTS: At the beginning of the study, mean serum 25-hydroxyvitamin D concentrations did not differ significantly among the groups (P = 0.280). When all 3 groups were considered, serum 25-hydroxyvitamin D concentrations showed different time-related changes among the groups during the study: group (P = 0.388), time (P = 0.000), and group x time (P = 0.001). When groups 1 and 2 were compared with group 3, serum 25-hydroxyvitamin D concentrations at 3 wk differed significantly between groups 1 and 3 (P = 0.032) as well as between groups 2 and 3 (P = 0.004). Serum 25-hydroxyvitamin D concentrations at 3 wk did not differ significantly between groups 1 and 2 (P = 0.317). CONCLUSIONS: We showed for the first time that ergocalciferol was well absorbed from lyophilized and homogenized mushrooms in humans and that vitamin D bioavailability can be studied in humans with such an experimental protocol.

Quantitation of antibody isotypes in solid-phase assays. Comparison of myeloma protein and monoisotypic antibody standards.

Two methods have been proposed for the standardization of isotype specific antibody assays. In one, myeloma proteins directly attached to plastic surfaces are used as standards, whereas the other method employs antigen coated surfaces followed by monoisotypic antibodies as standards. These standardization methodologies have been investigated by submitting 4 monoisotypic human antibodies to a solid-phase assay standardized by the myeloma method. Specific antibody concentrations of each were determined so that each could serve as a monoisotypic standard. Three purified monoclonal mouse antibodies were also tested which allowed use of the same preparation as a monoisotypic antibody standard or as a 'myeloma protein' standard. Ten times more myeloma protein than specific antibody is needed for the same level of binding of the anti-isotype antibody. Therefore, assays standardized with myeloma proteins give erroneously high concentrations for sample antibodies. The same concentration of antibodies of different specificities (used with different antigen coats) gave very comparable levels of binding of the labeled antibody. This supports the claim that for quantitation of antibodies an antibody standard can be used that is of different specificity to the sample antibody to be measured.

Evidence that lymphocyte traffic into rejecting cardiac allografts is CD11a- and CD49d-dependent.

Acute cardiac allograft rejection is characterized by infiltration of leukocytes into tissue parenchyma, but the site of entry and endothelial adhesion molecules involved are not yet defined. Lymphocyte binding to frozen sections prepared from day-3 rejecting cardiac allografts was significantly increased compared with sections made from normal hearts (number of bound lymphocytes, 983 +/- 216 per mm2 vs. 309 +/- 121, respectively, P < 0.001) or syngeneic grafts. The bound lymphocytes were located exclusively only on the top of the capillary structures and not on any other sites on the heart vasculature. We further wanted to analyze which of the cloned endothelial adhesion molecules and their counterreceptors would be involved in the increased lymphocyte binding. Lymphocyte pretreatment with mAb anti-CD11a or anti-CD49d inhibited this binding more than 50%. This inhibition on lymphocyte binding could not be increased by combining these two antibodies. Lymphocyte binding to endothelium has been shown to be at least partly organ specific; therefore, we asked whether increased lymphocytes adhere to cardiac allografts could be organ specific. Lymphocyte binding to lymph node high endothelial venules (HEV) has been shown to be inhibited by mannose-6-phosphate (M6P) and to kidney peritubular capillaries by mannose-1-phosphate (M1P). In the present study neither of these carbohydrates had any effect on lymphocyte binding to cardiac allograft endothelium. Monosaccharide inhibition studies demonstrate that the mechanism of lymphocyte adhesion to cardiac capillary endothelium differs from adhesion to kidney allografts or peripheral lymph node high endothelium.

Gamma-interferon induces prostacyclin and prostaglandin but not lipoxygenase product synthesis in rat endothelial cells.

The major arachidonic acid metabolites excreted by untreated rat heart endothelial cells were prostacyclin (PGI2), 5-hydroxyeicosatetraenoic acid (5-HETE) and 15-hydroxyeicosatetraenoic acid (15-HETE). Trace amounts of both prostaglandin E (PGE) and thromboxane B2 (TXB2) were also produced. The activity of leukotriene B4 (LTB4) was the same as baseline activity. Gamma-interferon (gamma-IFN) treatment increased the production of PGI2 and PGE more than three-fold compared to control values. Thromboxane production increased two-fold while the excretion of 5-HETE and 15-HETE was unaffected. No LTB4 excretion was seen after gamma-IFN stimulus. Methylprednisolone downregulated the 5-HETE, 15-HETE and prostacyclin production both in normal and gamma-IFN treated cells. Indomethacin decreased prostacyclin excretion in untreated cells and also decreased gamma-IFN-induced prostacyclin and PGE excretion. Taken together these results indicate that rat microvascular endothelial cells excrete prostaglandins and prostacyclin, but not leukotrienes.

Signal transduction during in vitro lymphocyte homing.

Lymphocyte binding to endothelium is a necessary prerequisite for lymphocyte homing through endothelium. This is mediated by the binding of ligands on endothelial cells to lymphocyte surface homing receptors. We show in this paper that the intracellular second messenger pathways involved in interferon gamma-induced intercellular adhesion molecule 1 upregulation on endothelial cells are protein kinase C and calcium dependent. Lymphocyte binding to endothelial cells is enhanced by both platelet activating factor and interleukin 1 alpha. Platelet activating factor added to endothelial cultures increases lymphocyte binding within 10 min and operates via protein kinase C but not via cAMP. On the other hand interleukin 1 alpha increases binding within 4 hr and operates via cAMP but not via protein kinase C. These results imply that different mediators of inflammation can activate different signal transduction pathways but lead to similar increases in lymphocyte binding.