Gastrointestinal helminths of wild hogs and their potential livestock and public health significance in Jamaica.

An investigation into the potential for transmission of gastrointestinal helminths from wild hogs to livestock and humans was prompted by concerns of recreational wild-hog hunting in the Caribbean region and the recent practice, by livestock farmers in Jamaica, of co-rearing wild and domesticated swine. Thirty-one wild hogs from the Hellshire Hills, a dry limestone forest in southern Jamaica, were necropsied during the period June 2004 to August 2006. Thirteen of the captured animals were male and 18 female. Four species of adult helminths were recovered from the gastrointestinal tracts of the wild hogs: Hyostrongylus rubidus (77%), Globocephalus urosubulatus (48%), Oesophagostomum dentatum (42%) and Macroacanthorhynchus hirudinaceus (77%). Two (6.2%), ten (32.2%) and 18 (58.0%) hogs harboured one, two and three species of helminths, respectively. Mean infection intensities varied from 8.1 for M. hirudinaceus, to 115.5 for O. dentatum. There was no association between any of the recovered helminths and sex of the host; however, a multivariate analysis indicated a positive association between the prevalence of G. urosubulatus and host age (odds ratio (OR) = 6.517). Domesticated hogs co-reared with wild hogs are potentially at risk of infection with all four helminths, while wild-hog hunters and pig farmers may be exposed to M. hirudinaceus.

Note: Time-gated 3D single quantum dot tracking with simultaneous spinning disk imaging.

We describe recent upgrades to a 3D tracking microscope to include simultaneous Nipkow spinning disk imaging and time-gated single-particle tracking (SPT). Simultaneous 3D molecular tracking and spinning disk imaging enable the visualization of cellular structures and proteins around a given fluorescently labeled target molecule. The addition of photon time-gating to the SPT hardware improves signal to noise by discriminating against Raman scattering and short-lived fluorescence. In contrast to camera-based SPT, single-photon arrival times are recorded, enabling time-resolved spectroscopy (e.g., measurement of fluorescence lifetimes and photon correlations) to be performed during single molecule/particle tracking experiments.

Macrophage and NK-mediated killing of precursor-B acute lymphoblastic leukemia cells targeted with a-fucosylated anti-CD19 humanized antibodies.

This work reports the tumoricidal effects of a novel investigational humanized anti-CD19 monoclonal antibody (Medi-551). An a-fucosylated antibody with increased affinity for human FcγRIIIA, Medi-551 is shown to mediate both antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). Medi-551/CD19 complexes internalize slowly (>5 h) and thus remain accessible to effector cells for prolonged periods. We evaluated in vitro ADCC and ADCP activities of primary human natural killer (NK) cells and macrophages against precursor-B (pre-B) acute lymphoblastic leukemia (ALL) cell lines and pediatric patient blasts. Fluorescent imaging studies document immunological synapses formed between anti-CD19-bound target leukemia cells and effector cells and capture the kinetics of both NK-mediated killing and macrophage phagocytosis. Genetic polymorphisms in FcγRIIIA-158F/V modulate in vitro activities of effector cells, with FcγRIIIA-158V homozygotes or heterozygotes showing the strongest activity. Medi-551 treatment of severe combined immunodeficiency (SCID) mice engrafted with human pre-B cells led to prolonged animal survival and markedly reduced disease burden in blood, liver and bone marrow. These data show that anti-CD19 antibodies effectively recruit immune cells to pre-B ALL cells and support a move forward to early phase trials in this disease.

Group VIA phospholipase A2 is a target for vasopressin signaling in the thick ascending limb.

Na(+)-K(+)-2Cl(-) cotransporter (NKCC2)-mediated NaCl reabsorption in the thick ascending limb (TAL) is stimulated by AVP via V2 receptor/PKA/cAMP signaling. This process is antagonized by locally produced eicosanoids such as 20-HETE or prostaglandin E(2), which are synthesized in a phospholipase A(2)-dependent reaction cascade. Using microarray-based gene expression analysis, we found evidence for an AVP-dependent downregulation of the calcium-independent isoform of PLA(2), iPLA(2)ß, in the outer medulla of rats. In the present study, we therefore examined the contribution of iPLA(2)ß to NKCC2 regulation. Immunoreactive iPLA(2)ß protein was detected in cultured mTAL cells as well as in the entire TAL of rodents and humans with the exception of the macula densa. Administration of the V2 receptor-selective agonist desmopressin (5 ng/h; 3 days) to AVP-deficient diabetes insipidus rats increased outer medullary phosphorylated NKCC2 (pNKCC2) levels more than twofold in association with a marked reduction in iPLA(2)ß abundance (-65%; P < 0.05), thus confirming microarray results. Inhibition of iPLA(2)ß in Sprague-Dawley rats with FKGK 11 (0.5 µM) or in mTAL cells with FKGK 11 (10 µM) or (S)-bromoenol lactone (5 µM) for 1 h markedly increased pNKCC2 levels without affecting total NKCC2 expression. Collectively, these data indicate that iPLA(2)ß acts as an inhibitory modulator of NKCC2 activity and suggest that downregulation of iPLA(2)ß may be a relevant step in AVP-mediated urine concentration.

GSI-I (Z-LLNle-CHO) inhibits γ-secretase and the proteosome to trigger cell death in precursor-B acute lymphoblastic leukemia.

Gamma secretase inhibitors (GSIs) comprise a growing class of compounds that interfere with the membrane-bound Notch signaling protein and its downstream intra-nuclear transcriptional targets. As GSI-I (Z-LLNle-CHO) is also a derivative of a widely used proteosome inhibitor MG-132, we hypothesized that this compound might be active in precursor-B acute lymphoblastic leukemia (ALL) cell lines and patient samples. We found that GSI-I treatment of precursor-B ALL blasts induced apoptotic cell death within 18-24 h. With confirmation using RNA and protein analyses, GSI-I blocked nuclear accumulation of cleaved Notch1 and Notch2, and inhibited Notch targets Hey2 and Myc. Microarray analyses of 207 children with high-risk precursor-B ALL demonstrate that Notch pathway expression is a common feature of these neoplasms. However, microarray studies also implicated additional transcriptional targets in GSI-I-dependent cell death, including genes in the unfolded protein response, nuclear factor-κB and p53 pathways. Z-LLNle-CHO blocks both γ-secretase and proteosome activity, inducing more robust cell death in precursor-B ALL cells than either proteosome-selective or γ-secretase-selective inhibitors alone. Using Z-LLNle-CHO in a nonobese diabetes/severe combined immunodeficiency (NOD/SCID) precursor-B ALL xenograft model, we found that GSI-I alone delayed or prevented engraftment of B-lymphoblasts in 50% of the animals comprising the experimental group, suggesting that this compound is worthy of additional testing.

Sensitivity analysis predicts that the ERK-pMEK interaction regulates ERK nuclear translocation.

Following phosphorylation, nuclear translocation of the mitogen-activated protein kinases (MAPKs), ERK1 and ERK2, is critical for both gene expression and DNA replication induced by growth factors. ERK nuclear translocation has therefore been studied extensively, but many details remain unresolved, including whether or not ERK dimerisation is required for translocation. Here, we simulate ERK nuclear translocation with a compartmental computational model that includes systematic sensitivity analysis. The governing ordinary differential equations are solved with the backward differentiation formula and decoupled direct methods. To better understand the regulation of ERK nuclear translocation, we use this model in conjunction with a previously published model of the ERK pathway that does not include an ERK dimer species and with experimental measurements of nuclear translocation of wild-type ERK and a mutant form, ERK1-4, which is unable to dimerise. Sensitivity analysis reveals that the delayed nuclear uptake of ERK1-4 compared to that of wild-type ERK1 can be explained by the altered interaction of ERK1-4 with phosphorylated MEK (MAPK/ERK kinase), and so may be independent of dimerisation. Our study also identifies biological experiments that can verify this explanation.

Revealing the topography of cellular membrane domains by combined atomic force microscopy/fluorescence imaging.

Simultaneous atomic force microscopy (AFM) and confocal fluorescence imaging were used to observe in aqueous buffer the three-dimensional landscape of the inner surface of membrane sheets stripped from fixed tumor mast cells. The AFM images reveal prominent, irregularly shaped raised domains that label with fluorescent markers for both resting and activated immunoglobin E receptors (FcepsilonRI), as well as with cholera toxin-aggregated GM1 and clathrin. The latter suggests that coated pits bud from these regions. These features are interspersed with flatter regions of membrane and are frequently surrounded and interconnected by cytoskeletal assemblies. The raised domains shrink in height by approximately 50% when cholesterol is extracted with methyl-beta-cyclodextrin. Based on composition, the raised domains seen by AFM correspond to the cholesterol-enriched dark patches observed in transmission electron microscopy (TEM). These patches were previously identified as sites of signaling and endocytosis based on their localization of activated FcepsilonRI, at least 10 associated signaling molecules, and the presence of clathrin-coated pits. Overall the data suggest that signaling and endocytosis occur in mast cells from raised membrane regions that depend on cholesterol for their integrity and may be organized in specific relationship with the cortical cytoskeleton.

Regulation of the very late antigen-4-mediated adhesive activity of normal and nonreleaser basophils: roles for Src, Syk, and phosphatidylinositol 3-kinase.

Normal human basophils express the integrin, VLA-4, and cross-linking their high-affinity IgE receptor, FcepsilonRI, increases their VLA-4-dependent adhesion to VCAM-1-transfected Chinese hamster ovary (CHO) cells. Here we show that the FcepsilonRI-mediated up-regulation of normal basophil VLA-4 adhesion is abolished by the Src inhibitor, PP1, the Syk inhibitor, ER-27319, and the phosphatidylinositol 3-kinase inhibitor, wortmannin. PP1, but not ER-27319 or wortmannin, also reduces basal adhesion and adhesion stimulated by chemotactic peptide, by Ca(++) ionophores, and by phorbol myristate acetate (PMA). Nonreleaser basophils (the consistently Syk-deficient, variably Lyn-deficient, severely degranulation-impaired cells found in about 10% of donors) share the PP1 phenotype of lowered basal adhesion, no FcepsilonRI-mediated adhesion up-regulation, and reduced adhesive responses to chemoattractant ionophores and PMA. These results implicate Src kinases in the control of basal VLA-4 activity and place Syk and phosphatidylinositol 3-kinase in the pathway linking FcepsilonRI cross-linking to VLA-4 up-regulation. Both Src and Syk-regulated components of adhesion may be impaired in nonreleaser basophils.

Overcoming the signaling defect of Lyn-sequestering, signal-curtailing FcepsilonRI dimers: aggregated dimers can dissociate from Lyn and form signaling complexes with Syk.

Clustering the tetrameric (alphabetagamma(2)) IgE receptor, FcepsilonRI, on basophils and mast cells activates the Src-family tyrosine kinase, Lyn, which phosphorylates FcepsilonRI beta and gamma subunit tyrosines, creating binding sites for the recruitment and activation of Syk. We reported previously that FcepsilonRI dimers formed by a particular anti-FcepsilonRI alpha mAb (H10) initiate signaling through Lyn activation and FcepsilonRI subunit phosphorylation, but cause only modest activation of Syk and little Ca(2+) mobilization and secretion. Curtailed signaling was linked to the formation of unusual, detergent-resistant complexes between Lyn and phosphorylated receptor subunits. Here, we show that H10-FcepsilonRI multimers, induced by adding F(ab')(2) of goat anti-mouse IgG to H10-treated cells, support strong Ca(2+) mobilization and secretion. Accompanying the recovery of signaling, H10-FcepsilonRI multimers do not form stable complexes with Lyn and do support the phosphorylation of Syk and phospholipase Cgamma2. Immunogold electron microscopy showed that H10-FcepsilonRI dimers colocalize preferentially with Lyn and are rarely within the osmiophilic "signaling domains" that accumulate FcepsilonRI and Syk in Ag-treated cells. In contrast, H10-FcepsilonRI multimers frequently colocalize with Syk within osmiophilic patches. In sucrose gradient centrifugation analyses of detergent-extracted cells, H10-treated cells show a more complete redistribution of FcepsilonRI beta from heavy (detergent-soluble) to light (Lyn-enriched, detergent-resistant) fractions than cells activated with FcepsilonRI multimers. We hypothesize that restraints imposed by the particular orientation of H10-FcepsilonRI dimers traps them in signal-initiating Lyn microdomains, and that converting the dimers to multimers permits receptors to dissociate from Lyn and redistribute to separate membrane domains that support Syk-dependent signal propagation.

High resolution mapping of mast cell membranes reveals primary and secondary domains of Fc(epsilon)RI and LAT.

In mast cells, cross-linking the high-affinity IgE receptor (Fc(epsilon)RI) initiates the Lyn-mediated phosphorylation of receptor ITAMs, forming phospho-ITAM binding sites for Syk. Previous immunogold labeling of membrane sheets showed that resting Fc(epsilon)RI colocalize loosely with Lyn, whereas cross-linked Fc(epsilon)RI redistribute into specialized domains (osmiophilic patches) that exclude Lyn, accumulate Syk, and are often bordered by coated pits. Here, the distribution of Fc(epsilon)RI beta is mapped relative to linker for activation of T cells (LAT), Grb2-binding protein 2 (Gab2), two PLCgamma isoforms, and the p85 subunit of phosphatidylinositol 3-kinase (PI3-kinase), all implicated in the remodeling of membrane inositol phospholipids. Before activation, PLCgamma1 and Gab2 are not strongly membrane associated, LAT occurs in small membrane clusters separate from receptor, and PLCgamma2, that coprecipitates with LAT, occurs in clusters and along cytoskeletal cables. After activation, PLCgamma2, Gab2, and a portion of p85 colocalize with Fc(epsilon)RI beta in osmiophilic patches. LAT clusters enlarge within 30 s of receptor activation, forming elongated complexes that can intersect osmiophilic patches without mixing. PLCgamma1 and another portion of p85 associate preferentially with activated LAT. Supporting multiple distributions of PI3-kinase, Fc(epsilon)RI cross-linking increases PI3-kinase activity in anti-LAT, anti-Fc(epsilon)RIbeta, and anti-Gab2 immune complexes. We propose that activated mast cells propagate signals from primary domains organized around Fc(epsilon)RIbeta and from secondary domains, including one organized around LAT.

p110beta and p110delta phosphatidylinositol 3-kinases up-regulate Fc(epsilon)RI-activated Ca2+ influx by enhancing inositol 1,4,5-trisphosphate production.

Fc(epsilon)RI-induced Ca2+ signaling in mast cells is initiated by activation of cytosolic tyrosine kinases. Here, in vitro phospholipase assays establish that the phosphatidylinositol 3-kinase (PI 3-kinase) lipid product, phosphatidylinositol 3,4,5-triphosphate, further stimulates phospholipase Cgamma2 that has been activated by conformational changes associated with tyrosine phosphorylation or low pH. A microinjection approach is used to directly assess the consequences of inhibiting class IA PI 3-kinases on Ca2+ responses after Fc(epsilon)RI cross-linking in RBL-2H3 cells. Injection of antibodies to the p110beta or p110delta catalytic isoforms of PI 3-kinase, but not antibodies to p110alpha, lengthens the lag time to release of Ca2+ stores and blunts the sustained phase of the calcium response. Ca2+ responses are also inhibited in cells microinjected with recombinant inositol polyphosphate 5-phosphatase I, which degrades inositol 1,4,5-trisphosphate (Ins(1,4,5)P3), or heparin, a competitive inhibitor of the Ins(1,4,5)P3 receptor. This indicates a requirement for Ins(1,4,5)P3 to initiate and sustain Ca2+ responses even when PI 3-kinase is fully active. Antigen-induced cell ruffling, a calcium-independent event, is blocked by injection of p110beta and p110delta antibodies, but not by injection of 5-phosphatase I, heparin, or anti-p110alpha antibodies. These results suggest that the p110beta and p110delta isoforms of PI 3-kinase support Fc(epsilon)RI-induced calcium signaling by modulating Ins(1,4,5)P3 production, not by directly regulating the Ca2+ influx channel.

Multiple defects in Fc epsilon RI signaling in Syk-deficient nonreleaser basophils and IL-3-induced recovery of Syk expression and secretion.

Human basophils respond to Ag-induced cross-linking of their high affinity IgE receptor, FcepsilonRI, by releasing histamine and other mediators from granules, producing IL-4 and other cytokines and, as shown in this study, by forming membrane ruffles and showing increased very late Ag-4 (VLA-4)-mediated adhesion to VCAM-1-expressing target cells. We have identified five blood donors whose basophils lack detectable levels of the FcepsilonRI-associated protein tyrosine kinase, Syk. Despite showing no obvious ultrastructural differences from normal basophils, nonreleaser basophils fail to form membrane ruffles, to show increased VLA-4-mediated adhesive activity, or to produce IL-4 in response to FcepsilonRI cross-linking. Although Syk protein levels are suppressed in basophils from all five donors, Syk mRNA is consistently present. Furthermore, culturing nonreleaser basophils for 4 days with IL-3 restores Syk protein expression and FcepsilonRI-mediated histamine release. Understanding the reversible suppression of Syk protein expression in nonreleaser basophils, and learning to replicate this property in patients with allergic inflammation could be a powerful and specific way to limit symptomatic disease.

Corneal scarring and vision in keratoconus: a baseline report from the Collaborative Longitudinal Evaluation of Keratoconus (CLEK) Study.

PURPOSE: The multicenter Collaborative Longitudinal Evaluation of Keratoconus (CLEK) Study is a prospective, observational study of 1,209 keratoconus patients. We report on the correlation of corneal scarring with clinical and patient-reported variables at the baseline visit. METHODS: Patients completed a questionnaire on their vision, effect of glare, contact lens wear, and work-related issues. Clinical examination included high- and low-contrast visual acuity, refraction, assessment of corneal scarring by the clinician and by photography, and measurement of corneal curvature. The correlation of central corneal scarring with visual acuity and patient-reported variables was analyzed using multiple regression analysis and generalized estimating equations. RESULTS: High- and low-contrast visual acuity with habitual and optimal correction is reduced in scarred eyes. Multiple regression analyses controlling for age, contact lens wear, and disease severity show that central scarring is associated with poorer visual acuity and increased patient-reported symptoms of glare. Restrictions on day-to-day activities do not appear to be associated with corneal scarring above and beyond the effects of keratoconus alone. CONCLUSIONS: Corneal scarring in keratoconus is significantly associated with decreased high- and low-contrast visual acuity.

Immunologically mediated signaling in basophils and mast cells: finding therapeutic targets for allergic diseases in the human FcvarepsilonR1 signaling pathway.

The high affinity IgE receptor, FcvarepsilonRI, plays key roles in an array of acute and chronic human allergic reactions including asthma, allergic rhinitis, atopic dermatitis, urticaria and anaphylaxis. In humans and rodents, this receptor is found at high levels on basophils and mast cells where its activation by IgE and multivalent antigen produces mediators and cytokines responsible for FcvarepsilonRI-dependent acute inflammation. Mast cells can additionally contribute to sustained inflammatory responses by internalizing antigen bound to IgE-FcvarepsilonRI complexes for processing to peptides and presentation to T cells. In humans, the FcvarepsilonRI is also expressed, at lower density, on monocytes, macrophages and dendritic cells (DC) where its likely functions again include both signaling to mediator and cytokine production and antigen presentation. Our laboratories have focused on defining the earliest steps in the FcvarepsilonRI signaling cascade in basophils and mast cells and on developing new routes to control allergic inflammation based on inhibiting these events. Here, we describe novel strategies to limit antigen-stimulated FcvarepsilonRI signaling by: (1) sequestering the FcvarepsilonRI-associated protein-tyrosine kinase, Lyn, that initiates FcvarepsilonRI signaling; (2) eliminating; or (3) inactivating the protein-tyrosine kinase, Syk, that propagates FcvarepsilonRI signaling; and (4) establishing inhibitory crosstalk between FcvarepsilonRI and a co-expressed receptor, FcgammaRII, that again limits FcvarepsilonRI-mediated Syk activation. These strategies may form the basis for new therapies for allergic inflammation.

Negative regulation of FcepsilonRI signaling by FcgammaRII costimulation in human blood basophils.

BACKGROUND: Signaling through the antigen receptors of human B and T cells and the high-affinity IgE receptor FcepsilonRI of rodent mast cells is decreased by cross-linking these receptors to the low-affinity IgG receptor FcgammaRII. The inhibition is thought to involve the tyrosine phosphorylation of immunoreceptor tyrosine-based inhibitory motifs (ITIMs) in the FcgammaRIIB cytoplasmic tail, creating binding sites for SH2-containing protein (Src homology domain containing protein tyrosine phosphatase 1 and 2 [SHP-1, SHP-2]) and/or lipid (SH2 domain-containing polyphosphatidyl-inositol 5-phosphatase) phosphatases that oppose activating signals from the costimulated antigen receptors. OBJECTIVE: In human basophils and mast cells FcepsilonRI signaling generates mediators and cytokines responsible for allergic inflammation. We proposed to determine whether FcepsilonRI signaling is inhibited by FcgammaRII costimulation in human basophils and to explore the underlying mechanism as an approach to improving the treatment of allergic inflammation. METHODS: FcgammaR expression on human basophils was examined using flow cytometry and RT-PCR analysis. FcgammaRII/FcepsilonRI costimulation was typically accomplished by priming cells with anti-dinitrophenol (DNP) IgE and anti-DNP IgG and stimulating with DNP-BSA. Phosphatases were identified by Western blotting, and their partitioning between membrane and cytosol was determined by cell fractionation. Biotinylated synthetic peptides and phosphopeptides corresponding to the FcgammaRIIB ITIM sequence were used for adsorption assays. RESULTS: We report that peripheral blood basophils express FcgammaRII (in both the ITIM-containing FcgammaRIIB and the immunoreceptor tyrosine-based activation motif-containing FcgammaRIIA forms) and that costimulating FcgammaRII and FcepsilonRI inhibits basophil FcepsilonRI-mediated histamine release, IL-4 production, and Ca(2+) mobilization. The inhibition of basophil FcepsilonRI signaling by FcgammaRII/FcepsilonRI costimulation is linked to a significant decrease in Syk tyrosine phosphorylation. Human basophils express all 3 SH2-containing phosphatases. CONCLUSIONS: Evidence that FcgammaRII/FcepsilonRI costimulation induces SHP-1 translocation from the cytosolic to membrane fractions of basophils and that biotinylated synthetic peptides corresponding to the phosphorylated FcgammaRIIB ITIM sequence specifically recruit SHP-1 from basophil lysates particularly implicates this protein phosphatase in the negative regulation of FcepsilonRI signaling by costimulated FcgammaRII.

Factors associated with corneal scarring in the Collaborative Longitudinal Evaluation of Keratoconus (CLEK) Study.

PURPOSE: The multicenter Collaborative Longitudinal Evaluation of Keratoconus (CLEK) Study is a prospective, observational study of 1,209 keratoconus patients. We report on the factors associated with corneal scarring at baseline. METHODS: We defined corneal scarring as scars that had been detected both by the clinician examining the patient with the slit-lamp biomicroscope and by masked readers of corneal photographs at the CLEK Photography Reading Center. We investigated associations between corneal scarring and patient variables including gender, ethnicity, a family history of keratoconus, a history of ocular trauma, eye rubbing, contact lens wear, rigid contact lens fitting relationships, and corneal findings (such as curvature, Vogt's striae, Fleischer's ring, and central/apical staining). Multiple logistic regression analysis using generalized estimating equations to adjust for the correlation between eyes was used for analysis. RESULTS: The following factors were found to increase the odds of corneal scarring at baseline in the CLEK Study: corneal staining (odds ratios (OR) = 3.40, 95% confidence interval 2.53-4.59), contact lens wear (OR = 3.51, 95% confidence interval 2.27-5.45), Fleischer's ring (OR = 1.63, 95% confidence interval 1.11-2.40), steeper first definite apical clearance lens base curve radius (per diopter, OR = 1.29, 95% confidence interval 1.25-1.33), and age (per decade, OR = 1.54, 95% confidence interval 1.35-1.75). CONCLUSIONS: These baseline data suggest that corneal scarring in keratoconus is associated with corneal staining, contact lens wear, Fleischer's ring, a steeper cornea, and increasing age. The factors that imply added risk for corneal scarring that may be affected by practitioner intervention are staining of the cornea, contact lens wear, and the contact lens fitting relationship.

Observing FcepsilonRI signaling from the inside of the mast cell membrane.

We have determined the membrane topography of the high-affinity IgE receptor, FcstraightepsilonRI, and its associated tyrosine kinases, Lyn and Syk, by immunogold labeling and transmission electron microscopic (TEM) analysis of membrane sheets prepared from RBL-2H3 mast cells. The method of Sanan and Anderson (Sanan, D.A., and R.G.W. Anderson. 1991. J. Histochem. Cytochem. 39:1017-1024) was modified to generate membrane sheets from the dorsal surface of RBL-2H3 cells. Signaling molecules were localized on the cytoplasmic face of these native membranes by immunogold labeling and high-resolution TEM analysis. In unstimulated cells, the majority of gold particles marking both FcepsilonRI and Lyn are distributed as small clusters (2-9 gold particles) that do not associate with clathrin-coated membrane. Approximately 25% of FcepsilonRI clusters contain Lyn. In contrast, there is essentially no FcepsilonRI-Syk colocalization in resting cells. 2 min after FcepsilonRI cross-linking, approximately 10% of Lyn colocalizes with small and medium-sized FcepsilonRI clusters (up to 20 gold particles), whereas approximately 16% of Lyn is found in distinctive strings and clusters at the periphery of large receptor clusters (20-100 gold particles) that form on characteristically osmiophilic membrane patches. While Lyn is excluded, Syk is dramatically recruited into these larger aggregates. The clathrin-coated pits that internalize cross-linked receptors bud from membrane adjacent to the Syk-containing receptor complexes. The sequential association of FcstraightepsilonRI with Lyn, Syk, and coated pits in topographically distinct membrane domains implicates membrane segregation in the regulation of FcstraightepsilonRI signaling.

Survival of Staphylococcus aureus inside neutrophils contributes to infection.

Neutrophils have long been regarded as essential for host defense against Staphylococcus aureus infection. However, survival of the pathogen inside various cells, including phagocytes, has been proposed as a mechanism for persistence of this microorganism in certain infections. Therefore, we investigated whether survival of the pathogen inside polymorphonuclear neutrophils (PMN) contributes to the pathogenesis of S. aureus infection. Our data demonstrate that PMN isolated from the site of infection contain viable intracellular organisms and that these infected PMN are sufficient to establish infection in a naive animal. In addition, we show that limiting, but not ablating, PMN migration into the site of infection enhances host defense and that repletion of PMN, as well as promoting PMN influx by CXC chemokine administration, leads to decreased survival of the mice and an increased bacterial burden. Moreover, a global regulator mutant of S. aureus (sar-) that lacks the expression of several virulence factors is less able to survive and/or avoid clearance in the presence of PMN. These data suggest that the ability of S. aureus to exploit the inflammatory response of the host by surviving inside PMN is a virulence mechanism for this pathogen and that modulation of the inflammatory response is sufficient to significantly alter morbidity and mortality induced by S. aureus infection.

Syk deficiency in nonreleaser basophils.

BACKGROUND: Peripheral blood basophils from 10% to 20% of donors fail to degranulate in response to cross-linking the high-affinity IgE receptor FcepsilonRI. The molecular mechanisms underlying the nonreleaser phenotype have not been established. OBJECTIVES: Our aim was to compare the expression of FcepsilonRI-associated protein tyrosine kinases between nonreleaser and releaser basophils. METHODS: With use of Western blotting we investigated Syk and Lyn protein levels in highly purified basophils from 3 anti-IgE nonreleasers and 2 releasers. RESULTS: We identified 3 healthy nonatopic donors whose nonreleaser basophils express FcepsilonRI normally but fail to express protein for the tyrosine kinase Syk, which is implicated in the initiation of FcepsilonRI-mediated secretion. Protein levels for the tyrosine kinase Lyn are somewhat reduced but not absent in nonreleaser basophils. Levels of Lyn and Syk protein are similar in B cells, eosinophils, and neutrophils from releaser and nonreleaser donors. During these studies one nonreleaser "converted" into a releaser with concomitant basophil Syk expression. CONCLUSION: The absence of detectable Syk could explain the nonreleaser phenotype of basophils from some donors.

Dissociation of airway hyperresponsiveness from immunoglobulin E and airway eosinophilia in a murine model of allergic asthma.

Nonspecific airway hyperresponsiveness (AHR) is a hallmark of human asthma. Both airway eosinophilia and high serum levels of total and antigen-specific immunoglobulin E (IgE) are associated with AHR. It is unclear, however, whether either eosinophilia or increased IgE levels contribute directly to, or predict, the development of AHR. Investigations conducted with various murine models of asthma and different mouse strains have resulted in conflicting evidence about the roles that IgE and airway eosinophilia play in the manifestation of AHR. We show that systemic priming with ovalbumin (OVA) in alum, followed by a single day of OVA aerosol challenge, is sufficient to induce AHR, as measured by increased pulmonary resistance in response to intravenously delivered methacholine in BALB/c, but not C57BL/6 or B6D2F1, mice. This was observed despite the fact that OVA-challenged BALB/c mice had less airway eosinophilia and smaller increases in total IgE than either C57BL/6 or B6D2F1 mice, and had less pulmonary inflammation and OVA-specific IgE than B6D2F1 mice. We conclude that airway eosinophilia, pulmonary inflammation, and high serum levels of total or OVA-specific IgE are all insufficient to induce AHR in C57BL/6 and B6D2F1 mice, whereas BALB/c mice demonstrate AHR in the absence of airway eosinophilia. These data confirm that the development of AHR is genetically determined, not only in naive mice, but also in actively immunized ones, and cannot be predicted by levels of airway eosinophilia, pulmonary inflammation, total IgE, or antigen-specific IgE.