Ontogeny of human IgE-expressing B cells and plasma cells.

BACKGROUND: IgE-expressing (IgE+ ) plasma cells (PCs) provide a continuous source of allergen-specific IgE that is central to allergic responses. The extreme sparsity of IgE+ cells in vivo has confined their study almost entirely to mouse models. OBJECTIVE: To characterize the development pathway of human IgE+ PCs and to determine the ontogeny of human IgE+ PCs. METHODS: To generate human IgE+ cells, we cultured tonsil B cells with IL-4 and anti-CD40. Using FACS and RT-PCR, we examined the phenotype of generated IgE+ cells, the capacity of tonsil B-cell subsets to generate IgE+ PCs and the class switching pathways involved. RESULTS: We have identified three phenotypic stages of IgE+ PC development pathway, namely (i) IgE+ germinal centre (GC)-like B cells, (ii) IgE+ PC-like 'plasmablasts' and (iii) IgE+ PCs. The same phenotypic stages were also observed for IgG1+ cells. Total tonsil B cells give rise to IgE+ PCs by direct and sequential switching, whereas the isolated GC B-cell fraction, the main source of IgE+ PCs, generates IgE+ PCs by sequential switching. PC differentiation of IgE+ cells is accompanied by the down-regulation of surface expression of the short form of membrane IgE (mIgES ), which is homologous to mouse mIgE, and the up-regulation of the long form of mIgE (mIgEL ), which is associated with an enhanced B-cell survival and expressed in humans, but not in mice. CONCLUSION: Generation of IgE+ PCs from tonsil GC B cells occurs mainly via sequential switching from IgG. The mIgEL /mIgES ratio may be implicated in survival of IgE+ B cells during PC differentiation and allergic disease.

Intrinsic properties of germinal center-derived B cells promote their enhanced class switching to IgE.

BACKGROUND: Research on the origins and development of human IgE-expressing (IgE(+) ) cells is required for understanding the pathogenesis of allergy and asthma. These studies have been thwarted by the rarity of IgE(+) cells in vivo and the low frequency of class switch recombination (CSR) to IgE ex vivo. To determine the main source of IgE(+) cells, we investigated the relation between the phenotypic composition of tonsil B cells and the CSR to IgE ex vivo. METHODS: Human tonsil B cells were analyzed by flow cytometry (FACS) and cultured with IL-4 and anti-CD40 to induce CSR to IgE. Naïve, germinal center (GC), early GC (eGC), and memory tonsil B cells were isolated by FACS, and their capacities for IL-4 and anti-CD40 signaling, cell proliferation, and de novo class switching to IgE were analyzed by RT-PCR and FACS. RESULTS: B cells from different tonsils exhibited varying capacities for CSR to IgE ex vivo. This was correlated with the percentage of eGC B cells in the tonsil at the outset of the culture. Despite relatively poor cell viability, eGC and GC B-cell cultures produced the highest yields of IgE(+) cells compared to naïve and memory B-cell cultures. The main factors accounting for this result were the strength of IL-4R and CD40 signaling and relative rates of cell proliferation. CONCLUSIONS: This study shows that the maturation state of tonsil B cells determines their capacity to undergo class switching to IgE ex vivo, with the GC-derived B cells yielding the highest percentage of IgE(+) cells.

Inhibition of allergen-dependent IgE activity by antibodies of the same specificity but different class.

IgG4 purified from patients undergoing specific allergen immunotherapy inhibits the activities of the serum IgE in in vitro assays and is thought to reduce the symptoms of the disease. However, it is not known whether this is related to an intrinsic property of this subclass or only the allergen specificity. We tested the hypothesis that allergen specificity is the critical determinant for this activity using a panel of antibodies with identical specificity but different subclasses. The different antibodies were all able to inhibit the activity of IgE to the same extent. We demonstrate that specificity is the dominant factor determining the ability of an antibody to block allergen-dependent IgE activity.

Downregulation of polymeric immunoglobulin receptor and secretory IgA antibodies in eosinophilic upper airway diseases.

BACKGROUND: Immunoglobulin (Ig) A represents a first-line defence mechanism in the airways, but little is known regarding its implication in upper airway disorders. This study aimed to address the hypothesis that polymeric Ig receptor (pIgR)-mediated secretory IgA immunity could be impaired in chronic upper airway diseases. METHODS: Nasal and ethmoidal biopsies, as well as nasal secretions, were collected from patients with chronic rhinosinusitis (CRS) with nasal polyps (CRSwNP) or without nasal polyps (CRSsNP), allergic rhinitis (AR) and controls, and assayed for IgA1/IgA2 synthesis, pIgR expression, production of secretory component (SC), IgA and relevant IgA antibodies, and correlated with local eosinophils and inflammatory features (IL-12, IL-13 and ECP). RESULTS: pIgR expression was decreased in the ethmoidal mucosa in patients with CRSwNP (P = 0.003) and in AR (P = 0.006). This pIgR defect was associated with reduced levels of SC (P = 0.007) and IgA antibodies to Staphylococcus aureus enterotoxin B (SAEB) (P = 0.003) in nasal secretions from patients with CRSwNP, and with increased IgA deposition in subepithelial areas. pIgR downregulation was selectively observed in patients with tissue eosinophilia, whilst no clear relation to smoking history was observed. CONCLUSION: Epithelial pIgR expression is decreased in patients with CRSwNP and AR and results in decreased SC and IgA antibodies to certain bacterial antigens (SAEB) in nasal secretions of patients with CRSwNP in parallel to subepithelial accumulation of IgA. This defect in mucosal immunity is associated with eosinophilic, Th2-related inflammation.

Genetic analysis of potassium use efficiency in Brassica oleracea.

BACKGROUND AND AIMS: Potassium (K) fertilizers are used in intensive and extensive agricultural systems to maximize production. However, there are both financial and environmental costs to K-fertilization. It is therefore important to optimize the efficiency with which K-fertilizers are used. Cultivating crops that acquire and/or utilize K more effectively can reduce the use of K-fertilizers. The aim of the present study was to determine the genetic factors affecting K utilization efficiency (KUtE), defined as the reciprocal of shoot K concentration (1/[K](shoot)), and K acquisition efficiency (KUpE), defined as shoot K content, in Brassica oleracea. METHODS: Genetic variation in [K](shoot) was estimated using a structured diversity foundation set (DFS) of 376 accessions and in 74 commercial genotypes grown in glasshouse and field experiments that included phosphorus (P) supply as a treatment factor. Chromosomal quantitative trait loci (QTL) associated with [K](shoot) and KUpE were identified using a genetic mapping population grown in the glasshouse and field. Putative QTL were tested using recurrent backcross substitution lines in the glasshouse. KEY RESULTS: More than two-fold variation in [K](shoot) was observed among DFS accessions grown in the glasshouse, a significant proportion of which could be attributed to genetic factors. Several QTL associated with [K](shoot) were identified, which, despite a significant correlation in [K](shoot) among genotypes grown in the glasshouse and field, differed between these two environments. A QTL associated with [K](shoot) in glasshouse-grown plants (chromosome C7 at 62.2 cM) was confirmed using substitution lines. This QTL corresponds to a segment of arabidopsis chromosome 4 containing genes encoding the K+ transporters AtKUP9, AtAKT2, AtKAT2 and AtTPK3. CONCLUSIONS: There is sufficient genetic variation in B. oleracea to breed for both KUtE and KUpE. However, as QTL associated with these traits differ between glasshouse and field environments, marker-assisted breeding programmes must consider carefully the conditions under which the crop will be grown.

Evidence of neutral transcriptome evolution in plants.

* The transcriptome of an organism is its set of gene transcripts (mRNAs) at a defined spatial and temporal locus. Because gene expression is affected markedly by environmental and developmental perturbations, it is widely assumed that transcriptome divergence among taxa represents adaptive phenotypic selection. This assumption has been challenged by neutral theories which propose that stochastic processes drive transcriptome evolution. * To test for evidence of neutral transcriptome evolution in plants, we quantified 18 494 gene transcripts in nonsenescent leaves of 14 taxa of Brassicaceae using robust cross-species transcriptomics which includes a two-step physical and in silico-based normalization procedure based on DNA similarity among taxa. * Transcriptome divergence correlates positively with evolutionary distance between taxa and with variation in gene expression among samples. Results are similar for pseudogenes and chloroplast genes evolving at different rates. Remarkably, variation in transcript abundance among root-cell samples correlates positively with transcriptome divergence among root tissues and among taxa. * Because neutral processes affect transcriptome evolution in plants, many differences in gene expression among or within taxa may be nonfunctional, reflecting ancestral plasticity and founder effects. Appropriate null models are required when comparing transcriptomes in space and time.

Control of cytokine gene transcription in Th1 and Th2 cells.

Summary Analysis of T-helper cell differentiation to T-helper type 1 (Th1) and Th2 lineages has begun to reveal a complex mechanism whereby transcription factors, enzymes that either deposit or remove covalent modifications from histone tails and DNA methylating enzymes are recruited to cytokine genes. Each resultant cell lineage subsequently displays a programme of transcriptional restrictions that firstly, facilitates expression of a particular subset of signature cytokines and secondly, silences expression of the cytokines normally recognized as being markers of the opposite differentiation limb. Some essential proteins in this differentiative paradigm, such as the transcription factors GATA3 and T-bet, are well studied; however, the types of enzymatic activities that these proteins recruit in order to implement differentiation are more obscure. Recent genome-wide studies of histone modifications have begun to clarify how specific modifications of histones impact upon both transcriptional regulation and chromatin organization. Here we review how this information has enlightened our knowledge of how Th1/Th2 differentiation is orchestrated.

Interactions between selenium and sulphur nutrition in Arabidopsis thaliana.

Selenium (Se) is an essential plant micronutrient, but is toxic at high tissue concentrations. It is chemically similar to sulphur (S), an essential plant macronutrient. The interactions between Se and S nutrition were investigated in the model plant Arabidopsis thaliana (L.) Heynh. Arabidopsis plants were grown on agar containing a complete mineral complement and various concentrations of selenate and sulphate. The Se/S concentration ratio in the shoot ([Se](shoot)/[S](shoot)) showed a complex dependence on the ratio of selenate to sulphate concentration in the agar ([Se](agar)/[S](agar)). Increasing [S](agar) increased shoot fresh weight (FW) and [S](shoot), but decreased [Se](shoot). Increasing [Se](agar) increased both [Se](shoot) and [S](shoot), but reduced shoot FW. The reduction in shoot FW in the presence of Se was linearly related to the shoot Se/S concentration ratio. These data suggest (i) that Se and S enter Arabidopsis through multiple transport pathways with contrasting sulphate/selenate selectivities, whose activities vary between plants of contrasting nutritional status, (ii) that rhizosphere sulphate inhibits selenate uptake, (iii) that rhizosphere selenate promotes sulphate uptake, possibly by preventing the reduction in the abundance and/or activity of sulphate transporters by sulphate and/or its metabolites, and (iv) that Se toxicity occurs because Se and S compete for a biochemical process, such as assimilation into amino acids of essential proteins.

c-Myc represses the murine Nramp1 promoter.

The Nramp1 (natural resistance-associated macrophage protein 1) gene modulates the growth of intracellular pathogens and encodes a divalent cation transporter within lysosomes/late endosomes of macrophages. Nramp1 modulates the cytoplasmic iron pool. Wu, Polack and Dalla-Favera [(1999) Science 283, 676-679] showed reciprocal control of H-ferritin and IRP2 by c-Myc, and suggest that c-Myc regulates genes to increase cytoplasmic iron. A role for c-Myc in Nramp1 regulation was evaluated. Co-transfection studies show that c-Myc represses Nramp1 promoter function. Five non-canonical Myc-max binding sites (E-box) identified within the Nramp1 5'-flanking sequence are not responsible for the inhibitory effects of c-Myc on Nramp1 expression. An initiator(s) adjacent to the transcription-initiation site is a candidate for the inhibition observed. Results are consistent with a role for Nramp1 removing iron from the cytosol and antagonizing c-Myc function.

Influx and accumulation of Cs(+) by the akt1 mutant of Arabidopsis thaliana (L.) Heynh. lacking a dominant K(+) transport system.

An extensive literature reports that Cs(+), an environmental contaminant, enters plant cells through K(+) transport systems. Several recently identified plant K(+) transport systems are permeable to Cs(+). Permeation models indicate that most Cs(+) uptake into plant roots under typical soil ionic conditions will be mediated by voltage-insensitive cation (VIC) channels in the plasma membrane and not by the inward rectifying K(+) (KIR) channels implicated in plant K nutrition. Cation fluxes through KIR channels are blocked by Cs(+). This paper tests directly the hypothesis that the dominant KIR channel in plant roots (AKT1) does not contribute significantly to Cs(+) uptake by comparing Cs(+) uptake into wild-type and the akt1 knockout mutant of Arabidopsis thaliana (L.) Heynh. Wild-type and akt1 plants were grown to comparable size and K(+) content on agar containing 10 mM K(+). Both Cs(+) influx to roots of intact plants and Cs(+) accumulation in roots and shoots were identical in wild-type and akt1 plants. These data indicate that AKT1 is unlikely to contribute significantly to Cs(+) uptake by wild-type Arabidopsis from 'single-salt' solutions. The influx of Cs(+) to roots of intact wild-type and akt1 plants was inhibited by 1 mM Ba(2+), Ca(2+) and La(3+), but not by 10 microM Br-cAMP. This pharmacology resembles that of VIC channels and is consistent with the hypothesis that VIC channels mediate most Cs(+) influx under 'single-salt' conditions.

Community exposures to chemical incidents: development and evaluation of the first environmental public health surveillance system in europe.

OBJECTIVE: To describe the frequency, nature and location of acute chemical incidents in Wales, and the morbidity in employees, emergency responders and the general public who were exposed. DESIGN: Active multi-agency community-based surveillance system. SETTING: Wales, 1993-5. MAIN OUTCOME MEASURES: Frequency, nature and location of incidents, populations potentially exposed and with symptoms. RESULTS: Most of the 402 incidents identified were not associated with sites governed by the Control of Industrial Major Accident Hazard Regulations but with smaller industrial sites and commercial premises. About two in every thousand of the estimated 236 000 members of the public considered to be at risk from exposure reported symptoms, which were mainly nausea, headaches, and irritation of the eye, skin and respiratory tract. The most commonly reported chemicals that members of the public were exposed to were smoke toxins, miscellaneous organics, toxic gases and flammable gases. A health authority was reported to be involved in only 34 (8%) of the incidents and in only 3 of the 29 incidents where more than 100 members of the public were exposed. CONCLUSION: A geographically defined, multi-agency surveillance system can identify high risk locations and types of incidents, together with the chemicals most likely to be involved. Such ongoing surveillance information is essential for appropriate policy making, emergency planning, operational management and training.

Nramp1: a link between intracellular iron transport and innate resistance to intracellular pathogens.

Nramp1 (natural resistance-associated macrophage protein one) regulates intracellular pathogen proliferation and macrophage inflammatory responses. Murine Nramp1 exhibits a natural polymorphism with alleles termed resistant and susceptible. Alleles restrict or allow the proliferation of intracellular pathogens, respectively. Structural predictions suggest that Nramp1 encodes the prototypic member of a transporter family. Nramp1 exhibits sequence identity to Nramp2, which regulates intestinal and reticulocyte iron uptake. Based on this sequence identity we have initiated experiments for Nramp1 to investigate its role in macrophage iron homoeostasis and using a transfection approach in the RAW264.7 murine macrophage-like cell line, which lacks a functional Nramp1 gene. Nramp1 expression supports increased acute cytoplasmic influx of iron, detected using the fluorescent iron sensor dye calcein. Analysis of the endogenous iron sensors, iron regulatory protein 1 and 2, reveals a greater flux of iron in Nramp1-expressing cells and in its exclusion from the cytoplasm. Other work supports the prediction that Nramp1 is a phosphoprotein and the extent of phosphorylation changes in response to inflammatory cytokines. Together these data support the hypothesis that control of intracellular iron homoeostasis is a vital element used by phagocytes to control the proliferation of intracellular pathogens.

Cell marking in Arabidopsis thaliana and its application to patch-clamp studies.

Ion transport processes at the plasma membrane of plant cells are frequently studied by applying membrane-patch voltage-clamp (patch-clamp) electrophysiological techniques to isolated protoplasts. As plants are composed of many tissues and cell types, and each tissue and cell type may be specialized to a particular function and possess a unique complement of transport proteins, it is important to certify the anatomical origin of the protoplasts used for patch-clamp studies. This paper describes a general molecular genetic approach to marking specific cell types for subsequent patch-clamp studies and presents a specific example: a comparison of the K+ currents in protoplasts from cortical and stelar cells of Arabidopsis roots. Transgenic Arabidopsis were generated in which the expression of green fluorescent protein (GFP) from Aequoria victoria was driven by the CaMV 35S promoter (line mGFP3). In roots of the transgenic mGFP3 line, visible fluorescence was restricted to the stele. Protoplasts were generated from roots of the mGFP3 line and K+ currents in non-fluorescent (cortical/epidermal) and fluorescent (stelar) protoplasts were assayed using patch-clamp techniques. It was found that both the frequency of observing inward rectifying K+ channel (IRC) activity and the relative occurrence of IRC compared to outward rectifying K+ channels were significantly lower in protoplasts from cortical/epidermal cells compared to cells of the stele. The presence of GFP did not affect the occurrence or biophysical properties of K+ channels. It is concluded that the generation of transgenic Arabidopsis expressing GFP in a cell-specific fashion is a convenient and reliable way to mark protoplasts derived from contrasting cell types for subsequent patch-clamp studies.

Oxidative changes associated with beta-carotene and alpha-tocopherol enrichment of human low-density lipoproteins.

OBJECTIVE: To determine what effects enrichment of human low-density lipoprotein (LDL) with combinations of alpha-tocopherol and beta-carotene would exert on LDL oxidation and attempt to define the nature of the effects. METHODS: Human plasma was pooled and alpha-tocopherol and beta-carotene was added in a four-by-four design resulting in the enrichment of LDL with alpha-tocopherol and beta-carotene in varying concentrations. Enriched and control LDL was oxidized in Cu2+ mediated oxidation system and resistance of LDL to oxidation was determined by lag time, thiobarbituric acid reactive substances (TBARS) activity, and rate of oxidation. RESULTS: Increasing LDL alpha-tocopherol concentration had a linear relationship with lag time and a negative correlation with rate of oxidation. LDL beta-carotene concentration was linearly correlated with the rate of LDL oxidation and beta-carotene loss, and exponentially related to TBARS concentration. CONCLUSIONS: These results support earlier findings for the protective effect of a-tocopherol against LDL oxidation, and suggest that beta-carotene participates as a prooxidant in the oxidative degradation of LDL under these conditions. Since high levels of alpha-tocopherol did not mitigate the prooxidative effect of beta-carotene, these result indicate that increased LDL beta-carotene may cancel the protective qualities of alpha-tocopherol.

Blood antioxidants changes in young women following beta-carotene depletion and repletion.

OBJECTIVE: This study was undertaken to investigate the relationship between beta-carotene intake and biochemical indices of antioxidant status in the blood of nine premenopausal women ages 18 to 42. METHODS: Nine healthy adult women were fed a low beta-carotene diet for 68 days. They were repleted with the same diet supplemented with beta-carotene (15 mg beta-carotene) for 28 days. During the last week of the study, they received an additional mixed carotenoid supplement. Indices of blood antioxidant status were measured on days 1, 29, 36, 43, 50, 64, 71, 92, and 99. RESULTS: We found significant increases of erythrocyte conjugated dienes between the 71st and 99th day of the study; increases of glutathione (GSH) peroxidase (GP) on day 43 and day 92 compared to a decrease on day 29; and decreases of GSH reductase throughout the treatment period. Erythrocyte catalase activities seemed to parallel GP activities. Erythrocyte oxidized glutathione (GSSG) levels were depressed both after beta-carotene depletion and repletion. beta-Carotene depletion/repletion had no effect on plasma vitamin E or GSH levels. Platelet GSH levels were depressed after beta-carotene depletion followed by elevated GSH levels after beta-carotene repletion. CONCLUSION: A diet low in beta-carotene and adequate in all other nutrients, including vitamin A, resulted in altered erythrocyte and platelet antioxidant indices; however, it had little impact on plasma GSH or vitamin E levels in young healthy women. Our results are consistent with the suggestion that carotenes may be important in the prevention of oxidative damage.

Effects of calcium phosphate solutions on dentin permeability.

Calcium phosphate solutions at various concentrations and pH levels were used to obstruct the dentinal tubules. The effects were evaluated by measurements of permeability through dentin discs and by scanning electron microscopy. Precipitation kinetics were followed by pH changes in the solutions and products were determined by X-ray powder diffraction. The solutions were applied in two ways: (a) calcium and phosphate solutions were mixed before application and (b) one solution (calcium or phosphate) was applied first followed by the other solution. Three kinds of human dentin discs were used; one with smear layer and the other two with tubules exposed by sonication or etched by acid. The high concentration calcium phosphate solutions at pH = 9.5 rapidly precipitated amorphous calcium phosphates that obstructed the dentinal tubules and decreased dentin permeability by 85% or more. At pH = 5.6, the calcium phosphate solutions precipitated large crystals of dicalcium phosphate dihydrate. In this case, the effectiveness in obstructing dentinal tubules was found to be procedure sensitive.

Fracture-surface analysis of dental ceramics.

This study demonstrated that quantitative fractography can be used to study failed aluminous and glass-ceramic central porcelains. Fracture surfaces of DICOR and Vitadur-N core porcelain modulous-of-rupture bars were studied to identify fracture mirror features useful in (1) locating the source of fracture and (2) calculating the stress at fracture in clinically failed restorations. The morphology of fracture surfaces results from events related to the initiation and propagation of the crack front during failure. Modulus-of-rupture testing was performed in four-point bending. Fracture surfaces were studied by scanning electron microscopy. The mean fracture stress for the Vitadur-N porcelain was 94.7 +/- 12.4 MPa (13,730 psi); for DICOR the fracture stress was 55.4 +/- 10.6 MPa (8,030 psi). The standard quantitative fractography relationship between in mirror radius and ln fracture stress was followed for both materials. This quantitative fractography relationship was used to calculate the in vivo stress at failure in a clinically fractured DICOR molar crown. Five clinically failed DICOR crowns were seen to fail from the internal surface.

Cross-gender behavior and psychopathology in boy psychiatric outpatients.

One hundred 5- to 12-year-old boys referred for outpatient psychiatric evaluation were assessed for cross-gender behavior using the Child Behavior and Attitude Questionnaire (CBAQ) and the Child Game Participation Questionnaire (CGPQ), and for possible associated psychopathology using the Child Behavior Checklist (CBCL) and clinical psychiatric (DSM-III) diagnoses. On the two feminine scales of the CBAQ and CGPQ, 30 to 50% scored within defined clinical ranges. High feminine scale scorers did not have higher Total CBCL scores than lower feminine scale scorers, and scores on the feminine scales correlated minimally with scores on the CBCL broad and narrow-band behavior problem scales, except for a significant positive correlation with the Delinquent subscale. No particular clinical psychiatric diagnoses were significantly associated with high feminine scorers: however, high feminine behavior scorers tended to have more conduct problems and mixed adjustment disorders and less anxious and depressive psychopathology. Clinicians were not alert to the degree of cross-gender behavior found, perhaps due to the concomitant externalizing psychopathology and masculine behavior in these same patients.