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.

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.

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.

Glucose turnover and disposal in maturity-onset diabetes.

The glucose turnover rate in maturity-onset diabetes in man has been variously reported as increased, normal, and decreased. The present experiments suggest that these discrepancies may have been due to methodology, and to nonrecognition of a circadian cycle in the glucose turnover rate that is present in health, and marked in diabetes. During the early morning hours the glucose turnover rate in maturity-onset diabetes is increased in proportion to the fasting blood glucose level. It may reach three to four times the rate found in health. During the evening hours the increments are about one-half as great. The glucose outflow rate constant, k, lower in diabetes than in health, is also lower in both groups in the evening than in the morning. An analysis of the relative contributions of glucose overproduction and underutilization to the development of hyperglycemia in maturity-onset diabetes indicates that overproduction is the greater factor. The relative role of underutilization appears to increase as the fasting blood glucose level increases. The circulating glucose oxidation rate in maturity-onset diabetes is only slightly lower than in health, but the fraction oxidized is markedly lower, and only a small fraction is excreted. The principal conclusion is that in maturity-onset diabetes there is a hypertrophied flux of endogenous glucose, most of which is neither oxidized nor excreted. The precursors and the qualitative and quantitative metabolic fates of this excess glucose are unknown.

Circulating alanine disposal in diabetes mellitus.

Alanine was selected for study as a representative circulating glucose precursor in relation to the question of the source of the excess circulating glucose in diabetes mellitus. U-14C alanine and U-14C glucose infusions were given to healthy subjects and to subjects with untreated mild maturity, severe maturity, and juvenile diabetes. Comparative studies after a 24-hour fast were made in healthy and in mildly diabetic subjects. The alanine production rate was unaltered by fasting or diabetes. The glucose production rate was unaltered by fasting but increased in diabetes in relation to the severity of the disease. The fractions of alanine-to-glucose and of glucose-from-alanine were increased by fasting. The effect of diabetes was different. The fraction of alanine-to-glucose was much less in mild maturity diabetes than in health, and it was increased only in juvenile diabetes. In all the diabetic groups the glucose-from-alanine fraction was much less than in health. In every group the change in the alanine oxidation rate was reciprocal to that in the alanine-derived glucogenesis rate. The results are consistent with the possibility that the principal source of the excess circulating glucose in diabetes is glycogen.

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.

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.

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.

The use of electrolyte measurements in the detection of cystic fibrosis.

Six tests recommended for use in the diagnosis of cystic fibrosis (CF) have been compared in the same subjects. The tests were carried out on 165 normal persons, 64 known cases of CF, their 67 parents and 18 sibs. The tests measured sodium in fingernails by activation, sodium in saliva and chloride in saliva and in thermal and pilocarpine-induced sweat by means of ion-specific electrodes, and chloride in pilocarpine-sweat by the standard titrimetric method. The tests ranged in complexity from simple screening methods to individual clinical procedures. None of the simpler tests matched the standard pilocarpine method in diagnostic efficiency, whether they were used singly or in combination.

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.

The use of reference materials in the elemental analysis of biological samples.

Reference materials (RMs) are useful to compare the accuracy and precision of laboratories and techniques. The desirable properties of biological reference materials are listed, and the problems of production, homogenization and storage described. At present there are only 10 biological RMs available compared with 213 geological and 520 metallurgical RMs. There is a need for more biological RMs including special materials for microprobe analysis and for in vivo activation analysis. A study of 650 mean values for elements in RM Kale, analysed by many laboratories, leads to the following conclusions. 61% of the values lie within +/- 10% of the best mean, and 80% lie within +/- 20% of the best mean. Atomic absorption spectrometry gives results that are 5-30% high for seven elements, while instrumental neutron activation analysis gives low and imprecise results for K. Other techniques with poor interlaboratory precision include neutron activation for Mg, polarography for Zn and arc-spectrometry for many elements. More than half the values for elements in Kale were obtained by neutron activation, confirming the importance of this technique and the need for RMs. As a rough estimate, 6 X 10(9) elemental analyses of biological materials are carried out each year, mostly by medical, agricultural and food scientists. It seems likely that a substantial percentage of these are inaccurate, a situation that might be improved by quality control using standard RMs.

Structure of Sendai viral proteins in plasma membranes of virus-infected cells.

Purified plasma membranes attached to polycationic polyacrylamide beads by their external surface were isolated from BHK cells infected with Sendai virus. Each of the viral proteins could be identified in the membranes of infected cells. Proteolysis with trypsin, which digests only the cytoplasmic surface of these membranes (because the external surface is protected by its attachment to beads), revealed that the internal proteins, L, P, NP, and M, were present on the cytoplasmic surface of the membrane and that small segments of the viral envelope glycoproteins, HN and F0, were partially exposed on the cytoplasmic surface. Since the major portions of HN and F0 are known to be present on the external membrane surface, these glycoproteins are transmembrane proteins before Sendai virus budding in infected cells.

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.

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.