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.

Transcription factors regulating B cell fate in the germinal centre.

Diversification of the antibody repertoire is essential for the normal operation of the vertebrate adaptive immune system. Following antigen encounter, B cells are activated, proliferate rapidly and undergo two diversification events; somatic hypermutation (followed by selection), which enhances the affinity of the antibody for its cognate antigen, and class-switch recombination, which alters the effector functions of the antibody to adapt the response to the challenge faced. B cells must then differentiate into antibody-secreting plasma cells or long-lived memory B cells. These activities take place in specialized immunological environments called germinal centres, usually located in the secondary lymphoid organs. To complete the germinal centre activities successfully, a B cell adopts a transcriptional programme that allows it to migrate to specific sites within the germinal centre, proliferate, modify its DNA recombination and repair pathways, alter its apoptotic potential and finally undergo terminal differentiation. To co-ordinate these processes, B cells employ a number of 'master regulator' transcription factors which mediate wholesale transcriptomic changes. These master transcription factors are mutually antagonistic and form a complex regulatory network to maintain distinct gene expression programs. Within this network, multiple points of positive and negative feedback ensure the expression of the 'master regulators', augmented by a number of 'secondary' factors that reinforce these networks and sense the progress of the immune response. In this review we will discuss the different activities B cells must undertake to mount a successful T cell-dependent immune response and describe how a regulatory network of transcription factors controls these processes.

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.

Local receptor revision and class switching to IgE in chronic rhinosinusitis with nasal polyps.

BACKGROUND: Chronic rhinosinusitis with nasal polyps (NP) and allergic rhinitis (AR) is characterized by local Th2 inflammation and up-regulation of IgE; however, IgE in NP is 'polyclonal' and allergen specific, whereas IgE in AR is 'oligoclonal' and allergen specific. Germinal center (GC) reactions occur in AR, while only the formation of GC-like structures in NP is described. The aim of this study was to investigate the involvement of local IgE production, class switch recombination, and receptor revision in NP. METHODS: We compared the levels of local IgE, germline gene transcripts, and mature Ig mRNA expression, recombination activating gene (RAG1 and RAG2), key markers of Th2 inflammation, and GC reactions in NP tissue vs AR and control tissue. Nasal mucosa was immunostained for the co-expression of RAG1 and RAG2 in B cells, plasma cells, and T cells, using dual or triple immunofluorescence (IF). RESULTS: In NP, local IgE level and key markers of local class switching are increased compared with AR and normal controls (NC). In NP, switch circle transcripts reveal ongoing local class switch recombination to IgE. Up to 30% of B cells, plasma cells, and T cells in nasal polyps re-express both RAG1 and RAG2, required for receptor revision. RAG1 and RAG2 mRNA concentrations are increased in NP and correlated with the magnitude of inflammation and the presence of S. aureus enterotoxin (superantigen)-specific IgE in the nasal polyp mucosa. CONCLUSION: Our results provide the first evidence of local receptor revision and class switching to IgE, and B-cell differentiation into IgE-secreting plasma cells in NP.

Seven genomic subtypes of chronic fatigue syndrome/myalgic encephalomyelitis: a detailed analysis of gene networks and clinical phenotypes.

AIM: Chronic fatigue syndrome/myalgic encephalomyelitis (CFS/ME) is a multisystem disease, the pathogenesis of which remains undetermined. The authors have recently reported a study of gene expression that identified differential expression of 88 human genes in patients with CFS/ME. Clustering of quantitative PCR (qPCR) data from patients with CFS/ME revealed seven distinct subtypes with distinct differences in Medical Outcomes Survey Short Form-36 scores, clinical phenotypes and severity. METHODS: In this study, for each CFS/ME subtype, those genes whose expression differed significantly from that of normal blood donors were identified, and then gene interactions, disease associations and molecular and cellular functions of those gene sets were determined. Genomic analysis was then related to clinical data for each CFS/ME subtype. RESULTS: Genomic analysis revealed some common (neurological, haematological, cancer) and some distinct (metabolic, endocrine, cardiovascular, immunological, inflammatory) disease associations among the subtypes. Subtypes 1, 2 and 7 were the most severe, and subtype 3 was the mildest. Clinical features of each subtype were as follows: subtype 1 (cognitive, musculoskeletal, sleep, anxiety/depression); subtype 2 (musculoskeletal, pain, anxiety/depression); subtype 3 (mild); subtype 4 (cognitive); subtype 5 (musculoskeletal, gastrointestinal); subtype 6 (postexertional); subtype 7 (pain, infectious, musculoskeletal, sleep, neurological, gastrointestinal, neurocognitive, anxiety/depression). CONCLUSION: It was particularly interesting that in the seven genomically derived subtypes there were distinct clinical syndromes, and that those which were most severe were also those with anxiety/depression, as would be expected in a disease with a biological basis.

Gene expression in peripheral blood mononuclear cells from patients with chronic fatigue syndrome.

BACKGROUND: Chronic fatigue syndrome (CFS) is a multisystem disease, the pathogenesis of which remains undetermined. AIMS: To test the hypothesis that there are reproducible abnormalities of gene expression in patients with CFS compared with normal healthy persons. METHODS: To gain further insight into the pathogenesis of this disease, gene expression was analysed in peripheral blood mononuclear cells from 25 patients with CFS diagnosed according to the Centers for Disease Control criteria and 25 normal blood donors matched for age, sex, and geographical location, using a single colour microarray representing 9522 human genes. After normalisation, average difference values for each gene were compared between test and control groups using a cutoff fold difference of expression > or = 1.5 and a p value of 0.001. Genes showing differential expression were further analysed using Taqman real time polymerase chain reaction (PCR) in fresh samples. RESULTS: Analysis of microarray data revealed differential expression of 35 genes. Real time PCR confirmed differential expression in the same direction as array results for 16 of these genes, 15 of which were upregulated (ABCD4, PRKCL1, MRPL23, CD2BP2, GSN, NTE, POLR2G, PEX16, EIF2B4, EIF4G1, ANAPC11, PDCD2, KHSRP, BRMS1, and GABARAPL1) and one of which was downregulated (IL-10RA). This profile suggests T cell activation and perturbation of neuronal and mitochondrial function. Upregulation of neuropathy target esterase and eukaryotic translation initiation factor 4G1 may suggest links with organophosphate exposure and virus infection, respectively. CONCLUSION: These results suggest that patients with CFS have reproducible alterations in gene regulation.

Bupivacaine 0.125% produces motor block and weakness with fentanyl epidural analgesia in children.

PURPOSE: Epidural infusions of fentanyl (2 micrograms.ml-1) alone or combined with bupivacaine 0.125% were compared for perioperative analgesia, motor block and other side-effects in children who underwent urological surgery. METHODS: In a prospective, double-blind study, 42 children, ASA I-II, 1-16 yr, were randomly allocated to receive either epidural F (fentanyl bolus 2 micrograms.kg-1 in 0.5 ml.kg-1 saline followed by 2 micrograms.ml-1 fentanyl infusion) or epidural F-B (fentanyl bolus 2 micrograms.kg-1 in 0.5 ml.kg-1 bupivacaine 0.25% followed by 2 micrograms.ml-1 fentanyl infusion in bupivacaine 0.125%) after induction of general anaesthesia. Adequacy of analgesia, lower limb motor block and side-effects were assessed four hourly postoperatively. RESULTS: Both infusion regimens provided excellent analgesia (median objective pain scores = 0). Epidural infusion rates were similar in the F (0.29 +/- 0.07 ml.kg-1.hr-1) and F-B (0.26 +/- 0.05 ml.kg-1.hr-1) groups. Three children in the F group and all children in the F-B group developed lower limb weakness. (P < 0.05) Bromage scores were different in the F group (median 0, range 0-0.66) compared with the F-B group (median 0.33, range 0-1) (P < 0.001). Other side-effects did not differ. CONCLUSION: Postoperative epidural fentanyl infusion provides equipotent analgesia to administration of a solution including both fentanyl and bupivacaine 0.125% and causes less lower limb weakness. No reduction in the fentanyl requirement resulted from the addition of bupivacaine 0.125%.

Post-tonsillectomy infiltration with bupivacaine reduces immediate postoperative pain in children.

Pain management after tonsillectomy in children remains a dilemma for the anaesthetist. A previous study demonstrated that the administration of lidocaine 1% topical spray to the peritonsillar fossae before tracheal extubation provided considerable immediate postoperative pain relief in infants and children. However, the pain relief was of short duration. We were hopeful that the use of bupivacaine would offer more prolonged pain relief because of its pharmacological characteristics. Therefore, this study was designed to compare the effects of bupivacaine 0.5% with 1:200,000 epinephrine administered after tonsillectomy either as topical spray or submucosal infiltration on postoperative pain in children. Forty-three patients aged two to ten years were randomized into three groups after tonsillectomy was performed. Group (1) received 0.5 ml.kg-1 normal saline spray; (2) received 2 mg.kg-1 bupivacaine 0.5% with 1:200,000 epinephrine peritonsillar infiltration in a similar volume to Group 1 and; (3) received 2 mg.kg-1 bupivacaine 0.5% with 1:200,000 epinephrine spray to both tonsillar beds. The patients in each group were compared postoperatively with regard to the quality of pain control using the Objective Pain Score, and their analgesic requirements. Peritonsillar infiltration of bupivacaine provided superior immediate postoperative analgesia as reflected by lower recovery room pain scores (P < 0.05) and opioid requirements (P < 0.01). Ward pain scores and analgesic requirements were similar among groups. Peritonsillar infiltration of bupivacaine 0.5% with 1:200,000 epinephrine provides better post-tonsillectomy pain control in the immediate postoperative period than bupivacaine spray or placebo.

Nuclear translocation of a maternal CCAAT factor at the start of gastrulation activates Xenopus GATA-2 transcription.

The transcription factor GATA-2 is present in blood cell precursors and plays a pivotal role in the control of erythroid differentiation. In Xenopus embryos, low levels of GATA-2 mRNA are maternally derived, while the onset of zygotic GATA-2 expression coincides with commitment to haematopoietic lineages. However, its initial transcriptional activation is not restricted to the presumptive blood islands, but occurs throughout ventral and lateral regions, in all three germ layers. In order to determine how this expression pattern is controlled, we have isolated and characterized the Xenopus GATA-2 gene. We show that 1.65 kb of 5' flanking sequences are sufficient to direct both correct transcriptional initiation in oocytes and appropriate temporal and spatial gene expression in early embryos. The transgene is activated during gastrulation and by neurula stages in predominantly expressed in the ventral hemisphere. We demonstrate that a CCAAT element is necessary for gene activity in both systems and that extracts prepared from oocytes and embryos contain a factor which specifically recognizes this element. We also show that cytoplasmic localization inhibits the function of this CCAAT factor until the beginning of gastrulation, when the zygotic GATA-2 gene is activated. These observations extend our understanding of the mechanisms by which maternal factors control the temporal activation of transcription in early vertebrate embryos.

Continuing medical education practices of community-based and university-affiliated anaesthetists in Ontario.

To remedy the lack of information about the continuing medical education (CME) practices of anaesthetists, we designed a survey to define and compare the CME activities of specialist anaesthetists in community-based and university-affiliated practices: 463 members of the Canadian Anaesthetists' Society in the Province of Ontario (263 community-based and 200 university-affiliated (University of Toronto) anaesthetists). Data from 304 (65.6%) respondents (172 community-based and 132 university-affiliated anaesthetists) were analyzed by non-parametric analysis (statistical significance P < 0.05). Most respondents spent between two to four hours per week on CME activities. Journal reading was the most commonly used method to obtain CME and was perceived to be the most efficient of the methods surveyed (P < 0.05). Formal teaching, including seminars, workshops, and annual society meetings, although the second most commonly used technique to obtain CME, was considered as effective as journal reading. Instructional media techniques were the least commonly used and considered the least effective (P < 0.05). Most community-based and university-affiliated anaesthetists obtained CME by a variety of techniques; of all respondents, 77% have no formal method of assessing their learning needs and 88% would consider participation in a formalized learning needs assessment programme.

Analgesic efficacy and safety of a caudal bupivacaine-fentanyl mixture in children.

The analgesic efficacy and safety of a single caudal injection of a bupivacaine-fentanyl mixture was investigated in this prospective, controlled, triple-blinded study of 34 children, aged 1-11 yr and of ASA physical status I-II undergoing urological surgery. After induction of anaesthesia and before surgery, the children were randomly assigned to receive a caudal injection of 1.0 ml.kg-1 bupivacaine 0.125% with epinephrine 1:400,000 and either fentanyl 1.0 microgram.kg-1 in 1.0 ml of normal saline or 1.0 ml of normal saline. After completion of surgery, patients were assessed in the recovery room for six hours from the time of the caudal injection and for a further 18 hr on the ward. While in the recovery room arterial oxygen saturation and respiratory rate were monitored continuously and recorded hourly together with end-tidal carbon dioxide, pain and sedation scores. Other complications were also recorded. While on the ward, pain and sedation scores, respiratory rate and side effects were recorded every two hours. Postoperative analgesia was provided by intravenous morphine. Analgesic requirements were recorded for the 24-hr study period. Pain and sedation scores did not differ between groups. Respiratory depression or hypoxia did not occur. The incidences of other side effects did not differ. There were no differences in the numbers of patients requiring morphine within eight hours, the time to first morphine administration or the total morphine requirements. We conclude that a single caudal injection of a bupivacaine-fentanyl mixture with epinephrine administered prior to surgery, while safe, offers no advantage over an injection of bupivacaine 0.125% with epinephrine for paediatric urological surgery.

Adequacy of caudal analgesia in children after penoscrotal and inguinal surgery using 0.5 or 1.0 ml.kg-1 bupivacaine 0.125%.

To determine the optimal volume of bupivacaine 0.125% for postoperative caudal analgesia, we compared the effectiveness of 0.5 ml.kg-1 and 1 ml.kg-1 of bupivacaine 0.125% with 1:200,000 epinephrine in 80 children undergoing penoscrotal and inguinal surgery. The adequacy of caudal analgesia and supplemental analgesic requirements did not differ between the two groups at any time during the first 12 hr after surgery. We conclude that 0.5 ml.kg-1 of bupivacaine 0.125% with 1:200,000 epinephrine is as effective as 1 ml.kg-1 of the same solution and recommend its use for penoscrotal surgery. The evidence for effectiveness of 0.5 ml.kg-1 of bupivacaine 0.125% for inguinal surgery, however, is inconclusive because of an insufficient number of patients studied.

Pharmacokinetic analysis of cloxacillin loss in children undergoing major surgery with massive bleeding.

To determine the magnitude of cloxacillin loss during surgical procedures involving significant blood loss and high fluid replacement, we compared the pharmacokinetics of cloxacillin in children during craniomaxillofacial surgery with the disposition of the drug in healthy young adult volunteers with intact circulation. Blood loss during craniofacial operations may exceed blood volume, in some cases by as much as three times. Hemodynamic replacement with electrolyte solutions and blood products, which do not contain the drug, further dilute cloxacillin concentrations. In the patients that we studied, mean drug loss was estimated at 71%. Cloxacillin concentrations in serum fell below the lower range of the MIC for Staphylococcus aureus during significant portions of the surgical procedures. Thus, the traditional dosing of cloxacillin during prolonged operations with massive blood loss is inadequate. A more frequent dosing interval or priming of all replacement fluids with the drug may be required to maintain therapeutic levels. Our findings suggest that massive blood loss is likely to have a dramatic effect on the level of any drug with a small distribution volume. If such a drug is essential to the patient's well-being (e.g., antibiotics, antiarrhythmics, and anticonvulsants), it must be replaced promptly.

Treatment of chronic drooling: a preliminary report.

Chronic drooling can be both psychologically and physically damaging. The technique of sialodochoplasty with sublingual gland resection is a viable treatment modality for this problem. The results of a series of eight patients who underwent the procedure are reported. A short-term success rate of 75% was achieved without morbidity.