Human brown adipose tissue as a target for obesity management; beyond cold-induced thermogenesis.

Elevating energy expenditure via adaptive thermogenesis in brown adipose tissue (BAT) is a potential strategy to reverse obesity. Much early enthusiasm for this approach, based on rodent studies, was tempered by the belief that BAT was relatively inconsequential in healthy adult humans. Interest was reinvigorated a decade ago when a series of studies re-identified BAT, primarily in upper thoracic regions, in adults. Despite the ensuing explosion of pre-clinical investigations and identification of an extensive list of potential target molecules for BAT recruitment, our understanding of human BAT physiology remains limited, particularly regarding interventions which might hold therapeutic promise. Cold-induced BAT thermogenesis (CIT) has been well studied, although is not readily translatable as an anti-obesity approach, whereas little is known regarding the role of BAT in human diet-induced thermogenesis (DIT). Furthermore, human studies dedicated to translating known pharmacological mechanisms of adipose browning from animal models are sparse. Several lines of recent evidence suggest that molecular regulation and physiology of human BAT differ to that of laboratory rodents, which form the majority of our knowledge base. This review will summarize knowledge on CIT and expand upon the current understanding and evidence gaps related to human adaptive thermogenesis via mechanisms other than cold.

Predictors of response to bronchial allergen challenge in 5- to 6-year-old atopic children.

BACKGROUND: The relationship between atopy and bronchial allergy in young children is not completely understood. OBJECTIVE: To examine the association between response to bronchial allergen challenge, immune markers of atopy and other clinical characteristics in 5- to 6-year-old children. METHODS: Children with positive skin test (SPT) to aeroallergen, together with a proportion of SPT negative children (as controls), were recruited from a birth cohort of 198 children at high risk of developing atopic disease and underwent allergen challenge. RESULTS: Thirty-seven children (26 atopic and 11 SPT negative), median age 74.5 months, were challenged: 31 with house dust mite and six with grass allergen. Only atopic children responded to challenge: n = 12/26 (46%). Wheal size [odds ratio (OR) 2.5 (1.2-5.3), P = 0.01], allergen-specific immunoglobulin E (IgE) [OR 3.4 (1.23-9.61), P = 0.02], total IgE [OR 8.6 (1.1-68.7), P = 0.04], current wheeze [OR 12 (1.7-81.7), P = 0.006] and persistent eczema [OR 11.0 (1.7-68.3), P = 0.006] emerged as the strongest independent predictors of response to allergen challenge. Prediction of response to allergen challenge was significantly improved when immune markers of atopy, and in particular wheal size, were combined with clinical characteristics. CONCLUSION: The relationship between atopy and bronchial allergy is quantitative at this age. There may be potential to create more powerful indicators of the presence of respiratory allergy in young children when immunological markers of atopy are considered quantitatively and when combined with clinical history of coexistent allergic disease.

Value of eosinophil cationic protein and tryptase levels in bronchoalveolar lavage fluid for predicting lung function impairment in anaesthetised, asthmatic children.

Bronchial hyperactivity, a key feature of active asthma in children, is a risk factor for respiratory adverse events in the peri-operative period. The presence of activated eosinophils in the lungs and mast cell degranulation can contribute to bronchial hyperreactivity. Eosinophil cationic protein is released by activated eosinophils and tryptase reflects mast cell degranulation. This study focused on the relationship of respiratory mechanics, eosinophil cationic protein and tryptase levels in bronchoalveolar lavage fluid in asthmatic and healthy children under general anaesthesia. We measured eosinophil cationic protein and tryptase levels in bronchoalveolar lavage fluid from 21 asthmatic and 21 healthy children following induction of general anaesthesia. Respiratory system resistance and dynamic compliance were measured during mechanical ventilation. Eosinophil cationic protein was more common in bronchoalveolar lavage fluid from asthmatics (12/21) than from controls (4/21, p = 0.01) and was present at higher levels (p = 0.002). Tryptase was also more common in the asthmatics (8/21 vs 1/21, p = 0.01). Respiratory resistance was significantly higher in asthmatic children with detectable eosinophil cationic protein levels than in those with undetectable eosinophil cationic protein levels (p = 0.019). Furthermore, 50% of the asthmatics with detectable eosinophil cationic protein exhibited bronchospasm after sampling their bronchoalveolar lavage fluid. These findings suggested that high levels of eosinophil cationic protein in the bronchoalveolar lavage fluid are associated with irritable airways, presumably secondary to airway inflammation, and this might be a useful marker for respiratory adverse events in the peri-operative period.

Latex allergy in an Australian population of children and adolescents with spinal dysfunction.

OBJECTIVES: To determine the prevalence of latex allergy in an Australian population of children and adolescents with spinal cord dysfunction and a comparison population of their siblings without spinal dysfunction. DESIGN AND SETTING: Cross-sectional study of all patients with spinal cord dysfunction attending the single tertiary spinal dysfunction clinic in Western Australia, and of their siblings closest in age. SUBJECTS: 104 patients with spinal dysfunction born 1978-1996 inclusive, and 50 siblings. MAIN OUTCOME MEASURES: Prevalence estimates and adjusted odds ratio estimates of the risk of having a history of latex allergy and of testing latex-specific IgE positive. RESULTS: Of the patients, 15.4% (95% CI, 9.1%-23.8%) had a history of latex allergy compared with none (95% CI, 0-5.8%) of the siblings. Of the 84 patients tested, 36.9% (95% CI, 26.6%-48.1%) were latex-specific IgE positive compared with 15.4% (95% CI, 4.4%-35.9%) of the 26 siblings tested. For patients, every operation after the first increased the risk of a positive IgE result cumulatively by 41% (odds ratio, 1.41; 95% CI, 1.18-1.68). CONCLUSIONS: The prevalence of latex sensitivity and clinical allergy in children and adolescents with spinal dysfunction in Australia is as high as that seen in the United States, and is related to latex exposure during surgery. The management policy regarding latex exposure for patients with spinal dysfunction requires urgent consideration.

Mechanisms of inhibition of IgE synthesis by nedocromil sodium: nedocromil sodium inhibits deletional switch recombination in human B cells.

IgE synthesis requires IL-4 and a T cell-B cell interaction that involves the B-cell antigen CD40 and its ligand expressed on activated T cells. Nedocromil sodium (NS), an effective prophylactic agent in asthma, inhibits IgE synthesis by human B cells. In this report we examined the mechanisms of this inhibition. NS targeted the B cells because it inhibited IgE synthesis induced by anti-CD40 and IL-4 in highly purified B cells (greater than 98% CD19+). NS had no effect on the induction of epsilon-germline transcripts by IL-4 but strongly inhibited CD40-mediated S mu --> S epsilon deletional switch recombination. The effect of NS was not specific for CD40 because it inhibited IgE synthesis in B cells stimulated with hydrocortisone plus IL-4. Moreover, the effect of NS was not specific for IgE because it inhibited CD40/IL-4-driven IgG4 synthesis by B cells sorted for lack of surface expression of IgG4. NS caused only modest inhibition of spontaneous IgE synthesis by B cells from patients with hyper-IgE syndrome, suggesting that it has little effect on B cells that have already undergone isotype switching. These results strongly suggest that NS inhibits IgE isotype switching by inhibiting deletional switch recombination and that NS has a novel potential mechanism for the prevention of asthma and other allergic diseases.

Inhalant allergen-specific T-cell reactivity is detectable in close to 100% of atopic and normal individuals: covert responses are unmasked by serum-free medium.

BACKGROUND: It is widely held that in vitro T cell responses to allergens are more prominent in atopic than in normal individuals, though this conclusion is based upon culture techniques which fail to detect proliferative responses in a significant minority of atopics and many normals. OBJECTIVES: Study allergen-specific proliferative responses of T cells cultured in serum-free medium (SFM). Examine associations between atopic status, age and T cell reactivity. METHODS: Initially, peripheral blood mononuclear cells were stimulated with allergens or antigens in SFM, and compared with cells cultured in RPMI + 10% fetal calf serum or human AB serum. Subsequently, T cell reactivity was studied in 34 adults (20-49 years), 27 children (2-13 years), and 19 infants (< or = 10 weeks) using SFM alone. RESULTS: Compared with serum-supplemented medium, SFM enhanced net T cell proliferation, both in bulk culture and when cloning at limiting dilution. In many subjects, SFM unmasked T cell reactivity to allergens which was not otherwise evident, and lowered the threshold allergen levels required for in vitro T cell triggering. For most allergens, T cell proliferative responses did not differ between adults who had specific IgE, and those who did not. The most vigorous responses observed were to ubiquitous inhalant allergens, which stimulated T cells from close to 100% of adults and children, and over 60% of infants. In contrast, responses to the 'vaccine' antigen tetanus toxoid were completely absent in the latter age group, but present in the majority of adults and children. CONCLUSIONS: These findings suggest that the extent of active T cell recognition of environmental allergens has been hitherto underestimated, and further that these responses may frequently be initiated in very early life. Additionally, these findings reinforce the notion that qualitative (as opposed to quantitative) variations in specific T cell reactivity ultimately determine allergen responder phenotype.

Role of protein tyrosine kinases and phosphatases in isotype switching: crosslinking CD45 to CD40 inhibits IgE isotype switching in human B cells.

Protein tyrosine kinases and protein tyrosine phosphatases play an important role in the transduction of signals via antigen receptors in T and B cells, and in CD40-dependent B-cell activation. To examine the role of tyrosine kinases and phosphatases in B-cell isotype switching, we examined the effects of the engagement of the transmembrane phosphatase CD45 on the synthesis of IgE induced by IL-4 and anti-CD40 monoclonal antibody (mAb). Crosslinking CD45 to CD40 using biotinylated mAbs and avidin strongly inhibited CD40-mediated IgE synthesis in IL-4-treated human B cells. CD40/CD45 crosslinking did not affect epsilon germline transcription in B cells stimulated with IL-4, but strongly inhibited induction of S mu/S epsilon switch recombination as detected by a nested primer polymerase chain reaction assay. The B-cell src-type tyrosine kinase lyn, which is activated following CD40 engagement, is a potential target for the effects of CD45 observed in our experiments, because CD45/CD40 crosslinking resulted in the inhibition of CD40-mediated lyn phosphorylation and activation. These results suggest an important role for protein tyrosine kinases and phosphatases in CD40-mediated induction of isotype switching to IgE.

Role of protein tyrosine kinases in CD40/interleukin-4-mediated isotype switching to IgE.

The B-cell antigen CD40 transduces signals, which synergize with interleukin (IL)-4 to induce IgE synthesis in human B cells. IL-4 induces epsilon germline transcription but not mature epsilon transcripts or IgE protein synthesis in B cells. Addition of anti-CD40 monoclonal antibody to IL-4-treated B cells results in deletional S mu--> S epsilon switch recombination, expression of mature epsilon transcripts, and IgE synthesis and secretion. Because both IL-4 and anti-CD40 induce protein tyrosine phosphorylation in B cells, we investigated the role of protein tyrosine kinase in IL-4/CD40-mediated IgE synthesis. The protein tyrosine kinase inhibitors genistein and herbimycin A, but not the protein kinase C inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7) or the protein kinase A inhibitor N-2-guanidinoethyl-5-isoquinolinesulfonamide, inhibited IgE synthesis in B cells stimulated with IL-4 and CD40. Genestein and herbimycin, but not H7, inhibited IL-4-driven epsilon germline transcription in B cells. Both genestein and herbimycin, but not H7, inhibited CD40-mediated IgE synthesis in B cells pretreated for 4 days with IL-4 to allow optimal expression of epsilon germline transcripts. Inhibition of IgE synthesis in these cultures was accompanied by inhibition of S mu--> S epsilon deletional switch recombination as assayed by nested polymerase chain reactions. These results suggest that activation of protein tyrosine kinase plays an important role in both the IL-4 and the CD40 signalling pathways that lead to IgE isotype switching in B cells.

Disodium cromoglycate inhibits S mu-->S epsilon deletional switch recombination and IgE synthesis in human B cells.

IgE synthesis requires interleukin 4 (IL-4) and a T-B cell interaction that involves the B cell antigen CD40 and its ligand expressed on activated T cells. IL-4 induces epsilon germline transcription whereas ligation of CD40 results in deletional S mu-->S epsilon switch recombination, expression of mature epsilon transcripts, and IgE synthesis and secretion. We demonstrate that disodium cromoglycate (DSCG), a drug commonly used for the prophylactic treatment of allergic disease, inhibits T cell-driven IgE synthesis by human B cells at concentrations readily achievable in the course of inhaled therapy for asthma. Inhibition of IgE synthesis by DSCG was not the result of drug toxicity because DSCG did not affect the viability of T and B cells or their proliferation to mitogens. DSCG did not interfere with CD40 ligand expression by T cells but clearly targeted the B cells because it inhibited IgE synthesis induced by anti-CD40 and IL-4 in populations of highly purified B cells. DSCG had no effect on the induction of epsilon germline transcripts by IL-4 but strongly inhibited CD40 mediated S mu-->S epsilon deletional switch recombination in IL-4-treated B cells as assayed by nested primer PCR. The effect of DSCG was not specific for CD40-mediated induction of IgE isotype switching because DSCG inhibited IgE synthesis as well as S mu-->S epsilon deletional switch recombination induced by hydrocortisone and IL-4 in B cells. Moreover, the effect of DSCG was not specific for IgE isotype switching because DSCG inhibited the synthesis of IgG4 by B cells sorted for lack of surface expression of IgG4 and stimulated with anti-CD40 and IL-4. DSCG caused only minimal inhibition (< 15%) of spontaneous IgE synthesis by lymphocytes from patients with the hyper-IgE syndrome and did not affect pokeweed mitogen-induced IgG and IgA synthesis by lymphocytes suggesting that it has little effect on B cells that have already undergone isotype switching. These results indicate that DSCG inhibits switching to IgE in B cells and suggest a novel potential mechanism for the prevention of allergic disease by DSCG.

Human erythrocyte membrane lipid asymmetry: transbilayer distribution of rapidly diffusing phosphatidylserines.

Human erythrocytes were incubated with sonicated vesicles composed of diheptanoyl-, dioctanoyl-, didecanoyl-, or dimyristoylphosphatidylserine, and the transbilayer distribution of the incorporated foreign lipid was examined by monitoring changes in cell morphology (Daleke & Huestis (1989) J. Cell. Biol. 108, 1375). Cells incubated with all phosphatidylserine homologs crenated initially and then reverted to discoid and stomatocytic morphology. Cells exposed to didecanoyl- or dimyristoylphosphatidylserine retained stable stomatocytic morphology during more than 10 h of incubation at 37 degrees C. Cells exposed to the diheptanoyl or dioctanoyl homologs reverted from stomatocytes to discocytes within 1-4 h. This reversion was more rapid for the shorter acyl chain diheptanoylphosphatidylserine. Reversion was accelerated in both cases by vanadate, an inhibitor of the aminophospholipid translocator. Heat denaturation of cytoskeletal proteins had no effect on phosphatidylserine-induced stomatocytosis or on the reversion to discoid shape of cells exposed to the short-chained homologs. These observations suggest that the aminophospholipid transporter rather than cytofacial lipid binding sites plays the primary role in maintenance of phosphatidylserine asymmetry in the erythrocyte membrane bilayer.

A multicentre randomized placebo-controlled double-blind study on the efficacy of Ketotifen in infants with chronic cough or wheeze.

The efficacy of Ketotifen was examined in the treatment of 113 infants between 6 and 36 months of age presenting with a history of cough and/or wheeze in a multicentre randomized placebo-controlled double-blind study. A 4 week no-medication baseline phase preceded the 16 week treatment phase in which infants took 2.5 mL twice daily of either placebo or Ketotifen (0.5 mg) syrup; this was followed by a 4 week wash-out phase. Diary card evaluation was performed by the parent or guardian for the duration of the study and recorded wheeze and cough twice daily as well as medication used. The percentage of symptom-free days decreased significantly in both groups (P < 0.005) with placebo-treated infants experiencing significantly more symptom-free days compared with the Ketotifen group (P < 0.01), although this difference was never more than 10% in any 4 week treatment period. Symptom severity scores and use of beta-agonist medication were also less in the placebo-treated infants but did not reach statistical significance. This study was unable to show a therapeutic advantage of Ketotifen over placebo in this group of infants with chronic cough and/or wheeze and the apparent statistical advantage of placebo is not a clinically relevant finding.

Deficiency of IgG subclasses and IgA, and elevation of IgE in children with a past history of bacterial meningitis.

Of 44 children who recovered from an attack of bacterial meningitis, 3 (7%) were found to have IgG subclass deficiency, 5 (11%) had IgA deficiency and 22 (50%) had raised IgE levels. These results suggest that immunoglobulin abnormalities may be an important predisposing factor in some cases of bacterial meningitis.

Immunoglobulin G subclass deficiency and predisposition to infection in Down's syndrome.

Serum immunoglobulins and IgG subclasses were measured in 26 children with Down's syndrome using an enzyme-linked immunosorbent assay and monoclonal antibodies. Eighteen (69%) of the children had increased susceptibility to infection. None of the children had deficiencies of total IgG and IgM, and only one had an IgA deficiency. IgG4 deficiency was diagnosed in 14 (54%) children. One child had a deficiency of IgG2. There were no children with deficiencies of either IgG1 or IgG3. There was a significant correlation between IgG subclass deficiency and predisposition to infection (P less than 0.05). Ninety percent of the patients with severe infections had low IgG4 whereas only 25% of those with no infections had low concentrations of IgG4. These results suggest that it is important to screen patients with Down's syndrome who have frequent systemic or respiratory infections for IgG subclass deficiencies because this may not be apparent from the assay of total IgG.

Evaluation of partially purified salivary gland allergens from the Australian paralysis tick Ixodes holocyclus in diagnosis of allergy by RIA and skin prick test.

Two diagnostic assays that detect IgE specific to I. holocyclus were developed using purified sources of allergens. Salivary gland extract was superior to whole body extract in both the radioimmunoassay and the skin prick test. Of the partially purified salivary gland allergens, the 28-KD protein fraction gave the most promising results.

Immunoglobulin G subclass deficiency in children with high levels of immunoglobulin E and infection proneness.

Of 32 unrelated children with serum IgE greater than 1,000 U/ml, 17 were found to have infection proneness according to standard clinical criteria, and 15 were not infection prone. There were no statistical differences between these 2 groups of children with regard to age, sex, serum IgE levels or prevalence of asthma. However, the prevalence of eczema was significantly lower in the infection-prone group (p = 0.035). Of greater interest was the finding that 7 children in the infection-prone group had IgG subclass and/or IgA deficiency compared with none in the non-infection-prone group (p = 0.006). These results suggest that IgG subclass studies may be warranted in children with markedly elevated levels of serum IgE and proneness to infection.

Allergic reactions to the Australian paralysis tick, Ixodes holocyclus: diagnostic evaluation by skin test and radioimmunoassay.

Allergic reactions to Ixodes holocyclus are well recognized but poorly defined. Tick-bite reactions in 42 individuals in this study fell into six classes. Skin-prick tests and radioimmunoassay (RIA) indicated that all systemic hypersensitivity (class 3) and atypical reactions (class 4) were IgE-mediated. Some 73% of the large local reactions (class 2) and only 12.5% of the small local reactions (class 1) were associated with IgE specific for tick allergens. Subjects who reported heavy exposure to tick-bite were more likely to have positive RIA values (P less than 0.05). There was an association between the individual's atopic status and tick allergy (P greater than 0.05).