Changes in severity of psychosocial difficulties in adolescents accessing specialist mental healthcare in England (2009-2014).

In England, clinicians and professional organisations report that higher numbers of adolescents with more severe psychosocial difficulties are accessing specialist services. A lack of national data on patterns of access to specialist services means there is limited information to inform policy. We examined whether severity of psychosocial difficulties in adolescents accessing mental healthcare has changed over time. Adolescents seen in specialist child mental healthcare in 2009 vs. 2014 were matched on demographics and problem types using propensity score matching; final sample N = 2776 adolescents. We found: 1) stability over time in overall severity of difficulties, 2) an increase in severity of young women's emotional problems, and 3) a decrease in adolescents' conduct problems. The findings suggest the intriguing possibility that the criteria for accessing mental healthcare are not universally rising, but rather the patterns in access to specialist services may mirror epidemiological changes in severity of psychosocial difficulties in the population.

Hypoxia causes IL-8 secretion, Charcot Leyden crystal formation, and suppression of corticosteroid-induced apoptosis in human eosinophils.

BACKGROUND: Inflamed environments are typically hypercellular, rich in pro-inflammatory cytokines, and profoundly hypoxic. While the effects of hypoxia on neutrophil longevity and function have been widely studied, little is known about the consequences of this stimulus on eosinophils. OBJECTIVE: We sought to investigate the effects of hypoxia on several key aspects of eosinophil biology, namely secretion, survival, and their sensitivity to glucocorticosteroids (GCS), agents that normally induce eosinophil apoptosis. METHODS: Eosinophils derived from patients with asthma/atopy or healthy controls were incubated under normoxia and hypoxia, with or without glucocorticoids. Activation was measured by flow cytometry, ELISA of cultured supernatants, and F-actin staining; apoptosis and efferocytosis by morphology and flow cytometry; and GCS efficacy by apoptosis assays and qPCR. RESULTS: Hypoxic incubation (3 kPa) caused (i) stabilization of HIF-2α and up-regulation of hypoxia-regulated genes including BNIP3 (BCL2/adenovirus E1B 19-kDa protein-interacting protein 3) and GLUT1 (glucose transporter 1); (ii) secretion of pre-formed IL-8, and Charcot Leyden crystal (CLC) formation, which was most evident in eosinophils derived from atopic and asthmatic donors; (iii) enhanced F-actin formation; (iv) marked prolongation of eosinophil lifespan (via a NF-κB and Class I PI3-kinase-dependent mechanism); and (v) complete abrogation of the normal pro-apoptotic effect of dexamethasone and fluticasone furoate. This latter effect was evident despite preservation of GCS-mediated gene transactivation under hypoxia. CONCLUSION AND CLINICAL RELEVANCE: These data indicate that hypoxia promotes an eosinophil pro-inflammatory phenotype by enhancing eosinophil secretory function, delaying constitutive apoptosis, and importantly, antagonizing the normal pro-apoptotic effect of GCS. As eosinophils typically accumulate at sites that are relatively hypoxic, particularly during periods of inflammation, these findings may have important implications to understanding the behaviour of these cells in vivo.

The psychometric properties of the Ages & Stages Questionnaires for ages 2-2.5: a systematic review.

BACKGROUND: Early identification of children with potential development delay is essential to ensure access to care. The Ages & Stages Questionnaires (ASQ) is used as population outcome indicators in England as part of the 2.5-year review. METHOD: The aim of this article was to systematically review the worldwide evidence for the psychometric properties of the ASQ third edition (ASQ-3TM ) and the Ages & Stages Questionnaires®: Social-Emotional (ASQ:SE). Eight electronic databases and grey literature were searched for original research studies available in English language, which reported reliability, validity or responsiveness of the ASQ-3TM or ASQ:SE for children aged between 2 and 2.5 years. Twenty studies were included. Eligible studies used either the ASQ-3TM or the ASQ:SE and reported at least one measurement property of the ASQ-3TM and/or ASQ:SE. Data were extracted from all papers identified for final inclusion, drawing on Cochrane guidelines. RESULTS: Using 'positive', 'intermediate' and 'negative' criteria for evaluating psychometric properties, results showed 'positive' reliability values in 11/18 instances reported, 'positive' sensitivity values in 13/18 instances reported and 'positive' specificity values in 19/19 instances reported. CONCLUSIONS: Variations in age or language versions used, quality of psychometric properties and quality of papers resulted in heterogeneous evidence. It is important to consider differences in cultural and contextual factors when measuring child development using these indicators. Further research is very likely to have an important impact on the interpretation of the ASQ-3TM and ASQ:SE psychometric evidence.

When is Sessional Monitoring More Likely in Child and Adolescent Mental Health Services?

Sessional monitoring of patient progress or experience of therapy is an evidence-based intervention recommended by healthcare systems internationally. It is being rolled out across child and adolescent mental health services (CAMHS) in England to inform clinical practice and service evaluation. We explored whether patient demographic and case characteristics were associated with the likelihood of using sessional monitoring. Multilevel regressions were conducted on N = 2609 youths from a routinely collected dataset from 10 CAMHS. Girls (odds ratio, OR 1.26), older youths (OR 1.10), White youths (OR 1.35), and youths presenting with mood (OR 1.46) or anxiety problems (OR 1.59) were more likely to have sessional monitoring. In contrast, youths under state care (OR 0.20) or in need of social service input (OR 0.39) were less likely to have sessional monitoring. Findings of the present research may suggest that sessional monitoring is more likely with common problems such as mood and anxiety problems but less likely with more complex cases, such as those involving youths under state care or those in need of social service input.

Realistic evaluation of Situation Awareness for Everyone (SAFE) on paediatric wards: study protocol.

INTRODUCTION: Evidence suggests that health outcomes for hospitalised children in the UK are worse than other countries in Europe, with an estimated 1500 preventable deaths in hospital each year. It is presumed that some of these deaths are due to unanticipated deterioration, which could have been prevented by earlier intervention, for example, sepsis. The Situation Awareness For Everyone (SAFE) intervention aims to redirect the 'clinical gaze' to encompass a range of prospective indicators of risk or deterioration, including clinical indicators and staff concerns, so that professionals can review relevant information for any given situation. Implementing the routine use of huddles is central to increasing situation awareness in SAFE. METHODS AND ANALYSIS: In this article, we describe the realistic evaluation framework within which we are evaluating the SAFE programme. Multiple methods and data sources are used to help provide a comprehensive understanding of what mechanisms for change are triggered by an intervention and how they have an impact on the existing social processes sustaining the behaviour or circumstances that are being targeted for change. ETHICS AND DISSEMINATION: Ethics approval was obtained from London-Dulwich Research Ethics Committee (14/LO/0875). It is anticipated that the findings will enable us to understand what the important elements of SAFE and the huddle are, the processes by which they might be effective and-given the short timeframes of the project-initial effects of the intervention on outcomes. The present research will add to the extant literature by providing the first evidence of implementation of SAFE and huddles in paediatric wards in the UK.

Thermographic imaging during nasal peanut challenge may be useful in the diagnosis of peanut allergy.

BACKGROUND: Double-blinded challenges are widely used for diagnosing food allergy but are time-consuming and cause severe reactions. Outcome relies on subjective interpretation of symptoms, which leads to variations in outcome between observers. Facial thermography combined with nasal peanut challenge was evaluated as a novel objective indicator of clinical allergy. METHODS: Sixteen children with positive blinded peanut challenge underwent nasal challenge with 10 µg peanut protein or placebo. Mean skin temperatures were recorded from the mouth and nose using infrared thermography over 18 min. RESULTS: The area under curve of nasal skin temperature was significantly elevated after peanut vs placebo (18.2 vs 4.8°Cmin). The maximum increase in temperature was also significantly greater after peanut: mean difference +0.9°C. CONCLUSION: This feasibility study shows thermography can detect inflammation caused by nasal challenges whilst employing one thousand-fold less peanut than an oral challenge. This novel technique could be developed to provide a rapid, safe and objective clinical allergy test.

A longitudinal study of resolution of allergy to well-cooked and uncooked egg.

BACKGROUND: Egg allergy is common and although resolution to uncooked egg has been demonstrated, there is lack of evidence to guide reintroduction of well-cooked egg. OBJECTIVES: To examine the rate of resolution to well-cooked, compared with uncooked egg in children, and safety of egg challenges. METHOD: A longitudinal study of egg-allergic children from 2004 to 2010, who underwent challenge with well-cooked and if negative, uncooked egg. Participants underwent repeat annual challenges and egg-specific IgE measurement. RESULTS: One hundred and eighty-one open egg challenges were performed in 95 children whose median age of allergy onset was 12 months. Fifty-three of 95 (56%) had at least one annual repeat challenge. Pre-study historical reactions occurred to baked egg in five (5%), lightly cooked in 58 (61%) and uncooked in nine (9%); respiratory reactions occurred in 11 (12%) and seven (7%) had anaphylaxis; adrenaline was used during five reactions. There were 77 well-cooked and 104 uncooked egg challenges. Tolerance was gained twice as rapidly to well-cooked than uncooked egg (median 5.6 vs. 10.3 years; P<0.0001) and continued to 13 years; hazard ratio 2.23 (95% confidence interval 1.6-3.9). Nearly 1/3 had resolved allergy to well-cooked egg at 3 years and 2/3 at 6 years. Of 28/77 (37%) positive well-cooked egg challenges, 65% had cutaneous symptoms, 68% gastrointestinal and 39% rhinitis, with no other respiratory reactions. Adrenaline was not required. CONCLUSIONS AND CLINICAL RELEVANCE RESOLUTION: of egg allergy takes place over many years, with children outgrowing allergy to well-cooked egg approximately twice as quickly as they outgrow allergy to uncooked egg. There were no severe reactions to well-cooked egg challenge, and adrenaline was not required. Our data support initiation of home reintroduction of well-cooked egg from 2 to 3 years of age in children with previous mild reactions and no asthma. Resolution continues to occur in older children, so that despite an earlier positive challenge, attempts at reintroduction should be continued.

Efficacy and safety of high-dose peanut oral immunotherapy with factors predicting outcome.

BACKGROUND: Peanut allergy is severe and rarely resolves. OBJECTIVE: To test the efficacy and safety of a new oral immunotherapy (OIT) protocol for peanut allergy. METHOD: Twenty-two peanut-allergic children underwent oral challenge. OIT was administered by gradual updosing with 2-weekly increments (8-38 weeks) to 800 mg of protein (5 peanuts/day) followed by 30-week maintenance. Oral challenge was repeated after 6 and 30 weeks maintenance. RESULTS: Twenty-two children (median 11 years) had positive challenges (threshold 1-110 mg). Nineteen of 22 (86%) tolerated updosing and maintenance at 800 mg protein/day. One of 22 dropped out; 2/22 tolerated updosing and maintenance at 200-400 mg protein. Reactions, mostly mild, occurred in 86% during immunotherapy, adrenaline was not required. Eight of 8 with pre-immunotherapy peanut IgE<27.3 kU/L required no dose adjustment compared with 5/13 with pre-immunotherapy peanut IgE≥27.3 kU/L. Twelve of 22 (54%) required a transient dose reduction because of reactions possibly related to extrinsic factors: tiredness, infection and exercise. After 6 weeks, 12/22 (54%) had no reaction to a 2.6 g protein challenge. After 30 weeks, 14/22 (64%) tolerated 6.6 g protein. The median tolerated peanut dose increased 1000-fold following immunotherapy, from 6 to 6459 mg of protein. CONCLUSIONS AND CLINICAL RELEVANCE: We used a novel protocol using gradual updosing, and higher maintenance dose resulting in a better outcome compared with rush protocols. There was a 1000-fold increase in the amount of peanut tolerated with a good safety profile. No serious adverse events occurred. Most subjects tolerated five peanuts and all were protected against amounts likely during accidental ingestion. New information is provided on 'extrinsic factors', updosing method and factors associated with success (trial registration http://ClinicalTrials.gov- ID number NCT01259804).

Successful oral tolerance induction in severe peanut allergy.

BACKGROUND: Peanut allergy is common, potentially severe and rarely resolves causing impaired quality of life. No disease-modifying treatment exists and there is therefore a need to develop a therapeutic intervention. AIMS OF THE STUDY: The aim of this study was to investigate whether peanut oral immunotherapy (OIT) can induce clinical tolerance to peanut protein. METHODS: Four peanut-allergic children underwent OIT. Preintervention oral challenges were performed to confirm clinical allergy and define the amount of protein required to cause a reaction (dose thresholds). OIT was then administered as daily doses of peanut flour increasing from 5 to 800 mg of protein with 2-weekly dose increases. After 6 further weeks of treatment, the oral challenge was repeated to define change in dose threshold and subjects continued daily treatment. RESULTS: Preintervention challenges confirmed peanut allergy and revealed dose thresholds of 5-50 mg (1/40-1/4 of a whole peanut); one subject had anaphylaxis during challenge and required adrenaline injection. All subjects tolerated immunotherapy updosing to 800 mg protein and i.m. adrenaline was not required. Each subject tolerated at least 10 whole peanuts (approximately 2.38 g protein) in postintervention challenges, an increase in dose threshold of at least 48-, 49-, 55- and 478-fold for the four subjects. CONCLUSIONS: We demonstrated a substantial increase in dose threshold after OIT in all subjects, including the subject with proven anaphylaxis. OIT was well tolerated and conferred protection against at least 10 peanuts, more than is likely to be encountered during accidental ingestion.

Patterns of immunoglobulin G responses to egg and peanut allergens are distinct: ovalbumin-specific immunoglobulin responses are ubiquitous, but peanut-specific immunoglobulin responses are up-regulated in peanut allergy.

BACKGROUND: The clinical significance of food-specific IgG subclasses in food allergy and tolerance remains unclear. Specific IgG titres are often reported in non-standardized units, which do not allow comparisons between studies or allergens. OBJECTIVE: To quantify, in absolute units, ovalbumin (OVA)- and peanut-specific IgG levels in children with peanut or egg allergy (active or resolved) and in non-allergic controls. Methods Children aged 1-15 years were recruited. Peanut allergy was diagnosed by convincing history and a 95% predictive level of specific IgE; egg allergy or resolution was confirmed by oral challenge. Serum IgG, IgG1 and IgG4 levels (microg/mL) to OVA and peanut extract were quantified by ELISA. RESULTS: OVA- and peanut-specific IgG was detected in all subjects. In non-allergic controls (n=18), OVA-specific IgG levels were significantly higher than peanut-specific IgG (median microg/mL IgG=15.9 vs. 2.2, IgG1=1.3 vs. 0.6, IgG4=7.9 vs. 0.7; P<0.01). There were no differences in OVA-specific IgG, IgG1 and IgG4 between egg-allergic (n=40), egg-resolved (n=22) and control (n=18) subjects. In contrast, peanut-specific IgG (median microg/mL IgG=17.0, IgG1=3.3, IgG4=5.2) were significantly higher in peanut-allergic subjects (n=59) compared with controls and with non-peanut-sensitized but egg-allergic subjects (n=26). Overall, the range of IgG4 was greater than IgG1, and IgG4 was the dominant subclass in >60% of all subjects. CONCLUSION: OVA-specific IgG levels of egg-allergic, egg-resolved or control groups are not distinguishable. Higher peanut-specific IgG levels are associated with clinical allergy, but the range of IgG titres of the allergic and control groups overlapped. Hence, OVA and peanut-specific IgG measurements do not appear to be of diagnostic value. Strong IgG responses to OVA may be a normal physiological response to a protein frequently ingested from infancy, whereas up-regulated IgG responses in peanut allergy may be indicative of a dysregulated immune response to peanut allergens.

T cell proliferation and cytokine responses to ovalbumin and ovomucoid detected in children with and without egg allergy.

BACKGROUND: The specific T cell responses in egg allergy and resolution have not been fully elucidated. OBJECTIVE: To characterize egg allergen-specific T cells of children with active and resolved egg allergy, in comparison with non-allergic controls. METHOD: We studied children with active (n=35) or resolved (n=20) egg allergy determined by oral challenge, and non-allergic controls (n=15). Peripheral blood mononuclear cells were labelled with carboxyfluorescein succinimidyl ester (CFSE) and stimulated with ovalbumin (OVA), ovomucoid (OM) or tetanus toxoid. Flow cytometry was used to detect divided CD3+ CFSE(lo) cells that expressed intra-cytoplasmic IL-4 or IFN-gamma. The cell division index (CDI) was calculated as a measure of allergen-specific proliferation. Peanut-specific T cells of a subgroup of children who also had peanut allergy were also studied. RESULTS: OVA-specific T cells were found in subjects with active (87%) or resolved (75%) egg allergy and in controls (67%), with a trend towards increased T cell proliferation in allergy. OM-induced weaker T cell responses than OVA, stimulating fewer responders (46% allergic, 50% resolved, 60% controls) and 10-fold less proliferation [CDI(OVA) 2.0 (median), 25.6 (maximum) vs. CDI(OM) 0.2 (median), 15.1 (maximum); P<0.01]. Both egg allergens induced significant IL-4+ (median 10%, range 1.4-58%) and IFN-gamma+ (median 28%, range 4.5-63%) cells in responders, including non-allergics. There were no significant differences in IFN-gamma+ or IL-4+ cells or in IFN-gamma/IL-4 ratios between groups. Peanut-specific T cell proliferation was significantly higher in peanut allergy [CDI(CPE) 16.5 (median), 24.8 (maximum)] compared with controls [CDI(CPE) 2.1 (median), 16.1 (maximum)] but cytokine profiles were not different. Tetanus-specific T cells were seen in 90% of the subjects, with no significant inter-group differences in responses. CONCLUSION: Egg allergen-specific T cells are readily detected in all groups and not restricted to egg allergy. In contrast, peanut-specific proliferation was significantly higher in peanut allergy. This suggests that T cell responses in peanut and egg allergy may differ. We did not find T helper type 2-deviated cytokine responses in egg or peanut allergy.

Differential effect of thalidomide and dexamethasone on the transcription factor NF-kappa B.

Thalidomide was initially used as a sedative during pregnancy but was withdrawn from the market due to its teratogenic effects. In vitro studies have shown that thalidomide inhibits tumour necrosis factor alpha (TNF-alpha) mRNA expression and protein production by mitogen-stimulated macrophages and activated T cells. Even at the highest concentration (10-1 mM) tested, however, TNF-alpha levels are inhibited only partially and the mechanism of action is unknown. In the present investigations, we have examined the influence of thalidomide on nuclear levels of NF-kappa B in human peripheral blood mononuclear cells (PBMC) following activation with mitogen or phorbol myristate acetate (PMA)/ionophore. Dexamethasone was used as a positive control due to its well-characterised mechanism of action and NF-kappa B-mediated effects on TNF-alpha expression. PBMC from healthy human volunteers were stimulated optimally with phytohemagglutinin (PHA) or PMA/ionophore in the presence of 10(-1)-10(-5) mM thalidomide or dexamethasone, concentrations that displayed a range of inhibitory effects on TNF-alpha production. Cells were harvested at varying time points and nuclear extracts prepared. Nuclear levels of NF-kappa B were measured using electrophoretic mobility shift assays (EMSA) with a radiolabelled DNA probe specific for NF-kappa B. Results were analysed using optical densitometry. Nuclear levels of NF-kappa B were found to be unaffected by thalidomide at all concentrations tested, including concentrations (10(-1)-10(-3) mM) that exhibited significant inhibition of TNF-alpha protein and mRNA expression. In concurrent experiments, dexamethasone was found to reduce NF-kappa B expression in a dose-dependent manner with maximal inhibition at the highest dose tested (10(-1) mM). TNF-alpha gene expression is controlled by at least three separate transcription factors that are involved in binding to the promoter region. These observations suggest that thalidomide does not act directly on NF-kappa B and therefore inhibits TNF-alpha production through another independent mechanism.

Selective down-regulation of T cell- and non-T cell-derived tumour necrosis factor alpha by thalidomide: comparisons with dexamethasone.

Both thalidomide and dexamethasone have been shown to inhibit the production of tumour necrosis factor alpha (TNF-alpha), but little is known of their cellular selectivity. Inhibition of monocyte TNF-alpha expression has been implicated in the clinical efficacy of thalidomide, and it has been suggested that the drug modulates only monocyte-derived cytokines. Given the importance of T lymphocyte responses in immunological disorders in which treatment with thalidomide has been successful, it is pertinent to study the effects of this drug on T cell-derived TNF-alpha. In the present investigations we have examined the influence of both thalidomide and dexamethasone on mitogen-induced elaboration of TNF-alpha by CD3+ peripheral blood mononuclear cells (PBMC) and the T cell line MOLT-4. PBMC from healthy human volunteers were stimulated optimally with phytohaemagglutinin (PHA) in the presence of varying concentrations of thalidomide or dexamethasone, and supernatants assayed for TNF-alpha and interleukin 2 (IL-2). Concurrently, PHA-stimulated PBMC were treated with 1 x 10(-1) mM thalidomide or dexamethasone and the cells fixed, permeabilised, stained with anti-CD3 and anti-TNF-alpha fluorescently labelled antibodies and analysed by flow cytometry. MOLT-4 cells were cultured in the presence or absence of the drugs following activation with phorbol myristate acetate (PMA)/ionophore, and supernatants analysed by enzyme-linked immunosorbent assay (ELISA) for cytokine expression. Thalidomide was found to inhibit PBMC-derived TNF-alpha, but not IL-2. In contrast, dexamethasone down-regulated both TNF-alpha and IL-2 in a dose-dependent manner. Thalidomide and dexamethasone both suppressed intracellular levels of TNF-alpha in CD3+ PBMC, reducing percentages of double positive staining cells by 28 and 52%, respectively, compared with controls. In addition, TNF-alpha production by CD3- PBMC was inhibited by 31% by thalidomide and by 47% by dexamethasone. In order to determine whether thalidomide was acting directly on T cells, or indirectly through effects on accessory cells, TNF-alpha production in the T cell line MOLT-4 was investigated. TNF-alpha secretion by PMA/ionophore activated MOLT-4 cells was reduced by 80% following thalidomide treatment and close to background levels following dexamethasone treatment. To verify that thalidomide was acting selectively to down-regulate TNF-alpha, IL-2 production by MOLT-4 cells was also measured and found to be unaffected by the drug. In contrast, dexamethasone reduced MOLT-4-derived IL-2 levels by 20%. These observations suggest that thalidomide, in addition to its known inhibitory effect on monocyte-derived TNF-alpha, is capable also of down-regulating T cell-derived TNF-alpha in a direct and selective manner. In addition, the inhibition of intracellular levels of TNF-alpha strengthens the evidence that the inhibitory effect of thalidomide is at the level of transcription and/or translation and does not reduce cellular TNF-alpha secretion. Such effects could explain the efficacy of thalidomide treatment in various immunological disorders where T cell activation plays an important role in the pathogenesis of the disease.

Differential regulation by thalidomide and dexamethasone of cytokine expression in human peripheral blood mononuclear cells.

Immunosuppressive drugs are used routinely to reduce the inappropriate production of cytokines in an immune response. Recent attention has focused on drugs that selectively inhibit specific cytokines. Both thalidomide and dexamethasone have been reported to exhibit immunomodulatory effects on cytokines in vitro. We wished to examine the effects of thalidomide and dexamethasone on the production of cytokines by peripheral blood mononuclear cells (PBMC), following mitogenic stimulation, at the level of both secreted product and mRNA production. PBMC from healthy human volunteers were stimulated optimally with phytohaemagglutinin (PHA) in the presence of varying concentrations of thalidomide and dexamethasone using dimethyl sulphoxide (DMSO) as the solvent. Analysis of supernatants by enzyme-linked immunosorbent assay (ELISA) showed that thalidomide caused a dose-dependent inhibition of the pro-inflammatory cytokines interleukin 6 (IL-6) and tumour necrosis factor alpha (TNF-alpha), maximally reducing production by 20 (P < 0.05) and 30% (P < 0.01), respectively, compared with controls. However, thalidomide did not affect either proliferation or the production of interleukin 2 (IL-2), interleukin 4 (IL-4) or interleukin 10 (IL-10). A slight bell shaped inhibition of interferon gamma (IFN-gamma) was seen which was statistically significant (P < 0.05). In contrast, dexamethasone inhibited markedly the expression of all cytokines tested (IL-2, IL-4, IL-6, IL-10, IFN-gamma and TNF-alpha) in dose-dependent fashion, reducing levels to near to background. Reverse transcription-polymerase chain reaction (RT-PCR) analyses showed that thalidomide inhibited selectively the expression of TNF-alpha and IL-6 mRNA, whereas dexamethasone inhibited mRNA levels of all cytokines examined. The data indicate that dexamethasone is a broad range immunosuppressant inhibiting all cytokines tested in a dose-dependent manner at the level of both secreted product and mRNA. Conversely, thalidomide selectively inhibits the production of IL-6 and TNF-alpha. Due to their markedly different effects on cytokine production, and the fact that both drugs act at the level of transcription, we believe they influence separate pathways involved in cytokine gene regulation.

Kinetics and functional implications of Th1 and Th2 cytokine production following activation of peripheral blood mononuclear cells in primary culture.

The importance of cytokine production in some disease processes is now widely recognized. To investigate temporal relationships between cytokines, we stimulated peripheral blood mononuclear cells (PBMC) in vitro using the T cell mitogen phytohemagglutinin (PHA) and various antigens chosen to induce predominantly Th1 (streptokinase: streptodornase or purified protein derivative) or Th2 (Dermatophagoides pteronyssinus, bee or wasp venom: allergens in sensitive subjects) responses. Cytokine production was measured by sensitive bioassays or enzyme-linked immunosorbent assays. Of the 30 subjects studied, 10 were normal and 20 individuals were allergic to either D. pteronyssinus (n = 10) or bee venom (n = 10) (examined before specific allergen immunotherapy). We examined the temporal profiles of a panel of cytokines produced in primary culture. In PHA-driven cultures, cytokines were found to be sequentially produced in the order interleukin (IL)-2, IL-4, IL-5, IL-3, interferon (IFN)-gamma, IL-10, IL-6, IL-12 and tumor necrosis factor (TNF)-alpha. The response to allergen in allergic patients was predominantly Th2 in nature, with the production of IL-4, IL-5, IL-6 and IL-10, but little or no IFN-gamma. IL-2, IL-3, TNF-alpha and IL-12 were also produced in low amounts. The response of both atopic and normal subjects to recall bacterial antigens was predominantly Th1, with high levels of IFN-gamma, IL-2 and TNF-alpha. The relevance of the order, amount and speed of production, characteristic kinetics (production, consumption, homeostatic regulation) and the cell source of the cytokines are discussed.

Manage marketing by the customer equity test.

Managers have recently begun to think of good marketing as good conversation, as a process of drawing customers into progressively more satisfying relationships with a company. And just as the art of conversation follows two steps--first striking up a conversation with a likely partner and then maintaining the flow--so the new marketing naturally divides itself into the work of customer acquisition and the work of customer retention. But how can managers determine the optimal balance between spending on acquisition and spending on retention? Robert Blattberg and John Deighton use decision calculus to help managers answer that question. That is, they ask managers to approach the large, complex problem through several smaller, more manageable questions on the same topic. Then they use a formal model to turn those smaller judgments into an answer to the larger question. The ultimate goal, the authors say, is to grow the company's customer equity the sum of all the conversations-to its fullest potential. Recognizing that managers must constantly reassess the spending points determined by the decision-calculus model, the authors also provide a series of guidelines and suggestions to help frame the issues that affect acquisition, retention, and customer equity. When managers strive to grow customer equity rather than a brand's sales or profits, they put a primary indicator of the health of the business at the fore front of their strategic thinking: the quality of customer relationships.

Bee venom immunotherapy induces a shift in cytokine responses from a TH-2 to a TH-1 dominant pattern: comparison of rush and conventional immunotherapy.

BACKGROUND: The mechanism of immunotherapy is unclear. Allergic disease is known to involve enhanced TH-2 cytokine responses to allergen. OBJECTIVE: In order to investigate the mechanisms of immunotherapy, we have examined changes in cytokine secretion before (13 patients) and during (nine patients) both rush and conventional venom immunotherapy (VIT) in bee venom allergic patients. METHODS: Peripheral blood mononuclear cells were stimulated in vitro with bee venom, non-specific antigen or mitogen and secretion of IL-4 (TH-2) and IFN gamma (TH-1) over the culture period measured. RESULTS: Untreated patients had TH-2 responses to venom and TH-1 responses to antigen and strong proliferative responses to venom. Controls showed no response (proliferation or cytokines) to venom and the normal TH-1 response to antigen. VIT resulted in marked changes in cytokine secretion to venom, with reduction of the abnormal TH-2 response and induction of a TH-1 response. The pattern differed in rush and conventional VIT. One day after rush VIT there was a significant fall in IL-4 secretion (P < 0.01), which rose by 3 weeks then declined. In conventional VIT there was a gradual reduction of IL-4 production significant after 2 months and undetectable by 6 months. IFN gamma secretion was induced by VIT. Proliferative responses mirrored the IL-4 changes. One day after rush VIT there was a loss of T cells, monocytes and NK cells from peripheral blood. CONCLUSION: This study shows that immunotherapy shifted cytokine responses to allergen from a TH-2 to a TH-1 dominant pattern, suggesting direct effects on T cells. How these cytokine changes relate to clinical desensitization is not clear. In the longer term they would result in an isotype switch from IgE to IgG. Early changes in cytokine or chemokine production might downregulate mast cell or basophil reactivity and explain the rapid desensitization in rush VIT.

The immunosuppressive drug thalidomide induces T helper cell type 2 (Th2) and concomitantly inhibits Th1 cytokine production in mitogen- and antigen-stimulated human peripheral blood mononuclear cell cultures.

Thalidomide is an effective immunomodulatory drug in man, but its mechanism of action remains unclear. We hypothesized that, in addition to its reported inhibitory effects on production of monocyte-derived tumour necrosis factor-alpha (TNF-alpha), thalidomide might be effective at the level of Th immunoregulation. In a comparative study with the immunosuppressant cyclosporin A, we have demonstrated a potent and specific effect of thalidomide on cytokine production relating to the distinct Th1 and Th2 subsets. It induced and enhanced the production of IL-4 and IL-5 and, at the same dose (1000 ng/ml), significantly inhibited interferon-gamma (IFN-gamma) production in phytohaemagglutinin (PHA)-stimulated human peripheral blood mononuclear cell (PBMC) cultures. Stimulation of PBMC with recall antigen (streptokinase:streptodornase (SKSD)) at 144 h in the absence of thalidomide resulted in a predominantly Th1 response, with the production of IFN-gamma and IL-2. Thalidomide switched this response from a Th1 to a Th2 type. The effect was most pronounced at 1000 ng/ml thalidomide, where inhibition of IFN-gamma and enhancement of IL-4 production was maximal. In unstimulated cultures thalidomide alone induced IL-4 production. Cyclosporin A, in contrast, inhibited both Th1 and Th2 cytokine production by PHA-stimulated PBMC. Time course data from thalidomide-treated cultures revealed that the augmented IL-4 production diminished as the culture time increased, whereas IFN-gamma production was significantly increased. This response might be due to activation-induced apoptosis of Th2 cells or the induction of Th2 cell anergy, in the continued presence of stimulating agents, with the emergence of IFN-gamma-secreting Th1 cells when Th2 antagonism declines. The effects of thalidomide and related compounds may enhance our understanding of the mechanisms of T helper cell selection, offer the possibility of controlled therapeutic switching between Th1 and Th2 responses, and may lead to a rational approach for the treatment of some T cell-mediated immunological disorders.

The profiles of interleukin (IL)-2, IL-6, and interferon-gamma production by peripheral blood mononuclear cells from house-dust-mite-allergic patients: a role for IL-6 in allergic disease.

We have developed a model to measure cytokine production by peripheral blood mononuclear cells (PBMC) in vitro. In this report, we examine the production of interleukin-2 (IL-2), IL-6, and interferon-gamma (IFN-gamma) by PBMC of house-dust-mite (Dermatophagoides pteronyssinus)-allergic subjects. When stimulated with specific allergen (D. pteronyssinus), PBMC of patients produced significant levels of IL-2 and high levels of IL-6, but little or no IFN-gamma. Nonatopic control PBMC also produced IL-6, although at lower levels, but no IL-2 or IFN-gamma. A ubiquitous antigen, streptokinase/streptodornase (SKSD), induced high levels of IL-2 in patients, but only low levels of IFN-gamma and IL-6. Nonatopic controls produced similar levels of IL-2 and IL-6, but high levels of IFN-gamma to SKSD. IL-2 and IFN-gamma levels induced by the T-cell mitogen phytohaemagglutinin (PHA) were similar in patient and control groups, but IL-6 levels were significantly lower in the patients. IgE synthesis in vitro was shown only in atopic PBMC cultures stimulated with specific allergen. The major points can be summarized as 1) IL-2 production by atopic patients in response to allergen; 2) IL-6 production to allergen by both atopic and nonatopic patients, but significantly increased in atopic patients; and 3) defective IFN-gamma production by atopic patients to both allergen and antigen.(ABSTRACT TRUNCATED AT 250 WORDS)