The serial millisecond crystallography instrument at the Australian Synchrotron incorporating the "Lipidico" injector.

A serial millisecond crystallography (SMX) facility has recently been implemented at the macromolecular crystallography beamline, MX2 at the Australian Synchrotron. The setup utilizes a combination of an EIGER X 16M detector system and an in-house developed high-viscosity injector, "Lipidico." Lipidico uses a syringe needle to extrude the microcrystal-containing viscous media and it is compatible with commercially available syringes. The combination of sample delivery via protein crystals suspended in a viscous mixture and a millisecond frame rate detector enables high-throughput serial crystallography at the Australian Synchrotron. A hit-finding algorithm, based on the principles of "robust-statistics," is employed to rapidly process the data. Here we present the first SMX experimental results with a detector frame rate of 100 Hz (10 ms exposures) and the Lipidico injector using a mixture of lysozyme microcrystals embedded in high vacuum silicon grease. Details of the experimental setup, sample injector, and data analysis pipeline are designed and developed as part of the Australian Synchrotron SMX instrument and are reviewed here.

Febrile respiratory illnesses in infancy and atopy are risk factors for persistent asthma and wheeze.

Severe viral respiratory illnesses and atopy are risk factors for childhood wheezing and asthma. The aim of this study was to explore associations between severe respiratory infections and atopy in early childhood with wheeze and asthma persisting into later childhood. 147 children at high atopic risk were followed from birth to age 10 yrs. Data on all respiratory infections occurring in infancy were collected prospectively and viral aetiology ascertained. Atopy was measured by skin prick tests at 6 months, and 2 and 5 yrs. History of wheeze and doctor-diagnosed eczema and asthma was collected regularly until 10 yrs of age. At 10 yrs, 60% of the cohort was atopic, 25.9% had current eczema, 18.4% current asthma and 20.4% persistent wheeze. 35.8% experienced at least one lower respiratory infection (LRI) associated with fever and/or wheeze in first year of life. Children who had wheezy or, in particular, febrile LRI in infancy and were atopic by 2 yrs, were significantly more likely to have persistent wheeze (RR 3.51, 95% CI 1.83-6.70; p<0.001) and current asthma (RR 4.92, 95% CI 2.59-9.36; p<0.001) at 10 yrs. Severe viral respiratory infections in infancy and early atopy are risk factors for persistent wheeze and asthma. The strongest marker of the asthmatogenic potential of early life infections was concurrent fever. The occurrence of fever during respiratory illnesses is an important marker of risk for wheeze and asthma later in childhood, suggesting it should be measured in prospective studies of asthma aetiology.

Antibiotic use in the first year of life and risk of atopic disease in early childhood.

BACKGROUND: Reduced post-natal microbial stimulation resulting from improvements in public health measures, smaller family size, and through increased antibiotic use has been postulated to account for the increasing prevalence of atopic diseases seen predominantly in developed countries. OBJECTIVE: To investigate use of antibiotics in the first year of life and subsequent development of atopic disease in early childhood. METHODS: A prospective birth cohort of 198 children at high atopic risk was recruited prenatally and followed for 5 years. Illnesses and antibiotic use were ascertained through daily diaries, and diagnoses of asthma and hayfever were collected by questionnaire interviews. The children were examined regularly for eczema, and atopic status was defined by skin prick tests and serum total IgE. The effect of antibiotic use on subsequent atopic disease was examined using logistic regression with propensity score adjustment. RESULTS: 54.0% (107/198) of children received at least one course of antibiotics, mainly for acute respiratory illnesses (ARI). Thirty-three percent (329/984) of the ARI involved the lower respiratory tract (LRI). Twenty-three percent (222/984) of ARI were treated with antibiotics, with LRI significantly more likely to receive antibiotics. Antibiotic use was associated with asthma (unadjusted odds ratio 2.3; 95% confidence interval 1.2-4.5; P=0.01) but this association was reduced after propensity score adjustment. No associations were found between antibiotic use and eczema, current wheeze, current asthma, atopic asthma, allergic rhinoconjunctivitis or atopy. CONCLUSION: Although this was a small study, systematic and careful monitoring of ARI, antibiotic use, and asthma and atopic diseases did not indicate that receipt of antibiotics early in life led to subsequent asthma or atopy at 5 years.

Differences in the antibody response to a mucosal bacterial antigen between allergic and non-allergic subjects.

BACKGROUND: The immune response to bacterial antigens on mucosal surfaces may be modified in individuals allergic to aeroallergens due to a maturational or genetic difference or from the interaction between inhaled allergens and bacteria at the mucosa. METHODS: Plasma from children and adults allergic (n = 97) and non-allergic (n = 54) to aeroallergens were initially tested for IgG1 (Th1) and IgG4 (Th2) reactivity to P6, a conserved outer membrane protein of Haemophilus influenzae. IgE binding was measured for some allergic donors. The development of the antibody responses to P6 was subsequently examined in the plasma from 35 children aged 1, 2 and 5 years taken from a prospective birth cohort. RESULTS: IgG4 antibodies to P6 were more readily detected in allergic subjects than in non-allergic subjects (p<0.001), with a strong bias to the male gender. Some allergic subjects (35%) also had IgE antibody (1-10 ng/ml) that was not associated with IgG4 or gender. In the cohort study of infants, subjects who developed skin prick test positivity to mite allergens by 5 years of age had an 85% reduction in the IgG1 anti-P6 antibody at year 2 (p<0.05) and, unlike skin test negative infants, this group had IgG4 anti-P6 antibodies at 5 years of age. CONCLUSIONS: The antibodies of subjects allergic to a bacterial antigen included IgE and IgG4 (particularly for males) compared with the almost exclusive IgG1 response of non-allergic subjects. The IgG1 responses of 2-year-old children who became skin test positive was markedly reduced and P6-specific IgG4 became detectable at 5 years of age.

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.

Specific patterns of responsiveness to microbial antigens staphylococcal enterotoxin B and purified protein derivative by cord blood mononuclear cells are predictive of risk for development of atopic dermatitis.

BACKGROUND: Mononuclear cells from children with active atopic dermatitis (AD) have been reported to be hyper-responsive to certain microbial stimuli, in particular staphylococcal enterotoxin B (SEB). However, it is not known whether this responsiveness is acquired during disease development, or is inherent. We investigated this question in a cohort of children at high risk of atopy followed prospectively from birth to age 3 years. We asked whether their cord blood mononuclear cell (CBMC) cytokine responses to SEB, to an unrelated microbial stimulus purified protein derivative (PPD), or to common allergens, were predictive of risk for subsequent AD development during infancy. METHODS: Children at high risk of developing atopy were randomly selected from an ongoing prospective cohort. Cord blood was collected at birth. The children were seen at 6 months, 1, 2 and 3 years and examined for the development of AD. IFN-gamma, IL-5, IL-10 and IL-13 production by CBMC cultured in the presence of SEB, PPD, PHA, house dust mite (HDM) allergen, ovalbumin (OVA) and cat allergen was determined. RESULTS: SEB-induced IL-5 production by CBMC was elevated in children who developed AD at 6 months (P = 0.01) and 2 years (P = 0.009). PPD-induced IL-5 responses were also elevated in CBMC from children who developed AD at 6 months, 2 years and 3 years (P = 0.05, P = 0.06 and P = 0.06, respectively), as were PPD-induced IL-10 responses (P = 0.05 at 1 years, P = 0.007 at 2 years, P = 0.003 at 3 years) and corresponding IFN-gamma responses (P = 0.05 at 6 months, P = 0.003 at 2 years, P = 0.0004 at 3 years). Increased IL-10 responses to HDM allergen were also observed throughout the observation period in CBMC from children who developed AD. CONCLUSION: Children who develop infantile AD appear to have a predisposition to respond to SEB in a Th2-dominant manner involving selective stimulation of IL-5 production. The increased IL-10 and IFN-gamma induced in response to PPD by children with AD may point to additional intrinsic differences in responses to microbial stimuli between those at high vs. those at low risk for AD, which merit more detailed investigations.

Barriers to immunisation in general practice.

OBJECTIVE: To identify barriers to immunisation in general practice. METHOD: The study was conducted in Perth, Western Australia, as a cross-sectional postal survey between November 1996 and January 1997. Questionnaires were sent to all known GPs in three of the seven metropolitan Divisions of General Practice, of whom 301 (72%) responded. RESULTS: When a child presented with a minor illness and there were no contraindications to immunisation, 62% of GPs said they would always or frequently offer immunisation. Immunisation would be withheld incorrectly because of an upper respiratory tract infection by 43% of GPs and because of antibiotics by 50%. Combined diphtheria-tetanus vaccine (CDT) would be substituted incorrectly for diphtheria-tetanus-pertussis vaccine (DTP) by 41% if there was an unexplained temperature of 38 degrees C following a previous dose of DTP. While more than half (56%) reported that vaccines were correctly stored, only 26% reported that the refrigerator temperature was checked daily. Eighty per cent reported that they completed an Australian Childhood Immunisation Register notification form. CONCLUSIONS: GPs require ongoing education about contra-indications to immunisation and when substitution of CDT for DTP is required. There is room for increased opportunistic immunisation and encouragement to notify the Australian Childhood Immunisation Register when they immunise a child. IMPLICATIONS: A major challenge is to find an innovative approach that would encourage and enable GPs to assess immunisation status and offer immunisation where appropriate at every clinical encounter.

The effect of heat-stable Escherichia coli enterotoxin, theophylline and forskolin on cyclic nucleotide levels and mucosal surface (acid microclimate) pH in rat proximal jejunum in vivo.

The normally acidic mucosal surface pH of 6.24 +/- 0.02(30) in rat proximal jejunum in vivo is effectively neutralised by 30 min exposure to heat-stable Escherichia coli (STa) enterotoxin (14 micrograms/ml) to 6.80 +/- 0.07 (n = 5) or to a forskolin/theophylline combination (1 mM:20 mM) to 7.10 +/- 0.07(7) while perfusion with Krebs-phosphate buffer alone without glucose left the mucosal surface pH unchanged at a pH of 6.21 +/- 0.02(9). Forskolin alone had no effect, and 20 mM theophylline moderately elevated the surface pH to 6.52 +/- 0.03(5). Theophylline, forskolin and their combination all elevated cAMP levels per mg tissue DNA above control values while STa enterotoxin was without effect. In contrast, all agents elevated cGMP levels per mg tissue DNA above control levels. These findings indicate that surface pH is only moderately affected by changes in cAMP levels and is affected to a much greater extent by altered cGMP levels.