Challenges and opportunities of remote public involvement and community engagement during a pandemic: refining the MapMe childhood healthy weight intervention.

AIMS: Including parents and other stakeholders in the development of interventions to address the sensitive public health issues such as childhood obesity, through public involvement is critical. However, the Covid-19 pandemic has created a challenge for public involvement and engagement activities (PICE). The aim of this paper is to describe the process and challenges of setting up, maintaining, evaluating, and recording impact of three public and stakeholder groups via remote methods in the context of the MapMe2 study during the Covid-19 pandemic. Parental reaction to result letters received as part of the National Child Measurement Programme (NCMP) informing parents of their child's overweight status is often one of hostility or disbelief. As a result, parents often do not act on these letters to address child overweight. The MapMe2 study is working in collaboration with the NCMP and local authorities, building on previous work (MapMe) and aims to support parents of primary school-aged children to recognise and maintain a healthy weight in their child. The existing MapMe Intervention includes an enhanced NCMP child weight result letter, supplemented with Body Image Scales (BIS), and an intervention website with material to support healthy eating, physical activity, and signposting supporting information. The intervention was to be refined and the evaluation informed with PICE input. METHODS: Covid-19 restrictions meant that planned face-to-face PICE methods had to be altered with all recruitment, all correspondence, and activities taking place remotely. A Parent Involvement Panel (PIP), a child panel, and an expert panel were established. Several adaptations were made to accommodate a new way of involving the public in research. RESULTS/CONCLUSIONS: Working remotely created many challenges and was a learning experience for all involved. However, an active group was successfully established. Using continuous assessment and evaluation methods, we were able to demonstrate successful involvement and engagement in the refinement of the MapMe2 study. Through the sharing of PICE methods practice, this paper adds to the literature, the value of partnership working.

Agreement in the reporting of General Practitioner requested musculoskeletal radiographs: Reporting radiographers and consultant radiologists compared with an index radiologist.

INTRODUCTION: This study assessed the inter-observer agreement of reporting radiographers and consultant radiologists compared with an index radiologist when reporting General Practitioner (GP) requested musculoskeletal radiographs. The potential effect of discordant reports on patient management and outcome was also examined. METHODS: Three reporting radiographers, three consultant radiologists and an index radiologist reported on a retrospective randomised sample of 219 GP requested musculoskeletal radiographs, in conditions simulating clinical practice. A speciality doctor in radiology compared the observers' reports with the index radiologist report for agreement and assessed whether any discordance between reports was clinically important. RESULTS: Overall agreement with the index radiologist was 47.0% (95% CI, 40.5-53.6) and 51.6% (95% CI, 45.0-58.1) for the consultant radiologists and reporting radiographers, respectively. The results for the appendicular and axial skeleton were 48.6% (95% CI, 41.3-55.9) and 40.9% (95% CI, 27.7-55.6) for the radiologists, and 52.6% (95% CI, 45.2-59.8) and 47.7% (95% CI, 33.8-62.1) for the radiographers, respectively. The difference in overall observer agreement between the two professional groups with the index radiologist was not statistically significant (p = 0.34). Discordance with the index radiologist's reports was judged to be clinically important in less than 10% of the observer's reports. CONCLUSION: Reporting radiographers and consultant radiologists demonstrate similar levels of concordance with an index radiologist when reporting GP requested musculoskeletal radiographs. IMPLICATIONS FOR PRACTICE: These findings contribute to the wider evidence base that selected radiographers with appropriate postgraduate education and training are proficient to report on musculoskeletal radiographs, irrespective of referral source.

Metam sodium reduces viability and infectivity of Eimeria oocysts.

Metam sodium (MS, sodium N-methyldithiocarbamate) is a widely used soil pesticide. Fumigation or chemical sterilization of poultry litter containing infectious oocysts could be an effective strategy to block the transmission of avian coccidia. In the current study, the effect of MS on the viability and infectivity of ocysts was investigated. The development of isolated, unsporulated oocysts of both Eimeria tenella and Eimeria maxima was inhibited, in a dose-related manner (IC(50) 8 to 14 microg/ml), by exposure to aqueous MS. Most treated oocysts failed to develop beyond early stages of sporulation. To determine the effect of MS on infectivity, isolated oocysts of E. tenella , Eimeria acervulina , and E. maxima were exposed for 24 hr to aqueous concentrations of MS ranging from 0 to 1,000 microg/ml. Treated oocysts were inoculated into chickens, and parameters of coccidiosis infection were compared to chickens inoculated with equal numbers of untreated oocysts. In a dose-related manner, MS significantly reduced the infectivity of oocysts with maximum effect observed at a dose of 300 microg/ml. When a mixture of oocysts containing 3 coccidian species was exposed to 300 microg/ml MS, from 0 to 24 hr, infectivity of oocysts was significantly reduced after a minimum of 12 hr of exposure. Treatment of aqueous slurries of litter samples obtained from commercial poultry houses, with 300 microg/ml MS for 24 hr, prevented the sporulation of eimerian oocysts in the litter samples relative to untreated control samples. The results indicate that MS could be used to reduce coccidial contamination of poultry litter.

Household characteristics and mite allergen levels in Manchester,UK.

BACKGROUND: Mite allergen levels vary enormously between different homes in the same geographical area. No large scale studies of mite levels in Manchester homes has been conducted to identify factors associated with higher levels. OBJECTIVES: To quantify exposure to mite allergens and to identify characteristics associated with higher Der p 1 levels in a large sample of homes in Manchester, UK. METHODS: Der p 1 was measured in dust from the living room floor, sofa, bedroom floor and mattress in 564 homes. Data on household characteristics were collected by administering a questionnaire. Univariate and multivariate analyses were performed to identify household characteristics associated with higher mite allergen levels. RESULTS: Der p 1 levels were highest in the mattress (GM 1.19 microg/g, 95% CI 0.98-1.45, P < 0.001). Two-thirds of homes contained Der p 1 levels > 2 microg/g in at least one dust reservoir, and 40.3% contained Der p 1 greater than 10 microg/g. There was a large range in Der p 1 levels between homes (> 10(3)-fold). Damp and condensation were common findings in homes. In the multivariate analyses, factors associated with higher Der p 1 levels in more than one dust reservoir were older homes, older living room carpets, damp, condensation and mixed glazing. Older mattresses were associated with higher mite allergen levels in the mattress where the age of the mattress was recorded. Twenty-four homes contained no detectable mite allergen, six of which reported damp. CONCLUSIONS: Mite allergen levels are high enough in two of every three homes to be associated with an increase in the risk of sensitization to mite. Factors such as older homes, carpets and mattresses, damp and condensation are associated with higher mite allergen. However, mite allergen levels are occasionally unpredictably very low in homes with risk factors for high levels.

A common oocyst surface antigen of Cryptosporidium recognized by monoclonal antibodies.

Two hybridoma clones, CMYL3 and CMYL30, were generated by immunizing Balb/c mice with excysted oocysts of Cryptosporidium muris. Both clones secreted monoclonal antibodies against an oocyst-wall antigen with apparent molecular mass of 250 kDa (called CM250) from C. muris and C. parvum. The epitope appeared to be periodate-sensitive, suggesting the involvement of a carbohydrate moiety. Immunofluorescence and confocal microscopy on purified oocysts and infected mouse tissues revealed staining confined to the oocyst wall of both Cryptosporidium species. Immunogold labeling further revealed the presence of the CM250 antigen in electron-dense vesicles and cytoplasm of developing macrogametocytes, and ultimately localized to the oocyst wall of mature oocysts. Both antibodies cross-reacted with C. serpentis oocysts but did not recognize the other enteropathogenic protozoans Giardia muris, Eimeria falciformis and E. nischulz. These antibodies may be valuable tools for the analysis of oocyst-wall formation in Cryptosporidium and characterization of the common antigen.

Lymphoproliferative responses in cord blood and at one year: no evidence for the effect of in utero exposure to dust mite allergens.

BACKGROUND: Maternal allergen exposure beyond the 22nd week of pregnancy may be important in foetal T cell priming. Allergen-specific cord blood mononuclear cell (CBMC) immunoproliferative responses without corresponding bacterial antigen responses (tetanus toxoid), have been suggested as evidence of in utero sensitization. OBJECTIVES: To investigate the relationship between lymphoproliferative responses at birth and at 1 year with maternal and 1-year infants house dust mite allergen exposure. METHODS: Home visits and dust sampling were performed by the 20th week of pregnancy, immediately after birth, and then at 1 years of age. Der p 1 was assayed using a two-site immunometric ELISA. CBMC immunoproliferative responses (AIM V serum-free medium; 1 x 105 cells/well) were measured for 225 neonates (171 had a high risk of atopy (HR)--both parents skin test positive; 59 had a low risk of atopy (LR) - both parents skin test negative, no history of atopy) by 3H-Thymidine (1microCi/well) incorporation after stimulation in primary culture with phytohaemagglutinin (PHA) (1 microg/mL), house dust mite [HDM] extract (30 microg/mL), immunopurified Der p 1 (30 microg/mL), Tetanus toxoid (TT) (aluma free, 30 Lf/mL) or vehicle. Blood was collected from 144 infants at the age of 1 years and stimulated proliferative responses were assessed using the same procedure. RESULTS: PHA-stimulated lymphoproliferative response was significantly lower in HR compared to LR neonates (mean difference 38%, 95% CI 15%-54%; P = 0.003); significantly lower proportion of positive CBMC responses to HDM occurred in LR than in HR neonates (30.4% vs. 46.6%; P = 0.034). There was no relationship between Der p 1 levels in maternal bed and CBMC immunoproliferative responses, despite the 21 000-fold range of maternal Der p 1 exposure. No significant differences in magnitude, or in proportion of positive responses to any stimulant were observed between the neonates at low, medium or high tertile of allergen exposure. Immunoproliferative responses at birth were not predictive of 1-year PBMC responses. There was no relationship between maternal allergen exposure in pregnancy and 1-year PBMC proliferative responses. However, the proportion of positive proliferative responses at 1 years significantly increased with increasing infant Der p 1 exposure at 1 years. CONCLUSION: These results indicate that the magnitude of immunoproliferative responses are unrelated to maternal mite allergen exposure and cannot be used as evidence for in utero sensitization to inhalant allergens. The immunoproliferative responses at 1 year seem to shift away from the genetically influenced responses at birth towards responses to specific stimulants which correlate with environmental exposure to those specific stimulants. These data support the concept of sensitization to inhalant allergens occurring in early life, but not in utero.

Urinary bladder hyporeflexia and reduced pain-related behaviour in P2X3-deficient mice.

Extracellular ATP is implicated in numerous sensory processes ranging from the response to pain to the regulation of motility in visceral organs. The ATP receptor P2X3 is selectively expressed on small diameter sensory neurons, supporting this hypothesis. Here we show that mice deficient in P2X3 lose the rapidly desensitizing ATP-induced currents in dorsal root ganglion neurons. P2X3 deficiency also causes a reduction in the sustained ATP-induced currents in nodose ganglion neurons. P2X3-null mice have reduced pain-related behaviour in response to injection of ATP and formalin. Significantly, P2X3-null mice exhibit a marked urinary bladder hyporeflexia, characterized by decreased voiding frequency and increased bladder capacity, but normal bladder pressures. Immunohistochemical studies localize P2X3 to nerve fibres innervating the urinary bladder of wild-type mice, and show that loss of P2X3 does not alter sensory neuron innervation density. Thus, P2X3 is critical for peripheral pain responses and afferent pathways controlling urinary bladder volume reflexes. Antagonists to P2X3 may therefore have therapeutic potential in the treatment of disorders of urine storage and voiding such as overactive bladder.

Detrusor myopathy in young beagle dogs.

Hematoxylin and eosin-stained sections of urinary bladder were examined microscopically from 449 male and female beagle dogs after 2- to 4-week toxicology studies. Degenerative lesions (detrusor myopathy) of the urinary bladder muscular tunic were present in 59 of 449 (13%) dogs. Myopathic lesions consisted of focal to multifocal areas of smooth muscle fiber atrophy with cytoplasmic basophilia and vacuolation, individual cell necrosis, enlarged smooth muscle nuclei and nucleoli, and occasional mitotic figures. Adjacent areas of arteritis and periarteritis were present in 10 of 59 (17%) beagles with detrusor myopathy suggesting a possible ischemic pathogenesis of the smooth muscle lesions. There was no significant difference in prevalence of myopathy in beagles administered vehicle or various test compounds. Prior urinary catheterization procedures appeared to augment the prevalence of myopathy; some lesions were adjacent to minor, iatrogenically traumatized urinary bladder mucosa. Muscle lesions were not observed in urinary bladders from mongrel dogs, monkeys, cats, rats, or microswine. When compared to crossbred dogs and other laboratory species, the beagle dog thus appears to be sensitive to development of detrusor myopathy.

Disubstituted indazoles as potent antagonists of the integrin alpha(v)beta(3).

A new series of indazole-containing alpha(v)beta(3) integrin antagonists is described. Starting with lead compound 18a, variations in a number of structural features were explored with respect to inhibition of the binding of beta(3)-transfected 293 cells to fibrinogen and to selectivity for alpha(v)beta(3) over GPIIbIIIa, another RGD-binding integrin. Indazoles attached to a 2-aminopyridine or 2-aminoimidazole by a propylene linker at the indazole 1-position and to a diaminopropionate derivative via a 5-carboxylate amide provided the best potency with moderate selectivity. Several differences in the SAR of the diaminopropionate moiety were observed between this series and a series of isoxazoline-based selective GPIIbIIIa antagonists. Compound 34a (SM256) was a potent antagonist of alpha(v)beta(3) (IC(50) 2.3 nM) with 9-fold selectivity over GPIIbIIIa.

Identification, characterization and expression of Toxocara canis nematode polyprotein allergen TBA-1.

We have cloned the cDNA of TBA-1, the Nematode polyprotein allergen (NPA) of Toxocara canis and found it to be most similar to ABA-1, the Ascaris NPA, on the basis of amino acid sequence. We could study the antigenic properties of an E-coli synthesized fusion protein prepared with the cloned gene since no glycosylation site was expected from the deduced amino acid sequence. Although no IgE responses to TBA-1 were detected, recombinant TBA-1 was differently recognized by serum IgG antibodies when the recombinant TBA-1 was directly adsorbed vs when immobilized via a streptavidin linkage on polystyrene microtitre wells. One group of sera recognized TBA-1 directly immobilized while the second only recognized TBA-1 immobilized via streptavidin linkage. The former were from rodents immunized with a Toxocara sp. adult worm extract while the latter were obtained from rodents infected with T. canis larva or immunized with a Anisakis simplex L3 larval extract. These observations suggest that the two in vivo forms of TBA-1 are expressed, but during different stages of the parasite's life cycle.

The effect of dry heat on mite, cat, and dog allergens.

BACKGROUND: Various techniques have been tried in an attempt to reduce allergen levels in homes. This study investigated the effect of dry heat on mite, cat, and dog allergens. METHODS: Samples (50 mg) of Dermatophagoides pteronyssinus and D. farinae cultures, and of house dust rich in the major cat and dog allergens Fel d 1 and Can f 1 were heated for 5, 10, 15, 30, and 60 min at 60 degrees, 80 degrees, 100 degrees, 120 degrees, and 140 degrees C. Control samples remained at room temperature. Extracts were assayed with the appropriate two-site mono- or mono/polyclonal sandwich ELISA. RESULTS: For Der p 1, the breakdown was proportional to temperature and heating time; after 30 min at 120 degrees C, allergen levels were reduced to < 1% of control. Der p 2 was more heat stable, requiring 140 degrees C for 30-60 min to achieve > 99% reduction. D. farinae groups 1 and 2 allergens showed results similar to those obtained with D. pteronyssinus. In contrast, Can f 1 and Fel d 1 were considerably more thermostable, with 50% and 70%, respectively, of allergen remaining after 60 min at 140 degrees C. CONCLUSIONS: The effect of dry heat on allergens increased with increasing time and temperature, cat and dog allergens demonstrating greater heat resistance than mite allergens. Dry heating methods may represent an alternative technique for removal of mite allergens; however, the greater stability of Fel d 1 and Can f 1 suggests that this procedure may not be appropriate for pet allergens.

The role of alternative splicing of the adhesion molecule, CD44, in lymphoid malignancy.

AIM: To investigate the expression of CD44 isoforms containing variant exon 6 (v6) in a well characterised cohort of patients with non-Hodgkin's lymphoma (NHL) and chronic lymphocytic leukaemia (CLL), and to correlate this with phenotype and disease course. METHODS: Cryostat sections of OCT embedded diagnostic nodal material from NHL patients and cryopreserved mononuclear preparations from CLL patients were used as sources of RNA. After reverse transcription, PCR was carried out with amplimers positioned at either side of the variant exon insertion site to amplify all possible CD44 isoforms. Those isoforms containing v6 were identified after Southern blotting and hybridisation with a radiolabelled oligonucleotide. RESULTS: Of 32 NHL samples analysed, 16 did not express CD44 isoforms containing v6, six expressed an isoform containing exon v6 alone, and 10 expressed v6 long isoforms which contained exon v6 in addition to other variant exons. These data did not correlate with lymphoma classification, disease staging, or the presence or absence of extranodal disease. However, those patients expressing v6 long CD44 isoforms had a worse overall survival than those that did not. The plateau of the survival curves was 50% compared with 82%. No v6 long isoforms were detected in the 21 CLL samples investigated. CONCLUSIONS: The expression of v6 long CD44 isoforms is associated with aggressive disease in NHL, independent of grade, stage, or presence of extranodal disease.

Discovery of an orally active series of isoxazoline glycoprotein IIb/IIIa antagonists.

Using isoxazoline XR299 (1a) as a starting point for the design of highly potent, long-duration GPIIb/IIIa antagonists, the effect of placing lipophilic substituents at positions alpha and beta to the carboxylate moiety was evaluated. Of the compounds studied, it was found that the n-butyl carbamate 24u exhibited superior potency and duration of ex vivo antiplatelet effects in dogs. Replacement of the benzamidin-4-yl moiety with alternative basic groups, elimination of the isoxazoline stereocenter, and reversal of the orientation of the isoxazoline ring resulted in lowered potency and/or duration of action.

Molecular characterization of Echinostoma caproni and E. paraensei by random amplification of polymorphic DNA (RAPD) analysis.

Genomic DNA from Echinostoma caproni and Echinostoma paraensei was amplified by the polymerase chain reaction using primers (5'-TCGTAGCCAA and 5'-TCACGATGCA), originally found to differentiate species and strain of Schistosoma. The 2 putative species of Echinostoma produced distinct banding patterns clearly distinguishable from one another, thereby suggesting that RAPD (random amplification of polymorphic DNA) analysis may be useful for the identification of echinostome strains and species previously misunderstood or undescribed, and that primers developed for species within a given genus, e.g., Schistosoma, may have broader application in identifying other trematodes.

OCT embedded sections of pathological specimens as a source of high quality RNA for reverse transcriptase/polymerase chain reaction.

OCT embedded cryostat sections of stored pathological specimens of non-Hodgkin's lymphoma were used to provide RNA. After reverse transcription to produce cDNA, the polymerase chain reaction was performed with primers for standard and variant forms of the CD44 molecule. Using Southern transfer and hybridisation with a probe specific for exon 4 of the CD44 gene, both standard and variant forms were visualised by autoradiography. This method was shown to be applicable to other gene products by using primers specific for the abl and bcr genes. This technique permits retrospective analysis of RNA from small amounts of stored pathological samples.

The immunosuppressive metabolite of leflunomide is a potent inhibitor of human dihydroorotate dehydrogenase.

The active metabolite of leflunomide. A771726, is a novel immunosuppressive compound that has been shown to be a powerful antiproliferative agent for mononuclear and T-cells. The molecular mechanism of action for this compound has not been clearly established. In vitro cellular and enzymatic assays, however, demonstrate that leflunomide is an inhibitor of several protein tyrosine kinases, with IC50 values between 30 and 100 microM. The in vivo properties of A771726 are reminiscent of another immunosuppressive agent, brequinar sodium, which has been shown to be a nonomolar inhibitor (Ki = 10-30 nM) of the enzyme dihydroorotate dehydrogenase (DHODase). On the basis, we have investigated the effects of leflunomide and A771726 on the activity of purified recombinant human DHODase. We find that A771726 is a potent inhibitor of DHODase (Ki = 179 +/- 19 nM), while the parent compound, leflunomide, had no inhibitory effect at concentrations as high as 1 microM. Studies of the dependence of inhibition on the concentrations of the substrates ubiquinone and dihydroorotate demonstrate that A771726 is a competitive inhibitor of the ubiquinone binding site and is noncompetitive with respect to dihydroorotate. The potency of A771726 as a DHODase inhibitor is thus 100-100-fold greater than that reported for its inhibition of protein tyrosine kinases. These data suggest that an alternative explanation for the immunosuppressive efficacy of A771726 may be the potent inhibition of DHODase by this compound.

Targeting gene expression to the vascular wall in transgenic mice using the murine preproendothelin-1 promoter.

To develop a system for overexpressing genes in the vascular wall, we created transgenic mice using the reporter gene luciferase and the murine preproendothelin-1 promoter. In vitro analysis suggested that the murine 5'-flanking region contained endothelial-specific elements in a 5.9-kb fragment. Five transgenic mice colonies established from independent founders all exhibited the highest level of luciferase activity in the aorta with up to 8,540 light units per microgram of protein. Immunohistochemistry with anti-luciferase antisera revealed high levels of expression in the endothelial cells of both large and small arteries and lower levels of expression in veins and capillaries. Significant expression was also seen in arterial smooth muscle cells and in select epithelial surfaces which is consistent with the known distribution of endothelin-1 in mammals. The further demonstrate the targeting capability of this system, we overexpressed the lipid-peroxidating enzyme, human 15-lipoxygenase, in the vessel wall of transgenic mice. As with luciferase, expression of active enzyme and immunohistochemical localization in vascular cells were documented in transgenic animals. Hence, this new system can be used to direct expression of molecules to the vascular wall for the purpose of examining the biological significance of either overexpression or inhibition of select proteins.

Molecular heterogeneity of variant Philadelphia translocations in childhood acute lymphoblastic leukaemia.

In acute lymphoblastic leukemia (ALL), it is unclear whether variant Philadelphia (Ph) translocations have the same molecular and clinical implications as the classical translocation. Two children with Ph+ ALL with variant translocations are described. One, in whom cytogenetic remission was not achieved, had evidence of translocation of c-abl to chromosome 22, rearrangement of minor breakpoint cluster region (mBCR) and expression of hybrid bcr/abl transcripts. In the other case, no gene rearrangement was found and complete remission was achieved. Variant Ph translocations in childhood ALL are heterogeneous at the molecular level. Molecular studies coupled with observations of clinical outcome are needed in larger numbers of such children to determine whether poor clinical response correlates with bcr/abl involvement and to allow planning of appropriate therapeutic strategies.