England's Healthy Living Pharmacy (HLP) initiative: Facilitating the engagement of pharmacy support staff in public health.

BACKGROUND: The concept of the Healthy Living Pharmacy (HLP) in England was first piloted in Portsmouth in 2010. HLPs proactively promote health and wellbeing, offering brief advice, services or signposting on a range of health issues such as smoking, physical activity, sexual health, healthy eating and alcohol consumption. OBJECTIVES: To explore the views and attitudes of pharmacy support staff on the Healthy Living Pharmacy (HLP) initiative. METHODS: Qualitative semi-structured, face-to-face interviews were conducted with pharmacy support staff recruited from community pharmacies involved in the HLP initiative in the Northumberland region of England. A topic guide was developed which underwent face validity testing and piloting with one participant. Interviews were audio recorded, transcribed verbatim and analyzed using framework technique. RESULTS: A total of 21 pharmacy support staff from 12 HLPs participated in the study. Results suggest that involving pharmacy support staff at very early stages of the HLP planning process drives their motivation for service delivery. Level of engagement with HLP services was often related to support staff roles within pharmacy. Integration of public health roles with routine pharmacy activities was perceived to be more suited to pharmacy counter based roles than dispensing roles. Further training needs were identified around how to proactively deliver public health advice, mainly in service areas perceived 'difficult' by the participants, such as weight management. A total of 19 facilitators/barriers were identified from the data including training, access to information, client feedback, availability of space and facilities within pharmacies, time and competing priorities. CONCLUSIONS: Pharmacy support staff engagement with the HLP initiative can be promoted by involving them from the outset of the service introduction process. Support staff might benefit from targeted training around certain public health areas within the HLP initiative. Facilitators/barriers identified in this study will inform development and further roll out of HLP initiative in wider areas.

Cytotoxicity of pilosulin 1, a peptide from the venom of the jumper ant Myrmecia pilosula.

The synthetic peptide pilosulin 1, corresponding to the largest defined allergenic polypeptide found in the venom of the jumper ant Myrmecia pilosula, inhibited the incorporation of [methyl-3H]thymidine into proliferating Epstein-Barr transformed (EBV) B-cells. The LD50 was four-fold lower in concentration than melittin, a cytotoxic peptide found in honey bee venom. Loss of cell viability was assessed by flow cytometry by measuring the proportion of cells that fluoresced in the presence of the fluorescent dye 7-aminoactinomycin D. Examination of proliferating EBV B-cells indicated that the cells lost viability within a few minutes exposure to pilosulin 1. Partial peptides of pilosulin 1 were less efficient in causing loss of cell viability and the results suggest that the 22 N-terminal residues are critical to the cytotoxic activity of pilosulin 1. Normal blood white cells were also labile to pilosulin 1. T- and B-lymphocytes, monocytes and natural killer cells, however, were more labile than granulocytes. Analysis of pilosulin 1 using circular dichroism indicated that, in common with melittin and other Hymenoptera venom toxins, it had the potential to adopt an alpha-helical secondary structure.

Flow cytometric analysis of cell killing by the jumper ant venom peptide pilosulin 1.

Pilosulin 1 is a synthetic 56-amino acid residue polypeptide that corresponds to the largest allergenic polypeptide found in the venom of the jumper ant Myrmecia pilosula. Initial experiments showed that pilosulin 1 lysed erythrocytes and killed proliferating B cells. Herein, we describe how flow cytometry was used to investigate the cytotoxicity of the peptide for human white blood cells. Cells were labeled with fluorochrome-conjugated antibodies, incubated with the peptide and 7-aminoactinomycin D (7-AAD), and then analyzed. The effects of varying the peptide concentration, serum concentration, incubation time, and incubation temperature were measured, and the cytotoxicity of pilosulin 1 was compared with that of the bee venom peptide melittin. The antibodies and the 7-AAD enabled the identification of cell subpopulations and dead cells, respectively. It was possible, using the appropriate mix of antibodies and four-color analysis, to monitor the killing of three or more cell subpopulations simultaneously. We found that 1) pilosulin 1 killed cells within minutes, with kinetics similar to those of melittin; 2) pilosulin 1 was a slightly more potent cytotoxic agent than melittin; 3) both pilosulin 1 and melittin were more potent against mononuclear leukocytes than against granulocytes; and 4) serum inhibited killing by either peptide.

Expression of jumper ant (Myrmecia pilosula) venom allergens: post-translational processing of allergen gene products.

N-terminal analyses of electrophoretically-separated allergenic polypeptides of the venom of the jumper ant M. pilosula showed that five out of the six allergenic polypeptides identified are homologous with the cloned major allergen Myr p I and may be derived from a single precursor polypeptide. The sixth polypeptide is homologous with a second cloned major allergen, Myr p II which is expressed as a single precursor polypeptide but exists in its native form as a disulphide bond-linked complex.

Molecular cloning and characterization of the major allergen Myr p II from the venom of the jumper ant Myrmecia pilosula: Myr p I and Myr p II share a common protein leader sequence.

A major allergen Myr p II of the Australian jumper ant Myrmecia pilosula has been cloned, immunocharacterized and nucleotide sequenced. An open reading frame of 225 bases was identified and found to encode a deduced amino acid sequence of 75 residues which contained a typical hydrophobic peptide leader sequence. Expressed fusion proteins of Myr p II in both phage and plasmid vectors bind high levels of ant venom-specific IgE and the expressed clones are recognised by 35% of ant venom-allergic individuals. IgE antibodies that recognise the expressed clone have been shown to recognise IgE-binding bands in blots of native venom after separation by SDS-PAGE. The amino acid sequence of Myr p II shares close structural homology with the other major jumper ant allergen Myr p I, differing by only three amino acids in the first 47 residues of both sequences. However, N-terminal analysis of IgE-binding bands derived from Tricine-SDS-PAGE gel blots indicates that both Myr p I and Myr p II undergo extensive post-translational proteolytic processing to unique peptides of 45 and 27 residues, respectively.

Glutathione S-transferase a major allergen of the house dust mite, Dermatophagoides pteronyssinus.

Recently, we cloned a new Dermatophagoides pteronyssinus allergen homologous with glutathione S-transferase. IgE radioimmunoassays against the Escherichia coli lysate of the recombinant clone reveal that 40% of mite allergic subjects recognize the mite glutathione S-transferase allergen. Of these sera, greater that 50% have reactivity with the recombinant mite glutathione S-transferase that is greater than 10 times the result observed with a normal control. Immunoblotting studies with sera from patients that recognize the recombinant protein reveal IgE binding to a 26-kDa protein on immunoblots of reduced mite protein extracts. The 26-kDa IgE-binding band observed on immunoblots of reduced mite proteins, corresponding to the cloned protein, is a separate and unique allergen from the 25-kDa Der p I as the apparent electrophoretic molecular mass of Der p I shifts from 25 to 30 kDa under conditions of reduction.

Separation of jumper ant (Myrmecia pilosula) venom allergens: a novel group of highly basic proteins.

The sting of the jumper ant (Myrmecia pilosula) causes severe allergic reactions, including anaphylaxis in sensitized individuals. Two of the major allergens, Myr p I and Myr p II, have been cloned, immunocharacterized and nucleotide-sequenced and they encode 112 and 75 residue polypeptides, respectively. Both allergens are highly basic proteins having isoelectric point values greater than 10. However, electrophoretic analysis has generated conflicting results as to the actual sizes of the allergens in the native venom. Electrophoretic, immunological and N-terminal analyses suggested that these allergens undergo extensive post-translational processing to final forms of 45 and 27 residues, respectively. The results highlight the difficulties in the study of small, basic proteins and polypeptides by electrophoretic techniques.

Cloning and characterization of a major allergen of the house dust mite, Dermatophagoides pteronyssinus, homologous with glutathione S-transferase.

A major allergen of the house dust mite, Dermatophagoides pteronyssinus, has been identified and characterized from a lambda gt11 cDNA library of the mite. IgE antibodies from the sera of allergic patients that recognise the cloned polypeptide bind to an approximately 26 kDa polypeptide on a Western blot of reduced mite polypeptides. Nucleotides sequencing of the clone revealed a 219 amino acid open reading frame encoding a protein with a derived molecular mass of 25,589 Da and a pI of 6.3. Comparison of the deduced amino acid sequence with amino acid sequence databanks revealed a strong homology with glutathione S-transferases. The nucleotide sequence of the clone displayed a strong homology with the active glutathione binding site of glutathione transferases and contained all but one of the 19 positionally conserved amino acid residues found in glutathione transferases. The cloned polypeptide was expressed in Escherichia coli and affinity-purified on glutathione agarose.

Immediate allergic reactions to Myrmecia ant stings: immunochemical analysis of Myrmecia venoms.

IgE antibody reactivities to the venoms of Myrmecia pilosula, Myrmecia nigrocincta, Myrmecia tarsata, Myrmecia pyriformis, Myrmecia simillima and Myrmecia gulosa have been identified in sera from subjects allergic to ant venom. Sera with IgE reactivity to only a single Myrmecia venom most often recognize M. pilosula venom although all six venoms appear capable of inducing IgE antibodies. The six different Myrmecia venoms were electrophoretically separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Western blotted to nitrocellulose and probed with sera from ant venom-allergic subjects. Four to six IgE-binding components ranging in size from 2 to 25 kDa were identified in each of the venoms. Similarities in molecular weights of the IgE-binding components exist and close taxonomic relationships between the species suggest that common or similar peptides may be present in the different venoms.

Identification of an IgE-binding determinant of the major allergen Myr p I from the venom of the Australian jumper ant Myrmecia pilosula.

The structure of the single allergenic determinant of the major ant venom allergen, Myr p I from the Australian jumper ant Myrmecia pilosula has been determined by inhibition studies with synthetic peptides. A 14 amino-acid C-terminal peptide sequence has been shown to constitute this determinant. Half-maximal inhibition of binding of ant venom-specific IgE antibodies to the native venom was obtained with this peptide at a concentration of 5 x 10(-8) M. This allergenic determinant was invariant for all ant venom-allergic subjects tested whose IgE antibodies recognized this allergen.

Immunoaffinity analysis of cross-reacting allergens by protein blotting.

IgE antibodies from sera having reactivity against ryegrass pollen protein allergens, wheat endosperm protein allergens and also several other cereal protein allergens were adsorbed with either ryegrass pollen or the wheat/globulin fraction immobilised on solid phases and subsequently eluted with low pH buffer. The eluted antibodies were reacted with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) blots of the different allergens. Antibodies adsorbed and subsequently eluted from the two allergen sources recognised different spectra of proteins in the ryegrass pollen and cereal allergen sources and indicated the degree of immunological cross-reactivity. Intra-species cross-reactivity of IgE antibodies was demonstrated employing similar methods to those used for the pollen and cereal allergens by using a recombinant allergen from the venom of the ant Myrmecia pilosula as the immunoadsorbent protein on the solid phase.

Isolation and characterization of wheat triticin cDNA revealing a unique lysine-rich repetitive domain.

Polyclonal antibodies were raised against a purified 22 kDa triticin polypeptide (delta) and were used to screen a wheat seed cDNA library in the Escherichia coli expression vector lambda gt11. The isolated cDNA clones were grouped into three families based on their cross-hybridization reactions in DNA dot-blot studies. Southern blots of genomic DNAs extracted from ditelocentric and nullisomic-tetrasomic lines of Chinese Spring wheat, probes with the excised cDNA inserts, indicated that one of the three families (9 clones) had triticin clones. This was finally confirmed by comparing the predicted amino acid sequences of two of these clones (lambda Tri-12, lambda Tri-25) with the published tryptic peptide sequences of triticin. The Southern blots also showed that there is at least one triticin gene located on the short arm of each of the homoeologous group 1 chromosomes (1A, 1B, 1D), although till now no triticin protein product has been identified for the chromosome 1B. The nucleotide sequence of the largest triticin cDNA clone lambda Tri-25 (1567 bp) is presented here, and its predicted amino acid sequence shows strong homology with the legumin-like proteins of oats (12S globulin), rice (glutelin) and legume seeds. A unique feature of the triticin sequence is that it contains a lysine-rich repetitive domain, inserted in the hypervariable region of the typical legumin-like genes. Northern blotting of total RNA extracted from different stages of the developing wheat seed revealed that the triticin gene expression is switched on 5-10 days after anthesis (DAA). There was a steady increase in the level of triticin mRNA until 20 DAA, after which it started decreasing. The maximum mRNA accumulation occurred between 17 and 20 DAA. These observations conform closely with the published data on triticin protein accumulation during grain development.

Molecular cloning and characterization of a major allergen (Myr p I) from the venom of the Australian jumper ant, Myrmecia pilosula.

Five IgE-binding components were identified in the venom of the Australian jumper ant, Myrmecia pilosula using SDS polyacrylamide gel electrophoresis and Western blotting. A cDNA clone which encodes the entire amino acid sequence of one of the major IgE-binding venom allergens has been nucleotide sequenced. The IgE-binding determinants of this allergen are located in its C-terminal domain. Database searches, however, did not reveal any homology with any other known nucleotide or protein sequence. The sequenced allergenic polypeptide has, according to the convention recommended by the International Union of Immunological Societies (IUIS), been named Myr p I.

Crossreactivity of IgE antibodies from sera of subjects allergic to both ryegrass pollen and wheat endosperm proteins: evidence for common allergenic determinants.

Positive RAST (greater than 5% radioactive uptakes) to wheat endosperm proteins were found in approximately one-quarter of subjects who had both a positive skin prick and RAST (greater than 10% radioactive uptake) to ryegrass pollen proteins. Immunoblotting of proteins electrophoretically transferred to nitrocellulose membrane after SDS-PAGE of ryegrass pollen and wheat endosperm proteins confirmed the crossreactive properties of the sera identified by RAST testing. Immunoadsorption of serum IgE onto nitrocellulose membrane, to which ryegrass pollen or wheat endosperm proteins had been adsorbed, removed IgE from crossreactive sera reactive to both ryegrass pollen and wheat endosperm proteins. Elution of the adsorbed IgE from the nitrocellulose membrane after immunoadsorption and probing blotted strips of both ryegrass pollen and wheat endosperm proteins supported the results obtained from the immunoadsorption experiments. This data provides evidence that the crossreactivity of IgE antibodies in sera reacting with both ryegrass pollen and wheat endosperm proteins involves common or related determinants and has implications for the clinical management of these allergic subjects.

The structural basis of allergenicity: recombinant DNA-based strategies for the study of allergens.

The use of recombinant DNA techniques for the study of allergenicity of proteins is a viable, and in many ways a preferred, alternative to the traditional procedures of protein purification, digestion and analysis of peptides for both allergenicity and amino acid sequence. The process of protein purification can be difficult and in many instances workers are forced to use only partially pure fractions that make the identification of the allergenic proteins uncertain. Furthermore, the purification and sequencing of peptides and their testing for retention of allergenic properties, represents a substantial and time-consuming work load. The synthesis of families of synthetic peptides to characterize the amino acids important for allergenic properties is also expensive and time-consuming. On the other hand, the preparation of a cDNA library from an allergen source is today a relatively easy and inexpensive task. The isolation and purification of cDNA clones is comparatively trivial compared to protein purification. Using the techniques described in this text, it can be seen that the molecular biological approach, although in some respects similar in principle to those of the protein chemist to study allergens, provides the capability to study several clones at the same time, and to compare clones for the presence of conserved regions corresponding to allergenic determinants. In addition, the techniques for generating mutant sequences provides perhaps the most powerful and simple set of procedures available for defining the amino acid structures essential for proteins or peptides to behave as allergens.