A turning point: Head and neck cancer patients' exercise preferences and barriers before and after participation in an exercise intervention.

This study examined the exercise barriers and preferences of head and neck cancer (HNC) survivors in relation to exercise experience. Participants (n = 22; 46.8% response rate) completed retrospective self-report questionnaires on demographic and medical information, exercise barriers and preferences. A subset of participants then completed semi-structured interviews (n = 18). Participants had previously engaged in the ENHANCE trial during, or immediately following, radiation treatment, an average of 22.1 ± 5.8 months before. Retrospective questionnaires revealed that before ENHANCE participation, lack of interest and time were the primary exercise barriers. After participation, there was a significant decrease in typical barriers including lack of interest (p = .008), exercise not a priority (p = .039) and exercise not in routine (p = .004). Number of barriers experienced after ENHANCE participation was negatively correlated with age, quality of life and minutes of resistance exercise training per week. After ENHANCE participation, significant increases were found in preference for exercising at a cancer centre (p = .031) and with other cancer survivors (p = .016). Four higher order themes emerged inductively from interview data analysis pertaining to preferences (i.e., class format) and three higher order themes regarding barriers (physical, psychological and external). By investigating participants' perspectives after ENHANCE participation, key factors for effective HNC exercise programme design were identified.

Prediction of adherence to a gluten-free diet using protection motivation theory among adults with coeliac disease.

BACKGROUND: Coeliac disease is a chronic autoimmune disease that requires strict adherence to a gluten-free diet. However, strict adherence to a gluten-free diet is difficult, with findings from a recent review suggesting that up to 42% of individuals with coeliac disease do not eat a strict gluten-free diet. METHODS: The present study aimed to examine psychosocial predictors of adherence (purposeful and accidental) to a gluten-free diet among adults with coeliac disease over a 1-month period. In this longitudinal study, 212 North American adults with coeliac disease completed online questionnaires at two time points, baseline and 1 month later. RESULTS: The results revealed that intentions partially mediated the effects of symptom severity, self-regulatory efficacy, planning and knowledge on purposeful gluten consumption. Intentions did not mediate the effects of severity, response cost, self-regulatory efficacy, planning and knowledge for accidental gluten consumption but, interestingly, self-regulatory efficacy directly predicted fewer accidental incidents of gluten-consumption. CONCLUSIONS: These findings delineate the differential psychological processes in understanding accidental and purposeful gluten consumption among adults with coeliac disease and emphasise the importance of bolstering self-regulatory efficacy beliefs to prevent accidental and purposeful consumption of gluten.

Motives for adherence to a gluten-free diet: a qualitative investigation involving adults with coeliac disease.

BACKGROUND: Currently , the only treatment for coeliac disease is life long adherence to a strict gluten-free diet. Strict adherence to a gluten-free diet is challenging, with recent reports suggesting that adherence rates range from 42% to 91%. The present study aimed to: (i) identify motives for adhering to a gluten-free diet and (ii) explore factors implicated in adherence and non-adherence behaviour in terms of accidental and purposeful gluten consumption among adults with coeliac disease. METHODS: Two hundred and three adults with coeliac disease completed an online questionnaire. Using a qualitative design, relationships were examined between reported adherence and motivation to follow a gluten-free diet, as well as the onset, duration and severity of symptoms. RESULTS: Feelings of desperation ('hitting rock bottom') and needing to gain or lose weight were associated with the strictest adherence to a gluten-free diet. Participants who accidentally consumed gluten over the past week developed symptoms the most quickly and reported the most pain over the past 6 months. Participants who consumed gluten on purpose over the past week reported a shorter duration of symptoms and less pain over the past 6 months. CONCLUSIONS: Hitting rock bottom and needing to gain or lose weight were factors associated with the strictest adherence, when considered in the context of both accidental and purposeful gluten consumption. Future research is warranted to develop resources to help people with coeliac disease follow a strict gluten-free diet.

Purification and identification of a 7.6-kDa protein in media conditioned by superinvasive cancer cells.

BACKGROUND: Selection of the human drug sensitive and invasive cell line (MDA-MB-435S-F) with the chemotherapeutic agent paclitaxel, resulted in the development of drug resistant cell lines displaying enhanced invasion-related characteristics. MATERIALS AND METHODS: Serum-free conditioned media from the human cancer drug-sensitive and invasive cell line (MDA-MB-435S-F) and its paclitaxel-resistant superinvasive variant (MDA-MB-435S-F/Taxol10p4pSI) were analyzed using Surface enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS). RESULTS: A differentially expressed protein was observed at 7.6 kDa, which was 4-fold up-regulated in MDA-MB-435S-F/Taxol10p4pSI. The differentially expressed protein was identified using matrix-assisted laser desorption ionization tandem time-of-flight mass spectrometry (MALDI-TOF/TOF MS), as a fragment of bovine transferrin. The transferrin receptor was also found to be overexpressed in the superinvasive cell line. CONCLUSION: Cleavage of serum proteins such as transferrin could provide a valuable source of markers for malignant tumours and could also play a role in aspects of cancer pathogenesis, such as tumour cachexia.

Patterns of LH and FSH release from perifused rat pituitaries in response to infusions of hypothalamic extract.

A continuous flow incubation (perufusion) system was developed in which the secretory responses of pools of hemipituitaries from adult male rats to hypothalamic extract (HE) were characterized by serial radioimmunoassay of LH and FSH in the effluent medium. There was in initial massive release of LH and FSH which, in the absence of HE, declined to low basal levels at a rate which depended on the flow rate. Thereafter, the baseline for LH continued to decline gradually while that for FSH was stable. The rate of LH and FSH release rose abruptly after addition of the HE to the medium and returned promptly to baseline after withdrawal of the HE. During continuous infusion of HE for five hours, LH secretion was maintained at a relatively constant, elevated level. The responses to repeated identical pulses of HE were highly reproducible. The variability between responses by any one pool of tissue was significantly less than between responses of separate pituitary pools including pools comprising right and left halves of the same glands. For any given pool of pituitaries of the relationships were linear between: 1) duration of HE pulses (concentration constant) and increases in LH output, and 2) log of concentration of HE (pulse duration constant) and increases inLH and in FSH output. Consistent responses were obtained for up to 12 hr, the maximum period tested.

Cathepsin L proteinase secreted by Fasciola hepatica in vitro prevents antibody-mediated eosinophil attachment to newly excysted juveniles.

Cathepsin L-like activity was demonstrated in the excretory/secretory (E/S) products of Fasciola hepatica newly excysted juveniles (NEJ), 3-week-old, 5-week-old and mature flukes using the fluorogenic substituted 7-amino-4-methylcoumarin substrates Z-phe-arg-AMC, Z-arg-arg-AMC and Z-arg-AMC. Gelatin-substrate polyacrylamide gel analysis revealed that the E/S from each of these stages contained multiple proteolytic enzymes; however, the pattern of proteinases obtained for NEJ E/S differed markedly from that of all other stages examined. The four NEJ proteinases identified were inhibited by leupeptin and Z-phe-ala-diazomethyl ketone indicating that each had cathepsin L-like activity. The E/S products of all four developmental stages contain an enzyme capable of cleaving immunoglobulin at the hinge region, the activity of which is also inhibited by Z-phe-ala-diazomethyl ketone. Using in vitro cell attachment assays we show that the cathepsin L-like proteinase purified from the E/S products of adult F. hepatica can prevent the antibody-mediated attachment of eosinophil to NEJ. These experiments indicate that this proteinase has an important biological function in immune evasion.

Isolation of a cDNA encoding Fasciola hepatica cathepsin L2 and functional expression in Saccharomyces cerevisiae.

Cathepsin L2 is a major cysteine proteinase secreted by adult Fasciola hepatica. The enzyme differs from other reported cathepsin Ls in that it can cleave peptide substrates that contain proline in the P2 position. A cDNA was isolated from an expression library by immunoscreening with antiserum prepared against purified native cathepsin L2. This cDNA was sequenced and shown to encode a complete preprocathepsin L proteinase. Functionally active recombinant cathepsin L proteinase was expressed and secreted by Saccharomyces cerevisiae transformed with the cDNA. The recombinant enzyme was purified from large-scale fermentation broths using ultrafiltration and gel filtration chromatography on Sephacryl S200 HR columns. NH2-terminal amino acid sequencing showed that the cleavage point for activation of the recombinant pro-enzyme is identical to that of the F. hepatica-produced cathepsin L2. The mature active recombinant proteinase behaved similarly to the native enzyme when analysed by SDS-PAGE, immunoblotting and zymography and also cleaved peptides containing proline in the P2 position. Finally, the recombinant cathepsin L2 cleaved fibrinogen to form a fibrin clot, a property we described for F. hepatica cathepsin L2.

Purification of a cathepsin L-like proteinase secreted by adult Fasciola hepatica.

A cysteine proteinase released in vitro by Fasciola hepatica was purified to homogeneity by Sephacryl S-200 gel filtration chromatography followed by QAE-Sephadex chromatography. The purified enzyme resolves as a single band with an apparent molecular size of 27 kDa on reducing SDS-polyacrylamide gel electrophoresis; however, under non-reducing conditions it migrates as multiple bands, each with enzymatic activity, in the apparent molecular size range 60-90 kDa. The sequence of the first 20 N-terminal amino acids of the enzyme shows considerable homology with cathepsin L-like proteinases. Immunolocalisation studies revealed that the cathepsin L-like proteinase is concentrated within vesicles in the gut epithelial cells of liver fluke.

Fasciola hepatica: a secreted cathepsin L-like proteinase cleaves host immunoglobulin.

Adult Fasciola hepatica secrete a cysteine proteinase capable of cleaving host IgG close to the papain cleaving site. The proteinase was separated by size permeation chromatography. Gelatin-substrate polyacrylamide gel electrophoresis analysis revealed that the proteinase migrates as 6 proteolytic bands in the apparent molecular size range 60-90 kDa. Based on pH profiles of activity, inhibition studies using diethylpyrocarbonate and the diazomethylketone Z-phe-ala-CHN2, and characterising the substrate specificity of the enzymes using fluorogenic peptide substrates we have shown that the 60-90-kDa proteinases are cathepsin L-like proteinases.

Secretion of cysteine proteinase activity by the zoonotic hookworm Ancylostoma caninum.

The zoonotic hookworm, Ancylostoma caninum, probably induces human eosinophilic enteritis by inducing allergic responses to its secretions. This species is already known to secrete metalloproteinases, but in other parasites, cysteine proteinases are involved in pathogenesis. We studied somatic extracts of A. caninum adults and infective larvae and adult excretory/secretory (ES) antigens for cysteine proteinase activity using fluorogenic peptide substrates and by gelatin and fluorogenic substrate polyacrylamide gel electrophoresis. Proteolytic activity was observed against the cathepsins L and B-specific substrate Z-phe-arg-AMC, against the plasmin substrate Boc-val-leu-lys-AMC, and against gelatin. The Z-phe-arg-AMC-hydrolyzing activity in ES antigens and in adult extracts was enhanced up to 15-fold by the reducing agent dithiothreitol (DTT), but was totally blocked by specific inhibitors of cysteine proteinases, including the peptidyl diazomethyl ketone Z-phe-ala-CHN2,E-64, leupeptin, and N-ethylmaleimide. In a similar fashion, gelatinolytic activity in ES antigens detected using substrate gels was enhanced by the addition of reducing agents and inhibited by Z-phe-ala-CHN2 and E-64. The DTT-enhanced, Z-phe-arg-AMC-hydrolyzing activity in ES antigens was active over a wide pH range (pH 5-9). Similar cysteine proteinase activity to that detected in ES antigens was present in extracts of adult and infective larvae of A. caninum. Because the substrate Z-phe-arg-AMC was specifically hydrolyzed, and because this hydrolysis was totally blocked by cysteine proteinase-specific inhibitors, ES antigens and tissue extracts of A. caninum clearly possess cysteine proteinase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Short report: Immunodiagnosis of human fascioliasis using recombinant Fasciola hepatica cathepsin L1 cysteine proteinase.

Our laboratory recently developed a diagnostic test (ELISA) for human fascioliasis based on the detection of serum IgG4 antibodies reactive with Fasciola hepatica cathepsin L1 (CL1). In the present study, we have used recombinant CL1, generated by functional expression of the cDNA in Saccharomyces cerevisiae, in this immunodiagnostic test and compared its performance with native CL1. Sera obtained from 64 individuals living in Cutusuma village in the northern Altiplano of Bolivia, a region with a high prevalence of human fascioliasis, were analyzed by the IgG4-ELISA. A highly statistically significant correlation (r2 = 0.751, P < 0.001) was demonstrated between the absorbances obtained using the recombinant and native proteins. These assays showed that 38 (59%) of the individuals tested were seropositive for fascioliasis, whereas only 26 of them were coprologically positive for F. hepatica eggs. All seronegative patients were also coprologically negative. Serum from individuals infected with schistosomiasis mansoni, cysticercosis, hydatidosis, and Chagas disease did not contain antibodies reactive with the recombinant or native CL1. Therefore, recombinant CL1 shows excellent potential for the development of the first standardized assay for the sensitive and specific diagnosis of human fascioliasis. Finally, our data supports earlier reports on the high prevalence of human fascioliasis in the Bolivian Altiplano, which collectively suggest that the disease has been endemic there for more than a decade.

Antihistamine blockade of the ovarian hyperstimulation syndrome. II. Possible role of antigen-antibody complexes in the pathogenesis of the syndrome.

Antihistamine can prevent the ovarian hyperstimulation syndrome in the rabbit. The mechanism underlying this experimental observation has not been elucidated. Our experiments were directed at the question of whether antigen-antibody complexes are a factor in the development of the ovarian hyperstimulation syndrome. The results do not support the theory that antigen-antibody complexes play a role in the pathogenesis of this syndrome.

Purification of a diagnostic, secreted cysteine protease-like protein from the hookworm Ancylostoma caninum.

The enteric infection of humans with the canine hookworm Ancylostoma caninum varies in its clinical presentation, ranging from asymptomatic to eosinophilic gastroenteritis requiring surgical intervention. Infections are not patent, but can be diagnosed immunologically by detecting antibodies to an immunodominant secreted hookworm protein termed Ac68. To characterise Ac68, we purified the native protein from A. caninum excretory/secretory products using size exclusion followed by anion exchange chromatography. The epitopes in the purified protein recognised by human infection sera were shown to be proteins and not carbohydrates. The N-terminal amino acid sequence of the purified Ac68 was determined and six of the 11 residues obtained were shared with a previously characterised cysteine protease of A. caninum, AcCP1.

Immune responses of cattle to experimental anti-Fasciola hepatica vaccines.

Fasciola hepatica infection of cattle and sheep is an important cause of clinical disease and production losses, and is controlled at present by a combination of chemotherapy and management measures. However, the prospects for the control of F. hepatica infection by vaccination are good, and we have previously shown substantial protection of cattle against experimental challenge infection following immunisation with a combination of the purified fluke-derived enzymes cathepsin L1 (CATL 1), cathepsin L2 (CATL 2) and fluke-derived Hb fraction (FHB). This and other recent studies have also demonstrated fundamental differences between protective and non-protective immune responses to liver fluke infection. In this present study we have further analysed the response of animals to liver fluke challenge following experimental vaccination. Calves were vaccinated with either CATL 2 plus FHB, or CATL 1 plus CATL 2. Partial protection against challenge infection was achieved in both vaccinated groups, with the greatest level of protection (55 per cent reduction in fluke burdens) recorded in the group vaccinated with CATL 1 plus CATL 2. This latter group also showed the greater level of lymphocyte proliferation and the greater production of gamma-INF in response to stimulation with fluke antigen in vitro following challenge. These results are significant in our attempts to characterise the elements within the immune response to vaccination which are protective.

Simple monitoring technique for muscle flaps.

A simple monitoring technique for the detection of postoperative arterial flow failure in muscle flaps is described. The technique consists of isolating a musculocutaneous perforator on elevation of a muscle flap. This cutaneous perforator is observed for pulsation in the postoperative period. Abrupt cessation prior to 48 hours should be an indicator for prompt clinical evaluation of the muscle flap by an experienced microsurgeon to rule out arterial thrombosis.

Fasciola hepatica cathepsin L proteinase cleaves fibrinogen and produces a novel type of fibrin clot.

A cathepsin L proteinase secreted by the parasitic helminth Fasciola hepatica can cleave fibrinogen and produce a fibrin clot with a specific activity of 4.7 National Institutes of Health thrombin-equivalent U/mg. This is the first report of a fibrinogen-clotting activity aside that of thrombin and the snake venom proteinases, which are all serine proteinases. Clot formation by cathepsin L is not inhibited by the thrombin inhibitor hirudin or by the anti-polymerant H-Gly-Pro-Arg-Pro-OH. The enzyme exerts its activity on fibrinogen in a unique manner. Although the cleavage of fibrinogen may involve the initial removal of fibrinopeptides, additional proteolysis of the alpha, beta and gamma fibrinogen polypeptides takes place. SDS/PAGE analysis of the cathepsin-L-produced clots revealed that cleavage of the alpha polypeptide (66 kDa) precedes that of the beta (52 kDa) and gamma (46.5 kDa) polypeptides. Concurrent with the cleavage of these polypeptides is the appearance of components of 120, 100 and 25 kDa. The appearance of higher molecular-sized components in the cathepsin L clots suggests that polymerisation involves the formation of molecular interactions that are resistant to boiling in mercaptoethanol and SDS.

Purification and characterisation of a second cathepsin L proteinase secreted by the parasitic trematode Fasciola hepatica.

A 29.5-kDa cysteine proteinase was purified from medium in which mature Fasciola hepatica parasites were maintained. The N-terminal sequence (14 residues) of the purified protein is similar to known cathepsin L proteinases, including a 27-kDa cathepsin L proteinase, also secreted by this parasite, which had been isolated previously in our laboratory [Smith, A. M., Dowd, A. J., Mc Gonigle, S., Keegan, P.S., Brennan, G., Trudgett, A. & Dalton, J.P. (1993) Mol. Biochem. Parasitol. 62, 1-8]. The N-terminal sequences of the 29.5-kDa and 27-kDa cathepsin L proteinases differ only in residue number seven (arginine and proline, respectively). Immunoblot studies, using antiserum that reacts with both cathepsin L proteinases, rule out the possibility of both enzymes arising from a higher molecular sized parent molecule. The reaction kinetics of the two F. hepatica cathepsin L proteinases on a variety of peptide substrates revealed that the two enzymes differ in their substrate specificity. Five peptide substrates that are cleaved with high affinity by the 29.5-kDa cathepsin L isolated in this study are not cleaved by the previously purified 27-kDa cathepsin L. The protein-modifying reagent, tetranitromethane, affected the 29.5-kDa cathepsin L proteinase only, causing inactivation of the enzyme and changing its migration in polyacrylamide gel electrophoresis. Our studies suggest that the two F. hepatica cysteine proteinases represent two distinct subclasses within the cathepsin L class.

Schistosoma mansoni: differential expression of cathepsins L1 and L2 suggests discrete biological functions for each enzyme.

Schistosoma mansoni cathepsins L1 (SmCL1) and L2 (SmCL2) were expressed as active recombinant proteinases in Saccharomyces cerevisiae. The recombinant enzymes exhibited substrate preferences characteristic of cathepsin-L-like cysteine proteinases. However, the enzymes differed in their substrate specificities; SmCL1 cleaved Boc-Val-Leu-Lys-NHMec with a higher efficiency than it cleaved Z-Phe-Arg-NHMec, whereas the opposite was true for SmCL2. The enzymes also differed in their pH profiles of activity; SmCL1 exhibited a broad pH profile with an optimum of pH 6. 5, while SmCL2 was active only in the acidic pH range with an optimum of 5.35. Immunoblot and RT-PCR analyses revealed that the native forms of both SmCL1 and SmCL2 are expressed in male and female worms, but at higher levels in adult female compared to male schistosomes. Additionally, both enzymes were observed in the excretory/secretory products of adult worms. The RT-PCR analysis indicated that neither enzyme is expressed in S. mansoni eggs or in miracidia, suggesting that the cathepsin-L-like activity that has been previously reported to be expressed in these stages may be the product of another gene(s). Cercariae do not express SmCL2, but appear to express SmCL1 in its inactive precursor form. Together with the findings of previous immunolocalization and phylogenetic analyses, the results reported here demonstrate that SmCL1 and SmCL2 are distinct cathepsin cysteine proteinases and strongly suggest that they play discrete biological roles.