Protocol: Using N-of-1 tests to identify responders to melatonin for sleep disturbance in Parkinson's disease.

BACKGROUND: 40% of Parkinson's Disease (PD) sufferers experience insomnia, impacting health and quality of life for patients and family members, especially carers. There is little evidence that current treatments are effective. OBJECTIVES: To determine the effectiveness of melatonin in reducing insomnia in 44 individuals with PD using N-of-1 trials. To aggregate group data to arrive at population estimates of effectiveness (measured by improvements in PDSS-2) and safety (measured by adverse events) of melatonin in improving insomnia in PD. To assess the feasibility of offering N-of-1 trials for insomnia in PD. METHODOLOGY: Participants will receive either immediate-release melatonin or placebo in random order in 3 paired two-week treatment periods (12 weeks total). Based on their response in a two-week run-in period on 3 mg daily, they will trial either 3 mg or 6 mg. Patients will keep daily sleep diaries and wear a MotionWatch throughout. After the trial patients will discuss their individual report with their doctor, which provides direct feedback about effectiveness and safety of melatonin for them. STATISTICAL METHODS: We will analyse N-of-1 tests 1) individually: effects of melatonin on PDSS-2 and safety will be reported; and 2) aggregated across individual N-of-1 studies, combined using a Bayesian multilevel random effects model, which will account for repeated measures on individuals over time, and will return posterior estimates of overall treatment effect, and effect in each individual. CLINICAL TRIAL REGISTRATION NUMBER: ACTRN12617001103358.

Auckland Stroke Outcomes Study. Part 1: Gender, stroke types, ethnicity, and functional outcomes 5 years poststroke.

BACKGROUND: Studying long-term stroke outcomes including body functioning (neurologic and neuropsychological impairments) and activity limitations and participation is essential for long-term evidence-based rehabilitation and service planning, resource allocation, and improving health outcomes in stroke. However, reliable data to address these issues is lacking. METHODS: This study (February 2007-December 2008) sourced its participants from the population-based incidence study conducted in Auckland in 2002-2003. Participants completed structured self-administered questionnaires, and a face-to-face interview including a battery of neuropsychological tests. Logistic regression analysis was used to analyze associations between and within functional outcomes and their potential predictors. RESULTS: Of 418 5-year stroke survivors, two-thirds had good functional outcome in terms of neurologic impairment and disability (defined as modified Rankin Score <3), 22.5% had cognitive impairment indicative of dementia, 20% had experienced a recurrent stroke, almost 15% were institutionalized, and 29.6% had symptoms suggesting depression. Highly significant correlations were found between and within various measurements of body functioning (especially neuropsychological impairments), activity, and participation. Age, dependency, and depression were independently associated with most outcomes analyzed. CONCLUSIONS: The strong associations between neuropsychological impairment and other functional outcomes and across various measurements of body functioning, activity, and participation justify utilizing a multidisciplinary approach to studying and managing long-term stroke outcomes. Observed gender and ethnic differences in some important stroke outcomes warrant further investigations.

Auckland Stroke Outcomes Study. Part 2: Cognition and functional outcomes 5 years poststroke.

BACKGROUND: Understanding the extent of long-term neuropsychological deficits poststroke and their contribution to functional outcomes is essential for evidence-based rehabilitation and resource planning, and could improve stroke outcomes. However, most existing neuropsychological stroke data are not population-based, examine limited outcomes, and have short-term follow-up. METHODS: This population-based long-term stroke follow-up study examined associations between neuropsychological deficits (memory, executive function, information processing speed [IPS], visuoperceptual/construction ability, language), depression, and a range of functional outcomes and their interrelationships 5 years poststroke. RESULTS: The greatest proportion of the 307 participants exhibited neuropsychological functioning within the average range, and about 30%-50% performed at lower levels on most measures; few performed above the average range. Deficits were most common in executive functioning and IPS, and 30.4% of participants were depressed. While correlation analyses indicate all cognitive domains are significantly related to functional outcomes, multiple regression analyses showed that only IPS and visuoperceptual ability made significant independent contributions to functional outcomes over and above age, depression, and current Barthel Index. Depression also made a significant and independent contribution to functional outcomes. CONCLUSION: A considerable proportion of 5-year stroke survivors experience neuropsychological deficits, with these being more likely to involve IPS and executive functioning. Visuoperceptual/construction abilities, visual memory, and IPS were independently associated with handicap, disability, and health-related quality of life over and above contributions made by age, depression, and stroke severity, suggesting these areas are important targets for rehabilitation to improve overall stroke recovery and should be evaluated in future randomized controlled trials.

Continuous affinity-based selection: rapid screening and simultaneous amplification of bacterial surface-display libraries.

A new method for continuous biopanning has been developed. We have combined the power of affinity chromatography with the fecundity of bacteria in a unique process that mimics clonal selection. Mixed populations of bacteria were applied to a fermenter containing the immobilized ligand of interest. Bacteria retained in this affinity fermenter were allowed to grow under continuous washout conditions, such that weakly bound organisms were selectively lost. Those initially rare founder bacteria expressing a receptor for the immobilized ligand (R+ve) were thus enriched and amplified simultaneously. From an initial culture containing 1 x 10(10) R-ve cells spiked with fewer than 30 R+ve bacteria (<1 in 10(8)), final ratios of R+ve/R-ve bacteria as high as 1 in 12 were observed, representing an enrichment factor of 55 million-fold. This technology has considerable potential for rapid screening of bacterial surface-display libraries and in facilitating directed-evolution studies.

Functional protective role for mucin glycosylated repetitive domains.

Mucins carry out a number of protective roles, some of which are more easily studied than others. One mucin function is believed to be the protection of the mucosal epithelium against acidic and proteolytic damage in the stomach and intestines. In the present work, a portion of stomach mucin tandem repeat sequence (Muc6) was joined to the catalytic domain of a reporter enzyme [human milk cholesterol esterase (CE)] to determine whether the former can protect the latter protein from damage. This Muc6 domain replaced a unique series of glycosylated C-terminal repeats normally present in CE. The chimeric protein (CE/Muc6) was expressed in two different cell lines and its properties compared to recombinant full-length CE and a truncated version of CE which contained only the catalytic domain (CE/trunc). Results showed that both CE and CE/Muc6 were resistant to denaturation by acid and to proteolysis by pepsin at low pH values or by pancreatic proteases compared to CE/trunc. Thus, a stomach Muc6 domain is sufficient to confer stability on the CE catalytic domain, demonstrating a protective effect by a glycosylated mucin sequence.

Bile salt activation of human cholesterol esterase does not require protein dimerisation.

Human milk cholesterol esterase (bile salt-activated lipase) plays a role in the dietary uptake of triacylglyceride and cholesteryl ester. The activities toward these substrates are mediated through a unique bile salt-activated mechanism. Previously, it has been proposed that a necessary step in this process is prior protein dimerisation in the presence of primary bile salts. In this study, we addressed the role of protein dimerisation by investigating bile salt interactions on full length and truncated recombinant forms, as analysed by size exclusion chromatography and concanavalin A Sepharose binding experiments. The present findings demonstrate that protein dimerisation is not an obligatory component of the bile salt-activated pathway. A new functional role for the glycosylated C-terminal domain in cholesterol esterase is also demonstrated in the prevention of non-specific hydrophobic interactions.

Neuropeptides and general neuronal marker in psoriasis--an immunohistochemical study.

Nerve fibres immunoreactive to antibodies to vasoactive intestinal polypeptide (VIP) and substance P (SP) were increased in lesional psoriatic skin when assessed semiquantitatively. Biopsies from psoriatic plaques on the arm were studied in 13 patients and compared with biopsies from non-lesional areas (in three of the same psoriatic subjects) and from normal skin in seven non-psoriatic controls. Immunohistochemical methods were used on cryocut skin sections to demonstrate the neuropeptides SP, VIP, calcitonin gene-related peptide and neuropeptide Y, and the general neuronal marker protein gene product (PGP) 9.5. The immunofluorescence was examined by semiquantitative and, for PGP 9.5, by quantitative methods. VIP reactive nerve fibres were increased at areas of eccrine sweat glands throughout the dermis, at the dermo-epidermal junction, and in the epidermis, in psoriasis lesional skin. SP reactive nerve fibres were increased at the dermo-epidermal junction, where the nerves ran parallel with and perpendicularly through the junction. PGP 9.5 reactive nerve fibres showed an increase at the dermo-epidermal junction, in the papillary dermis, and at the eccrine sweat glands in lesional psoriatic skin but not in non-lesional, or in control skin. These findings support the hypothesis that neuropeptides may be involved in the pathogenesis of psoriasis.

Neuropeptide and neuronal marker studies in vitiligo.

Neuropeptide and neuronal marker immunoreactivity was studied in skin biopsies from lesional and marginal areas in 12 patients with vitiligo, and in seven normal controls. The vitiligo was active in seven, static in two, and of unknown activity in three. Antibodies against general neuronal marker PGP 9.5 (PGP 9.5), substance P (SP), calcitonin gene-related peptide (CGRP), vasoactive intestinal polypeptide (VIP), and neuropeptide Y (NPY), were used. The epidermis, dermo-epidermal junction, papillary and reticular dermis, and appendages, were assessed semiquantitatively for reactivity with each antibody. Staining with PGP 9.5 in the upper dermis was assessed quantitatively by image analysis. An increase in reactivity against NPY antibody was seen in five of 10 cases (three with active vitiligo) in the marginal areas, and in three of 12 subjects (all with active vitiligo) in the lesional vitiligo areas. VIP antibody reactivity showed a minimal increase in the marginal and lesional vitiligo areas (in two cases each, both of whom had active vitiligo). SP and CGRP reactivities did not differ from normal. PGP 9.5 staining was minimally increased at the dermo-epidermal junction and lower Malpighian layer in biopsies from marginal areas in three of 10 subjects (all with active vitiligo). Quantitative analysis of PGP 9.5 reactivity in the upper dermis showed no difference between vitiligo and normal biopsies. These findings support the concept of neuronal or neuropeptide involvement in vitiligo, and in particular suggest that NPY may have a role in the pathogenesis of the disease.

The in vitro properties of dermal papilla cell lines established from human hair follicles.

The in vitro properties of cells cultured from the dermal papilla of human hair follicles were studied and compared with those of lines of dermal fibroblasts derived from the same material. In serial subcultures, the dermal papilla cells displayed a spread out, polygonal cellular morphology at stationary growth phases and a tendency to form multi-layered aggregates before reaching confluence. Aggregation was particularly marked when papilla cells were grown on collagen gels. In contrast, dermal fibroblasts grew as branching, parallel arrays of spindle-shaped cells which remained as monolayers until confluence. Compared with dermal fibroblasts, papilla cells also exhibited a shorter in vitro survival time. The properties of cultured human papilla cells are similar to those of rat vibrissa papilla cells.

Calmodulin activation of adenylate cyclase in the mouse B16 melanoma.

Calmodulin antagonists inhibited hormone-stimulated cyclic AMP accumulation in both cultured cells and cell lysates of mouse B16 melanoma. Particulate preparations of B16 melanoma contained 34-45% of total cell calmodulin, which could not be dissociated by extensive washing irrespective of the presence of EGTA in the buffer. The adenylate cyclase activity in such preparations was unaffected by the addition of exogenous calmodulin. However, the rare-earth-metal ion La3+, which can mimic or replace Ca2+ in many systems, produced an immediate inhibition of agonist-stimulated adenylate cyclase activity and preincubation of particulate preparations was La3+ followed by washing with La3+-free buffer dissociated calmodulin (96% loss) from particulate preparations. The loss of calmodulin from particulate preparations was associated with a decrease in agonist responsiveness (74%) and a marked change in the Ca2+-sensitivity of the enzyme, low concentrations of calcium (approx. 10 nM) now failing to stimulate enzyme activity, high concentrations of calcium (greater than or equal to 100 nM) producing greater-than-normal inhibition of enzyme activity. Direct activation of adenylate cyclase by the addition of pure calmodulin was now demonstrable in such calmodulin-depleted particulate preparations. Half-maximal stimulation of agonist-responsive adenylate cyclase occurred at 80 nM-calmodulin in the presence of 10 microM free Ca2+. Maximal stimulation by calmodulin (at 300-600 nM) restored enzyme activity to 89 +/- 5% (mean +/- S.E.M., n = 7) of the activity in untreated, calmodulin-intact, preparations.

Effects of extracellular calmodulin and calmodulin antagonists on B16 melanoma cell growth.

Two drugs known to inhibit the action of calmodulin, prochlorperazine offP) and N-(6-aminohexyl)-5-chloro-1-napthalene sulfonamide (W7), were investigated for their ability to control cell proliferation in murine B16 melanoma cells in culture. PCP and W7 inhibited [3H]thymidine uptake in these cells, 50% inhibition occurring with 13 microM PCP and 40 microM W7. In the presence of relatively high concentrations of fetal calf serum (FCS), cells withstood high concentrations of both drugs (100 microM PCP and 200 microM W7) and showed increased pigment production. Drug-inhibited DNA synthesis could be reversed by the addition of fresh medium containing FCS or by the addition of exogenous pure calmodulin. Extracellular calmodulin itself stimulated DNA synthesis. FCS was found to contain calmodulin-like activity at concentrations that may be relevant to the stimulation of [3H]thymidine uptake by cells in culture.

Calmodulin activation of cyclic AMP phosphodiesterase in the B16 mouse melanoma.

Mouse B16 melanoma extracts of both cultured cells and tumour tissue contain cyclic AMP phosphodiesterase activity, with 95% present in the soluble fraction. Although activation of the enzyme by added calmodulin did not occur, it was found that endogenous calmodulin was present at a level sufficient to activate fully the enzyme. The ability of Ca-calmodulin to stimulate cyclic AMP phosphodiesterase in this tissue was shown by the inhibitory effect of N-(6-aminohexyl)-5-chloronaphthalenesulphonamide (W7), a known calmodulin antagonist; by the activation of the enzyme with exogenous calmodulin observed in supernatants depleted of endogenous calmodulin by passage over fluphenazine-Sepharose 6B in the presence of Ca2+; by the Ca-dependent binding of the enzyme to calmodulin-agarose and its activation by Ca-calmodulin after elution from the column with EGTA-containing buffer. It was calculated that about 50% of the total cyclic AMP phosphodiesterase activity was calmodulin-activated in this tissue.