Accuracy, reproducibility, and variability of hand-held dynamometry in motor neuron disease.

A spring-loaded device that "breaks" at preset forces was used to assess readings obtained by hand-held dynamometry by three raters with varying experience in the method. Overall accuracy (3%), but not reproducibility or variability, was improved by greater experience. Readings obtained jointly by three raters had 53% greater variability than those obtained by a single rater. Nine muscle groups in 19 patients with motor neuron disease were assessed at 10 sessions (three replications per session) over six days by the experienced rater. Muscle force was expressed relative to that of 22 matched normal controls. The reproducibility was good with a mean % difference of 13.2 and repeatability coefficient of 2.17 kg-force for readings six days apart; the overall correlation coefficient was 0.98. The mean coefficient of variation (CV) of 10 readings was 9.9%. The poorer reproducibility and greater variability seen in clinically weaker muscles may account for differences in patients with bulbar palsy and classical amyotrophic lateral sclerosis; the degree of spasticity had no effect. The rater was estimated to contribute 37% of the total variability when testing patients. The use of a composite score by combining normalised dynamometry readings of eight limb muscles improved mean % difference to 6.7 and mean CV to 5.8%. The reproducibility and variability of hand-held dynamometry readings obtained by a single rater compare well with those of fixed devices. Readings from single raters, irrespective of experience, have similar reproducibility and variability. If, however, multiple raters are used in longitudinal assessments of individual patients, as occurs in clinical trials, the variability of their combined readings should be estimated when calculating the same size required.     

Increased detection of proliferating,polyfunctional, HIV‐1‐specific T cells in DNA‐modified vaccinia virus Ankara‐vaccinated human volunteers by cultured IFN‐γ ELISPOT assay

Induction of a long‐term immunological memory, which can expand and defend the host upon pathogen encounter, is the “holy grail” of vaccinology. Here, using a sensitive cultured IFN‐γ ELISPOT assay, we show that 50% (15 out of 30) of healthy, HIV‐1/2‐uninfected volunteers who received pTHr.HIVA DNA prime‐modified vaccinia virus Ankara. HIVA boost vaccine regimen 1 to 3 1/2 years ago had detectable HIV‐1‐specific T‐cell responses. These T cells, predominantly of the CD4⁺ subtype, could proliferate and produce multiple cytokines in response to in vitro peptide stimulation. Peptide mapping studies showed that the vaccine‐induced CD4⁺ T cells were mostly directed toward epitopes targeted in HIV‐1‐infected individuals. In addition, we used the same assay to re‐evaluate 51 volunteers from past vaccine trial IAVI‐006 and corrected the previously reported 10% of vaccine responders to 50%. Thus, we confirmed that cultured assays are a valuable tool for studying T‐cell memory. These results are discussed in the context of the current state‐of‐affairs of the HIV‐1 vaccine field.     

A DNA prime-modified vaccinia virus ankara boost vaccine encoding thrombospondin-related adhesion protein but not circumsporozoite protein partially protects healthy malaria-naive adults against Plasmodium falciparum sporozoite challenge

The safety, immunogenicity, and efficacy of DNA and modified vaccinia virus Ankara (MVA) prime-boost regimes were assessed by using either thrombospondin-related adhesion protein (TRAP) with a multiple-epitope string ME (ME-TRAP) or the circumsporozoite protein (CS) of Plasmodium falciparum. Sixteen healthy subjects who never had malaria (malaria-naive subjects) received two priming vaccinations with DNA, followed by one boosting immunization with MVA, with either ME-TRAP or CS as the antigen. Immunogenicity was assessed by ex vivo gamma interferon (IFN-gamma) enzyme-linked immunospot assay (ELISPOT) and antibody assay. Two weeks after the final vaccination, the subjects underwent P. falciparum sporozoite challenge, with six unvaccinated controls. The vaccines were well tolerated and immunogenic, with the DDM-ME TRAP regimen producing stronger ex vivo IFN-gamma ELISPOT responses than DDM-CS. One of eight subjects receiving the DDM-ME TRAP regimen was completely protected against malaria challenge, with this group as a whole showing significant delay to parasitemia compared to controls (P = 0.045). The peak ex vivo IFN-gamma ELISPOT response in this group correlated strongly with the number of days to parasitemia (P = 0.033). No protection was observed in the DDM-CS group. Prime-boost vaccination with DNA and MVA encoding ME-TRAP but not CS resulted in partial protection against P. falciparum sporozoite challenge in the present study.     

Changes in ocular torsion position produced by a single visual line rotating around the line of sight--visual "entrainment" of ocular torsion

A large- or full-field visual stimulus slowly rotating around the naso-occipital axis of an observer causes both eyes to tort, and many of the factors controlling this optokinetic torsional response have been identified. The present study reports that a single line rotating about the line of sight can cause both eyes to tort in the same direction as the stimulus but with a low gain. We have used the term 'entrainment' to describe this torsional response. This paper reports some of the factors associated with entrainment. Video measures of 3-d eye position were recorded while the subject made settings of a simple visual line to subjective visual horizontal (SVH) and vertical (SVV) using the standard method-of-adjustment paradigm. The visual line was composed of 11 light-emitting diodes; the line subtended a visual angle of 19 degrees, and moved at a constant speed of 4.8 degrees /s. Settings were made in an otherwise darkened room, and also in the light. Subjects were required to maintain fixation of the central LED while making settings from starting positions 10 or 20 degrees either side of gravitational horizontal or vertical. We show that entrainment of ocular torsion by the single moving visual line is low in gain but a reliable and repeatable effect and that (1) there are considerable individual differences between subjects but within-subject consistency, (2) all subjects show larger and more consistent torsional entrainment for lines moving to SVH than lines moving to SVV, (3) the strongest entrainment generally occurs within about 10 degrees of the target position, and (4) entrainment is also present in the light, though with slightly reduced gain.     

Cellular immune responses induced in cattle by heterologous prime-boost vaccination using recombinant viruses and bacille Calmette-Guérin

The development of novel vaccine strategies to replace or supplement bacille Calmette-Guérin (BCG) is urgently required. Here we study, in cattle, the use of heterologous prime-boost strategies based on vaccination with BCG and the mycobacterial mycolyl transferase Ag85A (Rv3804c) expressed either in recombinant modified vaccinia virus Ankara (MVA85A) or attenuated fowlpox strain FP9 (FP85A). Five different vaccination schedules were tested in the first experiment: MVA85A followed by BCG (group 1); BCG followed by MVA85A (group 2); BCG followed by FP85A and then MVA85A (group 3); MVA85A followed by MVA85A and then FP85A (group 4); and FP85A followed by FP85A and then MVA85A (group 5). Vaccine-induced levels of cellular immunity were assessed by determining interferon-gamma (IFN-gamma) responses in vitro. Prime-boost protocols, using recombinant MVA and BCG in combination (groups 1-3), resulted in significantly higher frequencies of Ag85-specific IFN-gamma-secreting cells than the two viral vectors used in combination (P=0.0055), or BCG used alone (groups 2 and 3, P=0.04). The T-cell repertoires of the calves in all five groups were significantly broader following heterologous booster immunizations than after the primary immunization. In a second experiment, the effects of BCG\MVA85A heterologous prime-boost vaccination were compared with BCG\BCG homologous revaccination. The results suggested a higher Ag85A-specific response with a wider T-cell repertoire in the MVA85A-boosted calves than in the BCG\BCG-vaccinated calves. In conclusion therefore, the present report demonstrates the effectiveness of heterologous prime-boost strategies based on recombinant MVA and BCG to induce strong cellular immune responses in cattle and prioritise such vaccination strategies for rapid assessment of protective efficacy in this natural target species of tuberculosis.     

Theophylline blood levels in Sri Lankan asthmatics: comparison of two methods of assay

OBJECTIVES: To assay theophylline blood levels in a sample of Sri Lankan chronic asthmatics taking oral theophylline, and to evaluate a simple and cost effective ultraviolet spectrophotometric assay for theophylline levels in blood. SETTING: Chronic asthmatics taking oral theophylline attending medical clinics at the National Hospital of Sri Lanka (NHSL) were recruited for the study. Blood samples were collected from recruited patients on their subsequent clinic visit. DESIGN AND METHODS: A cross-sectional study of theophylline blood levels. Blood samples were assayed for trough theophylline levels using two methods: an automated homogeneous enzyme immunoassay (EMIT), and a low cost ultraviolet spectrophotometric method. RESULTS: Only 2 patients of the 24 had theophylline blood levels in the accepted therapeutic range (10 to 20 micrograms/ml) (3.4); 19 patients had levels under 5 micrograms/ml. A correlation coefficient of 0.99 was obtained in the statistical comparison of the two methods, indicating that the spectrophotometric method has similar accuracy as the reference EMIT assay. CONCLUSIONS: The results signal a need for monitoring of theophylline in asthmatics when accepted clinical indications are present. The ultraviolet spectrophotometric method is ideal to initiate therapeutic drug monitoring (TDM) in the country because of its low cost (about Rs. 55 per assay), requiring only a UV recording spectrophotometer.     

Protective immunity against Mycobacterium tuberculosis induced by dendritic cells pulsed with both CD8(+)- and CD4(+)-T-cell epitopes from antigen 85A

Immunization with DNA followed by modified vaccinia virus Ankara strain, both expressing the antigen 85A, induced both CD4(+)- and CD8(+)-T-cell responses in BALB/c mice. Following challenge with Mycobacterium tuberculosis, this prime-boost regimen produced protection equivalent to that conferred by Mycobacterium bovis BCG. Following immunization with dendritic cells pulsed with an antigen 85A CD4(+)- or CD8(+)-restricted epitope, alone or in combination, copresentation of both epitopes on the same dendritic cell was required for protection, demonstrating that induced CD8(+) T cells can play a protective role against tuberculosis.     

Feasibility of proniosomes-based transdermal delivery of frusemide: formulation optimization and pharmacotechnical evaluation

The aim of the present study was to formulate non-ionic surfactant vesicles of frusemide to enhance its skin permeation and to develop a transdermal therapeutic system using provesicular approach. The effect of various formulation variables on the transdermal flux, amount of drug deposited in skin, and plasma level of drug were studied. The skin permeation studies were conducted on rat skin and human skin for quantification of permeation parameters. With PGS3 formulation [Span 40:soyalecithin:cholesterol (4.5:4.5:1)], the plasma level in the rats had reached to a level of 0.42 +/- 0.13 microg/mL at the sampling interval of 4 hr and remained within the therapeutic concentration range (1.66-0.3 microg/mL) for the next 12 hr. Results showed that proniosomal formulation was able to sustain the drug level in the blood and offer a promising means for non-invasive delivery of frusemide.