Preclinical assessment of an anti-HTLV-1 heterologous DNA/MVA vaccine protocol expressing a multiepitope HBZ protein.

BACKGROUND: Human T-lymphotropic virus 1 (HTLV-1) is associated with the development of several pathologies and chronic infection in humans. The inefficiency of the available treatments and the challenge in developing a protective vaccine highlight the need to produce effective immunotherapeutic tools. The HTLV-1 basic leucine zipper (bZIP) factor (HBZ) plays an important role in the HTLV-1 persistence, conferring a survival advantage to infected cells by reducing the HTLV-1 proteins expression, allowing infected cells to evade immune surveillance, and enhancing cell proliferation leading to increased proviral load. METHODS: We have generated a recombinant Modified Virus Vaccinia Ankara (MVA-HBZ) and a plasmid DNA (pcDNA3.1(+)-HBZ) expressing a multiepitope protein based on peptides of HBZ to study the immunogenic potential of this viral-derived protein in BALB/c mice model. Mice were immunized in a prime-boost heterologous protocol and their splenocytes (T CD4+ and T CD8+) were immunophenotyped by flow cytometry and the humoral response was evaluated by ELISA using HBZ protein produced in prokaryotic vector as antigen. RESULTS: T CD4+ and T CD8+ lymphocytes cells stimulated by HBZ-peptides (HBZ42-50 and HBZ157-176) showed polyfunctional double positive responses for TNF-α/IFN-γ, and TNF-α/IL-2. Moreover, T CD8+ cells presented a tendency in the activation of effector memory cells producing granzyme B (CD44+High/CD62L-Low), and the activation of Cytotoxic T Lymphocytes (CTLs) and cytotoxic responses in immunized mice were inferred through the production of granzyme B by effector memory T cells and the expression of CD107a by CD8+ T cells. The overall data is consistent with a directive and effector recall response, which may be able to operate actively in the elimination of HTLV-1-infected cells and, consequently, in the reduction of the proviral load. Sera from immunized mice, differently from those of control animals, showed IgG-anti-HBZ production by ELISA. CONCLUSIONS: Our results highlight the potential of the HBZ multiepitope protein expressed from plasmid DNA and a poxviral vector as candidates for therapeutic vaccine.

Sperm phospholipase Czeta from humans and cynomolgus monkeys triggers Ca2+ oscillations, activation and development of mouse oocytes.

Fusion with a fertilizing spermatozoon induces the mammalian oocyte to undergo a remarkable series of oscillations in cytosolic Ca(2+), leading to oocyte activation and development of the embryo. The exact molecular mechanism for generating Ca(2+) oscillations has not been established. A sperm-specific zeta isoform of phospholipase C (PLCzeta) has been identified in mice. Mouse PLCzeta triggers Ca(2+) oscillations in mouse oocytes and exhibits properties synonymous with the 'sperm factor' that has been proposed to diffuse into the oocyte after gamete fusion. The present study isolated the PLCzeta homologue from human and cynomolgus monkey testes. Comparison with mouse and monkey PLCzeta protein sequences indicates a shorter X-Y linker region in human PLCzeta and predicts a distinctly different isoelectric point. Microinjection of complementary RNA for both human and cynomolgus monkey PLCzeta elicits Ca(2+) oscillations in mouse oocytes equivalent to those seen during fertilization in mice. Moreover, human PLCzeta elicits mouse egg activation and early embryonic development up to the blastocyst stage, and exhibits greater potency than PLCzeta from monkeys and mice. These results are consistent with the proposal that sperm PLCzeta is the molecular trigger for egg activation during fertilization and that the role and activity of PLCzeta is highly conserved across mammalian species.

Patient-dependent beam-modifier physics in Monte Carlo photon dose calculations.

Model pencil-beam on slab calculations are used as well as a series of detailed calculations of photon and electron output from commercial accelerators to quantify level(s) of physics required for the Monte Carlo transport of photons and electrons in treatment-dependent beam modifiers, such as jaws, wedges, blocks, and multileaf collimators, in photon teletherapy dose calculations. The physics approximations investigated comprise (1) not tracking particles below a given kinetic energy, (2) continuing to track particles, but performing simplified collision physics, particularly in handling secondary particle production, and (3) not tracking particles in specific spatial regions. Figures-of-merit needed to estimate the effects of these approximations are developed, and these estimates are compared with full-physics Monte Carlo calculations of the contribution of the collimating jaws to the on-axis depth-dose curve in a water phantom. These figures of merit are next used to evaluate various approximations used in coupled photon/electron physics in beam modifiers. Approximations for tracking electrons in air are then evaluated. It is found that knowledge of the materials used for beam modifiers, of the energies of the photon beams used, as well as of the length scales typically found in photon teletherapy plans, allows a number of simplifying approximations to be made in the Monte Carlo transport of secondary particles from the accelerator head and beam modifiers to the isocenter plane.

Correlated histogram representation of Monte Carlo derived medical accelerator photon-output phase space.

We present a method for condensing the photon energy and angular distributions obtained from Monte Carlo simulations of medical accelerators. This method represents the output as a series of correlated histograms and as such is well-suited for inclusion as the photon-source package for Monte Carlo codes used to determine the dose distributions in photon teletherapy. The method accounts for the isocenter-plane variations of the photon energy spectral distributions with increasing distance from the beam central axis for radiation produced in the bremsstrahlung target as well as for radiation scattered by the various treatment machine components within the accelerator head. Comparison of the isocenter energy fluence computed by this algorithm with that of the underlying full-physics Monte Carlo photon phase space indicates that energy fluence errors are less than 1% of the maximum energy fluence for a range of open-field sizes. Comparison of jaw-edge penumbrae shows that the angular distributions of the photons are accurately reproduced. The Monte Carlo sampling efficiency (the fraction of generated photons which clear the collimator jaws) of the algorithm is approximately 83% for an open 10x10 field, rising to approximately 96% for an open 40x40 field. Data file sizes for a typical medical accelerator, at a given energy, are approximately 150 kB, compared to the 1 GB size of the underlying full-physics phase space file.

Monte Carlo calculations to characterize the source for neutron therapy facilities.

Modern radiation treatment planning for photons includes full 3D modeling of the adsorbed dose distribution, accurate inclusion of the patient anatomy, and consideration of significant changes in material density and composition. Such efforts are founded in an accurate description of the radiation source and the beam delivery system. Modern fast neutron therapy facilities employ highly penetrating beams and isocentric beam delivery. Treatment planning is largely based on analytic models adapted from photon codes and interaction cross sections normalized to macroscopic attenuation. However, the recent PEREGRINE initiative at Lawrence Livermore Laboratory offers the possibility of fully stochastic modeling if the neutron source can be adequately described. In this article we report neutron source modeling of three high energy facilities. Neutron production is based on the intra-nuclear cascade model of the LAHET code while neutron transport through the beam delivery system is managed by MCNP using cross section libraries extended to 100 MeV neutron energy. PEREGRINE is then used to transport the neutron beam through typical phantoms. The resulting neutron sources are in excellent agreement with the limited experimental information and the measured phantom data are well described by the PEREGRINE transport using the LAHET/MCNP determined neutron sources.

Food safety training for nutritionists.

A course on food safety for nutritionists has been developed in Indonesia through collaboration between government, industry, academia and international agencies. By teaching the basic principles of the subject it equips the participants to recommend foods that are safe as well as nutritious.

Optimization of haemolysis in enhanced haemolysis agar (EHA)--a selective medium for the isolation of Listeria monocytogenes.

The presence of Listeria monocytogenes in enrichment media can be masked by faster growth of other Listeria spp. Therefore, enhanced haemolysis agar (EHA) is a good alternative for another isolation media, because the presence of a few L. monocytogenes colonies can be detected in a majority of colonies of other listeriae on the basis of haemolysis. In this study the haemolysis reaction in EHA was optimized. In a collaborative study using reference samples, no significant differences in counts on EHA, Palcam and Oxford agar were shown.

Measurement of proinsulin and intermediates. Validation of immunoassay methods by high-performance liquid chromatography.

Human proinsulin and 32-33 split proinsulin have been measured in the peripheral circulation by immunoradiometric assays (IRMAs) and have been shown to be elevated in impaired glucose tolerance and non-insulin-dependent diabetes mellitus (NIDDM). The IRMA for 32-33 split proinsulin did not discriminate between this molecule and des-32 or des-31,32 split proinsulin. We describe the comparison of IRMA for human plasma proinsulin and 32-33 split proinsulins with assays combined with high-performance liquid chromatography (HPLC), which can discriminate between 32-33 split, des-32 split, and des-31,32 split proinsulin. Subjects were those with normal glucose tolerance (n = 8) and those with NIDDM (n = 17), who were studied while fasting and 30 min after a glucose load. After collection, blood was centrifuged promptly, and the serum/plasma was stored frozen until assay. Both IRMA and HPLC methods were calibrated against synthetic peptides. Interassay coefficients of variation for the IRMA for proinsulin and 32-33 split proinsulin were < 13% over the ranges 3.8-65 pmol/l and 6.4-65 pmol/l, respectively. The following regression lines were obtained: proinsulin IRMA = -0.143 + 1.066 HPLC, r = 0.860; 32-33 split proinsulin IRMA = 0.048 + 1.051 HPLC; and des-31,32 split proinsulin, r = 0.814. For both analytes, there was no significant difference in the relationship of IRMA to HPLC results between the various subject groups and various time points. Thus, the IRMA for proinsulin has been validated by an independent method.(ABSTRACT TRUNCATED AT 250 WORDS)

Undiagnosed glucose intolerance in the community: the Isle of Ely Diabetes Project.

The Isle of Ely Diabetes Project is a prospective population-based study of the aetiology and pathogenesis of Type 2 diabetes mellitus. Between 1990 and 1992, 1156 subjects aged between 40 and 65 years underwent a standard 75 g oral glucose tolerance test (OGTT). A total of 1122 individuals who were not known to have diabetes completed the test and were classified according to WHO criteria; 51 subjects (4.5%) had previously undiagnosed diabetes and 188 (16.7%) had impaired glucose tolerance. The subjects with newly diagnosed glucose intolerance were significantly older, more obese, and shorter than those with normal glucose tolerance. Blood pressure, cholesterol, triglyceride, and LDL-cholesterol concentrations were elevated and HDL-cholesterol levels were lower among those with abnormal rather than normal glucose tolerance. In multiple regression analyses stratified by gender and including age, body mass index, and the waist-hip ratio as covariates, there were significant differences between those with normal and abnormal glucose intolerance in blood pressure, triglyceride, and HDL-cholesterol, but not total or LDL-cholesterol. In both male and female subjects, height had a significant independent negative association with the plasma glucose at 120 min after administration of oral glucose (standardized beta coefficient = -0.12, p < 0.01).

Hypertriglyceridaemia in subjects with normal and abnormal glucose tolerance: relative contributions of insulin secretion, insulin resistance and suppression of plasma non-esterified fatty acids.

Although plasma insulin and triglyceride concentrations are positively correlated in many studies, the relationships between insulin resistance, insulin secretion and hypertriglyceridaemia remain unclear. To study these associations, subjects between the ages of 40 and 64 were randomly selected from a general practice register and invited to attend for a standard oral glucose tolerance test for measurement of insulin, triglyceride and non-esterified fatty acid concentrations. The study comprised 1122 subjects who were not previously known to have diabetes and who completed the test. Using the World Health Organisation criteria, 51 subjects were classified to have non-insulin-dependent diabetes mellitus, 188 had impaired glucose tolerance and 883 subjects had normal glucose tolerance. Triglyceride concentrations in subjects with glucose intolerance were elevated compared to those in control subjects, even after adjustment for age, obesity and gender (p < 0.001 for subjects with diabetes and p < 0.01 for those with impaired glucose tolerance compared to normal subjects). In separate multiple regression analyses for males and females, the most important determinants of the plasma triglyceride concentration were the area under the non-esterified fatty acid suppression curve (p < 0.001 in both genders) and the waist-hip ratio (p < 0.001 for men and < 0.01 for women). The fasting insulin concentration was independently associated with triglyceride concentration in women only (p < 0.01). The most important determinant of the area under the non-esterified fatty acid suppression curve in men was the 30-min insulin increment, a measure of insulin secretion, (p < 0.001) whereas for women age (p < 0.001) and the body mass index (p < 0.01) were the most important.

Effect of exogenous proline, betaine, and carnitine on growth of Listeria monocytogenes in a minimal medium.

Three Listeria monocytogenes strains isolated from food or food-processing environments were used to assess the response of this species to salinity in a chemically defined minimal medium. Growth in a minimal medium containing five essential amino acids and glucose as a carbon and energy source was comparable to growth in a rich medium (brain heart infusion broth). In the absence and presence of 3% NaCl the final cell numbers reached in minimal medium were 10(9) and 10(7) CFU/ml, respectively. Growth under the latter conditions could not be detected by spectrophotometry by measuring A660. Apparently, this technique was not suitable for these experiments since the detection level was > 10(7) CFU/ml. Exogenously added proline (10 mM), trimethylglycine (betaine) (1 mM), and beta-hydroxy-gamma-N-trimethyl aminobutyrate (carnitine) (1 mM) significantly stimulated growth under osmotic stress conditions in minimal medium at both 37 and 10 degrees C. Betaine and carnitine are present in foods derived from plants and animals, respectively. Therefore, these compounds can contribute significantly to growth of L. monocytogenes in various foods at high osmolarities.

Lithium chloride-sodium propionate agar for the enumeration of bifidobacteria in fermented dairy products.

Lithium chloride-sodium propionate agar has been developed for the enumeration of bifidobacteria in fermented dairy products. The medium contains lithium chloride and sodium propionate to inhibit the growth of other lactic acid bacteria. Pure cultures of bifidobacteria, lactobacilli, and streptococci were tested for growth in this medium. With one exception, all bifidobacteria were able to grow in this medium and in a nonselective agar with a difference not exceeding .4 log units. However, none of the lactobacilli tested and only one strain each of Streptococcus salivarius ssp. thermophilus and Lactococcus lactis ssp. cremoris grew in lithium chloride-sodium propionate agar. In those cases, the numbers of colonies were lower in lithium chloride-sodium propionate agar by 1.26 and 2.51 log units, respectively, compared with a nonselective agar. Bifidobacteria were also selectively isolated from all fermented milks and cheeses analyzed.

Fetal and infant growth and impaired glucose tolerance at age 64.

OBJECTIVE: To discover whether reduced fetal and infant growth is associated with non-insulin dependent diabetes and impaired glucose tolerance in adult life. DESIGN: Follow up study of men born during 1920-30 whose birth weights and weights at 1 year were known. SETTING: Hertfordshire, England. SUBJECTS: 468 men born in east Hertfordshire and still living there. MAIN OUTCOME MEASURES: Fasting plasma glucose, insulin, proinsulin, and 32-33 split pro-insulin concentrations and plasma glucose and insulin concentrations 30 and 120 minutes after a 75 g glucose drink. RESULTS: 93 men had impaired glucose tolerance or hitherto undiagnosed diabetes. They had had a lower mean birth weight and a lower weight at 1 year. The proportion of men with impaired glucose tolerance fell progressively from 26% (6/23) among those who had weighted 18 lb (8.16 kg) or less at 1 year to 13% (3/24) among those who had weighed 27 lb (12.25 kg) or more. Corresponding figures for diabetes were 17% (4/23) and nil (0/24). Plasma glucose concentrations at 30 and 120 minutes fell with increasing birth weight and weight at 1 year. Plasma 32-33 split proinsulin concentration fell with increasing weight at 1 year. All these trends were significant and independent of current body mass. Blood pressure was inversely related to birth weight and strongly related to plasma glucose and 32-33 split proinsulin concentrations. CONCLUSIONS: Reduced growth in early life is strongly linked with impaired glucose tolerance and non-insulin dependent diabetes. Reduced early growth is also related to a raised plasma concentration of 32-33 split proinsulin, which is interpreted as a sign of beta cell dysfunction. Reduced intrauterine growth is linked with high blood pressure, which may explain the association between hypertension and impaired glucose tolerance.

Impedimetric determination of activity of disinfectants and detergents on Listeria: preliminary study.

The efficiency of several disinfectants or detergents against three strains of Listeria monocytogenes, one strain of Listeria innocua and two strains of Streptococcus group D was tested in water as well as in the presence of milk, whey and salt by an impedimetric method using a Bactometer M120. Certain synergistic effects between active agents and matrix could be observed. Differences in sensitivities were noticed amongst the tested strains. Products containing iodine, peroxide or quaternary ammonium as active agents were shown to be efficient, even at relatively low concentrations.