The fluorolysis assay, a highly sensitive method for measuring the cytolytic activity of T cells at very low numbers

We have developed a highly sensitive cytolysis test, the fluorolysis assay, as a simple nonradioactive and inexpensive alternative to the standard Cr-51-release assay. P815 cells were stably transfected with a plasmid expressing the enhanced green fluorescent protein (EGFP) gene. These target cells were coated with or without cognate peptide or anti-CD3 Ab and then incubated with CD8(+) T cells to allow antigen-specific or nonspecific lysis. The degree of target cell lysis was measured using flow cytometry to count the percentage of viable propidium iodide(-) EGFP(+) cells, whose numbers were standardized to a reference number of fluorochrome-linked beads. By using small numbers of target cells (200-800 per reaction) and extended incubation times (up to 2 days), the antigen-specific cytolytic activity of one to two activated CD8(+) T cells of a CTL line could be detected. The redirected fluorolysis assay also measured the activity of very few ( greater than or equal to6) primary CD8(+) T cells following polyclonal activation. Importantly, antigen-specific lysis by small numbers ( greater than or equal to 25) of primary CD8(+) T cells could be directly measured ex vivo. This exquisite sensitivity of the fluorolysis assay, which was at least 8-33-folds higher than an optimized 51 Cr-release assay, allows in vitro and ex vivo studies of immune responses that would otherwise not be possible due to low CTL numbers or frequencies. (C) 2002 Elsevier Science B.V. All rights reserved.

A clonal culture system demonstrates that IL-4 induces a subpopulation of noncytolytic T cells with low CD8, perforin, and granzyme expression

Immune deviation of cytolytic T cell function, induced by type 2 cytokines like IL-4, is an attractive concept to explain failure of the immune system in some diseases. However, this concept is challenged by previous conflicting results on whether type 2 cytokine-producing CD8(+) T cells are cytolytic. Therefore, we have analyzed the relationship between cytolytic activity and cytokine production among large numbers of primary CD8(+) T cell clones. Single murine CD8(+) T cells of naive phenotype were activated at high efficiency with immobilized Abs to CD3, CD8, and CD11a in the presence of IL-2 (neutral conditions) or IL-2, IL-4, and anti-IFN-gamma Ab (type 2-polarizing conditions) for 8-9 days. Under neutral conditions, most clones produced IFN-gamma without IL-4 and were cytolytic. Under type 2-polarizing conditions, most clones produced IFN-gamma and IL-4 but displayed variable cytolytic activity and CD8 expression. Separation on the basis of surface CD8 levels revealed that, compared with CD8(high) cells from the same cultures, CD8(low) cells were poorly cytolytic and expressed low levels of perforin mRNA and protein and granzyme A, B, and C mRNA. A similar, smaller population of noncytolytic CD8(low) cells was identified among CD8(low) T cells activated in mixed lymphocyte reaction with IL-4. Variable efficiency of generation of the noncytolytic cells may account for the differing results of earlier studies. We conclude that IL-4 promotes the development of a noncytolytic CD8(low) T cell phenotype that might be important in tumor- or pathogen-induced immune deviation.

High-performance L-band series and parallel switches using low-cost p-i-n diodes

Low-cost UHF-band p-i-n diodes are used to develop high-performance L-band series and parallel switches. To stop the rectification of large RF, signals, the diodes are biased at a large reverse-bias voltage. Parasitic elements of the diodes are tuned out using LC circuits in biasing circuits without increasing the size of the switches. (C) 2002 John Wiley Sons, Inc.

Low intraspecific variation in the rRNA internal transcribed spacer 2 (ITS2) of the Australian paralysis tick, Ixodes holocyclus

Ixodes holocyclus has a narrow, discontinuous distribution along the east coast of Australia. We studied ticks from 17 localities throughout the geographic range of this tick. The ITS2 of I. holocyclus is 793 bp long. We found nucleotide variation at eight of the 588 nucleotide positions (1.4%) that were compared for all ticks. There were eight different nucleotide sequences. Most sequences were not restricted to a particular geographic region. However, sequences F, G and H, which had an adenine at position 197, were found only in the far north of Queensland - all other ticks had a guanine at this position. The low level of intraspecific variation in this tick (0.7%) contrasts with the sequence divergence between L holocyclus and its close relative, I. cornuatus (13.1 %). These data indicate that L holocyclus does not contain cryptic species despite possible geographic isolation of some populations. We conclude that variation in the ITS2 is likely to be informative about the phylogeny of the group.

Violation of multiparticle Bell inequalities for low- and high-flux parametric amplification using both vacuum and entangled input states

We show how polarization measurements on the output fields generated by parametric down conversion will reveal a violation of multiparticle Bell inequalities, in the regime of both low- and high-output intensity. In this case, each spatially separated system, upon which a measurement is performed, is comprised of more than one particle. In view of the formal analogy with spin systems, the proposal provides an opportunity to test the predictions of quantum mechanics for spatially separated higher spin states. Here the quantum behavior possible even where measurements are performed on systems of large quantum (particle) number may be demonstrated. Our proposal applies to both vacuum-state signal and idler inputs, and also to the quantum-injected parametric amplifier as studied by De Martini The effect of detector inefficiencies is included, and weaker Bell-Clauser-Horne inequalities are derived to enable realistic tests of local hidden variables with auxiliary assumptions for the multiparticle situation.

Characterization of [Fe(AMN3S3sarH)]3+ - a rigorously low-spin iron(II) complex

The iron(II) complex [Fe(AMN(3)S(3)sarH)](ClO4)(3).3H(2)O (AMN(3)S(3)sarH = 8-ammonio-1-methyl-3,13,16-trithia-6,10,19-triazabicyclo[6.6.6]icosane) has been synthesized and characterized by single crystal structure and spectroscopic methods. The Fe(II)-S(thiaether) bond lengths are short, indicative of a large degree of metal-ligand orbital mixing (pi-acceptor character) of the thiaether ligand. The complex is stable to metal centred oxidation. (C) 2002 Elsevier Science Ltd. All rights reserved.

The relation between the transversus abdominis muscles, sacroilac joint mechanics, and low back pain

Study Design. Two abdominal muscle patterns were tested in the same group of individuals, and their effects were compared in relation to sacroiliac joint laxity. One pattern was contraction of the transversus abdominis, Independently of the other abdominals; the other was a bracing action that used all the lateral abdominal muscles. Objectives. To demonstrate the biomechanical effect of the exercise for the transversus abdominis known to be effective in low back pain. Summary of Background Data. Drawing in the abdominal wall is a specific exercise for the transversus abdominis muscle (in cocontraction with the multifidus), which is used in the treatment of back pain. Clinical effectiveness has been demonstrated to be a reduction of 3-year recurrence from 75% to 35%. To the authors' best knowledge, there is not yet in vivo proof of the biomechanical effect of this specific exercise. This study of a biomechanical model on the mechanics of the sacroiliac joint, however, predicted a significant effect of transversus abdominis muscle force. Methods. Thirteen healthy individuals who could perform the test patterns were included. Sacroiliac joint laxity values were recorded with study participants in the prone position during the two abdominal muscle patterns. The values were recorded by means of Doppler Imaging of vibrations. Simultaneous electromyographic recordings and ultrasound imaging were used to verify the two muscle patterns. Results. The range of sacroiliac joint laxity values observed in this study was comparable with levels found in earlier studies of healthy individuals. These values decreased significantly in all individuals during both muscle patterns (P < 0.001). The independent transversus abdominis contraction decreased sacroiliac joint laxity (or rather increased sacroiliac joint stiffness) to a significantly greater degree than the general abdominal exercise pat-tern (P < 0.0260). Conclusions. Contraction of the transversus abdominis significantly decreases the laxity of the sacroiliac joint. This decrease in laxity is larger than that caused by a bracing action using all the lateral abdominal muscles. These findings are in line with the authors' biomechanical model predictions and support the use of independent transversus abdominis contractions for the treatment of low back pain.

Use of high and low velocity cervical manipulative therapy procedures by Australian manipulative physiotherapists

The use of cervical manipulation presents concerns because of a risk of devastating side effects of trauma to the vertebral artery. Little is known about the frequency of use of cervical manipulation versus passive mobilisation by physiotherapists. A recent national, multi-centre randomised clinical trial of the physiotherapy management of cervicogenic headache provided an opportunity to gain an insight into practices of a sample of manipulative physiotherapists across Australia. The treatment records for the 100 subjects who received only manipulative therapy, or manipulative therapy with exercise as per the trial protocol, were audited. The results revealed that cervical manipulation was used in 20.2% of the 1090 treatments provided to these subjects but cervical joint mobilisation only was used in the vast majority of treatments (77.6%). Nevertheless, 42% of subjects were treated with cervical manipulation at some time. In most instances, manipulation was accompanied by passive mobilisation in the same treatment session. Patients were manipulated on one to six occasions and this occurred predominantly in the latter half of the 12-treatment program. Cervical manipulation was used less frequently in the group who also received exercise. The data suggest that the physiotherapists participating in this study used cervical manipulation selectively and relatively conservatively considering the high use of cervical mobilisation techniques. This may reflect their due regard to safety in the treatment of the cervical region.

Nutritional strategies for the prevention of hypocalcaemia at calving for dairy cows in pasture-based systems

This review considers the current literature on the macro-mineral nutrition of the soon-to-calve, or transition, dairy cow. Calcium is the main focus, since milk fever (clinical hypocalcaemia) appears to be the most common mineral-related problem faced by the transition cow Australia-wide. The importance of minimising calcium intake and optimising the balance of the key dietary electrolytes, sodium, potassium, sulfate, and chloride, in the weeks before calving is highlighted. Excess dietary potassium can, in some situations, induce milk fever, perhaps even more effectively than excess calcium. Excess sodium remains under suspicion. In contrast, excess dietary chlorine and, to a lesser extent, sulfur can improve the ability of the cow to maintain calcium homeostasis. Diets that promote either a hypomagnesaemia or hyperphosphataemia have also the potential to precipitate milk fever at calving. Current prevention strategies focus on the use of forages with moderate to low levels of calcium, potassium, and sodium, and also rely on or utilise addition of chloride and sulfate in the form of 'anionic' feeds. Anionic salts are one example of an anionic feed. However, legitimate questions remain as to the effectiveness of anionic salts in pasture-feeding systems. The causes and prevention of milk fever are considered from the perspective of the variety of Australian feedbases. Impediments to the use of anionic feeds in Australia feeding systems are outlined. The potential for improving maternal reserves of calcium around calving to reduce the risk of milk fever is also discussed.

Dietary chromium tripicolinate supplementation reduces glucose concentrations and improves glucose tolerance in normal-weight cats

The effect of dietary chromium supplementation on glucose and insulin metabolism in healthy, non-obese cats was evaluated. Thirty-two cats were randomly divided into four groups and fed experimental diets consisting of a standard diet with 0 ppb (control), 150 ppb, 300 ppb, or 600 ppb added chromium as chromium tripicolinate. Intravenous glucose tolerance, insulin tolerance and insulin sensitivity tests with minimal model analysis were performed before and after 6 weeks of feeding the test diets. During the glucose tolerance test, glucose concentrations, area under the glucose concentration-time curve, and glucose half-life (300 ppb only), were significantly lower after the trial in cats supplemented with 300 ppb and 600 ppb chromium, compared with values before the trial. Fasting glucose concentrations measured on a different day in the biochemistry profile were also significantly lower after supplementation with 600 ppb chromium. There were no significant differences in insulin concentrations or indices in either the glucose or insulin tolerance tests following chromium supplementation, nor were there any differences between groups before or after the dietary trial. Importantly, this study has shown a small but significant, dose-dependent improvement in glucose tolerance in healthy, non-obese cats supplemented with dietary chromium. Further long-term studies are warranted to determine if the addition of chromium to feline diets is advantageous. Cats most likely to benefit are those with glucose intolerance and insulin resistance from lack of exercise, obesity and old age. Healthy cats at risk of glucose intolerance and diabetes from underlying low insulin sensitivity or genetic factors may also benefit from long-term chromium supplementation. (C) 2002 ESFM and AAFP.

Accurate orientation relationship between ferrite and austenite in low carbon martensite and granular bainite

Convergent beam Kikuchi diffraction was used to accurately determine the orientation relationships (ORs) between austenite and martensite, and between austenite and granular bainite in two Fe-Ni-Mn-C alloys. Both martensite and granular bainite have the same crystallographic characteristics with the OR: (111)(A)parallel to(101)(F), [1 (1) over bar0](A) 2.5degrees +/- 2degrees from [1 (1) over bar(1) over bar](B).

Relationship between stress-induced martensitic transformation and impact toughness in low carbon austenitic steels

The effect of test temperature, which controls the stability of austenite, on the impact toughness of a low carbon Fe-Ni-Mn-C austenitic steel and 304 stainless steel, has been investigated. Under impact conditions, stress-induced martensitic transformation occurred, in a region near the fracture surface, at test temperatures below 80degreesC for the Fe-Ni-Mn-C steel and below -25degreesC for 304 stainless steel. The former shows significant transformation toughening and the highest impact toughness was obtained at 10degreesC, which corresponds to the maximum amount of martensite formed by stress-induced transformation above the Ms temperature. The stress-induced martensitic transformation contributes negatively to the impact toughness in the 304 stainless steel. Increasing the amount of stress-induced transformation to martensite, lowered the impact toughness. The experimental results can be well explained by the Antolovich theory through the analysis of metallography and fractography. The different effect of stress-induced transformation on the impact toughness in Fe-Ni-Mn-C steel and 304 stainless steel has been further understood by applying the crystallographic model for stress-induced martensitic transformation to these two steels. (C) 2002 Kluwer Academic Publishers.