A novel approach to antigen-specific deletion of CTL with minimal cellular activation using alpha 3 domain mutants of MHC class I/peptide complex

In this study, we have compared the effector functions and fate of a number of human CTL clones in vitro or ex vivo following contact with variant peptides presented either on the cell surface or in a soluble multimeric format. In the presence of CD8 coreceptor binding, there is a good correlation between TCR signaling, killing of the targets, and Fast-mediated CTL apoptosis. Blocking CD8 binding using (alpha3 domain mutants of MHC class I results in much reduced signaling and reduced killing of the targets. Surprisingly, however, Fast expression is induced to a similar degree on these CTLs, and apoptosis of CTL is unaffected. The ability to divorce these events may allow the deletion of antigen-specific and pathological CTL populations without the deleterious effects induced by full CTL activation.

A parent-completed developmental questionnaire: Follow up of ex-premature infants

Objective: premature infants are at increased risk of developmental disability. Early identification of problems allows intervention to ameliorate or attenuate problems. A reliable screening tool allows triage of children in this high-risk population by identifying those unlikely to need full developmental assessment. To explore the test characteristics of an established parent-completed developmental assessment questionnaire 'Ages and Stages Questionnaire' (ASQ) in follow up of an Australian population of premature infants. Methodology: One hundred and sixty-seven children born prematurely with corrected ages 12- to 48-months attending the Growth and Development Clinic at the Mater Children's Hospital in Brisbane, Queensland, Australia; 136 questionnaires 'ASQ' were returned completed (81%) and were compared to formal psychometric assessment (Griffith Mental Development Scales for 12- and 24-months, Bayley Mental Development Intelligence Scale for 18-months, McCarthy General Cognitive Intelligence Scale for 18-months). Developmental delay was considered to be present if any of the above psychometric assessments fell below 1.0 standard deviations (SD). The ASQ cut-off used was 2.0 SD (US data derived means and SD). Results: Aggregate results for all age groups comparing ASQ to psychometric assessments as 'gold standards' found the ASQ to have the following test characteristics: sensitivity (90%); specificity (77%); positive predictive value (40%); negative predictive value (98%): % over-referred (20%); % under-referred (1%); % agreement (79%). likelihood ratio for children failing the ASQ was 3.8 and for passing the ASQ was 0.13. Twenty-one children with known disabilities were included in the study and in 14 of these, the ASQ overall score agreed with the psychometric assessment (67%). Conclusion: The high negative predictive value of the ASQ supports its use as a screening tool for cognitive and motor delays in the follow up of ex-premature infants. This would need to be combined with other strategies as part of a comprehensive follow up program for ex-premature infants.

Lipophilic methotrexate conjugates with glucose-lipoamino acid moieties: Synthesis and in vitro antitumor activity

To obtain methotrexate (MTX) derivatives with a balanced hydrolipophilic character, we synthesized a series of conjugates in which the drug was linked to lipoamino acid (LAA)-glucose residues (LAAG-MTX). These conjugates displayed increased solubility in polar media compared with the corresponding LAA-MTX conjugates previously described. In vitro biological testing of LAAG-MTX indicated that the introduction of the sugar moiety decreased the biological activity of these MTX conjugates. The tetradecyl derivative 6b, however, was effective in inhibiting the dihydrofolate reductase activity in vitro and showed an inhibitory effect on human lymphoblastoid cell growth. (C) 2001 Wiley-Liss, Inc.

Species and regional variations in the effectiveness of antivenom against the in Vitro neurotoxicity of death adder (Acanthophis) venoms

Although viperlike in appearance and habit, death adders belong to the Elapidae family of snakes. Systemic envenomation represents a serious medical problem with antivenom, which is raised against Acanthophis antarcticus venom, representing the primary treatment. This study focused on the major Acanthophis variants from Australia and islands in the Indo-Pacific region. Venoms were profiled using liquid chromatography-mass spectrometry, and analyzed for in vitro neurotoxicity (0.3-10 mug/ml), as well as the effectiveness of antivenom. (1-5 units/ml; 10 min prior to the addition of 10 mug/ml venom). The following death adder venoms were examined: A. antarcticus (from separate populations in New South Wales, Queensland, South Australia, and Western Australia), A. hawkei, A. praelongus, A. pyrrhus, A. rugosus, A. wellsi, and venom from an unnamed species from the Indonesian island of Seram. All venoms abolished indirect twitches of the chick isolated biventer cervicis nerve-muscle preparation in a dose-dependent manner. In addition, all venoms blocked responses to exogenous acetylcholine (1 m-M) and carbachol (20 muM), but not KCl (40 mM), suggesting postsynaptic neurotoxicity. Death adder antivenom (1 unit/ml) prevented the neurotoxic effects of A. pyrrhus, A. praelongus, and A. hawkei venoms, although it was markedly less effective against venoms from A. antarcticus (NSW, SA, WA), A. rugosus, A. wellsi, and A. sp. Scram. However, at 5 units/ml, antivenom was effective against all venoms tested. Death adder venoms, including those from A. antarcticus geographic variants, differed not only in their venom composition but also in their neurotoxic activity and susceptibility to antivenom. For the first time toxicological aspects of A. hawkei, A. wellsi, A. rugosus, and A. sp. Seram venoms were studied. (C) 2001 Academic Press.

Cardiostimulant effects of urotensin-II in human heart in vitro

The effects of the recently identified human peptide urotensin-II (hU-II) were investigated on human cardiac muscle contractility and coronary artery tone. In right atrial trabeculae from non-failing hearts, hU-II caused a concentration-dependent increase in contractile force (pEC(50)=9.5+/-0.1; E-max= 31.3+/-4.8% compared to 9.25 mM Ca2+; n = 9) with no change in contraction duration. In right ventricular trabeculae from explanted hearts, 20 nM hU-II caused a small increase in contractile force (7.8+/-1.4% compared to 9.25 mM Ca2+; n= 3/6 tissues from 2 out of 4 patients). The peptide caused arrhythmic contractions in 3/26 right atrial trabeculae from 3/9 patients in an experimental model of arrhythmia and therefore has less potential to cause arrhythmias than ET-1. hU-II (20 nM) increased tone (17.9% of the response to 90 mM KCI) in 7/7 tissues from 1 patient, with no response detected in 8/8 tissues from 2 patients. hU-II is a potent cardiac stimulant with low efficacy.

New methods for the assessment of in vitro and in vivo stress cracking in biomedical polyurethanes

This article describes a new test method for the assessment of the severity of environmental stress cracking of biomedical polyurethanes in a manner that minimizes the degree of subjectivity involved. The effect of applied strain and acetone pre-treatment on degradation of Pellethane 2363 80A and Pellethane 2363 55D polyurethanes under in vitro and in vivo conditions is studied. The results are presented using a magnification-weighted image rating system that allows the semi-quantitative rating of degradation based on distribution and severity of surface damage. Devices for applying controlled strain to both flat sheet and tubing samples are described. The new rating system consistently discriminated between. the effects of acetone pre-treatments, strain and exposure times in both in vitro and in vivo experiments. As expected, P80A underwent considerable stress cracking compared with P55D. P80A produced similar stress crack ratings in both in vivo and in vitro experiments, however P55D performed worse under in vitro conditions compared with in vivo. This result indicated that care must be taken when interpreting in vitro results in the absence of in vivo data. (C) 2001 Elsevier Science Ltd. All rights reserved.

Sexual recombination in Phytophthora cinnamomi in vitro and aggressiveness of single-oospore progeny to Eucalyptus

Fifty single oospore progeny were established from an in vitro mating of A1 and A2 mating type isolates of Phytophthora cinnamomi from South Africa. Forty-nine progeny were identified as F-1 hybrids using seven random amplified polymorphic DNA (RAPD) primers, and one was a selfed isolate of the A1 mating type parent. Among the hybrid progeny, 24 and 25 were A1 and A2 mating type, respectively. Aggressiveness of progeny and parental isolates was assessed on 1-year-old seedlings of Eucalyptus smithii. The mean aggressiveness of hybrid oosporic isolates, expressed as lesion length, was significantly (P = 0.0001) lower than that of the parental isolates. No significant difference in aggressiveness of A1 and A2 mating type F-1 hybrid isolates was observed. This is the first report demonstrating sexual recombination in vitro in P. cinnamomi.

In vitro evidence for a bacterial pathogenesis of equine laminitis

Utilizing an in vitro laminitis explant model, we have investigated how bacterial broth cultures and purified bacterial proteases activate matrix metalloproteinases (MMPs) and alter structural integrity of cultured equine lamellar hoof explants. Four Gram-positive Streptococcus spp. and three Gram-negative bacteria all induced a dose-dependent activation of MMP-2 and MMP-9 and caused lamellar explants to separate. MMP activation was deemed to have occurred if a specific MMP inhibitor, batimastat, blocked MMP activity and prevented lamellar separation. Thermolysin and streptococcal pyrogenic exotoxin B (SpeB) both separated explants dose-dependently but only thermolysin was inhibitable by batimastat or induced MMP activation equivalent to that seen with bacterial broths. Additionally, thermolysin and broth MMP activation appeared to be cell dependent as MMP activation did not occur in isolation. These results suggest the rapid increase in streptococcal species in the caecum and colon observed in parallel with carbohydrate induced equine laminitis may directly cause laminitis via production of exotoxin(s) capable of activating resident MMPs within the lamellar structure. Once activated, these MMPs can degrade key components of the basement membrane (BM) hemidesmosome complex, ultimately separating the BM from the epidermal basal cells resulting in the characteristic laminitis histopathology of hoof lamellae. While many different causative agents have been evaluated in the past, the results of this study provide a unifying aetiological mechanism for the development of carbohydrate induced equine laminitis. (C) 2001 Elsevier Science B.V. All rights reserved.

Dendritic cells rapidly undergo apoptosis in vitro following culture with activated CD4(+) V alpha 24 natural killer T cells expressing CD40L

Human V alpha 24 natural killer T (V alpha 24NKT) cells are activated by -glycosylceramide-pulsed dendritic cells (DCs) in a CDld-dependent and T-cell receptor-mediated manner. There are two major subpopulations of V alpha 24NKT cells, CD4(-) CD8(-) V alpha 24NKT and CD4(+) V alpha 24NKT cells. We have recently shown that activated CD4(-) CD8 V alpha 24NKT cells have cytotoxic activity against DCs, but knowledge of the molecules responsible for cytotoxicity of V alpha 24NKT cells is currently limited. We aimed to investigate whether CD4(+) V alpha 24NKT cells also have cytotoxic activity against DCs and to determine the mechanisms underlying any observed cytotoxic activity. We demonstrated that activated CD4(+) V alpha 24NKT cells [CD40 ligand (CD40L) -positive] have cytotoxic activity against DCs (strongly CD40-positive), but not against monocytes (weakly CD40-positive) or phytohaemagglutinin blast T cells (CD40-negative), and that apoptosis of DCs significantly contributes to the observed cytotoxicity. The apoptosis of DCs following culture with activated CD4(+) V alpha 24NKT cells, but not with resting CD4(+) V alpha 24NKT cells (CD40L-negative), was partially inhibited by anti-CD40L mAb, Direct ligation of CD40 on the DCs by the anti-CD40 antibody also induced apoptosis of DCs. Our results suggest that CD40-CD40L interaction plays an important role in the induction of apoptosis of DCs following culture with activated CD4+ Va24NKT cells. The apoptosis of DCs from normal donors. triggered by the CD40-CD40L interaction, may contribute to the homeostatic regulation of the normal human immune system, preventing the interminable activation of activated CD4(+) V alpha 24NKT cells by virtue of apoptosis of DCs.

TRAIL expression by activated human CD4(+)V alpha 24NKT cells induces in vitro and in vivo apoptosis of human acute myeloid leukema cells

Human V alpha 24NKT cells are activated by alpha -galactosylceramide (alpha -GalCer)-pulsed dendritic cells in a CD1d-dependent and a T-cell receptor-mediated manner. Here, we demonstrate that CD4(+)V alpha 24NKT cells derived from a patient with acute myeloid leukemia (AML) M4 are phenotypically similar to those of healthy donors and, in common with those derived from healthy donors, express tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) when the cells are activated by alpha -GalCer-pulsed dendritic cells but not prior to activation. We also show that myeloid that human activated CD4(+)V alpha 24NKT cells induced apoptosis of human leukemia cells in vivo. This is the first evidence that activated V alpha 24NKT cells express TRAIL and that TRAIL causes apoptosis of monocytic leukemia cells from patients with AML M4 in vitro and in vivo. Adoptive immune therapy with activated V alpha 24NKT cells, or other strategies to increase activated V alpha 24NKT cells in vivo, may be of benefit to patients with AML M4.

Antagonism of the two-needle pine stem rust fungi Cronartium flaccidum and Peridermium pini by Cladosporium tenuissimum in vitro and in planta

Selected isolates of Cladosporium tenuissimum were tested for their ability to inhibit in vitro aeciospore germination of the two-needle pine stem rusts Cronartium flaccidum and Peridermium pini and to suppress disease development in planta. The antagonistic fungus displayed a number of disease-suppressive mechanisms. Aeciospore germination on water agar slides was reduced at 12, 18, and 24 h when a conidial suspension (1.5 x 10(7) conidia per ml) of the Cladosporium tenuissimum isolates was added. When the aeciospores were incubated in same-strength conidial suspensions for 1, 11, 21, and 31 days, viability was reduced at 20 and 4 degreesC. Light and scanning electron microscopy showed that rust spores were directly parasitized by Cladosporium tenuissimum and that the antagonist had evolved several strategies to breach the spore wail and gain access to the underlying tissues. Penetration occurred with or without appressoria. The hyperparasite exerted a mechanical force to destroy the spore structures (spinules, cell wall) by direct contact, penetrated the aeciospores and subsequently proliferated within them. However, an enzymatic action could also be involved. This was shown by the dissolution of the host tell wall that comes in contact with the mycelium of the mycoparasite, by the lack of indentation in the host wall at the contact site, and by the minimal swelling at the infecting hyphal tip. Culture filtrates of the hyperparasite inhibited germination of rust propagules. A compound purified from the filtrates was characterized by chemical and spectroscopic analysis as cladosporol, a known beta -1,3-glucan biosynthesis inhibitor. Conidia of Cladosporium tenuissimum reduced rust development on new infected pine seedlings over 2 years under greenhouse conditions. Because the fungus is an aggressive mycoparasite, produces fungicidal metabolites, and can survive and multiply in forest ecosystems without rusts, it seems a promising agent for the biological control of pine stem rusts in Europe.

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.