Characterization of lymphotoxin-alpha beta complexes on the surface of mouse lymphocytes.

The lymphotoxin-alpha beta complex (LT alpha beta) is found on the surface of activated lymphocytes and binds to a specific receptor called the LT beta receptor (LT beta R). In the mouse, signaling through this pathway is important for lymph node development and splenic organization, yet the biochemical properties of murine LT alpha and LT beta are essentially unknown. Here we have used soluble receptor-Ig forms of LT beta R and TNF-R55 and mAbs specific for murine LT alpha, LT beta, and LT beta R to characterize the appearance of surface LT alpha beta complexes and LT beta R on several common murine cell lines. Cells that bound LT beta R also bound anti-LT alpha and anti-LT beta mAbs in a FACS analysis. The ability of these reagents to discriminate between surface TNF and LT was verified by analysis of surface TNF-positive, LPS-activated murine RAW 264.7 monocytic cells. Primary mouse leukocytes from spleen, thymus, lymph node, and peritoneum were activated in vitro, and CD4+ and CD8+ T cells as well as B cells expressed surface LT ligand but not the LT beta R. Conversely, elicited peritoneal monocytes/macrophages were surface LT negative yet LT beta R positive. This study shows that on mononuclear cells, surface LT complexes and receptor are expressed similarly in mice and man, and the tools described herein form the foundation for study of the functional roles of the LT system in the mouse.

Cytotoxic activities of recombinant soluble murine lymphotoxin-alpha and lymphotoxin-alpha beta complexes.

Human lymphotoxin-alpha (LT alpha) is found in a secreted form and on the surface of lymphocytes as a complex with a second related protein called lymphotoxin-beta (LT beta). Both secreted human LT alpha and TNF have similar biological activities mediated via the TNF receptors, whereas the cell surface LT alpha beta complex binds to a separate receptor called the LT beta receptor (LT beta R). The murine LT alpha and LT beta (mLT alpha and mLT beta) proteins have never been characterized. When recombinant mLT alpha was produced by either of several methods, the protein had a very low specific activity relative to that of human LT alpha in the conventional WEHI 164 cytotoxicity bioassay. The weak activity observed was inhibited by a soluble murine TNF-R55 Ig fusion protein (mTNF-R55-Ig), but not by mLT beta R-Ig. Coexpression of both mLT alpha and a soluble version of mLT beta in insect cells led to an LT alpha beta form that was cytotoxic in the WEHI 164 assay via the LT beta R. To determine whether natural mLT alpha-like forms with cytotoxic activity comparable to that of secreted human LT alpha were secreted from primary spleen cells, splenic lymphocytes were activated in various ways, and their supernatants were analyzed for cytotoxic activity. Using specific Abs to distinguish between mTNF and mLT, a TNF component was readily detected; however, there was no evidence for a secreted mLT alpha cytotoxic activity using this assay. Combined, these observations suggest that secreted mLT alpha may not play a role in the mouse via interactions with TNF-R55, and the ramifications of this hypothesis are discussed.

Preparation and characterization of soluble recombinant heterotrimeric complexes of human lymphotoxins alpha and beta.

The lymphotoxin (LT) protein complex is a heteromer of alpha (LT-alpha, also called tumor necrosis factor (TNF)-beta) and beta (LT-beta) chains anchored to the membrane surface by the transmembrane domain of the LT-beta portion. Both proteins belong to the TNF family of ligands and receptors that regulate aspects of the immune and inflammatory systems. The LT complex is found on activated lymphocytes and binds to the lymphotoxin-beta receptor, which is generally present on nonlymphoid cells. The signaling function of this receptor-ligand pair is not precisely known but is believed to be involved in the development of the peripheral lymphoid organs. To analyze the properties of this complex, a soluble, biologically active form of the surface complex was desired. The LT-beta molecule was engineered into a secreted form and co-expressed with LT-alpha using baculovirus/insect cell technology. By exploiting receptor affinity columns, the LT-alpha3, LT-alpha2/beta1, and LT-alpha1/beta2 forms were purified. All three molecules were trimers, and their biochemical properties are described. The level of LT-alpha3-like components in the LT-alpha1/beta2 preparation was found to be 0.02% by following the activity of the preparation in a WEHI 164 cytotoxicity assay. LT-alpha3 with an asparagine 50 mutation (D50N) cannot bind the TNF receptors. Heteromeric LT complexes were prepared with this mutant LT- alpha form, allowing a precise delineation of the extent of biological activity mediated by the TNF receptors. A LT-alpha3 based cytotoxic activity was used to show that the LT-alpha1/beta2 form cannot readily scramble into a mixture of forms following various treatments and storage periods. This biochemical characterization of the LT heteromeric ligands and the demonstration of their stability provides a solid foundation for both biological studies and an analysis of the specificity of the LT-bet a and TNF receptors for the various LT forms.

Metabolism of xenobiotics by Beauveria bassiana.

1. Diazepam, warfarin and testosterone were metabolized by whole resting cells of the fungus Beauveria bassiana IMI 12939 via oxidative reactions such as hydroxylation and N-demethylation. 2. Metabolism of each substrate was inhibited by the cytochrome P450 inhibitors SKF-525A and metyrapone, consistent with the involvement of this enzyme system in the metabolism of these drugs by B. bassiana. 3. Substrate concentration-dependent inhibition was observed during diazepam metabolism by this organism, as has been observed in some mammalian systems. 4. Unlike most mammalian P450 systems, the warfarin-metabolizing activity of B. bassiana could not be induced by growing the organism in the presence of phenobarbitone, beta-naphthoflavone, 3-methylcholanthrene, 1-benzylimidazole or warfarin. 5. Overall findings indicate that B. bassiana possesses an oxidative metabolizing system capable of producing metabolites found in mammalian systems.

Effect of 3'-azido-3'-deoxythymidine (AZT) on organic ion transport in rat renal brush border membrane vesicles.

3'-Azido-3'-deoxythymidine (AZT), a nucleoside analog which has potent activity against the acquired immunodeficiency virus, is actively secreted by the mammalian kidney. In order to study the mechanism of renal drug transport, the effect of AZT on the organic cation and organic anion transport systems in rat renal brush border membrane vesicles was examined by using a rapid filtration assay. The following prototypic substrates were used: [3H]N1-methylnicotinamide and [3H]tetraethylammonium for organic cations and p-[3H]aminohippurate for an organic anion. AZT inhibited pH-driven [3H]N'-methylnicotinamide transport (pHi = 6.0, pH0 = 7.5), but not as effectively as mepiperphenidol (MEPI), a known organic cation transport blocker; the corresponding IC50 values for AZT and MEPI were 2500 and 25 microM, respectively. Counterflow studies, which examined the capability of the drug to cross the plasma membrane, indicated that [3H] tetraethylammonium and MEPI trans-stimulated [3H]tetraethyl-ammonium uptake, but AZT did not. To clarify further the actions of AZT on the organic cation transporter, kinetic studies were undertaken. A Hanes-Woolf transformation of the data revealed that both AZT and MEPI inhibited [3H]N'-methylnicotinamide transport in a competitive manner. The specificity of competition was studied by looking at the effect of AZT on the organic anion transporter. Probenecid, a classical inhibitor of organic anion transport, blocked p-[3H]aminohippurate transport, but AZT did not. We conclude that AZT is a weak inhibitor of the renal brush border organic cation transport system.

The screening of selected microorganisms for use as models of mammalian drug metabolism.

Fifty fungi and two Streptomyces species were screened for their ability to metabolise the probe substrates aminopyrine, diazepam, testosterone, theophylline and warfarin. The metabolism of the 14C-labelled substrates by whole growing cells was compared with that by rat liver microsomes using TLC-autoradiography. Testosterone, warfarin and diazepam were readily metabolised by most microorganisms, and aminopyrine and theophylline were only metabolised by a few. A relationship between substrate lipophilicity and number of microorganisms able to biotransform the substrate was observed, lipophilic substrates being favoured for metabolism, analagous to mammalian cytochrome P-450. A wide variety of metabolites were produced by the screened cultures, with a significant number co-chromatographing with mammalian metabolites. Most microorganisms appeared to exhibit cytochrome P-450-type oxidative reactions such as hydroxylation and N-demethylation, similar to mammalian hepatic microsomal cytochrome P-450 systems.

Interaction of 3'-azido-3'-deoxythymidine with organic ion transport in rat renal basolateral membrane vesicles.

3'-Azido-3'-deoxythymidine (AZT), a nucleoside analog effective against the acquired immunodeficiency syndrome virus, is actively secreted by rat, rabbit and human kidney. The mechanism of AZT transport across the basolateral membrane was characterized by examining the effect of AZT on organic cation and organic anion transport systems in rat renal basolateral membrane vesicles (BLMV) by using a rapid filtration assay. The following prototypic substrates were used: N1-[3H]methylnicotinamide and [3H]tetraethylammonium (TEA) for organic cations and p-[3H]aminohippurate (PAH) for an organic anion. AZT was an effective inhibitor of PAH transport. The dose-response curves for AZT and probenecid, an organic anion inhibitor, revealed IC50 values of 225 and 15 microM, respectively. To clarify further the actions of AZT at the organic anion transporter, counterflow studies were performed. Preloading BLMV with AZT trans-stimulated the uptake of PAH. The specificity of transport was assessed by examining the effect of AZT on organic cation transport. AZT did not inhibit uptake of NMN or TEA (pHin = pHout = 7.5). However, AZT slightly inhibited uptake of TEA under optimized transport conditions (1 mM TEA load). We conclude that AZT transport in rat BLMV is mediated predominantly by the renal organic anion transport system which is consistent with the capability of an organic anion to reduce the renal clearance of AZT in vivo.

Vermicomposting in the management of pig-waste in Hong Kong.

The treatment and disposal of pig-waste in Hong Kong has received much attention in recent years but, following any of the presently used treatment processes, solids remain to be further stabilized. Vermicomposting is a waste stabilization technique which converts waste into potentially recyclable materials such as worm protein and worm casts. The earthworm, Pheretima asiatica, can stabilize most of the solids arising from the treatment of pig-waste, including raw pig manure, suggesting that vermicomposting has a high potential as a unit process in the management of pig-waste in Hong Kong.

Biochemical investigation of tissue growth: towards definition of "standard" tissue samples.

This paper presents an animal model [the kangaroo], a quantitative anatomical dissection procedure, and a mathematical model [two-phase linear regression] which illustrate that body tissues grow at varying rates relative to each other. An argument is developed that biochemists interested in tissue chemical activity need to be able to sample tissue of known [predicted] growth rate. It is assumed that the ability to select, say muscle tissue samples, from any one animal at a stage of its growth where the individual selected pieces of tissue have known [predicted] low, average and high growth rates would allow comparisons to be made between the sampled tissues that may elucidate the underlying biochemical mechanisms involved in the growth process. It is asserted that to establish standards for tissue samples used in biochemical growth studies, the growth rate of the sampled tissue should be one of the criteria incorporated into the definition of what is "standard" for a tissue sample.

Skin neoplasms of dogs in Sydney.

In a survey of dogs in Sydney, mastocytomas (16.1%) and histiocytomas (14.0%) were the most common in a total of 1,000 skin neoplasms. The basal cell and appendage group provided 25.5% of the neoplasms. The prevalence of the various neoplasms, the age of affected dogs, the proportion in the sexes, the common sites of occurrence and prevalence in the different breeds were broadly similar to findings in surveys in other countries, except that in the Syndeny dogs there was a greater prevalence of histiocytomas and haemangiopericytomas, a more common occurrence of histiocytomas in mature dogs, an occurrence of histiocytomas in similar numbers on the head, trunk and limbs, and a remarkably common development of squamous cell carcinomas in Dalmatians.

A genetic model describing the evolution of levamisole resistance in Trichostrongylus colubriformis, a nematode parasite of sheep.

Data from 21 generations of selection on a levamisole-resistant strain of Trichostrongylus colubriformis, either exposed to selection with the anthelmintics levamisole (LEV) or thiabendazole (TBZ), or unexposed, were used to fit a genetic model describing the evolution of LEV resistance in this parasite species. A statistical model describing the dose-response relationship for a mixed population of susceptible and resistant parasite eggs exposed to anthelmintic was fitted to egg-hatch assay data for each generation and for each selection regimen. Estimated parameters from the statistical model provided the input for the genetic model from which were obtained estimates of the relative fitness of susceptible and resistant genotypes under each selection regimen. The experimental data and the genetic models both indicated that, in this parasite strain, LEV resistance was determined by a single dominant gene, and that TBZ selects for LEV susceptibility. A variety of drug alternation programmes was simulated for this genetic system. The programme that minimized the development of LEV resistance involved alternating the drugs (LEV and TBZ) between each worm generation.

Storage mites culturing, sampling technique, identification and their role in housedust allergy in rural areas in the United Kingdom.

Patients with symptoms suggesting housedust mite allergy were tested to storage mites, housedust, and Dermatophagoides spp. Housedust was examined for mites. All patients responded to storage mites. Some were negative to Dermatophagoides. All houses proved to have storage mites in the dust as well as Dermatophagoides, mainly in food stores or pet beds. Storage mites, therefore, are involved in dust allergy.

The protection given by pilus and whole cell vaccines of Bacteroides nodosus strain 198 against ovine foot-rot induced by strains of different serogroups.

A highly purified pilus vaccine prepared from cells of Bacteroides nodosus strain 198 provided a high level of protection against homologous challenge and small, not statistically significant, levels of protection against challenge with 4 other strains each from different serogroups. In a second experiment, a partially purified pilus vaccine from strain 198 induced significant immunity to 1 of 4 heterologous strains which were different from those used in the first experiment. In a third experiment a strain 198 whole cell vaccine produced significant immunity against 3 of 6 heterologous strains used in the first 2 experiments. There was no obvious relationship between the colony type, degree of piliation and level of cross-protection obtained against a particular strain. The results provide further evidence that immunogens associated with, but distinct from, the pilus are involved in cross-protection and that cross-protective antigens are common to some, but not all, strains.

Housedust mites (D. pteronyssinus) in pets' beds and their relation to dust allergy.

Houses were surveyed by dust sampling to examine the occurrence and significance of storage mites in rural areas in the U.K. As a part of this project pets' beds were sampled, and it was discovered that not only do they contain storage mites, but that in nearly all instances the classical housedust mite (D. pteronyssinus) was present in significant numbers.

Tardive dyskinesia: fluctuating patient or fluctuating rater.

Tardive dyskinesia (TD) is usually described as fluctuating in its clinical manifestations. We attempted to quantify fluctuations in TD using the Abnormal Involuntary Movement Scale (AIMS). Three psychiatrists rated multiple video-recorded examinations of four outpatients with mild TD. The unexpected finding was that within-rater variability dominated within-patient variability.

Effect of pilus dose and type of Freund's adjuvant on the antibody and protective responses of vaccinated sheep to Bacteroides nodosus.

Groups of sheep were immunised twice with one or other of six vaccines consisting of purified pili from Bacteroides nodosus at three dose levels (10, 38 and 154 micrograms) and emulsified with either complete (CFA) or incomplete Freund's adjuvant (IFA). Beginning one month after vaccination the sheep were homologously challenged on irrigated pasture, with naturally transmitted foot rot for a period of 26 weeks. Statistical analyses of the number of feet per sheep with severe foot rot demonstrated that there was a significant effect of vaccinal dose but neither an adjuvant effect nor an interaction between dose and adjuvant. Similar conclusions were reached when the titres of antipilus agglutinins in the serum were analysed. By both criteria the responses to doses of 154 and 38 micrograms of pili were significantly better than to 10 micrograms, but not significantly different from each other. The IFA vaccines caused less reaction at the sites of injection than the CFA vaccines and within the former the vaccines containing 10 and 38 micrograms pilus produced less reaction than those containing 154 micrograms. Hence a vaccine containing 38 micrograms of purified pili in IFA is nearly optimal for homologous protection against severe foot rot and is acceptable in terms of the reaction at the injection site.

Importance of pilus-associated antigen in Bacteroides nodosus vaccines.

The performances of two cellular vaccines, one sparsely piliated and the other well piliated, were compared on irrigated pasture with those of vaccines containing their respective, purified pili. There were statistically significant differences among the four vaccines in the mean numbers of feet per sheep which developed severe foot rot during 27 weeks of exposure. The incidence of severe foot rot was significantly lower in the two pili-vaccinated groups than in the group vaccinated with well-piliated cells, which were in turn substantially more effective than the sparsely piliated. However, the two pili vaccines did not prevent infection of the interdigital skin so that the differences in vaccine performance were much less marked when interdigital skin lesions were included. The number of feet affected by blowfly strike was closely associated with the presence of the most severe lesions and consequently there were far fewer struck feet in the two pili vaccine groups than in the two cellular vaccine groups. Analysis of serum agglutinin titres led to the same assessment of vaccinal efficacy as that derived from the analysis of severe foot rot lesions. Individually the agglutinin response to vaccination was not universally associated with resistance or susceptibility to severe foot rot but mean titres were significantly higher in the two pili vaccine groups than in the highly piliated-cell vaccine group, which were all in turn significantly higher than in the poorly piliated-cell vaccine group.

Allometry and multivariate growth revisited.

Turner (1978) introduced a class of deterministic models to describe multivariate growth processes along with a least squares fitting procedure. Turner's fitting procedure is shown to be ill founded and the methods of inference associated with it dangerously misleading. An alternative class of stochastic models is introduced; this is largely based on Turner's generalization of allometry and the Lotka-Volterra equations. The associated fitting procedure is the method of maximum likelihood which allows valid inferences to be made. As an example of the superiority of this new class of models in describing multivariate growth processes one of the data sets analysed by Turner is reconsidered here. Some general comments are made on Turner's analysis of the famous lynx and hare data.