Parastrongyloides trichosuri, a nematode parasite of mammals that is uniquely suited to genetic analysis.

Commonly studied nematode parasites have not proven amenable to simple genetic analyses and this has significantly reduced the available research options. We introduce here a nematode parasite of mammals, Parastrongyloides trichosuri, which has features uniquely suited for genetic analysis. This parasite has the capacity to undergo multiple reproductive cycles as a free-living worm and thereby amplify the numbers of its infective L3s in faeces. Culture conditions are presented that permit facile laboratory maintenance of this worm for >90 free-living life cycles (to date) without the need for re-entry into a permissive host. Even after long maintenance as a free-living worm, culture conditions can be manipulated to favour development of infective L3 worms, which remain able to successfully infect their marsupial hosts. The switch to infective L3 development is triggered by a secreted factor contained in culture medium conditioned by multiple generations of free-living worm culture. It is simple to perform single pair crosses with P. trichosuri to carry out Mendelian genetics in the laboratory and this has been done multiple times with sibling pairs to generate highly inbred lines. Lines of worms can readily be cryopreserved and recovered. Over 7000 expressed sequence tags have been produced from cDNAs at different life cycle stages and used to identify single nucleotide polymorphisms and microsatellites as genetic markers. Free-living worms live only a few days on average while the patency of parasitic infections can last for several months. Since we show this is not the result of re-infection, we conclude that parasitic worms have a lifespan capacity at least 20-30 times longer than their free-living counterparts. We discuss how it should be possible to exploit these unique features of P. trichosuri as a model for future studies that explore the genetic basis of longevity and parasitism.

Characterisation and expression of an Hsp70 gene from Parastrongyloides trichosuri.

Parastrongyloides trichosuri is a nematode parasite of Australian brushtail possums that has an alternative free-living life cycle which can be readily maintained indefinitely in a laboratory setting. The ability to maintain this parasite in a free-living cycle and induce it to parasitism at the free-living L1 stage makes this an excellent model for the study of genes associated with parasitism. A 70kD protein from infective larvae of P. trichosuri that appears to be immunogenic in infected possums has been identified as a heat shock protein (Hsp)70 homologue. The complete gene for Pt-Hsp70 was cloned and sequenced. The protein encoded by the Pt-Hsp70 gene is the likely orthologue of the Caenorhabditis elegans protein, Hsp70A, also known as hsp-1. Reverse transcriptase-PCR data indicate that Pt-Hsp70 (designated Pt-hsp-1) is expressed at readily detectable levels in all developmental stages of both the parasitic and free-living P. trichosuri life cycles and the promoter is mildly inducible by heat shock. Bioinformatic analysis of expressed sequence tag databases indicates that C. eleganshsp-1 homologues, together with C. eleganshsp-3 homologues, are the predominant members of the Hsp70 superfamily that are normally expressed in parasitic stages of the Strongyloididae family. Promoter fusions to a beta-galactosidase coding sequence were prepared and introduced into wild type C. elegans to produce transgenic nematodes. Reporter gene expression was clearly present within embryonic cells and within intestinal cells of larval and adult stages. Thus, the expression of the Pt-hsp-1 promoter within P. trichosuri and transgenic C. elegans appears similar to the known expression of C. elegans hsp-1. This promoter should be of value in efforts to develop genetic manipulation tools for P. trichosuri.

Gram-negative bacteria can induce contact lens related acute red eye (CLARE) responses.

PURPOSE: Twelve volunteers participated in a study designed to measure the overnight corneal edema response with a variety of hydrogel contact lenses. During the study four subjects (5 eyes) experienced a contact lens related acute red eye (CLARE) reaction, which manifested as severe ocular pain, photophobia, corneal infiltration, and conjunctival hyperemia. An additional five subjects (7 eyes) developed corneal infiltrates only. Twelve eyes (of 9 subjects) showed no response. METHODS AND RESULTS: Upon microbiological examination of the contact lenses and storage solutions, gram-negative bacteria were isolated in large amounts. The bacteria were identified as Serratia marcescens, Pseudomonas putida, and Pseudomonas aeruginosa. Significantly greater numbers of bacteria were isolated from contact lenses of subjects who experienced CLARE than from the other subjects (P = 0.005) and from the contact lenses of subjects who experienced an adverse reaction (CLARE or infiltrates) than from the other subjects (P < 0.001). The contaminating bacteria are thought to have been introduced to the lens storage vials as a result of lens handling and subsequent failure to disinfect lenses. CONCLUSIONS: This study draws attention to the possible contribution of contaminated lenses and storage cases in contact lens related acute inflammation and specifically implicates gram-negative bacteria, in particular Pseudomonas spp. and Serratia spp., in the inducement of acute inflammatory reactions such as CLARE.

The Dk project: an interlaboratory comparison of Dk/L measurements.

The oxygen transmissibilities (Dk/L) of a set of 48 contact lenses made from 8 different materials were measured by 4 laboratories. The L/Dk measurements from each laboratory were compared and correlated. Samples which were not masked with a fixed front surface aperture during measurement were corrected for edge effects. This paper shows that provided L/Dk is calculated for each lens using the same technique and Dk is derived using a graphical method of calculation, similar results can be obtained by all laboratories. However, the agreement was less good for materials of Dk greater than 70 x 10(-11) (cm2/s) (ml O2/ml x mm Hg).

Acute effects of inhaled isoproterenol on the mechanical characteristics of the lungs in normal man.

We investigated the effects of isoproterenol on the pulmonary mechanics of eight healthy male subjects. We measured the flow-volume, pressure-volume, resistance-volume, and pressure-flow relationships of the lungs of our subjects in addition to the forced expiratory volume (FEV(1)). The results of this study confirm earlier observations that isoproterenol produces a considerable decrease in airway resistance but only small changes in maximum expiratory flow. Measurements of static pressure-volume curves showed that isoproterenol caused a temporary decrease in the elastic recoil pressure of the lungs. In five men there were mean falls in recoil pressure of 4.1 cm H(2)O at 85% total lung capacity (TLC), 2.6 cm H(2)O at 75% TLC, and 1.5 cm H(2)O at 50% TLC. We postulate that the reason for the relatively small increments in maximum expiratory flow after isoproterenol is primarily that the effects of airway dilatation are in large part negated by the reduction in lung recoil pressure, which results in a fall in the maximum effective driving force for expiratory air flow, and secondly that there is an increase in the compliance of the flow-limiting airways. These studies emphasize that tests of maximum flow and of airway resistance should not be regarded as invariably interchangeable in the assessment of airway reactions or mild disease of the airways.

Bronchial reactivity to cigarette and cigar smoke.

The change in specific airway conductance produced by smoking a cigarette under standard conditions was measured in 91 heavy smokers. Subsequently 19 of the most reactive subjects smoked two cigarettes with different filters and another containing cigar tobacco. The results indicated that reactivity to cigarette smoke was reduced significantly by increasing the retention efficiency of the filter and that reactivity to inhaled cigar-tobacco smoke was no less than that to cigarette smoke.