Eosinophilia in nude rats and nude mice after infection with Fasciola hepatica or injection with its E/S antigens.

Peripheral blood and bone marrow eosinophilia occurred in nude rats and nude mice following F. hepatica infection, with the magnitude of the response in nude rats greater than that in nude mice. Injection of E/S antigens induced eosinophilia in nude rats and a limited eosinophilia in nude mice. Bone marrow eosinophilia was greatly enhanced in Fasciola-infected nude rats, particularly 14 days after infection and later. In nude mice, bone marrow eosinophilia developed soon after infection and persisted for the duration of the experiment, but was not as marked as in nude rats. Bone marrow eosinophils in antigen-injected animals were also elevated, and again this was more marked in nude rats. The number of colonies formed in agar culture from bone marrow cells of both nude rats and nude mice also increased following infection and remained significantly elevated throughout the experiment. Bone marrow colonies in antigen-injected nude rats increased on day 8, while in injected nude mice, the number of colonies rose rapidly following injection with antigens. Thus, nude rats and nude mice develop T-cell-independent eosinophilia, which appears to originate in the bone marrow. The magnitude of eosinophilia is greater in nude rats and it has yet to be determined whether these effects have any relevance to the ability of rats, but not mice, to develop resistance to reinfection with F. hepatica.

Glutathione S-transferases as markers of salicylanilide resistance in isolates of Fasciola hepatica.

A possible link between the level of glutathione S-transferase (GST, E.C. 2.5.1.18) activity and the development of salicylanilide resistance in Fasciola hepatica was investigated. Various isolates of F. hepatica with varying susceptibilities to salicylanilides were isolated and maintained in the laboratory. Individual flukes of these isolates were surveyed for their level of GST activity and a correlation between the level of GST activity and drug efficacy was found. In contrast to most other studies, a decrease in GST activity was associated with an increase in drug resistance. Evidence was collected to show that this may be a selective process since flukes which had survived exposure to rafoxanide and closantel in vivo (in sheep) had lower activity levels of GST than flukes from untreated sheep. Treatment with other flukicides (oxyclozanide, luxabendazole and triclabendazole) did not have this effect. Furthermore, in vivo treatment with closantel induced selection of particular isoenzymes in different isolates of F. hepatica having different degrees of susceptibility to closantel. However, no single isoenzyme or isoenzyme profile was associated with resistance and, in total, up to 8 different isoenzymes could be present in a single isolate. Thus, GST has some potential as a marker enzyme for salicylanilide resistance in F. hepatica. However, the precise role of GST in resistance is unclear and the extensive inter- and intra-isolate variation in activity levels and isoenzyme characteristics of this enzyme indicate the need for considerably more study before application in field situations.

Eosinophil differentiation in response to Fasciola hepatica and its excretory/secretory antigens.

Bone marrow cells from mice infected with Fasciola hepatica, from mice injected with F. hepatica excretory/secretory (ES) antigens, and from uninfected or uninjected control animals were cultured in the presence of F. hepatica ES antigens or the eosinophil differentiation cytokine IL-5. Eosinophil maturation in cultures was assessed quantitatively by measuring eosinophil peroxidase (EPO) activity and qualitatively by visual appraisal in stained preparations over a week. It was found that the presence in all cultures (including those from control animals) of either ES antigens at an optimal concentration of 100 micrograms ml-1 (established in preliminary trials) or IL-5 at 500 units ml-1 led to enhanced EPO activity. EPO activity in cultures without IL-5 or ES antigens remained static or fell over the culture period. At day 3 in all cultures containing IL-5 or ES antigens, there was maintenance of or only a slight decline in, the number of eosinophils that were present when cultures were initiated, and more of them were mature than at day 0 as evidenced by their EPO activity. However, there was a marked fall in eosinophil numbers in all cultures in the absence of IL-5 or ES antigens. The results indicate that F. hepatica ES antigens, like IL-5, stimulate eosinophil maturation in bone marrow with a consequent rise in EPO activity in the cells. Whether the antigen(s) acts directly or indirectly on the eosinophils or their precursors has yet to be established. Nevertheless, it seems clear that F. hepatica produces a molecule with a functionally similar effect to that of IL-5.

Host effects on glutathione S-transferase activity in Fasciola hepatica.

Glutathione S-transferases (GST, E.C. 2.5.1.18) in Fasciola hepatica from sheep were previously found to be extremely variable with regard to specific GST activity and isoenzyme profile within and between parasite isolates. The effect of the host on GST activity and isoenzyme profile was examined by infecting mice, rats and cattle as well as sheep with one or the other of two isolates--either salicylanilide-resistant or salicylanilide-susceptible F. hepatica. In the case of both isolates, GST activity in hosts relatively resistant to reinfection--rats and cattle--was lower and more restricted in range compared with hosts susceptible to multiple infection--mice and sheep. In the case of the rat flukes, there was little variation in isozyme profiles whereas cattle flukes appeared to exhibit more variation than sheep flukes. In mice, despite the apparent variability in GST activity, only one GST band was found in the isoenzyme profiles. Therefore, the host appears to exert a pronounced effect on the activity and expression of GSTs in F. hepatica which may be related to variation in the immune responses of the different hosts during infection.

The surface coat of bloodstream forms of Trypanosoma musculi from mice.

Iodination and immunoprecipitation techniques together with indirect fluorescent antibody tests identified two polypeptides (SP) of molecular weights 88,000-92,000 and 66,000-70,000 in the surface coat of bloodstream forms of the mouse trypanosome, Trypanosoma musculi. As parasites multiply and enter the early plateau phase of infection the 88,000-92,000 SP is present while the 66,000-70,000 SP is only detectable after the mid-plateau phase. Western blotting of parasite extracts showed that the 88,000-92,000 SP was present throughout the course of infection, but it appears to become masked by the 66,000-70,000 SP or possibly immunoglobulin from about 16 days after infection. Based on results when Western blots of parasite extracts were probed with antibodies affinity purified against the 88,000-92,000 SP, the two SP appear to be immunologically related and the smaller may be a cleavage product of the larger. This would explain why affinity purified antibodies to each SP bound to trypanosomes collected 8 days after infection, when only the 88,000-92,000 is detectable in parasite extracts. However, the failure of antibodies affinity purified against the 66,000-70,000 SP to bind to the 88,000-92,000 SP in Western blots suggests that the smaller SP has some epitopes that are immunologically distinct from those of the larger SP.

Eosinophil responses to Fasciola hepatica in rodents.

Qualitative and quantitative cellular changes in the peripheral blood and bone marrow of resistant (rat) and susceptible (mouse) hosts of Fasciola hepatica have been examined. Eosinophil numbers in the peripheral blood and bone marrow of both hosts increased almost immediately following infection. Rats responded more rapidly than mice. Bone marrow colony formation in both rats and mice was greatly enhanced following F. hepatica infection. Injection of excretory/secretory (E/S) antigens of the fluke into rats and mice caused peripheral eosinophilia. Eosinophil levels in mice dropped by day 7 post-injection, but those in rats remained high. Eosinophil precursors in the bone marrow of injected animals also rose. Bone marrow colony formation in antigen-injected mice peaked sharply at day 7 but then fell rapidly. Rats injected with E/S antigens had about twice the level of bone marrow colonies as controls, 12 days post-injection. For most parameters measured, the magnitude of the responses of rats was greater than mice, which may be significant in the context of the rat's ability to acquire resistance to reinfection.

Characterization of cloned antigens of Taenia ovis.

Fusion proteins derived from recombinant clones of Escherichia coli carrying the pEX series of expression vectors with cDNA inserts of the tapeworm, Taenia ovis, were characterized by their antigenicity when administered to mice and sheep. Antisera derived from these hosts were shown to react with a number of T. ovis adult worm and oncospheral antigens in Western blots, and to bind to oncospheres and worm tissue in fluorescent antibody tests. Epitopes produced by two clones (designated T07 and T08) are found on several antigenic polypeptides of T. ovis and a related species T. hydatigena. Those produced by a third clone (T03) are specific to T. ovis. The immunological results were confirmed by Northern blotting of T. ovis and T. hydatigena RNA: RNA transcripts corresponding to T07 and T08 are found in both T. ovis and T. hydatigena, whereas RNA corresponding to T03 is found in T. ovis only. Cross reacting epitopes in fusion proteins from T07 and T08 appear to have different amino acid sequences, and T. ovis antigenic epitopes are shared by a number of polypeptides in and between adult worms and oncospheres.

Glutathione S-transferases in Fasciola hepatica.

Glutathione S-transferases (GST's) are widespread in the tissues of the liver fluke, Fasciola hepatica, and consist of multiple isozymes. Following purification to apparent homogeneity by affinity chromatography on glutathione agarose, fluke GST's were shown to comprise 2 components with molecular weights of about 25,000. Fluke GST's were immunogenic to rats, but when used as a vaccine conferred no protection on the animals against a challenge infection with F. hepatica metacercariae.

A simple enzymic method for the measurement of magnesium in serum and urine on a centrifugal analyser.

A method is described based on a reaction that requires magnesium-ATP as a co-factor for the activity of hexokinase (HK), coupled with glucose 6-phosphate dehydrogenase (G6PDH). Glucose and NADP are converted to D-gluconolactone 6-phosphate and NADPH, respectively. The rate of increase in absorbance at 340 nm due to the formation of NADPH is proportional to the magnesium concentration in the sample. Magnesium levels in serum and urine measured by the enzymic method compared well with those obtained by atomic absorption spectrometry. The within-batch precisions were 1.4% and 1.5% for the enzymic method and the atomic absorption method, respectively for a quality assurance sample with a magnesium concentration of 2 mmol/L. The enzymic method is accurate (recoveries of added magnesium to serum samples are 101-102%), reproducible (between batch CV 2.8%), and rapid (23 samples may be measured in 10 min). Data on accuracy, precision and correlation for the enzymic and atomic absorption methods are presented.

Cloning and expression of Taenia ovis antigens in Escherichia coli.

Double stranded DNA complementary to poly(A)+ mRNA from the tapeworm Taenia ovis was cut with Sau 3A to an average length of about 300 bp and inserted into the Bam HI site of the expression plasmids pEX1, pEX2 and pEX3. These plasmids express a hybrid protein derived from a fusion of the cro gene with the lac Z gene (truncated at its 5' end by 53 bp) of phage lambda. Cloning sites lie downstream from the gene fusion. Escherichia coli infected with another plasmid (pCI857) bearing the temperature sensitive repressor of phage lambda was transformed with the pEX plasmids into which T. ovis DNA had been inserted; recombinants were selected by growth at 30 degrees C in the presence of ampicillin at 100 micrograms ml-1. Replicas were made and hybrid protein expression induced in recombinants by transferring them to 42 degrees C. Several recombinants expressing antigenic determinants of T. ovis were detected with T. ovis infected sheep serum that had been absorbed to remove antibodies to E. coli. Of five selected for further study, three expressed hybrid proteins of between 165 and 170 kDa of which the T. ovis component contributed between 48 and 55 kDa; in the other two, the tapeworm contribution was between 0.5 and 1.5 kDa. These antigenic determinants may be of some interest with respect to vaccine development since they are expressed during the normal course of T. ovis infection in sheep, and they are also present in the oncosphere - the infective larva of the parasite which stimulates immunity in sheep. The native antigens in adult worms and oncospheres that correspond to the antigenic determinants produced by the recombinant clones comprise a number of species ranging from 92.5 to 180 kDa. Tests with affinity purified antibodies indicate that the expressed products of the clones represent different epitopes on the same subset of polypeptides in both adult worms and oncospheres.

Paired associate learning in reading-disabled children: evidence for a rule-learning deficiency.

Two experiments examined whether normal and disabled readers differed in the utilization of rules in a paired associate learning task. Experiment 1 required children to learn symbol-word associates. Children were assigned to one of three conditions: nonrule, consistent rule, or inconsistent rule. When present, the rule was based on semantic opposites. Subjects benefited from having the rule, but disabled readers showed less improvement across four test blocks than both chronological age (CA) controls and reading age (RA) controls, particularly in the inconsistent condition. Experiment 2 required subjects to learn symbol-symbol associations in one of three conditions: nonrule, consistent rule, or inconsistent rule. When present, the rule specified the locations of a subsidiary figure in each symbol according to the pattern top-right, bottom-left. Disabled and normal readers did not differ in the nonrule condition where reliance on visual memory would be an effective strategy. Normal readers were superior to disabled readers in both rule conditions. In addition, disabled readers in the inconsistent rule condition were less able than normal readers to apply the rule in a generalization task where memory demands were reduced. Results supported the hypothesis that disabled readers have greater difficulty than normal readers inducing and/or using rules, particularly when they are inconsistent. It is suggested that difficulties in acquiring or using complex and inconsistent rules may be one important source of problems learning spelling-sound correspondence rules, which in English are complex and inconsistent.

Immunoprecipitation of in vitro translation products of Fasciola hepatica RNA.

Incubation of in vitro translation products of RNA isolated from Fasciola hepatica with immune rat serum and sera from infected sheep resulted in the immunoprecipitation of a number of polypeptides, most of which had similar electrophoretic mobilities. Although only one of these (a 30 kDa species) correlated precisely in mobility with that of biosynthetically labelled excretory-secretory (ES) antigens, this polypeptide (along with several others) was also immunoprecipitated by sheep sera raised against authentic ES antigens. This verified that the isolated RNA contained mRNA species encoding ES antigens of the parasite. In addition, immune rat serum immunoprecipitated a 64 kDa polypeptide which was not immunoprecipitated by infected sheep serum or anti-ES antigen sheep serum. The possible significance of this finding in terms of the ability of rats to develop resistance to fascioliasis in contrast to sheep which apparently cannot is discussed.

Contrast agents and enzyme inhibition. II. Mechanisms.

The inhibition of enzymes by radiological contrast agents has been invoked to explain some of their clinical effects. In a previous paper, in-vitro studies of the inhibition of the enzyme acetylcholinesterase were presented and some clinical correlations were discussed. More detailed studies, described here, have now shed new light on the mechanisms involved in this important phenomenon. Because the findings restore the iodine atoms to a central role, they have important general implications for contrast agent pharmacology.