Toxocara canis larval excretory/secretory proteins impair eosinophil-dependent resistance of mice to Nippostrongylus brasiliensis.

Survival of parasitic helminths within a host requires immune evasion and excretory/secretory (ES) proteins may contribute to this process. Eosinophils are important effector cells in immunity of mice to the nematode Nippostrongylus brasiliensis and eosinophilic interleukin-5 transgenic (IL-5 Tg) mice are highly resistant to the earliest stages of primary infections. In contrast, Toxocara canis is largely resistant to eosinophils, with viable larvae encysted in tissues often surrounded by these and other leucocytes. The aim of this study was to investigate whether T. canis ES (TES) proteins inhibit eosinophil-dependent resistance to N. brasiliensis. Mouse serum pre-treated with TES had reduced capacity to mediate the adherence of leucocytes to N. brasiliensis infective-stage larvae (L3) and this correlated with reduced complement C3 deposition on the parasite. TES did not inhibit eosinophil survival or eotaxin-dependent eosinophil migration in vitro. Cellular inflammation and eosinophil degranulation in the skin in response to injection of L3 was also not impaired by TES. However, when TES was included with L3 in an inoculum given to IL-5 Tg mice, a greatly increased number of parasites migrated to the lung. This suggests that the early eosinophil-dependent resistance in these mice was suppressed, by mechanisms yet to be determined.

Characterisation of humoral immune responses in dogs vaccinated with irradiated Ancylostoma caninum.

Infection with Ancylostoma caninum, an intestinal hookworm of dogs, can cause debilitating and potentially life-threatening disease. In the current study, protective immunity to hookworm infection was induced in dogs following vaccination with irradiation-attenuated third-stage larvae (L3) with significant reductions in both worm (P<0.03) and faecal egg counts (P<0.0004) following a challenge infection. Vaccination with irradiated L3 and challenge with infective L3 stimulated a dominant antibody response to antigens of less than 20 kDa in an excretory/secretory extract from adult parasites. Immunoscreening of an adult A. caninum cDNA library with antisera from the vaccine trial identified a number of clones. The three clones with the strongest immunoreactivity proved to be identical and encoded a peptide with similarity to the N-terminal domain of the tissue matrix metalloproteinase inhibitor (TIMP)-2 mammalian tissue metalloproteinase inhibitor family.

Antibody-dependent cell-mediated cytotoxicity to newly excysted juvenile Fasciola hepatica in vitro is mediated by reactive nitrogen intermediates.

Passive intraperitoneal transfer of sera from Fasciola hepatica-infected sheep, cattle or rats can protect naive rats from F. hepatica infection, suggesting a parasite killing mechanism within the peritoneal cavity that is dependent on the presence of parasite-specific antibody. We investigated antibody-dependent cell-mediated cytotoxicity by resident peritoneal lavage cell populations, containing large numbers of monocytes/macrophages, as a potential host resistance mechanism by which juvenile flukes could be killed within the peritoneal cavity of naive rats. Comparative studies were conducted using cell populations containing large numbers of monocytes/macrophages from sheep. The results demonstrate that monocyte/macrophage-rich lavage cell populations from rat and sheep differ substantially in their ability to generate nitric oxide. Only resident rat peritoneal lavage cells were able to mediate antibody-dependent cell-mediated cytotoxicity against newly excysted juvenile liver fluke. The mechanism of cytotoxicity was dependent on, and directly proportional to, the production of nitric oxide and required attachment of effector cells to the newly excysted juvenile liver fluke tegument, which occurred following the addition of sera from F. hepatica-infected animals. This is the first report demonstrating a mechanism of cell-mediated cytotoxicity to newly excysted juvenile liver fluke.

Time-lapse video reveals immediate heterogeneity and heritable damage among human ileocecal carcinoma HCT-8 cells treated with raltitrexed (ZD1694).

BACKGROUND: Cellular heterogeneity in drug response has important clinical implications, and is believed to develop over many generations during clonal evolution in human tumors. The purpose of this study was to determine the level of heterogeneity exhibited by sister cells soon after their birth. METHODS: Human ileocecal carcinoma cells (HCT-8) were followed up to 11 days in vitro after a 2-h exposure to 1 microM raltitrexed (IC(95)) in a time-lapse video system. RESULTS: Over five experiments, 414 cells were followed after exposure to raltitrexed. Immediate sterility occurred in 74% of treated cells. Only 6% of cells could produce more than two generations of offspring, and heterogeneity in drug response was seen. Comparing sister cells < 24 h old, the more proliferative sibling produced up to 73 times more offspring, with a median ratio of 9.0 (control median = 1.19). Offspring of prolific drug-treated cells had a decreased probability of division (68% compared with 92%) and an increased average interdivision time (19.0 h compared with 15.1 h). CONCLUSIONS: Short-term exposure to raltitrexed resulted in increased interdivision times and production of sterile offspring extending seven generations. Cellular heterogeneity (difference in proliferation potential comparing drug-treated sister cells) was evident without a period of clonal evolution.

Juvenile Fasciola hepatica are resistant to killing in vitro by free radicals compared with larvae of Schistosoma mansoni.

Free radicals have previously been shown to kill the immature stages of the trematode, Schistosoma mansoni but their effect on newly excysted juvenile (NEJ) flukes of Fasciola hepatica has not been established. Using acetaldehyde and xanthine oxidase to chemically generate reactive oxygen intermediates (ROI), up to 61% of NEJ were killed but only when exposed to high levels of ROI. At low concentrations of acetaldehyde and xanthine oxidase as sources of reactive oxygen intermediates, only 6-29% of NEJ were killed compared with 70-92% of schistosomula. Incubation with lipopolysaccharide (LPS)-stimulated rat peritoneal lavage cells (PLCs) killed only 7-15% of NEJ whereas 78-87% of schistosomula were killed under the same conditions by a mechanism dependent on the production of reactive nitrogen intermediates. Relative to immature and adult parasites, NEJ expressed 2.5-20-fold lower levels of superoxide dismutase and glutathione S-transferase but no catalase activity was detected. Incubation of NEJ with inhibitors of peroxidases and glutathione metabolism increased the mean killing of NEJ by LPS-stimulated rat PLCs to 40-75%. These results demonstrate that, in comparison to schistosomula of S. mansoni, NEJ of F. hepatica are relatively resistant to killing by free radicals and this resistance could, in part, be due to the activity of oxidant scavenger enzymes of NEJ.

Interleukin-5 transgenic mice show enhanced resistance to primary infections with Nippostrongylus brasiliensis but not primary infections with Toxocara canis.

In this study, interleukin-5 (IL-5) transgenic mice with lifelong eosinophilia were assessed for resistance to primary infections with two tissue-invading nematodes, Nippostrongylus brasiliensis and Toxocara canis. Relative to nontransgenic littermates, three lines of IL-5 transgenic mice with varying degrees of eosinophilia all displayed enhanced resistance to N. brasiliensis. Although the timing of final worm expulsion was similar in transgenic and nontransgenic hosts, intestinal worms in transgenic mice were fewer in number throughout infection, failed to increase in size over the course of the infection, and were much less fecund. In contrast, T. canis larvae were recovered in similar numbers from tissues of transgenic mice with "low" or "high" eosinophilia and from nontransgenic mice. These results and other data suggest that eosinophils can contribute to host resistance to some parasite species. Parasite transit time through the host may correlate with relative sensitivity to eosinophils.

An assay for the quantitation of Photofrin in tissues and fluids.

A method for determining the concentration of Photofrin in tissues and biological fluids was developed. The procedure is based on the dissolution of biological material with Solvable, a commercially available tissue solubilizer, followed by porphyrin-specific fluorescence detection and measurement. It was found necessary to use a quadratic standard curve for the estimation of unknown Photofrin concentrations. While this method is limited to compounds that are stable in strong base, it has the advantages of being sensitive, rapid and low cost.

Fasciola hepatica: irradiation-induced alterations in carbohydrate and cathepsin-B protease expression in newly excysted juvenile liver fluke.

Irradiation has been successful in the attenuation of infective stage parasites for use as vaccines against a number of parasites including Fasciola spp. The mechanisms of action of irradiation-attenuated vaccines, however, are not clearly understood. In this study, we examined the effect of 3, 10, and 40 krad of gamma-irradiation on the expression of carbohydrates and cathepsin-B by newly excysted juvenile Fasciola hepatica (NEJ). Following irradiation of metacercariae, the expression of concanavalin A (ConA)-specific sugars was decreased on the surface of NEJ and the expression of wheat germ agglutinin (WGA)-specific sugars was increased in the gut and reduced on the surface of NEJ. Cathepsin proteases are a major component of liver fluke excretory/secretory material (ES) and can cleave host immunoglobulin (Ig). Cathepsin-B protease was localized in nonirradiated NEJ to the gut lumen and to secretory granules within the gut epithelia. Irradiation of fluke with 3, 10, and 40 krad of gamma-rays significantly reduced the tissue expression of cathepsin-B at 8 hr postirradiation in an apparently dose-dependent manner. After a further 24 hr culture tissue expression of cathepsin-B was significantly reduced in 10- and 40-krad-irradiated NEJ. Protease activity of ES samples collected over a 24-hr period from irradiated and nonirradiated NEJ cultured in vitro were tested using a rabbit Ig cleavage assay. The proteolytic activity of ES from 10- and 40-krad-irradiated NEJ was reduced during the initial 6 hr in culture and between 12 and 24 hr when compared to ES from nonirradiated controls. Biosynthetic labeling experiments using [35S]methionine and [35S]cysteine indicated that ES material was actively synthesised during 48 hr in vitro culture. Therefore, from this study, we conclude that gamma-irradiation of NEJ alters expression of cathepsin-B protease and WGA- and ConA-specific sugars which may be detrimental to parasite invasion and contribute to the protective immune responses generated in the host by irradiation-attenuated metacercariae of Fasciola spp.

The validation of a new vascular damage assay for photodynamic therapy agents.

The therapeutic effect of photodynamic therapy (PDT: photodynamic sensitizer + light) is partly due to vascular damage. This report describes a new vascular photodamage assay for PDT agents and a validation of the assay. The method described here quantitates changes in tissue blood perfusion based on the relative amount of injected fluorescein dye in treated and untreated tissues. A specially designed fluorometer uses chopped monochromatic light from an argon laser as a source for exciting fluorescein fluorescence. The fluorescent light emitted from the tissue is collected by a six element fiberoptic array, filtered and delivered to a photodiode detector coupled to a phase-locked amplifier for conversion to a voltage signal for recording. This arrangement permits a rather simple, inexpensive construction and allows for the simultaneous use of the argon laser by other investigators. The routine assay for characterizing a specific photosensitizer at a standard dose consists of the sequential allocation of eight mice to a set of different light doses designed to span the dose-response range of fluorescein fluorescence exclusion (measured 8-10 min after fluorescein injection). The assay validation experiment used an anionic photosensitizer, 2-[1-hexyloxyethyl]-2-devinyl pyropheophorbide-a at a dose of 0.4 mumol/kg. The parameter estimates (n = 34 mice) from fitting the standard Hill dose-response model to the data were: median fluorescence exclusion light dose FE50 = 275 +/- 8.3 J/cm2 and Hill sigmoidicity parameter m = -3.66 +/- 0.28. Subsets of the full data set randomly selected to simulate a standard eight mice experiment yielded similar parameter estimates. The new assay provides reliable estimates of PDT vascular damage with a frugal sequential experimental design.

Characterisation of a novel Kunitz-type molecule from the trematode Fasciola hepatica.

A low molecular mass monomeric protein termed Fh-KTM (Fasciola hepatica Kunitz-type molecule) was isolated from the trematode Fasciola hepatica. Fh-KTM is a single polypeptide of 58 amino acids and a Mr of 6751. The complete amino acid sequence of Fh-KTM was determined and revealed significant similarity to the Kunitz-type (BPTI) family of proteinase inhibitors. Several polymorphisms were observed suggesting that more than one Fh-KTM molecule may be expressed by this parasite. Modified proline residues were shown to occur at all four positions in this protein as 3-hydroxy derivatives. This is the first report of 3-hydroxyproline residues in a Kunitz-type molecule. Indirect immunofluorescence and immunogold labelling revealed that Fh-KTM is an abundant molecule within the parasite localised to the gut, the parenchymal tissue and the tegument of adult F. hepatica. Serine protease inhibition assays revealed that Fh-KTM exhibited little or no inhibition against chymotrypsin, kallikrein, urokinase or key serine proteases of the blood coagulation pathways. However, Fh-KTM was able to inhibit trypsin even though the P1 reactive amino acid of Fh-KTM was a leucine residue.

Fasciola hepatica: localisation of glutathione S-transferase isoenzymes in adult and juvenile liver fluke.

Four cDNA clones (GST-1, -7, -47, and -51) encoding isoenzymes of the detoxification enzyme glutathione S-transferase (GST) have previously been identified and characterised from Fasciola hepatica. In the present study, antisera were generated to synthetic peptides of regions unique to each of the four GST proteins predicted by the cDNAs. The antisera were characterised, and two were found to distinguish GST-1 from GST-7, GST-47, and GST-51 as a group. These two antisera were used to localise different GSTs in adult and newly excysted juvenile F. hepatica. The antiserum to GST-1 was specific and localised GST-1 to the parenchyma of adult fluke but not to the lamellae of the intestinal caeca. The antiserum to a GST-51 peptide, which cross-reacted with GST-7 and GST-47 but not GST-1, localised the other GSTs not only to the parenchyma but also to the intestinal lamellae of adult fluke. This appears to be the first evidence of tissue-specific expression of GST isoenzymes in trematodes. In contrast to adult fluke, immunolocalisation of the GSTs in juvenile F. hepatica revealed the binding of both the GST-1 and GST-51 antisera to the parenchymal cytoplasm, to cytoplasmic extensions of the parenchyma cells in the subtegumental area, as well as the excretory ducts. No labeling was observed in the intestinal epithelium of the juvenile fluke. These results demonstrate that adult F. hepatica, in contrast to juvenile flukes, contain a GST, which is not GST-1, associated with the lamellae of the gut and suggest that GSTs in adult fluke may play a role in the absorptive function of the adult gut.

Attempted immunisation of sheep against Fasciola hepatica using gamma-irradiated metacercariae.

The potential of gamma-irradiated Fasciola hepatica metacercariae to vaccinate sheep against fascioliasis was examined. The effect of the size of the inocula of irradiated metacercariae and the level of gamma-irradiation on the recovery of non-irradiated fluke was assessed following homologous challenge. Groups of Merino wethers were vaccinated with a single infection of either 500 or 2000 metacercariae, previously exposed to either 30, 100 or 400 Gy of gamma-irradiation. No significant reduction of fluke burdens were observed in any group, although a nonsignificant 20% reduction was observed in sheep vaccinated with 2000 metacercariae irradiated with 100 Gy. A second trial was conducted in which groups of sheep were vaccinated with 2 doses, given 4 weeks apart, of 2000 metacercariae, previously irradiated at either 70, 100 or 150 Gy. In both trials parasite viability was severely affected by doses of gamma-irradiation of 30 Gy or greater and no mature flukes were recovered from control sheep given metacercariae attenuated with 70 Gy or greater. A strong humoral immune response to somatic F. hepatica antigens was observed in all sheep. Only sera from sheep receiving 70 Gy irradiated metacercariae recognised the 2 candidate liver fluke vaccine molecules, F. hepatica glutathione S-transferase and cathepsin-L proteases. No reduction was observed in either the number of flukes or the production of fluke eggs in any vaccinated group. Vaccination appeared to affect the development of the challenge fluke population, resulting in reduced hepatic damage during migration, as measured by levels of serum glutamate dehydrogenase, and an increase in mean fluke weight.

Lack of reciprocity in drug and light dose dependence of photodynamic therapy of pancreatic adenocarcinoma in vitro.

Two human pancreatic cell lines, MIA PaCa 2 and Capan 2, were treated by photodynamic therapy in vitro with Photophrin (0.01-25 micrograms/ml; 24 h) and then light (1-50 J/cm2; lambda = 630 nm). The following model was fit to 6 datasets with weighted nonlinear regression: [sequence: see text] The symbols are: E, cell growth; Econ, control growth in the absence of the combination; B, background signal; m, slope parameter; gamma, interaction parameter; D, concentration of Photofrin; L, light dose; F, fraction of Photofrin not photobleached by the light dose; k, k1, k2, bleaching parameters; A, distribution parameter for biexponential bleaching equation. Simple reciprocity of photosensitizer concentration and light dose was not found; compensation for photobleaching was critical. MIA PaCa2 required the monoexponential bleaching factor, whereas Capan 2 required the biexponential bleaching factor. The greater photosensitivity of MIA PaCa2 over Capan 2 can be best explained not by differences in the interaction parameter but rather by differences in the photobleaching pattern and rate. It may be possible to further enhance the selectivity of photodynamic therapy if differences in photobleaching between different cell types can be exploited by adequate dosimetry.

Antibody to interleukin 5 prevents blood and tissue eosinophilia but not liver trapping in murine larval toxocariasis.

Mice previously sensitized by infective-stage larvae of the canine nematode, Toxocara canis, trap large numbers of challenge larvae within the liver; trapped larvae are found within eosinophilic granulomas. To investigate the role of eosinophils in this phenomenon we examined larval trapping in mice depleted of blood and tissue eosinophils by treatment with a monoclonal antibody (MoAb) (TRFK-5) produced against recombinant murine interleukin 5 (rmIL-5). Control mice received either an isotype-matched control MoAb or PBS. On day 0 test mice were given a sensitization dose of 125 infective T. canis eggs. Test and challenge control mice received 500 infective eggs on day 28. All mice were killed on day 42 and larval numbers within the liver were determined. Liver samples were also collected for histopathological and morphometric examination. When compared to test mice treated with PBS or the isotype control, the level of circulating eosinophils in anti-IL-5-treated test mice was reduced by 94-96% on days 14 and 27, 99% on day 35, and 100% on day 42; the level of tissue eosinophils within liver granulomas on day 42 was reduced by 92-95%. The total area of inflammation within the liver was similar among all test groups. However, the highly eosinophilic infiltrates, present in control sections, were replaced in anti-IL-5-treated mice by lymphocytes, macrophages, and foreign-body giant cells. No difference was found in larval trapping between antibody-treated groups. These findings suggest that the eosinophil is not necessary for liver trapping in murine larval toxocariasis.

Normal elastin content of aorta in bovine Marfan syndrome.

Samples from the ascending aortae from two calves affected by bovine Marfan syndrome were subjected to biochemical analyses of the connective tissue and were compared to age-matched controls. Elastin was extracted from the aortic samples with 5 M guanidine-HCl, bacterial collagenase digestion, and dithiothreitol reduction. Amino acid analysis revealed that desmosine and isodesmosine levels were the same in Marfan calves as in control animals. Gravimetric measurements of elastin, amino acid composition, soluble protein, and uronic acid values also showed no significant difference between Marfan and control tissue. In contrast to elastin, collagen in aortae of Marfan calves was significantly higher than the mean of several controls. These findings, along with other observations of this animal model, support the conclusion that the microscopic and biochemical lesions of aortic elastin in bovine Marfan syndrome likely result from defective microfibrillar metabolism. Absence of cystic medial necrosis in bovine Marfan aortae may explain normal elastin content in the animal model.

Quantitation of folic acid enhancement of antifolate synergism.

Trimetrexate (TMTX), 5,10-dideazatetrahydrofolate (DDATHF), and 10-propargyl-5,8-dideazafolate (PDDF, CB3717) are antifolates whose primary intracellular targets are dihydrofolate reductase, glycinamide ribonucleotide formyltransferase, and thymidylate synthase, respectively. Varying the medium folic acid (PteGlu) concentration over the range of 0.5 to 100 microM increasingly blocks the growth inhibitory effects of the individual antifolates in Manca human lymphoma cells, but increasingly enhances the synergistic interaction of both TMTX + DDATHF and TMTX+ PDDF combinations. Drug interactions were quantitated using the universal response surface approach, which consists of fitting a concentration-effect surface to experimental data with weighted nonlinear regression, enabling the estimation of the synergism parameter, alpha. Estimates for alpha are larger (more intense synergism) for the TMTX + DDATHF combination (alpha = 4.68 +/- 0.66 at 2 microM PteGlu; alpha = 53.6 +/- 3.7 at 40 microM PteGlu) than for the TMTX + PDDF combination (alpha = 0.690 +/- 0.25 at 2 microM PteGlu; alpha = 7.20 +/- 1.8 at 40 microM PteGlu). However, the relative increase brought about by increasing the PteGlu concentration from 2 microM to 40 microM is similar in each instance, 11- and 10-fold, respectively. These experiments suggest that the enhanced cytotoxic interaction brought about by increased PteGlu concentration with the TMTX + DDATHF combination and the TMTX + PDDF combination may share a common mechanism. The dramatic intensity of the synergism between DDATHF and TMTX at 40 microM PteGlu, along with the concept of modulation of antifolate synergism by folates, suggests future in vivo and/or clinical applications of combinations of these (or similar) compounds.

Chemoprophylactic effects of milbemycin oxime against larvae of Dirofilaria immitis during prepatent development.

Three studies were conducted to determine the efficacy of milbemycin oxime in the prevention of Dirofilaria immitis infection in dogs. Dogs were given single or multiple experimental inoculations with infective third-stage D immitis larvae and were treated with milbemycin oxime at a target dosage of 0.5 mg/kg of body weight either once or at monthly intervals at various times after inoculation. The compound was effective in preventing infection when 1 dose was administered 30 or 45 days after inoculation. Significant, but incomplete, protection was achieved when single treatments were administered 60 or 90 days after inoculation. Multiple monthly treatments beginning 60 days after inoculation appeared to provide additive effects that resulted in restoration of complete efficacy.

Kinetics of liver trapping of infective larvae in murine toxocariasis.

Mice sensitized by prior infection with Toxocara canis eggs trap many larvae of a challenge infection within the liver. In this study the distribution of challenge larvae in sensitized mice was examined to determine the earliest onset of liver trapping and to establish if the previously described phenomenon truly represented larval trapping. In all experiments, C57BL/6J mice were infected with a sensitization dose of 125 infective T. canis eggs on day 0 postinfection (PI) and challenged with 500 infective eggs on day 28 PI. In the initial experiments, larval numbers were determined within the intestinal contents, intestinal wall, mesenteric tissues, liver, lungs, skeletal muscle, and brain of each mouse on days 0.5, 1, 2, 3, 5, and 6 postchallenge (PC). Migration patterns were similar among the test and control groups except the peak of larval numbers in the liver, seen at 1 day PC in control mice, was delayed until 3 days PC in the test group. Larval trapping occurred within the liver of test mice at least by day 5 PC. In subsequent experiments, larval numbers were determined within the liver, skeletal muscle, brain of each mouse, and within the eyes of each mouse group at 4, 8, 12, and 16 wk PC. Larval numbers within the liver of test mice were similar both at 5 days PC and 16 wk PC, implying that larvae were trapped in this organ rather than delayed in their migration to other body sites. Liver trapping did not protect the eyes or brain of sensitized mice from larval migration, nor did it result in larval killing.