Molecular cloning, expression and enzymatic activity of a thioredoxin peroxidase from Dirofilaria immitis

A Dirofilaria immitis cDNA clone encoding a nucleic acid homolog of thioredoxin peroxidase (nDiTPx) was isolated from a fourth-stage larval cDNA library, using serum from dogs vaccinated by chemotherapeutically-abbreviated D. immitis larval infections. The protein encoded by nDiTPx had a predicted molecular mass of 22.1 kDa and the deduced amino acid sequence was homologous to thioredoxin peroxidase-like sequences described in other filarial nematodes, yeast, bacteria and mammals. As is true for other members of this peroxiredoxin family, the nDiTPx-encoded protein had the conserved cysteine near the amino terminus, considered to be essential for enzyme activity. nDiTPx was expressed in E. coli and the resulting recombinant fusion protein was shown to have thioredoxin peroxidase (TPx) activity, by its ability to protect DNA from oxidative-nicking in a metal-catalyzed oxidation system. A polyclonal antibody to the DiTPx fusion protein detected a 22-kDa native protein in D. immitis larval and adult parasite extracts.     

Echinococcus granulosus infection of farm dogs of Iran

The non-enzymatic metabolites of prostacyclin (PGI2) and thromboxane A2 (TXA2), 6-keto-prostaglandin F1alpha (6-keto-PGF1alpha) and thromboxane B2 (TXB2), and their 2,3-dinor metabolites, 2,3-dinor-6-keto-PGF1alpha and 2,3-dinor-TXB2, were measured in early morning urine samples in 24 in vitro fertilization (IVF) cycles in 24 women and in 27 women who became pregnant after IVF and embryo transfer (ET). The sum of the non-enzymatic metabolites and their 2,3-dinor metabolites was considered to be a reflection of total PGI2 and total TXA2 production in vivo. Both the ratio of 'total' PGI2/'total' TXA2 and the ratio of the 2,3-dinor metabolites were calculated. TXB2 concentrations showed virtually no change and the ratios of the non-enzymatic metabolites of PGI2 and TXA2 versus their 2,3-dinor metabolites remained relatively constant. As a consequence, the ratio of 2,3-dinor-6-keto-PGF1alpha/2,3-dinor-TXB2 was a close reflection of the ratio of 'total' PGI2/'total' TXA2, although the latter ratio was significantly higher all the time. We conclude that for comparative studies on the balance between PGI2 and TXA2 in IVF cycles and during gestation, the determination of the 2,3-dinor metabolites alone can replace the measurement of all four metabolites.     

Immunological assessment of exposure to Echinococcus granulosus in a rural dog population in Uruguay

An ELISA was used to screen a dog population in Uruguay (Sarandi Del Yi, Durazno District) for the prevalence of specific serum antibodies (IgG, IgA and IgE) to Echinococcus granulosus. The sensitivity (61%) and specificity (97%) of the ELISA were determined using well-defined serum groups. A total of 408 dogs from Sarandi del Yi and environs were screened serologically, and 29.7% (8.6-13.8% for each antibody class) of dogs had positive levels of antibody to E. granulosus. This antibody prevalence (exposure) was significantly higher than the percentage of dogs found to be positive for E. granulosus worms by arecoline purgation (7.6%). This level of exposure to E. granulosus determined by ELISA is considered unacceptable from a public health perspective. Measures will now focus on obtaining data on the true prevalence of current infection in this dog population and on determining the transmission patterns of the disease in this endemic region.     

Diagnosis and treatment of unilocular hydatid disease (Echinococcus granulosus infection)]

Unilocular hydatid disease occurs in humans after oral intake of eggs of the dog tapeworm, Echinococcus granulosus. Cysts develop mostly in the liver, but sometimes also in the lung. The diagnosis of Echinococcus infection is based on history-taking, physical examination, ultrasound and CT examination and serological testing; the diagnosis is confirmed by parasitological examination of cystic fluid. Treatment until some 15 years ago consisted in operation. Subsequently, treatment with initially mebendazole, later with albendazole or with percutaneous drainage (puncture, aspiration, injection of a scolicidal, respiration (PAIR)), or with combinations of the same, became accepted forms of management. The PAIR method is reported to give high proportions of success, low proportions of recurrence and few complications. However, adequate evaluation is not yet possible because of the short follow-up period. For the prevention of leakage it is recommended to perform the PAIR method with a transhepatic puncture under continuous ultrasonographic or CT guidance; for avoidance of recurrences, one week's pretreatment and 1-4 weeks' after-treatment with albendazole are recommended. The results of albendazole monotherapy are hard to predict and highly variable: success: 30-88% (median: 71%); failure: 22-32% (median: 25%); recurrences: 9.5-31% (median: 16%). Both albendazole therapy and the PAIR management should be followed by protracted follow-up to check regression of cysts and detect recurrences. It is not clear which treatment is the best one.     

Factors influencing the development and carbohydrate metabolism of Echinococcus granulosus in dogs

Echinococcus granulosus adult worms, 35 days postinfection, were measured for dispersion in the intestines of 10 dogs, a range of morphological characters, and the excreted end products of carbohydrate catabolism following 4 hr incubation in vitro. Most worms were found in the proximal sections of the small intestine, but the pattern of dispersion differed between dogs. Worm development varied both between dogs and between different regions of the small intestine of individual dogs. Overall there was a high level of variability with no simple patterns. Worm metabolism was related to worm development and, also independently, to local population density within the intestine. Larger, more mature worms produced less lactate and, at higher densities, worms tended to produce more acetate and succinate (pathways with a higher energy yield than lactate) and less ethanol. Thus, both more developed worms and high population density are associated with a shift from cytosolic to mitochondrial metabolism. The variation between worm populations along the small intestine along with the observed variation between worm populations from sibling dogs infected with genetically identical parasites suggests that the local host environment has a significant effect on parasite development.     

Molecular cloning and characterization of a mitochondrial selenocysteine-containing thioredoxin reductase from rat liver

A thioredoxin reductase (TrxR), named here TrxR2, that did not react with antibodies to the previously identified TrxR (now named TrxR1) was purified from rat liver. Like TrxR1, TrxR2 was a dimeric enzyme containing selenocysteine (Secys) as the COOH-terminal penultimate residue. A cDNA encoding TrxR2 was cloned from rat liver; the open reading frame predicts a polypeptide of 526 amino acids with a COOH-terminal Gly-Cys-Secys-Gly motif provided that an in-frame TGA codon encodes Secys. The 3'-untranslated region of the cDNA contains a canonical Secys insertion sequence element. The deduced amino acid sequence of TrxR2 shows 54% identity to that of TrxR1 and contained 36 additional residues upstream of the experimentally determined NH2-terminal sequence. The sequence of this 36-residue region is typical of that of a mitochondrial leader peptide. Immunoblot analysis confirmed that TrxR2 is localized almost exclusively in mitochondria, whereas TrxR1 is a cytosolic protein. Unlike TrxR1, which was expressed at a level of 0.6 to 1.6 microgram/milligram of total soluble protein in all rat tissues examined, TrxR2 was relatively abundant (0.3 to 0.6 microgram/mg) only in liver, kidney, adrenal gland, and heart. The specific localization of TrxR2 in mitochondria, together with the previous identification of mitochondria-specific thioredoxin and thioredoxin-dependent peroxidase, suggest that these three proteins provide a primary line of defense against H2O2 produced by the mitochondrial respiratory chain.     

Primary infection of dogs with Echinococcus granulosus: systemic and local (Peyer's patches) immune responses

Local and systemic lymphocyte proliferation and antibody production were tested in five dogs 35 days after primary experimental infection with Echinococcus granulosus. A significant cell proliferation was demonstrated by [3H] thymidine incorporation in mesenteric, popliteal and/or Peyer's patches (PPs) cells in response to E. granulosus protoscolex or adult worm antigen in three of five infected dogs, but not in five control animals. In contrast, blood mononuclear cells responded very weakly in only two of the infected dogs to parasite antigens. Elevated levels (compared with preinfection status) of protoscolex- and adult worm antigen-specific serum IgG were detected (ELISA) in four of the five dogs 35 days after infection. Furthermore, slightly elevated levels of parasite-specific IgE and IgA were observed in the sera of three and four in four infected dogs, respectively. Specific serum IgM was not significantly higher 35 days after infection than before infection. Local antibody production was studied in vitro using PPs, mesenteric and popliteal cells isolated from three infected and three uninfected dogs by ELISA using adult worm antigen. In two of three cultures of unstimulated PPs cells of infected dogs, parasite-specific IgG was detectable. Parasite-specific IgA and IgM were detected in one of the unstimulated PPs cell culture derived from an infected dog. Following in vitro stimulation with parasite antigen, PPs cells from two infected dogs showed increased parasite-specific IgG and PPs cells of all three infected dogs produced parasite-specific IgA. PPs cells from uninfected dogs did not produce significant quantities of parasite-specific antibodies and cells from mesenteric and popliteal lymph nodes of infected or uninfected dogs neither produced antibodies whilst in in vitro cultures.     

The homeobox-containing gene EgHbx3 from Echinococcus granulosus is expressed in the stalk of protoscoleces

A method is described for the determination of the antibacterial drug trimethoprim in tissues. Minced tissue is homogenised with chloroform-acetone (1 + 1 v/v), filtered, and the filtrate evaporated to an oily residue using a rotary evaporator. The residue is redissolved in methanol-water-acetic acid (50 + 48.7 + 1.3 v/v) and any fats present are partitioned into hexane. The aqueous phase is analysed by liquid chromatography-thermospray mass spectrometry in positive mode with the protonated molecular ion at m/z 291 being monitored. Recoveries ranged between 60% in liver and 79% in muscle. The limit of determination was 25 micrograms kg-1 and the limit of detection was approximately 4 micrograms kg-1. The method is suitable for monitoring tissues taken under national surveillance schemes for veterinary drug residues.     

T-cell activity associated with secondary infections and implanted cysts of Echinococcus granulosus in BALB/c mice

Selected cytokine profiles of lymphocytes were assessed in BALB/c mice infected with protoscoleces of Echinococcus granulosus. Late stages of the infection (three months+) were characterized by more dominant Th2 activity with elevated IL-4 and IL-10 and reduced IFN-gamma output by Con-A and antigen stimulated splenocytes. Circumparasitic leucocytes produced mainly IL-10 by five months post infection. A peak in IFN-gamma production in the first month of infection may suggest Th1 or Th0 activity at this time and this may be correlated with initial protoscolex death. In addition, cytokine profiles from mice implanted with intact hydatid cysts were also assessed. At two weeks post implantation all cysts were still viable and cytokine production was characterized mainly by elevated IL-10 production. However, at four months post implantation, some of the cysts from two mice had been killed whilst all cysts in the remaining mouse remained viable. In the mice where dead cysts were present, elevated levels of IFN-gamma were detected from splenocytes and circumparasitic cells. Elevated IL-4 was also evident with the splenocytes. In the mouse with viable cysts IFN-gamma production was reduced Results indicate that IFN-gamma(Th1) activity may be correlated with killing of both protoscoleces and established cysts of E. granulosus.     

Some studies on the growth and development of Echinococcus granulosus, camel origin in experimentally infected dogs

Development of Echinococcus granulosus of camel subspecies in 35 experimentally infected dogs was performed at 3, 7, 10, 13, 18, 23, 35 and 56 days post-infection (d.p.i.). Morphological characters of each developmental stage was studied and discussed. Generally, the results indicate that the segmentation firstly appeared at 18 d.p.i. Also, male and female genitalia could be detected at the same period. However, completely developed of genitalia appeared at 56 d.p.i. Histological examination of small intestine of experimentally infected dogs revealed that the parasites were found in distended and thin wall crypts of Liberkuhn at 3, 7, & 10 d.p.i, while the parasite scolices were found embedded in the mucosa at 13, 18 & 23 d.p.i. No significant pathological changes were encountered in both infected and control dogs. This is the first report of strobilar development of E. granulosus of camel origin in experimentally infected dogs.     

Rates of reinfection with Echinococcus granulosus, Taenia hydatigena, taenia ovis and other cestodes in a rural dog population in Uruguay

Forced thinking is an incompletely understood and rarely described epileptic aura. We studied three patients with forced thinking from left frontal lesions, two neoplastic and one vascular. All three experienced repetitive, intrusive thoughts at the onset of seizures. Their forced thinking was associated with the desire to vocalize, orobuccal movements, and speech arrest. The episodes occurred with other ictal manifestations and responded to antiseizure therapy. These patients suggest that epileptic forced thinking is a heterogeneous phenomenon; forced thinking from left frontal lesions is a manifestation of expressive language and is distinct from experiential thoughts arising from temporal limbic foci.     

Developing protoscoleces of Echinococcus granulosus on the outer surface of the brood capsule, detected by scanning electron microscopy

A controversy presently exists about the structure of lymphatic valves, the bicuspid structure vs. the funnel-like architechture. This present study, along with a review of the literature was undertaken in an attempt to clarify this controversy. Twenty lymphatic valves from the collecting lymphatic channels draining the back paw region in four dogs were examined and several were photographed. The bicuspid nature and the three dimensional anatomy of these valves is clearly demonstrated.     

Effect of mebendazole on free amino acid composition of cyst wall and cyst fluid of Echinococcus granulosus harbored in mice

Eighteen and 23 FAA components were detected in the cyst wall and cyst fluid of E granulosus, respectively, by using automatic amino acid analyzer. The concentrations of most of the determined FAA were higher in the cyst fluid than those in the cyst wall, especially the taurine was 5-fold higher. Mebendazole treatment resulted in an increase in the concentration of alanine, valine, lysine, and taurine in both cyst wall and cyst fluid, the most notable being the alanine in the cyst wall. The results are interpreted as a coupling of glycolysis and amino acid metabolism, suggesting an involvement of FAA metabolism in the mechanism of Meb action.     

Contrasting patterns of regulation of the antioxidant selenoproteins, thioredoxin reductase, and glutathione peroxidase, in cancer cells

There is strong evidence that selenium protects against certain human cancers, but the underlying mechanism is unknown. Glutathione peroxidase (GPX1) and thioredoxin reductase (TR), the most abundant antioxidant selenium-containing proteins in mammals, have been implicated in this protection. We analyzed the expression of TR and GPX1 in the following model cancer systems: (1) liver tumors in TGFalpha/c-myc transgenic mice; (2) human prostate cell lines from normal and cancer tissues; and (3) p53-induced apoptosis in a human colon cancer cell line. TR was induced while GPX1 was repressed in malignancies relative to controls in transgenic mice and prostate cell lines. In the colon cell line, p53 expression resulted in elevated GPX1, but repressed TR. The data indicate that TR and GPX1 are regulated in a contrasting manner in the cancer systems tested and reveal the p53-dependent regulation of selenoprotein expression. The data suggest that additional studies on selenoprotein regulation in different cancers are required to evaluate future implementation of selenium as a dietary supplement in individuals at risk for developing certain cancers.     

A mucosal IgA response, but no systemic antibody response, is evoked by intranasal immunisation of dogs with Echinococcus granulosus surface antigens iscoms

Current understanding of the pathogenesis of rheumatoid arthritis has provided evidence that therapeutic benefit can be achieved by using antagonists targeted to the inflammatory cytokines involved, mainly tumour necrosis factor-alpha and interleukin-1. Gene delivery of antagonists, which can inhibit the production or action of these cytokines and other mediators, has been achieved in experimental animal models. This new method of delivery can produce therapeutic effects at lower concentrations and in a local environment, overcoming the adverse effects that often accompany protein therapy. However, several technological and biological restraints preclude the immediate adaptation of this method to human treatment. Based on the experimental evidence, possible target therapeutic genes, cell types and vector systems that could be used are discussed in this article.