Determination of Growth-Phase Dependent Influences Exerted by Prions on Yeast Lipid Content Using HPTLC-Densitometry.

Prions of the baker's yeast Saccharomyces cerevisiae allow for the inheritance of complex traits based solely on the acquisition of cytoplasmic protein aggregates and confer distinctive phenotypes to the cells which harbor them, creating heterogeneity within an otherwise clonal cell population. These phenotypes typically arise from a loss-of-function of the prion-forming protein that is unable to perform its normal cellular function(s) while sequestered in prion amyloid aggregates, but the specific biochemical consequences of prion infection are poorly understood. To begin to address this issue, we initiated a direct investigation into the potential control that yeast prions exert over fungal lipid content by utilizing the prions [URE3] and [PSI+], the first two prions discovered in yeast. We utilized silica gel high-performance thin-layer chromatography (HPTLC)-densitometry to conduct pair-wise quantifications of the relative levels of free sterols, free fatty acids, and triacylglycerols [petroleum ether-diethyl ether-acetic acid (80:20:1) mobile phase, phosphomolybdic acid (PMA) detection reagent]; steryl esters and squalene (hexane-petroleum ether-diethyl ether-acetic acid (50:20;5:1), PMA]; and phosphatidylethanolamine, phosphatidylcholine, and phosphatidylinositol (chloroform-diethyl ether-acetic acid (65:25:4.5), cupric sulfate-phosphoric acid) in otherwise clonal prion-infected ([PSI+] or [URE3]) and prion-free ([psi-] or [ure-o]) cells in two growth phases: log-phase and stationary phase. Our analysis revealed multiple statistically significant differences (p < 0.00625) between prion-infected and prion-free cells. Interestingly, prion-induced changes varied dramatically by growth phase, indicating that prions exert differential influences on cell physiology between log and stationary growth. Further experimental replication and extension of the analysis to other prions is expected to resolve additional physiological effects of prion infection. This investigation demonstrates that HPTLC-densitometry is an effective method for studying prion-induced alterations in lipid content in yeast.

Partial resistance to homologous challenge infections of the digenean Echinostoma caproni in ICR mice.

In the present paper, we analyse the effect of a primary infection of ICR mice with Echinostoma caproni (Trematoda: Echinostomatidae) on the generation of resistance against homologous challenge infections. In ICR mice, E. caproni induces chronic infections concomitantly with strong responses characterized by the development of T-helper 1 (Th1)-type local immune responses with elevated levels of local interferon-gamma (IFN-γ) and inflammatory and antibody responses. Here, the effect of the response generated against a primary infection with E. caproni in the generation of resistance against subsequent homologous infections was analysed. For this purpose, ICR mice were challenged with metacercariae of E. caproni and the results obtained showed that primary infection induces partial resistance against subsequent homologous infections in ICR mice. This resistance was expressed as a reduced rate of infection, worm recovery and worm size, indicating that primary infection induces changes in the host, making a hostile environment for the development of the parasite.

Echinostoma caproni (Trematoda): differential in vivo mucin expression and glycosylation in high- and low-compatible hosts.

Enhanced mucus production and release appears to be a common mechanism for the clearance of intestinal helminths, and this expulsion is normally mediated by Th2-type immune responses. To investigate the factors determining the expulsion of intestinal helminths, we have analysed in vivo expression of mucin genes at the site of infection in two host species displaying different compatibility with Echinostoma caproni (Trematoda). Surprisingly, a general down-regulation on mucin mRNA expression was detected in low-compatible hosts (rats) coinciding with the development of Th2/Th17 responses and the early rejection of the worms from the intestinal lumen. This suggests the existence of a mechanism by which the parasites can modulate the mucus barrier to favour their survival. In highly compatible hosts (mice), some mucin genes were found to be up-regulated throughout the infection, probably, to protect the intestinal epithelium against the infection-induced inflammation developed in this host species. Moreover, infection-induced changes on mucin glycans were also studied by lectin histochemistry. Similar alterations were detected in the ileum of infected mice and rats, except with SNA lectin, indicating that sylated mucins might play an important role in determining the evolution of the infection in each host species.

Current status of food-borne trematode infections.

Food-borne trematodiases constitute an important group of the most neglected tropical diseases, not only in terms of research funding, but also in the public media. The Trematoda class contains a great number of species that infect humans and are recognized as the causative agents of disease. The biological cycle, geographical distribution, and epidemiology of most of these trematode species have been well characterized. Traditionally, these infections were limited, for the most part, in populations living in low-income countries, particularly in Southeast Asia, and were associated with poverty. However, the geographical limits and the population at risk are currently expanding and changing in relation to factors such as growing international markets, improved transportation systems, and demographic changes. The diagnosis of these diseases is based on parasitological techniques and only a limited number of drugs are currently available for treatment, most of which are unspecific. Therefore, in-depth studies are urgently needed in order to clarify the current epidemiology of these helminth infections and to identify new and specific targets for both effective diagnosis and treatment. In this review, we describe the biology, medical and epidemiological features, and current treatment and diagnostic tools of the main groups of flukes and the corresponding diseases.

Th17 responses in Echinostoma caproni infections in hosts of high and low compatibility.

In order to investigate the factors determining the expulsion of intestinal helminths, we have analyzed the in vivo expression of IL-17, TGF-ß and IL-23 in several tissues of two host species displaying different compatibility with Echinostoma caproni (Trematoda). We did not observe upregulation of these cytokines in any of the tissues of the high compatible host (mice). In contrast, the responses in the host of low compatibility (rats) with the parasite were markedly different. Significant increases in the expression of IL-17 and TGF-ß were observed in the Peyer's patches and the intestine from the 2 to 8 weeks post-infection. The expression of IL-23 was upregulated from 2 to 4 weeks post-infection in the spleen, Peyer's patches and the intestine. Considering together our results with those published previously the development of chronic infections appears to be related with the development of local Th1 responses, whereas the early rejection of the worms is mediated by the development a biased Th17/Th2 phenotype. The Th17 response generated in rats may facilitate the worm expulsion via the suppression of the inflammatory Th1 responses and the increase in intestinal contractility.

Longevity of Zygocotyle lunata in Balb/c mice and long-term survival of digeneans in vertebrate hosts.

This study used Balb/c mice to examine the longevity of Zygocotyle lunata in a murine host. Of 11 mice, each exposed to 20 Z. lunata cysts, six were infected with a total of 12 worms from 11 to 24 weeks postinfection (PI). Live worms recovered at 24 weeks PI had a mean body area of about 25 mm2. These worms produced viable eggs with well-developed miracidia following embryonation in artificial spring water for 2 weeks at 28°C. The Balb/c mouse is a useful model to study longevity of this paramphistomid trematode for at least 6 months PI. An additional aspect of this article is a review of the pertinent literature published from 1937 to 2007 on ageing and longevity of digeneans.

Encystment and excystment of the paramphistomid trematode Zygocotyle lunata.

This study reports further observations on encystment and excystment of the paramphistomid trematode Zygocotyle lunata. Of numerous substrates tested in the laboratory for cercarial encystment, i.e. plastic and glass dishes, Styrofoam™, aluminium foil, snail shells, and cheesecloth, all but cheesecloth allowed for 100% encystment within 4 h of cercarial release at 28°C. Numerous physical and chemical factors were tested to kill cysts, and their capacity to do so was determined by the subsequent inability of cysts to excyst within a chemical excystment medium. Vinegar, Italian salad dressing, soy sauce and 10% solutions of sucrose, acetic acid and sodium chloride were the most detrimental. Freezing, boiling and storage of cysts for more than 2 years also killed them. Several marinades such as lemon juice, Worcestershire sauce, and various concentrations of potassium permanganate, did not kill the majority of cysts tested. Since Z. lunata is a potential foodborne trematode for humans and animals, it is important to determine suitable ways to control or kill these cysts.

Autonomous public organization policy: a case study for the health sector in Thailand.

This paper describes factors affecting autonomous public organization (APO) policy agenda setting and policy formation through comparison of policy processes applied to one educational institute under the Ministry of Education and the other educational institute under the Ministry of Public Health in Thailand. This study employs mixed method including a qualitative approach through documentary research, in-depth interviews, and participant observation. Factors that facilitated the formulation of the APO policy were: (1) awareness of need; (2) clarity of strategies; (3) leadership, advocacy, and strategic partnerships, (4) clear organizational identity; (5) participatory approach to policy formulation, and (6) identification of a policy window. Factors that impeded the formulation of the APO policy were: (1) diverting political priorities; (2) ill-defined organizational identity; (3) fluctuating leadership direction, (4) inadequate participation of stakeholders; and (5) political instability. Although findings cannot be generalized, this case study does offer benchmarking for those in search of ways to enhance processes of policy formulation.

[Reexamination of the life cycle of Isthmiophora melis (Trematoda: Echinostomatidae) on material from southeast Europe].

The life cycle of Isthmiophora melis (Schrank, 1788) on material from Southeast Europe was experimentally reexamined. Thirteen names or combinations can be accepted as true synonyms of I. melis: Distoma melis (Schrank, 1788) Zeder, 1800; Echinocirrus melis (Schrank, 1788) Mendhaim, 1943; Isthmiophora spiculator (Dujardin, 1845); Echinostoma trigonocephalum (Rud., 1802) Cobbold, 1861; E. melis (Schrank, 1788) Dietz, 1909; E. spiculator Dujardin, 1845; Euparyphium jassyense Leon and Ciurea, 1922; E. melis (Schrank, 1788) Railliet, 1919; E. suinum Ciurea, 1921; Fasciola armata Rud., 1802; F. melis Schrank, 1788; F. putorii Gmelin, 1791; F. trigonocephala Rud., 1802. The first intermediate hosts are the pulmonate freshwater snail Lymnaea stagnalis. The second intermediate hosts are many amphibians and freshwater fishes. The list of definitive hosts includes more than 30 species of vertebrates including humans. I. melis occurs in Europe, Asia and North America.

Molecular cloning and characterization of Echinostoma caproni heat shock protein-70 and differential expression in the parasite derived from low- and high-compatible hosts.

We cloned and expressed Echinostoma caproni HSP70 in Escherichia coli. This molecule presents an open reading frame (ORF) of 655 amino acids, and a theoretical molecular weight of 71 kDa. E. caproni HSP70 protein showed a high homology to other helminth molecules, major differences being located in the C-terminal region of the molecule, with a hydrophobic portion. Studies of protein and messenger RNA (mRNA) expression revealed a distinct pattern, depending on the host (low- or high-compatible). Specific polyclonal antisera raised against the recombinant protein expressed in Escherichia coli demonstrated its selective presence in excretory/secretory products (ESP) of adult parasites obtained from high-compatible hosts. Immunological studies showed clearly the association of HSP70 with the parasite surface and other structures, including eggs.

Identification of antigenic proteins from Echinostoma caproni (Trematoda) recognized by mouse immunoglobulins M, A and G using an immunoproteomic approach.

Antigenic proteins of Echinostoma caproni (Trematoda) against mouse IgM, IgA, IgG, IgG1 and IgG2a were investigated by immunoproteomics. Excretory/secretory products (ESP) of E. caproni separated by two-dimensional (2D) gel electrophoresis were transferred to nitrocellulose membranes and probed with the different mouse immunoglobulin classes. A total of four proteins (enolase, 70 kDa heat-shock protein (HSP-70), actin and aldolase) were accurately identified. Enolase was recognized in eight different spots of which seven of them were detected in the expected molecular weight and were recognized by IgA, IgG or IgG and IgG1. Another spot identified as enolase at 72 kDa was only recognized by IgM. Digestion with N-glycosidase F of the 72 kDa band rendered a polypeptide with an apparent molecular weight similar to that expected for enolase recognized by Western immunoblotting using anti-enolase antibodies. This suggests that glycosylated forms of enolase may be involved in the early thymus-independent responses against E. caproni. Early IgM responses were also generated by actin and the HSP-70 which suggests that these proteins are exposed early to the host and may be of importance in the parasite establishment. The IgA responses also appear to be mediated by the HSP-70 and aldolase which could be related with the close contact of these proteins with the host mucosal surface after secretion.

Longevity of Echinostoma caproni in Balb/c mice.

This study used Balb/c mice to examine the longevity of Echinostoma caproni. Five mice each exposed to 75 encysted metacercariae (cysts) were necropsied at 23 weeks postinfection (PI) (160 days PI). Two of the 5 were infected with a total of 33 worms; 23 in one mouse and 10 in the other. Body and organ area measurements showed that these worms were robust and normal in appearance. No signs of atrophy of any of the genital structures were observed. The mean +/- SE of eggs/uterus per worm (n = 10) was 243 +/- 6. This strain of mouse will be suitable to study the effect of long-term survival on the host-parasite relationship of E. caproni in Balb/c mice.

Cercarial tail loss in Echinostoma caproni: the influence of in vivo encystment and copper sulphate.

Echinostoma caproni tail loss was studied in vitro in the presence of the toxicant copper sulphate (CuSO4) in concentrations ranging from 1 to 10 000 mg l(-1) in standardized artificial spring water (pH 7.4, osmolarity 34 mOsm kg(-1) H2O, Ca(2+) 20 mg l(-1)) at 23 degrees C. Tail loss was also studied in the absence of toxicants during in vivo encystment of the cercariae in juvenile Biomphalaria glabrata. As the concentration of CuSO4 increased, the percentage of cercarial tail loss increased. By 2 h in 10 000 mg l(-1), 1000 mg l(-1) and 100 mg l(-1) CuSO4, 50%, 23% and 13%, respectively, of the cercariae had lost their tails. In the in vivo studies, by 1 h PI, 59+/-5% of cercariae had lost their tails and only 4+/-1% of the cercariae were actively swimming in the multi-well dishes. At 3 h PI, 72+/-3% of the cercariae began to form cysts within the snails.

Kinetics of antibodies and antigens in serum of mice experimentally infected with Echinostoma caproni (Trematoda: Echinostomatidae).

The present study reports on the kinetics of antibodies and antigens in serum of mice experimentally infected with 75 metacercariae of Echinostoma caproni during the first 12 wk postinfection (wpi). Antibody titers in the serum of mice were determined by an indirect enzyme-linked immunosorbent assay (ELISA) using excretory/secretory (ES) antigens of E. caproni. The early detection of antibodies against ES antigens of E. caproni is feasible using indirect ELISA. Mice developed significant antibody responses at 2 wpi, and the values progressively increased until the end of the experiment. This may be related to the intestinal absorption of adult worm antigens that induces humoral responses. The presence of E. caproni circulating antigens was determined by a capture ELISA based on polyclonal rabbit antibodies against ES antigens of E. caproni. High levels of seroantigens in mice were detected by 1-2 wpi, probably because of the local inflammatory responses in mice induced by the adult worms. A drop in circulating antigen levels was observed at 9 wpi, which could reflect changes in the intestinal tissues over the course of the infection.

Effects of Schistosoma mansoni infection on inorganic elements in the snail Biomphalaria glabrata.

Inductively coupled plasma atomic emission spectrometry (ICP-AES) was used to study element ions in whole bodies of uninfected Biomphalaria glabrata snails and those experimentally infected with larval Schistosoma mansoni trematodes. Infected snails were analysed 8 weeks post-infection. Cohort snails that were left uninfected were analysed at the same time as the infected snails. Sixteen elements (aluminum, boron, barium, calcium, cadmium, copper, iron, potassium, magnesium, manganese, sodium, nickel, lead, selenium, tin and zinc) were found to be present in infected and uninfected whole bodies at concentrations above the detection limit of the ICP-AES analysis. Of these, calcium, cadmium, manganese and sodium were present in significantly higher amounts (Student's t-test, P<0.05) in whole infected versus whole uninfected snails. Variations in the present results compared with other studies reflect intrinsic differences in the larval trematode-snail systems used.

Effects of copper sulphate on in vitro encystment of the cercariae of Echinostoma caproni.

The effects of various concentrations of copper sulphate were studied on in vitro encystment of Echinostoma caproni in a Locke's-artificial spring water (ASW) (1:1) medium. Cercariae were killed in 10,000 mg l(-1) CuSO4 in Locke's-ASW (1:1) within 24 h and extruded cystogenous material to produce an abnormal cyst wall. The 'emergency response' of encystment to high concentrations of copper reported for Parorchis acanthus cercariae did not occur in E. caproni. Concentrations of 1000 mg l(-1) and 100 mg l(-1) CuSO4 in Locke's-ASW (1:1) also killed the cercariae without encystment by 48 h. A concentration of 10 mg l(-1) CuSO4 in Locke's-ASW (1:1) allowed for normal in vitro encystment within 48 h and these cysts were capable of excystation in a trypsin-bile salts medium.

Effects of snail size on encystment of Echinostoma caproni in juvenile Biomphalaria glabrata (NMRI strain) and observations on the survival of infected snails.

The effects of snail size on encystment of Echinostoma caproni cercariae in neonatal and juvenile Biomphalaria glabrata (NMRI strain) snails were studied. Encystment in neonatal (0.7-1.1 mm shell diameter) and juvenile (2-3 mm shell diameter) snails was compared 24 h post-infection (PI) following individual exposure of snails of each size to 1, 5, 10, 25 and 50 cercariae. Significantly more cysts were recovered from juveniles exposed to 1, 5, 10 and 50 cercariae than from neonatals with comparable exposure. Size of B. glabrata was a major factor in determining cyst burden in this planorbid. Survival of infected versus uninfected neonatals and juveniles was also examined for 7 days. Neonatals exposed to 10 cercariae showed a significant decrease in survival at 3, 6 and 7 days PI when compared to the uninfected controls. There was no significant decrease in the survival of juveniles exposed to 10 cercariae compared to uninfected controls at any time point. Snail size was a factor in mortality associated with echinostome cercarial penetration and encystment.

Effects of glucose on survival, infectivity and linear movement of the cercariae of Echinostoma caproni.

The effects of glucose in artificial spring water (ASW) on the survival, infectivity, and linear movement of Echinostoma caproni cercariae were studied. Cercariae maintained at 23 degrees C in 1% glucose in ASW (ASWG) or ASW alone, reached 50% survival at 26 and 23 h, respectively. All cercariae in ASWG and ASW were dead by 50 and 32 h, respectively. Infectivity to juvenile Helisoma trivolvis (Colorado strain) snails was significantly less for cercariae aged 16 h in ASWG compared to cercariae aged 16 h in ASW. Linear movement, i.e. the ability of cercariae to traverse a 1-cm radius, ceased at 16 and 20 h for cercariae maintained in ASWG and ASW, respectively. Glucose added to ASW extended the survival time of E. caproni cercariae but decreased their ability to infect snails or move in a linear direction.

Effects of temperature on survival, infectivity and in vitro encystment of the cercariae of Echinostoma caproni.

The effects of temperature on survival, infectivity and in vitro encystment of Echinostoma caproni cercariae in artificial spring water (ASW) were studied. Effects of aging cercariae in ASW at various temperatures showed that at 23 degrees C cercariae achieved 50% survival in 24 h, compared to 92 h at 12 degrees C. Cercariae aged in ASW at 28 and 37.5 degrees C showed 50% survival at 16 and 10 h, respectively. Cercariae aged at different temperatures for various times were used to infect juvenile Helisoma trivolvis (Colorado strain) snails maintained in ASW at 23 degrees C. Index of infectivity was based on counting encysted metacercariae in the snails at 8 to 12 h post-infection. Cercariae aged at 23, 28 and 37.5 degrees C showed 50% encystment at 6, 8 and 4 h, respectively. Cercariae aged at 4 degrees C showed 50% encystment in 10 h and cercariae aged at 12 degrees C showed 50% encystment beyond 16 h. Cercariae showed maximal longevity and infectivity in snails when aged at 12 degrees C in ASW. For E. caproni, as in other digeneans, the infective period of cercariae is markedly shorter than the maximal life-span at any given temperature. Studies on in vitro encystment of E. caproni cercariae in Locke's solution:ASW (1:1) showed that encystment was optimal at 23 degrees C (78% encystment) and that it declined to 44% at 28 degrees C and became almost nil (0.02%) at 12 or 37.5 degrees C.