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.

Back to work more quickly after an inguinal hernia repair.

Tension-free hernia repair plus recovery expectancy statements return personnel to work more quickly. On the day of primary inguinal hernia repair, patients were given statements about their likelihood of returning from convalescent leave after 7 days and performing nonstrenuous work. Similar statements were given to them by telephone at 72 hours postoperatively and at a 1-week follow-up appointments. Seventy-four percent of the 73 patients returned to nonstrenuous work within 7 days, and 90% returned to strenuous work within 30 days. In this small sample, 385 work days were saved from the Navy's recommended 14 days of convalescent leave. By combining recovery expectancy statements with an effective surgical procedure, it is possible to avoid prolonged convalescence, thereby enhancing military readiness.

Self-hypnosis training and captivity survival.

In February and March, 1973, 566 U.S. military prisoners (POWs) were released from North Vietnam. These men had been POWs for a period of time between 2 months and 9 years, with a mean incarceration of 4.44 years. They had faced physical and psychological stress similar to that experienced by POWs from previous wars: starvation, disease, inadequate shelter, lack of medical care, interrogations and torture (Deaton, Burge, Richlin & Latrownik, 1977; Mitchell, 1991). By definition, such prison conditions constituted a traumatic experience (Deaton et al., 1977). However, a unique stress for our POWs in North Vietnam was the additional trauma of solitary confinement. This paper reviews the coping and "time killing" activities of U.S. Navy Vietnam POWs who experienced solitary confinement and tortuous interrogation. This paper also reports the physical and psychological adjustment of our POWs following their release from captivity. Suggestions are made regarding the revision of the curriculum for captivity survival training programs such as Survival, Evasion, Resistance, and Escape (SERE) school.

Crystallization, structural determination and analysis of a novel parasite vaccine candidate: Fasciola hepatica glutathione S-transferase.

Glutathione S-transferases (GSTs) represent the major class of detoxifying enzymes from parasitic helminths. As a result, they are candidates for chemotherapeutic and vaccine design. Indeed, GSTs from Fasciola hepatica have been found to be effective for vaccinating sheep and cattle against fasciolosis. This helminth contains at least seven GST isoforms, of which four have been cloned. The cloned isoforms (Fh51, Fh47, Fh7 and Fh1) all belong to the mu class of GSTs, share greater than 71% sequence identity, yet display distinct substrate specificities. Crystals of Fh47 were obtained using the hanging drop vapour diffusion technique. The crystals belong to space group I4122, with one monomer in the asymmetric unit, which corresponds to a very high solvent content of approximately 75%. The physiological dimer is generated via a crystallographic 2-fold rotation. The three-dimensional structure of Fh47 was solved by molecular replacement using the Schistosoma japonicum glutathione S-transferase (Sj26) crystal structure as a search model. The structure adopts the canonical GST fold comprising two domains: an N-terminal glutathione-binding domain, consisting of a four-stranded beta-sheet and three helices whilst the C-terminal domain is entirely alpha-helical. The presence of Phe19 in Fh47 results in a 6 degrees interdomain rotation in comparison to Sj26, where the equivalent residue is a leucine. Homology models of Fh51, Fh7 and Fh1, based on the Fh47 crystal structure, reveal critical differences in the residues lining the xenobiotic binding site, particularly at residue positions 9, 106 and 204. In addition, differences amongst the isoforms in the non-substrate binding site were noted, which may explain the observed differential binding of large ligands. The major immunogenic epitopes of Fh47 were surprisingly found not to reside on the most solvent-exposed regions of the molecule.

Protection of cattle against Fasciola hepatica infection by vaccination with glutathione S-transferase.

Glutathione S-transferase (GST) from the liver fluke Fasciola hepatica was assessed as a vaccine immunogen in cattle in a number of immunological adjuvants. Significant reductions in fluke burdens (49-69%) were only observed in cattle vaccinated with GST in Quil Alsqualene Montanide (SM) and PLG microspheres in SM but there was no correlation between anti-GST IgG titres and protection. In separate experiments, animals vaccinated with GST in Quil AlSM were still significantly protected (48%, P < 0.05) 6 months after boosting and no significant differences in protection were seen when the metacercarial challenge was given over 1 month instead of as a single bolus. Inhibition of GST enzyme activity in vitro by cattle antisera did not correlate with reduced fluke burdens.

Basic science training in psychopharmacology. How much is enough?

Training psychologists to administer psychotropic medication will require acquisition of a unique knowledge base and set of skills that are generally not components of graduate education in psychology. Nevertheless, the current level of basic science training in graduate education in psychology is substantial and should, with minor modification, allow adequate preparation for students to enter into specialized training to prescribe. The direct provision of psychopharmacology requires psychologists to demonstrate competencies in addition to those required in the general provision of psychological services. Such competencies are perhaps best taught at the postdoctoral level. The authors argue that all curricula training professional psychologists should be able to train psychologists capable of practicing as independent, full-fledged health care providers.

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.

Biochemical analysis of recombinant glutathione S-transferase of Fasciola hepatica.

Four cDNAs encoding GST (rGST1, rGST7, rGST47 and rGST51) of Fasciola hepatica were expressed in Escherichia coli and the rGST proteins purified for biochemical analyses. The rGST proteins are 95% pure as indicated by Coomassie staining of proteins separated by SDS-PAGE. Molecular sieving by HPLC infers that, like the native protein, the rGST proteins form homodimers under non-denaturing conditions. The rGST proteins are recognised by antisera raised to the native GST of F. hepatica. All four rGST proteins from F. hepatica actively conjugate glutathione to the universal substrate, 1-chloro-2,4-dinitrobenzene. The activity of the rGSTs was also measured for substrates which have been shown to have partial specificity for the Alpha, Mu or Pi classes of mammalian GSTs (trans-4-phenyl-3-buten-2-one, ethacrynic acid), for substrates known to be products of lipid peroxidation (trans-2-nonenal, trans,trans-2,4-decadienal) and for epoxy-3-(p-nitrophenoxy)-propane (EPNP), a known substrate for the theta class of GST. No rGST were active with EPNP. rGST47 and 51 showed activity with the other four substrates. rGST7 was active with three substrates whereas rGST1 showed relatively low activity with all substrates except trans,trans-2,4-decadienal. The sensitivity of the rGST activity to inhibition by the GST inhibitors triphenyltin chloride and bromosulphophthalein also varied among the rGSTs with rGST1 showing a 800-fold difference in sensitivity between the inhibitors. These results show that F. hepatica expresses a family of GST isoenzymes which exhibit unique substrate and inhibitor profiles.

Vaccination of sheep against Fasciola hepatica with glutathione S-transferase. Identification and mapping of antibody epitopes on a three-dimensional model of the antigen.

The glutathione S-transferases (FhGST) of the liver fluke Fasciola hepatica have been identified as novel vaccine candidates that protect sheep against a fluke infection. With the use of overlapping peptides covering the predicted amino acid sequences of four FhGST cDNAs, we have defined the linear epitopes recognized by polyclonal antibody from sheep vaccinated with FhGST. Dominant and minor epitopes were found to be present on all four of the sequences although some epitopes were shown to be specific to particular FhGST. A high percentage of the FhGST peptides were found to be antigenic although considerable variability in response to the peptides was observed among the animals. This analysis was extended to the IgG1 and IgG2 response at the peptide level. Based on the recently solved crystal structure of the rat mu-class GST 3-3, a three-dimensional model of one of the FhGST sequences was generated that allowed the predicted spatial localization of defined epitopes. Most epitopes were localized on regions of high flexibility and accessibility. A comparison of epitopes on FhGST with the B cell epitopes on Sm28, a 28-kDa GST from Schistosoma mansoni, has found few similarities. There was no correlation between an antibody response to linear peptide epitopes and the level of protection induced in sheep by vaccination with FhGST.

Primary sequence heterogeneity and tissue expression of glutathione S-transferases of Fasciola hepatica.

Glutathione S-transferases (GSTs) from Fasciola hepatica have been purified by glutathione affinity chromatography. Two closely migrating species of Mr 26,000 and 26,500 were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and several species resolved by two-dimensional gel analysis, indicating substantial heterogeneity among the GSTs. N-terminal amino acid sequencing revealed one core sequence containing three polymorphisms, whereas the sequence of GST peptides implied a minimum of three different GSTs. The amino acid sequence data assigned the F. hepatica GSTs to the mu class of GSTs with high similarities to these proteins in other helminths and mammals. The native GSTs of F. hepatica appeared to behave as dimers as determined by molecular sieving chromatography. The observation that the GSTs of F. hepatica are heterogeneous in sequence and behave as dimers in the native state suggest that these isoenzymes may exhibit considerable functional heterogeneity which may be of importance to the parasite. Immunocytochemical studies suggest that the main source of GST in F. hepatica are the parenchymal cells and peripheral tissues of the parasite. Some extracellular GST is associated with the lamellae of the intestinal epithelium. The identification of an intestinal GST is unique among trematodes studied to date.

Fasciola hepatica: immunoprecipitation analysis of biosynthetically labelled antigens using sera from infected sheep.

The antigenicity of the biosynthetically labelled somatic and excretory/secretory proteins of adult Fasciola hepatica was investigated over 20 weeks of an infection with F. hepatica in sheep. The antibody response was initially detected by ELISA 2 weeks after infection, and was sustained at this level for the remainder of the infection. Immunoprecipitation analysis indicated that a large number of proteins were recognized by the sheep, with several dominant antigens occurring in the 29-31 kilodalton molecular weight range. No differences were found between the antigens recognized by sheep in the early or late stages of infection suggesting that there are similarities between the antigens of the immature and mature forms of the parasite.

Glutathione S-transferase. Novel vaccine against Fasciola hepatica infection in sheep.

The potential of GST as a vaccine candidate against liver fluke infection in ruminants was studied by vaccinating sheep (n = 9) with GST purified from adult worms of Fasciola hepatica and challenging with 500 F. hepatica metacercariae. The immunization induced a high antibody response to GST in contrast to the poor or undetectable response to this Ag observed in naturally infected sheep. Throughout the trial, the progress of the fluke infection was monitored by measuring RBC hemoglobin levels, the extent of liver damage and the fecal egg output in the sheep. This analysis indicated that a subpopulation (n = 4) of the GST vaccinated animals exhibited no anemia, reduced liver damage and a lower mean fecal egg count relative to the infected control group suggesting a lower fluke burden in these animals. Worm burdens in the livers of the GST vaccine group (107 +/- 22) were 57% lower than in the infected control group (250 +/- 25). The subpopulation of the GST vaccine group demonstrated a 78% reduction in mean worm burdens relative to the control group. These results show that GST of adult F. hepatica is a novel Ag that can significantly protect sheep against liver fluke infection. The results suggest that the immune response to GST is directed to the juvenile worm reducing the number of worms that can establish in the liver of the vaccinated animals.

Characterization of interferons produced by peripheral blood mononuclear cells from healthy subjects in response to Corynebacterium parvum or poly I: poly C.

The biological significance of acid labile interferon alpha is presently unknown. We examined the putative production of acid labile interferon in vitro from human peripheral blood mononuclear cells induced with Corynebacterium parvum or poly I: poly C. Both agents induced up to 1200 IU/ml interferon, and the interferon was 80 to 90% acid labile. The interferons were typed by antibody neutralization of their antiviral activity. Contrary to previous reports, C. parvum induced predominantly interferon gamma, which is normally acid labile, whereas poly I: poly C induced an acid labile interferon alpha activity with characteristics similar to those of acid labile interferon alpha reported in serum in certain human diseases.