D. Janssen

Serologic recognition of hydatid cyst antigens using different purification methods

The specificity and sensitivity of enzyme-linked immunosorbent assays (ELISA) and Western immunoblot assays in detecting antibodies in serum from patients suffering cystic hydatid disease (Echinococcus granulosus) are compared using either crude antigen preparations (total sheep hydatid fluid and homogenates of protoscoleces), purified fractions enriched in Antigens 5 and B, and glycoproteins from hydatid fluid. Polyprotein bands of 12-14, 20, and 34 kDa, when purified from hydatid fluid by applying changes in the ionic strength, yielded a sensitive (95%) immunodiagnostic test that was also extremely specific (100%) when assayed with sera from noninfected humans and from patients suffering from other parasitic diseases. However, subjecting hydatid fluid to chromatography through a concanavilin A column rendered a 42 kDa band that was sensitive (95%) as well as highly specific (100%) for hydatidosis. Therefore purification procedures can strongly affect the diagnostic value of antigens with identical electrophoretic behavior in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

Specific recognition of hydatid cyst antigens by serum IgG, IgA using Western blot

Diagnosis of hydatid disease in humans relies on the detection of specific antibodies against antigens of the matacestode from Echinococcus granulosus. The specificity and sensitivity of current immunological techniques based on specific serum IgG rely on the way antigens are purified. We used Western immunoblotting to detect specific IgG, IgE, and IgA antibodies in serum from patients with hydatid disease using either crude antigen preparations (total hydatid fluid), purified fractions enriched in Antigens 5 and B, and glycoproteins from hydatid fluid. Depending on whether crude HF or purified antigen fractions were used, IgG and IgE recognized specifically low‐to‐medium MW bands between 12 and 42 kDa. IgA recognized specifically 110 kDa band in crude hydatid fluid and in the glycoprotein fraction of hydatid fluid, and a 42 kDa band in all antigen samples used. Besides the advantage of detecting specific IgA in crude hydatid fluid, these results offer the possibility of simplifying future immunological tests if specific secretory IgA can be similarly detected. J. Clin. Lab. Anal. 11:154–157, 1997.

IMMUNOMODULATION BY HYDATID CYST FLUID TOXIN (ECHINOCOCCUS GRANULOSUS)

Since the experimental infection by hydatid cysts (Echinococcus granulosus) in mice causes immunomodulation of the host, the effects of hydatid fluid (HF) and fractions of HF were compared in vitro and in vivo. Fractions of HF were obtained using ammonium sulphate precipitation, chloroform/methanol extraction and thin‐layer chromatography (TLC). HF proved to be toxic to murine peritoneal macrophages in vitro, and when macrophages were incubated with the different fractions of HF, most toxicity was found in a single TLC‐purified fraction with an adjuvant‐like effect on the production of specific antibodies against bovine albumin and human red blood cells in mice. Treatment of mice with the toxin caused a drop in the percentage of peripheral blood lymphocytes. Flow‐cytometric analysis showed that T‐cells from toxin‐treated mice had lower membrane‐CD3, CD4 and CD8 density, and had higher percentages of CD8+ splenocytes and CD4+ thymocytes expressing CD25. The toxin caused a down‐regulation of CD4 and CD8 expression on thymocytes in vitro, that was dependent on the presence of macrophages. The results may attribute to these toxins a role in the host‐parasite relationship of hydatidosis.

Changes in T-cell subpopulations in mice during prolonged experimental secondary infection with Echinococcus granulosus

Balb/c mice were infected intraperitoneally with protoscoleces ofEchinococcus granulosus. After 15 months of infection, and by means of flow cytometry, the expression of T-cell markers CD3, CD4, and CDS on T cells from peripheral blood, spleen, and thymus was analyzed and compared with that of age-matched controls. Infected mice had higher percentages of CD3+, and CD4+ cells in peripheral blood, and higher percentages of CD8+ cells in the spleen, when compared with control mice. CD4+ and CD8+ cells in peripheral blood and CD8+ cells in thymus also showed higher percentages of expression of interleukin-2 receptor. The results infer a role for interleukin-2 in experimental secondary echinococcosis.

Copper-zinc superoxide dismutase from Ascaris suum (Nematoda): purification and characterization.

Copper-zinc superoxide dismutase fromAscaris suum (Nematoda) was purified in a new, more efficient, and faster manner. The process included differential centrifugation, fractionation with ammonium sulfate, and sodium dodecyl sulfate-polyacrylamide electrophoresis, yielding a 340-fold purification (specific activity of 47 units/mg). Optimal storage conditions, optimal pH range, thermostability, molecular weight and ultravioltet-visible absorption spectrum of the enzyme are described, and a new enzymatic model for pharmacological screening is suggested.