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The inhibitory effects of L-type Ca2+ channel antagonists on Na cholate-induced in vitro excystment (CIIE) of Paragonimus ohirai metacercariae were studied. At concentrations of 10 microM, nicardipine and nimodipine inhibited CIIE completely and by approximately 92%, respectively. Nitrendipine and (+/-)-verapamil inhibited CIIE by about one half and one third, respectively. Nifedipine and diltiazem did not inhibit CIIE significantly. At higher concentrations, nitrendipine at 20 microM completely inhibited CIIE, and (+/-)-verapamil at 40 microM inhibited CIIE by 93%. Nifedipine and diltiazem inhibited CIIE only slightly and little, respectively, even at 40 microM. Complete inhibition by nicardipine at 10 microM required preincubation of metacercariae with the antagonist for 15 min. The inhibitory effects of nicardipine and nimodipine were reversible, and most of the nimodipine-treated metacercariae could excyst within 1 h after being washed, but the nicardipine-treated ones started to excyst 1 h after washing. Nicardipine suppressed the active movement of encysted juveniles evoked by Na cholate, whereas nimodipine did not suppress this significantly. These results suggested that L-type Ca2+ channels appeared to be involved in CIIE of P. ohirai metacercariae and that the inhibitory effect of the channels was due primarily to factors other than the inhibition of muscular activity, probably involving the secretion and release of enzymes lytic against the metacercarial cyst wall.

An enzyme-linked immunsorbent assay (ELISA) using worm extract antigens from lung flukes of Paragonimus westermani provided good sensitivity to sera from patients with paragonimiasis westermani but high cross-reactivity with sera from most fascioliasis patients and some patients with onchocerciasis or clonorchiasis. To improve the specificity, we tested an ELISA using fluke cysteine proteinases as antigens. Cysteine proteinases were partially purified from the excretory/secretory products of P. westermani by 40-75% ammonium sulfate fractionation, hydrophobic chromatography, and arginine affinity chromatography. An ELISA using the enzyme preparation not only had increased sensitivity to paragonimiasis westermani sera but also reduced cross-reactivity with the fascioliasis, onchocerciasis, and clonorchiasis sera to negligible levels. The reactivity of the ELISA to paragonimiasis miyazakii sera was similar to that of paragonimiasis westermani sera. A proteinase preparation from P. ohirai, which can be obtained easily from infected rats, provided similar results. Therefore, the ELISA using cysteine proteinases of Paragonimus could not distinguish the parasite species with which patients were infected, but it is a valuable assay with which to immunodiagnose paragonimiasis even when the proteinases are prepared from nonhuman species.

An IgM monoclonal antibody (mAb) that reacted with the gut epithelium and luminal contents of adult Paragonimas obirai was prepared from a mouse immunized with the excretory/secretory (ES) products of the parasite. The mAb reacted with the same sites not only of immature worms and newly excysted juveniles of P. ohirai , but also of adult worms of other Paragonimus species, such as P. westermani and P. miyazakii . The mAb did not react with adult antigens prepared from any other helminth parasites. Thus, the mAb is a genus-specific antibody against the gut-associated antigen. Immunoblotting revealed that two antigens with molecular masses (M r s) of 27 and 29 kDa were present in the ES products of adult P. ohirai , whereas four antigens with M r 27, 29, 36, and 38 kDa were detected in adult worm extract. Partially purified fractions containing acidic and neutral cysteine proteinases have 27 and 29 kDa protein molecules reactive with the mAb. The possibilities that the 36 and 38 kDa antigens may be precursors of these proteinases are discussed.

The tegumental glycocalyx of excysted juvenile (EJ) of Paragonimus ohirai was immunobiochemically characterized using a monoclonal antibody (MS-Mab). HPLC gel filtration showed that the antigens detected by two-site ELISA had a molecular weight of greater than or equal to 2 x 10(6) Da (dextran marker). On reduced SDS-PAGE, the glycocalyx antigen retained in the stacking gel was cleaved into several much smaller antigens after pronase treatment. The antigenic activity of the glycocalyx was stable in two-site ELISA to heat and acid treatments, but sensitive to alkali, periodate, base/borohydride, and pronase treatments. Precipitin formation in immunodouble diffusion between MS-Mab and EJ crude antigen was inhibited only by two monosaccharides: galactose and N-acetylgalactosamine. The purified glycocalyx bound strongly to PNA lectin, fairly well to RCA120 lectin, and slightly to SBA lectin, but not to Con A, WGA, UEA-1, DBA, or LFA lectins. Exo-beta-galactosidase treatment increased SBA binding, whereas it decreased PNA binding. PNA was observed to strongly bind to the body surface of living EJ. The antigenic activity of the glycocalyx was remarkably lost by incubation with exo-beta-galactosidase and O-glycanase. The glycocalyx was reactive with sera of P. ohirai-infected rats, and its reactivity was remarkably reduced by O-glycanase treatment. The ELISA level was higher in sera at an early stage of infection than in a late one. These studies show that the EJ tegumental glycocalyx is antigenic in infection, a marked, high molecular weight glycoprotein containing antigenic O-linked sugars, and that the sugar epitope is at the nonreducing terminal of the O-linked sugars and is composed of galactose and N-acetylgalactosamine.

Non-specific suppression of the immune response was investigated in Brugia pahangi-infected Lewis rats. The proliferative response of peripheral blood lymphocytes or splenic non-adherent cells to mitogens was significantly reduced by B. pahangi infection. The degree of hyporesponsiveness of splenic non-adherent cells to mitogens was comparable between microfilaremic and non-microfilaremic animals. The suppressed proliferative response of splenic non-adherent cells was restored by blocking with anti-CD8 monoclonal antibody. After separation of T cells into CD4+ and CD8+ subpopulations, only CD8+ T cells from B. pahangi-infected rats suppressed the proliferative response of normal spleen cells to concanavalin A. CD8+ T cells from normal rats had no suppressive effect. On the other hand, the proliferative response of CD4+ T cells to concanavalin A was comparable between normal and infected rats. These results suggest that CD8+ T cells participate in the non-specific suppression of immune response in experimental filariasis.

The inhibitory effects of various ion channel blockers were examined on in vitro excystment of Paragonimus ohirai metacercariae induced by a bile salt, sodium cholate. At a concentration of 10 microM, bepridil, a non-selective Ca(2+) channel blocker, completely inhibited in vitro excystment, whereas TEA, lidocaine, and R(+)-IAA-94, channel blockers against K(+), Na(+) and Cl(-) ions, respectively, benzamil, an Na(+)/H(+) and Na(+)/Ca(2+) ion exchanger blocker, and R(+)-DIOA, a [K(+), Cl(-)] cotransporter inhibitor, did not. Considering the previous result that Ca(2+) ionophores are also efficient inducing factors for in vitro excystment of P. ohirai metacercariae and the present result, bile salts appear to induce the excystment of P. ohirai metacercariae through evoking the Ca(2+) channels of target cells within the metacercarial juveniles.

Ca2+ ionophores (A23187 and ionomycin) induced in vitro excystment of Paragonimus ohirai metacercariae, whereas Na+ ionophore (monensin) and K+ ionophores (nigericin and valinomycin) did not. The effect of A23187 on in vitro excystment was dependent on the concentration of the ionophore. A high concentration of 1 microM or more induced rapid excystment, but resulted in the death of metacercarial juveniles. The appropriate concentration of 0.2 microM induced in vitro excystment by 5 h after incubation with little or no damage to the juveniles.

Monoclonal antibodies against infective third-stage larvae (L3) of Brugia pahangi were generated from mice immunized with L3 antigens. The monoclonal antibodies were L3 stage-specific or stage-nonspecific. A BpG1 monoclonal antibody (IgG1 subclass) showing L3 stage-specificity was examined in detail. BpG1 recognized the surface of B. pahangi L3 and also reacted with the surface of Brugia malayi L3 but not with the surface of filarial worms of other genera, such as Acanthocheilonema viteae and Litomosoides carinii. BpG1 promoted cellular adhesion to the surface of B. pahangi L3. BpG1 bound on living L3 was shed but the shedding rate was relatively slow. The surface antigen recognized by BpG1 had a molecular weight of 58 kDa. It was stable to heat and periodate treatments but sensitive to trypsin digestion and was released from living L3 by SDS but not by Triton X-100 or CTAB. Preincubation of L3 with BpG1 significantly reduced the recovery rate of worms compared with the preincubation with a monoclonal antibody (IgG1 subclass) against the inner tissues of B. pahangi L3 or control supernatant of P3U1 myeloma cells. This result suggests that the antigen containing the BpG1 epitope may be one of the targets of a protective immune response against Brugia infection.

The allergens of the lung fluke Paragonimus westermani were localized by indirect immunostaining in adult fluke sections using pleural exudates from 3 patients with P. westermani. Immunostaining performed by using pleural exudate with the highest level of specific IgE revealed that the P. westermani major allergen (or allergens) was located in the gut epithelium and luminal contents and that minor allergens were in the tegument and parenchyma. The antigens recognized by specific IgG were located at various sites including those recognized by specific IgE. Paragonimus westermani-specific IgE cross-reacted with only the gut of 2 other Paragonimus species, Paragonimus miyazakii and Paragonimus ohirai. The major allergen in the gut also was recognized by the other 2 pleural exudates. These results indicate that the substance present in and secreted from the gut is not only a major allergen but is also a common allergen among Paragonimus species.

Ikeda T. , Oikawa Y. and Fujita K. 1982. Kinetics and localization of parasite-specific IgE in Paragonimus ohirai -infected rats. International Journal for Parasitology 12: 395–398. In Wistar rats infected with Paragonimus ohirai (P.O.), P.O. -specific IgE responses of the mesenteric and mediastinal lymph nodes and spleen were determined by homologous adoptive cutaneous anaphylaxis (ACA) assay since P.o. -specific IgGa was not detected by either 2 h or 4 h PCA. Intraperitoneal (i.p.) infection with metacereariae elicited similar patterns of ACA response in the three lymphoid tissues examined, with the mediastinal lymph node giving the highest response. ACA positive cells were detected 2 weeks after infection, peaked at 3 weeks and then declined. These kinetics of ACA responses nearly paralleled the kinetics of serum P.o. -specific IgE titre. In intrapleural infection with metacereariae, on the other hand, the mediastinal lymph node gave a high ACA response comparable to the lymph node in i.p. infection, but the mesenteric lymph node and spleen gave negligible ACA responses. In infection established by i.p. transplantation of 4–5-week-old worms, only the mediastinal lymph node of the three lymphoid tissues responded and its response was at a low ACA level. The level of serum P.O. -specific IgE was much lower in the above two infections than in i.p. infection with metacercariae.

In Guatemala, an indirect hemagglutination (IHA) test for onchocerciasis, performed with a crude saline extract of Onchocerca volvulus and blood samples taken on filter paper, showed a high level of sensitivity and specificity. IHA titers of blood samples from the ear lobe taken on filter paper and of sera obtained by venipuncture showed a high correlation.

An epidemiological survey for onchocerciasis was carried out in the San Vicente Pacaya area of Guatemala. A total of 2,153 inhabitants were examined by a single skin snip, and 664 (30.8%) were positive for microfilariae. Slit lamp examination of the anterior chamber of the eye revealed microfilariae in 6.2%; the positive rate for microfilariae in the anterior chamber was proportional to the microfilarial density in the skin. The altitude-dependent character of onchocercal infection was shown in this mountainous area, suggesting that transmission occurs principally between 600 and 1,300 m. Of 1,217 persons examined simultaneously by skin snip and by palpation for nodules, 587 were positive by one or both methods; 101 infections were detected by onchocercal nodules only and these were usually in children or persons living in areas of low endemicity. It was concluded that both the skin snip and a search for nodules are required for accurately determining the prevalence of onchocerciasis in Guatemala., American Journal of Tropical Medicine and Hygiene, 28(1), pp.67-71; 1979

In seven patients with paragonimiasis westermani, parasite-specific IgE and IgG levels in sera and pleural effusion were determined by an enzyme-linked immunosorbent assay (ELISA). The sensitivity to adult excretory-secretory (E-S) antigen was compared with the sensitivity to whole worm extract antigen, and the former was more sensitive in both an IgE-ELISA and IgG-ELISA. Both parasite-specific IgE and IgG could be detected by ELISA at levels much higher than those in control subjects using E-S antigen. When specific IgE and IgG levels in sera and pleural effusion of individual patients were compared, the latter had higher values. The difference between levels of specific IgE in pleural effusion and serum did not correlate with that of specific IgG. These results indicate that specific IgE and IgG antibodies form locally, i.e., in the lung, and that pleural effusions from patients with paragonimiasis are more suitable than serum for immunodiagnosis.

Once organisms entered the alveolar macrophages the growth of these organisms was similar, independent of the animal species. To our knowledge it has not been determined whether adult rats are more susceptible to Toxoplasma after intranasal and pulmonary inoculation than after intraperitoneal inoculation. Differences in alveolar and peritoneal macrophages have been observed also in mice; alveolar macrophages may exert a stronger antibacterial action than the peritoneal exudate macrophages (Walker, 1976. In Immunobiology of the macrophages, D. S. Nelson (ed.). Academic Press, pp. 91-111). It would be of interest to determine whether the rat peritoneal macrophages could protect rat alveolar macrophages. It is not clear whether the low multiplication of Toxoplasma in peritoneal cells of rats is associated with killing or whether this is merely an inhibition of rate or multiplication. Ultrastructural studies may clarify this problem. This work was supported in part by the Vicerrectoria de Investigaci6n, Universidad de Costa Rica, and the Consejo Nacional de Investigaciones Cientificas y Tecnolo6gicas (CONICIT). We thank Aleida Quir6s and Zaida Umafia for their excellent secretarial assistance and Fabio Camacho for his concerned care of the laboratory animals.

A sero-epidemiological study of onchocerciasis was carried out with the IHA test in Guate- mala. In the endemic area, 94.8% of 191 subjects with microfilariae, 82.1% of 172 cases with onchocercal nodules, and 22.5% of236 cases without either microfilariae or nodules gave positive reactions in the IHA test. On the other hand, the positive rate was 3.4% and 2.0% in each of 2 nonendemic populations. A follow-up study of reactors in the IHA with neither microfilariae nor nodules showed that 11 (42%) of 26 cases were microfilaria positive 6-7 mo after the first examination. A clear correlation was found between the results of the IHA test and skin biopsy in surveyed communities. The age distribution curve of the IHA test was closely associated with skin biopsy although the former was higher. IHA titers rose in proportion to microfilarial density. A sex-related difference was evident in the IHA positive rate in the subjects from medium and low endemic areas, but little difference was shown in individuals from a higher endemic area. Although onchocerciasis can be diagnosed by detecting microfilariae in the skin, skin biopsy is not sensitive, especially in light or early stages of infection. Therefore, a stan- dardized immunological test may offer a pref- erable method of diagnosis and for epidemi- ological studies. The immunodiagnosis of onchocerciasis has been attempted by various workers using the skin test and complement fixation test (as reviewed by Kagan, 1963), im- munofluorescent antibody test (Lucasse, 1962; Lucasse and Hoeppli, 1963; Woodruff and

In rats infected with Paragonimus ohirai (P.o.), IgE response against the parasite was influenced by both the site of metacercarial inoculation and the age of the inoculated worms, whereas the IHA response was not influenced by these variables. Intraperitoneal infection and oral infection with 10 metacercariae induced a high level of P.o.-specific IgE (maximum PCA titer of 1:128 and 1:111, respectively). In contrast, intrapleural infection induced little IgE (1:496, maximum titer). Thus migration of the parasite in the peritoneal cavity and the liver, or the former alone, was an important event in the production of IgE, because intrapleurally inoculated metacercariae normally develop into mature worms without migrating into the abdominal cavity. Intraperitoneal transplantation of five immature or adult worms produced significantly lower IgE levels and correlated with the age of the transplanted worms. Four-week-old worms were observed to induce negligible or no P.o.-specific, IgE antibody. The stage-specific, IgE resonse observed in infections established by transplantation appears to be partly dependent on migration of the parasite in the abdominal cavity. Worms in the pleural cavity may have suppressed IgE production.