Tsuneji Araki

Immunodiagnosis of human sparganosis mansoni by micro-chemiluminescence enzyme-linked immunosorbent assay

We compared a microcolorimetric enzyme-linked immunosorbent assay (colorimetric ELISA) and a microchemiluminescence enzyme-linked immunosorbent assay (chemiluminescence ELISA) for the detection of specific immunoglobulin G (IgG) in the serum of 9 patients with sparganosis mansoni and 9 healthy controls. The chemiluminescence ELISA was able to measure serum levels of specific IgG over a far wider range than the colorimetric assay, and its detection limit was at least 10-fold lower. An additional 5 sera from sparganosis patients and 5 more from healthy controls, together with sera from 28 patients with other parasitic diseases, were also examined by the chemiluminescence ELISA. All 14 patients with sparaganosis mansoni showed high levels of chemiluminescence (21,302 +/- 18,907 counts per second [cps]). All sera from the 14 healthy controls (1580 +/- 569 cps) and sera from 27 of the 28 patients with other parasites (4 with taeniasis saginata [1767 +/- 501 cps], 11 with diphyllobothriasis latum [1479 +/- 501 cps], 13 with cysticercosis cellulosae [2376 +/- 1437 cps]) showed chemiluminescence levels lower than those of any of the sparganosis mansoni patients. The exception was a patient with cysticercosis (5980 cps), who may have had a dual infection with Cysticercus cellulosae and Sparganum mansoni. Thus, the chemiluminescence ELISA demonstrated high sensitivity and specificity for human sparganosis mansoni.

A SPECTRUM OF ANTIBODY RESPONSE WITH TIME AFTER TRICHINELLA SPIRALIS INFECTION IN RATS

A chronology of class-specific antibody response against the Trichinella spiralis infection in Fischer rats was investigated. G-class antibodies against the cuticle inner layer(s), hypodermis, hemolymph, glycogen aggregates, discrete areas in genital primordial cells, intestinal gland cell granules, and cytoplasmic granules in the cords were detectable 2 wk after infection (the rapid-responding group), whereas G-class antibodies against the cuticle surface, stichocyte granules, and the esophagus-occupying substance were detected 6 wk after infection (the slow-responding group). M-class antibodies recognized a narrower spectrum of antigens than did G-class antibodies; M-class antibodies against hemolymph, cord granules, and intestinal gland cell granules were not detectable. M-class antibodies tended to decrease in titer with time after infection. This tendency was more striking with antibodies against the rapid-responding group than with those against the slow-responding group. This information sheds light upon antibody response against many antigenic components of T. spiralis muscle larvae.

Ultrastructural localization of antigenic substances in Trichinella spiralis

The in situ localization of antigenic substances inTrichinella spiralis muscle larvae was demonstrated at the subcellular level. Larvae recovered from mouse muscle were fixed with halfstrength Karnovsky fixative, dehydrated with alcohol, and embedded in LR White resin. Ultrathin sections were incubated with sera from infected Wistar rats and, subsequently, protein A-gold complex. The specificity of the immunostaining was confirmed by a control experiment. Positively immunostaining structures included the stichocyte granules, body cuticle, hindgut cuticle, hypodermis, hemolymph, glycogen aggregates, esophagusoccupying substance (EOS), midgut-occupying substance (MOS), brush border, cytoplasmic granules in the cord, intestinal gland cell granules, and discrete areas in the genital primordial cell. However, the esophageal cuticle, nucleus, nucleolus, mitochondria, rough endoplasmic reticulum, and muscle fibers were negative by immunostaining.

The morphology ofTrichinella spiralis: ultrastructural study of the mid- and hindgut of the muscle larvae

The ultrastructure of the gut ofTrichinella spiralis muscle larvae is comprehensively described, based on extensive observations. The midgut was composed of a single cell layer of epithelium over the basal lamina, the brush border, the septate junction, prominent glycogen aggregates, and other common cell organelles. The epithelial cells of the midgut were basically uniform, but the lumen presented a remarkably diversified appearance. In the ampullar portion, the midgut accommodated an amorphous substance of medium electron density, the middle portion was collapsed, and the terminal portion again dilated, but without any particular contents. The mid- and hindgut were devoid of muscle cells that could be responsible for the peristaltic movement of the gut.

Immunocytolocalization study of the external covering of Trichinella spiralis muscle larva.

The antibody-binding sites of the muscle larva of Trichinella spiralis were investigated by immunogold staining on the ultrathin sections of LR white resin. The antibodies, which were produced in the course of T. spiralis infection in rats, specifically bound to the inner layers of the body cuticle and the cuticle of the hindgut, but not to the cuticle of the esophagus. This is the first report that reveals the antigenic nature of the inner layers of the external coverings of T. spiralis larva.

Immunocytochemical localization of antigens in adult worms of Trichinella spiralis recognized by Fischer rats.

We demonstrate the tissue localization, in adultTrichinella spiralis, of antigens recognized by Fischer rat sera at 32 weeks postinfection. Immunodominant antigens were located in a wide variety of tissues, including type 1 stichocyte granules, stichocyte cytoplasm, the canalicular tree, hemolymph, hypodermis, hypodermal glands, cord cytoplasm, intestinal-gland cell granules, membranous structures in the midgut epithelium, midgut-occupying substance, brush border, hindgut epithelial cytoplasm, hindgut cuticle, vaginal cuticle, epithelial cytoplasm of the female genital tract, microvilli and discrete areas of the ovum, embryo sheath, intersperm space, discrete areas in immature sperm, small granules and cup-shaped membrane structures of sperm, and exocrine granules in the seminal vesicle and ejaculatory duct. A small amount of antigen was located in the inner layers of the genital portion of the body cuticle. The precise localization of antigens in adult worms should form a basis for better analysis ofT. spiralis-related immunology.

Morphology of the alimentary tract of Trichinella spiralis muscle larvae with emphasis on the esophagus

This study was designed to provide a comprehensive description of the ultrastructure of the esophagus ofTrichinella spiralis muscle larvae. Although the esophagus exhibited basically the same structure throughout its entire length, being composed of a single cell-layered epithelium, the basal lamina, and the cuticle, some morphological diversity was observed, depending on the level of sectioning. The upper esophagus, devoid of a muscular sheath, was equipped with myofilamentous cytoplasm and a thick cuticle. The middle and lower esophagus was surrounded by the muscular sheath on the basal side and thin cuticle on the luminal side. The cytoplasm usually contained glycogen, ribosomes, and mitochondria. The presence of an amorphous substance in the lumen of the esophagus is reported for the first time. It was completely homogeneous or finely granular and always devoid of any substructure.

Direct evidence that the cuticle surface of Trichinella spiralis muscle larvae shares antigenicity with stichocyte α-granules and the esophagus-occupying substance.

Antibodies against the cuticle surface of Trichinella spiralis muscle larvae were purified by means of immunoaffinity chromatography and incubated with ultrathin sections of muscle larvae. Major constituents of the parasite reactive with the purified antibodies included the cuticle surface, stichocyte alpha-granules, and the esophagus occupying substance of the muscle larvae. Thus the present data suggest that the cuticle surface is an antigenically different entity from the cuticle inner layers and its origin is likely stichocyte alpha-granules.

Detection of Soluble T Cell Receptor-Releasing Cells by ELISPOT Assay

A specific and sensitive enzyme-linked immunospot (ELISPOT) assay has been developed for the detection and enumeration of soluble T cell receptor (TCR)-releasing cells. Using this method, we readily detected at the single cell level the release of soluble TCR by living T lymphoma cells (MT-2 and HSB-2) but not by human B lymphoma cells (DAKIKI), mouse hepatoma cells (MH134) and dead MT-2. Furthermore, distinct spots in MT-2 cell culture were not visualized using several monoclonal antibodies against antigens unrelated to TCRs as a primary antibody. The specific and quantitative detection of soluble TCR-releasing cells using ELISPOT assay will certainly provide a valuable tool to better characterize soluble TCRs and their relationship to immune regulation and a number of diseases.

Trichinella spiralis: Antigenic substances associated with the alimentary tract

Postembedding immunogold and immunoperoxidase staining methods revealed that substances occupying the lumen of the esophagus and the midgut were antigenic for both Wistar rats and humans. Specificity of the alimentary tract-associated antigen was assessed by reacting these substances with a panel of serum pools from patients with non-Trichinella helminth infections, including anisakiasis, paragonimiasis, gnathostomiasis, fascioliasis, dirofilariasis, and trichuriasis. The substances had no, or negligible, cross-reactivity among the serum pools with the only exception of severe trichuriasis serum. Cytochemical staining revealed that the substances are PAS positive and are stained red by azan, and the midgut-occupying substance was equipped with exposed concanavalin A-binding sites. The present data suggest the alimentary tract-associated antigen can be purified by lectin affinity chromatography and may be used as a fairly specific antigen for immunodiagnostic purposes.