Yoshitsugu Matsumoto

Yeast Glucan in the Cyst Wall of Pneumocystis carinii

Ultrastructurally, the cyst wall of Pneumocystis carinii consists of an electron-dense outer layer, an electron-lucent middle layer, and an innermost plasmalemma. This is similar in appearance to the cell wall of some yeasts, e.g. Saccharomyces cerevisiae, which consists of an outer dense layer of mannan, a middle lucent layer of beta-1,3-glucan (yeast glucan) and an innermost plasmalemma. The cyst wall of P. carinii, as well as the cell wall of S. cerevisiae, can be labeled by a variety of methods which stain polysaccharides, such as Gomoris methenamine silver (GMS) and by Aniline blue, a dye which selectively stains beta-1,3-glucan. The treatment of P. carinii cysts with Zymolyase, which the key enzyme is beta-1,3-glucan laminaripentaohydrolase, results in lysis of the outer 2 layers of the cyst wall and the loss of positive staining by both GMS and Aniline blue. The lysis of elements of the cyst wall of P. carinii is achieved under the same conditions and concentration at which Zymolyase lyses the outer 2 layers of the cell wall of viable cells of S. cerevisiae. These observations indicate that a major component of the cyst wall of P. carinii is beta-1,3-glucan.

Light microscopical study of Blastocystis spp. in monkeys and fowls

An investigation of a Blastocystis species obtained from several species of monkeys and fowls was conducted to clarify the morphology of the organism, using light microscopical techniques including Giemsa, Heidenhain iron hematoxylin, Trichrome stains, iodine mount and phase-contrast microscopy. A comparison was made with Blastocystis hominis from humans. Blastocystis spp. were found in 15 out of 26 monkeys and in all of 12 fowls (10 chickens and 2 ostriches) examined. The behaviour of the parasites in the bowels was also examined, using paraffin-embedded sections. Microscope examination of the lumen contents at death revealed the organism in the caecum of monkeys and fowls. The organisms from faeces, cultures and the lumen contents of the caecum of humans, monkeys and fowls were similar, except for variations in the size and contents of the central vacuoles, which occupied the centre of the organisms.

Light-microscopical appearance and ultrastructure of Blastocystis hominis, an intestinal parasite of man*

A study on Blastocystis hominis was undertaken for the purpose of clarifying the morphology of the organism using the following techniques; Giemsa, Heidenhain Iron Hematoxylin, and DAPI stains, phase-contrast microscopy, and transmission- and scanning-electron microscopy. Microscopic examinations of the lumen fluids aspirated at the endoscopical examination revealed the habitation of B. hominis in the lower ileum and cecum of a patient. When examined light-microscopically, organisms from stool materials, cultures, and aspirated intestinal lumen contents of a patient showed morphological resemblances to each other except for variations in size. Vacuolated cells, which were spherical in shape and characteristically had a large central vacuole and a narrow rim of cytoplasm containing nuclei and some inclusions, were the only form of the organism observed in this study, although the contents of the vacuole notably varied. DAPI stains clearly revealed the nucleus and the possible mitochondrion in the narrow rim of cytoplasm. Phase-contrast microscopy of fresh material prepared with physiological saline was recommended for diagnosis. When examined electron-microscopically, the organisms were coated with a capsule that was composed of fine filamentous materials. All the organisms contained a central vacuole although the contents of it varied considerably. The cytoplasm gave the organism a signet ring appearance and contained cristate mitochondria, a great number of ribosomes, Golgis apparatuses, cytoplasmic microtubules, and nuclei with a nucleolus. Very few of the ultrastructures are those that would be expected of a yeast. Recent occurrences of B. hominis infection in Kyoto City, Japan, during a two-year period (June 1983 to August 1985) were also reported.

Variations in egg size ofTrichuris trichiura

In patients infected withTrichuris trichiura, large eggs as well as more typical eggs were frequently found in the feces; these large eggs have been attributed to human infection byT. vulpis. To clarify whether the female worms laying the large eggs wereT. trichiura orT. vulpis, we examined the morphology of adult worms and the size of whipworm eggs obtained from a patient and from several domestic dogs. Adult femaleT. trichiura worms were easily and quickly distinguished from those ofT. vulpis by their convoluted vaginal tracts and uteri. Eggs obtained from the uteri ofT. trichiura were of two sizes, with approximately 80% being of the smaller variety (57×26 μm) and 20%, the larger (78×30 μm). These percentages were about equal to those found the feces of the patient.T. vulpis uterine eggs were slightly larger (82×39 μm) than the large eggs ofT. trichiura. The distribution of the 2 populations of eggs in 30 adult femaleT. trichiura was as follows: 1 worm had only large eggs, 14 had only the typical smaller eggs, and 15 had both varieties in their uteri.

Pneumocystis carinii: electron microscopic investigation on the interaction of trophozoite and alveolar lining cell.

The habitat, behaviour of Pneumocystis carinii, and the interaction between the organisms and host alveolar cells were studied electron microscopically in corticosteroid-treated rats. The trophozoites of P. carinii used to line up along the type I alveolar epithelial cells at the early stage of infection. No organism was found being adhere to type II cells which have numerous microvilli on their surface. By adding tannic acid to the fixative we could preserve the alveolar lining layer, in which the organisms were found all the time. Therefore P. carinii is not exposed to alveolar air but is living in the liquid layer. Those findings are important in considering nutrition of the organism as well as pathogenicity to the host. The trophozoites and type I cells were in close contact mostly with their smooth surface rather than with tubular expansions of the organism. The cytoplasm of type I cell was often found protruded along the pellicle of the organism as if it surrounded the organism. Phagocytosis of P. carinii by alveolar macrophage and neutrophil was often seen in the alveolar lumen when the infection advanced. Sometimes the trophozoites were found sub- and intra- epithelially . Those trophozoites seemed alive from their ultrastructures. However, it remained unsolved, whether those figures were the resultants of active invasion of the trophozoite or of being surrounded by the epithelial cells.

Pneumocystis infection in macaque monkeys: Macaca fuscata fuscata and Macaca fascicularis

Retrospective examination of lungs from 128 monkey necropsies was attempted for Pneumocystis infection using special stains, including toluidine blue-O and Gomoris methenamine silver nitrate. Four Japanese monkeys (7.7%), Macaca fuscata fuscata, and one crab-eating monkey (7.7%), Macaca fascicularis, were found to have Pneumocystis infection. The organism was found in young and infant animals. At the time of death, one infant and two young monkeys were debilitated and/or emaciated. Pneumocystis infection was considered an important lesion which could have caused reduced respiratory function in two of the Japanese monkeys, but constituted only an incidental finding in the others.

Bromodeoxyuridine labeling studies on the proliferation of intestinal mucosal mast cells in normal and athymic rats

The proliferation of mucosal‐type mast cells (MMC) in rat small intestine was studied using a bromodeoxyuridine (BrdU)‐labeling method. After 24‐h cumulative injections of BrdU into adult SD rats, 3.5% of MMC were labeled, while only 0.3% and 0.1% of mast cells were labeled in back skin and ear respectively. From the results, it was concluded that MMC division occurred more than 10 times as frequently as the division of skin mast cells. Similar results were obtained in athymic adult rats (F344/N Jcl‐rnu) in which the number of MMC was similar to that in heterologous animals. Thus, thymic factor(s) or T cells may not have an important role in MMC division in normal states. When SD rats were infected with Nippostrongylus brasiliensis, vigorous proliferation of MMC was brought about 13 to 15 days after infection. At that period, 40% of MMC were labeled by a single injection of BrdU, and 85% of MMC were labeled by 9‐h cumulative injections of BrdU, with the result that most MMC rapidly proliferated in the intestinal mucosa during this period. Mitotic figures of MMC were sometimes observed. On the contrary, hyperplasia of MMC was not observed in athymic rats infected with nematodes. Therefore, MMC hyperplasia after nematode infection is dependent on thymic factor or T cells, and its mechanism is different from that of MMC division in normal states, in which thymic factor(s) or T cells are not essential.

Mouse spleen cell-derived toxoplasma growth inhibitory factor: its effect on toxoplasma multiplication in the mouse kidney cells.

When Toxoplasma tachyzoites were inoculated into normal kidney cell monolayers, they multiplied in the cells and further causing cell rupture. However, when Toxoplasma immune lymphokines (LKs) was added to normal kidney cells, the intracellular multiplication of the tachyzoites was inhibited remarkably; particularly, in the case in which the tachyzoites were exposed to immune fresh serum prior to inoculation into the cells. Toxoplasma growth inhibitory factor (Toxo-GIF) in LKs, with a m.w. of 30,000 to 40,000 was found to be contained in LKs-II or MIF-I fraction after Sephadex G-100 gel filtration. LKs collected from Balb/c mice inhibited tachyzoite multiplication in ICR-JCL normal mice kidney cells, suggesting that LKs activity did not show mouse-strain specificity. In contrast, the addition of Toxoplasma immune serum to infected cells had no effect on the intracellular multiplication of Toxoplasma and also did not prevent cell-to-cell spread. However, when extracellular tachyzoites were exposed to Toxoplasma immune fresh or heat-inactivated immune serum, especially to the former, at 37 degrees C for 30 min, their active penetration into the kidney cells were extremely inhibited and their subsequent multiplication in the cells were almost totally inhibited within 24 h as compared with those exposed to normal serum.

Production of a monoclonal antibody with specificity for the pellicle ofPneumocystis carinii by hybridoma

This study reports the establishment of cloned hybridomas between mouse myeloma cells (P3-X63-Ag8, 6·5·3) and spleen cells from BALB/c mice immunized with lung homogenates of nude mice (BALB/c nu/nu) heavily infected withPneumocystis carinii. A hybridoma subclone, designated 1E12-8, produced antibodies of an IgG1 subclass. Using indirect immunofluorescent antibody techniques, the monoclonal antibody was found to be directed toward the pellicle antigen of air-driedP. carinii both in trophozoite and cyst forms, and to recognize theP. carinii antigen not only from nude mice but also from rats. The antibody did not cross-react with other components of the infected host lung tissue and showed little cross-reactivity with fungi examined. The monoclonal antibody should be useful in the isolation and identification of corresponding antigenic substances ofP. carinii and may provide a useful tool in epidemiology, taxonomy, and diagnosis of this organism.

Further light microscopic studies on morphology and development of Pneumocystis carinii.

In order to add more advance in light microscopic investigation of P. carinii, phase contrast microscopy partly followed by wet giemsa stain and semiultrathin section of the lungs embedded in JB-4 plastic were studied. In phase contrast microscopy, small and large sized trophozoites of P. carinii were clearly recognized. Although movement of trophozoite was not found, rhythmic movement of intracystic bodies with filopodia was often seen in mature cyst. Those living organisms were then directly stained with Giemsa by infiltrating under the coverglass. Thus the organism could be investigated both in unstained and stained conditions. It is noticed with interest that 8 intracystic bodies seem to fill up the cavity of cyst when cell division is completed, then they liberate and become independent into spherical bodies, followed by banana-shaped or amoeboid forms with motility. An emphasis was done that semiultrathin section made from JB-4 plastic embedded lungs was quite useful for investigation of P. carinii infection. Several sizes of mononuclear thin-walked trophozoites, mature and immature cysts, and empty cysts were more clearly distinguished than any other light microscopical method ever reported.