Yoichiro Horii

Selective effector mechanisms for the expulsion of intestinal helminths

In the middle of the era of molecular biology, much less attention is paid to in vivo phenomena. However, carefully designed experimental systems in vivo still can provide valuable information as to the mechanisms underlying the establishment and maintenance of host‐parasite relationships. In this review we describe the advantage of using concurrent infections with appropriately chosen combinations of different genera or different maturation stages of parasites to segregate the cellular responses of the host. By means of simple experimental approaches we have found that mucosal mast cells and goblet cells, both of which have long been considered as non‐specific effectors, are in fact highly selective and specific effector cells of the host defence mechanisms capable of acting on the establishment and the expulsion of intestinal helminths.

Mucosal mast cells and the expulsive mechanisms of mice against Strongyloides venezuelensis

The possible importance of mucosal mast cells in the expulsive mechanisms of mice against Strongyloides venezuelensis was examined. After a primary infection by subcutaneous inoculation with various doses into C57BL/6 mice, about 50% of the initial dose of infective larvae (L3) became adult worms and, regardless of the dose of infection, they were completely expelled by Day 12 with similar kinetics. Intestinal mastocytosis at the time of expulsion was comparable among groups given different doses of infection. A kinetic study after infection with 2000 L3 in C57BL/6 mice revealed that mastocytosis started from Day 8, rapidly reached a peak on Day 12, and then gradually decreased. The strongest mastocytosis was observed in the upper one sixth of the small intestine where the majority of adult worms parasitized. Over 80% of mast cells induced by the infection were located in the intestinal epithelial layer. When mast cell-deficient W/Wv and their normal littermate +/+ mice were infected with 1000 L3, expulsion was significantly delayed in W/Wv mice, though adult worms were eventually expelled by Day 18 in W/Wv mice. Delayed expulsion as well as defective mast cell responses of W/Wv mice were completely restored by bone marrow grafting 10 weeks prior to infection. These results show that, like S. ratti infection, intestinal mucosal mast cells are important in causing expulsion of S. venezuelensis.

Intestinal Intraepithelial Lymphocytes Sustain the Epithelial Barrier Function against Eimeria vermiformis Infection

ABSTRACT Eimeria spp. are intracellular protozoa that infect intestinal epithelia of most vertebrates, causing coccidiosis. Intestinal intraepithelial lymphocytes (IEL) that reside at the basolateral site of epithelial cells (EC) have immunoregulatory and immunoprotective roles against Eimeria spp. infection. However, it remains unknown how IEL are involved in the regulation of epithelial barrier during Eimeria sp. infection. Here, we demonstrated two distinct roles of IEL against infection with Eimeria vermiformis, a murine pathogen: production of cytokines to induce protective immunity and expression of junctional molecules to preserve epithelial barrier. The number of IEL markedly increased when oocyst production reached a peak. During infection, IEL increased production of gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α) and decreased transforming growth factor β (TGF-β) production. Addition of IFN-γ and TNF-α or supernatants obtained from cultured IEL from E. vermiformis-infected mice reduced transepithelial electrical resistance (TER) in a confluent CMT93 cell monolayer, a murine intestine-derived epithelial line, but antibodies against these cytokines suppressed the decline of TER. Moreover, TGF-β attenuated the damage of epithelial monolayer and changes in TER caused by IFN-γ and TNF-α. The expression of junctional molecules by EC was decreased when IEL produced a high level of IFN-γ and TNF-α and a low level of TGF-β in E. vermiformis-infected mice. Interestingly, IEL constantly expressed junctional molecules and a coculture of EC with IEL increased TER. These results suggest that IEL play important multifunctional roles not only in protection of the epithelium against E. vermiformis-induced change by cytokine production but also in direct interaction with the epithelial barrier when intra-EC junctions are down-regulated.

Persistent infection of Strongyloides venezuelensis and normal expulsion of Nippostrongylus brasiliensis in Mongolian gerbils, Meriones unguiculatus, with reference to the cellular responses in the intestinal mucosa

The kinetics of daily faecal egg count, worm burdens, and intestinal cellular responses were examined in Mongolian gerbils after infection with either Strongyloides venezuelensis or Nippostrongylus brasiliensis alone, or concurrently with both parasites. The results show that, both in individual and concurrent infections, S. venezuelensis infection persisted for over 10 weeks and elicited a gradual increase in number of mast cells in the jejunal mucosa. On the other hand, N. brasiliensis worms were expelled by 3 weeks in association with goblet cell hyperplasia. These results suggest that effector/regulator cells involved in worm expulsion are different and highly selective depending on the genus of intestinal helminths.

Identification of tissue-embedded ascarid larvae by ribosomal DNA sequencing.

Polymerase chain reaction (PCR) was applied to identify tissue-embedded ascarid nematode larvae. Two sequences of the internal transcribed spacer (ITS) regions of ribosomal DNA (rDNA), ITS1 and ITS2, of the ascarid parasites were amplified and compared with those of ascarid-nematodes registered in a DNA database (GenBank). The ITS sequences of the PCR products obtained from the ascarid parasite specimen in our laboratory were compatible with those of registered adult Ascaris and Toxocara parasites. PCR amplification of the ITS regions was sensitive enough to detect a single larva of Ascaris suum mixed with porcine liver tissue. Using this method, ascarid larvae embedded in the liver of a naturally infected turkey were identified as Toxocara canis. These results suggest that even a single larva embedded in tissues from patients with larva migrans could be identified by sequencing the ITS regions.

Morphological and molecular identification of two Paragonimus spp., of which metacercariae concurrently found in a land crab, Potamiscus tannanti, collected in Yenbai Province, Vietnam

Paragonimosis is an important food-borne zoonosis especially in Asian countries. Among Paragonimus species, Paragonimus westermani followed by P. skrjabini complex are the major pathogens for human paragonimosis in Asia. In addition, P. heterotremus is an important pathogen in southern China and the Indochina Peninsula and is the only proven species to cause human paragonimosis in Vietnam. During a recent survey in Yenbai Province in northern Vietnam, we found small and large types of Paragonimus metacercariae often concurrently in mountainous crabs, Potamiscus tannanti. Adult worms from those small and large metacercariae were obtained separately by experimental infection in dogs and cats. Morphological and molecular phylogenetic study based on sequences of ITS2 and a part of CO1 genes were performed for the identification of small and large metacercariae and their adults. The results showed that small metacercariae and their adults are completely identical with P. heterotremus in morphology and molecular genetic profiles. In contrast, large metacercariae and their adults have some morphological similarities with P. skrjabini and P. harinasutai, but are unidentifiable from each other by morphology alone. Molecular phylogenetic tree analyses on ITS2 and CO1 genes revealed that large metacercariae and their adults were grouped in the same clade and different from any known Paragonimus species. Although they share the same ancestor with P. skrjabini complex, their genetic distance was considerably different from two other known subspecies, P. skrjabini skrjabini and P. skrjabini miyazakii. Our results provide a new insight on the phylogeny of the genus Paragonimus.

Histochemical and Cytological Characterizations of Mucosal and Connective Tissue Mast Cells of Mongolian Gerbils (Meriones unguiculatus)

Mast cells in jejunum, skin, and tongue of Mongolian gerbils (Meriones unguiculatus) were examined in terms of their histochemical, enzyme-histochemical, and ultrastructural properties. When glycosaminoglycans of mast cells in jejunum and tongue were characterized by measuring the critical electrolyte concentration, the salt concentration at which 50% of mast cells could be stained was > 1.0 M for those in the jejunum and also in the tongue, indicating that mast cells in both sites contained heparin. Enzyme-histochemical study revealed that mast cells in the jejunum of Mongolian gerbils were strongly positive for chymase and tryptase, whereas those in tongue and skin were essentially negative for both proteases. By electron microscopy, granular morphology and distribution of surface microfolds of mast cells in the jejunum were different from those in the ear skin. After in vivo stimulation with compound 48/80, however, mast cells in both sites showed remarkable degranulation. From these results, although Mongolian gerbils also have distinct mast cell subsets, their phenotypic properties are different from those of the previously known mast cell subsets of other animals.

Effect of testosterone on the susceptibility of C57BL/6 mice to infection with Brugia pahangi with reference to inflammatory cell response.

Effects of testosterone on the susceptibility and inflammatory cell responses of C57BL/6 mice infected intraperitoneally with Brugia pahangi larvae were examined. On day 15 postinfection, female mice showed significantly greater resistance than did males, and peritoneal cell responses (lymphocytes, macrophages, and eosinophils) were great in females. Castration of highly susceptible male mice increased their resistance and peritoneal cell responses to the level of female mice; whereas, castration of female mice did not affect the susceptibility and cell responses. Furthermore, testosterone treatment at a physiological dose in the castrated male mice or a pharmacological dose in female mice suppressed resistance and inflammatory cell responses. These results suggest that male sex hormone, testosterone, but not female sex hormone has a regulatory role in the susceptibility and cellular response of C57BL/6 mice to infection with B. pahangi, and it causes differences between sexes in susceptibility.

Clindamycin in the Treatment of Babesia gibsoni Infections in Dogs

This report examines the effectiveness of clindamycin for the treatment of babesiosis in dogs (n=10) experimentally infected with Babesia gibsoni (B. gibsoni). Clindamycin (25 mg/kg body weight, per os, q 12 hours for 14 days) gradually reduced parasitemia levels and induced morphological changes that indicated degeneration of parasites (e.g., segmentation; size reduction; localization in the cell limbic and/or torn state of the nucleus; and swelling, decrease, or disappearance of the cytoplasm) in the majority of dogs. Clindamycin treatment reduced the clinical symptoms characteristic of Babesia infection, including anemia, anorexia, and listlessness. Clindamycin might be useful as a medicine for treatment of B. gibsoni infection.

Plasma anti-Müllerian hormone as a biomarker for bovine granulosa-theca cell tumors: Comparison with immunoreactive inhibin and ovarian steroid concentrations

Granulosa-theca cell tumors (GTCTs) are the most frequently reported ovarian tumors in cattle. Clinically, GTCTs could be confused with other ovarian abnormalities; therefore, the only definitive diagnosis for such tumors is histopathology of a biopsy from the affected ovary. However, this is an invasive technique and unsuitable for farm conditions. As a result, the key aim of this study was to evaluate the diagnostic value of anti-Müllerian hormone (AMH), a glycoprotein hormone that is synthesized exclusively by ovarian granulosa cells, as a sensitive noninvasive biomarker for diagnosing GTCTs in cattle. To achieve this aim, we conducted two experiments. In experiment 1, four clinically healthy Japanese Black cows had blood samples taken daily over one estrous cycle to characterize their AMH profiles throughout the estrous cycle. Additionally, single blood samples were collected from 21 cyclic cows to clarify the physiological range of AMH. In experiment 2, cows with histologically confirmed GTCT (GTCT group, n = 9) and cows affected with cystic ovarian disease (COD group, n = 8) had one blood sample taken before extraction of the tumorous ovary or therapeutic treatment for the COD. Blood samples (n = 105) from cyclic cows (n = 25) in experiment 1 were assigned as a physiologically cyclic group (PC group). Plasma AMH, immunoreactive inhibin (ir-INH), estradiol-17β (E2), testosterone (T), and progesterone (P4) concentrations were assayed in all samples. In experiment 1, the mean plasma AMH concentration was 0.09 ± 0.003 ng/mL and did not show substantial fluctuation throughout the estrous cycle. In experiment 2, plasma AMH, ir-INH, and E2 concentrations were significantly elevated in the GTCT group in comparison with the PC group; among these parameters, only the AMH concentrations were significantly higher in the GTCT group than in the COD group. The area under the receiver operating characteristic curve of AMH for diagnosis of GTCT was 0.99 and was significantly higher than that of ir-INH (P < 0.001) and E2 (P < 0.01). Moreover, the AMH at a cutoff point of ≥0.36 ng/mL had the highest diagnostic accuracy (99.2%), sensitivity (100%), and specificity (99.1%) compared with the other tested parameters. In conclusion, plasma AMH concentration is probably a more reliable and sensitive biomarker for bovine GTCTs than the concentrations of ir-INH or ovarian steroids.