Mizuki Hirata

Chromosomal differentiation of the Schistosoma japonicum complex

The C-banding pattern, location of telomere sequence and chiasma frequency of four species of the Schistosoma japonicum complex were compared with those of two African species, Schistosoma mansoni and Schistosoma haematobium. In the six species, C-banding patterns of seven autosomes and the two sex chromosomes (Z and W) showed relatively species-specific and geographical (Asian and African) differences. Particularly, a plausible pathway of alteration of chromosome 2 revealed a direction from the A-chromosome to the M- chromosome in terms of rearrangements of pericentric inversion and elimination of constitutive heterochromatin (AM inversion). This chromosome change suggested hypothetically that the S. japonicum complex is the original type, and the African species represents the derived type. Moreover, the mosaic construct of the Asian and African types in Schistosoma sinensium chromosomes prompted us to propose that the species might have been formed by hybrid speciation of the genomes of Asian and African species. Localisation of telomeric repeats enabled Asian and African schistosomes to be distinguished clearly by simple terminal location and by terminal and interstitial locations, respectively. Change of chiasma frequency in the S. japonicum complex might be caused by the reduction of interstitial chiasmate (Xi) in the larger chromosomes, 1 and Z (or W), and the change seems to have progressed to Japan from South East Asia. These data enabled us to predict a tentative evolutionary pathway of schistosomes at the cytogenetic level.

Schistosoma japonicum egg granuloma formation in the interleukin-4 or interferon-gamma deficient host.

The roles of interleukin (IL)‐4 and interferon (IFN)‐γ in Schistosoma japonicum egg granuloma formation were investigated in cercariae‐infected (infection model) or after implantation of laid parasite eggs (egg implantation model) in cytokine deficient mice. Two weeks after hepatic egg‐implantation, a markedly decreased mononuclear cell infiltration and lack of multinuclear cell formation were characteristic features in IL‐4 deficient mice. By 4 weeks (late stage), the cellular reactions around the eggs were negligible in the deficient mice. Compared to the controls, there was a drastic reduction in the production of the Th2 cytokines, IL‐4, IL‐5 and IL‐13. MCP‐1 levels were also significantly lowered. In mice experimentally infected with cercariae, granuloma cellularity in both the wild‐type and IL‐4 deficient mice at 45 days and 10 weeks postinfection was analogous to the egg implantation model at 2 and 4 weeks. Overall, the effects of IFN‐γ deficiency on granuloma induction differed markedly from the IL‐4 results. Two weeks after egg implantation, IFN‐γ deficient mice showed suppressed neutrophil response and hepatic necrosis with confluent mononuclear cell infiltration along the outer layer of granulomas. By 4 weeks, there was a decrease in cell infiltration, fibrosis and MCP‐1 production while IL‐10 production increased. While these early characteristic features for IFN‐γ deficiency were common to both the egg implantation (at 2 and 4 weeks) and cercariae infection model (at 45 days), there was a surprising difference, i.e. marked fibrosis was found in the late stages (at 10 weeks postinfection) of cercariae‐infected mice, but not in parasite egg implanted mice. Furthermore, while IL‐13 levels were unchanged, both MCP‐1 and IL‐4 production were significantly lower at 10 weeks in comparison with wild‐type. The present study clearly demonstrates the importance of both Th1 and Th2 cytokine responses in S. japonicum egg‐induced granuloma formation.

Relationships between Schistosoma malayensis and other Asian schistosomes deduced from DNA sequences.

At least three Schistosoma species can infect humans in South-East Asia. The most widespread of these is S. japonicum Katsurada, 1904 which may represent a species complex and occurs in many countries including China, Japan and the Philippines. The second species is S. mekongi Voge, Bruckner and Bruce, 1978 which is endemic to a small area near the junction of Laos, Cambodia and Thailand. Most recently described is S. malayensis Greer, Ow-Yang and Yong, 1988 from a restricted area of peninsular Malaysia. This is primarily a parasite of rats but has also been found in people Davis, on the basis of snail intermediate host phylogeny and biogeography, proposed that S. malayensis and S. mekongi are sister taxa relative to S. japonicum. The three studies on allozymes among these taxa also support this hypothesis.

Comparative analysis of hepatic, pulmonary, and intestinal granuloma formation around freshly laidSchistosoma japonicum eggs in mice

Hepatic, pulmonary, and intestinal granuloma formation was comparatively studied in mice implanted with freshly laidSchistosoma japonicum eggs. The liver and the lung showed similar kinetics of tissue reactivity, with the magnitude in the lung being of a significantly lower degree. When the footpad-swelling test was performed, the delayed-type hypersensitivity (DTH) response during hepatic granuloma formation was found to be significantly increased from the 28th day after egg implantation onward, whereas pulmonary granuloma formation showed a peak response at 14–21 days, suggesting differing kinetics of granulomatous reaction. In histologic analysis, the temporal infiltration of monocytes was revealed to correspond to the increase in the DTH response. During intestinal granuloma formation, cosinophil infiltration was the most marked feature. The present study demonstrates that throughout the course of reaction, cellular components participating in egg-granuloma formation differ greatly according to the tissues involved.

Induction of experimental murine granuloma formation againstSchistosoma japonicum eggs produced by in vitro ova deposition, in vitro tissue extraction, or lyophilization

Fresh eggs, laid bySchistosoma japonicum adult worms in vitro or recovered from tissue after trypsin digestion, as well as non-viable lyophilized eggs were injected into mice via the cecal vein. The freshly laid eggs induced a marked, maximal reaction at 2 weeks after implantation. This reaction was indistinguishable from that seen early in naturally infected mice. Lyophilized eggs induced granulomas only one-tenth the size of those formed around freshly laid eggs. Maximal granulomatous reactivity was not seen until the 4th week after implantation and the intensity of cellular reactivity and associated histopathological change was much lower than that observed with freshly laid eggs. Reactions against live, tissue-extracted eggs were quantitatively and temporally intermediate between those observed against laid and those surrounding lyophilized eggs. The results suggests that these differences in granuloma formation are due to variable quantities of locally produced cytotoxic materials and/or antigen that stimulate immune reactions of different intensities.

Effect of nitric oxide synthase inhibition on Schistosoma japonicum egg‐induced granuloma formation in the mouse liver

Nitric oxide (NO) plays diverse roles in a variety of pathological processes. We investigated the role of NO in Schistosoma japonicum egg‐induced granuloma formation in a mouse hepatic model. Immunohistological analysis revealed that there is the most intense and extensive inducible nitric oxide (iNOS) expression 2 weeks after egg implantation, and thereafter it decreased considerably with time. Treatment with nitric oxide synthase inhibitors, NIL (lN6‐ (iminoethyl)‐lysine) or Nω‐nitro‐l‐arginine methyl ester (l‐NAME), resulted in two different types of unusual granulomas at 2 weeks. One type showed suppressed fibrosis, while another showed foreign body‐type multinuclear cell formation which frequently appeared particularly when 50 µg/ml NIL was given. At 3 weeks following treatment, fibrotic granulomas with scanty peripheral cellularity was obvious. However, there were no apparent changes after this period (at 4 weeks). Cytokine analysis in NIL‐treated mice showed a significant increase of IL‐4 and IL‐13 production at 2 weeks. These findings indicated that nitric oxide contributes to granuloma development during the early stages, probably through the regulation of Th2 cytokine production.

Heme oxygenase1 (HSP-32) is induced in myelin-phagocytosing Schwann cells of injured sciatic nerves in the rat.

Schwann cells participate in myelin phagocytosis in the early stage of Wallerian degeneration, prior to the recruitment of macrophages. This is the first report that Schwann cells induce heme oxygenase‐1 (HO‐1), a 32‐kDa heat shock protein, only when they have transformed into myelin‐phagocytosing cells from myelinating cells (days 2–3) immediately after crush injury of rat sciatic nerves. Double immunofluorescent labelling for HO‐1 and transferrin receptors revealed that HO‐1‐immunoreactive Schwann cells also expressed transferrin receptors suggesting activation of iron metabolism. The transient induction of HO‐1 in Schwann cells may contribute to the adaptive function in an altered environment when the cells have lost contact with axons, and may play a crucial role in the ensuing regeneration.

DIFFERENT COURSES OF GRANULOMATOUS REACTIONS AROUND SCHISTOSOMA JAPONICUM EGGS IN THREE STRAINS OF MICE

Tissue reaction around Schistosoma japonicum eggs was studied after implantation into 3 strains of mice. When freshly laid eggs were implanted into the livers of C57BL/6, CBA/J, and BALB/c mice, the tissue reaction, which could be divided into 3 stages, i.e., abscess formation, inflammatory stage, and fibrous stage, differed in persistence and reactivity according to the strain. This was true especially during the later 2 stages. In BALB/c mice, the inflammatory stage ended earlier. In CBA/J mice, it continued for the longest period and the subsequent fibrosis was the most marked. In C57BL/6 mice, the period of persistence of this inflammatory stage was intermediate, but these mice showed the highest degree of inflammatory reaction. When footpad reactions were tested, the delayed-type hypersensitivity reaction was generally comparable with the morphometric analysis. In presensitized mice implanted with lyophilized eggs, tissue reactivity was analogous to the fibrous stage of freshly laid egg-implanted mice. In addition, there was an inverse relationship between antibody level and granuloma size, particularly at later reactions. The analysis of experimental granuloma formation presented in this report contributes to the study of stage-specific regulation.

Cytokine regulation in experimentally-induced Schistosoma japonicum egg granuloma formation

The formation of granulomas in host tissues in response to trapped Schistosoma japonicum eggs is central to the etiology of schistosomiasis. However, analysis of the host hypersensitivity reactions that result in granuloma formation, in schistosome infection, is not without difficulty. This is due, in part, to the fact that the parasites continuously deposit their eggs as clusters. In order to synchronize host reactions, we established an experimental model of hepatic granuloma formation whereby in vitro laid schistosome eggs are implanted directly into normal and cytokine-deficient mice livers. This model, validated by comparison with an infection model, was used to analyze cytokine regulation of granuloma formation around S. japonicum eggs. Combined models of implantation and cercarial infection were also studied. With special reference to IL-4, IL-13, IFN-gamma and IL-18, our in vitro schistosome egg implantation model has shed new light on the roles of cytokines in both the acute and chronic stages of schistosome egg-induced granuloma formation.

Neutropenia augments experimentally induced Schistosoma japonicum egg granuloma formation in CBA mice, but not in C57BL/6 mice

The present study was designed to investigate the role of neutrophils during the development of Schistosoma japonicum egg granulomas, in C57BL/6 and CBA mice. Laid eggs were implanted into the liver and monoclonal antibody, RB6‐8C5, was used to eliminate neutrophils. After daily antibody treatment between days 9 and 13 of egg implantation, both strains of mice showed a marked decrease in neutrophil infiltration and coagulative hepatocyte necrosis at 2 weeks. At 4 weeks, after antibody administration every other day between days 16 and 26, granuloma formation in C57BL/6 mice was not affected by the treatment, whereas CBA mice exhibited a significant increase of reactions. Neutropenia augmented the Th2 cytokine response (IL‐4, IL‐13 and IL‐5), but not for IFN‐γ at any time point examined and in either strain of mice. Higher levels of IL‐4 and IL‐13 were noted in CBA mice at early and late stages of granuloma formation, compared to C57BL/6 mice. There was also a striking difference in IL‐13 production between the two strains. Our results indicate that neutropenia is associated with a significant augmentation of S. japonicum egg‐induced granuloma formation in CBA mice, probably through increase in Th2 cytokines, however, the effects differ between early and late stages and between high and low responders.