Alternative Activation of Macrophages in Mice Peritoneal Cavities and Diaphragms by Newborn Larvae of Trichinella spiralis.

BACKGROUND: Trichinellosis is a serious zoonosis with a worldwide distribution. Fecund adult worms in the intestine release newborn larvae (NBL) that enter the general circulation from 4 days post infection (dpi). Alternatively activated macrophages in the peritoneal cavities and the diaphragms in Trichinella spiralis infected mice have been reported. However, a role of newborn larvae is poorly understood. METHODS: The total numbers of peritoneal macrophages in mice infected with 500 muscle-stage larvae were counted during early infection and then total RNA was extracted. Peritoneal macrophages from uninfected C57BL/6 mice were incubated with IL-4 or LPS as a control, or co-cultured with live NBL, and peritoneal macrophages were obtained from mice injected with live or frozen dead NBL into peritoneal cavity. Total RNA was extracted from these macrophages. Two types of gene expression, classical and alternative activation, were examined in the macrophages and diaphragms of the infected mice using semi-quantitative reverse transcription-PCR. RESULTS: The number of peritoneal macrophages in T. spiralis infected mice increased significantly. mRNA peak expression of alternative activation markers, Ym1 and arginase-1 (Arg1), was confirmed in the peritoneal macrophages and in diaphragm of mice around 15 dpi, while mRNA expression of classical activation markers, TNFα, IP-10, and iNOS was not detected. Injection of live NBL into the peritoneal cavities induced mRNA expression of Ym1 and Arg1 in the peritoneal macrophages of mice 9 dpi. However, dead NBL did not induce such gene expression. Alternative activation was not detected in the peritoneal macrophages co-cultured with NBL in vitro. CONCLUSION: Gene expression of alternative activation makers, Ym1 and Arg1, was confirmed in the peritoneal macrophages and diaphragms of mice infected with T. spiralis. However, gene expression of classical activation markers was not detected. Live NBL induced an alternative activation of peritoneal macrophages in vivo, but not in vitro.

Molecular cloning and characterization of plerocercoid-immunosuppressive factor from Spirometra erinaceieuropaei.

A platyhelminth, Spirometra erinaceieuropaei, belonging to the class Cestoda, causes human sparganosis, and infection with its larva results in subtle inflammation in the body of its host. We previously reported the purification of a glycoprotein, plerocercoid-immunosuppressive factor (P-ISF) from the excretory/secretory products of S. erinaceieuropaei plerocercoids that may be involved in immuno-modification. We determined the sequence of P-ISF from the N-terminal and the internal 10 amino acids of P-ISF using degenerate PCR and 5'- and 3'-RACE methods. The putative gene encoding P-ISF was 1443 bp long and the gene contained 10 exons and 9 introns in a genomic DNA of size 5205 bp. P-ISF consists of 480 amino acids including the N-terminal signal peptide sequence, and has two unknown domains,-cestoda cysteine-rich domains (CCDs) and a fibronectin type III domain between the two CCDs. All cysteine residues were conserved in the two CCDs, which shared 62% amino acid identities. Homologous analysis revealed that the CCDs were homologous with an unknown protein of Diphyllobothrium latum. To produce specific antibodies, we expressed recombinant P-ISF (rP-ISF) using wheat germ protein synthetic system. P-ISF was localized in the sub-cutaneous tissues and the parenchymal tissues of plerocercoids. Transcription of P-ISF was detected only in plerocercoid stage, but not in adult stage. Western blotting also showed a band in plerocercoide stage but not in adult. The rP-ISF did not suppress nitrite production in RAW 264.7 cells stimulated with LPS, and this might be due to lack of carbohydrate chains in the recombinant protein.

Suppression of IP-10/CXCL10 gene expression in LPS- and/or IFN-γ-stimulated macrophages by parasite-secreted products.

T helper (Th)2 polarized immune responses are characteristically dominant in helminth infections. The gene expression of interferon (IFN)-γ-inducible protein 10 (IP-10/CXCL10), which promotes Th1 responses, in mouse macrophages stimulated with lipopolysaccharide (LPS) and/or IFN-γ was suppressed by excretory/secretory (ES) products of Spirometra erinaceieuropaei plerocercoids. ES products suppressed LPS- and/or IFN-γ-induced transcriptional activities of a luciferase reporter gene under the control of a 243-bp fragment of the IP-10 gene promoter/enhancer, which contains an IFN-stimulated response element (ISRE) and two κB elements. Consistent with this result, ES products inhibited ISRE-dependent heterologous promoter activities and LPS- or IFN-γ-induced ISRE-binding activity. ES products also suppressed LPS-induced IFN-ß gene expression. Furthermore, ES products suppressed nuclear factor (NF)-κB RelA (p65)-dependent transcriptional activity, whereas ES products had no effect on the κB-binding activity. These results suggest that ES products suppress the IP-10 gene expression by inhibiting the ISRE- and RelA-dependent transcriptional activities in mouse macrophages.

Diplogonoporiasis in Japan: genetic analyses of five clinical isolates.

Infection of the whale tapeworm Diplogonoporus balaenopterae (Diphyllobothriidae) is occasionally found in humans, especially among Japanese. In the present study, we analysed the nucleotide sequences of the 18S rDNA, ITS1 and cox1 genes of the immature and mature proglottids of Diplogonoporus species recovered from five Japanese patients. The nucleotide sequences of 18S rDNA, ITS1 and cox1 showed little, if any, intraspecific divergence. Phylogenetic analyses of several diphyllobothriid species revealed a close relationship of Diplogonoporus isolates with the cetacean tapeworm Diphyllobothrium stemmacephalum. The results suggest that the genus Diphyllobothrium is paraphyletic and raise a question regarding the validity of the genus Diplogonoporus.

A glycoprotein from Spirometra erinaceieuropaei plerocercoids suppresses osteoclastogenesis and proinflammatory cytokine gene expression.

Various parasites modify the immune-reactions of the host. We have previously shown that crude excretory/secretory (ES) products from plerocercoids of Spirometra erinaceieuropaei, the plerocercoids of which cause sparganosis in humans, suppress the expression of tumor necrosis factor (TNF)-alpha and IL-1beta in lipopolysaccharide (LPS)-stimulated macrophages. As osteoclasts are cells of the monocyte/macrophage lineage, we hypothesised that ES products might suppress receptor activator of nuclear factor kappaB ligand-induced osteoclastogenesis. Crude ES products from plerocercoids suppressed osteoclastogenesis, judged by tartrate-resistant acid phosphatase (TRAP)-positive multinuclear cell counting, and the mature osteoclast-specific gene expression (calcitonin receptor and TRAP). Second, we purified the inhibitory factor for osteoclastogenesis from the crude ES products. The factor was a trypsin-sensitive glycoprotein and had a relative molecular mass of 90 kDa. The glycoprotein, plerocercoid-immunosuppressive factor, from crude ES products could suppress the gene expression of TNF-alpha, IL-1beta and NO synthesis in LPS-stimulated RAW264.7 macrophages.

eNOS Glu298Asp polymorphism and hypertension in a cohort study in Japanese.

BACKGROUND: Some recent case-control association studies have suggested negative and positive relationship between Glu298Asp (the substitution of aspartic acid for glutamic acid at amino acid position 298) polymorphism of the endothelial nitric oxide synthase (eNOS) gene and hypertension. To investigate whether the Glu298Asp polymorphism of the eNOS gene affects the incidence of hypertension, a retrospective cohort study was performed. METHODS: The baseline data among Japanese workers in Shimane Prefecture, Japan, were obtained at regular health examination in 1992, and a retrospective cohort study was performed to analyze the influence of Glu298Asp polymorphism on the incidence of hypertension in 1998. RESULTS: The incidences of Glu298Glu, Glu298Asp, and Asp298Asp genotypes in the subjects were 86.4%, 12.6% and 1.1%, respectively. The risk ratios of Glu298Asp and Asp298Asp against Glu298Glu for the incidence of hypertension by single variance analysis were 0.830 in total subjects [95% confidence interval (CI) 0.474-1.452], 0.596 in subjects 20-39 years old (95% CI; 0.207-1.717), and 0.915 in subjects 40-59 years old (95% CI; 0.464-1.805). The risk ratios of Glu298Asp and Asp298Asp against Glu298Glu for the incidence of hypertension by multiple variance analysis adjusted for sex, BMI, serum total cholesterol, serum high-density lipoprotein (HDL) cholesterol, fasting glucose, cigarette smoking, drinking habits, eating habits, and exercise in 1992 were 0.750 in total subjects (95% CI; 0.421-1.335), 0.505 in subjects 20-39 years old (95% CI; 0.170-1.496), and 0.873 in subjects 40-59 years old (95% CI; 0.434-1.757). CONCLUSION: These results suggested no association between the Glu298Asp gene polymorphism and the incidence of hypertension in this selected population.

The lack of relationship between an endothelin-1 gene polymorphism (Ala288ser) and incidence of hypertension: a retrospective cohort study among Japanese workers.

BACKGROUND: Some case-control association studies revealed the relationship between some endothelin-1 (ET-1) gene polymorphisms and blood pressure. Because no report was available about the relationship between any ET-1 gene polymorphism and incidence of hypertension, we examined the relationship between novel ET-1 gene polymorphism (G862T / Ala288Ser in exon 5) and incidence of hypertension by a retrospective cohort study. METHODS: The subjects were Japanese workers at a company in Shimane Prefecture in Japan. The polymorphism with genome DNA extracted from the blood of the workers was analyzed using the polymerase chain reaction confronting two pair primers method. According to the results of two regular health checkups with a 6-year interval, the study population was divided into two groups by blood pressure and antihypertensive treatment in 1998, after excluding people who had hypertension in 1992. RESULTS: There were 133 (93 males and 40 females) incidences of hypertension observed among the study population of 922 (540 males and 382 females). In the univariate analysis, odds ratios of Ala/Ser and Ser/Ser against Ala/Ala were 0.98 (95% confidence interval [CI]): 0.7-1.4) and 0.79 (95% CI: 0.4-1.6), respectively. In the multivariate analysis adjusted for sex, age, body mass index, serum total cholesterol, fasting blood sugar, and smoking and drinking habits, odds ratios for Ala/Ser and Ser/Ser against Ala/Ala were 0.97 (95% CI: 0.7-1.4) and 0.75 (95% CI: 0.4-1.5), respectively. CONCLUSIONS: The ET-1 gene polymorphism in this study did not seem to be associated with the incidence of hypertension among the Japanese workers.

Excretory/secretory products from plerocercoids of Spirometra erinaceieuropaei suppress interleukin-1beta gene expression in murine macrophages.

The present study shows that ES products from plerocercoids of Spirometra erinaceieuropaei suppressed interleukin-1beta mRNA expression in lipopolysaccharide-stimulated RAW 264.7 macrophages in the absence or presence of a cyclic AMP analogue, dibutyryl cyclic AMP. Investigation using the inhibitors of mitogen-activated protein kinase (MAPK) pathways revealed that extracellular signal-regulated protein kinase 1/2 and p38 mitogen-activated protein kinase pathways are crucial for full induction of interleukin-1beta mRNA expression. ES products additionally suppressed interleukin-1beta mRNA expression in the cells treated with p38 mitogen-activated protein kinase inhibitor (SB203580) or extracellular signal-regulated protein kinase 1/2 inhibitor (PD98059). Western blot analysis showed that dibutyryl cyclic AMP enhanced lipopolysaccharide-induced phosphorylation of extracellular signal-regulated protein kinase 1/2, p38 mitogen-activated protein kinase and cyclic AMP responsive element binding protein (CREB) and, in turn, we demonstrated that ES products reduced the lipopolysaccharide and dibutyryl cyclic AMP-induced phosphorylation of extracellular signal-regulated protein kinase 1/2 and p38 mitogen-activated protein kinase, but not cyclic AMP responsive element binding protein. These data demonstrate that ES products from the plerocercoids of S. erinaceieuropaei may evade induction of interleukin-1beta mRNA by inhibiting extracellular signal-regulated protein kinase 1/2 and p38 mitogen-activated protein kinase pathways in lipopolysaccharide and/or dibutyryl cyclic AMP-stimulated macrophages.

Excretory/secretory products from plerocercoids of Spirometra erinaceieuropaei suppress the TNF-alpha gene expression by reducing phosphorylation of ERK1/2 and p38 MAPK in macrophages.

We previously reported that excretory/secretory products from plerocercoids of Spirometra erinaceieuropaei suppress gene expression and production of tumour necrosis factor-alpha in murine macrophages stimulated with lipopolysaccharide. The present study investigated the suppressive mechanisms of tumour necrosis factor-alpha mRNA by excretory/secretory products in lipopolysaccharide-stimulated murine macrophages. Electrophoretic mobility shift assay and supershift assay revealed that neither nuclear translocation of nuclear factor-kappa B nor conformation of the p50/p65 nuclear factor-kappa B subunits was affected by the treatment of excretory/secretory products in lipopolysaccharide-stimulated macrophages. Inhibition of extracellular signal-regulated protein kinase 1/2 with PD98059 or p38 mitogen-activated protein kinase with SB203580 partially reduced tumour necrosis factor-alpha mRNA expression, and a combination of the two inhibitors additionally suppressed the level of tumour necrosis factor-alpha mRNA, revealing that both pathways are crucial for full induction of the gene. Northern blot analysis showed that excretory/secretory products additionally suppressed tumour necrosis factor-alpha mRNA expression in cells treated with PD98059 or SB208530 and, in turn, we found that excretory/secretory products reduced phosphorylation of extracellular signal-regulated protein kinase 1/2 and p38 mitogen-activated protein kinase in lipopolysaccharide-stimulated macrophages by Western blot analysis. This is the first report demonstrating that excretory/secretory products from parasites suppress tumour necrosis factor-alpha mRNA expression by reducing phosphorylation of extracellular signal-regulated protein kinase 1/2 and p38 mitogen-activated protein kinase without any effect on nuclear factor-kappa B activity in macrophages stimulated with lipopolysaccharide. We hypothesise that excretory/secretory products may enable this parasite to survive within the host.