A delayed diagnosis of fascioliasis: The importance of appropriate fecal diagnostic method.

Fascioliasis, a zoonotic helminthiasis, occurs sporadically in Japan. In this report, we describe a case of fascioliasis that was initially difficult to diagnose because the fecal examination method was negative for the Fasciola sp. eggs. A 64-year-old man living in Shimonoseki City, Japan, presented with fatigue and anorexia. Laboratory tests showed hepatic dysfunction and eosinophilia. Abdominal dynamic contrast-enhanced computed tomography and magnetic resonance cholangiopancreatography suggested intrahepatic biliary cysts. Thereafter, fever and night sweats persisted, and positron emission tomography and biopsy of the porta hepatis lymph node were performed on suspicion of malignancy. However, histopathological diagnosis found non-specific inflammation. As fascioliasis was suspected due to eosinophilia and the multiple hepatic masses, fecal egg examination was performed by an external private laboratory, which adopted the flotation method and reported the absence of parasite eggs. However, fecal examination was retried in our laboratory using the formalin-ether concentration method, and we detected Fasciola sp. eggs. This case suggests that misdiagnosis may occur depending on the fecal examination method; thus, it is necessary to choose a suitable method for certain parasite species.

Helminth-induced impairment of humoral immunity differently contribute to their anti-arthritic effects in mice: Comparison of Schistosoma mansoni and Trichinella spiralis.

AIMS: We have previously reported reduction of anti-type II collagen (IIC) IgG levels in collagen-induced arthritis (CIA) by Schistosoma mansoni (Sm) and Trichinella spiralis (Ts). To clarify the contribution of the impairment of humoral immunity to their anti-arthritic activities, we herein investigated the relationship between anti-IIC IgG levels and arthritic swelling in Sm- or Ts-infected mice. METHODS AND RESULTS: Male DBA/1J mice were infected with Sm cercariae or Ts muscle larvae prior to the IIC immunization. In the Sm-infected mice, paw swelling and anti-IIC IgG levels were continuously lower than those of non-infected control group. In contrast, arthritic swelling in the Ts-infected mice only decreased in the early phase of CIA progression, despite the continued impairment of anti-IIC IgG production throughout the experimental period. Correlation coefficients between residual paw swelling and anti-IIC IgG titers were similar or higher in the Sm group than in the control group, but were similar or lower in the Ts group than in the control group. CONCLUSION: The down-modulations of anti-IIC IgG levels by the two parasitic infections and the correlation analyses suggest that the anti-arthritic activity of Sm was primarily attributed to the modulation of IgG-independent arthritogenic mechanisms and secondarily to the impairment of anti-IIC IgG production. In contrast, Ts could alleviate CIA mainly via the impairment of antibody production.

Comprehensive lipidomics of lupus-prone mice using LC-MS/MS identifies the reduction of palmitoylethanolamide that suppresses TLR9-mediated inflammation.

Lipid mediators are known to play crucial roles not only in the onset of the inflammatory response but also in the induction of resolution of inflammation. Here, we report that palmitoylethanolamide (PEA), an endogenous N-acylethanolamine, can suppress the inflammation induced by Toll-like receptor (TLR) signaling both in vitro and in vivo. PEA was found to be significantly reduced in the serum and spleen of lupus-prone MRL/lpr mice analyzed by lipidomics. PEA suppressed pro-inflammatory cytokine production in a mouse macrophage cell line stimulated with TLR ligands such as lipopolysaccharide, peptidoglycan, poly (I:C), imiquimod, and CpG-ODN. PEA also inhibited both mRNA and protein levels of IL-6 in bone marrow-derived dendritic cells (BMDCs) and B cells stimulated with CpG-ODN. Augmentation of cell surface CD86 and CD40 on BMDCs and B cells, IgM production, and cell proliferation of B cells in response to CpG-ODN were attenuated by PEA. Moreover, PEA treatment significantly reduced mortality and serum IL-6 levels in mice injected with CpG-ODN plus D-galactosamine. Taken together, PEA ameliorates inflammation induced by TLR signaling, which could be a novel therapeutic target for inflammatory disorders.

Non-lethal rodent malarial infection prevents collagen-induced arthritis in mice via anti-arthritic immunomodulation.

AIMS: Immunomodulatory effects of parasitic infections on the outcomes of allergic or autoimmune disorders have been addressed in many experimental studies. We examined the effects of Plasmodium yoelii 17X NL (Py) infection on collagen-induced arthritis (CIA). METHODS AND RESULTS: Male DBA/1J mice were immunized with bovine type II collagen (IIC). Py inoculation was induced at three different time points (1, 4 weeks after or 4 weeks before the immunization). Only the inoculation at 4 weeks after IIC immunization significantly inhibited arthritis development. Non-malarial anaemia induced by phenylhydrazine hydrochloride (PHZ) did not affect arthritis development. In the infected mice, anti-IIC IgG levels were transiently reduced. In addition, splenic production of pro-arthritic cytokines (IL-17 and TNF-α) and IFN-γ decreased, whereas IL-10 production increased. Flow cytometric analysis clarified that the main IL-10 producers in Py-infected mice had the CD4+ CD25- Foxp3- phenotype, presumably Tr1 cells. CONCLUSION: We demonstrated that experimental malarial infection alleviated autoimmune arthritis via immunomodulation, suggesting the importance of malaria in the hygiene hypothesis and the significance of searching for therapeutic immunomodulatory molecules from malarial parasites.

Th2 signals are not essential for the anti-arthritic effects of Trichinella spiralis in mice.

AIMS: Many parasitic helminths are known to alter host immune responses and consequently affect the progression of autoimmune and allergic diseases. The parasitic nematode Trichinella sp has been reported to suppress several experimental diseases in rodents, including experimental autoimmune encephalomyelitis, type 1 diabetes, colitis, airway inflammation and autoimmune arthritis. We tried to clarify requirement of Th2 cytokines in the anti-arthritic effects of Trichinella spiralis (Ts) against collagen-induced arthritis (CIA). METHODS AND RESULTS: We infected Ts and then induced CIA in STAT6KO DBA/1 mice, comparing the disease progression with that in wild-type (WT) DBA/1 mice, Ts significantly mitigated arthritis in WT mice, in addition to the impairment of anti-type II collagen (IIC) IgG production in a subclass-independent manner. The genetic absence of STAT6 in the mice did not abrogate the anti-arthritic effects of Ts. Alteration of splenic cytokines was not related to the anti-arthritic effects of the parasite. Moreover, lack of IL-10 did not abrogate the anti-arthritic effects of Ts. CONCLUSION: Our results suggest that the anti-arthritic effects of Ts do not require host Th2 signals.

Dual genetic absence of STAT6 and IL-10 does not abrogate anti-hyperglycemic effects of Schistosoma mansoni in streptozotocin-treated diabetic mice.

Schistosoma mansoni (Sm) is known to exert protective effects against various allergic and autoimmune disorders. It has been reported that this parasite protects NOD mice from spontaneous type 1 diabetes (T1D) and ameliorates streptozotocin (STZ)-induced T1D in wild-type mice. Here, we tried to clarify the anti-diabetic mechanisms of Sm in the latter model. Sm infection partially prevented the degradation of pancreatic islets and hyperglycemia in multiple low-dose (MLD) STZ-treated mice. Neither Treg cell depletion nor genetic absences of IL-10 and/or STAT6 abrogated the anti-hyperglycemic effects of Sm. Among M2 macrophage markers, Arg-1 and Ym1, but not Retnla, remained up-regulated in the pancreatic lymph nodes and in the spleens of STAT6/IL-10 double deficient (DKO) mice. Collectively, it is suggested that Sm exerts anti-diabetic effects on this experimental T1D model via Treg/IL-4/IL-13/IL-10-independent mechanisms. Augmented expressions of Arg-1 and Ym1 in the lymphoid organs adjacent to pancreas may be relevant to the anti-diabetic effects of Sm.

Use of cell-free circulating schistosome DNA in serum, urine, semen, and saliva to monitor a case of refractory imported schistosomiasis hematobia.

This case of imported refractory schistosomiasis has highlighted the usefulness of cell-free parasite DNA as a diagnostic marker to assess active schistosome infection. In contrast to the rapid disappearance of ova in urine, parasite DNA remained persistent in several other specimen types even after the fourth treatment with praziquantel. This result was consistent with the presence of morphologically intact ova in bladder biopsy samples and with the corresponding symptoms.

Heligmosomoides polygyrus infection reduces severity of type 1 diabetes induced by multiple low-dose streptozotocin in mice via STAT6- and IL-10-independent mechanisms.

Some parasitic helminths are known to protect their hosts from allergic and autoimmune disorders. Here, we tested the effects of a gastrointestinal nematode, Heligmosomoides polygyrus (Hp), on streptozotocin (STZ)-induced type 1 diabetes (T1D) in mice. Hp infection significantly suppressed hyperglycemia induced by multiple low-dose administration of STZ, but did not affect hyperglycemia induced by single high-dose administration of STZ. In the multiple low dose model, Hp infection prevented a decrease in pancreatic islet size. The augmentation of TNF-α and IL-1ß expression in the pancreas was abrogated by Hp infection. The genetic absence of IL-10 or STAT6 did not abrogate the anti-hyperglycemic effect of Hp. Hp has a suppressive effect on immune mechanism-mediated experimental T1D via Th2 polarization-independent mechanisms.

Origin of a novel protein-coding gene family with similar signal sequence in Schistosoma japonicum.

BACKGROUND: Evolution of novel protein-coding genes is the bedrock of adaptive evolution. Recently, we identified six protein-coding genes with similar signal sequence from Schistosoma japonicum egg stage mRNA using signal sequence trap (SST). To find the mechanism underlying the origination of these genes with similar core promoter regions and signal sequence, we adopted an integrated approach utilizing whole genome, transcriptome and proteome database BLAST queries, other bioinformatics tools, and molecular analyses. RESULTS: Our data, in combination with database analyses showed evidences of expression of these genes both at the mRNA and protein levels exclusively in all developmental stages of S. japonicum. The signal sequence motif was identified in 27 distinct S. japonicum UniGene entries with multiple mRNA transcripts, and in 34 genome contigs distributed within 18 scaffolds with evidence of genome-wide dispersion. No homolog of these genes or similar domain was found in deposited data from any other organism. We observed preponderance of flanking repetitive elements (REs), albeit partial copies, especially of the RTE-like and Perere class at either side of the duplication source locus. The role of REs as major mediators of DNA-level recombination leading to dispersive duplication is discussed with evidence from our analyses. We also identified a stepwise pathway towards functional selection in evolving genes by alternative splicing. Equally, the possible transcription models of some protein-coding representatives of the duplicons are presented with evidence of expression in vitro. CONCLUSION: Our findings contribute to the accumulating evidence of the role of REs in the generation of evolutionary novelties in organisms' genomes.

Pentacyclic triterpenoid ursolic acid induces apoptosis with mitochondrial dysfunction in adult T-cell leukemia MT-4 cells to promote surrounding cell growth.

We investigated the antitumor effects of oleanolic acid (OA) and ursolic acid (UA) on adult T-cell leukemia cells. OA and UA dose-dependently inhibited the proliferation of adult T-cell leukemia cells. UA-treated cells showed caspase 3/7 and caspase 9 activation. PARP cleavage was detected in UA-treated MT-4 cells. Activation of mTOR and PDK-1 was inhibited by UA. Autophagosomes were detected in MT-4 cells after UA treatment using electron microscopy. Consistently, mitophagy was observed in OA- and UA-treated MT-4 cells by confocal microscopy. The mitochondrial membrane potential in MT-4 cells considerably decreased, and mitochondrial respiration and aerobic glycolysis were significantly reduced following UA treatment. Furthermore, MT-1 and MT-4 cells were sorted into two regions based on their mitochondrial membrane potential. UA-treated MT-4 cells from both regions showed high activation of caspase 3/7, which were inhibited by Z-vad. Interestingly, MT-4 cells cocultured with sorted UA-treated cells showed enhanced proliferation. Finally, UA induced cell death and ex vivo PARP cleavage in peripheral blood mononuclear cells from patients with adult T-cell leukemia. Therefore, UA-treated MT-4 cells show caspase activation following mitochondrial dysfunction and may produce survival signals to the surrounding cells.

Cerebral schistosomiasis due to Schistosoma haematobium confirmed by PCR analysis of brain specimen.

The case of a 25-year-old Japanese male who had cerebral schistosomiasis caused by Schistosoma haematobium is reported here. Although serum antibody tests showed a cross-reaction with other helminths and no ova were excreted in urine or feces, the existence of Schistosoma haematobium in the brain was confirmed by PCR analysis.

Schistosome: its benefit and harm in patients suffering from concomitant diseases.

Schistosomiasis is an important tropical disease affecting approximately 200 million people worldwide. Because of its chronicity and robust immunomodulatory activity, the effects of schistosomes on other diseases, such as allergies, autoimmunity, and infectious diseases, have been studied extensively in both epidemiological and experimental settings. In this paper, we summarize the beneficial and harmful effects of schistosomes. The importance of controlling schistosomiasis is also discussed.

Parasitic helminths: new weapons against immunological disorders.

The prevalence of allergic and autoimmune diseases is increasing in developed countries, possibly due to reduced exposure to microorganisms in childhood (hygiene hypothesis). Epidemiological and experimental evidence in support of this hypothesis is accumulating. In this context, parasitic helminths are now important candidates for antiallergic/anti-inflammatory agents. Here we summarize antiallergic/anti-inflammatory effects of helminths together along with our own study of the effects of Schistosoma mansoni on Th17-dependent experimental arthritis. We also discuss possible mechanisms of helminth-induced suppression according to the recent advances of immunology.

Suppressive effect of azithromycin on Plasmodium berghei mosquito stage development and apicoplast replication.

BACKGROUND: Azithromycin (AZM) is a macrolide antibiotic that displays an excellent safety profile even in children and pregnant women and has been shown to have anti-malarial activity against blood stage Plasmodium falciparum. This study evaluated the transmission-blocking effect of AZM using a rodent malaria model. METHODS: AZM-treated mice infected with Plasmodium berghei were exposed to Anopheles stephensi mosquitoes, followed by the observation of parasite development at different phases in the mosquito, i.e., ookinetes in the midgut, oocysts on the midgut, and sporozoites in the midgut and salivary glands. Furthermore, to evaluate the effect on organelle replication of each stage, quantitative real-time PCR analysis was performed. RESULTS: The inhibitory effect of AZM was noticeable in both gametocyte-ookinete transformation in the midgut and sporozoite production in the oocyst, while the latter was most remarkable among all the developmental phases examined. Real-time PCR analysis revealed that AZM suppressed apicoplast replication at the period of sporozoite production in oocysts. CONCLUSIONS: AZM inhibits parasite development in the mosquito stage, probably through the same mechanism as in the liver and blood stages. Such a multi-targeting anti-malarial, along with its safety, would be ideal for mass drug administration in malaria control programmes.

Peroxiredoxin-1 from Schistosoma japonicum functions as a scavenger against hydrogen peroxide but not nitric oxide.

Three peroxiredoxins (Prxs) are expressed during most of the developmental stages in the schistosome. Prx-1 is localized on the surface of the schistosomula and adults of Schistosoma japonicum, while Prx-2 is localized in the sub-tegumental tissues, parenchyma, vitelline glands, and gut epithelium, but not on the surface of the worms. We applied RNA interference techniques to suppress the specific genes of S. japonicum Prxs. Schistosomula of S. japonicum were cultured together with long-dsRNA encoding Prx-1 and Prx-2 of S. japonicum (the soaking method). The transcription level of each Prx gene was reduced by an RNA interference (RNAi)-mediated effect specifically. Although neither Prx was the essential protein for survival of S. japonicum schistosomula, Prx-1 dsRNA-treated larvae were susceptible to hydrogen peroxide. Moreover, these larvae were also susceptible to t-butyl hydroperoxide and cumene-hydroperoxide. However, the knockdown of neither Prx-1 nor Prx-2 influenced the resistance against nitric oxide generated from DETA/NO. Prx-1 may work as a scavenger against reactive oxygen species (ROS) generated outside of the schistosomes to prevent the oxidation of the bodies and/or the attack by immune cells producing the ROS. These findings suggest that Prx-1 may become a novel target of drugs and vaccines for schistosomiasis.

[Effectiveness of learning of nursing-work as early exposure by the 1st year medical students at a university hospital].

We conducted a half day program included in the subject of "nursing and care" as early exposure at clinical sites for the 1st year medical students at a university hospital. This program aimed at understanding what nursing is through visit for study and what patients expect through doctor-patient communication. In order to evaluate the program and to clarify problems to be solved, comments and impressions reported by the medical students were analyzed qualitatively and inductively. As a result, we found that the students recognized the importance of communication with patients and of mental care for them. As for nursing, the students also realized the characteristics and significance of nursing. It is noteworthy that they acquired clear images of a medical doctor, including their roles in team-based medical care. We conclude that this program of early exposure to clinical sites is instructive for 1st year medical students.

5-Aminolevulinic Acid Suppresses Prostaglandin E2 Production by Murine Macrophages and Enhances Macrophage Cytotoxicity Against Glioma.

BACKGROUND: 5-Aminolevulinic acid (5-ALA) induces the accumulation of a large amount of protoporphyrin IX (PpIX) in tumors, which has been used in the treatment of several cancers. 5-ALA is commonly used for fluorescence-guided tumor resection in clinical neurosurgery and for photodynamic therapy based on the generation of cytotoxic oxygen. OBJECTIVE: The purpose of this study was to identify the mechanisms of 5-ALA-induced immune response in macrophages in malignant glioma. METHODS: Intracellular levels of 5-ALA-induced PpIX in C3H/HeN murine peritoneal macrophages were measured by the median fluorescence intensity using flow cytometry and confocal laser scanning microscopy. Macrophages were cultured in vitro with or without 0.5 mM 5-ALA, 0.1 µg/mL lipopolysaccharide, and 20% glioma-conditioned medium. Levels of immunosuppressive prostaglandin E2 (PGE2), interleukin-10, and transforming growth factor ß were measured using enzyme immunoassay in the culture supernatant. In addition, macrophages and RSV-M mouse glioma cells were co-cultured in vitro with cell culture inserts with or without 5-ALA (0.1 and 0.5 mM) and lipopolysaccharide (0.1 µg/mL). RESULTS: We found that 5-ALA-induced PpIX accumulated in macrophages and significantly suppressed PGE2 production and expression of both cyclooxygenase-2 and microsomal prostaglandin E synthase-1. 5-ALA treatment also suppressed PGE2 production by glioma-conditioned medium. 5-ALA suppressed RSV-M glioma cell proliferation in a concentration-dependent manner. CONCLUSIONS: These results indicate that 5-ALA suppressed PGE2 production by macrophages via the downregulation of cyclooxygenase-2 and microsomal prostaglandin E synthase-1 expression levels. This is a novel mechanism to induce effective immune response against glioma in macrophages.

2-Cys peroxiredoxins from Schistosoma japonicum: the expression profile and localization in the life cycle.

Peroxiredoxin (Prx) is known to be an antioxidant protein that protects the organisms against various oxidative stresses and functions as a signal transductor. Here, we determined the full-length cDNA sequences of three types of Prx from an Asian blood fluke, Schistosoma japonicum: Prx-1, Prx-2 and Prx-3. According to the deduced amino acid sequences, only Prx-3 had a mitochondria-targeting sequence. Using RT-PCR, it was shown that these Prx genes were constitutively expressed in the eggs, cercariae and adult worms of the schistosome. Western blot analysis using antisera specific for each Prx revealed that all the three Prx proteins existed in these developmental stages. By immunolocalization analysis, Prx-1 existed on the surface of a miracidium and in the space between a miracidium and an eggshell. Furthermore, Prx-1 was deposited in the host tissues around the eggs. In adult worms, Prx-1 was not only expressed in the tegument, but also contained in their excretory/secretory products. The surface of the 7 day-schistosomula was stained with anti-Prx-1 antiserum. On the other hand, Prx-2 only existed inside the miracidia in eggs. In addition, Prx-2 was mainly detected in the sub-tegumental tissues, parenchyma, vitelline gland and gut epithelium of the adult worms, but was not detected in the tegument of adults and schistosomula. Taken together with previous reports by other investigators, these data suggest that Prx-1 acts to protect the parasite against the ROS produced by host immune cells, and that Prx-2 plays important roles in intracellular redox signaling and/or in the reduction of ROS generated through the hemoglobinolytic process in the digestive tract.

Early detection of Schistosoma mansoni infection by touchdown PCR in a mouse model.

A detection assay for Schistosoma mansoni DNA in mouse serum samples based on touchdown PCR was developed and evaluated. The serum samples could be assayed directly without the need to extract DNA. No cross reactions between S. mansoni and related species inducing human schistosomiasis were observed. After the infection, mouse sera and feces were collected for 8 weeks. Anti-worm antigen IgG and anti-soluble egg antigen IgG were detected in the sera at 6 weeks post-infection by ELISA. The parasite's eggs were detected in the feces at 8 weeks. In contrast, S. mansoni DNA was detected in the sera at 2 weeks post-infection. These data suggest that touchdown PCR is a potential tool for the early diagnosis of S. mansoni infection.

Mutagenicity evaluation of Schistosoma spp. extracts by the umu-test and V79/HGPRT gene mutation assay.

Schistosomiasis has been suspected of being a risk factor for various types of cancers for sometime, e.g., bladder cancer, colorectal cancer and hepatic cancer. Among them, the etiological relationship between urinary schistosomiasis and bladder cancer is now widely accepted. However, mechanisms of the carcinogenesis are still unclear. Here, we tested the mutagenicity of the parasite extracts by the umu-test and hypoxanthine guanine phosphoribosyltransferase (HGPRT) gene mutation assay, which both overcome disadvantages of the Ames plate assay. Adult worm extracts and egg extracts of Schistosoma haematobium and Schistosoma mansoni were tested. Under our experimental conditions, neither worm nor egg extracts were shown to have any mutagenicity in both tests even in the presence of S9 mix. Our results suggest that there is very little possibility of immediate gene mutation due to the parasite-derived substances in schistosomiasis-related carcinogenesis.