Omega-Class Glutathione Transferases Protect DNA from Oxidative Stress in Pathogenic Helminth Reproductive Cells.

Pathogenic helminths have evolved mechanisms to preserve reproductive function while surviving long-term in the host via robust protective responses. A protective role of antioxidant enzymes in preventing DNA degradation has long been proposed, but little evidence has been provided. Here, we show that omega-class glutathione transferases (GSTOs) are critical for maintaining viability by protecting the reproductive cell DNA of the carcinogenic liver fluke, Clonorchis sinensis. Clonorchis sinensis GSTO (CsGSTO) activities modified by changes in the GSH/GSSG and NADPH/NADP+ molar ratios suppressed the overproduction of reactive oxygen species. CsGSTO1 and CsGSTO2 catalyzed deglutathionylation under physiologic and low-stress conditions (GSH/GSSG ratio of 6:1 or higher) but promoted glutathionylation under high-stress conditions (GSH/GSSG ratio of 3:1 or lower). Gliotoxin-induced functional disruption of CsGSTOs in living C. sinensis reduced the GSH/GSSG molar ratio and increased the production of protein glutathionylation (PSSG) under physiologic and low-stress conditions, indicating that suppression of GSTO function did not affect deglutathionylation. However, the perturbation of CsGSTOs decreased the GSH/GSSG ratio but also reduced PSSG production under high oxidative stress, demonstrating that glutathionylation was impeded. In response to oxidative stimuli, C. sinensis decreased GSTO-specific dehydroascorbate reductase and thiol transferase activities and the GSH/GSSG ratio, while it increased the NADPH/NADP+ ratio and PSSG. CsGSTOs utilized GSH to regulate GSH/GSSG and NADPH/NADP+ recycling and triggered a redox signal leading to nuclear translocation. Nuclear-imported CsGSTOs were modified by glutathionylation to prevent DNA damage. Antibodies specific to CsGSTOs dose-dependently inhibited this process. Disruption of CsGSTOs or the depletion of GSH caused glutathionylation defects, leading to DNA degradation. Our results demonstrate that CsGSTOs and the GSH system play a previously unappreciated role in protecting DNA from oxidative stress.

Echinococcus granulosus Protoscolex DM9 Protein Shows High Potential for Serodiagnosis of Alveolar Echinococcosis.

Alveolar echinococcosis (AE) caused by infection with E. multilocularis metacestode, represents one of the most fatal helminthic diseases. AE is principally manifested with infiltrative, proliferating hepatic mass, resembling primary hepatocellular carcinoma. Sometimes metastatic lesions are found in nearby or remote tissue. AE diagnosis largely depends on imaging studies, but atypical findings of imaging features frequently require differential diagnosis from other hepatic lesions. Serological tests may provide further evidence, while obtaining reliable AE materials is not easy. In this study, alternative antigens, specific to AE were identified by analyzing E. granulosus protoscolex proteins. An immunoblot analysis of E. granulosus protoscolex showed that a group of low-molecular-weight proteins in the range from 14 kDa to 16 kDa exhibited a sensitive and specific immune response to AE patient sera. Partial purification and proteomic analysis indicated that this protein group contained myosin, tubulin polymerization promoting protein, fatty-acid binding protein, uncharacterized DM9, heat shock protein 90 cochaperone tebp P-23, and antigen S. When the serological applicability of recombinant forms of these proteins was assessed using enzyme-linked immunosorbent assay, DM9 protein (rEgDM9) showed 90.1% sensitivity (73/81 sera tested) and 94.5% specificity (172/181 sera tested), respectively. rEgDM9 showed weak cross-reactions with patient sera from the transitional and chronic stages of cystic echinococcosis (3 to 5 stages). rEgDM9 would serve as a useful alternative antigen for serodiagnosis of both early- and advanced-stage AE cases.

Omega-Class Glutathione Transferases of Carcinogenic Liver Fluke, Clonorchis sinensis, Modulate Apoptosis and Differentiation of Host Cholangiocytes.

The small liver fluke Clonorchis sinensis causes hepatobiliary ductal infections in humans. Clonorchiasis is characterized histopathologically by ductal dysplasia, hyperplasia and metaplasia, which closely resembles cholangiocarcinoma (CCA). The disruption of programmed cell death is critical for malignant transformation, while molecular events underlying these phenomena have poorly been understood in clonorchiasis-related CCA tumorigenesis. We incorporated recombinant C. sinensis omega-class glutathione transferase (rCsGSTo) 1 or 2 into human intrahepatic biliary epithelial cells (HIBECs) and analyzed pathophysiological alterations of HIBECs upon the application of oxidative stress. rCsGSTos partially but significantly rescued HIBECs from cell death by inhibiting oxidative stress-induced apoptosis (p < 0.01). rCsGSTos modulated transcriptional levels of numerous genes. We analyzed 13 genes involved in programmed cell death (the upregulation of five antiapoptotic and two apoptotic genes, and the downregulation of one antiapoptotic and five apoptotic genes) and 11 genes associated with cell differentiation (the increase in seven and decrease in four genes) that showed significant modifications (p < 0.05). The induction profiles of the mRNA and proteins of these differentially regulated genes correlated well with each other, and mostly favored apoptotic suppression and/or cell differentiation. We detected increased active, phosphorylated forms of Src, PI3K/Akt, NF-κB p65, MKK3/6 and p38 MAPK, but not JNK and ERK1/2. CsGSTos were localized in the C. sinensis-infected rat cholangiocytes, where cytokeratin 19 was distributed. Our results demonstrated that CsGSTos excreted to the biliary lumen are internalized and accumulated in the host cholangiocytes. When cholangiocytes underwent oxidative stressful condition, CsGSTos appeared to be critically involved in both antiapoptotic process and the differentiation of host cholangiocytes through the regulation of target genes following the activation of responsible signal molecules.

Spectrum of pleuropulmonary paragonimiasis: An analysis of 685 cases diagnosed over 22 years.

OBJECTIVES: Paragonimiasis is a global foodborne zoonosis. Overlapping clinical and imaging features with other lung pathologies hamper correct diagnosis and require differential diagnosis. METHODS: During 1982-2003, 49,012 samples were referred for immunodiagnosis of helminthiases. We detected paragonimiasis cases by enzyme-linked immunosorbent assay (ELISA). We assessed clinical, radiographical and laboratory characteristics, and diagnostic dilemmas associated with delayed diagnosis. RESULTS: We analyzed 685 pleuropulmonary paragonimiasis cases. ELISA-positive was 665. Eggs were detected in 50. Symptom duration correlated well with the appearance of chest radiographs; 359 pleural, 33 pleuroparenchymal, and 264 parenchymal lesions (P < 0.001). Twenty-nine had normal chest images. Eosinophilia, seen in 304, was common in pleural and pleuroparenchymal patients (P < 0.05). Chest pain and dyspnea were characteristic for pleurisy patients. Sputum (odds ratios [OR]: 6.79; 95% CI: 4.41-10.47), blood-tinged sputum (OR: 5.62; 95% CI: 3.75-8.42), and foul-odor (OR: 2.70; 95% CI: 1.42-5.16) were significant in parenchymal patients. Delayed diagnosis (119) for ≥ 25 weeks was attributed mainly to misdiagnosis as tuberculosis, malignancy, or chronic obstructive pulmonary disease (COPD) (OR: 111.75; 95% CI: 43.25-288.74). CONCLUSIONS: Variable symptoms and radiographs of pleuropulmonary paragonimiasis depended on the stage of infection. Suspicion of tuberculosis, malignancy, or COPD was major cause of delayed diagnosis.

Survey of echinococcoses in southeastern Qinghai Province, China, and serodiagnostic insights of recombinant Echinococcus granulosus antigen B isoforms.

BACKGROUND: Echinococcoses, caused by metacestodes of Echinococcus granulosus (cystic echinococcosis; CE) and E. multilocularis (alveolar echinococcosis; AE), represent major emerging parasitic diseases. These enzootic helminthiases invoke significant public health concerns and social burdens in endemic areas. The diseases are prevalent in the Qinghai-Tibetan Plateau, China, while community-based epidemiological studies have been scarcely reported. We surveyed echinococcosis patients in the southeastern Qinghai Province, China, to better understand the concurrent epidemiological situation in this area. METHODS: During July and August of 2013 and 2014, we screened echinococcosis patients at Yushu and Golog Prefectures, Qinghai Province, China, in a diagnostic campaign. A total of 2856 people (male:female ratio, 1:1.12; mean age, 34.6 years; age range, 6-88 years) were ultrasonographically examined for the presence of hepatic echinococcal cysts. We also collected serum samples from patients and analyzed antibody reactivity against recombinant forms of diverse E. granulosus antigen Bs (rEgAgB1-5) by enzyme-linked immunosorbent assay. RESULTS: We detected 134 patients whose imaging scans were compatible with CE (115 cases) and AE (20 patients). One patient might have been infected with both CE and AE. The overall incidence was 4.7% (CE, 4.0%; AE, 0.7%). A large proportion (67.5%) of CE patients was diagnosed at active and transitional CE1-CE3 stages in their late 30s. The AE cases were generally detected at advanced stage in patients at early 20s (60%). Analysis of the receiver operating characteristic curve and Youden's index indicated that rEgAgB2 was the most promising biomarker, followed by rEgAgB3 and rEgAgB1. Overall, sensitivity and specificity of rEgAgB1-3 were 84.5-92.7% and 91.9-94.6%, respectively. rEgAgB4 and 5 showed low sensitivity with high cross-reactivity. CONCLUSIONS: Our results strongly suggest that disability-adjusted life years related to echinococcoses in Qinghai-Tibetan areas might be more serious than previously considered. Control and prevention strategy against CE and AE are highly required in these areas. In addition to ultrasonography, serological tests might provide supportive data. However, serological data should be carefully interpreted for differential diagnosis, especially in areas where both CE and AE are co-endemic.

Advances in Serological Diagnosis of Taenia solium Neurocysticercosis in Korea.

Cysticercosis, a parasitic disease caused by Taenia solium metacestode (TsM), has a major global public health impact in terms of disability-adjusted life years. The parasite preferentially infects subcutaneous tissue, but may invade the central nervous system, resulting in neurocysticercosis (NC). NC is an important neglected tropical disease and an emerging disease in industrialized countries due to immigration from endemic areas. The prevalence of taeniasis in Korea declined from 0.3%-12.7% during the 1970s to below 0.02% since the 2000s. A survey conducted from 1993 to 2006 revealed that the percentage of tested samples with high levels of specific anti-TsM antibody declined from 8.3% to 2.2%, suggesting the continuing occurrence of NC in Korea. Modern imaging modalities have substantially improved the diagnostic accuracy of NC, and recent advances in the molecular biochemical characterization of the TsM cyst fluid proteome also significantly strengthened NC serodiagnosis. Two glycoproteins of 150 and 120 kDa that induce strong antibody responses against sera from patients with active-stage NC have been elucidated. The 150 kDa protein showed hydrophobic-ligand binding activities and might be critically involved in the acquisition of host-derived lipid molecules. Fasciclin and endophilin B1, both of which play roles in the homeostatic functions of TsM, showed fairly high antibody responses against calcified NC cases. NC is now controllable and manageable. Further studies should focus on controlling late-onset intractable seizures and serological diagnosis of NC patients infected with few worms. This article briefly overviews diagnostic approaches and discusses current issues relating to NC serodiagnosis.

Biliary Taeniasis with Cholecystitis: An Unusual Case of Taenia solium Infection with a Literature Review.

Taeniasis is a cosmopolitan helminthic disease caused by Taenia species, which included Taenia solium, Taenia saginata, and Taenia asiatica. These parasites typically infect the small intestine, but cases of aberrant migration have been reported. We treated a 70-year-old man who presented with vomiting and colicky abdominal pain. On physical examination, Murphy's sign was positive, and laboratory findings indicated severe inflammation. Computed tomography and magnetic resonance cholangiopancreatography revealed typical features of cholecystitis. An 82-cm-long, slender and degenerated, parasite-like organism was aspirated through a percutaneous transhepatic gallbladder drainage tube. After extensive washing of the organism, we detected yellowish-brown colored, spherical 37.9 × 33.8-µm-sized taenid eggs with thick transverse striations. Hematoxylin-eosin-stained worm sections also contained Taeniidae eggs. Polymerase chain reaction amplification of DNA extracted from the worm with species-specific cytochrome c1 (cox1) primer sets detected a T. solium-specific fragment. Because of sustained high fever combined with inflammatory signs, the patient underwent laparoscopic cholecystectomy and inflamed gallbladder removal. A histopathologic specimen demonstrated chronic reactive cholecystitis. The patient's fever and leukocytosis rapidly resolved after surgery. We experienced an uncommon case of biliary taeniasis representing cholecystitis caused by adult worm of T. solium.

Comparison of Echinococcus multilocularis and Echinococcus granulosus hydatid fluid proteome provides molecular strategies for specialized host-parasite interactions.

Alveolar and cystic echinococcoses, caused by the metacestodes of Echinococcus multilocularis and E. granulosus, are prevalent in several regions and invoke deleterious zoonotic helminthiases. Hydatid fluid (HF), which contains proteinaceous and non-proteinaceous secretions of the parasite- and host-derived components, critically affects the host-parasite interplay and disease progression. We conducted HF proteome profiling of fully mature E. multilocularis vesicle (nine months postinfection) and E. granulosus cyst (stage 2). We identified 120 and 153 proteins, respectively, in each fluid. Fifty-six and 84 proteins represented distinct species; 44 and 66 were parasites, and 12 and 18 were host-derived proteins. The five major parasite protein populations, which included antigen B isoforms, metabolic enzymes, proteases and inhibitors, extracellular matrix molecules (ECMs), and developmental proteins, were abundantly distributed in both fluids and also exclusively in one sample or the other. Carbohydrate-metabolizing enzymes were enriched in E. granulosus HF. In the E. multilocularis HF, proteins that constitute ECMs, which might facilitate adhesion and cytogenesis, were highly expressed. Those molecules had physical and functional relationships along with their biochemical properties through protein-protein interaction networks. Twelve host-derived proteins were largely segregated to serum components. The major proteins commonly and uniquely detected in these HFs and their symbiotic interactome relationships might reflect their biological roles in similar but distinct modes of maturation, invasion, and the longevity of the parasites in the hosts.

Fasciclin-calcareous corpuscle binary complex mediated protein-protein interactions in Taenia solium metacestode.

BACKGROUND: Neurocysticercosis (NC) caused by Taenia solium metacestode (TsM) is a serious neurological disease of global concern. Diverse bioactive molecules involved in the long-term survival of TsM might contribute to disease progression. Fasciclin (Fas) is an extracellular protein that mediates adhesion, migration and differentiation of cells by interacting with other molecules. We hypothesized that TsMFas might bind to calcareous corpuscle (CC) through its adhesive property and participate in crucial protein-protein interactions, thus contributing to the creation of a symbiotic interactome network. METHODS: Two paralogous TsMFas (TsMFas1 and TsMFas2) were isolated, and their molecular properties were characterized. The co-localization pattern of TsMFas1 and TsMFas2 with CC was determined. CC-TsMFas binary complex was generated by incubating CC with recombinant proteins (rTsMFas1 and 2). In vitro binding assay of CC-rTsMFas1 or CC-rTsMFas2 binary complex with TsM cellular proteins extracted from scolex and neck was conducted. Their binding partners were identified through proteomic analysis. Integrated protein-protein interaction networks were established. RESULTS: TsMFas1 (6072 bp long) was composed of 15 exons (841 amino acid polypeptide) interrupted by 14 introns. TsMFas2 (5201 bp long) comprised of 11 exons (597 amino acids) and 10 intervening introns. These proteins displayed 22% amino acid sequence identity to each other, but tightly conserved Fas-related domains. Several isoforms of Fas1 and Fas2 proteins might have been expressed through post-translational modifications. They showed adhesion activity with other cells. TsMFas proteins were largely distributed in parenchymal regions of the scolex and bladder wall. These molecules were co-localized with CC, a unique organelle found in platyhelminths. Subsequent proteome analysis of CC-Fas binary complex mediated protein-protein interactions revealed seven protein ligands in the TsM cellular proteins. Their functions were mainly segregated into carbohydrate metabolism (enolase, phosphoenolpyruvate carboxykinase, phosphoglycerate kinase and glyceraldehyde 3-phosphate dehydrogenase) and cytoskeleton/cellular motility (actin, paramyosin and innexin nuc-9). Those proteins had direct (physical) and/or indirect (functional) relationships along with their biochemical properties and biological roles. CONCLUSION: Protein repertoires strongly suggest that TsMFas and CC may symbiotically mediate protein-protein interactions during biological processes to maintain efficacious homeostatic functions and ensure the prolonged survival of TsM in the host.

Biochemical Characterization of Echinococcus multilocularis Antigen B3 Reveals Insight into Adaptation and Maintenance of Parasitic Homeostasis at the Host-Parasite Interface.

Alveolar echinococcosis (AE) caused by Echinococcus multilocularis metacestode is frequently associated with deleterious zoonotic helminthiasis. The growth patterns and morphological features of AE, such as invasion of the liver parenchyme and multiplication into multivesiculated masses, are similar to those of malignant tumors. AE has been increasingly detected in several regions of Europe, North America, Central Asia, and northwestern China. An isoform of E. multilocularis antigen B3 (EmAgB3) shows a specific immunoreactivity against patient sera of active-stage AE, suggesting that EmAgB3 might play important roles during adaptation of the parasite to hosts. However, expression patterns and biochemical properties of EmAgB3 remained elusive. The protein profile and nature of component proteins of E. multilocularis hydatid fluid (EmHF) have never been addressed. In this study, we conducted proteome analysis of EmHF of AE cysts harvested from immunocompetent mice. We observed the molecular and biochemical properties of EmAgB3, including differential transcription patterns of paralogous genes, macromolecular protein status by self-assembly, distinct oligomeric states according to individual anatomical compartments of the worm, and hydrophobic ligand-binding protein activity. We also demonstrated tissue expression patterns of EmAgB3 transcript and protein. EmAgB3 might participate in immune response and recruitment of essential host lipids at the host-parasite interface. Our results might contribute to an in depth understanding of the biophysical and biological features of EmAgB3, thus providing insights into the design of novel targets to control AE.

Spatiotemporal Expression Patterns and Antibody Reactivity of Taeniidae Endophilin B1.

Larval Taeniidae, such as metacestodes of Taenia solium, Echinococcus granulosus, and Echinococcus multilocularis, produce chronic and fatal helminthic diseases. Proper identification of these zoonotic cestodiases is often challenging and is hampered in some clinical settings. Endophilin B1 plays critical roles in the maintenance of membrane contours and endocytosis. We isolated proteins homologous to endophilin B1 from T. solium, Taenia saginata, and Taenia asiatica The three Taeniidae endophilin B1 proteins shared 92.9 to 96.6% sequence identity. They harbored a Bin1/amphiphysin/Rvs (BAR) domain and residues for a dimeric interface but lacked a SRC homology 3 (SH3) domain. Endophilin B1 showed a unique immunological profile and was abundantly expressed in the tegumental syncytium of Taeniidae metacestodes and adults. Bacterially expressed recombinant T. solium endophilin B1 (rTsMEndoB1) demonstrated a sensitivity of 79.7% (345/433 cases) for serodiagnosis of larval Taeniidae infections. The protein showed strong immune recognition patterns against sera from patients with chronic neurocysticercosis, cystic echinococcosis, or advanced-stage alveolar echinococcosis. Adult Taeniidae infections exhibited moderate degrees of positive antibody responses (65.7% [23/35 samples]). rTsMEndoB1 showed some cross-reactivity with sera from patients infected with Diphyllobothriidae (23.6% [25/106 samples]) but not with sera from patients with other parasitic diseases or normal controls. The specificity was 91.7% (256/301 samples). The positive and negative predictive values were 93.6% and 73.4%, respectively. Our results demonstrate that Taeniidae endophilin B1 may be involved in the control of membrane dynamics, thus contributing to shaping and maintaining the tegumental curvature. rTsMEndoB1 may be useful for large-scale screening, as well as for individual diagnosis and follow-up surveillance of Taeniidae infections.

Clonorchis sinensis omega-class glutathione transferases play major roles in the protection of the reproductive system during maturation and the response to oxidative stress.

BACKGROUND: Clonorchis sinensis causes a major food-borne helminthic infection. This species locates in mammalian hepatobiliary ducts, where oxidative stressors and hydrophobic substances are profuse. To adapt to the hostile micromilieu and to ensure its long-term survival, the parasite continuously produces a diverse repertoire of antioxidant enzymes including several species of glutathione transferases (GSTs). Helminth GSTs play pertinent roles during sequestration of harmful xenobiotics since most helminths lack the cytochrome P-450 detoxifying enzyme. METHODS: We isolated and analyzed the biochemical properties of two omega-class GSTs of C. sinensis (CsGSTo1 and CsGSTo2). We observed spatiotemporal expression patterns in accordance with the maturation of the worm's reproductive system. Possible biological protective roles of CsGSTos in these organs under oxidative stress were investigated. RESULTS: The full-length cDNAs of CsGSTo1 and 2 constituted 965 bp and 1,061 bp with open reading frames of 737 bp (246 amino acids) and 669 bp (223 amino acids). They harbored characteristic N-terminal thioredoxin-like and C-terminal α-helical domains. A cysteine residue, which constituted omega-class specific active site, and the glutathione-binding amino acids, were recognized in appropriate positions. They shared 44 % sequence identity with each other and 14.8-44.8 % with orthologues/homologues from other organisms. Bacterially expressed recombinant proteins (rCsGSTo1 and 2) exhibited dehydroascorbate reductase (DHAR) and thioltransferase activities. DHAR activity was higher than thioltransferase activity. They showed weak canonical GST activity toward 1-chloro-2,4-dinitrobenzene. S-hexylglutathione potently and competitively inhibited the active-site at nanomolar concentrations (0.63 and 0.58 nM for rCsGSTo1 and 2). Interestingly, rCsGSTos exhibited high enzyme activity toward mu- and theta-class GST specific substrate, 4-nitrobenzyl chloride. Expression of CsGSTo transcripts and proteins increased beginning in 2-week-old juveniles and reached their highest levels in 4-week-old adults. The proteins were mainly expressed in the elements of the reproductive system, such as vitelline follicles, testes, seminal receptacle, sperm and eggs. Oxidative stressors induced upregulated expression of CsGSTos in these organs. Regardless of oxidative stresses, CsGSTos continued to be highly expressed in eggs. CsGSTo1 or 2 overexpressing bacteria demonstrated high resistance under oxidative killing. CONCLUSIONS: CsGSTos might be critically involved in protection of the reproductive system during maturation of C. sinensis worms and in response to oxidative conditions, thereby contributing to maintenance of parasite fecundity.

Phylogenetic characterization of Clonorchis sinensis proteins homologous to the sigma-class glutathione transferase and their differential expression profiles.

Glutathione transferase (GST) is one of the major antioxidant proteins with diverse supplemental activities including peroxidase, isomerase, and thiol transferase. GSTs are classified into multiple classes on the basis of their primary structures and substrate/inhibitor specificity. However, the evolutionary routes and physiological environments specific to each of the closely related bioactive enzymes remain elusive. The sigma-like GSTs exhibit amino acid conservation patterns similar to the prostaglandin D synthases (PGDSs). In this study, we analyzed the phylogenetic position of the GSTs of the biocarcinogenic liver fluke, Clonorchis sinensis. We also observed induction profile of the GSTs in association with the parasite's maturation and in response to exogenous oxidative stresses, with special attention to sigma-class GSTs and PGDSs. The C. sinensis genome encoded 12 GST protein species, which were separately assigned to cytosolic (two omega-, one zeta-, two mu-, and five sigma-class), mitochondrial (one kappa-class), and microsomal (one membrane-associated proteins in eicosanoid and glutathione metabolism-like protein) GST families. Multiple sigma GST (or PGDS) orthologs were also detected in Opisthorchis viverrini. Other trematode species possessed only a single sigma-like GST gene. A phylogenetic analysis demonstrated that one of the sigma GST lineages duplicated in the common ancestor of trematodes were specifically expanded in the opisthorchiids, but deleted in other trematodes. The induction profiles of these sigma GST genes along with the development and aging of C. sinensis, and against various exogenous chemical stimuli strongly suggest that the paralogous sigma GST genes might be undergone specialized evolution to cope with the diverse hostile biochemical environments within the mammalian hepatobiliary ductal system.

An Echinococcus multilocularis Antigen B3 Proteoform That Shows Specific Antibody Responses to Active-Stage Alveolar Echinococcosis.

Alveolar echinococcosis (AE), caused by the Echinococcus multilocularis metacestode, represents one of the most frequently fatal zoonoses. Early diagnosis significantly reduces morbidity and mortality associated with AE. Diagnosis of AE largely depends on a combination of imaging and serological tests due to its minimal clinical manifestations. Several antigens derived from the whole worm and protoscolex have been targeted for AE serodiagnosis, while the antigenic properties of E. multilocularis hydatid fluid (EmHF) are unclear. We observed two AE-specific 6- and 8-kDa antigen proteoforms through an immunoproteome array of the EmHF. We identified these proteins as representing an E. multilocularis antigen B3 (EmAgB3) isoform, and the proteins were shown to be encoded by the same gene. We cloned the gene and expressed the recombinant EmAgB3 protein (rEmAgB3) in Escherichia coli. rEmAgB3 exhibited sensitivity of 90.9% (80/88 cases) and specificity of 98.5% (597/606 samples) by immunoblotting. The positive and negative predictive values were 89.9% and 98.6%, respectively. The protein did not show antibody responses to 33 AE sera collected during posttreatment follow-up monitoring. Mouse sera experimentally infected with AE protoscoleces began to demonstrate specific antibody responses to native and recombinant EmAgB3 6 months after infection. At that stage, fully mature metacestode vesicles that harbored the brood capsule, primary cell, and protoscolex were observed within an AE mass(es). The response declined along with worm degeneration. Our results demonstrate that the immune responses to this EmAgB3 isoform were highly correlated with worm viability accompanied with AE progression. rEmAgB3 is a promising biomarker for serological assessment of AE patients.

Expression characteristics and specific antibody reactivity of diverse cathepsin F members of Paragonimus westermani.

Paragonimiasis, caused by the lung fluke Paragonimus, is a major food-borne helminthic disease. Differential diagnosis of paragonimiasis from tuberculosis and other infectious granulomas in the lung is a prerequisite to proper management of patients. Cysteine proteases of Paragonimus westermani (PwCPs) invoke specific antibody responses against patient sera, while antibody capturing activity of different PwCPs has not been comparatively analyzed. In this study, we observed the expressional regulation of 11 species of different PwCPs (PwCP1-11). We expressed recombinant PwCPs and assessed diagnostic reliability employing sera from patients with P. westermani (n=138), other trematodiases (n=80), cestodiases (n=60) and pulmonary tuberculosis (n=20), and those of normal controls (n=20). PwCPs formed a monophyletic clade into cathepsin F and showed differential expression patterns along with developmental stages of worm. Bacterially expressed recombinant PwCPs (rPwCPs) exhibited variable sensitivity of 38.4-84.5% and specificity of 87.2-100% in diagnosing homologous infection. rPwCPs recognized specific antibodies of experimental cat sera as early as 3 or 6weeks after infection. Patient sera of fascioliasis, Schistosomiasis japonicum and clonorchiasis demonstrated weak cross-reactions. Our results demonstrate that diverse PwCPs of the cathepsin F family participate in inducing specific antibody responses. Most P. westermani cathepsin F, except for PwCP2 (AAF21461), which showed negligible antibody responses, might be applicable for paragonimiasis serodiagnosis.

Aspergillus nidulans translationally controlled tumor protein has a role in the balance between asexual and sexual differentiation and normal hyphal branching.

Translationally controlled tumor protein (TCTP) is a highly conserved and ubiquitously expressed protein present in all eukaryotes. Cellular functions of TCTP include growth promoting, allergic response and responses to various cellular stresses, but the functions in filamentous fungi have not been reported. In this report, we characterized an Aspergillus nidulans TCTP (TcpA) with high similarity to TCTP. The level of tcpa mRNA was relatively high, both during vegetative growth stage and at early phases of development. TcpA was found predominantly in the nucleus during germination and mycelial growth, and was localized in cytoplasm and nuclei of vesicles on stipes during conidia development. Deletion of tcpA resulted in abnormal hyphal branch formation during vegetative growth. The tcpA deletion inhibited sexual development, but enhanced asexual development via induction of brlA expression. These results imply that TcpA is involved in normal hyphal branch establishment during vegetative growth and also has a role in the balance between asexual and sexual differentiation.

Stem cell treatment for patients with autoimmune disease by systemic infusion of culture-expanded autologous adipose tissue derived mesenchymal stem cells.

Prolonged life expectancy, life style and environmental changes have caused a changing disease pattern in developed countries towards an increase of degenerative and autoimmune diseases. Stem cells have become a promising tool for their treatment by promoting tissue repair and protection from immune-attack associated damage. Patient-derived autologous stem cells present a safe option for this treatment since these will not induce immune rejection and thus multiple treatments are possible without any risk for allogenic sensitization, which may arise from allogenic stem cell transplantations. Here we report the outcome of treatments with culture expanded human adipose-derived mesenchymal stem cells (hAdMSCs) of 10 patients with autoimmune associated tissue damage and exhausted therapeutic options, including autoimmune hearing loss, multiple sclerosis, polymyotitis, atopic dermatitis and rheumatoid arthritis. For treatment, we developed a standardized culture-expansion protocol for hAdMSCs from minimal amounts of fat tissue, providing sufficient number of cells for repetitive injections. High expansion efficiencies were routinely achieved from autoimmune patients and from elderly donors without measurable loss in safety profile, genetic stability, vitality and differentiation potency, migration and homing characteristics. Although the conclusions that can be drawn from the compassionate use treatments in terms of therapeutic efficacy are only preliminary, the data provide convincing evidence for safety and therapeutic properties of systemically administered AdMSC in human patients with no other treatment options. The authors believe that ex-vivo-expanded autologous AdMSCs provide a promising alternative for treating autoimmune diseases. Further clinical studies are needed that take into account the results obtained from case studies as those presented here.

Clinical analysis of surgical approaches for orbital floor fractures.

OBJECTIVE: To identify the optimal surgical method for managing blowout fractures of the inferior orbital wall by analyzing the location and type of fracture based on computed tomographic findings and medical records. METHODS: Medical records of 102 patients with pure inferior blowout fractures who were treated between June 1996 and December 2005 were reviewed regarding fracture type and location and surgical approach. RESULTS: Ocular symptoms persisted in 14 of the 102 cases after surgery, and revision procedures were performed in 11 of those cases. Cases with persistent symptoms were analyzed in terms of fracture location and type of surgery. For anterior orbital floor fractures, symptoms persisted in 2 of the 4 cases treated using a transantral approach, while no symptoms persisted in any of the 15 cases treated using a transorbital approach or in either of the 2 cases treated using a combined approach. For posterior orbital floor fractures, symptoms persisted in 2 of the 31 cases treated using a transantral approach, in 4 of the 6 cases treated using a transorbital approach, and in 1 of the 19 cases treated using a combined approach. For anteroposterior orbital floor fractures, symptoms persisted in 2 of the 5 cases treated using a transorbital approach and in 3 of the 20 cases treated using transantral and combined approaches. CONCLUSION: Patients with large orbital floor fractures or posterior half fractures of the orbit should undergo surgery via a transantral or a combined approach, while patients with trapdoor fractures or anterior half fractures of the orbit should undergo surgery via a transorbital or a combined approach.

Integrin signaling and cell spreading mediated by phorbol 12-myristate 13-acetate treatment.

Spreading of SNU16mAd gastric carcinoma cells was previously shown to be regulated via a signaling network from transforming growth factor beta1 (TGFbeta1) to integrins signaling, through a mediation of protein kinase C delta (PKCdelta). However, in the previous study, the roles of PKCdelta appeared complicated. In this study to clarify the roles of PKCdelta in the spreading of the gastric carcinoma cells, we questioned if PKC activation via phorbol 12-myristate 13-acetate (PMA) treatment could mimic the TGFbeta1 effects. An acute PMA treatment increased phosphorylations of focal adhesion (FA) kinase, paxillin, c-Src, and cofilin, just as TGFbeta1 did. Furthermore, cell spreading mediated by TGFbeta1- or acute PMA treatment correlated with activation of RhoA, which regulates actin reorganization and FA formation. However, stress fiber formation was prominent in TGFbeta1-treated cells, compared to cortical actin organization in PMA-treated cells. Altogether, these observations indicate that acute PMA treatment could mimic the TGFbeta1 mechanisms for cell spreading through subtly different effects on actin reorganization.