Intermittent schedules of the oral RAF-MEK inhibitor CH5126766/VS-6766 in patients with RAS/RAF-mutant solid tumours and multiple myeloma: a single-centre, open-label, phase 1 dose-escalation and basket dose-expansion study.

BACKGROUND: CH5126766 (also known as VS-6766, and previously named RO5126766), a novel MEK-pan-RAF inhibitor, has shown antitumour activity across various solid tumours; however, its initial development was limited by toxicity. We aimed to investigate the safety and toxicity profile of intermittent dosing schedules of CH5126766, and the antitumour activity of this drug in patients with solid tumours and multiple myeloma harbouring RAS-RAF-MEK pathway mutations. METHODS: We did a single-centre, open-label, phase 1 dose-escalation and basket dose-expansion study at the Royal Marsden National Health Service Foundation Trust (London, UK). Patients were eligible for the study if they were aged 18 years or older, had cancers that were refractory to conventional treatment or for which no conventional therapy existed, and if they had a WHO performance status score of 0 or 1. For the dose-escalation phase, eligible patients had histologically or cytologically confirmed advanced or metastatic solid tumours. For the basket dose-expansion phase, eligible patients had advanced or metastatic solid tumours or multiple myeloma harbouring RAS-RAF-MEK pathway mutations. During the dose-escalation phase, we evaluated three intermittent oral schedules (28-day cycles) in patients with solid tumours: (1) 4·0 mg or 3·2 mg CH5126766 three times per week; (2) 4·0 mg CH5126766 twice per week; and (3) toxicity-guided dose interruption schedule, in which treatment at the recommended phase 2 dose (4·0 mg CH5126766 twice per week) was de-escalated to 3 weeks on followed by 1 week off if patients had prespecified toxic effects (grade 2 or worse diarrhoea, rash, or creatinine phosphokinase elevation). In the basket dose-expansion phase, we evaluated antitumour activity at the recommended phase 2 dose, determined from the dose-escalation phase, in biomarker-selected patients. The primary endpoints were the recommended phase 2 dose at which no more than one out of six patients had a treatment-related dose-limiting toxicity, and the safety and toxicity profile of each dosing schedule. The key secondary endpoint was investigator-assessed response rate in the dose-expansion phase. Patients who received at least one dose of the study drug were evaluable for safety and patients who received one cycle of the study drug and underwent baseline disease assessment were evaluable for response. This trial is registered with ClinicalTrials.gov, NCT02407509. FINDINGS: Between June 5, 2013, and Jan 10, 2019, 58 eligible patients were enrolled to the study: 29 patients with solid tumours were included in the dose-escalation cohort and 29 patients with solid tumours or multiple myeloma were included in the basket dose-expansion cohort (12 non-small-cell lung cancer, five gynaecological malignancy, four colorectal cancer, one melanoma, and seven multiple myeloma). Median follow-up at the time of data cutoff was 2·3 months (IQR 1·6-3·5). Dose-limiting toxicities included grade 3 bilateral retinal pigment epithelial detachment in one patient who received 4·0 mg CH5126766 three times per week, and grade 3 rash (in two patients) and grade 3 creatinine phosphokinase elevation (in one patient) in those who received 3·2 mg CH5126766 three times per week. 4·0 mg CH5126766 twice per week (on Monday and Thursday or Tuesday and Friday) was established as the recommended phase 2 dose. The most common grade 3-4 treatment-related adverse events were rash (11 [19%] patients), creatinine phosphokinase elevation (six [11%]), hypoalbuminaemia (six [11%]), and fatigue (four [7%]). Five (9%) patients had serious treatment-related adverse events. There were no treatment-related deaths. Eight (14%) of 57 patients died during the trial due to disease progression. Seven (27% [95% CI 11·6-47·8]) of 26 response-evaluable patients in the basket expansion achieved objective responses. INTERPRETATION: To our knowledge, this is the first study to show that highly intermittent schedules of a RAF-MEK inhibitor has antitumour activity across various cancers with RAF-RAS-MEK pathway mutations, and that this inhibitor is tolerable. CH5126766 used as a monotherapy and in combination regimens warrants further evaluation. FUNDING: Chugai Pharmaceutical.

Immunodiagnosis of alveolar echinococcosis using urine samples.

Alveolar echinococcosis (AE) is one of the most lethal zoonotic parasitic infections. The diagnosis is based on the combination of the abdominal imaging including CT, MRI and PET, and serology. To develop a new diagnostic tool for AE with urine as samples, mouse-Echinococcus multilocularis (Em) model and then human cases were studied. The antibody levels of urine and serum samples from the infected mice and AE cases were well correlated with each other. The sensitivity and specificity of the method with urine were 91% and 98%, respectively, when IgG4 to crude Em was examined. Comparing with serum samples, the collection of urine is easier and safer and the urine diagnostic tool makes surveys of this silent disease easier.

Effect of exercise on kidney function, oxidative stress, and inflammation in type 2 diabetic KK-A(y) mice.

Exercise is recommended for the management of type 2 diabetes, but its effects on diabetic nephropathy (DN) are still unknown. We hypothesized that appropriate exercise improves early DN via attenuation of inflammation and oxidative damage. Type 2 diabetic KK-A(y) mice, a spontaneous DN model, underwent two different kinds of exercise (i.e., moderate and low intensity). Sedentary mice or those undergoing an exercise regimen causing no significant body weight loss were used. We examined the urinary excretion of albumin, number of podocytes and macrophages, renal expressions of HIF-1α and MCP-1, and biomarkers of oxidative stress such as urinary 8-OHdG and serum SOD. Exercise reduced urinary levels of albumin and also maintained the number of podocytes in the exercised KK-A(y) mice independently of improvements of overweight and hyperglycemia, although moderate-intensity exercise increased expression of HIF-1α. Sedentary KK-A(y) mice showed increased expression of MCP-1 and infiltration of macrophage, increased urinary 8-OhdG, and decreased serum SOD levels compared with exercised KK-A(y) mice. On the whole, low-intensity exercise attenuates progression of early DN without affecting marked renal ischemia. Reduction rates of urinary albumin and maintained podocyte numbers, with parallel improvements in oxidative damage and inflammation, are related to beneficial effects of exercise in diabetic kidney disease.

Environmental protection: researches in National Institute of Radiological Sciences.

Some studies for radiological protection of the environment have been made at the National Institute of Radiological Sciences (NIRS). Transfer of radionuclides and related elements has been investigated for dose estimation of non-human biota. A parameter database and radionuclide transfer models have been also developed for the Japanese environments. Dose (rate)-effect relationships for survival, growth and reproduction have been investigated in conifers, Arabidopsis, fungi, earthworms, springtails, algae, duckweeds, daphnia and medaka. Also genome-wide gene expression analysis has been carried out by high coverage expression profiling (HiCEP). Effects on aquatic microbial communities have been studied in experimental ecosystem models, i.e., microcosms. Some effects were detected at a dose rate of 1 Gy day(-1) and were likely to arise from interspecies interactions. The results obtained at NIRS have been used in development of frameworks for environmental protection by some international bodies, and will contribute to environmental protection in Japan and other Asian countries.

Histopathological, serological, and molecular confirmation of indigenous alveolar echinococcosis cases in Mongolia.

Alveolar echinococcosis cases diagnosed histopathologically in 2002, 2006, 2007, and 2009 in Ulaanbaatar, Mongolia were reconfirmed by evaluating the cytochrome c oxidase subunit I gene of mitochondrial DNA. The most recent three cases using paraffin-embedded and ethanol-fixed specimens revealed that one was of the "Asian" haplotype, whereas two others were of the "Inner Mongolian" type. All patients were born in the western provinces of Mongolia, they never resided outside of Mongolia, and they were given a preliminary diagnosis of malignant hepatic tumor or abscess. The most recent two cases were also confirmed serologically to be active alveolar echinococcosis.

The safety, tolerability, pharmacokinetics, and pharmacodynamics of single oral doses of CH4987655 in healthy volunteers: target suppression using a biomarker.

PURPOSE: CH4987655 (RO4987655) is an orally active and highly selective small-molecule MEK inhibitor. It potently inhibits mitogen-activated protein kinase signaling pathway activation and tumor cell growth, with an in vitro IC(50) of 5.2 nmol/L for inhibition of MEK1/2. Single-agent oral administration of CH4987655 resulted in complete tumor regressions in xenograft models. EXPERIMENTAL DESIGN: All 40 subjects received a single oral dose followed by 72 hrs of pharmacokinetic, pharmacodynamic, and safety/tolerability assessments. The pharmacodynamics were measured by changes in phosphorylated extracellular signal-regulated kinase (pERK) levels in a surrogate tissue peripheral blood mononuclear cells ex vivo stimulated by PMA. RESULTS: Doses of 0.5, 1, 2, 3, and 4 mg were safe and well tolerated. No clinically significant safety event was observed. A total of 26 adverse events (n = 15) were reported: 21 mild, 5 moderate, and none severe. Moderate adverse events were experienced by one subject at 1 mg (autonomic nervous system imbalance) and three subjects at 4 mg (diarrhea, abdominal pain, autonomic nervous system and acne). CH4987655 was rapidly absorbed with a t(max) of approximately 1 h. Exposures were dose proportional from 0.5 to 4 mg. The disposition was biphasic with a terminal t(1/2) of approximately 25 hr. Intersubject variability was low, 9% to 23% for C(max) and 14% to 25% for area-under-the-curve (AUC). pERK inhibition was exposure dependent and was greater than 80% inhibition at higher doses. The pharmacokinetic-pharmacodynamic relationship was characterized by an inhibitory E(max) model (E(max) approximately 100%; IC(50) 40.6 ng/mL) using nonlinear mixed-effect modeling. CONCLUSIONS: A significant extent of pERK inhibition was achieved for a single dose that was considered to be safe and well tolerated in healthy volunteers.

Live imaging of radiation-induced apoptosis by yolk injection of Acridine Orange in the developing optic tectum of medaka.

To observe the sequential radiation-induced apoptosis in a living embryo, we injected Acridine Orange (AO) solution into the yolk of embryo and visualized radiation-induced apoptosis in developing optic tectum (OT). Medaka embryos at stage 28, when neural cells proliferate rapidly in the OT, were irradiated with 5 Gy X-rays which is a non-lethal dose for irradiated embryos at hatching. The irradiated embryos hatched normally without morphological abnormalities in their brains, even though a large number of apoptotic cells were induced transiently in OT. By yolk injection, apoptotic cells in OT were distinguished as AO-positive small nuclei at 3 h after irradiation. At 8-10 h after irradiation, AO-positive rosette-shaped clusters were obviously distinguished in marginal tectal regions of OT where cells are proliferating intensely. The AO-positive clusters became bigger and more obvious, but the number did not increase up to 24 h after irradiation and completely disappeared up to 49 h after irradiation. This characteristic appearance of the AO-positive nuclei/clusters is in good agreement with our previous results, based on the examination of fixed specimens stained with AO by injection into the peri-vitelline space, suggesting that the AO-yolk injection method is highly reliable for detecting apoptotic cells in living embryos. The live imaging of apoptotic cells in developing Medaka embryos by AO-yolk injection method is expected to reveal more of the details of the dynamics of apoptotic responses in the irradiated brain and other tissues.

Serological monitoring of progression of alveolar echinococcosis with multiorgan involvement by use of recombinant Em18.

Two cases of alveolar echinococcosis (AE) with multiple-organ involvement (the liver, lungs, and bone) were monitored by imaging and serology for 20 years. Resection of the bone lesion was complete in one case but incomplete in the other case. Albendazole treatment was markedly to moderately effective against hepatic and pulmonary AE lesions in both cases, whereas it had almost no effect against the bone lesion in one case. The results of the serological tests with recombinant Em18 antigen coincided with the clinical findings in each case. An enzyme-linked immunosorbent assay for the detection of immunoglobulin G (IgG) responses, especially IgG4 responses, is expected to be a real-time indicator of the dynamics of active AE.

Combination effects of enalapril and losartan on lipid peroxidation in the kidneys of KK-Ay/Ta mice.

BACKGROUND: Adenosine monophosphate activated protein kinase (AMPK) has a protective effect on lipid peroxidation. Adiponectin and AMPK might have a role in the pathogenesis of diabetic nephropathy. Blockade of the renin-angiotensin system (RAS) increases adiponectin levels and reduces oxidative stress. The objective of the present study was to examine lipid peroxidation via adiponectin and AMPK activation in the kidneys of KK-A(y)/Ta mice by RAS inhibitors, such as enalapril and/or losartan. METHODS: KK-A(y)/Ta mice were given enalapril (2.5 mg/kg/day) and/or losartan (25 mg/kg/day), or hydralazine (25 mg/kg/day) in the drinking water for 8 weeks starting at 8 weeks of age. They were divided into 5 groups as follows: enalapril 2.5 mg/kg/day treatment group (n = 5), losartan 25 mg/kg/day treatment group (n = 5), enalapril 2.5 mg/kg/day + losartan 25 mg/kg/day combination treatment group (n = 5), hydralazine 25 mg/kg/day treatment group (n = 5) and tap water group as the untreated group (n = 5). The urinary albumin/creatinine ratio (ACR), serum adiponectin and systemic blood pressure were measured as test parameters. Expressions of adiponectin, phospho-AMPKalpha (p-AMPKalpha) and phospho-acetyl CoA carboxylase(beta) (p-ACC(beta)) in the kidneys were evaluated by Western blot analyses. Pathological changes of glomeruli were evaluated by light microscopy. Accumulations of N(epsilon)-(carboxymethyl) lysine (CML), malondialdehyde (MDA) and 4-hydroxy-2-nonenal (4-HNE) in glomeruli were evaluated by immunohistochemical analyses. RESULTS: Enalapril and/or losartan improved levels of urinary ACR with activation of adiponectin, p-AMPKalpha and p-ACC(beta) in the kidneys. CML, MDA and 4-HNE expressions in glomeruli were significantly suppressed by enalapril and/or losartan, especially in the combination treatment group. CONCLUSIONS: It appears that enalapril and/or losartan, especially in combination, inhibited accumulation of CML/MDA/4-HNE in diabetic renal tissues. These effects might be related to lipid peroxidation via tissue-specific activation of adiponectin and AMPK.

Rapid and simple method for quantitative evaluation of neurocytotoxic effects of radiation on developing medaka brain.

We describe a novel method for rapid and quantitative evaluation of the degree of radiation-induced apoptosis in the developing brain of medaka (Oryzias latipes). Embryos at stage 28 were irradiated with 1, 2, 3.5, and 5 Gy x-ray. Living embryos were stained with a vital dye, acridine orange (AO), for 1-2 h, and whole-mount brains were examined under an epifluorescence microscope. From 7 to 10 h after irradiation with 5 Gy x-ray, we found two morphologically different types of AO-stained structures, namely, small single nuclei and rosette-shaped nuclear clusters. Electron microscopy revealed that these two distinct types of structures were single apoptotic cells with condensed nuclei and aggregates of apoptotic cells, respectively. From 10 to 30 h after irradiation, a similar AO-staining pattern was observed. The numbers of AO-stained rosette-shaped nuclear clusters and AO-stained single nuclei increased in a dose-dependent manner in the optic tectum. We used the number of AO-stained rosette-shaped nuclear clusters/optic tectum as an index of the degree of radiation-induced brain cell death at 20-24 h after irradiation. The results showed that the number of rosette-shaped nuclear clusters/optic tectum in irradiated embryos exposed to 2 Gy or higher doses was highly significant compared to the number in nonirradiated control embryos, whereas no difference was detected at 1 Gy. Thus, the threshold dose for brain cell death in medaka embryos was taken as being between 1-2 Gy, which may not be so extraordinarily large compared to those for rodents and humans. The results show that medaka embryos are useful for quantitative evaluation of developmental neurocytotoxic effects of radiation.

Recombinant human hexamer-dominant IgM monoclonal antibody to ganglioside GM3 for treatment of melanoma.

PURPOSE: L612, a human IgM monoclonal antibody produced by an EBV-transformed human B-cell line, binds to ganglioside GM3 and kills GM3-positive human melanoma cells in the presence of complement. It has been shown to be effective in some patients with late-stage melanoma. L612 consists of hexameric IgM (about 20%), pentameric IgM (about 74%), and other minor IgM molecules. Because hexameric IgM activates complement more effectively than pentameric IgM, we developed and evaluated a hexamer-dominant recombinant IgM for clinical applications. EXPERIMENTAL DESIGN: Chinese hamster ovary (CHO) cells were transfected with heavy- and light-chain genes of L612, with or without the joining-chain gene. Antitumor effects of the recombinant IgM secreted from CHO cells were evaluated in vitro and in vivo. RESULTS: Recombinant IgM secreted from CHO cells without the joining chain (designated CA19) was approximately 80% hexameric, whereas recombinant IgM from CHO cells transfected with heavy-, light-, and joining-chain genes (designated CJ45) was about 90% pentameric. Both CA19 and CJ45 recombinant IgMs caused complement-dependent cytotoxicity against human and mouse melanoma cell lines, but the amount of CA19 required for 50% specific cytotoxicity was 5 to 10 times smaller. I.v. injection of CA19 compared with CJ45 or native L612 elicited more profound antitumor activity in nude rats bearing a GM3-positive mouse melanoma xenograft. CONCLUSIONS: A hexamer-dominant human IgM against GM3 may provide a more potent treatment option for patients with GM3-positive melanoma.

Radiation-induced brain cell death can be observed in living medaka embryos.

Medaka (Oryzias latipes) embryos at 25-26 and 28-30 stages were irradiated with a single acute dose of 10 Gy of X-ray, which is lower than the LD(50 )of the embryos. The effects on developing brains were examined under a stereomicroscope in living embryos until hatching. All the irradiated embryos survived; however, from 6 to 35 h after X-ray irradiation, massive clusters of optically opaque and round cells were observed either in the entire brain region (when irradiated at 25-26 stages) or mainly in the optic tectum (when irradiated at 28-30 stages). Histological examination and TUNEL showed that these cells are clusters of dead cells. These dead cell clusters disappeared thereafter, and the irradiated embryos continued to develop apparently normally. The grown irradiated embryos, however, had smaller brains and eyes than the nonirradiated control embryos. At hatching, the irradiated embryos exhibited histological abnormalities in the brain, particularly in the torus longitudinalis, and in the retina, although most of them hatched normally and survived. The results indicate that brain cell death and a reduced brain size can be observed in living irradiated embryos, and suggest that the medaka embryo is useful for screening the developmental neurotoxicity effects of various hazardous factors.

Recent advances in characterization of Echinococcus antigen B.

Antigen B (AgB) in hydatid cyst fluid of Echinococcus granulosus is a polymeric lipoprotein of 160 kDa and a highly immunogenic major antigen in echinococcal infection. The antigen is comprised of a group of subunit monomers of approximately 8 kDa in molecular size. Recent studies have revealed that the E. granulosus AgB (EgAgB) shows a high degree of genetic variability and the genes encoding the EgAgB 8-kDa subunit monomers that have been identified to date could be grouped into four clades, corresponding to the genes EgAgB8/1, EgAgB8/2, EgAgB8/3 and EgAgB8/4. It has been suggested that the recombinant EgAgB8/2 (rEgAgB8/2) provides better performance in serodiagnosis of human cystic echinococcosis (CE) than does the recombinant EgAgB8/1 (rEgAgB8/1). The EgAgB has been identified as a protease inhibitor with an ability to inhibit recruitment of neutrophils and exploit activation of T helper cells by eliciting a non-protective Th2 cell response, predominantly in patients with progressive CE. Recently it has been revealed that AgB also exists in the cyst fluid of Echinococcus multilocularis. Five different cDNAs encoding the EgAgB homologues have been identified in vesicles, protoscoleces and/or immature adult worms of E. multilocularis and named as EmAgB8/1, EmAgB8/2, EmAgB8/3, EmAgB8/4 and EmAgB8/5. These genes appeared to be expressed in a developmentally regulated manner in the parasite life cycle. This review focuses on recent advances in molecular biological and immunological characterization of AgB from both of E. granulosus and E. multilocularis.

Usefulness of severe combined immunodeficiency (scid) and inbred mice for studies of cysticercosis and echinococcosis.

The topics in this review are the usefulness of immunodeficient and inbred mice for studies of developmental biology, drug efficacy and host specificity in cysticercosis and echinococcosis. In non-obese diabetic severe combined immunodeficiency (NOD/Shi-scid) mice of both sexes, in vitro hatched oncospheres of all three human taeniid species (Taenia solium, Taenia saginata and Taenia asiatica) developed into cysticerci comparable to or bigger than those developed in their known intermediate host animals, whereas only females were susceptible to these infections in other scid mice of BALB/c, C57BL or C.B-17 inbred strains. Detailed morphological observation from post-oncospheral to cysticercus developmental stages is expected to be easy when we use NOD/Shi-scid mice experimentally inoculated with in vitro hatched oncospheres. Metacestocidal effect of oxfendazole was evaluated in NOD/Shi-scid mice experimentally inoculated with oncospheres of T. solium. In Echinococcus multilocularis infection, larval tissue proliferated without induction of inflammatory host responses in scid mice, thus facilitating isolation of the larval vesicles and protoscoleces for biochemical and molecular biological studies. Trans portal inoculation of metacestode tissues resulted in proliferation of secondary echinococcal foci localized exclusively in the liver without metastasis to other tissues or organs. The advantages of a mouse model for Echinococcus granulosus are also described.

Usefulness of recombinant Em18-ELISA to evaluate efficacy of treatment in patients with alveolar echinococcosis.

Alveolar echinococcosis (AE) is a rare parasitic disease caused by Echinococcus multicularis and most commonly involves the liver. Early diagnosis is essential to improve the prognosis of patients with AE of the liver. Em18, an 18-kD diagnostic antigen from Echinococcus multilocularis, is highly specific and sensitive to detect AE. We previously reported that an enzyme-linked immunosorbent assay (ELISA) system using a recombinant Em18 antigen (RecEm18) was highly useful in the differential serodiagnosis of AE. In this report, we present seven AE patients who showed dynamic changes in RecEm18-ELISA values in the course of long-term follow up of albendazole (ABZ) chemotherapy, and/or resections of the liver or bone metastasis. All seven AE patients revealed positive values, over the cutoff level, of the RecEm18-ELISA before the treatments. The values in six patients fell below the cutoff level after the treatments, but the value in a patient with recurrence never fell below the cutoff level, and increased again. From these results, it seems that the RecEm18-ELISA is useful to evaluate the efficacy of treatment and predict recurrence in patients with AE. RecEm18-ELISA may be an important examination for: (a) the mass screening of AE in Japan, (b) the confirmative diagnosis of AE prior to surgical and/or chemotherapeutic treatments, (c) the follow up of AE patients after treatments, and (d) for deciding on the discontinuation of chemotherapy in patients with an appropriate response.

The medaka fish Tol2 transposable element can undergo excision in human and mouse cells.

Tol2 is an active DNA-based transposable element identified in the medaka fish, Oryzias latipes. Originating from a vertebrate and belonging to the hAT ( hobo/ Activator/ Tam3) transposable element family, featuring a wide distribution among organisms, Tol2 would be expected to be active if introduced into mammals. We, therefore, examined if excision, one part of the transposition reaction, can occur in human and mouse culture cells. A Tol2 clone was introduced into cells and, after incubation, recovered. PCR and sequencing analysis provided evidence for precise and near precise excision in these cells. Tol2 can thus be expected to serve as a material for developing a gene transfer vector and other genetic tools applicable to mammals. It was also suggested that an intact Tol2 element could retain autonomy as a transposable element in mammalian cells.

Development of Em18-immunoblot and Em18-ELISA for specific diagnosis of alveolar echinococcosis.

Extensive experience has documented that Em2(plus)-ELISA, Em10-ELISA and Em18-immunoblot and Em18-ELISA are reliable serologic methods for detection of alveolar echinococcosis (AE) caused by the metacestodes of Echinococcus multilocularis. Among these, tests based on detection of antibodies to the specific Em18 antigen, either immunoblot or ELISA, appears to be the most specific for AE. Between 90 and 97% of AE cases with characteristic hepatic lesions detectable by image analysis have been positive in Em18-serology. In contrast Antigen B (8 kDa)-immunoblot is the most sensitive for all forms of echinococcosis, although it can not differentiate AE from cystic echinococcosis (CE). Primary serologic screening for echinococcosis, especially for CE using hydatid cyst fluid of Echinococcus granulosus appears to be highly sensitive in endemic areas. Glycoproteins (GPs) purified from cyst fluid of Taenia solium are highly specific for diagnosis of T. solium neuorcysticercosis (NCC). Using currently available antigens it is not difficult to differentiate these three larval cestodiases serologically. We recommend that (1) primary screening of CE in endemic areas should be carried out using hydatid cyst fluid of E. granulosus prepared from cysts in either sheep, human or mouse for immunoblot and from sheep or mouse for ELISA, (2) both primary screening and confirmation of AE in endemic areas should be carried out using Em18-ELISA, Em18-immunoblot or Em2(plus)-ELISA. Serodiagnosis in areas where both AE and CE are endemic, such as in China, should be carried out as a combination of (1) and (2), and (3) serology of NCC should be carried out using GP-ELISA or GP-immunoblot. All samples showing antibody to Em18 are exclusively from echinococcosis cases. There have been no false positive test reactions with sera from other diseases. Strongest Em18 responders are all from patients with AE but some weaker responses may be found in sera of persons with advanced complex lesions of CE. These highly reliable serodiagnostic methods using native, recombinant and synthetic antigens are briefly summarized and experiences with these methods in Japan is reviewed. We believe that use of these specific antigens in screening and confirmation programs for AE in Japan will improve specificity and reduce the confusion, anxiety and expense in persons whose sera give false positive reactions with crude echinococcal antigens.

Echinococcosis and cysticercosis in Asia: evaluation of the modern technology for epidemiological study.

The recent emergence of zoonotic parasitic diseases of public health importance represents a growing global concern. Among zoonotic helminthic diseases, both echinococcosis and cysticercosis are the most serious diseases threatening human life. Neurocysticercosis (NCC) caused by ingestion of eggs of the pork tapeworm, Taenia solium, is spreading worldwide and not rare even in Muslim or Jewish communities. Alveolar echinococcosis (AE) caused by the proliferation of metacestodes of the fox tapeworm, Echinococcus multilocularis, is the most potentially lethal parasitic infection of the non-tropical northern hemisphere, whereas cystic echinococcosis (CE) caused by the proliferation of metacestodes of the dog tapeworm, E. granulosus, has rather a cosmopolitan distribution. As the life cycles of T. solium, E. multilocularis and E. granulosus are completed through predator-prey interactions, including humans, it is crucial to interrupt the cycle for control of these zoonotic cestodiases. Both NCC and CE are expected to be eradicable, since the principal life cycles of T. solium and E. granulosus are maintained between humans and pigs and between dogs and herbivorous domestic animals, respectively. In contrast, AE is perhaps not eradicable, since the life cycle of E. multilocularis is maintained between wild foxes and rodents. Modern technologies, including imaging, immunology and molecular biology, have been applied for epidemiological surveys. In the present review, we introduce such technologies applied in Japan, China and Indonesia, and point out the problems that need to be solved for control of these three zoonotic cestodiases.

Evaluation of an enzyme-linked immunosorbent assay (ELISA) with affinity-purified Em18 and an ELISA with recombinant Em18 for differential diagnosis of alveolar echinococcosis: results of a blind test.

Alveolar echinococcosis (AE) is the most potentially lethal parasitic zoonosis of the nontropical areas in the northern hemisphere, where cystic echinococcosis (CE) is also endemic. Both AE and CE are highly endemic in China, and both serologic detection of echinococcosis, either AE or CE, and differentiation of AE from CE are crucial problems. Evaluation of Western blot analysis (WB) and enzyme-linked immunosorbent assay (ELISA) for the Em18 antigen, using affinity-purified and recombinant Em18, was carried out "blindly" using 60 human sera from patients diagnosed in France. The results were compared with those obtained using a commercially available Echinococcus WB immunoglobulin G (IgG) kit developed in France. The Em18 WB and Echinococcus WB IgG showed very similar results for detection of AE. Both affinity-purified Em18 or a recombinant Em18 WB and Echinococcus WB IgG seem useful for identification of AE, and the latter seems appropriate for both AE and CE, whereas affinity-purified Em18 ELISA and the newly developed recombinant Em18 ELISA appear to be suitable for detection of AE, especially for epidemiological surveys.