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Although Lp(a) have been thought to be a cardiovascular risk factor, it is unclear whether lowering Lp(a) levels reduces the risk of cardiovascular diseases. No pharmacological agents which selectively reduce serum Lp(a) levels, and Lp(a) is present in primate but absent in common laboratory animals such as mice and pigs. In the present study we used transgenic mice of human Lp(a) and tested effect a novel Lp(a) lowering drug D-47 on neointima formation after vascular injury. D-47 successfully decreased plasma levels of Lp(a) and possibly inhibited neointima formation in Lp(a) transgenic mice. The results indicate that we can modulate plasma Lp(a) levels by pharmacologic agents and inhibit atherogenic properties of Lp(a) by reducing plasma levels of Lp(a). J. Med. Invest. 64: 64-67, February, 2017.

Trematodes in the genus Metagonimus occur naturally in the small intestines of a variety of mammalian and avian hosts. At present, a total of 6 Metagonimus species are recognized in Japan: M. yokogawai (Katsurada, 1912), M. takahashii Suzuki, 1930, M. katsuradai Izumi, 1935, M. otsurui Saito et Shimizu, 1968, M. miyatai Saito et al., 1997 and M. hakubaensis Shimazu, 1999. Metacercarial infection by Metagonimus spp. has been confirmed in a variety of fresh and barackish water fishes [3]. Metagonimus hakubaensis was originally described in the adult flukes obtained from laboratory rats that had been experimentally fed metacercariae isolated from the sand lamprey, Lethenteron reissneri (Dybowski), collected in Nagano Prefecture, Japan [11]. No further information on natural and experimental final hosts of the flukes has been reported to date. The present study was therefore conducted to estimate the suitability of several mammals and birds to experimental infection with M. hakubaensis, on the basis of recovery rates, development and fecundity of the flukes. Further, since physiological aspects, such as predilection site of adult Metagonimus spp. in final hosts, are considered useful for differentiating among members of the genus Metagonimus[1, 10], we compared the results from this study with previous studies on other Metagonimus spp. Metagonimus hakubaensis metacercariae were obtained from naturally infected lampreys captured in irrigation ditches in Temmabayashi, Aomori Prefecture, Japan (Fig. 1). The lampreys were cut into small pieces and digested in artificial gastric juice (pepsin, 1:10,000, 7 g; HCl, 7 ml in 1,000 ml distilled water) at 37°C for 30 min. After digestion, the metacercariae were collected from the digested fluid under a dissecting microscope and used to infect the following laboratory animals: 4 Syrian golden hamsters (4-week-old males), 6 Wistar rats (4-week-old males), 3 ddY mice (4-week-old males), 4 dogs (puppies), 4 cats (kittens), 12 White Leghorn chickens (12- or 15-day-old) and 6 quails (adults). Each animal was orally inoculated with 200–1,000 encysted metacercariae and sacrificed under ether anesthesia at 8 and 15 days post-infection (DPI). The small intestines (divided into upper, middle and lower sections) and large intestines were opened, the contents were removed, and the mucous membrane was exfoliated with pointed forceps. The obtained samples were then washed by repeated sedimentation with saline solution. The flukes recovered by examining the washed sediments under a dissecting microscope were placed between a glass slide and a cover slip and fixed in 70% alcohol, stained with alum carmine and then mounted in Canada balsam for examination under a camera lucida. All research was conducted according to the Guidelines for the Care and Use of Laboratory Animals of Kitasato University. The experimental protocols were approved by the Animal Care and Use Committee of Kitasato University. Fig. 1. Encysted metacercariae of Metagonimus hakubaensis isolated from Lethenteron reissneri. Bar=100 µm. The results of fluke recovery from the experimentally infected animals at 8 and 15 DPI are summarized in Table 1. These findings suggest that a variety of mammals and birds could potentially serve as natural final hosts for M. hakubaensis, which has remained unknown to date. However, the recovery rates at 8 DPI were highest in dogs (88.8%) and hamsters (87.8%), followed by cats (56.3%), rats (53.7%), chickens (27.1%) and quails (2.1%). Similarly, the recovery rates at 15 DPI were highest in hamsters (76.8%) and dogs (73.8%) and lower in the other animal hosts tested (0–23.9%). The rates of fluke recovery in dogs and hamsters were significantly higher than those in mice, chickens and quails at 8 and 15 DPI (P 80%), followed by hamsters (17.8–38.5%), and then ddY mice (3.4%) [5, 6, 13]. In addition, the fluke recovery rate at 8 DPI in chickens experimentally infected with M. takahashii was 16% [7]. Thus, with the exception of the high M. hakubaensis recovery rates in hamsters, the findings of the present study are similar to those of the previous reports. Table 1. Susceptibility of several mammalian and avian hosts to experimental infection with Metagonimus hakubaensis At 8 and 15 DPI, most of the flukes in the experimentally infected animals used in this study were found in the lower small intestine, and the remaining flukes were mainly recovered from the middle portion of the small intestine. Several studies examining the distribution of adult Metagonimus spp. in definitive hosts have reported differences in the predilection site among Metagonimus spp. [4, 5, 8,9,10, 12]. For example, M. miyatai, M. katsuradai and M. otsurui exhibited a predilection for the lower small intestine, while M. yokogawai and M. takahashii exhibited a preference for the upper small intestine. The predilection site of M. hakubaensis in this study was thus similar to that of M. miyatai, M. katsuradai and M. otsurui. All of the approximately 900 flukes that were randomly collected from the different host species for morphological observation were sexually mature with numerous uterine eggs. These flukes were identified as M. hakubaensis based on morphological characteristics that are considered typical for the species: the oral sucker is slightly smaller than, as large as, or slightly larger than the acetabulum, the intestinal ceca and vitellaria do not extend posteriorly beyond the right testis, and the uterus hardly enters the post-testicular region. The morphometric measurement results for flukes recovered from different hosts at 8 and 15 DPI are summarized in Table 2. The body size of M. hakubaensis differed between hosts (Fig. 2); for example, at 8 DPI, the body length of flukes recovered from hamsters and dogs was significantly larger than that of flukes from the other hosts (P

Fecal egg count reduction tests (FECRT) and larval migration inhibition tests (LMIT) were conducted to assess the efficacy of ivermectin (IVM) against gastrointestinal nematodes on 2 cattle farms in northern Japan in 2009 and 2010. Twelve to 20 calves on each farm were treated topically with 0.5 mg IVM/kg 2 (Farm 2) or 4 times (Farm 1) during the grazing season (May–October). On Farm 1, fecal egg count (FEC) reduction at 14 days post-treatment ranged from 16 to 87% in 2009 and from 24 to 96% in 2010, with relatively low reductions in August and October (16–53%). Conversely, IVM treatment on Farm 2 reduced FEC by 97% in September 2009. Larvae obtained from fecal cultures and identified by PCR-RFLP analysis revealed that the dominant species on both farms prior to IVM administration was Cooperia oncophora. In 2009, the FEC reduction of C. oncophora on Farm 1 decreased from 85% in May to 56% in August. In 2010, the reduction in C. oncophora in August was 28%. In the LMIT using larvae collected from the fecal cultures on Farm 1 in May and August 2009, the EC50 value of IVM in C. oncophora in August (0.892 µg/ml) was 3 times higher than that in May (0.296 µg/ml). The results of the LMIT corroborated the FECRT data, indicating the presence of IVM-resistant C. oncophora on Farm 1, at least in August. This is the first report of IVM-resistant nematodes in Japanese cattle.

A total of 611 preserved adult Metagonimus spp. specimens recovered from 32 of 53 Japanese water shrews (Chimarrogale platycephala) that had previously been collected in Aomori Prefecture between June 1994 and August 1996, were examined in this study. Morphological examination revealed that 603 of these flukes were identical to M. hakubaensis Shimazu, 1999, and that the others were M. takahashii Suzuki, 1930 (n=4), M. otsurui Saito et Shimizu, 1968 (n=2), and M. miyatai Saito et al., 1997 (n=2). Each of the 32 Japanese water shrews infected with M. hakubaensis contained between 1 and 83 flukes. This is the first record of the natural final host for M. hakubaensis, since this fluke species was described.

A composite 2,206 nucleotide DNA sequence encoding a putative immunoglobulin-binding protein (BiP) was constructed from a sequence obtained from Babesia caballi cDNA library clones. The 1,962 nucleotide open reading frame predicts a 72 kD protein with extensive homology with BiPs from Apicomplexa parasites. The BiP gene had a predicted N-terminal signal sequence of 18 amino acids and a C-terminal tetrapeptide sequence (Ser-Asp-Glu-Leu) for signaling in the endoplasmic reticulum lumen. The recombinant protein expressed in baculovirus showed an apparent mass of 72 kD, which is identical to that of the native B. caballi protein. Monoclonal antibodies (MAbs) against B. caballi BiP reacted strongly with extracellular merozoites, but not in early intraerythrocytic stage. Detailed observation showed that the reaction of MAbs against pear-shaped forms was markedly irregular, with either no reaction, or reaction with one or two brightly fluorescent pear-shaped forms (two parasites) of B. caballi.

Third-stage larvae of Gongylonema pulchrum from naturally infected dung beetles were inoculated orally into 24 rabbits. Worm recovery ranged from 54 to 91% (mean = 67.5%) during the period from 24 hr to 52 wk postinoculation (PI). Two hours PI, the larvae entered the mucosa at the junction of the stomach and esophagus and migrated upward. Early development occurred primarily in pharyngeal mucosa, tongue, and buccal mucosa. The third molt took place 11 days PI and the final molt at 36 days PI. Male worms reached sexual maturity at 7 wk PI and females at 9 wk PI. Adult worms were found mainly in the esophagus but also occurred in the tongue and the wall of the oral cavity after 30 wk PI. Embryonated eggs appeared in the feces of 3 rabbits inoculated with 50 or 100 larvae on days 72-81 PI. Morphologically, the cuticle in young fourth-stage larvae exhibited bosses on the anterior portion on day 11 PI, and the left spicule length : total body length exhibited no remarkable change between 9 and 52 wk PI. The latter finding confirms the utility of the ratio for identification of the nematode.

Detection and analysis of Babesia gibsoni infection were performed with whole-blood samples collected between July 2002 and July 2003 from 945 and 137 dogs from the Aomori and Okinawa Prefectures of Japan, respectively, by PCR and loop-mediated isothermal amplification (LAMP). On the basis of the criterion for positivity by PCR, 3.9% (37 of 945) and 10.9% (15 of 137) of the dogs had B. gibsoni DNA. All 37 positive animals from Aomori Prefecture were male Tosa dogs (Japanese mastiff). The 15 dogs from Okinawa Prefecture with positive PCR assay results were of various breeds, ages, and sexes. The 18S ribosomal DNA (18S rDNA) sequences from all samples showed 100% homology to each other and to published B. gibsoni sequences. The limits of detection of B. gibsoni parasitemia by the PCR and LAMP methods with an 18S rDNA-based primer set were 0.0005% each. A comparison of the PCR and LAMP methods with microscopic examination for the detection of B. gibsoni infections in blood samples from 945 field dogs in Aomori Prefecture and 137 field dogs in Okinawa Prefecture showed that 37 and 15 dogs, respectively, were positive by the PCR and LAMP methods and that 16 and 12 dogs, respectively, were positive by light microscopic examination. All samples found to be positive by microscopic examination were also positive by the PCR and LAMP methods. The results of the PCR and LAMP methods agreed for samples with positive results by either method. Moreover, nonspecific reactions were not observed by the LAMP method. These results suggest that the LAMP method provides a useful tool for the detection of B. gibsoni infections in dogs.

Japanese White rabbits, Wistar rats, ddY mice, Suffolk sheep, and a domestic cat were each orally inoculated with 20-140 third-stage larvae (L3) of Gongylonema pulchrum, isolated from naturally infected dung beetles captured in Aomori Prefecture. Worm recovery rates were 40.0-72.0% in rabbits at 7, 14, and 19 weeks post-infection (PI) and 3.3-25.0% in rats at 19 weeks PI. Those in 2 sheep at 7 weeks PI showed 53.6% and 29.3%. No worms were recovered from the mice and the cat. In the susceptible animals, many worms were found in the esophagus, and a few were present in the pharyngeal mucosa, tongue, buccal mucosa, and cardiac portion of the stomach wall. No distinct morphological differences were observed in the worms from rabbits and sheep. These results indicate that rabbits are very suitable experimental definitive hosts for G. pulchrum.

Gongylonema pulchrum is an important parasite of captive primates. Twelve rabbits were infected with 30 third-stage larvae of G. pulchrum. At 4–7 months post-infection, animals were administered levamisole at a single dose of 12 mg/kg, levamisole at 8 mg/kg three times at 2-day intervals, levamisole at a single dose of 8 mg/kg after administration of mebendazole at 70 mg/kg for 3 days or 8 ml of distilled water for 3 days (control). Necropsy at 14 days after treatment revealed that single and multiple dosages of levamisole reduced nematode burdens by 68.4% and 89.5%, respectively. The combined regimen of mebendazole and levamisole exhibited high efficacy for treating G. pulchrum located widely within the upper digestive tract, with a reduction of 98.2%. These results suggest that this combined chemotherapy treatment may be effective against G. pulchrum infection, including buccal and lingual gongylonemiasis in primates.

ABSTRACT. A total of 611 preserved adult Metagonimus spp. specimens recovered from 32 of 53 Japanese water shrews (Chimarrogale platycephala) that had previously been collected in Aomori Prefecture between June 1994 and August 1996, were examined in this study. Morphological examination revealed that 603 of these flukes were identical to M. hakubaensis Shimazu, 1999, and that the others were M. takahashii Suzuki, 1930 (n=4), M. otsurui Saito et Shimizu, 1968 (n=2), and M. miyatai Saito et al., 1997 (n=2). Each of the 32 Japanese water shrews infected with M. hakubaensis contained between 1 and 83 flukes. This is the first record of the natural final host for M. hakubaensis, since this fluke species was described.

A case of direct-antiglobulin-test (DAT)-negative auto-immune haemolytic anaemia (AIHA) and immune thrombocytopenia (ITP) associated with Hodgkin’s disease (HD) is reported. A 52-year-old male was admitted with anaemia, thrombocytopenia, and lymphadenopathy. The patient was DAT negative, although he exhibited the clinical features of warm-type AIHA and elevated levels of red-blood-cell-associated IgG (RBC-IgG). The serum level of platelet-associated IgG (PA-IgG) was markedly increased. A biopsy specimen of the inguinal lymph nodes showed HD of mixed cellularity. Marked improvement of subjective symptoms, normalization of haematological values and a decrease in the level of both RBC- and PA-IgG were observed after the start of combination chemotherapy for HD. Although the association of HD, ITP, and/or AIHA has been infrequently reported, the measurement of RBC-IgG is recommended in cases of HD with anaemia even though DAT is negative, since HD is known to be associated with various protean immunological abnormalities.

An epidemiological study of Babesia canis in dogs in Nigeria was performed. Four hundred blood samples collected from dogs in Nigeria were investigated using nested PCR and sequence analysis. On nested PCR screening, nine samples (2.3%) produced a band corresponding to a 698-bp fragment indicative of B. canis infection. Sequence analysis of the PCR products identified eight samples (2.0%) as B. canis rossi and the ninth (0.3%) as B. canis vogeli. This is the first report of the prevalence of B. canis rossi and B. canis vogeli in dogs in Nigeria.

From June 1993 to November 1996, a total of 977 gobiid fish consisting of three species (Tridentiger brevispinis, Chaenogobius urotaenia, and Rhinogobius brunneus) collected from eastern Aomori Prefecture, were examined for Gnathostoma nipponicum larvae infection. Only one species, C. urotaenia was infected with advanced third-stage larvae (AdL3), and a total of 22 larvae were recovered from 17 (3.4%) of 500 fish. The infected fish were larger than 12 cm in body length and collected in the May-June and September-November seasons. Experimental studies confirmed that C. urotaenia was susceptible to both the early third-stage larvae (EaL3) obtained from Eucyclops serrulatus and AdL3 from Misgurnus anguillicaudatus. Eight of 10 C. urotaenia inoculated orally with 10 EaL3 were positive, and 36 AdL3 were recovered from them (recovery rate: 36.0%) at 30 days postinoculation (PI). All 10 C. urotaenia inoculated with 10 AdL3 were also positive, and a total of 63 AdL3 were recovered (recovery rate: 63.0%) at 10 days PI. The main location of the larvae was the body muscles of the fish. No morphological alterations or death of the larvae were observed in this study. From these results, it seems that the C. urotaenia has characteristics suitable to be the host to the larvae and they may serve as the second intermediate and paratenic host in the natural life cycle of this nematode.

To investigate the factors associated with recurrence following remission of at least 12 months after photodynamic therapy in patients with age-related macular degeneration (AMD).We analyzed 42 eyes with AMD in 41 patients who did not require additional treatment within 12 months of their photodynamic therapy (PDT). Additional treatment for recurrence beyond 12 months after the last PDT was required in 20 eyes in the recurrent group (n = 20). The remaining eyes (controls; n = 22) showed no recurrence during this period. We statistically analyzed the factors associated with recurrence by comparing the findings of the 2 groups.Compared to the controls, the recurrent group showed significantly improved baseline visual acuity (VA) (p = 0.005), but required significantly more PDT applications to achieve remission (p = 0.010). Age (p = 0.026), multiple PDT sessions (p = 0.001), and high baseline VA (p = 0.003) were factors significantly related to the recurrence following remission maintenance with PDT for AMD (R = 0.63; R2 = 0.39; p0.0001).Age, preoperative VA and number of PDT sessions were associated with a significant risk of recurrence after remission maintenance with PDT.

From September 1993 to November 1995, to clarify the natural infection of Gnathostoma nipponicum (G.n.) larvae in small wild mammals, a total of 313 animals consisted of three species of rodents and two species of insectivores, collected from an endemic area of this nematode in Aomori Prefecture, were investigated. Two species, Rattus norvegicus (R.n.) and Chimarrogale himalayica (C.h.) were infected with G.n. larvae, and the infection rate was extremely high with 27.2% and 72.7% in the former and latter. The infected animals harbored the larvae of 2-10 (mean: 6.0) in R.N. and 2-40 (10.6) in C.h., and a total of 273 larvae was recovered. The all larvae (18) from R.n. and the vast majority of the larvae (216/255: 82.4%) from C.h. were recovered from the musculature, and many encapsulated larvae were found in these portions of C.h. Taxonomically, all the larvae were identified as the advance third-stage larvae (AdL3) of G.n. The body length was 1,056-2,110 microns, and was larger than those of the naturally-infected AdL3 reported from cold-blooded animals. No morphological alterations or death of the larva was observed. From these results, it would seem that R.n. and C.h. are highly susceptible to G.n. larvae, and has very suitable characteristics as the hosts. Furthermore, a hypothesis is advanced that these mammals serves as the plausible paratenic hosts in the life cycle of G.n. This report is the first record of R.n. (Rodentia) and C.h. (Insectivora) naturally-infected with AdL3 of G.n.

Thirteen Sika deer (Cervus nippon Temminck) showing ataxia on a stock farm in the north eastern district of China were examined histopathologically. The principal pathological changes were spongy vacuolation and myelin deficiency in the white matter of the spinal cord and brain stem, fibrosis and rupture of the elastic lamina of the spinal arterioles, and mesothelial hyperplasia in the spinal arachnoid. Other findings included defective formation of the elastic laminae of the aorta, and the blood vessels in the kidney and lung, hemosiderosis in the spleen and liver, and lymphocyte depletion in the systemic lymph nodes. Copper concentrations were low in the serum and liver. In the white matter of the spinal cord and brain stem, demyelination appeared to coexist with dysmyelination and secondary myelin breakdown. It was inferred that decreased activity of copper containing enzyme induced various lesions. The possible role of copper deficiency in the pathogenesis of the ataxic conditions is discussed.

Des-γ-carboxy prothrombin (DCP) is an established tumor marker for hepatocellular carcinoma (HCC), but the precise mechanism of its production remains unknown. We have recently demonstrated that cytoskeletal rearrangement during the phenotypic changes involved in epithelial mesenchymal transition (EMT) plays a crucial role in DCP production through the impairment of vitamin K uptake. However, DCP production in long-lasting severe hypoxic conditions with nutrient deprivation–such as transarterial embolization–remains unknown. We examined the effects of long-lasting hypoxia with nutrient deprivation, as well as the constitutive expression of hypoxia-inducible factor (HIF)-1α, on EMT status, DCP production, and protein synthesis in human hepatoma cell lines by enzyme-linked immunosorbent assay, immunofluorescent studies, and western blotting. Immunohistochemistry findings for DCP, anti-hepatocyte paraffin 1 (Hep Par 1), and vimentin were examined using human resected HCC samples. Both severe hypoxia with nutrient deprivation and HIF-1α transfection led to the cessation of DCP production, by attenuating protein synthesis through the hypophosphorylation of mammalian target of rapamycin and its effector proteins, indicative of a further phenotypic shift involving impaired liver-specific protein synthesis. In immunohistochemistry, the distribution of DCP- and Hep Par 1-positive HCC cells was mostly similar and vimentin-positive HCC cells were frequently observed in the areas that were negative for Hep Par 1 and/or DCP. HCC cells produce DCP when they undergo mild phenotypic changes. However, when HCC cells adopt mesenchymal properties they lose their capacity for protein synthesis, and the production of DCP is attenuated. Building upon our previous works, it appears that DCP could be a unique tumor marker that reflects the stepwise phenotypic changes of HCC.

A sensitive immunoradiometric assay (IRMA) was used to quantitate the amount of red blood cell-associated IgG (RBC-IgG). The mean RBC-IgG of 91 healthy volunteers was 33±13 (SD) molecules per one RBC. The correlation between the agglutination strength in antiglobulin test and the number of IgG were very satisfactry. We also tested the amount of RBC-IgG of 27 patients with immune hemolytic anemia of warm type (AIHA). (AIHA, n=17; RBCs with positive antiglobulin test, n=2; RBCs with negative antiglobulin test, n=8; eluates from patients' RBCs). The mean RBC-IgG of 17 patients with antiglobulin test positive AIHA was 998±930 (SD) molecules per one RBC. It was ranged from 257 to 3000 molecules per one RBC. Two patients with antiglobulin test negative AIHA (7.4%, 2/27) had 138 and 172 IgG molecules per one RBC, respectively. The indirect antiglobulin tests of RBCs sensitized with 8 eluates showed positive. The RBC-IgG of sensitized RBCs was higher than that of RBCs from healthy volunteers. Using this assay, quantitative studies of RBC-IgG was extremely useful for the detection of small amount of IgG on RBC in case of the antiglobulin test negative AIHA.

Des-gamma-carboxy prothrombin (DCP) is an established HCC tumor marker, but the precise mechanism of its production is still unclear. Recently, we demonstrated that cytoskeletal changes during epithelial-to-fibroblastoid conversion (EFC) or epithelial mesenchymal transition (EMT) induced by chemicals plays a critical mechanistic role in DCP production via impairment in vitamin K uptake. Our proposed mechanism of DCP production is consistent with substantial clinical evidence. Supplementary vitamin K2 analogues reduced serum DCP levels in hepatocellular carcinoma (HCC) patients. HCC patients with high serum DCP are associated with vascular invasion, metastasis and tumor recurrence. On the other hand, hypoxia has been reported to induce EMT or cytoskeletal changes. Therefore, we examined whether hypoxia induced DCP production during EFC or EMT in HCC cells. Indeed, hypoxic stimulation induced hepatoma cell lines (HepG2 or PLC/PRF/5 cells) to undergo EFC or EMT and these cells produced DCP. Immunofluorescence study demonstrated that hypoxic stimulation impaired labeled low-density lipoprotein uptake, which was a surrogate for vitamin K uptake. In addition, fine filamentous actin network, which has crucial role for clathrin-mediated endocytosis of vitamin K, was disrupted in DCP producing cells by hypoxic stimulation. Thus, hypoxic stimulation induced HCC cells to produce DCP in the same mechanism as chemicals. Furthermore, immunohistochemical study using surgically resected HCC samples showed that a positive staining of nuclear hypoxia inducible factor (HIF)-1alpha was more frequently observed in HCC cells with stronger staining intensity of DCP. Importantly, clinical observations that DCP as an HCC tumor marker was more useful in larger tumors, which is likely to be exposed with hypoxia during tumor development, support our results.

Des-gamma-carboxy prothrombin (DCP) is an established HCC tumor marker, but the precise mechanism of its production is still unclear. Recently, we demonstrated that cytoskeletal changes during epithelial-to-fibroblastoid conversion (EFC) or epithelial mesenchymal transition (EMT) induced by chemicals plays a critical mechanistic role in DCP production via impairment in vitamin K uptake. Our proposed mechanism of DCP production is consistent with substantial clinical evidence. Supplementary vitamin K2 analogues reduced serum DCP levels in hepatocellular carcinoma (HCC) patients. HCC patients with high serum DCP are associated with vascular invasion, metastasis and tumor recurrence. On the other hand, hypoxia has been reported to induce EMT or cytoskeletal changes. Therefore, we examined whether hypoxia induced DCP production during EFC or EMT in HCC cells. Indeed, hypoxic stimulation induced hepatoma cell lines (HepG2 or PLC/PRF/5 cells) to undergo EFC or EMT and these cells produced DCP. Immunofluorescence study demonstrated that hypoxic stimulation impaired labeled low-density lipoprotein uptake, which was a surrogate for vitamin K uptake. In addition, fine filamentous actin network, which has crucial role for clathrin-mediated endocytosis of vitamin K, was disrupted in DCP producing cells by hypoxic stimulation. Thus, hypoxic stimulation induced HCC cells to produce DCP in the same mechanism as chemicals. Furthermore, immunohistochemical study using surgically resected HCC samples showed that a positive staining of nuclear hypoxia inducible factor (HIF)-1alpha was more frequently observed in HCC cells with stronger staining intensity of DCP. Importantly, clinical observations that DCP as an HCC tumor marker was more useful in larger tumors, which is likely to be exposed with hypoxia during tumor development, support our results.

Hereditary spherocytosis (HS) is a genetic disorder of the red blood cell membrane clinically characterized by anemia, jaundice and splenomegaly. Evans' syndrome is a clinical syndrome characterized by autoimmune hemolytic anemia (AIHA) accompanied by immune thrombocytopenic purpura (ITP). It results from a malfunction of the immune system that produces multiple autoantibodies targeting at least red blood cells and platelets. HS and Evans' syndrome have different mechanisms of pathophysiology one another. We reported the quite rare case of an infant who had these diseases concurrently. Possible explanations of the unexpected complication are discussed.

To clarify whether measurement of serum haptoglobin (Hp) has impact on understanding pathophysiology in bone marrow failure (BMF) syndromes, we investigated concentrations of serum Hp by nephelometric procedure in 156 Japanese patients with BMF, including 54 aplastic anemia (AA), 50 paroxysmal nocturnal hemoglobinuria (PNH), and 52 myelodysplastic syndromes (MDS) patients. The frequencies with low concentrations of serum Hp (

Direct antiglobulin test (DAT)-negative autoimmune hemolytic anemia (Coombs-negative AIHA) is characterized by laboratory evidence of in vivo hemolysis, together with a negative DAT performed by conventional tube technique (CTT) in clinically suspected AIHA patients. The immunoradiometric assay (IRMA) for red-blood-cell-bound immunoglobulin G (RBC-IgG) can be used to diagnose patients in whom CTT does not detect low levels of red cell autoantibodies. We investigated the diagnostic cutoff value of the IRMA for RBC-IgG in Coombs-negative AIHA and calculated its sensitivity and specificity. Of the 140 patients with negative DAT by CTT referred to our laboratory with undiagnosed hemolytic anemia, AIHA was clinically diagnosed in 64 patients (Coombs-negative AIHA). The numbers of Coombs-negative AIHA and non-AIHA patients changed with age and gender. The cutoff values were determined from receiver operating characteristic (ROC) curve according to age and gender. The IRMA for RBC-IgG proved to be sensitive (71.4%) and specific (87.8%) when using these cutoffs. Using these cutoffs for 41 patients with negative DAT referred to our laboratory in 2006, all the pseudonegative cases were treated with steroids before the test. The 31 untreated cases could be grouped using one cutoff value of 78.5 and showed 100% sensitivity and 94% specificity, independent of gender and age. Results indicate that RBC-IgG could become a standard approach for the diagnosis of Coombs-negative AIHA, when measured before treatment.

The prevalence of Hepatozoon canis infections in dogs in Nigeria was surveyed using molecular methods. DNA was extracted from blood samples obtained from 400 dogs. A primer set that amplified the Babesia canis 18S rRNA gene, which has high similarity to the H. canis 18S rRNA gene, was used for the PCR. As a result, samples from 81 dogs (20.3%) produced 757 bp bands, which differed from the 698 bp band that corresponded to B. canis infection. The sequence of the PCR products of 10 samples were determined, all of which corresponded with the H. canis sequence.

In vitro and in vivo studies were conducted to evaluate the effects of thiabendazole, mebendazole, levamisole and ivermectin against Gongylonema pulchrum. For in vitro assays, third-stage larvae (L3) incubated with the drugs were administered orally to mice and the ability of larvae to invade the gastric mucosa of the animals was examined. After incubation, only those larvae treated with high concentrations of levamisole (1 and 10 microg/ml) were tightly coiled with intestines exhibiting morphological abnormalities. Good dose-response data for the drugs tested was observed at the time of worm recovery from mice, with no worms recovered at the two highest concentrations of levamisole. In vivo efficacy of the drugs against adult worms was evaluated in six groups of three rabbits, each of which was infected with 30 L3 of G. pulchrum and treated with thiabendazole at 100 mg/kg for 3 days, mebendazole at 70 mg/kg for 3 days, levamisole as a single dose of 8 mg/kg, and subcutaneously injected ivermectin as a single dose of 0.2 mg/kg or vehicles of the drugs (control) at 4 months post-infection. Necropsy 14 days after treatment revealed that levamisole, mebendazole and ivermectin reduced worm burdens by 63.2%, 22.8% and 25.8%, respectively, with no reductions in worms observed with thiabendazole. The surviving worms were principally found in the esophagus with the remainder distributed among the buccal mucosa, the tongue, and/or pharyngeal mucosa in all groups. A number of morphologically abnormal eggs were observed within the uterus and ovijector in female worms recovered from the thiabendazole-treated group. These findings suggest that levamisole exhibits in vivo efficacy against G. pulchrum infection and that the larval invasion tests using mice could be used to screen for anthelmintic susceptibility of nematodes.

An epidemiological survey of bovine Setaria collected from the abdominal cavities was performed morphologically on the cattle in Aomori and Kumamoto Prefectures, Japan, between August 2005 and July 2006. Fifty Setaria worms were collected from the cattle in Aomori Prefecture and 847 from those in Kumamoto Prefecture. Of these worms, 35 were identified as Setaria digitata, 14 as S. marshalli, and one as S. labiatopapillosa in Aomori Prefecture, while 816 were identified as S. digitata and 31 as S. marshalli in Kumamoto Prefecture.

We studied the tick, Haemaphysalis longicornis, to determine the possibility of both transovarial and transstadial transmission of Babesia equi. We also studied the usefulness of the needle injection method for pathogenic tick-transmitted organisms including Babesia parasites. Erythrocytes infected with B. equi were injected into the midgut of engorged adults or nymphs using a hypodermic needle passed through the integument. DNA of B. equi in ticks was detected using nested polymerase chain reaction (PCR). B. equi DNA was present in adults, eggs, and larvae, indicating that transovarial transmission occurred. B. equi DNA was present in adults that developed from infected nymphs, and the B. equi antigen was present in their salivary glands, indicating that transstadial transmission occurred. These findings suggest that H. longicornis may play a role in the transmission of B. equi.

From June 1993 to September 1997, a survey was carried out for the prevalence of larval Gnathostoma nipponicum infection in several kinds of frogs, toads, and their tadpoles collected from an endemic area of this nematode in Aomori Prefecture. Two frog species, one of 436 (0.2%) Rana nigromaculata and 51 of 147 (34.7%) R. catesbeiana were infected, and a total of 446 advanced third-stage larvae (AdL3) of G. nipponicum were recovered. These results confirmed that two frog species which can serve as the second intermediate and/or paratenic hosts in the life cycle of G. nipponicum exist in nature. This report is the first record of spontaneous infection of frogs with AdL3 of G. nipponicum.

Babesia caballi infected erythrocytes were collected from the blood of an experimentally infected horse and could be continuously cultivated in vitro with parasitemia ranging from 2-4% in RPMI 1640 medium supplemented with 2 mM L-glutamine, 20 mM HEPES and 40% adult horse serum in a low oxygen atmosphere (2% O2, 5% CO2 and 93% N2). All attempts to increase parasitemia failed using other culture media, serum concentrations and culture vessels. However, parasite growth was enhanced by transfer of cultures from a low oxygen to 5% CO2 in air, with parasitemia ranging from 8-10%.

A composite 2,206 nucleotide DNA sequence encoding a putative immunoglobulin-binding protein (BiP) was constructed from a sequence obtained from Babesia caballi cDNA library clones. The 1,962 nucleotide open reading frame predicts a 72 kD protein with extensive homology with BiPs from Apicomplexa parasites. The BiP gene had a predicted N-terminal signal sequence of 18 amino acids and a C-terminal tetrapeptide sequence (Ser-Asp-Glu-Leu) for signaling in the endoplasmic reticulum lumen. The recombinant protein expressed in baculovirus showed an apparent mass of 72 kD, which is identical to that of the native B. caballi protein. Monoclonal antibodies (MAbs) against B. caballi BiP reacted strongly with extracellular merozoites, but not in early intraerythrocytic stage. Detailed observation showed that the reaction of MAbs against pear-shaped forms was markedly irregular, with either no reaction, or reaction with one or two brightly fluorescent pear-shaped forms (two parasites) of B. caballi.

Lactoferrin (LF) is an important biologic molecule with many functions, one of which is antimicrobial defense. We evaluated the growth-inhibiting effects of four types of LF (native LF, Fe(+3)-bound [holo] LF, Fe(+3)-free [apo] LF, and LF hydrolyzate) on the in vitro growth of Babesia caballi and B. equi. The growth of B. caballi was significantly suppressed in media containing apo LF, but was not inhibited in media containing native LF, holo LF, or LF hydrolyzate. The growth of B. equi was not inhibited by media containing native LF, holo LF, or apo LF. These data indicate that apo LF had the strongest inhibitory effect on B. caballi. This may have been caused by inactivation or inhibition of a growth factor in the culture medium.

A case of pulmonary dirofilariasis in a Japanese hare, Lepus brachyurus angustidens captured in Aomori Prefecture, was first reported in Japan. Two dead worms recovered from the pulmonary artery and detected within an organized arterial thrombus in the lung, were identified as a male and a female young adult Dirofilaria immitis, restiectively. Principal lesions in the lung were associated with pulmonary arterial degeneration and consisted of rugose and villous intimal fibrosis, endarteritis with infiltration by numerous eosinophils and mononuclear inflammatory cells, and thrombosis.

Antibodies to Babesia caballi and Babesia equi were examined on a total of 2,019 horse serum samples that had been collected in 1971-1973 by the National Institute of Animal Health by enzyme-linked immunosorbent assay (ELISA) using recombinant proteins and by Western-blot analysis. Based on the criterion for positivity by ELISA, 5.4% (109/ 2,019) and 2.2% (44/ 2,019) had antibodies against B. caballi and B. equi, respectively. The ELISA-positive sera were further examined by Western blot; 30/ 109 for B. caballi and 2/ 44 for B. equi were positive for native B. caballi or B. equi, but none of them was seropositive for both infections. Based on the results of this study, further investigations should be required to survey horses that have arrived in Japan relatively recently and tick vectors of equine Babesia using ELISA with some recombinant protein, a parasite detection method in an in vitro culture of equine Babesia, and PCR testing.

A case of wildlife trichinosis was found in a red fox (Vulpes vulpes japonica) captured at Rokkasho, Aomori Prefecture on November 27, 1998. Trichinella larvae were obtained from almost all of the muscle tissues except for the masseter. The highest number of larvae per gram of tissue was found in the muscles of the gluteal region and throat. The lowest number was found in the diaphragm and tongue. Trichina cysts within the muscle fibers had groups of fatty cells at the poles, and minimal tissue reaction was observed around the cyst. No calcification was found in the cyst. These morphological findings suggested that the considerable time had elapsed since the invasion. This is the first case of trichinosis in a red fox in Japan.

Susceptibility of five species of Cyprinidae and Salmonidae freshwater fish to the early third-stage larvae (EaL3) and advanced third-stage larvae (AdL3) of Gnathostoma nipponicum infection were examined. Two fish species inoculated orally with EaL3 were infected, and AdL3 were recovered from them with rate of 21.0% in Tribolodon hakonensis and 0.5% in Cyprinus carpio at 30 days postinoculation (PI). Attempts to infect five fish species with AdL3 were all successful. The recovery rate of AdL3 was 69.0% in T. hakonensis, 47.5% in Carassius auratus subsp., 35.0% in C. carpio, 53.0% in Oncorhynchus masou, and 32.0% in O. mykiss at 10 days PI. These results confirmed that the Cyprinidae and Salmonidae fish species reported here were susceptible to larval G. nipponicum infection and AdL3 had higher infectivity to them than the EaL3.

(1) The polymerase chain reaction (PCR) was used to amplify Rh-related cDNAs from erythroid cells cultured by the selective two-phase liquid culture system for human erythroid progenitors in peripheral blood. Two Rh polypeptide cDNAs have been isolated from the PCR products and tentatively designated RhPI cDNA and RhPII cDNA. Both cDNA clones have an open reading frame composed of 1251 nucleotides. The RhPI cDNA clone shows a single nucleotide substitution with no amino acid substitution compared with the published sequence. The RhPII cDNA clone differs from the above by 41 nucleotide substitutions in the open reading frame, resulting in 31 amino acid substitutions. Besides these cDNA clones, eleven and five truncated isoforms of the RhPI and RhPII cDNAs, have been isolated, respectively. (2) The promoter region of the Duffy gene was cloned by IPCR of 1.1 kb SacI fragment and the 3' flanking sequence was cloned by IPCR of 1.9 kb EcoRI fragment. The IPCR products contained the known Duffy cDNA sequence without introns. By comparing the coding area of the Duffy gene in 28 Duffy positive individuals, we elucidated that one base change that results in an amino acid substitution (GAT(Asp44)-->GGT(Gly)) is in accordance with the Fya/Fyb polymorphism. This fact proves that the Duffy cDNA and its gene encode the Duffy blood group system. (3) Two common alleles in Esterase D (EsD) polymorphism, EsD1 and EsD2 were characterized by the substitution of one amino acid (Gly-Glu) caused by the point mutation of one nucleotide (G-A). The point mutation between cDNAs of EsD1 and EsD2 alleles was detectable as restriction fragment length polymorphism (RFLP) using Ssp1. The RFLP makes it possible to determine the EsD phenotypes using DNA samples from forensic materials without EsD enzymatic activity. (4) The authors report studies on 19 pairs of donors and recipients in bone marrow transplantation. A broad range of genetic markers at 42 gene loci, including one DNA marker 11 red blood cell markers, five human lymphocyte antigen types, 12 serum protein markers, five red cell enzyme markers, and eight salivary markers was evaluated before and after BMT over about 2 months. As a result, 11 out of 42 gene loci of genetic markers in recipients were transformed into the donor type.

The canine C blood group was determined by an agglutination reaction with a plant agglutinin extracted from the seeds of Clerodendron trichotomum (CTL). A positive agglutination reaction was classified as type C and a negative reaction as type c. In this study, the C phenotype and the gene frequency of C were examined in 377 dogs (224 purebred dogs of 29 breeds and 153 mongrel dogs) that were bred in Utsunomiya-city, Tochigi Prefecture. Phenotype C was less frequent than phenotype c in every purebred dog. A variation in C gene frequency was observed among the purebred dogs. The highest frequency of C allele was in the Yorkshire Terrier (0.202), followed by Beagle, Shiba (0.091), Maltese (0.085), Shi Tzu (0.051) and Shetland Sheepdog (0.036). The C blood group system as a genetic marker may provide useful information for bleed identification, genetic studies between breeds, and pedigree certification.

The phenotypes and gene frequencies of genetic markers in 8 blood group systems were studied in Japanese crossbred cats. The gene frequency was 0.051 for Ca, 0.949 for ca, 0.484 for TFF, 0.5133 for TFs, 0.279 for GCF, 0.721 for GCs, 0.945 for PGDA, 0.055 for PGDB, 0.544 for ESD1 and 0.456 for ESD2. Genetic polymorphism was not detected in the PGM, ACP and GLO systems.

The mammary tissues of 6 cows with bovine leukemia virus (BLV) antibody and subclinical mastitis were investigated histopathologically, and their organ cultures were ultrastructurally observed. Numerous BLV particles, 110 to 120 nm in diameter, were seen around lymphocytes, which had infiltrated into mammary alveoli and showed blastogenesis under culture. Particles budding from the cell membrane were also found.

A case of direct antiglobulin test(DAT)-negative autoimmune hemolytic anemia (AIHA) associated with autoimmune hepatitis (AIH) is presented. A 54-year-old female was admitted with liver dysfunction and anemia. AIH was diagnosed based on the diagnostic criteria of the International Autoimmune Hepatitis Group. The patient was DAT-negative, and exhibited all the clinical features of warm type AIHA and elevated levels of red blood cell-associated IgG. Marked improvement of subjective symptoms, the return to normal of hematological and liver test values, and a decrease in the level of RBC-associated IgG were observed after the start of corticosteroid therapy. Although an association of AIH and AIHA is rarely reported, the measurement of RBC-associated IgG is recommended in cases of AIH with DAT-negative anemia, as both disorders are autoimmune in nature.

A pathologic study was performed on parasitic lesions found in the urinarybladder of cattle. Grossly, these lesions were curved, linear antd sclerotic. Microscopically, they were nodular, granulomatous lesions with parasites at their center, presenting featurescharacteristic of exudative inflammation with marked infiltration of eosinophils, in rela-Lively fresh cases. In contrast, they were characterized by tire infiltration of giant cellsand phagocytes, as well as by the follicular proliferation of lymphocytes, in long-standingcases.The parasite found in tlae tissue of the urinary bladder bore morphological resemblancein manay respects to Selaria digitata isolated from the abdominal cavity of cattle in a districtwhere intra-abdominal infection with this worm showed an incidence as high as 55%.The worms isolated from lesions in the urinary bladder of two of the infected cattle wereidentified as Setaria digitata.These results indicate that the lesions were caused by adult worms of Setaria digitalaoriginally harbored in the abdominal cavity and accidentally entering the wall of tlaeurinary bladder.

Histopathogenesis of intestinal tumors in 178 rats induced by oral administration of bracken was studied. The tumors was noted to be epithelial and non-epithelial being mainly localized at ileum (93.2%). The epithelial ones occurred at a high rate, and they were diagnosed as tubular adenocarcinoma (178 cases), papillary adenocarcinoma (22 cases) and adenoma (6 cases). Tubular adenocarcinoma with most frequences was presumed to occur from the proliferative zone in crypts of the small intestine. On the other hand, the non-epithelial tumors occurred being closely related to the stroma or adjacent tissue of carcinoma. They were noted to be leiomyosarcoma (62 cases), fibrosarcoma (14 cases), fibroma (10 cases), leiomyoma (8 cases), lymphangioendothelioma (2 cases), hemangioma (1 case) and hemangioendothelioma (1 case).