[Background factors of recurrence after remission maintenance with photodynamic therapy for age-related macular degeneration].

PURPOSE: 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). SUBJECTS AND METHODS: 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. RESULTS: 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; p < 0.0001). CONCLUSIONS: Age, preoperative VA and number of PDT sessions were associated with a significant risk of recurrence after remission maintenance with PDT.

Cut-off value of red-blood-cell-bound IgG for the diagnosis of Coombs-negative autoimmune hemolytic anemia.

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.

Efficacy of thiabendazole, mebendazole, levamisole and ivermectin against gullet worm, Gongylonema pulchrum: in vitro and in vivo studies.

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.

A PCR-based epidemiological survey of Hepatozoon canis in dogs in Nigeria.

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.

Molecular evidence of Babesia equi transmission in Haemaphysalis longicornis.

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.

Molecular survey of Babesia canis in dogs in Nigeria.

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.

Morphological survey of bovine Setaria in the abdominal cavities of cattle in Aomori and Kumamoto Prefectures, Japan.

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.

A novel lipoprotein (a) lowering drug, D-47, decreases neointima thickening after vascular injury.

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.

Short report: molecular cloning and characterization of a putative binding protein of Babesia caballi.

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.

Inhibitory effect of lactoferrin on in vitro growth of Babesia 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.

Further observations on the development of Gongylonema pulchrum in rabbits.

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.

Efficacy of levamisole alone and in combination with mebendazole against Gongylonema pulchrum infection in rabbits.

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.

Experimental infection of laboratory animals and sheep with Gongylonema pulchrum in Japan.

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.

Seroepidemiologic studies on Babesia caballi and Babesia equi infections in Japan.

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.

Discovery of natural infection by Metagonimus hakubaensis Shimazu, 1999 (Trematoda: Heterophyidae) in Japanese water shrews (Chimarrogale platycephala) in Japan.

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.

Experimental final hosts of Metagonimus hakubaensis (Trematoda: Heterophyidae) and their suitability to the fluke.

Seven laboratory mammal and bird species were orally inoculated with 200-1,000 encysted Metagonimus hakubaensis metacercariae that had been isolated from naturally infected lampreys (Lethenteron reissneri) captured in Aomori Prefecture. At 8 and 15 days post-infection, adult flukes were recovered from all of the laboratory animals tested, and therefore, hamster, rat, mouse, dog, cat, chicken and quail were considered as final hosts of M. hakubaensis. Recovery rates of the fluke were higher in dogs and hamsters than in cats, rats, mice, chickens and quails. The flukes recovered from dogs and hamsters showed increased body length and higher fecundity than those recovered from the other hosts. These results indicate that the suitability of dogs and hamsters for M. hakubaensis infection is higher than that of the other laboratory animals.

Reduced efficacy of ivermectin treatments in gastrointestinal nematode infections of grazing cattle in Japan.

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.

Phenotype-dependent production of des-γ-carboxy prothrombin in hepatocellular carcinoma.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Hypoxia-induced des-gamma-carboxy prothrombin production in hepatocellular carcinoma.

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.