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Abstract Background In the Philippines, malaria morbidity and mortality have decreased since the 1990s by effective malaria control. Several epidemiological surveys have been performed in the country, but the characteristics of the Plasmodium falciparum populations are not yet fully understood. In this study, the genetic structure of P. falciparum populations in the Philippines was examined. Methods Population genetic analyses based on polymorphisms of 10 microsatellite loci of the parasite were conducted on 92 isolates from three provinces (Kalinga, Palawan, and Davao del Norte) with different malaria endemicity. Results The levels of genetic diversity and the effective population sizes of P. falciparum in the Philippines were similar to those reported in the mainland of Southeast Asia or South America. In the low malaria transmission area (Kalinga), there was a low level of genetic diversity and a strong linkage disequilibrium (LD) when the single-clone haplotype (SCH) was used in the multilocus LD analysis, while in the high malaria transmission areas (Palawan and Davao del Norte), there was a high level of genetic diversity and a weak LD when SCH was used in the multilocus LD analysis. On the other hand, when the unique haplotypes were used in the multilocus LD analysis, no significant LD was observed in the Kalinga and the Palawan populations. The Kalinga and the Palawan populations were, therefore, estimated to have an epidemic population structure. The three populations were moderately differentiated from each other. Conclusion In each area, the level of genetic diversity correlates with the local malaria endemicity. These findings confirm that population genetic analyses using microsatellite loci are a useful tool for evaluating malaria endemicity.

Infections by gastrointestinal parasites are found in a variety of animals worldwide. For the diagnosis of such infections, the flotation method is commonly used to detect parasitic microorganisms, such as oocysts or eggs, in feces. Instead of adding a flotation solution after the final centrifugation step and using a cover slip to collect the parasites, the method using a wire loop for the recovery of the organisms has been reported as one of alternative methods. However, the recovery rates of microorganisms from the flotation method have not been analysed. In the present study, the utility of a flotation method with the use of a wire loop of 8 mm in diameter (the loop method) was evaluated using different numbers of E. tenella oocysts and Heterakis gallinarum eggs, and chicken fecal samples collected at the farms. Consequently, we found that the oocysts and eggs in tubes could be collected at a ratio of 2.00 to 3.08. Thus, our results indicate that the loop method is a simple and time saving method, implicating the application for the estimated OPG/ EPG (Oocysts/Eggs per gram) of the samples., (© 2024 The Author(s). Veterinary Medicine and Science published by John Wiley & Sons Ltd.)

The Japanese rock ptarmigan ( Lagopus muta japonica ) is an herbivorous species of partridges that inhabits only alpine zones. Alpine plants are their main source of food. These alpine plants contain toxic compounds to deter herbivores from consuming them. A previous analysis of the alpine plants frequently consumed by Japanese rock ptarmigans revealed the presence of a unique mixture of secondary metabolites and a novel compound. Additionally, wild Japanese rock ptarmigans are often infected by two species of Eimeria parasites. When these parasites were experimentally administered to Svalbard rock ptarmigans ( Lagopus muta hyperborean ), which do not feed on alpine plants, the birds exhibited symptoms, such as diarrhea and depression, and in some cases, they died. Although little is known about the pathogenesis of these parasites in wild Japanese rock ptarmigans, it was hypothesized that compounds found in alpine plants, their main food source, may reduce the pathogenicity of Eimeria parasites. In the present study, we evaluated the anticoccidial activity of the compounds derived from alpine plants in vitro using Eimeria tenella , which infects chickens belonging to the same pheasant family, as an experimental model. Twenty-seven natural components were extracted from eight alpine plants. The natural components were added to E. tenella sporozoites and incubated for 24 h to evaluate their direct effect. Additionally, Madin-Darby bovine kidney cells were incubated with sporozoites and natural components for 24 h to evaluate the inhibitory effect of the components on sporozoite cell invasion. Six compounds from four alpine plants decreased sporozoite viability by up to 88.3%, and two compounds inhibited sporozoite invasion into the cells. Although further studies are needed to evaluate the effects of these components against Eimeria infections in vivo , our findings suggest that these alpine plants may reduce the degree of infection by decreasing the number of sporozoites in the intestinal tract., (© 2024 The Authors.)

Acoel flatworms possess epidermal sensory-receptor cells on their body surfaces and exhibit behavioral repertoires such as geotaxis and phototaxis. Acoel epidermal sensory receptors should be mechanical and/or chemical receptors; however, the mechanisms of their sensory reception have not been elucidated. We examined the three-dimensional relationship between epidermal sensory receptors and their innervation in an acoel flatworm, Praesagittifera naikaiensis. The distribution of the sensory receptors was different between the ventral and dorsal sides of worms. The nervous system was mainly composed of a peripheral nerve net, an anterior brain, and three pairs of longitudinal nerve cords. The nerve net was located closer to the body surface than the brain and the nerve cords. The sensory receptors have neural connections with the nerve net in the entire body of worms. We identified five homologs of polycystic kidney disease (PKD): PKD1-1, PKD1-2, PKD1-3, PKD1-4, and, PKD2, from the P. naikaiensis genome. All of these PKD genes were implied to be expressed in the epidermal sensory receptors of P. naikaiensis. PKD1-1 and PKD2 were dispersed across the entire body of worms. PKD1-2, PKD1-3, and PKD1-4 were expressed in the anterior region of worms. PKD1-4 was also expressed around the mouth opening. Our results indicated that P. naikaiensis possessed several types of epidermal sensory receptors to convert various environmental stimuli into electrical signals via the PKD channels and transmit the signals to afferent nerve and/or effector cells., (© 2024. The Author(s).)

The centrohelid heliozoan Raphidocystis contractilis has many radiating axopodia, each containing axopodial microtubules. The axopodia show rapid contraction at nearly a video rate (30 frames per second) in response to mechanical stimuli. The axopodial contraction is accompanied by cytoskeletal microtubule depolymerization, but the molecular mechanism of this phenomenon has not been elucidated. In this study, we performed de novo transcriptome sequencing of R. contractilis to identify genes involved in microtubule dynamics such as the rapid axopodial contraction. The transcriptome sequencing generated 7.15-Gbp clean reads in total, which were assembled as 31,771 unigenes. Using the obtained gene sets, we identified several microtubule-severing proteins which might be involved in the rapid axopodial contraction, and kinesin-like genes that occur in gene duplication. On the other hand, some genes for microtubule motor proteins involved in the formation and motility of flagella were not found in R. contractilis, suggesting that the gene repertoire of R. contractilis reflected the morphological features of nonflagellated protists. Our transcriptome analysis provides basic information for the analysis of the molecular mechanism underlying microtubule dynamics in R. contractilis., (© 2022 International Society of Protistologists.)

The present study aimed to evaluate the effects of 5-aminolevulinic acid (5-ALA) on Eimeria tenella infection in laying hens. Oocyst shedding and histopathology were evaluated. A reduced oocyst shedding was observed 5 and 7 days post-infection (dpi) in the 5-ALA-administered group, but the total number of oocysts during the first infection period was not different between control and 5-ALA-treated groups. After E. tenella attack infection, the period of oocyst shedding in the 5-ALA-administered group lasted less long than that in controls. During the attack infection period, the total number of fecal oocysts in the 5-ALA-treated group was significantly lower than that in the control group. However, the parasite burden score in hens receiving 5-ALA was higher than that in controls after E. tenella attack infection. The lesion scores at 5 and 30 dpi in the control group were significantly lower than those in the 5-ALA-administered group. Therefore, 5-ALA administration might be beneficial against E. tenella infection in laying hens., (© 2023 Japanese Society of Animal Science.)

This study aimed to evaluate the benefits of oral administration of Lactobacillus acidophilus strain L-55 (LaL-55) to chickens inoculated with a Newcastle disease virus (NDV)-based live-attenuated vaccine by examining the mRNA expression of several genes related to viral infection in the spleen and ileum by quantitative reverse transcription polymerase chain reaction. In the spleen, interferon (IFN)-α was significantly higher in the low- and middle-dose LaL-55 groups at 6 weeks than at 4 weeks. IFN regulatory factor (IRF)-3 and IRF-7 expression was significantly higher in the low-dose LaL-55 group than in the middle- and high-dose LaL-55 groups. In the ileum, melanoma differentiation-associated gene 5 showed a dose-dependent increase at 4 weeks. IFN-γ and IRF-7 showed dose-dependent increases at 6 weeks. These results suggested that LaL-55 boosts the immune response to the NDV vaccine, albeit by different mechanisms in the spleen and ileum., (©2022 BMFH Press.)

This study aimed to evaluate the disease severity and local immune responses in macrophage-depleted chicks with Eimeria tenella. Macrophages were reduced by intraperitoneal injection of a carrageenan solution at 12, 13, and 16 days old, whereas the control group received intraperitoneal phosphate-buffered saline. Both chick groups were orally inoculated with E. tenella sporulated oocysts at 14 days old. Feces were collected daily, which were then quantified for oocysts. The chicks were sacrificed on day 5, and the ceca were collected for histopathological observation. The gene expression levels were measured using real-time quantitative reverse transcription-polymerase chain reaction. Macrophage-depleted chicks have been observed to shed a significantly reduced number of fecal oocysts compared to the infected control group. The parasite burden score in cecum specimens of macrophage-depleted chicks was significantly lower than those of infected control on day 5 after infection. Furthermore, macrophage reduction yielded significantly lower cecum histopathological scores and CD4 expression than those of the infected control group. The expression of interleukin (IL)-18, IL-22, interferon-γ, and inducible nitric oxide synthase was also noted to be significantly upregulated in both infected control and macrophage-depleted chicks compared to uninfected chicks. IL-4, IL-13, IL-17, and perforin expressions were also higher with macrophage depletion than in both control groups. These results suggest that macrophages serve as an invasive gate or a transporting vehicle to the site of first merogony. Furthermore, mononuclear phagocytes may play an important role in local immune responses, thus contributing to parasite development during early E. tenella infection., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The major clinical signs of coccidiosis in chickens due to Eimeria parasite are diarrhea and bloody feces. Previous studies showed that the impairment of the intestinal epithelial barrier and the elevation of the intestinal permeability are causes of clinical signs associated with coccidia challenges. Nevertheless, the information about molecular changes of the epithelial barrier at the early stage of the infection with a specific Eimeria species has not been mentioned. Hence, this study aims to elucidate the temporal relationships between epithelial barrier conditions and clinical signs in chickens infected with Eimeria tenella over the time from the earliest stages of infection. White Leghorn chickens were inoculated with 1 × 10 4 oocysts of E. tenella. Thereafter the chickens were monitored for their daily clinical signs through observation, and between 5 dpi to 10 dpi, feces were collected for oocysts counting. Chickens were then administrated with fluorescein isothiocyanate-dextran (FITC-d) for gastrointestinal permeability test and tissues were collected each day for histopathological observation and total RNA extraction. Finally, the mRNA expression levels of the tight and adherens junction genes and cytokine genes were evaluated using the quantitative real-time polymerase chain reaction (qRT-PCR). In this study, clinical signs such as diarrhea and bloody feces were observed concurrently from 3 to 8 dpi. Histopathology changes such as severe inflammation, hemorrhage, and epithelial desquamation were identified in the cecum specimens. The FITC-d level in the E. tenella-infected group was significantly higher than in the control group. In the infected group, the expression of claudin-2 gene was also upregulated, whereas the expressions of claudin-3 and E-cadherin genes were decreased as compared to the control group. These results implied that clinical signs of avian coccidiosis were associated with the intestinal barrier disruption via changes in expression levels of claudins and E-cadherin at the intestine., (Copyright © 2021 Elsevier B.V. All rights reserved.)

A high rate of glycolysis, one of the most common features of cancer, is used in positron emission tomography (PET) imaging to visualize tumor tissues using 18 F-fluorodeoxyglucose ( 18 F-FDG). Heterogeneous intratumoral distribution of 18 F-FDG in tissues has been established in some types of cancer, and the maximum standardized uptake value (SUVmax) has been correlated with poor prognosis. However, the phenotype of cells that show high 18 F-FDG accumulation in tumors remains unknown. Here, we combined quantitative micro-autoradiography with fluorescence immunohistochemistry to simultaneously visualize 18 F-FDG distribution, the expression of multiple proteins, and hypoxic regions in the cancer microenvironment of a human A431 xenograft tumor in C.B-17/Icr-scid/scid mice. We found that the highest 18 F-FDG accumulation was in cancer-derived cells undergoing epithelial-mesenchymal transition (EMT) in hypoxic regions, implicating these regions as a major contributor to increased glucose metabolism, as measured by 18 F-FDG-PET.

After parturition, cows frequently develop uterine bacterial infections, resulting in the onset of endometritis. To eliminate the bacteria, bovine endometrial cells secrete chemokines, such as IL-6 and MCP1, which attract macrophages (MΦs) to the subepithelial stroma. These attracted MΦs are not only involved in bacterial elimination but also the orchestration of inflammation and tissue repair. These immune responses aid in the recovery from endometritis; however, the recovery from endometritis takes longer in summer than in any other season. Based on these findings, we hypothesized that heat stress (HS) affects the chemokine production in endometrial cells. To confirm this hypothesis, we compared IL-6 and MCP1 production induced by lipopolysaccharide (LPS) in bovine endometrial epithelial and stromal cells under normal (38.5°C) and HS conditions (40.5°C). In the endometrial epithelial cells, IL-6 production stimulated by LPS was significantly (p < .05) suppressed under HS conditions. MCP1 production in endometrial epithelial cells was not detected under both the control and HS conditions regardless of the presence of LPS. Moreover, LPS significantly (p < .05) stimulated IL-6 and MCP1 production in endometrial stromal cells. Moreover, HS significantly (p < .05) enhanced their production compared to that under the control conditions. In addition, HS did not affect the migration ability of MΦs; however, the supernatant of the endometrial stromal cells cultured under the HS condition significantly (p < .05) attracted the MΦs when compared to the control condition. These results suggest that HS disrupts chemokine production in two types of endometrial cells and alters the distribution of MΦs in the endometrium during the summer., (© 2020 The Authors. Physiological Reports published by Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society.)

There have been no reports of the prevalence of Eimeria spp. in poultry breeding farms in Japan unlike those of broiler farms. From 2017 to 2018, we examined the prevalence of Eimeria spp. on breeding farms in Japan by oocyst morphology and PCR analyses. A total of 143 samples was collected from 37 breeding farms in 21 prefectures of Japan. We detected oocysts of seven species at 34 of 37 breeding farms by PCR, and we identified E. brunetti at 51.5% of farms found to be positive for Eimeria. The differences in the identification of Eimeria spp. between the morphology and PCR assay methods of oocysts were pronounced for E. maxima and E. necatrix. We confirmed that molecular tools were more suitable for accurately estimating prevalence of Eimeria spp., and these findings suggest that E. brunetti could be widespread in Japan.

Aging is a natural biological process that results in progressive loss of cell, tissue, and organ function. One of the causing factors of the aging process is the decrease in muscle mass, which has not been fully verified in Drosophila. Apoptotic cell death may result in aberrant cell loss and can eventually diminish tissue function and muscle atrophy. If so, inhibition of apoptosis may prolong longevity and reduce motor function and muscle mass decline with age in Drosophila flies. Here, we used Drosophila melanogaster as study material, and induced the overexpression of Drosophila inhibitor of apoptosis protein 1 gene to inhibit apoptosis, and investigated the effect of apoptosis inhibition on the longevity and age-related declines in flight and climbing ability and muscle mass. As a result, the inhibition of apoptosis tended to mitigate the aging effects and prolonged longevity and reduced climbing ability decline with age. The current study suggests that apoptosis inhibition could mitigate the aging effects in D. melanogaster. Although such effects have already been known in mammals, the current results suggest that the apoptosis may play a similar role in insects as well.

Probiotic supplements containing living bacteria have attracted interest as a potential source of health benefits for humans and livestock. The aim of this study was to determine whether administration of Lactobacillus acidophilus strain L-55 (LaL-55) enhances the immune response among chicks exposed to a Newcastle disease virus (NDV)-based live attenuated vaccine. Oral administration of LaL-55 augmented the elevation in the total numbers of leukocytes and lymphocytes following inoculation with the NDV-based live attenuated vaccine. Monocyte counts increased after LaL-55 administration independent of inoculation with the NDV vaccine. Among chicks that were administered LaL-55, there was a dose-dependent increase in the NK cell activity measured by a 51 Cr release assay at 2 weeks after the secondary NDV vaccine inoculation. Two weeks after the secondary inoculation with the NDV vaccine, interferon (IFN)-γ-mRNA expression was significantly elevated in mononuclear splenocytes from chicks that were administered LaL-55. Meanwhile, LaL-55 administration did not change the mRNA levels of IFN-α, IFN-β, and interleukin-1β. These results may suggest that coadministration of LaL-55 with an NDV vaccine augments the immune response against the virus. Therefore, LaL-55 may help protect against viral diseases in poultry., (©2020 BMFH Press.)

The aim of this study was to investigate the effects of egg yolk powder enriched with astaxanthin (ASX-E) on blood pressure in spontaneously hypertensive rats (SHR) and to verify the benefits of ASX-E as a functional food. To investigate the antihypertensive effect, SHR were fed with an ASX-E mixed diet before hypertension development. Blood pressures were determined periodically during the study by the tail-cuff method. At the end of the study, animals were euthanized, and their thoracic aortas were collected to determine vascular conductance. The thoracic aorta tension was measured with a force displacement transducer. Concentration-dependent response relationships were determined by cumulative addition of 10 -9 -10 -4 M Carbamoylcholine (Cch). Blood pressures of the SHR in the ASX-E mixed diet group were ASX-dose-dependently lower than that of those in the control group. In SHR fed with an ASX-E mixed diet, Cch induced vasorelaxation in the thoracic aorta with endothelium lining but not without endothelium. However, the antihypertensive effect of ASX-E was not observed on blood pressures in SHR that were fed with ASX-E only after the development of hypertension. Results suggest that ASX-E protects endothelial function and thereby prevents the development of hypertension. Hence, the results of our research indicate that daily consumption of ASX-E has a potential benefit on human health.

Attenuated strains of avian Eimeria parasites, generated by the selection of precocious lines through serial passaging in chicks, have been used widely as live vaccines. Detailed morphological transitions including their life cycle depending on the passages remain poorly understood. Here, we showed early development and acceleration of transitions in morphological forms of the asexual schizonts of E. tenella that had been attenuated for virulence by serial passaging. Our results may be helpful in understanding parasitism, facilitating further molecular analyses such as comparative genomic or transcriptomic tests., (Copyright © 2019 Elsevier B.V. All rights reserved.)

This study aimed to evaluate the microbial compositions and gene expression related to inflammation in dextran sodium sulfate (DSS)-induced acute colitis and the effect of mulberry supplementation. Male BALB/c mice received a diet supplemented with mulberry juice freeze-dried powder (MFP) or not for 3 weeks. After 3 weeks, the mice received water containing 5% (w/v) DSS or not for 1 week. The disease activity index score in mice fed MFP was significantly decreased. A significant decrease in Bifidobacterium spp. and the Clostridium perfringens subgroup was observed in mice not fed MFP. The number of goblet cell and NLRP6 expression were observed in mice fed a diet supplemented with MFP compared with mice not fed MFP. These results may indicate that mulberry mitigates DSS-induced acute colitis by a changing the gut microbial flora and by improving mucosal conditions.

Previous intervention studies have shown that the most effective agents used in the treatment of malaria were isolated from natural sources. Plants consumed by non-human primates serve as potential drug sources for human disease management due to the similarities in anatomy, physiology and disease characteristics. The present study investigated the antiplasmodial properties of the primate-consumed plant, Schima wallichii ( S. wallichii) Korth. (family Theaceae ), which has already been reported to have several biological activities. The ethanol extract of S. wallichii was fractionated based on polarity using n -hexane, ethyl acetate and water. The antiplasmodial activity was tested in vitro against chloroquine-resistant Plasmodium falciparum ( P. falciparum ) at 100 μg/ml for 72 h. The major compound of the most active ethyl acetate fraction was subsequently isolated using column chromatography and identified by nuclear magnetic resonance. The characterized compound was also tested against chloroquine-resistant P. falciparum in culture to evaluate its antiplasmodial activity. The ethanol extract of S. wallichii at 100 μg/ml exhibited a significant parasite shrinkage after 24 h of treatment. The ethyl acetate fraction at 100 μg/ml was the most active fraction against chloroquine-resistant P. falciparum . Based on the structural characterization, the major compound isolated from the ethyl acetate fraction was kaempferol-3- O -rhamnoside, which showed promising antiplasmodial activity against chloroquine-resistant P. falciparum with an IC 50 of 106 μM after 24 h of treatment. The present study has provided a basis for the further investigation of kaempferol-3- O -rhamnoside as an active compound for potential antimalarial therapeutics.

In chickens, although estrogen receptors (ER) are reported to be associated with the immunological processes, detailed information about the differences in ER expression in the tissues related to the development of lymphocytes is not fully known, especially during the developmental stage. To learn more about this immunological relationship, we used semi-quantitative polymerase chain reaction method to detect the ER expression levels in the thymus tissues of chicks during the developmental stage. Furthermore, ER-expressing cells were detected by immunohistochemistry. The results of this study show that the expression level of ER increased on embryonic day 16 and decreased on day 20. Furthermore, ER expression was significantly higher in male than in female chickens at day 16. The increased expression on day 16 and decreased level on day 20 were also reproduced in the incidence of immunoreactive cells, although there was a 1-day delay in the elevated incidence of the cells. This study revealed the changes in ER expression and the incidence of ER-positive cells in the thymus of chickens during the developmental stage., (© 2013 Japanese Society of Animal Science.)

Chagas disease (human American trypanosomiasis), which is caused by the protozoan parasite Trypanosoma cruzi, is responsible for numerous deaths each year; however, established treatments for the disease are limited. Differentiation-inducing factor-1 (DIF-1) and DIF-3 are chlorinated alkylphenones originally found in the cellular slime mold Dictyostelium discoideum that have been shown to possess pharmacological activities. Here, we investigated the effects of DIF-3 derivatives on the infection rate and growth of T. cruzi by using an in vitro assay system utilizing host human fibrosarcoma HT1080 cells. Certain DIF-3 derivatives, such as butoxy-DIF-3 (Bu-DIF-3), at micro-molar levels strongly suppressed both the infection rate and growth of T. cruzi in HT1080 cells and exhibited little toxicity for HT1080 cells. For example, the IC50 of DIF-3 and Bu-DIF-3 versus the growth of T. cruzi in HT1080 cells were 3.95 and 0.72μM, respectively, and the LD50 of the two compounds versus HT1080 cells were both greater than 100μM. We also examined the effects of DIF-3 and Bu-DIF-3 on T. cruzi activity in C57BL/6 mice. Intraperitoneally administered Bu-DIF-3 (50mg/kg) significantly suppressed the number of trypomastigotes in blood with no apparent adverse effects. These results strongly suggest that DIF-3 derivatives could be new lead compounds in the development of anti-trypanosomiasis drugs., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Plasmodium falciparum has for some time been developing resistance against known anti-malarial drugs, and therefore a new drug is urgently needed. Selenium (Se), an essential trace element, in the form of inorganic Se, selenite (SeO32-), has been reported to have an anti-plasmodial effect, but its mechanism is still unclear. In the present study, we evaluated the anti-plasmodial effect of several Se compounds against P. falciparum in vitro. The anti-plasmodial effect of several Se compounds was analysed and their apoptosis-inducing activity was evaluated by morphological observation, DNA fragmentation assay and mitochondrial function analysis. SeO32-, methylseleninic acid, selenomethionine and selenocystine have anti-plasmodial effects with 50% inhibition concentration at 9, 10, 45, and 65 μm, respectively, while selenate and methylselenocysteine up to 100 μm have no effect on parasite growth. The effective Se compounds caused the parasites to become shrunken and pyknotic and significantly increased mitochondrial damage against P. falciparum compared to the untreated control. In conclusion, SeO32-, methylseleninic acid, selenomethionine and selenocystine have anti-plasmodial activities that induce apoptosis-like cell death in P. falciparum, and the anti-plasmodial effects of Se seem to be based on its chemical forms. The apoptosis-like cell-death mechanism in P. falciparum can be beneficial to respond to the growing problem of drug resistance.

VAMP7 or tetanus neurotoxin-insensitive vesicle- associated membrane protein (TI-VAMP) has been proposed to regulate apical transport in polarized epithelial cells, axonal transport in neurons and lysosomal exocytosis. To investigate the function of VAMP7 in vivo, we generated VAMP7 knockout mice. Here, we show that VAMP7 knockout mice are indistinguishable from control mice and display a similar localization of apical proteins in the kidney and small intestine and a similar localization of axonal proteins in the nervous system. Neurite outgrowth of cultured mutant hippocampal neurons was reduced in mutant neurons. However, lysosomal exocytosis was not affected in mutant fibroblasts. Our results show that VAMP7 is required in neurons to extend axons to the full extent. However, VAMP7 does not seem to be required for epithelial cell polarity and lysosomal exocytosis., (© 2011 John Wiley & Sons A/S.)

We conducted a 28-day follow-up of 17 Laotian patients diagnosed with uncomplicated Plasmodium falciparum malaria treated with mefloquine (Mephaquine, MQ) alone to determine the efficacy. All patients were completely cured with MQ, without reappearance of asexual stage parasitemia at follow-up. Of the 7 isolates tested for genotypic analysis, one isolate was a Y86 mutant type of the pfmdr1 gene, the others were N86 wild. These findings suggest no MQ resistance in the study area possibly because the drug is rarely used in southern Lao PDR.

Specific mutations in the pfcrt and pfmdr1 genes have been reported to be associated with chloroquine-resistant falciparum malaria parasites worldwide. These genetic markers are considered to be useful tools for the elucidation of several aspects of the epidemiology of drug resistant malaria. In this study, Plasmodium falciparum isolates from three distinct areas of the Philippines were analyzed for drug-resistance-associated genetic mutations, and their association with the in vitro chloroquine (CQ) response. Two novel pfcrt 72-76 allelic types, CVMDT and SVMDT, were detected. The frequency of the pfcrt K76T mutation in the isolates that were successfully tested for in vitro CQ susceptibility was found to be 100% in Kalinga, 80% in Palawan, and 87% in Mindanao. The frequency of the pfmdr1 N86Y mutation was 39% in Kalinga, 35% in Palawan, and 93% in Mindanao isolates. No mutations were found at positions 1042 and 1246 of pfmdr1. However, there were no significant associations found between polymorphisms in these genes and in vitro CQ susceptibility. The results of this study indicate that mutations in pfcrt and pfmdr1 are not predictive of in vitro CQ resistance in Philippine isolates and may therefore not be suitable as molecular markers for surveillance.

Background: In the Philippines, malaria morbidity and mortality have decreased since the 1990s by effective malaria control. Several epidemiological surveys have been performed in the country, but the characteristics of the Plasmodium falciparum populations are not yet fully understood. In this study, the genetic structure of P. falciparum populations in the Philippines was examined., Methods: Population genetic analyses based on polymorphisms of 10 microsatellite loci of the parasite were conducted on 92 isolates from three provinces (Kalinga, Palawan, and Davao del Norte) with different malaria endemicity., Results: The levels of genetic diversity and the effective population sizes of P. falciparum in the Philippines were similar to those reported in the mainland of Southeast Asia or South America. In the low malaria transmission area (Kalinga), there was a low level of genetic diversity and a strong linkage disequilibrium (LD) when the single-clone haplotype (SCH) was used in the multilocus LD analysis, while in the high malaria transmission areas (Palawan and Davao del Norte), there was a high level of genetic diversity and a weak LD when SCH was used in the multilocus LD analysis. On the other hand, when the unique haplotypes were used in the multilocus LD analysis, no significant LD was observed in the Kalinga and the Palawan populations. The Kalinga and the Palawan populations were, therefore, estimated to have an epidemic population structure. The three populations were moderately differentiated from each other., Conclusion: In each area, the level of genetic diversity correlates with the local malaria endemicity. These findings confirm that population genetic analyses using microsatellite loci are a useful tool for evaluating malaria endemicity.

Malaria is one of the major infectious diseases in the Philippines. It is being targeted for control through sustained early diagnosis, treatment and mosquito control. It is in this light that understanding the genetic background of the parasite population is important not only for basic biology of the organism but also for epidemiology and control of the disease. In the present study, molecular phylogenetic relationships of the 3 Plasmodium falciparum populations in the Philippines with the other populations in the world were inferred based on polymorphisms of 9 highly polymorphic microsatellite DNA loci in the parasite genome. A total of 92 P. falciparum isolates collected from 3 provinces (Kalinga, Palawan and Davao del Norte) in the Philippines, and 8 from other populations (3 African, 2 South American, 2 Papua New Guinean, and 1 Thai) that were previously reported, were used for the analysis. The phylogenetic tree showed that the 3 Philippine populations were genetically divergent from each other as compared to the other populations. The branching pattern of the tree suggests that the 3 Philippine populations were relatively close to the Thai population, rather than the Papua New Guinean populations, indicating that the ancestor of the 3 Philippines populations were introduced from Indochina peninsula, and not from countries located south of the Philippines such as Papua New Guinea or Indonesia.

The in vitro antimalarial activity of the fungal metabolite gliotoxin (GTX) was evaluated, and its mechanism of action was studied. GTX showed plasmodicidal activity against both Plasmodium falciparum chloroquine-resistant strain K-1 and chloroquine-susceptible strain FCR-3. GTX cytotoxicity was significantly lower against a normal liver cell line (Chang Liver cells). The intracellular reduced glutathione level of parasitized and of normal red blood cells was not affected by GTX treatment. However, GTX decreased the chymotrypsin-like activity of parasite proteasomes in a time-dependent manner. The results of this study indicate that GTX possesses plasmodicidal activity and that this effect is due to inhibition of parasite proteasome activity, suggesting that GTX may constitute a useful antimalarial therapy.

A 28-day in vivo treatment trial to evaluate the efficacy of pyrimethamine/sulfadoxine (Fansidar, PS) was conducted in 21 Lao patients with uncomplicated Plasmodium falciparum malaria. Sixteen patients (76%) were completely cured with PS without any reappearance of asexual stage parasitemia during the follow-up examination. On the other hand, 5 patients (24%) failed to respond to this trial medication, resulting in recrudescence of asexual stage P. falciparum malaria. PS resistance resulted in higher prevalence of post-treatment gametocytemia, 25% gametocyte carriers among PS sensitive cases versus 75% of the resistant cases. These findings suggest that although the level of PS resistance is still valid for treatment of malaria in the study area of Lao PDR, post-treatment induction of gametocytemia among resistant cases may result an increase in transmission rate of PS resistant falciparum malaria.

The emergence and spread of drug-resistant Plasmodium falciparum continue to pose problems in malaria chemotherapy. Therefore, it is necessary to identify new antimalarial drugs and therapeutic strategies. In the present study, the activity of a heat-treated form of amphotericin B (HT-AMB) against P. falciparum was evaluated. The efficacy and toxicity of HT-AMB were also compared with those of the standard formulation (AMB). HT-AMB showed significant activity against a chloroquine-resistant strain (strain K-1) and a chloroquine-susceptible strain (strain FCR-3) in vitro. The 50% inhibitory concentrations of HT-AMB were 0.32 +/- 0.03 mug/ml for strain K-1 and 0.33 +/- 0.03 mug/ml for strain FCR-3. In the presence of 1.0 mug of HT-AMB per ml, only pyknotic parasites were observed after 24 h of incubation of early trophozoites (ring forms). However, when late trophozoites and schizonts were cultured with 1.0 mug of HT-AMB per ml, those forms multiplied to ring forms but the number of infected erythrocytes did not increase. These results indicate that HT-AMB possesses potent antiplasmodial activity and that the drug is more effective against the ring-form stage than against the late trophozoite and schizont stages. HT-AMB was observed to have little cytotoxic effect against a human liver cell line (Chang liver cells). In conclusion, the results suggest that HT-AMB has promising properties and merits further in vivo investigations as a treatment for falciparum malaria.

In vitro drug susceptibility to chloroquine (CQ) and mefloquine (MF) were assessed in 39 P. falciparum isolates from the Thai-Myanmar border area. To further characterize CQ- and MF-resistance profiles in this area, we also analyzed pfcrt K76T mutation that is critical for CQ resistance, and pfmdr1 polymorphism that has an association with MF resistance. Eighteen isolates were successfully examined by in vitro tests for CQ, and 17 of them had resistance to the drug. Geometric mean concentration of CQ that inhibited the growth of parasites at 50% (IC50) was 371 +/- 227 nM (105-971 nM). Sixteen isolates were successfully examined by in vitro tests for MF, and 8 of them were resistant to the drug. Geometric mean of IC50 for MF was 41 +/- 31 nM (4-125 nM). Genotypes of drug resistance, such as pfcrt and pfmdr1 mutations, were also analyzed. All the 39 isolates had the same haplotype (CVIET) for PfCRT at its 72-76th amino acids. A pfmdr1 Y86 mutation was found in 95% of isolates. A pfmdr1 D1042 mutation was also present in 7 isolates, while no pfmdr1 Y1246 mutation was observed. These results indicated a correlation between CQ resistance and the pfcrt T76 and pfmdr1 Y86 mutations.

The in vitro antimalarial activity of sodium selenite (NaSe) was investigated and the mechanism of its action was studied. NaSe had antimalarial activity against both the chloroquine-susceptible strain FCR-3 and chloroquine-resistant strain K-1 of Plasmodium falciparum. The shrunken cytoplasm of the parasite was observed in a smear 12 h after treatment with NaSe. Co-treatment with copper sulfate (CuSO(4)) in culture did not affect the antimalarial activity of NaSe, but NaSe cytotoxicity against the mammalian cell line Alexander was decreased significantly. The intracellular reduced glutathione level of parasitized red blood cells was decreased significantly by treatment with NaSe, and the decrease was consistent with their mortality. Treatment with NaSe had a strong inhibitory effect on plasmodial development, and NaSe cytotoxicity to human cells was decreased by co-treatment with CuSO(4). These results suggest that co-treatment with NaSe and CuSO(4) may be useful as a new antimalarial therapy.

Plasmodium falciparum-infected erythrocytes (pRBCs) adhere to the endothelium via receptors expressed on the surface of vascular endothelial cells (EC) and sequester in the microvasculature of several organs and block the blood circulation. The sequestration, which involves receptors, may be related to the severity of malaria. Here, we report that pRBCs bind to the membrane-bound form of Fractalkine/CX3CL1 (FKN), which is expressed on the surface of vascular EC in various organs. pRBCs adhered to FKN on the surface of FKN cDNA-transfected Chinese hamster ovary cells (CHO-FKN cells). Both the recombinant human FKN-chemokine domain (FKN-CD) and anti-FKN-CD antibody efficiently blocked adherence of pRBCs to CHO-FKN cells. Similar to binding between FKN and FKN receptor on blood mononuclear cells, two amino acid residues, Lys-7 and Arg-47 within FKN-CD, were critical for FKN-pRBC binding. Immunohistological analysis revealed the expression of FKN on EC at the site of sequestration in the brain of a patient with cerebral malaria. These results suggest that the membrane-bound form of FKN acts as a receptor for pRBCs, and this may contribute to furthering our present understanding of cytoadherence in the pathology of falciparum malaria.

Although the presence of multi-drug-resistant falciparum malaria has been reported in the Philippines, the distribution of drug-resistant malaria parasites has not yet been determined in Mindanao Island. In vitro susceptibility of P. falciparum to both chloroquine and mefloquine was assessed to forecast the spread of drug-resistant parasites in various foci in southeastern Mindanao Island. Of the 33 isolates of P. falciparum successfully tested, 10 (30%) were susceptible, 12 (36%) showed decreased susceptibility (80 nM < or = IC50 < 114 nM), and 11 (33%) were resistant (IC50 > or = 114 nM) to chloroquine. Ten (91%) of the resistant isolates and 9 (75%) of those with decreased susceptibility were from northern and northwestern Davao del Norte Province. Chloroquine-susceptible isolates were found among patients in the eastern parts of Davao del Norte and Davao Oriental provinces. Seven isolates from several foci in the study area were all mefloquine- susceptible (IC50 < 10 nM). This is the first report indicating the potential emergence of chloroquine-resistant P. falciparum on Mindanao Island, which is presently regarded as a drug-susceptible area.

Spread of multi-drug resistant malaria in the endemic areas has made malaria control more difficult. Thus, WHO recommends combination therapy for the treatment of malaria. The aim of combination therapy is to improve efficacy and to reduce the incidence of resistance development to the each component of the combination. Particularly, the combination with artemisinin derivatives shows good outcome in Thailand where high resistance for mefloquine has already been found. We report the first case of falciparum malaria, successfully treated with Artemether-Lumefantrine in Japan. Artemether-Lumefantrine is a newly developed artemisinin-based combination agent for the treatment of uncomplicated multi-drug resistant malaria. This drug has proved highly effective and well tolerated by some clinical trials abroad. This Japanese female case showed a good clinical course without any side effect.

We report a 68-year-old woman with severe falciparum malaria contracted in Tanzania. She presented high parasitemia and was treated successfully with intravenous artesunate, a qinghaosu derivative, and aggressive supportive therapy. She developed hemolytic anemia and jaundice on day 11 and blood transfusion was required. This case illustrates that intravenous artesunate has excellent antimalarial activity with rapid efficacy and that no severe adverse effect but conventional aggressive supportive therapy is still important in the treatment of severe falciparum malaria.

It has been reported that Leishmania promastigotes have ability to express foreign genes on drug selectable plasmids. To investigate further abilities of the recently described expression vector, P6.5, in the transfection of Leishmania organisms (Chen D-Q, Kolli BK, Yadava N et al. Episomal expression of specific sense and antisense mRNAs in Leishmania amazonensis: modulation of gp63 levels in promastigotes and their infection of macrophages in vitro. Infect Immun 2000;68:80--86), the constructed expression vector, which contains canine interleukin-8 (cIL-8) coding cDNA, was introduced by electroporation to promastigotes of four species of the genus Leishmania: Leishmania amazonensis, L. equatorensis, L. donovani and L. infantum. Extrachromosomal DNAs and total RNAs from the transfected promastigotes were subjected to polymerase chain reaction (PCR) and reverse transcriptase-PCR, respectively, using cIL-8 gene specific primers, and a predicted product of 330 bp was detected. Western blot analysis using a mouse monoclonal antibody raised against cIL-8 demonstrated the successful expression of cIL-8 in the transfectants and culture supernatants. Culture supernatants of the transfected L. amazonensis and L. equatorensis promastigotes showed a high chemotactic activity to both dog and mouse polymorphonuclear leukocytes. These results indicate that Leishmania promastigotes transfected with the expression vector P6.5 containing cIL-8 cDNA are capable of producing biologically active cIL-8. The Leishmania expression system using the P6.5 vector might be a useful alternative for the production of biologically active recombinant cytokines.

The adhesion of Plasmodium falciparum-infected erythrocytes to vascular endothelium and to uninfected red blood cells (RBCs) plays a key role in the pathology of severe malaria. Adhesion is known to be mediated in part by the antigenically-variant erythrocyte membrane protein-1 (PfEMP-1), which is encoded by the var-gene family of P. falciparum. It has recently been reported that in vitro a single parasite simultaneously transcribes multiple var-genes but that, through a developmentally regulated process, the parasite selects only one PfEMP-1 that will to reach the surface of the host RBC. Were this to be true in vivo, one would expect a correlation between the type of var/PfEMP-1 that is expressed on the parasite-infected RBC and the severity of clinical disease. In order to test this assumption, we determined the sequence of the var-gene that was expressed by the parasites in patients' blood samples. Seven blood samples were collected from patients with or without severe clinical symptoms (cerebral malaria): two samples were from patients diagnosed as having imported falciparum malaria at the International Medical Center of Japan (IMCJ); the five others were from patients of the Davao Regional Hospital in Davao, the Philippines. The parasites (ring stage) in the blood samples were cultured for 24 hours; the matured trophozoites, in which the var-gene selection had taken place, served as material for mRNA isolation. The cDNA corresponding to the Duffy-binding-like (DBL)-1 domain of the var-gene was amplified by RT-PCR, using a region-specific primer set. The amplified cDNAs were cloned into the plasmid vector; the resultant clones (32) were sequenced on both strands. The results indicated that there was considerable diversity in the sequence of the DBL-1 domain among the clones, even among those from a single patient. In conclusion, it was difficult to demonstrate the correlation between the type of var-gene transcripts found in the RBCs of malaria patients and the severity of their symptoms.

The case was a 47-year-old Nigerian male who was thought to have contracted malaria in Nigeria and then manifested fever with chill, arthralgia and diarrhea in Japan. The blood test at International Medical Center of Japan revealed thrombocytopenia and anemia. Ring forms of 0.03% of his RBCs and ICT Malaria P.f/P.v test was also positive for Plasmodium falciparum. We prescribed mefloquine to him, but the number of the paresites in his peripheral blood did not decrease, and, in fact, they came to increase (maximum 6.66%) 20 hours after the drug treatment. As clinical condition of malaria were liable to change seriously, intravenous Artesunate (a qinghaosu derivative) was decided to be given additionally to the patient. Consequently the parasites disappeared in 20 hours from his blood but a low grade fever still continued possibly because of cholecystitis. At the same time of Artesunate treatment, hemoglobinuria started and anemia worsened partly because of his G-6-PD deficiency. All pending problems were improved by the time he left Japan and those parasites were finally found to be susceptible for mefloquine by the in vitro susceptibility test. This is the first reported case of falciparum malaria successfully treated with intravenous Artesunate in Japan.

Leishmania amazonensis is an intracellular protozoan parasite of macrophages. Cutaneous leishmaniasis in an immunocompetent host begins as papules or nodules followed by ulceration at the site of promastigote inoculation. In this study, the pathological changes of cutaneous leishmaniasis lesions in T cell deficient nude mice were examined. When infected with L. amazonensis promastigotes, nude mice developed non-ulcerative cutaneous nodules. By histological examination of cutaneous lesions, massive accumulation of vacuolated histiocytes containing amastigotes was observed in all the nude mice. Although infiltration of mononuclear and polymorphonuclear cells was seen in the lesions of immunocompetent mice, few such cells were observed in the lesions of nude mice. These results indicate the importance of T cells on the ulcer formation in cutaneous leishmaniasis.