Takahisa Furuta

Antimalarial activity of extracts of Malaysian medicinal plants.

In vitro and in vivo studies revealed that Malaysian medicinal plants, Piper sarmentosum, Andrographis paniculata and Tinospora crispa produced considerable antimalarial effects. Chloroform extract in vitro did show better effect than the methanol extract. The chloroform extract showed complete parasite growth inhibition as low as 0.05 mg/ml drug dose within 24 h incubation period (Andrographis paniculata) as compared to methanol extract of drug dose of 2.5 mg/ml but under incubation time of 48 h of the same plant spesies. In vivo activity of Andrographis paniculata also demonstrated higher antimalarial effect than other two plant species.

Protective Roles of Mast Cells and Mast Cell-Derived TNF in Murine Malaria

TNF plays important roles in the protection and onset of malaria. Although mast cells are known as a source of TNF, little is known about the relationship between mast cells and pathogenesis of malaria. In this study, mast cell-deficient WBB6F1-W/Wv (W/Wv) and the control littermate WBB6F1+/+ (+/+) mice were infected with 1 × 105 of Plasmodium berghei ANKA. +/+ mice had lower parasitemia with higher TNF levels, as compared with W/Wv mice. Diminished resistance in W/Wv mice was considered to be due to mast cells and TNF. This fact was confirmed by experiments in W/Wv mice reconstituted with bone marrow-derived mast cells (BMMCs) of +/+ mice or of TNF−/− mice. W/Wv mice with BMMCs of +/+ mice exhibit lower parasitemia and mortality accompanying significantly higher TNF levels than those of W/Wv mice. Parasitemia in W/Wv mice with BMMCs of TNF−/− mice was higher than that in +/+ mice. Activation of mast cells by anti-IgE or compound 48/80 resulted in release of TNF and decrease of parasitemia. In addition, splenic hypertrophy and increased number of mast cells in the spleen were observed after infection in +/+ mice and W/Wv mice reconstituted with BMMCs of +/+ mice as compared with W/Wv mice. These findings propose a novel mechanism that mast cells and mast cell-derived TNF play protective roles in malaria.

Association of mast cell-derived VEGF and proteases in Dengue shock syndrome.

Background Recent in-vitro studies have suggested that mast cells are involved in Dengue virus infection. To clarify the role of mast cells in the development of clinical Dengue fever, we compared the plasma levels of several mast cell-derived mediators (vascular endothelial cell growth factor [VEGF], soluble VEGF receptors [sVEGFRs], tryptase, and chymase) and -related cytokines (IL-4, -9, and -17) between patients with differing severity of Dengue fever and healthy controls. Methodology/Principal Findings The study was performed at Childrens Hospital No. 2, Ho Chi Minh City, and Vinh Long Province Hospital, Vietnam from 2002 to 2005. Study patients included 103 with Dengue fever (DF), Dengue hemorrhagic fever (DHF), and Dengue shock syndrome (DSS), as diagnosed by the World Health Organization criteria. There were 189 healthy subjects, and 19 febrile illness patients of the same Kinh ethnicity. The levels of mast cell-derived mediators and -related cytokines in plasma were measured by ELISA. VEGF and sVEGFR-1 levels were significantly increased in DHF and DSS compared with those of DF and controls, whereas sVEGFR-2 levels were significantly decreased in DHF and DSS. Significant increases in tryptase and chymase levels, which were accompanied by high IL-9 and -17 concentrations, were detected in DHF and DSS patients. By day 4 of admission, VEGF, sVEGFRs, and proteases levels had returned to similar levels as DF and controls. In-vitro VEGF production by mast cells was examined in KU812 and HMC-1 cells, and was found to be highest when the cells were inoculated with Dengue virus and human Dengue virus-immune serum in the presence of IL-9. Conclusions As mast cells are an important source of VEGF, tryptase, and chymase, our findings suggest that mast cell activation and mast cell-derived mediators participate in the development of DHF. The two proteases, particularly chymase, might serve as good predictive markers of Dengue disease severity.

Severe pulmonary pneumocystosis in simian acquired immunodeficiency syndrome induced by simian immunodeficiency virus: its characterization by the polymerase-chain-reaction method and failure of experimental transmission to immunodeficient animals

Pneumocystis carinii (Pc) infection was observed in three of five rhesus monkeys infected with simian immunodeficiency virus (SIVmac251). They showed severe symptoms similar to those associated with human acquired immunodeficiency syndrome (AIDS). Histopathology revealed severe pulmonary pneumocystosis in one of three Pc-positive monkeys, and anti-Pc antibodies were detected in sera from two of the three monkeys. Localization of Pc organisms in various organs of the monkeys was examined by the polymerase-chain-reaction (PCR) method, and Pc-specific bands of DNA amplification were detected in the liver, kidney, spleen, adrenal gland, testis, brain, and other organs examined, but no Pc organism was found in these organs by histopathologic examination. These results suggest that the activation of a latent infection of Pc occurs in SIV-infected rhesus monkeys as well as in human AIDS. Experimental transmission of Pc derived from a simian was attempted in severe combined immunodeficiency (SCID) mice and athymic nude (rnu/rnu, F344) rats. These animals were inoculated intranasally with 104 Pc cysts, but neither histopathologic changes nor Pc organisms were detected in SCID mice at 4 months after inoculation or in nude rats at 2 months postinoculation, suggesting that simian Pc is species-specific.

Mast cell-mediated immune responses through IgE antibody and Toll-like receptor 4 by malarial peroxiredoxin

In this study, 2‐Cys Plasmodium berghei ANKA (PbA) peroxiredoxin (Prx) was identified as an antigenic protein recognized by an anti‐PbA IgE antibody using two‐dimensional polyacrylamide gel electrophoresis and proteomic analysis. Innate immune responses to PbAPrx were examined using cells from mice deficient in Toll‐like receptors (TLR) or related molecules, and it was demonstrated that responses were severely impaired in TLR4–/–, MyD88–/– and MD‐2–/– mice, but not in Toll/IL‐1 receptor domain‐containing adaptor inducing IFN‐γ (TRIF)–/–, TLR2–/– or radioprotective 105 (RP105)–/– mice. An association between PbAPrx and TLR4 was observed following immunoprecipitation and immunoblotting, suggesting that PbAPrx was associated with TLR4/MD‐2. Interactions between Prx and TLR4/MD‐2 were also examined by flow cytometry using TLR4/MD‐2‐ or TLR2‐expressing cells. NFκB/GFP activity was observed in TLR4/MD‐2‐ but not in TLR2‐expressing cells following stimulation with Prx. However, this effect was not observed after treatment with proteinase K, suggesting that PbAPrx is a protein ligand for TLR4 and that the PbAPrx activity observed in this study is not due to contamination with LPS. These findings indicate that malarial Prx induces IgE‐mediated protection through FcϵRI on mast cells and innate immunity through TLR4 with MyD88 and MD‐2, suggesting a novel function for malarial Prx in innate and acquired immune responses in malaria.

Agonistic Antibody to TLR4/MD-2 Protects Mice from Acute Lethal Hepatitis Induced by TNF-α

LPS is recognized by a heterodimer consisting of TLR4 and its coreceptor MD-2. LPS signal causes excessive inflammation and tissue damage. In this study, we show that a mAb to TLR4/MD-2 protected mice from acute lethal hepatitis caused by LPS/d-galactosamine. The protective effect of the mAb was not due to inhibition of LPS response, because serum TNF-α, which was induced by LPS and caused lethal hepatitis, was 10 times up-regulated by the mAb pretreatment. Moreover, this mAb induced antiapoptotic genes in liver in a TLR4/MD-2-dependent manner. These results demonstrated that an agonistic mAb to TLR4/MD-2 protected mice from LPS/d-galactosamine-induced acute lethal hepatitis by delivering a protective signal activating NF-κB through TLR4/MD-2.

Elevated Levels of Vascular Endothelial Growth Factor (VEGF) and Soluble Vascular Endothelial Growth Factor Receptor (VEGFR)-2 in Human Malaria

In cerebral malaria, the binding of parasitized erythrocytes to the cerebral endothelium and the consequent angiogenic dysregulation play a key role in pathogenesis. Because vascular endothelial growth factor (VEGF) is widely regarded as a potent stimulator of angiogenesis, edema, inflammation, and vascular remodeling, the plasma levels of VEGF and the soluble form of the VEGF receptor (sVEGFR)-1 and -2 in uncomplicated malaria patients and healthy adults were measured by enzyme-linked immunosorbent assay (ELISA) to examine their roles in malaria. The results showed that VEGF and sVEGFR-2 levels were significantly elevated in malaria patients compared with healthy adults. Moreover, it was confirmed that malarial parasite antigens induced VEGF secretion from the human mast cell lines HMC-1 or KU812 cell. This is the first report to suggest that the interaction of VEGF and sVEGFR-2 is involved in the host immune response to malarial infection and that malarial parasites induce VEGF secretion from human mast cells.

FR131535, a novel water-soluble echinocandin-like lipopeptide: synthesis and biological properties

The synthesis and biological properties of a novel water-soluble echinocandin-like lipopeptide, FR131535, are described. This compound displayed potent in vitro and in vivo antifungal activities. The hemolytic activity of FR901379 was reduced by replacing the acyl side chain. This compound showed good water-solubility, comparable to the natural product FR901379.

Membrane localization and demonstration of isoforms of nucleoside triphosphate hydrolase from Toxoplasma gondii.

Toxoplasma gondii has a unique enzyme, a NTPase, which has a wide specificity toward NTP. In the present study, we produced a monoclonal antibody (IgG1, 6C6) against the enzyme which could recognize NTPase isozymes among several strains of T. gondii. Three avirulent strains of T. gondii, ME49, Beverley and Nakayama, were found to have 1 NTPase (63 kDa, pI 6.0), while a virulent strain RH and an avirulent strain Fukaya had 2 isozymes (63 kDa) with different pIs (pIs 6.0 and 6.5 for the former, and pIs 6.2 and 6.4 for the latter, respectively), suggesting that this monoclonal antibody recognizes a common epitope of NTPase among T. gondii strains. Furthermore, 6C6 could inhibit NTPase activity in the presence of dithiothreitol in a dose-dependent manner, and immuno-EM study of NTPase revealed that this molecule is located on the surface membrane of T. gondii tachyzoites. When Vero cells were co-cultured with tachyzoites pre-treated with 6C6, the number of infected cells significantly decreased, suggesting that 6C6 inhibits invasion of the parasites to host cells. These data suggest that the molecule recognized by 6C6 might be considered a potential candidate antigen for vaccines against T. gondii tachyzoites.

Detection of antibodies to Pneumocystis carinii by enzyme-linked immunosorbent assay in experimentally infected mice.

Pulmonary pneumocystosis, caused by Pneumocystis carinii is one of the most important opportunistic infections in man and animals under immunosuppressed state (Arean, 1972. Pathology of protozoal and helminthic disease. Williams & Wilkins Co., Baltimore, 291 p.; Frenkel et al., 1966, Laboratory Investigation 15: 1559-1577). In such immunosuppressive hosts, serum antibody to P. carinii is generally produced at a low level (Kagan and Norman, 1976, National Cancer Institute Monograph 43: 121125). Therefore the development of a highly specific and sensitive method to measure antibody would be required to elucidate the relationship between progression of infection and antibody response and to evaluate the plausibility of serodiagnosis (Pifer et al., 1978, Pediatrics 61: 3541) as well as for epidemiological survey. In the present study we developed an enzyme-linked immunosorbent assay (ELISA) to detect serum antibodies to P. carinii, and the sensitivity was compared with an indirect immunofluorescence (IF) test routinely used in our laboratory (Furuta et al., 1984, Japanese Journal of Experimental Medicine 54: 57-64). The P. carinii, isolated from a naturally infected nu/nu mouse, has been maintained by the intranasal (i.n.) passage in nu/nu mice in our laboratory (Furuta et al., 1984, loc. cit.) and organisms from the sixth passage were used in this experiment. The standard ELISA technique for various viruses was modified for P. carinii as follows (Voller et al., 1976. Microplate enzyme immunoassays for immunodiagnosis of virus infection. American Society of Microbiology, Washington, D.C., 506 p.). Pneumocystis carinii cyst suspension was prepared from infected mouse lungs as described previously (Furuta et al., 1984, loc. cit.). The sample was sonicated at 200 W (Kubota Insonator, Model 200) for 50 min, and diluted to 5 ,tg/ml with phosphate buffered saline (PBS) pH 7.2. Fifty 1l of the cyst suspension was added to each well in a polystyrene microplate (Nunc-Immuno Plate 96-F, Nunc Inter Med, Roskilde) and the plate was incubated at room temperature for 1 hr. The plate was washed 3 times with 0.05% Tween 20 in PBS (Tween 20-PBS), and 50 ,ul of serum dilution was added to each well. After further incubation at room temperature for 1 hr, the plate was washed 3 more times with Tween 20-PBS. Subsequently, 50 ,l of 1:1,000 rabbit anti-mouse IgG or IgM serum which were both conjugated with horseradish peroxidase (specific to gamma or mu chain, respectively; Cappel Labs., Cochranville, Pennsylvania) was added to the well. After incubation at room temperature for 1 hr, the residual conjugate was removed by washing 3 times with Tween 20-PBS, and 200 ,dl of substrate solution consisting of 40 mg o-phenylenediamine dihydrochloride and 10 ,l H202 in 100 ml of 0. 1 M citrate buffer, pH 4.7 was added to each well. The plate was incubated in the dark for 1 hr at 37 C, and 50 ,1 of 6 N sulfuric acid was added to each well to stop the enzyme reaction. The absorbance of the reaction mixture was measured at 492 nm by a spectrophotometer (Titertek Multiskan; Flow Labs., Inc., Rockville, Maryland) and the reading of a given diluted serum higher than 1.5 times of equivalent dilution of normal serum was taken as a positive reaction. Outbred ICR mice were obtained from a commercial breeder (Charles River Japan Co., Atsugi). The colony had no detectable antibodies to P. carinii by our routine IF test. The mice were maintained in cages with a filter cap (Sankikagaku Co., Tokyo) and placed in laminar airflow racks. Twenty mice were inoculated i.n. with 104 P. carinii cysts in 0.05 ml PBS (Furuta et al., 1984, loc. cit.) and 5 mice were killed at weekly intervals. The number of cysts in the lung preparations stained with toluidine blue-O (Chalvardjian and Grawe, 1963, Journal of Clinical Pathology 16: 383-384) was counted under the microscope (Furuta et al., 1984, loc. cit.). As