[Show abstract][Hide abstract] ABSTRACT: House dust mites produce serine and cysteine proteases. Mite-derived proteases have been suggested to be involved in the pathogenesis of allergies; however, whether mite-derived serine protease activity can stimulate keratinocytes remains unknown.
We examined the activation of primary human keratinocytes by serine protease-rich extract of whole mite culture and compared with that by recombinant group 1 allergens (rDer f 1 and rDer p 1), which exclusively exhibit cysteine protease activity.
Protease activity of whole mite culture extract (WCE), rDer f 1 and rDer p 1 induced the release of IL-8 and granulocyte-macrophage colony-stimulating factor. Protease activity of WCEs induced a significant upregulation of their mRNA expression but rDer f 1 had much less effect. Protease activity of the WCE stimulated intracellular Ca(2+) mobilization but rDer f 1 and rDer p 1 did not. The mobilization induced by agonists for the human protease-activated receptor (PAR)-2, an agonist peptide or trypsin, was diminished by pre-incubation of keratinocytes with WCE. rDer f 1 inefficiently cleaved a synthetic N-terminal peptide of PAR-2 at different sites from trypsin, but the resultant peptides did not stimulate the release of interleukin-8.
The results suggest that mite-derived serine protease activity may contribute to the pathogenesis of atopic dermatitis by activating keratinocytes via PAR-2 activation but cysteine protease activity of Der f 1 and Der p 1 acts via another mechanism.
[Show abstract][Hide abstract] ABSTRACT: House dust mites are a major source of allergens associated with allergic diseases including allergic conjunctivitis. Here, we demonstrate that mite-derived serine protease activity induces the release of cytokines from human ocular conjunctival epithelial cells in vitro and innate antiproteases, secretory leukocyte protease inhibitor (SLPI) and alpha1-antitrypsin, can inhibit the response. An extract prepared from a whole-mite culture induced the release of IL-6 and IL-8 and upregulated their gene expression in the human conjunctival epithelial cell line Chang, responses which were inhibited not only by a synthetic serine protease-specific inhibitor, AEBSF, but also by SLPI and alpha1-antitrypsin at a physiologically relevant concentration. The findings suggest a homeostatic role for SLPI and alpha1-antitrypsin against the proteases contained in allergen sources in the ocular conjunctiva and that exposure to house dust particles containing mite-derived serine protease activity could be involved in the initiation of sensitization through the ocular conjunctival epithelium and/or exacerbation of allergic conjunctivitis.
Biochemical and Biophysical Research Communications 01/2009; 379(3):681-5. · 2.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Anopheline mosquitoes play an essential role in malaria transmission. The mosquito salivates copiously when probing for the location of a blood vessel. We found that the saliva of anopheline mosquitoes has chemotactic activity for naive eosinophils or neutrophils. The major eosinophil chemotactic component in saliva was shown to be one of the chitinase family proteins. A similar chitinase family protein was found also in the midgut of the anopheline mosquito. Production of antibodies to the chitinase family protein was generally observed in the sera of residents of a malaria endemic area. Both Plasmodium falciparum-infected and uninfected individuals had antibodies to chitinases. These results suggest that the chitinase family protein in mosquito saliva contributes to eliciting an inflammatory response of eosinophils in the host skin followed by antibody production in the host.
Parasitology Research 03/2008; 102(3):357-63. · 2.85 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We evaluated the ICT Malaria P.f./P.v. immunochromatographic test for the detection of the panmalarial antigen (PMA) using a rodent malaria model. Mice were infected with Plasmodium berghei by mosquito bite, and blood was examined by microscopy and the ICT test. Treatment with artemether was started when the parasite density exceeded 70,000/microL. The ICT PMA band appeared when the parasite density was more than 2,000/microL, but it continued to be positive after the parasitemia became negative in response to the drug treatment. When all the test results were divided into increasing phase (IP) and declining phase (DP), the sensitivity in the DP was significantly higher than that in the IP, suggesting that the reactivity of the ICT PMA is significantly influenced by persistent and accumulated PMA after drug treatment and longer duration of infection in the DP. Recognizing that the patient population in a clinical situation would be a mixture of individuals in the IP and DP, it should be emphasized that the individual history of recent fever, duration of illness, and drug treatment must be considered carefully for the interpretation of the ICT results.
The American journal of tropical medicine and hygiene 03/2004; 70(2):139-43. · 2.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The display of foreign proteins on the surface of baculovirus virions has provided a tool for the analysis of protein-protein interactions and for cell-specific targeting in gene transfer applications. To evaluate the baculovirus display system as a vaccine vehicle, we have generated a recombinant baculovirus (AcNPV-CSPsurf) that displays rodent malaria Plasmodium berghei circumsporozoite protein (PbCSP) on the virion surface as a fusion protein with the major baculovirus envelope glycoprotein gp64. The PbCSP-gp64 fusion protein was incorporated and oligomerized on the virion surface and led to a 12-fold increase in the binding activity of AcNPV-CSPsurf virions to HepG2 cells. Immunization with adjuvant-free AcNPV-CSPsurf virions induced high levels of antibodies and gamma interferon-secreting cells against PbCSP and protected 60% of mice against sporozoite challenge. These data demonstrate that AcNPV-CSPsurf displays sporozoite-like PbCSP on the virion surface and possesses dual potentials as a malaria vaccine candidate and a liver-directed gene delivery vehicle.
[Show abstract][Hide abstract] ABSTRACT: In vitro growth of Plasmodium falciparum is restricted in glucose-6-phosphate dehydrogenase (G6PD)-deficient erythrocytes (RBC), as a result of oxidative stress. Bathocuproine disulphonate (BCS), a copper chelator, as well as cysteine have been shown to synergistically stimulate the in vitro growth of various mammalian cells and Trypanosoma under oxygenated conditions. We examined the effects of these two chemicals on the in vitro growth of P. falciparum in G6PD-deficient RBC, and found that addition of BCS and cysteine synergistically enhanced the growth of the P. falciparum FCR-3 strain in these RBC to the same level as in normal RBC. However, BCS or cysteine alone had no stimulatory effect. To explain this synergistic enhancement, changes in thiol, NADPH and glutathione contents were investigated. After addition of cysteine alone, thiol content in the medium decreased rapidly, but when BCS was added, it was maintained at about 35% at 24 hours after incubation, suggesting that BCS stimulates parasite growth in G6PD-deficient RBC by inhibiting copper-mediated oxidation of cysteine in the medium. In these RBC, no increase in NADPH level, but a slight increase in glutathione, was observed in the presence of both BCS and cysteine. The slight increase of glutathione, was probably due to incorporation of cysteine from the medium, although this could not fully explain the synergistic growth enhancement. These findings taken together suggest that cysteine incorporated into G6PD-deficient RBC may help maintain the thiol groups in many proteins, such as membrane proteins, hemoglobin and enzymes, and plays an important role in maintaining an appropriate culture state necessary for parasite growth. We also examined the effects of BCS and cysteine on adaptation of wild isolates of P. falciparum to in vitro cultivation using the candle jar method. Although there was no drastic effect on growth enhancement, the presence of BCS and cysteine accelerated the appearance of schizonts in many isolates.
The Southeast Asian journal of tropical medicine and public health 07/2003; 34(2):301-9. · 0.61 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Direct immunization via epithelial surfaces has been considered for many vaccine approaches, including DNA vaccines. It remains to be determined, however, which body site is suitable for genetic vaccination.
To characterize the effects of the oral mucosa-mediated genetic vaccination, we compared antigen-specific immune responses of the oral mucosal DNA vaccine to the flank skin vaccination against influenza virus and malaria parasite.
DNA vaccines against the influenza A/WSN/33 (H1N1) hemagglutinin and the malaria Plasmodium berghei circumsporozoite protein were administered respectively three times at 3-week intervals into the oral mucosa, skin, or liver of hamsters. The effects of their vaccine were evaluated by antigen-specific antibody production and cell-mediated killing activity. Furthermore, the in vivo malaria challenge test was also performed after the vaccination.
Significant specific antibody production was not observed in each case, but interferon-gamma production and cell-mediated killing activity were strongly induced in splenic lymphocytes from hamsters with the oral vaccination. The in vivo malaria challenge after the oral mucosal vaccination significantly delayed the blood-appearance day of the parasites in comparison with other immunization sites (P<0.05).
These results suggest that gene immunization via the oral mucosa may induce cell-mediated immunity more efficiently than via the skin or liver, and that the oral mucosa may be one of the most suitable tissues for gene gun-based DNA vaccination against infectious diseases.
Journal of Dermatological Science 05/2003; 31(3):203-10. · 3.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We have proposed a mathematical model for the transmission of Plasmodium vivax malaria quantitatively, which is adjusted to the infected region, Guadalcanal, in the Solomon Islands. The simulation of a transmission model will be instrumental in planning the malaria control strategy. A characteristic of the life cycle of P. vivax is that a sporozoite injected into the blood stream by a mosquito bite may sometimes stay in a hepatocyte as a hypnozoite. Therefore, we have incorporated a phenomenon of renewed infections caused by a relapse into the transmission model. Also through the simulations we have attempted to evaluate the decline in prevalence caused by the programs of selective mass drug administration (MDA) and vector control such as the distribution of permethrin-treated bednets. The simulations have indicated that the concentrated repetition of MDA at 1-week intervals would reduce the prevalence of vivax malaria swiftly in the beginning and would keep the parasite rate below 1% for a few years but the prevalence would increase thereafter. In contrast, the parasite rate would remain below 1% for a long time if a trial of 1 or 2 times MDA is accompanied with some reduction of the vectorial capacity by the enforcement of vector control. In any case, it is important to beware of relapse cases because even after the execution of MDA it takes a long time to decrease the proportion of hypnozoite carriers.
Parasitology International 04/2003; 52(1):81-93. · 2.30 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A novel bispecific single-chain antibody fragment (biscFv) has been constructed to address the possibility of a new approach to malaria therapeutic drug development. The biscFv consists of 2 different single-chain antibody fragments linked by a flexible peptide linker (Gly(4)-Ser)(3). Of the 2 scFv fragments, one is directed against a conserved epitope of the 19-kDa C-terminal fragment of the major surface protein of human malignant malaria parasite, Plasmodium falciparum, and the other is directed against the CD3 antigen of human T cells. The biscFv expressed by a recombinant baculovirus retained the antigen-binding properties of the corresponding univalent single-chain antibody fragments and formed a bridge between P falciparum and T cells. In cooperation with T cells, the biscFv specifically induced not only interferon gamma and tumor necrosis factor alpha, but also a significant increase of merozoite phagocytosis and growth inhibition of P falciparum in vitro. Thus, the biscFv possesses highly selective malaria-targeting properties and stimulates T cells to induce cytokines, presumably resulting in activation of macrophages, neutrophils, and natural killer cells, and parasite killing in vivo.
[Show abstract][Hide abstract] ABSTRACT: In Nepal, we tested 300 males for glucose-6-phosphate dehydrogenase (G6PD) activity. Two subjects were G6PD deficient (0.67%). Compared with normal controls, G6PD activity was 12% and 26%, respectively. The hemoglobin concentration of these two subjects was normal. We extracted genomic DNA from whole blood and read all sequences of G6PD. Both subjects had the same replacement of 563C>T, which was classified as G6PD Mediterranean. The amino acid might change from Ser to Phe at codon 188. These subjects also had a replacement of 1311C>T, which caused no replacement of an amino acid. A similar replacement pattern of G6PD Mediterranean is described from persons living in Mediterranean countries and Middle East countries. However, G6PD Mediterranean found in India and Pakistan has no replacement at nucleotide 1311. Thus, these two subjects in Kathmandu, Nepal, would be closer to people in Middle East countries than people in India. This is the first study of molecular analysis for G6PD deficiency in Nepal.
Journal of Human Genetics 01/2003; 48(5):275-7. · 2.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The presence of Plasmodium ovale has never been previously reported in Myanmar. Using blood samples obtained in many villages across the country between 1996 and 2000, molecular diagnosis of Plasmodium species was made with semi- or full-nested polymerase chain reaction (PCR) with species-specific primers, followed by agarose gel electrophoresis to detect amplification products. The presence of P. ovale was also confirmed with the another PCR-based diagnosis, the microtiterplate hybridization (MPH) method using species-specific probes. Both methods target the A type of the small subunit ribosomal RNA gene of the four human malaria parasites. Plasmodium ovale DNA was amplified in samples from 65 (4.9%) of 1323 PCR-positive patients, with perfect agreement between results obtained by nested PCR and MPH. Only four P. ovale-infected patients had single-species infection; all others were coinfected with P. falciparum, P. vivax and/or P. malariae. Quadruple infections were observed in six subjects. Parasites with typical P. ovale morphology were found in only 19 patients by conventional microscopy of Giemsa-stained thin smears or fluorescence microscopy of acridine orange-stained thin smears. Plasmodium ovale infections were found in villages situated in the southern, central and western regions of Myanmar, suggesting that P. ovale may be widely distributed in this country.
Tropical Medicine & International Health 04/2002; 7(3):231-9. · 2.94 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We found that infection of a rodent malaria, Plasmodium berghei, occurred when the sporozoites were injected into the skin, the muscle, the peritoneal cavity and the tail end. Mice, which were injected with sporozoites in the tail end and had the site cut 5 min later, did not develop malaria. We also found that mice developed malaria when malaria infective mosquitoes, Anopheles stephensi, were forced not to take blood but only to probe into the skin. Moreover, the mice probed by the infective mosquitoes were protected from malaria infection if the site was treated with Kyu (heat treatment) after the mosquitoes had probed. These findings indicate that malaria infection occurs not only by blood feeding of the infective mosquito but also by probing of the mosquito. Sporozoites injected into the skin remain at the injected site for at least 5 min, then migrate to the blood vessels and invade into the blood stream. At present, the mechanism is not clear, although we propose here the existence of the skin stage of malaria parasites before the liver stage and the blood stage.
Parasitology International 04/2002; 51(1):17-23. · 2.30 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Superoxide plays a crucial role in innate immunity to various pathogens. We examined the role of superoxides in the transmission of malaria using gp91phox knockout (X-CGD) mice that lack the ability to produce superoxide. Mosquitoes that fed on X-CGD mice infected intraperitoneally with Plasmodium berghei NK65 ANKA formed more oocysts than did those that fed on control mice at any day after infection. The number of oocysts peaked on day 5 post-infection in X-CGD and control mice and then decreased significantly after day 5 post-infection. However, on day 7 post-infection, the infectivity of gametocytes in X-CGD mice was significantly higher than that in control mice. These results show that two pathways, superoxide-dependent and -independent, are involved in the host systems regulating the transmission of malaria and inhibiting gametocyte development.
Parasitology Research 09/2001; 87(8):605-8. · 2.85 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a heterogeneous enzyme abnormality with high frequency in tropical areas. We performed population screening and molecular studies of G6PD variants to clarify their distribution and features in Southeast Asia. A total of 4317 participants (2019 males, 2298 females) from 16 ethnic groups in Myanmar, Lao in Laos, and Amboinese in Indonesia were screened with a single-step screening method. The prevalence of G6PD-deficient males ranged from 0% (the Akha) to 10.8% (the Shan). These G6PD-deficient individuals and 12 G6PD-deficient patients who had been diagnosed at hospitals in Indonesia and Malaysia were subjected to molecular analysis by a combination of polymerase-chain-reaction-based single-strand conformation polymorphism analysis and direct sequencing. Ten different missense mutations were identified in 63 G6PD-deficient individuals (50 hemizygotes, 11 heterozygotes, and 2 homozygotes) from 14 ethnic groups. One missense mutation (1291 G-->A) found in an Indonesian Chinese, viz., G6PD Surabaya, was previously unknown. The 487 G-->A (G6PD Mahidol) mutation was widely seen in Myanmar, 383 T-->C (G6PD Vanua Lava) was specifically found among Amboinese, 871 G-->A (G6PD Viangchan) was observed mainly in Lao, and 592 C-->T (G6PD Coimbra) was found in Malaysian aborigines (Orang Asli). The other five mutations, 95 A-->G (G6PD Gaohe), 1003 G-->A (G6PD Chatham), 1360 C-->T (G6PD Union), 1376 G-->T (G6PD Canton), and 1388 G-->A (G6PD Kaiping) were identified mostly in accordance with distributions reported previously.
Human Genetics 07/2001; 108(6):445-9. · 4.63 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Mosquito bites can elicit dermal hypersensitivity reactions, but little is known about the chemotactic factors for host leukocytes in mosquito saliva. In this study, we determined that saliva from a malarial vector mosquito, Anopheles stephensi, possesses intense neutrophil chemotactic activity. In contrast, the midgut extract had only marginal neutrophil chemotactic activity. Eosinophil chemotactic activity was detected in the midgut but not in the saliva. According to the results of size-exclusion HPLC on a G3000SW column and Western blot analysis, the apparent molecular weight (MW) of the main neutrophil chemotactic factor (NCF) was estimated to be 200 kDa. NCF could bind with IgG from the pooled serum of Solomon islanders, whereas not with that of healthy Japanese. NCF activity was increased upon heating to 56 degrees C for 30 min or protease digestion, whereas it was affected by periodate treatment. Protease-digested NCF and naive NCF bound to lentil lectin-Sepharose, and both were eluted with a competitive sugar, methyl-alpha-D-glucoside. These results indicate that A. stephensi saliva-derived NCF is a high MW glycoprotein, and its protein moiety is important for neutrophil chemotactic activity. This NCF is thought to contribute to the inflammatory reactions through the accumulation of neutrophils at the site of the mosquito bite.
Parasitology Research 06/2001; 87(5):376-82. · 2.85 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Single-chain immunotoxins are ideal tools to selectively kill infectious agents. In applying this technology to block transmission of malaria parasites in the mosquito vector, we have constructed a single-chain immunotoxin composed of a single-chain antibody fragment (scFv) directed to Pbs2l on the surface of Plasmodium berghei ookinetes linked to a lytic peptide, Shiva-1. The single-chain immunotoxin was expressed in Escherichia coli, and the protein was purified by a Ni-NTA column. The single-chain immunotoxin was initially shown to exhibit greater killing properties for P. berghei ookinetes in vitro compared with the scFv or synthetic Shiva-1 peptide alone. In an attempt to block malaria transmission by genetically engineered bacteria, recombinant E. coli harboring the single-chain immunotoxin gene were introduced into the mosquito midgut by membrane feeding. The number of infected mosquitoes and their oocyst densities were significantly reduced when the mosquitoes were subsequently allowed to feed on P. berghei-infected mice. These results indicate not only that a single-chain immunotoxin with enhanced parasiticidal activity could form a basis for the development of more effective malaria therapeutic agents, but also that introduction of genetically engineered bacteria into anopheline mosquitoes may offer a practical approach to the regulation of malaria transmission.
Molecular and Biochemical Parasitology 04/2001; 113(1):89-96. · 2.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We developed a sensitive polymerase chain reaction (PCR) method for the detection of Plasmodium falciparum DNA from mosquitoes collected in the field. Plasmodium falciparum was detected from 15.2% of 1-parous mosquitoes, Anopheles farauti, in the Solomon Islands through use of the PCR method. A novel mathematical model was developed to estimate the sporozoite rate based on the malaria-positive rate of 1-parous mosquitoes. Using this model, the sporozoite rate of Anopheles farauti in the Solomon Islands was calculated to be 0.09%. This method enables estimation of the sporozoite rate based on a relatively small number (100-200) of mosquitoes compared with the number needed for the ELISA method.
[Show abstract][Hide abstract] ABSTRACT: The liver is the first target organ for malaria parasites immediately after the bite of an infected mosquito. We studied local immunization of malaria DNA vaccines at the site of the liver using a gene gun as a useful tool for in vivo transfection of foreign genes. A malaria DNA vaccine consisting of the Plasmodium berghei circumsporozoite protein (PbCSP) gene plus the mouse IL-12 gene was bombarded directly by a gene gun into mouse liver once or into the skin twice. A marked protective effect was induced by gene bombardment into the liver (more than 71%) compared with that into the skin (less than 33%). A Th1-type immune response and high production of iNOS were observed in the hepatic lymphocytes from mice bombarded into the liver, resulting in more effective protection compared with those bombarded into the skin. These results provide an important implication on the development of efficient malaria vaccine strategies.
Biochemical and Biophysical Research Communications 05/2000; 271(1):107-15. · 2.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Binding capacities of synthetic peptides to HLA-DR molecules were tested on filter papers to identify putative helper T-cell epitopes on a malarial protein. The antigen tested was the merozoite surface glycoprotein 1 (MSP1) of Plasmodium falciparum, a vaccine candidate targeting the asexual erythrocytic stage. Bindings between synthetic oligopeptides and HLA-DR molecules were tested. Such bindings were not non-specific, and a known helper T-cell epitope peptide showed positive binding to the restricting HLA-DR molecule. By using this screening system, we observed the unequal distribution of HLA-DR-binding peptides in 10 out of 17 MSP1 blocks tested. Block #6 of MSP1 seemed to show the highest frequency in the positive binding; on the other hand, blocks #1 and #17, both of which were thought to be vaccine candidate regions, contained fewer HLA-DR binding peptides. This was not inconsistent with the results that block #17 was less stimulatory to peripheral T cells than block #6. The peptides with positive binding to HLA-DR showed actual epitope activities when we tested peptide-driven proliferation of human bulk T-cell lines, and association between the two parameters was statistically significant (P<0.001). For more detailed information for vaccine development, peptides with both IgG- and HLA-DR binding activities were mapped in block #17 of MSP1. Together with these results, we demonstrate that our simple screening system seems to provide essential information for vaccine development through uncovering locations of putative epitopes for human helper T cells.
Microbiology and Immunology 02/2000; 44(4):249-57. · 1.55 Impact Factor