[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.
No preview · Article · Feb 2009 · Biochemical and Biophysical Research Communications
[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.
No preview · Article · Mar 2008 · Parasitology Research
[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.
Preview · Article · Mar 2004 · The American journal of tropical medicine and hygiene
[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.
Full-text · Article · Jul 2003 · The Southeast Asian journal of tropical medicine and public health
[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.
Preview · Article · May 2003 · Journal of Human Genetics
[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.
No preview · Article · May 2003 · Journal of Dermatological Science
[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: 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.
Preview · Article · Apr 2003 · Parasitology International
[Show abstract][Hide abstract] ABSTRACT: The single-step screening method (SSS) is a qualitative rapid screening test for glucose-6-phosphate dehydrogenase (G6PD) deficiency based on blue formazan formation on anion-exchanger.The reaction mixture contains equal volumes of anion exchanger, substrate mixture, coloring mixture (MTT-PMS mix) and distilled water. We assessed the stability of the reaction mixture and evaluated its reliability with two anion exchangers, DEAE-Sephadex A50™ and DEAE-Sephacel™, for applications in tests under field conditions. The reaction mixture was sufficiently stable under conditions of incubation at 70°C for 6 hours or vigorous shaking for 24 hours at room temperature. The reaction mixture could be kept at 30-35°C for 14 days under indoor conditions without shielding if it contained no MTT-PMS mix. The coloring was detectable even in diluted blood with hemoglobin concentration as low as 1.6 g/dl. Under laboratory conditions, the proportion of the samples with 10% of the normal level of activity that were diagnosed as 'low activity' was higher with DEAE-Sephacel (92%) than with DEAE-Sephadex A-50 (81%) (p=0.023). The proportion of the samples with normal activity that were diagnosed as 'normal' was 98% with DEAE-Sephacel and 100% with DEAE-Sephadex A-50. In field samples obtained in Myanmar and Indonesia, the sensitivity was lower (P=0.03 using DEAE-Sephadex and p< 0.001using DEAE-Sephacel) when we used the blue formazan spot test (BFST) as the standard. Twenty-three of 27 G6PD-deficient individuals subjected to genetic analysis were found to have mutations. All individuals who had concordant results between the SSS and the blue formazan spot test (BFST) carried molecular mutations. One case of G6PD mutation was detected among four cases diagnosed as G6PD-deficient by SSS with DEAE-Sephacel™, but diagnosed as 'normal' by BFST. The costs of one test with the DEAE-Sephadex A-50™ and the DEAE-Sephacel™ system were 0.15 US dollar and 0.30 US dollar, respectively. (297 words)
[Show abstract][Hide abstract] ABSTRACT: Kynurenine 3-hydroxylase (K3H) is a NADPH-dependent flavin monooxygenase involved in the tryptophan pathway. Xanthurenic acid (XA) is a metabolite of this pathway and has recently been identified as a gamete activating factor (GAF) of the malarial parasite. We cloned K3H cDNA from Anopheles stephensi (AsK3H), because anopheline mosquitoes are a vector of the human malaria parasite, Plasmodium falciparum and the catalytic function of AsK3H in XA production. Recombinant AsK3H protein was expressed in Sf-9 cells using the baculovirus system and its enzymatic properties were characterized. The specific activities of crude cell lysate and affinity purified protein were 94.9 +/- 6.2 and 865.6 +/- 10.5 nmol/min/mg protein, respectively. The optimum pH of AsK3H was 7.0. Analysis of AsK3H gene expression using RT-PCR revealed that AsK3H was constitutively expressed in egg, larva, pupa and adult.
No preview · Article · Nov 2002 · Insect Molecular Biology
[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.
No preview · Article · Apr 2002 · Parasitology International
[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.
Full-text · Article · Apr 2002 · Tropical Medicine & International Health
[Show abstract][Hide abstract] ABSTRACT: Peripheral blood mononuclear and polymorphonuclear cells have been demonstrated to kill and inhibit malaria parasite proliferation in vitro, but most of the reports required activation of the cells by cytokines or presence of immune sera or opsonins. In our study, peripheral blood leukocytes from non-immune donor efficiently inhibited Plasmodium falciparum growth, depending on the effecter/target ratio. Moreover, these cells produced a large amount of interlenkin-8 (IL-8) under stimulation with infected erythrocytes or supernatant of the Plasmodium falciparum culture. IL-8 secretion in the culture supernatant of polymorphonuclear cells was noted from 6-9 hr of stimulation with the parasites, with a substantial increase over 24 hours of culture. Attempt to elucidate whether IL-8 was involved in malaria suppression was done, and the result suggested that IL-8 was not directly involved in the anti-malarial activity of the mononuclear and polymorphonuclear cells. The other mechanism besides IL-8 may work for inhibiting parasite growth in the culture system of malaria parasites associated with leukocytes.
[Show abstract][Hide abstract] ABSTRACT: Gamete activation factor (GAF) induces exflagellation of Plasmodium microgametes. We found GAF in the salivary glands of female mosquitoes, Anopheles stephensi. The exflagellation was induced in a concentration-dependent manner in the supernatant of salivary gland's crude homogenate. The exflagellation-inducing activity in the salivary gland was higher than that in the midgut and the head. GAF in the salivary glands was found to be heat stable and low molecular weight (<3000 molecular weight). Analysis of the supernatant by capillary electrophoresis and UV absorbance profile showed that the salivary glands contained xanthurenic acid, which was previously identified as GAF in the head of A. stephensi. The exflagellation-inducing activity in the salivary gland declined immediately after a blood meal, implying that GAF was in the saliva, and was delivered into the midgut together with the blood and induced exflagellation in the midgut.
No preview · Article · Oct 2001 · Biochemical and Biophysical Research Communications
[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.
No preview · Article · Sep 2001 · Parasitology Research
[Show abstract][Hide abstract] ABSTRACT: We summarized the consultation cases of parasitic diseases and entomological cases presented at the Department of Medical Zoology, Jichi Medical School. Among 173 consultations from January 1996 to December 2000, 64 cases were parasitologically or entomologically positive. We primarily diagnosed the cases morphologically and intermittently used sero-diagnosis methods. Ascariasis and diphyllobothriasis due to foods comprised the major consultation cases. Cases of malaria, second in frequency, were all imported from tropical countries. Some rare but important cases, including entomological cases, are discussed.
Preview · Article · Sep 2001 · Japanese journal of infectious diseases
[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.