Induction of ssDNA-binding autoantibody secreting B cell immunity during murine malaria infection is a critical part of the protective immune responses.

Although it has been hypothesized that autoimmune-like phenomena may play a critical role in the protective immune responses to both human and animal malaria, there are still no evidence-based data to support this view. In this study we demonstrate that the majority of anti-single stranded (ss) DNA autoantibody secreting B cells were confined to B220(+)CD21(+)CD23(-) cells and that these cells expanded significantly in the spleen of C57BL/6 mice infected with Plasmodium yoelii 17X non-lethal (PyNL). To determine the role of ssDNA-binding autoantibody secreting B cell responses in murine malaria, we conjugated generation 6 (poly) amidoamine dendrimer nanoparticles with ssDNA to deplete ssDNA-binding autoreactive B cells in vivo. Our data revealed that 55.5% of mice died after DNA-coated nanoparticle-mediated in vivo depletion of ssDNA-specific autoreactive B cells and subsequent challenge using PyNL. Adoptive transfer of B cells with ssDNA specificity to mice, followed by PyNL infection, caused a later appearance and inhibition of parasitemia. The possible mechanism by which the ssDNA-binding autoantibody secreting B cells is involved in the protection against murine malaria has also been demonstrated.

Time-resolved catch and release of an optical pulse from a dynamic photonic crystal nanocavity.

We perform time-domain measurements of the interaction between light and silicon photonic crystal nanocavities under dynamic Q factor control. Time-resolved evidence of optical pulse capture and release on demand is demonstrated and compared for samples with dynamic Q ranges from ~3,000 to 26,000 and from 18,500 to 48,000. Observing the energy behaviour in response to dynamic control provides insight not available with time-integrated measurements into factors influencing device performance such as carrier absorption and pulse capture efficiency.

Statistical studies of photonic heterostructure nanocavities with an average Q factor of three million.

We have measured the quality (Q) factors and resonant wavelengths for 80 photonic crystal nanocavities with the same heterostructure. In this statistical evaluation, the Q factors varied according to a normal distribution centered at 3 million and ranging between 2.3 million and 3.9 million. The resonant wavelengths also fluctuated but with a standard deviation of only 0.33 nm. Such a high average Q factor and highly controlled resonant wavelength will be important for the development of advanced applications of photonic crystal nanocavities. Comparing the experimental values with calculated values suggests that factors other than structural variations of air holes, which decrease the Q factor, are indeed present in the fabricated nanocavities.

Characterization of a novel proteinous toxin from sea anemone Actineria villosa.

The sea anemone Actineria villosa expresses a lethal protein toxin. We isolated a novel 120-kDa protein, Avt120, from partially purified toxin and found it to possess extremely strong lethal activity. The 3,453-bp Avt120 gene translates to a 995-amino acid protein. The 50% lethal dose (LD(50)) of purified Avt120 in mice was 85.17 ng. Among several tested cell lines, Colo205 cells were most sensitive to Avt120: 50% of them were damaged by 38.4 ng/mL Avt120. Avt120 exerted ATP degradation activity (10 µmol ATP h(-1) mg(-1)), which was strongly inhibited by ganglioside GM1 to decrease the cytotoxicity of Avt120.

Efficacy of mefloquine treatment and genetic profiles in uncomplicated Plasmodium falciparum malaria in southern Lao PDR.

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.

Molecular characterization on the genome structure of hemolysin toxin isoforms isolated from sea anemone Actineria villosa and Phyllodiscus semoni.

We recently identified the existence of new isoforms of Avt-I (from sea anemone Actineria villosa) and Pstx20 (from sea anemone Phyllodiscus semoni) hemolytic toxins, and named them Avt-II and Pst-I. Avt-II and Pst-I differ in length by 14 and 7 bp, respectively, as compared to their corresponding isoform genes. Both newly found isoform genes have the coding regions with the identical length of 1033 bp. The restriction fragment length polymorphism analysis with endonuclease HphI was able to clearly distinguish between the two Avt isoforms, but not Pstx isoforms, and based on the densitometric analysis of DNA bands, it indicated that relative expression levels of Avt-I and Avt-II genes were 18.3% and 81.7%, respectively. PCR amplification of the two Avt isoform genes using the genomic DNA as template indicated the existence of two introns within each toxin isoform gene. The first intron with the identical 242 bp in length for both Avt isoform was found within the 5'-untranslated region, and the second intron with lengths of 654 bp and 661 bp in Avt-I and Avt-II isoforms, respectively, was found within the signal sequence coding region. This is for the first time to identify the existence of introns within hemolysin genes of sea anemone. Having several unique characteristics that have identified only for a new member of actinoporin family of A. villosa and P. semoni, e.g., strong toxicity and genes with introns, it is plausible to speculate that these toxins have a unique genetic evolutionary linage differed from that for other sea anemone hemolytic toxins.

Intranasal administration of Schistosoma japonicum paramyosin induced robust long-lasting systemic and local antibody as well as delayed-type hypersensitivity responses, but failed to confer protection in a mouse infection model.

To investigate intranasal (i.n.) immunization efficacy of Schistosoma japonicum 97-kDa myofibrillar protein paramyosin (PM), a vaccine candidate for Asian schistosomiasis, BALB/c mice were i.n. immunized with Escherichia coli-expressed recombinant PM (rPM). I.n. immunization using rPM mixed with cholera toxin (CT) was more potent than subcutaneous (s.c.) immunization with rPM emulsified in incomplete Freund's adjuvant for induction of serum (IgG, IgE, and IgA) and mucosal (IgA in nose, lung, and intestine) antibody and delayed-type hypersensitivity (DTH) responses. The second i.n. immunization was sufficient to induce maximal serum IgG and DTH responses, which were almost completely maintained for more than 6 months. Next, to evaluate protective efficacy of the rPM against S. japonicum infection, immunized mice were infected with S. japonicum cercariae at 2 weeks after the second immunization. At 7 weeks after infection, we observed no reduction in worm burden or fecundity in both i.n. and s.c. immunized groups. Results showed that i.n. immunization with rPM/CT failed to provide protection against parasite infection, albeit the antigen was a very potent mucosal immunogen. These results may emphasize the need to innovate new mucosal adjuvants or delivery molecules to overcome such hurdles in the construction of a mucosal antiparasite vaccine platform.

Design and demonstration of high-Q photonic heterostructure nanocavities suitable for integration.

We have investigated the localized design of heterostructure photonic crystal nanocavities in order to make them more suitable for integration. While retaining theoretical quality factors of more than ten million, the total length of the heterostructure nanocavity can be reduced to ~5 mum and the shifted air holes comprising the heterostructure can be restricted to the two rows nearest the nanocavity on each side. Though the area for the heterostructure nanocavity investigated thus far was larger than 10 x 10 microm(2) in the photonic crystal slab, heterostructure nanocavities of this new design have sizes of approximately 3 x 5 microm(2), thus allowing dense integration.

A molecular epidemiologic study of point mutations for pyrimethamine-sulfadoxine resistance of Plasmodium falciparum isolates from Lao PDR.

To understand the current condition of pyrimethamine-sulfadoxine (PS) resistant falciparum malaria in Lao PDR, the frequency of point mutations in dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS) genes of Plasmodium falciparum were examined in 50 blood samples collected from the patients with P. falciparum infection in Southern Lao PDR. Point mutations in 5 codons of the DHFR gene, which is known to be related to pyrimethamine resistance, were detected in 15 out of the 50 samples (30%). Among the 15 samples, 10 samples showed a double mutation of codons 59 and 108 (Cys59Arg with Ser108Asn). In the remaining 5 samples, an additional mutation was observed in codon 51 (Asn51 lle), providing a triple mutation of codons 51, 59 and 108. On the other hand, point mutations in the 4 codons of DHPS gene related to sulfadoxine resistance were observed only in 2 samples (4.0%), namely in codon 437 (Ala437Gly). Only one sample showed mutations in both DHFR and DHPS genes. From the results, it should be considered that the frequency of PS resistant malaria is still low in Lao PDR. Continuous monitoring for the PS resistant malaria, however, is necessary because of the increasing use of PS in this country.

Biochemical and physiological analyses of a hemolytic toxin isolated from a sea anemone Actineria villosa.

A species of venomous sea anemone Actineria villosa was recently found inhabiting the coastal areas of Okinawa, Japan. This marine animal produces various proteinous toxins, so that a local health organization was called for medical treatment for those who had accidental contact with this animal. In this study we analyzed the biochemical and physiological properties of hemolytic protein from A. villosa. The toxin purified from the tentacles of the animals was found to be a protein with a molecular weight of approximately 19 kDa. We named this newly found hemolytic toxin of A. villosa, Avt-I. Incubation of the toxin with sphingomyelin inhibited hemolytic activity by up to 85%, showing that Avt-I may target sphingomyelin on the erythrocyte membrane. The hemolytic activity was stably maintained at temperatures below 45 degrees C, however, a sharp linear decrease in heat stability was observed within the range of 45-55 degrees C. Our results provide the first evidence that A. villosa produces a toxin with strong hemolytic activity similar in biochemical and physiological properties to other members of actinoporin family previously isolated from related species of sea anemones.

Molecular cloning and functional expression of hemolysin from the sea anemone Actineria villosa.

The full-length cDNA that encodes the hemolytic toxin Avt-I, with 226 amino acids, from the venomous sea anemone Actineria villosa has been cloned using the oligo-capping method. The cDNA contains 681bp open reading frame and its predicted amino acid sequences revealed that Avt-I was basic polypeptides without cysteine residues and Arg-Gly-Asp (RGD) motif sequence. The mature Avt-I has a predicted molecular weight of 19.6 kDa and its theoretical isoelectric point is 9.3. The Avt-I revealed 99, 61, 57, and 57% amino acid similarity with hemolytic toxins Pstx20, EqtII, StII, and HmT from Phyllodiscus semoni, Actinia Equina, Stichodactyla helianthus, and Heteractis magnifica, respectively. The characteristic amphiphilic alpha-helix structure was found at the N-terminal region of the mature Avt-I. Recombinant Avt-I (rAvt-I) was expressed in Escherichia coli BL21 (DE3) strain as a biologically active form and purified rAvt-I caused 50% hemolytic activity against 1% sheep erythrocytes at a concentration of 6.3 ng/ml (0.32 nM). M9Y medium led to more than 2-fold increase in rAvt-I yield than cultivation in Luria-Bertani medium.

Pyrimethamine-sulfadoxine treatment of uncomplicated Plasmodium falciparum malaria in Lao PDR.

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.

Association of a sex-related difference of Strongyloides stercoralis-specific IgG4 antibody titer with the efficacy of treatment of strongyloidiasis.

It is difficult to completely eradicate strongyloidiasis, a human intestinal nematode infection with Strongyloides stercoralis with drugs, especially in males. To find host factors involved in the response to treatment, patients infected with S. stercoralis were examined for S. stercoralis-specific antibody titers and the effect of treatment with albendazole on these titers were determined. The cure rate was slightly but not significantly lower in males than in females (P = 0.108). However, a significantly higher titer of S. stercoralis-specific IgG4 antibody was observed in males than in females (P = 0.0097), and the S. stercoralis-specific IgG4 antibody titer was significantly higher in the male non-cured group than in the cured group (P = 0.035). These results suggest that elevation of the S. stercoralis-specific IgG4 antibody titer is associated with resistance to treatment of S. stercoralis infection, especially in males.

Intranasal sensitization with Blomia tropicalis antigens induces allergic responses in mice characterized by elevated antigen-specific and non-specific serum IgE and peripheral blood eosinophil counts.

In order to evaluate the potential allergenicity of Blomia tropicalis (Bt) antigen, IgE production of both specific and non-specific for Bt antigen was monitored in BALB/c mice after exposure to the antigen by nasal route. It was evidenced that B. tropicalis contains a functional allergen in its components. The allergenic components, however, when administered intranasally without any adjuvant, did not function to induce IgE response within a short period. On the other hand, intranasal inoculation of Bt antigens augmented serum IgE responses in mice pretreated by a subcutaneous priming injection of the same antigens. Inoculation of Bt antigen without subcutaneous priming injections induced IgE antibody production only when the antigen was continuously administered for a long period of over 24 weeks. Even when the priming injection was absent, the Bt antigen inoculated with cholera toxin (CT) as a mucosal adjuvant also significantly augmented the Bt antigen-specific IgE responses depending on the dose of CT co-administered. The present study also demonstrated that Bt antigen/CT-inoculated mice showed increased non-specific serum IgE level and peripheral blood eosinophil rates without noticeable elevations of the total leukocyte counts. The immunoblot analysis demonstrated 5 main antigenic components reactive to IgE antibodies induced. These components at about 44-64 kDa position were considered to be an important candidate antigen for diagnosis of the mite-related allergy.

Intranasal sensitization with Blomia Tropicalis antigens induces allergic responses in mice characterized by elevated antigen-specific and non-specific serum ige and peripheral blood eosinophil counts

Para avaliar a capacidade alergizante do antígeno da Blomia tropicalis (Bt) a produção de IgE específica e não específica a antígeno Bt foi monitorada em camundongos BALB/c após exposição ao antígeno por via nasal. Foi evidenciado que Bt contem um alérgeno funcional em seus componentes. Os componentes alergênicos entretanto, quando administrados por via intra-nasal, sem qualquer adjuvante, não induzem resposta IgE durante um pequeno período. Por outro lado, a inoculação intra-nasal de antígenos Bt aumentou a resposta sérica de IgE em camundongos pré-tratados por uma injeção inicial sensibilizante sub-cutânea aos mesmos antígenos. A inoculação do antígeno Bt sem as injeções sensibilizantes iniciais induziu a produção de anticorpos IgE somente quando o antígeno foi administrado de maneira contínua, por um período longo de mais de 24 semanas. Mesmo quando as injeções sensibilizantes iniciais foram ausentes, o antígeno Bt inoculado com a toxina de cólera (CT) como adjuvante mucoso também aumentou de maneira significante a resposta IgE antígeno específica do Bt dependendo da dose de CT administrada conjuntamente. O presente estudo também demonstrou que camundongos inoculados com antígeno Bt/CT mostram aumento do nível IgE não específico no soro e médias de eosinófilos no sangue periférico sem qualquer elevação da contagem total de leucócitos. A análise por Immunoblot demonstrou cinco principais componentes antigênicos reativos aos anticorpos IgE induzidos. Estes componentes na posição 44-64 kilodaltons foram considerados importantes antígenos-candidatos para o diagnóstico da alergia relacionada ao ácaro.

Malaria parasite developmental analyses by the nested polymerase chain reaction method: an implication for the evaluation of mosquito infection rates in epidemiological studies.

A malaria mosquito vector, Anopheles saperoi, and a non-vector, Aedes albopictus, were allowed to feed on mice infected with murine malaria, Plasmodium yoelii nigeriensis, and were subsequently monitored for the development of parasites by the nested polymerase chain reaction (PCR) method, using Plasmodium genus-specific primer pairs. The mosquitos were divided into two parts, head/thorax and abdomen, for DNA analyses. The parasite DNA and murine DNA for each mosquito were examined in parallel. In both groups of mosquitos, murine DNA was detected up to 4 days post-blood meal in both the head/thorax and abdomen. After 4 days, the murine DNA fell below detectable limits. Murine DNA and parasite DNA remained undigested for the first 4 days post-blood meal. Parasite DNA was detected in the abdomen of 25% (3/12) of Ae. albopictus on day five and 10% (1/10) on day six, after murine DNA had fallen below detectable limits. Parasite DNA was not detected in the head/thorax of Ae. albopictus on those days or afterwards in either the head/thorax or abdomen, demonstrating that the parasite detected on days 5 and 6 in the abdomen degenerated and did not develop into mature oocysts or sporozoites. In the vector An. saperoi, parasite DNA was detected continuously in the head/thorax and abdomen for many days after the murine DNA had fallen below detectable limits. The detection rate of parasite DNA in the head/thorax of An. saperoi increased gradually from day 8 post blood meal until it reached a maximum level of 71.4% (15/21 12 days post-infection. Parasite DNA in abdomen reached its maximum level of 81% (17/21) 10 days post-blood meal. The implications of these results for the design and interpretation of epidemiological surveys is discussed.

Field application and evaluation of a rapid immunochromatographic test for detection of Plasmodium falciparum infection among the inhabitants of Lao PDR.

Field application and evaluation of a rapid immunochromatographic test (ICT) for detection of Plasmodium falciparum infection were performed in 13 villages in a southern province of Lao PDR in 1999. More than 2,000 inhabitants, accounting for 61.8% of the total estimated population, were examined. Malaria infection was confirmed in all villages surveyed by ICT and microscopic diagnosis. The positive rates of P. falciparum malaria by microscopy ranged from 9.7% to 59.2% (mean 27.2%), whereas by ICT they were from 11.6% to 64.5% (mean 29.8%). The positive rates by ICT were generally higher in 8 out of 13 villages. However, a significant difference between the positive rates by microscopy and ICT was not observed in all villages. Plasmodium falciparum infection was actually confirmed by microscopy in 84.1% of specimens that tested positive by ICT. The results by ICT were consistent with those of the microscopic diagnosis, the discrepancy of the results was less than 10% (141/2,066). The ICT was falsely-positive in 4.7% and falsely-negative in 2.1% of the test cases. These results showed the efficacy of ICT not only in the diagnosis of the respective cases, but also in the mass-examination in the field.

Serum antibodies induced by intranasal immunization of mice with Plasmodium vivax Pvs25 co-administered with cholera toxin completely block parasite transmission to mosquitoes.

Transmission-blocking vaccines (TBVs) targeting ookinete surface proteins expressed on sexual-stage malaria parasites are considered one promising strategy for malaria control. To evaluate the prospect of developing non-invasive and easy-to-administer mucosal malaria transmission-blocking vaccines, mice were immunized intranasally with a Plasmodium vivax ookinete surface protein, Pvs25 with a mucosal adjuvant cholera toxin (CT). Immunization induced significant serum IgG with high IgG1/IgG2a ratio (indicative of Th-2 type immune response). Feeding Anopheles dirus mosquitoes with mixtures of immune sera and gametocytemic blood derived from vivax-infected volunteer patients in Thailand significantly reduced both the number of midgut oocysts as well as the percentage of infected mosquitoes. The observed transmission-blocking effect was dependent on immune sera dilution. This study demonstrates for the first time that the mucosally induced mouse immune sera against a human malaria ookinete surface protein can completely block parasite transmission to vector mosquitoes, suggesting the possibility of non-invasive mucosal vaccines against mucosa-unrelated important pathogens like malaria.

Expansion of unconventional T cells with natural killer markers in malaria patients.

Immunological states during human malarial infection were examined. In parallel with parasitemia and anemia, granulocytosis was induced in the blood of patients, especially those infected with Plasmodium (P.) falciparum. At that time, the level of lymphocytes remained unchanged or slightly increased in the blood. However, the distribution of lymphocyte subsets was modulated, showing that the proportion of CD56(+)T cells, CD57(+)T cells, and gammadeltaT cells (i.e. all unconventional T cells) had increased in patients infected with P. falciparum or P. vivax. This phenomenon occurred at the early phase of infection and disappeared in the course of recovery. The data from patients with multiple attacks of P. vivax infection showed that there was no augmentation of these responses. In adult cases, the increase in the proportion of unconventional T cells seemed to closely parallel disease severity. However, all these responses were weak in children, even those infected with P. falciparum. In conjunction with accumulating evidence from mouse malaria experiments, the present results suggest that the immunological state induced by malarial infection might mainly be an event of unconventional T cells and that the immunological memory might not be long-lasting, possibly due to the properties of unconventional T cells.