Control of Schistosoma mekongi in Cambodia: results of eight years of control activities in the two endemic provinces.

In Cambodia, schistosomiasis is transmitted in the provinces of Kratie and Stung Treng where approximately 80000 individuals are estimated to be at risk of infection. The baseline prevalence of infection was estimated to be between 73% and 88%, and cases of severe morbidity (hepatosplenomegaly, puberty retardation) and mortality were very common. In 1994, the Ministry of Health of Cambodia started schistosomiasis control applying universal chemotherapy with praziquantel (40mg/kg). The coverage of the programme was between 62% and 86% for 8 years. This simple control measure resulted in the control of the disease: no cases were reported in 2004 and only three cases were reported in 2005. In addition, there are no longer reports of cases of severe morbidity due to schistosomiasis. Since the beginning of the control programme, a single dose of mebendazole (500mg) has been combined with praziquantel during the mass chemotherapy; as a result the prevalence of Ascaris lumbricoides and hookworms dropped from 74.5% to 10% and from 86% to 40% respectively. The experience in Cambodia demonstrates that, with political commitment, control of parasitic diseases is achievable even in a situation of minimal resources. The programme represents a successful model for other developing countries.

Horizontal and vertical transmission of mouse class I MHC sequence in Schistosoma mansoni.

The mouse major histocompatibility complex (MHC) class I sequence was detected in 8-week-old Schistosoma mansoni by in situ polymerase chain reaction (in situ PCR). The signals to the mouse class I MHC sequence were observed in the nuclei of the mesenchymal and reproductive cells of S. mansoni. Signals were also observed in the cytoplasm of the tegumental tubercles. This finding suggested the possibility of MHC gene transfer from the host to schistosomes. Furthermore, the class I MHC sequence was detected in the DNA extracted from the cercariae of S. mansoni by nested PCR. Neither the nucleotide sequence of class I MHC detected in adult worm DNA nor that of class I MHC detected in the host (mouse) DNA was identical with that of class I MHC detected in the cercarial DNA. From the data we assumed that S. mansoni may have retained their own mouse class I MHC sequence in their genome throughout their life-cycle.

The role of saliva of Anopheles stephensi in inflammatory response: identification of a high molecular weight neutrophil chemotactic factor.

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.

Variation of incubation time in an in vitro drug susceptibility test of Plasmodium falciparum isolates studied in the Solomon Islands.

A study on chloroquine resistance of falciparum malaria was conducted in the Solomon Islands. Both in vitro and clinical tests were performed. In our regular studies of in vitro chloroquine susceptibility tests on Plasmodium falciparum from non-immuners in Japan, the threshold point to differentiate resistant and susceptible isolates was set at a 0. 114 microM chloroquine in the semi-micro culture system, and this point was also applicable in the study of the malaria parasite taken in the highly endemic malarious area with good coincidence with clinical observation. Variation in the incubation time (24-63) to reach the schizont stage of the isolated parasites were noted. It appeared that chloroquine resistant P. falciparum showed traits to reach the schizont stage within a shorter incubation period.

Improved preparation and crystallization of 25 kDa human fibroblast growth factor-9.

We prepared 25 kDa human fibroblast growth factor-9 (hFGF-9 N33) on a large scale after overproduction in Escherichia coli MM294 (DE3)/pTG931. The purification was performed by a combination of hydrophobic chromatography and HPLC with an ion exchange column, a heparin affinity column and a gel filtration column. This improved procedure was rapid and simple, and the purified hFGF-9 N33 was found to be homogeneous as judged by various criteria, such as amino acid analysis, N-terminal amino acid sequence, C-terminal amino acid analysis and biological activity. Furthermore, as determined by low endotoxin and DNA content, the protein was of high purity. In addition, the hFGF-9 N33 prepared in the present study was easily crystallized by the vapour diffusion method.

Horizontal and vertical transfer of mouse endogenous retroviral DNA sequences in schistosomes.

The in situ polymerase chain reaction (PCR) results revealed that mouse type A and type C retroviral sequences were transmitted horizontally from the host to schistosomes. The signals to these retroviral sequences were observed in the nuclei of the mesenchymal and reproductive cells of 8-week Schistosoma japonicum. These signals were also detected in the nuclei of the mesenchymal and reproductive cells and in the cytoplasm of the tegumental tubercles of 24-week S. mansoni. Furthermore, mouse type A retroviral sequence was detected in the DNA extracted from the cercariae of both species. However, mouse type C retroviral sequence and mouse type 2 Alu sequence (B2) were difficult to detect in the cercarial DNA of either species. These findings may indicate that some host sequences are propagated in the schistosome progeny, that is to say, not only horizontal but also vertical transfer of the host gene may occur in schistosomes.

Epitope-specific impairment of production of antibody against merozoite surface glycoprotein 1 of Plasmodium falciparum in symptomatic patients with malaria.

We analyzed the relationships between levels of antibody specific for merozoite surface glycoprotein-1 (MSP1) of Plasmodium falciparum and clinical manifestations in humans. We prepared recombinant MSP1 proteins representing block 3 (M3), block 6 (M6), blocks 1-6 (M1/6), and block 17. When we divided the slide-positive individuals in Guadalcanal into symptomatic and asymptomatic groups, the former group showed lower IgG levels against M6 and block 17, but not against M3, than did the asymptomatic group (P < 0.01). The possibility of nonspecific suppression was unlikely, given that the levels of antibody against poliomyelitis virus observed in the two groups were almost the same. Among the IgG subclasses tested, production of cytophilic IgG3 seemed to be dominant. When we analyzed epitopes recognized by antibodies against block 17, a peptide (SSSNFLGIS) was preferentially recognized by sera from asymptomatic individuals. These results suggest that clinical symptoms occurring during falciparum malaria seem to be associated with the development of levels of antibody against particular epitopes on MSP1, which is under the control of an immunoregulatory mechanism.

Sequence diversity of serine repeat antigen gene exon II of Plasmodium falciparum in worldwide collected wild isolates.

Field isolates of Plasmodium falciparum collected from endemic areas of Southeast Asia, Solomon Islands, tropical African countries and Brazil were analyzed for the genetic diversity of the exon II of serine repeat antigen gene (SERA) by sequencing of genomic DNA. Of sixty-nine isolates, as compared to the reported FCR3, K1 and Honduras-1 types of exon II sequences, 5, 9 and 20 new allelic forms were found in 23 isolates of the FCR3 type, 36 of the K1 type and 10 of the Honduras-1 type. A group of novel non-synonymous substitutions, 4 new insertions and 3 new deletions of octamer units were found in the octamer repeat region (OR) of the exon II, and most of them clustered within a 40-residues domain. An octamer "SNPVSSEP" revealed in the OR was confirmed as a new repeat unit. Based on the sequences of the serine repeat region (SR) of the exon II, the allelic forms of the Honduras-1 type were conjectured to be the recombinant forms between the K1 type and FCR3 type. The allelic forms of K1 type with less or more repeat serine residues in the serine stretch of the SR than the reported 21 serine residues had most of the variations in the OR. Moreover, a biased geographical distribution of allelic forms was observed. Isolates from African and Southeast Asian countries accounted for most of the new allelic forms (29/33). All of the three types were detected in Southeast Asia but none of the FCR3 type in Africa. One of two groups of FCR3 new allelic forms was found solely in Brazil while another was mainly in Solomon Islands.

Reduction of tyrosine kinase B and tyrosine kinase C inductions by treatment with neurotrophin-3 after transient middle cerebral artery occlusion in rat.

Infarct volume and immunoreactivities for trkB and trkC in rat brain were compared at 24 h after 90 min of transient middle cerebral artery occlusion (MCAO) between animal groups with or without neurotrophin-3 (NT-3, 10 microg/250 g animal). Treatment of rat brain with topical application of NT-3 significantly reduced infarct volume (P = 0.02) and trkB and trkC inductions. These data suggest that NT-3 reduced the ischemic injury along with the reduction of trkB and trkC inductions.

Topical application of neurotrophin-3 attenuates ischemic brain injury after transient middle cerebral artery occlusion in rats.

In order to examine the effect of neurotrophin-3 (NT-3) on ischemic brain injury, NT-3 was topically applied to brain surface just after 90 min of middle cerebral artery occlusion (MCAO) in rats. NT-3 significantly reduced the infarct size at 24 h of reperfusion. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin in situ nick labeling (TUNEL) staining and immunohistochemical study for caspase-3 and heat shock protein 72 (HSP72) showed that NT-3 treatment decreased the number of cells with DNA fragmentation and caspase-3 and HSP72 expressions. These data suggest that NT-3 protects neuronal cells from ischemic injury, and it is possibly associated with inhibition of DNA fragmentation.

Localization of mouse type 2 Alu sequence in schistosomes.

Localization of the type 2 Alu sequence (B2), a highly repetitive DNA sequence in the mouse genome, was examined by in situ polymerase chain reaction (in situ PCR) in schistosomes. The signals to the B2 sequence were detected in the cytoplasm of the tegumental membrane and in the nuclei of the mesenchymal, testicular, ovarian and vitelline cells of 8-week Schistosoma japonicum. In contrast, it was difficult to detect any signals of this sequence in 8-week S. mansoni, whereas in 24-week male S. mansoni the signals were observed in the cytoplasm of the tegumental tubercles and in the nuclei of the mesenchymal and testicular cells. On the other hand, in 24-week female S. mansoni the signals were found in the nuclei of the mesenchymal, ovarian and vitelline cells but not found in the tegument. On the contrary, no hybridization band of the B2 sequence was detected in the amplified DNA of 3-week schistosomula of either species. These observations proved that the host DNA sequences existed in restricted schistosome cells and were accumulated in the schistosome body during their development.

Renaturation of recombinant human neurotrophin-3 from inclusion bodies using a suppressor agent of aggregation.

Escherichia coli has been widely used in the production of recombinant proteins. One of the drawbacks inherent in this method is that the proteins produced in the cells often form inactive inclusion bodies. Usually, the inclusion bodies can be separated from other cell components, solubilized by denaturants such as guanidine hydrochloride or urea, and then renatured through a refolding process such as dilution or dialysis. However, it has been shown that biologically active recombinant human neurotrophin-3 cannot be obtained at high yield by this procedure due to aggregation and precipitation of the protein. We applied the refolding process using the aggregation suppressor L-arginine in the renaturation of neurotrophin-3, and obtained biologically active neurotrophin-3 at high yield from the inclusion bodies. Consequently, about 10 mg of purified neurotrophin-3 was prepared from 1 litre of culture broth.

[Spontaneous rupture of malignant fibrous histiocytoma arising from the renal capsule: a case report].

We report a case of spontaneous rupture of malignant fibrous histiocytoma. A 50-year-old male with right flank pain was referred to our hospital. Computed tomography (CT) showed a heterogeneous space-occupying lesion on the upper pole of the right kidney. Selective right renal arteriography revealed a hypovascular mass. Preoperative clinical diagnosis was spontaneous rupture of renal cell carcinoma. Radical nephrectomy was performed. Histopathological diagnosis was malignant fibrous histiocytoma arising from the renal capsule.

Trehalose-induced destabilization of interdigitated gel phase in dihexadecylphosphatidylcholine.

Trehalose is believed to have the ability to protect some organisms against low temperatures. To clarify the cryoprotective mechanism of trehalose, the structure and the phase behavior of fully hydrated dihexadecylphosphatidylcholine (DHPC) membranes in the presence of various concentrations of trehalose were studied by means of differential scanning calorimetry (DSC), static x-ray diffraction, and simultaneous x-ray diffraction and DSC measurements. The temperature of the interdigitated gel (Lbeta(i))-to-ripple (Pbeta') phase transition of DHPC decreases with a rise in trehalose concentration up to approximately 1.0 M. Above a trehalose concentration of approximately 1.0 M, no Lbeta(i) phase is observed. In this connection, the electron density profile calculated from the lamellar diffraction data in the presence of 1.6 M trehalose indicates that DHPC forms noninterdigitated bilayers below the P beta' phase. It was concluded that trehalose destabilizes the Lbeta(i) phase of DHPC bilayers. This suggests that trehalose reduces the area at the interface between the lipid and water. The relation between this effect of trehalose and a low temperature tolerance was discussed from the viewpoint of cold-induced denaturation of proteins.

epsilon-N-acetylation in the production of recombinant human basic fibroblast growth factor mutein.

During large scale preparation of recombinant human basic fibroblast growth factor (hbFGF) mutein CS23(CS23) produced in Escherichia coli (E. coli), three species, named Mf-1, Mf-2 and Mf-3 in the order of their elution, were isolated from the early fraction of Sulfated Cellulofine chromatography by high performence liquid chromatography (HPLC) on heparin-5pw. Structural analysis through HPLC peptide mapping, mass spectrometry, sequencing and amino acid composition revealed that epsilon-N-acetylation occurred at positions Lys135, Lys26 and Lys119 of CS23. Together with this observation, the results on large scale preparation of CS23 were also presented.

Relationship of haptoglobin polymorphism to malaria in the Solomon Islands.

Haptoglobin (Hp) polymorphism and its relationship to malaria infections was examined in Solomon Islanders under 25 years of age. The slide positive rate for malaria was 48% (53/111): Plasmodium vivax (Pv) 21 cases, Plasmodium falciparum (Pf) 16 cases and the remaining 16 were a mixture of Pv/Pf/P. malariae. Of 111 subjects examined, 17% (19 subjects) were ahaptoglobinemic (HpO). No association was observed between HpO and parasitemia, gender, anemia, serum insulin-like growth factor-1 (IGF-1) level, blood glucose level and enlargement of the spleen. These results suggest that malaria may not be the major cause of HpO in the people of the Solomon Islands.

Prevalence of malaria and its relationship to anemia, blood glucose levels, and serum somatomedin c (IGF-1) levels in the Solomon Islands.

As part of establishing effective methods for malaria control, the malaria-associated nutritional status was surveyed on Guadalcanal Island in the Solomon Islands in 1993. A total of 506 residents participated in this study. The slide positive rate for malaria was 54% (275/506) in all ages, with a high of 79% for children aged 4-6 years. Plasmodium falciparum was the most common species (52%), followed by P. vivax (29%). Splenomegaly in children from infants to age 15 was detected at the rate of 30% (104/343) by the palpation method. Body mass index was lower in Solomon Islanders than for the Japanese population up to 15 years old in both genders. Mean values for serum insulin-like growth factor-1 (IGF-1) were also lower in Solomon Islanders in children under 18 years old. The hemoglobin distribution curves were almost identical in the malaria-positive (P(+)) and -negative (P(-)) groups. The percentage of cases with less than 80 mg/dl of blood glucose and those with less than 50 ng/ml of IGF-1 were higher in the P(+) group than for the anti-malaria drug-untreated malaria-negative (P(-)D(-)) group. It is suggested that low blood glucose and low IGF-1 levels may have some relationship with the malaria infection.

Integrated information-processing system in clinical orthodontics: an approach with use of a computer network system.

A computer network system has been developed in the Orthodontic Clinic, Osaka University Dental Hospital, to improve treatment efficiency and patient service. The system consists of a 32-bit host computer, its peripheral units, and personal computers connected to the host computer by data-transmission circuits, making up a local area network (LAN). It is possible in this system to integrate various types of data, such as the patient's basic information, treatment records, image data, and diagnostic analysis results to construct a relational database. It has been shown that the computer system developed in the orthodontic clinic has various clinical advantages.

Ultrasonographic and serologic abnormalities in Schistosoma japonicum infection in Leyte, the Philippines.

We have identified specific ultrasonographic changes in Schistosoma japonicum-infected patients associated with serologic indicators of general liver function. An ultrasonographic examination concomitant with hematologic and biochemical serum analyses was performed on 102 patients at the Schistosomiasis Hospital in Leyte, The Philippines. The ultrasonographic liver images were classified into four patterns, according to the development of periportal fibrosis and the patterns of echogenic bands. Eleven cases with a long-term infection showed typical septal formation (network pattern). Other ultrasonographic changes in the portal system, such as the severity of splenomegaly, did not correlate with the age of the study patients or the duration of their infection; however, the production of collateral vessels was clear in the group of older patients. Among various hematologic and biochemical serum indicators of liver damage, the serum levels of total bile acid (TBA) and procollagen-III-peptide (P-III-P) strongly correlated with the development of hepatic fibrosis and protal hypertension. These findings suggest that the ultrasonographic liver patterns classified here, along with the changes in serum levels of TBA and P-III-P, provide useful indicators for field monitoring of S. japonicum infection.