Recombinant soluble form of the human high-affinity immunoglobulin E (IgE) receptor inhibits IgE production through its specific binding to IgE-bearing B cells.

A recombinant soluble form of the alpha subunit of the human high-affinity receptor for IgE (rsFc epsilon RI alpha), one of the potent IgE-binding molecules, was tested for its ability to regulate IL-4-induced IgE synthesis by human lymphocytes. Addition of rsFc epsilon RI alpha to cultures induced a dose-dependent inhibition of the T cell-dependent and independent synthesis of IgE. The suppression of IgE synthesis was observed at the protein and the mRNA levels, and it was IgE class specific. By flow cytometry, specific binding of rsFc epsilon RI alpha was detected on surface IgE-bearing B cells as well as on U266 cells, and it was completely blocked by preincubation with IgE. rsFc epsilon RI alpha bound to the cell surface IgE could be effectively dissociated not only by a large excess of IgE, but also by an anti-rsFc epsilon RI alpha mAb that competes with IgE for the binding to rsFc epsilon RI alpha. This mAb abolished the rsFc epsilon RI alpha-mediated suppression of IgE synthesis. These data suggest that rsFc epsilon RI alpha may have a function in selectively suppressing IgE synthesis through its interaction with the membrane-bound form of IgE.

Solid dispersion of spironolactone with porous silica prepared by the solvent method.

Solid dispersions of spironolactone (SPI) with porous silica (Sylysia 730 and Sylysia 350) were prepared by the solvent method. The physicochemical properties of the prepared solid dispersions were evaluated by powder X-ray diffractometry (PXRD), differential scanning calorimetry (DSC), scanning electron microscopy (SEM), and atomic force microscopy (AFM). In the SEM study, no differences in the surface condition between Sylysia 350 and the solid dispersion of a Sylysia 350:SPI system in a weight ratio of 1:1 were observed. However, AFM phase images showed that the surface of the solid dispersion of the Sylysia 350:SPI system (weight ratio of 1:1) was rather smooth due to the adsorption of SPI as compared with that of a Sylysia 350 intact. The results of PXRD and DSC data in the solid dispersion of the Sylysia 350:SPI system (weight ratio of 1:1) indicated that the molecular state of the adsorbed SPI changed from crystalline to amorphous. Although the decrease in the SPI concentration increased with the amorphous fraction in the solid dispersion, the diffraction peaks due to SPI crystals still remained in the solid dispersion of a Sylysia 730:SPI system (weight ratio of 1:1), indicating that the mean pore diameter and specific surface area of an additive are some of the important factors for the amorphization of SPI crystals. The dissolution property of the SPI from the solid dispersions was remarkably improved in comparison with that of SPI crystals. The dissolution rate of the SPI from the solid dispersions with Sylysia 350 was faster than that of the SPI from the solid dispersions with Sylysia 730. The difference in the dissolution properties of SPI from both the solid dispersions was attributed to the difference in the molecular state of the SPI in both the solid dispersions. In the stability test, the amorphous state of the SPI in the solid dispersion of the Sylysia 350:SPI system (weight ratio of 1:1) was maintained for 2 weeks at 25 degrees C and 0% RH, while the amorphous SPI without Sylysia 350 crystallized under the same conditions.

Biological activities of chemically synthesized N-acetylneuraminic acid-(alpha 2----6)-monosaccharide analogs of lipid A.

The mitogenicity and lethal toxicity of chemically synthesized lipid A analogs, in which 2,3-acyloxyacylglucosamine-4-phosphate linked to tetraacetyl-N-acetylneuraminic acid (compound A-207) or to N-acetylneuraminic acid (compound A-307), were examined. Although the mitogenic activity of the synthetic compounds was weaker than that of bacterial LPS, doses of 10-50 micrograms/ml of A-207 and 5-10 micrograms/ml of A-307 were capable of increasing incorporation of [3H]thymidine into cultured spleen cells of C57BL/6 mice. Lethal toxicity of A-207 was observed at 10 micrograms/mouse in C57BL/6 mice sensitized with D-galactosamine hydrochloride. However, the attachment of tetraacetyl-N-acetylneuraminic acid or N-acetylneuraminic acid does not appear to enhance the biological activity of acyloxyacylglucosamine-4-phosphate.

Direct action of cyclosporin A on human B-lymphocytes with regard to immunoglobulin production.

Highly purified preparations of human B-lymphocytes were cultured with or without cyclosporin A (CyA; 1 microgram/ml) for 8 days with pokeweed mitogen (PWM) in the presence of helper factors or with Epstein-Barr virus (EBV). The amounts of IgG, IgM, IgA and IgE produced in cultures were measured by radioimmunoassays or by reverse hemolytic plaque-forming cell assays. The results demonstrate that whereas CyA had a strong suppressive effect on the production of immunoglobulins (Ig) by PWM-activated B-cells, it had an enhancing effect on EBV-activated B-cells. It is concluded that CyA has a direct effect on human B-lymphocytes and that it may suppress or enhance their activation depending on the stimulant employed to trigger the cells.

Elevation of serum IgE level and peripheral eosinophil count during T lymphocyte-directed gene therapy for ADA deficiency: implication of Tc2-like cells after gene transduction procedure.

We have successfully carried out T-cell-directed gene therapy for a boy with severe combined immunodeficiency due to adenosine deaminase deficiency (ADA SCID) and unexpectedly found an elevation of serum IgE level and peripheral eosinophil count during the course. More than 90% of transduced cells cultured for 7-11 days before infusion into the patient were positive for CD8 and expressed Th2-type cytokine genes such as IL-4, IL-5 and IL-13. Furthermore, CD4(+) T-depleted PBMC (peripheral blood mononuclear cells) from the patient synthesized IgE in vitro by stimulation with IL-4. Collectively, these results suggested that Tc2-like cells in the transduced cells have distinct immunological functions to help IgE synthesis and activate eosinophils.

Comparison of the biological activity of synthetic N-acylated asparagine or serine linked monosaccharide lipid A analogs.

The mitogenicity, lethal toxicity, induction of tumor necrosis factor (TNF), production of nitric oxide (NO) and antitumor activity against Meth A fibrosarcoma by chemically synthesized N-acylated asparagine-linked (A-701, A-702 and A-703) or N-acylated serine-linked (A-607) nonphosphorylated acylglucosamine and 4-0-phosphorylated acylglucosamine (A-103) derived lipid A analogs were determined. compound A-607 (with tetradecanoyl and (R)-3-tetradecanoyloxytetradecanoyl at the C-2 and C-3 positions) induced a significant incorporation of 3H-thymidine into splenocytes of C3H/He mice at concentrations ranging from 3.13 to 50 microM, but the mitogenic activity of A-701 (2-N-acetylglucosamine), A-702 (tetradecanoyl at the C-2), and A-703 (with (R)-tetradecanoyloxytetradecanoyl and tetradecanoyl at the C-2 and C-3) was very weak. The lethality of A-703 and A-103 (with (R)-3-tetradecanoyloxytetradecanoyl at the C-2 and C-3) was weaker than that of A-607 at doses of 300 and 750 nmol/kg in C57BL/6 mice loaded with D-galactosamine. Peritoneal macrophages, stimulated with A-701-A-703, caused production of TNF which induce L929 cell lysis in vitro, and A-703 showed a high production of TNF. The compounds, except for A-607, exhibited little NO production by macrophages, but did induce the NO production in the presence of interferon gamma. Induction of TNF and NO inducible activity by A-703 was lower than that of A-607. A-703, A-607 and A-103 showed antitumor activity against Meth A fibrosarcoma in BALB/c mice. When A-703 or A-103 with muramyl dipeptide was administered, A-703 failed to show combined effects, but A-103 did. We concluded from these findings that the biological potency of asparagine compounds appears to be placed between serine- and amino-free compounds.

Allergic bronchopulmonary aspergillosis due to Aspergillus oryzae.

A 19-year-old female student with allergic bronchopulmonary aspergillosis (ABPA) due to Aspergillus oryzae is reported. This organism was used for fermentation starter to make soybean paste in her father's workshop adjacent to the home where she lived. ABPA might be considered an occupational disease in certain situations.

Measurement of distensibility of blood vessels using cineangiograms.

We developed a new, cineangiographic method to accurately measure the dynamic changes in the internal diameter of human arteries in vivo. Cine films were digitized at a spatial resolution of 4 microns/pixel, using a line image sensor. The vessel edges, with a Gaussian fit to a unilateral profile curve of the vessel, were determined with the aid of a computer program. We measured contrast-filled cylinder vessel models (2 to 7 mm in diameter) and evaluated precision, accuracy and linearity of the diameter measurement. A pulsatile vessel model of about 3.9 mm in internal diameter was used to examine the reliability of our method for detecting arterial wall motion. If the coefficient of variation of the vessel diameter determined cineangiographically was less than 2.2% we considered the cineangiograms sufficiently accurate to determine the internal vessel diameter and evaluate arterial distensibility.

3-Cyano-2,6-dihydroxypyridine (CNDP), a new potent inhibitor of dihydrouracil dehydrogenase.

3-Cyano-2,6-dihydroxypyridine (CNDP) was identified as a potent inhibitor (IC50 value, 4.4 nM) of dihydrouracil dehydrogenase (DHUDase) [EC 1.3.1.2], a rate-limiting enzyme in 5-fluorouracil (5-FU) degradation. The inhibitory activity of CNDP was about 2,000 times that of uracil under our assay conditions. Kinetic analyses with partially purified enzyme from rat liver revealed that the mechanism of inhibition of DHUDase by CNDP was of mixed type with an inhibition constant (Ki) of 1.51 nM. CNDP had less effect on 5-FU phosphorylation than on 5-FU degradation. The inhibitory effect of CNDP on ribosylation of 5-FU was 600 to 1,000 times less than that on DHUDase. Moreover, CNDP did not inhibit uridine kinase, thymidine kinase, or pyrimidine phosphoribosyltransferase. Coadministration of CNDP with 1-ethoxymethyl-5-fluorouracil (EM-FU) to rats with Yoshida sarcoma elevated the level of 5-FU in both the blood and the tumor and enhanced the antitumor effect of EM-FU. These findings indicated that CNDP would be a useful chemical modulator in chemotherapy with 5-FU or its prodrugs.

Characterization of Borrelia garinii isolated from Lyme disease patients in Hokkaido, Japan, by sequence analysis of OspA and OspB genes.

The outer surface proteins OspA and OspB genes of clinical Borrelia garinii isolates (JEM1-8) from Hokkaido, Japan were sequenced. One strain, JEM4, has a single ospA gene which is similar to European B. garinii strains PBr (sequence homology value: 94.1%) and T25 (91.2%). Five of the other seven strains exhibit a homologous C-terminus (300 bp) on both ospA and ospB genes (88.7-97.3%). The other two strains seem to be derived from the five strains by ospA or ospB alterations. In a phylogenetic analysis based on ospA, these strains could be classified into B. garinii, but formed independent branches and separated from the typical B. garinii isolates from Europe and Russia.

Relationship between infectivity and OspC expression in Lyme disease Borrelia.

Borrelia burgdorferi sensu stricto strain 297 and B. garinii strains HP1 and 12-92 were serially subcultured for 36-50 passages in vitro for 1 year. All low-passage strains showed abundant expression of outer surface protein C (OspC) in the 22-23-kDa range, but the high-passage strains lost or showed reduced expression of OspC in comparison with the low-passage strains. The low-passage strains efficiently infected outbred ddY mice when inoculated into the hind footpad or peritoneal cavity. In contrast, the incidence of infection with the high-passage strains was low. Isolates from the bladders of mice inoculated with the high-passage strains expressed large amounts of OspC in comparison with those originally inoculated. These results indicate that OspC expression is related to the infectivity of Lyme disease borreliae.

Relationship between pathogenicity of Coxiella burnetii isolates and gene sequences of the macrophage infectivity potentiator (Cbmip) and sensor-like protein (qrsA).

Coxiella burnetii, the Q fever agent, is an obligate intracellular bacterium and survival in phagolysosomes is an important virulence factor. The present study was performed to determine the relationship between its pathogenicity and genes related to its survival in macrophages, i.e. macrophage infectivity potentiator and Q fever agent regulatory sensor-like protein. The sequence similarity was more than 99% among Japanese, European and American strains, and no relationship was found between pathogenicity in guinea pigs and these genes.

Homogeneity of Borrelia japonica and heterogeneity of Borrelia afzelii and 'Borrelia tanukii' isolated in Japan, determined from ospC gene sequences.

Borrelia afzelii, B. japonica, and 'B. tanukii' isolated from various sources and geographical origins in Japan were characterized by restriction fragment length polymorphism (RFLP) analysis and sequencing analysis of the outer surface protein C (OspC) amplicon. B. afzelii and 'B. tanukii' generated variable RFLP patterns and differences in ospC gene sequence were confirmed. In contrast, 26 isolates of B. japonica generated one OspC RFLP type, and sequence similarity between B. japonica ranged from 96.4 to 99.7%. These finding suggests that B. japonica is unique in comparison with other members of B. burgdorferi sensu lato species with respect to homogeneity of the ospC gene.

Characterization of Borrelia sp. isolated from Ixodes tanuki, I. turdus, and I. columnae in Japan by restriction fragment length polymorphism of rrf (5S)-rrl (23S) intergenic spacer amplicons.

Borrelia isolated from various sources in Japan, including rare species of ixodid ticks, Ixodes tanuki, I. turdus, and I. columnae, were characterized by restriction fragment length polymorphism analysis and sequencing analysis of the 5S-23S rRNA intergenic spacer amplicon. Borrelia sp. isolated from I. tanuki, I. turdus and I. columnae generated restriction fragment length polymorphism patterns different from those of known B. burgdorferi sensu lato isolates previously reported. Furthermore, some B. afzelii and B. garinii isolated in Japan showed unique RFLP patterns which were not observed among European B. afzelii and B. garinii.

Vi-deficient and nonfimbriated mutants of Salmonella typhi agglutinate human blood type antigens and are hyperinvasive.

We generated nonfimbriated mutants from both Vi-positive and -negative Salmonella typhi to analyze the role of type 1 fimbriae and Vi-antigen in bacterial invasion. A Vi-defective mutant of S. typhi GIFU 10007-3 was more invasive than the wild-type strain GIFU 10007. The wild-type strain expressing Vi-antigen did not agglutinate both Saccharomyces cerevisiae and human erythrocytes but Vi-defective mutants were able to agglutinate S. cerevisiae and human erythrocytes. Nonfimbriated mutants from Vi-negative GIFU 10007-3 lost the ability to adhere to S. cerevisiae but still could agglutinate human erythrocytes. The Vi-negative mutant increased secreted proteins and became 5-fold more invasive than the wild-type strain. Nonfimbriated Vi mutants became 50-120-fold more invasive than the wild-type GIFU 10007. To determine why nonfimbriated Vi mutants still agglutinate human red blood cells, we searched bacterial proteins that could bind human blood-type antigens. We finally identified a candidate 37 kDa outer membrane protein that recognized fucosyl-galactose, a structure common to blood type A, B and H antigens.

Time-course detection of HIV-1 proviral DNA and genomic RNA by polymerase chain reaction in sera from seropositive and seronegative hemophiliacs treated with clotting factor concentrates.

The detection of HIV-1 proviral DNA and genomic RNA was performed by polymerase chain reaction (PCR) in hemophiliacs treated with non-heated clotting factor concentrates. Reamplification with double PCR was performed on those samples that were negative for single PCR. Primer pairs of the gag, env, and pol regions were used for the amplification of HIV-1 proviral DNA sequences. Amplification of the gag region by the SK38/SK39 primer pair was useful for the detection of proviral DNA sequences. With double PCR, 44 of 47 seropositive samples (93.6%) were PCR-positive. All 23 seronegative samples were PCR-negative. Reverse transcription and PCR amplification (RT-PCR) according to the primer pair of the gag region were performed to detect HIV-1 genomic RNA sequences. Double RT-PCR analysis of the HIV-1 RNA sequence in frozen-preserved sera revealed that 49 of 55 seropositive sera (89.1%) were PCR-positive. Although quantification of the PCR method was not performed in this study, we concluded that, in patients in whom proviral DNA or genomic RNA sequences are detected with difficulty with PCR, the onset and progression of HIV-1 infection is delayed.

Column-switching liquid chromatographic method for the simultaneous determination of iothalamic acid and creatinine in biological fluids.

A column-switching liquid chromatographic method for simultaneous determination of iothalamate and creatinine in human serum and urine was developed. Iothalamate and creatinine were separated on a weakly acidic ion-exchange column (C1) by ion-exclusion chromatography and iothalamate excluded from the column was purified by gel chromatography on a hydrophilic gel column (C2) and then by ion-exchange chromatography on a weakly basic ion-exchange column (C3). Creatinine that was eluted from C1 after iothalamate was transferred to a hydrophilic gel column (C4) and then to a strongly acidic ion-exchange column (C5). The mobile phase for C1-C4 was a pH 3.8 propionate buffer (propionic acid-NaOH = 0.35 + 0.035 mol/kg in water) and a pH 5.6 propionate buffer (propionic acid-NaOH = 0.04 + 0.035 mol/kg in water) was used for C5. Diluted serum and urine samples could be injected directly on to C1, as the matrix of C1 is hydrophilic and C1 is backflushed after the transfer of the creatinine fraction from C1 to C4. Iothalamate and creatinine in the eluates were determined by measuring their ultraviolet absorption at 245 and 234 nm, respectively. The precision (R.S.D.) of the chromatographic method was 1.6% (n = 7) and 0.36% (n = 6) for diluted serum and urine with iothalamate concentrations of 1.0 and 10.0 mumol/l, respectively, and 0.85% (n = 7) and 0.55% (n = 7) for diluted serum and urine with creatinine concentrations of 5.77 and 272 mumol/l, respectively.

Screening for yeasts incorporating the exogenous eicosapentaenoic and docosahexaenoic acids from crude fish oil.

The screening for yeasts incorporating exogenous eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) into their cellular lipids was conducted. Two percent of oil from fish scraps was added to a yeast isolation medium as a sole carbon source. From 143 soil samples, we isolated 23 yeast strains, nine of them were found to be capable of quickly assimilating the scrap fish oil. These nine strains, in addition to four previously isolated triglyceride-assimilating and lipase-producing strains, Yarrowia lipolytica and three Geotrichum species (FO274A, FO347-2 and FO401B), were cultured in a growth medium at 30 degrees C. Strains FO726A, FO765A and FO347-2 were selected on the basis of dry cell weight production and ability to store EPA and DHA in their cells, and their performance was further compared by varying cultivation temperature and time. From 1 g of the scrap fish oil, FO726A yielded 620 mg of dry cells, containing 47.1% lipid, 38.1% triglyceride, 3.3% EPA, and 4.9% DHA, when cultured at 25 degrees C for 36 h. Strain FO726A apparently has the highest ability to incorporate EPA and DHA into its cellular lipids. Results from further experiments showed that the incorporated EPA and DHA mainly existed in the form of triglyceride in the FO726A cell. These results suggest that FO726A is suitable for the production of cell mass rich in EPA and DHA for feed.

Effect of rush immunotherapy in house-dust-mite (HDM)-sensitive adult bronchial asthma: changes in in vivo and in vitro responses to HDM.

An open study was conducted to evaluate the changes in in vivo and in vitro responses to house-dust-mite (HDM) after rush immunotherapy (RI). A 7-day RI protocol using an extract containing HDM allergen was administered to 12 subjects with HDM-sensitive asthma, and the effects on bronchial responsiveness and serum antibody levels were evaluated up to 16 or 20 weeks after RI. The levels of HDM-specific IgG, IgG1 and IgG4 antibodies were significantly elevated from 4 or 8 weeks after RI. Provocative doses causing a 20% fall in FEV1 (PD20) by allergen inhalation were elevated in all subjects at 16 to 20 weeks after RI. There was a high correlation between the increase in log-PD20 and the increase in the ratio of HDM-specific IgG4 to IgG1 (r = 0.68, p < 0.05). The results suggest that RI elicits the improvement of allergen-specific bronchial responsiveness and the increase in serum antibody levels within a relatively short period.

A case of Lyme disease diagnosed serologically.

A 57-year-old Japanese male developed erythema migrans, arthralgia, and neurological symptoms after mountain climbing in Nagano Prefecture. On ELISA, his serum reacted to Borrelia isolated from various sources. The patient's serum reacted with a 41 kilodalton protein which was reactive with monoclonal antibody H9724 specific to flagellin of Borrelia on Western blotting. He recovered after taking minocycline 200 mg daily for 2 weeks.