Adoptive transfer of T-helper cell type 1 clones attenuates an asthmatic phenotype in mice.

T-helper cell type 1 (Th1) cells have been postulated to have a significant role in protective immunity against allergic diseases. However, recent studies using polarised Th1 cells showed conflicting effects on both airway responsiveness and eosinophilic inflammation in a mouse asthma model. The current study explored the effects of adoptive transfer of established Th1 clones on a murine model of atopic asthma. Mice (BALB/c) were sensitised with ovalbumin (OVA) and challenged with aerosolised OVA (5%, 20 min) for 5 days. Just before starting the first challenge, Th1 clones (5x10(6) x body(-1)) or PBS alone were injected via the tail vein. After assessment of airway responsiveness to methacholine, bronchoalveolar lavage fluid (BALF) was obtained. Histological examination, including morphometric analysis, measurement of cytokines in the BALF and Northern blotting of lung chemokines, was also performed. Adoptive transfer of Th1 clones showed a significantly increased total number of cells, whereas significantly decreased eosinophils were found in the BALF, when compared with mice with injection of vehicle alone or splenic mononuclear cells. Administration of Th1 clones significantly decreased the infiltration of eosinophils but increased mononuclear cells in the peribronchial area. Goblet cell hyperplasia and peribronchial fibrosis were also suppressed by Th1 clones. The transfer of Th1 cells significantly decreased airway responsiveness. Th1 injection significantly increased interferon gamma in the BALF, but significantly decreased interleukin (IL)-5 and IL-13. Eotaxin mRNA was predominantly expressed in the lungs of asthma model mice, whereas RANTES (regulated on activation, normal T-cell expressed and secreted) predominates in such mice with Th1 transfer. In conclusion, results suggest that the adoptive transfer of T-helper cell type 1 clones can suppress both lung eosinophilia and airway responsiveness, but increase noneosinophilic inflammation in a mouse model of asthma.

T-helper 1 cells induce alveolitis but do not lead to pulmonary fibrosis in mice.

T-helper (Th)1 cells have a pivotal role in the pathogenesis of hypersensitivity pneumonitis. Continued low-level exposure to the antigens may induce chronic hypersensitivity pneumonitis with lung fibrosis. Although such exposure may activate Th1 cells in the lung, it is not known whether activation of Th1 cells per se can lead to pulmonary fibrosis. To determine this, the lung pathology induced by Th1 clones was investigated. Mice (BALB/c) were injected intraperitoneally with Th1 clones 1-4 times. Each injection was performed 4 days apart and was followed by repeated exposure to aerosolised ovalbumin (OVA) once a day for 5 days. The number of macrophages and lymphocytes in bronchoalveolar lavage fluids (BALF) increased as the number of Th1 transfers increased. The number of neutrophils also increased but peaked in the second transfer and then decreased following further transfers. Increased cell infiltration, thickness of alveolar walls and number of type II cells in the lung occurred. However, histological findings showed no evidence of fibrosis and hydroxyproline levels did not increase. Findings of histology and BALF were ameliorated 2 weeks after the discontinuation of OVA exposure, indicating the reversibility of the Th1-induced pathology. In conclusion, adoptive transfer of T-helper 1 cells results in reversible alveolitis but does not lead to pulmonary fibrosis.

Presence of an SAR-like sequence in junction regions between an introduced transgene and genomic DNA of cultured tobacco cells: its effect on transformation frequency.

A 12.5-kb DNA fragment with junction regions between the transgene and genomic DNA was cloned from a transgenic tobacco cell line obtained by microprojectile bombardment of plasmid pCaMVNEO. Nucleotide sequence analysis of the fragment (DDBJ accession no. D84238) showed that it carried a 7.7-kb core sequence (concatemer of a complete pCaMVNEO and a partial pCaMVNEO) and two identical 1.3-kb junction sequences that flanked both the 5' and 3' ends of the core sequence and had inverted orientations. These sequences had topoisomerase II (Topo II) cleavage sites and adenine and thimine-rich sequences known to be specific to nuclear scaffold-attachment regions (SARs). An in vitro binding assay showed that a 507-bp fragment (designated TJ1) from the 1.3-kb sequence had the ability to bind to nuclear scaffold preparations of cultured tobacco cells, confirmation that the 1.3-kb sequence is an SAR. Insertion of TJ1 at the 5' and 3' sides of the expression cassette for the npt II gene increased transformant yields 5- to 10-fold and the NPT II enzyme activity per copy of the gene 5-fold. TJ1 enhances the integration or expression of the transgene, or both. Clearly, TJ1 is very useful for producing transgenic plants. This is the first report on an SAR-like sequence that is located in the transgene locus and enhances transformation efficiency in eukaryotic cells. The possible role of TJ1-SAR in the molecular evolution of plant genome is discussed.

Stable transformation of Eustoma grandiflorum by particle bombardment.

Explants (7.5±2.5 mm) cut from stems and roots of 3-week-old Eustoma grandiflorum Grise, (lisianthus) cv. Glory White seedlings were bombarded with plasmid pBI221, which harbors the uidA gene encoding ß-glucuronidase (GUS) driven by the cauliflower mosaic virus (CaMV) 35S promoter. More than 800 blue spots of GUS-expressing cells were observed per 90 explants. Explants bombarded with pARK22 harboring the bar gene encoding phosphinothricin acetyltransferase driven by the CaMV 35S promoter were selected for bialaphos resistance. Putative transgenic plants were obtained about 3 months after bombardment. Southern blot analysis of putative transgenic plants revealed the presence of the bar gene in their genome.

Molecular and cytological characterization of a highly repeated DNA sequence in Raphanus sativus.

A highly repeated DNA sequence with a repeat unit of ca. 180 bp was found in genomic DNA HindIII-digests of Raphanus sativus. The repeating units of six isolated, independent clones were sequenced. These units have 177 or 178 bp, are 36% G+C in their DNA base composition, and show 90% sequence homology. The copy number of this 180-bp repeat unit is about 0.5 x 10(6) per diploid genome. In situ hybridization analysis with the repeating units as the probe and C-banding analysis indicated that the repeated DNA sequence of R. sativus is closely associated with the major C-heterochromatins in the proximal regions of all 18 chromosomes at mitotic metaphase.

Successful expression in pollen of various plant species of in vitro synthesized mRNA introduced by particle bombardment.

Gold particles coated with beta-glucuronidase (GUS) mRNA with a 5' cap structure that had been synthesized in vitro were introduced, by use of a pneumatic particle gun, into pollen grains of lily (Lilium longiflorum), freesia (Freesia refracta) and tulip (Tulipa gesneriana). A fluorometric assay for the GUS activity indicated that in vitro synthesized GUS mRNA introduced into these pollen cells by particle bombardment was successfully expressed. GUS activity in extracts of the bombarded lily pollen became detectable fluorometrically within 30 min after bombardment, peaked at 6 h, then gradually decreased. This activity changed as a function of the developmental stage of the pollen cell of lily.

[A report to 2 cases where Stenotrophomonas maltophilia with a mucoid phenotype was isolated from the sputum].

Mucoid Stenotrophomonas maltophilia was isolated from the sputum of 2 women. Case 1 which we reported recently was primary pneumonia caused by S. maltophilia. However, in Case 2, mucoid S. maltophilia represented part of the transient flora. Interestingly, colonization of mucoid Pseudomonas aeruginosa on respiratory tract occurred in both cases after mucoid S. maltophilia was isolated from their sputum.

Nucleotide sequence of a highly repeated DNA sequence and its chromosomal localization in Allium fistulosum.

A highly repeated DNA sequence with a repeating unit of approximately 380bp was found in EcoRV digests of the total genomic DNA of Allium fistulosum. Three independent clones containing this unit were isolated, and their repeating units sequenced. These units showed more than 94% sequence homology, and the copy number was estimated to be about 2.8×10(6) per haploid genome. In situ hybridization, with the repeating unit as a probe, and C-banding analyses indicated that the repeated DNA sequence of A. fistulosum is closely associated with the major C-heterochromatin in the terminal regions of all 16 chromosomes at mitotic metaphase. The characters of the repeating unit are similar to those of the A. cepa unit, which is taxonomically closely related to A. fistulosum.

In-vitro and in-vivo activity of a new quinolone AM-1155 against Mycoplasma pneumoniae.

We investigated the in-vitro and in-vivo activity of a new quinolone AM-1155 against Mycoplasma pneumoniae, and compared it with ofloxacin, ciprofloxacin, lomefloxacin, tosufloxacin, erythromycin and minocycline. AM-1155 was the most potent agent in vitro of the quinolones tested. Its pre-treatment minimal inhibitory concentrations for 90% of the 41 strains (MIC90) was 0.06 mg/L. In contrast, pre-treatment MIC90 values for ofloxacin, ciprofloxacin, lomefloxacin, tosufloxacin, erythromycin, and minocycline were 1, 1, 2, 0.5, 0.0156, and 0.5 mg/L, respectively. Post-treatments MIC90s, which may reflect mycoplasmacidal potency, of AM-1155, ofloxacin, ciprofloxacin, lomefloxacin, tosufloxacin, erythromycin and minocycline were 0.125, 1, 2, 4, 0.5, 0.125 and 4 mg/L, respectively. In-vitro activities of antimicrobial agents were assessed in an experimental pulmonary infection model in Syrian golden hamsters. AM-1155 was the most effective agent among five antimicrobial agents (AM-1155, ofloxacin, tosufloxacin, erythromycin, minocycline) tested in terms of reduction in viable M. pneumoniae cells and in reducing macroscopic lung lesions. These results suggest that AM-1155 will be a useful antimicrobial agent for the treatment of M. pneumoniae infections.

Pneumonia caused by Stenotrophomonas maltophilia with a mucoid phenotype.

We describe the first known case of pneumonia caused by a mucoid Stenotrophomonas maltophilia (Xanthomonas maltophilia) strain in a patient with bronchiectasis. The patient was admitted because of mild hemoptysis and productive cough with infiltrative shadow in the right lower lung field on chest X ray. The clinical symptoms were mild, and treatment with minocycline was effective.

In vitro and in vivo activities of sparfloxacin against Mycoplasma pneumoniae.

The in vitro and in vivo activities of sparfloxacin against Mycoplasma pneumoniae were compared with those of erythromycin, levofloxacin, ofloxacin, and minocycline. The MICs of sparfloxacin, erythromycin, levofloxacin, ofloxacin, and minocycline for 90% of the 43 M. pneumoniae strains tested were 0.063, 0.016, 0.5, 1, and 0.5 microgram/ml, respectively. In the experimental pulmonary M. pneumoniae infection model in Syrian golden hamsters, sparfloxacin was as effective as erythromycin when orally administered at 15 mg/kg twice daily for 5 days and more effective than erythromycin when orally administered at 10 mg/kg once daily for 5 days. Sparfloxacin was more effective than levofloxacin and ofloxacin in both dosing regimens. The peak concentrations of sparfloxacin in hamster sera after administration of single oral doses of 15 mg/kg were almost the same as those in human sera after administration of single oral doses of 200 mg (the usual clinical dose), and the half-life of sparfloxacin in hamster serum was shorter than that in human serum after administration of a single oral dose of 200 mg. These results suggest that sparfloxacin may be clinically useful for the treatment of M. pneumoniae infections.

In vitro and in vivo activities of macrolides against Mycoplasma pneumoniae.

We investigated the in vitro and in vivo activities of macrolides against Mycoplasma pneumoniae. In vitro MICs of azithromycin, erythromycin, clarithromycin, and roxithromycin were determined. Azithromycin was the most potent antimicrobial agent tested in vitro. Its MIC for 90% of the strains was 0.00024 micrograms/ml. MICs for 90% of the strains of erythromycin, clarithromycin, and roxithromycin were 0.0156, 0.0078, and 0.03125 micrograms/ml, respectively. In vivo activities were assessed in a pulmonary infection model with Syrian golden hamsters. We evaluated the in vivo effects on reduction of viable M. pneumoniae cell counts and on reduction of microscopic and macroscopic histopathologies for azithromycin, erythromycin, and clarithromycin given at 10 mg/kg once daily for 1 and 3 days and given at 15 mg/kg twice daily for 2.5 and 5 days. Azithromycin was significantly more effective than erythromycin or clarithromycin in the same regimens. Especially at 10 mg/kg once daily for 1 day, only azithromycin was significantly effective in the reduction of viable M. pneumoniae cells and histopathologies. These results show that azithromycin is more efficacious than the other drugs tested against M. pneumoniae pneumonia in hamsters. These data suggest that clinical studies of macrolides in human patients are warranted.

Nucleotide sequence of a gene for nitrite reductase from Arabidopsis thaliana.

A nitrite reductase (NiR) gene was recovered from Arabidopsis thaliana genomic library by the homology with a cDNA of spinach NiR and sequenced. Based on the comparison with the spinach cDNA, the Arabidopsis NiR gene was concluded to contain 4 exons [exon 1 of 376 bp (beginning with ATG start codon), exon 2 of 355 bp, exon 3 of 289 bp and exon 4 of 741 bp (ending at TGA stop codon)] and 3 introns (intron 1 of 196 bp, intron 2 of 81 bp and intron 3 of 77 bp). This conclusion was confirmed by the analysis using the RT-PCR method. The deduced amino acid sequence of the coding region of the Arabidopsis NiR gene had high similarities with those of NiR genes of other plants including spinach.

Expression of the [beta]-Glucuronidase Gene in Pollen of Lily (Lilium longiflorum), Tobacco (Nicotiana tabacum), Nicotiana rustica, and Peony (Paeonia lactiflora) by Particle Bombardment.

A [beta]-glucuronidase (GUS) gene that is under the control of the anther-specific LAT52 promoter of tomato (Lycopersicon esculentum) and the nopaline synthetase polyadenylation terminator was successfully expressed in pollen of Lilium longiflorum, Nicotiana tabacum, Nicotiana rustica, and Paeonia lactiflora using a pneumatic particle gun. The GUS gene in plasmid pBI221 was also expressed, to a lesser extent, in pollen of all of these species. The presence of methanol in the substrate solution for histochemical GUS assay and the incubation time in this solution influenced successful detection of GUS expression in bombarded pollen. Cytological analysis of GUS-expressing pollen of lily showed that introduced gold particles were seen in intracellular compartments of pollen, including the vegetative cytoplasm, vegetative nucleus, and generative cytoplasm.

Fluorescent banding pattern analysis of eight taxa of Phaseolus and Vigna in relation to their phylogenetic relationships.

Phylogenetic relationships among eight taxa of seven species of Phaseolus and Vigna (Phaseolus angularis, P. aureus, P. calcaratus, P. coccineus, P. vulgaris, Vigna sesquipedalis and V. sinensis; 2n = 22 each) were studied by the fluorescent chromosome banding technique. Preparations of somatic metaphase chromosomes of each taxon were sequentially stained with Giemsa, GC-specific fluorochrome chromomycin A3 (CMA) and AT-specific fluorochrome 4'-6-diamidino-2-phenylindole (DAPI). On the basis of the fluorescent banding patterns of the 22 chromosomes of each taxon, P. angularis, P. coccineus (from China and Korea) and P. vulgaris were grouped into one group ("Phaseolus group"), P. aureus and two Vigna species were grouped into another ("Vigna group") and P. calcaratus was grouped in an independent group.

[Role of capsular polysaccharide and lipopolysaccharide of Klebsiella pneumoniae in experimental mice pneumonia model].

In this study, role of capsular polysaccharide (CPS) and lipopolysaccharide (LPS) of Klebsiella pneumoniae was investigated in experimental mice pneumonia model. Inoculation with K. pneumoniae mucoid strain DT-S into mice lung induced expansive, voluminous lethal pneumonia characterized with thickening of the alveolar septa caused by infiltration of inflammatory cell and packing of bacteria within alveolar spaces. On the other hand, mice lung inoculated with K. pneumoniae DT-X, which was non-mucoid mutant isolated from DT-S during natural passage, showed infiltration of inflammatory cell into alveolar spaces but there was no death of mice during the course of this pneumonia. Inoculation of CPS 100 micrograms of DT-S strain into mice lung induced lesser extent of accumulation of inflammatory cell than that of LPS 4 micrograms of this strain. Stimulation of alveolar and peritoneal macrophage with CPS, even at a concentration of 100 micrograms/ml, induced weaker Interleukin-1 (IL-1) activity than stimulation with LPS 4 micrograms/ml. These results suggest that since CPS of K. pneumoniae DT-S encapsulate bacteria including LPS, CPS may inhibit chemotaxis of inflammatory cell and IL-1 production of macrophage to be induced by LPS during course of pneumonia. It is speculated that existence of CPS have important role in modulating host response to bacterial LPS, and this effect of CPS may be related with difference of pathological findings of lung and lethality between K. pneumoniae DT-S and DT-X.

Congenital mesenteric arterio-portal fistula: report of a case.

A male patient with an arterio-portal fistula resulting from a mesenteric arteriovenous malformation, who developed portal hypertension and liver cirrhosis, is presented herein. The malformation was considered to be congenital in origin and its location made any ablative surgical procedure impossible. Such alternative treatments as ligation of the afferent arteries, followed by transarterial embolization were therefore given, but both were unsuccessful. We also present a review of the literatures of mesenteric arteriovenous fistula. Radical surgical approach for this rare entity is proposed. The case reported here as related to mesenteric arteriovenous communications of congenital origin is the seventh such case published, and the first which was ever found to be located in the trunk of the superior mesenteric artery.

Alveolar destruction in experimental Klebsiella pneumonia.

Sequential histological changes of the lungs were studied in experimental Klebsiella pneumonia, using untreated control mice, cyclophosphamide-treated mice, and carrageenan-treated mice. Cyclophosphamide was used to deplete polymorphonuclear leukocytes and monocytes, and carrageenan was used to deplete mononuclear phagocytes selectively. At 72 hours, varying degree of alveolar necrosis could be seen in untreated control mice. However, the lung lesions of cyclophosphamide- or carrageenan-treated mice were significantly different from those of the control mice. The lung lesions of cyclophosphamide-treated mice indicated that destruction of the alveolar septa was not induced by K. pneumoniae itself but by inflammatory cells, because the alveolar walls were preserved very well in spite of considerable bacterial multiplication in alveolar lumina until infiltration of inflammatory cells occurred. The lung lesions of carrageenan-treated mice showed that alveolar spaces were packed with polymorphonuclear leukocytes, but the alveolar walls were preserved very well as far as the authors could tell after examining the lung lesions by silver impregnation staining. These results suggest that macrophages rather than polymorphonuclear leukocytes and organisms play an important role in alveolar injury in experimental Klebsiella pneumonia.

Rapid identification of Klebsiella pneumoniae in the lung tissue by fluorescence microscopy.

Klebsiella pneumoniae is stained weekly with hematoxylin and eosin, moderately with periodic acid Schiff (PAS), and strongly by the Warthin-Starry method. Staining by the Warthin-Starry method, however, takes more than one hour. Therefore, we developed a new procedure for rapid identification of K. pneumoniae in paraffin sections of the lung under a fluorescence microscope.