Appearance of B220low autoantibody-producing B-1 cells at neonatal and older stages in mice.

In this study, normal adult mice carried B220(high) conventional B cells in the spleen and liver, but carried both B220(high) and B220(low) in the bone marrow. However, at the neonatal stage, only B220(low) unconventional B cells were found in all these organs. This pattern continued up to 2 weeks after birth, and at this stage autoantibodies were detected in the sera. This phenomenon was seen in all tested young mice (1-2 weeks), irrespective of their gender. Furthermore, at older stages (more than 20 weeks), B220(low) cells reappeared in the spleen and liver, and these B220(low) cells became dominant in the bone marrow. Autoantibodies also reappeared in the sera of these older mice. Cell-sorting experiments revealed that B220(low) cells were able to produce autoantibodies upon lipopolysaccharide stimuli in vitro. These results suggest that B220(low) cells appear at both neonatal and older stages as physiological responses and eventually produce autoantibodies.

Usefulness of sparfloxacin against Chlamydia pneumoniae infection in patients with bronchial asthma.

The efficacy of sparfloxacin (SPFX) for the control of bronchial asthma was evaluated in 26 patients with suspected Chlamydia pneumoniae infection. Patients were randomly allocated to receive SPFX 200 mg/day (n = 14) or control treatment (n = 12) for 21 days. Significant improvements in serum C-reactive protein levels, and significant decreases in peripheral eosinophil counts, serum eosinophil cationic protein (ECP) and sputum ECP were observed in the SPFX-treated group at day 21. SPFX-treated patients also had a significantly reduced frequency of asthma symptoms, reduced inhalant beta2-stimulant use, and significant increases in morning peak expiratory flow. At the end of the study, C. pneumoniae was undetectable in two SPFX-treated patients who underwent polymerase chain reaction testing, but one control patient who was tested still had detectable levels of C. pneumoniae. These results suggest that SPFX could be used to control bronchial asthma in patients with suspected persistent C. pneumoniae infection.

Effects of suplatast tosilate on airway inflammation and airway hyperresponsiveness.

Suplatast tosilate (IPD) is a Th2 cytokine inhibitor that lowers the titer of the IgE antibody through specific inhibition of the production of IL (interleukin)-4 and IL-5 by T cells and inhibits tissue infiltration by eosinophils. In this clinical trial, suplatast tosilate (300 mg/day) was administered orally for 4 weeks to 25 patients (13 patients with atopic asthma, 12 patients with nonatopic asthma) whose bronchial asthma was staged in step 1 or step 2 according to the Guidelines for Prevention and Management of Bronchial Asthma, 1998. Before and after administration, the parameters of airway inflammation, that is, peripheral blood eosinophils count, serum level of eosinophil cationic protein (ECP), ECP level in induced sputum, airway hyperresponsiveness (Dmin), and morning peak expiratory flow (PEF), were measured. The peripheral blood eosinophil count, serum level of ECP, and ECP level in induced sputum decreased significantly. Of these parameters, the ECP level in induced sputum was the most sensitive. Furthermore, suplatast tosilate significantly inhibited Dmin. These results were especially significant in patients with atopic asthma. Suplatast tosilate was considered to have inhibited airway eosinophilic inflammation through decreases in peripheral blood eosinophils counts and in ECP levels in induced sputum, which resulted in inhibition of airway hyperresponsiveness.

[Clinical utility and safety of diagnostic thoracoscopy].

The increasing use of thoracoscopy performed under local anesthesia has made contributions to the diagnosis of pleural disease with effusion. During the past 7 years, we have performed 100 such thoracoscopy procedures using a flexible fiberoptic bronchoscope. On the basis of our clinical findings, we are able to discuss the utility and safety of this procedure. The causes of pleural effusion were carcinomatous pleurisy in 72 cases, tuberculosis pleurisy in 15 cases, infection without tuberculosis in 4 cases, malignant pleural mesothelioma in 8 cases and one case of asbestosis. The success rate of thoracoscopic pleural biopsies were 97% for carcinomatous pleurisy, 100% for malignant pleural mesothelioma and 86% for tuberculosis pleurisy. This procedure was performed with no serious effect on blood pressure, oxygen saturation, monitored ECG or BGA data, and with no serious complications. Therefore, we concluded that this method is very useful for the diagnosis of pleural effusions and has few complications.

[Clinical significance of serum KL-6 in pulmonary sarcoidosis].

We investigated whether the level of serum KL-6 could be an activity marker for pulmonary sarcoidosis. In 33 patients with pulmonary sarcoidosis, the relationships between serum KL-6 levels and diagnostic imaging, serum angiotensin-converting enzyme (ACE) levels, serum lysozyme levels, steroid therapy, and prognosis were evaluated. There were no significant differences in the level of serum KL-6 when the patients were divided on the basis of radiographic findings, but the level of serum KL-6 was markedly elevated in some patients with stage-II pulmonary sarcoidosis. There was a significant correlation between serum KL-6 levels and the following two parameters: serum ACE and lysozyme levels. Among patients with a high initial level of serum KL-6, pulmonary sarcoidosis tended to become exacerbated within one year. Steroid therapy significantly decreased the level of serum KL-6, suggesting that the level of serum KL-6 could be an activity indicator for pulmonary sarcoidosis. Immunohistochemical staining by anti-KL-6 antibody revealed that KL-6 was localized in proliferating type-II alveolar epithelial cells.

Usefulness of serum carboxy-terminal telopeptide of type I collagen (ICTP) as a marker of bone metastasis from lung cancer.

BACKGROUND: Serum pyridinoline cross-linked carboxy-terminal telopeptide of type I collagen (ICTP) is a metabolite of type I collagen comprising 90% or more of organic substances in bone. Its usefulness as a marker of bone metastasis from malignant tumors is expected. METHOD: We measured ICTP to evaluate its clinical usefulness for diagnosis of bone metastasis in 140 patients with lung cancer. For comparison, serum carcinoembryonic antigen (CEA), cytokeratin 19 fragment (CYFRA 21-1), gastrin-releasing peptide precursor (ProGRP), alkaline phosphatase and calcium were simultaneously measured. ICTP was measured by double-antibody radioimmunoassay. RESULTS: ICTP was significantly higher in patients with bone metastasis from lung cancer than in the group without bone metastasis, patients with other pulmonary diseases or healthy control subjects and showed excellent sensitivity and specificity, indicating that this marker is highly useful for complementary diagnosis of bone metastasis from lung cancer. Moreover, the survival duration was significantly shorter in the ICTP-positive group than in the ICTP-negative group, suggesting that ICTP can be a prognostic factor in lung cancer. CONCLUSION: It is suggested that measurement of ICTP is worthwhile as a serological diagnostic method of bone metastasis from lung cancer. Moreover, since repeated measurements are possible, this measure was considered very helpful in complementary diagnosis of bone metastasis and also as a standard to determine the timing of examinations such as bone scintigraphy.

Developmental competence of frozen-thawed blastocysts from fair-quality bovine embryos cultured with beta-mercaptoethanol.

Two-hundred-and-thirty-one fair-quality embryos at the compacted morula stage collected from 89 superovulated cows were cultured in TCM199 or Brinster's BMOC-3 medium with or without 100 microM beta-mercaptoethanol (beta-ME). After 24 h culture, a total of 142 fair-quality embryos developed to the blastocyst stage, of which 106 were subsequently frozen with 1.8 M ethylene glycol. The mean cell number and development rates of frozen-thawed blastocysts from the fair-quality embryos cultured in TCM199 containing beta-ME were higher than those of the fair-quality embryos directly frozen without culture. The pregnancy rates obtained with frozen blastocysts from fair-quality embryos tended to be lower than those of non-cultured fresh fair-quality embryos and cultured fresh blastocysts. These results indicate that the inclusion of beta-ME in pre-freezing culture media improve the development of frozen-thawed blastocysts from fair-quality embryos, but not the pregnancy rate.

Clinical studies on bronchial asthma caused by contact with hamsters.

Bronchial asthma induced by contact with hamsters and other small rodents is receiving higher attention from the medical profession not only because of the problem of laboratory animal allergies (LAA), but also because of increasing household allergens for asthma, since keeping these pets has become more common in Japanese homes. The present report describes our studies on the backgrounds of nine patients with asthma who kept Dzungarian Dwarf hamsters as household pets. The following features were recognized among patients with bronchial asthma induced by contact with hamsters: 1) earlier onset of symptoms than for keeping other household pets, at an average of 14.7 months or within 12 months in 78% of the cases following the start of pet keeping; 2) adults ranging from their late 30s to 40s who have children of primary school age; 3) dwelling in apartments; 4) relatively high level of serum IgE and ECP; 5) positive for both immediate and late type asthmatic responses on an inhaling induction test; and 6) rapid remission after the cessation of pet keeping.

Relationship between dead cells and DNA fragmentation in bovine embryos produced in vitro and stored at 4 degrees C.

DNA fragmentation and its relationship with dead cells were examined in bovine blastocysts produced in vitro and stored at 4 degrees C for 1-5 days. Survival and development to the hatching and hatched blastocyst stage decreased with increasing storage time. Both were significantly lower at 72 hr than at 48 hr. None of the embryos stored for 120 hr developed to the hatching or hatched blastocyst stage. The proportion of dead cells per embryo increased progressively as the time of storage increased, until 69% of embryonic cells were dead after 120 hr of storage. There was no significant difference between the proportions of DNA fragmentation per embryo stored for 0 and 24 hr (12% vs 16%). However, the proportion of DNA fragmentation in embryos stored for longer than 48 hr was significantly greater than that in embryos stored for less than 24 hr. There were no significant differences among those stored for longer than 48 hr (28-33%). These results suggest that the reduced developmental competence of bovine embryos stored at 4 degrees C is characterized by necrotic change rather than apoptotic change.

Fine structure of bovine morulae and blastocysts in vivo and in vitro.

The ultrastructure of bovine morulae and blastocysts developed from in vitro-matured and -fertilized oocytes in a serum-supplemented medium was compared with that of morulae and blastocysts collected non-surgically from superovulated cows. In the in vivo-derived morulae, two characteristic cells types could be identified by the electron-density of their cytoplasm and by their ultrastructural features. One type appeared light in color with low electron-dense cytoplasm. These cells were located in the peripheral layer of the cluster of blastomeres, possessed numerous cellular organelles such as mitochondria and Golgi apparatus and had microvilli projecting into the perivitelline space. The other cell type was distinguished by cytoplasm that stained more densely than that of the lighter-appearing cells. The darker-appearing cells generally possessed fewer organelles than the lighter cells, but many lysosome-like structures were present in the cytoplasm. The in vitro-developed morulae also contained two types of cells similar to those observed in the in vivo morulae. However, most of the in vitro-developed cells possessed numerous lipid droplets and contained fewer lysosome-like structures than the cells of the in vivo-derived morulae. The blastocysts, both in vivo and in vitro, showed a clear differentiation of trophoblast cells and inner cell mass (ICM)-cells. In the in vivo-derived blastocyst, the apical membrane of trophoblast cells was covered with large, numerous microvilli and well-developed junctional complexes were observed. Lipid droplets were present in the cytoplasm of trophoblast and ICM-cells but were not abundant. In vitro-developed blastocysts showed less well-developed junctional complexes between trophoblast cells, less well-developed apical microvilli on the trophoblast cells, and contained large numbers of lipid droplets. This accumulation of lipid droplets was higher in the trophoblast cells than in the ICM-cells. The zonae pellucidae of in vitro-developed embryos were thinner than that of the in vivo-derived embryos. This study demonstrates conspicuous differences in the ultrastructural features between the in vivo-derived and in vitro-developed embryos, suggesting that the ultrastructure may reflect the various physiological anomalies observed in previous studies.

Suppression of airway inflammation by theophylline in adult bronchial asthma.

BACKGROUND: Chronic continuous airway inflammation caused by eosinophils has been noted to play critical roles in the pathophysiology of bronchial asthma, in addition to reversible obstruction and hypersensitivity of the respiratory tract. Therefore, suppression of chronic airway inflammation has become more important in asthma treatment. Although theophylline has been a conventionally used bronchodilator, it has been recently reported to have concurrent anti-inflammatory effects. OBJECTIVE: Accordingly, we studied the effects of a slow-release theophylline preparation, Theolong, on airway inflammation. METHODS: Administration of Theolong 400 mg/day to 24 patients with mild or moderate asthma and measuring eosinophil cationic protein (ECP), a marker of airway inflammation, and eosinophils in sputum and peripheral blood at 4 and 8 weeks. RESULTS: As a result, sputum ECP, serum ECP and sputum eosinophil count (%) were significantly lowered after 4 and 8 weeks. CONCLUSION: Thus, in the theophylline-administered group, slow-release theophylline, Theolong, was effective in treating asthma, with anti-inflammatory effects on inflammatory cells besides its bronchodilator action.

Effects of pranlukast hydrate on serum eosinophil cationic protein levels in patients with adult bronchial asthma.

Pranlukast hydrate (CAS 103177-37-3, ONO-1078) was administered to 20 patients with bronchial asthma, and examined the effects of this agent on serum eosinophil cationic protein (ECP) levels and peripheral blood eosinophil counts. The mean serum ECP level in the group comprised of patients with bronchial asthma was significantly higher than that in the control group comprised of healthy adults. In responders in whom improvement in asthmatic symptoms was observed, serum ECP levels were significantly decreased compared to the pretreatment values 4 weeks after commencement of pranlukast hydrate administration. However, there were no changes in non-responders. There was no correlation between serum ECP level and peripheral blood eosinophil count before administration of pranlukast hydrate. However, there was a correlation 4 weeks after administration was started. It is suggested that pranlukast hydrate inhibits eosinophilic activity in patients with bronchial asthma.

Development to live young from bovine small oocytes after growth, maturation and fertilization in vitro.

Bovine oocytes (90 to 99 microns in diameter) were isolated from early antral follicles (0.5 to 0.7 mm in diameter). Cumulus-oocyte complexes (COC) with pieces of parietal granulosa were embedded in collagen gels and cultured for 14 d. After in vitro growth culture, oocytes recovered from the collagen gels were further matured, fertilized and cultured in vitro, and then were transferred to recipient cows. After 14 d of growth culture, 37% of the oocytes (203/556) showed normal morphology in the collagen gels. The mean diameter of the oocytes was 110.1 +/- 6.0 microns, significantly larger (P < 0.01) than before growth culture (94.8 +/- 2.7 microns), and 77% were at the germinal vesicle stage while 23% had undergone germinal vesicle breakdown. After 24 h of maturation culture followed by insemination, 27% of in vitro-grown oocytes reached the second metaphase, and 42% of the oocytes were normally fertilized. After insemination, 18.2% of in vitro-grown oocytes cleaved and 3.7% developed to the blastocyst stage. Three blastocysts obtained from in vitro-produced 90- to 99-micron oocytes were transferred to 3 recipients. One recipient subsequently became pregnant and delivered a live calf on Day 277. These results demonstrated for the first time that 90 to 99-micron oocytes from early antral follicles can complete growth and acquire full developmental competence in vitro so that live young can be produced after maturation, fertilization, subsequent culture in vitro, and transfer to recipient cows.

Superovulatory responses in Japanese black beef cows following largest follicle aspiration or human chorionic gonadotrophin (hCG) treatment.

The effect of largest follicle aspiration or hCG administration before induction of superovulation on the ovarian response of Japanese Black beef cows was investigated using a crossover design in which induction of superovulation was attempted in every cow. The superovulatory response of cows whose largest follicle had been aspirated from the ovaries by ultrasound-guided follicular aspiration 1 day before induction of superovulation, did not differ from the response in non-treated control cows. In contrast, in cows given 5,000 IU of hCG 3 days before induction of superovulation, the proportions of fertilized ova and transferable embryos significantly decreased compared with the other groups.

Cryopreservation of mature bovine oocytes by vitrification in straws.

Experiments were conducted to determine optimal conditions for vitrification of in vitro matured bovine oocytes in straws. In the first series of experiments, the effects of stepwise addition before exposure of oocytes to vitrification solution consisting of 30% (v/v) ethylene glycol (EG) with 0.35 M sucrose was tested. The rates of morphological survival and cleavage of oocytes vitrified by the three-step addition procedure were higher than those vitrified by the one-step addition procedure. In the second series, the effect of the concentration of the vitrification solution was tested (20, 30, 40, and 50% EG, all with 0.35 M sucrose). The survival rates of oocytes vitrified in 20 and 30% EG were lower than those in 40 and 50% EG. The rates of cleavage and development to blastocysts of oocytes vitrified in 40% EG were the highest among the four groups. In the third series, the effect of duration of exposure of oocytes to 0.5 M sucrose (1, 5, and 10 min) at the first step during the three-step dilution was tested. Although there were no significant differences among the groups with respect to developmental competence of oocytes vitrified in 40% EG, the highest development rate (10%) to blastocysts was observed when oocytes were exposed to sucrose for 5 min. These data demonstrate that improvements to the concentration of cryoprotectant and addition procedures have critical effects on the developmental competence of oocytes vitrified in straws.

Clinical efficacy of sparfloxacin in mycoplasmal pneumonia: a pilot study.

Sparfloxacin, a new synthetic quinolone antimicrobial agent, was administered to 20 patients with mycoplasmal pneumonia to evaluate its clinical efficacy and adverse effects. The efficacy rate was 100%. In all cases, the subjective symptoms, chest x-ray findings and inflammatory response improved within 5 days after sparfloxacin administration. There were no adverse events including abnormal laboratory values in any of the patients during the treatment period, and no adverse effects that were thought to be due to sparfloxacin were encountered. Since sparfloxacin displays an excellent activity against Mycoplasma pneumoniae and has a broad spectrum of antimicrobial activity against not only Gram-negative bacteria but also Gram-positive bacteria, Legionella, Chlamydia and acid-fast bacteria, it appears to be useful as the drug of first choice in the treatment of respiratory tract infections.

Cryopreservation of mature bovine oocytes following centrifugation treatment.

In vitro matured bovine oocytes were frozen slowly in 1.6 M 1,2-propanediol following centrifugation treatment for polarization of lipid droplets in the cytoplasm. After thawing, the survival of the oocytes was assessed morphologically and also by in vitro fertilization and culture. The polarization of cytoplasmic lipid droplets had a negative effect on the survival of frozen-thawed oocytes. Thus, this treatment did not improve the frequency of normal fertilization and development to blastocysts, compared with that of frozen control oocytes. However, the frequency of polyspermy and activation of lipid-polarized oocytes that survived after freezing-thawing and subsequent in vitro fertilization tended to be less than those of surviving control oocytes. In addition, the effect of centrifugation treatment was to produce a small but significant increase in the cleavage rate of oocytes that survived after freezing-thawing and the development rates to blastocysts of surviving lipid-polarized oocytes tended to increase, compared with those of surviving control oocytes. These results suggest that the freezing tolerance of the spindle and other organelles of in vitro matured bovine oocytes is associated with lipid droplets and may be improved by the polarization of cytoplasmic lipid droplets before cryopreservation.

Mucosa-associated lymphoid tissue lymphoma coexisting with epithelioid granulomas in the stomach of a patient with systemic sarcoidosis.

Malignant lymphoma arising in the stomach of a 23-year-old Japanese man with systemic sarcoidosis is presented. The patient was followed because of systemic sarcoidosis involving the lungs, eyes, and lymph nodes. Biopsy specimens from the stomach were repeated because of recurrent epigastralgia and multiple ulcerations. Some of the specimens revealed epithelioid granulomas with no caseous necrosis, which confirmed gastric involvement of sarcoidosis. Three years after the initial diagnosis, biopsy specimens taken from the stomach were diagnosed as malignant lymphoma of the large cell type. The resected stomach revealed multicentric mucosa-associated type malignant lymphoma of low-grade B cell type, with foci of high-grade transformation coexisting with numerous epithelioid granulomas with no caseous necrosis. Epithelioid granulomas were observed in all the respected lymph nodes, liver, and appendix, which had been obtained at operation, whereas malignant lymphoma was limited to the stomach. Helicobacter pylori (H. pylori) infection was also observed in the stomach. Consequently, the present report is a rare case of coexistence of malignant lymphoma and involvement of sarcoidosis in the stomach. Both H. pylori infection and active sarcoid nodules may play a role in the development of malignant lymphoma, although the exact mechanism remains unclear.

Bovine oocyte diameter in relation to developmental competence.

This study was conducted to determine the diameter of bovine oocytes that were able to attain their full developmental competence to blastocysts. Oocytes were recovered by aspiration of surface-visible follicles (1 to 7 mm in diameter) from slaughterhouse ovaries. Only healthy-looking cumulus-oocyte complexes were used for in vitro maturation, and they were divided into six groups based on diameter: < 110 microm, 110 to < 115 microm, 115 to < 120 microm, 120 to < 125 microm, 125 to < 130 microm and >/= 130 microm. Oocytes were processed through standard procedures for in vitro maturation, fertilization and culture. Following in vitro maturation or fertilization, some oocytes were stained to assess nuclear maturation and penetration rates. The numbers of embryos that cleaved at 42 h post insemination and developed to blastocysts and hatched blastocysts after 8 days of culture were recorded. The mean oocyte diameters were 114.0 +/- 4.8 microm. The oocytes displayed size-related ability to undergo meiotic maturation. The rates of nuclear maturation of oocytes in the greater than 115-microm size range were significantly higher than those of oocytes with diameters < 115 microm. In the < 120 microm diameter groups, the polyspermic fertilization rates of oocytes < 115 microm were significantly higher than those of oocytes 115 to < 120 microm in diameter. The rates of cleavage and development to blastocysts and hatched blastocysts rose as oocyte diameter increased. Among oocytes with a diameter >110 microm, oocytes < 120 microm were found to have significantly lower developmental competence than oocytes 120 to < 130 microm in diameter. These results suggest that bovine oocytes have acquired full meiotic competence at a diameter of 115 microm but not yet attained full developmental competence to blastocysts, and that oocytes have acquired full developmental competence at a diameter of 120 microm.