Comparison of the amino acid and nucleotide sequences between human and two guinea pig major basic proteins.

By means of reverse-phase HPLC, 2 different proteins were obtained from apparently purified pig eosinophil major basic protein (MBP) and these proteins were named GMPB1 and GMBP2. It was revealed that these 2 components of MBP have similar molecular weights and pI values, although the amino acid compositions were slightly different. In the previous study, we cloned and sequenced GMPB1 cDNA. Here we obtained another clone by plaque hybridization using a screening probe synthesized by means of polymerase chain reaction. After sequencing, it became apparent that this clone corresponded to GMBP2. As in the case of GMBP1, the cDNA of GMBP2 encoded pre-proGMBP2 with 3 domains; signal peptide, acidic pro-portion, and mature GMBP2. By comparing the sequences of GMBP1 and GMBP2, it was revealed that the proteins were quite similar to each other. In addition, their sequences also resembled those of human MBP, especially in the basic domain of mature protein; but no such similarity existed in the pro-portion. Although the molecular weights determined by SDS-PAGE of guinea pig and human MBPs were 11,000 and 9,300, respectively, the calculated molecular weights of these 3 MBPs were all 13.8 kDa. The calculated pI values of GMBP1, GMBP2 and human MBP were 11.7, 11.3 and 11.6, respectively. By means of Harr plot analysis, it was revealed that the amino acid sequences, not only in signal peptides but also in the basic domains of mature proteins, were well conserved between guinea pig and human MBPs.

Sequencing and cloning of the cDNA of guinea pig eosinophil major basic protein.

Major basic protein (MBP) purified from guinea pig eosinophils elicited histamine release from rat peritoneal mast cells at concentrations higher than 3 micrograms/ml both in the presence and in the absence of extracellular Ca2+. After reverse-phase high-performance liquid chromatography, it was revealed that MBP was composed of two different proteins with quite similar molecular weights and pI values, although the amino acid compositions were slightly different. The partial amino acid sequence of one of these MBPs was determined and the primers for the polymerase chain reaction (PCR) were synthesized according to the partial amino acid sequence. Using these primers and the cDNAs obtained from guinea pig eosinophils, the PCR was carried out in order to synthesize the hybridization probe of MBP for screening the cDNA library. After screening with 8 x 10(5) clones, a positive clone, which encoded a full length of pre-proMBP, was obtained. According to the sequencing data of this clone, it was revealed that pre-proMBP was composed of 3 domains; signal peptide, acidic domain and mature MBP. The predicted pI value of mature MBP was 11.7, though that of proMBP was 7.8. The homology in the amino acid sequence between guinea pig proMBP and human proMBP was 49.4%, while guinea pig mature MBP was more homologous (58%) to human mature MBP.

Novel thyrotropin (TSH)-TSH antibody complex in a healthy woman and her neonates.

A novel high-molecular-weight (MW) form of immunoreactive TSH [35,000 Da on Sephacryl S-200HR gel chromatography (S-200 chromatography)] was documented in a 32-year-old healthy woman who delivered two neonates with transient hyperthyrotropinemia. Her TSH levels ranged from 21.2 to 53.9 mU/L on different days or from 11.0 to 48.1 mU/L by the different immunoradiometric assay methods. The IgG fractions showed specific 125I-labeled hTSH binding and inhibited in vitro cAMP increase induced by hTSH but not that induced by bTSH. On protein G Superose HR affinity chromatography (protein G chromatography) equilibrated with 10 mmol/L sodium/potassium phosphate buffer (PB) followed by elution with 0.1mol/L glycine buffer, 95-99% of her TSH immunoreactivity eluted in the latter (bound) fraction while almost all was in the former (unbound) fraction in the control serum containing authentic hTSH. However, after dialysis of this bound fraction overnight with PB adding 0.5 mol/L NaCl (PB/NaCl), which exhibited greater ionic strength than PB, almost all TSH immunoreactivity changed from the bound fraction into the unbound fraction on the protein G chromatography equilibrated with PB/NaCl. These data indicate that the novel immunoreactive TSH was due to hTSH and hTSH-specific antibody complex, and dissociation of the complex may be incomplete on direct S-200. The immunoreactive TSH showed high MW form (35,000 Da). The dissociation may be almost complete during dialysis with greater ionic strength; the native TSH then appeared to be of formal size.

Serum antibody responses of foals to virulence-associated 15- to 17-kilodalton antigens of Rhodococcus equi.

Humoral immune responses in 16 foals to virulence-associated 15- to 17-kDa antigens of Rhodococcus equi were studied during the first fourteen weeks of life on two horse-breeding farms with a persistent incidence of R. equi infection. Serum antibody levels specific for 15- to 17-kDa antigens were measured by enzyme-linked immunosorbent assay and Western immunoblotting. Immunoglobulin G (IgG) antibodies specific to 15- to 17-kDa antigens were detected by all the foals. R. equi was found in the feces of foals during week 1 of life, and the number of fecal R. equi rapidly increased to the highest level. Virulent R. equi were isolated from the feces of the foals at a high frequency and from their environmental soil on the farms. Evidence that serum antibody response to 15- to 17-kDa antigens of virulent R. equi occurred naturally in every foal in correlation with the quantitative changes of fecal R. equi during the first 1 to 3 months of life suggests that intestinal virulent R. equi might be the most important source of antigenic stimulation in foals from contaminated farms.

Pock-forming plasmids isolated from streptomyces roseochromogenus.

A series of high copy number plasmids designated pSRC were isolated from Streptomyces roseochromogenus S264. The pSRC series were found to be self-transmissible by conjugation and to elicit lethal zygosis (Ltz). Using the Ltz phenotype to detect plasmid transformants, the pSRC plasmids were shown to have a wide host range. Among them pSRC1 consisted of two different plasmids with the same molecular weight, pSRC1a and 1b. Information regarding restriction sites suitable for the insertion of DNA was obtained by cloning the thiostrepton resistance gene from pIJ702 into pSRC1b. The single Bgl II site of pSRC1b was nonessential for replication and pock-formation. The pSRC plasmids may be suitable as cloning vectors in Streptomyces.

Modulation of human basophil growth in vitro by xiao-qing-long-tang (syo-seiryu-to), chai-pu-tang (saiboku-to), qing-fei-tang (seihai-to), baicalein and ketotifen.

Our previous study showed the inhibitory effect of Qing-Fei-Tang (Q.F.T.) and baicalein on the leukotriene (LT)B4 synthesis of human alveolar macrophages. It has recently been demonstrated that LTs support various cell growth, and basophil and its precursor numbers increase in atopic patients. Therefore, we examined the effect of anti-allergic drugs, including Q.F.T., Xiao-Qing-Long-Tang (X.Q.L.T.), Chai-Pu-Tang (C.P.T.), baicalein and ketotifen which have been used for treatment of bronchial asthma, on human basophil growth in vitro using cord blood mononuclear cells as a basophil precursor source and conditioned medium of T cell leukemia cell line Mo as a growth factor. Two-week cultured basophil numbers identified by alcian blue-safranin staining and those histamine contents assayed fluorometrically were inhibited by Q.F.T. (1.0 mg/ml), X.Q.L.T. (0.01-1.0 mg/ml), C.P.T. (0.01-1.0 mg/ml), baicalein (1-100 microM) or ketotifen (1-100 microM) in a dose-dependent manner while low dose (0.01-0.1 mg/ml) of Q.F.T. showed an enhancing effect on the basophil growth and the histamine content. However, LTB4 or LTC4 failed in restoring the basophil growth reduced by 1 mg/ml of C.P.T. or 100 microM of ketotifen. These results suggest that anti-allergic drugs may modulate basophil growth and differentiation in vitro and/or in vivo and therefore be useful and reasonable for controlling allergic diseases including bronchial asthma.

Effects of qing-fei-tang (seihai-to) and baicalein, its main component flavonoid, on lucigenin-dependent chemiluminescence and leukotriene B4 synthesis of human alveolar macrophages.

A traditional Chinese remedy, Qing-Fei-Tang (Seihai-to, T90), has been used for treatment of chronic respiratory diseases with long-lasting cough and sputum, e.g. chronic bronchitis. We examined the effect of T90 and its main component flavonoid, baicalein, on the lucigenin-dependent chemiluminescence (CL) and leukotriene B4 (LTB4) synthesis of human alveolar macrophages (AM). AM were obtained by bronchoalveolar lavage from patients with various respiratory diseases, including sarcoidosis, idiopathic pulmonary fibrosis, bronchial asthma, chronic bronchitis and lung cancer. CL were observed by stimulating 1 x 10(5) AM with phorbol myristate acetate in the presence of lucigenin. LTB4 were generated by incubating 1 x 10(6)/ml AM with Ca ionophore A23187 for 30 min and determined by reverse phase high performance liquid chromatography and radioimmunoassay. T90 (0.2-2.0 mg/ml) and baicalein (0.1-100 microM) inhibited both CL and LTB4 production of AM in a dose-dependent fashion. These inhibitory effects were not due to cytotoxic effects of the procedure because neither 2 mg/ml T90 nor 100 M baicalein affected the viability of AM nor lactate dehydrogenase release from AM. These results suggest that T90 exerts its effect on inflammatory lung diseases through the anti-inflammatory action, i.e. inhibiting the oxidative and arachidonate metabolism of local inflammatory lung cells.

Wegener's granulomatosis with relapsed bleeding of gastric ulcers: a case report.

We treated a 55-year-old male patient with Wegener's granulomatosis (WG) associated with frequent gastric bleeding from multiple ulcerative lesions. Only a few cases of frequent hemorrhaging of peptic ulcers associated with WG have been reported. In our case, a gastric biopsy showed mononuclear cell infiltration in the submucosal area, without granulomas or vasculitis. An endoscopic maneuver, as well as administration of immunosuppressive agents, combined with an H2 receptor antagonist and proton pump inhibitor successfully eliminated the gastrointestinal bleeding. In this case, proof that the gastrointestinal involvement was pathologically related to WG could not be demonstrated because neither granulomas nor vasculitis were observed in the insufficient biopsy specimen of the stomach. It is also possible that the uremic state and cytotoxic agents worsened the gastrointestinal involvement. However, immunosuppressive therapy combined with routine antiulcer treatment was very effective in repairing the ulcerative lesions. The gastrointestinal involvement was considered a possible complication of the WG.

Effects of interleukin 3, interleukin 4, and fibroblasts on cultures of human lung mast cells in bronchoalveolar lavage fluids.

The effects of T cell factors, including interleukin (IL)-3 and IL-4, and fibroblasts on the growth and differentiation of human lung mast cells (MCs) obtained by bronchoalveolar lavage (BAL) were examined. The number of MCs identified by alcian blue-safranin staining was twice that of the control culture without conditioned medium (CM) when BAL cells were cultured for 2 weeks in RPMI 1640 containing 10% fetal calf serum and partially purified CM derived from PHA-stimulated lymphocytes. In the presence of both recombinant (r) IL-3 and rIL-4, the number of MCs was twice as high as the control without increase in the per-cell histamine content after 2 weeks' culture. In umbilical cord blood cultures, IL-3 plus IL-4 augmented basophilic cells about 20-fold more than the control when cultured for 2 weeks. In some cases, the percentage of safranin-positive MCs was about 2-5 fold greater, with 2-7 fold higher histamine content, when cultured for 10 days with CM and fibroblasts derived from human embryonic lung. However, in all BAL experiments, there was no increase in the total number of MCs after culture compared with the initial number of MCs, unlike the umbilical cord blood cultures. These results suggest that T cell factors, including IL-3 and IL-4, and fibroblasts may influence the phenotype and the survival of lung mast cells in BAL, whereas there was no evidence for the presence of MC precursors in BAL fluids.

Effect of antibiotics on immediate hypersensitivity reactions in vitro: suppression of IgE-mediated histamine release from peripheral blood basophils by fosfomycin.

The effect of antibiotics on allergic reactions was studied in vitro using the release of histamine from human peripheral blood leukocytes (basophils) after incubation with anti-IgE. For the several antibiotics we tested, including beta-lactams and aminoglycosides, none had the capacity to enhance antigen-induced histamine release, but some of them (minocycline, polymyxin B, and fosfomycin) suppressed the release of histamine in a dose-dependent manner. Since fosfomycin has proved to be capable of suppressing IgE-mediated histamine release non-cytotoxically, the effect of fosfomycin on histamine release induced by other secretagogues was further studied. The suppression of histamine release was also demonstrated when the leukocytes, preincubated with fosfomycin, were challenged with either Ca ionophore A 23187 or a synthetic peptide, formyl-methionyl-leucyl-phenylalanine (FMLP). We concluded that some antibiotics, particularly fosfomycin, have the capacity to suppress histamine release mediated by various secretagogues, suggesting they may possess an anti-allergic property as well as a bactericidal activity.

Identification of the minimal replication region of the multicopy Streptomyces plasmid pSL1.

The 1.52-kb minimal replication origin of the 3.9-kb Streptomyces plasmid pSL1 was determined using a bifunctional derivative, pMCP44, of pSL1. Plasmids with linker insertions into the pSL1 part of pMCP44 were isolated from Escherichia coli. The sites of insertion were determined by restriction enzyme analysis and the ability of the mutant plasmids to replicate in S. lividans 66 was determined. All except one of the inserts in the 1.52-kb essential region inactivated replication. A 104-bp segment from this region could function as a replication origin in the presence of a helper plasmid containing a nonoverlapping pSL1 fragment. The sequence of this 104-bp fragment shows similarities to those of known plasmid replication origins.

Characterization of the incompatibility region of Streptomyces plasmid pSL1.

The incompatibility region of the Streptomyces plasmid pSL1 was identified as a 240-bp segment, though some other function from the vector plasmid was also necessary. A 540-bp fragment including the 240-bp region was enough for full expression of incompatibility. Inserted mutation analysis led to a more detailed location of the region essential for replication.

Morphological characterization of basophilic cells in bronchoalveolar lavage fluids from patients with bronchial asthma and idiopathic pulmonary fibrosis.

A marked increase in the number of basophilic cells (BCs) was found in the bronchoalveolar lavage (BAL) fluids from the patients with bronchial asthma and idiopathic pulmonary fibrosis (IPF). Histochemical analysis revealed that basophils were the major components of BCs in asthmatic patients, while formalin-insensitive BCs, which are presumed to be connective tissue mast cells, were observed in BAL fluids from IPF patients. In control subjects, almost all of BCs were mucosal mast cells.

Interleukin-12 prevents diaphragm muscle deterioration in a septic animal model.

The effects of an intravenous injection of Interleukin-12 (IL-12) after endotoxin administration and without endotoxin administration on diaphragm muscle were studied using Wistar rats. Three treatment groups, namely a control (Saline+endotoxin) group, an IL-12+endotoxin group and an IL-12 only group were studied. E. coli endotoxin (30 mg/kg) was injected intraperitoneally 5 min after Saline or IL-12 (0.25 microg) injection. In the control group, the force-frequency curves, twitch tension (TT) and slope during contraction time (TT/CT) were significantly lower at 4 h than those at 0 h due to endotoxin (P<0.001, P<0.01 and P<0.01, respectively), and NO production was increased at 4 h as shown by NADPH diaphorase staining. In the IL-12+endotoxin group, the decrement of the force-frequency curves, TT and TT/CT induced by endotoxin at 4 h were significantly prevented compared with those of the control group (P<0.001, P<0.05 and P<0.05, respectively), and NO production was blocked at 4 h. In the IL-12 only group, the force-frequency curves were decreased in the range of high frequency and IL-12 resulted in NO production. Furthermore, the positive muscle fibers detected by NADPH diaphorase staining were classified as type I and IIa muscle fibers by ATPase staining in the control and IL-12 only groups. It is concluded that IL-12 prevents the deterioration of diaphragm muscle contraction induced by endotoxin by reducing NO production in type I and IIa muscle fibers. These results suggest that IL-12 and endotoxin may interfere with each other.

[Chronic eosinophilic pneumonia associated with high concentrations of interleukin-5, -6, and granulocyte colony stimulating factor in serum and in pleural fluid].

A 44-year-old Japanese man who had suffered from bronchial asthma since childhood was given the diagnosis of chronic eosinophilic pneumonia because of his symptoms, chest roentgenographic findings, and the results of a transbronchial lung biopsy. At the time of the onset of the disease, the pleural effusion contained 73% eosinophils. Symptoms were relieved and the laboratory findings returned forward normal after a short course of high-dose corticosteroids. The concentrations of IL-5, IL-6, and G-CSF in pleural fluid and in serum were very high; the concentrations of these cytokines were 3 times to 35 times higher in pleural fluid than in serum. In contrast, no IL-3 or GM-CSF was detected in any of these samples. The precise etiology of chronic eosinophilic pneumonia is still unclear, but this case suggests that inappropriate production of IL-5, IL-6 and G-CSF in the lung play a pivotal role in this disease. Inhibition of the production of these cytokines may be another therapeutic approach to this disease.

Mast cells in bronchoalveolar lumen of patients with bronchial asthma.

Bronchoalveolar lavage was performed on patients with stable bronchial asthma to elucidate the existence and significance of mast cells in human bronchoalveolar lumen. The percentage of total mast cell population, in which 2 different types of mast cells could be identified morphologically in lavage fluids, was 0.25 +/- 0.19% (mean +/- SD) in asthmatic patients and 0.08 +/- 0.02% in control subjects. The number of mast cells in asthmatic patients was greater than in control subjects (p less than 0.05). The histamine content per mast cell in bronchoalveolar lumen was calculated to be 8.2 +/- 6.0 pg/cell in asthmatic patients and 15.3 +/- 10.8 pg/cell in control subjects, which suggests that the " releasability " of histamine is greater in asthmatic patients during asymptomatic periods than it is in control subjects, although the differences were not statistically significant. Our results suggest that an increase in the mast cell population of bronchoalveolar lumen is significant in the pathogenesis of bronchial asthma.

Concentrically arranged endoplasmic reticulum containing some lamellae (bar-like structure) in alveolar type II cells of rat lung.

We examined in vivo the effect of pilocarpine (a cholinergic agent) and cycloheximide (an inhibitor of protein synthesis) on the "bar-like structures" in alveolar type II cells of rat lung to clarify their origin and significance in pulmonary surfactant production and secretion. Lungs were examined with an electron microscope using ultrathin sectioning, freeze-fracture technique, and morphometry. The bar-like structures in type II cells consisted of a concentrically arranged endoplasmic reticulum containing some amount of osmiophilic periodic material similar to the lamellae of lamellar bodies. Pilocarpine induced the accumulation of lamellar bodies of normal size which paralleled the increase in the number of bar-like structures in the cytoplasm of the type II cells. Cycloheximide induced a decrease in size of the lamellar bodies and an enlargement of the bar-like structures. Our morphological findings suggest that: The phospholipid that would normally be incorporated into the lamellar bodies might be sequestered instead in the concentrically arranged endoplasmic reticulum, forming the bar-like structures, and The enlargement and the increased number of bar-like structures may be responsible in part for the changed metabolic process of surfactant production by alveolar type II cells.

[A case of Goodpasture's syndrome with myeloperoxidase specific anti-neutrophil cytoplasmic autoantibody (MPO-ANCA) during chronic interstitial pneumonia].

A 55-year-old man was admitted to our hospital with of hemoptysis, progression of anemia and renal failure in February, 1996. Idiopathic interstitial pneumonia had been diagnosed and he had been followed at a regional hospital since 1988. On the third day after admission, he suffered from sudden and massive hemoptysis. Goodpasture's syndrome was diagnosed because anti-GBM antibody was detected in serum. A high titer of MPO-ANCA was also recognized simultaneously. Steroid pulse therapy, immunosuppressive therapy, and plasmapheresis were begun, but he died on the 28th hospital day because of severe hypoxemia and multi-organ failure. Histological examination after autopsy revealed crescentic glomerulonephritis with linear deposition of IgG in the glomerular capillary wall, and interstitial pneumonia accompanied by massive alveolar hemorrhage. It was suggested that in this patient, not only anti-GBM antibody but also circulating MPO-ANCA might have participated in the progression of the crescentic glomerulonephritis and alveolar hemorrhage observed in Goodpasture's syndrome.

Cellular analysis in bronchoalveolar lavage fluids in infiltrative and fibrotic stages of idiopathic pulmonary fibrosis.

We performed cellular analysis of bronchoalveolar lavage fluids (BALF) from 20 patients in different stages of idiopathic pulmonary fibrosis (IPF) and compared the results with those from 16 controls and 10 sarcoidosis patients. Patients with IPF were divided into infiltrative and fibrotic groups as diagnosed by transbronchial lung biopsy and chest x-rays. Total cell numbers counted from BALF in IPF patients were not different from those from controls and were lower than in sarcoidosis patients (p less than 0.01). Lymphocytes in BALF were significantly higher in the infiltrative type IPF than in the fibrotic type (p less than 0.05), but there was no significant difference in fibrotic type IPF and control subjects. Numbers of neutrophils and eosinophils counts were not significantly different between infiltrative type and fibrotic type. The neutrophil count in fibrotic type IPF was higher than in control subjects (p less than 0.01) but not in infiltrative type IPF. 67Ga scintiscanning uptake correlated with the lymphocyte population (r = 0.65, p less than 0.01) but with neutrophils. These findings suggest that lymphocytes play a major role in the pathogenesis of the infiltrative type of IPF and that neutrophils are related to the development of fibrotic changes in IPF.