Surfactant protein C G100S mutation causes familial pulmonary fibrosis in Japanese kindred.

Several mutations in the surfactant protein C (SP-C) gene (SFTPC) have been reported as causing familial pulmonary fibrosis (FPF). However, the genetic background and clinical features of FPF are still not fully understood. We identified one Japanese kindred, in which at least six individuals over three generations were diagnosed with pulmonary fibrosis. We examined the patients radiologically and histopathologically and sequenced their SFTPC and ABCA3 genes. We also established a cell line stably expressing the mutant gene. All the patients had similar radiological and histopathological characteristics. Their histopathological pattern was that of usual interstitial pneumonia, showing numerous fibroblastic foci even in areas without abnormal radiological findings on chest high-resolution computed tomography. No child had respiratory symptoms in the kindred. Sequencing of SFTPC showed a novel heterozygous mutation, c.298G>A (G100S), in the BRICHOS domain of proSP-C, which co-segregated with the disease. However, in the ABCA3 gene, no mutation was found. In vitro expression of the mutant gene revealed that several endoplasmic reticulum stress-related proteins were strongly expressed. The mutation increases endoplasmic reticulum stress and induces apoptotic cell death compared with wild-type SP-C in alveolar type II cells, supporting the significance of this mutation in the pathogenesis of pulmonary fibrosis.

Th1-Th2 cytokine kinetics in the bronchoalveolar lavage fluid of mice infected with Cryptococcus neoformans of different virulences.

Th1 immune response plays an important role in protection against infection with Cryptococcus neoformans in mice. We investigated the effect of virulence of C. neoformans on cytokine production in the lung of a mouse model of pulmonary cryptococcosis. BALB/c mice were inoculated intratracheally with a high or low virulence strain of C. neoformans, followed by serial measurements of Th1 and Th2 cytokine concentrations in the bronchoalveolar lavage (BAL) fluid using appropriate enzyme-linked immunosorbent assay kits. The number of colony-forming units (CFU) increased with time, and all mice infected with the highly virulent strain were dead at 28 days after inoculation. In contrast, the number of microorganisms diminished with time in the mice infected with the low virulence strain during the 4-week study. The numbers of neutrophils and lymphocytes in the BAL fluid paralleled those of CFU. High neutrophil counts were observed in the BAL fluid of mice infected with the highly virulent strain, while lymphocyte counts were increased only in the later part of the study in mice infected with the high and low virulence strains. The concentrations of Th2 cytokine, interleukin (IL)-4 were significantly higher in mice infected with the highly virulent strain at days 14 and 21 of infection, whereas the level of Th1 cytokine, interferon-gamma, was significantly higher in the latter strain at days 7 and 14. Our results suggest that strain-specific difference in the organism's ability to induce (or evade) the host immune system contributes to the outcome of infection.

[Evaluation of CYFRA 21-1 and ProGRP in serum and bronchoalveolar lavage fluid of patients with benign lung disease].

CYFRA 21-1 and ProGRP have recently been established as new tumor markers for lung cancer. However, there are few reports evaluating concentrations in their bronchoalveolar lavage (BAL) fluid. In this study, we examined 81 patients with benign lung disease. The mean values of CYFRA 21-1 in the BAL fluid of each lung disease were as follows: bronchiolitis obliterans organizing pneumonia (BOOP), 3.9 +/- 2.1 ng/ml (positive rate 50%); collagen vascular disease associated interstitial pneumonia (CVD-IP), 10.7 +/- 15.7 ng/ml (positive rate 50%); diffuse panbronchiolitis (DPB), 4.2 +/- 6.4 ng/ml (positive rate 29%); idiopathic pulmonary fibrosis (IPF), 1.5 +/- 2.1 ng/ml (positive rate 17%); pulmonary infiltration with eosinophilia, 6.3 +/- 7.1 ng/ml (positive rate 44%); sarcoidosis, 4.6 +/- 6.2 ng/ml (positive rate 27%); and healthy volunteer (HV), 0.6 +/- 0.6 ng/ml; and total, 4.4 +/- 5.6 ng/ml (positive rate 32%). The mean values of ProGRP in the BAL fluid were as follows: DPB, 5.0 +/- 7.6 pg/ml (positive rate 0%); IPF, 6.4 +/- 10.6 pg/ml (positive rate 0%); HV, 12.4 +/- 8.3 pg/ml; and total, 5.6 +/- 8.7 pg/ml (positive rate 0%). These results indicate that the two tumor markers have no disease specificity in benign lung disease.

[A case of radiation lung injury with characteristics of bronchiolitis obliterans organizing pneumonia after radiation therapy for breast carcinoma].

We report a case of sputum and dyspnea with patchy migratory air space infiltrates that developed in a 49-year old woman after she started breast radiation therapy following surgery for breast carcinoma. Our case clearly differed from ordinary radiation pneumonitis. Chest roentogenography and computed tomographic (CT) scanning demonstrated alveolar opacities. Bronchoalveolar lavage showed a moderate elevation of the total cell concentration and a considerable increase of lymphocytes, and transbronchial lung biopsies revealed a histological pattern of bronchiolitis obliterans organizing pneumonia with intra-alveolar granulation tissue. The present case suggests that breast irradiation may contribute to the development of a histological pattern of bronchiolitis obliterans organizing pneumonia.

Possible role of L-selectin in T lymphocyte alveolitis in patients with active pulmonary sarcoidosis.

A number of adhesion molecules participate in the recruitment of inflammatory cells to the site of inflammation, and selectins together with their ligands are important in the early transient adhesion phase. In this study, we evaluated the role of L-selectin in T lymphocyte alveolitis in patients with active pulmonary sarcoidosis. We measured serum and bronchoalveolar lavage fluid (BALF) concentrations of soluble (s)L-selectin using an ELISA. Serum and BALF concentrations of sL-selectin were significantly elevated in patients with sarcoidosis compared with control healthy subjects and idiopathic pulmonary fibrosis (IPF) patients (P < 0.05 and P < 0. 01, respectively). The lymphocyte surface marker was also examined in peripheral blood and BALF by flow cytometric analysis. The percentage of CD3+CD62L+ cells (L-selectin-bearing T lymphocytes) was significantly lower in peripheral blood of sarcoidosis than in that of healthy subjects (P < 0.01). In contrast, the percentage of CD3+CD62L- cells (L-selectin-negative T lymphocytes) in BALF of patients with sarcoidosis was significantly higher than in healthy subjects (P < 0.05) and IPF patients (P < 0.01). Furthermore, there was a significant correlation between serum concentrations of sL-selectin and the number of L-selectin-negative T lymphocytes in BALF (r = 0.535, P < 0.01). Our results suggest that L-selectin may be involved in T lymphocyte alveolitis in patients with active pulmonary sarcoidosis.

Involvement of collagen-binding heat shock protein 47 and procollagen type I synthesis in idiopathic pulmonary fibrosis: contribution of type II pneumocytes to fibrosis.

The most common pathologic form of idiopathic pulmonary fibrosis is usual interstitial pneumonia, which is characterized by patchy fibrotic areas, marked increase in the number of fibroblasts and type II pneumocytes, and excessive deposition of extracellular matrix proteins, especially collagen. Heat shock protein 47 is a collagen-binding stress protein and has a specific role in intracellular processing of procollagen molecules as a collagen-specific molecular chaperone. However, its role in the causation of fibrosis in usual interstitial pneumonia is unknown. In this study, we examined the expression of heat shock protein 47 and type I procollagen in 12 patients with usual interstitial pneumonia by immunohistochemistry on sequential sections. Heat shock protein 47 was localized predominantly in alpha-smooth muscle actin-positive myofibroblasts and surfactant protein-A-positive type II pneumocytes in active fibrotic areas of usual interstitial pneumonia. Type I procollagen was also expressed in those cells. In contrast, heat shock protein 47 and type I procollagen were weakly or not at all expressed in myofibroblasts and type II pneumocytes in bronchiolitis obliterans organizing pneumonia and normal lung tissue samples obtained from excised lung cancer tissues. The numbers of heat shock protein 47- and type I procollagen-positive cells to type II pneumocytes or myofibroblasts were significantly higher in usual interstitial pneumonia than in bronchiolitis obliterans organizing pneumonia and normal lung tissue specimens. Our results suggest that myofibroblasts and type II pneumocytes play an important role in the progression of fibrosis through the induction of heat shock protein 47, which regulates the synthesis/assembly of type I procollagen in usual interstitial pneumonia. HUM PATHOL 31:1498-1505.

Immunological function and nutritional status in patients with hepatocellular carcinoma.

BACKGROUND/AIMS: Infection is a major complication associated with increased morbidity and mortality in patients with hepatocellular carcinoma. We compared the immunological function and nutritional status in 16 patients with hepatocellular carcinoma (13 patients had liver cirrhosis) with those of 21 normal healthy subjects. METHODOLOGY: The immunological function was assessed by chemotaxis and superoxide anion production by neutrophils, phagocytosis and killing activities of neutrophils and monocytes, absolute and relative number of peripheral blood lymphocytes, the percentage of peripheral lymphocyte subsets and serum concentrations of immunoglobulins. RESULTS: Although the phagocytic and bactericidal activities of monocytes and superoxide production of neutrophils were not different between the groups, the phagocytic and bactericidal activities of neutrophils and the percentage of natural killer cells were significantly reduced in patients with hepatocellular carcinoma. In the latter group, the prognostic nutrition index was significantly high compared with normal subjects, indicating a poor nutritional status. The phagocytic and bactericidal activities of neutrophils were low in patients with a poor nutritional status compared to those with a good nutritional status. CONCLUSIONS: Our results suggest that impaired immunological competence and undernourishment may be one of the mechanisms causing increased susceptibility of patients with hepatocellular carcinoma to infection.

[Cytokines produced by cells in bronchoalveolar lavage fluid from a patient with primary pulmonary cryptococcosis].

Cytokines in the culture supernatant of concanavalin A-stimulated macrophages/lymphocytes isolated from bronchoalveolar lavage (BAL) fluid in a 29-year-old patient with primary pulmonary cryptococcosis were evaluated to study the immune reaction against Cryptococcus neoformans in the lung. Before fungicidal therapy, levels of interferon-gamma and interleukin-10 were markedly elevated, and declined after therapy. There were no changes in interleukin-2 or interleukin-4 throughout the clinical course. This result suggests that IFN-gamma and IL-10 may be involved in the immune reaction against pulmonary cryptococcosis.

[Analysis of cases allowed to cease erythromycin therapy for diffuse panbronchiolitis--comparative study between patients with cessation of the therapy and patients continuing the therapy].

We investigated the effect of erythromycin (EM) in patients with diffuse panbronchiolitis (DPB) who were divided into 2 groups, the short term group with 8 patients who were treated for at least 2 years and the long term group with 7 patients who were treated for more than 3 years. Each mean value of %VC, FEV1.0, FEV1.0% and PaO2 two years after administration to these patients was improved than before administration of EM, with the exception of PaO2 in the long term group. There was no change in %VC, FEV1.0, FEV1.0% or PaO2 in each group between one year after the therapy and thereafter, with the exception of the fact that FEV1.0% in the short term group, except patients restarting EM therapy, 2 years after the therapy was significantly higher than in the long term group. One patient was readministered EM because of recurrence of DPB after cessation of EM therapy, whose %VC, FEV1.0 and PaO2, but not FEV1.0%, one year after the therapy were increased in those before the therapy. The Neutrophil proportion in the bronchoalveolar lavage (BAL) fluid in this patient was still high after the therapy (94.5%), while that in the patients with cessation of the therapy was improved to the level of less than 8%. Neutrophil proportion in BAL fluids in the patients with continuation of the therapy revealed a high or low level after the therapy. These results suggest that the patients, whose %VC, FEB1.0% and PaO2 were improved and whose proportion of neutrophils in BAL fluid reduced to a normal level compared with those before EM therapy, can be allowed to cease therapy after 2 years or more of EM therapy.

Significance of IL-1beta and IL-1 receptor antagonist (IL-1Ra) in bronchoalveolar lavage fluid (BALF) in patients with diffuse panbronchiolitis (DPB).

We evaluated the effect of erythromycin therapy on pulmonary function tests and the airway inflammatory response of patients with DPB. The number of neutrophils in BALF obtained from DPB patients was significantly higher than that of healthy volunteers. Treatment with erythromycin (600 mg/day for 12.9+/-9.5 months (mean +/- s.d.)) significantly reduced the total number of cells and neutrophils in the airway, and significantly improved pulmonary function tests. The levels of IL-1beta and IL-8 were significantly higher in DPB compared with healthy volunteers (P<0.05, P<0.05, respectively). IL-1Ra in patients is considered to have a weak inhibitory activity for IL-1beta, with approximately five-fold concentration of IL-1beta compared with that in healthy volunteers (approx. nine-fold concentration of IL-1beta). Erythromycin therapy significantly reduced these cytokines to levels comparable to those of healthy volunteers, and produced a trend toward reduction in the level of IL-1Ra in BALF. The level of IL-1beta correlated significantly with the concentration of neutrophils in BALF (r=0.72, P<0.01), as well as with the level of IL-1Ra (r=0.688, P<0.05) and IL-8 (r=0.653, P<0.05). A nearly significant or significant correlation was observed between the concentration of neutrophils and levels of IL-1Ra or IL-8 in BALF (r=0.526, P=0.053 or r=0.776, P<0.01, respectively). There was also a significant relationship between FEV(1) and the concentration of neutrophils in BALF (r=0.524, P<0.05). Our results suggest that the relative amounts of IL-1beta and IL-1Ra or IL-8 may contribute, at least in part, to the neutrophil-mediated chronic airway inflammation in patients with chronic airway disease, and long-term erythromycin therapy may down-regulate the vigorous cycle between the cytokine network and neutrophil accumulation, with resultant reduction of neutrophil-mediated inflammatory response.

Sorbitol production in charged membrane bioreactor with coenzyme regeneration system: I. Selective retainment of NADP(H) in a continuous reaction.

The concept of a charged membrane bioreactor (CMBR) has been proposed for continuous reactions of enzymatic reduction dependent upon the nicotinamide coenzyme NADP(H). It was found that a composite membrane with a negative charge, NTR 7410, could retain NADP(H) selectively without any chemical modification. Several permeation experiments have revealed that the retainment of a coenzyme is based on electrostatic repulsion of negative charges between the membrane and the phosphate moiety of NADP(H). The retainment ratio was reduced by the addition of inorganic salt, although it could be restored to 0.8 in the presence of albumin. A reactor equipped with a charged membrane as the coenzyme separator module was constructed and used in the continuous production of sorbitol. NADPH-dependent aldose reductase isolated from Candida tropicalis IAM 12202 was used for the production of sorbitol from glucose. The coenzyme oxidized in this reaction was enzymatically regenerated by conjugation with glucose dehydrogenase, together with the coproduction of gluconic acid from glucose. With a substrate conversion of 85%, 100 g/L sorbitol was produced and equimolar gluconic acid was coproduced for more than 800 h, indicating that the reaction was efficiently coupled to the enzymatic regeneration. The initial high retainment ratio of the membrane was almost maintained throughout the entire reaction. Consequently, the turnover number of the coenzyme reached 106,000.

Sorbitol production in charged membrane bioreactor with coenzyme regeneration system: II. Theoretical analysis of a continuous reaction with retained and regenerated NADPH.

A theoretical model was constructed in order to study charged membrane bioreactors (CMBRs). In this model, it was postulated that a native nicotinamide coenzyme NADP(H) can be partially retained by a charged membrane in continuous operation. A multienzyme system composed of NADPH-dependent aldose reductase (AR) and glucose dehydrogenase (GDH) was used for the production of sorbitol and gluconic acid from glucose and for the conjugated enzymatic regeneration of NADP(H). Both enzymes were studied with respect to their reaction kinetics. AR was determined to obey the Theorell-Chance mechanism. GDH reaction was approximated by the initial velocity equation of the sequential Bi-Bi mechanism since the reverse reaction could be neglected. Significant inhibitions of both enzymes by sorbitol, gluconic acid, and glucose were observed, and the mode of inhibition was estimated to modify the velocity equations. The differential equation system for each component was derived and numerically analyzed according to the model. The theoretical model elucidated several features of the CMBR. (1) When compared at the same productivity, higher retainment was found to bring about a higher coenzyme turnover number, indicating that the feed coenzyme concentration can be reduced. (2) Under constant conversion, a contradictory relationship between turnover number and residence time arises if the feed concentration of a coenzyme varies. The theoretical model predicts that there is a practically optimal concentration for using NADP(H) efficiently. This concentration was consistent with that yielding the estimated minimum total cost. (3) In this system, excess-GDH-to-AR activity was required because of differences in their kinetic constants. The amount of regeneration enzyme required can be reduced by the accumulation of excels NADPH due to coenzyme retainment. (4) Comparison with an ideal repeated batch reaction revealed that the continuously operated CMBR was vastly superior with respect to productivity as well as operation ability.

The membrane bioreactor with coenzyme recycling system.

Much attention has been paid to membrane bioreactors, especially to the newly developed charged membrane bioreactor (CMBR) for recycling the native form of the coenzyme NAD(P)H. Charged membranes with anionic groups effectively retained the free nicotinamide coenzyme due to the electrostatic repulsion between membrane and coenzyme. The capability of the CMBR was demonstrated by the continuous production of sorbitol using a multi-enzyme system of NADPH-dependent aldose reductase and glucose dehydrogenase. Several important features of the CMBR were elucidated by a theoretical model of coenzyme turnover.

Macrophage colony-stimulating factor purified from normal human urine. Amino-terminal sequence and amino acid composition.

A macrophage colony-stimulating factor (M-CSF) was purified to homogeneity from a large amount of normal human urine. Microanalysis of the N-terminal amino acid sequence up to residue 44 revealed only a single residue difference from that deduced by other workers from the nucleotide sequence of M-CSF cDNA clones. The amino acid composition of the present preparation suggested that the M-CSF which we purified possessed a structure fitting the sequence 1-190 of TPA30-1 cell M-CSF deduced by Wong et al. [(1987) Science 235, 1504-1508].