Association between Reduction of Muscle Mass and Faster Declines in Global Cognition among Older People: A 4-Year Prospective Cohort Study.

OBJECTIVES: A few studies reported that both decrease and increase in body mass index (BMI) were associated with the development of dementia in later life. However, it is unclear what changes in body composition are associated with cognitive decline. This study investigated the longitudinal influences of changes in body composition on cognitive function among community-dwelling adults. DESIGN, SETTING AND PARTICIPANTS: This longitudinal study included older adults aged ≥60 years without cognitive impairment who participated in National Institute for Longevity Sciences - Longitudinal Study of Aging. MEASUREMENTS: Cognitive function was assessed using the MMSE. Body composition was measured by a dual-energy X-ray absorptiometry system. Then, BMI, fat mass index (FMI), fat-free mass index (FFMI), and muscle mass index (MMI) were calculated. The changes in body composition over 6 years (second wave to fifth wave) were calculated, and three groups were created: decreased group, decrease of >5%; stable group, change within 5%, and increased group, increase of >5%. In statistical analysis, a linear mixed model was applied by sex to investigate the influences of body composition changes on cognitive function over 4 years (fifth wave to seventh wave). RESULTS: This study analyzed 515 participants (mean age, 67.05 years; 53.4% men). Men with decreased group in FFMI and MMI exhibited faster declines in MMSE scores than those with stable group (ß [95% CI]: FFMI, -0.293 [-0.719 to -0.020]; MMI, -0.472 [-0.884 to -0.059]). In women, there was no significant association between body composition changes and cognitive functions. CONCLUSIONS: Decrease in fat-free mass and muscle mass is associated with faster cognitive declines in men. These results suggest the importance of continuous monitoring of muscle mass to prevent cognitive decline in later life.

Cross-Sectional Associations of Sarcopenia and Its Components with Neuropsychological Performance among Memory Clinic Patients with Mild Cognitive Impairment and Alzheimer's Disease.

BACKGROUND: The association of sarcopenia with cognitive function in its specific domains remains poorly understood. OBJECTIVES: To investigate the association of sarcopenia and its components with neuropsychological performance among patients with mild cognitive impairment (MCI) and Alzheimer's disease (AD). DESIGN: Cross-sectional design. SETTING: A memory clinic in Japan. PARTICIPANTS: The study included 497 MCI/684 AD patients aged 65-89 years. MEASUREMENTS: Patients were assessed for muscle mass by bioelectrical impedance analysis, muscle strength by hand grip strength (HGS), and physical performance by timed up and go test (TUG). Sarcopenia was defined as presence of both low muscle strength and low muscle mass. The patients underwent neuropsychological tests, including logical memory, frontal lobe assessment battery, word fluency test, Raven's colored progressive matrices, digit span, and the Alzheimer's disease assessment scale-cognitive subscale (ADAS-cog). RESULTS: The prevalence of sarcopenia in men and women was 24.1% and 19.5%, respectively. In multiple regression analyses adjusting for confounders, unlike in men, sarcopenia was associated with memory function in women (ADAS-cog, memory domain, coefficient = 1.08, standard error (SE) = 0.36), which was thought likely due to the relationship between HGS and memory function (immediate recall of logical memory, coefficient = 0.07, SE = 0.03; ADAS-cog, memory domain, coefficient = -0.10, SE = 0.03). Of the components of sarcopenia in both sexes, HGS and TUG were associated with visuospatial function and frontal lobe function, respectively. CONCLUSIONS: The specific association of sarcopenia and its components with cognitive domains may provide the key to elucidating the muscle-brain interactions in AD.

Phagocytosis of alveolar macrophages after conagenin injection to rats.

Phagocytic functions of rat alveolar macrophages (AM) following intraperitoneal injection of conagenin (CNG) and of AM sub-populations fractionated by Percoll discontinuous gradient centrifugation were investigated. Phagocytosis of opsonized-sheep red blood cells (SRBC) following in vitro incubation with CNG showed a significant increase in a higher density of AM (fraction IV). In addition, phagocytosis was also increased in lower density ones (fractions I and II) by macrophage-activating factor (MAF) co-cultivation. CNG-injected rats for 5 consecutive days showed a dose-dependent increase in phagocytosis of AM compared to the control rats. Although the distribution of AM sub-population in rats injected CNG was not significantly different compared to the control rats, phagocytosis was significantly increased in AM of a lower density fraction (fraction II). These results suggest that CNG directly increases phagocytosis of AM in a higher density fraction, and indirectly enhances phagocytosis in AM of a lower density fraction via increasing MAF-like material production.

Effect of amino acid mixtures on nasal allergic responses induced by toluene diisocyanate in mice.

We studied the effect of various amino acid mixtures on nasal allergy induced by the intranasal application of toluene diisocyanate (TDI) in mice. In Experiment 1 (Exp. 1), mice were fed a 25% casein, soy protein isolate (SPI), egg white protein (EW) or gluten diet. In Experiment 2 (Exp. 2), mice were fed a 25% amino acid mixture diets patterned after casein (AA-casein), SPI (AA-SPI), EW protein (AA-EW) or gluten (AA-gluten). In Experiment 3 3 (Exp. 3) we modified the glutamine/glutamic acid (Gln/Glu) concentrations in the amino acid mixtures. Mice were fed a 25% AA-SPI, low Gln/Glu AA-SPI (LG-AA-SPI), AA-EW or high Gln/Glu AA-EW (HG-AA-EW) diet. At the 5th week, mice were divided into sensitized (sen-) and non-sensitized (ns-) groups. The mice in sensitized groups were treated with two courses of intranasal application of toluene diisocyanate (TDI) in ethyl acetate for 5 consecutive days, separated by 9 days rest. The non-sensitized groups of mice were treated with a vehicle. Nine days after the second sensitization, all mice were provoked by TDI. Nasal responses and serum IgE concentration were studied. The findings of Exp. 1 showed that the sen-EW group exhibited a lower body weight gain, higher nasal symptom score and higher IgE concentration than the other sensitized groups. The findings dings of Exp. 2 showed that the sen-EW group had a lower body weight gain, higher nasal symptom score and higher IgE concentration than the other sensitized groups. In Exp. 3, the AA-EW group showed a higher total nasal score and IgE concentration than the HG-AA-EW group, however, the findings of LG-AA-SPI and AA-SPI were similar. These findings demonstrated that amino acid mixtures affect nasal allergy induced by the intranasal application of TDI in mice.

Hormonal and dietary regulation of lysosomal cysteine proteinases in liver under gluconeogenesis conditions.

The activities of cathepsin B, L, J and H in rat liver were significantly increased by starvation if compared with normal diet rats. Furthermore, the activity of cathepsin L increased with glucagon treatment, and the activities of cathepsin L and H decreased significantly with insulin treatment. The changes in cathepsin B and J activities showed the same tendencies as those of cathepsin L and H, but the differences were not statistically significant. The changes in the activities of cathepsin B and L on starvation corresponded with the changes of enzyme protein amounts judged from Western blotting analysis. The levels of the lysosomal cysteine proteinases and amino acid deaminases in the liver changed in parallel with the hormonal and dietary conditions. The increases of alanine amino transferase activity (AAT) started from a much earlier stage than those of cathepsins under the starvation condition. Although administration of prednisolone caused marked induction of the deamination enzymes such as AAT, the levels of cathepsins in the liver were not changed.

Levels of pulmonary surfactant protein A in fetal lung and amniotic fluid from protein-malnourished pregnant rats.

Surfactant protein A (SP-A) is a major apo-protein of pulmonary surfactant, which lines the alveolar walls, lowering the surface tension to prevent lung collapse. Pregnant rats were divided into two groups which received a diet with either 5% or 20% protein from gestational day 9. By a sensitive immunoassay, SP-A levels in the fetal lungs and the amniotic fluid showed a dramatic increase with advancing gestation after the initial appearance on gestational day 18 in both diet groups. Significantly lower levels of SP-A in pregnant rats fed 5% protein diet than those in pregnant rats fed 20% protein diet were observed in the fetal lungs on gestational day 21 and in the amniotic fluid on gestational days 20 and 21. The profiles of increased SP-A levels in the amniotic fluid reflected those in the fetal lungs during gestation. Immunohistochemical examination with anti-rat SP-A antibody at 21 days of gestation showed that the immunoreactive staining of bronchiolar epithelial Clara cells and alveolar type II cells were weaker in the fetal lung sections from pregnant rats fed 5% protein diet than in those from pregnant rats fed 20% protein diet. It is concluded that protein malnutrition in pregnant rats affects the biosynthesis of SP-A in the fetal lungs, which may have important consequences for prematurity and decreased respiratory functions in the neonatal lungs at birth.

Body mass index (BMI) is a reliable index to estimate obesity as a risk factor for deteriorating health.

The objective of this study was to find out what index is appropriate to evaluate obesity, by measuring body fat using bioelectrical impedance analysis (BIA). Subjects in this study were 74 women, aged 44 to 73, living in Tokushima prefecture. The means +/- standard deviation (SD) of Broca index, Body mass index (BMI) and body fat were 103 +/- 14.3%, 23.0 +/- 2.8 kg/m2 and 28.1 +/- 5.5%, respectively. In addition, their clinical data such as blood pressure, glutamate-pyruvate transaminase activity (GPT), triglycerides (TG) and fasting blood sugar (FBS) were within normal ranges. When compared with correlation between obesity indices (Broca index or BMI) and height, there was a negative correlation between Broca index and height (= -0.447). Furthermore, the number of obese subjects estimated by Broca index was less than that of obese subjects estimated by BMI. Although both Broca index and BMI showed higher correlations with body fat estimated by BIA, BMI (r = 0.927) showed a higher correlation compared to that of Broca index (r = 0.875). These results suggest that BMI is a reliable index to evaluate the body fat.

Immunohistochemical localization of surfactant protein A in N-bis (2-hydroxypropyl) nitrosamine-induced lung tumors in rats.

Surfactant protein A (SP-A) is most abundant protein associated with pulmonary surfactant which is synthesized by alveolar type II cells in the alveoli. In this study, we localized SP-A in experimentally induced pulmonary hyperplasias and tumors in rats, by immunohistochemistry. When rats were given a single intraperitoneal injection of N-bis (2-hydroxypropyl) nitrosamine (BHPN) followed by exposed to mixture gases of O3 and NO2, hyperplastic alveolar type II cells stained with the antibody against SP-A were located in the alveolar walls near the alveolar ducts. Adenomas and adenocarcinomas were stained with the anti-SP-A antibody in the lung parenchyma. These immunohistochemical findings suggested that the lung tumors induced in rats treated with BHPN and additionally exposed to mixture gases of O3 and NO2 are derived from mainly alveolar type II cells.

The role of macrophages in increased mitogen response of rat splenic lymphocytes following in vitro incubation with vitamin E.

The role of macrophages (M phi) in the enhancement of lymphocyte proliferation by alpha-tocopherol (VE) was investigated using rat splenocytes. The proliferation of whole splenocytes was significantly higher than that of M phi-depleted splenocytes at all concentrations of concanavalin A (Con A; 0.5-10 micrograms/ml). When whole and M phi-depleted splenocytes were preincubated with VE (2 micrograms/ml) for 24 h, the proliferation of whole splenocytes was significantly enhanced compared to that of whole splenocytes preincubated with medium alone. In contrast, M phi-depleted splenocytes did not show any increase of proliferation following in vitro pretreatment with VE. When the splenic: M phi pretreated with both VE (2 micrograms/ml) and Con A (10 micrograms/ml) for 24 h were further incubated with splenic lymphocytes, their proliferation was significantly enhanced compared to that of splenic lymphocytes cultured with splenic M phi pretreated with Con A. In this experiment, the medium containing 2-mercaptoethanol (2-ME) had the ability to enhance splenic lymphocyte proliferation, which masked the enhanced effect of VE on splenic: lymphocyte proliferation. Furthermore, in vitro treatment of VE could not decrease the production of prostaglandin E2, but could enhance the production of interleukin 1 from splenic M phi. These results suggest that M phi play an important role in the proliferation of splenic lymphocytes following in vitro incubation with VE, which is closely associated with the action of VE as an immunomodulator rather than antioxidant.

Glutamine supplementation prevents the decrease of mitogen response after a treadmill exercise in rats.

The effect of glutamine (Gln)-supplemented diet on mitogen response decreased immediately after a treadmill exercise was examined by measuring the proliferations of peripheral blood lymphocytes (PBL) with phytohemagglutinin (PHA) and concanavalin A (ConA) in male Fisher rats. Although the plasma Gln concentration was significantly decreased in the control group immediately after a treadmill exercise (20 m/min, 60 min) compared to rested rats, plasma Gln concentration of rats fed Gln-supplemented diet for 3 weeks was significantly higher than that of control group in resting and was not significantly decreased even immediately after a treadmill exercise. In addition, proliferation of PBL with PHA or ConA and interleukin 2 (IL2) production were also significantly decreased immediately after a treadmill exercise in control group. On the contrary, their functions were almost maintained in Gln-supplemented group even immediately after a treadmill exercise. PBL from rats fed Gln-supplemented diet showed a higher response to mitogens such as PHA and ConA compared to the control group. Furthermore, their PBL showed higher incorporation of [3H]Gln compared to that of the control group irrespective of treadmill exercise. These results indicate that the preventive effect of Gln-supplemented diet on mitogen response decreased after a treadmill exercise is due to an increased response to mitogen and increased uptake of Gln as sources of fuel and nucleotides to the immune cells.

Morphological changes in rat aortic endothelial cell line stimulated by endothelin.

We have reported that endothelin (ET)-1 activates interleukin (IL)-6 production in rat aortic endothelial cell line (WAE-1 cell). In this experiment, we investigated the morphological changes induced by ET-1 in cultured WAE-1 cells. The cells were treated with ET-1 for 8 h or 24 h, and then compared with untreated cells by light and electron microscopies. The WAE-1 cells treated with ET-1 for 8 h showed remarkable alterations as following: by light microscopic observation, the cells were enlarged and filled with many vesicles in the cytoplasm, and by electron microscopic observation, the cells showed the increases of nuclear membrane infoldings, increased density of chromatin granules just inside the nuclear membrane, and multivesicular bodies in the cytoplasm. These morphological changes were hardly observed in WAE-1 cells-treated with ET-1 for 24 h. It was suggested that ET-1 stimulated DNA synthesis and secretion of cytoplasmic products in WAE-1 cells.

Ultracytochemistry of thymic epithelial cells in rats given organotin.

A combined light-and electron microscopic study of thymic changes in the adult rats after di-n-butyltin dichloride (DBTC) administration has been made. A rapid depletion of thymocytes in the cortex of thymus and subsequent rapid recovery of the number of thymocytes occurred. In this process, at 1-3 days after DBTC treatment the number of necrotic thymocytes was maximal. At 3-6 days, reticular epithelial cells were predominantly phagocytizing and acid phosphatase-positive in the cortex and cortico-medullary regions, where they appeared to develop from macrophage-like cells. During acute involution, it was likely that reticular epithelial cells were phagocytic and remove the necrotic thymocytes. The proportion of CD4+ CD8+ cells in the cortex of thymus was maximally reduced from day 3 onwards and reached the lowest level at 6 days after single oral dose of DBTC. On these days, the proportions of CD4- CD8- and single positive cell (CD4- CD8+ or CD4+ CD8-) subsets were relatively increased. These data suggest that DBTC preferentially causes an initial depletion of CD4+ CD8+ cells in thymus, and both macrophages and reticular epithelial cells of the cortex may be involved in the rapid removal of damaged thymocytes from thymus.

Arginine-enriched solution induces a marked increase in muscle glutamine concentration and enhances muscle protein synthesis in tumor-bearing rats.

Using a transplantable Yoshida sarcoma in a rat model of total parenteral nutrition (TPN), we measured the effectiveness of an arginine-enriched amino acid solution (AI-82) on muscle glutamine concentration and muscle protein synthesis compared with that of a conventional amino acid solution (Proteamin12). After tumor-bearing rats had been given one of two isocaloric TPN regimens for 6 days, [15N]glycine (99 atom %) containing TPN solution was infused into animals at a constant rate of 8 mg of [15N]glycine per hour for 18 hours, after which the liver, skeletal muscle (gastrocnemius muscle), and tumor protein synthesis rates were measured. A significantly increased whole muscle protein synthesis rate was observed in the AI-82 group; there was no difference in the whole liver and tumor protein synthesis rates between the two groups. When each TPN solution was administered for 1 week, muscle concentrations of arginine, ornithine, glutamine, and glutamate were considerably higher in the AI-82 group than in the Proteamin12 group, and these differences were also accompanied by a decrease in the plasma branched-chain amino acid (BCAA) (leucine, isoleucine, and valine) levels in the AI-82 group. The high levels of muscle glutamine concentration in the AI-82 group were investigated in connection with the high use of exogenous branched-chain amino acids.

Specific assay method for the activities of cathepsin L-type cysteine proteinases.

We have established a new differential assay method for cathepsin L-type proteinases using specific inhibitors, E-64 for all cysteine proteinases, CA-074 for cathepsin B, and PLCPI for cathepsin L-type proteinases with Z-Phe-Arg-MCA as the substrate. The value of cathepsin B calculated by this method did not coincide with value assayed directly in terms of the hydrolysis of Z-Arg-Arg-MCA, a specific substrate for cathepsin B. The activities of cathepsin L-type proteinases, cathepsins B and J in rat liver and kidney were assayed at the same time using this new assay method as a representative example.

Sendai virus infection changes the subcellular localization of tryptase Clara in rat bronchiolar epithelial cells.

Tryptase Clara activates the infectivity of Sendai and influenza viruses proteolytically. In this study, we investigated changes in the subcellular localization of tryptase Clara in rat bronchioles with progression of Sendai virus infection. Tryptase Clara and Sendai virus F2 antigen were localized by light and electron immunohistochemical studies. In the uninfected rat lung, tryptase Clara was specifically localized in the secretory granules of respiratory bronchiolar epithelial nonciliated cells, but not in bronchiolar ciliated, or alveolar cells. In the initial stage of Sendai virus infection with slight pathological changes, however, anti-tryptase Clara was highly reactive in luminal peripheral membranes of both nonciliated and ciliated epithelial cells of the bronchioles together with some Sendai virus envelope glycoprotein, F2 antigen. In the progressed stage, tryptase Clara was hard to detect, with heavy accumulation of F2 antigen in the epithelial cells. These immunohistochemical results support our previous findings that in the bronchial lavage fluid tryptase Clara is significantly increased both in amount and activity after viral infection. These results suggest that Sendai virus stimulates the secretion of tryptase Clara from nonciliated bronchiolar epithelial cells to the airway lumen. Accumulation of tryptase Clara on the luminal surface of the bronchiolar epithelial cells and/or in the airway lumen may produce favourable conditions for proteolytic viral activation and multiplication.

A non-glycosylated form of pulmonary surfactant protein A appears in rat amniotic fluid.

Surfactant protein A (SP-A) is a family of glycoproteins that have a triplet with 26, 32 and 36 kDa under reducing conditions in rat lung. We wanted to evaluate the SP-A forms in amniotic fluid of pregnant rats compared to those found in rat lungs. By Western blot analysis, glycosylated SP-A, was not found in the amniotic fluid in contrast to the pulmonary surfactant triplet SP-A, which comprises a 26 kDa protein and its glycosylated 32 and 36 kDa forms. The SP-A concentration in amniotic fluid was barely detectable at 18 days of gestation (20 +/- 12 ng.ml-1), and then increased and reached 700 +/- 333 ng.ml-1 at the final gestational day 21, as determined by an enzyme-linked immunoabsorbent assay. Immunohistochemically, SP-A was found in some epithelial cells of larger respiratory bronchi, but not, or to a lesser degree, in smaller respiratory bronchi at gestational day 18. At 21 days of gestation, SP-A was detected in bronchial and bronchiolar nonciliated epithelial Clara cells, alveolar epithelial type II cells and some alveolar macrophages. The ratio of the 26, 32 and 36 kDa SP-A forms in bronchoalveolar, bronchobronchiolar and tracheal lavage fluids prepared from adult rats was 6:29:65, 84:5:11 and 100:0:0, respectively. These findings show the presence of a non-glycosylated SP-A in rat amniotic fluid. This may reflect the increased ratio of non-glycosylated SP-A to bronchoalveolar, bronchobronchiolar and tracheal lavage fluids, respectively.

Vitamin E is an important factor in T cell differentiation in thymus of F344 rats.

The effect of vitamin E (dl-alpha-tocopheryl acetate) on T cell differentiation in thymus of F344 rats was examined in this study. The rats were divided into three groups: vitamin E-free, regular and high vitamin E groups and fed a diet containing various levels of vitamin E (0, 50, and 585 mg/kg diet) for 7 weeks. The number of thymocytes was significantly lower in the vitamin E-free group relative to the regular group. Although the proportions of both CD4+CD8- and CD4-CD8+ T cells in thymocytes were significantly greater in the high vitamin E group, the proportion of CD4+CD8- T cells inversely decreased in vitamin E-free group compared to that of the regular group. The ratio of CD4+CD8-/CD4-CD8+ T cells increased in the high vitamin E group (p < 0.01) and significantly decreased in the vitamin E-free group (p < 0.001) compared to that of the regular group. Although the marked changes of T cell subsets were not seen in peripheral blood lymphocytes (PBL), the ratio of CD4+CD8-/CD4-CD8+ T cells was significantly lower in the vitamin E-free group and significantly greater in the high vitamin E group compared to that of the regular group. Production of interleukin (IL) 2 by thymocytes following the stimulation with Con A for 48 h increased about threefold in the high vitamin E group compared to the regular group. Conversely, thymocytes from rats fed the vitamin E-free diet showed a significant decrease of IL2 production compared to that of the regular group. Prostaglandin E2 (PGE2) production from thymocytes was significantly lower in the high vitamin E group compared to that of the regular group, whereas thymocytes of rats fed the vitamin E-free diet showed a significant increase of PGE2 production compared to that of rats fed the regular diet. Furthermore, in vitro addition of indomethacin provided a restoration of IL2 production from thymocytes of rats fed the vitamin E-free diet to the level of rats fed the regular diet. These results suggest that vitamin E plays an important role in T cell differentiation in thymus, which may be related to the action of vitamin E as antioxidant.

Pulmonary surfactant is a potential endogenous inhibitor of proteolytic activation of Sendai virus and influenza A virus.

The pathogenicities of influenza viruses and paramyxoviruses have been proposed to be primarily determined by a host cell protease(s) that activates viral infectivity by proteolytic cleavage of the envelope glycoproteins. We recently isolated a trypsin-type endoprotease, named tryptase Clara, from rat bronchial and bronchiolar epithelial Clara cells, which is secreted into the airway lumen and activates Sendai virus and influenza A virus proteolytically. We report here that surfactant in the bronchial fluid inhibited tryptase Clara specifically, having a Ki value of 0.13 microM, and inhibited the proteolytic activations by tryptase Clara in vitro and in organ cultures of rat lung. Intranasal infection of rats with Sendai virus was shown to stimulate secretion of tryptase Clara without changing the amount of surfactant in the bronchial lumen, resulting in a preferable condition for proteolytic viral activation and multiplication.

Radical resection of primary malignant melanoma of the gallbladder with multiple metastases: report of a case.

We present herein an usual case of primary malignant melanoma of the gallbladder in a 51-year-old man in whom an exploratory laparotomy for melena revealed six malignant melanoma lesions located in the gallbladder, main pancreatic duct, stomach, duodenum, jejunum, and a mesenteric lymph node. Total pancreatectomy was performed and histologically, junctional activity was seen only in the gallbladder, suggesting that this was the primary site. No melanotic lesions were found on the skin or eyes. The metastases to the main pancreatic duct and gastrointestinal tract appeared likely to have occurred as a consequence of the mucosal dissemination of the tumor cells shed into the bile. The post-operative course was uneventful and combined chemotherapy was administered for 16 months. No new metastatic lesions were found until 21 months postoperatively, when metastases were detected in the brain and thoracic spinal cord. These metastatic tumors were removed surgically, but the patient died from cerebral disturbance 26 months after the initial operation. Thus, we consider that aggressive surgical therapy was effective for extending the survival time and improving the quality of life of this patient.

Electron immunohistochemical localization in rat bronchiolar epithelial cells of tryptase Clara, which determines the pneumotropism and pathogenicity of Sendai virus and influenza virus.

The intracellular localization in rat bronchiolar epithelial cells of a novel trypsin-like protease named tryptase Clara, a possible activator of inactive viral fusion glycoprotein of influenza A and wild-type Sendai virus in the respiratory tract, was examined by electron microscopy. In thin sections embedded in LR White, gold particles indicating immunoreactivity of tryptase Clara were detected specifically in secretory granules of Clara cells. No immunoreactivity was detected in bronchiolar ciliated cells, alveolar cells including epithelial Type I and II cells, or alveolar macrophages. Some granules enveloped in a thin membrane and labeled intensely with immunogold particles were seen protruding from peripheral and submembrane regions and a few were observed free in the airway lumen. Scanning electron microscopy also revealed smooth droplets along the main body of Clara cells. These data suggest that tryptase Clara is secreted into the bronchiolar lumen. These findings are the first to show the subcellular localization of tryptase Clara in rat bronchioles and suggest the site of proteolytic activation of the progeny of enveloped pneumotropic viruses, such as Sendai virus and influenza virus.