Bovine κ-casein inhibits human rotavirus (HRV) infection via direct binding of glycans to HRV.

Human rotavirus (HRV) is a major etiologic agent of severe infantile gastroenteritis. κ-Casein (κ-CN) from both human and bovine mature milk has been reported to have anti-HRV activity; however, the mechanism of this activity is poorly understood. The present study examined the molecular basis for the protective effect of bovine κ-CN derived from late colostrum (6-7 d after parturition) and from mature milk. Among the components of casein, κ-CN is the only glycosylated protein that has been identified. Therefore, we investigated whether the glycan residues in κ-CN were involved in the anti-HRV activity. Desialylated CN obtained by neuraminidase treatment exhibited anti-HRV activity, whereas deglycosylated CN obtained by o-glycosidase treatment lacked antiviral activity, indicating that glycans were responsible for the antiviral activity of CN. Furthermore, an evanescent-field fluorescence-assisted assay showed that HRV particles directly bound to heated casein (at 95°C for 30 min) in a viral titer-dependent manner. Although the heated κ-CN retained inhibitory activity in a neutralization assay, the activity was weaker than that observed before heat treatment. Our findings indicate that the inhibitory mechanism of bovine κ-CN against HRV involves direct binding to viral particles via glycan residues. In addition, heat-labile structures in κ-CN may play an important role in maintenance of κ-CN binding to HRV.

Skimmed, sterilized, and concentrated bovine late colostrum promotes both prevention and recovery from intestinal tissue damage in mice.

Bovine colostrum is a rich source of tissue repair and growth factors, and inhibits gastrointestinal injury induced by the side effects of nonsteroidal antiinflammatory drugs (NSAID), such as indomethacin. Nonsteroidal antiinflammatory drugs are drugs with analgesic and antipyretic effects, but in higher doses they have inflammatory effects. The pathogenesis of small intestinal damage caused by NSAID is unclear. The present study was performed to investigate the antiinflammatory effects of skimmed, sterilized, and concentrated bovine late colostrum on intestinal injury induced by side effects of NSAID, and then to identify the active ingredient in the colostrum for intestinal tissue. In Japan, the sale of bovine colostrum within 5 d after parturition is prohibited by law. Therefore, we focused on bovine late colostrum obtained from healthy lactating cows 6 to 7 d after parturition. Proliferation of small intestine epithelial cells was stimulated in mice fed the colostrum for 1 wk. With regard to indomethacin-induced enteropathy, both prefeeding and postfeeding with colostrum facilitated growth of the intestinal villi, indicating preventive and healing effects. Furthermore, to identify the active ingredient in the colostrum responsible for this effect, the casein and whey fractions were prepared from the colostrum and fed to normal mice. Only the colostrum casein fraction stimulated intestinal villus elongation, whereas the whey fraction and mature milk casein showed no such effect. Taken together, these observations indicate that the skimmed, sterilized, and concentrated bovine late colostrum, especially the casein fraction, could be used to treat the injurious effects of NSAID in the intestine and could be effective for treatment of other ulcerative conditions in the bowel, suggesting that the colostrum has therapeutic potential for intestinal inflammation.

Sustained-release microcapsules of a bone formation stimulant, TAK-778, for local injection into a fracture site.

The benzothiepin derivative (2R,4S)-(-)-N-(4-diethoxyphosphorylmethylphenyl)-1,2,4,5-tetrahydro-4-methyl-7,8-methylenedioxy-5-oxo-3-benzothiepin-2-carboxamide (TAK-778) is a potent bone formation stimulant. A sustained release formulation was prepared by encapsulating the drug into biodegradable microcapsules for local application to fracture repair in rats. The microcapsules consisted of TAK-778 (10% w/w) and a biodegradable polymer, copoly (d,l-lactic/glycolic acid), with a copolymer ratio of 85:15 (mol/mol) and an average molecular weight of 15 k. The TAK-778 amount at the injection site progressively diminished for 4 weeks after administration, and the serum level of TAK-778 was sustained over the same period. The local concentration of TAK-778 after administration of the microcapsules was simulated by a two-compartment open model. In the model, a first-order release rate constant and a transfer rate constant were obtained from the release profile of the microcapsules and the serum level of TAK-778 after administration of the TAK-778 solution, respectively. Localization at the injection site was examined by radiography using microcapsules in which iodoform was encapsulated as a contrast agent. The microcapsules formed a clot at the injection site, and their spread was narrowly restricted. The local concentration was calculated to be maintained within the range 10(-3)-10(-6) M over 4 weeks on the assumption that the dose and spread volume were 5 mg of TAK-778/site and 3 mL, respectively.

Enhancement of fracture repair in rats with streptozotocin-induced diabetes by a single injection of biodegradable microcapsules containing a bone formation stimulant, TAK-778.

The feasibility of using microcapsules containing a bone formation stimulant, (2R,4S)-(-)-N-(4-diethoxyphosphorylmethylphenyl)-1,2,4, 5-tetrahydro-4-methyl-7, 8-methylenedioxy-5-oxo-3-benzothiepin-2-carboxamide (TAK-778) to enhance fracture repair was assessed in rats with streptozotocin-induced diabetes. The release profile of the microcapsules was designed to mimic a dosing regimen of multiple injections of TAK-778 solution. The solution was injected locally every third day from day 0 (the day of operation) to day 27 according to several dosing regimens, and fracture repair was assessed at day 28. The production of callus was most prominent when TAK-778 solution was injected so that 50-75% of the total dose (5 mg TAK-778/site) was administered during the first half of the treatment period. Thus, injectable microcapsules of 30 micrometer in mean diameter were prepared in order to release TAK-778 over 4 weeks using a biodegradable polymer, poly(d,l-lactic/glycolic) acid, with a copolymer ratio of 85:15 (mol/mol) and an average molecular weight of 14,000. A single local injection of the microcapsules markedly enhanced fracture repair, which resulted in recovery of destructive bending strength of the bone at day 28. Histologically, the injection of TAK-778 microcapsules stimulated both fibrous and cartilaginous proliferation and periosteal ossification in the callus at day 7; bony bridge formation was observed at day 28. At day 56, the callus was remodeled and cortical bony union was evidenced in the microcapsule-treated fractures compared with the controls, which showed only fibrous union.

Enhancement of osteogenesis in vitro and in vivo by a novel osteoblast differentiation promoting compound, TAK-778.

TAK-778 [(2R,4S)-(-)-N-(4-diethoxyphosphorylmethylphenyl)-1,2,4, 5-tetrahydro-4-methyl-7, 8-methylenedioxy-5-oxo-3-benzothiepin-2-carboxyamide; mw 505.53], a novel osteoblast differentiation promoting compound, was characterized in vitro and in vivo models. TAK-778 at doses of 10(-6) M and higher promoted potently bone-like nodule formation in the presence of dexamethasone in rat bone marrow stromal cell culture. This was accompanied by increases in cellular alkaline phosphatase activity, soluble collagen release, and osteocalcin secretion. Under the culture conditions, TAK-778 also stimulated the secretion of transforming growth factor-beta and insulin-like growth factor-I, indicating that TAK-778 may exert regulatory effects on osteoblast differentiation via autocrine/paracrine mechanisms. Furthermore, the in vivo osteogenic potential of TAK-778 was studied in bony defect and osteotomy animal models, using sustained release microcapsules consisted of a biodegradable polymer, poly (dl-lactic/glycolic) acid (PLGA). Single local injection of TAK-778/PLGA-microcapsules (PLGA-MC) (0.2-5 mg/site) to rat skull defects resulted in a dose-dependent increase in new bone area within the defects after 4 weeks. When the pellet containing TAK-778/PLGA-MC (4 mg/pellet) was packed into place to fill the tibial segmental defect in rabbit, this pellet induced osseous union within 2 months, whereas the placebo pellet did not. In addition, single local application of TAK-778/PLGA-MC (10 mg/site) to rabbit tibial osteotomy site enhanced callus formation accompanied by an increase in breaking force after 30 days. These results reveal for the first time that a nonendogenous chemical compound promotes potently osteogenesis in vitro and enhances new bone formation during skeletal regeneration and bone repair in vivo and should be useful for the stimulation of fracture healing.

Uptake of low molecular weight fractionated [3H]heparin by rat hepatocytes in the primary culture.

The uptake of low molecular weight fractionated [3H]heparin (LMWFH: 7000 Da) was examined, for comparison with that of high molecular weight fractionated [3H]heparin (HMWFH:20000 Da), in a primary culture of rat hepatocytes. The uptake of LMWFH increased almost linearly with time up to 60 min (extended uptake), although a faster uptake was observed in the initial 2 min (initial uptake). Both the initial and extended uptake were saturable, and the maximum uptake velocity (Vmax) and the Michaelis constant (Km) were estimated to be 10.7 pmol/min/mg protein and 398 nM, respectively, for the initial uptake and 0.34 pmol/min/mg protein and 116 nM, respectively, for the extended uptake. The Km for the extended uptake was 5 times larger than that of 21 nM for HMWFH, but the other parameters were comparable with those for HMWFH. Thus, an increase in Km, or a decrease in the apparent affinity, with a decrease in molecular weight in the extended uptake may be responsible for the reported lower hepatic uptake of low molecular weight heparin, compared with unfractionated heparin. It was also shown that both the initial and the extended uptake of LMWFH were inhibited by several analogs of heparin, including HMWFH, and anionic compounds such as 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), suggesting that LMWFH and HMWFH, in spite of a large difference in the molecular weight, share the same specialized uptake mechanism, in which an anionic moiety and/or heparin-like structure plays an important role.

Multiple pulmonary nodules manifested in a patient with NK cell granular lymphocyte proliferative disorder.

An 18-year-old man was admitted to our hospital with high temperature and dyspnea. A chest radiograph revealed the presence of multiple round nodules compatible with a metastatic lung cancer. The peripheral white blood cell count was 22,000/mm3 and more than 85 percent were atypical large lymphocytes with azurophilic granules. He was diagnosed as having natural killer (NK)-cell granular lymphocyte proliferative disorder (NK-GLPD) as the lymphocytes were positive with CD56, a cell surface marker characteristic for NK cells. The major pathologic finding of the tissue collected from the pulmonary nodules by transbronchial lung biopsy was infiltration of mostly large granular lymphocytes.

Dose-dependent uptake of radioactivity by liver parenchymal and non-parenchymal cells after intravenous administration of fractionated 3H-heparin to rats.

The dose-dependent uptake of fractionated 3H-heparin in the subpopulations of liver cells, parenchymal and non-parenchymal cells, was characterized in rats in vivo. Following the intravenous administration of fractionated 3H-heparin, the radioactivity in plasma was eliminated according to the first order kinetics at each dose. However, the elimination rate constant decreased with dose over the dose range of 0.3 to 100 U/kg, suggesting nonlinear elimination. In accordance with the delay in the plasma elimination, the uptake rate constant of radioactivity by parenchymal as well as non-parenchymal cells of liver, the major distribution organ, also decreased. Although heparin has long been considered to be taken up by a reticuloendothelial system (RES) such as non-parenchymal cells in the liver, the uptake of fractionated 3H-heparin by parenchymal cells was found to be comparable with that by non-parenchymal cells at the lowest dose of 0.3 U/kg, and even larger than that by non-parenchymal cells at the highest dose of 100 U/kg. The uptake clearances of fractionated 3H-heparin at the dose of 0.3 U/kg were 86.4 and 504 ml/10(8) cells/d, respectively, for parenchymal and non-parenchymal cells. These values were much larger than those reported for polyvinylpyrrolidone, which has been suggested to be taken up by fluid phase endocytosis. Thus, the present study revealed the significant contribution of parenchymal cells in the hepatic uptake of fractionated 3H-heparin. The dose-dependent uptake with high clearance values in both parenchymal and non-parenchymal cells provides an in vivo suggestion of the specialized transport of fractionated heparin in these two subpopulations of liver cells.

Macromolecule-macromolecule interaction in drug distribution. III. Kinetic characterization of the uptake of fractionated [3H]heparin and the effect of plasma proteins in the perfused rat liver.

The concentration-dependent hepatic uptake of fractionated [3H]heparin, a macromolecular model drug, was kinetically characterized and the effect of plasma proteins, albumin and alpha-globulin, was evaluated in the perfused rat liver as part of an ongoing effort to elucidate the mechanism of interaction of macromolecular drugs with biological macromolecules and the role of this interaction in the drugs' distribution. In the absence of proteins, the uptake of fractionated [3H]heparin was saturable with the maximum uptake velocity (Vmax) of 7.6 pmol/min/g liver and the Michaelis constant (Km) of 32.2 nM, suggesting the involvement of a specialized transport. alpha-Globulin (8.0 mg/ml) reduced the uptake of fractionated [3H]heparin at lower heparin at lower heparin concentrations. However, albumin (40 mg/ml) did not affect the uptake of fractionated [3H]heparin, suggesting an insignificant interaction. Assuming that fractionated [3H]heparin bound to alpha-globulin cannot be uptaken and that the reduction in uptake was solely attributable to the saturable Scatchard-type binding of fractionated [3H]heparin to alpha-globulin, the dissociation constant (Kd) and the binding capacity (n) were estimated to be 2.1 nM and 0.002, respectively. In in vitro binding experiments by ultrafiltration, Kd and n were estimated as 168 nM and 0.5, respectively, for alpha-globulin and 1021 nM and 0.02, respectively, for albumin, suggesting lower affinity and higher capacity in vitro for each protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Uptake of fluorescein isothiocyanate (FITC)-fractionated heparin by rat parenchymal hepatocytes in primary culture.

The distribution of fractionated heparin in a primary culture of rat parenchymal hepatocytes was investigated optically using the fluorescence labelled drug and confocal imaging system with an inverted fluorescence microscope. The cell-associated fluorescein isothiocyanate (FITC)-fractionated heparin was observed to increase in conjunction with incubation time and also to localize, suggesting an internalization to cell organella with the exception of the nuclei.

Uptake mechanism of fractionated [3H]heparin in rat parenchymal hepatocytes in primary culture: effect of transport inhibitors on the uptake.

In order to elucidate the uptake mechanism of fractionated [3H]heparin by rat parenchymal hepatocytes, the concentration dependent uptake was kinetically analyzed in primary culture of rat parenchymal hepatocytes, and the effects of established transport inhibitors and heparin analogues on the uptake were also examined. The uptake rate of heparin measured over an extended period of 60 min was saturable, with the maximum uptake velocity (Vmax) of 0.36 +/- 0.05 pmol/min/mg protein and the Michaelis constant (Km) of 21.2 +/- 5.4 nM. The uptake was inhibited by the addition of 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), an inhibitor of the anion transport system, and rose bengal, an organic anion. Heparin analogues (pentosan polysulphate, or heparan sulphate) also inhibited the uptake of fractionated heparin. However, the uptake was not inhibited by the inhibitors of receptor-mediated endocytosis (phenylarsine oxide). These results suggest that fractionated heparin may be taken up by anion transport system, rather than by receptor-mediated endocytosis, though the fractionated [3H]heparin is a compound with the high molecular weight of about 20000 Da. At least the negative charge or sulphate group in the drug structure is supposed to play an important role in the uptake of fractionated heparin by parenchymal hepatocytes.

Macromolecule-macromolecule interaction in drug distribution. II. Effect of alpha-globulin on saturable uptake of fractionated [3H]heparin by rat parenchymal hepatocytes in primary culture.

The uptake of fractionated [3H]heparin was investigated in rat parenchymal hepatocytes in primary culture. The initial uptake of fractionated [3H]heparin was found to be saturable with the maximum uptake velocity (Vmax) of 10.1 +/- 1.46 pmol/min/mg protein and the Michaelis constant (Km) of 284 +/- 47.9 nM. The effect of alpha-globulin, the major protein binding to fractionated [3H]heparin, on the saturable uptake profile of fractionated [3H]heparin was also investigated. The uptake clearance was reduced, depending on the concentration of fractionated [3H]heparin, by the addition of 1 mg/ml alpha-globulin. We assumed that fractionated 3H-heparin bound to alpha-globulin was not available for uptake and that the reduction in the uptake clearance was solely attributable to the saturable binding of fractionated [3H]heparin to alpha-globulin. The uptake clearance versus concentration profile was analyzed to obtain the dissociation constant (Kd) of 31.8 nM and the capacity (n) of 0.047 for the binding of fractionated [3H]heparin to alpha-globulin. The saturable binding of fractionated [3H]heparin to alpha-globulin was supported by in vitro binding experiments using gel chromatography, in which bound fractionated [3H]heparin decreased with the concentration of fractionated [3H]heparin in the presence of alpha-globulin. In conclusion, the present study demonstrated the saturable uptake of fractionated [3H]heparin by rat parenchymal hepatocytes and the saturable binding of fractionated [3H]heparin to alpha-globulin. The saturable uptake may suggest the involvement of a specific transport system such as receptor-mediated endocytosis.

Stereotactic radiosurgery using a linear accelerator.

A basic and clinical study of radiosurgery using the linear accelerator (Linac) system for unremovable deep-seated brain tumors is reported. A Komai stereotactic ring was used to locate the target coordinates. The patient was laid on the Linac treatment table and held in the head fixation system. Irradiation was given in five positions. The dose profile by film dosimetry and Rando phantom was satisfactory. Seventeen tumors in 14 patients were treated. Clinical or histological diagnoses were nine metastases, one benign and two malignant gliomas, one meningioma, and one craniopharyngioma. Tumor sizes were between 8 and 30 mm. Doses were between 12 and 30 Gy. Computed tomographic evaluation after 3 months of 12 tumors in 11 survivors showed one complete remission, three partial remission, six no change, and two partial deterioration. For progressive tumors, Linac radiosurgery results are excellent.

Uptake of fractionated [3H]heparin by rat parenchymal hepatocytes in primary culture: effects of alpha-globulin, temperature, and pH.

The mechanism of uptake of fractionated [3H]heparin in conjunction with the effect of alpha-globulin (the major binding protein of heparin) was investigated in primary cultures of rat parenchymal hepatocytes. The uptake clearances were estimated from the initial linear phase, up to 1 min, of the uptake-versus-time profile. The uptake clearance for fractionated [3H]heparin was 11.0 mL/min/g of liver at 7.5 nM fractionated [3H]heparin in the absence of alpha-globulin. This value decreased with increasing concentration of alpha-globulin in the incubation solution, a fact suggesting that the rate of uptake of alpha-globulin-bound, fractionated [3H]heparin is lower than that of unbound [3H]heparin, as generally assumed for hepatic drug elimination. However, the uptake clearance in the presence of alpha-globulin at 8 mg/mL, where free, fractionated [3H]heparin was supposed to be negligible according to the results of our in vitro study, was approximately 12% of that in the absence of alpha-globulin. These results suggest that alpha-globulin-bound, fractionated [3H]heparin also contributes to the uptake of fractionated [3H]heparin. This effect on uptake could be explained by the protein-mediated transport concept rather than the traditional assumption that protein-bound drugs are not transported. The uptake clearance was reduced significantly by reducing the incubation temperature from 37 to 4 degrees C. However, neither the pH of the incubation solution (6.4-8.4) nor several inhibitors of active transport had any clear effects on the uptake clearance.

Simultaneous measurements of adenosine deaminase activity and tuberculostearic acid in pleural effusions for the diagnosis of tuberculous pleuritis.

Adenosine deaminase (ADA) activity and tuberculostearic acid (TSA) levels in pleural effusions were measured in 18 patients with active tuberculous pleuritis, 16 patients suspected of having tuberculous pleuritis, 14 patients with carcinomatous pleuritis, and 19 patients suffering from pleuritis of non-malignant and non-tuberculous etiology. In the patients with active tuberculous pleuritis, ADA was elevated in 56% and TSA was positive in 78%. In 83% of these patients, either ADA was elevated or TSA was positive. ADA was elevated together with a positive TSA in 50%. In contrast, TSA was positive in only 6% and ADA was elevated in 24% of the patients with non-tuberculous pleuritis, and none of these patients showed the combination of an elevation of ADA and a positive TSA. These results suggest that simultaneous measurements of both ADA and TSA in pleural effusions are useful for the diagnosis of tuberculous pleuritis.

Time-dependent alterations of leukotriene production and catabolism in rat peritoneal macrophages following intraperitoneal injection of thioglycollate broth.

Alterations of leukotriene (LT) productivity in peritoneal macrophages (PM) from untreated rats (control) as well as from rats treated i.p. with thioglycollate broth (TG) were investigated on days 3, 7 and 14 after TG administration. The resident PM from the untreated rats produced mainly LTB4 and 5-HETE with small amounts of 12-HETE and LTD4 with only a trace of LTC4 when stimulated with the calcium ionophore A23187. The PM elicited from rats on days 3 and 7 produced more LTC4 than did the resident PM but fewer other lipoxygenase metabolites. On day 14, however, the elicited PM resembled the resident PM in terms of lipoxygenase metabolite production. Similar results were achieved in the presence of arachidonic acid and A23187. A decrease in lipoxygenase metabolism in the elicited PM was also suggested by using opsonized zymosan. Catabolism studies indicated a reduction in r-glutamyl transpeptidase activity in the elicited PM and suggested a reduction in catabolism for LTB4 in the former cells. The authors conclude that the TG-elicited PM generate fewer lipoxygenase metabolites than the resident PM following stimulation, but show a preferential conversion of LTA4 to sulfidopeptide LTs rather than to LTB4. The elicited PM also show a reduced catabolism for LTC4 and LTB4.

Macromolecule-macromolecule interaction in drug distribution: effect of alpha-globulin concentration on the hepatic uptake of fractionated 3H-heparin by perfused rat liver.

The effect of alpha-globulin, the dominant binding protein for fractionated 3H-heparin, on the hepatic uptake of 3H-heparin was studied by liver perfusion experiments in rats. Fractionated 3H-heparin concentration in the recirculated perfusate decline one-exponentially with time for each of six initial concentration levels of alpha-globulin. The hepatic uptake clearance of fractionated 3H-heparin was 0.154 ml/min/g liver in the absence of alpha-globulin, and it decreased with increasing alpha-globulin concentrations. This result indicates that the hepatic uptake rate of alpha-globulin-bound fractionated 3H-heparin is lower than that of unbound fractionated 3H-heparin. On the other hand, it was indicated that almost all fractionated 3H-heparin binds to alpha-globulin at 8 mg/ml of alpha-globulin in in vitro study. However, the hepatic uptake clearance of the heparin at the concentration was of a certain value that could not be ignored. It was suggested that alpha-globulin-bound fractionated 3H-heparin also contributed to the hepatic uptake of fractionated 3H-heparin. Therefore, a protein-mediated transport system, which has been reported for some low molecular weight drugs, may also exist in the hepatic uptake of such a high molecular weight compound as fractionated 3H-heparin.

Enhanced leukotriene C4 synthase activity in thioglycollate-elicited peritoneal macrophages.

The utilization of LTA4 by peritoneal macrophages (MO) obtained from untreated rats (control) as well as by those elicited from rats was investigated at designated intervals (on days 3, 7, and 14) following the intraperitoneal injection of thioglycollate (TG). On day 7 following the injection the elicited MO converted LTA4 to LTC4 at the highest rate while the resident MO showed the lowest rate. The conversion of LTA4 to LTC4 and LTB4 was next examined by using each MO lysate. The apparent LTC4 synthase activity was significantly higher in the MO lysate both on day 3 and day 7, with the latter being the highest value obtained. The GSH S-transferase activity in each lysate using as the substrate, DNCB was significantly lower on day 3 but significantly higher on day 7 as compared to control values. However, this elevated activity was less variable than that observed with LTC4 synthase. The possible implication for these observations is discussed.

Measurement of tuberculostearic acid in sputa, pleural effusions, and bronchial washings. A clinical evaluation for diagnosis of pulmonary tuberculosis.

Gas chromatography/mass spectrometry combined with selected ion monitoring was used to measure tuberculostearic acid (TSA), in sputum, pleural effusion, and bronchial washing. The detection limit corresponded to the amount of TSA eluted from as low as 10(3) tubercle bacilli. Sputa were collected from 169 patients with active pulmonary tuberculosis, 35 clinically suspected to be active, 53 with obsolete pulmonary tuberculosis, and 160 with pulmonary diseases other than tuberculosis. TSA was positive in 90% of the patients with active pulmonary tuberculosis (152/169) and 71% of the clinically suspected cases (25/35), respectively. In contrast, less than 10% of the patients with obsolete tuberculosis or other pulmonary diseases had a positive TSA. Pleural effusions and bronchial washings were also collected from patients with active tuberculosis and from those with other diseases, as the controls. TSA in pleural effusions and bronchial washings was detected in 24 of 32 patients and 15 of 22 patients with active tuberculosis, respectively. In those with pulmonary diseases other than tuberculosis, only 8.7% of pleural effusion (4/46) and 4.3% of bronchial washing samples (3/69) showed a positive TSA. Therefore, the measurement of TSA is useful as a rapid and sensitive method for diagnosing pulmonary tuberculosis.