3-1. Chemicals and reagents
The Monoclonal Anti-b-Actin IgG, glyceraldehyde-3-phosphate-dehydrogenase antibody (AC-15; #A5441), horseradish peroxidase (HRP) labeled secondary antibodies (donkey anti-goat IgG and goat anti-rabbit IgG; #sc2020 and #sc2004), and the rabbit polyclonal IgG, Ikaros antibody (H-100; #sc13039), were purchased from Santa Cruz Biotechnology Inc.
(Santa Cruz, CA, USA). Anti-ACE2 (#ab59351) antibodies were purchased from Abcam (Cambridge, MA, USA). Bleomycin and Tetracycline were purchased from Sigma-Aldrich (St.Louis, MO, USA). The ACE2 overexpression lentiviruses were purchased from Vectorite Biomedica Inc. (Vectorite Biomedica, Taipei, Taiwan). The ACE2 inhibitor, DX600, ACE and ACE2 fluorescence substrate, Mca-APK (Dnp), were purchased from Ana Spec (Fremont, CA, USA). All other reagents were obtained from Sigma-Aldrich.
3-2. Pleural effusion samples collection and analyses
Pleural effusions from 125 patients were processed in our laboratory from August 2010 to December 2011. All pleural effusions were designated as transudates or exudates according to Light’s criteria. Definitive diagnosis of tuberculous, pneumonia, or adenocarcinoma
effusions for the exudate was verified by examining effusion biochemistry, cytology, acid-fast staining, and clinical follow-up. The study was performed with the approval of the
Institutional Review Board of Mackay Memorial Hospital. Informed consent was obtained from all patients.
According to Light’s criteria, pleural effusions were divided into 45 transudative pleural effusions and 80 exudative pleural effusions. The exudative pleural effusions were further divided into tuberculous pleural effusions (20 patients), pneumonia pleural effusions (32 patients), and malignant pleural effusions (28 patients). Fresh pleural fluid was collected in sterile tubes without anticoagulant reagents to prevent the release of gelatinases during platelet activation, and the tubes were immediately centrifuged at 3,000 × g for 30 min to separate the supernatants and cell pellets. The supernatants were aliquoted and stored with the cell pellets at −80°C until further use.
For each pleural effusion sample, the routine pleural analyses included total protein, lactate dehydrogenase (LDH), glucose, and white blood cell (WBC) count. In addition, the activities of ACE, ACE2, ADA , gelatinases (MMP-2 and MMP-9), TGF-β1 and TNF-α in the pleural effusions were measured.
3-3. ADA activity
ADA activity was measured or determined in the pleural fluids with a commercial colorimetric assay kit (Bio Quant, San Diego, CA, USA) (Valdés et al., 1996). Adenosine deaminase (ADA) isoenzyme analysis in pleural effusions: Diagnostic role and relevance to the origin of increased ADA in tuberculous pleurisy. The absorbance of the quinone dye generated in the last step of the ADA reaction series was monitored by absorbance at 550 nm with an ELISA reader (Bio-Rad model 550; Hercules, CA, USA).
3-4. ACE and ACE2 activity assay
ACE and ACE2 activities were assayed with the fluorogenic substrates Mca-YVADAPK and Mca-APK-Dnp (AnaSpec, San Jose, CA, USA), according to Vickers et al. with slight modifications (Vickers et al., 2002). The assay was performed in a microquartz cuvette with 20 μL pleural fluid, 50 μM fluorogenic substrate and protease inhibitor cocktail (1: 200;
Sigma-Aldrich) in a final volume of 100 μL in ACE or ACE2 assay buffer. The reaction was followed kinetically for 1 hour using a fluorescence reader at an excitation wavelength of 330 nm and an emission wavelength of 390 nm. All samples were fitted and plotted using Grafit v.
4.0 (Sigma-Aldrich), and enzyme activity was expressed as RFU/hour/mL. Parallel samples were incubated with the above mentioned reaction mixture in the presence of 1 μM captopril (Sigma-Aldrich), a specific ACE inhibitor for determining specific ACE activity, or 1 μM DX600 (AnaSpec), a specific ACE2 inhibitor for determining specific ACE2 activity.
3-5. Gelatin zymography assay
The MMP-2 and -9 activities were detected by gelatin zymography utilized
gelatin-containing gels as our previous report (Chang et al., 2011). Plasma was mixed with 2x
zymography sample buffer (0.125 M Tris-HCl, pH 6.8, 20% (v/v) glycerol, 4% (w/v) SDS, and 0.005% bromophenol blue) incubated for 10 min at room temperature, and then loaded into SDS-PAGE which was performed in 8% acrylamide gels containing 0.1% (w/v) gelatin (Sigma-Aldrich). After electrophoresis under power supply of 100 V, the gel was washed twice for 30 min in zymography renaturing buffer (2.5% Triton X-100) with gentle shake at room temperature to remove SDS, then incubated 18 hour at 37°C in reaction buffer (50 mM Tris-HCl, pH 7.4, 200 mM NaCl, 5 mM CaCl2, and 0.02% Brij35). The gels were then stained with Coomossie blue for 30 min prior to destain with destain buffer (50% methanol, 10%
acetic acid, and 40% ddH2O). The presence of enzyme activity was evident by clear or unstained zones, indicating the action of the enzyme on the gelatin substrate (Stawowy et al., 2004). Gelatinase activities in the gel slabs were quantified by Scion Image software (NIH, Bethesda, MD, USA), which quantifies the area of bands hydrolyzed by gelatinase. A MMP-2 or MMP-9 positive controls (Chemicon, Temecula, CA, USA) was contained in each gel as a standard intensity value to normalize sample intensity and express in arbitrary units.
3-6. Enzyme-linked immunosorbent assay (ELISA)
Pleural fluids were analyzed for TGF-β1 or TNF-α using sandwich ELISA. Each recombinant human TGF-β1 or recombinant human TNF-α were used as a standard. Pleural fluids were incubated in ELISA plates in which wells had been coated with anti-human TGF-β1 or anti-human TNF-α primary antibodies. Following addition of biotinylated antibodies, the plates were washed and reacted with HRP-conjugated streptavidin.
Tetramethylbenzidine (TMB) one- step substrate tablets were used to detect TGF-β1 or TNF-α activity and the product was measured at 450 nm using a micro-plate reader (Bio-Rad Laboratories).
3-7. ACE2 knockout mice
ACE2 knockout mice were established by Gurley et al. (2006). The ace2 gene consists of 18 exons, and the exon 1 was targeted by homologous recombination. The exon containing nucleotides +1069 to +1299 encoding the active site of the ACE2 enzyme (including the Zn-binding signature motif, HEMGH) was replaced with a NEO/URA3 cassette to obtain the targeting vector which disrupted ace2 gene (Fig. 3-1). The targeting construct was
electroporated into MPI1-12D ES cells that had been derived from 129/SvEvfBRTac mice and then injected into C57BL/6H blastocysts to generate chimeras.
The male chimeras were crossed with C57BL/6J female mice to obtain male hemizygous mutants and female heterozygous and homozygous females mutants. The ACE2 KO mice utilized in this study was named B6;129S5-Ace2tm1Lex/Mmcd (MMRRC:31665) and obtain from Mutant Mouse Regional Resource Centers (MMRRC). The first generation of ACE2 KO mice we obtained from MMRRC was bred in National laboratory animal center (NLAC) and distinguished between hemizygous, heterozygous and homozygous mutants by DNA genotyping.
Fig. 3-1. Strategy for producing targeted disruption of the ace2 gene. Strategy for producing targeted disruption of the ace2 gene. In the targeting vector, the exon containing nucleotides +1069 to +1299 encoding the active site of the ACE2 enzyme (including the Zn-binding signature motif, HEMGH) was replaced with a NEO/URA3 cassette from YCpLac22 plasmid. (Gurley et al, 2006)
3-8. RNA isolation and quantification
Total tissue RNA was extracted using TRIzol Plus RNA Purification System (Invitrogen) following the manufacturer’s recommendations and procedures reported by Pan et al. (Pan et al., 2008). Briefly, 1 mL of TRIzol reagent was added to one part of lung tissue. The mixture was vigorously agitated for 30 sec and incubated at room temperature for 5 min. Next, 200 μL chloroform was added and the solution was centrifuged at 12,000 x g for 15 min. The aqueous phase was transferred to a clean tube, precipitated with 500 μL of isopropyl alcohol, and centrifuged at 12,000 x g for 15 min. The resulting RNA pellet was washed with 1 mL of 75%
cold ethanol (−20°C) and centrifuged at 12,000 x g at 4°C for 5 min. The pellet was dried at room temperature, resuspended in 25 μL of diethylpyrocarbonate (DEPC)-treated water, and stored at −80°C. RNA was quantified by measuring the absorbance at 260 nm and 280 nm, and was electrophoresed on a denaturing 1% agarose gel.
3-9. Reverse transcription-polymerase chain reaction (RT-PCR) and Real time polymerase chain reaction
The cDNA was synthesized using ReverTra Ace Set (Toyobo, Osaka, Japan). For cDNA synthesis, 3 μg of RNA was reverse transcribed in a total reaction volume of 20 μL with 1 x reverse transcription buffer, 0.5 mM of dNTPs, 2.5 μM of oligo-dT (TOYOBO, Osaka, Japan), 1 U/μL of RNase inhibitor (TOYOBO), and 5 U/μL of ReverTra AceTM reverse transcriptase (TOYOBO). After incubation for 60 min at 42°C, the mixture was incubated for 5 min at 95
°C to denature the products. The PCR reactions contained 2 μL of cDNA, 2 μL of each primer (10 μM), 5 μL of 10 x PCR buffer, 2 μL of 10 mM of dNTPs, 1 μL of 5 U/μL Taq polymerase (Promega, Madison, WI, USA), and 36 μL distilled water in a total volume of 50 μL. Thermal cycler (MiniCyclerTM; MJ Research, Waltham, MA, USA) conditions were 5 min at 94°C followed by 18-36 cycles of denaturation (94°C for 30 sec), annealing (55°C for 30 sec), and elongation (72°C for 45 sec). The resulting PCR products were visualized on 2% agarose gels stained with ethidium bromide. The stained image was recorded by an image analyzer (Kodak DC290 Digital Camera SystemTM; Eastman Kodak, Rochester, NY, USA). Band intensity was quantified using densitometric analysis by ImageJTM. The relative mRNA expression of the determined gene was normalized as a ratio to GAPDH expression.
Semi-quantitative real-time (RT) PCR was performed using SYBR Green Realtime PCR
Master Mix Plus (Toyobo) with 20 pM of each primer and 5 μL cDNA, in a total volume of 25 μL and monitored using Prism 7000 Sequence Detection System (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s recommendations. Specificity of the real-time PCR was confirmed by routine agarose gel electrophoresis and melting-curve analysis, according to a published method (Livak et al., 2001). Expression of the GAPDH (GenBank ID: NM_002046.3) gene was used as an internal standard. The primers for ACE2 (GenBank ID: AF291820 and NM002046.3), were: ACE2 forward, hACE2-F,
5’-CATTGGAGCAAGTGTTGGATCTT-3’, and, ACE2 reverse, hACE2-R, 5′
-GAGCTAATGCATGCCATTCTCA-3′; GAPDH forward, hGAPDH-F, 5′
-ACAGTCAGCCGCATCTTCTT-3′, and, GAPDH reverse, hGAPDH-R, 5′
-GTTAAAAGCAGCCCTGGTGA-3′.
3-10. Animal model, induction of lung fibrosis and treatment of mice with Lenti-ACE2
Control animals were wild-type B6;129S5 and ACE2 KO mice (ACE2-/y, ACE2 +/-andACE2-/-) weighing 20-25 g obtained from the National laboratory animal center (NLAC), and mice were randomly assigned into 9 groups of 3-5 animals per group. They were house in a plastic suspended cage placed in a well-ventilated mice house, provided mice pellets and water ad libitum, and subject to a natural photoperiod of 12 hour light and 12 hour dark cycle.
After the animals were anaesthetised by the intraperitoneal injection of 40mg/kg pentobarbital, the right chest was cleansed with an Et-OH solution and a 25-gauge needle attached to a 1-mL syringe containing the solution was inserted through the skin and chest muscles, 1 cm lateral to the right parasternal line. The plunger of the syringe was removed and the needle was slowly advanced until it reached the pleural space, where the subatmospheric intrapleural pressure allowed the fluid to enter the pleural cavity spontaneously. Received 0.1 mL of intrapleural 1 U/kg bleomycin or 20 mg/kg tetracycline. A Control animals group received PBS. The mice were monitored after the procedure until they completely awakened. Mice treated with PBS, bleomycin and tetracycline were sacrificed after 3 days, 7 days and 28 days.
The animals were injected of pentobarbital and the thorax was dissected in order excise the right lung. Empty virus (Control), lenti-ACE2 viral particles (3 x 106 U in 100 μL of phosphate-buffered saline) were injected through the tail vein of ACE2 KO mice, using a
27-gauge syringe needle. One week after lentiviral treatment, animals were subjected to bleomycin or tetracycline administration. All experimental procedures were carried out in accordance with the guidelines of the Institutional Animal Care and Use Committee of National Chiao Tung University, Every effort was made to minimize the suffering of the animals and the number of animals used.
3-11. Sample preparation
3-11-1. Protein extraction
The organ samples were supplied by B6;129S5 and ACE2 KO mice (ACE2-/y, ACE2 +/-andACE2-/-) mice and used for further analyses was prepared as described. The organ samples were collected and were weighted 100 mg, and then homogenous for 3 to 5 times by lysis buffer PRO-PREPTM Protein Extraction Solution (iNtRON Biotechnology, Inc., Kyungki-Do, Korea). Samples were centrifuged at 13,000 x g for 10 min to separate the supernatants and pellets. The total amount of protein in homogeneous extract was measured by the Bradford dye binding assay (Bio-Rad Laboratories, Hercules, CA, USA) and bovine serum albumin as the standard. The supernatants were aliquoted and stored at - 80°C until further use.
3-11-2. Histological determination of fibrosis
The organ samples were supplied by B6;129S5 and ACE2 KO mice (ACE2-/y, ACE2 +/-andACE2-/-) mice and a part of right lung were excised and encased in 10% formaldehyde prepared for Masson's Trichrome and hematoxylin-eosin staining. Inflation fixed lungs were stained with Masson's Trichrome; muscles and cells are stained red, nuclei black and collagen blue. The stained sections were photographed using a digital camera mounted on a
microscope. Manual planimetry was performed on the microscope using PALM
RoboSoftware v2.2 using hematoxylin-eosin-stained slices.A computerized microscope equippedwith a high-resolution video camera (BX 51, Olympus, Tokyo, Japan,magnification 100 x) was used for morphometric analysis.
3-12. Western blotting
The Western blot for TIMP1 and ß-actin was carried out as our previous report. Aliquots containing 20 μg protein were electrophoresed on 12% SDS-PAGE gels and then transferred electrophoretically to polyvinylidene fluoride membranes (Immobilon-PTM; Millipore, Bedford, MA, USA) by semi-dry electro-blotting (HoeferTM). Briefly, nonspecific binding sites were blocked by incubating the membranes in 5% non-fat milk in Tris-buffered saline.
Primary antibodies against proteins were diluted 1:1,000 for TIMP1 and ß-actin. The secondary antibodies were applied using a dilution of 1:2,000. Substrates were visualized using enhanced chemiluminescence detection (Western Lightning Plus-ECL, Enhanced
Chemiluminescence Substrate; PerkinElmer, Boston, MA, USA) and exposing the membranes to X-ray film (Fujifilm). The bands on the film were detected at the anticipated location, based on size. Band intensity was quantified by densitometric analysis using Scion Image software (Scion, Frederick, MD, USA). The amounts of TIMP-1 were expressed relative to the amount of ß-actin (as the internal standard) in each sample.
3-13. Statistical analysis
All values were expressed as mean ± standard deviation (SD). Data were compared with one-way analysis of variance (ANOVA) test to evaluate differences among multiple groups.
The Student’s t-test was used for comparisons involving two groups. All results are expressed as the mean ± standard deviation (SD). Differences were considered statistically significant when p < 0.05. Statistical analysis was performed using statistical software (SPSS, Chicago, IL, USA).
IV. Results
Part I: MMPs and ACE/ACE2 in pleural effusion
4-1-1. General characteristics of pleural effusions
The pleural fluid characteristics of 125 patient s included in the study are presented in Table 4-1-1 and Table 4-1-2. Generally, total protein, LDH, and the number of WBC in exudative effusions (n = 80) were significantly higher than those in transudative effusions (n
= 45) (p < 0.001), whereas a lower glucose level was detected in the pleural fluid of exudates compared with that in transudates (p < 0.001).
4-1-2. ACE and ACE2 activities in transudates and exudates
ACE and ACE2 activities in the pleural effusions of all patients were determined. ACE activity in exudative effusions was higher than that in transudative effusions (0.74 (0.43–1.47) RFU/hour/mL vs. 0.57 (0.32–0.9) RFU/hour/mL, respectively, p < 0.01) (Fig. 4-1-2A). On the contrary, ACE2 activity in exudative effusions was lower than that in transudative effusions (1.58 (1.02–2.54) RFU/hour/mL vs. 1.98 (1.27–3.12) RFU/hour/mL, respectively) (Fig. 4-1-2B), but this difference was not statistically significant (p = 0.485). The ratio of ACE/ACE2 activity in the pleural effusions was significantly higher in exudative effusions than in transudative effusions (median 0.49 (0.27–0.91) vs. 0.27 (0.19–0.43), respectively, p <
0.001) (Fig. 4-1-2C). In transudates, a significant positive correlation was found between ACE and ACE2 activities (r = 0.456, p < 0.001) (Fig. 4-1-3A). However, the ACE level was not correlated with the ACE2 level in exudative effusions (r = 0.020, p = 0.214) (Fig.
4-1-3B).
4-1-3. MMP-2 and MMP-9 activities in transudates and exudates
The MMP-2 and MMP-9 activities in the pleural effusions of all patients were
determined with gelatin zymography. The activity level of MMP-2 in exudative effusions was comparable with that in transudative effusions (median, 1375 (944–1841) ng/mL vs. 1148 (806–1730) ng/mL, respectively) (Fig. 4-1-4A). However, MMP-9 activity in exudative
effusions was significantly higher than that in transudative effusions (median, 30 (19–52) ng/mL vs. 11 (8–23) ng/mL, respectively, p < 0.001) (Fig. 4-1-4B). In transudates, a
significant positive correlation was found between MMP-2 and MMP-9 activities (r = 0.544, p < 0.001). However, no strong correlation was found between MMP-2 and MMP-9 activities in exudates (r = 0.125, p < 0.01).
4-1-4. ACE and ACE2 levels in exudates from patients with different diseases
Elevated ACE activity and an elevated ACE/ACE2 ratio were observed in exudative effusions. We then differentiated both ACE and ACE2 enzyme levels in the exudates
according to different diseases, including tuberculosis, pneumonia, and adenocarcinoma. The level of ACE activity in the tuberculous pleural effusions was significantly higher than in pneumonia and adenocarcinoma effusions, by 2.89 (p < 0.001) and 2.62 (p < 0.001) folds, respectively (Fig. 4-1-5A). In contrast, ACE2 activity in the tuberculous effusions was significantly lower than in pneumonia and adenocarcinoma effusions, by 0.68 (p < 0.05) and 0.58 (p < 0.05) folds, respectively (Fig. 4-1-5B). According to the changes we detected in ACE and ACE2, a significantly higher difference in the ACE/ACE2 ratio in tuberculous effusions (median, 1.72 (0.15–2.52)) compared with ratios in pneumonia (0.32 (0.20–0.50)) and adenocarcinoma (0.43 (0.26–0.67)) effusions was also observed (p < 0.001) (Fig.
4-1-5C).
4-1-5. MMP-2, MMP-9, and ADA levels in exudates from patients with different diseases
In tuberculous pleural effusions, MMP-2 activity was 1.48 (p < 0.01) and 1.36 fold (p <
0.05) higher than MMP-2 activity in pneumonia and adenocarcinoma effusions, respectively (Fig. 4-1-6A). Similar to MMP-2, MMP-9 activity in tuberculosis effusions was significantly higher than MMP-9 activity in pneumonia and adenocarcinoma effusions by 1.62 (p < 0.01) and 1.77 fold (p < 0.01), respectively (Fig. 4-1-6B). In tuberculous effusions, significantly higher ADA activity was also detected compared with ADA activity in pneumonia and adenocarcinoma effusions (p < 0.001) (Fig. 4-1-6C).
4-1-6. ELISA for TGF-β1 concentration in pleural transudative and exudative effusions
The concentration of TGF-β1 in pleural transudative and exudative effusion (including TB, Pn and Ad effusions) was determined with ELISA. The concentration level of TGF-β1in exudative effusions was comparable with that in transudative effusions (median, 0.103 (0.007–1.113) ng/mL vs. 0.155 (0.007–1.314) ng/mL, respectively) (Fig. 4-1-7A). The
concentration of TGF-β1 was no differentiation between transudative and exudative effusions.
Even the concentration of TGF-β1 in different exudative effusions was no significant difference in each other’s.
4-1-7. ELISA for TNF-α concentration in pleural transudative and exudative effusions
The concentration of TNF-α in pleural transudative and exudative effusion (including TB, Pn and Ad effusions) was determined with ELISA. The concentration level of TNF-α in
exudative effusions was comparable with that in transudative effusions (median, 35.3 (6.3–
98.6) pg/mL vs. 30.3 (2.3–138.3) pg/mL, respectively) (Fig. 4-1-8A). The concentration of TNF-α was no differentiation between transudative and exudative effusions. However, a significantly higher difference in the concentration of TNF-α in pneumonia (39.9 (2.3–138.3)) compared with the concentration in adenocarcinoma (24.6 (6.3–76.6)) effusions was also observed (p < 0.01) (Fig. 4-1-8B).
Table 4-1-1. Pleural fluid characteristics of the study population
Transudates (n = 45) Exudates (n = 80)
Age (years) 74 12 66 18
Male/Female 29/16 49/31
Pleural fluid
White blood cells (cell/mm3) 305 326 927 1090 ***
Glucose (mmol/L) 176 77 130 58 ***
Total protein (g/L) 2.11 1.02 4.01 1.02 ***
Lactate dehydrogenase (U/L) 86 32 265 222 ***
Transudates were from patients with heart failure or liver cirrhosis.
Exudates were from patients with tuberculous, pneumonia, or adenocarcinoma effusions.
Data are the means SD. *** indicates p < 0.001 compared with transudates.
Table 4-1-2. Pleural fluid characteristics of tuberculous, pneumonia and adenocarcinoma effusions.
Pleural fluid Tuberculosis (n = 20)
Pneumonia (n = 32)
Adenocarcinoma (n = 28)
White blood (cell/mm3) 1,201 ± 1,284 825 ± 905 848 ± 1,099
Glucose (mmol/L) 126 ± 62 135 ± 53 126 ± 59
Total protein (g/L) 4.56 ± 1.25 3.75 ± 0.86* 4.14 ± 0.74
Lactate dehydrogenase (U/L) 313 ± 255 237 ± 239 264 ±164
Data are the means ±SD, * indicates p < 0.05.
Fig. 4-1-2. Enzymatic activity of ACE and ACE2 in pleural transudative and exudates effusions. The specific activities of ACE (A) and ACE2 (B) in pleural transudative (n = 45) and exudative (n = 80) effusions from 125 patients. The ratio of ACE/ACE2 in the pleural effusions (C). Pleural effusion (20 μL) was assayed for the ability to cleave the fluorescent substrate at 37°C for 1 hour with a specific ACE inhibitor or a specific ACE2 inhibitor. Each symbol represents one individual, and horizontal bars represent median values. ** and *** indicate p < 0.01 and p < 0.001, respectively, compared with transudates.
Fig. 4-1-3. Correlations between ACE and ACE2 activity in pleural effusion. ACE and ACE2 activities were measured in each sample of pleural transudative effusions (A; n = 45) and exudative effusions (B; n = 80). For correlation analysis, Pearson’s correlation analysis (SPSS statistics package, Chicago, IL) was applied. Statistically significant differences were established at p < 0.05. ACE and ACE2 positively correlate with each other in the group of transudative effusions (F(1,43) = 36.052, r = 0.456, p < 0.001) (A), but not in the group of exudative effusions (F(1,78) = 1.567, r = 0.020, p > 0.05) (B).
Fig. 4-1-4. MMP-2 and MMP-9 activities in pleural transudative and exudative effusions.
The activities of MMP-2 (A) and MMP-9 (B) in pleural transudative and exudative effusions from 125 patients were determined with zymography. The gelatinase activities detected in this study were based on pro-MMP-2 (72 kDa) and pro-MMP-9 (95 kDa). Each symbol represents one individual, and horizontal bars represent median values. *** indicates p < 0.001
compared with transudates.
Fig. 4-1-5. ACE and ACE2 activities in exudative effusions from patients with tuberculosis (TB), pneumonia (Pn), and
adenocarcinoma (Ad). The activities of ACE (A), ACE2 (B), and the ratio of ACE/ACE2 (C) in TB (n = 20), Pn (n = 32), and Ad (n = 28) effusions. Each symbol represents one individual, and horizontal bars represent median values. * and *** indicate p < 0.05 and p < 0.001, respectively, compared with values measured in Pn and/or Ad effusions.
Fig. 4-1-6. MMP-2, MMP-9, and ADA levels in exudative effusions from patients with tuberculosis (TB), pneumonia (Pn), and adenocarcinoma (Ad). The activities of MMP-2 (A), MMP-9 (B), and ADA (C) in TB (n=20), Pn (n=32), and Ad (n=28) effusions. Each symbol represents one individual, and horizontal bars represent median values. *, ** and *** indicate p < 0.05, p
< 0.01 and p < 0.001, respectively, compared with values measured in Pn and/or Ad effusions.
Fig. 4-1-7. ELISA for TGF-β1 concentration in pleural transudative and exudative effusions. The activities of TGF-β1 in pleural transudative and exudative effusions (A) and in TB, Pn and Ad effusions (B) were determined with ELISA. The concentration of TGF-β1 was no differentiation between transudative and exudative effusions. Each symbol represents one individual, and horizontal bars represent median values.
Fig. 4-1-8. ELISA for TNF-α concentration in pleural transudative and exudative effusions. The activities of TNF-α in pleural transudative and exudative effusions (A) and in TB, Pn and Ad effusions (B) were determined with ELISA. Each symbol represents one individual, and horizontal bars represent median values. Each symbol represents one
individual, and horizontal bars represent median values. ** indicates p < 0.01, Pn compared with values measured in Ad effusions.