Cystic fibrosis alpha 2-macroglobulin protease interaction in vitro

alpha 2-Macroglobulin was purified from plasma of five cystic fibrosis patients and five normal controls. SDS gel electrophoresis of native alpha 2-macroglobulin from cystic fibrosis patients and normal donors showed identical subunit molecular weights, as did trypsin cleavage products. Cystic fibrosis and control alpha 2-macroglobulins were indistinguishable by isoelectric focusing and exhibited appropriate shifts in isoelectric point following binding of trypsin. The trypsin-binding capacities of control and cystic fibrosis alpha 2-macroglobulins did not differ, nor did the esterolytic activity of the trypsin-alpha 2-macroglobulin complexes.     

Activity levels and properties of acid α-glucosidase from liver and neutral α-glucosidase from sera of cystic fibrosis patients and controls

The average activity levels of acid α-glucosidase are comparable in liver supernatant fluids for 15 cystic fibrosis patients and 12 controls (401 ± 131 and 347 ± 109 nmol/h/mg protein, respectively) and no significant differences were found for the cystic fibrosis and control liver acid α-glucosidases in their (a) apparent K_m values for the 4-methylumbelliferyl substrate (1.1 mmol/1), (b) pH optima (4.2) and thermostability curves and (c) isoelectric profiles (one form with an isoelectric point of 4.5 ± 0.2).In contrast, average neutral α-glucosidase activity levels were significantly increased (p < 0.0002) in sera from 21 cystic fibrosis patients compared to 15 controls (10.7 and 2.7 nmol/h/ml). This increased activity is not due to (a) different stability upon storage at ?20°C, (b) the presence of activators in cystic fibrosis sera or inhibitors in normal sera (as determined by mixing studies), (c) altered K_m values or (d) altered pH optima curves. Cystic fibrosis serum neutral α-glucosidase appears to be more thermostable and has a consistently altered isoelectric profile (greater percentage of activity above pI 4.8) when compared to the normal serum enzyme. This altered isoelectric composition may reflect changes in neutral α-glucosidase which contribute to its increased activity in cystic fibrosis sera.     

The contribution of α1-antitrypsin to the total antitrypsin activity of human serum. a study of two phenotype pi oo individuals

Comparison of the levels of α₁AT, α₂-M, inter α-AT, C₁ inactivator and antiplasmin and global antitrypsin activity in a group of normal phenotype PI MM individuals, a group of normal individuals with phenotypes with intermediate α₁AT activities and two α₂-AT-deficient persons show that α₁AT contributes more than 90% of the total antitrypsin activity of normal plasma.AT III and fast reacting antiplasmin are shown to contribute to the remaining activity. It can be assumed that due to test conditions the antitrypsin activity of α₂-M is not assessed. C₁ inactivator and inter α₁-AT do not contribute to a perceptible extent to the overall antitrypsin activity estimated according to the method of Eriksson (Eriksson, S. (1965) Acta Med. Scand. 177, 1).     

Calcium-alginate beads for the oral delivery of transforming growth factor-β1 (TGF-β1): stabilization of TGF-β1 by the addition of polyacrylic acid within acid-treated beads

The susceptibility of the gastrointestinal tract to the toxic effects of chemotherapeutic drugs remains a complication in chemotherapy. Recent studies have suggested that transforming growth factor-β₁ (TGF-β₁) can be used as a cytoprotectant against cell cycle specific drugs. This work describes the use of alginate beads as a potential oral delivery system for TGF-β₁ designed to release the drug in the lumen of the small intestine. TGF-β₁ encapsulation and extent of release from alginate beads approached 100% as determined by ¹²⁵I-labelled TGF-β₁. However, when assayed by ELISA and a growth inhibition assay, nearly all immunoreactivity and bioactivity was lost, apparently due to a very high affinity of the alginate for TGF-β₁. This limitation was overcome by two novel methods: (1) incorporation of selected polyanions within the alginate beads to ‘shield’ TGF-β₁ from interaction with alginate and (2) exposure of the alginate beads containing TGF-β₁ to 0.1 N HCl (acid treatment) to simultaneously reduce the molecular weight of the alginate and its ability to interact with TGF- β₁. If the beads were only acid treated, just 8% of the immunoreactivity ofTGF-β₁ was retained. If polyacrylic acid (90 kDa) was added to the beads, 50% of the immunoreactivity of TGF-β₁ was retained. However, when TGF-β₁ was released from acid-treated beads also containing polyacrylic acid, more than 80% of the TGF-β₁ remained immunoreactive and bioactive. The retained TGF-β₁ activity after release from the beads was found to continue to increase with increasing concentrations of polyacrylic acid, until a concentration was reached where beads would not form. The dramatic increase in retained TGF-β₁ activity is attributed to the ability of polyacrylic acid to shield TGF-β₁ from interaction with lower molecular fragments of alginate.     

Elastase binding capacity of α2-macroglobulin and its association with glucocorticoid concentration in southern African black patients with hepatocellular carcinoma

Thirty-three Southern African black patients with hepatocellular carcinoma (HCC) (7 women) and 43 black control individuals (14 women), all in the age group 18–45 years, were investigated for plasma α₂-macroglobulin (α₂M) elastase binding capacity (EBC). Cortisol levels were measured in 15 (3 women) of the HCC patients and 10 (5 women) of the control subjects. A significant difference in EBC was found between the HCC patients and the control subjects (P < 0.001). A significant difference was also found in cortisol levels between the two groups (P < 0.001). A significant correlation between EBC and cortisol levels was obtained (r = 0.57; P < 0.042). The significant increase in EBC of α₂M in HCC patients could be due to an increase in circulating cortisol.     

β-N-Acetylhexosaminadases in human cerebrospinal fluid and serum of patients with multiple sclerosis

β-N-Acetylhexosaminidase activity and isoenzyme have been investigated in normal human cerebrospinal fluid and that of patients with multiple sclerosis. β-N-acetylhexosaminidase activity in normal cerebrospinal fluids has been resolved into five components. The major component was in a form that eluted from DEAE cellulose at the same salt concentration as hexosaminidase As, the isoenzyme previously identified in human serum. Cerebrospinal fluid from patients exhibited a different isoenzyme profile, showing a remarkable increase in a form having a pI which was more acidic than that of As. These changes have a potential use in the diagnosis and further biochemical characterization of multiple sclerosis.     

THE FORMATION AND PROPERTIES OF POLIOVIRUS-NEUTRALIZING ANTIBODY : IV. NORMAL ANTIBODY AND EARLY IMMUNE ANTIBODY OF RABBIT ORIGIN: A COMPARISON OF BIOLOGICAL AND PHYSICOCHEMICAL PROPERTIES

Rabbit sera were found to possess neutralizing activity (normal antibody) to polioviruses and Coxsackie B viruses. This normal antibody showed high specificity in cross-neutralization and absorption tests. It was associated with heat-stable, mercaptan-sensitive, 19S γ₁-β-macroglobulins, which formed weak complexes with the viral antigen. In rare instances, sera with normal macroglobulin antibody, also contained very low activity which was due to 7S γ₂-globulins. The neutralization of poliovirus by normal 19S γ₁-β-antibody appeared to follow first order kinetics, and the thermodynamic parameters of this reaction were the same as those of serological reactions employing immune antibody. The electrophoretic mobility, sedimentation properties, sensitivity to mercaptan, thermostability, and avidity of normal and early (up to day 3) immune antibodies to poliovirus were similar, but differed in several respects from those of late immune antibodies. Thus, the available evidence suggested, that earlier reported differences between normal and immune antibodies reflected differences between antibodies of diverse physicochemical properties rather than between normal and immune antibodies per se. It is proposed that the normal macroglobulin antibody is associated with an immunological response to repeated stimulation with minute amounts of antigen.     

Cardiovascular magnetic resonance of cardiomyopathy in limb girdle muscular dystrophy 2B and 2I

Background

Limb girdle muscular dystrophies (LGMD) are inclusive of 7 autosomal dominant and 14 autosomal recessive disorders featuring progressive muscle weakness and atrophy. Studies of cardiac function have not yet been well-defined in deficiencies of dysferlin (LGMD2B) and fukutin related protein (LGMD2I). In this study of patients with these two forms of limb girdle muscular dystrophy, cardiovascular magnetic resonance (CMR) was used to more specifically define markers of cardiomyopathy including systolic dysfunction, myocardial fibrosis, and diastolic dysfunction.

Methods

Consecutive patients with genetically-proven LGMD types 2I (n = 7) and 2B (n = 9) and 8 control subjects were enrolled. All subjects underwent cardiac magnetic resonance (CMR) on a standard 1.5 Tesla clinical scanner with cine imaging for left ventricular (LV) volume and ejection fraction (EF) measurement, vector velocity analysis of cine data to calculate myocardial strain, and late post-gadolinium enhancement imaging (LGE) to assess for myocardial fibrosis.

Results

Sixteen LGMD patients (7 LGMD2I, 9 LGMD2B), and 8 control subjects completed CMR. All but one patient had normal LV size and systolic function; one (type 2I) had severe dilated cardiomyopathy. Of 15 LGMD patients with normal systolic function, LGE imaging revealed focal myocardial fibrosis in 7 (47%). Peak systolic circumferential strain rates were similar in patients vs. controls: ε_endo was -23.8 ± 8.5vs. -23.9 ± 4.2%, ε_epi was -11.5 ± 1.7% vs. -10.1 ± 4.2% (p = NS for all). Five of 7 LGE-positive patients had grade I diastolic dysfunction [2I (n = 2), 2B (n = 3)]. that was not present in any LGE-negative patients or controls.

Conclusions

LGMD2I and LGMD2B generally result in mild structural and functional cardiac abnormalities, though severe dilated cardiomyopathy may occur. Long-term studies are warranted to evaluate the prognostic significance of subclinical fibrosis detected by CMR in these patients.     

Conformational state and receptor recognition of the C-terminal domain of human α2-macroglobulin after dissociation into half-molecules

Background: Dissociation of native human α₂-macroglobulin (α₂M) by sodium thiocyanate generates stable half-molecules with intact thiol esters. Significant conformational changes occur by the dissociation, which are similar to those occurring by transformation from native to methylamine-treated α₂-macroglobulin. Methods: The conformational state of the receptor-binding domain of the half-molecules was investigated by receptor binding and clearance studies, and by use of a panel of 11 monoclonal antibodies (mAbs) specific for the 18-kDa C-terminal receptor-binding fragment of α₂-macroglobulin. Results: The half-molecules simultaneously express epitopes specific for native, as well as epitopes specific for transformed α₂-macroglobulin. While it is possible to immunochemically discriminate between the different forms of tetrameric protein, the half-molecules retain a conformational state with no observed conformational changes in the C-terminal domain following cleavage of thiol esters or bait regions. The in vivo clearance rate in mice was consequently significantly slower for the half-molecules than for the tetrameric receptor-recognized forms of α₂-macroglobulin. Furthermore, half-molecules demonstrate lower affinity for binding to mouse macrophages than methylamine-treated tetrameric α₂-macroglobulin in competition studies. Conclusions: It is suggested that contact zones are functionally important for mediating conformational switches, which result in trapping and exposure of the receptor-binding sites.     

SUBSTRATES OF HAGEMAN FACTOR : I. Isolation and Characterization of Human Factor XI (PTA) and Inhibition of the Activated Enzyme by α1-Antitrypsin

Unactivated partial thromboplastin antecedent (PTA) has been purified by sequential chromatography of plasma on quaternary aminoethyl Sephadex, sulphoprophyl Sephadex, Sephadex G-150, and passage over an anti-IgG immunoadsorbant. The preparation gave a single band after alkaline disc gel electrophoresis, sodium dodecyl sulfate (SDS) gel electrophoresis and isoelectric focusing in acrylamide gels and was found to have a mol wt of 175,000 by gel filtration, 163,000 by SDS gel electrophoresis, and an isoelectric point of 8.8–9.4 (peak 9.0–9.1). Pre-PTA was activated directly by activated Hageman factor or by Hageman factor prealbumin fragments. Its coagulant activity was inhibited by DFP, soybean trypsin inhibitor and trasylol but not by lima bean trypsin inhibitor or ovomucoid trypsin inhibitor indicating that activated PTA possesses the same inhibition profile utilizing these reagents as does plasma kallikrein. A major plasma inhibitor of activated PTA was found to be a 65,000 mol wt α-globulin which was isolated free of α₁-chymotrypsin inhibitor, inter α-trypsin inhibitor, α₂-macroglobulin, and the other known inhibitors of activated PTA, the activated first component of complement (C1 INH), and antithrombin III. Its physicochemical properties were identical to α₁-antitrypsin, and it was absent in α₁-antitrypsin-deficient plasma thereby identifying this PTA inhibitor as α₁-antitrypsin.     

Susceptibility to neuraminidase of α-l-fucosidase and N-acetyl-β-d-glucosaminidase of cystic fibrosis, I-cell and neuraminidase-deficient fibroblasts

Intracellular α-l-fucosidase and hexosaminidase showed similar isoelectrofocusing patterns in control, cystic fibrosis and neuraminidase-deficient fibroblasts and were unaffected by neuraminidase treatment. An I-cell strain excreted these two enzymes at 3–4 times the rate of the three other cell types. I-cell and neuraminidase-deficient cells excreted more of the electronegative forms of these enzymes than control and cystic fibrosis cells. Extracellular hexosaminidase A and B were both sensitive to neuraminidase for the four cell types. Extracellular α-l-fucosidase consisted of a pH 6.1 form insensitive to neuraminidase and other forms that were sensitive and changed to a pI 7.0–7.1 form. Cystic fibrosis extracellular a-l-fucosidase and hexosaminidase behaved as for control fibroblasts.     

Impact of Different Etiologies of Bronchiectasis on the Pulmonary Function Tests

Background

Bronchiectasis develops along the natural course of several respiratory and systemic conditions and induces significant changes in the morphofunctional structure of airways. Our objective was to assess the impact of various causes of bronchiectasis on clinical data, pulmonary function tests, and high-resolution computed tomography (HRCT).

Methods

The present report was a cross-sectional study that was conducted with 112 consecutive patients with bronchiectasis, who were allocated to five groups, as follows: sequelae of tuberculosis, history of non-tuberculosis infection, cystic fibrosis (CF), primary ciliary dyskinesia (PCD), and rheumatoid arthritis. All of the participants underwent spirometry, whole-body plethysmography, measurement of the diffusing capacity for carbon monoxide (DLco), and HRCT.

Results

The highest HRCT score was exhibited in patients with CF (6.03±1.03). The values of forced expiratory volume in 1 second (FEV₁) (52.2±17.7%) and DLco (74.1±15.2%) were lower in patients with sequelae of tuberculosis. The increase in the residual volume was more accentuated in the patients with CF (193.5 ± 39.5%) and PCD (189 ± 36.4%). By the multivariate analysis, the cause of FEV₁ and bronchiectasis, HRCT score, and degree of dyspnea behaved as independent predictors of DLco.

Conclusion

In individuals with bronchiectasis, the pulmonary function abnormalities are associated with the etiology of the underlying disease.     

The β3 subunit of the Na,K-ATPase mediates variable nociceptive sensitivity in the formalin test

It is widely appreciated that there is significant inter-individual variability in pain sensitivity, yet only a handful of contributing genetic variants have been identified. Computational genetic mapping and quantitative trait locus analysis suggested that variation within the gene coding for the β₃ subunit of the Na⁺,K⁺-ATPase pump (Atp1b3) contributes to inter-strain differences in the early phase formalin pain behavior. Significant strain differences in Atp1b3 gene expression, β₃ protein expression, and biophysical properties of the Na⁺,K⁺ pump in dorsal root ganglia neurons from resistant (A/J) and sensitive (C57BL/6J) mouse strains supported the genetic prediction. Furthermore, in vivo siRNA knockdown of the β₃ subunit produced strain-specific changes in the early phase pain response, completely rescuing the strain difference. These findings indicate that the β₃ subunit of the Na⁺,K⁺-ATPase is a novel determinant of nociceptive sensitivity and further supports the notion that pain variability genes can have very selective effects on individual pain modalities.     

HUMAN PLASMA ALPHA 2-MACROGLOBULIN : AN INHIBITOR OF PLASMA KALLIKREIN

Activation of plasma kallikrein arginine esterase activity by kaolin resulted in peak activity at 1 min of incubation and a 50% reduction in activity at 5 min in normal plasma, and 30% reduction in the plasma of patients with hereditary angioneurotic edema who lacked the C1 inactivator. The peak esterolytic activity was inhibited by soybean trypsin inhibitor whereas the 5 min activity was resistant to this inhibitor. Acid treatment of normal and hereditary angioneurotic edema plasma destroyed the factor responsible for the fall in esterase activity at 5 min and the factor which rendered the esterase resistant to soybean trypsin inhibitor. Purified α₂-macroglobulin inhibited approximately 50% of the TAMe esterase activity of purified plasma kallikrein without changing its activity toward basic amino acid esters. The interaction between the α₂-macroglobulin and kallikrein resulted in alterations in the gel filtration chromatographic pattern of the TAMe esterase and biologic activity of kallikrein, indicating that kallikrein was bound to the α₂-macroglobulin. The TAMe esterase activity of this complex, isolated by column chromatography, was resistant to C1 inactivator and SBTI. Studies of incubation mixtures of kallikrein, α₂-macroglobulin and C1 inactivator suggested that these inhibitors compete for the enzyme, and that the α₂-macroglobulin partially protects the esterase activity of kallikrein from C1 inactivator. The α₂-macroglobulin isolated from kaolin-activated plasma possessed 240 times the esterolytic activity of the α₂-macroglobulin purified from plasma treated with inhibitors of kallikrein and of its activation. The α₂-macroglobulin blocked the uterine-containing activity and vascular permeability-inducing effects of plasma kallikrein. These studies suggest that the α₂-macroglobulin is a major plasma inhibitor of kallikrein and provide a new example of an interrelationship between the coagulation, fibrinolytic, and kallikrein enzyme systems.     

FACTORS CONTROLLING SERUM γ-GLOBULIN CONCENTRATION

Both synthetic and catabolic processes determine the serum γ-globulin level. The rate of γ-globulin synthesis appears to be the primary factor determining the amount of serum γ-globulin. Increase of γ-globulin synthesis (as may occur following immunization or development of plasma cell tumor) elevates the serum γ-globulin level. This, in turn, accelerates the fractional rate of γ-globulin catabolism. The change in catabolic rate reduces the dimensions of the serum change from that which would occur if synthesis alone determined the serum γ-globulin level. The present studies indicate the existence of a homeostatic mechanism controlling the rate of γ-globulin catabolism. The mechanisms of γ-globulin catabolism are specific and selective. Marked serum increase of other immunoglobulin components (β_2A-globulins and γ₁-macroglobulins) do not accelerate γ-globulin catabolism. Similarly, serum albumin increases do not influence γ-globulin catabolism. The site determining γ-globulin catabolism is restricted to a part of the γ-globulin molecule; i.e., on the F piece obtained by papain digestion and, by inference, on the H chains obtained by reduction and alkylation of γ-globulin molecules.     

Immunochemiluminometric assays (ICMA) specific for growth hormone releasing hormone 1–44 NH2 and 1–40 OH

We describe specific two-site immunochemiluminometric assays able to directly measure human growth hormone-releasing hormone 1–44 NH₂ and 1–40 OH concentrations in unextracted plasma. A common N-terminal antibody was purified from polyclonal rabbit antisera to growth hormone-releasing hormone 1–44 NH₂ on a growth hormone-releasing hormone 1–29 NH₂ linked affinity column and labelled with chemiluminescent acridinium ester. C-terminal specific monoclonal antibodies to growth hormone-releasing hormone 1–44 NH₂ and 1–40 OH were raised in Balb/C mice and used as solid phase antibodies. Assay of fasting specimens from normal individuals gave medians (and ranges) of 23 pg/ml (2–200) and 30 pg/ml (3–134) for growth hormone-releasing hormone 1–44 NH₂ and 1–40 OH, respectively. Samples from a series of acromegalics showed that most have values in the normal range though median values were higher, 56 pg/ml for growth hormonereleasing hormone 1–44 NH₂ (P < 0.001) and 52 pg/ml for 1–40 OH (P < 0.001). Using these assays it will be possible for the first time to directly study the physiology and pathophysiology of these two peptides.     

Inflammatory cytokines TNF-α and IL-17 enhance the efficacy of cystic fibrosis transmembrane conductance regulator modulators

Without cystic fibrosis transmembrane conductance regulator–mediated (CFTR-mediated) HCO₃^– secretion, airway epithelia of newborns with cystic fibrosis (CF) produce an abnormally acidic airway surface liquid (ASL), and the decreased pH impairs respiratory host defenses. However, within a few months of birth, ASL pH increases to match that in non-CF airways. Although the physiological basis for the increase is unknown, this time course matches the development of inflammation in CF airways. To learn whether inflammation alters CF ASL pH, we treated CF epithelia with TNF-α and IL-17 (TNF-α+IL-17), 2 inflammatory cytokines that are elevated in CF airways. TNF-α+IL-17 markedly increased ASL pH by upregulating pendrin, an apical Cl^–/HCO₃^– exchanger. Moreover, when CF epithelia were exposed to TNF-α+IL-17, clinically approved CFTR modulators further alkalinized ASL pH. As predicted by these results, in vivo data revealed a positive correlation between airway inflammation and CFTR modulator–induced improvement in lung function. These findings suggest that inflammation is a key regulator of HCO₃^– secretion in CF airways. Thus, they explain earlier observations that ASL pH increases after birth and indicate that, for similar levels of inflammation, the pH of CF ASL is abnormally acidic. These results also suggest that a non-cell-autonomous mechanism, airway inflammation, is an important determinant of the response to CFTR modulators.     

Impaired 24,25-Dihydroxyvitamin D Production in Anephric Human and Pig

Plasma 25-hydroxyvitamin D and 24, 25-dihydroxy-vitamin D [24,25-(OH)₂D] concentrations were measured in normal and chronically dialyzed anephric humans and pigs. Measurement of the 24, 25-(OH)₂D was preceded by three purification steps involving one Sephadex LH-20 column and two high-pressure liquid chromatographic columns. The final high-pressure liquid chromatography step involved resolution of 25-hydroxy-vitamin D₃-26,23 lactone and 25,26-dihydroxy-vitamin D₂ from 24,25-dihydroxyvitamin D₂ and 24,25-dihydroxyvitamin D₃ [24,25-(OH)₂D₃]. The total 25-hydroxyvitamin D [25-hydroxyvitamin D₂ plus 25-hydroxyvitamin D₃ (25-OHD₃)] was 31.7±3.6 ng/ml in the plasma of eight anephric human subjects and 40.1±3.7 ng/ml in five normal human subjects. Six of the eight anephric patients had undetectable (<0.2 ng/ml) 24,25-(OH)₂D concentrations. Two of the eight patients had very low (0.51 and 0.41 ng/ml), but detectable, 24,25-dihydroxyvitamin D₂. The normal human volunteers had plasma 24,25-(OH)₂D concentrations of 2.8±0.7 ng/ml. Chronically dialyzed anephric and normal pigs were given intramuscular injections of massive amounts (5 × 10⁶ IU) of vitamin D₃ immediately after surgery (day 0) and again on day 7. In anephric pigs, plasma 25-OHD₃ progressively rose from 12±4 ng/ml on day 0 to 705±62 ng/ml on day 10. The 25-OHD₃ concentrations in normal pigs rose from 8±2 ng/ml on day 0 to 439±64 ng/ml on day 10. Plasma 25-OHD₃ was higher in anephrics throughout the experiment, and concentrations were significantly higher (P < 0.05) on days 9 and 10. Plasma 24,25-(OH)₂D₃ concentrations declined progressively in anephric pigs from 3.6±0.6 ng/ml on day 0 to 3.2±0.7 ng/ml on day 2. During days 4-10, plasma 24,25-(OH)₂D₃ was not apparent until plasma 25-OHD₃ was >400 ng/ml. In control pigs, plasma 24,25-(OH)₂D₃ was elevated from 4.3±0.6 ng/ml on day 0 to 178±2.7 ng/ml on day 3. Plasma 24,25-(OH)₂D₃ was significantly higher (P < 0.05) in controls on days 1-8. At the end of the experiment (day 10), 24,25-(OH)₂D₃ concentrations were similar and not significantly different in both groups (87.0±18.4 ng/ml in anephric and 110.3±32.1 ng/ml in normal pigs). The identity of the 24,25-(OH)₂D₃ isolated from anephric pig plasma was confirmed by mass spectroscopy. Our data suggest that anephric humans receiving normal dietary levels of vitamin D₃ have little or no ability to produce 24,25-(OH)₂D. However, we have shown that pigs produce 24,25-(OH)₂D₃ when plasma 25-OHD₃ is extremely high (>400 ng/ml).     

Attenuation effects of heparin–superoxide dismutase conjugate on bleomycin-induced lung fibrosis in vivo and radiation-induced inflammatory cytokine expression in vitro

In this study, the effects of heparin–superoxide dismutase conjugate (heparin–SOD) on bleomycin-induced pulmonary fibrosis in vivo and on inflammatory cytokine expression in vitro were evaluated. To investigate the effects of heparin–SOD on pulmonary fibrosis, heparin–SOD was administered to bleomycin (BLM)-treated mice by intraperitoneal injection once a day and the hydroxyproline content in lung was determined per 7 days. The degree of fibrosis was assessed quantitatively using histopathologic features. The results showed that heparin–SOD inhibited BLM-induced lung fibrotic lesions as reflected by the decrease of lung hydroxyproline content and lung fibrosis grade 28 days after BLM instillation. The in vitro effects on the cytokine level expressed by irradiated 3T3 fibroblasts showed that heparin–SOD significantly lowered the levels of transforming growth factor-β1 and interleukin-1β. These results strongly demonstrated that heparin–SOD might be useful in the prevention and treatment of pulmonary fibrosis.