Lung tissue engineering and preservation of alveolar microstructure using a novel casting method.

We used a rat model to decellularize and seed alveolar cells on a three-dimensional lung scaffold to preserve alveolar microarchitecture. We verified the preservation of terminal respiratory structure by casting and by scanning electron microscopy (SEM) of the casts after decellularization. Whole lungs were obtained from 12 healthy Sprague-Dawley rats, cannulated through the trachea under sterile conditions, and decellularized using a detergent-based method. Casting of both natural and decellularized lungs was performed to verify preservation of the inner microstructure of scaffolds for further cell seeding. Alveolar cell seeding was performed using green fluorescent protein (GFP) lung cells and non-GFP lung cells, and a peristaltic pump. We assessed cell seeding using histological and immunohistochemical staining, and enzymatic evaluation. All cellular components were removed completely from the scaffolds, and histological staining and SEM of casts were used to verify the preservation of tissue structure. Tensile tests verified conservation of biomechanical properties. The hydroxyproline content of decellularized lungs was similar to native lung. Histological and immunohistochemical evaluations showed effective cell seeding on decellularized matrices. Enzymatic measurement of trypsin and alpha 1 antitrypsin suggested the potential functional properties of the regenerated lungs. Casts produced by our method have satisfactory geometrical properties for further cell seeding of lung scaffolds. Preservation of micro-architecture and terminal alveoli that was confirmed by SEM of lung casts increases the probability of an effective cell seeding process.

Spectroscopic studies of the interaction of aspirin and its important metabolite, salicylate ion, with DNA, A·T and G·C rich sequences.

Among different biological effects of acetylsalicylic acid (ASA), its anticancer property is controversial. Since ASA hydrolyzes rapidly to salicylic acid (SA), especially in the blood, interaction of both ASA and SA (as the small molecules) with ctDNA, oligo(dA·dT)15 and oligo(dG·dC)15, as a possible mechanism of their action, is investigated here. The results show that the rate of ASA hydrolysis in the absence and presence of ctDNA is similar. The spectrophotometric results indicate that both ASA and SA cooperatively bind to ctDNA. The binding constants (K) are (1.7±0.7)×10(3) M(-1) and (6.7±0.2)×10(3) M(-1) for ASA and SA, respectively. Both ligands quench the fluorescence emission of ethidium bromide (Et)-ctDNA complex. The Scatchard plots indicate the non-displacement based quenching (non-intercalative binding). The circular dichroism (CD) spectra of ASA- or SA-ctDsNA complexes show the minor distortion of ctDNA structure, with no characteristic peaks for intercalation of ligands. Tm of ctDNA is decreased up to 3°C upon ASA binding. The CD results also indicate more distortions on oligo(dG·dC)15 structure due to the binding of both ASA and SA in comparison with oligo(dA·dT)15. All data indicate the more affinity for SA binding with DNA minor groove in comparison with ASA which has more hydrophobic character.

Mechanism of inhibition of cytochrome P450 C21 enzyme activity by autoantibodies from patients with Addison's disease.

OBJECTIVE: To study possible mechanisms for the inhibition of cytochrome P450 C21 (steroid 21-hydroxylase) enzyme activity by P450 C21 autoantibodies (Abs) in vitro. DESIGN: Two possible mechanisms for the inhibition of P450 C21 enzyme activity by P450 C21 Abs were studied: (a) conformational changes in the P450 C21 molecule induced by Ab binding and (b) the effects of Ab binding to P450 C21 on the electron transfer from the nicotinamide adenine dinucleotide phosphate reduced (NADPH) cytochrome P450 reductase (CPR) to P450 C21. METHODS: The effect of P450 C21 Ab binding on the conformation of recombinant P450 C21 in yeast microsomes was studied using an analysis of the dithionite-reduced CO difference spectra. The effect of P450 C21 Abs on electron transfer was assessed by analysis of reduction of P450 C21 in the microsomes in the presence of CO after addition of NADPH. RESULTS: Our studies confirmed the inhibiting effect of P450 C21 Abs on P450 C21 enzyme activity. Binding of the Abs did not induce significant change in the P450 C21 peak at 450nm (native form) and did not produce a detectable peak at 420 nm (denatured form) in the dithionite-reduced CO difference spectra. This indicated that conformation of P450 C21 around the heme was not altered compared with the native structure. However, incubation of the P450 C21 in yeast microsomes with P450 C21 Ab inhibited the fast phase electron transfer from the CPR to P450 C21. CONCLUSIONS: Our observations suggested that the mechanism by which P450 C21 Abs inhibit P450 C21 enzyme activity most likely involves inhibition of the interaction between the CPR and P450 C21.

Oral manifestations in selective IgA deficiency.

Selective immunoglobulin A (IgA) deficiency is the most common of the primary immunodeficiencies with a frequency of 1/300-1/3000, depending on the screened population. As secretory IgA (SIgA) has a protective role in mucosal surfaces from invasion of microorganisms, it is thought that IgA-deficient subjects are susceptible to periodontal diseases and oral manifestations. Previous studies show contradictory results, concerning the involvement of the individuals' periodontium with IgA deficiency. The aim of this study was to investigate and compare the oral manifestations in IgA-deficient subjects with controls. Eleven selective IgA-deficient subjects aged 3-18 years with serum IgA levels <10 mg dl(-1) and 11 age-sex-matched healthy children as the controls entered the study. Oral mucosal investigation, dental caries, plaque accumulation and periodontal status were assessed. Serum immunoglobulin levels were measured by single radial immunodiffusion (SRID) method. Saliva immunoglobulins and secretory component levels were measured by enzyme linked immunosorbent assay (ELISA) methods. IgA-deficient patients had serum and saliva IgA levels less than 10 mg dl(-1) and 10 microg ml(-1), respectively, but other serum immunoglobulin levels were normal and saliva immunoglobulin M (IgM) levels were increased, compared with controls. There were no significant differences in oral manifestations between IgA-deficient subjects and controls, which may be a result of compensatory increase of saliva IgM or other non-immunological defence factors in saliva. Thus, it is not necessary to evaluate IgA and SIgA in all the patients with oral and dental lesions and it is thought that it is better to investigate other factors.