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Perceval S is a self-expandable, stent-mounted bioprosthetic valve (BPV), with glutaraldehyde treated bovine pericardium, processed with homocysteic acid as an anti-calcification treatment. The stent is crimpable but the valve insertion is done surgically via a shorter procedure which does not require sutures. OBJECTIVES: MATERIAL AND METHODS: RESULTS: CONCLUSIONS: Collapsing and ballooning do not alter cusp collagen periodicity. Structural valve deterioration with stenosis, due to dystrophic calcification and fibrous tissue overgrowth, seldom occurred in the mid-term. Glutaraldehyde fixed pericardium has the potential to undergo structural valve deterioration with time, similar to well-known BPV failure. This supports the recommendation to pursue improvement of tissue valve treatment with enhanced durability.

Background Limited mid-term durability of 12A/LX Mitroflow bioprosthesis has been reported. Aim of the study was to ascertain the pathologic substrates and possible mechanisms of structural valve deterioration in explants from animals and humans. Methods Nine aortic 12A/LX Mitroflow bioprostheses preserved in hypotonic solution and three aortic 12A/LX bioprostheses, preserved in isotonic solution, were explanted from juvenile sheep, mean time from implant 95.66 ± 36.04 days and 132.33 ± 28.88 days from implant respectively. One stented unimplanted 12A/LX Mitroflow preserved in isotonic colution before glutaraldeyde fixation served as control. Ten aortic 12A/LX Mitroflow bioprostheses were explanted from humans because of severe dysfunction: five children, (3 females and 2 males, mean age 14.19 ± 4.77 years, range 11-21), 26 ± 8.24 months from implant and 5 adults (4 females and 1 male, mean age 57.4 ± 19.85 years, range 31-72), 64.4 ± 26.94 months from implant. X-ray, histology, and transmission electron microscopy were carried out as well as spectroscopy for calcium (Ca++) and phosphorus (P) content in human explants. Results Explants, from both animals and humans, showed cusp folding and stiffness, with coarse calcific deposits at gross examination and X-ray. Severe collagen denaturation, plasma insudation and massive calcification, involving both collagen and cell debris, were observed microscopically. Mean Ca++ content of 183.27 ± 62.48 and P content of 94.35 ±33.76 mg/g dry weight was found in children and Ca++ content of 205.49 ± 2.23 and P content of 99.75 ± 0.11 mg/g dry weight in adults. Obstructive fibrous tissue overgrowth was detected in 6 human cases. Conclusions Collagen denaturation was observed in pericardial Mitroflow 12A/LX bioprosthesis with premature structural valve deterioration. Optimal collagen fixation and preservation as well as phospholipids reduction by removing cell debris, as employed in the novel CROWN PRT Mitroflow bioprosthesis, are expected to solve the flaw and achieve long-term durability.

Background Aortic stentless bioprosthetic valve (SLBPV), either porcine or pericardial, minimizes transvalvular gradient and favors regression of left ventricular hypertrophy. The drawback consists of longer time for suturing. While structural valve deterioration (SVD) in stented porcine and pericardial BPVs has been extensively investigated, less information is available on SLBPVs. Material and Methods We studied 82 SLBPVs explants, either porcine (Toronto SPV, [St. Jude Medical, MN, USA], CryolifeO'Brien Model 300 and CryoLife-O'Brien [Cryolife International, GA, USA], BioCor PVS [St. Jude Medical, MN, USA] Prima and Prima Plus [Edwards Lifesciences Corp. One Edwards Way, CA, formerly Baxter Inc, CA, USA]) or pericardial ([Pericarbon Freedom and Freedom Solo [Sorin-Biomedica, S.p.A., Saluggia, Italy]). Results By excluding cases with leak and endocarditis, we focused the investigation on 46 SLBPVs, which failed because of SVD. Gender was male in 29 (63%). Mean age of patients at time of implant was 59.8 years. Postoperative time of SVD was 115.0 months for porcine and 79.0 months for pericardial SLBPVs. Dysfunction requiring reoperation was mainly incompetence for porcine and stenosis for pericardial SLBPVs. Even pinpoint mineralization at the commissures resulted in sudden cusp tearing and incompetence. Cuspal atheromasia accounted for cusp tearing even in the absence of calcification. Mineralization showed progression with time in pericardial but not in porcine SLBPVs. Conclusions Tissue mineralization remains the nightmare also of SLBPVs, with the peculiar features of pinpoint calcific deposits at commissures, tearing and abrupt incompetence in porcine SLBPVs and of massive cuspal mineralization and stenosis in pericardial SLBPVs.

Amiodarone (AMI) is a potent antiarrhythmic agent; however, its clinical use is limited due to numerous side effects. In order to investigate the structure--cytotoxicity relationship, AMI analogues were synthesized, and then, using rabbit alveolar macrophages, were tested for viability and for the ability to interfere with the degradation of surfactant protein A (SP-A) and with the accumulation of an acidotropic dye. Our data revealed that modification of the diethylamino-beta-ethoxy group of the AMI molecule may affect viability, the ability to degrade SP-A and vacuolation differently. In particular, PIPAM (2d), an analogue with a piperidyl moiety, acts toward the cells in a similar manner to AMI, but is less toxic. Thus, it would be possible to reduce the cytotoxicity of AMI by modifying its chemical structure.

Amiodarone may induce lung damage by direct toxicity or indirectly through inflammation. To clarify the mechanism of direct toxicity, we briefly exposed rabbit alveolar macrophages to amiodarone and analyzed their morphology, synthesis, and degradation of dipalmitoylphosphatidylcholine (DPPC); distribution of lysosomal enzymes; and uptake of diphtheria toxin and surfactant protein (SP) A used as tracers of the endocytic pathway. Furthermore, in newborn rabbits, we studied the clearance of DPPC and SP-A instilled into the trachea together with increasing amounts of amiodarone. We found that in vitro amiodarone decreases the surface density of mitochondria and lysosomes while increasing the surface density of inclusion bodies, increases the incorporation of choline into DPPC, modifies the distribution of lysosomal enzymes, and does not affect the uptake and processing of diphtheria toxin but inhibits the degradation of SP-A. In vivo amiodarone inhibits the degradation of SP-A but not of DPPC. We conclude that the acute exposure to amiodarone perturbs the endocytic pathway acting after the early endosomes, alters the traffic of lysosomal enzymes, and interferes with the turnover of SP-A.

Reperfusion injury represents a key event leading to graft nonfunction. Maintaining adequate nitric oxide levels and stimulating vasodilator synthesis can probably minimize endothelial damage. The aim of this study was to investigate the effect of L-arginine, a substrate of nitric oxide synthesis, and oligotide, a promoter of prostacyclin synthesis, on liver function and morphology after warm ischemia-reperfusion injury. After constructing a side-to-side portacaval shunt, ischemia was induced by clamping the hepatic hilum for 2 h above the shunt, in 19 female pigs divided into a control group (n = 7), an L-arginine treatment group (n = 6), and an oligotide treatment group (n = 6). Liver function tests and measurements of serum and red blood cell malondialdehyde and plasma nitric oxide levels were performed before reperfusion and at 1, 10, 60, and 120 min after reperfusion. Liver biopsies, taken before reperfusion and at 30 min and 7 days after reperfusion, were analyzed for tissue malondialdehyde, histological-ultrastructural features, and apoptosis evaluation. Thirty minutes after reperfusion, liver malondialdehyde, sinusoidal congestion, necrosis, and apoptosis were significantly lower in the L-arginine group than in the controls (p.05). On postoperative day 7, tissue malondialdehyde decreased, while plasma nitric oxide and hepatocyte glycogen content were increased in the L-arginine group compared to controls (p.05). This study demonstrates the protective effect of L-arginine on hepatic lipoperoxidation and liver morphology in a pig model of warm ischemia-reperfusion injury. The increased plasma levels of nitric oxide a week after ischemia-reperfusion injury support the hypothesis that it has a role in preventing liver damage. The same beneficial effect was not confirmed for oligotide.

Graphical abstract Amiodarone, having basic pKa and high water solubility at acidic pH, accumulates within late endocytic compartments, blocking fluid-phase endocytosis, proteolysis and lipid trafficking and inducing a Niemann-Pick C-like phenotype., Patients treated with amiodarone accumulate lysobisphosphatidic acid (LBPA), also known as bis(monoacylglycero)phosphate, in airway secretions and develop in different tissues vacuoles and inclusion bodies thought to originate from endosomes. To clarify the origin of these changes, we studied in vitro the effects of amiodarone on endosomal activities like transferrin recycling, Shiga toxin processing, ESCRT-dependent lentivirus budding, fluid phase endocytosis, proteolysis and exosome secretion. Furthermore, since the accumulation of LBPA might point to a broader disturbance in lipid homeostasis, we studied the effect of amiodarone on the distribution of LBPA, unesterified cholesterol, sphingomyelin and glycosphyngolipids. Amiodarone analogues were also studied, including the recently developed derivative dronedarone. We found that amiodarone does not affect early endosomal activities, like transferrin recycling, Shiga toxin processing and lentivirus budding. Amiodarone, instead, interferes with late compartments of the endocytic pathway, blocking the progression of fluid phase endocytosis and causing fusion of organelles, collapse of lumenal structures, accumulation of undegraded substrates and amassing of different types of lipids. Not all late endocytic compartments are affected, since exosome secretion is spared. These changes recall the Niemann-Pick type-C phenotype (NPC), but originate by a different mechanism, since, differently from NPC, they are not alleviated by cholesterol removal. Studies with analogues indicate that basic pKa and high water-solubility at acidic pH are crucial requirements for the interference with late endosomes/lysosomes and that, in this respect, dronedarone is at least as potent as amiodarone. These findings may have relevance in fields unrelated to rhythm control.

Amiodarone interferes with the endocytic pathway, inhibits proteolysis, and causes the formation of vacuoles, but uptake and intracellular distribution of the drug, origin of vacuoles, and functional consequences of amiodarone accumulation remain unclear. Our objective was to study amiodarone uptake, clarify the origin of vacuoles, and investigate the effect of amiodarone on the life cycle of the coronavirus responsible for the Severe Acute Respiratory Syndrome (SARS), which, to enter cells, relies on the proteolytic cleavage of a viral spike protein by the endosomal proteinase cathepsin L. Using alveolar macrophages, we studied uptake of (125)I-amiodarone and (125)I-B2, an analog lacking the lateral group diethylamino-beta-ethoxy, and analyzed the effects of amiodarone on the distribution of endosomal markers and on the uptake of an acidotropic dye. Furthermore, using Vero cells, we tested the impact of amiodarone on the in vitro spreading of the SARS coronavirus. We found that (1) amiodarone associates with different cell membranes and accumulates in acidic organelles; (2) the diethylamino-beta-ethoxy group is an important determinant of uptake; (3) vacuoles forming upon exposure to amiodarone are enlarged late endosomes; (4) amiodarone inhibits the spreading in vitro of SARS coronavirus; and (5) trypsin cleavage of the viral spike protein before infection, which permits virus entry through the plasma membrane, does not impair amiodarone antiviral activity. We conclude that amiodarone alters late compartments of the endocytic pathway and inhibits SARS coronavirus infection by acting after the transit of the virus through endosomes.

The responses of the adult rabbit lung to multiple doses of surfactant after intratracheal injections of either natural calf surfactant or Surfactant-TA were evaluated. For each surfactant, four groups of 1.4-kg rabbits were studied: group 1 received 100 mg of surfactant containing isotopically-labeled dipalmitoylphosphatidylcholine; group 2 received the same labeled surfactant and then three tracheal injections of vehicle; group 3 received labeled surfactant and then three doses (100 mg) of unlabeled surfactant; group 4 was treated in the same way as group 3 except that the final dose was of the labeled surfactant. All rabbits were killed, and alveolar washes were recovered 24 h after the labeled surfactant dose had been given. The amount of labeled palmitate-saturated phosphatidylcholine (Sat PC) in alveolar washes did not change after multiple doses of calf surfactant, indicating that subsequent doses did not alter the clearance of previous doses. The four doses of calf surfactant increased the alveolar Sat PC pool size by a factor of 2.5 only when measured 6 h after the last dose, but the total lung Sat PC pool size doubled, indicating a loss of most of the surfactant Sat PC to the lung tissue. In contrast, Surfactant-TA increased the alveolar pool size by a factor of 4 after the single dose and by a factor of 11 after the multiple doses, and the percentage clearance of labeled Sat PC from the lungs decreased with multiple doses, indicating an effect of subsequent doses on the initial dose. The quantity of Sat PC cleared from the lungs increased by about a factor of 2 after the multiple doses of Surfactant-TA. Although repetitive surfactant doses changed alveolar and lung Sat PC pool sizes the quantity of Sat PC cleared from the lungs increased, and the lungs accommodated the large amount of surfactant without short-term adverse effects.

Objective: Anticalcification strategies of glutaraldehyde-fixed xenograft tissue aim to extract lipids or to neutralize toxic aldehyde residuals. The purposeof this studywasto evaluatethe efficacyof octanediolcompared to standardtreatmentsof glutaraldehyde-fixedbovinepericardium in the subdermal rat model. Octanediol treatment is an ethanolic solution (40%) containing a long chain aliphatic alcohol (5% 1,2-octanediol) that removes lipids without diminishing the stability of collagen. Methods: Octanediol and standard glutaraldehyde fixed bovine pericardium were both implanted in 24 Sprague-Dawley rats, explanted after 30—75 days (12 animals each) and submitted to X-ray (score 0—4), histology, electron microscopy and elemental analysis by spectroscopy (Ca and P content). Unimplanted octanediol and standard glutaraldehyde fixed pericardium servedascontrol.Results:At30daysoctanediol-treatedpericardiumshowedcalciumcontentof0.20 0.1vs20.07 36.79 mg/gdryweightfor standard pericardium. The difference was also evident at 75 days: calcium content of 2.36 7.38 mg/g dry weight for octanediol vs 165.61 23.35 mg/g dry weight for standard (p < 0.0001). Differences were also detected at X-ray (mean score 0.7 0.6 octanediol vs 3.8 0.4 standard at 75 days). Equally, mean P content was 11.69 21.33 mg/g dry weight for standard vs 0.60 1.45 mg/g dry weight for octanediol samples at 30 days, and 90.90 12.61 mg/g dry weight for standard vs 1.42 4.34 mg/g dry weight for octanediol at 75 days (p < 0.0001). At electron microscopy collagen appeared well preserved regardless of the type of treatment; in octanediol treated pericardium cell membranes almost disappeared and only few profiles of endoplasmic reticulum and rare mitochondria were visible. Conclusions: Treatment with octanediol strongly prevents calcification of glutaraldehyde fixed bovine pericardium in rat subdermal model, even in the long-term. Evidence of octanediol efficacy may entail important implications for new generation bioprosthetic valves. # 2008 European Association for Cardio-Thoracic Surgery. Published by Elsevier B.V. All rights reserved.

Introduction A new flexible annuloplasty ring (Sovering, Sorin Biomedica Cardio), both closed and open, has been designed and consists of radiopaque silicone core impregnated with barium sulfate and covered by a knitted polyester fabric coated with Carbofilm. The aim of the study was to test the biological compatibility of the new device in large animals in orthotopic position. Methods Ten Sovering rings were implanted in 10 female adult sheep, 7 mitral (3 open, 4 closed) and 3 tricuspid (all open). The size was 23–25 mm in the mitral and 28–30 mm in the tricuspid position, and the time in place varied from 63 to 110 days (mean=89±14) and from 58 to 63 days (mean=61±3), respectively. The morphological analysis consisted of gross, histological, immunohistochemical and ultrastructural investigations. Results The prosthetic ring appeared well implanted in the valve atrioventricular (AV) junction, encapsulated by a thin fibrous sheath without any evidence of thrombus deposition, fibrinous lining or exuberant fibrous tissue overgrowth. No adverse inflammatory reaction was observed, but rare lymphocytes, macrophages and foreign body giant cells. At electron microscopy, the fibrous tissue appeared to permeate deeply within the fabric network; reendothelization on the surface was noted and confirmed by immunohistochemistry. Sirius red staining at polarized light revealed a higher content of collagen type III in the mitral than in the tricuspid position. Conclusions Sovering annuloplasty rings implanted in the AV valves of adult sheep showed excellent biocompatibility, fibrous encapsulation and reendothelization. The absence of thrombosis and exuberant fibrous tissue reaction supports the effective use of Carbofilm covering in annuloplasty devices.

Amiodarone, an antiarrhythmic drug toxic toward the lung, is metabolized through sequential modifications of the diethylaminoethoxy group to mono- N-desethylamiodarone (MDEA), di- N-desethylamiodarone (DDEA), and amiodarone-EtOH (B2-O-EtOH), whose effects on lung cells are unclear. To clarify this, we exposed rabbit alveolar macrophages to analogs with different modifications of the diethylaminoethoxy group and then searched for biochemical signs of cell damage, formation of vacuoles and inclusion bodies, and interference with the degradation of surfactant protein A, used as a tracer of the endocytic pathway. The substances studied included MDEA, DDEA, and B2-O-EtOH, analogs with different modifications of the diethylaminoethoxy group, fragments of the amiodarone molecule, and the antiarrhythmic agents dronedarone (SR-33589) and KB-130015. We found the following: 1) MDEA, DDEA, and B2-O-EtOH rank in order of decreasing toxicity toward alveolar macrophages, indicating that dealkylation and deamination of the diethylaminoethoxy group represent important mechanisms of detoxification; 2) dronedarone has greater, and KB-130015 has smaller, toxicity than amiodarone toward alveolar macrophages; and 3) the benzofuran moiety, which is toxic to liver cells, is not directly toxic toward alveolar macrophages.

BIOSA is a single sheet, two-cuspal-shaped ("bileaflet") glutaraldehyde fixed bovine pericardial valve prosthesis (BP), designed to minimize stress and prevent mechanical failure. We tested this device in the adult sheep model.Seven BIOSA and two Baxter Carpentier-Edwards (Perimount) pericardial BPs, 25 mm in size, were implanted in the tricuspid position of adult sheep. A mid term BIOSA explant (81 days) died of infective endocarditis. The remaining BPs were divided in three early explants (0-3 days: two BIOSA and one Perimount) and five late explants (162-189 days: four BIOSA and one Perimount). Protocol of the study included gross examination, mammography X-ray (score 0-4), histology, scanning (SEM) and transmission (TEM) electron microscopy and atomic absorption spectroscopy.(a) Early explants: The mesothelium was detached, collagen-elastic fibers and pericardiocytes of the fibrosa showed optimal preservation. Calcium content in BIOSA BPs was 6.22 mg/g dry weight (mean) versus 7.75 mg/g of the Perimount BP. (b) Late explants: At naked eye all BPs showed regular cusp pliability and coaptation, without tears, perforations, fibrous pannus or calcific deposits. X-ray was either negative (three BPs) or exhibited score 1 (two BPs). Microscopic features were excellent both in terms of collagen-elastic fibers preservation and absence of inflammation. The calcium content was 4.95 mg/g dry weight (mean) in BIOSA BPs versus 5.29 mg/g in the Perimount BP.Tissue characteristics of BIOSA BPs were optimal, without difference with Perimount BPs and in this animal model no case of structural valve deterioration occurred in the long term.

Background Primary tissue failure, which is mainly caused by calcification, is still the limiting factor in the long-term outcome of heart valve bioprostheses. Even though the precise nature of this process is not fully understood, in vitro tests have been developed to reproduce and predict calcification for individual bioprostheses. Methods In vitro calcification testing was performed by using an accelerated pulsatile valve tester which was adapted for testing stented as well as stentless bioprostheses with physiological fluid dynamics. A total of 84 bioprostheses (porcine, pericardial and stentless porcine of different manufacturers) were cyclically loaded at a test rate of 300/min at 37 degrees C within a rapid calcification fluid with CaxP = 130(mg/dl)2 at pH 7.4. Calcification was assessed by microradiography after 12 x 10(6) cycles. In a previous step, holographic interferometry was performed to identify irregularities of valve leaflets in order to predict later calcification. Selected specimens of calcified bioprostheses underwent histology, transmission (TEM) and scanning (SEM) electron microscopy. Tissue mineralization was investigated by coupling SEM, electron microprobe analysis (EMPA) and X-ray powder diffraction (XRPD) methods. Results For all tested bioprostheses, a significant calcification was achieved within 4 to 6 weeks of ongoing testing, and the degree of calcification increased with time. A significant correlation between calcification and leaflet irregularities (detected by holographic interferometry) was found (r = 0.80, p = 0.001). Calcification varied between individual bioprostheses, and significant differences were detected for different groups (calculated as percentage of total leaflet area, mean +/- SD): porcine stented (37.3 +/- 12.0%), bovine stented (23.0 +/- 8.9%), porcine stentless (16.2 +/- 7.6%). Histological and ultrastructural investigation showed intrinsic calcification involving both the spongiosa and fibrosa with collagen fibrils, interfibrillar spaces and cells as early sites of calcification. There was clear evidence of apatite crystallization, and observations made with in vitro calcification were quite similar to those occurring with in vivo implanted bioprostheses. Conclusion In vitro tests can reproduce intrinsic calcification of bioprostheses even in the absence of viable biologic host factors. Moreover, degree and sites of calcification have become predictable. This enables the development and evaluation of bioprostheses with reduction of animal experiments. From our results obtained with a broad range of available bioprostheses, stented bovine and stentless porcine valves seem to be superior to conventional stented porcine bioprostheses with regard to leaflet calcification.

This paper examines the removal from the airways of Curosurf, a commercial surfactant derived from porcine lungs, administered at pharmacological concentrations to newborn or adult animals. Curosurf was labelled by the addition of radioactive dipalmitoyl phosphatidylcholine (DPPC) and administered intratracheally to newborn and adult rabbits at a dose of 200 mg x kg(-1) body weight. The disappearance of DPPC from the airways and its appearance in alveolar macrophages, lung parenchyma, lamellar bodies, serum, liver, kidneys and brain was then studied for 24-48 h. The in vitro degradation of Curosurf DPPC by alveolar macrophages was also studied. During the first 3 h after instillation, large amounts of Curosurf left the airways and became associated with tissue, indicating that it mixed rapidly with the endogenous pools of surfactant. A fraction of administered DPPC became associated with the lamellar bodies, suggesting that Curosurf can be recycled. Curosurf administration did not stop the secretion of endogenous surfactant. Very little intact radioactive DPPC could be recovered at any time in alveolar macrophages, however, macrophages have the ability, in vitro, to degrade Curosurf. Newborn rabbits lose Curosurf from the lungs at a slower rate than adult rabbits. One and two days after instillation, organic extracts from the liver, kidney, brain and serum contained small but measurable amounts of radioactivity. These results indicate that Curosurf rapidly enters the pathways of surfactant metabolism and that alveolar macrophages may play an important role in the catabolism of Curosurf.

Hepatic ischaemia/reperfusion is characterized by circulatory and metabolic derangement, liver dysfunction, and tissue damage. To evaluate the role of L-arginine, a substrate of nitric oxide, in ischaemia/reperfusion injury, total liver ischaemia was induced for 120 min in 22 Landrace x Large White female pigs, which were randomly assigned to a treatment group (10 animals) or a control group (12 animals). An L-arginine bolus (540 mg/kg i.v.) was administered to the treatment group 1 h before clamping the hepatic hilum, at clamping, at reperfusion, and at 1 and 2 h after reperfusion. The control animals received normal saline and an i.v. infusion. Liver function tests and analysis of serum, erythrocyte, and tissue malondialdehyde contents were performed at commencement of laparotomy, before reperfusion, and at 30 min and 7 days after reperfusion. Liver biopsies were taken at laparotomy, at 30 min, and at 7 days after reperfusion for histological and ultrastructural examination. Assessment of apoptosis included in situ end-labelling analysis and DNA gel electrophoresis. Survival at 7 days was better in the treated animals than in the controls (9/10 vs. 7/12). Tissue malondialdehyde content, aspartate aminotransferase, and lactate dehydrogenase levels were lower in the treatment group, in which morphological changes were significantly less evident than in the controls 30 min after reperfusion. At 7 days, differences between the groups with respect to cell integrity were apparent only on ultrastructural analysis. Glycogen content, 7 days after reperfusion, was higher in the treatment group than in the controls: 70.25 per cent vs. 21.66 per cent positive hepatocytes (score 3 vs. score 1). Multiparametric analysis showed fewer apoptotic cells in the treatment group at all times. Our data show that the administration of L-arginine reduces damage to liver tissue after ischaemia/reperfusion injury in a pig model. This may be explained not only by the known vasodilator, anti-aggregation, and superoxide inactivation effects of increased nitric oxide release, but possibly also by some other action of L-arginine, such as its influence on cellular metabolism.

This paper reports on some pharmacological and biological properties of 22-mer antisense oligodeoxynucleotides which contain an L-deoxyribonucleoside at each terminus. Compared with natural compounds, of which they retain the DNA hybridizing ability and the cell uptake mechanism, the L-22-mers exhibited an increased resistance to phosphodiesterase degradation, an apparent higher intracellular concentration and a longer intracellular half life. Antiviral activity was not prominent.

The effect of profound repetitive prenatal asphyxial insults on the cardiopulmonary function of premature ventilated lambs was studied. Twenty-nine fetal lambs (approximately 138 days gestational age) were exteriorized. In 16 of these lambs, the umbilical cord was occluded for 4 min then released for 10 min. This asphyxial episode was repeated until the arterial pH was approximately 7.00, and the mean arterial blood pressure was less than 40 mmHg and falling. The 13 control lambs were simply exteriorized with the umbilical circulation intact. The lambs were then ventilated for 3-4 h. There were no differences between the control vs. asphyxiated lambs in pulmonary compliances (0.57 and 0.58 ml.cmH2O-1.kg-1) wet-to-dry weight ratios (8.18 and 7.55), cardiac outputs (177.8 and 141.8 ml.kg-1.min-1), surfactant-saturated phosphatidylcholine pool sizes, or atrial and/or ductal shunts. Asphyxia did not interfere with the redirection of blood away from atelectatic lung segments created by bronchial obstruction with balloon catheters. Also, although the bidirectional flux of protein into and out of the airways of these preterm lambs was large relative to term lambs, there was no effect of asphyxia on this protein leak. In this animal model, prenatal asphyxia did not impact negatively on the severity of the respiratory failure.

Fetal rabbits were treated with corticosteroids by maternal administration for 48 h before delivery at 27 days gestational age. Both corticosteroid-treated and control animals then received exogenous natural rabbit surfactant at birth at doses of 0–75 mg lipid/kg. After 10 min of ventilation at tidal volumes of 12–15 ml/kg, static pressure-volume measurements were made. At all surfactant doses there was a significantly higher maximal lung volume, higher dynamic compliance, and lower pressure requirement in the corticosteroid-treated than in the control rabbits (P less than 0.01). Control animals showed incremental improvements in dynamic compliances and maximal lung volumes up to a dose of 50 mg/kg, whereas corticosteroid treated animals improved to a maximum at the low dose of 15 mg/kg (P less than 0.01). However, surface tension as assessed by lung stability index improved with increasing surfactant dose but was not significantly different between corticosteroid-treated and control animals at a given dose. The results imply that maternal corticosteroid treatment potentiates surfactant replacement by a change in lung structure that is independent of surface tension effects.

Tracer quantities of 3H-labeled lysoPC and 32P-labeled natural rabbit surfactant were given intratracheally via a bronchoscope and [14C]palmitate was given intravenously to 25 rabbits with labeled PC and lysoPC measured in the alveolar wash, lung homogenate, lamellar bodies and microsomes at five times from 10 min to 6 h after tracheal injection. Surprisingly, only 31% of the administered lysoPC remained in its original form in the total lungs (alveolar wash + lung homogenate) by 10 min, of which 77% was in the alveolar wash. Meanwhile, by 10 min an additional 37% was already converted to PC, of which more than 98% was in the lung homogenate. LysoPC continued to be rapidly and efficiently converted to PC, with 62% conversion measured at 3 h. The converted lysoPC initially appeared with high specific activity in microsomes, then in lamellar bodies, and finally in the alveolar wash. The intravascular palmitate labeled lung PC had similar specific activity-time profiles in the subcellular fractions, while intratracheally administered natural rabbit surfactant had a constantly low specific activity in microsomes and much higher specific activities in lamellar bodies and alveolar wash. Another 25 rabbits received intratracheal lysoPC labeled in both the choline and palmitate moieties and then were studied from 1 to 24 h after tracheal injection. The ratio of the palmitate to choline labels indicated uptake and conversion to PC primarily by direct acylation rather than transacylation and by intact reuptake and conversion rather than breakdown and resynthesis. LysoPC is an attractive 'metabolic probe' of surfactant metabolism which undergoes very rapid and efficient intracellular conversion to PC via a subcellular pathway that parallels the remodeling and de novo synthetic pathways.

Preterm lambs were delivered at 138 days gestational age and ventilated for periods up to 24 h in order to study surfactant metabolism and surfactant function. The surfactant-saturated phosphatidylcholine pool in the alveolar wash was 13 +/- 4 mumol/kg and did not change from 10 min to 24 h after birth. Trace amounts of labeled natural sheep surfactant were mixed with fetal lung fluid at birth. By 24 h, 80% of the label had become lung-tissue-associated, yet there was no loss of label from phosphatidylcholine in the lungs when calculated as the sum of the lung tissue plus alveolar wash. De novo synthesized phosphatidylcholine was labeled with choline given by intravascular injection at 1 h of age. Labeled phosphatidylcholine accumulated in the lung tissue linearly to 24 h, and the labeled phosphatidylcholine moved through lamellar body to alveolar pools. The turnover time for alveolar phosphatidylcholine was estimated to be about 13 h, indicating an active metabolic pool. A less surface-active surfactant fraction recovered as a supernatant after centrifugation of the alveolar washes at 40,000 x g increased from birth to 10 min of ventilation, but no subsequent changes in the distribution of surfactant phosphatidylcholine in surfactant fractions occurred. The results were consistent with recycling pathway(s) that maintained surface-active surfactant pools in preterm ventilated lambs.

Rabbits were given various doses of rabbit surfactant and treatment doses of approximately 100 mg/kg body wt of calf surfactant and Surfactant TA by tracheal injection. The linear loss of radiolabeled phosphatidylcholine from the total lung (alveolar wash and lung tissue) was 3.1, 1.5, and 1.8%/h for rabbit surfactant, calf surfactant, and Surfactant TA, respectively. After 24 h only 6% rabbit, 19% calf, and 9.7% Surfactant TA phosphatidylcholine were recovered by alveolar wash, and alveolar macrophage fractions contained less than 1% of the injected labeled phosphatidylcholine. The loss of rabbit surfactant phosphatidylcholine 24 h after tracheal injection did not change for doses in the range of 0.5-70 mumol phosphatidylcholine per kilogram, indicating nonsaturable clearance pathways. Very little of the labeled rabbit surfactant phosphatidylcholine lost from the lungs could be recovered in other organs, and 90% of the recovered labeled phosphatidylcholine in the liver was unsaturated, implying de novo synthesis using precursors from degraded phosphatidylcholine. The surfactant did not change endogenous lung phosphatidylcholine synthesis or its secretion to the alveolus. There were no adverse effects of the surfactant treatments noted in healthy rabbits.

ObjectivesDurability remains the main problem of all bioprosthetic valves, and calcification is the major cause of failure. New tissue treatment processes are expected to reduce mineralization. A comparative animal study was undertaken to evaluate the behavior of a new-generation porcine bioprosthesis in contrast with a first-generation porcine bioprosthesis. The primary goal was to evaluate the efficacy of α-amino-oleic acid as an anticalcification treatment.MethodsSeventeen Targhee sheep (aged 4.5-7 months) had a mitral valve replacement with a Mosaic or Hancock Standard. The animals were followed up to 20 weeks (144.1 ± 4.0 days vs 144.3 ± 8.2 days) and then euthanized as scheduled. After gross examination, the explants were radiographed for the presence of calcification. The central portions were preserved for histologic examination, and the remainder of the sample was analyzed for quantitative calcium content by atomic absorption spectroscopy.ResultsFour Mosaic sheep were excluded because of perioperative surgical mortality. The remaining 13 were enrolled in the study (9 Mosaic and 4 Hancock Standard). The mean calcium content was 1.97 ± 2.21 μg/mg tissue weight for Mosaic versus 8.36 ± 4.12 μg/mg for Hancock Standard valves (P < .01). Mild fibrous tissue overgrowth and fibrinous lining were observed regardless the xenograft type.ConclusionsThe low level of calcification in the Mosaic versus Hancock Standard xenografts confirms the efficacy of α-amino-oleic acid treatment in mitigating mineralization. A longer durability is expected with the clinical use of the Mosaic porcine valve.

Pregnant does were treated with betamethasone, T3, TRH, or combinations of betamethasone plus T3 or TRH using doses of each agent known to effect lung maturation. Preterm rabbits were then delivered at 27 days gestational age, half of each group was treated with Surfactant TA, and the rabbits were ventilated in a ventilator-plethysmograph system such that tidal volumes were regulated to mean values of 12 to 13 ml/kg. At 30 min of age when mean PCO2 values for the study groups were between 33 and 42 mm Hg, lung function of each rabbit was evaluated by the peak inspiratory pressure needed to achieve the desired tidal volume, by compliance, and by the ventilation efficiency index. These measures of lung function showed no benefit of corticosteroids or T3 in the absence of surfactant, whereas TRH significantly improved lung function. Although surfactant treatment improved lung function in all groups, the best effect after a single hormone treatment was with corticosteroids. The combined use of corticosteroids and TRH in surfactant-treated animals resulted in the best overall responses. The leak of protein into and out of the lungs was measured with radiolabeled albumins. The leak decreased as peak ventilatory pressures decreased in all groups and decreased with surfactant treatments of all groups. Corticosteroids decreased the protein leak into the airways more than could be accounted for by the effects of corticosteroids on ventilatory pressures alone (p less than 0.01). None of the hormone treatments significantly increased the saturated phosphatidylcholine pool sizes from the low values noted in the control animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Fetal rabbits were treated with corticosteroids by maternal administration for 48 h before delivery at 27 d gestational age. The treated and control rabbits were placed on ventilator-plethysmographs so that ventilation could be adjusted by regulation of tidal volumes to 10-13 ml/kg body wt. [125I]albumin was mixed with fetal lung fluid at birth, alternate rabbits from each litter were treated with Surfactant-TA, and [131I]albumin was injected intravascularly. The movement of the labeled albumins into and out of the alveolar wash and lung tissue was measured after 30 min of ventilation. Corticosteroid treatment (total dose, 0.2 mg/kg betamethasone) significantly decreased the protein leak across the endothelium (P less than 0.001) but increased the protein leak across the epithelium (P less than 0.001). Surfactant treatment decreased both the endothelial and epithelial leaks, and the combination of surfactant and corticosteroid treatments decreased endothelial leaks to 29% of control values and increased compliance more than either treatment alone. The 48-h corticosteroid treatment did not increase alveolar surfactant pool sizes. Corticosteroids significantly changed lung protein leaks independently of surfactant, and improved the response of the preterm lung to surfactant treatments.

Rabbits were given by tracheal instillation liposomes, liposomes carrying metaproterenol sulfate (MPS) and suspended in a MPS solution, rabbit surfactant, or rabbit surfactant suspended in a MPS solution. The lipid suspensions were labeled with [14C]cholesterol and [3H]phosphatidylcholine. The percent recoveries of the labels were measured over 24 h in alveolar wash, lung tissue after alveolar wash, and the total lungs. All clearance curves for both phosphatidylcholine and cholesterol from liposomes were the same in the presence or absence of MPS. Alveolar clearance curves for both labels from rabbit surfactant were the same; however, the surfactant-associated labels were cleared to the lung tissue more rapidly than were the liposome-derived labels. Despite different alveolar clearance curves, all clearance curves for cholesterol from the total lungs were similar at a rate of 20 to 30%/24 h of the injected labeled cholesterol. Phosphatidylcholine was cleared from the total lungs more rapidly, at rates from 35 to 56%/24 h. Although MPS did not change labeled liposomal lipid clearance, the beta-agonist increased surfactant lipid clearance. The different responses to beta-agonist and the different alveolar clearance curves indicated distinct alveolar-to-lung tissue metabolism for liposomal versus surfactant phosphatidylcholine and cholesterol, although overall lung clearance rates were similar.

To clarify the metabolic cycle of a low-molecular-weight protein of surfactant (SP-C), we obtained alveolar surfactant from 3 day old rabbits killed 24 h after the tracheal administration of 32 P or l -[ 35 S]methionine (donors). Aliquots of this naturally labelled surfactant were administered into trachea to 3-day-old rabbits (recipients) which were killed after 1 min or 3, 8 or 24 h. We then analyzed the radioactivity associated with SP-C and with saturated phosphatidylcholine in fractions of lung lavage fluid and in lung homogenate. We found that alveolar SP-C is turned over faster than saturated phosphatidylcholine, that alveolar macrophages do participate in the removal of SP-C and that SP-C does not enter the fraction of alveolar surfactant that remains unsedimented after ultracentrifugation. Considering the whole lung, SP-C and saturated phosphatidylcholine are turned over at a comparable speed.

Premature rabbits delivered by cesarean section at 28 d of gestation were each given intratracheally 75 mg/kg of radiolabeled preparation of either natural rabbit surfactant, natural calf surfactant, or surfactant-TA. Each newborn rabbit was ventilated for up to 6 h in a ventilator-plethysmograph with individual adjustments of peak inspiratory pressures to attain tidal vol of 12-15 mL/kg body wt. Dynamic compliances were about 0.7-0.9 mL/cm H2O. kg after treatment with the three surfactants and did not deteriorate during the 6-h study. Rabbits were randomly studied at 0.5, 1.5, 3, 4.5, and 6 h of age for the recovery of the labeled surfactant phosphatidylcholine in the total lungs (alveolar wash plus postlavage lung tissue). The labeled phosphatidylcholine was cleared from the total lungs of rabbits treated with natural rabbit or calf surfactants at comparable rates of about 25%/6 h. In contrast, the clearance rate of surfactant-TA phosphatidylcholine from the total lungs was not significantly different from 0. Lipids from rabbit surfactant that had been administered intratracheally were only minimally present in the blood and liver. In other similarly treated rabbits, the lipids from radiolabeled rabbit surfactant and liposomes of dipalmitoylphosphatidylcholine that had been injected intravenously were recovered in blood and liver in substantial quantities. These studies documented significant losses of rabbit and calf surfactant phosphatidylcholine but not surfactant-TA phosphatidylcholine from the lungs of preterm ventilated rabbits. The losses were not explained by surfactant losses to the vascular compartment.

Constant distending pressure when applied to the immature fetal lamb lung was reported to improve compliance and prevent the subsequent development of the respiratory distress syndrome after delivery. These experiments were designed to repeat those observations and identify the mechanism(s) responsible for the effects of constant distending pressure on subsequent lung function. The compliance of the lungs of exteriorized fetal lambs at 130 to 133 days gestational age increased 2.5-fold after 1 h of 15 cm of distending pressure. However, subsequent ventilation of the lambs exposed to distending pressure and control lambs resulted in comparable sequential compliance and blood gas and pH measurements. Severity of lung disease as reflected by the peak inspiratory pressure needed to normalize Pco2 values decreased as surfactant-saturated phosphatidylcholine pool sizes increased (r values greater than 0.90) and minimum surface tensions of alveolar washes decreased, but the distention procedure did not change these relationships. Distention of the fetal lung did result in an apparent increase in pulmonary blood flow in the fetus and an increased leak of labeled albumin from the vascular space to the lung interstitium and airways during the 1-h period of ventilation after delivery. However, the leak of protein into the lungs exposed to the distending pressures was not increased during the period of exteriorization and distention, suggesting that distention sensitized the preterm lung to leak protein with subsequent mechanical ventilation. The leak of labeled albumin out of the airways was not changed by distention, and total lung water was not changed.(ABSTRACT TRUNCATED AT 250 WORDS)

Surfactants were labeled in vivo with [3H] choline and the large aggregates of the surfactant were recovered by alveolar wash and centrifugation. The labeled natural surfactants from rabbit, sheep, cat, and pig were injected into the airways of 3-day-old rabbits, and the percent recoveries of the labeled surfactant-associated phosphatidylcholine were measured in alveolar washes, lung tissue after alveolar wash, and in the lungs (alveolar wash plus lung tissue). The rabbit surfactant-associated phosphatidylcholine was cleared from the lungs at a constant 15.6 ± 1.8% per 24 h (mean ± SE) of the injected doses of surfactant that contained from 0.41 to 10.2 µmol phosphatidylcholine. At all times following injection, approximately 50% of the labeled phosphatidylcholine remaining within the lungs was recovered in the alveolar wash and 50% with the lung tissue. The percent clearances for sheep, cat, and pig surfactant phosphatidylcholine in rabbits were 12.5, 16.6, and 16.3% per 24 h, respectively, values not different from that measured for rabbit surfactant. The results documented a slow clearance of exogenously administered surfactant phosphatidylcholine as a fixed percent of the injected dose that was unchanged by species source of the surfactant.

A possible route of clearance of surfactant phosphatidylcholine from the lungs is via the airways. To quantify surfactant loss via this pathway, latex bags were surgically placed into the abdomens of adult rabbits such that secretions cleared via the esophagus could be collected. The rabbits then were given treatment or trace doses of radiolabeled phosphatidylcholine-surfactant by tracheal injection and/or intravascular radiolabeled precursors of phosphatidylcholine. Labeled saturated phosphatidylcholine was measured in all fluids that were collected from the bags at 2-h intervals for 24 h and in alveolar washes and lung tissues at 24 h. No more than 7% of either treatment or trace doses of intratracheal surfactant-saturated phosphatidylcholine was lost via clearance up the airways over 24 h. Clearances of endogenously synthesized and secreted saturated phosphatidylcholine were estimated to be no more than 3% of the flux of labeled saturated phosphatidylcholine through the alveolar pool. These experiments demonstrate that surfactant phosphatidylcholine clearance via movement up the airways is not a major pathway leading to surfactant catabolism.

Rabbit fetuses were delivered by hysterotomy on day 27 or 28 of gestation. Immediately after birth, the animals were tracheotomized and received by intravenous injection 0.2 µCi radiolabeled albumin and 11 mg iron dextran in 0.2 ml saline. The newborn rabbits then were ventilated artificially with a tidal vol of 12 ml/kg for 5–20 min. One group of nonventilated animals served as controls. At the end of the experiment, one lung was lavaged via the airways and the other was fixed for histologic examination. The recovery of labeled albumin and iron dextran in the lavage fluid was quantified. Iron dextran complexes were easily identified in the lung sections by staining with Prussian blue. Iron dextran accumulated in the airspaces of animals delivered on day 27 (about 4% of the injected dose during 10–20 min of ventilation). The albumin leakage was slightly higher than that of the dextran, a result consistent with different mol wt of the markers. The vol density of leaking alveoli in histologic sections increased with time, from 0 at birth to a mean value of 0.36 after 20 min of ventilation. The leakage starts as a focal event, gradually involving more and more terminal airspaces. In the histologic sections, there was no indication of a significant leakage at the bronchiolar level, although the epithelium of terminal and preterminal airways was clearly injured in all ventilated animals.

We tested whether maternal administration of corticosteroids, thyrotropin-releasing hormone (TRH), triiodothyronine (T3) or their combinations, would improve lung function of ventilated preterm newborn rabbits. Maternal corticosteroids and T3 treatments did not improve lung compliance; TRH did. The major effect observed was a large improvement in lung compliance following maternal treatment with corticosteroids plus TRH in animals treated with surfactant. These agents made the lung "receptive" to the surfactant treatment. T3 did not improve lung function and no augmented response to surfactant was seen. None of the treated groups had surfactant pool sizes significantly different from controls. The mechanisms of action of corticosteroids and/or TRH seemed to be independent of changes in surfactant pool sizes.

Three-day-old rabbits were given intratracheal injections with a variety of surfactants at doses of about 100 mg lipid/kg, doses commonly used in clinical trials of surfactant for respiratory distress syndrome. Calf and sheep natural surfactants isolated by centrifugation of alveolar washes were compared with Surfactant-TA and two aggregate sizes of lipid solvent extracted sheep surfactant by measuring the percent recoveries of labeled phosphatidylcholine in alveolar washes and lung tissue at times to 48 h after surfactant injection. Surfactant-TA and the lipid extracted surfactants did not contain the 28 to 35-kdalton surfactant protein. All surfactants had similar linear clearance rates from the total lung (alveolar wash plus lung tissue), independent of species source, extraction with lipid solvents, or aggregate sizes of the phospholipids in suspension. There were no metabolic consequences to lipid extraction, the loss of 28-35 kdaltons of protein, or changes in aggregate sizes of surfactant lipids when injected in treatment doses into the airways of 3-day-old rabbits.

The loss of radiolabelled phosphatidylcholine associated with surfactants and lipid extracts of surfactants from different species sources was measured following tracheal injection into the lungs of adult and 3 day old rabbits. Clearance was more rapid from the lungs of adult than 3 day old rabbits. The percent labelled phosphatidylcholine cleared per 24 h did not change independently of dose injected indicating that clearance and catabolic pathways were not saturable in either group of rabbits. Different species sources or lipid extraction of natural surfactants did not alter clearance rates very much in the 3 day old rabbits. Small differences in clearance rates were identified by comparing rabbit surfactant with calf surfactant or Surfactant-TA in the adult rabbits. These results indicate that the lungs of developing and adult rabbits can clear large doses of surfactants from multiple sources at rates comparable to the species common natural surfactant.

Transformations of surfactant after secretion are incompletely understood. To clarify them, we lavaged lungs in fetuses and in newborn rabbits, fractionated the lavage fluid by differential and density gradient centrifugation, and analyzed the distribution of surfactant protein (SP) phospholipids, SP-A, SP-B, and SP-C. Furthermore, we administered into trachea of newborn rabbits labeled surfactant and compared the alveolar clearance of SP-A, SP-B, SP-C and saturated phosphatidylcholine. We found that, in the fetus, secreted lamellar bodies contain all components of surfactant, except a small amount of SP-A. As breathing starts and new surfactant subtypes are generated, the proteins are mostly associated with dense subtypes, but SP-B and SP-C are especially concentrated in dense materials that contain minor amounts of phospholipids and SP-A. Furthermore, we found that, during breathing, alveolar surfactant is degraded into more than one type of remnant, that the lavage fluid contains a pool of SP-A not associated with membranes, and that SP-A, SP-B, and SP-C are all turned over at a faster rate than saturated phosphatidylcholine.

To clarify the steps following surfactant secretion, we injected adult rabbits with radioactive palmitate, lavaged the airways, removed the cells, separated by ultracentrifugation lavage components into two fractions (B and C), and followed the labeling of phospholipids of these fractions. The results were compatible with the view that total and saturated phosphatidylcholine are transferred from B to C. Furthermore, the fluxes of total and saturated phosphatidylcholine through fraction C (0.45 and 0.30 mumol . h-1 . g lung-1, respectively) were compatible with the actual estimates of surfactant recycling. The labeling of phosphatidylglycerol ruled out a simple precursor-product relationship between B and C but was compatible with a nonideal first-order relationship. The labeling of phosphatidylinositol, cardiolipin, and phosphatidylethanolamine was incompatible with the existence of a direct precursor-product relationship between B and C. The labeling of total and saturated phosphatidylcholine suggests that fraction B may be made by active surfactant, whereas fraction C may contain surfactant modified for reuptake or for reuptake and catabolism.

Glutaraldehyde is considered a promoter of calcification by the action of toxic aldehyde group residuals from cross-linking. Post-fixation treatment with homocysteic acid (HA), besides bonding aldehyde groups and neutralizing toxicity, should enhance biocompatibility due to the strongly electronegative sulfonic group. The aim of this investigation was to evaluate HA efficacy on tissue preservation and dystrophic calcification mitigation in glutaraldehyde cross-linked bovine pericardium (BP) using a subcutaneous rat model.Four samples of BP, two with glutaraldehyde-HA and two with glutaraldehyde treatment, were implanted in each of 24 male Sprague-Dawley rats. Three rats were killed at 14 days, eight at 28 days, eight at 56 days and five at 84 days. Unimplanted glutaraldehyde-HA- and glutaraldehyde-treated samples served as controls. All samples were studied by gross examination, mammography, light transmission and scanning electron microscopy, and atomic absorption spectroscopy. The nature of mineralization was investigated by coupling techniques of scanning electron microscopy, electron microprobe analysis and X-ray powder diffraction.No histological and ultrastructural differences were found between glutaraldehyde-HA- and glutaraldehyde-treated BP, whether implanted or unimplanted. In both groups, calcification progressed with time, but significantly less after glutaraldehyde-HA treatment than after glutaraldehyde alone and at all time intervals (14.63 +/- 21.34 versus 43.17 +/- 15.99 at 28 days, p = 0.003; 56.42 +/- 40.20 versus 90.59 +/- 32.90 at 56 days, p = 0.008; 91.68 +/- 67.68 versus 156.23 +/- 17.85 at 84 days, p = 0.01). Differences were evident by mammography and histology (von Kossa stain). Electron microprobe analysis in both groups showed the composition of calcified nuclei to be calcium phosphate, stoichiometrically close to apatite (Ca5(PO4)3(OH)). The occurrence of crystallized apatite was supported by X-ray powder diffraction findings, the amount of crystallized apatite being higher in glutaraldehyde-treated samples.Post-fixation treatment with HA preserves BP structural properties and significantly mitigates mineralization of long-term subcutaneous implants.

Biodegradable D,L-Lactide-ε-caprolactone copolymer was used in substitution to bovine collagen to seal porosity in nine Dacron vascular Sorin Carbografts®. One served as control and 8 were implanted in mini-pigs as vascular by-pass in the thoracic aorta. The grafts were explanted at 7 days (4 animals), 30 (2 animals) and at 90 days (2 animals), and submitted to gross examination, X-ray, histology and electron microscopy. Aim of the study was to assess the safety and the reliability of these polyester vascular prostheses impregnated with the copolymer in terms of containment of the bleeding in the perioperative period, host inflammatory response, copolymer biodegradation and prostheses “healing”. All the grafts were patent at angiographic and X-ray examination. At 7 days blood infiltration between Dacron and copolymer lining was detected. Inflammatory granulocyte infiltrates and granulomatous reaction with polymer degradation was observed at 30 days and fibrous tissue healing at 90 days. Luminal surface was covered by thin thrombi at 7 and by a neointima at 30 and 90 days. We conclude that D,L-Lactide-ε-caprolactone copolymer is effective in preventing perigraft bleeding, even though an early hematoma between Dacron and the copolymer coating occurs. Copolymer is degraded through a mild inflammatory reaction, with eventual evolution to fibrous healing.

Glutaraldehyde may promote calcification in xenograft tissue by the action of toxic aldehyde group residues involved in the cross-link process. Post-fixation treatment with homocysteic acid (HA) neutralizes this toxicity by bonding aldehyde groups, and enhances biocompatibility on the basis of strongly electronegative sulfonic groups. Previous studies in a rat subcutaneous model showed significant long-term mitigation of mineralization of glutaraldehyde-fixed pericardium treated with HA. This study aimed to assess the anticalcific efficacy of HA in a valvular implant in growing sheep, and establish if the tricuspid position is suitable for testing replacement bioprosthetic valves.Eleven stented 25 mm Pericarbon bioprostheses (seven HA-treated, four standard) were implanted in the tricuspid position of growing sheep. Infective endocarditis occurred in four prostheses. Among the remaining seven, three (two HA-treated, one standard) were explanted at 91 days (mid-term), and four (two HA-treated, two standard) at 140-141 days (long-term). All explants were studied by gross, X-ray, light, transmission and scanning electron microscopy, as well as by atomic absorption spectroscopy.No histological and ultrastructural difference in tissue preservation were observed between HA-treated and standard Pericarbon bioprostheses, either in the mid or long term. The mean calcium content of mid-term HA-treated explants was 9.55 mg/g compared with 16.26 mg/g in mid-term standard explants. Only one late standard explant failed as a result of severe stenosis caused by massive dystrophic calcification. Among four late explants, two showed significant increase in mineralization (HA-treated, 87.45 mg/g; standard, 181.20 mg/g), while two showed calcium contents similar to those in mid-term explants (HA-treated, 11.96 mg/g; standard, 17.32 mg/g).Post-fixation treatment with HA preserves structural properties after tricuspid implantation in growing sheep. The tricuspid implant in the sheep model failed to reproduce remarkable accelerated progressive calcification in all xenografts so as to demonstrate a significant difference between HA and standard explants. The tricuspid position for testing replacement bioprosthetic valves should be abandoned, and investigations repeated with the prosthesis in the mitral position.

To clarify the life cycle of surfactant protein C (SP-C), we obtained alveolar surfactant from 3-day-old rabbits killed 15 h after the tracheal administration of 32P and L-[35S]methionine. Small amounts of this surfactant were administered into the tracheas of 3-day-old rabbits, which were killed after 1 min and 2 and 4 h. We then analyzed the radioactivity associated with SP-C and with saturated phosphatidylcholine (DPPC) in lung lavage fluid and in a lamellar body-rich fraction isolated from the lung homogenate. We found that SP-C and DPPC are both taken up from the alveolar spaces and incorporated into lamellar bodies, but the time courses of reuptake are different. Our results indicate that in the alveoli SP-C is turned over faster than DPPC.

Jaundiced babies undergoing phototherapy often develop diarrhoea. The cause of it is still uncertain. Increasing evidence supports a role of a secretory mechanism for the diarrhoea. We therefore studied the effects of bile from congenitally jaundiced rats undergoing phototherapy and of unconjugated bilirubin on rat small intestine in vivo and in vitro. Results suggest that: (1) the bile from homozygous Gunn rats under phototherapy has an anti-absorptive effect when tested in the perfused jejunum of normal Wistar rats; (2) unconjugated bilirubin has a dose dependent secretory effect on the intestinal transport of water and electrolytes, when tested in the same system. Alteration of cyclic AMP or cyclic GMP, known intracellular mediators of secretion, was not observed. We conclude that free bilirubin is an intestinal secretagogue acting by an as yet unknown mechanism, that may mediate the secretory type of diarrhoea in jaundiced neonates undergoing phototherapy.

Nonviral vectors might represent a safe alternative to adenovirus for gene therapy of lung disorders, in particular cystic fibrosis (CF). Cationic lipids have been shown to correct the CF defect both in vitro and in vivo, but more efficient vectors are needed to improve the low gene transfer efficiency. Here, we show that the cationic polymer ExGen 500, a linear polyethylenimine derivative, is more efficient than cationic lipids in transferring reporter genes to lung epithelial cells in vitro. In vivo ExGen 500 was able to mediate gene transfer into both newborn and adult rabbit lungs with comparable efficiencies. The best levels of transfection were obtained using neutral complexes. Under such conditions, luciferase activities corresponding to about 10(3) RLU/10 s/mg of protein were reproducibly obtained 2 days after transfection throughout the four lung lobes of newborn and adult rabbits. A nlslacZ reporter gene showed transfected cells around the lumen of large and small bronchi. No signs of acute toxicity (inflammation, cellular infiltration etc) were detected by direct histopathological analysis. Within 1 week after instillation, transgene expression decreased by two orders of magnitude.