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Biomedical subjects

C Pederzolli

Publications and source records attributed to C Pederzolli.

34 records · Page 2Linked to original sources
PubMed1995

Successful treatment of aortic dissection after heterotopic heart transplantation.

Heterotopic heart transplantation is a valid option when there is a large donor-recipient size mismatch. However, the presence of the diseased native heart can jeopardize the medium-term and long-term outcome. The problems stemming from this most commonly described in the literature are thromboembolism, angina, and arrhythmias. In this report, we describe the case of a type A aortic dissection in the native aorta that occurred 30 months after heterotopic heart transplantation and the surgical technique successfully applied for its repair. We also discuss some of the alternative techniques.

Aortic Dissection↗
PubMed1995

Different results of cardiac transplantation in patients with ischemic and dilated cardiomyopathy.

We retrospectively analyzed 275 consecutive transplanted patients, dividing them into group A (128 patients) affected by ischemic cardiomyopathy and group B (147 patients) affected by dilated cardiomyopathy. The difference in demographic, clinical and hemodynamic preoperative and postoperative data between the groups was studied; group A patients presented at transplantation with a less compromised hemodynamic picture, requiring inotrope infusion and mechanical assistance less frequently. The influence of etiology on early postoperative complications was also analyzed: group A patients needed postoperative mechanical assistance, inotrope, infusion and prolonged mechanical ventilation more often, therefore requiring a longer stay in the intensive care unit (ICU). Hospital mortality was twice as high in group A. The older age of group A patients per se did not influence these results significantly. The long-term follow-up was then studied with particular attention to parenchymal functions, hemodynamics, coronary artery disease, metabolic and surgical complications, and survival. The complication rate was higher in group A, with more severe hypertension and higher cholesterol levels at 1 year, a higher prevalence of accelerated coronary artery disease (CAD) and a more frequent onset of insulin-dependent diabetes. Surgical and vascular complications were also more frequent. The final result was a better 5-year actuarial survival rate for group B patients. Donor and recipient ages at the time of transplant did not influence this result. We conclude that ischemic patients, even if they are transplanted in better condition and operated more electively, have a more critical early and long-term postoperative course and a worse survival rate. These findings are not explained by advanced age, but could be due to the impact of atherosclerosis and metabolic impairments associated with ischemic disease.

Actuarial Analysis↗
PubMed1994

Primary and secondary structure of a pore-forming toxin from the sea anemone, Actinia equina L., and its association with lipid vesicles.

The complete amino acid sequence of equinatoxin II, a potent pore-forming toxin with hemolytic, cytotoxic and cardiotoxic activity from the venom of the sea anemone, Actinia equina L., is reported. In addition, circular dicroism was used to estimate the secondary structure of this toxin either in the water-soluble or in the membrane-anchored form. Equinatoxin II when in water was found to contain about 29-33% of alpha-helical structure, 53-58% of beta-strand+beta-turn and 10-16% of random structure. Upon association with phospholipids, in particular with sphingomyelin, a rearrangement of the secondary structure occurs resulting in an increase of the alpha-helix content. An amphiphilic alpha-helical segment is predicted at the N-terminus, which shares structural homology with membrane active peptides like melittin and viral fusion peptides. In analogy to the behaviour of these peptides we propose that at least part of the alpha-helix content increase of equinatoxin II is due to the insertion of its N-terminus into the lipid bilayer. As in the case of melittin, association of 3-4 equinatoxin molecules is necessary to induce membrane permeabilisation.

Amino Acid Sequence↗
PubMed1994

Mechanism of action of equinatoxin II, a cytolysin from the sea anemone Actinia equina L. belonging to the family of actinoporins.

Actinia equina equinatoxin II (EqT-II) is a representative of a family of pore-forming, basic, polypeptide toxins from sea anemones, now called actinoporins. This family comprises at least 27 members, which are all hemolytic at rather low concentrations. Red blood cell (RBC) hemolysis by EqT-II is the result of a colloid-osmotic shock caused by the opening of toxin-induced pores. Using osmotic protectants of different size the functional radius of the lesion was estimated to be approximately 1.1 nm. These pores are most probably constituted by oligomeric aggregates of cytolysin molecules, whose presence on the membrane of lysed RBC was directly demonstrated by polyacrylamide gel electrophoresis (PAGE) after covalent cross-linking. EqT-II is active also against a variety of mammalian cells including leukocytes, platelets and cardiomiocytes. An increased permeability of the plasma membrane after Eq-II attack is compatible with the notion that the toxin forms pores also on these cells. Eq-II permeabilises even purely lipidic model membranes, suggesting a protein receptor is not necessary. Using calcein-loaded unilamellar vesicles (UVs) comprised of phosphatydylcholine (PC) mixed with other lipids we observed that the rate and extent of permeabilization greatly increases when sphingomyelin (SM) or the ganglioside GM1 were introduced, particularly in the case of large UVs (which are more sensitive to the toxin than small UVs). PAGE indicated that the increased effect of Eq-II on SM containing vesicles is due to an increased level of toxin binding to such vesicles. The formation of cation-selective channels by EqT-II was directly demonstrated using planar lipid membranes where the toxin induced discrete increases of the film conductivity. The conductance of the channel was consistent with the estimated size of the lesion formed in RBC. Several factors can affect toxin activity: serum, low pH, low ionic strength and multivalent cations are potent inhibitors. pH Dependence is bell shaped, optimum activity being between pH 8 and 9. Similarly the action of Ca2+ is also bivalent: up to a concentration of approximately 2 mM it stimulates hemolysis, but above this concentration it inhibits (with 50% inhibition occurring at approximately 10 mM). When the known amino acid sequences of actinoporins are examined a common trait emerges; the presence of a well conserved, amphiphilic, putative alpha-helix at the N-terminus, which might be involved in the insertion of EqT-II in lipid membranes.

Animals↗
PubMed1993

Pore formation by the sea anemone cytolysin equinatoxin II in red blood cells and model lipid membranes.

The interaction of Actinia equina equinatoxin II (EqT-II) with human red blood cells (HRBC) and with model lipid membranes was studied. It was found that HRBC hemolysis by EqT-II is the result of a colloid-osmotic shock caused by the opening of toxin-induced ionic pores. In fact, hemolysis can be prevented by osmotic protectants of adequate size. The functional radius of the lesion was estimated to be about 1.1 nm. EqT-II increased also the permeability of calcein-loaded lipid vesicles comprised of different phospholipids. The rate of permeabilization rised when sphingomyelin was introduced into the vesicles, but it was also a function of the pH of the medium, optimum activity being between pH 8 and 9; at pH 10 the toxin became markedly less potent. From the dose-dependence of the permeabilization it was inferred that EqT-II increases membrane permeability by forming oligomeric channels comprising several copies of the cytolysin monomer. The existence of such oligomers was directly demonstrated by chemical cross-linking. Addition of EqT-II to one side of a planar lipid membrane (PLM) increases the conductivity of the film in discrete steps of defined amplitude indicating the formation of cation-selective channels. The conductance of the channel is consistent with the estimated size of the lesion formed in HRBC. High pH and sphingomyelin promoted the interaction even in this system. Chemical modification of lysine residues or carboxyl groups of this protein changed the conductance, the ion selectivity and the current-voltage characteristic of the pore, suggesting that both these groups were present in its lumen.

Animals↗
PubMed1993

Emergency and elective cardiac retransplantation.

Among 265 patients transplanted at our Institution, 7 underwent cardiac retransplantation. There were five emergency retransplantations, the indication being graft failure in one case and acute rejection in four cases. Two patients, retransplanted because of acute rejection, had a positive panel reactivity antibody and a negative donor crossmatch. In the rejection cases immunosuppression was enhanced by perioperative plasmapheresis and a postoperative 1-month course of cyclophosphamide. In two cases emergency retransplantations were successfully performed despite a highly positive prospective crossmatch. Two patients underwent elective retransplantations for chronic rejection 12 and 41 months, respectively, after the primary transplants. The overall early and late survival rates are 71% and 57%, respectively, with a mean follow-up of 48.5 months. The early and late mortality for elective retransplantation is zero. Our experience confirms both the high operative risk for emergency retransplantation and the excellent results for elective retransplantation. The use of plasmapheresis and cyclophosphamide allowed us to undertake retransplantation successfully in 2 cases with positive donor crossmatch. Both hyperimmunized patients in our series were retransplanted because of irreversible acute rejection despite a negative crossmatch with the primary donor. The meaning of negative crossmatch in patients with preformed cytotoxic antibodies is therefore questionable.

Adult↗
PubMed1992

A fluorimetric assay for the effects of cytolytic toxins on the transport properties of resealed erythrocyte ghosts.

We prepared resealed erythrocyte ghosts loaded with SPQ and chloride. We demonstrated that these membranes were still functional, as they were capable of exchanging anions, most probably through the band-3 protein. When cytolytic toxins (Escherichia coli hemolysin and Staphylococcus aureus alpha-toxin) were offered to the resealed ghosts, the internal SPQ was released. This could be attributed to the formation of toxin-induced ion channels into the ghost membrane that were so large that SPQ could escape through them. This release was actually independent of the anion-exchanging protein, since DIDS had no inhibitory effect on it. Due to their simplicity, and because they do not lyse, erythrocyte ghosts may serve as useful models to study the action of cytolytic pore-forming toxins. To assess the validity of these model membranes we compared results obtained using RBC and resealed erythrocyte ghosts as targets for the toxin, finding complete consistency. Pre-assembled toxin channels could also be studied on the ghosts. Applying different proteolytic enzymes to the external compartment after channel formation, we found that performed E. coli hemolysin pores were at least partially destroyed by enzymatic digestion.

Erythrocyte Membrane↗
PubMed1992

Membrane damage: common mechanisms of induction and prevention.

Common features in the induction of pores by various agents are as follows: induction is stochastic and progressive; damage by different agents is often synergistic and limited. The prevention of membrane damage is affected by trivalent and divalent cations, by low pH, by low ionic strength and by high osmotic pressure. The inhibitory role of protons and divalent cations is considered in greater detail: pore-forming agents can be classified into two groups: channels across planar lipid bilayers induced by the first group display voltage-sensitive, reversible inhibition by divalent cations; channels of the second group show voltage-insensitive, irreversible inhibition by divalent cations. A search for the ligands to which divalent cations and protons bind has proved elusive. Comparison with the phenomenon of 'surface conductance' through narrow apertures, that is manifest in the absence of any pore-forming agent, may prove fruitful.

Animals↗
PubMed1991

Chemical modification of Staphylococcus aureus alpha-toxin by diethylpyrocarbonate: role of histidines in its membrane-damaging properties.

Staphylococcus aureus alpha-toxin causes cell damage by forming an amphiphilic hexamer that inserts into the cell membrane and generates a hydrophilic pore. To investigate the role of the three histidine residues of this toxin we modified them with diethylpyrocarbonate, obtaining N-carbethoxy-histidine whose appearance may be followed spectrophotometrically. Despite the statistical nature of random chemical modification, it was possible to establish that modification of any one of the three histidines was enough to impair alpha-toxin activity on red blood cells and platelets. Two out of three histidines were essential for the interaction of the toxin with model membranes such as lipid vesicles and planar bilayers. Loss of lytic activity in both natural and model membranes was due both to defective binding and to defective oligomerization. When alpha-toxin hexamers inserted into lipid vesicles were assayed for chemical modifiability two histidines per monomer were found to be protected from diethylpyrocarbonate modification, whereas only one was protected after delipidation of the oligomer with a detergent. A possible model for the role of each histidine in the monomer is presented.

Animals↗
PubMed1991

Modification of lysine residues of Staphylococcus aureus alpha-toxin: effects on its channel-forming properties.

Staphylococcus aureus alpha-toxin opens an ion channel in planar phospholipid bilayers, which is selective for anions over cations, supposedly because of the presence of positively charged groups along the ion pathway. To remove some positive charges of this protein toxin, we chemically modified part of its lysine residues either with diethylpyrocarbonate, followed by histidine regeneration with hydroxylamine, or with trinitrobenzenesulfonic acid. The extent of chemical modification can be followed accurately by native polyacrylamide gel electrophoresis and isoelectric focusing. Ethoxyformilation of two to three lysine residues per toxin monomer does not impair hemolysis of rabbit red blood cells nor formation of pores in model membranes. It reduces the conductance and the anion selectivity of the channel and changes the shape of its current-voltage characteristic. This indicates that positively charged lysine residues are actually important in determining the electrical properties of the pore. Ethoxyformilation of channels preassembled in planar bilayers produces the same changes as modification of toxin monomers before channel formation. Furthermore, it can be performed by adding diethylpyrocarbonate on either side of the bilayer. This suggests that the lysine residues relevant for the electrical properties of the pore are located inside its lumen where they can be reached by diethylpyrocarbonate diffusing from either entrance of the channel.

Bacterial Toxins↗
PubMed1991

Lipid interaction of Pseudomonas aeruginosa exotoxin A. Acid-triggered permeabilization and aggregation of lipid vesicles.

We have investigated the interaction of Pseudomonas exotoxin A with small unilamellar vesicles comprised of different phospholipids as a function of pH, toxin, and lipid concentration. We have found that this toxin induces vesicle permeabilization, as measured by the release of a fluorescent dye. Permeabilization is due to the formation of ion-conductive channels which we have directly observed in planar lipid bilayers. The toxin also produces vesicle aggregation, as indicated by an increase of the turbidity. Aggregation and permeabilization have completely different time course and extent upon toxin dose and lipid composition, thus suggesting that they are two independent events. Both time constants decrease by lowering the pH of the bulk phase or by introducing a negative lipid into the vesicles. Our results indicate that at least three steps are involved in the interaction of Pseudomonas exotoxin A with lipid vesicles. After protonation of one charged group the toxin becomes competent to bind to the surface of the vesicles. Binding is probably initiated by an electrostatic interaction because it is absolutely dependent on the presence of acidic phospholipids. Binding is a prerequisite for the subsequent insertion of the toxin into the lipid bilayer, with a special preference for phosphatidylglycerol-containing membranes, to form ionic channels. At high toxin and vesicle concentrations, bound toxin may also induce aggregation of the vesicles, particularly when phosphatidic acid is present in the lipid mixture. A quenching of the intrinsic tryptophan fluorescence of the protein, which is induced by lowering the pH of the solution, becomes more drastic in the presence of lipid vesicles. However, this further quenching takes so long that it cannot be a prerequisite to either vesicle permeabilization or aggregation. Pseudomonas exotoxin A shares many of these properties with other bacterial toxins like diphtheria and tetanus toxin.

ADP Ribose Transferases↗
PubMed1991

Activation energy of the cardiac Na+/Ca2+ exchanger in sarcolemmal vesicles and reconstituted proteoliposomes.

Sarcolemmal membrane vesicles isolated from bovine ventricular tissue accumulate Ca2+ through the Na+/Ca2+ exchanger when exposed to an outwardly directed Na+ gradient. This Ca2+ is then released by the same mechanism if the vesicles are transferred to a Ca(2+)-depleted Na+ buffer. Using the Ca+ indicator, arsenazo III, and a stopped-flow spectrophotometer, we can directly follow the kinetics of Ca2+ extrusion. We can thus measure the activity of the Na+/Ca2+ exchanger by the initial rate of Ca2+ release. We found that it depends upon the external Na+ concentration in a cooperative way, with a Hill coefficient of 2. By studying the temperature dependence of Na+/Ca2+ exchange, we found that it can be described by a single activation energy: Ea = 8.3 +/- 0.4 Kcal/mol. When the Na+/Ca2+ exchanger is reconstituted into lipid vesicles of defined composition, we observe a higher activity if cholesterol is among the lipids. The activation energy becomes 6.1 +/- 0.1 Kcal/mol in this system, but the Arrhenius plot shows a decreased slope for temperatures above 33 degrees C.

Animals↗
PubMedDate not supplied

Biochemical and cytotoxic properties of conjugates of transferrin with equinatoxin II, a cytolysin from a sea anemone.

Transferrin, a serum glycoprotein, is a major regulator of cellular growth via its cellular receptor. Because transferrin receptors are absent from the plasma membranes of most normal adult resting cells, but are present on transformed, activated, and malignant cells, it can be used to address a toxin toward these cells. The cytolysin equinatoxin II, isolated from the sea anemone Actinia equina L., was coupled to human apo or diferric transferrin by using a heterobifunctional cross-linking reagent, N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP). The conjugates were separated by column chromatography, and their composition was demonstrated by electrophoresis, antibody staining, and determination of the hemolytic activity in the absence or presence of a reducing agent. The average molar ratio of equinatoxin II to transferrin for the studied conjugates was found to be approximately 3.4. The activity of the conjugates against human erythrocytes and human tumor cells (Raji and Jurkat) was assessed. The conjugate is very active on tumor cells in vitro; however, the hybrid molecule maintains an unspecific hemolytic activity. This unspecific toxicity is due to the fact that transferrin-bound toxin partially retains its original ability to bind to the cell membrane directly. It could be strongly reduced (and even eliminated) by pretreating the conjugates with sphingomyelin, the natural ligand of sea anemone cytolysins. These conjugates were stable versus temperature (up to at least 40 degrees C), versus time (up to several weeks at 4 degrees C and at least 1 year at -80 degrees C), and versus repeated freeze-thaw cycles with liquid nitrogen (but not with -80 degrees C).

Animals↗
PubMedDate not supplied

Echo-controlled endomyocardial biopsy.

Endomyocardial biopsy is an essential procedure for the diagnosis and grading of rejection in heart transplant patients. Direct control of the bioptome positioning has classically been obtained by fluoroscopy. Starting in June 1988, at our institution an alternative approach involving the use of two-dimensional echocardiography was introduced in clinical practice. In 125 patients 1591 biopsies have been performed: 445 under echographic control and 1146 under fluoroscopic control with 3.6 and 4.5 samples/biopsy, respectively. The percentages of inadequate samples caused by biopsy site sampling were 0.4% and 1.3%, respectively, in the two groups. Cardiac perforation has occurred twice in the fluoroscopic group; it has not been observed in the echographic group. One case of iatrogenic tricuspid regurgitation was detected in each group. We now consider echocardiography the method of choice to guide the bioptome. We prefer it to fluoroscopy because it eliminates the risks of x-ray exposure, increases the number of sampling sites in cases of echocardiographic evidence of rejection, can be easily performed as a bedside procedure, allows choice and variation of sampling sites, and permits monitoring of cardiac complications during and after the procedure. A randomized clinical trial is probably needed to assess with statistical significance the superiority of the echographic-controlled biopsy.

Biopsy↗