Biomedical subjects

C Pederzolli

Publications and source records attributed to C Pederzolli.

At least 19 recordsLinked to original sources

PubMed2004

Role of chemical interactions in bacterial adhesion to polymer surfaces.

Development of biomaterial-related infections is attracting an increasing interest due to the significant percentage of implant failure in the hospital care. Recent literature puts in evidence the dependence of the infection risk on the different biomaterials used, because of the different interactions between material surface and micro-organisms. Despite this, the mechanisms underlying the adhesion of bacteria to the biomaterial surface are still unclear. Aim of this work is to study the initial events of the processes responsible for the bacterial adhesion on polymers in order to prevent the development of bacterial infections and the consequent failure and replacement of biomedical devices. Electrostatic and Lifshitz-van der Waals forces are usually considered responsible for the interactions at the biomaterial interface. A new term that involves Lewis acid-base interactions is here introduced to better describe the bacterial adhesion to the polymer surface. Two requirements are needed to test this hypothesis: the development of an ideal polymeric surface in terms of chemical and morphological properties and the choice of a specific bacterial strain to be utilized as "probe". Experiments were worked out using an Escherichia coli (Gram-) strain that represent one of the principal isolates from infected biomaterial implants and its adhesion was investigated on polymers having different acid/basic character. The findings indicate that the bacterial adhesion is influenced by the chemical properties of the polymeric surface. These results may be interpreted taking into account a mechanism in which the acid/base (Lewis) interaction plays an important role.

Bacterial Adhesion↗

PubMed2002

Continuous monitoring of myocardial acid-base status during intermittent warm blood cardioplegia.

OBJECTIVE: Intermittent warm blood cardioplegia (IWBC) is a well-established technique for myocardial protection during cardiac operations. According to standardized protocols, IWBC administration is currently performed every 15-20 min regardless of any individual variable and in the absence of any instrumental monitoring. We devised a new system for continuous measurement of the acid-base status of coronary sinus blood for on-line evaluation of myocardial oxygenation during IWBC. METHODS: In 19 patients undergoing cardiac surgery for coronary artery bypass graft and/or valve surgery and receiving IWBC (34-37 degrees C) by antegrade induction (3 min) and retrograde or antegrade maintenance (2 min) every 15 min, continuous monitoring of myocardial oxygenation and acid/base status was performed by means of a multiparameter PO(2), PCO(2), pH, and temperature sensor (Paratrend7 (R), Philips Medical System) inserted into the coronary sinus. RESULTS: Mean cross-clamping time was 76+/-26 min; ischemic time was 13+/-0.2 min. pH decline was not linear, showing an initial fast decline, a point of flexus, and a progressive slow decline. After every ischemic period, the pH adaptation curve showed a complex pattern reaching step-by-step lower minimum levels (7.28+/-0.14 during the first ischemic period, to 7.16+/-0.19 during the third ischemic period - P=0.003). PO(2) decreased rapidly at 90% in 5.0+/-1.2 min after every reperfusion. During ischemia, PCO(2) increased steadily at 1.6+/-0.1 mmHg per minute, with progressively incomplete removal after successive reperfusion, and progressive increase of maximal level (42+/-12 mmHg during the first ischemic period, to 53+/-23 mmHg during the third ischemic period - P=0.05). CONCLUSIONS: Myocardial oxygen, carbon dioxide, and pH show marked changes after repeated IWBC. Myocardial ischemia is not completely reversed by standardized reperfusions, as reflected by steady deterioration of PCO(2) and pH after each reperfusion. Progressive increase of reperfusion durations or direct monitoring of myocardial oxygenation could be advisable in cases of prolonged cross-clamping time.

Acid-Base Equilibrium↗

PubMed2000

Orthotopic heart transplantation: standard versus bicaval technique.

We compared orthotopic heart transplantation (HT) by bicaval technique with the standard technique. Between January 1995 and December 1997, 117 patients underwent 118 HTs; 71 patients (15 women and 56 men) had 72 HTs by standard technique and 46 patients (9 women, 37 men) underwent HT using bicaval procedures. Preoperative parameters were similar in both groups; 5 patients who underwent the standard technique and no patients who underwent bicaval procedures required permanent pacemakers (p = NS). Isoproterenol infusion was significantly longer in the standard technique. Major perioperative arrhythmias (ventricular tachycardia and fibrillation, asystole) appeared in 8.2% and 7.0% of standard and bicaval HTs, respectively; atrial fibrillation appeared in 13.1% and 4.6%, respectively (p = NS). At 1 month, mitral and tricuspid regurgitation rates were higher in the standard group (p = NS); at 1 year only tricuspid regurgitation was still higher (p = NS). Right atrial pressure, Wood units, cardiac output, and cardiac index were examined (p = NS). At multivariate analysis, interaction between preoperative Wood units and transplant type was elicited for Wood units at 1 month and for right atrial pressure at 1, 3, and 6 months. In the high resistance subgroup, the patients who underwent bicaval procedures had higher resistances at 1 month. In the low resistance subgroup, right atrial pressure was higher in patients who underwent standard techniques at 1, 3, and 6 months follow-up. Thus, bicaval HT was found to be safe, without surgically related complications, it provoked significantly less blood loss, and required less isoproterenol use. No significant advantages were observed in conduction disturbances and major arrhythmias or regarding the need for temporary or permanent pacemakers.

Adolescent↗

PubMed1999

The spectrum of aortic complications after heart transplantation.

BACKGROUND: The connection between the donor and the recipient aorta is a potential source of early and late complications as a result of infection, compliance mismatch, and technical and hemodynamic factors. Moreover, the abrupt change in systolic pressure after heart transplantation involves the entire thoracic aorta in the risk of aneurysm formation. The aim of this study was to analyze the types of aortic complications encountered in our heart transplantation series and to discuss etiology, diagnostic approach, and modes of treatment. METHODS: Of the 442 patients having orthotopic heart transplantation and the 11 patients having heterotopic heart transplantation at our center, 9 (2%) sustained complications involving the thoracic aorta. These 9 patients were divided into four groups according to the aortic disease: acute aortic rupture (2 patients); infective pseudoaneurysm (3 patients); true aneurysm and dissection of native aorta (2 patients); and aortic dissection after heterotopic heart transplantation (2 patients). Surgical intervention was undertaken in 8. RESULTS: Five (83%) of 6 patients who underwent surgical treatment for noninfective complications survived the operation, and 4 are long-term survivors. One patient who underwent a Bentall procedure 71/2 years after heterotopic heart transplantation died in the perioperative period of low-output syndrome secondary to underestimated chronic rejection of the graft. One patient with pseudoaneurysm survives without surgical treatment but died several years later of cardiac arrest due to chronic rejection. Both patients operated on for evolving infective pseudoaneurysm died in the perioperative period. CONCLUSIONS: Infective pseudoaneurysms of the aortic anastomosis are associated with a significant mortality. In noninfective complications, an aggressive surgical approach offers good long-term results. The possibility of retransplantation in spite of complex surgical repair should be considered in the late follow-up after heart transplantation, due to the increasing incidence of chronic rejection.

Adult↗

PubMed1999

Heart transplantation in ischemic heart disease when recipients are older than 55 years and donors older than 50.

The purpose of this article is to analyze patients affected with ischemic cardiomyopathy, older than 55 years, who have undergone heart transplantation. We conducted a retrospective analysis comparing clinical course and outcome in patients whose donor age was > or = 50 years (Group A) with patients who had younger donor heart (Group B). Group A was composed of 25 patients, 55 to 68 years old (27.4% of the patients), 20 males and 5 females; Group B was composed of 68 patients, 55 to 66 years old, 65 males and 3 females. Mean donor age in Group A was 54.7 years old (range 51-61), while in Group B it was 29.5 years old (range 9-49). Operative mortality was 16% (4 cases) and 12% in Group B (8 cases) p = ns. Total mortality in Group A was 24%, or 6 cases: 2 graft failures, 1 infection, 1 neoplasm, 1 multiorgan failure, 1 ischemic heart disease; in Group B it was 27%, or 18 cases: 2 cerebrovascular accidents, 4 graft failures, 3 infections, 5 neoplasms, 3 multiorgan failures, 1 acute rejection, p = ns. Coronarography was performed in 51 patients, 14 in Group A (10 cases normal, 3 with irregularities, and 1 case with a critical stenosis of the circumflex artery; 37 in Group B (32 cases were normal, 3 had irregularities and 2 had critical stenosis in a coronary artery). In conclusion, we emphasize that extending donor age in recipients older than 55 years of age does not determine a higher risk and mortality.

Age Factors↗

PubMed1998

Risk factors for early death in patients awaiting heart-lung or lung transplantation: experience at a single European center.

BACKGROUND: Our purpose was to establish whether patients on the waiting list for heart-lung or lung transplantation had different survival rates according to diagnosis and to determine the specific variables responsible for early death. METHODS: Between 1988 and 1996, 278 patients were placed on the waiting list for organ transplant. Diagnoses were pulmonary vascular disease in 128, parenchymal disease in 141, and retransplantation in 9 patients. Eighty patients received transplants, 100 patients died awaiting transplantation, and 98 patients are still awaiting transplantation. Univariate and multivariate analyses of risk factors for early death on the waiting list were performed. Patients still listed < or =6 months (n=24), transplanted < or =6 months (n=37), or in the retransplantation group (n=9) were excluded. Of the remaining 208 patients, 52 died < or =6 months and 156 survived >6 months. RESULTS: Patients with primary pulmonary hypertension, pulmonary fibrosis, or cystic fibrosis had statistically significantly lower survival rates at 6, 12, and 24 months (31%, 36% and 26%, respectively, at 24 months) than patients with Eisenmenger's syndrome and chronic obstructive pulmonary disease (76% and 71%). Patients with Eisenmenger's syndrome who died < or =6 months had significantly higher systolic pulmonary artery pressure (134+/-39 vs. 108+/-25 mmHg) and pulmonary vascular resistance (1928+/-1686 vs. 1191+/-730 dyn/sec/cm(-5)) than those who survived longer. Patients with pulmonary fibrosis who died < or =6 months had significantly lower forced vital capacity (36+/-15 vs. 47+/-13% predicted), forced expiratory volume (37+/-14 vs. 48+/-14% predicted), room air PO2 (42+/-11 vs. 50+/-11 mmHg), and room air O2-saturation (78+/-10 vs. 84+/-8%) than those who survived longer. In the multivariate analysis, only the type of pathology was a significant risk factor for death after being on the waiting list < or =6 months. CONCLUSIONS: Certain pathologies and variables are risk factors for early death in patients on the waiting list. This information may be used to allocate specific donor organs to patients in greater need.

Adult↗

PubMed1998

A conserved tryptophan in pneumolysin is a determinant of the characteristics of channels formed by pneumolysin in cells and planar lipid bilayers.

Pneumolysin is one of the family of thiol-activatable, cytolytic toxins. Within these toxins the amino acid sequence Trp-Glu-Trp-Trp is conserved. Mutations made in this region of pneumolysin, residues 433-436 inclusive, did not affect cell binding or the formation of toxin oligomers in the target cell membrane. However, the mutations did affect haemolysis, leakage of low-molecular-mass metabolites from Lettre cells and the induction of conductance channels across planar lipid bilayers. Of eight modified pneumolysins examined, Trp-433-->Phe showed the smallest amount of haemolysis or leakage (less than 5% of wild type). Pneumolysin-induced leakage from Lettre cells was sensitive to inhibition by bivalent cations but the extent of inhibition varied depending on the modification. Leakage by the mutant Trp-433-->Phe was least sensitive to cation inhibition. The ion-conducting channels formed across planar lipid bilayers exhibit small (less than 30 pS), medium (30 pS-1 nS) and large (more than 1 nS) conductance steps. Small- and medium-sized channels were preferentially closed by bivalent cations. In contrast with wild-type toxin, which formed predominantly small channels, the modified toxin Trp-433-->Phe formed large channels that were insensitive to cation-induced closure. Polysaccharides of molecular mass more than 15 kDa inhibited haemolysis by wild-type toxin, but polysaccharide of up to 40 kDa did not prevent haemolysis by Trp-433-->Phe. Electron microscopy revealed that Trp-433-->Phe formed oligomeric arc and ring structures with dimensions identical with those of wild-type toxin, and that the ratio of arcs to rings formed was the same for wild-type toxin and the Trp-433-->Phe variant. We conclude that the change Trp-433-->Phe affects channel formation at a point subsequent to binding to the cell membrane and the formation of oligomers, and that the size of arc and ring structures revealed by electron microscopy does not reflect the functional state of the channels.

Animals↗

PubMed1998

Echo-guided endomyocardial biopsy in heterotopic heart transplantation. Case report.

Right ventricular endomyocardial biopsy is difficult to perform in patients who underwent heterotopic heart transplantation because of the complex vascular anatomy. The procedure is usually performed under fluoroscopic control. We present a case of a 59-year-old woman that after heterotopic heart transplantation underwent echo-guided endomyocardial biopsy. We report the technique discussing data obtained in other 11 patients heart transplanted in the heterotopic way. In conclusion we believe that echocardiographic guidance during endomyocardial biopsy allows a better choice of bite sites, reduces the risk of free wall perforation. Moreover is diminished the risk of X-ray exposure to both patient and operator.

Biopsy↗

PubMed1998

Cardiac lipomas. Description of 3 cases.

Cardiac lipomas, extremely rare benign tumours, can develop in the pericardial surface or inside the cardiac chambers. We report three cases, 2 intracardiac (one in the left and right atrium at the level of the interatrial septum and the other in the right ventricle) and one epicardial at the level of the left atrial roof. All patients underwent surgery and are now asymptomatic.

Adult↗

PubMed1997

[Heart transplant over 55 years].

BACKGROUND: The age of recipient has been thought for several years to be one of the most important predictors of survival after heart transplantation. Therefore patients older than age 50 years were usually excluded from heart transplantation. The marked improvement in survival after clinical introduction of cyclosporine made a critical revision of selection criteria for heart recipients. In this article we retrospectively analyze the outcome of heart transplantation dividing the patients into two groups: Group A < or = 55 years, Group B > 55 years respectively. METHODS: We analyzed 437 patients who underwent heart transplantation, immunosuppressive protocol for all the patients was cyclosporine, azathioprine and prednisone. Group A included 284 patients, Group B 153 patients. We studied demographic, clinical and haemodynamic data pre- and post-operatory in both groups. RESULTS: In Group A was more frequent dilated cardiomyopathy (p < 0.01) and the patients arrived at heart transplantation in worse haemodynamic conditions requiring more frequently inotropic infusion and/or mechanical support (intraaortic balloon pump, left ventricular assist device). Post-operatory course was more complicated in Group B patients where ischemic cardiomyopathy was more frequent (p < 0.001); intensive care stay, inotropic drugs infusion, mechanical ventilation and hospital mortality were higher in Group B, although non significantly. The donor age was significantly higher in Group B (p < 0.001), anyway the statistical analysis did not show a correlation between donor age and more complicated post-operative course. Follow-up did not show significative differences in the two groups considering haemodynamic data, metabolic disorders, graft coronary disease, infection and rejection rates. On Group B is significantly higher tumor incidence (p < 0.05) and peripheral vascular complications (p < 0.01). Actuarial survival at 5 and 7 years is respectively in Group A 80.3 and 72.9%, in Group B 75.4 and 71% (p = ns). CONCLUSIONS: Heart transplantation in patients older than 55 years with end-stage heart disease is a valid therapeutic option with excellent long-term survival; pre-operative screening must be particularly accurate and older donors should be considered.

Aged↗

PubMed1997

Echo-controlled endomyocardial biopsy in orthotopic heart transplantation with bicaval anastomosis.

Starting in January 1995, we performed heart transplantation, randomly using standard and bicaval techniques. In the latter technique, the anatomy of the right atrium is maintained, since the venae cavae are anastomosed. In 38 patients who received heart transplantation with bicaval anastomosis, 339 endomyocardial biopsies (EMB) were performed. EMB was done under echocardiographic control in 309 cases, whereas the remaining 30 were done under fluoroscopy. When EMB was echo-guided there was one major complication, namely right hemothorax in a 29-year-old man, who had had heart transplantation one week before, and this required surgical exploration. Other complications, correlated to venipuncture were: left hemothorax in a 65-year-old woman determined by arterial puncture, treated by means of chest tube drainage; pneumothorax (1 case). Echocardiographic guidance during EMB allows a better choice of biopsy site, reduces the risk of damaging cardiac structures and allows immediate monitoring of heart performance. Moreover the risk of X-ray exposure to both patient and operators is reduced. In any case, because the superior vena cava suture line is not visualized by two-dimensional echocardiography, if the bioptome cannot be introduced easily through superior vena cava, fluoroscopic control should be immediately applied, particularly in the early post-operative period when cicatrization is not complete.

Adult↗

PubMed1996

Human cytomegalovirus early infection, acute rejection, and major histocompatibility class II expression in transplanted lung. Molecular, immunocytochemical, and histopathologic investigations.

The present study aimed to investigate the relationship between acute rejection and human cytomegalovirus (HCMV) infection, as well as the coexpression of HLA-DR and immediate-early (IE) viral antigens, in 143 transbronchial biopsies and bronchoalveolar lavage fluids of 32 lung transplant recipients. We investigated the occurrence of morphologically overt viral infection with conventional histopathology, the expression of IE antigens with single labeling immunohistochemistry, the coexpression of IE antigens and HLA-DR molecules with double labeling techniques, and the presence of viral IE genes with polymerase chain reaction. Histopathologic study showed overt viral infections (12.6%) in 18 of the 143 biopsies; 8 were in a context of pneumonia and 10 were localizations without surrounding inflammatory cells; immunohistochemistry showed IE viral antigen expression in 31 (21.67%); PCR detected viral IE genes in 73/143 lavage fluids and biopsies (51%). The double labeling immunohistochemical technique showed that most IE antigen-expressing, noncytopathic cells were either HLA-DR negative in areas without infiltrates, or HLA-DR positive in those areas where inflammatory infiltrates were consistent, in the absence of viral cytopathy, with acute rejection. The results indicate that, in transplanted lung, the frequency of morphologically occult HCMV infection (as detected by immunohistochemically and/or PCR) is much higher than that of morphologically overt viral infection. The occurrence of inflammatory infiltrates (consistent with acute rejection) around morphologically occult infected cells and the possible lack of inflammation around both early- and late-infected cells suggest that in biopsies with occult infection the infiltrates should be attributed to allograft reaction. This conclusion would be in keeping with the coexpression of HLA-DR and HCMV IE in infiltrate-rich biopsies that are consistent with acute rejection, as well as with the absence of HLA-DR expression in IE antigen-positive cells in infiltrate-free-areas.

Acute Disease↗

PubMed1996

Permeability increase induced by Escherichia coli hemolysin A in human macrophages is due to the formation of ionic pores: a patch clamp characterization.

Escherichia coli hemolysin is known to cause hemolysis of red blood cells by forming hydrophilic pores in their cell membrane. Hemolysin-induced pores have been directly visualized in model systems such as planar lipid membranes and unilamellar vesicles. However this hemolysin, like all the members of a related family of toxins called Repeat Toxins, is a potent leukotoxin. To investigate whether the formation of channels is involved also in its leukotoxic activity, we used patch-clamped human macrophages as targets. Indeed, when exposed to the hemolysin, these cells developed additional pores into their membrane. Such exogenous pores had properties very different from the endogenous channels already present in the cell membrane (primarily K+ channels), but very similar to the pores formed by the toxin in purely lipidic model membranes. Observed properties were: large single channel conductance, cation over anion selectivity but weak discrimination among different cations, quasilinear current-voltage characteristic and the existence of a flickering pre-open state of small conductance. The selectivity properties of the toxin channels appearing in phospholipid vesicles were also investigated, using a specially adapted polarization/depolarization assay, and were found to be completely consistent with that of the current fluctuations observed in excised macrophage patches.

Bacterial Proteins↗

PubMed1996

Pore formation by S. aureus alpha-toxin in liposomes and planar lipid bilayers: effects of nonelectrolytes.

Nonelectrolytes such as polyethylene glycols (PEG) and dextrans (i) promote the association of S. aureus alpha-toxin with liposomes (shown by Coomassie staining) and (ii) enhance the rate and extent of calcein leakage from calcein-loaded liposomes; such leakage is inhibited by H+, Zn2+ and Ca2+ to the same extent as that of nonPEG-treated liposomes. Incubation of liposomes treated with alpha-toxin in the presence of PEG with the hydrophobic photo-affinity probe 3-(trifluoromethyl)-3-m-[125I]iodophenyl) diazirine (125I-TID) labels monomeric and-predominantly-hexameric forms of liposome-associated alpha-toxin; in the absence of PEG little labeling is apparent. At high concentrations of H+ and Zn2+ but not of Ca(2+)-all of which inhibit calcein leakage-the distribution of label between hexamer and monomer is perturbed in favor of the latter. In alpha-toxin-treated planar lipid bilayers from which excess toxin has been washed away, PEGs and dextrans strongly promote the appearance of ion-conducting pores. The properties of such pores are similar in most regards to pores induced in the absence of nonelectrolytes; the differ only in being more sensitive to "closure" by voltage (as are pores induced in cells). In both systems, the stimulation by nonelectrolytes increase with concentration and with molecular mass up to a maximum around 2,000 Da. We conclude (i) that most of the alpha toxin that becomes associated with liposome or planar lipid bilayers does not form active pores and (ii) that the properties of alpha-toxin-induced pores in lipid bilayers can be modulated to resemble those in cells.

Animals↗

PubMed1996

[Lung transplantation in Pavia. Clinical experience].

Single and bilateral lung transplantation represents the most recent success in the field of organ replacement. The procedure underwent a very long period of laboratory and clinical experimentation and it is still a high-risk procedure to be performed by a few highly specialized centers. At our Department of Cardiac Surgery, the preliminary experience in lung replacement was obtained by clinically applying heart-lung transplantation in patients with both vascular and parenchymal lung disease during 1991. In the following years, the indications for isolated lung transplantation were enlarged by some centers with good results; this consideration, together with the scarce availability of integral heart-lung blocks, led us to use single and bilateral lung transplantation initially for parenchymal disease and subsequently also for vascular disease. Since February 1992, 12 single lung transplantations and 12 bilateral lung transplantations have been performed at our Center. In this report the selection criteria, the preoperative characteristics and the perioperative variables of donors and recipients are discussed. Particular emphasis is given to immunosuppression treatment, diagnosis of rejection and airways suture complications. The conclusions summarize the present indications for the different types of lung transplantations according to the different preoperative diagnosis.

Adolescent↗

PubMed1995

Intrinsic tryptophan fluorescence of equinatoxin II, a pore-forming polypeptide from the sea anemone Actinia equina L, monitors its interaction with lipid membranes.

Equinatoxin II is a cytolytic polypeptide from the sea anemone Actinia equina L. which forms pores in natural and artificial membranes. The intrinsic fluorescence of its five tryptophanyl residues was used to monitor the conformational changes induced by denaturing agents, pH and lipids. In the presence of denaturants, the emitted fluorescence peak, normally occurring at 335 nm, was reduced in height by about 65% and red-shifted to 354 nm indicating unfolding. The toxin fluorescence intensity reversibly decreased by increasing the pH, whereas lipid vesicles, at every pH, caused an increase and a blue shift. The amount of toxin binding to the lipid vesicle was increased by the presence of sphingomyelin. With sphingomyelin-containing vesicles half-saturation occurred at a lipid/toxin molar ratio of about 40, whereas with phosphatidylcholine no saturation appeared up to a ratio of 300. One hydrophilic neutral quencher (acrylamide) and two lipid-confined phosphatidyltype quenchers [bis(9,10-dibromostearoyl)-sn-glycero-3-phosphocholine and 1-palmitoyl-2-(1-pyrenedecanoyl)-sn-glycero-3-phosphocholine] were used to assess the exposure of the emitting centres to the solvent and/or to the lipid. Most of the indolyl residues were found to be solvent-exposed in the water-soluble form of the toxin, as inferred from acrylamide quenching. Upon association with lipid vesicles, the fraction accessible to acrylamide dropped considerably, meanwhile the toxin became sensitive to lipid-soluble quenchers. Taken together these results suggest that insertion of equinatoxin II into sphingomyelin-containing bilayers is facilitated by high pH and results in the transfer of one or more exposed tryptophanyl residues into the liquid phase. Calcein-loaded vesicles, with or without a lipid quencher, were used to monitor simultaneously the formation of pores and the transfer of the tryptophans to the lipid phase. We found that the rate constants for vesicles permeabilization and for changes of intrinsic tryptophanyl fluorescence had a different dependence on the lipid/toxin ratio suggesting they correspond to separate steps in the toxin lipid interaction.

Animals↗

PubMed1995

Binding of antibodies to functional epitopes on the pore formed by Escherichia coli hemolysin in cells and model membranes.

Escherichia coli hemolysin (HlyA) inserts into target membranes producing a cation-selective pore. We approached the problem of determining which portions of this protein remain exposed on the side of attack by applying specific antibodies. Results obtained with resealed erythrocyte ghosts and planar phospholipid membranes were compared. The effects of one polyclonal and four monoclonal anti-hemolysin antibodies (mAbs) were studied. Using ghosts we found one mAb which strongly reduced the ion-permeability through the preinserted HlyA channels and one which clearly increased it. Experiments with planar bilayers corroborated these results by showing that the former mAb effectively promoted the closed state of the channel whereas the latter forced the HlyA channel into an open configuration. Anti-hemolysin polyclonal antibodies initially stimulated but then prevented channel opening, indicating they contained clones able to act on both these channel determinants. They were effective only when applied on the same side as the hemolysin indicating that the epitopes were exposed to that side. Finally, the antigenic epitopes of three of the mAbs were localised on the HlyA molecule by using different mutants (amber and frame shift mutants and hemolysin gene hybrids).

Animals↗

PubMed1995

Bacterial hemolysins and leukotoxins affect target cells by forming large exogenous pores into their plasma membrane: Escherichia coli hemolysin A as a case example.

Many bacteria include among their virulence factors exoproteins which exert leukocidal and cytolytic functions and have the ability to form pores in model membranes. We show that, at least in the case of the RTX hemolysin produced by Escherichia coli (HlyA), formation of pores in planar lipid membranes is parallelled by opening of strikingly similar channels in the plasma membrane of exposed macrophages. Formation of such lesions in leukocytes can give rise to a variety of effects leading altogether to a diminished immune response towards the invasive bacteria.

Bacterial Proteins↗