Mechanisms of polymyxin B endotoxin removal from extracorporeal blood flow: molecular interactions.

The outer leaflet of Gram-negative bacteria membrane contains a great amount of lipopolysaccharides, also known as endotoxins, which play a central role in the pathogenesis of septic shock. It has been demonstrated that the polymyxin B (PMB) molecule has both antibacterial and antiendotoxin capabilities; in fact, it is able to compromise the bacterial outer membrane and bind lipopolysaccharides, thereby neutralizing its toxic effects. Extracorporeal hemoperfusion treatments based on cartridges containing PMB-immobilized fibers (Toraymyxin PMX-F; Toray Industries, Tokyo, Japan) are used to remove endotoxins circulating in the blood flow. In this study, we focused on the characterization of the interactions occurring in the formation of the PMB-endotoxin complex at the molecular level. In particular, the molecular mechanics approach was used to evaluate the interaction energy and eventually the interaction force between the two molecules. PMB was faced with five molecular portions of lipopolysaccharides differing in their structure. The interaction energy occurring for each molecular complex was calculated at different intermolecular distances and the binding forces were estimated by fitting interaction energy data. Results show that the short-range interactions between PMB and endotoxins are mediated mainly by hydrophobic forces, while in the long term, the complex formation is driven by ionic forces only. Maximum binding forces calculated via molecular mechanics for the PMB-endotoxin complex are in the range of 1.39-3.79 nN. Understanding the interaction mechanism of the single molecular complex is useful both in order to figure out the molecular features of such interaction and to perform higher scale level analysis, where such nanoscale detail is impractical but could be used to account for molecular behavior at a coarse level of discretization.

Mechanisms of polymyxin B endotoxin removal from extracorporeal blood flow: hydrodynamics of sorption.

The removal of blood endotoxins with the Toraymyxin extracorporeal sorption device exploits the capability of immobilized polymyxin B (PMB) to bind endotoxins stably with a high specificity. Although adsorption is a molecular-scale mechanism, it involves hydrodynamic phenomena in the whole range from the macroscopic down to the supramolecular scales. In this paper we summarize our experience with a computational, multiscale investigation of this device's hydrodynamic functionality. 3D computational fluid dynamics models were developed for the upper-scale studies. The flow behavior in the sorbent material was either modeled as a homogeneous Darcy's flow (macroscale study), or described as the flow through realistic geometrical models of its knitted fibers (mesoscale study). In the microscale study, simplified 2D models were used to track the motion of modeled endotoxin particles subjected to the competition of flow drag and molecular attraction by the fiber-grafted PMB. The results at each scale level supplied worst-case input data for the subsequent study. The macroscale results supplied the peak velocity of the flow field that develops in the sorbent. This was used in the mesoscale analysis, yielding a realistic range for the shear stresses in the fluid next to the fiber surface. With wall shear stresses in this range, endotoxin particle tracking was studied both in the vicinity of a single immobilized PMB molecule, and in the presence of a layer of PMB molecules evenly distributed at the fiber surface. Results showed that the capability to seize endotoxin molecules extends at least at a distance of 10-20 nm from the surface, which is one order of magnitude greater than the stable intermolecular bond characteristic distance. We conclude that a multiscale approach has the power to provide a comprehensive understanding, shedding light both upon the physics involved at each scale level and the mutual interactions of phenomena occurring at different scales.

Multi-scale analysis of the toraymyxin adsorption cartridge. Part I: molecular interaction of polymyxin B with endotoxins.

Endotoxins or lipopolysaccharides are the main constituents of the outer leaflet of Gram-negative bacteria membrane and play a central role in the pathogenesis of the septic shock. Polymyxin B has both antibacterial and antiendotoxin capability; indeed it is able to destroy the bacterial outer membrane and bind endotoxin neutralizing its toxic effects. Cartridges containing polymyxin B-immobilized fibers (Toraymyxin PMX-F, Toray Industries, Japan) are used in extracorporeal hemoperfusion to remove circulating endotoxin. The aim of this study is the characterization of the polymyxin B-endotoxin system at the molecular level, thus providing quantitative evaluation of the binding forces exerted in the molecular complex. Polymyxin B was interfaced with five molecular models of lipopolysaccharides differing in their structure and molecular mechanics simulations were performed at different intermolecular distances aimed at calculating the interaction energies of the complex. Binding forces were calculated by fitting interaction energies data. Results show that in the short range the polymyxin B-endotoxin complex is mediated by hydrophobic forces and in the long range the complex is driven by ionic forces only. From a mechanical standpoint, polymyxin B-endotoxin complex is characterized by maximum binding forces ranging between 1.39 nN to 3.79 nN. The knowledge of the binding force behavior at different intermolecular distances allows further investigations at higher scale level (Part II).

Multi-scale analysis of the toraymyxin adsorption cartridge. Part II: computational fluid-dynamic study.

Extracorporeal endotoxin removal by means of the Toraymyxin device is based on the ability of polymyxin B to bind endotoxins with a high specificity. The endotoxins/polymyxin molecular interactions were computationally analyzed in a parallel work (Part I). In this paper we investigate with a multi-scale approach the phenomena involving blood and plasma fluid dynamics inside the device. The macro- and mesoscale phenomena were studied by means of 3D models using computational fluid dynamics. The flow behavior in the sorbent material was focused, modeling the sorbent as a homogeneous porous medium at the macroscale level, or accounting for the realistic geometry of its knitted fibers at the mesoscale level. A microscale model was then developed to analyze the behavior of endotoxin molecules subjected to the competition of flow drag and molecular attraction by fiber-grafted polymyxin B. The macroscale results showed that a very regular flow field develops in the sorbent, furthermore supplying the peak velocity to be input in the lower-scale model. The mesoscale analysis yielded the realistic range for wall shear stresses (WSSs) acting on fiber walls. With WSS values in the entire range, the results of the microscale analysis demonstrated that the capability of polymyxin B to capture endotoxin molecules from the flow extends at distances one order of magnitude greater than the characteristic distance of the stable intermolecular bond. We conclude that the use of an integrated, multi-scale analysis allows for a comprehensive understanding of the complex mechanisms involved in endotoxin sorption phenomena with immobilized polymyxin B.

Biological performances of collagen-based scaffolds for vascular tissue engineering.

Collagen is widely used for biomedical applications and it could represent a valid alternative scaffold material for vascular tissue engineering. In this work, reconstituted collagen films were prepared from neutralized acid-soluble solutions for subsequent haemocompatibility and cell viability performance assays. First, haemoglobin-free, thrombelastography and platelet adhesion tests were performed in order to investigate the blood contact performance. Secondly, specimens were seeded with endothelial cells and smooth muscle cells, and cell viability tests were carried out by MTT and SEM. Results show that neutralized acid-soluble type I collagen films do not enhance blood coagulation, do not alter normal viscoelastic properties of blood and slightly activate platelet adhesion and aggregation. Cell culture shows that the samples are adequate substrates to support the adhesion and proliferation of endothelial and smooth muscle cells.

Endothelial cell adhesion force estimation at the nanoscale.

Endothelial cell adhesion to a synthetic surface includes a definite set of molecular interactions. Cell adhesion is managed by fibronectin and vitronectin in extracellular matrix (ECM) that binds the receptor site of the trans-membrane protein dimers, the integrins. These proteins contain one of the binding sites (I-like domain receptor) for the Arg-Gly-Asp (RGD) peptides that are the established adhesion receptor sites in the ECM. A molecular approach can quantify the adhesion strength by ligand-receptor force computation. The molecular interaction energy between a polyethylene (PE) surface covalently grafted with the adhesion sites (RGD-containing) and trans-membrane integrin receptor (I-domain) was evaluated through a molecular model of a single ligand-receptor complex. The aims of this work were: (i) the generation of the receptor molecular model: the I-like domain; (ii) the evaluation of the greatest binding chemical affinity between the I-like domain and three RGD-containing peptides; (iii) the development of the molecular model of crystalline lamellae of PE; and (iv) the evaluation of the interac-tion energies and the interaction force between the I-like domain and the grafted biomaterial. The calculation of the interaction energies can provide an estimation of the adhesion force of the ligand-receptor complex and, finally, of the endothelial cell adhesion force. The calculated cell adhesion force is in agreement with experimental data.(Journal of Applied Biomaterials and Biomechanics 2005; 3: 42-9).

Possible role of decorin glycosaminoglycans in fibril to fibril force transfer in relative mature tendons--a computational study from molecular to microstructural level.

Experimental studies on immature tendons have shown that the collagen fibril net is discontinuous. Manifold evidences, despite not being conclusive, indicate that mature tissue is discontinuous as well. According to composite theory, there is no requirement that the fibrils should extend from one end of the tissue to the other; indeed, an interfibrillar matrix with a low elastic modulus would be sufficient to guarantee the mechanical properties of the tendon. Possible mechanisms for the stress-transfer involve the interfibrillar proteoglycans and can be related to the matrix shear stress and to electrostatic non-covalent forces. Recent studies have shown that the glycosaminoglycans (GAGs) bound to decorin act like bridges between contiguous fibrils connecting adjacent fibril every 64-68 nm; this architecture would suggest their possible role in providing the mechanical integrity of the tendon structure. The present paper investigates the ability of decorin GAGs to transfer forces between adjacent fibrils. In order to test this hypothesis the stiffness of chondroitin-6-sulphate, a typical GAG associated to decorin, has been evaluated through the molecular mechanics approach. The obtained GAG stiffness is piecewise linear with an initial plateau at low strains (<800%) and a high stiffness region (3.1 x 10(-11)N/nm) afterwards. By introducing the calculated GAG stiffness in a multi-fibril model, miming the relative mature tendon architecture, the stress-strain behaviour of the collagen fibre was determined. The fibre incremental elastic modulus obtained ranges between 100 and 475 MPa for strains between 2% and 6%. The elastic modulus value depends directly on the fibril length, diameter and inversely on the interfibrillar distance. In particular, according to the obtained results, the length of the fibril is likely to play the major role in determining stiffness in mature tendons.

Risk of death from acute pancreatitis. Role of early, simple "routine" data.

CONCLUSIONS: The analysis of all the data available in 192 patients at 24 h from admission shows that only serum glucose above 250 mg/dL (13.88 mmol/L) and serum creatinine above 2 mg/dL (176.8 mumol/L) are prognostic factors of death (P < 0.0001). When, however, pathological chest X-rays are also considered in a subset of 149 patients, these and serum creatinine are prognostic factors of death with odd ratios of 2.9 (95% CL 1.3-6.3) and 9.4 (95% CL 2.2-40.7), respectively (P < 0.0001). BACKGROUND: In patients suffering from acute pancreatitis, neither Ranson scores nor Glasgow criteria evaluation at 24 h yield a sufficiently reliable prognosis of the risk of death from the first acute attack. METHODS: After excluding posttraumatic, postsurgical, and post-ERCP acute pancreatitis, we selected 192 consecutive patients admitted in the first instance to our center for a first attack, distinguishing between patients who died and patients who survived. We used Cox's model to analyze the prognostic weight of variables available within 24 h of admission (sex, age, alcohol intake, smoking habits, 17 biochemical tests, body mass index, chest X-rays, body temperature, and shock status). RESULTS: Seventeen (8.8%) patients died; mortality showed a decreasing trend over the period of years considered and was correlated, among other things, with necrotizing type of pancreatitis, idiopathic etiology, and shock status on admission.

Behavior of antibiotics during human necrotizing pancreatitis.

The aim of the study was to verify whether antibiotics excreted by the normal pancreas are also excreted in human necrotizing pancreatitis, reaching the tissue sites of the infection. Twelve patients suffering from acute necrotizing pancreatitis were treated with imipenem-cilastatin (0.5 g), mezlocillin (2 g), gentamicin (0.08 g), amikacin (0.5 g), pefloxacin (0.4 g), and metronidazole (0.5 g). Serum and necrotic samples were collected simultaneously at different time intervals after parenteral drug administration by computed tomography-guided needle aspiration, intraoperatively, and from surgical drainages placed during surgery. Drug concentrations were determined by microbiological and high-performance liquid chromatography assays. All antibiotics reached the necrotic tissues, but with varying degrees of penetration, this being low for aminoglycosides (13%) and high in the case of pefloxacin (89%) and metronidazole (99%). The concentrations of pefloxacin (13.0 to 23 micrograms/g) and metronidazole (8.4 micrograms/g) in the necrotic samples were distinctly higher than the MICs for the organisms most commonly isolated in this disease; the concentrations in tissue of imipenem (3.35 micrograms/g) and mezlocillin (8.0 and 15.0 micrograms/g) did not always exceed the MICs for 90% of strains tested, whereas the aminoglycoside concentrations in necrotic tissue (0.5 microgram/g) were inadequate. Repeated administration of drugs (for 3, 7, 17, and 20 days) seems to enhance penetration of pefloxacin, imipenem, and metronidazole into necrotic pancreatic tissue. The choice of antibiotics in preventing infected necrosis during necrotizing pancreatitis should be based on their antimicrobial activity, penetration rate, persistence, and therapeutic concentrations in the necrotic pancreatic area. These requisites are provided by pefloxacin and metronidazole and to a variable extent by imipenem and mezlocillin.

Prophylaxis of complications after pancreatic surgery: results of a multicenter trial in Italy. Italian Study Group.

An Italian prospective multicentre study evaluated the efficacy of octreotide, a synthetic somatostatin analogue, in preventing the complications of elective pancreatic surgery. 303 patients with tumours of the pancreas or the ampullary region, in whom ultrasonography and computed tomography scan had shown a resectable lesion, or with chronic pancreatitis, were randomized in a double-blind fashion to treatment with octreotide 100 micrograms t.i.d. s.c. starting at least 1 h before surgery and continued till the 7th postsurgical day, or with matching placebo. Unresectable lesions were found at laparatomy in 31 patients (15% of those with tumours). In 14 others, procedures not anticipated in the study protocol had to be performed, and in 6 additional cases there were other protocol violations so that these 20 patients were excluded from the study analysis. Considering the 252 evaluable patients, the complication rate was significantly higher in the 130 placebo-treated patients than in the 122 who received octreotide (29.2 vs. 15.6%; p = 0.01). We therefore suggest that octreotide may substantially reduce the risk of complications after elective pancreatic surgery.

Effects of choline-esterase inhibitor in experimental acute pancreatitis in rats. Preliminary results.

Choline-esterase inhibitor (C1-INH), a regulatory alpha-glycoprotein, was administered at different dosages and intervals to rats with induced acute pancreatitis. When compared to controls, treated rats showed no significant differences in the severity of histopathological lesions, such as edema and single cell necrosis. On the other hand, both mortality and extent of massive necrosis were significantly affected by C1-INH administration regardless of the dosages.

Prospective comparison of C-reactive protein level, Ranson score and contrast-enhanced computed tomography in the prediction of septic complications of acute pancreatitis.

Randomized clinical trials of antibiotic prophylaxis in acute pancreatitis are now warranted in the light of recent evidence of pancreatic penetration of certain antibiotics at therapeutic minimal inhibitory concentrations. The aim of the present prospective clinical study was to investigate whether there are detectable risk factors for pancreatic sepsis in acute pancreatitis that would allow better selection of patients for inclusion in clinical trials. Fifty-nine consecutive patients with acute pancreatitis were recruited and submitted to admission baseline and 48-h determinations of Ranson score, and assay of C-reactive protein at admission and weekly intervals thereafter. Contrast-enhanced computed tomography (CT) was also performed within 24 h of admission. Pancreatic sepsis, defined as infection of pancreatic and/or peripancreatic collections, was demonstrated in all cases by culture of samples obtained by needle aspiration and at laparotomy. Although all prognostic indices correlated significantly with sepsis, multivariate logistic regression analysis showed that the only variables predictive of the risk of subsequent sepsis were the presence and extent of necrosis. Early detection of pancreatic necrosis by CT should be the primary inclusion criterion in future clinical trials of antibiotic prophylaxis in acute pancreatitis.

A randomized multicenter clinical trial of antibiotic prophylaxis of septic complications in acute necrotizing pancreatitis with imipenem.

Recent evidence of pancreatic penetration of several antibiotics active against the usual flora found in pancreatic sepsis, at therapeutic minimal inhibitory concentration, prompted the authors to perform a randomized, multicenter, clinical trial on imipenem prophylaxis in acute pancreatitis. Seventy-four patients with computed tomographic (CT) scans demonstrating necrotizing pancreatitis within 72 hours of onset were randomly assigned to two groups receiving no antibiotic treatment or 0.5 gram of prophylactic imipenem administered intravenously every eight hours for two weeks. Pancreatic sepsis was always detected by means of cultures (percutaneous CT or ultrasound-guided needle aspiration and intraoperative samples). The incidence of pancreatic sepsis was much less in treated patients (12.2 versus 30.3 percent, p < 0.01). Therefore, the authors recommend prophylactic use of imipenem in patients with acute necrotizing pancreatitis.

Antibacterial and mezlocillin-enhancing activity of pure human pancreatic fluid.

The majority of deaths in severe pancreatitis are the result of superinfection of necrotic tissue. The pathogen most commonly responsible for such infections is Escherichia coli. Antibiotic prophylaxis would appear a logical precaution. The antibacterial drugs of choice should possess two basic characteristics: they must be active against the flora responsible for the infections and must be capable of penetrating into the pancreas at adequate minimum inhibitory concentrations (MICs). Mezlocillin--which is active against E. coli--has been shown to possess the latter requisite, but achieving therapeutic concentrations requires administration at high doses. In the present study, pure human pancreatic fluid showed properties similar to those observed in the dog against E. coli (bacterial colony growth 100 times lower than in a control culture) and produced a 75% reduction in mezlocillin MICs against this organism. These enhancing characteristics might make the commonly used doses sufficient for prophylactic purposes.

Early graft injuries after pancreatic transplantation in syngeneic rats. Cytoprotective effects of gabexate-mesilate.

Failures in experimental and human pancreatic transplantation are mainly attributable to rejection, graft thrombosis, and technical problems. There are, however, problems related to other causes, such as preservation injuries, which we found to exhibit, at least within the first 6 h, the same histological patterns seen in experimental acute pancreatitis. We performed pancreatic transplantation in 110 syngeneic rats under different preservation techniques and administration of gabexate mesilate, a synthetic protease inhibitor. The results showed that antiprotease treatment reduces graft preservation injuries significantly.

Necrosectomy by lavage in the surgical treatment of severe necrotizing pancreatitis. Results in 263 patients.

A conservative surgical technique for treatment of necrotizing pancreatitis is described. Standardized since 1976, the technique is based on washout mechanical necrosectomy accomplished by lavages via intraoperatively placed wide-bore drainage tubes. The overall mortality rate from necrotizing pancreatitis was thereby reduced from 61% (with resective technique) to 18%, and in 106 cases observed from the onset of the disease the mortality fell to only 6.6%. The series included also patients with fulminant acute pancreatitis and multiorgan failure.

Pancreatic abscess and other pus-harboring collections related to pancreatitis: a review of 108 cases.

This is a report on 108 cases collected from 1970 to 1987, in the same department, of surgically-detected pancreatic abscesses or pus-harboring collections. The purulent areas were either of a spreading pattern or represented a clearly localized mass. To the spreading pattern belong 47 cases of necrotizing pancreatitis, without discontinuity in the clinical course from the early toxic to the late septic phase, 4 cases of acute pancreatitis, initially in remission and later complicated by septic collections, and 4 cases which developed after an acute attack of chronic pancreatitis. The abscess pattern was made up of 19 each of pseudocysts and predisposing pancreatitis, 10 cases of chronic pancreatitis, and only 5 necrotizing "nonstop" pancreatitis. The surgical treatment in all cases consisted of multiple drainages and postoperative irrigation. We exclude 3 cases of associated open packing. The etiological, clinical, and biochemical features of each group of patients are reported and discussed. Computed tomography availability seems to be the most important improvement reported as regards diagnosis and surgical tactics. The overall mortality rate was 15.7% with a significant difference between the 2 patterns (23.6% for the spreading pattern versus 7.5% for the abscess pattern). On the basis of this experience, it is possible to establish a relationship between the gross appearance of the collection and the underlying pancreatic disease with differences in terms of prognosis, morbidity, and mortality. Finally, a simple nomenclature can be chosen which is capable of distinguishing between the diverse pancreatic purulent collections. While the presence of pus may characterize the course of severe acute pancreatitis in many cases, the low incidence of "true" pancreatic abscess is emphasized.

Retroperitoneal and peritoneal drainage and lavage in the treatment of severe necrotizing pancreatitis.

In severe necrotizing pancreatitis, the retroperitoneum is the main site both of autodigestion and of the production of toxins. With the aim of removing necrotic tissues and active enzymes from the retroperitoneum, we developed a surgical approach based on a wide exposure of the pancreas and on the insertion of multiple drainages with postoperative irrigations with hypertonic solutions and antiproteases. We treated 191 patients, and our results correlated with the timing of the operation. The operative mortality rate ranges from 8.1 in patients undergoing our procedure within 48 hours from the onset of the disease to 28.4 per cent when the operation was delayed for more than 96 hours. Our technique is detailed and the indications as to the timing of surgical treatment in instances of pancreatitis are discussed.

Continuous peritoneal dialysis in acute experimental pancreatitis in dogs. Effect of aprotinin in the dialysate medium.

In 27 beagle dogs, acute necrotizing pancreatitis was induced by retrograde injection of autologous bile and trypsin into the main pancreatic duct. Animals were randomly assigned to the following treatments: group 1--(9 dogs) aprotinin 600,000 KIU/d by i.v. route; group 2--(9 dogs) peritoneal dialysis for 6 d plus 500,000 KIU/L of aprotinin in the dialysate fluid; group 3--(9 dogs) peritoneal dialysis without aprotinin in the dialysate fluid. All dogs of the group 1 died within 16 h following the induction of pancreatitis and extensive necrotizing and hemorrhagic changes were seen in the pancreatic and peripancreatic areas. Six dogs of the group 2 survived and no necrotizing changes were observed 30 or 50 d after the induction of pancreatitis. Three dogs of the group 3 survived, but slight necrotizing lesions were found at the autopsy. The survival rate was higher in dogs with peritoneal lavage (p = 0.0129) or with peritoneal lavage plus aprotinin (p less than 0.0001) than in those receiving i.v. aprotinin, indicating that the latter treatment has no beneficiary effect on the course of acute pancreatitis.