A rapid and versatile method for harnessing scFv antibody fragments with various biological effector functions.

A versatile expression vector is described for the rapid construction and evaluation of bispecific scFvs and scFv-based fusion proteins. An important feature of this vector is the presence of two multiple cloning sites (MCS) separated by an in frame linker sequence. The first MCS was specifically designed to contain unique SfiI and NotI restriction enzyme sites that can be used for directional and in frame insertion of scFvs (or potentially any molecule) selected from established phage-display systems. Using this new vector, a functional bs-(scFv)(2) (2C11-MOC31) was constructed for retargeted T-cell cytotoxicity towards EGP2 positive tumor cells. The vector was also used for grafting of a number of promising biological effector principles onto scFv MOC31, including the prodrug converting enzyme cytosine deaminase, the anti-angiogenic factor angiostatin, and the thrombogenic molecule tissue factor. We aimed at producing biologically active fusion proteins by directing them through the endoplasmic reticulum-based protein folding machinery of eukaryotic cells (COS-7) using a kappa light chain leader, thereby taking advantage of the associated quality control mechanisms that allow only fully folded and processed fusion proteins to be secreted into the medium. Supernatants derived from fusion protein transfected COS-7 cells, which were transiently transfected at low transfection rates, were directly assayed for the biological and/or targeting activity of the excreted fusion proteins without any prior purification steps. This procedure might help to identify those fusion proteins that have favourable characteristics like stability and biological activity in the presence of serum and at low protein concentrations. Targeted delivery of all effector principles was subsequently assessed in an in vitro model system. The method we devised is both rapid and versatile and can be useful to construct and identify series of new chimeric proteins with enhanced therapeutic potential in human cancer therapy.

Effects of hyperthermic isolated limb perfusion with recombinant tumor necrosis factor alpha and melphalan on the human fibrinolytic system.

This study was undertaken to determine the effects on systemic fibrinolysis of hyperthermic isolated limb perfusion with recombinant tumor necrosis factor alpha (r-TNF-alpha) and melphalan, with or without pretreatment with recombinant IFN-gamma (r-IFN-gamma). Twenty patients were treated with r-TNF-alpha and melphalan; four patients, treated with melphalan only, served as controls. Of the twenty patients treated with both r-TNF-alpha and melphalan, eight received r-IFN-gamma for two days before the perfusion and as a bolus into the perfusion circuit. A significant leak of r-TNF-alpha from the perfusion circuit to the systemic circulation was observed in all r-TNF-alpha-treated patients (mean maximum TNF-alpha, 87,227 ng/liter versus 31 ng/liter in controls; P < 0.002). In these patients, but not in controls, there was an almost instantaneous rise in systemic tissue plasminogen activator activity (from 0.26 to 5.28 IU/ml in 90 min), causing activation of fibrinolysis. After a delay of 90 min, plasminogen activator inhibitor-1 (PAI-1) antigen rose to high levels in the r-TNF-alpha-treated group (mean maximum PAI-1, 1652 ng/ml versus 211 ng/ml in controls; P < 0.02), associated with a sharp decrease of tissue plasminogen activator activity and a slower decrease of plasminogen-antiplasminogen complexes (from 5.28 to 0.02 IU/ml in 2 h and from 1573 to 347 micrograms/liter in 22 h, respectively). No additional effect of IFN-gamma pretreatment on fibrinolysis could be demonstrated. These results suggest that in isolated limb perfusion with r-TNF-alpha and melphalan an initial activation of systemic fibrinolysis, induced by leakage of r-TNF-alpha from the perfusion circuit, is set off by a subsequent inhibition of the fibrinolytic system by PAI-1. This large increase in PAI-1 could place the patient at risk for deposition of microthrombi in the systemic circulation.

Coagulation and fibrinolytic responses of human peritoneal fluid and plasma to bacterial peritonitis.

Significantly higher (P < 0.05) thrombin-antithrombin III complex levels were found in the abdominal exudate of patients with peritonitis (median 5500 ng/ml) than in that of controls (median 89 ng/ml). In patients, peritoneal fluid concentrations of tissue and urokinase-type plasminogen activator were increased by factors of 65 and 10 respectively (P < 0.05). The concentration of plasminogen activator inhibitor (PAI) 1 was increased by a factor of about 800 (median 395 versus 0.5 ng/ml, P < 0.05). Despite markedly raised concentrations of PAI, peritoneal fluid displayed fibrinolytic activity as demonstrated by significantly increased (P < 0.05) concentrations of plasmin-alpha 2-antiplasmin complex (median 10,952 versus 57 ng/ml) and fibrin degradation products (median 40,360 versus 126 ng/ml). There was no correlation between plasma and peritoneal fluid concentrations. Intraabdominal coagulation and fibrinolysis are stimulated in the abdominal cavity of patients with bacterial peritonitis.

Association of smooth muscle cell tissue factor with caveolae.

There is still no satisfactory explanation for the low catalytic activity of tissue factor (TF)/factor VII(a) complexes towards coagulation factor X, as found on the apical surface side of cell layers. It has been hypothesized that TF exists in a latent form. Layers of cultured human smooth muscle cells, constitutively expressing TF, were immunogold-labeled for TF in situ and processed for electron microscopy. We showed that, besides internalization and accumulation in lysosomal-like structures, TF remained associated with noncoated, flask-shaped microinvaginations of the plasma membrane. These invaginations were identified as caveolae. In regions in which intercellular contacts were interrupted, more TF-positive caveolae were observed. Enzymatically detached smooth muscle cells exhibited a similar enlargement of caveolar structures. Concomitantly, an increase of catalytic activity of apically formed TF/VIIa complexes towards factor X was found on the suspended cells. We speculate that caveolae-associated TF may function as a latent pool of procoagulant activity, which can rapidly be activated at sites in which vessel wall integrity is lost.

Pharmacokinetics of human recombinant tissue-type plasminogen activator, administered intra-abdominally, in a rat peritonitis model.

Human recombinant tissue-type plasminogen activator (rtPA), administered intraperitoneally, may promote intra-abdominal fibrinolysis in peritonitis, thereby preventing adhesion and abscess formation. The pharmacokinetics of a single intraperitoneal dose of 0.5 or 2.0 mg/ml human rtPA were assessed in rats with fecal peritonitis and related to their endogenous tPA and plasminogen activator inhibitor (PAI) activity. Endogenous PAI activity was strongly elevated both in peritoneal fluid and in plasma, whereas tPA activity was only slightly increased in the peritoneal fluid. Both doses of rtPA significantly enhanced fibrinolytic activity of the peritoneal fluid up to 24 h in a dose-dependent manner. However, tPA activity was lower than calculated from the tPA antigen levels and the specific activity of rtPA in the fluid of rats with peritonitis. Plasma tPA activity was low at both doses. A single dose of 0.5 or 2.0 mg/ml rtPA can raise fibrinolytic activity in the abdominal cavity up to 24 h. High endogenous peritoneal PAI levels may adversely effect intra-abdominal use of human rtPA in peritonitis.

Basal tissue factor expression in endothelial cell cultures is caused by contaminating smooth muscle cells. Reduction by using chymotrypsin instead of collagenase.

A discrepancy exists between basal tissue factor (TF) expression found in endothelial cell cultures and the failure to detect TF in unpertubated endothelial cells in vivo. We demonstrated that basal TF expression in endothelial cell cultures originated from contaminating cells. These cells were ultrastructurally and flowcytometrically identified as smooth muscle cells. The cell cultures had been obtained from collagenase-treated human umbilical cord vessels. Histologic studies revealed that after collagenase treatment the basement membrane was digested and underlying structures were disrupted at some areas of the vein. We selected chymotrypsin as an alternative for the isolation of endothelial cells. Using chymotrypsin, the endothelial lining was selectively lost leaving the basement membrane undisturbed. Furthermore, use of chymotrypsin instead of collagenase minimized the level of basal TF activity. Taken together, we demonstrated that basal TF expression in endothelial cell cultures is caused by contaminating smooth muscle cells. This contamination can strongly be reduced using chymotrypsin instead of collagenase for isolation of endothelial cells.

Plasma levels of protein S, protein C, and factor X: effects of sex, hormonal state and age.

We measured total and free protein S (PS), protein C (PC) and factor X (FX) in 393 healthy blood donors to assess differences in relation to sex, hormonal state and age. All measured proteins were lower in women as compared to men, as were levels in premenopausal women as compared to postmenopausal women. Multiple regression analysis showed that both age and subgroup (men, pre- and postmenopausal women) were of significance for the levels of total and free PS and PC, the subgroup effect being caused by the differences between the premenopausal women and the other groups. This indicates a role of sex-hormones, most likely estrogens, in the regulation of levels of pro- and anticoagulant factors under physiologic conditions. These differences should be taken into account in daily clinical practice and may necessitate different normal ranges for men, pre- and postmenopausal women.

Sensitivity to activated protein C; influence of oral contraceptives and sex.

We investigated sensitivity to activated protein C (APC) in 43 men, 42 women not using oral contraceptives (OC) and 38 women using OC containing 30.0-37.5 micrograms ethinyl estradiol. A commercially available kit was used. Men were more sensitive to APC as compared with women not using OC (p < 0.005), but this difference seems not to be of clinical importance. Women using OC showed to be significantly less sensitive to APC, reflected by a lower APC-ratio, as compared with men (p < 0.005) and women not using OC (p < 0.05). So, in normal individuals the APC-sensitivity differs according to sex and estrogen intake. This should be taken into account when interpreting APC-ratios.

PIP: The authors investigated sensitivity to activated protein C (APC) in 43 men, 42 women not using oral contraceptives (OCs), and 38 women using OCs containing 30.0-37.5 mcg ethinyl estradiol. A commercially available kit was used. Men were more sensitive to APC as compared with women not using OCs (p 0.005), but this difference seems not to be of clinical importance. Women using OCs were found to be significantly less sensitive to APC, reflected by a lower APC ratio, as compared with men (p 0.005) and women not using OCs (p 0.05). So, in normal individuals the APC sensitivity differs according to sex and estrogen intake. This should be taken into account when interpreting APC ratios.

Effect of recombinant tissue plasminogen activator on intra-abdominal abscess formation in rats with generalized peritonitis.

BACKGROUND: During generalized peritonitis, intraabdominal fibrin deposition is stimulated whereas fibrinolytic activity is reduced, which predisposes intra-abdominal abscess formation. We investigated the effects of increasing the intra-abdominal fibrinolytic activity on abscess formation by intra-abdominal administration of recombinant tissue plasminogen activator (rt-PA). Potential side effects, such as bacteremia and bleeding, were also assessed. STUDY DESIGN: A rat model of generalized peritonitis, induced by intraperitoneal injection of sterile feces contaminated with 10(4) cfu per mL Escherichia coli (E. coli) and 10(4) cfu per mL Bacteroides fragilis, was used. RESULTS: Rats treated with rt-PA dissolved in methyl hydroxy propyl cellulose (MHPC) gel (0.5 mg per mL), had significantly less intra-abdominal abscesses than rats in the control group, treated with either Ringer's lactate solution or MHPC gel alone (p < 0.01). Other than E. coli, cultures of abscesses revealed species originating from the intestine, demonstrating bacterial translocation. The mortality rate was significantly higher in the rats treated with rt-PA as compared with rats in the control group (p < 0.01), which was surprising considering the absence of bacteremia. By challenging the rats with a higher dose of E. coli, early bacteremia was observed in the rats treated with rt-PA, not related to increased mortality rates. Intraabdominal use of rt-PA was not associated with an increased incidence of bleeding events. CONCLUSIONS: Recombinant tissue plasminogen activator prevents abscess formation in rats with generalized peritonitis. However, early bacteremia and increased mortality rates are serious drawbacks of the intra-abdominal use of rt-PA in this rat model.

Tumor necrosis factor alpha-induced endothelial tissue factor is located on the cell surface rather than in the subendothelial matrix.

Because there is no consensus regarding the precise distribution of induced endothelial tissue factor (TF), we studied TF activity in and on tumor necrosis factor alpha-stimulated cultured human umbilical vein endothelial cells (ECs) and their underlying matrix. TF was mainly expressed on the cell surface. Only small traces were found on the apical surface suggesting that TF is predominantly located on the basolateral side of the cell membrane. The presence of TF on the cell surface was confirmed by flow cytometry. Subendothelial TF activity appeared to be dependent upon the procedure used to remove the stimulated EC monolayer. Whereas ammonium hydroxide or hypotonic lysis resulted in relatively high levels of matrix-associated TF, virtually no TF was found on the matrix after mild enzymatic detachment of stimulated ECs. Cell removal with EDTA resulted in intermediate levels of matrix-associated TF. Neither the enzymatic treatment nor EDTA degraded or removed this TF activity. Similar patterns were observed for matrix-associated TF antigen and EC surface markers. Electron microscopic analysis showed cell fragments on the matrix after monolayer lysis. The findings strongly suggest that induced endothelial TF associated with the subendothelial matrix actually represents TF on EC remnants.

Hemoperfusion during coronary angioplasty: first European experience with a new hemoperfusion pump.

Hemolysis tests with fresh human blood were performed in vitro with a new 5 ml, piston-type hemoperfusion pump, designed to prevent myocardial ischemia during coronary angioplasty. Despite driving pressures greater than 3 atmospheres, shear stress greater than 200 Pa, turbulent pump flow, and the presence of occlusive valves, hemolysis proved to be minimal. This effect is explained by the short amount of time that blood is subjected to mechanical forces that cause hemolysis in the system and by the small volumes of blood involved. During clinical application of the system, angina pectoris, electrocardiographic changes, and systemic blood pressure were used as parameters for myocardial ischemia. There was an effective reduction of ischemia during prolonged (10 min) balloon inflation, demonstrated by the absence of angina, minimal electrocardiographic changes, and normal blood pressure. In addition, the system proved to be safe and effective during high-risk angioplasty.

Fibrinolytic activity in the abdominal cavity of rats with faecal peritonitis.

Generalized peritonitis causes a reduction in abdominal fibrinolytic activity, resulting in persistence of intraabdominal fibrin with subsequent adhesion and abscess formation. The activities of tissue plasminogen activator (tPA) and plasminogen activator inhibitor (PAI) were measured in the peritoneal fluid of rats with faecal peritonitis and correlated with the extent of peritoneal damage to determine the cause of decreased fibrinolysis. Activity of tPA was low during the study period of 8 days, but higher in rats with peritonitis than in controls. The activity of PAI in rats with peritonitis was significantly increased compared with that of controls during the whole study period (P < 0.001). Histological signs of damage to the peritoneum were similar in rats with peritonitis and controls. There was no correlation between the extent of peritoneal damage and tPA or PAI activity. The increased activity of PAI in the peritoneal fluid of rats with faecal peritonitis may be the main cause of reduced fibrinolysis in the abdominal cavity. Activities of tPA and PAI may originate not only from the mesothelium but from other sources.

In-vivo evaluation of the "HIA-VAD": a new German ventricular assist device.

Helmholtz Ventricular Assist Devices (VAD) are pneumatically driven polyurethane membrane pumps with various volumes. The pumps are placed paracorporeally and connected with commercially available cannulas between the left atrium and aorta (left ventricular assist device) and/or right atrium and pulmonary artery (right ventricular assist device, bi-ventricular assist device). The pumps can be driven with a stand-alone driving system or with a Helmholtz IABP-console interface. Seventeen animal experiments (on calves) with Helmholtz VAD's were performed to evaluate experimental protocols, to optimize surgical techniques, and to improve design and manufacturing techniques. Blood chemistry and cell counts demonstrated that the tested HIA-70 produces low mechanical blood damage. In the course of the animal experiments the Helmholtz VAD's were made totally transparent, whereby they became easy to de-air, efficient, and affordable.

Severe bleeding caused by an inhibitor to coagulation factor V: a case report.

A 67-year-old man with a severe bleeding due to a high level of factor V inhibitor (maximum level of 350 Bethesda units) is described. Coagulation abnormalities improved initially during treatment with prednisolone in combination with cyclophosphamide. Subsequent treatment with either cyclophosphamide or cyclosporin alone was ineffective. After more than 2 years the inhibitor became undetectable after a prolonged period of high dose steroid therapy, but the patient remained steroid dependent. Therapeutic strategies for patients having a factor V inhibitor are discussed.

Rapid enzyme immunoassay of anti-streptokinase antibodies in human plasma.

A simple enzyme immunoassay for determination of anti-streptokinase antibodies (aSKa) in plasma is described. Commercially available reagents have been used for the assay, which is calibrated with a reference preparation of aSKa containing 100 AU/ml. The assay is specific and reproducible with a variation coefficient of 4.8%. In healthy individuals a broad range of values between 4 and 291 AU/ml was observed with a large difference between the mean and median value (55 AU/ml and 27 AU/ml, respectively). Data from a study on 21 patients with myocardial infarction treated with the streptokinase derivative antistreplase suggest that a high titre of aSKa before treatment is associated with failure of thrombolytic therapy. The assay procedure can be shortened to 0.5 h to screen patients for a high aSKa level. This assay allows a more routine assessment of aSKa in the clinic.

The clinical expression of hereditary protein C and protein S deficiency: a relation to clinical thrombotic risk factors and to levels of protein C and protein S?

We investigated 103 first-degree relatives of 13 unrelated protein C or protein S deficient patients to assess the role of additional thrombotic risk factors and of protein C and protein S levels in the clinical expression of hereditary protein C and protein S deficiency. Fifty-seven relatives were protein C (37) or protein S deficient (20). Thromboembolic events occurred in 30% of protein C deficient and in 35% of protein S deficient persons, compared with 3% and 0% in their normal controls respectively (P < 0.05). In protein C deficient persons, the median thromboembolic event-free survival was 55 years, while in protein S deficiency this interval was 33 years (P = 0.047). In the protein C deficient group 64% of the initial events occurred spontaneously, as did 71% in the protein S deficient group. Recurrent thromboembolic events were more often associated with concomitant risk factors than the initial events: 64% and 50% in persons with protein C or protein S deficiency respectively. These findings suggest a substantial role for these risk factors in triggering thromboembolic events in deficient persons. Protein C antigen and protein S antigen levels were similar in symptomatic and asymptomatic deficient persons. Total, but not free, protein S antigen levels were significantly higher in symptomatic protein C deficient persons, as were protein C antigen and activity levels in symptomatic protein S deficient ones. The clinical implication of this finding is not yet clear.