Bispecific monoclonal antibodies produced by somatic cell fusion increase the potency of tissue plasminogen activator

Bispecific monoclonal antibodies that bind simultaneously to human fibrin and tissue plasminogen activator (tPA) enhance the fibrinolytic potency of tPA. Two bispecific antibodies (F36.23 and F32.1) were generated by somatic cell fusion. Antibody F36.23 derives its tPA binding from monoclonal anti-tPA antibody TCL8 and its fibrin binding from monoclonal antifibrin antibody 59D8. After purification from cell supernatants and ascites by two steps of affinity chromatography, hybrid-hybridoma bispecific antibody F36.23 simultaneously bound tPA and fibrin in solution and in solid-phase assays. In an assay for the lysis of human fibrin monomer, F36.23 increased the fibrinolytic potency of tPA by 5 to 10 fold, regardless of whether the bispecific antibody had been combined with the tPA before or during the assay. Bispecific F36.23 F(ab')2 also bound tPA and fibrin simultaneously, and the enhancement in fibrinolysis in the presence of F36.23 F(ab')2 was identical to that in the presence of intact F36.23. The second bispecific antibody, F32.1, was produced by an alternative strategy that has a wider potential for application in other systems. Hybridoma bispecific antibody F32.1 was derived from the fusion of immune splenocytes (in mice immunized with a synthetic oligopeptide representing the amino terminus of the alpha-chain of human fibrin) with the anti-tPA cell line TCL8. The properties of hybridoma bispecific antibody F32.1 and its F(ab')2 were indistinguishable from those of hybrid-hybridoma bispecific antibody F36.23 in solid-phase binding assays and in assays of fibrinolysis. Bispecific antibodies produced by somatic cell fusion, particularly in the form of F(ab')2, may have potential for use in clinical thrombolysis.     

High-dose applications of gadolinium chelates in magnetic resonance imaging.

Administration of gadolinium chelates at doses greater than 0.1 mmol/kg IV can potentially improve both lesion detection and the assessment of tissue perfusion. Preliminary results are presented in clinical patients and two animal models. In human intracranial metastatic disease, administration of 0.3 (cumulative dose) mmol/kg gadoteridol (Gd HP-DO3A) has permitted detection of additional lesions not visualized at 0.1 mmol/kg. In a rabbit model of focal liver disease, 0.5 mmol/kg IV provided superior enhancement of both normal parenchyma and lesion rim compared to doses of 0.25 and 0.1. Dynamic imaging (T1-weighted turbo-FLASH) immediately following bolus injection of 0.5 mmol/kg permitted direct visualization (on unsubtracted images) of an acute perfusion defect in the cat brain not visible on conventional T1- and T2-weighted scans.     

Preload-responsive, pulsatile-flow, externally valved pump: cardiopulmonary bypass

Currently two pumps are used for cardiopulmonary bypass, the roller pump and the centrifugal or vortex pump. Both are steady-flow pumps. The procedure of cardiopulmonary bypass possesses a finite morbidity and mortality. The degree to which steady flow is responsible for this morbidity and mortality remains to be clarified, but investigators have established the fact that a physiologic degree of pulsatile flow must be achieved before its beneficial results, such as normal systemic resistance and absence of lactate production, can be demonstrated. Availability of a satisfactory pulsatile pump for cardiopulmonary bypass has been a problem in the past but the pump presented here may satisfy this need. It produces physiologic pulsatility with rate dependent ejection time equal to or less than that of humans (413 microseconds minus 1.7 times heart rate), and it is preload-responsive, varying its pumping rate and output with filling pressure. The pump is externally valved to minimize hemolysis, which has been demonstrated in two laboratory studies to be significantly less than with the roller pump. It produces pulsatile flow through membrane oxygenators. The pump is thought to have potential for several clinical applications in addition to (1) pulsatile-flow cardiopulmonary bypass, including (2) left, right, or combined transthoracic QRS synchronized ventricular assist, (3) femoral vein to femoral artery QRS synchronized left ventricular assist, (4) adult or infant ECMO, (5) pulsatile flow hemodialysis. In the latter, spallation and embolization of hemodialysis tubing particles should not be a problem as has proved to be the case with the present hemodialysis pump.     

Obesity-related excess mortality rate in an adult intensive care unit: A risk-adjusted matched cohort study

OBJECTIVE: To evaluate the obesity-related mortality rate in an intensive care unit. DESIGN: An exposed/unexposed matched cohort study. SETTING: An 18-bed adult medical-surgical intensive care unit in a 1,100-bed regional and teaching hospital in France. PATIENTS: From January 1, 1999, to December 31, 2001, 170 mechanically ventilated exposed patients (obese patients with body mass index of >30 kg/m) were matched with 170 mechanically ventilated unexposed patients (with ideal body mass index of 18.5-24.9 kg/m). INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: The matching process was conducted according to eight criteria: cause of admission, indication for ventilatory support, immunologic status, cardiac status, probability of death (+/-5%), age (+/-7 yrs), gender, and acquisition of severe events appearing within 24 hrs before admission (defined as resuscitated cardiac arrest, acute respiratory distress syndrome, or septic shock). The mortality rate between exposed and unexposed patients was compared by univariate analysis and then was adjusted for other possible confounding factors by multivariate analysis, using conditional logistic regression. The matching process was successful for 1,360 of 1,360 criteria. Obesity was significantly associated with intensive care unit mortality (odds ratio, 2.1; 95% confidence interval, 1.2-3.6). Obesity-related excess mortality was verified mainly for the youngest patients (odds ratio, 2.5; 95% confidence interval, 1.6-6.1) and for the patients with a probability of intensive care unit death of 11-50% (odds ratio, 2.6; 95% confidence interval, 1.2-5.5). This excess mortality rate could be explained by the higher risk of intensive care unit acquired complications among obese patients than among the unexposed ones (odds ratio, 4; 95% confidence interval, 1.4-11.8). CONCLUSIONS: Obesity is an independent risk factor for intensive care unit death and should be regarded as a severe comorbidity in such units.     

Toxicity, uptake kinetics and efficacy of new transfection reagents: increase of oligonucleotide uptake

Human arterial smooth muscle cell (haSMC) proliferation is stimulated by platelet-derived growth factor (PDGF) release of human arterial endothelial cells (haEC) whereas transforming growth factor-beta(1) (TGF-beta(1)) secretion by haSMC promotes extracellular matrix formation. Inhibitory concepts with antisense oligonucleotides (ASO) against those growth factors might be promising, requiring, however, sufficient transfection efficacy. Thus, toxicity and efficacy of new transfection reagents were examined. MTT tests showed that high doses >1.6 microg/ml of the liposome Cytofectin GSV((R)) (CF) and the dendrimer SuperFect (SF) reduced mitochondrial activity of haEC after > or =4 h transfection whereas viability of haSMC was not influenced. DAC-30((R)) showed significant toxic effects on haEC and haSMC at each dose after > or =4 h and Lipofectin((R)) (LF) caused complete detachment of haEC and haSMC in medium containing 10% serum. Uptake studies demonstrated that 'naked' ASO were not incorporated intracellularly whereas transfection within CF or SF resulted in a strong cytoplasmic and nuclear labeling after 2-5 h. With DAC-30, only a slight cytoplasmic fluorescence was found. SF caused an unexpected stimulation of endothelial PDGF-AB synthesis. Thus, CF was favored for inhibition studies. ELISA, Western and Northern blotting showed a significant inhibition of endothelial PDGF-B and smooth muscle TGF-beta(1) mRNA expression and synthesis after transfection for 3-5 h using 0.1-1.0 microM ASO versus control oligonucleotides. We conclude that Cytofectin GSV is superior to the other transfection reagents, predominantly at haEC, showing an improved efficacy and less toxicity than the classical liposome Lipofectin. Cytofectin GSV might offer a promising tool for antisense strategies in the treatment of vascular disorders.