Rapid screening and selection of monoclonal antibodies by bivariate flow cytometric analyses.

We describe a bivariate flow cytometric assay to rapidly identify hybridomas producing new monoclonal antibodies recognizing subpopulations that are unreactive with existing immunological reagents. In this screen, whole cells in microtiter wells are labeled first with a red-linked test antibody, and then with a green-linked cocktail of existing monoclonal antibody reagents. The multiply-stained fluorescent cells are analyzed flow cytometrically and bivariate distributions of red vs. green-linked antibody fluorescence are generated. Test antibodies that recognize different subpopulations than those labeled by antibodies in the cocktail are readily identified. The use of an antibody cocktail conjugated with a single fluorophore allows comparison of the reactivity of the test antibody with multiple existing antibodies in a single analysis. This screen allows rapid (approximately 100 test antibodies can be evaluated in 40 min) identification of potentially interesting new antibodies for discrimination of subpopulations in heterogeneous tissues. We describe application of this assay to identify antibodies useful to mark hemopoietic subpopulations.

Endogenous caloric sources and nitrogen balance: regulation in postoperative patients.

Twenty-three postoperative patients were divided into three groups to evaluate the peripheral vein administration of solutions containing glucose, amino acids, or glucose and amino acids. Serum insulin, glucose, and nitrogen balances were monitored in each patient. Serum insulin concentrations rose on the first postoperative day in all three groups, then fell to near preoperative levels by the third day after surgery. Negative nitrogen balance was most pronounced in patients recieving glucose only. Patients receiving only amino acids had a reduction in nitrogen balance, but some protein catabolism was present. The mean nitrogen balance in patients who received a combination of these solutions was positive on days one and two after surgery and slightly negative on the third postoperative day. These changes were not significantly better than the amino acid group. However, the combination group had 12 to 21 days of positive balance, as compared to seven of 20 days in the amino acid group. Since starvation adaptation accurs gradually, it is concluded that the simplest and safest way to reduce protein catabolism in the immediate postoperative period is by the peripheral intravenous administration of both glucose and amino acids.

Acute management of the upper airway in facial burns and smoke inhalation.

Among 740 patients with acute burns who were admitted to our burn center from 1972 through, 1975, thirty-six required upper airway access within the first 24 hours after burn for oral and facial burns or smoke inhalation. Nasotracheal intubation was initially used. Twelve survived; 11 were successfully extubated and one required a tracheostomy. If the patient had not sustained major smoke inhalation, extubation was usually possible without tracheostomy when edema subsided between one and six days after the burn. It is concluded that endotracheal intubation is a satisfactory method of gaining airway control in severe oral and facial burns and in smoke inhalation. The mortality associated with orofacial burns or smoke inhalation is related to the degree of lung damage, patients' s age, and the extent of the burn; it is not related to the method of upper airway control.

Extracorporeal life support outcome for 128 pediatric patients with respiratory failure.

PURPOSE: The aim of this study was to describe a single-center experience with pediatric extracorporeal life support (ECLS) and to determine variables predictive of outcome in pediatric patients, both before the institution of ECLS and while on support. METHODS: From October 1985 to September 1998 the authors supported 128 children with severe acute hypoxemic respiratory failure(n = 121, Pao2/FIo2 ratio = 58+/-29) or acute hypercarbic respiratory failure (n = 7, Paco2 = 128+/-37), despite maximal conventional ventilation. Mode of access included venoarterial bypass (VA, n = 64), venovenous bypass (VV, n = 53), and VV to VA bypass (n = 11). The techniques used included lung rest, pulmonary physiotherapy, diuresis to dry weight using hemofiltration if needed, minimal anticoagulation, and optimal systemic oxygen delivery. RESULTS: The median age was 1.4 years (range, 2 weeks to 17 years). The mean duration of ECLS was 288+/-240 hours (range, 4 to 1148 hours or 0.2 to 47.8 days). Lung compliance increased from 0.32+/-0.02 mL/cm H2O/kg to 0.59+/-0.03 mL/cm H2O/kg in survivors, but only increased from 0.34+/-0.02 mL/cm H2O/kg to 0.35+/-0.02 mL/cm H2O/kg in nonsurvivors (P<.002 comparing change between survivors and nonsurvivors). Mean body weight decreased from 9%+/-2% over dry weight to 4%+/-2% in survivors, whereas in nonsurvivors the mean body weight increased from 25%+/-5% over dry weight to 35%+/-7% (P<.001). Outcome results by diagnosis were pneumonia, 73%; acute respiratory distress syndrome, 67%; and airway support, 60%, with overall lung recovery occurring in 77%, and hospital survival in 71%. Multivariate logistic regression modelling of patients with hypoxemic respiratory failure found the only pre-ECLS variable significantly associated with outcome to be pH (P<.05). Variables during the course of ECLS significantly associated with decreased survival were the presence of creatinine greater than 3.0 (P<.01), the need for inotropes (P<.04), failure to return the patient to dry weight (P<.04), and lung compliance that did not improve significantly. (P<.01). CONCLUSIONS: ECLS provides life support in severe respiratory failure in children, allowing time for injured lungs to recover. Pre-ECLS predictors, such as pH and variables during ECLS, such as presence of renal failure, improvement in compliance, return to dry weight, and the need for inotropes on ECLS, may be useful for predicting outcome.

Extraction of rIL-2 inclusion bodies from Escherichia coli using cross-flow filtration.

A cross-flow membrane filtration process was developed for the recovery of rIL-2 inclusion bodies from homogenized Escherichia coli. The membrane extraction process was comprised of a two-step diafiltration followed by an extraction with 7 M GuHCl and a 40-fold dilution of the solubilized inclusion bodies into 0.01 M Tris-HCl, 0.035 M NaCl, pH 7.9. The first diafiltration was with a 0.03 M Tris-HCl, 5 mM ethylenediaminetetraacetic acid (EDTA), pH 8, followed by a diafiltration with 1.75 M GuHCl. All of the insoluble rIL-2 was retained behind the membrane, whereas a GuHCl wash solubilized approximately 15% of the rIL-2. The membrane process increased the yield of rIL-2 in the diluted extract by threefold as compared to a similar centrifuge process with a significant increase in purity as determined by reverse-phase high-performance liquid chromatography (HPLC). (c) 1994 John Wiley & Sons, Inc.

Reverse-micelle model: pH, electromagnetic field and inhibitor enzyme interaction.

The reverse micelle is one of many models thought to have properties more nearly resembling the biological cellular environment, than does the traditional dilute-solution biochemical reaction system. In order to evaluate the results of EMF perturbation of enzyme-catalyzed reactions, the description of the AOT reverse-micelle model, with respect to its internal pH, effect of chemical inhibitors, temperature, and electromagnetic-field perturbation has herein been extended. Acetylcholinesterase and NADPH cytochrome-P450 reductase, reacting within the AOT reverse-micelle, exhibit a temperature vs. activity profile equivalent to the same reaction in a buffered dilute-solution environment. In reverse micelles, some inhibitors of AChE (propidium, and d-tubocurarine) have much less effect upon indophenol-acetate hydrolysis than they do in a dilute solution environment. Other inhibitors act in the same manner within the structured environment of the reverse micelle as in the conventional dilute solution reaction model. These differences are explicable in terms of mechanism of action of the individual inhibitors. Perturbation by low-intensity microwave fields has a similar inhibitory effect upon dilute-solution reactions, as those in the 'low-water-activity' environment of the reverse micelle. However, the interactions between physical and chemical perturbants are differently limited by the structure of the aqueous phase of the reverse micelle. pH of the 'internal' reverse-micelle environment is a function of the availability of H-ions supplied by system components. Use of indicator dyes show that the low-molarity buffers which are compatible with reverse-micelle stability, are often insufficient to maintain a constant pH. Too, in the reverse micelle, reaction rate, for proton yielding reactions, is dramatically greater than the rate of the same reaction in dilute solution at the same acidic pH.

Venovenous extracorporeal life support in neonates using a double lumen catheter.

After satisfactory development and testing of a polyurethane 14 Fr double lumen catheter, we used this device for venovenous extracorporeal life support in neonates who had respiratory failure. This catheter was designed for single site cannulation of the internal jugular vein, thereby sparing the carotid artery from ligation. Cannulation was successful in 17 of 21 neonates, with 15 successful venovenous runs, whereas 2 of the 17 patients were converted to venoarterial bypass because of inadequate support. Oxygenation and CO2 removal were adequate in the remaining patients. Average time on bypass was 111 hours. All 15 patients survived, and exploration of the cannulation site for bleeding was required in three patients. Preoxygenator pressure, recirculation of oxygenated blood, and hemolysis were all within acceptable levels during each run. Venovenous extracorporeal life support with the double lumen catheter can replace venoarterial access in most cases of neonatal respiratory failure.