Direct quantitation of IgG subclasses 1, 2, and 3 bound to red cells by Rh1 (D) antibodies

The authors developed quantitative radioimmunoassays to allow direct measurement of total human IgG and individual IgG subclasses among antibodies bound to cell surfaces. The assays use four mouse monoclonal radioiodinated antibodies, one that reacts equally well with all four human IgG subclasses and three that are specific for human IgG subclasses 1, 2, or 3. The assays were used to analyze IgG subclass composition in 21 high-titer anti-D samples from Rh-negative volunteers immunized for Rh immunoglobulin production. Anti-D activity was restricted primarily to the IgG1 and IgG3 subclasses. Eleven of 21 sera demonstrated red cell antibodies with a marked predominance of IgG1 (87 +/- 3.6% of total IgG antibody, +/- SEM) and low levels of IgG3 (1.4 +/- 0.73%). In the remaining 10 sera, IgG3 made up a greater proportion of total IgG antibody (32 +/- 3.8%), although IgG1 was still predominant (61 +/- 4.1%). This observed dichotomy in the IgG subclass profiles of different anti-D sera may be a consideration in the selection of anti-D sera for the production of the immunoglobulin used in the prophylaxis of Rh-incompatible pregnancies.     

Addition of L-carnitine to additive solution-suspended red cells stored at 4 degrees C reduces in vitro hemolysis and improves in vivo viability

BACKGROUND: The role of L-carnitine (LC) as the requisite carrier of long-chain fatty acids into mitochondria is well established. Human red cells (RBCs), which lack mitochondria, possess a substantial amount of LC and its esters. In addition, carnitine palmitoyl transferase, an enzyme that catalyzes the reversible transfer of the acyl moiety from acyl-coenzyme A to LC is found in RBCs. It has recently been shown that LC and carnitine palmitoyl transferase play a major role in modulating the pathway for the turnover of membrane phospholipid fatty acids in intact human RBCs, and that LC improved the membrane stability of RBCs subjected to high shear stress. RBC membrane lesions occur during storage at 4 degrees C; this study investigated whether the addition of LC (5 mM) to a standard RBC preservative solution (AS-3) affected cellular integrity with 42 days' storage. STUDY DESIGN AND METHODS: A paired (n = 10) crossover design was used for RBCs stored in AS-3 with and without LC. Both in vitro RBC properties reflective of metabolic and membrane integrity and in vivo measures of cell viability (24-hour percentage of recovery and circulating lifespan) were measured at the end of the storage. In addition, the turnover of membrane phospholipid and long-chain acylcarnitine fatty acids and the carnitine content of control and LC-stored RBCs were measured. RESULTS: It was shown that LC was irreversibly taken up by RBCs during storage, with a fourfold increase at 42 days. Furthermore, as found by the use of radiolabeled palmitate, the stored RBCs were capable of generating long-chain acylcarnitine. The uptake of LC during storage was associated with less hemolysis and higher RBC ATP levels and by a significantly greater in vivo viability for LC-stored RBCs than for control-stored RBCs: a mean 24-hour percentage of recovery of 83.9 +/? 5.0 vs. 80.1 +/? 6.0 percent and a mean lifespan of 96 +/? 11 vs. 86 +/? 14 days, respectively (p < 0.05). CONCLUSION: A beneficial effect of the addition of LC to RBCs stored at 4 degrees C was evident. This effect may be related to both biophysical and metabolic actions on the cell membrane.     

Electrocardiographic abnormalities after radiofrequency catheter ablation of accessory bypass tracts in the Wolff-Parkinson-White syndrome.

Repolarization abnormalities on surface electrocardiograms have been described after loss of ventricular preexcitation in some patients with the Wolff-Parkinson-White syndrome. Radiofrequency catheter ablation of overt accessory pathways provides a unique opportunity to study this phenomenon. In this study, serial electrocardiograms were obtained before and after radiofrequency ablation of manifest accessory pathways in 19 patients, of concealed accessory pathways in 6 and after radiofrequency atrioventricular nodal modification in 12. Seven patients undergoing manifest right-sided accessory pathway ablation had left superior frontal plane T-wave axis deviations after ablation (-42 +/- 13 degrees). No patient with a manifest left-sided or concealed accessory pathway, or atrioventricular nodal modification had T-wave abnormalities after ablation; however, left anterior fascicular block and incomplete right bundle branch block each occurred in 1 patient with left accessory pathway ablation. Repolarization abnormalities observed after ablation were similar to T-wave abnormalities during the absence of preexcitation before ablation and persisted up to 5 weeks after the procedure. Patients with repolarization abnormalities after ablation had significantly longer preexcited QRS durations than those without such changes, suggesting that the initial contribution of the pathway to ventricular activation is an important determinant of T-wave changes after ablation. The proposed mechanism for repolarization abnormalities after ablation is the phenomenon of T-wave "memory."     

Surgical ablation of ventricular tachycardia with sequential map-guided subendocardial resection: electrophysiologic assessment and long-term follow-up

A new operative technique of sequential map-guided subendocardial resection (SER) was used in 45 consecutive patients for the treatment of sustained ventricular tachycardia due to coronary artery disease. This technique is characterized by map-guided SER or cryothermic ablation during normothermic cardiopulmonary bypass, followed by repeated sequences of programmed stimulation to assess adequacy of resection. The patients' mean age was 59 +/- 10 years and the mean left ventricular ejection fraction was 34 +/- 12%. Twenty-five (56%) patients had a history of myocardial infarction within the previous 2 months. After ventriculotomy, 34 patients (76%) had inducible monomorphic ventricular tachycardia. These patients underwent repeated sequences of ventricular tachycardia induction and mapping during normothermic bypass followed by successive SER or cryothermic ablation until sustained monomorphic ventricular tachycardia was no longer inducible. Twenty-seven patients had a total of 60 discrete, mappable tachycardias induced and seven patients had 10 discrete tachycardias that were too fast to accurately map. In the remaining 11 patients, no ventricular tachycardia was inducible after ventriculotomy and SER, which included all visually identifiable scar, was performed. The mean cardiopulmonary bypass time was 102 +/- 27 min. Forty-one of 45 patients (91%) survived to hospital discharge, and 35 of 41 patients (85%) had no inducible ventricular tachycardia at postoperative electrophysiologic evaluation performed in the absence of all antiarrhythmic drugs. The remaining six patients had no inducible ventricular tachycardia with drug therapy. All four operative nonsurvivors had refractory cardiac collapse preoperatively. Over 19 +/- 12 months of follow-up, there were four sudden cardiac deaths and no nonfatal recurrences of ventricular tachycardia. There were seven additional cardiac deaths. Actuarial cardiac survival was 0.57, and freedom from arrhythmic events was 0.76 at 42 months. Thus, in the absence of cardiogenic shock, the technique of sequential map-guided SER achieves: (1) a high operative survival with acceptable perfusion times, (2) excellent long-term arrhythmia control, and (3) survival comparable to that in patients with similar left ventricular function and no history of ventricular tachyarrhythmia.     

Restriction of cell lysis by homologous complement: II. Protection of erythrocytes against lysis by newly activated complement

Our previous work revealed that homologous complement (C) was ineffective in lysing antibody-sensitized erythrocytes (EA) even at high concentrations. It was also shown that activation of complement on homologous EA resulted in the binding of C9 and the formation of EA bearing complement proteins C1 through C9 (EAC1-9), yet few hemolytic sites were formed. Instead, as shown here, the formation of homologous EAC1-9 caused the cells to become resistant to lysis even by heterologous complement during a second incubation. In contrast, when homologous EAC1-8 were produced by incubating EA with C9-depleted serum, such intermediates were not protected against lysis by heterologous complement during a second incubation. Furthermore, homologous C9 on EAC1-9 was able to reduce the hemolytic efficiency of heterologous complement without blocking C activation and the formation of new C5b-9 complexes. Protection was not modified when homologous EAC1-9 were produced in one step, by incubation of EA with serum, or sequentially by adding C9 to EAC1-8. The minimum number of 9-sites required to confer a protective effect on EAC1-9 was less than 200 per cell. Thus, in addition to its known effect in heterologous cell killing, homologous C9 is capable of protecting homologous cells against inadvertent complement lysis.     

Recombinant human interleukin-3 stimulation of hematopoiesis in humans: loss of responsiveness with differentiation in the neutrophilic myeloid series

Recombinant human (rh) interleukin-3 (IL-3) stimulated the proliferation and differentiation of erythroid, granulocyte, macrophage, eosinophil (Eo), and mixed colonies as well as megakaryocytes from human bone marrow cells. rh IL-3 was a weaker stimulus than rh granulocyte-macrophage colony-stimulating factor (GM- CSF) for day 14 myeloid cell colonies. At day 7 of incubation, rh IL-3 stimulated a few G, M, and Eo clusters but no colonies. This loss of responsiveness of myeloid cells to rh IL-3 was accentuated with further differentiation of the cells. rh IL-3 stimulated very few or no clones after five-day incubation with enriched promyelocytes and myelocytes, whereas rh GM-CSF was an efficient stimulus. Responsiveness to rh IL-3 was completely lost in postmitotic mature neutrophils. Incubation of these cells with rh IL-3 did not result in enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) of tumor cells or superoxide anion production after stimulation with formyl-methyl-leucyl-phenylalanine (FMLP), although they could be stimulated by rh GM-CSF. In addition, preincubation of neutrophils with different concentrations of rh IL-3 failed to increase or decrease their response to rh GM-CSF. In contrast to neutrophils, mature Eos could be stimulated by rh IL-3 to kill antibody-coated tumor cells. These results show that cells of the neutrophilic myeloid series lose their responsiveness to h IL-3 as they differentiate and suggest that although h IL-3 may be an important therapeutic agent to use for hematopoietic regeneration in vivo, the lack of stimulation of mature neutrophil function makes it an unlikely sole candidate as adjunct therapy for treatment of infectious diseases.     

Oncogenic potential of erbB-2 in human mammary epithelial cells.

Introduction of the normal erbB-2 gene into immortalized human mammary epithelial cells (184B5) by transfection conferred a growth advantage to these cells both in vitro and in vivo. The 184B5 cells overexpressing erbB-2 formed colonies in semi-solid medium, frequently induced transient nodules in athymic mice and produced progressive tumors in vivo at a low frequency. Those tumors which did arise from erbB-2-transfected cells displayed substantially higher levels of normal gp185erb-2 protein when compared to the original transfectants, consistent with their selection for increased erbB-2 expression. Introduction of genes encoding genetically altered erbB-2 molecules into 184B5 cells increased their colony-forming efficiency and converted the cells to a tumorigenic phenotype at a high frequency. When the biological and biochemical properties of human mammary carcinoma cell lines known to overexpress erbB-2 were compared to the transfected 184B5 lines, they behaved most like those overexpressing the normal erbB-2 protein. Results indicate that overexpression of normal erbB-2 may directly contribute to the transformation of human mammary epithelium if sufficient levels of erbB-2 protein are expressed or if the erbB-2 gene is genetically altered.     

Combination of ECMO and cytokine adsorption therapy for severe sepsis with cardiogenic shock and ARDS due to Panton–Valentine leukocidin—positive <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> pneumonia and H1N1

Sepsis-induced cardiogenic shock in combination with severe acute respiratory failure represents a life-threatening combination that is often refractory to the conventional methods of treatment. We describe the case of a 33-year-old patient who developed acute cardiovascular collapse and ARDS secondary to superinfection of Panton–Valentine leukocidin—positive Staphylococcus aureus and H1N1 pneumonia who underwent successful combination therapy for severe sepsis-related cardiomyopathy and respiratory failure using extracorporeal membrane oxygenation and cytokine adsorption therapy.     

Functional magnetic stimulation of the respiratory muscles in dogs

This study assessed the ability of functional magnetic stimulation (FMS) to activate the respiratory muscles in dogs. With the animal supine, FMS of the phrenic nerves using a high-speed magnetic stimulator was performed by placing a round magnetic coil (MC) at the carotid triangle. Following hyperventilation-induced apnea, changes in volume (ΔV) and airway pressure (ΔP) against an occluded airway were determined. FMS of the phrenic nerves produced substantial inspired function (ΔV = 373 ± 20.5 mL and ΔP = −20 ± 2.0 cm H₂O). After bilateral phrenectomies, maximal inspired ΔV (219 ± 12.2 mL) and ΔP (−10 ± 1.0 cm H₂O) were produced when the MC was placed near the C6–C7 spinous processes, while maximal expired ΔV (−199 ± 22.5 mL) and ΔP (11 ± 2.3 cm H₂O) were produced following stimulation near the T9–T10 spinous processes. We conclude: (1) FMS of either the phrenic or upper intercostal nerves results in inspired volume production; (2) FMS of the lower intercostal nerves generates expired volume production; and (3) FMS of the respiratory muscles may be a useful noninvasive tool for artificial ventilation and assisted cough in patients with spinal cord injuries or other neurological disorders. © 1998 John Wiley & Sons, Inc. Muscle Nerve 21:1048–1057, 1998.