The enhanced in vitro hematopoietic activity of leridistim, a chimeric dual G-CSF and IL-3 receptor agonist.

The in vitro activity of leridistim was characterized for cell proliferation, generation of colony-forming units (CFU) and differentiation of CD34+ cells. In AML-193.1.3 cells, leridistim exhibited a significant increase in potency compared to rhG-CSF, SC-65303 (an IL-3 receptor agonist) or an equimolar combination of rhG-CSF and SC-65303. CFU-GM assays demonstrated that at 50% of the maximum response, the relative potency of leridistim was 12-fold greater than the combination of rhG-CSF and rhIL-3 and 44-fold more potent than rhG-CSF alone. In multi-lineage CFU assays, a combination of erythropoietin (rhEPO) and leridistim resulted in greater numbers of BFU-E, CFU-GEMM and CFU-Mk than rhEPO alone. Ex vivo culture of peripheral blood or bone marrow CD34+ cells with leridistim substantially increased total viable cells over cultures stimulated with rhG-CSF, SC-65303, or a combination of rhG-CSF and SC-65303. Culture with leridistim, resulted in a greater increase in myeloid (CD15+/CD11b+), monocytic (CD41-/CD14+) and megakaryocytic (CD41+/CD14-) precursor cells without depleting the progenitor pool (CD34+/CD15-/CD11b-). These results demonstrate that leridistim is a more potent stimulator of hematopoietic proliferation and differentiation than the single receptor agonists (rhG-CSF and SC-65303) either alone or combined. These unique attributes suggest that leridistim may enhance hematopoietic reconstitution following myelosuppressive chemotherapy.

Progenipoietins: biological characterization of a family of dual agonists of fetal liver tyrosine kinase-3 and the granulocyte colony-stimulating factor receptor.

The progenipoietins, a class of engineered proteins containing both fetal liver tyrosine kinase-3 and granulocyte colony-stimulating factor receptor agonist activities, were functionally characterized in vitro and in vivo. Four representative progenipoietins were evaluated for receptor binding, receptor-dependent cell proliferation, colony-forming unit activity, and their effects on hematopoiesis in the C57BL/6 mouse.The progenipoietins bound to fetal liver tyrosine kinase-3 and the granulocyte colony-stimulating factor receptor with affinities within twofold to threefold of the native ligands, and each progenipoietin bound simultaneously to both fetal liver tyrosine kinase-3 and the granulocyte colony-stimulating factor receptor. The progenipoietins exhibited different levels of activity in receptor-dependent cell proliferation assays. The fetal liver tyrosine kinase-3-dependent cell proliferation activity of three of four progenipoietins was decreased sixfold to 33-fold relative to native fetal liver tyrosine kinase-3 ligand, while granulocyte colony-stimulating factor receptor-dependent activity of the progenipoietins was within twofold to threefold of native granulocyte colony-stimulating factor. At nonsaturating concentrations, the progenipoietins stimulated colony formation to a greater extent than the equimolar combination of fetal liver tyrosine kinase-3 and granulocyte colony-stimulating factor. Treatment of mice with the progenipoietins yielded dramatic increases in peripheral blood and splenic white blood cells, polymorphonuclear leukocytes, and dendritic cells. These preclinical results demonstrate that the progenipoietins are potent hematopoietic growth factors that stimulate cells in a receptor-dependent manner. When administered in vivo, the progenipoietins effectively promote the generation of multiple cell lineages. Thus, in both in vitro and in vivo settings, the progenipoietins as single molecules exhibit the synergistic activity of the combination of fetal liver tyrosine kinase-3 and granulocyte colony-stimulating factor.

Work of breathing associated with pressure support ventilation in two different ventilators.

The purpose of this study was to compare the work of breathing during pressure support ventilation (PSV) with positive end expiratory pressure (PEEP) utilizing the Siemens SV300 (SV300) and Dräger Evita 4 (EV4) ventilators. Our hypothesis was that patients' work of breathing (WOB(P)) would be unchanged in PSV utilizing flow triggering (FT) in both the SV300 and EV4. We compared two ventilators using six healthy, intubated, sedated, spontaneously breathing pigs weighing approximately 10 kg each. WOB(P) (j/L) and ventilator work of breathing (WOB(V)) (j/L) were measured using a portable monitor which utilizes an esophageal balloon and flow transducer. Each breath was further analyzed for duration of inspiratory effort and negative deflection of pressure needed to trigger PSV. Animals were studied with the SV300 and EV4 on a pressure support of 5 cmH(2)O and PEEP settings of 0 and 5 cmH(2)O. Data were analyzed using the Wilcoxon signed rank test with significance set at P

Comparison of work of breathing between two neonatal ventilators utilizing a neonatal pig model.

OBJECTIVE: The purpose of this study was to determine whether variations in the delivery systems of continuous positive airway pressure between two ventilators would lead to differences in patient work of breathing (WOBp). DESIGN: Comparison of two neonatal ventilators with a neonatal pig model. SETTING: Animal laboratory. SUBJECTS: Thirty healthy, intubated, sedated, spontaneously breathing neonatal piglets weighing 1.0-2.0 kg. INTERVENTIONS: Patient work of breathing (WOBp) (gm cm/kg) was measured by using measurements based on an esophageal balloon and a flow transducer. Each breath was analyzed for ventilator response times (in msecs) and negative deflection of pressure. Each animal was studied with the Siemens SV300 and Drager Babylog 8000, on continuous positive airway pressure settings of 0, 3, and 5 cm H2O. Data were analyzed by using Wilcoxon's Signed Rank Test with significance of p 1 cm H2O negative pressure before flow was available with the Babylog. CONCLUSIONS: In intubated patients, maximum energy expenditure occurs at the initiation of ventilator breaths. WOBp in neonatal pigs was significantly increased. The response time of the ventilators may explain the differences in initiation of flow times and patient work. These differences may have important implications for energy kinetics, weight gain, and duration of mechanical ventilation in preterm neonates.

An evaluation of Automode, a computer-controlled ventilator mode, with the Siemens Servo 300A ventilator, using a porcine model.

BACKGROUND: Weaning of mechanical ventilation in patients optimally includes meeting their needs by making frequent ventilator adjustments. The Siemens Servo 300A mechanical ventilator is designed to allow the ventilator to be interactive with the patient's needs by making breath-by-breath adjustments in both control and support modes. We undertook the following experiment to validate that the Automode algorithm responded appropriately using a pediatric animal model when apnea occurred and if there was any impact on work of breathing. METHODS: We ventilated 6 sedated spontaneously-breathing piglets using Automode in pressure-regulated volume control/volume support (PRVC/VS) mode, pressure control/pressure support (PC/PS) mode, and volume control/volume support (VC/VS) mode. Data were collected using both a computerized respiratory monitor and data acquisition system that recorded and analyzed individual animal breaths for response time, effort of triggering, and work of breathing. Data collection began with the animals breathing spontaneously in each support mode, followed by the administration of a short-acting neuromuscular blocker (succinylcholine) to induce apnea, thus allowing the ventilator to switch between modes automatically. Data collection was continued before, during, and after apnea to observe the duration of inspiratory effort, trigger response time, and any significant pressure or flow variances of the Automode feature. In addition, patient work of breathing (WOB(P)) and ventilator work of breathing (WOB(V)) were measured before and after each phase. RESULTS: We found no instances of failure of Automode to follow the predetermined algorithms. There was a difference in both the amount of change in pressure and most negative deflection of pressure by each animal during triggering in the post-paralysis phase (p < 0.05). Response time for individual breaths was shorter from initiation of breath to most negative deflection of pressure during the post-paralysis phase (p < 0.05). Maximum flow reached was lower in the post-paralysis phase for VC/VS and PC/PS (p < 0.05). We also found WOB(P) decreased and WOB(V) increased in the post-paralysis phase for all modes tested. CONCLUSIONS: The Automode algorithm performed as expected in this animal experiment. We conclude that differences in response time and negative deflection of pressure, as an indication of animal effort, and maximum flow reached were due to continued weakness from the neuro-muscular blocker. However, the ventilator continued to trigger despite decreased effort by the animal.

Circular permutation of granulocyte colony-stimulating factor.

The sequence of granulocyte colony-stimulating factor (G-CSF) has been circularly permuted by introducing new chain termini into interhelical loops and by constraining the N- and C-terminal helices, either by direct linkage of the termini (L0) or by substitution of the amino-terminal 10-residue segment with a seven-residue linker composed of glycines and serines (L1). All the circularly permuted G-CSFs (cpG-CSFs) were able to fold into biologically active structures that could recognize the G-CSF receptor. CD and NMR spectroscopy demonstrated that all of the cpG-CSFs adopted a fold similar to that of the native molecule, except for one [cpG-CSF(L1)[142/141]] which has the new termini at the end of loop 34 with the shorter L1 linker. All of the cpG-CSFs underwent cooperative unfolding by urea, and a systematically lower free energy change (DeltaGurea) was observed for molecules with the shorter L1 linker than for those molecules in which the original termini were directly linked (the L0 linker). The thermodynamic stability of the cpG-CSFs toward urea was found to correlate with their relative ability to stimulate proliferation of G-CSF responsive cells. Taken together, these results indicate that the G-CSF sequence is robust in its ability to undergo linear rearrangement and adopt a biologically active conformation. The choice of linker, with its effect on stability, seems to be important for realizing the full biological activity of the three-dimensional structure. The breakpoint and linker together are the ultimate determinants of the structural and biological profiles of these circularly permuted cytokines. In the following paper [McWherter, C. A., et al. (1999) Biochemistry 38, 4564-4571], McWherter and co-workers have used circularly permuted G-CSF sequences to engineer chimeric dual IL-3 and G-CSF receptor agonists in which the relative spatial orientation of the receptor agonist domains is varied. Interpreting the differences in activity for the chimeric molecules in terms of the connectivity between domains depends critically on the results reported here for the isolated cpG-CSF domains.