Differences between rabbit sinoatrial pacemakers in their response to Mg, Ca and temperature.

The rabbit sinoatrial node is functionally inhomogeneous with respect to its response to changes in Mg concentration (0.6 to 6.0 mmol X litre-1) and in Ca concentration (1.1 to 2.2 mmol X litre-1) and to changes in experimental temperature (30 to 38 degrees C). High Mg (6.0 mmol X litre-1) stabilises the position of the leading pacemaker. This pacemaker decelerates under high Mg, but the subsidiary ones decelerate even more. Consequently when a subsidiary pacemaker turns dominant--eg under low Ca or at low temperature--an enhanced chronotropic response to high Mg is observed. The superior (cranial) part of the rabbit sinoatrial node is more responsive to changes in Ca concentration than the inferior (caudal) part. The same holds true for changes in temperature.

Inactivation of factor Xia in vivo: studies in chimpanzees and in humans.

C1-inhibitor (C1Inh), antithrombin III (ATIII), alpha 1-antitrypsin (a1AT), and alpha 2-antiplasmin (a2AP) are known inhibitors of factor XIa (FXIa). However, their precise contribution to FXIa inactivation in vivo is not known. We investigated FXIa inactivation in chimpanzees and assessed the contribution of these inhibitors to FXIa inactivation in patients with presumed FXI activation. Chimpanzees were infused with FXIa and the various FXIa-FXIa inhibitor complexes formed were measured. Most of FXIa was complexed to C1Inh (68%), followed by a2AP (13%), a1AT (10%), and ATIII (9%). Analysis of the plasma elimination kinetics revealed a half-life time of clearance (t1/2) for the FXIa-FXIa inhibitor complexes of 95 to 104 min, except for FXIa-a1AT, which had a t1/2 of 349 min. Due to this long t1/2, FXIa-a1AT complexes were predicted to show the highest levels in plasma samples from patients with activation of FXI. This was indeed shown in patients with disseminated intravascular coagulation, recent myocardial infarction or unstable angina pectoris. We conclude from this study that in vivo C1Inh is the predominant inhibitor of FXIa, but that FXIa-a1AT complexes due to their relatively long t 1/2 may be the best parameter to assess FXI activation in clinical samples.

Detection of erythrocyte membrane components in hemoglobin-based blood substitutes.

Two methods for the detection of membrane components in human stroma-free hemoglobin solutions are described. The first is a phospholipid assay with a detection limit of 0.5-1 nmol phospholipid/ml hemoglobin-solution. For the detection of membrane proteins an immunoassay with a monoclonal antibody against glycophorin alpha was developed (detection limit 0.01% of the original amount). These methods were used to determine the purity of Hb solutions prepared in two different ways. Hb solutions prepared by filtration of red blood cells, gradually swollen in hypotonic buffer, contained 0.25% of the original amount of phospholipid and no detectable glycophorin alpha. For Hb solutions prepared in a similar way from red blood cells lysed in water, the values for phospholipid and glycophorin alpha were 2.5% and 0.06%, respectively. The determination of both glycophorin alpha and phospholipid gives a useful indication of the purity of Hb solutions.

The potentiation of human C1-inhibitor by dextran sulphate is transient in vivo: studies in a rat model.

C1-inhibitor (C1-Inh) is an important regulator of inflammatory reactions because it is a potent inhibitor of the contact and complement system. C1-Inh application in inflammatory disease is, however, restricted because of the high doses required. The glycosaminoglycan-like molecule dextran sulphate (DXS) enhances C1-Inh function in vitro. Hence, we investigated whether co-administration with dextran sulphate reduces the amount of C1-Inh required, through enhancement in vivo. C1-Inh potentiation was measured in a newly developed C1s-inactivation assay that is based on activation of C4 by purified C1s. Activated C4 in rat plasma was quantified with a newly developed ELISA. Human C1-Inh (2.5 microM) inhibited C1s in rat plasma 55-fold faster in the presence of dextran sulphate (15 kDa, 5 microM). To study the stability of the complex in vivo, rats were given a mixture of C1-Inh (10 mg/kg) and dextran sulphate (3 mg/kg). C1-Inh activity during 5 h was analyzed ex vivo with the C1s inactivation assay. The noncovalent C1-Inh-dextran sulphate complex resulted in a transient enhancement of the inhibitory capacity of C1-Inh, lasting for 60-90 min. Dextran sulphate did not affect plasma clearance of C1-Inh. We conclude that the enhanced inhibitory capacity of C1-Inh complexed to dextran sulphate is transient in vivo. Hence, co-administration of these compounds seems a feasible approach to achieve short-term inhibition of complement in vivo.

Oxygen affinity of hemoglobin solutions modified by coupling with NFPLP and the effects on tissue oxygenation in the isolated perfused rat liver.

From these liver perfusions with Hb and Hb/HbNFPLP solutions the following conclusions can be drawn: In spite of the chemical modification of the hemoglobin molecule, no rheological differences are seen. All parameters measured were sensitive to hypoxia induced by a decrease in perfusion flow rate. The NFPLP-induced decrease in oxygen affinity was reflected in a higher venous PO2. These in-vivo observations are in agreement with the in-vitro measured oxygen dissociation curves. The difference in PO2 did not result in a change in the other oxygen-sensitive parameters in this model under the chosen conditions. Possible causes for these observations are: the level of hypoxia was too low the oxygen supply in the perfusions with the modified hemoglobin solutions was lower than the oxygen supply in the perfusions with normal hemoglobin. Whether or not this observation is due to an intrinsic property of the modified hemoglobin molecule remains to be established.

Accumulation of human EGF in nectar of transformed plants of Nicotiana langsdorffii x N. sanderae and transfer to honey by bees.

Honey has been used successfully in wound healing for thousands of years. The peptide hormone human epidermal growth factor (hEGF) is also known to have a beneficial effect in various wound healing processes via mechanisms that differ from those for honey. In this study, we show that hEGF can be incorporated into honey via nectar. Plants of Nicotiana langsdorffii x N. sanderae were transformed with the gene for hEGF, equipped with a nectary-targeted promoter and a signal sequence for secretion to nectar. These plants accumulated hEGF in the nectar. The maximum hEGF concentration recorded with ELISA in these plants is 2.5 ng·ml⁻¹. There is a significant linear relationship (P<0.001) between hEGF concentration and induction of hEGF-receptor phosphorylation. Since the flower morphology of these plants did not allow production of honey from their nectar, we used feeding solutions, spiked with synthetic hEGF, to study transfer of this peptide into honey through bee activity. Transfer of hEGF from a feeding solution to honey by bees occurred with retention of the hEGF concentration and the capacity to induce hEGF-receptor phosphorylation. These observations indicate that plants can function as a production platform for honey containing biologically active peptides, which may enhance wound healing and other biological processes.

Human kappa light chain targeted Pseudomonas exotoxin A--identifying human antibodies and Fab fragments with favorable characteristics for antibody-drug conjugate development.

Antibody-drug conjugates (ADC) represent promising agents for targeted cancer therapy. To allow rational selection of human antibodies with favorable characteristics for ADC development a screening tool was designed obviating the need of preparing individual covalently linked conjugates. Therefore, α-kappa-ETA' was designed as a fusion protein consisting of a human kappa light chain binding antibody fragment and a truncated version of Pseudomonas exotoxin A. α-kappa-ETA' specifically bound to human kappa light chains of human or human-mouse chimeric antibodies and Fab fragments. Antibody-redirected α-kappa-ETA' specifically inhibited proliferation of antigen-expressing cell lines at low toxin and antibody concentrations. Selected antibodies that efficiently delivered α-kappa-ETA' in the novel assay system were used to generate scFv-based covalently linked immunotoxins. These molecules efficiently triggered apoptosis of target cells, indicating that antibodies identified in our assay system can be converted to functional immunoconjugates. Finally, a panel of human epidermal growth factor receptor (EGFR) antibodies was screened--demonstrating favorable characteristics with antibody 2F8. These data suggest that antibodies with potential for Pseudomonas exotoxin A-based ADC development can be identified using the novel α-kappa-ETA' conjugate.

The plasma membrane of leading pacemaker cells in the rabbit sinus node. A qualitative and quantitative ultrastructural analysis.

We studied the fine structure of the plasma membrane of electrophysiologically identified leading pacemaker cells from the rabbit sinus node, using both ultrathin sections of fixed tissue and replicas of freeze-cleaved material. We found that differences exist between sinus node and working myocardial membranes, but these are only quantitative. The caveolae or sarcolemmal invaginations are present in very large numbers; they increase the surface area of the plasma membrane by about 100%. The small macular nexuses that are present represent 0.2% of the membrane surface area. Nexuses are therefore about 10 times less numerous in leading sinus node cells than in working myocardium cells. A single equivalent electrical representation of the sinus node shows, nevertheless, that an appreciable electrical coupling may be expected.

Accelerated autoantibody clearance by intravenous immunoglobulin therapy: studies in experimental models to determine the magnitude and time course of the effect.

Recently, it has been postulated that the beneficial effect of intravenous immunoglobulins (IVIGs) in antibody-mediated autoimmune disorders is based on accelerated catabolism of autoantibodies. In the current study, in vivo experiments were performed with mice in which autoantibody production was mimicked by continuous infusion of monoclonal antibodies. In this model, a single dose of IVIG reduced the plasma concentrations of the infused immunoglobulin (Ig)G1 monoclonal antibody (mAb) by approximately 40% after 3 days, whereas the concentration of an IgA mAb was not affected. To extrapolate these findings to humans, a computational model for IgG clearance was established that accurately predicted the time course and magnitude of the decrease in IgG plasma levels observed in mice. Adapted for humans, this model predicted a gradually occurring decrease in autoantibody levels after IVIG administration (2 g/kg), with a maximum reduction of approximately 25% after 3 to 4 weeks and a continued decrease of several months. In conclusion, a single high dose of IVIG induces a relatively small but long-lasting reduction of autoantibody levels by accelerated IgG clearance. This mechanism has clinical relevance in the sense that it can fully explain, as the sole mechanism, the gradual decrease in autoantibody levels observed in several patient studies. However, in some clinical studies, larger or more rapid effects have been observed that cannot be explained by accelerated clearance. Hence, IVIG can also reduce autoantibody levels through mechanisms such as down-regulation of antibody production or neutralization by anti-idiotypic antibodies.

Chronotropic effects of calcium and magnesium ions at different temperatures.

In the isolated right atrium of rabbit hearts, excess calcium causes an increase of heart rate at 38 degrees C; this effect is absent or even reversed at a temperature of 30 degrees C. From microelectrode studies it seems that when the calcium concentration is increased the pacemaker shifts within the sinoatrial node to cells in which excess calcium causes an acceleration of diastolic depolarization (type A). In fibers where the impulse originates at low calcium (type B), excess calcium causes a deceleration of diastolic depolarization. At low temperature, the effects on type A fibers are completely absent. Excess magnesium has a negative chronotropic effect at both temperatures, mainly by a deceleration of diastolic depolarization in type B fibers.

Protective effect of antithrombin III in acute experimental pancreatitis in rats.

In the present study we investigated the therapeutic action of antithrombin III (AT III) in taurocholate-induced experimental pancreatitis with high lethality in rats. High-dose AT III treatment greatly improved the survival rate not only when given as pretreatment but also when given 2 hr after induction. No favorable effect on survival rate was observed on administration after 5 hr. Both intravascular and intraperitoneal AT III administration locally restored decreased AT III levels in the peritoneal cavity and increased plasma AT III to supranormal levels. The primary pancreatic insult seemed to be unaffected by the treatment, because neither the rise in plasma lipase nor the development of ascites or the extension of the pancreatic necrosis were diminished. Because heparin pretreatment of the rats was also effective, the mechanism of the beneficial action was probably mediated by inhibition of the proteases of the coagulation cascade, thereby preventing intravascular coagulation in the pancreas and distant organs and subsequent systemic complications. The high efficacy of AT III treatment in this experimental model may stimulate clinical studies evaluating the efficacy of AT III treatment in an early stage of acute pancreatitis.

Safety evaluation of a polymerized hemoglobin solution in a murine infection model.

Several investigators have observed that free hemoglobin may increase the mortality rate in experimental Escherichia coli peritonitis in animals. This effect is probably mediated by the heme moiety of hemoglobin, but the mechanism remains controversial. Free hemoglobin might impair neutrophil function, and it might serve as a source of iron, which is necessary for bacterial replication. Several modified hemoglobin solutions, developed as blood substitutes, are currently being tested in clinical studies, but concern exists that these solutions may have the potential to exacerbate a bacterial infection. At the Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, a blood substitute based on modified hemoglobin (PolyHbXl) has been developed that has improved oxygen affinity and prolonged vascular retention. In the present study the potential risk of this solution on the promotion of infections has been evaluated. PolyHbXl was intravenously injected into mice in a clinically relevant dose of 1.5 gm/kg body weight 1 hour before intravenous administration of a sublethal number of Listeria monocytogenes, Salmonella typhimurium, E. coli, or Candida albicans organisms. PolyHbXl did not promote the proliferation of any of these microorganisms in the liver and spleen, nor did it lead to an increased mortality rate in the mice. Also, the in vitro proliferation of L. monocytogenes, S. typhimurium, and E. coli was not increased by PolyHbXl. In conclusion, PolyHbXl does not affect the course of infection with various microorganisms in mice, and no indication was found that this new blood substitute compromises the host defense against infections.

Endotoxin in blood products: correlation between the Limulus assay and the rabbit pyrogen test.

The possibility of replacing the rabbit pyrogen test by the Limulus (LAL) test, as a final release test for plasma products, was investigated. The LAL test measured the endotoxin content quantitatively, using a chromogenic substrate. The samples were boiled and centrifuged to remove inhibiting substances, which represent a major problem when testing plasma protein samples. In order to correlate the LAL test to the rabbit test, parallel assays were performed on 85 batches of two different human albumin preparations. For both products, a positive correlation was observed between the two tests. However, the pass/fail limit of the rabbit test was found at different endotoxin levels, corresponding to about 2 and 20 ng/kg, respectively. This discrepancy could be removed by extracting the endotoxin before administration to rabbits. It is concluded that the endotoxin, detected in plasma products by the LAL test, may be present in a non-pyrogenic state.

Prolonged vascular retention of a hemoglobin solution modified by cross-linking with 2-nor-2-formylpyridoxal 5'-phosphate.

The usefulness of hemoglobin solutions as plasma expanders with oxygen-carrying capacity is limited by a high oxygen affinity and a rapid clearance from the circulation. A large amount of the hemoglobin is cleared by the kidneys, because the hemoglobin can pass the glomeruli after dissociation into dimers. This dissociation can be prevented by cross-linking the beta chains with 2-nor-2-formylpyridoxal 5'-phosphate (NFPLP), a modification that also diminishes the oxygen affinity. In the present study, the vascular retention of modified hemoglobin (HbNFPLP) compared with unmodified hemoglobin (Hb) was investigated in rats and rabbits by replacing half the blood volume with a mixture of Hb and HbNFPLP (7 gm/100 ml). The amount of free hemoglobin in the circulation was determined from the plasma concentration, corrected for the decrease in plasma volume. The decrease in plasma volume was calculated from the increase in hematocrit (in the rats, 30% to 40% in 3 hours). The ratio Hb/HbNFPLP was determined by high-performance anion-exchange chromatography. The half-disappearance times for HbNFPLP and Hb were found to be 3 hours and 1 hour in rats and 7 hours and 2.5 hours in rabbits, respectively. In the rats, one third of the unmodified Hb was found in the urine 5 hours after the exchange, against only 5% of the HbNFPLP. In rats without kidneys, the ratio of Hb/HbNFPLP in the circulation remained constant. The results demonstrate that intramolecular cross-linking of hemoglobin with NFPLP prevents excretion by the kidneys, but does not influence other clearance mechanisms.

Key role of macrophages in hypotensive side effects of immunoglobulin preparations. Studies in an animal model.

Intravenous administration of certain immunoglobulin preparations may cause severe adverse reactions, especially in hypogammaglobulinaemic patients. Because the exact mechanism of the adverse reactions is still unknown, we investigated the severe, prolonged hypotension induced in anaesthetized rats on rapid i.v. infusion of standard immunoglobulin preparations. The hypotensive response was previously shown to be associated with IgG aggregates in the preparations but independent of complement activation. We found that the hypotension could be prevented by treating the rats with a specific receptor antagonist of platelet-activating factor; or by depletion of the macrophages of the rats; or by pretreatment with monomeric IgG. This provided evidence that the hypotension is initiated by interaction of IgG-aggregates with Fc-receptors on macrophages, leading to the production of platelet-activating factor. We conclude that the rat model provides a sensitive and reproducible test system for macrophage-activating properties of immunoglobulin preparations for i.v. administration which may lead to vasoactive side effects.

An animal model for the detection of hypotensive side effects of immunoglobulin preparations.

Intravenous administration of certain immunoglobulin preparations may cause severe adverse reactions, especially in immunodeficient patients. These reactions are generally assumed to be related to the anticomplementary activity of the preparations, caused by IgG aggregates. Because the exact mechanism of the adverse reaction is unknown, we investigated the reactions induced in anesthetized rats on rapid intravenous administration of different human immunoglobulin preparations. The most conspicuous observation was a severe, long-lasting hypotension, induced by standard immunoglobulin preparations (for intramuscular use), which appeared to be independent from the concomitant complement and neutrophil activation. The long-lasting hypotension was not related to the presence of prekallikrein activator, which induced a transient hypotensive reaction only after sensitizing the rats to bradykinin. The reactions appeared to be associated with IgG aggregates. It was found that certain aggregates induced mainly complement activation, whereas others had mainly a hypotensive effect or no effect at all. It was concluded that the rat model provides a sensitive and reproducible test system for vasoactive properties of immunoglobulin preparations for intravenous administration that cannot be predicted from in vitro measurements, such as anticomplementary activity, aggregate content or prekallikrein activator activity. It is suggested that the test may also be used for other plasma products for intravenous administration.

Pacemaker cell types in the rabbit sinus node: a correlative ultrastructural and electrophysiological study.

In isolated preparations of the rabbit sinus node, we have investigated the fine structure of the tissue at many sites which had been electro-physiologically identified by means of microelectrode recordings. For each of these sites we quantified the myofilament density of the cells, since this appeared to be a useful parameter for characterizing cell types in the sinus node region; and because myofilaments were present both in isolated form and organized in myofibrils, the degree of organization was also measured in a semi-quantitative fashion. The electrical activity of cells at a given site was characterized by the activation moment relative to the cardiac cycle and furthermore by the rate of diastolic depolarization and the maximum rate of rise of the action potential. From the centre of the node toward the periphery a very gradual increase in myofilament density was observed in all directions. It was found that the rate of diastolic depolarization, which feature is generally accepted as being basic to the automaticity of the sinus node, was inversely related to the volume percentage of myofilaments. This means that a relation exists between the pacemaker action and the cell type. The anatomically less developed cells, i.e. the cells with the lowest density of organelles, which are located in the central portion of the node, are the most specialized pacemakers. No clear relation was found between the myofilament density and the rate of rise of the action potential. In the direction of the crista terminalis we observed an increase in the rate of rise and an increase in conduction velocity concomitant with the increase in myofilament density. Toward the interatrial septum, however, the increase in myofilament density was not accompanied by an increase in rate of rise; in this direction the impulse conduction was blocked. A correlation between cell type and impulse conduction could thus not be established.

Little-excitable transitional cells in the rabbit sinoatrial node: a statistical, morphological and electrophysiological study.

It is demonstrated that systolic and diastolic depolarization rate are correlated with the percentage of myofilaments in the cells of the rabbit sinoatrial node. It appears that, in the rabbit sinoatrial node, little-excitable transitional cells exist in the zone of propagation at the septal side of the typical nodal cells.