Assessing immune competence in pigs by immunization with tetanus toxoid.

Immune competence can be tested by challenging organisms with a set of infectious agents. However, disease control requirements impose restrictions on the infliction of infections upon domestic pigs. Alternatively, vaccinations induce detectable immune responses that reflect immune competence. Here, we tested this approach with tetanus toxoid (TT) in young domestic pigs. To optimize the vaccination protocol, we immunized the pigs with a commercial TT vaccine at the age of 21 or 35 days. Booster immunizations were performed either 14 or 21 days later. TT-specific antibodies in plasma as well as lymphoproliferative responses were determined both 7 and 14 days after booster immunization using ELISA and lymphocyte transformation tests, respectively. In addition, general IgG and IgM plasma concentrations and mitogen-induced proliferation were measured. The highest TT-specific antibody responses were detected when blood samples were collected 1 week after a booster immunization conducted 21 days after primary immunization. The pigs' age at primary immunization did not have a significant influence on TT-specific antibody responses. Similarly, the TT-specific proliferative responses were highest when blood samples were collected 1 week after booster immunization, while age and time of primary and booster immunization were irrelevant in our setup. While general IgG and IgM plasma levels were highly age dependent, there were no significant age effects for TT-specific immune responses. In addition, mitogen-induced proliferation was independent of immunization as well as blood sampling protocols. In summary, our model of TT vaccination provides an interesting approach for the assessment of immune competence in young pigs. The detected vaccination effects were not biased by age, even though our data were acquired from immune systems that were under development during our tests.

Two evolutionary strategies of influenza viruses to escape host non-specific inhibitors: alteration of hemagglutinin or neuraminidase specificity.

The porcine serum inhibitor alpha 2-macroglobulin prevents influenza virus from entering host cells by competing for the SA alpha 2, 6Gal-binding site of the hemagglutinin (HA). We studied a series of inhibitor-sensitive and inhibitor-resistant human and porcine influenza virus isolates of the H3N2 subtype, all of which contained HAs, which initially bound only to SA alpha 2, 6Gal oligosaccharides. When their neuraminidase was inhibited, the naturally resistant viruses, as a result of no longer being able to elute from the inhibitor, became sensitive. Evidently it is the neuraminidase which enabled these viruses to grow in hosts which possess the inhibitor. Escape-mutants selected under laboratory conditions in the presence of porcine serum became inhibitor-resistant by two alternative mechanisms: they changed either their HA-specificity or their neuraminidase-specificity. The study thus disclosed two evolutionary strategies for acquiring resistance to a host neuraminidase-sensitive inhibitor: (i) acquisition of an HA able to bind to oligosaccharides not present on the inhibitor; or (ii) acquisition of a neuraminidase able to cleave the oligosaccharide bound by the HA.

New light-scattering and field-trapping methods access the internal electric structure of submicron particles, like influenza viruses.

A variety of AC-electrokinetic field effects can be exploited for handling or electric characterization of microscopic and submicroscopic particles, like cells, organelles, supramolecular structures, and artificial colloids. Despite the fact that dielectric spectroscopy methods by AC-electrokinetics, like common impedance methods, are based on the impedance properties of the different constituents of the particles, the first methods yield higher parameter resolutions. A drawback of the electrokinetic methods was that they required microscopic observability of field-induced particle movements. New AC-electrokinetic methods like electrorotational light scattering (ERLS), dielectrophoretic phase-analysis light scattering (DPALS), and dielectrophoretic field trapping (DFT) solve this problem and access the submicroscopic particle range. This paper gives an introduction to the new methods and presents measurements on influenza viruses. To develop a dielectric virus model, experiments of ERLS were combined with DFT of viruses in microstructured electric-field cages. The model assumes a spherical virus with a radius of 50 nm and a single-shell dielectric structure. The shell thickness of 18 nm summarizes the dimensions of the lipid and viral surface protein layers. For this model, the conductivities of core and shell of 0.1 mS/m and 0.1 microS/m, respectively, and the relative permittivities of 30 and 80, respectively, were obtained.

A comprehensive approach to electro-orientation, electrodeformation, dielectrophoresis, and electrorotation of ellipsoidal particles and biological cells.

Suspended cells may respond to AC polarization by orienting, deforming, moving or rotating. For modeling of ellipsoidal cells, a new dipole approach is proposed. Along each of the principal axis of the model, three finite elements of arbitrary but equal cross-sectional area for the interior, low conductive membrane shell and exterior are assumed. The length of the external medium elements is defined by influential radii which are related to the depolarizing factors. The model predicts the potential at the ellipsoid's surface leading to the induced dipole moment. The moment obtained is identical to the Laplace approach for homogeneous ellipsoids; in the single-shell case, it is slightly different. The reason is the constant shell thickness which overcomes the confocal thickness necessary for the Laplace solution. Expressions for electro-orientation, deformation, dielectrophoresis, and electrorotation are derived. In linearly and circularly polarized fields, different orientation spectra are predicted to occur. While in linearly polarized AC fields, particles are oriented along their axis of highest polarizability, in circularly polarized fields, the axis of lowest polarizability is oriented perpendicular to the plane of field rotation. Based on this finding, a new electro-orientation method is proposed. In dielectrophoresis and electrorotation, reorientations are predicted which lead to discontinuous spectra.

A decrease of intracellular ATP is compensated by increased respiration and acidification at sub-lethal parathion concentrations in murine embryonic neuronal cells: measurements in metabolic cell-culture chips.

We present a label-free in vitro method for testing the toxic potentials of chemical substances using primary neuronal cells. The cells were prepared from 16-day-old NMRI mouse embryos and cultured on silicon chips (www.bionas.de) under the influence of different parathion concentrations with sensors for respiration (Clark-type oxygen electrodes), acidification (pH-ISFETs) and cell adhesion (interdigitated electrode structures, IDES). After 12 days in vitro, the sensor readouts were simultaneously recorded for 350 min in the presence of parathion applying a serial 1:3 dilution. The parathion-dependent data was fitted by logistic functions. IC(50) values of approximately 105 µM, 65 µM, and 54 µM were found for respiration, acidification, and adhesion, respectively. An IC(50) value of approximately 36 µM was determined from the intracellular ATP-levels of cells, which were detected by an ATP-luminescence assay using micro-well plates. While the intracellular ATP level and cell adhesion showed no deviation from a simple logistic decay, increases of approximately 29% in the respiration and 15% in the acidification rates above the control values were found at low parathion concentrations, indicating hormesis. These increases could be fitted by a modified logistic function. We believe that the label-free, continuous, multi-parametric monitoring of cell-metabolic processes may have applications in systems-biology and biomedical research, as well as in environmental monitoring. The parallel characterization of IC(50) values and hormetic effects may provide new insights into the metabolic mechanisms of toxic challenges to the cell.

Attachment of rod-like (BAR) proteins and membrane shape.

Previous studies have shown that cellular function depends on rod-like membrane proteins, among them Bin/Amphiphysin/Rvs (BAR) proteins may curve the membrane leading to physiologically important membrane invaginations and membrane protrusions. The membrane shaping induced by BAR proteins has a major role in various biological processes such as cell motility and cell growth. Different models of binding of BAR domains to the lipid bilayer are described. The binding includes hydrophobic insertion loops and electrostatic interactions between basic amino acids at the concave region of the BAR domain and negatively charged lipids. To shed light on the elusive binding dynamics, a novel experiment is proposed to expand the technique of single-molecule AFM for the traction of binding energy of a single BAR domain.

Do band 3 protein conformational changes mediate shape changes of human erythrocytes?

The bilayer-couple model predicts a reversible membrane crenation for an increasing ratio of external to internal monolayer area. This was comprehensively proven. However, individual erythrocytes may undergo dramatic shape changes within seconds when the suspension medium is changed. In contrast, under physiological conditions with no addition of membrane active compounds, active phospholipid translocation and passive flip-flops are comparatively slow. We propose that conformational changes of the anion-exchange protein, band 3, may rapidly alter the monolayer area ratio. Band 3 occupies about 10% of the total membrane area of human erythrocytes. Under physiological conditions, its conformers are asymmetrically distributed with about 90% of the transport sites facing the cytoplasm. This distribution is altered when external conformations are recruited by changing the transmembranous Cl- gradient, the external pH, or by the application of inhibitors. In experiments, recruitment by low ionic strength caused a rapid, temporary formation of echinocytes. This suspension effect could also be found at high ionic concentrations, when Cl- was replaced by SO4(2-). Inhibitors known to recruit the external band 3 conformation, like DIDS, SITS and flufenamic acid, are echinocytogenic. For inhibitors not recruiting a certain conformation, e.g. phenylglyoxal and niflumic acid, no shape effect was found. Since band 3 ensures a fast equilibrium of internal and external anions these ions are usually distributed according to the transmembrane potential (TMP). In the literature, a correlation of TMP and band 3 conformation, as well as a correlation of TMP and red cell shape, is described. In the proposed model, low external Cl- concentrations, inhibitors, or a negative TMP may recruit the transport sit outwards.(ABSTRACT TRUNCATED AT 250 WORDS)

Determination of viral neuraminidase specificity for membrane-bound sialic acids by cell electrophoresis.

The ability of the influenza virus neuraminidase (NA) to cleave specific sialic acids was measured by cell electrophoresis. Most of the surface charge of human erythrocytes can be attributed to sialic acids. Therefore cleavage of sialic acids reduces the surface charge density which is measurable as a reduced cell electrophoretic mobility (EPM). For experiments specifically sialylated, erythrocytes were used. Their EPM was significantly decreased after incubation with virus strains possessing the corresponding NA specificity, even when the viral haemagglutinin (HA) was unable to bind to the erythrocyte's surface. Thus, the limited applicability of elution experiments, which requires virus binding, is overcome. An additional advantage of this procedure is that it is non-radioactive. In our model system the erythrocyte's surface resembles the natural situation of viral interaction with membrane-bound receptors.

A polarization model overcoming the geometric restrictions of the laplace solution for spheroidal cells: obtaining new equations for field-induced forces and transmembrane potential.

We present a new model for a variety of electric polarization effects on oblate and prolate homogeneous and single-shell spheroids. For homogeneous spheroids the model is identical to the Laplace model. For single-shell spheres of cell-like geometry the calculated difference of the induced dipole moments is in the thousandths range. To solve Laplace's equation for nonspherical single-shell objects it is necessary to assume a confocal shell, which results in different cell membrane properties in the pole and equator regions, respectively. Our alternative model addresses this drawback. It assumes that the disturbance of the external field due to polarization may project into the medium to a characteristic distance, the influential radius. This parameter is related to the axis ratio of the spheroid over the depolarizing factors and allows us to determine the geometry for a finite resistor-capacitor model. From this model the potential at the spheroid's surface is obtained and, consequently, the local field inside a homogeneous spheroid is determined. In the single-shell case, this is the effective local field of an equivalent homogeneous spheroid. Finally, integration over the volume yields the frequency-dependent induced dipole moment. The resistor-capacitor approach allowed us to find simple equations for the critical and characteristic frequencies, force plateaus and peak heights of deformation, dielectrophoresis and electrorotation for homogeneous and single-shell spheroids, and a more generalized equation for the induced transmembrane potential of spheroidal cells.

A unified resistor-capacitor model for impedance, dielectrophoresis, electrorotation, and induced transmembrane potential.

Dielectric properties of suspended cells are explored by analysis of the frequency-dependent response to electric fields. Impedance (IMP) registers the electric response, and kinetic phenomena like orientation, translation, deformation, or rotation can also be analyzed. All responses can generally be described by a unified theory. This is demonstrated by an RC model for the structural polarizations of biological cells, allowing intuitive comparison of the IMP, dielectrophoresis (DP), and electrorotation (ER) methods. For derivations, cells of prismatic geometry embedded in elementary cubes formed by the external solution were assumed. All geometrical constituents of the model were described by parallel circuits of a capacitor and a resistor. The IMP of the suspension is given by a meshwork of elementary cubes. Each elementary cube was modeled by two branches describing the current flow through and around the cell. To model DP and ER, the external branch was subdivided to obtain a reference potential. Real and imaginary parts of the potential difference of the cell surface and the reference reflect the frequency behavior of DP and ER. The scheme resembles an unbalanced Wheatstone bridge, in which IMP measures the current-voltage behavior of the feed signal and DP and ER are the measuring signal. Model predictions were consistent with IMP, DP, and ER experiments on human red cells, as well as with the frequency dependence of field-induced hemolysis. The influential radius concept is proposed, which allows easy derivation of simplified equations for the characteristic properties of a spherical single-shell model on the basis of the RC model.

Analytical description of the transmembrane voltage induced on arbitrarily oriented ellipsoidal and cylindrical cells.

We present an analytical equation for the transmembrane voltage (Deltaphi) induced by a homogeneous AC field on arbitrarily oriented cells of the general ellipsoidal shape. The equation generalizes the Schwan equation for spherical cells and describes the dependence of Deltaphi on field frequency, cell size and shape, membrane capacitance, conductivities of cytoplasm, membrane and external medium, the location of the membrane site under consideration, and on the orientation of the cell with respect to the field. The derivation is based on the fact that the cytoplasm and the Maxwellian equivalent body of the whole cell are both of a general ellipsoidal shape and must thus exhibit constant local fields. The constant fields allow for a relatively simple description of the potentials on the internal and external membrane sides, leading to Deltaphi. For this, the properties of cytoplasm, membrane, and external medium have been introduced into a special, finite element model. We found that Deltaphi can be unambiguously defined for non-spherical cells, provided that the membrane thickness is thin in comparison to the cell dimensions.

On the analytical description of transmembrane voltage induced on spheroidal cells with zero membrane conductance.

We present analytical equations for the transmembrane voltage (delta phi) induced by a homogeneous field on oriented cells of spheroidal shape in spherical coordinates. For simplicity, a nonconductive membrane and a highly polarizable cytoplasm were assumed. Under these conditions, the cell's polarizability is determined by the nonconductive membrane. For symmetry reasons the surface of the highly polarizable cytoplasm can be assumed to be at 0 V. Since the cell is of ellipsoidal shape its effective local field, i.e. the field of its Maxwellian equivalent body, must be constant. This allows for a simple description of the potential at the external membrane side, directly leading to delta phi. The dependence of delta phi on cell size and shape as well as on the location of the considered membrane site is described for both possible orientations of the symmetry axis, parallel and perpendicular to the external field, respectively.

Introducing phase analysis light scattering for dielectric characterization: measurement of traveling-wave pumping.

Phase analysis light scattering (PALS) was applied to characterize a high-frequency traveling-wave (TW) micropump. Field strength and frequency characteristics were measured for aqueous solutions up to 40 MHz and conductivities of 16 mS/m. The TW field was generated by an ultramicroelectrode array of intercastellated electrodes, which were driven by square-topped signals. Pumping exhibited one major relaxation peak, which strongly increased for conductivities above 4 mS/m. The conductivity dependence of the peak frequency showed an unexpected nonlinear behavior. Around 20 MHz an additional peak caused by electronic resonance was found. Additional coils or capacitors shifted the resonance peak and allowed us to determine the electronic properties of the array. Analysis of distortions in the pump spectra caused by the harmonic content of the driving signals showed that the pump direction is determined by the traveling direction of the field. For measurement of AC-field-induced particle translations, the advantage of PALS over the commonly used microscopic analysis is that it offers an objective method for statistically significant, computerized registration of extremely slow motions. Thus, for dielectric characterization, low field strengths can be used, which is advantageous not only for analyzing liquid pumping, but also for measuring particle translations induced by dielectrophoresis or TW dielectrophoresis.

Critical analysis of the impedance method for the evaluation of permittivity and conductivity of the plasma membrane.

We report a critical analysis of a typical method of dielectric spectroscopy consisting in impedance measurements as a function of frequency. Experimental data were obtained by measuring impedance on human erythrocyte suspensions. Since these cells do not have a nucleus they represent an ideal material for the application of the well established single shell model. This allows the evaluation of permittivity and conductivity of the plasma membrane. We discuss the influence on the reliability of results of parameters such as fractional volume, average dimensions and membrane thickness of cells.

Dielectric spectroscopy of human erythrocytes: investigations under the influence of nystatin.

When placed in rotating electric fields red blood cells show a typical electrorotation spectrum with antifield rotation in the lower and cofield rotation in the higher frequency range. Assuming a spherical cell geometry, however, dielectrical parameters were obtained that differ from those measured by independent methods. Dielectrophoresis and, in particular, electrorotation yielded lower membrane capacitance values than expected. Introduction of an ellipsoidal model with an axis ratio of 1:2 allowed a description that proved to be consistent with dielectrophoresis and electrorotation data. For control cells an internal conductivity of 0.535 S/m, a specific membrane capacitance of 0.82 x 10(-2) F/m2, and a specific conductance of 480 S/m2 were obtained. The first characteristic frequency (frequency of fastest antifield rotation) and the related rotation speed can be measured quite quickly by means of a compensation method. Thus it was possible to follow changes of dielectric properties on individual cells after nystatin application. Ionophore-membrane interaction caused cell shrinkage in parallel to a decrease of the first characteristic frequency and rotation speed. Analysis of data revealed a decrease of the internal conductivity that is not only caused by ion loss but also, to a large extent, by a strong increase of hindrance because of shrinkage. Ionophore-induced membrane permeabilities can be calculated from volume decrease as well as from electrorotational data. In no case can these permeabilities count for the high membrane-AC conductivity that is attributed to the band-3 anion exchanging protein. The membrane-AC conductance was found not to be decreased for cells in Donnan equilibrium, which had leaked out almost completely.

Dielectrophoresis and electrorotation of neurospora slime and murine myeloma cells.

Dielectrophoresis and electrorotation are commonly used to measure dielectric properties and membrane electrical parameters of biological cells. We have derived quantitative relationships for several critical points, defined in Fig. A 1, which characterize the dielectrophoretic spectrum and the electrorotational spectrum of a cell, based on the single-shell model (Pauly, H., and H.P. Schwan, 1959. Z. Naturforsch. 14b:125-131; Sauer, F.A. 1985. Interactions between Electromagnetic Field and Cells. A. Chiabrera, C. Nicolini, and H.P. Schwan, editors. Plenum Publishing Corp., New York. 181-202). To test these equations and to obtain membrane electrical parameters, a technique which allowed simultaneous measurements of the dielectrophoresis and the electrorotation of single cells in the same chamber, was developed and applied to the study of Neurospora slime and the Myeloma Tib9 cell line. Membrane electrical parameters were determined by the dependence of the first critical frequency of dielectrophoresis (fct1) and the first characteristic frequency of electrorotation (fc1) on the conductivity of the suspending medium. Membrane conductances of Neurospora slime and Myeloma also were found to be 500 and 380 S m-2, respectively. Several observations indicate that these cells are more complex than that described by the single-shell model. First, the membrane capacities from fct1 (0.81 x 10(-2) and 1.55 x 10(-2) F m-2 for neurospora slime and Myeloma, respectively) were at least twice those derived from fc1. Second, the electrorotation spectrum of Myeloma cells deviated from the single-shell like behavior. These discrepancies could be eliminated by adapting a three-shell model (Furhr, G., J. Gimsa, and R. Glaser. 1985. Stud. Biophys. 108:149-164). Apparently, there was more than one membrane relaxation process which could influence the lower frequency region of the beta-dispersion. fct1 of Myeloma in a medium of given external conductivity were found to be similar for most cells, but for some a dramatically increased fct1 was recorded. Model analysis suggested that a decrease in the cytoplasmatic conductivity due to a drastic ion loss in a cell could cause this increase in fct1. Model analysis also suggested that the electrorotation spectrum in the counter-field rotation range and fc1 would be more sensitive to conductivity changes of the cytoplasmic fluid and to the influence of internal membranes than would fct1, although the latter would be sensitive to changes in capacitance of the cytoplasmic membranes.

Traveling-wave dielectrophoresis of microparticles.

The traveling-wave-induced linear transfer of dielectric particles like living cells and artificial objects of microscopic dimensions is analyzed. It is shown that the electrode geometries must correspond to particle sizes to allow an effective manipulation of particles immersed in weakly electrolytic solutions by high frequency traveling waves. The theoretical model elaborated in this paper is in good agreement with experimental results obtained in microfabricated chambers of linearly arranged electrodes. It explains the behavior of homogeneous cellulose spheres as well as that of membrane-covered pine polls. The traveling-wave-driven electrodes are described by a superposition of time-dependent point charges. Subsequently, each of these point charges has to be considered as polarizing the dielectric particle and interacting with the polarized particle. This results in forces which effectively translocate the particle.

Dielectric spectroscopy of single human erythrocytes at physiological ionic strength: dispersion of the cytoplasm.

Usually dielectrophoretic and electrorotation measurements are carried out at low ionic strength to reduce electrolysis and heat production. Such problems are minimized in microelectrode chambers. In a planar ultramicroelectrode chamber fabricated by semiconductor technology, we were able to measure the dielectric properties of human red blood cells in the frequency range from 2 kHz to 200 MHz up to physiological ion concentrations. At low ionic strength, red cells exhibit a typical electrorotation spectrum with an antifield rotation peak at low frequencies and a cofield rotation peak at higher ones. With increasing medium conductivity, both electrorotational peaks shift toward higher frequencies. The cofield peak becomes antifield for conductivities higher than 0.5 S/m. Because the polarizability of the external medium at these ionic strengths becomes similar to that of the cytoplasm, properties can be measured more sensitively. The critical dielectrophoretic frequencies were also determined. From our measurements, in the wide conductivity range from 2 mS/m to 1.5 S/m we propose a single-shell erythrocyte model. This pictures the cell as an oblate spheroid with a long semiaxis of 3.3 microns and an axial ratio of 1:2. Its membrane exhibits a capacitance of 0.997 x 10(-2) F/m2 and a specific conductance of 480 S/m2. The cytoplasmic parameters, a conductivity of 0.4 S/m at a dielectric constant of 212, disperse around 15 MHz to become 0.535 S/m and 50, respectively. We attribute this cytoplasmic dispersion to hemoglobin and cytoplasmic ion properties. In electrorotation measurements at about 60 MHz, an unexpectedly low rotation speed was observed. Around 180 MHz, the speed increased dramatically. By analysis of the electric chamber circuit properties, we were able to show that these effects are not due to cell polarization but are instead caused by a dramatic increase in the chamber field strength around 180 MHz. Although the chamber exhibits a resonance around 180 MHz, the harmonic content of the square-topped driving signals generates distortions of electrorotational spectra at far lower frequencies. Possible technological applications of chamber resonances are mentioned.

Measurement of inherent particle properties by dynamic light scattering: introducing electrorotational light scattering.

Common dynamic light scattering (DLS) methods determine the size and zeta-potential of particles by analyzing the motion resulting from thermal noise or electrophoretic force. Dielectric particle spectroscopy by common microscopic electrorotation (ER) measures the frequency dependence of field-induced rotation of single particles to analyze their inherent dielectric structure. We propose a new technique, electrorotational light scattering (ERLS). It measures ER in a particle ensemble by a homodyne DLS setup. ER-induced particle rotation is extracted from the initial decorrelation of the intensity autocorrelation function (ACF) by a simple optical particle model. Human red blood cells were used as test particles, and changes of the characteristic frequency of membrane dispersion induced by the ionophore nystatin were monitored by ERLS. For untreated control cells, a rotation frequency of 2 s-1 was induced at the membrane peak frequency of 150 kHz and a field strength of 12 kV/m. This rotation led to a decorrelation of the ACF about 10 times steeper than that of the field free control. For deduction of ERLS frequency spectra, different criteria are discussed. Particle shape and additional field-induced motions like dielectrophoresis and particle-particle attraction do not significantly influence the criteria. For nystatin-treated cells, recalculation of dielectric cell properties revealed an ionophore-induced decrease in the internal conductivity. Although the absolute rotation speed and the rotation sense are not yet directly accessible, ERLS eliminates the tedious microscopic measurements. It offers computerized, statistically significant measurements of dielectric particle properties that are especially suitable for nonbiological applications, e.g., the study of colloidal particles.

Ion channel enzyme in an oscillating electric field.

To explain the electrical activation of several membrane ATPases, an electroconformational coupling (ECC) model has previously been proposed. The model explained many features of experimental data but failed to reproduce a window of the field intensity for the stimulated activity. It is shown here that if the affinities of the ion for the two conformational states of the transporter (one with binding site on the left side and the other on the right side of the membrane) are dependent on the electric field, the field-dependent transport can exhibit the observed window. The transporter may be described as a channel enzyme which opens to one side of the membrane at a time. It retains the energy-transducing ability of the earlier ECC models. Analysis of the channel enzyme in terms of the Michaelis-Menten kinetics has been done. The model reproduced the amplitude window for the electric field-induced cation pumping by (Na,K)-ATPase.