Enhanced ability of regulatory T cells in chronic hepatitis C patients with persistently normal alanine aminotransferase levels than those with active hepatitis.

In hepatitis C virus (HCV) infection, the Th1-type immune response is involved in liver injury. A predominance of immunosuppressive regulatory T cells (Treg) is hypothesized in patients with persistently normal alanine aminotransferase (PNALT). Our aim was to clarify the role of Treg in the pathogenesis of PNALT. Fifteen chronically HCV-infected patients with PNALT, 21 with elevated ALT (CH) and 19 healthy subjects (HS) were enrolled. We determined naturally-occurring Treg (N-Treg) as CD4+CD25high+FOXP3+ T cells. The expression of FOXP3 and CTLA4 in CD4+CD25high+ cells was quantified by real-time reverse transcriptase-polymerase chain reaction. Bulk or CD25-depleted CD4+ T cells cultured with HCV-NS5 loaded dendritic cells were assayed for their proliferation and cytokine release. We examined CD127-CD25-FOXP3+ cells as distinct subsets other than CD25+ N-Treg. The frequencies of N-Treg in patients were significantly higher than those in HS. The FOXP3 and CTLA4 transcripts were higher in PNALT than those in CH. The depletion of CD25+ cells enhanced HCV-specific T cell responses, showing that co-existing CD25+ cells are suppressive. Such inhibitory capacity was more potent in PNALT. The frequency of CD4+CD127-CD25-FOXP3+ cells was higher in CH than those in PNALT. Treg are more abundant in HCV-infected patients, and their suppressor ability is more potent in patients with PNALT than in those with active hepatitis.

Immunological agglutination kinetics of latex particles with physically adsorbed antigens.

The influence of the properties of antigens and particles on the immunological agglutination kinetics of the antigen-coated latex particles was studied. Horse cytochrome c, hen egg-white lysozyme (HEL), bovine serum albumin (BSA), and Aspergillus sp. glucose oxidase were physically adsorbed onto the surfactant free latices of styrene-methacrylic acid (MAA) copolymer (P (S/MAA)) and polystyrene (PS). The initial rates of the immunological agglutination of these protein-coated particles initiated by the addition of antibodies were quantified by the absorbance change at a wavelength of 680 nm. The initial agglutination rates of the particles covered with smaller antigens were lower. This effect of the molecular size of antigens was larger in P(S/MAA), because small antigens are probably buried in the hydrous polymethacrylic acid layer on the surface of particles. Thus, both the molecular size of antigens and the surface properties of particles affect the sensitivity of the immunological agglutination. On the other hand, the dependence of the initial rate of the immunological agglutination on the ionic strength and pH was similar irrespective of antigen-particle systems. The initial agglutination rates were largest at an ionic strength of approximately 0.05 at pH 7.0 and decreased with increasing pH. This dependence of the sensitivity on the pH and ionic strength is attributed to the electrostatic interactions of particle-particle and antibody-particle.

Adsorption of gamma-globulin, a model protein for antibody, on colloidal particles.

The effect of surface properties on the adsorption of bovine gamma-globulin, a model protein for antibody, was studied. Polystyrene latex (PS), hydrophilic copolymer lattices of styrene/2-hydroxyethyl methacrylate [P(S/HEMA)], styrene/ methacrylic acid [P(S/MAA)] and methyl methacrylate/ 2-hydroxyethyl methacrylate [P(MMA/HEMA)], and colloidal silica were used. The adsorption isotherms of gamma-globulin on these colloidal particles were measured as a function of pH and ionic strength. The hydrophilic particles showed low affinities for gamma-globulin at alkaline pH, while PS showed high affinities for gamma-globulin over the whole range of pH and ionic strength. The gamma-globulin adsorption on hydrophilic particles was highly reversible with respect to the pH and ionic strength compared with that on PS. These differences indicate that the dominant driving forces of adsorption are related to the hydrophilicity of particles. The adsorption isotherms of all colloidal particles showed the plateau values, and the order of maximum values of plateau adsorption was P(S/MAA) > PS or P(S/HEMA), silica > P(MMA/HEMA). Thus, they were also affected by the charged groups and the hydrophilicity of the surfaces. On the other hand, the plateau values of all colloidal particles were more or less symmetrical with a maximum at around the isoelectric point of gamma-globulin at an ionic strength of 0.01. This behavior is attributed to the important role of the lateral interaction between the adsorbed molecules at low ionic strength.

Development and application of thermo-sensitive immunomicrospheres for antibody purification.

The latex particles composed of poly(styrene/N-isopropylacrylamide/glycidyl methacrylate) [P(St/NIPAM/GMA)] and poly(styrene/N-isopropylacrylamide/methacrylic acid) [P(St/NIPAM/MAA)] were prepared by emulsifier-free emulsion polymerization. These latex particles with submicrometer size showed the thermosensitivity originated from the thermo-sensitive nature of NIPAM. That is, the minimum NaCI concentration for flocculation of these latex particles [critical flocculation concentration (CFC)] decreased significantly with increasing temperature and reached constant values at above the critical temperature [critical flocculation temperature (CFT)]. At a certain NaCl concentration, the thermo-sensitive latex particles were flocculated by raising temperature, and conversely, the flocculated thermo-sensitive latex particles were completely dispersed by lowering temperature. Bovine serum albumin (BSA) was covalently immobilized onto the P(St/NIPAM/GMA) and P(St/NIPAM/MMA) latex particles with high efficiency. The BSA-immobilized P(St/NIPAM/GMA) and P(St/NIPAM/MAA) latex particles (immunomicrospheres) showed the similar dependencies of CFC on temperature to the bare latex particles. These thermo-sensitive immunomicrospheres were successfully used for the immunoaffinity purification of anti-BSA antibodies from antiserum.

Development and application of thermo-sensitive magnetic immunomicrospheres for antibody purification.

Ultrafine magnetite particles were prepared by a co-precipitation method. The poly-(styrene/N- isopropylacrylamide/methacrylic acid) latex particles containing ultrafine magnetite [magnetic P(St/NIPAM/MAA)] were prepared by two-step emulsifier-free emulsion polymerization. The minimum NaCl concentration for flocculation of these magnetic latex particles (critical flocculation concentration, CFC) decreased with increasing temperature. These temperature dependence of CFC, namely its thermo-sensitivity, originated from NIPAM. At a certain NaCl concentration, some of the magnetic latex particles showed reversible transition between flocculation and dispersion by controlling the temperature, and the thermo-flocculated magnetic latex particles were separated quickly in a magnetic field. Bovine serum albumin (BSA) was covalently immobilized onto the magnetic P(St/NIPAM/MAA) latex particles with high efficiency by the carbodiimide method. These thermo-sensitive magnetic immunomicrospheres were effective for the immunoaffinity purification of anti-BSA antibodies from antiserum.

Purification of fusion proteins using affinity microspheres in aqueous two-phase systems.

Affinity microspheres were prepared by immobilizing human gamma-globulin (H gamma Gb) onto carboxylated poly (styrene/acrylamide) latex particles [P(St/AAm)-H; average diameter 0.33 microns], which were prepared by emulsifier-free emulsion polymerization. H gamma GB was covalently immobilized onto the latex particles with high efficiency by the carbodiimide method. A fusion protein (ZZB1B2) of immunoglobulin G and albumin-binding domains (ZZ and B1B2, respectively) was expressed intracellularly and extracellularly in Escherichia coli and was purified by the affinity microspheres. In poly (ethylene glycol) (PEG)/potassium phosphate aqueous two-phase system, the affinity microspheres were partitioned into the PEG-rich top phase, while cells and cell debris of E. coli were displaced into the salt-rich bottom phase. Therefore, ZZB1B2 was directly purified from cell disintegrate or culture broth by combining the affinity microspheres with the aqueous two-phase partitioning, and its purity was almost the same as that purified by conventional affinity chromatography. Therefore, by this purification method, the primary purification process and the subsequent high resolution purification process are combined, and the number of purification steps can be reduced.

Circular dichroism studies on conformational changes in protein molecules upon adsorption on ultrafine polystyrene particles.

The conformational changes in well-characterized model proteins [bovine ribonuclease A (RNase A), horseradish peroxidase, sperm-whole myoglobin, human hemoglobin, and bovine serum albumin (BSA)] upon adsorption on ultrafine polystyrene (PS) particles have been studied using circular dichroism (CD) spectroscopy. These proteins were chosen with special attention to molecular flexibility. The ultrafine PS particles were negatively charged and have average diameters of 20 or 30 nm. Utilization of these ultrafine PS particles makes it possible to apply the CD technique to determine the secondary structure of proteins adsorbed on the PS surface. Effects of protein properties and adsorption conditions on the extent of the changes in the secondary structure of protein molecules upon adsorption on ultrafine PS particles were studied. The CD spectrum changes upon adsorption were significant in the "soft" protein molecules (myoglobin, hemoglobin, and BSA), while they were insignificant in the "rigid" proteins (RNase A and peroxidase). The soft proteins sustained a marked decrease in alpha-helix content upon adsorption. Moreover, the native alpha-helix content, which is given as the percentage of the alpha-helix content in the free proteins, of adsorbed BSA was found to decrease with decreasing pH and increase with increasing adsorbed amount. These observations confirm some well-known hypotheses for the confirmational charges in protein molecules upon adsorption.

Kinetic and circular dichroism studies of enzymes adsorbed on ultrafine silica particles.

Negatively charged ultrafine silica particles (average diameter 20 nm) were used as support materials for adsorption immobilization of porcine trypsin, horseradish peroxidase, and bovine catalase under various conditions, and the changes in the enzyme activities and the circular dichroism (CD) spectra of these enzymes upon adsorption were measured. Since the light scattering intensity of the ultrafine particles was very low, the activities and the CD spectra of the enzymes adsorbed on the particle surfaces could be measured. The enzymes adsorbed at pH around and above their isoelectric points (pI) showed high activities. On the other hand, the enzymes adsorbed at pHs below their pI had significantly diminished activities and showed large CD spectral changes upon adsorption. The extent of CD spectral changes in the enzymes upon adsorption correlated very closely with that of the activity reduction. Therefore, the conformational changes in enzymes upon adsorption are one of the important factors that reduce the activities of adsorbed enzymes. These results demonstrate that the ultrafine particles are not only a novel support for enzyme immobilization but also are helpful for the molecular understanding of the immobilized enzymes.

Two-Dimensional Simulation of the Breakup Process of Aggregates in Shear and Elongational Flows.

A modified discrete element method in which the hydrodynamic contribution is taken into account is proposed to simulate the deformation and breakup process of coagulated particles in two-dimensional shear and elongational flows. The simulation was performed for aggregates of various sizes, constitutive particles and fractal dimensions, and the followings were found: (i) the average number of particles in broken fragments is related with the intensity of flow field Gamma by ~ Gamma-P, where the value of P for aggregates of fractal dimension 1.8 is about 0.86 in the shear flow and about 1.0 in the elongational flow, (ii) aggregates are fragmented in the same fashion if their fractal dimension is the same, and a scaling law for fragmentation will hold if their fractal dimension, particle number and ratio of the minimum gap between neighboring particles to the particle size are the same among aggregates, (iii) aggregates in flow fields are broken by splitting into the smaller fragments but not by eroding particles one by one from their surface, and (iv) the elongational flow is more effective to break up aggregates than the shear flow under usual flow conditions. Copyright 1998 Academic Press.

Magnetic Effects on Thickness of Adsorbed Layer in Aqueous Solutions Evaluated Directly by Atomic Force Microscope.

The short-range interaction force between a mica surface and an AFM probe tip in electrolyte solutions was measured by atomic force microscopy (AFM) to directly evaluate the thickness of the adsorbed layer on the surface. The magnetic effects were estimated by comparing the thickness in the magnetized solution with that in nonmagnetized solution. It was found that (i) the magnetic exposure thickens the adsorbed layer on the surface in electrolyte solutions, (ii) the magnetic effects are easily destroyed by the external disturbance, (iii) there exists a memory in the magnetic effects, which remains for at least a day, (iv) the magnetic effects appear mostly in the solutions of structure-disordering cations, such as Cs+, Rb+, and K+, and (v) the results are consistent with those obtained in preceding macroscopic experiments. It is hypothesized from these results that the water molecules weekly bound around the structure-disordering ions are quasistabilized and structured by the magnetic exposure and that the magnetic thickening of the adsorbed layer is caused by the adsorption of those structured ions on the surface. Copyright 1998 Academic Press.

Evaluation of Interaction Forces between Macroparticles in Simple Fluids by Molecular Dynamics Simulation.

The present article provides the description of the solvation forces between large spheres in a fluid. The molecular dynamics (MD) method was applied to the relatively simple systems in which a pair of structureless macroparticles, either solvophobic or solvophilic, is immersed in a simple fluid of two types, either a soft-sphere or a Lennard-Jones fluid. When a pair of solvophobic macroparticles was in the attractive Lennard-Jones fluid, no dense layer of the solvent particles formed near the surface of the macroparticles and the strong attractive forces were induced between them. In the other combinations of macroparticles and fluids, the dense layers formed and the solvation forces oscillated, exhibiting the attraction and repulsion, whose periodic distance was about the diameter of solvent particles. Our results agreed well with those of the other simulation and theoretical studies with respect to the solvent density profile near a macroparticle and the force-distance profile between macroparticles. The benefit of our approach would be the simplicity in specifying or finding the bulk condition that is in equilibrium with the thin film of molecules between large surfaces. The present method can be applied straightforward to macroparticles immersed in mixtures and complex fluids described by the bead-spring model, to which the conventional grand canonical ensemble Monte Carlo (GCEMC) method is hardly accessible. Copyright 1999 Academic Press.

Magnetic Effects on Electrolyte Solutions in Pulse and Alternating Fields.

The contribution of the pulse and alternating magnetic fields on the magnetic effects is examined and compared with that of the static field, using a rotational device by which the pulse and alternating fields are able to be applied to the stationary sample. The followings are found: (i) the substantial time required to reach the maximum magnetic effect in the pulse and alternating fields is much smaller than the time in the static field, (ii) the magnetic effect does depend on the frequency of magnetic field, and (iii) the pulse and alternating magnetic fields make the quasi-stable structure more stable than the static field. The results are discussed and compared with the magnetic effects in flow loops reported elsewhere. Copyright 1999 Academic Press.

Adhesive Force between Hydrophilic Surfaces in Alcohol-Water Solutions.

The interaction and adhesive forces between a mica plate and SiO(2) surfaces in water-alcohol (ethanol, 1-propanol, 1-butanol, 1-pentanol, 1-hexanol) mixtures were investigated on the molecular scale, using an atomic force microscope. The following results were obtained: (i) alcohols higher than 1-propanol adsorb standing on the hydrophilic surface vertically to form a structured monolayer, when the alcohol concentration is high enough; (ii) the adhesive force between surfaces depends on how closely two surfaces can be brought by breaking the adsorbed layers; (iii) the adhesive force between surfaces is maximized at w(w)/w(ws) approximately 0.25, independent of the kind of alcohol, where w(w) and w(ws) are the weight fractions of water and saturated water, respectively; and (iv) the adhesive force for a particle of rough surface is much smaller than the adhesive force predicted for a particle of smooth surface. It is hypothesized that this adhesive force much greater than the van der Waals attractive force originates from the water bridging between surfaces, and this hypothesis is confirmed by the predictions given by the Laplace equation and the Kelvin relation. Copyright 1999 Academic Press.

Adhesion between Silica Particle and Mica Surfaces in Water and Electrolyte Solutions.

An atomic force microscope (AFM) is used to study the adhesion between a silica sphere and a mica plate in pure water and solutions of monovalent cations (LiCl, NaCl, KCl, and CsCl). It is found that the adhesive force depends not only on the electrolyte concentration but also on the hydration enthalpy of cations and the contact time of the particle on the surface. Possible mechanisms by which the observed phenomena can be explained consistently are discussed extensively. It is suggested that the adhesive force is closely related to the structure of the layer of cations and water molecules adsorbed on the surfaces: the strong adhesive force is obtained when highly hydrated cations (Li(+), Na(+)) are adsorbed to form a thick but weakly adsorbed layer, while the weak adhesive force is observed when poorly hydrated cations (Cs(+), K(+)) are adsorbed to form a thin but strongly adsorbed layer. Copyright 2000 Academic Press.

Complete transection of the pancreas due to a single stamping injury: a case report.

Complete transection of the pancreas due to a single stamping injury is reported. A 47-year-old female was stamped on in the epigastric region by her common-law husband when lying on her back. Immediately after the stamping, she complained of severe abdominal pain. In an emergency hospital, the abdominal CT (computed tomography) scan showed a large haematoma around the pancreas head. In the surgical operation that followed, a complete transection of the pancreas between the head and body was unexpectedly discovered. Furthermore, severe traumatic changes were found in the liver, mesentery, splenic artery and vein. The medico-legal autopsy revealed marked haemorrhaging in the abdominal subcutaneous adipose tissue and rectus muscle in the epigastric region. The external blunt force that acted on the abdomen had forcibly compressed the pancreas against the spinal column. Blunt pancreatic injury is relatively uncommon, but pancreatic injuries should always be considered in patients or autopsy cases after external blunt force to the abdomen.

Magnetic Effect on Ion-Exchange Kinetics.

Effects of magnetic exposure on an ion-exchange process are investigated, employing ion-exchange resins. The electrolyte solution, resin suspension, and water are mixed in a container, and the mass transfer coefficient is determined by measuring the change of the ionic concentration in the bulk. Two methods of magnetic exposure are performed: case (1) in which only the electrolyte solution is exposed to the magnetic field and case (2) in which only the resin suspension is exposed to the magnetic field. The following features are found in both cases: (i) the film mass transfer rate reduces by about 5% when the exposure time is greater than 25 min, (ii) the magnetic effect is reduced by adding the alcohol, (iii) the magnetic effect remains for about 3 days but disappears after 6 days, and (iv) the magnetic effect is observable in the solutions of structure-disordering ions, but not in the solutions of structure-ordering ions. As for the temperature dependence, the magnetic effect decreases with temperature in case (1) but it stays constant in case (2). It is postulated that the magnetic effects are attributable to the stabilization of water molecules around the structure-ordering ion and those on the resin surface. Copyright 1999 Academic Press.

Effects of Hydrophobizing Methods of Surfaces on the Interaction in Aqueous Solutions.

The hydrophobic interaction between the surfaces hydrophobized by the adsorption of STAC in a STAC solution (system I) and the interaction between the surfaces hydrophobized by the reaction with OTS in water (system II) were investigated using an atomic force microscope, and their difference was compared. Clear differences of the interaction between systems I and II were found with respect to the force curves and the morphology of the surfactants adsorbed on the surfaces. It is postulated that the hydrophobic interaction for system II is attributable to the cavity bridging between the surfaces, but that for system I it is originated from a different mechanism. It is also implied that the existence of the surfactants in the bulk will play an important role for the hydrophobic interaction. Copyright 1999 Academic Press.