Erythrocyte sedimentation rate. I. Volume fraction dependence in saline solution

We have measured volume fraction dependence of the sedimentation curve of swine erythrocytes in a physiological saline solution at 10 degrees C, 20 degrees C, 30 degrees C and 40 degrees C. The sedimentation curves were found to consist of initial constant velocity region and final plateau region at the lower temperatures of 10 degrees C and 20 degrees C, while modified S-shaped curves were observed at the higher temperatures of 30 degrees C and 40 degrees C. The volume fraction dependence of the initial slope v of the sedimentation curve was fitted well to the following exponential type equation at all the temperatures: v = vs,exp (1 - H)exp[-(BH + CH2)] where vs,exp is the velocity in infinite dilution corresponding to the Stokes velocity and H is the volume fraction of erythrocytes. The volume fraction dependence of the relative velocity v/vs,exp was in close agreement with a semi-empirical equation derived for slurrys in the field of chemical engineering at the lower temperatures, while a small deviation between the observed and calculated curves was found at the higher temperatures. The volume fraction dependence of v at 20 degrees C was also analyzed on a theory recently developed by Oka. The explicit functional form of the medium up-flow factor phi (H) and the deformability factor f in the theory were determined using the experimental data.     

Extracorporeal immunosuppressive effect of papain]

Exposure of allogenic erythrocytes to papain induced their immunosuppressing properties within relatively narrow ranges of the incubation medium temperature (42 but not 37 or 40 degrees C) and the papain concentration (10 but not 2 or 50 micrograms/ml). Markedly pronounced immunosuppressing properties were acquired by the erythrocyte light fraction after heating and exposure to papain. The supernatant layer of adhesive spleen cells incubated in the presence of erythrocytes heat treated and exposed to papain suppressed development of the humoral immune response and DTH during the allogenic transfer and accelerated and increased excretion of the antigen specific immunosuppressing factor by the nonadhesive spleen cells of hyperimmunized sheep red blood cells.     

The titration of tetanus antitoxin I. Factors affecting the sensitivity of the indirect haemagglutination test

Various factors affecting the indirect HA test for the titration of tetanus antitoxin have been evaluated with a view to obtaining maximum sensitivity in tests using unfixed sheep erythrocytes and sheep erythrocytes fixed with glutaraldehyde, formaldehyde and pyruvic aldehyde. The optimal concentration of tannic acid has been found to be 1/40 000 for tanning both fixed and unfixed sheep erythrocytes. Tanned sheep erythrocytes sensitized with 50 Lf/ml of tetanus toxoid at pH 7.2 for one hour were the most sensitive. Although the optimal temperature of sensitization was found to be 56 degrees C, unfixed cells tended to clump and lyse at this temperature. Thus a temperature of 37 degrees C was used to sensitize unfixed sheep erythrocytes. Sheep erythrocytes from different animals and the final concentration of sensitized sheep erythrocytes both had great effects on sensitivity. A final concentration of 0.5% of sensitized sheep erythrocytes was found suitable as a compromise between sensitivity and readability. The loss of sensitivity of fixed and sensitized erythrocytes was investigated by storing these cells at 4-8 degrees C for six to nine months.     

Dielectric spectroscopy on aqueous electrolytic solutions

In this work, detailed dielectric measurements are presented on aqueous electrolytic solutions of NaCl and KCl in a broad frequency range, typical for modern telecommunication techniques. The complex dielectric permittivity or equivalently the complex conductivity are systematically studied as function of frequency (100 MHz-40 GHz), temperature (10-60 degrees C) and molar concentration (0.001-1 mol/l). By a detailed analysis of the dielectric results using an asymmetrically broadened Cole-Davidson distribution of relaxation times, in addition to dc conductivity, the dielectric response as function of frequency, temperature, and molar concentration was fully parameterized by a total of 13 parameters. This model ansatz and the 13 parameters include an enormous predictive power, allowing a reasonable estimation of the dielectric constant, loss, and the conductivity for any set of external variables frequency, temperature and concentration. The proposed method is not only useful for rather simple electrolytic solutions, but also for cell suspensions and biological matter, if additional processes, especially at low frequencies, are adequately taken into account.     

Dielectric and electric properties of synthetic melanin: the effect of europium ions

Detailed studies on dielectric and electric properties of synthetic pirocatechol and indolederived melanin, pure and doped with Eu³⁺, have been performed, D.C. and a.c. electrical conductivity as well as dielectric permittivity and loss angle tg have been investigated. Activation energy of d.c. conductivity for the investigated temperature range (0°C3+ doped to the samples do not influence the values of activation energy, but the addition of Eu³⁺ ions decreases the conductivity values. On the basis of depolarization current curves the energy of trap level referred to Eu³⁺ has been calculated. It equals 0.58 eV for pirocatechol and 0.60 eV for indolemelanin.     

Nucleotide profiles of normal human blood cells determined by high-performance liquid chromatography

An anion-exchange high-performance liquid chromatography method has been used to quantitate the intracellular purine and pyrimidine nucleotides in extracts of pure lymphocytes, monocytes, neutrophils, eosinophils, erythrocytes, and platelets isolated from the blood of healthy human donors. For accurate and reproducible measurements of the nucleotide profiles in different types of pure leukocytes, the cell suspensions have to be free of platelets and erythrocytes. Incubation of the purified leukocytes for 1 h at 0 degrees C did not alter the nucleotide concentrations but reduced the interdonor variation to 10%. Incubation of purified lymphocytes for 1 h at 37 degrees C caused considerable changes in the relative concentrations of the adenine, guanine, uracil, and cytosine nucleotides. During this incubation the cell viability, the cell number, and the ATP:ADP ratio decreased. Incubation of monocytes and granulocytes for 1 h at 37 degrees C caused considerable loss of cells and/or cell death. For erythrocytes and platelets reproducible nucleotide concentrations were obtained after extraction of freshly isolated cells. During storage of erythrocytes, both at 0 degrees C and at 37 degrees C, a decrease in the ATP:ADP ratio was detected. In all cell types the predominant nucleotides were purine nucleotides, especially adenosine triphosphate. The relative concentrations of the adenine, guanine, uracil, and cytosine nucleotides were very reproducible per cell type and appeared to be characteristic for each cell type. The total nucleotide content was nearly the same for all cell types except erythrocytes, when expressed per microgram of protein. The described methods for purification and storage of blood cells will be useful for comparison of blood cells from healthy donors with those of patients, for example, leukemia patients, in which deviations of the purine and pyrimidine metabolic enzymes have already been described.     

Factors affecting the ability of isolated Plasmodium knowlesi merozoites to attach to and invade erythrocytes

Plasmodium knowlesi merozoites were prepared by the polycarbonate sieving method of Dennis, Mitchell, Butcher & Cohen (1975). Merozoite function was assayed by their attachment to and invasion of rhesus erythrocytes at 37 degrees C. The early merozoites from the culture chamber were the most invasive, although maximum numbers of merozoites appeared later. Merozoites were most stable when incubated at room temperature (23 degrees C). At 37 and 0 degrees C invasiveness rapidly declined to zero. Attachment was rapidly lost at 37 degrees C but was retained at 0 degrees C. Attachment was unchanged in the pH range 6.8--7.9 but invasion was reduced at pH 7.9. The presence of L-fucose, D-galactose, D-glucose, D-mannose, N-acetyl-D-galactosamine or N-acetyl-D-glucosamine did not reduce invasion. Attachment and invasion were greatly reduced or abolished by the presence of 2.5 mM EDTA or EGTA, by lactoperoxidase-catalysed iodination of the merozoites, or by treatment of the merozoites with trypsin at a concentration of 1 micrograms/ml or greater for 10 min at 23 degrees C.     

Influence of the serum of rats subjected to the effects of moderately high temperatures on the immunogenicity of sheep erythrocytes]

A study was made of the properties of the blood of rats subjected to the action of a moderately high external temperature (40 degrees C); it was shown that the immunogenicity of sheep erythrocytes increased as a result of treatment with the serum at 37 degrees C. Preliminary incubation of the sera with a polyvalent proteinase inhibitor -- trasilol eliminated this effect completely. Immunogenicity of sheep erythrocytes did not increase with the treatment by the serum at a temperature of 2 degrees C. It can be supposed that the action of the serum of the heated rats was caused by an increase in them of the proteinase activity.     

Deformability of stored erythrocytes as a criteria for their in vivo survival rate

The loss of deformability observed in erythrocytes stored as whole blood for 36 days (ACD-AG) or as buffy-coat free erythrocyte concentrate (EK) was characterized by measuring their filterability. During the first 3 weeks the index of filterability for ACD-AG erythrocytes increased only slightly and rose to about 140% of its initial value on the 36th day. In contrast, a heavy loss of deformability (increase of the filterability index to more than 600%) was detected for erythrocytes from EK, which, from a rheological point of view, is apt to raise doubts of using this stored blood. An incubation of 1 hour at 37 degrees C in fresh plasma did not result in improving the deformability. A cell volume loss of more than 20% connected with an increase of the inner viscosity to more than 400% was found to be the cause of this decrease of deformability. These rheological differences are also reflected in the 24 hours in vivo survival rate (SR), if the "early loss" of damaged erythrocytes immediately after transfusion is taken into account. Whereas the SR values of 80% for whole blood erythrocytes do not change significantly, the SR values for EK values can be found to reach 54% approximately.     

Enhanced susceptibility to suicidal death of erythrocytes from transgenic mice overexpressing erythropoietin

Eryptosis, a suicidal death of mature erythrocytes, is characterized by decrease of cell volume, cell membrane blebbing, and breakdown of cell membrane asymmetry with phosphatidylserine exposure at the cell surface. Triggers of eryptosis include increased cytosolic Ca(2+) activity, which could result from activation of Ca(2+)-permeable cation channels. Ca(2+) triggers phosphatidylserine exposure and activates Ca(2+)-sensitive K(+) channels, leading to cellular K(+) loss and cell shrinkage. The cation channels and thus eryptosis are stimulated by Cl(-) removal and inhibited by erythropoietin. The present experiments explored eryptosis in transgenic mice overexpressing erythropoietin (tg6). Erythrocytes were drawn from tg6 mice and their wild-type littermates (WT). Phosphatidylserine exposure was estimated from annexin binding and cell volume from forward scatter in fluorescence-activated cell sorting (FACS) analysis. The percentage of annexin binding was significantly larger and forward scatter significantly smaller in tg6 than in WT erythrocytes. Transgenic erythrocytes were significantly more resistant to osmotic lysis than WT erythrocytes. Cl(-) removal and exposure to the Ca(2+) ionophore ionomycin (1 microM) increased annexin binding and decreased forward scatter, effects larger in tg6 than in WT erythrocytes. The K(+) ionophore valinomycin (10 nM) triggered eryptosis in both tg6 and WT erythrocytes and abrogated differences between genotypes. An increase of extracellular K(+) concentration to 125 mM blunted the difference between tg6 and WT erythrocytes. Fluo-3 fluorescence reflecting cytosolic Ca(2+) activity was larger in tg6 than in WT erythrocytes. In conclusion, circulating erythrocytes from tg6 mice are sensitized to triggers of eryptosis but more resistant to osmotic lysis, properties at least partially due to enhanced Ca(2+) entry and increased K(+) channel activity.     

Microwave dielectric relaxation in muscle. A second look.

The dielectric permittivity and conductivity of muscle fibers from the giant barnacle, Balanus nubilus, have been measured at 1, 25, and 37 degrees C, between 10 MHz and 17 GHz. The dominant microwave dielectric relaxation process in these fibers is due to dipolar relaxation of the tissue water, which shows a characteristic relaxation frequency equal to that of pure water, ranging from 9 GHz (1 degree C) to 25 GHz (37 degree C). The total permittivity decrease, epsilon 0 -- epsilon infinity, due to this process accounts for approximately 95% of the water content of the tissue; thus, the major fraction of tissue water is dielectrically identical to the pure fluid on a picosecond time scale. A second dielectric process contributes significantly to the tissue dielectric properties between 0.1 and 1--5 GHz, and arises in part form Maxwell-Wagner effects due to the electrolyte content of the tissue, and in part from dielectric relaxation of the tissue proteins themselves.     

Loss of heat-shock acquisition of thermotolerance in yeast is not correlated with loss of heat-shock proteins

Yeast cells when subjected to a primary heat shock, defined as a temperature shift from 23 to 37 degrees C for 30 min, acquired tolerance to heat stress (52 degrees C/5 min). Primary heat shocked cells incubated at 23 degrees C for up to 3 h, progressively lost thermotolerance but retained high levels of the major heat-shock proteins as observed on polyacrylamide gels. On the other hand, a temperature shift back up to 37 degrees C for 30 min fully restored thermotolerance. The major high-molecular-mass heat-shock proteins (hsp) identified were of approximate molecular mass 100 kDa (hsp 100), 80 kDa (hsp 80) and 70 kDa (hsp 70). The results indicate that loss of heat-shock acquisition of thermotolerance is not correlated with loss of heat-shock proteins.     

Dynamic BTPS correction factors for spirometric data

Because it is often difficult to completely control ambient temperature, a study was conducted to investigate dynamic body temperature pressure saturated (BTPS) correction factors for spirometric data. A forced expiratory simulator system was heated to 37 degrees C and loaded with air saturated with water vapor. This air was then forced from the simulator into a dry rolling-seal spirometer maintained at various ambient temperatures from 3 to 32 degrees C. Errors in forced expiratory volume in 1 s (FEV1) and peak flow from assuming a constant BTPS correction ranged from 7.7 and 14.1% at 3 degrees C to 2.1 and 4.6% at 23 degrees C. Differences between errors observed when saturated and dry air were forced into the spirometer indicate that water vapor condensation introduces an added heat load to the spirometer, adding approximately one percent to the error in FEV1 at lower temperatures. By use of a model to estimate the dynamic BTPS correction factor, errors in FEV1 at all temperatures between 3 and 32 degrees C were reduced to less than 1.5%.     

Mechanism of the effect of sera from rats subjected to high external temperature on rosette formation by splenic lymphocytes from non-immune animals]

Serum of rats subjected to the action of moderately high external temperature increased the immunogenicity of heterologous erythrocytes, but became inactive at a temperature of 2 degrees C and after the treatment with a polyvalent proteinase inhibitor--trasilol. Incubation of sheep erythrocytes with the serum of heated rats intensified the reaction of rosette formation by the lymphocytes of the spleen of intact rats. Incubation of the spleen cells with the sera of intact and heated rats failed to influence the rosette formation. After trasilol treatment the serum of heated rats completely lost the property to intensify the reaction of rosette formation. Phenomenon of intensification of rosette formation was revealed in case of treatment of erythrocytes with the sera at a temperature of 37 degrees C and was not reproduced at a temperature of 2 degrees C. Investigations carried out indicated that the increase in the immunogenicity of sheep erythrocytes and the intensification of their binding with the lymphocyte receptors of intact rats under the effect of the serum of heated animals were caused by the action of the same factor.     

Monitoring of temperature effects on animal cell metabolism in a packed bed process

Animal cell (Chinese Hamster Ovary) concentration was determined on-line in a packed bed process using dielectric spectroscopy. This enabled the evaluation of the effect of temperature on specific metabolic rates during 3 months of continuous culture. The effect of low cultivation temperature on cell growth and metabolism was monitored, and the data were used for process development. At 37 degrees C cells grew exponentially with a specific growth rate of 0.038 d-1 and specific glucose uptake and lactate production rates increased continually. Reduction of the temperature to 33.5 degrees C resulted in a lowering of these metabolic rates while having no effect on cell proliferation. Subsequent reduction of the temperature to 32 degrees C resulted in stabilization of the cell concentration at a high density (3.6 x 10(7) cell per mL of packed bed). In addition, the specific production rate of the protein of interest increased by a factor of 6 compared to the value at 37 degrees C. During the stationary phase at 32 degrees C, all other specific metabolic rates could be controlled to low and constant levels.     

Effect of Temperature on the Morphometrical and Physical Parameters of Erythrocytes and Polymorphonuclear Leucocytes in <Emphasis Type="Italic">Carassius gibelio</Emphasis> (Bloch)

Dynamics of the morphometric and physical properties of hemocytes of the Prussian carp Carassius gibelio (Bloch) under the influence of a temperature factor has been studied with atomic force microscopy in experiments in vitro. It is found that, at a low incubation temperature (5°C), as opposed to room temperature (20°C), morphometric parameters change in erythrocytes; at a high temperature (40°C) they change in polymorphonuclear leucocytes. The low incubation temperature reduces the adhesion and elasticity of polymorphonuclear leucocytes and erythrocytes of C. gibelio, whereas a high incubation temperature leads to a decrease in adhesion in polymorphonuclear white blood cells.     

The effect of chlorpromazine on the temperature and osmotic sensitivity of erythrocytes]

The effect of chlorpromazine on the development of cold shock in erythrocytes exposed to sodium chloride was shown to depend on the tonicity of the medium in which the cells were cooled from 37 degrees C down to 0 degrees C as well as on the amphipate concentration. After cooling of erythrocytes in a NaCl (0.75-1.5 M)-containing medium with chlorpromazine (7 x 10(-5) M, 2.1 x 10(-4) M and 3.5 x 10(-4) M) the hypertonic cold shock was inhibited, the protective effect of the amphipate being less pronounced at its increasing concentrations. After cooling of cells under conditions of moderate hypertonicity (0.3-0.6 M NaCl) no modifying effect of chlorpromazine on the sensitivity of erythrocytes to the temperature decrease from 37 degrees C down to 0 degrees C was manifested. However, under iso- and hypertonic conditions chlorpromazine used at 2.1 x 10(-4) M and 3.5 x 10(-4) M stimulated the cold shock development in erythrocytes. A sharp increase in the medium tonicity (up to 1.8-3.0 M and higher) the cells underwent isothermal hemolysis which was more expressed at 0 degrees C than at 37 degrees C. These data suggest that chlorpromazine significantly activates the hemolytic process at low temperatures.     

Koi herpesvirus disease

Koi herpesvirus (KHV) disease emerged at the late 1990s, and has rapidly spread to the world. In Japan, KHV disease first occurred at October 2003. The disease resulted in mass mortality of wild carp as well as cultured carp. Until now, KHV-infected carp were found in 42 out of 47 prefectures in Japan. Only carp Cyprinus carpio is susceptible to KHV, while goldfish, closely-related species to carp, is not. The affected carp swim lethargically. Sunken eyes and gill necrosis are frequently noticed, but no marked internal signs are observed. Optimal water temperature for the disease is 18-23 degrees C. Under 13 degrees C or over 28 degrees C, no death occurs. Keep at over 30 degrees C cures KHV disease, but can make the fish latent carriers. Because the fish do not get acquired immunity against KHV disease under low water temperature, the disease recurs with increase of water temperature. Isolation of KHV is difficult. KHV disease is diagnosed through epidemiological investigation, disease signs and PCR detection of KHV DNA. Vaccine development is ongoing for restart of culturing carp at KHV-contaminated places.     

Permeability of human blood platelets to glycerol

The permeability of human platelets to glycerol was determined at 37 degrees C, 25 degrees C, and 0 degrees C from the rate of change of cell volume after abrupt addition of 0.5 mol/liter glycerol in phosphate-buffered saline. Intracellular water volume was measured employing both tritiated water and a photometric method. Intracellular glycerol was measured employing tritiated glycerol. The glycerol permeability coefficient derived from the tracer cell volume data was 4.0 +/- 0.7 X 10(-7) cm/s at 37 degrees C, and 1.1 +/- 0.4 X 10(-7) cm/s at 25 degrees C, and the photometric data gave a permeability coefficient of 5.4 +/- 0.4 X 10(-7) cm/s at 37 degrees C. The activation energy between 23 degrees C and 37 degrees C for glycerol permeation was 19.8 kcal/mol. The cells were virtually impermeable to glycerol at 0 degrees C. The minimum intracellular water volume attained after the addition of 0.5 mol/liter glycerol at 37 degrees C determined by the photometric method was 47.8% of normal water volume, whereas the minimum water volume calculated assuming that glycerol exerted its full osmotic effect (i.e., sigma = 1) was 45.6%. The reflexion coefficient was therefore assumed to be unity. Neither method of cell volume determination could be used with 1 or 2 mol/liter glycerol: adequate separation of the cells from the labeled medium could not be achieved in the tracer method; in the photometric method, it was apparent that transmittance (660 nm) was influenced by one or more variables in addition to cell volume.     

Temperature-dependent specificity of cis-trans isomeric fatty acid interaction with the erythrocyte membrane

Stabilization of red cells against hypotonic haemolysis by cis-trans isomeric free C18 fatty acids occurs with pronounced specificity which is strongly temperature-dependent, but in a distinctly different manner for the two configurational isomers. Oleic acid (cis-18:1) stabilizes very efficiently at 0 degrees C, even at the highest concentrations. Elaidic acid (trans-18:1) causes neither stabilization nor haemolysis at this temperature. At room temperature (23 degrees C), elaidic acid acquires the ability to protect, without turning haemolytic at high concentrations. At 37 degrees C elaidic acid also becomes haemolytic. The protecting effect of oleic acid at 0 degrees C is the result of a rapid reaction. The characteristic, temperature-dependent specificity of cis-trans isomeric C18 fatty acid interaction with the red cell membrane appears to be a general phenomenon, since it was observed alike with erythrocytes of different species.