Porcine Red Blood Cells Research Articles

Identification of non-αGal xenoantigens on porcine red blood cells

Identification of non-αGal xenoantigens on porcine red blood cells

Effects of storage conditions and hemolysis on vitamin E concentrations in porcine serum and liver.

Vitamin E (alpha-tocopherol) is an antioxidant vitamin important in protecting unsaturated fatty acids in lipid membranes from peroxidation. Variation in collection, storage, and shipping conditions of samples can potentially lead to breakdown of vitamin E prior to analysis. Therefore, the purposes of this project were 1) to determine the stability of vitamin E in refrigerated and frozen porcine liver and serum and 2) to evaluate the effects of red blood cell (RBC) hemolysis on porcine serum vitamin E concentrations. Porcine liver and nonhemolyzed serum were collected and stored refrigerated or frozen. Samples were analyzed for vitamin E immediately or on days 2, 3, 7, or 14. In addition, porcine RBCs were added to normal serum at concentrations from 1 x 10(6) to 1 X 10(9) RBC/ml and hemolyzed by freeze-thaw prior to analysis for vitamin E or products of lipid peroxidation.

Introduction to porcine red blood cells: Implications for xenotransfusion

Advances in the field of xenotransplantation raise the intriguing possibility of using porcine red blood cells ( pRBCs) as an alternative source for blood transfusion. The domestic pig is considered the most likely donor species for xenotransplantation. However, identification of xenoantigens on porcine erythrocytes and elucidation of their possible roles in antibody-mediated RBC destruction are necessary for developing clinical strategies to circumvent immunological incompatibility between humans and pigs. Although the αGal epitope (Galα1,3Galβ1,4GlcNAc-R) is the major xenoantigen on porcine erythrocytes and is responsible for the binding of the majority of human natural antibodies, other non-αGal xenoantigens have been identified. The importance of these non-αGal xenoantigens in binding human natural antibodies and subsequently triggering immunological responses cannot be underestimated. Our data suggest that non-αGal xenoantigen(s) identified on the porcine erythrocyte membrane are not only recognized by xenoreactive human natural antibodies but are also involved in complement-mediated hemolysis.

In situ measurements of Doppler power vs. flow turbulence intensity in red cell suspensions

Whereas previous studies have shown that ultrasonic backscatter and Doppler power from blood are affected by flow turbulence, turbulence level has only been inferred from the flow Reynolds number and not directly measured. In this study, both ultrasonic Doppler power and flow turbulence intensity were measured in situ to quantify the relationship between Doppler power and flow turbulence. Three grid meshes of different geometries were used in a steady-flow mock loop to generate controlled levels of flow turbulence in porcine red blood cell saline suspensions. Doppler power was measured by a 10-MHz PW Doppler flowmeter, and the turbulence intensity by using constant-temperature hot film anemometry. We showed that Doppler power is affected by turbulence and hematocrit in a complex way. At a fixed hematocrit, Doppler power increases nonlinearly with turbulence intensity and, at fixed turbulence intensity, Doppler power peaks at an optimal hematocrit level that increases with turbulence level. The shape factor, introduced by Lucas and Twersky (1987) to take into account effects of shape and orientation of the scatterers in a dense distribution of small and tenuous scatterers, was estimated by fitting the experimental data to the theoretical model. The results indicate that shape factor decreases with increasing turbulence intensity.

Variation in porcine red blood cell α-galactosyl expression and agglutination by human serum

Variation in porcine red blood cell α-galactosyl expression and agglutination by human serum

Functional properties of porcine blood globin decolourized by different methods

The purpose of this study was to compare the functional properties of freeze‐dried porcine blood globin isolates, which were obtained after decolourization with sodium carboxymethylcellulose (Na‐CMC), acid‐acetone (A‐A), hydrogen peroxide (H2O2), or alcalase (an endopeptidase). Porcine red blood cell concentrates (RBCCs) without decolourization were freeze‐dried as controls. The proximate composition, solubility, emulsifying activity index, emulsifying stability, foaming capability, foam stability and gel strength of the globin isolates were determined. The solubility of the globin isolates was affected by decolourization treatment and pH (within the range 2–9). The globin isolates obtained by A‐A decolourization were particularly affected by pH. The solubility of the alcalase‐treated globin isolate was the highest of all the globin isolates, followed by the Na‐CMC‐ and H2O2‐treated globin isolates. The emulsifying activity index of A‐A‐treated globin isolate was unaffected by pH adjustment. The emulsifying stability of all isolates was not significantly different over the pH range 2–9. The foaming capacity was significantly different among treatments and pH levels (P < 0.01). The foaming capacity at pH 2–3 was greater than at pH 4–9, with the exception of the alcalase‐treated globin isolates. A‐A‐treated globin isolates had poor foaming capacity at pH 4 and 5, and foams produced at pH 8 and 9 from alcalase‐treated isolates had poor stability. The 10% solution of RBCCs (control) showed the greatest gel strength, followed by A‐A‐treated, Na‐CMC‐treated and H2O2‐treated globin isolates. The alcalase‐treated globin isolate solutions did not form gels. The decolourization methods surveyed in this study were all at least partially successful in ameliorating the dark‐brownish colour of RBCCs. However, the functional properties of the isolates, excluding gel strength, were generally comparable with those of RBCCs.

Porcine natural-killer-enhancing factor-B: oligomerisation and identification as a calpain substrate in vitro

Natural-killer-enhancing factor-B (NKEF-B) (monomeric mass=21.82 kDa) was purified from the cytosol of porcine red blood cells and its identity was established by microsequencing. NKEF-B oligomerisation was investigated by gel filtration and small-angle X-ray scattering (SAXS). Native NKEF-B readily forms disulphide-linked dimers, but when fully reduced, the protein forms discrete oligomers containing 16±1 monomers. A total of 40% of the purified enzyme was deduced to be cysteinylated, which is consistent with the modification of one or both of two putative active site cysteine residues. In vitro, NKEF-B was found to be a specific substrate of μ- and m-calpains, the calcium-dependent cysteine proteases. The cleavage events were followed by SDS-PAGE and the cleavage sites pinpointed by N-terminally sequencing the resulting digestion fragments. This in vitro cleavage data provides support to the hypothesis that calpromotin (NKEF-B), an erythron peroxiredoxin involved in the regulation of calcium-dependent potassium transport across the plasma membrane, is cleaved by calpain in vivo.

Effect of shipping and chromium supplementation on performance, immune response, and disease resistance of steers.

Forty-eight Angus crossbred steers (263 +/- 2 kg initial BW) were blocked by weight and randomly assigned within weight group to treatment. Treatments consisted of control or .4 mg of supplemental Cr as Cr-nicotinic acid complex/kg of DM. Steers were fed diets containing 90% corn silage (DM basis) and 10% of a soybean meal-mineral-vitamin supplement. After 56 d on the dietary treatment, half of the steers in each treatment were transported 343 km and unloaded in an unfamiliar location. The next day, d 58, shipped steers were returned to the feedlot (50 km). On d 58 after shipped steers were returned to the feedlot, all steers were inoculated with infectious bovine rhinotracheitis virus (IBRV) intranasally. Average daily gain from d 0 to 80 was increased (P < .10) by supplemental Cr. There was a shipping x time interaction for serum cortisol concentrations. Shipping increased (P < .02) serum cortisol on d 58, but 7 d after transport there were no effects of shipping on serum cortisol. Transportation increased (P < .05) the ratio of neutrophils to lymphocytes. Supplemental Cr did not affect rectal temperature after the IBRV challenge or the antibody response to IBRV or porcine red blood cells. Immunoglobulin G antibody response to porcine red blood cells was decreased (P < .09) by shipping. Supplemental Cr as Cr-nicotinic acid improved ADG of growing steers, regardless of whether they had been stressed by shipping. Supplemental Cr did not affect any of the immune responses that were measured.

Immune Response and Disease Resistance of Calves Fed Chromium Nicotinic Acid Complex or Chromium Chloride

Twenty-one Holstein bull calves (<7 d of age at the initiation of the experiment) fed a milk replacer diet were used to assess the effects of supplemental Cr on immune response. Treatments consisted of milk replacer without supplemental Cr (control) or milk replacer with 0.4ppm of supplemental Cr from CrCl3 or a Cr-nicotinic acid complex. On d 64, increases in skinfold thickness after an intradermal injection of phytohemagglutinin were measured to evaluate cell-mediated immune response. Calves supplemented with Cr-nicotinic acid complex had a greater response than did controls at 6, 12, 24, and 48h after injection. Calves supplemented with CrCl3 had a greater response than did controls at 24 and 48h after injection. In vitro blastogenic responses of lymphocytes to phytohemagglutinin or pokeweed mitogen and antibody response to porcine red blood cells were not affected by treatment. Following a disease challenge with an intranasal dose of infectious bovine rhinotracheitis on d 75, body temperature tended to be lower for calves supplemented with Cr-nicotinic acid complex than for control calves. Calves supplemented with either Cr source had lower serum cortisol concentrations at 5 d after challenge. Chromium supplementation enhanced cell-mediated immune function.

Cell membrane permeability and mitochondrial dysfunction-inducing activities in cell-free supernatants from Serpulina hyodysenteriae serotypes 1 and 2

Membrane permeability (MP) and mitochondrial dysfunction-inducing (MDI) activities were detected in cell-free supernatants (CFS) of Serpulina hyodysenteriae, using either hemoglobin release from porcine red blood cells (RBC) or cytoplasmic lactate dehydrogenase release from porcine peripheral blood lymphocytes (PBL), and reduction of the 3-(4,5-dimethylthiazoyl-2-yl)-2,5-diphenyltetrazolium bromide dye by porcine PBL. The MP and MDI activities of CFS correlated with each other for serotype 1 and 2 isolates taken at different population densities; however, the kinetics of toxin production varied between each serotype. The loss of enteropathogenicity of two field isolates with nonpathogenic phenotypes and pathogenic isolates passaged up to 45 times in vitro was not attributable to a loss of either membrane permeability or mitochondrial dysfunction-inducing activity of cell-free supernatants. Results from this study suggested the potential for two separate toxins being involved in the pathogenesis of swine dysentery, with the MDI activity correlating with age susceptibility to clinical disease.

Effects of Various Viscosity Enhancers and pH on Separating Haem from Porcine Red Blood Cells

Haem derived from animal blood is a valuable source of organic iron which may be used as a supplement in foods. The purpose of this study was to examine the effects of sodium carboxymethyl cellulose (NaCMC), propylene glycol alginate (PGA) and sodium alginate (SA) as viscosity enhancers on the separation of haem from porcine red blood cells, and on the distribution of iron between the haem and non-haem fractions of the extracts. Iron determinations were carried out spectrophotometrically on the supernatants obtained by centrifuging the cell suspensions and on two successive washings of the cells. In the first washings the total extracted iron content using NaCMC was highest when a high-viscosity grade was used ; there were no significant differences (P > 0.05) between the results obtained with this material and those obtained using PGA or SA. There were also no significant differences (P > 0.05) among the three treatments in terms of non-haem content. The absorbances at 540 nm of the first washings were lower when the washing was carried out at pH 1.4 rather than at pH 1.0 or 2.0. There were no significant differences associated with changes in the viscosity enhancer in the absorbances of the second washings, but the absorbances at pH 1.4 were generally higher than those at pH 1.0 or 2.0. Enhancement of the absorbances on the addition of 1,10-o-phenanthroline indicated that the extracted iron was present in the ferrous form.

Effects of Various Viscosity Enhancers and pH on Separating Haem from Porcine Red Blood Cells

Effects of Various Viscosity Enhancers and pH on Separating Haem from Porcine Red Blood Cells

Effects of Various Viscosity Enhancers and pH on Separating Haem from Porcine Red Blood Cells

Effects of Various Viscosity Enhancers and pH on Separating Haem from Porcine Red Blood Cells

CHARACTERIZATION OF HUMAN IgG-BINDING XENOANTIGENS EXPRESSED BY PORCINE AORTIC ENDOTHELIAL CELLS

Xenoreactive antibodies (XAb) play a major role in the rejection of xenografts. In this study, human IgG XAb that bind to xenoantigens expressed by porcine aortic endothelial cells (PAEC) were characterized, together with their corresponding xenoantigens. Using an ELISA with both fixed and unfixed confluent monolayers of PAEC, XAb of both IgG and IgM classes in pooled and individual normal human serum were identified. The binding of these IgG XAb to the endothelium is mediated by F(ab')2 and the only detectable subclasses that bind to the endothelium are IgG1 and IgG2. On the basis of direct binding experiments, inhibition and antibody adsorption studies, and enzymatic digestions, it is shown that only a minor component of the XAb binding is directed against galactose in an alpha 1,3 linkage with galactose on PAEC surfaces. There is some cross-reactivity with antigens expressed on porcine lymphocytes, but not porcine red blood cells. Histological examination of sections of porcine aortae, snap-frozen and stained using immunoperoxidase techniques, confirmed interaction with the vascular endothelium. Labeling of the PAEC with 125I, followed by cell lysis and immunoprecipitation under reducing conditions, showed binding of IgG XAb to several components on the endothelial cell surface, the most prominent of which have apparent molecular masses of 75 kDa, 110 kDa, 180 kDa, and 210 kDa. The 110-kDa component and the 180-kDa component were sensitive to digestion with endoglycosidase F, which suggests the participation of N-linked carbohydrate structures. These studies demonstrate that human IgG XAb recognize multiple determinants expressed by PAEC, a minor population of which contain alpha 1,3-linked galactose residues. Cross-reactive determinants are expressed on porcine lymphocytes but not porcine red blood cells.

Ultrasound scattering from blood with hematocrits up to 100%.

The backscattering coefficient of saline suspensions of porcine red blood cells was measured for hematocrits up to about 90%. It was found that the coefficient peaks at approximately 15%, but then, contrary to what a simple "gap theory" might suggest, it decays smoothly to zero, without showing another peak at high hematocrits. A one-dimensional (1-D) slab scattering model, in which the number of slabs per unit length represents the hematocrit and whose thickness and acoustical properties are similar to red cells/plasma, was also used to investigate the relation between the backscattered power and hematocrit. Monte-Carlo simulations performed for randomized boundary conditions show a similar relation to that of the 3-D system. The experimental data is compared to the Percus-Yevick theory for the packing of hard spheres, and the simulated data is compared to the Percus-Yevick theory for infinite slabs.

酵素分解による血液タンパクの培地素材への応用 （第２報） 培地用ペプトンとしての豚血球分解物の特性

酵素分解による血液タンパクの培地素材への応用 （第２報） 培地用ペプトンとしての豚血球分解物の特性

Effects of Dietary Leucine, α-Ketoisocaproate and Isovalerate on Antibody Production and Lymphocyte Blastogenesis in Growing Lambs

The chronic effects of oral leucine and leucine metabolites on sheep immune function were determined in two experiments. In replicate experiments, 30 mixed-breed ram lambs were individually fed diets supplemented with approximately 0.05% ruminally protected limestone (control), alpha-ketoisocaproate (KIC), isovalerate (IVA) or leucine (Leu). Serum titers of antibodies produced in response to Brucella abortus antigen and porcine red blood cells were determined. Mitogen-stimulated lymphocyte blastogenesis was determined in experiment 2 by adding phytohemagglutinin (PHA), concanavalin A (Con A) or pokeweed mitogen (PWM) to isolated lymphocytes and measuring [3H]thymidine incorporation. In both experiments, in lambs fed Leu, antibody production to porcine red blood cells was approximately 80% (P less than 0.05) of that in control animals. When KIC was fed, antibody titers to porcine red blood cells were approximately 120% (P less than 0.05) of that of controls. Compared to controls background lymphocyte blastogenesis was higher when KIC was fed, whereas background blastogenesis was lower when Leu was fed (KIC vs. Leu; P less than 0.05). IVA did not significantly affect either measurement. These data indicate that feeding Leu may adversely affect immune function by suppressing lymphocyte activity, whereas oral administration of KIC has a positive influence on immune function in sheep by increasing lymphocyte activity.

Canine parvovirus infection in Nigeria

ABSTRACTPups of Nigerian mongrel dogs randomly bought from two village markets for experimental purposes at the University of Nigeria, Nsukka, were observed to be ill within 4 days of purchase. By 13 days after purchase 52.5 per cent of all the animals had died. All sick animals were anorectous and suffered from diarrhoea. The faeces were mostly dark yellow to light brown and a few contained spots of blood. Few animals responded to treatment with fluids, antibiotics and vitamins and death occurred within 24 hours of the onset of clinical signs. In most cases, post mortem and histopathological lesions were similar to those characteristic of canine parvovirus. The sera of the surviving animals tested for haemagglutination‐inhibition antibodies to canine parvovirus, using porcine red blood cells, were positive.

Evaluation of the Hemagglutinating Activity of Low‐temperature Cooked Kidney Beans

ABSTRACTMethodology was developed that allowed sensitive measurement of phytohemagglutinin (PHA), the lectin of kidney beans. Trypsinated porcine red blood cells were treated with saline extracts of kidney beans, incubated, and the nonagglutinated cells were quantitated using a Coulter Counter. This hemagglutination activity (HA) assay was then used to monitor the PHA in cooked kidney beans. The thermal treatment (minutes @°C) required to reduce HA by 1 log cvcle was: 12 @ 100°C: 62 @ 93°C: 136 @ 88°C: 160 @ 82°C. Beans were prepared in commercially available low‐temperature cookers and evaluated for tenderness and residual hemagglutinating activity.

Autoantibodies to zona pellucida in tubectomized women

The study was conducted to detect autoantibodies to zona pellucida by indirect immunofluorescence technique. A total of 60 human sera was examined, which included 15 tubectomized, 15 pregnant, 15 nonpregnant fertile women, and 15 fertile men. In the unabsorbed sera, anti-zona activity was observed in 53.3% of the tubectomized women and in 40.0% of the nonpregnant fertile women, 20.0% of the pregnant women and 26.7% of the fertile men. All the positive sera were then absorbed with porcine red blood cells and retested to exclude the false positive reaction due to nonspecific serum components. After absorption, immunological response was retained in 26.7% of the tubectomized women and lost in all the other women and men. The positive sera were further absorbed with zona-coated eggs and were retested. Fluorescence was lost in all the positive sera. This observation demonstrates the presence of antibodies, specific to zona antigens, in tubectomized women. To date, however, antibodies to zona antigen have not been demonstrated in the tubectomized women. Presence of anti-zona activity in tubectomized women may be an autoimmune response, possibly due to absorption and degradation of the ova into the peritoneal cavity or in the reproductive tract and the subsequent exposure of the degradation products to the immune system.