Swine Erythrocytes Research Articles

Deletion of the EP402R Gene from the Genome of African Swine Fever Vaccine Strain ASFV-G-∆I177L Provides the Potential Capability of Differentiating between Infected and Vaccinated Animals.

The African swine fever virus (ASFV) mutant ASFV-G-∆I177L is a safe and efficacious vaccine which induces protection against the challenge of its parental virus, the Georgia 2010 isolate. Although a genetic DIVA (differentiation between infected and vaccinated animals) assay has been developed for this vaccine, still there is not a serological DIVA test for differentiating between animals vaccinated with ASFV-G-∆I177L and those infected with wild-type viruses. In this report, we describe the development of the ASFV-G-∆I177L mutant having deleted the EP402R gene, which encodes for the viral protein responsible for mediating the hemadsorption of swine erythrocytes. The resulting virus, ASFV-G-∆I177L/∆EP402R, does not have a decreased ability to replicates in swine macrophages when compared with the parental ASFV-G-∆I177L. Domestic pigs intramuscularly (IM) inoculated with either 102 or 106 HAD50 of ASFV-G-∆I177L/∆EP402R remained clinically normal, when compared with a group of mock-vaccinated animals, indicating the absence of residual virulence. Interestingly, an infectious virus could not be detected in the blood samples of the ASFV-G-∆I177L/∆EP402R-inoculated animals in either group at any of the time points tested. Furthermore, while all of the mock-inoculated animals presented a quick and lethal clinical form of ASF after the intramuscular inoculation challenge with 102 HAD50 of highly virulent parental field isolate Georgia 2010 (ASFV-G), all of the ASFV-G-∆I177L/∆EP402R-inoculated animals were protected, remaining clinically normal until the end of the observational period. Most of the ASFV-G-∆I177L/∆EP402R-inoculated pigs developed strong virus-specific antibody responses against viral antigens, reaching maximum levels at 28 days post inoculation. Importantly, all of the sera collected at that time point in the ASFV-G-∆I177L/∆EP402R-inoculated pigs did not react in a direct ELISA coated with the recombinant EP402R protein. Conversely, the EP402R protein was readily recognized by the pool of sera from the animals immunized with recombinant live attenuated vaccine candidates ASFV-G-∆I177L, ASFV-G-∆MGF, or ASFV-G-∆9GL/∆UK. Therefore, ASFV-G-∆I177L/∆EP402R is a novel, safe and efficacious candidate with potential to be used as an antigenically DIVA vaccine.

Prevalence and characteristics of a feline parvovirus-like virus in dogs in China

In this study, 192 diarrheal fecal samples were collected from 2019 to 2021 for monitoring the molecular prevalence of canine parvovirus 2 (CPV-2) among dogs in Southwest China, and 113 samples were detected as Carnivore protoparvovirus 1-positive. Surprisingly, 28/113 (24.8%) strains were identified as feline parvovirus (FPV)-like viruses based on the key amino acid (aa) residues in VP2. Further, 6 FPV-like strains were successfully isolated and genome sequenced, and phylogenetic trees based on the genome, VP2 and NS1 sequences showed that the 6 FPV-like strains were most genetically related with FPV instead of CPV-2. Interestingly, the VP2 proteins of the FPV-like virus contained all key aa residues typical for FPV and can be 100% identical to that of FPV, but the VP1 intron and NS1 aa sequences exhibited some unique molecular characteristics. The FPV-like isolate could hemagglutinate swine erythrocyte at pH values between 6 and 8, and replicated efficiently in MDCK cell line; moreover, the virus could cause canine systemic infection via oral administration. Further analysis based on VP2 sequences of FPV and CPV-2 in GenBank revealed that the FPV-like virus had already existed among dogs in 4 Asian countries, and have circulated widely in China. This study first confirmed that the FPV-like isolates could efficiently infect dogs, and has been prevalent among dogs in China. Moreover, this study first reported the genome characteristics of the FPV-like virus in dogs, which may represent a novel evolution pattern involving in the cross-species transmission of the virus from cats to dogs.

A unique combination of glycoside hydrolases in Streptococcus suis specifically and sequentially acts on host-derived αGal-epitope glycans

Infections by many bacterial pathogens rely on their ability to degrade host glycans by producing glycoside hydrolases (GHs). Here, we discovered a conserved multifunctional GH, SsGalNagA, containing a unique combination of two family 32 carbohydrate-binding modules (CBM), a GH16 domain and a GH20 domain, in the zoonotic pathogen Streptococcus suis 05ZYH33. Enzymatic assays revealed that the SsCBM-GH16 domain displays endo-(β1,4)-galactosidase activity specifically toward the host-derived αGal epitope Gal(α1,3)Gal(β1,4)Glc(NAc)-R, whereas the SsGH20 domain has a wide spectrum of exo-β-N-acetylhexosaminidase activities, including exo-(β1,3)-N-acetylglucosaminidase activity, and employs this activity to act in tandem with SsCBM-GH16 on the αGal-epitope glycan. Further, we found that the CBM32 domain adjacent to the SsGH16 domain is indispensable for SsGH16 catalytic activity. Surface plasmon resonance experiments uncovered that both CBM32 domains specifically bind to αGal-epitope glycan, and together they had a KD of 3.5 mm toward a pentasaccharide αGal-epitope glycan. Cell-binding and αGal epitope removal assays revealed that SsGalNagA efficiently binds to both swine erythrocytes and tracheal epithelial cells and removes the αGal epitope from these cells, suggesting that SsGalNagA functions in nutrient acquisition or alters host signaling in S. suis Both binding and removal activities were blocked by an αGal-epitope glycan. SsGalNagA is the first enzyme reported to sequentially act on a glycan containing the αGal epitope. These findings shed detailed light on the evolution of GHs and an important host-pathogen interaction.

A multistage-dialysis microdevice for extraction of cryoprotectants.

In this study, we present a multistage-dialysis microdevice (MDM) for extraction of cryoprotectants (CPAs) from a CPA-laden cell suspension. We confirmed the functions of the key designs of the MDM using a fluorescence solution, we assessed the performance of the MDM by using the MDM to unload glycerin from glycerin-loaded swine erythrocytes, and we investigated the effects of the cell suspension flow rate, glycerin concentration, cell density, and membrane pore size on the clearance efficiency of glycerin (CG), the survival rate of cells (SC), and the recovery rate of cells (RC). Under the designed conditions, CG, SC, and RC reached ~60%, ~90%, and ~70%, respectively. In addition, a high flow rate causes high SC and RC but a low CG. For a low glycerin concentration, CG, SC, and RC are all high. If a low cell density or a large pore membrane is used, CG is high, whereas both SC and RC are low. This work provides insight into the development of microfluidic devices for the inline extraction of cryoprotectants from a small volume of cryopreserved cells prior to the use of the cells in lab-on-a-chip applications.

Erythrocyte aggregation under high pressure studied by laser photometry and mathematical analysis

The effects of hydrostatic pressure on erythrocyte aggregation have been studied by laser photometry and analysis based on a phenomenological theory. Samples were prepared by suspending swine erythrocytes in their own plasma. A high-pressure vessel consisting of a stainless-steel block with a hole to hold a sample cell and two sapphire windows to allows the passage of a He–Ne laser beam was used in the experimental setup. The suspension was stirred at 1500rpm to disperse the erythrocytes homogeneously. Immediately after reducing the stirring rate from 1500rpm to 300rpm, the transmitted light intensity (I) was recorded every 10ms under a high pressure of 40–200MPa. The value of I increased with time (t) owing to erythrocyte aggregation. From the phenomenological theory, the equation ΔI(t)=ΔIeq[1−e−Kt/(1−B(1−e−Kt))] was derived for the change in the transmitted light intensity (ΔI) due to erythrocyte aggregation, where ΔIeq is the transmitted light intensity in the steady state, K is a time constant and B is a constant that represents the ratio of the number of interaction sites on erythrocyte aggregates at time t to that in the steady state. The observed time courses of ΔI obtained at all pressures could be closely fitted to the theoretical equation. ΔIeq roughly increased with increasing pressure. On the other hand, K and B abruptly decreased above 120MPa. The growth rate of aggregates decreased above 120MPa. These results suggest a change in the mechanism of erythrocyte aggregation at approximately 120MPa. We discuss the physical meaning of the parameters.

In vitro and in vivo validation of stored swine erythrocyte viability to establish an experimental model of homologous red blood cell transfusion: a pilot study.

Objective To develop experimental models of erythrocyte transfusion, the first step is to ensure the viability of the red blood cells transfused. In this pilot study, we assessed the viability of transfused red blood cells with validation in vitro and in vivo of homologous swine erythrocytes stored for 14 days.Methods Blood collected from one Agroceres® swine was stored in two red blood cell units. In vivo validation was performed by labeling the red blood cells with Na251CrO4 and recovering the viable erythrocytes after 24 hours of infusion in one autologous and four homologous animals. In vitro validation was performed at baseline and after 14 days in sixteen red blood cell units by measuring hemoglobin, hematocrit, hemolysis index and free hemoglobin. A post-mortem splenectomy was performed to evaluate the splenic sequestration of erythrocytes, and the radioactivity of the supernatant samples was counted to evaluate intravascular hemolysis.Results After 14 days of storage, the red blood cell units had lower volumes and equivalent total concentrations of hemoglobin and hematocrit compared to human standards. The free hemoglobin concentration increased from 31.0±9.3 to 112.4±31.4mg/dL (p<0.001), and the hemolysis index increased from 0.1±0.1 to 0.5±0.1% (p<0.001). However, these tests were within the acceptable range for human standards. The percentage of radioactivity in supernatant samples was similar at baseline and after 24 hours, thus excluding significant hemolysis. No evidence of splenic sequestration of radioactive erythrocytes was found.Conclusion Swine red blood cells stored for 14 days are viable and can be used in experimental studies of transfusion. These validation experiments are important to aid investigators in establishing experimental models of transfusion.

The CD2v protein enhances African swine fever virus replication in the tick vector, Ornithodoros erraticus

The NH/P68 non-haemadsorbing (non-HAD) African swine fever virus (ASFV) isolate contains frameshift mutations in the EP402R and adjacent EP153R genes. These encode, respectively, the protein (CD2v) that is required for the haemadsorption (HAD) of swine erythrocytes to ASFV-infected cells and a C-type lectin protein. Two recombinant HAD viruses were constructed in this parental strain. In one of these the intact EP153R gene sequence was restored. Although restoration of the HAD phenotype did not increase virus virulence in pigs, a significant increase was observed in the number of pigs which developed viraemia. These HAD recombinant viruses replicated to titres approximately 1000-fold higher than the parental non-HAD isolate when membrane fed to Ornithodoros erraticus ticks. Inoculation of the non-HAD isolate across the gut wall increased viral replication to levels comparable to that of the HAD recombinant viruses. These results demonstrate a novel role for the CD2v protein in virus replication in ticks.

Activation of the alternative complement pathway in canine normal serum by Paracoccidioides brasiliensis

The dimorphic fungus Paracoccidioides brasiliensis is the etiological agent of paracoccidioidomycosis, a human granulomatous disease. Recently the first case of natural disease in dogs was reported. The complement system is an important effector component of humoral immunity against infectious agents. Therefore, the aim of this study was to evaluate the activation of the dog alternative complement pathway by P. brasiliensis. Initially, the ability of erythrocytes of guinea pig, rabbit, sheep, chicken and swine to activate the dog alternative pathway was evaluated. The guinea pig erythrocytes showed the greatest capacity to activate dog alternative pathway. The alternative (AH50) hemolytic activity was evaluated in 27 serum samples from healthy dogs and the mean values were 87.2 AH50/ml. No significant differences were observed in relation to sex and age. The alternative pathway activation by P. brasiliensis was higher in serum samples from adult dogs when compared to puppies and aged dogs (p ≤ 0.05). This is the first report of dog alternative complement pathway activation by P. brasiliensis and suggests that it may play a protective role in canine paracoccidioidomycosis.

Feasibility of stored red blood cell transfusion in pigs

The mechanisms associated with immunomodulation after red blood cell transfusion are not completely understood, possibly due to methodological biases in the clinical studies and presence of comorbidities such as sepsis. Therefore, a controlled animal model of blood cell transfusion may be a more appropriate approach to minimize these issues. We designed this pilot study in order to validate in vitro and in vivo the survival of swine erythrocytes stored for 13 days.

Relationship between Clostridium septicum Alpha-Toxin Activity and Binding to Erythrocyte Membranes

The activity of Clostridium septicum alpha-toxin was determined in erythrocytes of various animals, with sensitivities observed in the order of mouse, rat, canine, equine, rabbit, chicken, bovine, swine and ovine. Temperature and protease treatment affected the sensitivity of erythrocytes to alpha-toxin. Proteinase K treatment decreased the sensitivity of murine, canine, equine and bovine erythrocytes, but ovine erythrocytes did not change the sensitivity to alpha-toxin activity. On the other hand, the activity of alpha-toxin on swine erythrocytes increased after treatment with proteinase K, trypsin, chymotrypsin or lysyl endopeptidase. Toxin overlay assay showed that alpha-toxin bound to erythrocyte membrane proteins with a molecular mass of 30 to 45-kDa in mouse, equine, bovine, swine and chicken, whereas in rat erythrocyte membranes the toxin reacted with 100-kDa protein. The treatment of murine and swine erythrocyte membranes with phosphatidylinositol-specific phospholipase C resulted in liberation of the toxin-binding protein from the individual membranes in a native state. These results show that alpha-toxin associates with specific erythrocyte membrane proteins in any animal species, and are subsets of glycosylphosphatidylinositol-anchored proteins in various animal species. These results may reflect distinct characteristics of the hemolytic activity of alpha-toxin in response to various erythrocytes.

Tritrichomonas foetus: a scanning electron microscopy study of erythrocyte adhesion associated with hemolytic activity

The in vitro hemolytic activity of Tritrichomonas foetus was investigated. The parasite was tested against human erythrocytes of groups A, B, AB, and O, and against erythrocytes of nine adult animals of different species (the rabbit, rat, chicken, cat, dog, swine, horse, bovine, and sheep). The results showed that T. foetus strains (ATCC KV1, K, PAL, 5022, RJ, 90) did not present any hemolytic activity against any human erythrocyte group nor against rabbit, rat, chicken, cat, dog and swine erythrocytes. T. foetus strains, however, lysed horse, bovine, and sheep erythrocytes. No hemolysin released by the parasites could be identified. Hemolysis did not occur with trichomonad culture supernatants, with sonicated extracts of T. foetus, nor with killed organisms. Scanning electron microscopy (SEM) showed that human erythrocytes did not adhere to the trophozoites, in contrast horse erythrocytes adhered to the surface of the parasites and were phagocytosed for up to 90 min. The parasites are able to exert their cytopathic effects through: (a) physical contact established between the two cell surfaces, (b) toxins released from parasites into the interaction media, or (c) the association of both mechanisms. Further studies are necessary to clarify the importance of the hemolytic activity in the biology of T. foetus.

Viscosity of Animal Erythrocyte Suspensions mixed with a Perflurocarbon Emulsion

Artificial blood substitutes (ABS) offer an alternative to donated blood. Increasing the oxygen carrying capacity of blood plasma through the addition of a Perfluorocarbon emulsion (PFE) is one approach in creating a blood substitute. The peripheral resistance of the circulatory system may be altered depending on the rheological properties of the ABS. Measurements of the rheological behavior of mixtures of a PFE, OxygentTM, and erythrocyte suspensions were conducted at room temperature at different hematocrits using sheep (nonaggregating) and swine (aggregating) erythrocytes. The pure PFE was found to be shear thinning. Adding 6 and 12 g per 100 mL of sheep blood at the various hematocrits increased the viscosity of the suspension from as low as 4% (6 g PFE/40% Hct) to as high as 26.5% (12 g PFE/plasma). The nonaggregating sheep erythrocyte and PFE mixtures exhibited Newtonian behavior. Shear thinning behavior continued upon addition of 6 and 12 g per 100 mL of swine blood at the various hematocrits, with a slight increase in viscosity in most cases. It was observed that adding 12 g of PFE (≈ 3 × intended clinical dose) to 40% Hct swine blood at room temperature led to a significant decrease in viscosity.

Viscosity of Animal Erythrocyte Suspensions mixed with a Perflurocarbon Emulsion

Artificial blood substitutes (ABS) offer an alternative to donated blood. Increasing the oxygen carrying capacity of blood plasma through the addition of a Perfluorocarbon emulsion (PFE) is one approach in creating a blood substitute. The peripheral resistance of the circulatory system may be altered depending on the rheological properties of the ABS. Measurements of the rheological behavior of mixtures of a PFE, Oxygent, and erythrocyte suspensions were conducted at room temperature at different hematocrits using sheep (nonaggregating) and swine (aggregating) erythrocytes. The pure PFE was found to be shear thinning. Adding 6 and 12 g per 100 mL of sheep blood at the various hematocrits increased the viscosity of the suspension from as low as 4% (6 g PFE/40% Hct) to as high as 26.5% (12 g PFE/plasma). The nonaggregating sheep erythrocyte and PFE mixtures exhibited Newtonian behavior. Shear thinning behavior continued upon addition of 6 and 12 g per 100 mL of swine blood at the various hematocrits, with a slight increase in viscosity in most cases. It was observed that adding 12 g of PFE (approximately 3 x intended clinical dose) to 40% Hct swine blood at room temperature led to a significant decrease in viscosity.

Effects of contrast media on the erythrocyte mobility from sedimentation rate at different osmotic pressures

X-ray contrast media (CMs) influence various systems in blood circulation. Mobility of erythrocyte μ has been determined from the erythrocyte sedimentation rate s at different osmotic pressure π, to compare effects of ionic CM to those of non-ionic CM. Tow kinds of CMs, ionic Urografin® 76% (URO) and non-ionic Iopamiron® 300 (IOP), were employed. The density ρ c and the volume V of erythrocyte, and s were measured at 25 °C by using a vibrating densimeter and the Coulter counter, and the Westergren tube, respectively. Swine erythrocytes were suspended in saline solutions without CMs (SAL) and with CMs at various π, where the hematocrit was ca. 10% and the CM concentrations were ca. 6.7 wt.%. ρ c and V behaved as a single function of π both without CMs and with CMs, meaning RBC to be a micro-osmometer. While μ increased with V vigorously for SAL at the highest level, μ varied slightly for URO at the lowest level and moderately for IOP. Deformability of erythrocyte may be decreased more seriously for ionic CM than for non-ionic CM at the physiological condition of π, which may disturb blood microcirculation.

Erythrocyte Aggregation Enhanced by Hydrostatic Pressure.

The effects of pressure on the aggregation of swine erythrocytes were investigated by monitoring the erythrocyte sedimentation rate under high pressure, up to 40 MPa. The aggregation of erythrocytes in plasma was propor-tionally enhanced by pressure, whereas aggregation of erythrocytes was not observed in saline solution up to 40 MPa. These results suggest that the adhesion energy between erythrocytes is not directly affected by pressure, but through a change in the interaction energy between plasma components and erythrocytes.

Correct assembly of human normal adult hemoglobin when expressed in transgenic swine: chemical, conformational and functional equivalence with the human-derived protein.

Structural and functional investigations of recombinant human hemoglobin A (HbA) isolated from the erythrocytes of transgenic swine coexpressing human alpha- and beta-globins have been carried out to authenticate its correct expression, post-translational processing and assembly. The HbA expressed in transgenic swine (TgHbA) is indistinguishable from the human-derived HbA in terms of its isoelectric pH, mass and elution pattern on a Mono S column. The chemical identity of the alpha- and beta-globin chains of TgHbA with the corresponding chains from human-derived HbA has been established by tryptic peptide mapping and amino acid sequencing. The proton NMR spectra of TgHbA have demonstrated that the conformational aspects of the protein around the heme pocket are indistinguishable from those of the control sample of HbA. The equivalence of the hydrogen bond pattern of TgHbA (in particular the inter-subunit surfaces) with that of authentic HbA has also been established by NMR studies. Consistent with these structural and conformational analyses, the TgHbA also exhibits complete functional equivalence with the human-derived HbA with respect to oxygen affinity, cooperativity, Bohr effect and allostery. Hence the studies presented here demonstrate that the transgenic swine system correctly transcribes the alpha- and beta-globin transgenes, translates the respective alpha- and beta-globin mRNA to generate the corresponding globin chains, carries out the correct cotranslational processing of the translated globin chains, inserts the heme into the globin chains in the same orientation as in the human-derived HbA and assembles the alpha- and beta-subunits into a functionally cooperative tetramer that exhibits a response to allosteric effectors identical with that of human-derived HbA. Thus, in the transgenic swine system, in vitro chemical manipulation steps such as those needed in the Escherichia coli and the yeast systems, to convert the rHbA expressed in these systems into forms functionally identical with that of the human-derived protein, are not needed. An additional advantage of the transgenic swine system is the stability of the transgenes over many generations. Hence the transgenic swine could serve as an excellent system for the production of human HbA (or its variants) for structure-function studies and for therapeutic applications.

A phenomenological study of aggregation of erythrocytes in dilute suspension under gravity

The growth process of swine erythrocyte aggregate was studied using microscopic observations and mathematical analysis. Both the cross-sectional area S and velocity V of an arbitrarily chosen aggregate in dilute suspension increased exponentially with time: S = S 0 exp( t τ s ) , V = V 0 exp( t τ v ) with S 0 = 160 μm 2, V 0 = 1.12 μm s −1, τ s = 0.20 s and τ v = 0.17 s. The average values of V and S for aggregates in suspension were proportional to each other and the proportionality constant deviates from the calculated value when aggregates are assumed to comprise homogeneous closely packed spheres. On the basis of these results and simple assumptions, a differential equation was derived for the number of aggregates consisting of n erythrocytes at time t, f( n, t): ∂f(n, t) ∂t = − k[ ∂f(n, t) ∂n + ( ln 2)f(n, t)] The solution of this equation indicates that f( n, t) is a function of n − kt.

Hypericin-induced phototoxicity in cultured fibroblasts and swine erythrocytes.

Hypericin is a naturally occurring photosensitizer, whose presence in plants has been responsible for cutaneous phototoxicity in grazing animals. The photosensitizing properties of this agent have recently been exploited in models for anti-tumor and anti-viral activity. The cytotoxicity of hypericin and light was assessed in 3T3 mouse fibroblasts using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide)] assay and the lactate dehydrogenase (LDH) leakage assay. Membrane damage was assessed in swine erythrocytes using hemolysis, potassium (K+) leakage and formation of lipid hydroperoxides. Concentration- and light-dependent decreases in fibroblast viability were seen starting at hypericin concentrations of 1.25 microM and light power flux levels of 24 J/cm2 using a visible light source and at 0.417 microM hypericin and a similar light dose using a solar simulator. No LDH leakage was observed at hypericin concentrations up to 30 microM and visible light up to 144 J/cm2. Light-and/or concentration-dependent increases in hemolysis, K+ leakage and formation of lipid hydroperoxides in red blood cell (RBC) membranes were observed, but at concentrations and light doses much greater than those required to induce cytotoxicity in fibroblasts. Lipid peroxidation and hemolysis occurred at 15 microM hypericin and 24 J/cm2 (visible light source). Potassium ion leakage occurred at concentrations and light levels as low as 5 microM and 12 J/cm2 or 15 microM and 4.8 J/cm2 (visible light source) but was still a less sensitive indicator than fibroblast cytotoxicity. Evidence for both type I and type II reactions was shown in RBC membranes by TLC analysis of cholesterol products. In the absence of light, hypericin appears to be relatively nontoxic in the models tested.

A monoclonal antibody to swine erythrocytes recognizes the B blood group on the major glycophorin

Recently monoclonal antibodies (mAbs) to swine red blood cells have been described. One of them (1AC11) was specific for the major swine glycoprotein with a molecular weight of 45 kDa and another mAb, 2G2, recognized the Ba allele in the B system of swine blood groups. Immunoblotting experiments to characterize the mAb 2G2 indicated that it reacts with an antigen of 45 kDa, present on the aqueous phase, glycophorin fraction, of swine red blood cells with the Ba allele and does not react with BbBb homozygous cells. The antigen recognized by 2G2 has the same characteristics as the major glycophorin recognized by 1AC11, so we can conclude that the B system of the swine blood group is on the major glycophorin of swine erythrocyte membranes.

Test for Ability of Decay‐Accelerating Factor (DAF, CD55) and CD59 to Alleviate Complement‐Mediated Damage of Xeno‐Erythrocytes

We investigated the susceptibility to human complement (C) of xeno-erythrocytes into which phosphatidylinositol (PI)-anchored human C regulatory protein, decay-accelerating factor (DAF) or CD59 had been incorporated. Erythrocytes of sheep (Esh), swine (Esw), dog (Edg), and guinea pig (Egp), unsensitized with human natural antibody (Ab), were used as xeno-target. C-mediated lysis of erythrocytes (E) was induced in both classical and alternative pathways in parallel with the density of the sensitized Ab, except for Egp. The efficacy of DAF/CD59-mediated protection of the xeno E from human C, however, differed among these E species. In both classical and alternative pathways, Esh or Esw, which are non-activator surfaces, were protected by the incorporated DAF or CD59, DAF being more effective than CD59. On the other hand, CD59 was more effective than DAF in both pathways in protection of Egp, which is an alternative pathway activator. To elucidate this different behaviour of DAF and CD59, C3 step inhibition by the incorporated DAF or CD59 was measured. DAF was effective in the suppression of classical pathway-mediated C3 deposition in Esh, Esw and Egp, but not in Edg, while CD59 exhibited negligible effects in this regard. Next, inhibition of the lysis by CD59 was tested by haemolytic assay. CD59 did not block the C5b-8-mediated lysis in any xeno E. It also barely blocked C5b-9-mediated lysis, except in the case of Egp, in which CD59 partly blocked C9 attack. Membrane constituents on targets other than the incorporated complement inhibitors may be a crucial factor in the induction of cytolysis and, presumably, in hyperacute rejection.