Pig Red Blood Cells Research Articles

Ultraviolet light and riboflavin accelerates red blood cell dysfunction in vitro and in a guinea pig transfusion model.

Quality assessment of modified or processed red blood cell (RBC) components, such as pathogen-reduced RBCs, using only in vitro testing may not always be predictive of in vivo performance. Mouse or rat in vivo models are limited by a lack of applicability to certain aspects of human RBC biology. Here, we used a guinea pig model to study the effects of riboflavin combined with UV light on the integrity of RBCs in vitro and following transfusion in vivo. Guinea pig RBCs were collected from whole blood (WB) treated with varying UV doses (10, 20, 40 or 80 J/mL) in the presence of riboflavin (UVR-RBCs). In vitro tests for UVR-RBCs included hemolysis, osmotic fragility, and cellular morphology by scanning electron microscopy. Guinea pigs transfused with one-day post-treatment UVR-RBCs were evaluated for plasma hemoglobin (Hb), non-transferrin bound iron (NTBI), total iron and Perls-detectable hemosiderin deposition in the spleen and kidney, and renal uptake of Hb. Acute RBC injury was dose dependently accelerated after treatment with UV light in the presence of riboflavin. Aberrant RBC morphology was evident at 20, 40, and 80 J/mL, and membrane lysis with Hb release was prominent at 80 J/mL. Guinea pigs transfused with 40 and 80 J/mL UVR-RBCs showed increased plasma Hb levels, and plasma NTBI was elevated in all UVR-RBC groups (10-80 J/mL). Total iron levels and Perls-hemosiderin staining in spleen and kidney as well as Hb uptake in renal proximal tubules were increased 8 hours post-transfusion with 40 and 80 J/mL UVR-RBCs. UVR-RBCs administered to guinea pigs increased markers of intravascular and extravascular hemolysis in a UV dose-dependent manner. This model may allow for the discrimination of RBC injury during testing of extensively processed RBCs intended for transfusion.

Influence of BOVAMINE DEFEND Plus on growth performance, carcass characteristics, estimated dry matter digestibility, rumen fermentation characteristics, and immune function in finishing beef steers.

One hundred and eighty crossbred beef steers (406.0 ± 2.2kg) were used to determine the impact of a novel direct-fed microbial (DFM) on growth performance, carcass characteristics, rumen fermentation characteristics, and immune response in finishing beef cattle. Steers were blocked by body weight (BW) and randomly assigned, within block, to 1 of 2 treatments (3 replicates/treatment: 30 steers/replicate). Treatments included: (1) no DFM (control) and (2) DFM supplementation at 50 mg ∙ animal-1 ∙ d-1 (BOVAMINE DEFEND Plus). All steers were fed a high-concentrate finishing diet and individual feed intake was recorded daily via the GrowSafe system. BWs were collected every 28 d. On day 55, 10 steers per pen were injected with ovalbumin (OVA). Jugular blood samples were collected from each steer on days 0, 7, 14, and 21 post injection. On day 112, the same steers were injected again with OVA and intramuscularly with a pig red blood cell solution. Jugular blood samples were collected from each steer on days 0, 7, 14, and 21 post injection. On day 124, rumen fluid was collected from 3 steers per treatment and used to estimate in vitro rumen fermentation characteristics. Equal numbers of steers per treatment were transported to a commercial abattoir on days 145, 167, and 185 of the experiment, harvested, and carcass data were collected. Initial BW was similar across treatments. On days 28 and 55, steers receiving DFM had heavier BW (P < 0.01) compared to controls. The average daily gain was greater in DFM-supplemented steers from days 0 to 28 (P < 0.01) and days 0 to 55 (P < 0.01) of the experiment compared to controls. Overall dry matter intake (DMI) was greater (P < 0.04) and overall feed efficiency was similar in DFM-supplemented steers compared to controls. Dressing percentage (P < 0.02) was greater in steers receiving DFM compared to controls. Antibody titers to injected antigens were similar across treatments. However, red blood cell superoxide dismutase activity was greater (P < 0.05) in DFM-supplemented steers compared to controls. In vitro molar proportions of isobutyric and butyric acid were greater (P < 0.01) and dry matter (DM) digestibility tended (P < 0.07) to be greater in rumen fluid obtained from steers supplemented with DFM. These data suggest that BOVAMINE DEFEND Plus supplementation improves growth performance during the initial period of the finishing phase, increases overall DMI and dressing percentage, and may impact antioxidant status in beef cattle.

Efficient in vitro and in vivo transfection of self-amplifying mRNA with linear poly(propylenimine) and poly(ethylenimine-propylenimine) random copolymers as non-viral carriers.

Messenger RNA (mRNA) based vaccines have been introduced worldwide to combat the Covid-19 pandemic. These vaccines consist of non-amplifying mRNA formulated in lipid nanoparticles (LNPs). Consequently, LNPs are considered benchmark non-viral carriers for nucleic acid delivery. However, the formulation and manufacturing of these mRNA-LNP nanoparticles are expensive and time-consuming. Therefore, we used self-amplifying mRNA (saRNA) and synthesized novel polymers as alternative non-viral carrier platform to LNPs, which enable a simple, rapid, one-pot formulation of saRNA-polyplexes. Our novel polymer-based carrier platform consists of randomly concatenated ethylenimine and propylenimine comonomers, resulting in linear, poly(ethylenimine-ran-propylenimine) (L-PEIx-ran-PPIy) copolymers with controllable degrees of polymerization. Here we demonstrate in multiple cell lines, that our saRNA-polyplexes show comparable to higher in vitro saRNA transfection efficiencies and higher cell viabilities compared to formulations with Lipofectamine MessengerMAX™ (LFMM), a commercial, lipid-based carrier considered to be the in vitro gold standard carrier. This is especially true for our in vitro best performing saRNA-polyplexes with N/P 5, which are characterised with a size below 100 nm, a positive zeta potential, a near 100% encapsulation efficiency, a high retention capacity and the ability to protect the saRNA from degradation mediated by RNase A. Furthermore, an ex vivo hemolysis assay with pig red blood cells demonstrated that the saRNA-polyplexes exhibit negligible hemolytic activity. Finally, a bioluminescence-based in vivo study was performed over a 35-day period, and showed that the polymers result in a higher and prolonged bioluminescent signal compared to naked saRNA and L-PEI based polyplexes. Moreover, the polymers show different expression profiles compared to those of LNPs, with one of our new polymers (L-PPI250) demonstrating a higher sustained expression for at least 35 days after injection.

Investigation of the efficacy and safety of wild- type and triple-gene knockout pig RBC transfusions in nonhuman primates.

Decreasing rates of blood donation and close margins between blood supply and demand pose challenges in healthcare. Genetically engineered pig red blood cells (pRBCs) have been explored as alternatives to human RBCs for transfusion, and triple-gene knockout (TKO) modification improves the compatibility of pRBCs with human blood in vitro. In this study, we assessed the efficacy and risks of transfusing wild-type (WT)- and TKO-pRBCs into nonhuman primates (NHPs). Blood from O-type WT and TKO pigs was processed to produce pRBCs for transfusion, which were transfused or not into NHPs (n=4 per group: WT, TKO, and control) after 25% total blood volume withdrawal: their biological responses were compared. Hematological, biochemical, and immunological parameters were measured before, immediately after, and at intervals following transfusion. Two months later, a second transfusion was performed in three NHPs of the transfusion group. Transfusion of both WT- and TKO-pRBCs significantly improved RBC counts, hematocrit, and hemoglobin levels up to the first day post-transfusion, compared to the controls. The transfusion groups showed instant complement activation and rapid elicitation of anti-pig antibodies, as well as elevated liver enzyme and bilirubin levels post-transfusion. Despite the higher agglutination titers with WT-pRBCs in the pre-transfusion crossmatch, the differences between the WT and TKO groups were not remarkable except for less impairment of liver function in the TKO group. After the second transfusion, more pronounced adverse responses without any hematological gain were observed. WT- and TKO-pRBC transfusions effectively increased hematologic parameters on the first day, with rapid clearance from circulation thereafter. However, pRBC transfusion triggers strong antibody responses, limiting the benefits of the pRBC transfusion and increasing the risk of adverse reactions.

PSXI-29 Influence of a Direct Fed Microbial on Growth Performance, Carcass Data, and Immune Function in Feedlot Finishing Steers

Abstract One hundred and eighty crossbred beef steers (405.9 ± 2.3 kg) were used to determine the impact of a direct fed microbial (DFM) on growth performance, carcass characteristics, and immune response in finishing beef cattle. Steers were blocked by body weight and randomly assigned, within block, to 1 of 2 treatments (3 replicates/treatment: 30 steers/replicate). Groups within a replicate were randomly assigned to treatments. Treatments included: 1) no DFM (control) and 2) DFM supplementation at 50 mg/animal/d [BOVAMINE DEFEND Plus containing Lactobacillus animalis, Propionibacterium freudenreichii, Bacillus licheniformis, and Bacillus subtilis]. All steers were fed a high concentrate, steam-flaked corn-based finishing diet. Individual feed intake was recorded daily via the GrowSafe system. Body weights were collected every 28 d. Jugular blood samples were collected from all steers on day 0 and 112. On d 55 of the experiment, 10 steers per pen (30 steers per treatment) were randomly selected and injected, subcutaneously with 4.0 mg of ovalbumin (OVA). Jugular blood samples were collected from each steer on d 0, 7, 14, and 21 post injection. On d 112 of the experiment, the same steers were injected with the same dose of OVA and intramuscularly with 5 mL of a 25 % pig red blood cell (PRBC) solution. Jugular blood samples were collected from each steer on day 0, 7, 14, and 21 post injection. After receiving the finishing diet for 185 d, cattle were transported to a commercial abattoir and slaughtered. Hot carcass weights were collected at the time of slaughter. Carcasses were allowed to chill for 24 h before carcass data were collected. Data were analyzed as a completely randomized block design and individual animal within pen was considered the experimental unit. Initial body weight, final body weight, and overall feed efficiency were similar across treatments. Steers receiving DFM had greater (P &amp;lt; 0.01) body weights and average daily gains on d 28 and 55 and greater (P &amp;lt; 0.04) dry matter intakes over the entire experiment compared with controls. Hot carcass weight, longissimus muscle area, quality grade, and marbling score were similar across treatments. However, dressing percentage (P &amp;lt; 0.02) was greater in steers receiving DFM compared with controls. Antibody titers to OVA and PRBC and d 112 interferon gamma concentrations were similar across treatments. However, red blood cell superoxide dismutase activity was greater (P &amp;lt; 0.05) in DFM-supplemented cattle compared with controls on d 112 of the experiment. These data suggest that BOVAMINE DEFEND Plus supplementation improves growth performance during certain periods of the finishing phase, increases overall dry matter intake and carcass yield, and may impact antioxidant status in beef cattle.

301.1: What further steps are required to enable the successful clinical transfusion of pig red blood cells? Experience in the TKO pig-to-capuchin monkey model

301.1: What further steps are required to enable the successful clinical transfusion of pig red blood cells? Experience in the TKO pig-to-capuchin monkey model

Virus-inhibitory activity of the antigen complex of opportunistic pathogenic bacteria against SARS-CoV-2

Introduction. The antigen complex of opportunistic pathogenic bacteria (ACOPB) has a protective effect against avian influenza viruses, herpes virus type 2, and other viruses that cause acute respiratory viral infections. In the context of the COVID-19 pandemic, an important task is to find out whether ACOPB has a protective effect against SARS-CoV-2.&#x0D; The purpose of the study was to evaluate in vitro the ACOPB virus-inhibitory activity against the Dubrovka laboratory strain of SARS-CoV-2.&#x0D; Materials and methods. The study was performed using Vero cell line CCL-81, human peripheral blood mononuclear cells (PBMCs), mouse monoclonal anti-idiotypic antibodies structurally mimicking biological effects of human interferons (IFNs), the Dubrovka laboratory strain of SARS-CoV-2. The infectivity of the virus was assessed by two methods: by virus titration using cell cultures and the limiting dilution method when the results are assessed by a cytopathic effect; the second method was a plaque assay. The in vitro virus inhibition test was performed using the cell culture susceptible to SARS-CoV-2; the mixture containing a specific dose of the virus and a two-fold dilution of ACOPB was transferred to the cell culture after the ACOPB medication had interacted with the virus at 4C for 2 hours. The ACOPB virus-inhibitory activity against SARS-CoV-2 was assessed by the functional activity of / and IFN receptors (RIFN) in human PBMCs induced in vitro by ACOPB and the ACOPB mixture with the specific dose of SARS-CoV-2. The RIFN expression level was measured by the indirect membrane immunofluorescence test.&#x0D; Results. Hemagglutination assay using chicken, mouse, guinea pig, and human red blood cells was performed for detection of the SARS-CoV-2 inhibitory protein. The lysate of Vero CCL-81 cells infected with SARS-CoV-2 Dubrovka demonstrated the highest hemagglutination activity with guinea pig red blood cells and low titers of hemagglutination in the virus-containing fluid. The virus inhibition test in the Vero CCL-81 cell culture demonstrated that ACOPB inhibited 10 doses of SARS-CoV-2 Dubrovka with the titer 1 : 32, providing 100% protection of the cell culture for 8 days (the monitoring period). ACOPB induced / and RIFN expression on membranes of human PBMCs in in vitro cultures and decreased RIFN / and expression after its interaction with SARS-CoV-2 Dubrovka.&#x0D; Conclusion. The experimental studies including the virus inhibition test in the cell culture susceptible to SARS-CoV-2 Dubrovka and the indirect membrane immunofluorescence assay using monoclonal anti-idiotypic antibodies mimicking IFN-like properties demonstrated that ACOPB had both an immunomodulatory and a virus-inhibitory effect.

H3N2 influenza hemagglutination inhibition method qualification with data driven statistical methods for human clinical trials.

Hemagglutination inhibition (HAI) antibody titers to seasonal influenza strains are important surrogates for vaccine-elicited protection. However, HAI assays can be variable across labs, with low sensitivity across diverse viruses due to lack of standardization. Performing qualification of these assays on a strain specific level enables the precise and accurate quantification of HAI titers. Influenza A (H3N2) continues to be a predominant circulating subtype in most countries in Europe and North America since 1968 and is thus a focus of influenza vaccine research. As a part of the National Institutes of Health (NIH)-funded Collaborative Influenza Vaccine Innovation Centers (CIVICs) program, we report on the identification of a robust assay design, rigorous statistical analysis, and complete qualification of an HAI assay using A/Texas/71/2017 as a representative H3N2 strain and guinea pig red blood cells and neuraminidase (NA) inhibitor oseltamivir to prevent NA-mediated agglutination. This qualified HAI assay is precise (calculated by the geometric coefficient of variation (GCV)) for intermediate precision and intra-operator variability, accurate calculated by relative error, perfectly linear (slope of -1, R-Square 1), robust (<25% GCV) and depicts high specificity and sensitivity. This HAI method was successfully qualified for another H3N2 influenza strain A/Singapore/INFIMH-16-0019/2016, meeting all pre-specified acceptance criteria. These results demonstrate that HAI qualification and data generation for new influenza strains can be achieved efficiently with minimal extra testing and development. We report on a qualified and adaptable influenza serology method and analysis strategy to measure quantifiable HAI titers to define correlates of vaccine mediated protection in human clinical trials.

Use of the particle agglutination/particle agglutination inhibition test for antigenic analysis of SARS-CoV-2.

The antigenicity of SARS-CoV-2 is a critical issue for the effectiveness of the vaccine, and thus, it should be phenotypically evaluated by serological assays as new field isolates emerge. The hemagglutination/hemagglutination inhibition (HA/HI) tests are well known as a representative method for antigenic analysis of influenza viruses, but SARS-CoV-2 does not agglutinate human or guinea pig red blood cells. Therefore, the antigenic analysis requires complicated cell-based assays using special equipment such as plate reader or ELISPOT analyzer. Based on the HA/HI tests for influenza viruses, we developed the particle agglutination/particle agglutination inhibition (PA/PAI) test to easily and rapidly quantify the virus and antibody using human angiotensin-converting enzyme 2 (hACE2)-bound latex beads. The virus titers were determined by mixing the beads and the virus from culture supernatant, settling it overnight, and then observing the sedimentation/agglutination pattern (PA test). The neutralization antibody titers were determined by mixing virus-infected hamster antisera in addition to the beads and virus (PAI test). The PA titer was positively correlated with the plaque-forming units. The PAI titer using the hamster antisera clearly revealed the antigenic difference between the omicron and previous variants. The antigenic differences were supported by the results shown in other methods. The PAI test is an easy and rapid method to analyze the antigenicity of SARS-CoV-2.

Will previous palliative surgery for congenital heart disease be detrimental to subsequent pig heart xenotransplantation?

IntroductionPig heart xenotransplantation might act as a bridge in infants with complex congenital heart disease (CHD) until a deceased human donor heart becomes available. Infants develop antibodies to wild-type (WT, i.e., genetically-unmodified) pig cells, but rarely to cells in which expression of the 3 known carbohydrate xenoantigens has been deleted by genetic engineering (triple-knockout [TKO] pigs). Our objective was to test sera from children who had undergone palliative surgery for complex CHD (and who potentially might need a pig heart transplant) to determine whether they had serum cytotoxic antibodies against TKO pig cells. MethodsSera were obtained from children with CHD undergoing Glenn or Fontan operation (n = 14) and healthy adults (n = 8, as controls). All of the children had complex CHD and had undergone some form of cardiac surgery. Seven had received human blood transfusions and 3 bovine pericardial patch grafts. IgM and IgG binding to WT and TKO pig red blood cells (RBCs) and peripheral blood mononuclear cells (PBMCs) were measured by flow cytometry, and killing of PBMCs by a complement-dependent cytotoxicity assay. ResultsAlmost all children and adults demonstrated relatively high IgM/IgG binding to WT RBCs, but minimal binding to TKO RBCs (p < 0.0001 vs WT), although IgG binding was greater in children than adults (p < 0.01). All sera showed IgM/IgG binding to WT PBMCs, but this was much lower to TKO PBMCs (p < 0.0001 vs WT) and was greater in children than in adults (p < 0.05). Binding to both WT and TKO PBMCs was greater than to RBCs. Mean serum cytotoxicity to WT PBMCs was 90% in both children and adults, whereas to TKO PBMCs it was only 20% and < 5%, respectively. The sera from 6/14 (43%) children were cytotoxic to TKO PBMCs, but no adult sera were cytotoxic. ConclusionsAlthough no children had high levels of antibodies to TKO RBCs, 13/14 demonstrated antibodies to TKO PBMCs, in 6 of these showed mild cytotoxicity. As no adults had cytotoxic antibodies to TKO PBMCs, the higher incidence in children may possibly be associated with their exposure to previous cardiac surgery and biological products. However, the numbers were too small to determine the influence of such past exposures. Before considering pig heart xenotransplantation for children with CHD, testing for antibody binding may be warranted.

Screening of Faecal Samples of Pet Dogs for the Presence of Canine Parvoviral Infection by using Haemagglutination Assay and Polymerase Chain Reaction Test

Background: Canine parvovirus-2 causes inflammation of the gastrointestinal tract in dogs. Dogs from Mhow and Indore area suffering from symptoms related to gastroenteritis were investigated for the presence of Canine parvovirus-2 infection. Methods: A total of 50 faecal samples from dogs (33 male and 17 female) were collected individually in 5 ml of phosphate buffer saline solution in sterile containers. These samples were tested in haemagglutination assay using pig red blood cells for the presence of Canine parvovirus-2 specific antigen and in a molecular test, polymerase chain reaction, using primers to amplify Canine parvovirus-2 specific product of 681 base pairs. Result: A total of 5 faecal samples (10%) tested positive in haemagglutination assay indicative of presence of Canine parvoviral-2 antigen in the faecal material. Haemagglutinating titre for positive samples ranged from 32 to 1024. Only one faecal sample found positive in polymerase chain reaction test for amplification of Canine parvovirus-2 specific product. The results of the present study indicate presence of Canine parvoviral-2 infection in pet dogs suffering from gastroenteritis, however at a low level.

A desirable transgenic strategy using GGTA1 endogenous promoter-mediated knock-in for xenotransplantation model

Pig-to-human organ transplantation is a feasible solution to resolve the shortage of organ donors for patients that wait for transplantation. To overcome immunological rejection, which is the main hurdle in pig-to-human xenotransplantation, various engineered transgenic pigs have been developed. Ablation of xeno-reactive antigens, especially the 1,3-Gal epitope (GalT), which causes hyperacute rejection, and insertion of complement regulatory protein genes, such as hCD46, hCD55, and hCD59, and genes to regulate the coagulation pathway or immune cell-mediated rejection may be required for an ideal xenotransplantation model. However, the technique for stable and efficient expression of multi-transgenes has not yet been settled to develop a suitable xenotransplantation model. To develop a stable and efficient transgenic system, we knocked-in internal ribosome entry sites (IRES)-mediated transgenes into the α 1,3-galactosyltransferase (GGTA1) locus so that expression of these transgenes would be controlled by the GGTA1 endogenous promoter. We constructed an IRES-based polycistronic hCD55/hCD39 knock-in vector to target exon4 of the GGTA1 gene. The hCD55/hCD39 knock-in vector and CRISPR/Cas9 to target exon4 of the GGTA1 gene were co-transfected into white yucatan miniature pig fibroblasts. After transfection, hCD39 expressed cells were sorted by FACS. Targeted colonies were verified using targeting PCR and FACS analysis, and used as donors for somatic cell nuclear transfer. Expression of GalT, hCD55, and hCD39 was analyzed by FACS and western blotting. Human complement-mediated cytotoxicity and human antibody binding assays were conducted on peripheral blood mononuclear cells (PBMCs) and red blood cells (RBCs), and deposition of C3 by incubation with human complement serum and platelet aggregation were analyzed in GGTA1 knock-out (GTKO)/CD55/CD39 pig cells. We obtained six targeted colonies with high efficiency of targeting (42.8% of efficiency). Selected colony and transgenic pigs showed abundant expression of targeted genes (hCD55 and hCD39). Knocked-in transgenes were expressed in various cell types under the control of the GGTA1 endogenous promoter in GTKO/CD55/CD39 pig and IRES was sufficient to express downstream expression of the transgene. Human IgG and IgM binding decreased in GTKO/CD55/CD39 pig and GTKO compared to wild-type pig PBMCs and RBCs. The human complement-mediated cytotoxicity of RBCs and PBMCs decreased in GTKO/CD55/CD39 pig compared to cells from GTKO pig. C3 was also deposited less in GTKO/CD55/CD39 pig cells than wild-type pig cells. The platelet aggregation was delayed by hCD39 expression in GTKO/CD55/CD39 pig. In the current study, knock-in into the GGTA1 locus and GGTA1 endogenous promoter-mediated expression of transgenes are an appropriable strategy for effective and stable expression of multi-transgenes. The IRES-based polycistronic transgene vector system also caused sufficient expression of both hCD55 and hCD39. Furthermore, co-transfection of CRISPR/Cas9 and the knock-in vector not only increased the knock-in efficiency but also induced null for GalT by CRISPR/Cas9-mediated double-stranded break of the target site. As shown in human complement-mediated lysis and human antibody binding to GTKO/CD55/CD39 transgenic pig cells, expression of hCD55 and hCD39 with ablation of GalT prevents an effective immunological reaction in vitro. As a consequence, our technique to produce multi-transgenic pigs could improve the development of a suitable xenotransplantation model, and the GTKO/CD55/CD39 pig developed could prolong the survival of pig-to-primate xenotransplant recipients.

ZOOMICS: Comparative Metabolomics of Red Blood Cells From Guinea Pigs, Humans, and Non-human Primates During Refrigerated Storage for Up to 42 Days.

Unlike other rodents, guinea pigs (Cavia porcellus) have evolutionarily lost their capacity to synthesize vitamin C (ascorbate) de novo and, like several non-human primates and humans, rely on dietary intake and glutathione-dependent recycling to cope with oxidant stress. This is particularly relevant in red blood cell physiology, and especially when modeling blood storage, which exacerbates erythrocyte oxidant stress. Herein we provide a comprehensive metabolomics analysis of fresh and stored guinea pig red blood cell concentrates (n = 20), with weekly sampling from storage day 0 through 42. Results were compared to previously published ZOOMICS studies on red blood cells from three additional species with genetic loss of L-gulonolactone oxidase function, including humans (n = 21), olive baboons (n = 20), and rhesus macaques (n = 20). While metabolic trends were comparable across all species, guinea pig red blood cells demonstrated accelerated alterations of the metabolic markers of the storage lesion that are consistent with oxidative stress. Compared to the other species, guinea pig red blood cells showed aberrant glycolysis, pentose phosphate pathway end product metabolites, purine breakdown products, methylation, glutaminolysis, and markers of membrane lipid remodeling. Consistently, guinea pig red blood cells demonstrated higher end storage hemolysis, and scanning electron microscopy confirmed a higher degree of morphological alterations of their red blood cells, as compared to the other species. Despite a genetic inability to produce ascorbate that is common to the species evaluated, guinea pig red blood cells demonstrate accelerated oxidant stress under standard storage conditions. These data may offer relevant insights into the basal and cold storage metabolism of red blood cells from species that cannot synthesize endogenous ascorbate.

Assessment of the hemagglutinating activity of the Porcine orthorubulavirus

Blue eye disease (BED) in pigs is caused by Porcine orthorubulavirus (PRV) of the Paramyxoviridae family. It is an endemic disease in swine production in the central region of Mexico and causes nervous signs and high mortality in suckling pigs, pneumonia in growing pigs, orchitis in boars and mummification during gestation. PRV hemagglutinates most red blood cells (RBCs) of domestic species. For serological diagnosis, the hemagglutination inhibition test is used, and in this test, guinea pig, bovine and chicken RBCs have been commonly used. In this investigation, hemagglutination with PRV was evaluated using the RBCs of seven domestic species (chicken, bovine, horse, pig, dog, guinea pig and rabbit). In the hemagglutination test, the following parameters were evaluated: temperature (25 °C and 37 °C), bottoms of the wells (V and U), erythrocyte concentration (0.5%, 0.75%, and 1%), and reading time (15, 30, 45, 60 and 90 min). Significant differences (P < 0.001) were found in most of the evaluated treatments. The best hemagglutination results were obtained with chicken, bovine and horse RBCs. The hemagglutination titer is higher (2 dilutions) when using chicken RBCs than when using bovine or horse RBCs. If chicken RBCs are used in the inhibition of hemagglutination, the test will be more sensitive, while it is more specific when bovine or horse RBCs are used. The hemagglutination readings are imprecise when using RBCs from dogs, pigs, guinea pigs and rabbits. RBCs from these species should not be used for the diagnosis or investigation of PRV.

Initial experimental experience of triple-knockout pig red blood cells as potential sources for transfusion in alloimmunized patients with sickle cell disease.

Blood transfusion remains important in the treatment of patients with sickle cell disease (SCD). However, alloimmunization after blood transfusion is associated with patient morbidity and mortality. Triple-knockout (TKO) pigs (i.e., pigs in which the three known xenoantigens to which humans have anti-pig antibodies have been deleted) may be an alternative source of RBCs for these patients because many humans have no preformed antibodies to TKO pig RBCs (pRBCs). In an in vitro study, plasma from alloimmunized (n=12) or non-alloimmunized (n=12) SCD patients was used to determine IgM/IgG binding to, and CDC of, TKO pRBCs. In an in vivo study, after an estimated 25% of blood volume was withdrawn from two capuchin monkeys, CFSE-labeled TKO pRBCs were transfused. Loss of TKO pRBCs was monitored by flow cytometry, and 7 weeks later, 25% of blood was withdrawn, and CFSE-labeled monkey RBCs were transfused. The in vitro study demonstrated that plasma from neither alloimmunized nor non-alloimmunized SCD patients bound IgM/IgG to, or induced CDC of, TKO pRBCs. In the in vivo study, survival of TKO pRBCs in the two capuchin monkeys was of 5 and 7 days, respectively, whereas after allotransfusion, survival was >28 days. In conclusion, (1) in the present limited study, no antibodies were detected that cross-reacted with TKO pRBCs, and (2) TKO pigs may possibly be an alternate source of RBCs in an emergency if no human RBCs are available.

Anti-pig IgE and IgA Antibodies in Naive Primates and Nonhuman Primates With Pig Xenografts

Natural preformed anti-pig IgM/IgG antibodies in primates play an important role in xenograft rejection. As it is not clear how IgE and IgA engage in the immune system in xenotransplantation, we investigated natural preformed and elicited anti-pig IgE/IgA in naive primates and after xenotransplantation in nonhuman primates. The binding of IgM/IgG/IgE/IgA antibodies to red blood cells (RBCs) from wild-type (WT), α1,3-galactosyltransferase gene-knockout (GTKO), and GTKO/cytidine monophospho-N-acetylneuraminic acid hydroxylase gene-knockout/β-1,4 N-acetylgalactosaminyltransferase 2 gene-knockout (ie, triple-knockout pigs) pigs were measured by flow cytometry in naive human (n = 50) and baboon (n = 14) sera. Antibody binding to WT and GTKO pig RBCs (pRBCs) was also measured in the sera of baboons (nonsensitized n = 7, sensitized n = 2) and rhesus monkeys (nonsensitized n = 2, sensitized n = 11) following WT or GTKO pig organ/tissue xenotransplantation. Deposition of IgM/IgG/IgE/IgA in the grafts was detected by immunohistochemistry. The majority of humans had natural preformed IgM/IgG/IgE/IgA to WT and GTKO pRBCs. In contrast, IgM/IgG/IgE/IgA to triple-knockout pRBCs were present at lower levels and frequency (P < 0.01). Baboons also had IgM/IgG/IgE/IgA antibodies against WT pRBCs, but fewer to GTKO and triple-knockout (P < 0.01). After xenotransplantation into nonhuman primates, when IgM/IgG increased, IgE/IgA also increased, but to a lesser extent. In addition to IgM/IgG, IgE or IgA deposition was observed in rejected pig xenografts. Primates develop serum anti-pig IgE/IgA antibodies both naturally and during xenograft rejection. The pathophysiological role, if any, of anti-pig IgE/IgA antibodies remains unknown.

NEW WORLD MONKEYS AS MODELS FOR TESTING TRIPLE-KNOCKOUT (TKO) PIG RED BLOOD CELL OR SKIN TRANSPLANTS

Background: TKO pigs are an optimal source of organs for transplantation into human recipients, who do not commonly have high levels of preformed antibodies against TKO pig cells. However, Old World monkeys (OWMs), e.g., baboons, do have antibodies directed to TKO pig cells, associated with significant serum cytotoxicity. New World monkeys (NWMs), e.g., capuchins, have low or no antibodies to TKO pig cells. Our aims were (i) to measure anti-pig IgM/IgG binding and (ii) complement-dependent cytotoxicity (CDC) to GTKO and TKO pig cells using sera from humans, baboons, and capuchins, and (iii) to measure the efficacy of anti-thymocyte globulin (ATG) and Rituximab in these three species. Methods: (i) Sera from humans (n=9), baboons (n=6), and capuchins (n=9) were tested by flow cytometry for binding of IgM and IgG to peripheral blood mononuclear cells (PBMCs) from GTKO and TKO pigs. Binding intensity was presented as relative geometric mean (rGM). (ii) The sera (final concentration 25%) were also tested for CDC to the same pig cells by flow cytometry. (iii) The effect of ATG and Rituximab on human (n=2), baboon (n=2), and capuchin (n=3) PBMCs or B cells deletion was measured by flow cytometry. Results: Binding of serum IgM/IgG to pig cells (Figure1) Mean serum anti-GTKO IgM and IgG binding were both significantly lower in baboons than in humans and capuchins (p<0.01). Mean baboon anti-TKO IgM binding was significantly higher than in humans and capuchins (p<0.01). Mean capuchin anti-TKO IgG was significantly lower than in humans and baboons (p<0.01). Serum CDC to pig cells (Figure1) Mean serum CDC of GTKO PBMCs did not differ between the three species. Baboon serum CDC of TKO PBMCs was significantly greater than in humans and capuchins (p<0.01), but there was no difference between humans and capuchins. The CDC of ATG and Rituximab on PBMCs and B cells (Figure2) ATG (25-50µg/ml) depleted fewer PBMCs in capuchins and baboons than in humans, but the depletion in capuchins was not significantly different from that in baboons, except at high concentration (100µg/ml). In contrast, the effect of Rituximab in depleting B cells in capuchins was very limited, and significantly less than in humans and baboons (p<0.05 or 0.01). Conclusions: When TKO pig organs are transplanted, OWMs, e.g., baboons, would be less than ideal as recipients as they do not mimic the human immune response. NWMs, e.g., capuchins, are to be preferred. However, their small size and the reduced effect of ATG, and, particularly, Rituximab, limit their use as recipients of organs, though they would be ideal for studies of TKO pig red blood cell transfusion or skin transplantation, where immunosuppressive therapy is not required.Work on xenotransplantation at the University of Alabama at Birmingham is supported in part by NIH NIAID U19 grant AI090959 and in part by a grant to UAB from United Therapeutics.

Comparison of antigenic mutation during egg and cell passage cultivation of H3N2 influenza virus.

PurposeWhen influenza viruses are cultured in eggs, amino acid mutations of the hemagglutinin may occur through egg adaptation. On the other hand, when influenza viruses are cultured in animal cells, no antigenic mutation occurs unlike in eggs. Therefore, we examined whether the antigenic mutations actually occurred after passage of H3N2 (A/Texas/50/2012) virus up to 15 times in eggs and MDCK-Sky3851 cells.Materials and MethodsPrototype A/Texas/50/2012 (H3N2) influenza virus which was isolated from clinical patient, not passaged in egg, was obtained and propagated using the specific pathogen free egg and the MDCK-Sky3851 cell line up to 15 passage, and the changes in the antigen sequence of the influenza viruses were confirmed by gene sequencing and protein structure analysis.ResultsIn term of the hemagglutination titer of influenza virus, the reactivity to chicken and guinea pig red blood cell showed different results between egg propagated and cell propagated viruses. In the sequence analysis results for hemagglutinin and neuraminidase, no antigenic mutation was observed throughout all passages when cultured in MDCK-Sky3851 cells. On the other hand, mutations occurred in three amino acid sequences (H156R, G186S, S219F) in hemagglutinin up to 15 passages when cultured in eggs.ConclusionH3N2 influenza virus cultured in eggs could lead mutations in amino acid sequence of hemagglutinin, distinct from the corresponding virus cultured in cells for which no antigenic mutation was observed. These findings suggest that cell culture is a more stable and effective way of production with lower risk of antigenic mutations for the manufacture of influenza vaccines.

Carboxylic Acids with Certain Molecular Structures Decrease Osmotic Fragility against Osmotic Pressure in Cattle Erythrocytes In Vitro: Appearance of a Wedge-like Effect Similar to RBCs in Other Animal Species

Osmotic fragility (OF) in red blood cells (RBCs) is a useful tool for evaluating the actions of various chemicals on the cell membrane in vitro. The effects of monocarboxylic and dicarboxylic acids on OF were evaluated in cattle RBCs. Isolated cattle RBCs were immersed in various carboxylic acids at 0-100 mM in a buffer solution for 1 hr and the 50% hemolysis was then determined by soaking in 0.1-0.8% NaCl solution. Although n-caprylic acid at 100 mM induced hemolysis, the other monocarboxylic acids possessing straight hydrocarbons did not affect OF. The dicarboxylic acids possessing straight hydrocarbons, except for glutaric acid, decreased OF in a dose-dependent manner. Some monocarboxylic acids with branched hydrocarbons tended to decrease OF, but these changes were not statistically significant. Although cyclopentanecarboxylic and cyclohexanecarboxylic acids at 100 mM decreased OF, other monocarboxylic acids with cyclic hydrocarbons did not affect OF. Among the dicarboxylic acids with cyclic hydrocarbons tested, only 1,2-cyclohexanedicarboxylic acid and phthalic acid with a benzene ring significantly decreased OF. There is no clear correlation between the effect of monocarboxylic or dicarboxylic acids on OF, and their partition coefficients. Thus, the partition coefficient is not a suitable parameter for explaining the effect of both groups of carboxylic acids on OF in cattle RBCs. With regard to the effect of monocarboxylic acids on OF, although an increase in OF was demonstrated in rat RBCs, no effect or rather a decrease in OF was demonstrated in cattle RBCs, similar to the results observed for guinea pig and sheep RBCs. With regard to the effect of dicarboxylic acids, decreases in OF were already demonstrated in rat, guinea pig and sheep RBCs. We have proposed that dicarboxylic acids exhibit a common stabilizing effect on the RBC membrane in various animals, which we termed a “wedge-like effect”. We clarified that cattle RBCs also show a similar OF response to dicarboxylic acids in this experiment.

Porcine signal regulatory protein alpha binds to human CD47 to inhibit phagocytosis: Implications for human hematopoietic stem cell transplantation into severe combined immunodeficient pigs.

Severe combined immunodeficient (SCID) pigs are an emerging animal model being developed for biomedical and regenerative medicine research. SCID pigs can successfully engraft human-induced pluripotent stem cells and cancer cell lines. The development of a humanized SCID pig through xenotransplantation of human hematopoietic stem cells (HSCs) would be a further demonstration of the value of such a large animal SCID model. Xenotransplantation success with HSCs into non-obese diabetic (NOD)-derived SCID mice is dependent on the ability of NOD mouse signal regulatory protein alpha (SIRPA) to bind human CD47, inducing higher phagocytic tolerance than other mouse strains. Therefore, we investigated whether porcine SIRPA binds human CD47 in the context of developing a humanized SCID pig. Peripheral blood mononuclear cells (PBMCs) were collected from SCID and non-SCID pigs. Flow cytometry was used to assess whether porcine monocytes could bind to human CD47. Porcine monocytes were isolated from PBMCs and were subjected to phagocytosis assays with pig, human, and mouse red blood cell (RBC) targets. Blocking phagocytosis assays were performed by incubating human RBCs with anti-human CD47 blocking antibody B6H12, non-blocking antibody 2D3, and nonspecific IgG1 antibody and exposing to human or porcine monocytes. We found that porcine SIRPA binds to human CD47 in vitro by flow cytometric assays. Additionally, phagocytosis assays were performed, and we found that porcine monocytes phagocytose human and porcine RBCs at significantly lower levels than mouse RBCs. When human RBCs were preincubated with CD47 antibodies B6H12 or 2D3, phagocytosis was induced only after B6H12 incubation, indicating the lower phagocytic activity of porcine monocytes with human cells requires interaction between porcine SIRPA and human CD47. We have shown the first evidence that porcine monocytes can bind to human CD47 and are phagocytically tolerant to human cells, suggesting that porcine SCID models have the potential to support engraftment of human HSCs.