Baboon Sera Research Articles

Erythrocytes from GGTA1/CMAH knockout pigs: implications for xenotransfusion and testing in non-human primates.

Pig erythrocytes are potentially useful to solve the worldwide shortage of human blood for transfusion. Domestic pig erythrocytes, however, express antigens that are bound by human preformed antibodies. Advances in genetic engineering have made it possible to rapidly knock out the genes of multiple xenoantigens, namely galactose α1,3 galactose (aGal) and N-glycolylneuraminic acid (Neu5Gc). We have recently targeted the GGTA1 and CMAH genes with zinc finger endonucleases resulting in double knockout pigs that no longer express aGal or Neu5Gc and attract significantly fewer human antibodies. In this study, we characterized erythrocytes from domestic and genetically modified pigs, baboons, chimpanzees, and humans for binding of human and baboon natural antibody, and complement-mediated lysis. Distribution of anti-Neu5Gc IgG and IgM in pooled human AB serum was analyzed by ELISA. Erythrocytes from domestic pigs (Dom), aGal knockout pigs (GGTA1 KO), aGal and Neu5Gc double knockout pigs (GGTA1/CMAH KO), baboons, chimpanzees, and humans were analyzed by flow cytometry for aGal and Neu5Gc expression. In vitro comparative analysis of erythrocytes was conducted with pooled human AB serum and baboon serum. Total antibody binding was accessed by hemagglutination; complement-dependent lysis was measured by hemolytic assay; IgG or IgM binding to erythrocytes was characterized by flow cytometry. The pooled human AB serum contained 0.38 μg/ml anti-Neu5Gc IgG and 0.085 μg/ml anti-Neu5Gc IgM. Both Gal and Neu5Gc were not detectable on GGTA1/CMAH KO erythrocytes. Hemagglutination of GGTA1/CMAH KO erythrocytes with human serum was 3.5-fold lower compared with GGTA1 KO erythrocytes, but 1.6-fold greater when agglutinated with baboon serum. Hemolysis of GGTA1/CMAH KO erythrocytes by human serum (25%) was reduced 9-fold compared with GGTA1 KO erythrocytes, but increased 1.64-fold by baboon serum. Human IgG binding was reduced 27-fold on GGTA1/CMAH KO erythrocytes compared with GGTA1 KO erythrocytes, but markedly increased 3-fold by baboon serum IgG. Human IgM binding was decreased 227-fold on GGTA1/CMAH KO erythrocytes compared with GGTA1 KO erythrocytes, but enhanced 5-fold by baboon serum IgM. Removal of aGal and Neu5Gc antigens from pig erythrocytes significantly reduced human preformed antibody-mediated cytotoxicity but may have complicated future in vivo analysis by enhancing reactivity from baboons. The creation of the GGTA1/CMAH KO pig has provided the xenotransplantation researcher with organs and cells that attract fewer human antibodies than baboon and our closest primate relative, chimpanzee. These finding suggest that while GGTA1/CMAH KO erythrocytes may be useful for human transfusions, in vivo testing in the baboon may not provide a direct transplantation to the clinic.

Rituximab Treatment Prevents the Early Development of Proteinuria following Pig-to-Baboon Xeno-Kidney Transplantation

We previously reported life-supporting α1,3-galactosyltransferase knockout (GalTKO) thymokidney xenograft survival of >2 months in baboons. However, despite otherwise normal renal function, recipients developed proteinuria with morphologic changes (podocyte effacement), a condition that presents a major obstacle to long-term studies in this model. A recent clinical study showed that rituximab therapy after allogeneic transplant prevented proteinuria possibly associated with loss of sphingomyelin phosphodiesterase acid-like 3b (SMPDL-3b). Here, we demonstrate that rituximab prevents the disruption of pig podocytes in an SMPDL-3b-dependent manner in vitro and the early development of proteinuria after xenogeneic kidney transplantation in baboons. Immunofluorescence showed SMPDL-3b expression in pig glomerular epithelium; immunoprecipitation demonstrated rituximab binding to SMPDL-3b in glomeruli. Culture of isolated pig podocytes with naive baboon sera, which has preformed antipig natural antibodies, reduced SMPDL-3b expression, disrupted podocyte morphology, and decreased podocyte proliferation, whereas pretreatment with rituximab prevented these effects. Six baboons received rituximab before transplantation to deplete B cells and again in the peri-transplant period; 18 baboons treated only before transplantation served as historical controls. The onset of post-transplant proteinuria was significantly delayed in a B cell-independent manner in the animals that received peri-transplant rituximab treatment. Although further optimization of this protocol is required, these data provide intriguing clues to the mechanisms of post-transplant proteinuria in xenogeneic kidney transplantation and a potential strategy for its prevention.

Optimization of cytotoxicity assay by real-time, impedance-based cell analysis

This paper presents an optimized procedure for assessing an immune-mediated cytotoxicity, produced after the addition of human and baboon serum to transgenic porcine fibroblasts. This procedure is performed with the xCELLigence Real-Time Cell Analyzer (RTCA). The xCELLigence system measures the impedance variations in the culture media of a 96-well microelectronic plate, and shows the changes in cell number and morphology in a real-time plot. However, different factors need to be optimized before developing an RTCA assay. Thus, we studied the influence of several variables, such as the number of cells seeded, the time the cells were allowed to grow before the tests, the serum concentration and the addition of rabbit complement. The findings were confirmed by the WST-1 classical cytotoxicity test. The results showed that 7.5 × 10(3) cells seeded per well produced the adequate CI in 10h. The area under the curve and the CImin versus concentration values showed a very high correlation index (r(2) = 0.966 and r(2) = 0.92 for the first 50h after challenge, respectively), proving that CI variations are directly proportional to the quantity of serum added. The addition of complement resulted in lower CImin values. Therefore, both the cytolysis level with and without exogenous complement addition had to be assessed. There was a high correlation between the relative cytotoxicity assessed by WST-1 and the CI obtained by RTCA when exogenous complement was not added (r(2) = 0.827; p < 0.001). The correlation was average when rabbit complement was added (r(2) = 0.523; p = 0.046). In conclusion, culture conditions have an important influence on RTCA cytotoxicity assays.

Effect of 30 % nutrient restriction in the first half of gestation on maternal and fetal baboon serum amino acid concentrations

Mechanisms linking maternal nutrient restriction (MNR) to intra-uterine growth restriction (IUGR) and programming of adult disease remain to be established. The impact of controlled MNR on maternal and fetal amino acid metabolism has not been studied in non-human primates. We hypothesised that MNR in pregnant baboons decreases fetal amino acid availability by mid-gestation. We determined maternal and fetal circulating amino acid concentrations at 90 d gestation (90dG, term 184dG) in control baboons fed ad libitum (C, n 8) or 70% of C (MNR, n 6). Before pregnancy, C and MNR body weights and circulating amino acids were similar. At 90dG, MNR mothers had lower body weight than C mothers (P< 0·05). Fetal and placental weights were similar between the groups. MNR reduced maternal blood urea N (BUN), fetal BUN and fetal BUN:creatinine. Except for histidine and lysine in the C and MNR groups and glutamine in the MNR group, circulating concentrations of all amino acids were lower at 90dG compared with pre-pregnancy. Maternal circulating amino acids at 90dG were similar in the MNR and C groups. In contrast, MNR fetal β-alanine, glycine and taurine all increased. In conclusion, maternal circulating amino acids were maintained at normal levels and fetal amino acid availability was not impaired in response to 30% global MNR in pregnant baboons. However, MNR weight gain was reduced, suggesting adaptation in maternal-fetal resource allocation in an attempt to maintain normal fetal growth. We speculate that these adaptive mechanisms may fail later in gestation when fetal nutrient demands increase rapidly, resulting in IUGR.

Assessment of In Vitro Heparin Complement Regulation Capacity During Real-Time Cell Analyzer Antibody-Mediated Cytolysis Assay: Compatibility Studies for Pig-to-Baboon Xenotransplantation

ObjectiveTo assess the effect of sodium heparin concentrations on antibody- and complement-mediated cytolysis by means of a real-time cell analyzer system (RTCA) investigating the complement regulation ability of heparin to reduce or prevent hyperacute in an in vitro model of pig-to-baboon xenotransplantation. Materials and methodsFibroblasts isolated from the skin of two transgenic pigs were cultured in microelectronic 96-well plates for 9 hours. Then, we added 20 μL of normal sera from two healthy adult olive baboons (Papio anubis) or two volunteer healthy humans. Simultaneous cultures had added heparin at 3.5, 5, 7.5, 15, and 30 IU. Moreover, rabbit complement was added for the exogenous complement group (ExC) versus the other group only with the complement present in the sera as an endogenous complement group (EnC). Cellular cultures were monitored over 150 hours after challenge. With cellular index (CI) data recorded by the xCELLigence software system, we calculate area under the curve versus concentration (AUC) and minimum CI (CImin) versus concentration. ResultsAll cultures showed decreased CI after challenge with human or baboon sera. There was a high correlation for AUC (r2 > 0.90) and CImin versus concentration (r2 > 0.970) during the first 40 hours postchallenge among the EnC group, regardless of human or baboon sera. However, there was no correlation for AUC and CImin for the ExC group. There was a reduction of CImin related to increased heparin concentrations. ConclusionsThe addition of heparin did not reduce antibody- and complement-mediated cytolysis assessed in vitro by RTCA in pig-to-baboon compatibility assays.

Validation of xCELLigence Real-Time Cell Analyzer to Assess Compatibility in Xenotransplantation With Pig-to-Baboon Model

Objective To validate the use of a microelectronic real-time cell analyzer system (RTCA) we developed a complement-mediated antibody cytotoxicity assay to investigate the compatibility of a graft and a recipient in pig-to-baboon xenotransplantation. Materials and methods Fibroblasts isolated from the skin of five hCD55, hCD59, and hCD46 transgenic pigs (TP) were cultured in 96 microelectronic well plates for 17 hours. Then, we added to each microwell 20 μL of normal sera from nine healthy adult olive baboons ( Papio anubis)—three males and six females. The evolution of the cell culture was assessed every 3 minutes during the pretreatment period, at 11 hours postaddition, and every 30 minutes from 12 to 96 hours. Simultaneously, we performed a 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Fibroblasts from wild-type (WT) pigs were used as positive controls and microwells without serum addition from each TP as negative controls. The RTCA results were expressed as a normalized cellular index (NCI). Results Differences were observed between the five TP fibroblasts and the WT fibroblasts, with greater cytotoxicity on WT cells. Among TP, a higher cytolytic level was observed in males than females. The MTT results correlated with NCI at different times, with the minimum NCI and with the time to for NCI recovery before serum addition. The correlation was lower than that previously reported in environmental toxicity assays. Conclusions RTCA allows a long-term assessment of the immunocytotoxic effect of baboon sera on pig cells, providing a suitable tool to perform compatibility tests for xenotransplantation.

Atorvastatin or transgenic expression of TFPI inhibits coagulation initiated by anti-nonGal IgG binding to porcine aortic endothelial cells.

Intravascular thrombosis remains a barrier to successful xenotransplantation. Tissue factor (TF) expression on porcine aortic endothelial cells (PAECs), which results from their activation by xenoreactive antibodies (Abs) to Galα1,3Gal (Gal) and subsequent complement activation, plays an important role. The present study aimed to clarify the role of Abs directed against nonGal antigens in the activation of PAECs to express functional TF and to investigate selected methods of inhibiting TF activity. PAECs from wild-type (WT), α1,3-galactosyltransferase gene-knockout (GT-KO) pigs, or pigs transgenic for CD46 or tissue factor pathway inhibitor (TFPI), were incubated with naïve baboon serum (BS) or sensitized BS (with high anti-nonGal Ab levels). TF activity of PAECs was assessed. Only fresh, but not heat-inactivated (HI), naïve BS activated WT PAECs to express functional TF. Similarly, PAECs from CD46 pigs were resistant to activation by naïve BS, but not to activation by fresh or HI sensitized BS. HI sensitized BS also activated GT-KO PAECs to induce TF activity. TF expression on PAECs induced by anti-nonGal Abs was inhibited if serum was pretreated with (i) an anti-IgG Fab Ab or (ii) atorvastatin, or (iii) when PAECs were transgenic for TFPI. Anti-nonGal IgG Abs activated PAECs to induce TF activity through a complement-independent pathway. This implies that GT-KO pigs expressing a complement-regulatory protein may be insufficient to prevent the activation of PAECs. Genetic modification with an 'anticoagulant' gene (e.g. TFPI) or a therapeutic approach (e.g. atorvastatin) will be required to prevent coagulation dysregulation after pig-to-primate organ transplantation.

Investigation of potential carbohydrate antigen targets for human and baboon antibodies

The continued presence of a primate antibody-mediated response to cells and organs from alpha1,3-galactosyltransferase gene-knockout (GTKO) pigs indicates that there may be antigens other than Gal alpha 1,3Gal (alpha Gal) against which primates have xenoreactive antibodies. Human and baboon sera were tested for reactivity against a panel of saccharides that might be potential antigen targets for natural anti-non-alpha Gal antibodies. Human sera (n = 16) and baboon sera (n = 15) of all ABO blood types were tested using an enzyme-linked immunoadsorbent assay for binding of IgM and IgG to a panel of synthetic polyacrylamide-linked saccharides (n = 15). Human sera were also tested after adsorption on alpha Gal immunoaffinity beads. Sera from healthy wild-type (WT, n = 6) and GTKO (n = 6) pigs and from baboons (n = 4) sensitized to GTKO pig organ or artery transplants (of blood type O) were also tested. Forssman antigen expression on baboon and pig tissues was investigated by immunohistochemistry. Both human and baboon sera showed high IgM and IgG binding to alpha Gal saccharides, alpha-lactosamine, and Forssman disaccharide. Human sera also demonstrated modest binding to N-glycolylneuraminic acid (Neu5Gc). When human sera were adsorbed on alpha Gal oligosaccharides, there was a reduction in binding to alpha Gal and alpha-lactosamine, but not to Forssman. WT and GTKO pig sera showed high binding to Forssman, and GTKO pig sera showed high binding to alpha Gal saccharides. Baboon sera sensitized to GTKO pigs showed no significant increased binding to any specific saccharide. Staining for Forssman was negative on baboon and pig tissues. We were unable to identify definitively any saccharides from the selected panel that may be targets for primate anti-non-alpha Gal antibodies. The high level of anti-Forssman antibodies in humans, baboons, and pigs, and the absence of Forssman expression on pig tissues, suggest that the Forssman antigen does not play a role in the primate immune response to pigs.

Human Immune Reactivity against Liver Sinusoidal Endothelial Cells from GalTα(1,3)GalT-Deficient Pigs

Elimination of galactose-α(1,3)galactose (Gal) expression in pig organs has been previously shown to prevent hyperacute xenograft rejection. However, naturally present antibodies to non-Gal epitopes activate endothelial cells, leading to acute humoral xenograft rejection. Still, it is unknown whether xenogeneic pig liver sinusoidal endothelial cells (LSECs) from α(1,3)galactosyltransferase (GalT)-deficient pigs are damaged by antibody and complement-mediated mechanisms. The present study examined the xeno-antibody response of LSECs from GalT-deficient and wild pigs. Isolated LSEC from wild-type and GalT pigs were expose to human and baboon sera; IgM and IgG binding was analyzed by flow cytometry. Complement activation (C3a and CH50) was quantified in vitro from serum-exposed LSEC cultures using Enzyme-Linked ImmunoSorbent assay (ELISA). Levels of complement-activated cytotoxicity (CAC) were also determined by a fluorescent Live-Dead Assay and by the quantification of LDH release. IgM binding to GalT knockout (KO) LSECs was significantly lower (80% human and 87% baboon) compare to wild-type pig LSEC. IgG binding was low in all groups. Moreover, complement activation (C3a and CH50) levels released following exposure to human or baboon sera were importantly reduced (42% human and 52% baboon), CAC in GalT KO LSECs was reduced by 60% in human serum and by 72% in baboon serum when compared to wild-type LSECs, and LDH release levels were reduced by 37% and 57%, respectively. LSECs from GalT KO pigs exhibit a significant protection to humoral-induced cell damage compared to LSECs from wild pigs when exposed to human serum. Although insufficient to inhibit xenogeneic reactivity completely, transgenic GalT KO expression on pig livers might contribute to a successful application of clinical xenotransplantation in combination with other protective strategies.

Prophylactic and curative activities of extracts from Warburgia ugandensis Sprague (Canellaceae) and Zanthoxylum usambarense (Engl.) Kokwaro (Rutaceae) against Plasmodium knowlesi and Plasmodium berghei

Ethnopharmacological relevance This work reports the anti-plasmodial activities of Warburgia ugandensis and Zanthoxylum usambarense commonly used as phytomedicines against malaria by some Kenyan communities. Aim of study To determine the anti-plasmodial activities of extracts from Warburgia ugandensis and Zanthoxylum usambarense against Plasmodium knowlesi and Plasmodium berghei. Materials and methods Eight plant extracts were screened for in vitro anti-plasmodial activity against Plasmodium knowlesi, in a 96-well plate incubated at 37 °C on a RPMI culture medium supplemented with baboon serum. Of the eight, three were investigated for prophylactic and curative activities in BALB/c mice against drug-sensitive Plasmodium berghei in a 4-day test at a dose rate of 200 mg/kg/day. Results Inhibitory concentrations (IC 50) values of between 3.14 and 75 μg/ml, up to 69% chemosuppression of parasites growth and over 80% survivorship of treated mice were observed. Conclusion The two medicinal plants, Warburgia ugandensis and Zanthoxylum usambarense possess bioactive compounds against malaria parasites and could be exploited for further development into malaria therapy.

Structural characterization of α1,3-galactosyltransferase knockout pig heart and kidney glycolipids and their reactivity with human and baboon antibodies

alpha1,3-galactosyltranferase knockout (GalT-KO) pigs have been established to avoid hyperacute rejection in GalT-KO pig-to-human xenotransplantation. GalT-KO pig heart and kidney glycolipids were studied focusing on elimination of Gal-antigens and whether novel antigens would appear. Non-human primates are used as pre-clinical transplantation experimental models. Therefore, sera from baboons transplanted with GalT-KO hearts were compared with human serum regarding reactivity with pig glycolipids. Neutral and acidic glycolipids were isolated from GalT-KO and WT pig hearts and kidneys. Glycolipid immune reactivity was tested on TLC plates using human affinity-purified anti-Gal Ig, anti-blood group monoclonal antibodies, lectins, and human serum as well as baboon serum collected before and after GalT-KO pig heart transplantations. Selected glycolipid fractions, isolated by HPLC, were structurally characterized by mass spectrometry and proton NMR spectroscopy. GalT-KO heart and kidney lacked alpha3Gal-terminated glycolipids completely. Levels of uncapped N-acetyllactosamine precursor compounds, blood group H type 2 core chain compounds, the P1 antigen and the x(2) antigen were increased. Human serum antibodies reacted with Gal-antigens and N-glycolylneuraminic acid (NeuGc) in WT organs of which only the NeuGc reactivity remained in the GalT-KO tissues. A clear difference in reactivity between baboon and human antibodies with pig glycolipids was found. This was most pronounced for acidic, not yet identified, compounds in GalT-KO organs which were less abundant or lacking in the corresponding WT tissues. GalT-KO pig heart and kidney completely lacked Gal glycolipid antigens whilst glycolipids synthesized by competing pathways were increased. Baboon and human serum antibodies showed a different reactivity pattern to pig glycolipid antigens indicating that non-human primates have limitations as a human pre-clinical model for immune rejection studies.

A sensitive and specific liquid chromatography/tandem mass spectrometry method for quantification of nevirapine and its five metabolites and their pharmacokinetics in baboons

A highly sensitive and specific LC-MS/MS assay was developed and validated to quantify nevirapine (NVP) and its five metabolites [2-, 3-, 8-, 12-hydroxyl NVP (OHNVP) and 4-carboxyl NVP (CANVP)] simultaneously in baboon serum and the assay was used to characterize their pharmacokinetic studies of an oral-dose escalation study in baboon. The lower limit of quantification (LLOQ) for NVP and its four hydroxyl nevirapine metabolites was 1.0 ng/mL and for 4-CANVP was 5.0 ng/mL. The between-run and within-run precisions and accuracies at four quality control concentrations (1, 5, 50 and 500 ng/mL) were evaluated in baboon serum with less than 14% variation and 93-114% accuracies (n = 6), except for the LLOQ for 2-OHNVP, which had an accuracy of 115.8% for between-run validation. The pharmacokinetics of NVP and its five metabolites in non-pregnant baboons by a single-dose escalation study were also profiled. The major metabolites detected were 4-CANVP and 12-OHNVP. 3-OHNVP and 2-OHNVP were the minor metabolites with only a trace amount of 2-OHNVP detected in some pharmacokinetic samples. No 8-OHNVP was observed in all of the pharmacokinetic samples. In addition, the fragmentation for the four hydroxyl metabolite isomers is also discussed.

Baboon serum is superior to human or bovine serum albumin for baboon sperm capacitation and zona binding

Baboon in vitro fertilization requires capacitated sperm inappropriate media. In this study, we compared the effect of baboon serum (Bas), human serum albumin (HSA) and bovine serum albumin (BSA) on baboon sperm capacitation. Five males (n = 5) were electroejaculated and 43 oocytes retrieved from super-ovulated female baboons (n = 10). Each sperm sample was assessed for initial motility and concentration before and after swim-up. For swim-up, each sperm sample was incubated separately in Biggers-Whitten-Whittingham media containing either BaS, HSA, BSA or without protein supplementation (control). After swim-up, each sperm aliquot was incubated with two to three oocytes. The number of sperm bound to the zona was evaluated after overnight incubation. Sperm motility and zona binding was significantly higher after capacitation in media supplemented with BaS than in HSA or BSA or in media without protein supplementation (P < 0.05). Baboon serum is superior to HSA or BSA for baboon sperm capacitation and zona binding.

In vitroinvestigation of pig cells for resistance to human antibody-mediated rejection

Although human complement-dependent cytotoxicity (CDC) of alpha1,3-galactosyltransferase gene-knockout (GTKO) pig cells is significantly weaker than that of wild-type (WT) cells, successful xenotransplantation will require pigs with multiple genetic modifications. Sera from healthy humans were tested by (i) flow cytometry for binding of IgM/IgG, and (ii) CDC assay against peripheral blood mononuclear cells and porcine aortic endothelial cells from five types of pig - WT, GTKO, GTKO transgenic for H-transferase (GTKO/HT), WT transgenic for human complement regulatory protein CD46 (CD46) and GTKO/CD46. There was significantly higher mean IgM/IgG binding to WT and CD46 cells than to GTKO, GTKO/HT, and GTKO/CD46, but no difference between GTKO, GTKO/HT, and GTKO/CD46 cells. There was significantly higher mean CDC to WT than to GTKO, GTKO/HT, CD46, and GTKO/CD46 cells, but no difference between GTKO and GTKO/HT. Lysis of GTKO/CD46 cells was significantly lower than that of GTKO or CD46 cells. CD46 expression provided partial protection against serum from a baboon sensitized to a GTKO pig heart. GTKO/CD46 cells were significantly resistant to lysis by human serum and sensitized baboon serum. In conclusion, the greatest protection from CDC was obtained by the combination of an absence of Gal expression and the presence of CD46 expression, but the expression of HT appeared to offer no advantage over GTKO. Organs from GTKO/CD46 pigs are likely to be significantly less susceptible to CDC.

Late onset of development of natural anti-nonGal antibodies in infant humans and baboons: implications for xenotransplantation in infants

If an ABO-incompatible heart is transplanted into an infant before natural antibodies have developed to the specific donor carbohydrate A/B antigen(s), then B-cell tolerance to the donor A/B antigen is achieved, and these antibodies never develop. Anti-carbohydrate antibodies play a role in the rejection of wild type (WT) and alpha1,3-galactosyltransferase gene-knockout (GT-KO) pig xenografts. We investigated development of these antibodies in infant baboons and humans. Serum samples from infant baboons (n = 42) and humans (n = 42) were tested by flow cytometry for immunoglobulin M and immunoglobulin G binding to peripheral blood mononuclear cells from WT and GT-KO pigs, and for complement-dependent cytotoxicity. The presence of anti-blood group antibodies was tested in baboon serum. In infant baboons and humans, cytotoxic anti-Galalpha1,3Gal antibodies develop during the first 3 months, and steadily increase with age, whereas cytotoxic anti-nonGal antibodies are either absent or minimal in the majority of cases throughout the first year of life. Anti-blood group antibodies were not detected before 16 weeks of age. Our data suggest GT-KO pig organ/cell transplants could be carried out in early infancy in the absence of preformed cytotoxic anti-nonGalalpha1,3Gal antibodies.

A liquid chromatography/atmospheric pressure ionization tandem mass spectrometry quantitation method for nevirapine and its two oxidative metabolites, 2‐hydroxynevirapine and nevirapine 4‐carboxylic acid, and pharmacokinetics in baboons

A rapid highly sensitive and specific electrospray ionization (ESI) liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for quantification of nevirapine (NVP) and its two metabolites, 2-hydroxynevirapine (2-OHNVP) and nevirapine 4-carboxylic acid (4-CANVP), in baboon serum was developed and validated. Nevirapine, 2-OHNVP, 4-CANVP, and the internal standard, hesperetin, were extracted from baboon serum with ethyl acetate. Components in the extract were separated on a 50 x 2.1 mm Aquasil C(18) 5 microm stainless steel column by isocratic elution with 40% acetonitrile/0.1% formic acid at a flow rate of 0.2 mL/min. The liquid flow was passed through a pre-source splitter and 5% of the eluant was introduced into the atmospheric pressure ionization (API) source. The components were analyzed in the multiple-reaction monitoring (MRM) mode as the precursor/product ion pair of m/z 267.2/226.2 for NVP, 283.0/161.2 for 2-OHNVP, 297.2/279.2 for 4-CANVP, and 303.2/177.2 for hesperetin. Linear calibration curves were obtained in the range of 1-1000 ng/mL for NVP and 2-OHNVP and 5-1000 ng/mL for 4-CANVP, using 0.2 mL baboon serum, respectively. The within-day and between-day precisions were <10% for NVP and 2-OHNVP, and <11.5% for 4-CANVP. Due to the similar structures and fragmentation patterns of 2-OHNVP and 3-OHNVP, it is not expected that the LC/MS/MS can differentiate 2-OHNVP and 3-OHNVP and they were assayed as a composite. The method was applied to a single-dose escalation study of NVP in non-pregnant baboons (Papio anubis) to characterize the pharmacokinetics of NVP, 2-OHNVP plus 3-OHNVP, and 4-CANVP, and to determine the appropriate dose necessary to achieve comparable peak serum concentration of NVP as reported in healthy human adults.

A preliminary study of the baboon prostate pathophysiology

Prostate cancer, benign prostatic hyperplasia, and prostatitis frequently affect men worldwide. At present there are no suitable animal models for these diseases. This study explores the potential use of the baboon as a model for prostatic diseases. Prostates of 48 baboons of different ages were studied. Prostate specific antigen (PSA) and alpha-methyl-acyl-CoA racemase (AMACR) were localized in the different lobes of the prostate by Western blotting and immunohistochemistry. PSA in baboon serum was demonstrated by radioimmunoassay and western blotting. Baboon AMACR cDNA was cloned and its expression assayed in baboon tissues. The baboon prostate is anatomically and histologically similar to its human counterpart, with cranial and caudal lobes corresponding to central and peripheral zones of the human prostate. We found lymphocytic infiltration (91%), and sclerosing/atrophic lesions (34%). PSA tissue immunostaining intensity and alpha-methyl-acyl-CoA racemase (AMACR) gene expression levels differed between the cranial and caudal lobes of the prostate. The cloned baboon AMACR cDNA showed 96% homology with its human counterpart. Anti-human AMACR, PSA and basal keratin antibodies stained intracellular and basement membrane structures in the baboon prostate. The sclerosing/atrophic lesions were comparable to their human counterparts. The similarity of baboon prostate to its human counterpart and the fact that human antibodies (AMACR, PSA, basal keratin) are reactive to baboon prostatic proteins indicates that the baboon is a promising model for human prostatic diseases.

Measurement of anti‐CD154 monoclonal antibody in primate sera by competitive inhibition ELISA

Anti-human CD154 monoclonal antibody (mAb)-based regimens have been demonstrated to prevent T cell-dependent elicited antibody response in baboon recipients of pig hematopoietic progenitor cells, organs and islets. Monitoring of anti-CD154 mAb in serum is important to ensure maintenance of adequate levels and for adjusting dosage of the anti-CD154 mAb. We describe a method for measuring the level in primate sera. The anti-CD154 mAb level in primate serum was measured with a competitive inhibition enzyme linked immunosorbent assay in which the extent of inhibition of binding by anti-CD154 mAb conjugated to horseradish peroxidase (anti-CD154-HRP) to soluble CD154 was used to determine the serum level. Briefly, a 96-well maxisorb plate coated with soluble human CD154, and blocked with bovine serum albumin, was loaded with graded doses of anti-CD154 mAb or primate sera containing anti-CD154 mAb. Both were mixed with a known dosage of anti-CD154-HRP before loading. Bound anti-CD154-HRP was detected by color developed using 3,3',5,5' tetramethyl-benzidine as substrate. Absorbance was measured in a Synergy HT Multi-Detection Microplate Reader at a wavelength of 450 nm. Data analysis was carried out using BioTek's KC4 Data Analysis Software. The standard curve was generated from the wells loaded with the mixture of anti-CD154 mAb and anti-CD154-HRP. The assay has been used successfully to measure anti-CD154 mAb levels in the serum of both baboons and monkeys.

Effect of an Anti-C5a Monoclonal Antibody Indicates a Prominent Role for Anaphylatoxin in Pulmonary Xenograft Dysfunction

In contrast to renal or cardiac xenografts, the inhibition of complement using cobra venom factor (CVF) accelerates pulmonary xenograft failure. By activating C3/C5 convertase, CVF depletes complement while additionally generating C5a and other anaphylatoxins, to which pulmonary xenografts may be uniquely susceptible. The current study investigates the role of C5a in pulmonary xenograft failure in baboons. Left orthotopic pulmonary xenografts using swine lungs expressing human CD46 were performed in baboons receiving: I) no other treatment (n=4), II) immunodepletion (n=5), and III) immunodepletion plus a single dose of mouse anti-human C5a monoclonal antibody (anti-C5a, 0.6 mg/kg administered intravenously) (n=3). The extent to which anti-C5a inhibits baboon C5a was assessed in vitro using a hemolytic reaction involving baboon serum and porcine red blood cells and by ELISA. Baboons in Group III exhibited significantly prolonged xenograft survival (mean=722+/-121 min, P=0.02) compared to baboons in Group I (mean=202+/-24 min) and Group II (mean=276+/-79 min). Furthermore, baboons in Groups I and II experienced pronounced hemodynamic compromise requiring inotropic support whereas those in Group III remained hemodynamically stable throughout experimentation without the need for additional pharmacologic intervention. These findings indicate that C5a exacerbates pulmonary xenograft injury and compromises recipient hemodynamic status. Moreover, blockade of anaphylatoxins, such as C5a, offers a promising approach for future investigations aimed at preventing pulmonary xenograft injury in baboons.

The serum level of xenoantibodies, and hDAF or αGAL expression on pig cells, modulate in vitro the protection given by hDAF to primate complement-mediated damage

The ability of human complement regulatory molecules to prevent xenograft rejection following pig-to-primate xenotransplantation is limited. We assayed the efficacy of transgenic human decay accelerating factor (hDAF) expressed on porcine cells to inhibit the in vitro complement activity of primate sera. We measured the cytotoxic activity of baboon or human sera against peripheral blood lymphocytes (PBLs) from hDAF or nontransgenic pigs using a flow cytometry complement-mediated cytotoxicity assay (FCCA). We also analyzed the anti–Galα1-3Gal (αGal) antibody titer of the baboon sera by ELISA and the expression of hDAF and αGal on the PBL surface by immunofluorescence. Transgenic hDAF expression was capable of protecting pig cells against injury produced by both baboon and human serum. However, the hDAF molecule was more efficient against human than baboon sera. The humoral cytotoxicity capacity correlated with the level of both IgG and IgM anti-αGal antibodies. In addition, inhibition of complement-mediated cytotoxicity of hDAF pig cells correlated with the expression of hDAF and αGal molecules on target cells. These results confirm in vitro the protective role of hDAF in pig cells to heterologus complement mediated damage, but they also suggest that protection decreases in the presence of high levels of anti-porcine antibodies in serum, low expression of hDAF, or high expression of αGal on pig cells.