Baboon Tissues Research Articles

Porcine cytomegalovirus: A very unwelcome stowaway.

The announcement of the first pig-to-human heart transplantation early this year was the culmination of a very long and arduous path. The groundbreaking first life-sustaining pig-to man heart transplantation by the team of University of Maryland at Baltimore under the lead of Professor Griffith provided the long-awaited proof that it can work–even if survival was cut short by a hitherto still to be defined process. In a fascinating webinar, Professor Griffith described in detail the course of the patient.1 One potential element in the deterioration was the increase of porcine cytomegalovirus (PCMV) DNAemia, detected in the blood of the patient. The exact role of PCMV in the sequence of events is unclear and currently analyzed in detail, and will undoubtedly increase our knowledge. As a young researcher in the laboratory of Jay Fishman, I had the privilege to play a small role in early pig-to-baboon xenotransplantation experiments done in the Transplantation Biology Research Center at MGH, headed by Professor David Sachs. Organ- and recipient survival was short in the early 2000s, and reasons were explored on many different angles. From the experience in human-to-human solid organ transplantation, one avenue was to look for activation or primary infection of latent viruses, primarily from the herpesvirus family. PCMV had been identified as a potential zoonotic agent before.2 Using quantitative PCR assays, we demonstrated activation of PCMV and baboon cytomegalovirus (BCMV) in a series of pig-to primate organ kidney xenotransplantations. High copy numbers of PCMV DNAemia were detected in recipient baboon tissue and particularly in the transplanted porcine graft tissues.3, 4 In a model of primary porcine aortic endothelial cells infected in vitro with PCMV, an increase in porcine tissue factor was observed, and we hypothesized that this was a potential cofactor in the development of an often fatal consumptive coagulopathy observed in pig-to baboon kidney xenotransplantation.5 Using PCMV-free pig donors, which was achieved by early weaning of piglets, consumptive coagulopathy was prevented in pig-to-baboon heart transplantation, resulting in an improved graft survival.6 Given the many modifications in the protocols over time, it was not possible to establish PCMV as the sole cause of this coagulopathy. Nevertheless, at this point elimination of PCMV was the clear goal given the harmful potential of this virus.7 Eradication protocols were developed and successfully tested.8 This was all the more important, as ganciclovir failed to control PCMV activation.9 BCMV activation in the recipient baboons, on the other hand, by then was controlled by prophylactic use of ganciclovir in the majority of recipients and had become part of all protocols. The most comprehensive study on the role of PCMV was recently published by Denner et al. in 2020. In a series of orthotopic pig-to-baboon heart transplantations, transmission of PCMV was observed from PCMV-positive donor pigs in some but not all baboon recipients, whereas no transmission events occurred for a number of other potentially harmful latent porcine viruses tested, including porcine lymphotropic herpesviruses, and porcine endogenous retroviruses. In baboons transplanted with a heart from a PCMV-positive donor pig, survival was reduced, inflammatory cytokines such as IL-6 and TNF-α were increased and tPA-PAI-1 complexes circulated in high concentrations in the recipients, pointing to substantial alterations of the coagulation system. This detailed report provided additional evidence for the deleterious effects of PCMV, and reiterated the need for elimination of PCMV from the pig donor.10 Why then are PCMV-positive donor pigs still an issue? Breeding pathogen-free pigs was shown to be feasible, but the effort to achieve this is considerable. In a publication on virus prevalence in different minipig breeds in 2021, PCMV was still detected in some but not all breeds.11 Recent work did primarily focus on porcine endogenous retroviruses, as elimination of these viruses is incomparably more challenging than that of traditional latent viruses. Testing for elimination requires not only the use of PCR for detection PCMV DNAemia, but also by serology, as PCR can be negative in swabs from a healthy swine. Validated serological assays for PCMV are not readily available. Efforts did understandably concentrate on the genetic modifications of donor pigs. Given the potential unfavorable role in the first human case of orthotopic heart transplantation, the focus must be redirected to the elimination of PCMV. It is a feasible and necessary step. The amazing initial clinical course of the patient is a huge incentive to keep the momentum going and continue on this path.

Diurnal and circadian rhythmicity of the human blood transcriptome overlaps with organ- and tissue-specific expression of a non-human primate

BackgroundTwenty-four-hour rhythmicity in mammalian tissues and organs is driven by local circadian oscillators, systemic factors, the central circadian pacemaker and light-dark cycles. At the physiological level, the neural and endocrine systems synchronise gene expression in peripheral tissues and organs to the 24-h-day cycle, and disruption of such regulation has been shown to lead to pathological conditions. Thus, monitoring rhythmicity in tissues/organs holds promise for circadian medicine; however, most tissues and organs are not easily accessible in humans and alternative approaches to quantify circadian rhythmicity are needed. We investigated the overlap between rhythmic transcripts in human blood and transcripts shown to be rhythmic in 64 tissues/organs of the baboon, how these rhythms are aligned with light-dark cycles and each other, and whether timing of tissue-specific rhythmicity can be predicted from a blood sample.ResultsWe compared rhythmicity in transcriptomic time series collected from humans and baboons using set logic, circular cross-correlation, circular clustering, functional enrichment analyses, and least squares regression. Of the 759 orthologous genes that were rhythmic in human blood, 652 (86%) were also rhythmic in at least one baboon tissue and most of these genes were associated with basic processes such as transcription and protein homeostasis. In total, 109 (17%) of the 652 overlapping rhythmic genes were reported as rhythmic in only one baboon tissue or organ and several of these genes have tissue/organ-specific functions. The timing of human and baboon rhythmic transcripts displayed prominent ‘night’ and ‘day’ clusters, with genes in the dark cluster associated with translation. Alignment between baboon rhythmic transcriptomes and the overlapping human blood transcriptome was significantly closer when light onset, rather than midpoint of light, or end of light period, was used as phase reference point. The timing of overlapping human and baboon rhythmic transcriptomes was significantly correlated in 25 tissue/organs with an average earlier timing of 3.21 h (SD 2.47 h) in human blood.ConclusionsThe human blood transcriptome contains sets of rhythmic genes that overlap with rhythmic genes of tissues/organs in baboon. The rhythmic sets vary across tissues/organs, but the timing of most rhythmic genes is similar in human blood and baboon tissues/organs. These results have implications for development of blood transcriptome-based biomarkers for circadian rhythmicity in tissues and organs.

Non-accidental structural chromosome aberrations in established monkey B-lymphocyte cell lines

Established primate lymphocyte cell lines obtained from tumour samples and from EBV-positive monkeys served us as the model system for studying the role of genetic factors and chromosomal abnormalities in malignization. The investigation of chromosome regions and genes involved in chromosomal aberrations leading to malignization in these lines was the aim of our work. Cytogenetic analysis was performed at different stages of cultivation in vitro. To determine the oncogenes and tumour suppressor genes located on aberrant chromosomes, data on mapping rhesus macaque genes, and high similarity of human and monkey karyotypes were used. We found that, in the line obtained from lymphomatous baboon tissue, the inactivation of tumour suppressor gene RB1 on chromosome 17 after chromosomal rearrangement is one of the most probable causes of in vivo malignization. Chromosomal aberrations at the region of oncogene c-Ki-ras and tumour suppressor gene TP53 change the proliferative status and differentiation in established cell lines obtained from healthy but EBV-seropositive primates. The other cause of malignization in these lines is an increase in expression of the oncogene c-myc caused by trisomy of chromosome 8 where c-myc is located. Structural aberrations in established primate cell lines affecting several chromosomal loci were identified as: (1) causing the proto-oncogene activation – the central event in the tumour clone occurrence, and (2) deactivating tumour suppressor genes. The change in the chromosome number leads to increase in oncogenic products and to damage of regulatory functions associated with cell proliferation.

A Landscape of Circadian and Ultradian Alternative Splicing Events in Mammalian Tissues

Mounting evidence points to a role of the circadian clock in the temporal regulation of alternative splicing (AS). In this study, we carried out a systematic analysis of circadian and ultradian transcriptional rhythms to characterize the landscape of rhythmic AS events in 76 tissues from mouse and baboon. We found a bimodal phase distribution of splicing factors with 24-h rhythmic expression across tissues and species and identified putative oscillating AS events in murine tissues and pairs of differentially rhythmic splice isoforms of the same gene in baboon tissues that peaked at opposing times of the day. Genes with phase-shifted isoforms included oncogenes and tumor suppressors. We further explored these findings using a new circadian data-set of human colorectal cancer cell lines. Rhythmic isoform expression patterns differed between the primary tumor and the metastatic cell line, indicating a functional role of rhythmic AS that might be implicated in tumor progression.

Molecular cloning of the myo-inositol oxygenase gene from the kidney of baboons.

The enzyme myo-Inositol oxygenase (MIOX) is also termed ALDRL6. It is a kidney-specific member of the aldo-keto reductase family. MIOX catalyzes the first reaction involved in the myo-inositol metabolism signaling pathway and is fully expressed in mammalian tissues. MIOX catalyzes the oxidative cleavage of myo-Inositol and its epimer, D-chiro-Inositol to D-glucuronate. The dioxygen-dependent cleavage of the C6 and C1 bond in myo-Inositol is achieved by utilizing the Fe2+/Fe3+ binuclear iron center of MIOX. This enzyme has also been implicated in the complications of diabetes, including diabetic nephropathy. The MIOX gene was amplified with reverse transcription-polymerase chain reaction from baboon tissue samples, and the product was cloned and sequenced. MIOX expression in the baboon kidney is described in the present study. The percentages of nucleotide and amino acid similarities between baboons and humans were 95 and 96%, respectively. The MIOX protein of the baboon may be structurally identical to that of humans. Furthermore, the evolutionary changes, which have affected these sequences, have resulted from purifying forces.

Therapeutic regulation of systemic inflammation in xenograft recipients.

Inflammation is known to preclude tolerance after transplantation. We have previously shown that systemic inflammation in xenograft recipients (SIXR) precedes activation of coagulation in the absence of T cell responses. Accordingly, SIXR may amplify innate and adaptive immune responses against xenografts after pig-to-primate xenotransplantation, even with efficient immunosuppressive therapy. We evaluated the impact of anti-inflammatory agents on pro-inflammatory cytokines and chemokines in pig artery patch and heart xenograft recipients. Baboons received an artery patch (Group1, n=8) or heart (Group2, n=4) from genetically engineered pigs. All baboons received lymphodepletion with thymoglobulin (ATG) and costimulation blockade-based immunosuppression (anti-CD40 and/or CTLA4Ig). In Group1, baboons received either (i) no anti-inflammatory agents (n=2), (ii) cobra venom factor (CVF, n=2), (iii) α1-antitrypsin (AAT, n=2), or (iv) interleukin (IL)-6 receptor antagonist (IL-6RA, n=2). In Group2, all baboon received corticosteroids, either without (n=2) or with (n=2) IL-6RA. Serum IFN-γ, TNF-α, IL-1β, IL-17, IL-6, IL-8, MCP-1, and sCD40L levels were measured by Luminex. Fibrinogen, D-dimers, and C-reactive protein (C-RP) were also measured. Recipient baboon T cell proliferation was evaluated by mixed lymphocyte reaction (MLR) before and after transplantation. Pig and baboon tissue factor (TF) mRNA levels in heart xenografts were measured by RT-PCR. In no recipient was a marked increase in T cell response to pig cells observed after transplantation. In Groups 1 and 2, post-transplantation levels of IFN-γ, TNF-α, IL-1β, and IL-17 remained comparable to or lower than pre-transplant levels, except in one heart recipient that succumbed to CMV infection. In Group1, when no anti-inflammatory agent was administered, post-transplant levels of IL-6, IL-8, and MCP-1 were elevated. After CVF, IL-6, IL-8, and MCP-1 remained low. After IL-6RA, IL-6 and MCP-1 were elevated. After AAT, IL-8 was elevated. sCD40L became elevated intermittently in most recipients irrespective of the administered anti-inflammatory agent. In Group2, IL-6 was transiently elevated, particularly after IL-6RA administration. MCP-1 gradually increased by 2months in Group2 recipients. sCD40L generally remained low except in one recipient. In Group1 and Group2 recipients, C-RP levels were elevated except after IL-6RA administration, while D-dimers were elevated regardless of administration of anti-inflammatory agent. In Group2, pig TF mRNA levels were increased in heart xenografts compared to naive pig hearts, irrespective of IL-6 receptor antagonist administration. Additionally, baboon TF mRNA levels were detectable in heart xenografts, but not in naive pig hearts. Some pro-inflammatory cytokines and chemokines are elevated in xenograft recipients, even with efficient T cell-directed immunosuppressive therapy. Persistent elevation of D-dimers, and individual cytokines and chemokines suggest a continuous inflammatory response, despite administration of anti-inflammatory agents. Systemic administration of combined anti-inflammatory agents as well as complement regulation may be essential to prevent SIXR after xenotransplantation.

Olfactomedin-like 2 A and B (OLFML2A and OLFML2B) expression profile in primates (human and baboon).

BackgroundThe olfactomedin-like domain (OLFML) is present in at least four families of proteins, including OLFML2A and OLFML2B, which are expressed in adult rat retina cells. However, no expression of their orthologous has ever been reported in human and baboon.ObjectiveThe aim of this study was to investigate the expression of OLFML2A and OLFML2B in ocular tissues of baboons (Papio hamadryas) and humans, as a key to elucidate OLFML function in eye physiology.MethodsOLFML2A and OLFML2B cDNA detection in ocular tissues of these species was performed by RT-PCR. The amplicons were cloned and sequenced, phylogenetically analyzed and their proteins products were confirmed by immunofluorescence assays.ResultsOLFML2A and OLFML2B transcripts were found in human cornea, lens and retina and in baboon cornea, lens, iris and retina. The baboon OLFML2A and OLFML2B ORF sequences have 96% similarity with their human’s orthologous. OLFML2A and OLFML2B evolution fits the hypothesis of purifying selection. Phylogenetic analysis shows clear orthology in OLFML2A genes, while OLFML2B orthology is not clear.ConclusionsExpression of OLFML2A and OLFML2B in human and baboon ocular tissues, including their high similarity, make the baboon a powerful model to deduce the physiological and/or metabolic function of these proteins in the eye.

The endocannabinoid system in the baboon (Papio spp.) as a complex framework for developmental pharmacology

IntroductionThe consumption of marijuana (exogenous cannabinoid) almost doubled in adults during last decade. Consumption of exogenous cannabinoids interferes with the endogenous cannabinoid (or “endocannabinoid” (eCB)) system (ECS), which comprises N-arachidonylethanolamide (anandamide, AEA), 2-arachidonoyl glycerol (2-AG), endocannabinoid receptors (cannabinoid receptors 1 and 2 (CB1R and CB2R), encoded by CNR1 and CNR2, respectively), and synthesizing/degrading enzymes (FAAH, fatty-acid amide hydrolase; MAGL, monoacylglycerol lipase; DAGL-α, diacylglycerol lipase-alpha). Reports regarding the toxic and therapeutic effects of pharmacological compounds targeting the ECS are sometimes contradictory. This may be caused by the fact that structure of the eCBs varies in the species studied. ObjectivesFirst: to clone and characterize the cDNAs of selected members of ECS in a non-human primate (baboon, Papio spp.), and second: to compare those cDNA sequences to known human structural variants (single nucleotide polymorphisms and haplotypes). Materials and methodsPolymerase chain reaction-amplified gene products from baboon tissues were transformed into Escherichia coli. Amplicon-positive clones were sequenced, and the obtained sequences were conceptually translated into amino-acid sequences using the genetic code. ResultsAmong the ECS members, CNR1 was the best conserved gene between humans and baboons. The phenotypes associated with mutations in the untranslated regions of this gene in humans have not been described in baboons. One difference in the structure of CNR2 between humans and baboons was detected in the region with the only known clinically relevant polymorphism in a human receptor. All of the differences in the amino-acid structure of DAGL-α between humans and baboons were located in the hydroxylase domain, close to phosphorylation sites. None of the differences in the amino-acid structure of MAGL observed between baboons and humans were located in the area critical for enzyme function. ConclusionThe evaluation of the data, obtained in non-human primate model of cannabis-related developmental exposure should take into consideration possible evolutionary-determined species-specific differences in the CB1R expression, CB2R transduction pathway, and FAAH and DAGLα substrate-enzyme interactions.

Olfactomedin‐like 3 (OLFML3) gene expression in baboon and human ocular tissues: cornea, lens, uvea, and retina

Olfactomedin-like is a family of polyfunctional polymeric glycoproteins. This family has at least four members. One member of this family is OLFML3, which is preferentially expressed in placenta but is also detected in other adult tissues including the liver and heart. However, its orthologous rat gene is expressed in the iris, sclera, trabecular meshwork, retina, and optic nerve. OLFML3 messenger amplification was performed by RT-PCR from human and baboon ocular tissues. The products were cloned and sequenced. We report OLFML3 expression in human and baboon eye. The full coding DNA sequence has 1221bp, from which an open reading frame of 406 amino acid was obtained. The baboon OLFML3 gene nucleotidic sequence has 98% and amino acidic 99% similarity with humans. OLFML3 gene expression in human and baboon ocular tissues and its high similarity make the baboon a powerful model to deduce the physiological and/or metabolic function of this protein in the eye.

DSCR9 gene simultaneous expression in placental, testicular and renal tissues from baboon (papio hamadryas)

BackgroundIn 2002 Takamatsu and co-workers described the human DSCR9 gene and observed that it was transcriptionally active in human testicular tissue, but no protein was identified as a product of this transcript. Similar results were obtained in chimpanzee tissue. This gene has not been detected in species other than primates, suggesting that DSCR9 is exclusively found in these mammals.ResultsWe report evidence of DSCR9 expression in placenta, testis and kidney of baboon (Papio hamadryas). We used primers specific for DSCR9 to amplify transcripts through reverse transcription (RT) coupled to polymerase chain reaction (PCR). Furthermore, PCR was used to amplify the complete DSCR9 gene from genomic DNA from three baboons. We amplified and sequenced five overlapping segments that were assembled into the 3284 bp baboon DSCR9 gene, including the putative promoter and the entire transcriptional unit (5'-UTR, CDS and 3'-UTR).ConclusionsThe baboon DSCR9 gene is highly similar to the human counterpart. The isolated transcripts from baboon tissues (placenta, testis and kidney) of three different baboons correspond to the human orthologous gene.

Dynamic testing of old and young baboon cortical bone with numerical validation

Cortical bone tensile mechanical properties at quasistatic and high rates (∼300s −1 ) were determined ex vivo using the right femurs of 12 female baboons, ( Papio hamadryas spp.) from the Texas Biomedical Research Institute/Southwest National Primate Research Center in San Antonio, Texas. The animals were divided into two age groups: a young age group (6.63 ± 0.6 years) and an old age group (26.96 ± 1.3 years). Seven specimens per group were monotonically loaded to failure to determine their mechanical properties. The quasistatic strength of the bone for the old group was just a little (but not significantly) lower than the young group. High strain rate tests performed with the Hopkinson bar indicate that baboon bone from the older group was significantly weaker under impact loads than that from the younger group. This observation is particularly important due to the similarities between baboon and human bone tissue. Typical strain rates for these tests ranged from 130s −1 to 250s −1 . A full-size 3-D simulation of the Hopkinson bar test was performed to confirm that the bone specimen was under stress equilibrium and to evaluate the consistency of the modulus and strength inferred from the tests. Simulations were performed in which the modulus, strength and failure strain were varied to see the sensitivity of the results. Additionally, simplified simulations were performed to estimate the strain rate environment of a femur during a fall at an impact velocity of 5 m/s, similar to a free fall velocity from a height of 1.3 meters. The simulations confirm that strain rates obtained in the Hopkinson bar are relevant because they are similar to those expected inr such a fall.

The polypyrimidine tract binding protein regulates desaturase alternative splicing and PUFA composition

The Δ6 desaturase, encoded by FADS2, plays a crucial role in omega-3 and omega-6 fatty acid synthesis. These fatty acids are essential components of the central nervous system, and they act as precursors for eicosanoid signaling molecules and as direct modulators of gene expression. The polypyrimidine tract binding protein (PTB or hnRNP I) is a splicing factor that regulates alternative pre-mRNA splicing. Here, PTB is shown to bind an exonic splicing silencer element and repress alternative splicing of FADS2 into FADS2 AT1. PTB and FADS2AT1 were inversely correlated in neonatal baboon tissues, implicating PTB as a major regulator of tissue-specific FADS2 splicing. In HepG2 cells, PTB knockdown modulated alternative splicing of FADS2, as well as FADS3, a putative desaturase of unknown function. Omega-3 fatty acids decreased by nearly one half relative to omega-6 fatty acids in PTB knockdown cells compared with controls, with a particularly strong decrease in eicosapentaenoic acid (EPA) concentration and its ratio to arachidonic acid (ARA). This is a rare demonstration of a mechanism specifically altering the cellular omega-3 to omega-6 fatty acid ratio without any change in diet/media. These findings reveal a novel role for PTB, regulating availability of membrane components and eicosanoid precursors for cell signaling.

Alternative splicing of fatty acid desaturases is associated with altered fatty acid composition

The delta-6 desaturase, encoded by FADS2, plays a crucial role in omega-3 and omega-6 fatty acid synthesis. We have recently identified an alternative transcript of FADS2 (FADS2 AT1) and 7 alternative transcripts for the putative desaturase FADS3. Here, we investigated regulation of desaturase alternative splicing, and fatty acid changes associated with altered splicing patterns. We hypothesized a role for the splicing repressor PTB/hnRNP I based on bioinformatics predictions of splicing factor binding sites. PTB and FADS2AT1 were inversely correlated in infant baboon tissues, implicating PTB as a major regulator of tissue-specific FADS2 splicing. In human liver-derived HepG2 cells, siRNA knockdown of PTB led to upregulation of FADS2 AT1 and FADS3 AT7. This splicing pattern was associated with a specific decrease in the ratio of total cellular omega-3 to omega-6 fatty acids, including a nearly 2-fold decrease in eicosapentaenoic acid (EPA) content, and a 43% reduction in the ratio of EPA with arachidonic acid (ARA). These findings reveal a novel mechanism controlling availability of eicosanoid precursors for cell signaling. H. R. was supported by a Ruth L. Kirchstein-NRSA predoctoral fellowship in reproductive sciences and genomics (Grant Number T32HD052471) from the National Institutes of Child Health and Human Development.

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.

Novel fatty acid desaturase 3 ( FADS3) transcripts generated by alternative splicing

Fatty acid desaturase 1 and 2 ( FADS1 and FADS2) code for the key desaturase enzymes involved in the biosynthesis of long chain polyunsaturated fatty acids in mammals. FADS3 shares close sequence homology to FADS1 and FADS2 but the function of its gene product remains unknown. Alternative transcripts (AT) generated by alternative splicing (AS) are increasingly recognized as an important mechanism enabling a single gene to code for multiple gene products. We report the first AT of a FADS gene, FADS3, generated by AS. Aided by ORF Finder, we identified putative coding regions of eight AT for FADS3 with 1.34 kb (classical splicing), 1.14 (AT1), 0.77 (AT2), 1.25 (AT3), 0.51 (AT4), 0.74 (AT6), and 1.11 (AT7). In addition we identified a 0.51 kb length transcript (AT5) that has a termination codon within intron 8–9. The expression of each AT was analyzed in baboon neonate tissues and in differentiated and undifferentiated human SK-N-SH neuroblastoma cells. FADS3 AT are expressed in 12 neonate baboon tissues and showed reciprocal increases and decreases in expression changes in response to human neuronal cell differentiation. FADS3 AT, conserved in primates and under metabolic control in human cells, are a putative mediator of LCPUFA biosynthesis and/or regulation.

Novel FADS3 Alternative Transcripts Generated By Alternative Splicing

ObjectiveWe report the first multiple transcript variants of a fatty acid desaturase (FADS), FADS3, generated by alternative splicing, and tissue and cell specific expression.BackgroundFatty acid desaturases are enzymes that catalyze the introduction of cis double bonds at specific positions in a fatty acid chain. FADS1, FADS2 and FADS3 are thought to be the key desaturase enzymes involved in the biosynthesis of long chain polyunsaturated fatty acids. Functions of FADS1 and FADS2 are known, however, the function of FADS3 gene product remains unknown.ProcedureThe protein coding region of baboon FADS3 and FADS3 alternative transcripts (AT), were generated from primers synthesized using human and monkey FADS3 cDNA sequences. To analyze the expression levels of each AT, primers bridging the deleted parts of the exons were designed, tested by RT‐PCR and the products were confirmed by sequencing. The expression was analyzed in baboon tissues and in differentiated and undifferentiated human SK‐N‐SH neuroblastoma cells (NB).ResultsAided by ORF Finder, we identified eight AT for FADS3 with 1.34 kb (classical splicing), 1.14 (AT1), 0.77 (AT2), 1.25 (AT3), 0.51 (AT4), 0.74 (AT6), and 1.11 (AT7). In addition we identified an AT of 0.51 kb (AT5) length that has termination codon within the intron 8‐9. FADS3 AT expression is tissue dependent in 12 baboon tissues. NB cells showed reciprocal increases and decreases in expression.ConclusionThis is the first finding of AT of FADS genes. The FADS3 AT are expressed in many tissues and showed changes in response to neuronal cell differentiation.Support: NIH grant GM071534

Trypanosoma cruzi in non‐human primates with a history of stillbirths: a retrospective study (Papio hamadryas spp.) and case report (Macaca fascicularis)

Congenital transmission of Trypanosoma cruzi has been described in humans and experimental work has been conducted with mice, but not with non-human primates (NHPs). We conducted a retrospective study of female baboons (Papio hamadryas spp.) naturally seropositive or seronegative for T. cruzi with history of fetal loss, and we report a stillbirth in a cynomolgus macaque (Macaca fascicularis) with placental T. cruzi amastigotes. There were no differences in menstrual cycle parameters and the number of fetal losses between seropositive and seronegative baboons with history of fetal loss. The amount of parasite DNA detected using quantitative polymerase chain reaction (Q-PCR) in M. fascicularis placenta was within the range detected in infected baboon tissues. There is no evidence that chronic maternal T. cruzi infection causes fetal loss in baboons. Q-PCR is a useful diagnostic tool to study archived NHP placentas.

Developmental expression of Toll-like receptors-2 and -4 in preterm baboon lung

Preterm babies are susceptible to respiratory infection due to immature lung and immune system. Immune cells express Toll-like receptors (TLRs), which may be important in local host defense of preterm infants. We studied the expression of TLR2 and TLR4 in lung tissues of fetal baboons delivered at 125, 140, and 175 days of gestation (dGA; term=185+/-2 days) and preterm baboons that became naturally infected with bacterial/fungal pathogens. The TLR-mRNA and protein were quantified by Northern and Western blotting, respectively. The expression of both TLRs was significantly low at 125 and 140dGA. At 175dGA, the levels reached equivalent to those in adult baboons. However, in naturally infected baboons, the TLR4-mRNA was reduced (p<0.05); TLR2-mRNA expression remained unaltered. The protein expression of both TLRs was found increased in naturally infected baboons. Our results suggest that the lung TLR expression is developmentally regulated and altered during respiratory infection in preterm babies.

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.

Expression of TLR2 and TLR4 in Preterm Baboon Lung (B165)

Abstract Respiratory infections can cause significant morbidity and mortality in prematurely delivered babies. The increased susceptibility of preterm babies to infection is related to immaturity of the immune system. The Toll-like receptors (TLR) are expressed by antigen-presenting cells and play a key role in recognition of pathogens and regulation of immune response. However, their role in preterm babies is not well-understood. In this study, we investigated the expression of TLR2 and TLR4 proteins in lung tissues of fetal (delivered at 125, 140, 175 days of gestation age, dGA; complete term 185 days) and naturally-infected, prematurely delivered (125dGA ventilated with PRN O2) baboons. The expression of TLR2 and TLR4 was studied at protein level by Western blotting using anti-human-TLR2 and TLR4 antibodies. We found that the expression of TLR2 and TLR4 protein increased with the gestational age; the expression was found low in lung tissues of 125dGA baboon which increased to adult level at 175dGA. But in the lung tissues of naturally-infected preterm baboons, the protein levels of both TLR2 and TLR4 were significantly increased as compared to non-infected controls. Our results suggest that the expression of TLR2 and TLR4 is developmentally regulated and it may play a significant role in regulation of immune responses during infection in preterm babies. Research Support by American Lung Association