ABSTRACT
The development of safe crop protection products is a complex process that traditionally relies on intensive animal use for hazard identification. Methods that capture toxicity in early stages of agrochemical discovery programs enable a more efficient and sustainable product development pipeline. Here, we explored whether the zebrafish model can be leveraged to identify mammalian-relevant toxicity. We used transgenic zebrafish to assess developmental toxicity following exposures to known mammalian teratogens and captured larval morphological malformations, including bone and vascular perturbations. We further applied toxicogenomics to identify common biomarker signatures of teratogen exposure. The results show that the larval malformation assay predicted teratogenicity with 82.35% accuracy, 87.50% specificity, and 77.78% sensitivity. Similar and slightly lower accuracies were obtained with the vascular and bone assays, respectively. A set of 20 biomarkers were identified that efficiently segregated teratogenic chemicals from nonteratogens. In conclusion, zebrafish are valuable, robust, and cost-effective models for toxicity testing in the early stages of product development.
Subject(s)
Agrochemicals , Spine , Zebrafish , Embryo, Nonmammalian , Agrochemicals/toxicity , Spine/drug effects , RNA/genetics , Larva/genetics , Gene Expression Regulation, Developmental/drug effects , Animals, Genetically Modified , Genetic MarkersABSTRACT
OBJECTIVE: To evaluate the clinically relevant anti-CD40 antibody iscalimab for baseline immunosuppression in a preclinical pig-to-rhesus renal xenograft model. SUMMARY BACKGROUND DATA: CD40/CD40L co-stimulation blockade-based immunosuppression has been more successful than calcineurin-based protocols in prolonging xenograft survival in preclinical models. METHODS: GGTA1 knockout/CD55 transgenic pig kidneys were transplanted into rhesus monkeys (n = 6) receiving an iscalimab-based immunosuppressive regimen. RESULTS: Two grafts were lost early (22 and 26 days) because of ectatic donor ureters with otherwise normal histology. The other recipients survived 171, 315, 422, and 439 days with good renal function throughout the posttransplant course. None of the recipients experienced serious infectious morbidity. CONCLUSIONS: It may be reasonable to evaluate an iscalimab-based immunosuppressive regimen in clinical renal xenotransplantation.
Subject(s)
Graft Survival , Heterografts , Immunosuppressive Agents , Kidney Transplantation , Macaca mulatta , Transplantation, Heterologous , Animals , Transplantation, Heterologous/methods , Swine , Graft Survival/immunology , Graft Survival/drug effects , Kidney Transplantation/methods , Immunosuppressive Agents/pharmacology , Heterografts/immunology , Graft Rejection/immunology , Graft Rejection/prevention & control , Antibodies, Monoclonal, Humanized/therapeutic use , Antibodies, Monoclonal, Humanized/pharmacology , Animals, Genetically Modified , Antibodies, Monoclonal/pharmacology , Humans , Galactosyltransferases/geneticsABSTRACT
BACKGROUND: The number of multigene-modified donor pigs for xenotransplantation is increasing with the advent of gene-editing technologies. However, it remains unclear which gene combination is suitable for specific organ transplantation. METHODS: In this study, we utilized CRISPR/Cas9 gene editing technology, piggyBac transposon system, and somatic cell cloning to construct GTKO/hCD55/hTBM/hCD39 four-gene-edited cloned (GEC) pigs and performed kidney transplantation from pig to rhesus monkey to evaluate the effectiveness of these GEC pigs. RESULTS: First, 107 cell colonies were obtained through drug selection, of which seven were 4-GE colonies. Two colonies were selected for somatic cell nuclear transfer (SCNT), resulting in seven fetuses, of which four were GGTA1 biallelic knockout. Out of these four, two fetuses had higher expression of hCD55, hTBM, and hCD39. Therefore, these two fetuses were selected for two consecutive rounds of cloning, resulting in 97 live piglets. After phenotype identification, the GGTA1 gene of these pigs was inactivated, and hCD55, hTBM, and hCD39 were expressed in cells and multiple tissues. Furthermore, the numbers of monkey IgM and IgG binding to the peripheral blood mononuclear cells (PBMCs) of the 4-GEC pigs were markedly reduced. Moreover, 4-GEC porcine PBMCs had greater survival rates than those from wild-type pigs through complement-mediated cytolysis assays. In pig-to-monkey kidney xenotransplantation, the kidney xenograft successfully survived for 11 days. All physiological and biochemical indicators were normal, and no hyperacute rejection or coagulation abnormalities were found after transplantation. CONCLUSION: These results indicate that the GTKO/hCD55/hTBM/hCD39 four-gene modification effectively alleviates immune rejection, and the pig kidney can functionally support the recipient monkey's life.
Subject(s)
Animals, Genetically Modified , Galactosyltransferases , Gene Editing , Kidney Transplantation , Transplantation, Heterologous , Animals , Transplantation, Heterologous/methods , Kidney Transplantation/methods , Swine , Gene Editing/methods , Galactosyltransferases/genetics , CRISPR-Cas Systems , Macaca mulatta , Nuclear Transfer Techniques , Heterografts , Humans , Graft Survival/immunology , Graft Rejection/immunology , Apyrase , Antigens, CDABSTRACT
Mechanical stress during muscle contraction is a constant threat to proteome integrity. However, there is a lack of experimental systems to identify critical proteostasis regulators under mechanical stress conditions. Here, we present the transgenic Caenorhabditis elegans model OptIMMuS (Optogenetic Induction of Mechanical Muscle Stress) to study changes in the proteostasis network associated with mechanical forces. Repeated blue light exposure of a muscle-expressed Chlamydomonas rheinhardii channelrhodopsin-2 variant results in sustained muscle contraction and mechanical stress. Using OptIMMuS, combined with proximity labeling and mass spectrometry, we identify regulators that cooperate with the myosin-directed chaperone UNC-45 in muscle proteostasis. One of these is the TRIM E3 ligase NHL-1, which interacts with UNC-45 and muscle myosin in genetic epistasis and co-immunoprecipitation experiments. We provide evidence that the ubiquitylation activity of NHL-1 regulates myosin levels and functionality under mechanical stress. In the future, OptIMMuS will help to identify muscle-specific proteostasis regulators of therapeutic relevance.
Subject(s)
Animals, Genetically Modified , Caenorhabditis elegans Proteins , Caenorhabditis elegans , Optogenetics , Proteostasis , Stress, Mechanical , Ubiquitin-Protein Ligases , Ubiquitination , Caenorhabditis elegans/metabolism , Caenorhabditis elegans/genetics , Animals , Caenorhabditis elegans Proteins/metabolism , Caenorhabditis elegans Proteins/genetics , Ubiquitin-Protein Ligases/metabolism , Ubiquitin-Protein Ligases/genetics , Myosins/metabolism , Myosins/genetics , Muscle Contraction/physiology , Muscles/metabolism , Molecular ChaperonesABSTRACT
Glucocorticoids (GCs) have a wide spectrum of effects on animal behavior. A recently suggested effect involves determining the structure of individual differences, that is how the behavioral traits of an individual covary, forming the so-called behavioral syndromes. As GCs can exert their action in multiple ways, e.g., via rapid non-genomic effects or via the activation of two highly homologous members of the steroid receptor family acting as transcription factors, it is unclear how the GC modulation of behavioral syndromes takes place. We exploited a zebrafish line with a frameshift mutation in the gene encoding the GC receptor (Gr), to investigate this question. We found that lack of Gr altered the average score of several behavioral traits in the mutant line, determining reduced boldness, and increased activity and sociability. Critically, the pattern of covariation between these traits was also substantially affected by the loss of Gr. The most evident effect was an association of traits involved in boldness in the gr mutant line. This study reveals that, in zebrafish, Gr is not only involved in the modulation of the average value of behavioral traits, but also in how the behavioral traits of an individual are interrelated and determine the behavioral syndromes.
Subject(s)
Behavior, Animal , Receptors, Glucocorticoid , Zebrafish , Animals , Receptors, Glucocorticoid/genetics , Receptors, Glucocorticoid/metabolism , Behavior, Animal/physiology , Frameshift Mutation , Male , Animals, Genetically Modified , Social Behavior , Female , Zebrafish Proteins/genetics , Zebrafish Proteins/metabolismABSTRACT
Adult zebrafish have an innate ability to recover from severe spinal cord injury. Here, we report a comprehensive single nuclear RNA sequencing atlas that spans 6 weeks of regeneration. We identify cooperative roles for adult neurogenesis and neuronal plasticity during spinal cord repair. Neurogenesis of glutamatergic and GABAergic neurons restores the excitatory/inhibitory balance after injury. In addition, a transient population of injury-responsive neurons (iNeurons) show elevated plasticity 1 week post-injury. We found iNeurons are injury-surviving neurons that acquire a neuroblast-like gene expression signature after injury. CRISPR/Cas9 mutagenesis showed iNeurons are required for functional recovery and employ vesicular trafficking as an essential mechanism that underlies neuronal plasticity. This study provides a comprehensive resource of the cells and mechanisms that direct spinal cord regeneration and establishes zebrafish as a model of plasticity-driven neural repair.
Subject(s)
Neurogenesis , Neuronal Plasticity , Single-Cell Analysis , Spinal Cord Injuries , Spinal Cord Regeneration , Spinal Cord , Zebrafish , Animals , Spinal Cord Injuries/metabolism , Neuronal Plasticity/physiology , Neurogenesis/genetics , Spinal Cord/metabolism , Neurons/metabolism , Neurons/physiology , CRISPR-Cas Systems , GABAergic Neurons/metabolism , Recovery of Function , Disease Models, Animal , Nerve Regeneration/physiology , Animals, Genetically ModifiedABSTRACT
One of the greatest strengths of Drosophila genetics is its easily observable and selectable phenotypic markers. The mini-white marker has been widely used as a transgenic marker for Drosophila transgenesis. Flies carrying a mini-white construct can exhibit various eye colors ranging from pale orange to intense red, depending on the insertion site and gene dosage. Because the two copies of the mini-white marker show a stronger orange color, this is often used for selecting progenies carrying two transgenes together in a single chromosome after chromosomal recombination. However, some GAL4 lines available in the fly community originally have very strong red eyes. Without employing another marker, such as GFP, generating a recombinant chromosome with the strong red-eyed GAL4 and a desired UAS-transgene construct may be difficult. Therefore, we decided to change the red eyes of GAL4 lines to orange color. To change the eye color of the fly, we tested the CRISPR/Cas9 method with a guide RNA targeting the white gene with OK371-GAL4 and elav-GAL4. After a simple screening, we have successfully obtained multiple lines of orange-eyed OK371-GAL4 and elav-GAL4 that still maintain their original expression patterns. All of these simple experiments were performed by undergraduate students, allowing them to learn about a variety of different genetic experiments and genome editing while contributing to the fly research community by creating fruit fly lines that will be used in real-world research.
Subject(s)
CRISPR-Cas Systems , Drosophila Proteins , Eye Color , Gene Editing , Animals , Gene Editing/methods , Drosophila Proteins/genetics , Eye Color/genetics , Animals, Genetically Modified , Transcription Factors/genetics , Drosophila/genetics , Students , Drosophila melanogaster/genetics , RNA, Guide, CRISPR-Cas Systems/genetics , Eye Proteins , ATP-Binding Cassette TransportersABSTRACT
The 18th Transgenic Technology Meeting, held in Houston, Texas from November 12-15, 2023, was a vibrant international forum. It brought together nearly 400 delegates to discuss advances in transgenic technologies and the science these technologies support. Among them were 329 in-person and 70 remote delegates, representing 26 countries from 5 continents. The event, hosted by the International Society for Transgenic Technologies (ISTT), was set against the backdrop of the Hyatt Regency's panoramic views, reflecting the innovative spirit of the conference. A notable precursor to the main conference was the Allele Design Pre-conference Workshop, which fostered in-depth discussions on state-of-the-art methodologies. The main conference encompassed ten sessions, delving into diverse topics from Precision Animal Models of Human Disease to the use of transgenic animals in Space Biology. Eighty posters provided for a lively exchange of ideas, while the ISTT Prize and other awards highlighted the event's commitment to excellence. Beyond the conference halls, attendees had the opportunity to venture into Houston's Museum District, home to 19 museums in the downtown area, or indulge in unique dining experiences.
Subject(s)
Animals, Genetically Modified , Animals , Humans , Animals, Genetically Modified/genetics , United StatesABSTRACT
Transplantation remains the preferred treatment for end-stage kidney disease but is critically limited by the number of available organs. Xenografts from genetically modified pigs have become a promising solution to the loss of life while waiting for transplantation. However, the current clinical model for xenotransplantation will require off-site procurement, leading to a period of ischemia during transportation. As of today, there is limited understanding regarding the preservation of these organs, including the duration of viability, and the associated molecular changes. Thus, our aim was to evaluate the effects of static cold storage (SCS) on α1,3-galactosyltransferase knockout (GGTA1 KO) kidney. After SCS, viability was further assessed using acellular sub-normothermic ex vivo perfusion and simulated transplantation with human blood. Compared to baseline, tubular and glomerular interstitium was preserved after 2 days of SCS in both WT and GGTA1 KO kidneys. Bulk RNA-sequencing demonstrated that only eight genes were differentially expressed after SCS in GGTA1 KO kidneys. During sub-normothermic perfusion, kidney function, reflected by oxygen consumption, urine output, and lactate production was adequate in GGTA1 KO grafts. During a simulated transplant with human blood, macroscopic and histological assessment revealed minimal kidney injury. However, GGTA1 KO kidneys exhibited higher arterial resistance, increased lactate production, and reduced oxygen consumption during the simulated transplant. In summary, our study suggests that SCS is feasible for the preservation of porcine GGTA1 KO kidneys. However, alternative preservation methods should be evaluated for extended preservation of porcine grafts.
Subject(s)
Galactosyltransferases , Kidney Transplantation , Kidney , Organ Preservation , Transplantation, Heterologous , Animals , Transplantation, Heterologous/methods , Kidney Transplantation/methods , Galactosyltransferases/genetics , Galactosyltransferases/deficiency , Swine , Organ Preservation/methods , Humans , Animals, Genetically Modified , Perfusion/methods , Heterografts , Cryopreservation/methods , Gene Knockout Techniques/methods , MiceABSTRACT
Hepatocyte transplantation and bioartificial liver (BAL) systems hold significant promise as less invasive alternatives to traditional transplantation, providing crucial temporary support for patients with acute and chronic liver failure. Although human hepatocytes are ideal, their use is limited by ethical concerns and donor availability, leading to the use of porcine hepatocytes in BAL systems due to their functional similarities. Recent advancements in gene-editing technology have improved porcine organ xenotransplantation clinical trials by addressing immune rejection issues. Gene-edited pigs, such as alpha-1,3-galactosyltransferase (GGTA1) knockout pigs, offer a secure source of primary cells for BAL systems. Our research focuses on optimizing the safety and functionality of porcine primary hepatocytes during large-scale cultivation. We achieved this by creating GGTA1 knockout pigs through one-step delivery of CRISPR/Cas9 to pig zygotes via oviduct injection of rAAV, and enhancing hepatocyte viability and function by co-culturing hepatocytes with Roof plate-specific spondin 1 overexpressing HUVECs (R-HUVECs). Using a Rocker culture system, approximately 1010 primary porcine hepatocytes and R-HUVECs rapidly formed organoids with a diameter of 92.1 ± 28.1 µm within 24 h. These organoids not only maintained excellent functionality but also supported partial hepatocyte self-renewal during long-term culture over 28 days. Gene-edited primary porcine hepatocyte organoids will significantly advance the applications of hepatocyte transplantation and BAL systems.
Subject(s)
Galactosyltransferases , Gene Editing , Hepatocytes , Liver, Artificial , Organoids , Transplantation, Heterologous , Animals , Galactosyltransferases/genetics , Swine , Transplantation, Heterologous/methods , Organoids/metabolism , Gene Editing/methods , Humans , Animals, Genetically Modified , CRISPR-Cas Systems , Gene Knockout Techniques/methods , Coculture Techniques/methodsABSTRACT
Flusilazole is a well-known triazole fungicide applied to various crops and fruits worldwide. Flusilazole residues are frequently detected in the environment, and many researchers have reported the hazardous effects of flusilazole on non-target organisms; however, the developmental toxicity of flusilazole has not been fully elucidated. In this study, we investigated flusilazole-induced developmental defects in zebrafish, which are used in toxicology studies to assess the toxic effects of chemicals on aquatic species or vertebrates. We confirmed that flusilazole exposure affected the viability and hatching rate of zebrafish larvae, and resulted in morphological defects, reduced body length, diminished eye and head sizes, and inflated pericardial edema. Apoptosis, oxidative stress, and inflammation were also observed. These factors interrupted the normal organ formation during early developmental stages, and transgenic models were used to identify organ defects. We confirmed the effects of flusilazole on the nervous system using olig2:dsRed transgenic zebrafish, and on the cardiovascular system using cmlc2:dsRed and fli1:eGFP transgenic zebrafish. Our results demonstrate the developmental toxicity of flusilazole and its mechanisms in zebrafish as well as the detrimental effects of flusilazole.
Subject(s)
Animals, Genetically Modified , Apoptosis , Fungicides, Industrial , Oxidative Stress , Triazoles , Zebrafish , Animals , Oxidative Stress/drug effects , Apoptosis/drug effects , Triazoles/toxicity , Fungicides, Industrial/toxicity , Embryo, Nonmammalian/drug effects , SilanesABSTRACT
Spring viremia of carp virus (SVCV) has a broad fish host spectrum and is responsible for a disease that generally affects juvenile fishes with a mortality rate of up to 90%. In the absence of treatments or vaccines against SVCV, the search for prophylactic or therapeutic solutions is thus relevant, particularly to identify solutions compatible with mass vaccination. In addition to being a threat to aquaculture and ecosystems, SVCV is a unique pathogen to study virus-host interactions in the zebrafish model. Establishing the first reverse genetics system for SVCV and the design of recombinant SVCV (rSVCV) expressing fluorescent or bioluminescent proteins adds a new dimension for the study of these interactions using innovative imaging techniques. The infection by bath immersion of zebrafish larvae with rSVCV expressing mCherry allows us to define the first SVCV replication sites and the host innate immune responses using different transgenic lines of zebrafish. The fins were found as the main initial sites of infection in both zebrafish and carp, its natural host. Hence, new insights into the physiopathology of SVCV infection have been described. We report that neutrophils are recruited at the sites of infection and persist up to the death of the animal leading to an uncontrolled inflammation correlated with the expression of the pro-inflammatory cytokine IL1ß. Tissue damage was observed at the site of initial replication, a likely consequence of virus-induced injury or the pro-inflammatory response. Interestingly, SVCV infection by bath immersion triggers a persistent pro-inflammatory response rather than activation of the antiviral IFN signaling pathway as observed following intravenous injection, highlighting the importance of the route of infection on the progression of pathogenicity. Thus, this model of zebrafish larvae infection by rSVCV offers new perspectives to study in detail virus-host interactions and to discover new prophylactic or therapeutic solutions.
Subject(s)
Carps , Fish Diseases , Rhabdoviridae Infections , Rhabdoviridae , Zebrafish , Animals , Zebrafish/virology , Rhabdoviridae/physiology , Fish Diseases/virology , Rhabdoviridae Infections/virology , Rhabdoviridae Infections/immunology , Carps/virology , Animals, Genetically Modified , Disease Models, Animal , Immunity, Innate , ViremiaABSTRACT
The central and peripheral nervous systems (CNS and PNS, respectively) exhibit remarkable diversity in the capacity to regenerate following neuronal injury with PNS injuries being much more likely to regenerate than those that occur in the CNS. Glial responses to damage greatly influence the likelihood of regeneration by either promoting or inhibiting axonal regrowth over time. However, despite our understanding of how some glial lineages participate in nerve degeneration and regeneration, less is known about the contributions of peripheral satellite glial cells (SGC) to regeneration failure following central axon branch injury of dorsal root ganglia (DRG) sensory neurons. Here, using in vivo, time-lapse imaging in larval zebrafish coupled with laser axotomy, we investigate the role of SGCs in axonal regeneration. In our studies we show that SGCs respond to injury by relocating their nuclei to the injury site during the same period that DRG neurons produce new central branch neurites. Laser ablation of SGCs prior to axon injury results in more neurite growth attempts and ultimately a higher rate of successful central axon regrowth, implicating SGCs as inhibitors of regeneration. We also demonstrate that this SGC response is mediated in part by ErbB signaling, as chemical inhibition of this receptor results in reduced SGC motility and enhanced central axon regrowth. These findings provide new insights into SGC-neuron interactions under injury conditions and how these interactions influence nervous system repair.
Subject(s)
Axotomy , Ganglia, Spinal , Nerve Regeneration , Zebrafish , Animals , Nerve Regeneration/physiology , Animals, Genetically Modified , Spinal Cord , Satellite Cells, Perineuronal/physiology , Neuroglia/physiology , Zebrafish Proteins/metabolism , Axons/physiologyABSTRACT
Introduction: 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). Methods: 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. Results: 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. Conclusions: 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.
Subject(s)
Erythrocyte Transfusion , Erythrocytes , Gene Knockout Techniques , Animals , Erythrocyte Transfusion/adverse effects , Erythrocyte Transfusion/methods , Swine , Erythrocytes/immunology , Erythrocytes/metabolism , Animals, Genetically Modified , Hemoglobins/metabolism , Galactosyltransferases/genetics , Galactosyltransferases/deficiency , Hematocrit , Female , Male , PrimatesABSTRACT
In zebrafish, like in mammals, radial glial cells (RGCs) can act as neural progenitors during development and regeneration in adults. However, the heterogeneity of glia subpopulations entails the need for different specific markers of zebrafish glia. Currently, fluorescent protein expression mediated by a regulatory element from the glial fibrillary acidic protein (gfap) gene is used as a prominent glia reporter. We now expand this tool by demonstrating that a regulatory element from the mouse Fatty acid binding protein 7 (Fabp7) gene drives reliable expression in fabp7-expressing zebrafish glial cells. By using three different Fabp7 regulatory element-mediated fluorescent protein reporter strains, we reveal in double transgenic zebrafish that progenitor cells expressing fluorescent proteins driven by the Fabp7 regulatory element give rise to radial glia, oligodendrocyte progenitors, and some neuronal precursors. Furthermore, Bergmann glia represent the almost only glial population of the zebrafish cerebellum (besides a few oligodendrocytes), and the radial glia also remain in the mature cerebellum. Fabp7 regulatory element-mediated reporter protein expression in Bergmann glia progenitors suggests their origin from the ventral cerebellar proliferation zone, the ventricular zone, but not from the dorsally positioned upper rhombic lip. These new Fabp7 reporters will be valuable for functional studies during development and regeneration.
Subject(s)
Animals, Genetically Modified , Fatty Acid-Binding Protein 7 , Zebrafish , Animals , Zebrafish/genetics , Zebrafish/metabolism , Fatty Acid-Binding Protein 7/metabolism , Fatty Acid-Binding Protein 7/genetics , Neuroglia/metabolism , Cerebellum/metabolism , Cerebellum/cytology , Oligodendroglia/metabolism , Oligodendroglia/cytology , Mice , Fatty Acid-Binding Proteins/genetics , Fatty Acid-Binding Proteins/metabolism , Zebrafish Proteins/metabolism , Zebrafish Proteins/geneticsABSTRACT
INTRODUCTION: Inflammatory responses and coagulation disorders are a relevant challenge for successful cardiac xenotransplantation on its way to the clinic. To cope with this, an effective and clinically practicable anti-inflammatory and anti-coagulatory regimen is needed. The inflammatory and coagulatory response can be reduced by genetic engineering of the organ-source pigs. Furthermore, there are several therapeutic strategies to prevent or reduce inflammatory responses and coagulation disorders following xenotransplantation. However, it is still unclear, which combination of drugs should be used in the clinical setting. To elucidate this, we present data from pig-to-baboon orthotopic cardiac xenotransplantation experiments using a combination of several anti-inflammatory drugs. METHODS: Genetically modified piglets (GGTA1-KO, hCD46/hTBM transgenic) were used for orthotopic cardiac xenotransplantation into captive-bred baboons (n = 14). All animals received an anti-inflammatory drug therapy including a C1 esterase inhibitor, an IL-6 receptor antagonist, a TNF-α inhibitor, and an IL-1 receptor antagonist. As an additive medication, acetylsalicylic acid and unfractionated heparin were administered. The immunosuppressive regimen was based on CD40/CD40L co-stimulation blockade. During the experiments, leukocyte counts, levels of C-reactive protein (CRP) as well as systemic cytokine and chemokine levels and coagulation parameters were assessed at multiple timepoints. Four animals were excluded from further data analyses due to porcine cytomegalovirus/porcine roseolovirus (PCMV/PRV) infections (n = 2) or technical failures (n = 2). RESULTS: Leukocyte counts showed a relevant perioperative decrease, CRP levels an increase. In the postoperative period, leukocyte counts remained consistently within normal ranges, CRP levels showed three further peaks after about 35, 50, and 80 postoperative days. Analyses of cytokines and chemokines revealed different patterns. Some cytokines, like IL-8, increased about 2-fold in the perioperative period, but then decreased to levels comparable to the preoperative values or even lower. Other cytokines, such as IL-12/IL-23, decreased in the perioperative period and stayed at these levels. Besides perioperative decreases, there were no relevant alterations observed in coagulation parameters. In summary, all parameters showed an unremarkable course with regard to inflammatory responses and coagulation disorders following cardiac xenotransplantation and thus showed the effectiveness of our approach. CONCLUSION: Our preclinical experience with the anti-inflammatory drug therapy proved that controlling of inflammation and coagulation disorders in xenotransplantation is possible and well-practicable under the condition that transmission of pathogens, especially of PCMV/PRV to the recipient is prevented because PCMV/PRV also induces inflammation and coagulation disorders. Our anti-inflammatory regimen should also be applicable and effective in the clinical setting of cardiac xenotransplantation.
Subject(s)
Animals, Genetically Modified , Heart Transplantation , Inflammation , Papio , Transplantation, Heterologous , Animals , Transplantation, Heterologous/methods , Heart Transplantation/methods , Swine , Inflammation/immunology , Blood Coagulation/drug effects , Anti-Inflammatory Agents/pharmacology , Anti-Inflammatory Agents/therapeutic use , Humans , Heterografts/immunology , Galactosyltransferases/genetics , Immunosuppressive Agents/pharmacology , Cytokines/metabolismABSTRACT
BACKGROUND: Xenotransplantation using pig organs is now a clinical reality. However, the process for xenograft recipient screening lacks clarity and scientific rigor: no established thresholds exist to determine which levels of preformed antipig natural antibodies (Nabs) will be safe for clinical xenograft transplantation, and hyperacute rejection (HAR) or acute humoral xenograft rejection (AHXR), which still impacts pig-to-primate kidney xenograft survivals, may impede broader application of pig-to-human clinical xenograft transplantation. METHODS: We retrospectively examined 28 cases of pig-to-baboon kidney xenotransplantation using GalTKO±human complement regulatory protein (hCRP)-transgenic (Tg) pig donors, as well as 6 cases of triple-KO multi-Tg (10GE) pig donors, and developed screening algorithms to predict risk of HAR/AHXR based on recipient antipig Nab levels. Preformed Nabs were evaluated using both complement-dependent cytotoxicity and antibody (IgM and IgG) binding flow-cytometry assays. RESULTS: High complement-dependent cytotoxicity was associated with HAR/AHXR as expected. However, we also found that high levels of IgG were independently associated with HAR/AHXR, and we developed 2 indices to interpret and predict the risk of IgG-mediated HAR/AHXR. CONCLUSIONS: Based on the data in this study, we have established a new 2-step screening, which will be used for future clinical kidney xenotransplantation trials.
Subject(s)
Animals, Genetically Modified , Graft Rejection , Graft Survival , Kidney Transplantation , Transplantation, Heterologous , Animals , Transplantation, Heterologous/adverse effects , Kidney Transplantation/adverse effects , Graft Rejection/immunology , Graft Rejection/prevention & control , Retrospective Studies , Swine , Risk Factors , Immunoglobulin G/blood , Galactosyltransferases/genetics , Galactosyltransferases/immunology , Galactosyltransferases/deficiency , Heterografts , Immunity, Humoral , Immunoglobulin M/blood , Humans , Male , Antibodies, Heterophile/immunology , Acute DiseaseABSTRACT
Leucine-rich repeat kinase-2 (LRRK2), a gene mutated in familial and sporadic Parkinson's disease (PD), controls multiple cellular processes important for GLIA physiology. Interestingly, emerging studies report that LRRK2 is highly expressed in oligodendrocyte precursor cells (OPCs) compared to the pathophysiology of other brain cells and oligodendrocytes (OLs) in PD. Altogether, these observations suggest crucial function(s) of LRRK2 in OPCs/Ols, which would be interesting to explore. In this study, we investigated the role of LRRK2 in OLs. We showed that LRRK2 knock-out (KO) OPC cultures displayed defects in the transition of OPCs into OLs, suggesting a role of LRRK2 in OL differentiation. Consistently, we found an alteration of myelin basic protein (MBP) striosomes in LRRK2 KO mouse brains and reduced levels of oligodendrocyte transcription factor 2 (Olig2) and Mbp in olig2:EGFP and mbp:RFP transgenic zebrafish embryos injected with lrrk2 morpholino (MO). Moreover, lrrk2 knock-down zebrafish exhibited a lower amount of nerve growth factor (Ngf) compared to control embryos, which represents a potent regulator of oligodendrogenesis and myelination. Overall, our findings indicate that LRRK2 controls OL differentiation, affecting the number of mature OLs.
Subject(s)
Cell Differentiation , Leucine-Rich Repeat Serine-Threonine Protein Kinase-2 , Oligodendroglia , Zebrafish Proteins , Zebrafish , Animals , Zebrafish/metabolism , Zebrafish/genetics , Leucine-Rich Repeat Serine-Threonine Protein Kinase-2/genetics , Leucine-Rich Repeat Serine-Threonine Protein Kinase-2/metabolism , Oligodendroglia/metabolism , Oligodendroglia/cytology , Cell Differentiation/genetics , Mice , Zebrafish Proteins/genetics , Zebrafish Proteins/metabolism , Mice, Knockout , Myelin Basic Protein/metabolism , Myelin Basic Protein/genetics , Animals, Genetically ModifiedABSTRACT
Gephyrin is thought to play a critical role in clustering glycine receptors at synapses within the central nervous system (CNS). The main in vivo evidence for this comes from Gephyrin (Gphn)-null mice, where glycine receptors are depleted from synaptic regions. However, these mice die at birth, possibly due to impaired molybdenum cofactor (MoCo) synthesis, an essential role Gephyrin assumes throughout an animal. This complicates the interpretation of synaptic phenotypes in Gphn-null mice and raises the question whether the synaptic and enzymatic functions of Gephyrin can be investigated separately. Here, we generated a gephyrinb zebrafish mutant, vo84, that almost entirely lacks Gephyrin staining in the spinal cord. gephyrinbvo84 mutants exhibit normal gross morphology at both larval and adult stages. In contrast to Gphn-null mice, gephyrinbvo84 mutants exhibit normal motor activity and MoCo-dependent enzyme activity. Instead, gephyrinbvo84 mutants display impaired rheotaxis and increased mortality in late development. To investigate what may mediate these defects in gephyrinbvo84 mutants, we examined the cell density of neurons and myelin in the spinal cord and found no obvious changes. Surprisingly, in gephyrinbvo84 mutants, glycine receptors are still present in the synaptic regions. However, their abundance is reduced, potentially contributing to the observed defects. These findings challenge the notion that Gephyrin is absolutely required to cluster glycine receptors at synapses and reveals a new role of Gephyrin in regulating glycine receptor abundance and rheotaxis. They also establish a powerful new model for studying the mechanisms underlying synaptic, rather than enzymatic, functions of Gephyrin.