Composition and Bioactivity of a Placental Tissue Particulate (PTP-001) Indicate Greater Potential than Platelet-Rich Plasma for the Treatment of Osteoarthritis.

OBJECTIVE: This study was conducted to compare therapeutically relevant properties of platelet-rich plasma (PRP), a commonly used autologous intra-articular treatment for osteoarthritis (OA), with those of a novel placental tissue particulate, PTP-001, which is in development as a regulated biologic treatment for knee OA. DESIGN: Quantitative immunoassays were performed to determine the content of key growth/regulatory biofactors in PTP-001, and in leukocyte-rich (LR)-PRP or leukocyte-poor (LP)-PRP. An anti-inflammatory bioassay was used to evaluate the effects of each treatment on pro-inflammatory cytokine (tumor necrosis factor (TNF)-α) production in a macrophage cell culture system. Gene expression experiments were conducted using a co-culture system of human synoviocytes (pre-stimulated with interleukin (IL)-1ß) and articular chondrocytes, with quantitative polymerase chain reaction analyses of the separate cellular compartments. RESULTS: The concentrations of several biofactors (e.g., basic fibroblast growth factor, tissue inhibitor of metalloproteases-3, interleukin-1 receptor antagonist) representative of diverse disease-relevant mechanisms of action were significantly higher for PTP-001 relative to LR-PRP or LP-PRP. PTP-001 and PRP preparations were able to reduce TNF-α production in macrophage cell cultures; however, greater variability was observed for PRP in comparison with PTP-001. In the chondrocyte/synoviocyte co-culture experiments, PTP-001 and LR-PRP (but not LP-PRP) significantly reduced chondrocyte MMP13 expression in cultures containing IL-1-pretreated synoviocytes. In addition, ADAMTS5 expression was reduced in the chondrocyte compartment following treatment with PTP-001 relative to PRP. CONCLUSION: These findings support evidence of a potent, multifactorial mechanism of action for a consistently manufactured biologic (PTP-001), which may be of greater therapeutic benefit in comparison with more heterogeneous preparations of PRP which may be generated at the time of treatment.

T Helper 17-Like Regulatory T Cells in Equine Synovial Fluid Are Associated With Disease Severity of Naturally Occurring Posttraumatic Osteoarthritis.

BACKGROUND: Infiltration of cluster of differentiation (CD) 3+ (CD3+) T cells into the synovium and synovial fluid occurs in most patients with posttraumatic osteoarthritis. During disease progression, proinflammatory T helper 17 cells and anti-inflammatory regulatory T cells infiltrate the joint in response to inflammation. This study aimed to characterize the dynamics of regulatory T and T helper 17 cell populations in synovial fluid from equine clinical patients with posttraumatic osteoarthritis to determine whether phenotype and function are associated with potential immunotherapeutic targets. HYPOTHESIS: An imbalance of the ratio of regulatory T cells and T helper 17 cells would be associated with disease progression in posttraumatic osteoarthritis, suggesting opportunities for immunomodulatory therapy. STUDY DESIGN: Descriptive laboratory study. METHODS: Synovial fluid was aspirated from the joints of equine clinical patients undergoing arthroscopic surgery for posttraumatic osteoarthritis resulting from intra-articular fragmentation. Joints were classified as having mild or moderate posttraumatic osteoarthritis. Synovial fluid was also obtained from nonoperated horses with normal cartilage. Peripheral blood was obtained from horses with normal cartilage and those with mild and moderate posttraumatic osteoarthritis. Synovial fluid and peripheral blood cells were analyzed by flow cytometry, and native synovial fluid was analyzed by enzyme-linked immunosorbent assay. RESULTS: CD3+ T cells represented 81% of lymphocytes in synovial fluid, which increased in animals with moderate posttraumatic osteoarthritis to 88.3% (P = .02). CD14+ macrophages were doubled in those with moderate posttraumatic osteoarthritis compared with mild posttraumatic osteoarthritis and controls (P < .001). Less than 5% of CD3+ T cells found within the joint were forkhead box P3 protein+ (Foxp3+) regulatory T cells, but a 4- to 8-times higher percentage of nonoperated and mild posttraumatic osteoarthritis joint regulatory T cells secreted interleukin (IL)-10 than peripheral blood Tregs (P < .005). T regulatory-1 cells that secreted IL-10 but did not express Foxp3 accounted for approximately 5% of CD3+ T cells in all joints. T helper 17 cells and Th17-like regulatory T cells were increased in those with moderate posttraumatic osteoarthritis (P < .0001) compared with mild and nonoperated patients. IL-10, IL-17A, IL-6, chemokine (C-C motif) ligand (CCL) 2 (CCL2), and CCL5 concentrations detected by enzyme-linked immunosorbent assay in synovial fluid were not different between groups. CONCLUSIONS: An imbalance of the ratio of regulatory T cells and T helper 17 cells and an increase in T helper 17 cell-like regulatory T cells in synovial fluid from joints with more severe disease provide novel insights into immunological mechanisms that are associated with posttraumatic osteoarthritis progression and pathogenesis. CLINICAL RELEVANCE: Early and targeted use of immunotherapeutics in the mitigation of posttraumatic osteoarthritis may improve patient clinical outcomes.

Interleukin-6 neutralization and regulatory T cells are additive in chondroprotection from IL-1ß-induced inflammation.

Anti-inflammatory Regulatory T cells (Tregs) are enriched in the joints of patients with osteoarthritis (OA) compared to healthy joints. Tregs maintain homeostasis through secretion of anti-inflammatory cytokines and cell-to-cell interactions including immune checkpoint signaling. Interleukin-6 (IL-6) is a pleiotropic cytokine secreted by inflamed synoviocytes and chondrocytes that can inhibit or alter Treg function. This study tested the hypothesis that neutralization of IL-6 would enable Treg anti-inflammatory function to resolve inflammation and catabolism elicited by IL-1ß in an equine chondrocyte/synoviocyte/Treg tri-culture OA model. Synoviocyte/chondrocyte co-cultures were stimulated with IL-1ß, and treated with αIL-6 neutralizing antibody. Activated Tregs secreting IL-10 were added in direct contact with synoviocytes to create a tri-culture. Neutralization of IL-6 partially restored Treg anti-inflammatory functions and, in combination, reduced IL-1ß-stimulated synoviocyte MMP13 expression to control levels and restored Acan expression in chondrocytes. IL-6 neutralization alone decreased Il6 expression in chondrocytes and synoviocytes, mitigating IL-6 positive feedback loop. Although Tregs were the primary producers of anti-inflammatory IL-10 and IL-4, they also produced pro-inflammatory IL-17A, as detected by ELIA, which may have been responsible for incomplete rescue of synoviocyte/chondrocyte homeostasis following IL-1ß stimulation. Treg secretion of IL-10, IL-4, and IL-17A was not altered by tri-culture conditions or presence of αIL-6, therefore, it was unlikely that Treg phenotype instability occurred. The significant effect of chondrocyte/synoviocyte donor, but not Treg donor, on gene expression and IL-6 concentration in conditioned media, indicated that personalized therapy considering the patient's OA status might be needed for successful implementation of immunotherapy in the context of OA.

Synovium secretome as a disease-modifying treatment for equine osteoarthritis.

OBJECTIVE: To identify chondroprotective factors as potential disease-modifying osteoarthritis treatments using an unbiased, bottom-up proteomics approach. SAMPLES: Paired equine cartilage explants and synovial membrane were collected postmortem from 4 horses with no history of lameness and grossly normal joints at necropsy. PROCEDURES: Six groups were established: cartilage, synoviocytes, and cartilage + synoviocytes (coculture), all with or without interleukin (IL)-1ß. The catabolic effect of IL-1ß was verified by glycosaminoglycan (GAG) released from cartilage into media by 1,9-dimethyl-methylene blue assay and cartilage toluidine blue histochemistry. Conditioned media from cocultures with or with IL-1ß were submitted for bottom-up proteomic analysis. Synoviocyte gene expression was evaluated using reverse transcription-quantitative PCR (RT-qPCR) for proteins of interest identified in the proteomics scan. RESULTS: GAG content was retained in cartilage when in cocultures treated with IL-1ß. Fourteen proteins of interest were selected from the proteomic analysis. From these 14 proteins, metalloproteinase inhibitor 3 precursor (TIMP3), tumor necrosis factor receptor superfamily member 11B (TNFRSF11B), insulin-like growth factor-binding protein 2 (IGFBP2), and alpha-2 macroglobulin (A2M) were selected for synoviocyte gene expression analysis by RT-qPCR. Gene expression of TIMP3 (P = .02) and TNFRSF11B (P = .04) were significantly increased in synoviocytes from cocultures treated with IL-1ß compared to controls. Contrary to expectations based on protein expression, IGFBP2 gene expression (P = .04) was significantly decreased in IL-1ß-stimulated coculture synoviocytes compared to control coculture synoviocytes. A2M gene expression in synoviocytes was not different between coculture groups. CLINICAL RELEVANCE: The secretome from synoviocytes could provide a milieu of bioactive factors to restore joint homeostasis in osteoarthritis.

Proteomic Analysis and Cell Viability of Nine Amnion, Chorion, Umbilical Cord, and Amniotic Fluid-Derived Products.

OBJECTIVE: Amnion products are used in various musculoskeletal surgeries and as injections for joint pain with conflicting reports of cell viability and protein contents. The objective of this study was to determine the full proteome and examine cell viability in 9 commercial amnion products using an unbiased bottom-up shotgun proteomics approach and confocal microscopy. DESIGN: Products were subjected to liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis and searched against a UniProt Homo sapiens database. Relative protein abundance was determined for each sample. Based on proteomics results, lumican was measured by enzyme-linked immunosorbent assay (ELISA) and Western blot analysis was performed for interleukin-1 receptor antagonist (IL-1Ra) and tissue inhibitor of matrix metalloproteinase-2 (TIMP-2). Cell viability was determined by calcein AM (live) and ethidium homodimer (dead) staining and confocal microscopy. RESULTS: Proteomic analysis revealed 919 proteins in the nine products. Proteins were primarily collagens, keratin, and albumin. Lumican, a small leucine-rich proteoglycan (SLRP) was found in all samples. Western blot analysis for IL-1Ra and TIMP-2 indicated presence of both proteins, with nonspecific antibody binding also present in all samples. No live cells were identified in any product. CONCLUSIONS: Several novel proteins were identified through proteomics that might impart the beneficial effects of amnion products, including SLRPs, collagens, and regulators of fibroblast activity. IL-1Ra and TIMP-2 were identified, but concentrations measured by ELISA may be falsely increased due to nonspecific antibody binding. The concept that the amnion tissues provide live cells to aid in tissue regeneration cannot be supported by the findings of this study.

Regulatory T cells provide chondroprotection through increased TIMP1, IL-10 and IL-4, but cannot mitigate the catabolic effects of IL-1ß and IL-6 in a tri-culture model of osteoarthritis.

Objective: To gain insight into Treg interactions with synovial tissues in early OA, an equine tri-culture model of OA was used to test the hypothesis that Tregs, in the absence of T Helper 17 â€‹cells, are sufficient to resolve inflammation elicited by IL-1ß. Methods: To model normal and OA joints, synoviocytes were co-cultured with chondrocytes in a transwell system and ± stimulated with IL-1ß. Tregs were activated and enriched, then added to co-cultures, creating tri-cultures. At culture end, synoviocytes and chondrocytes were analyzed for gene expression, Treg Foxp3 expression was reexamined by flow cytometry, and conditioned media were evaluated by ELISA. Results: Tregs increased IL-10 and IL-4 in tri-culture media and increased TIMP1 gene expression in synoviocytes and chondrocytes. Tregs increased IL-6 in conditioned media and Il6 gene expression in synoviocytes, which was additive with IL-1ß. In chondrocytes, addition of Tregs decreased Col2b gene expression while Acan gene expression was decreased by IL-1ß and addition of Tregs. IL-17A was detected in tri-cultures. CCL2 and CCL5 were increased in tri-cultures. Conclusions: In a tri-culture model of OA, addition of Tregs resulted in conditions conducive to chondroprotection including increased concentration of IL-10 and IL-4 in conditioned media and increased gene expression of TIMP1 in both chondrocytes and synoviocytes. However, there was increased concentration of the catabolic cytokine IL-6, and decreased gene expression of Col2b and Acan in IL-1ß-stimulated chondrocytes. These results suggest that blocking IL-6 could enhance Treg function in mitigating OA progression.

Disease-Modifying Osteoarthritis Treatment With Interleukin-1 Receptor Antagonist Gene Therapy in Small and Large Animal Models.

OBJECTIVE: Gene therapy holds great promise for the treatment of osteoarthritis (OA) because a single intraarticular injection can lead to long-term expression of therapeutic proteins within the joint. This study was undertaken to investigate the use of a helper-dependent adenovirus (HDAd)-mediated intraarticular gene therapy approach for long-term expression of interleukin-1 receptor antagonist (IL-1Ra) as sustained symptomatic and disease-modifying therapy for OA. METHODS: In mouse models of OA, efficacy of HDAd-IL-1Ra was evaluated by histologic analysis, micro-computed tomography (micro-CT), and hot plate analysis. In a horse OA model, safety and efficacy of HDAd-IL-1Ra were evaluated by blood chemistry, analyses of synovial fluid, synovial membrane, and cartilage, and gross pathology and lameness assessments. RESULTS: In skeletally immature mice, HDAd-IL-1Ra prevented development of cartilage damage, osteophytes, and synovitis. In skeletally immature and mature mice, treatment with HDAd-interleukin-1 receptor antagonist post-OA induction resulted in improved-albeit not significantly-cartilage status assessed histologically and significantly increased cartilage volume, cartilage surface, and bone surface covered by cartilage as assessed by micro-CT. Fewer osteophytes were observed in HDAd-IL-1Ra-treated skeletally immature mice. Synovitis was not affected in skeletally immature or mature mice. HDAd-IL-1Ra protected against disease-induced thermal hyperalgesia in skeletally mature mice. In the horse OA model, HDAd-IL-1Ra therapy significantly improved lameness parameters, indicating efficient symptomatic treatment. Moreover, macroscopically and histologically assessed cartilage and synovial membrane parameters were significantly improved, suggesting disease-modifying efficacy. CONCLUSION: These data from OA models in small and large animals demonstrated safe symptomatic and disease-modifying treatment with an HDAd-expressing IL-1Ra. Furthermore, this study establishes HDAd as a vector for joint gene therapy.

AAV-mediated Overexpression of IL-10 Mitigates the Inflammatory Cascade in Stimulated Equine Chondrocyte Pellets.

BACKGROUND: Following joint trauma, a posttraumatic inflammatory cascade drives degeneration of the joint. We aimed to assess whether transduction of chondrocytes with AAV5 overexpressing the immunomodulatory cytokine IL-10 would have protective effects in pellet cultures stimulated with IL-1ß. METHODS: Chondrocytes were isolated from 3 healthy horses and were transduced with AAV5-IL-10 at a dose of 1 x 105vg/cell. Chondrocyte pellets were formed by centrifugation and were stimulated with IL-1ß starting 48 hours following transduction. After 2, 6 and 14 days in culture, supernatants were collected for cytokine analysis and RNA was isolated from cells for gene expression analysis. Pellets were also collected for biochemical analysis. RESULTS: Transduction of chondrocytes led to significant increases in IL-10 expression. IL-10 expression was further enhanced by IL-1ß stimulation. IL-10 overexpression led to significantly decreased expression of IL-1ß and ADAMTS4. PGE2 synthesis was also significantly decreased. IL-1ß mediated suppression of GAG synthesis was not rescued by IL-10. CONCLUSIONS: Overexpression of IL-10 modulates the inflammatory response in chondrocytes, which may mitigate some of the deleterious effects of pro-inflammatory cascades in the posttraumatic joint. AAV5-IL-10 led to efficient and sustained overexpression of IL-10 in chondrocytes and could represent a viable treatment option for preventing osteoarthritis.

Matrix-Induced Autologous Chondrocyte Implantation (MACI) Using a Cell-Seeded Collagen Membrane Improves Cartilage Healing in the Equine Model.

BACKGROUND: Autologous chondrocyte implantation (ACI) using a collagen scaffold (matrix-induced ACI; MACI) is a next-generation approach to traditional ACI that provides the benefit of autologous cells and guided tissue regeneration using a biocompatible collagen scaffold. The MACI implant also has inherent advantages including surgical implantation via arthroscopy or miniarthrotomy, the elimination of periosteal harvest, and the use of tissue adhesive in lieu of sutures. This study evaluated the efficacy of the MACI implant in an equine full-thickness cartilage defect model at 1 year. METHODS: Autologous chondrocytes were seeded onto a collagen type-I/III membrane and implanted into one of two 15-mm defects in the femoral trochlear ridge of 24 horses. Control defects either were implanted with cell-free collagen type-I/III membrane (12 horses) or were left ungrafted as empty defects (12 horses). An additional 3 horses had both 15-mm defects remain empty as nonimplanted joints. The repair was scored by second-look arthroscopy (12 weeks), and necropsy examination (53 weeks). Healing was assessed by arthroscopic scoring, gross assessment, histology and immunohistology, cartilage matrix component assay, and gene expression determination. Toxicity was examined by prostaglandin E2 formation in joint fluid, and lymph node morphology combined with histologic screening of organs. RESULTS: MACI-implanted defects had improved gross healing and composite histologic scores, as well as increases in chondrocyte predominance, toluidine blue-stained matrix, and collagen type-II content compared with scaffold-only implanted or empty defects. There was minimal evidence of reaction to the implant in the synovial membrane (minor perivascular cuffing), subchondral bone, or cartilage. There were no adverse clinical effects, signs of organ toxicity, or evidence of chondrocytes or collagen type-I/III membrane in draining lymph nodes. CONCLUSIONS: The MACI implant appeared to improve cartilage healing in a critical-sized defect in the equine model compared with collagen matrix alone. CLINICAL RELEVANCE: These results indicate that the MACI implant is quick to insert, provides chondrocyte security in the defect, and improves cartilage healing compared with ACI.

Implantation of rAAV5-IGF-I transduced autologous chondrocytes improves cartilage repair in full-thickness defects in the equine model.

Cartilage injury often precipitates osteoarthritis which has driven research to bolster repair in cartilage impact damage. Autologous chondrocytes transduced with rAAV5-IGF-I were evaluated in chondral defects in a well-established large animal model. Cartilage was harvested from the talus of 24 horses; chondrocytes were isolated and stored frozen. Twenty million cells were cultured and transduced with 10(5) AAV vg/cell prior to implantation. Chondrocytes from eight horses were transduced with rAAV5-IGF-I, chondrocytes from eight horses with rAAV5-GFP, and chondrocytes from eight horses were not transduced. A 15 mm full-thickness chondral defect was created arthroscopically in the lateral trochlear ridge of the femur in both femoropatellar joints. Treated defects were filled with naive or gene-enhanced chondrocytes, in fibrin vehicle. Control defects in the opposite limb received fibrin alone. rAAV5-IGF-I transduced chondrocytes resulted in significantly better healing at 8 week arthroscopy and 8 month necropsy examination when compared to controls. At 8 months, defects implanted with cells expressing IGF-I had better histological scores compared to control defects and defects repaired with naive chondrocytes. This included increased chondrocyte predominance and collagen type II, both features of hyaline-like repair tissue. The equine model closely approximates human cartilage healing, indicating AAV-mediated genetic modification of chondrocytes may be clinically beneficial to humans.

Autologous chondrocyte implantation drives early chondrogenesis and organized repair in extensive full- and partial-thickness cartilage defects in an equine model.

Autologous chondrocyte implantation (ACI) has been used clinically for over 15 years and yet definitive evidence of chondrocyte persistence and direct impact on cartilage repair in full-thickness lesions is scant and no data are available on ACI in partial-thickness defects in any animal model. This study assessed the effect of chondrocytes secured using periosteal overlay in partial- and full-thickness cartilage defects in the equine model. Paired cartilage defects 15 mm in diameter were made in the patellofemoral joint of 16 horse and repaired with ACI or periosteal flap alone. Response was assessed at 8 weeks by clinical, microradiographic, and histologic appearance, and by collagen type II immunohistochemistry, and proteoglycan and DNA quantification. ACI improved histologic scores in partial- and full-thickness cartilage defects, including defect filling, attachment to the underlying subchondral bone, and presence of residual chondrocyte accumulations. For partial-thickness defects chondrocyte predominance, collagen type II content, and toluidine stained matrix were enhanced, and attachment to the surrounding cartilage improved. DNA and PG content of grafted partial-thickness defects was improved by chondrocyte implantation. Periosteal patches alone did not induce cartilage repair. This study indicated implantation of chondrocytes to cartilage defects improved healing with a combination of persisting chondrocyte regions, enhanced collagen type II formation, and better overall cartilage healing scores. Use of ACI in the more challenging partial-thickness defects also improved histologic indices and biochemical content. The equine model of cartilage healing closely resembles cartilage repair in man, and results of this study confirm cell persistence and improved early cartilage healing events after ACI.

Fasting-induced reduction in locomotor activity and reduced response of orexin neurons in carnitine-deficient mice.

We found reduced locomotor activity (LA) under fasting in systemic carnitine-deficient juvenile visceral steatosis (jvs(-/-)) mice. When food was withdrawn at 8:00 a.m. (lights-off at 7:00 p.m., 12h/cycle), the nocturnal LA of jvs(-/-) mice was much less than the control (jvs(+/+) and jvs(+/-)) mice. LA recovered under carnitine or sucrose administration, but not under medium-chain triglyceride. In addition, fasted jvs(-/-) mice, without any energy supply, were activated by modafinil, a stimulator of the dopamine pathway. These results suggest that the reduced LA is not adequately explained by energy deficit. As the fasted jvs(-/-) mice showed lower body core temperature (BT), we examined the central nervous system regulating LA and BT. We found lower percentage of c-Fos positive orexin neurons in the lateral hypothalamus and reduced orexin-A concentration in the cerebrospinal fluid of fasted jvs(-/-) mice. Sleep analysis revealed that fasted jvs(-/-) mice had disruption of prolonged wakefulness, with a higher frequency of brief episodes of non-REM sleep during the dark period than fasted jvs(+/+) mice. These results strongly suggest that the reduced LA in fasted jvs(-/-) mice is related to the inhibition of orexin neuronal activity.

A case of adult-onset type II citrullinemia--deterioration of clinical course after infusion of hyperosmotic and high sugar solutions.

BACKGROUND: Adult-onset type II citrullinemia (CTLN2) is an autosomal recessive disorder caused by mutations of SLC25A13 gene encoding citrin and is characterized by recurrent encephalopathy with hyperammonemia. Factors affecting disease progression remain unknown. We report a case with CTLN2, whose clinical course was rapidly worsened by the administration of Glyceol, a hyperosmotic diuretic solution consisting of 10% glycerol and 5% fructose in saline. CASE REPORT: A 34-year-old man was admitted in coma after repeated episodes of altered consciousness. His plasma ammonia level was markedly elevated without any evidence of liver diseases. Brain MRI revealed high signal intensities at the bilateral cingulate gyri and insular cortices, suggesting hepatic encephalopathy. We administered Glyceol. intravenously to alleviate brain edema, however, he developed intractable seizures along with steep increase in plasma ammonia levels (from 808 to 2210 microg/dL) and died. The diagnosis of CTLN2 was confirmed by elevations of plasma citrulline level (384.3 nmol/mL; normal 17-43 nmol/mL) and serum pancreatic secretory trypsin inhibitor (PSTI) (110 ng/mL; normal 4.6-12.2 ng/mL), decrease in hepatic argininosuccinate synthetase activity (5.5% of control), lack of hepatic citrin protein expression and mutations in SLC25A13 gene (compound heterozygote with S225X and Ex1-1G>A). CONCLUSIONS: Physicians should take CTLN2 into consideration as a differential diagnosis in Asian patients with a history of repeated unconsciousness with hyperammonemia and use D-mannitol but not glycerol to treat brain edema in patients with CTLN2.

Attenuation of cardiac hypertrophy in carnitine-deficient juvenile visceral steatosis (JVS) mice achieved by lowering dietary lipid.

We examined the development of cardiac hypertrophy in juvenile visceral steatosis (JVS) mice, a model of systemic carnitine deficiency, by varying the amount of lipid in the diet. Cardiac hypertrophy was markedly attenuated by decreasing soy bean oil (SBO) from 5% (w/w) to 1%. Triglyceride contents of the ventricles of JVS mice fed 1% SBO were significantly lower than in JVS mice fed 5% SBO. The addition of medium-chain triglycerides metabolically utilized by JVS mice did not affect the development of cardiac hypertrophy. On the other hand, the mRNA levels of atrial natriuretic peptide and skeletal alpha-actin, which are related to cardiac hypertrophy, were also attenuated by decreasing lipid in the diet. Adenylate energy charge and creatine phosphate in the heart of JVS mice at the early stage of hypertrophy were not significantly different from control mice given the same laboratory chow (4.6% of lipid). Although urinary prostaglandin F(2alpha) levels were found to be increased in JVS mice at 15 days of age when they developed cardiac hypertrophy, administration of aspirin was not efficacious. We, therefore, propose that the proportion of lipid in the diet is important in the development of cardiac hypertrophy in carnitine-deficient JVS mice, and that this is not related to prostaglandin formation.

Reduced N-acetylaspartate levels in mice lacking aralar, a brain- and muscle-type mitochondrial aspartate-glutamate carrier.

Aralar is a mitochondrial calcium-regulated aspartate-glutamate carrier mainly distributed in brain and skeletal muscle, involved in the transport of aspartate from mitochondria to cytosol, and in the transfer of cytosolic reducing equivalents into mitochondria as a member of the malate-aspartate NADH shuttle. In the present study, we describe the characteristics of aralar-deficient (Aralar-/-) mice, generated by a gene-trap method, showing no aralar mRNA and protein, and no detectable malate-aspartate shuttle activity in skeletal muscle and brain mitochondria. Aralar-/- mice were growth-retarded, exhibited generalized tremoring, and had pronounced motor coordination defects along with an impaired myelination in the central nervous system. Analysis of lipid components showed a marked decrease in the myelin lipid galactosyl cerebroside. The content of the myelin lipid precursor, N-acetylaspartate, and that of aspartate are drastically decreased in the brain of Aralar-/- mice. The defect in N-acetylaspartate production was also observed in cell extracts from primary neuronal cultures derived from Aralar-/- mouse embryos. These results show that aralar plays an important role in myelin formation by providing aspartate for the synthesis of N-acetylaspartate in neuronal cells.

Adult-onset type II citrullinemia and idiopathic neonatal hepatitis caused by citrin deficiency: involvement of the aspartate glutamate carrier for urea synthesis and maintenance of the urea cycle.

Citrin is a mitochondrial aspartate glutamate carrier primarily expressed in the liver, heart, and kidney. We found that adult-onset type II citrullinemia is caused by mutations in the SLC25A13 gene that encodes for citrin. In this report, we describe the frequency of SLC25A13 mutations, the roles of citrin as a member of the urea cycle and as a member of the malate-aspartate shuttle, the relationship between its functions and symptoms of citrin deficiency, and therapeutic issues.

Slc25a13-knockout mice harbor metabolic deficits but fail to display hallmarks of adult-onset type II citrullinemia.

Adult-onset type II citrullinemia (CTLN2) is an autosomal recessive disease caused by mutations in SLC25A13, the gene encoding the mitochondrial aspartate/glutamate carrier citrin. The absence of citrin leads to a liver-specific, quantitative decrease of argininosuccinate synthetase (ASS), causing hyperammonemia and citrullinemia. To investigate the physiological role of citrin and the development of CTLN2, an Slc25a13-knockout (also known as Ctrn-deficient) mouse model was created. The resulting Ctrn-/- mice were devoid of Slc25a13 mRNA and citrin protein. Liver mitochondrial assays revealed markedly decreased activities in aspartate transport and the malate-aspartate shuttle. Liver perfusion also demonstrated deficits in ureogenesis from ammonia, gluconeogenesis from lactate, and an increase in the lactate-to-pyruvate ratio within hepatocytes. Surprisingly, Ctrn-/- mice up to 1 year of age failed to show CTLN2-like symptoms due to normal hepatic ASS activity. Serological measures of glucose, amino acid, and ammonia metabolism also showed no significant alterations. Nitrogen-loading treatments produced only minor changes in the hepatic ammonia and amino acid levels. These results suggest that citrin deficiency alone may not be sufficient to produce a CTLN2-like phenotype in mice. These observations are compatible, however, with the variable age of onset, incomplete penetrance, and strong ethnic bias seen in CTLN2 where additional environmental and/or genetic triggers are now suspected.

Identification of 16 novel mutations in the argininosuccinate synthetase gene and genotype-phenotype correlation in 38 classical citrullinemia patients.

Classical citrullinemia (CTLN1), a rare autosomal recessive disorder, is caused by mutations of the argininosuccinate synthetase (ASS) gene, localized on chromosome 9q34.1. ASS functions as a rate-limiting enzyme in the urea cycle. Previously, we identified 32 mutations in the ASS gene of CTLN1 patients mainly in Japan and the United States, and to date 34 different mutations have been described in 50 families worldwide. In the present study, we report ASS mutations detected in 35 additional CTLN1 families from 11 countries. By analyzing the entire coding sequence and the intron-exon boundaries of the ASS gene using RT-PCR and/or genomic DNA-PCR, we have identified 16 novel mutations (two different 1-bp deletions, a 67-bp insertion, and 13 missense) and have detected 12 known mutations. Altogether, 50 different mutations (seven deletion, three splice site, one duplication, two nonsense, and 37 missense) in 85 CTLN1 families were identified. On the basis of primary sequence comparisons with the crystal structure of E. coli ASS protein, it may be concluded that any of the 37 missense mutations found at 30 different positions led to structural and functional impairments of the human ASS protein. It has been found that three mutations are particularly frequent: IVS6-2A>G in 23 families (Japan: 20 and Korea: three), G390R in 18 families (Turkey: six, U.S.: five, Spain: three, Israel: one, Austria: one, Canada: one, and Bolivia: one), and R304W in 10 families (Japan: nine and Turkey: one). Most mutations of the ASS gene are "private" and are distributed throughout the gene, except for exons 5 and 12-14. It seems that the clinical course of the patients with truncated mutations or the G390R mutation is early-onset/severe. The phenotype of the patients with certain missense mutations (G362V or W179R) is more late-onset/mild. Eight patients with R86H, A118T, R265H, or K310R mutations were adult/late-onset and four of them showed severe symptoms during pregnancy or postpartum. However, it is still difficult to prove the genotype-phenotype correlation, because many patients were compound heterozygotes (with two different mutations), lived in different environments at the time of diagnosis, and/or had several treatment regimes or various knowledge of the disease.

Novel mRNA molecules are induced in hypertrophied ventricles of carnitine-deficient mice and belong to a family of up-regulated gene in cells overexpressing c-erbB-2.

To clarify the pathogenesis of cardiac hypertrophy in carnitine-deficient juvenile visceral steatosis (JVS) mice, we performed differential mRNA display analysis with the ventricles of control and JVS mice. We found a novel up-regulated gene, designated as carnitine deficiency-associated gene expressed in ventricle (CDV)-3. Northern blot analysis with a cDNA probe derived from the novel gene revealed two substantial mRNA species of prominent 4.1- and faint 3.5-kb in examined tissues of control and JVS mice. In spite of their widely expressed features, up-regulation of the gene was found predominantly in the ventricles and slightly in the auricles and skeletal muscles of JVS mice. The up-regulation of CDV-3 gene in the ventricles of JVS mice was significantly relieved by carnitine administration within 6 h. The entire cDNA nucleotide sequences showed that two kinds of cDNA, long and short versions (CDV-3A and -3B), corresponding to the detected mRNAs, are different in a 711 base fragment. Analysis of genomic DNA revealed that the two mRNAs were derived from a single CDV-3 gene with five exons by alternative splicing. The deduced amino acid sequences indicated that the isoforms consist of 236 and 281 residues, differing at regions near the carboxy-terminus but sharing 231 residues of the amino-terminal regions. A BLAST search revealed that they show a high similarity to a human predicted nuclear protein (H41), which has been reported to be up-regulated in breast cancer cells overexpressing cellular-erythroblastosis B-2 (c-erbB-2, a kind of tyrosine kinase).We report the identification and characterization of novel transcripts that may be involved in the development of cardiac hypertrophy caused by carnitine deficiency.

Expression of three mitochondrial solute carriers, citrin, aralar1 and ornithine transporter, in relation to urea cycle in mice.

The present report describes the expression profiles of different tissues and developmental changes of mouse aspartate/glutamate carrier (AGC) genes, Slc25a13 and Slc25a12, and an ornithine transporter gene, Ornt1, in relation to urea cycle enzyme genes, carbamoylphosphate synthetase I (CPS) and argininosuccinate synthetase (ASS). Slc25a13 encodes citrin, recently found to be deficient in adult-onset type II citrullinemia and to function as AGC together with its isoform and product of Slc25a12, aralar1. Citrin was broadly distributed, but mainly in the liver, kidney and heart. Aralar1 was expressed in diaphragm, skeletal muscle, heart, brain and kidney, but not in the liver. These distribution profiles are different from the restricted of Ornt1, ASS and CPS. Citrin, ASS, CPS and Ornt1 showed similar patterns of developmental changes in the liver and small intestine, where they play a role in urea and arginine synthesis. Dietary, hormonal and physical manipulations caused varied changes of CPS, ASS and Ornt1 in the liver, but the change of citrin was not so marked as that of the others. Analysis using RT-PCR and restriction enzyme digestion revealed that the ornithine transporter most expressed is Ornt1, although Ornt2 is detectable at a minute level. All these results suggest that citrin as AGC plays a role in urea synthesis as well as many fundamental metabolic pathways in the liver, and shares metabolic functions with aralar1 in other tissues, and that Ornt1 is an important component in urea synthesis in the liver and in arginine synthesis in the small intestine during the neonatal period.