Sustainable production of multimeric and functional recombinant human adiponectin using genome-edited chickens.

BACKGROUND: Adiponectin (ADPN) plays a critical role in endocrine and cardiovascular functions, but traditional production methods, such as Escherichia coli and mammalian systems, have faced challenges in generating sufficiently active middle molecular weight (MMW) and high molecular weight (HMW) forms of recombinant human ADPN (hADPN). In our previous study, we proposed genome-edited chickens as an efficient platform for producing multimeric hADPN. However, the consistency of multimeric hADPN expression in this system across generations had not been further investigated. RESULTS: In this study, subsequent generations of ovalbumin (OVA) ADPN knock-in chickens showed stable multimeric hADPN production, yielding ~ 26% HMW ADPN (0.59 mg/mL) per hen. Comparative analysis revealed that egg white (EW)-derived hADPN predominantly consisted of hexameric and HMW forms, similar to serum-derived hADPN. In contrast, hADPN obtained from human embryonic kidney (HEK) 293 and High-Five (Hi-5) cells also exhibited the presence of trimers, indicating variability across different production systems. Furthermore, transcriptional expression analysis of ADPN multimerization-associated endoplasmic reticulum chaperone genes (Ero1-Lα, DsbA-L, ERP44, and PDI) indicated upregulation in the oviduct magnum of ADPN KI hens, suggesting the chicken oviduct magnum as the optimal site for HMW ADPN production. Lastly, the functional analysis demonstrated that EW-derived hADPN significantly reduced lipid droplets and downregulated lipid accumulation-related genes (LOX-1, AT1R, FAS, and FABP4) in human umbilical vein endothelial cells (HUVECs). CONCLUSION: In summary, stable and functional multimeric hADPN can be produced in genome-edited chickens even after generations. This highlights the potential of using chicken bioreactor for producing various high-value proteins.

Modification of surface glycan by expression of beta-1,4-N-acetyl-galactosaminyltransferase (B4GALNT2) confers resistance to multiple viruses infection in chicken fibroblast cell.

Introduction: Infectious viruses in poultry, such as avian influenza virus (AIV) and Newcastle disease virus (NDV), are one of the most major threats to the poultry industry, resulting in enormous economic losses. AIVs and NDVs preferentially recognize α-2,3-linked sialic acid to bind to target cells. The human beta-1,4-N-acetyl-galactosaminyltransferase 2 (B4GALNT2) modifies α-2,3-linked sialic acid-containing glycan by transferring N-acetylgalactosamine to the sub-terminal galactose of the glycan, thus playing a pivotal role in preventing viruses from binding to cell surfaces. However, chickens lack a homolog of the B4GALNT2 gene. Methods: Here, we precisely tagged the human B4GALNT2 gene downstream of the chicken GAPDH so that the engineered cells constitutively express the human B4GALNT2. We performed a lectin binding assay to analyze the modification of α-2,3-linked sialic acid-containing glycan by human B4GALNT2. Additionally, we infected the cells with AIV and NDV and compared cell survivability, viral gene transcription, and viral titer using the WST-1 assay, RT-qPCR and TCID50 assay, respectively. Results: We validated human B4GALNT2 successfully modified α-2,3-linked sialic acid-containing glycan in chicken DF-1 cells. Following viral infection, we showed that human B4GALNT2 reduced infection of two AIV subtypes and NDV at 12-, 24-, and 36-hours post-infection. Moreover, cells expressing human B4GALNT2 showed significantly higher cell survivability compared to wild-type DF-1 cells, and viral gene expression was significantly reduced in the cells expressing human B4GALNT2. Discussion: Collectively, these results suggest that artificially expressing human B4GALNT2 in chicken is a promising strategy to acquire broad resistance against infectious viruses with a preference for α-2,3-linked sialic acids such as AIV and NDV.

Production of recombinant human IgG1 Fc with beneficial N-glycosylation pattern for anti-inflammatory activity using genome-edited chickens.

Intravenous immunoglobulin (IVIG) is a plasma-derived polyclonal IgG used for treatment of autoimmune diseases. Studies show that α-2,6 sialylation of the Fc improves anti-inflammatory activity. Also, afucosylation of the Fc efficiently blocks FcγRIIIA by increasing monovalent affinity to this receptor, which can be beneficial for treatment of refractory immune thrombocytopenia (ITP). Here, we generated genome-edited chickens that synthesize human IgG1 Fc in the liver and secrete α-2,6 sialylated and low-fucosylated human IgG1 Fc (rhIgG1 Fc) into serum and egg yolk. Also, rhIgG1 Fc has higher affinity for FcγRIIIA than commercial IVIG. Thus, rhIgG1 Fc efficiently inhibits immune complex-mediated FcγRIIIA crosslinking and subsequent ADCC response. Furthermore, rhIgG1 Fc exerts anti-inflammatory activity in a passive ITP model, demonstrating chicken liver derived rhIgG1 Fc successfully recapitulated efficacy of IVIG. These results show that genome-edited chickens can be used as a production platform for rhIgG1 Fc with beneficial N-glycosylation pattern for anti-inflammatory activities.

Sequential verification of exogenous protein production in OVA gene-targeted chicken bioreactors.

The chicken has potential as an efficient bioreactor system because of its outstanding protein production capacity and low cost. The CRISPR/Cas9-mediated gene-editing system enables production of highly marketable exogenous proteins in transgenic chicken bioreactors. However, because it takes approximately 18 mo to evaluate the recombinant protein productivity of the bioreactor due to the generation interval from G0 founders to G1 egg-laying hens, to verification of the exogenous protein at the early stage is difficult. Here we propose a system for sequential validation of exogenous protein production in chicken bioreactors as in hatching female chicks as well as in egg-laying hens. We generated chicken OVALBUMIN (OVA) EGFP knock-in (KI) chicken (OVA EGFP KI) by CRISPR/Cas9-mediated nonhomologous end joining at the chicken OVA gene locus. Subsequently, the estrogen analog, diethylstilbestrol (DES), was subcutaneously implanted in the abdominal region of 1-wk-old OVA EGFP KI female chicks to artificially increase OVALBUMIN expression. The oviducts of DES-treated OVA EGFP KI female chicks expressed OVA and EGFP at the 3-wk-old stage (10 d after DES treatment). We evaluated the expression of EGFP protein in the oviduct, along with the physical properties of eggs and egg white from OVA EGFP KI hens. The rapid identification and isolation of exogenous protein can be confirmed at a very early stage and high-yield production is possible by targeting the chicken oviduct.

Dissecting chicken germ cell dynamics by combining a germ cell tracing transgenic chicken model with single-cell RNA sequencing.

Avian germ cells can be distinguished by certain characteristics during development. On the basis of these characteristics, germ cells can be used for germline transmission. However, the dynamic transcriptional landscape of avian germ cells during development is unknown. Here, we used a novel germ-cell-tracing method to monitor and isolate chicken germ cells at different stages of development. We targeted the deleted in azoospermia like (DAZL) gene, a germ-cell-specific marker, to integrate a green fluorescent protein (GFP) reporter gene without affecting endogenous DAZL expression. The resulting transgenic chickens (DAZL::GFP) were used to uncover the dynamic transcriptional landscape of avian germ cells. Single-cell RNA sequencing of 4,752 male and 13,028 female DAZL::GFP germ cells isolated from embryonic day E2.5 to 1 week post-hatch identified sex-specific developmental stages (4 stages in male and 5 stages in female) and trajectories (apoptosis and meiosis paths in female) of chicken germ cells. The male and female trajectories were characterized by a gradual acquisition of stage-specific transcription factor activities. We also identified evolutionary conserved and species-specific gene expression programs during both chicken and human germ-cell development. Collectively, these novel analyses provide mechanistic insights into chicken germ-cell development.

Efficient production of recombinant human adiponectin in egg white using genome edited chickens.

The prevalence of obesity-related metabolic diseases caused by insulin resistance is rapidly increasing worldwide. Adiponectin (ADPN), a hormone derived from adipose tissue, is a potential therapeutic agent for insulin resistance. Chickens are considered efficient bioreactors for recombinant protein production because they secrete large amounts of high-concentration proteins from the oviduct. Additionally, chickens express high levels of high-molecular-weight (HMW) ADPN, which is considered the active form in the body. Therefore, in this study, a gene-targeted chicken model was produced in which the gene encoding human ADPN was inserted into Ovalbumin (OVA) using the CRISPR/Cas9 system, and the characteristics of the resulting recombinant ADPN protein were evaluated. As a result, human ADPN was expressed in G1 hen oviducts and egg whites of OVA ADPN knock-in (KI) chickens. The concentration of ADPN in egg white ranged from 1.47 to 4.59 mg/mL, of which HMW ADPN accounted for ∼29% (0.24-1.49 mg/mL). Importantly, egg white-derived ADPN promoted expression of genes related to fatty acid oxidation and activated the 5'-AMP-activated protein kinase (AMPK) signaling pathway in muscle cells. In summary, the OVA gene-targeted chicken bioreactor proved to be an advantageous model for production of human ADPN, and the resulting protein was of sufficient quantity and efficacy for industrial use.

DMRT1 gene disruption alone induces incomplete gonad feminization in chicken.

Compared with the well-described XY sex determination system in mammals, the avian ZW sex determination system is poorly understood. Knockdown and overexpression studies identified doublesex and mab-3-related transcription factor 1 (DMRT1) as the testis-determining gene in chicken. However, the detailed effects of DMRT1 gene disruption from embryonic to adult development are not clear. Herein, we have generated DMRT1-disrupted chickens using the clustered regularly interspaced short palindromic repeats-associated protein 9 system, followed by an analysis of physiological, hormonal, and molecular changes in the genome-modified chickens. In the early stages of male chicken development, disruption of DMRT1 induced gonad feminization with extensive physiological and molecular changes; however, functional feminine reproductivity could not be implemented with disturbed hormone synthesis. Subsequent RNA-sequencing analysis of the DMRT1-disrupted chicken gonads revealed gene networks, including several novel genes linearly and non-linearly associated with DMRT1, which are involved in gonad feminization. By comparing the gonads of wild type with the genome-modified chickens, a set of genes were identified that is involved in the ZW sex determination system independent of DMRT1. Our results extend beyond the Z-dosage hypothesis to provide further information about the avian ZW sex determination system and epigenetic effects of gonad feminization.

In vivo enrichment of busulfan-resistant germ cells for efficient production of transgenic avian models.

Most transgenic animals are generated using a genome-modified stem cell system and genome modification directly in embryos. Although this system is well-established in the development of transgenic animals, donor cell-derived transgenic animal production is inefficient in some cases. Especially in avian models such as chickens, the efficiency of transgenic animal production through primordial germ cells (PGCs) is highly variable compared with embryonic manipulation of mammalian species. Because germ cell and germline-competent stem cell-mediated systems that contain the transgene are enriched only at the upstream level during cell cultivation, the efficiency of transgenic animal production is unreliable. Therefore, we developed an in vivo selection model to enhance the efficiency of transgenic chicken production using microsomal glutathione-S-transferase II (MGSTII)-overexpressing PGCs that are resistant to the alkylating agent busulfan, which induces germ cell-specific cytotoxicity. Under in vitro conditions, MGSTII-tg PGCs were resistant to 1 µM busulfan, which was highly toxic to wild-type PGCs. In germline chimeric roosters, transgene-expressing germ cells were dominantly colonized in the recipient testes after busulfan exposure compared with non-treated germline chimera. In validation of germline transmission, donor PGC-derived progeny production efficiency was 94.68%, and the transgene production rate of heterozygous transgenic chickens was significantly increased in chickens that received 40 mg/kg busulfan (80.33-95.23%) compared with that of non-treated germline chimeras (51.18%). This system is expected to significantly improve the efficiency of generating transgenic chickens and other animal species by increasing the distribution of donor cells in adult testes.

Efficacy and safety of GV1001 in patients with moderate-to-severe Alzheimer's disease already receiving donepezil: a phase 2 randomized, double-blind, placebo-controlled, multicenter clinical trial.

BACKGROUND: Our previous studies showed that GV1001 has various protective effects against ß-amyloid and other stressors. Based on these findings, we hypothesized that GV1001 might have beneficial effects in patients with Alzheimer's disease (AD). METHODS: A phase 2, double-blind, parallel-group, placebo-controlled, 6-month randomized clinical trial was performed to evaluate the safety and efficacy of subcutaneously administered GV1001. Between September 2017 and September 2019, 13 centers in South Korea recruited participants. A total of 106 patients were screened, and 96 patients with moderate-to-severe AD were randomized 1:1:1 to the placebo (group 1, n = 31), GV1001 0.56 mg (group 2, n = 33), and 1.12 mg (group 3, n = 32) groups. GV1001 was administered every week for 4 weeks (4 times), followed by every 2 weeks until week 24 (10 times). The primary endpoint was the change in the Severe Impairment Battery (SIB) score from baseline to week 24. The key secondary efficacy endpoints were the change in the Clinical Dementia Rating Sum of Box (CDR-SOB), Alzheimer's Disease Cooperative Study-Activities of Daily Living (ADCS-ADL), Neuropsychiatric Inventory (NPI), Mini-Mental State Examination, and Global Deterioration Scale scores. The safety endpoints were also assessed based on adverse events, laboratory test results, vital signs, and other observations related to safety. RESULTS: Group 3 showed less decrease in the SIB score at 12 and 24 weeks compared with group 1 (P < 0.05). These were not significantly observed in group 2. Among the secondary endpoints, only the NPI score showed significantly better improvement in group 2 than in group 3 at week 12; however, there were no other significant differences between the groups. Although the ADCS-ADL and CDR-SOB scores showed a pattern similar to SIB scores, a statistically significant result was not found. Adverse events were similar across all three groups. CONCLUSIONS: The results indicate that GV1001 1.12 mg met the primary endpoint of a statistically significant difference. GV1001 was well tolerated without safety concerns. This study warrants a larger clinical trial. TRIAL REGISTRATION: ClinicalTrials.gov NCT03184467 . Registered on June 12, 2017.

Highly elevated base excision repair pathway in primordial germ cells causes low base editing activity in chickens.

Base editing technology enables the generation of precisely genome-modified animal models. In this study, we applied base editing to chicken, an important livestock animal in the fields of agriculture, nutrition, and research through primordial germ cell (PGC)-mediated germline transmission. Using this approach, we successfully produced two genome-modified chicken lines harboring mutations in the genes encoding ovotransferrin (TF) and myostatin (MSTN); however, only 55.5% and 35.7% of genome-modified chickens had the desired base substitutions in TF and MSTN, respectively. To explain the low base-editing activity, we performed molecular analysis to compare DNA repair pathways between PGCs and the chicken fibroblast cell line DF-1. The results revealed that base excision repair (BER)-related genes were significantly elevated in PGCs relative to DF-1 cells. Subsequent functional studies confirmed that the editing activity could be regulated by modulating the expression of uracil N-glycosylase (UNG), an upstream gene of the BER pathway. Collectively, our findings indicate that the distinct DNA repair property of chicken PGCs causes low editing activity during genome modification, however, modulation of BER functions could promote the production of genome-modified organisms with the desired genotypes.

Precise Genome Editing in Poultry and Its Application to Industries.

Poultry such as chickens are valuable model animals not only in the food industry, but also in developmental biology and biomedicine. Recently, precise genome-editing technologies mediated by the CRISPR/Cas9 system have developed rapidly, enabling the production of genome-edited poultry models with novel traits that are applicable to basic sciences, agriculture, and biomedical industry. In particular, these techniques have been combined with cultured primordial germ cells (PGCs) and viral vector systems to generate a valuable genome-edited avian model for a variety of purposes. Here, we summarize recent progress in CRISPR/Cas9-based genome-editing technology and its applications to avian species. In addition, we describe further applications of genome-edited poultry in various industries.

Production of quail (Coturnix japonica) germline chimeras by transfer of Ficoll-enriched spermatogonial stem cells.

Due to the absence of long-term in vitro germline competent stem cell maintenance systems and efficient methods for germline transmission, efforts to develop an effective transgenic system in quail has remained limited. To overcome this limitation, here we produced germline chimeric quails through transplantation of spermatogonial stem cells (SSCs) enriched by density gradient methods utilizing Ficoll-Paque PLUS (Ficoll), Percoll and sucrose solution as a practical strategy for germline transmission in quail. For all gradient methods, testicular cells were separated as two fractions, and the expression levels of SSC-specific genes (GFRA1, ITGA6, ITGB1) and pluripotency genes (NANOG, POUV) were examined. As a result, quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and RNA probe hybridization analysis revealed that the upper fraction that was separated by Ficoll showed the highest expression of SSC-specific and pluripotency genes among all fractions. Cells in the upper Ficoll gradient fraction also displayed reduced heterochromatin distribution, as observed in differentiated spermatogonia using transmission electron microscopy (TEM). These results indicate that SSCs were enriched in the upper fraction by Ficoll density gradient centrifugation. Subsequent transplantation experiments revealed that the efficiency of germline transmission to donor-derived gametes in the germline chimeras with transplanted SSCs and whole testicular cells was 0-13.2% and 0-4.4%, respectively. Collectively, these results demonstrate that quail SSCs were easily enriched with a density gradient method and that this method is a feasible and practical way to preserve the germplasm of quail. Furthermore, we can expect to apply this method in research examining the production of transgenic quail and preservation of avian species.

Targeted gene insertion into Z chromosome of chicken primordial germ cells for avian sexing model development.

Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) have facilitated the production of genome-edited animals for use as models. Because of their unique developmental system, avian species offer many advantages as model vertebrates. Here, we report the development of novel chicken models using the CRISPR/Cas9-mediated nonhomologous end joining repair pathway in chicken primordial germ cells (PGCs). Through the introduction of a donor plasmid containing short guide RNA recognition sequences and CRISPR/Cas9 plasmids into chicken PGCs, exogenous genes of donor plasmids were precisely inserted into target loci, and production of transgenic chickens was accomplished through subsequent transplantation of the Z chromosome-targeted PGCs. Using this method, we successfully accomplished the targeted gene insertion to the chicken sex Z chromosome without detected off-target effects. The genome-modified chickens robustly expressed green fluorescent protein from the Z chromosome, which could then be used for easy sex identification during embryogenesis. Our results suggest that this powerful genome-editing method could be used to develop many chicken models and should significantly expand the application of genome-modified avians.-Lee, H. J., Yoon, J. W., Jung, K. M., Kim, Y. M., Park, J. S., Lee, K. Y., Park, K. J., Hwang, Y. S., Park, Y. H., Rengaraj, D., Han, J. Y. Targeted gene insertion into Z chromosome of chicken primordial germ cells for avian sexing model development.

The transgenic chicken derived anti-CD20 monoclonal antibodies exhibits greater anti-cancer therapeutic potential with enhanced Fc effector functions.

Modern genetic techniques, enable the use of animal bioreactor systems for the production and functional enhancement of anti-cancer antibodies. Chicken is the most efficient animal bioreactor for the production of anti-cancer antibodies because of its relatively short generation time, plentiful reproductive capacity, and daily deposition in the egg white. Although several studies have focused on the production of anti-cancer antibodies in egg white, in-depth studies of the biological activity and physiological characteristics of transgenic chicken-derived anti-cancer antibodies have not been fully carried out. Here, we report the production of an anti-cancer monoclonal antibody against the CD20 protein from egg whites of transgenic hens, and validated the bio-functional activity of the protein in B-lymphoma and B-lymphoblast cells. Quantitative analysis showed that deposition of the chickenised CD20 monoclonal antibody (cCD20 mAb) from transgenic chickens increased in successive generations and with increasing transgene copy number. Ultra-performance liquid chromatography (UPLC) tandem mass spectrometry (LC/MS/MS) analysis showed that the cCD20 mAb exhibited 14 N-glycan patterns with high-mannose, afucosylation and terminal galactosylation. The cCD20 mAb did not exhibit significantly improved Fab-binding affinity, but showed markedly enhanced Fc-related functions, including complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) compared to commercial rituximab, a chimeric mAb against CD20. Our results suggest that the transgenic chicken bioreactor is an efficient system for producing anti-cancer therapeutic antibodies with enhanced Fc effector functions.

Characteristic Motor and Nonmotor Symptoms Related to Quality of Life in Drug-Naïve Patients with Late-Onset Parkinson Disease.

BACKGROUND/AIMS: Unlike young-onset Parkinson disease (YOPD), characteristics of late-onset PD (LOPD) have not yet been clearly elucidated. We investigated characteristic features and symptoms related to quality of life (QoL) in LOPD patients. METHODS: We recruited drug-naïve, early PD patients. The patient cohort was divided into 3 subgroups based on patient age at onset (AAO): the YOPD group (AAO <50 years), the middle-onset PD (MOPD) group, and the LOPD group (AAO ≥70 years). Using various scales for motor symptoms (MS) and non-MS (NMS) and QoL, we compared the clinical features and impact on QoL. RESULTS: Of the 132 enrolled patients, 26 were in the YOPD group, 74 in the MOPD group, and 32 in the LOPD group. Among parkinsonian symptoms, patients in the LOPD group had a lower score on the Korean version of the Montreal Cognitive Assessment than the other groups. Logistic regression analysis showed genitourinary symptoms were related to the LOPD group. Linear regression analysis showed both MS and NMS were correlated with QoL in the MOPD group, but only NMS were correlated with QoL in the LOPD group. Particularly, anxiety and fatigue affected QoL in the LOPD group. CONCLUSION: LOPD patients showed different characteristic clinical features, and different symptoms were related with QoL for LOPD than YOPD and MOPD patients.

Weight Change Is a Characteristic Non-Motor Symptom in Drug-Naïve Parkinson's Disease Patients with Non-Tremor Dominant Subtype: A Nation-Wide Observational Study.

Despite the clinical impact of non-motor symptoms (NMS) in Parkinson's disease (PD), the characteristic NMS in relation to the motor subtypes of PD is not well elucidated. In this study, we enrolled drug-naïve PD patients and compared NMS between PD subtypes. We enrolled 136 drug-naïve, early PD patients and 50 normal controls. All the enrolled PD patients were divided into tremor dominant (TD) and non-tremor dominant (NTD) subtypes. The Non-Motor Symptom Scale and scales for each NMS were completed. We compared NMS and the relationship of NMS with quality of life between normal controls and PD patients, and between the PD subtypes. Comparing with normal controls, PD patients complained of more NMS, especially mood/cognitive symptoms, gastrointestinal symptoms, unexplained pain, weight change, and change in taste or smell. Between the PD subtypes, the NTD subtype showed higher total NMS scale score and sub-score about weight change. Weight change was the characteristic NMS related to NTD subtype even after controlled other variables with logistic regression analysis. Even from the early stage, PD patients suffer from various NMS regardless of dopaminergic medication. Among the various NMS, weight change is the characteristic NMS associated with NTD subtype in PD patients.

Cortical Morphology in Patients with Orthostatic Intolerance.

BACKGROUND: We evaluated the cortical morphology in patients with orthostatic intolerance. METHODS: Thirty patients with orthostatic intolerance, as well as age- and sex-matched normal controls, were enrolled in this study. We divided the patients into orthostatic hypotension (n = 22) and postural tachycardia syndrome (n = 8) groups based on their response to a head-up tilt table test. We analyzed whole-brain T1-weighted MRI images using FreeSurfer 5.1. The measures of cortical morphology were compared between the groups. RESULTS: The cortical thickness in the right hemisphere, including the medial orbitofrontal, peri-calcarine, post-central, inferior temporal, and lateral occipital cortex, and in the peri-calcarine cortex of the left hemisphere was thinned in patients with orthostatic hypotension compared to normal controls. The other measures of cortical morphology, including the surface area, volume, and curvatures, did not differ between patients with orthostatic hypotension and normal controls. However, none of the measures of cortical morphology differed between patients with postural tachycardia syndrome and normal controls. CONCLUSIONS: We demonstrated that the cortical morphology significantly changed in patients with orthostatic hypotension but not in patients with postural tachycardia syndrome compared to normal controls. These findings support the hypothesis that orthostatic intolerance is a heterogeneous syndrome.

A Case of Acute Vestibular Neuritis Visualized by Three-Dimensional FLAIR-VISTA Magnetic Resonance Imaging.

A 72-year-old man presented with vertigo and unsteady gait. The three-dimensional fluid-attenuated inversion recovery-volume isotopic turbo spin echo acquisition (3D-FLAIR-VISTA) magnetic resonance imaging (MRI) showed high signal intensity in the cisternal segment of the right vestibular nerve. The video-based oculography with caloric test revealed spontaneous left-beating nystagmus and canal paresis in the right ear. This case suggests that 3D-FLAIR-VISTA images are useful for the visualisation of acute vestibular neuritis.

Refinement of reference limits for alanine aminotransferase in the Korean elderly population.

OBJECTIVES: For the geriatric population, upper reference limits (URLs) for alanine aminotransferase (ALT) levels have not been established. The aim of the study was to determine the URLs for ALT and to describe the factors associated with elevated ALT level in an elderly population. DESIGN AND METHODS: We set the URLs for ALT for participants aged 65 years and over (n=3646) based on the 2007-2009 Korea National Health and Nutrition Examination Survey at the 97.5th percentile of that population who were determined to be at low risk for liver disease (n=896; low risk was defined as testing negative for the HBs antigen, low alcohol intake, and the absence of metabolic syndrome, medical comorbidity, or medication use). RESULTS: The URLs for ALT were 39 IU/L for men and 30 IU/L for women. The weighted prevalence for the elevated ALT level was 8.0% (95% confidence interval [CI], 7.1% to 8.9%) in the sample, 5.8% (95% CI, 4.7% to 7.1%) in men and 9.5% (95% CI, 8.3% to 10.8%) in women. CONCLUSIONS: The URLs for ALT are established for the first time in the elderly, and our results may be useful for monitoring trends in the burden of liver disease, in addition to guiding the evaluation of ALT abnormalities in clinical practice.