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Glycogen storage disease type Ia (GSD-Ia) is an inherited metabolic disease caused by a deficiency in glucose-6-phosphatase-α (G6Pase-α or G6PC) which plays a critical role in blood glucose homeostasis by catalyzing the hydrolysis of glucose-6-phosphate (G6P) to glucose and phosphate in the terminal step of glycogenolysis and gluconeogenesis. Patients with GSD-Ia manifest life-threatening fasting hypoglycemia along with the excessive accumulation of hepatic glycogen and triglycerides which results in hepatomegaly and a risk of long-term complications such as hepatocellular adenoma and carcinoma (HCA/HCC). The etiology of HCA/HCC development in GSD-Ia, however, is unknown. Recent studies have shown that the livers in model animals of GSD-Ia display impairment of autophagy, a cellular recycling process which is critical for energy metabolism and cellular homeostasis. However, molecular mechanisms of autophagy impairment and its involvement in pathogenesis in GSD-Ia are still under investigation. Here, we summarize the latest advances for signaling pathways implicated in hepatic autophagy impairment and the roles of autophagy in hepatic tumorigenesis in GSD-Ia. In addition, recent evidence has illustrated that autophagy plays an important role in hepatic metabolism and liver-directed gene therapy mediated by recombinant adeno-associated virus (rAAV). Therefore, we highlight the possible role of hepatic autophagy in metabolic control and rAAV-mediated gene therapy for GSD-Ia. In this review, we also provide potential therapeutic strategies for GSD-Ia on the basis of molecular mechanisms underlying hepatic autophagy impairment in GSD-Ia.

Hepatocellular adenoma/carcinoma (HCA/HCC) is a long-term complication of the metabolic disorder glycogen storage disease type Ia (GSD-Ia) deficient in glucose-6-phosphatase-α (G6PC or G6Pase-α). We have shown previously that hepatic G6Pase-α deficiency leads to autophagy impairment, mitochondrial dysfunction, enhanced glycolysis, and augmented hexose monophosphate shunt, all of which can contribute to hepatocarcinogenesis. However, the mechanism underlying HCA/HCC development in GSD-Ia remains unclear. We now show that G6Pase-α deficiency-mediated hepatic autophagy impairment leads to sustained accumulation of an autophagy-specific substrate p62 which can activate tumor-promoting pathways including nuclear factor erythroid 2-related factor 2 (Nrf2) and mammalian target of rapamycin complex 1 (mTORC1). Consistently, the HCA/HCC lesions developed in the G6Pase-α-deficient livers display marked accumulation of p62 aggregates and phosphorylated p62 along with activation of Nrf2 and mTORC1 signaling. Furthermore, the HCA/HCC lesions exhibit activation of additional oncogenic pathways, β-catenin and Yes-associated protein (YAP) which is implicated in autophagy impairment. Intriguingly, hepatic levels of glucose-6-phosphate and glycogen which are accumulated in the G6Pase-α-deficient livers were significantly lower in HCC than those in HCA. Conversely, compared to HCA, the HCC lesion display increased expression of many oncogenes and the M2 isoform of pyruvate kinase (PKM2), a glycolytic enzyme critical for aerobic glycolysis and tumorigenesis. Collectively, our data show that hepatic G6Pase-α-deficiency leads to persistent autophagy impairment and activation of multiple tumor-promoting pathways that contribute to HCA/HCC development in GSD-Ia.

Viral mediated gene therapy has progressed after overcoming early failures, and gene therapy has now been approved for several conditions in Europe and the USA. Glycogen storage disease (GSD) type Ia, caused by a deficiency of glucose-6-phosphatase-α, has been viewed as an outstanding candidate for gene therapy. This follow-up report describes the long-term outcome for the naturally occurring GSD-Ia dogs treated with rAAV-GPE-hG6PC-mediated gene therapy. A total of seven dogs were treated with rAAV-GPE-hG6PC-mediated gene therapy. The first four dogs were treated at birth, and three dogs were treated between 2 and 6 months of age to assess the efficacy and safety in animals with mature livers. Blood and urine samples, radiographic studies, histological evaluation, and biodistribution were assessed. Gene therapy improved survival in the GSD-Ia dogs. With treatment, the biochemical studies normalized for the duration of the study (up to 7 years). None of the rAAV-GPE-hG6PC-treated dogs had focal hepatic lesions or renal abnormalities. Dogs treated at birth required a second dose of rAAV after 2–4 months; gene therapy after hepatic maturation resulted in improved efficacy after a single dose. rAAV-GPE-hG6PC treatment in GSD-Ia dogs was found to be safe and efficacious. GSD-Ia is an attractive target for human gene therapy since it is a monogenic disorder with limited tissue involvement. Blood glucose and lactate monitoring can be used to assess effectiveness and as a biomarker of success. GSD-Ia can also serve as a model for other hepatic monogenic disorders.

Cellular metabolism generally refers to biochemical processes that produce or consume energy within the cell. Recent studies have established that aberrant metabolic states caused by internal or external stresses and genetic mutations are intertwined with several human pathologies. Gaining insight into these metabolic alterations is, therefore, essential for understanding the pathophysiology of various diseases. Glycogen storage disease type Ib (GSD-Ib) is an autosomal recessive disorder characterized by hypoglycemia, excessive glycogen accumulation in the liver and kidney, neutropenia, neutrophil dysfunction, and inflammatory bowel disease. GSD-Ib is caused by a deficiency of glucose-6-phosphate transporter (G6PT). Recently, it was reported that deficiency of G6PT also leads to the aberrant proliferation and differentiation of mesenchymal stem cells and impaired regulatory T-cell function. This review describes the broad impact of altered cellular metabolism resulting from a lack of G6PT activity on cellular function and considers the prospects of developing novel approaches for GSD-Ib treatment.

Mutations in the liver glycogen phosphorylase (Pygl) gene are associated with the diagnosis of glycogen storage disease type VI (GSD-VI). To understand the pathogenesis of GSD-VI, we generated a mouse model with Pygl deficiency (Pygl -/-). Pygl -/- mice exhibit hepatomegaly, excessive hepatic glycogen accumulation, and low hepatic free glucose along with lower fasting blood glucose levels and elevated blood ketone bodies. Hepatic glycogen accumulation in Pygl -/- mice increases with age. Masson's trichrome and picrosirius red staining revealed minimal to mild collagen deposition in periportal, subcapsular, and/or perisinusoidal areas in the livers of old Pygl -/- mice (>40 weeks). Consistently, immunohistochemical analysis showed the number of cells positive for alpha smooth muscle actin (α-SMA), a marker of activated hepatic stellate cells, was increased in the livers of old Pygl -/- mice compared with those of age-matched wild-type (WT) mice. Furthermore, old Pygl -/- mice had inflammatory infiltrates associated with hepatic vessels in their livers along with up-regulated hepatic messenger RNA levels of C-C chemokine ligand 5 (Ccl5/Rantes) and monocyte chemoattractant protein 1 (Mcp-1), indicating inflammation, while age-matched WT mice did not. Serum levels of aspartate aminotransferase and alanine aminotransferase were elevated in old Pygl -/- mice, indicating liver damage. Conclusion: Pygl deficiency results in progressive accumulation of hepatic glycogen with age and liver damage, inflammation, and collagen deposition, which can increase the risk of liver fibrosis. Collectively, the Pygl-deficient mouse recapitulates clinical features in patients with GSD-VI and provides a model to elucidate the mechanisms underlying hepatic complications associated with defective glycogen metabolism.

Hepatocellular adenoma/carcinoma (HCA/HCC) is a long-term complication of glycogen storage disease type-Ia (GSD-Ia) which is caused by a deficiency in glucose-6-phosphatase-α (G6Pase-α or G6PC), a key enzyme in gluconeogenesis. Currently, there is no therapy to address HCA/HCC in GSD-Ia. We have previously shown that a recombinant adeno-associated virus (rAAV) vector-mediated G6PC gene transfer to 2-week-old G6pc−/− mice prevents HCA development. However, it remains unclear whether G6PC gene transfer at the tumor developing stage of GSD-Ia can prevent tumor initiation or abrogate the pre-existing tumors. Using liver-specific G6pc-knockout (L-G6pc−/−) mice that develop HCA/HCC, we now show that treating the mice at the tumor-developing stage with rAAV-G6PC restores hepatic G6Pase-α expression, normalizes glucose homeostasis, and prevents de novo HCA/HCC development. The rAAV-G6PC treatment also normalizes defective hepatic autophagy and corrects metabolic abnormalities in the non-tumor liver tissues of both tumor-free and tumor-bearing mice. However, gene therapy cannot restore G6Pase-α expression in the HCA/HCC lesions and fails to abrogate any pre-existing tumors. We show that the expression of 11 β-hydroxysteroid dehydrogenase type-1 that mediates local glucocorticoid activation is down-regulated in HCA/HCC lesions, leading to impairment in glucocorticoid signaling critical for gluconeogenesis activation. This suggests that local glucocorticoid action downregulation in the HCA/HCC lesions may suppress gene therapy mediated G6Pase-α restoration. Collectively, our data show that rAAV-mediated gene therapy can prevent de novo HCA/HCC development in L-G6pc−/− mice at the tumor developing stage, but it cannot reduce any pre-existing tumor burden.

Background This study evaluated the impact of lymph node-related factors on the risk of and site of recurrence in patients who had papillary thyroid carcinoma with lymph node metastasis in the lateral compartment (classified as pN1b). Methods Patients underwent total thyroidectomy with unilateral modified radical neck dissection for classical papillary thyroid carcinoma. Risk factors for recurrence were evaluated according to the pattern of recurrence. Results A total of 324 patients were included in the study. The median follow-up was 63 (range 14–181) months. Recurrence was detected in 47 patients (14·5 per cent). In the multivariable analysis, a maximum diameter of metastatic lymph nodes larger than 2·0 cm (hazard ratio (HR) 1·15, 95 per cent c.i. 1·06 to 1·25; P = 0·033) and a central compartment metastatic lymph node ratio of more than 0·42 (HR 3·35, 1·65 to 6·79; P < 0·001) were identified as independent risk factors for locoregional recurrence. Age 45 years or older (HR 5·69, 1·24 to 26·12; P = 0·025) and extranodal extension of metastasis (HR 12·71, 1·64 to 98·25; P = 0·015) were risk factors for distant metastasis. In subgroup analysis of locoregional recurrence, several lymph node-related factors affected the risk of recurrence according to the specific site of metastasis. Conclusion Lymph node-related factors are of importance for the risk of recurrence in patients with classical papillary thyroid carcinoma classified as pN1b.

Glycogen storage disease type Ia (GSD-Ia), characterized by impaired glucose homeostasis and chronic risk of hepatocellular adenoma (HCA), is caused by a deficiency in glucose-6-phosphatase-α (G6Pase-α or G6PC) activity. In a previous 70–90 week-study, we showed that a recombinant adeno-associated virus (rAAV) vector-mediated gene transfer that restores more than 3% of wild-type hepatic G6Pase-α activity in G6pc−/− mice corrects hepatic G6Pase-α deficiency with no evidence of HCA. We now examine the minimal hepatic G6Pase-α activity required to confer therapeutic efficacy. We show that rAAV-treated G6pc−/− mice expressing 0.2% of wild-type hepatic G6Pase-α activity suffered from frequent hypoglycemic seizures at age 63–65 weeks but mice expressing 0.5–1.3% of wild-type hepatic G6Pase-α activity (AAV-LL mice) sustain 4–6 h of fast and grow normally to age 75–90 weeks. Despite marked increases in hepatic glycogen accumulation, the AAV-LL mice display no evidence of hepatic abnormalities, hepatic steatosis, or HCA. Interprandial glucose homeostasis is maintained by the G6Pase-α/glucose-6-phosphate transporter (G6PT) complex, and G6PT-mediated microsomal G6P uptake is the rate-limiting step in endogenous glucose production. We show that hepatic G6PT activity is increased in AAV-LL mice. These findings are encouraging for clinical studies of G6Pase-α gene-based therapy for GSD-Ia., Highlights • Establish the minimal hepatic G6Pase-α activity restoration required to provide a blood glucose level that enables GSD-Ia mice grow to old age (75–90 weeks). • Establish the minimal hepatic G6Pase-α activity restoration required that enables GSD-Ia mice grow to old age (75-90 weeks). • Define the lowest dose for G6Pase-α gene therapy that provides efficacy in mice, which informs a phase I/II clinical trial design in human GSD-Ia.

Glycogen storage disease type Ib (GSD-Ib) is caused by mutations of the glucose-6-phosphate transporter (G6PT) and characterized by disrupted glucose homeostasis, neutropenia, and neutrophil dysfunction. To investigate the role of G6PT in human adipose-derived mesenchymal stem cells (hMSCs), the G6PT gene was mutated by CRISPR/Cas9 technology and single cell-derived G6PT-/- hMSCs were established. G6PT-/- hMSCs have significantly increased cell proliferation but impaired adipogenesis and osteogenesis. These phenotypes are associated with two mechanisms: i) metabolic reprogramming in G6PT-/- hMSCs causing a metabolic shift toward glycolysis rather than oxidative phosphorylation and ii) increased cyclooxygenase-2-derived prostaglandin E2 secretion in G6PT-/- hMSCs. This study demonstrates that G6PT is essential for proliferation and differentiation of MSCs, providing important insights into the GSD-Ib phenotypes.

Glycogen storage disease type-Ib (GSD-Ib), deficient in the glucose-6-phosphate transporter (G6PT), is characterized by impaired glucose homeostasis, myeloid dysfunction, and long-term risk of hepatocellular adenoma (HCA). We examined the efficacy of G6PT gene therapy in G6pt-/- mice using recombinant adeno-associated virus (rAAV) vectors, directed by either the G6PC or the G6PT promoter/enhancer. Both vectors corrected hepatic G6PT deficiency in murine GSD-Ib but the G6PC promoter/enhancer was more efficacious. Over a 78-week study, using dose titration of the rAAV vectors, we showed that G6pt-/- mice expressing 3-62% of normal hepatic G6PT activity exhibited a normalized liver phenotype. Two of the 12 mice expressing

Glycogen storage disease type Ia (GSD-Ia), characterized by impaired glucose homeostasis and chronic risk of hepatocellular adenoma (HCA) and carcinoma (HCC), is caused by a deficiency in glucose-6-phosphatase-α (G6Pase-α or G6PC). We have previously shown that G6pc−/− mice receiving gene transfer mediated by rAAV-G6PC, a recombinant adeno-associated virus (rAAV) vector expressing G6Pase-α, and expressing 3–63% of normal hepatic G6Pase-α activity maintain glucose homeostasis and do not develop HCA/HCC. However, the threshold of hepatic G6Pase-α activity required to prevent tumor formation remained unknown. In this study, we constructed rAAV-co-G6PC, a rAAV vector expressing a codon-optimized (co) G6Pase-α and showed that rAAV-co-G6PC was more efficacious than rAAV-G6PC in directing hepatic G6Pase-α expression. Over an 88-week study, we showed that both rAAV-G6PC- and rAAV-co-G6PC-treated G6pc−/− mice expressing 3–33% of normal hepatic G6Pase-α activity (AAV mice) maintained glucose homeostasis, lacked HCA/HCC, and were protected against age-related obesity and insulin resistance. Of the eleven rAAV-G6PC/rAAV-co-G6PC-treated G6pc−/− mice harboring 0.9–2.4% of normal hepatic G6Pase-α activity (AAV-low mice), 3 expressing 0.9–1.3% of normal hepatic G6Pase-α activity developed HCA/HCC, while 8 did not (AAV-low-NT). Finally, we showed that the AAV-low-NT mice exhibited a phenotype indistinguishable from that of AAV mice expressing ≥ 3% of normal hepatic G6Pase-α activity. The results establish the threshold of hepatic G6Pase-α activity required to prevent HCA/HCC and show that GSD-Ia mice harboring less than 2% of normal hepatic G6Pase-α activity are at risk of tumor development.

Glycogen storage disease type Ib (GSD-Ib), characterized by impaired glucose homeostasis, neutropenia, and neutrophil dysfunction, is an inherited autosomal recessive disorder caused by a deficiency in the glucose-6-phosphate transporter (G6PT). Neutrophils play an essential role in the defense against invading pathogens. The recruitment of neutrophils towards the inflammation sites in response to inflammatory stimuli is a tightly regulated process involving rolling, adhesion, and transmigration. In this study, we investigated the role of G6PT in neutrophil adhesion and migration using in vivo and in vitro models. We showed that the GSD-Ib (G6pt(−/−)) mice manifested severe neutropenia in both blood and bone marrow, and treating G6pt(−/−) mice with granulocyte colony-stimulating factor (G-CSF) corrected neutropenia. However, upon thioglycolate challenge, neutrophils from both untreated and G-CSF-treated G6pt(−/−)mice exhibited decreased ability to migrate to the peritoneal cavity. In vitro migration and cell adhesion of G6PT-deficient neutrophils were also significantly impaired. Defects in cell migration were not due to enhanced apoptosis or altered fMLP receptor expression. Remarkably, the expression of the β2 integrins CD11a and CD11b, which are critical for cell adhesion, was greatly decreased in G6PT-deficient neutrophils. This study suggests that deficiencies in G6PT cause impairment in neutrophil adhesion and migration via aberrant expression of β2 integrins, and our finding should facilitate the development of novel therapies for GSD-Ib.

Background: Understanding the burden of disease is important to establish cost-effective treatment strategies and to allocate healthcare resources appropriately. However, little reliable information is available regarding the overall economic burden imposed by chronic obstructive pulmonary disease (COPD) in Korea. Methods: This study is a multicenter observational research on the COPD burden in Korea. Total COPD costs were comprised of three categories: direct medical, direct non-medical, and indirect costs. For direct medical costs, institutional investigation was performed at 13 medical facilities mainly based on the claims data. For direct non-medical and indirect costs, site-based surveys were administered to the COPD patients during routine visits. Total costs were estimated using the COPD population defined in the recent report. Results: The estimated total costs were approximately 1,245 million US dollar (1,408 billion Korean won). Direct medical costs comprised approximately 20% of the total estimated costs. Of these, formal medical costs held more than 80%. As direct non-medical costs, nursing costs made up the largest percentage (39%) of the total estimated costs. Costs for COPD-related loss of productivity formed four fifths of indirect costs, and accounted for up to 33% of the total costs. Conclusion: This study shows for the first time the direct and indirect costs of COPD in Korea. The total costs were enormous, and the costs of nursing and lost productivity comprised approximately 70% of total costs. The results provide insight for an effective allocation of healthcare resources and to inform establishment of strategies to reduce national burden of COPD.

Glycogen storage disease type-Ia (GSD-Ia) patients deficient in glucose-6-phosphatase-α (G6Pase-α or G6PC) manifest impaired glucose homeostasis characterized by fasting hypoglycemia, growth retardation, hepatomegaly, nephromegaly, hyperlipidemia, hyperuricemia, and lactic acidemia. Two efficacious recombinant adeno-associated virus pseudotype 2/8 (rAAV8) vectors expressing human G6Pase-α have been independently developed. One is a single-stranded vector containing a 2864-bp of the G6PC promoter/enhancer (rAAV8-GPE) and the other is a double-stranded vector containing a shorter 382-bp minimal G6PC promoter/enhancer (rAAV8-miGPE). To identify the best construct, a direct comparison of the rAAV8-GPE and the rAAV8-miGPE vectors was initiated to determine the best vector to take forward into clinical trials. We show that the rAAV8-GPE vector directed significantly higher levels of hepatic G6Pase-α expression, achieved greater reduction in hepatic glycogen accumulation, and led to a better toleration of fasting in GSD-Ia mice than the rAAV8-miGPE vector. Our results indicated that additional control elements in the rAAV8-GPE vector outweigh the gains from the double-stranded rAAV8-miGPE transduction efficiency, and that the rAAV8-GPE vector is the current choice for clinical translation in human GSD-Ia.

Background and objective: The neurotransmitter, 5-hydroxytryptamine, acts as an immunomodulator by stimulating the release of inflammatory cytokines and regulating the function of dendritic cells and monocytes. The 5-hydroxytryptamine receptor 4 (HTR4) gene is located in a region previously linked to an increased risk of asthma and atopy. The aim of this study was to investigate the association between HTR4 and asthma. Methods: Thirty-two single nucleotide polymorphisms (SNP) in HTR4 were investigated by direct sequencing of 24 DNA samples from unrelated Korean subjects. Results: The 32 genetic variants comprised 22 intronic SNP, two SNP in the 3′-untranslated region (exon 7) and eight SNP in the 3′-downstream region. Logistic regression analysis showed that two intronic polymorphisms were significantly associated with the risk of asthma. Two minor HTR4 alleles, +142828G > A and +122769G > A, occurred at significantly higher frequencies in the asthmatic group than in the healthy control group (49.59% vs 42.29%, P = 0.003, and 47.99% vs 40.35%, P = 0.008, respectively), and these differences remained significant after correction for multiple testing (P = 0.05, dominant mode of inheritance; and P = 0.03, dominant mode, respectively). Haplotype analysis revealed three haplotype blocks. The frequency of haplotype 1 in block 2 was significantly higher in asthmatics (P = 0.003, dominant mode), whereas the frequency of haplotype 4 in block 3 was significantly lower in asthmatics (P = 0.0009, dominant mode). Conclusions: SNP and haplotypes of the HTR4 gene were associated with the asthma phenotype and genetic variation of HTR4 may affect susceptibility to the development of asthma.

A canine model of Glycogen storage disease type Ia (GSDIa) is described. Affected dogs are homozygous for a previously described M121I mutation resulting in a deficiency of glucose-6-phosphatase-α. Metabolic, clinicopathologic, pathologic, and clinical manifestations of GSDIa observed in this model are described and compared to those observed in humans. The canine model shows more complete recapitulation of the clinical manifestations seen in humans including “lactic acidosis”, larger size, and longer lifespan compared to other animal models. Use of this model in preclinical trials of gene therapy is described and briefly compared to the murine model. Although the canine model offers a number of advantages for evaluating potential therapies for GSDIa, there are also some significant challenges involved in its use. Despite these challenges, the canine model of GSDIa should continue to provide valuable information about the potential for generating curative therapies for GSDIa as well as other genetic hepatic diseases.

Chronic cough is common in the community and causes significant morbidity. Several factors may underlie this problem, but comorbid conditions located at sensory nerve endings that regulate the cough reflex, including rhinitis, rhinosinusitis, asthma, eosinophilic bronchitis, and gastroesophageal reflux disease, are considered important. However, chronic cough is frequently non-specific and accompanied by not easily identifiable causes during the initial evaluation. Therefore, there are unmet needs for developing empirical treatment and practical diagnostic approaches that can be applied in primary clinics. Meanwhile, in referral clinics, a considerable proportion of adult patients with chronic cough are unexplained or refractory to conventional treatment. The present clinical practice guidelines aim to address major clinical questions regarding empirical treatment, practical diagnostic tools for non-specific chronic cough, and available therapeutic options for chronic wet cough in children and unexplained chronic cough in adults in Korea.

We recently developed bimodal germline chimera production approaches by transfer of primordial germ cells (PGCs) or embryonic germ cells (EGCs) into embryos and by transplantation of spermatogonial stem cells (SSCs) or germline stem cells (GSCs) into adult testes. This study was undertaken to investigate the reversible developmental unipotency of chicken germ cells using our established germline chimera production systems. First, we transferred freshly isolated SSCs from adult testis or in vitro cultured GSCs into stage X and stage 14–16 embryos, and we found that these transferred SSCs/GSCs could migrate to the recipient embryonic gonads. Of the 527 embryos that received SSCs or GSCs, 135 yielded hatchlings. Of 17 sexually mature males (35.3%), six were confirmed as germline chimeras through testcross analysis resulting in an average germline transmission efficiency of 1.3%. Second, PGCs/EGCs, germ cells isolated from embryonic gonads were transplanted into adult testes. The EGC transplantation induced germline transmission, whereas the PGC transplantation did not. The germline transmission efficiency was 12.5 fold higher (16.3 vs 1.3%) in EGC transplantation into testis (EGCs to adult testis) than that in SSC/GSC transfer into embryos (testicular germ cells to embryo stage). In conclusion, chicken germ cells from different developmental stages can (de)differentiate into gametes even after the germ cell developmental clock is set back or ahead. Use of germ cell reversible unipotency might improve the efficiency of germ cell-mediated germline transmission.

The present study was conducted to apply an interspecies germ cell transfer technique to wild bird reproduction. Pheasant (Phasianus colchicus) primordial germ cells (PGCs) retrieved from the gonads of 7-day-old embryos were transferred to the bloodstream of 2.5-day-old chicken (Gallus gallus) embryos. Pheasant-to-chicken germline chimeras hatched from the recipient embryos, and 10 pheasants were derived from testcross reproduction of the male chimeras with female pheasants. Gonadal migration of the transferred PGCs, their involvement in spermatogenesis, and production of chimeric semen were confirmed. The phenotype of pheasant progenies derived from the interspecies transfer was identical to that of wild pheasants. The average efficiency of reproduction estimated from the percentage of pheasants to total progenies was 17.5% .I n conclusion, interspecies germ cell transfer into a developing embryo can be used for wild bird reproduction, and this reproductive technology may be applicable in conserving endangered bird species. assisted reproductive technology, chicken, developmental biology, gamete biology, interspecies germ cell transfer, pheasant, primordial germ cell, seasonal reproduction, spermatogenesis, wild bird conservation

Rationale: Severe respiratory syncytial virus (RSV) bronchiolitis has been associated with deficient IFN-γ production in humans, but the role of this cytokine in determining the outcome of reinfection is unknown. Objectives: To define the role of IFN-γ in the development of RSV-mediated airway hyperresponsiveness (AHR) and lung histopathology in mice. Methods: Wild-type (WT) and IFN-γ knockout mice were infected with RSV in the newborn or weaning stages and reinfected 5 weeks later. Airway responses were assessed on Day 6 after the primary or secondary infection. Measurements and Main Results: Both WT and IFN-γ knockout mice developed similar levels of AHR and airway inflammation after primary infection. After reinfection, IFN-γ knockout mice, but not WT mice, developed AHR, airway eosinophilia, and mucus hyperproduction. Intranasal administration of IFN-γ during primary infection but not during reinfection prevented the development of these altered airway responses on reinfection in IFN-γ knockout mice. Adoptive transfer of WT T cells into IFN-γ knockout mice before primary infection restored IFN-γ production in the lungs and prevented the development of altered airway responses on reinfection. Treatment of mice with IFN-γ during primary neonatal infection prevented the enhancement of AHR and the development of airway eosinophilia and mucus hyperproduction on reinfection. Conclusions: IFN-γ production during primary RSV infection is critical to the development of protection against AHR and lung histopathology on reinfection. Provision of IFN-γ during primary infection in infancy may be a potential therapeutic approach to alter the course of RSV-mediated long-term sequelae.

Background: Chitinase may play a role in regulating allergic diseases. Objective: We studied the role of chitinase in a mouse model exposed to diesel exhaust particles (DEP). Mice were exposed to intranasal DEP (0.6 mg/mL) for 5 days and challenged with aerosolized DEP (6 mg/m3) on days 6–8. Enhanced pause (Penh), as an airway obstruction marker, was measured on day 9, and bronchoalveolar lavage (BAL) fluid and lung tissues were collected on day 10. The expression of Ym1 and Ym2 mRNA was assessed in lung tissue extracts by reverse transcription-polymerase chain reaction. Results: DEP induced significant increases in methacholine-induced Penh and IL-4 levels in BAL fluid relative to the control group. Peribronchial and perivascular inflammatory cell infiltrates were prominent in the DEP group. DEP induced Ym1 and Ym2 mRNA expression in lung tissue extracts relative to the control group. Conclusion: These results demonstrate that DEP induced airway hyperresponsiveness and Ym mRNA expression via a Th2 cell-biased response, suggesting that chitinase may play an important role in airway inflammation and responsiveness upon exposure to DEP in a mouse model, and may therefore be involved in regulating allergic diseases. © 2008 Wiley Periodicals, Inc. Environ Toxicol, 2008.

Transient receptor potential vanilloid-1 (TRPV1), a heat-gated channel, was recently found on human keratinocytes and the activation of epidermal TRPV1 was known to induce release of proinflammatory mediators. However, the functional consequences of TRPV1 activation in cutaneous physiology and pathology have not been elucidated clearly. In this study, we investigated the role of TRPV1 on the matrix metalloproteinase (MMP)-1 expression induced by heat shock in human epidermal keratinocytes. Heat shock induced the expression of MMP-1 mRNA and protein in a temperature-dependent manner in an immortalized human keratinocyte cell line (HaCaT) and normal human epidermal keratinocytes (NHK). Heat-shock-induced MMP-1 expression was decreased by treatment of the TRPV1 inhibitors (capsazepine and ruthenium red) or knockdown of TRPV1 using RNA interference in HaCaT cells. Overexpression of TRPV1 greatly increased heat-shock-induced MMP-1 promoter activity in HEK 293 cells. Furthermore, direct activation of TRPV1 by capsaicin, a TRPV1 agonist, increased MMP-1 expression. We found that heat shock induced calcium influx through TRPV1 and that extracellular calcium was necessary for heat-shock-induced MMP-1 expression in HaCaT cells. Taken together, our results suggest that heat-shock-induced MMP-1 expression is mediated by activation of TRPV1 and is dependent on a calcium-dependent signaling process in human epidermal keratinocytes.

Glycogen storage disease type-Ia (GSD-Ia) is caused by a lack of glucose-6-phosphatase-α (G6Pase-α or G6PC) activity. We have shown that gene therapy mediated by a recombinant adeno-associated virus (rAAV) vector expressing human G6Pase-α normalizes blood glucose homeostasis in the global G6pc knockout (G6pc(-/-)) mice for 70-90 weeks. The treated G6pc(-/-) mice expressing 3-63% of normal hepatic G6Pase-α activity (AAV mice) produce endogenous hepatic glucose levels 61-68% of wild-type littermates, have a leaner phenotype and exhibit fasting blood insulin levels more typical of young adult mice. We now show that unlike wild-type mice, the lean AAV mice have increased caloric intake and do not develop age-related obesity or insulin resistance. Pathway analysis shows that signaling by hepatic carbohydrate response element binding protein that improves glucose tolerance and insulin signaling is activated in AAV mice. In addition, several longevity factors in the calorie restriction pathway, including the NADH shuttle systems, NAD(+) concentrations and the AMP-activated protein kinase/sirtuin 1/peroxisome proliferator-activated receptor-γ coactivator 1α pathway are upregulated in the livers of AAV mice. The finding that partial restoration of hepatic G6Pase-α activity in GSD-Ia mice not only attenuates the phenotype of hepatic G6Pase-α deficiency but also prevents the development of age-related obesity and insulin resistance seen in wild-type mice may suggest relevance of the G6Pase-α enzyme to obesity and diabetes.

In this study, we proposed a testis-mediated germline chimera production system based on the transplantation of testicular cells directly into heterologous testes. The testicular cells of juvenile (4-wk-old) or adult (24-wk-old) Korean Ogol chickens with a recessive pigmentation inhibitory gene, with or without prior culture, were injected (2 x 10(7) cells/head) into the seminiferous tubules of juvenile or adult recipients with White Leghorn with a dominant pigmentation inhibitory gene in a 2 x 2 factorial arrangement. The localization of transplanted cells into the inner space of the seminiferous tubules was confirmed within 24 h after injection. Subsequent testcross analyses showed that 7.8% (5/64) of the recipients had chimeric status in their testes. The periods of time from transfer to hatching of the first progeny with black feathers were 38 and 45 days for adult cells transplanted into an adult recipient, 188 days for adult cells into a juvenile recipient, and 137 days for juvenile cells into a juvenile recipient. Culture of the testicular cells derived both colony-forming and monolayer-forming cells. The colony-forming cells were stained positively for periodic acid Schiff solution, and further reacted with anti-SSEA-1, anti-SSEA-3, and anti-SSEA-4 antibodies both before and after culture for 15 days. In conclusion, it may be possible to develop the testis-mediated germline chimera production technique, which extends the feasibility of genetic manipulations in avian species.

Landau-Kleffner syndrome is characterized by epileptic aphasia associated with electrical status epilepticus of slow wave sleep. A 5-year-old female, who had manifested normal developmental progress, was referred with principal complaints of fluctuating sensory aphasia and bizarre behavior during the preceding 4 months. Landau-Kleffner syndrome was confirmed by clinical and electroencephalographic features; in addition, the patient's mitochondrial respiratory chain-complex I deficiency was confirmed by fibroblast culture with the evidence of energy metabolism disorder. This patient's seizures were intractable to many antiepileptic drugs, adrenocorticotrophic hormone, and intravenous immunoglobulin, with catastrophic cognitive and behavioral decline, but the seizures were successfully controlled by ketogenic diet with supplementary mitochondrial cocktail including coenzyme Q10, riboflavin, L-carnitine, and high-dose multivitamins. The patient finally regained fully normal cognitive functioning. Landau-Kleffner syndrome with mitochondrial respiratory chain-complex I deficiency was controlled in this case by ketogenic diet and supplementary mitochondrial cocktail therapy.

The developmental similarity between the chicken and pheasant (Phasianus colchicus) allows the novel biotechnologies developed in the chicken to be applied to the production of transgenic pheasants and interspecies germline chimeras. To detect pheasant primordial germ cells (PGCs) efficiently, which is important for inducing germline transmission, the ultrastructure of PGCs and their reactivity to several antibodies (2C9, QB2, anti-SSEA-1, and QCR1) and periodic acid-Schiff's solution (PAS) were examined. To obtain PGCs, blood was taken from embryos incubated for 62-72 h or from gonads from embryos incubated for 156-216 h. The PGCs collected from both sources had the typical ultrastructure of pluripotent cells: a large nucleus with a distinct nucleolus, a high ratio of nuclear to cytoplasmic volume, and a distinct cytoplasmic membrane. In comparing the morphology of PGCs collected from different sites, more mitochondria and better-developed membrane microvilli were found in gonadal PGCs than in circulating PGCs. The nucleus of gonadal PGCs was flattened and had a large eccentrically positioned nucleolus. Of the antibodies tested, only QCR1 antibody reacted with an epitope in pheasant PGCs, and no specific signal was detected to other antibodies. The temporal change in the PGC populations in the blood and gonads of embryos was examined. In blood, the population was greater (P < 0.0001) in embryos incubated for 64 h than in embryos incubated for 62 or 66-72 h (31.4 versus 5.6-16.2 microL(-1)). In embryonic gonads, the number of PGCs increased continuously from 156 to 216 h of incubation (193-2,718 cells/embryo), although the ratio of PGCs to total gonadal cells did not change significantly (0.50-0.61%). In conclusion, pheasant PGCs have typical germ cell morphology and possess the QCR1 epitope. Circulating blood and the gonads of embryos incubated for 64 and 216 h, respectively, are good sources of PGCs.

The possibility of producing quail germline chimeras by the transfer of gonadal primordial germ cells (gPGCs) into recipient embryos was investigated. Japanese quail of the black (D: homozygous for the autosomal incomplete dominant gene D) and wild-type plumage (WP: d+/d+) strains were used as donors and recipients, respectively. Gonadal cells were retrieved from the gonads of 5-day-old D embryos, and gPGCs were enriched by magnetism-activated cell sorting. Fresh (noncultured) gPGCs or those isolated after culture for 3 days with gonadal stromal cells present in the mixed cell population were introduced into the dorsal aorta of 2-day-old recipient WP embryos. Hatchability of the recipient embryos was 23.7% (31/131) and 34.4% (31/90) for those transfused with cultured or noncultured gPGCs, respectively. Of the hatched quail, 28 acquired sexual maturity; among these animals, 7.1% (1/14) and 21.4% (3/14) of those that received cultured or noncultured gPGCs, respectively, were proved to be germline chimeras. The percentage of germline transmission to the donor-derived gametes in the chimeras that received cultured and noncultured gPGCs were 1.9 and 2.2-4.7%, respectively. In conclusion, quail gPGCs retrieved from 5-day-old embryos were thus transmitted in the germline after their transfer to quail embryos of a different strain. This property of the gPGCs was not adversely affected by culture for up to 3 days.

Study objectives: Extensive airway inflammation and excessive mucus production are implicated in deaths from asthma. High-resolution CT (HRCT) can be used to image both large and small airway abnormalities in asthmatics. The aims of this study were to clarify the distinction of HRCT features between near-fatal asthma (NFA) and non-NFA, and to evaluate serial follow-up HRCT scans of patients with NFA. Patients and design: Abnormalities of the large airway (bronchial wall thickness) and small airways (prominence of centrilobular structures and air trapping) were measured semiquantitatively on HRCT scans of 24 non-NFA, 16 NFA, and 16 control subjects. In addition, these abnormalities were reevaluated after intensive and relatively long-term (> 6 months) treatment with inhaled corticosteroids. Results: Prominence of centrilobular structures was observed in 36% of mild asthma cases, in 70% of moderate-to-severe asthma cases, and in 100% of NFA cases. Prominence of centrilobular structures, but neither bronchial wall thickness nor the area of air trapping, was significantly increased in NFA, as compared with mild or moderate-to-severe asthma (p < 0.05). In the seven non-NFA and five NFA patients who underwent follow-up HRCT scans, only bronchial wall thickness was decreased significantly in the NFA cases (p < 0.05), while bronchial wall thickness and the prominence of centrilobular structures were significantly decreased in the non-NFA cases. These small airway abnormalities were partially reversible in the both groups. Residual prominence of centrilobular structures after long-term inhaled corticosteroid treatment was significantly higher in NFA than non-NFA patients. Conclusions: The results of our study indicate that extensive small airway abnormalities may be associated with NFA, and that these abnormalities are partially reversible after the successful control of asthma symptoms. (CHEST 2004; 126:1840–1848)

Study objectives Eosinophilic bronchitis (EB) presents as a chronic cough and sputum eosinophilia without airflow limitation or bronchial hyperreactivity. Its long-term clinical course remains unknown. This study evaluated how frequently EB recurs and whether it develops chronic airway obstruction. Design This study was a prospective analysis. Methods Cough severity, FEV 1 , provocative concentration of methacholine causing a 20% fall in FEV 1 , and sputum eosinophil percentages were serially measured in 36 subjects for up to 48 months. All subjects inhaled corticosteroids until cough subsided. Results Five of the twenty four follow-up subjects (21%) had a recurrent episode of EB 4 to 6 months after disappearance of the first episode of EB (recurrent eosinophilic bronchitis). Progressive FEV 1 reduction > 20% was observed in three of the subjects, including a subject with asthma developing at the ninth month. Nineteen subjects had no recurrence of cough (nonrecurrent eosinophilic bronchitis) and no progressive FEV 1 reduction > 20%. However, sputum eosinophilia recurred between 4 months and 24 months in 10 subjects. Mean values of FEV 1 at the ninth and 12th months of the study were significantly lower in the recurrent eosinophilic bronchitis group than in the nonrecurrent eosinophilic bronchitis group (p Conclusion These results suggest that repeated episode of EB is associated with the development of chronic airflow obstruction, including asthma.

Cholesterol sulfate is a highly amphipathic molecule that is present in a relatively high concentration in the epidermis of human skin, particularly in the granular layer. The physiologic significance of this finding, however, is not well-understood. Therefore, we examined expression of the gene encoding for the enzyme that sulfonates cholesterol (SULT2B1b). Of the three enzymes known to sulfonate steroids/sterols, only the SULT2B1b isozyme was detected in cultures of normal human epidermal keratinocytes (NHEK) in response to Ca 2+ -induced terminal differentiation as well as by normal human epidermal tissue. Immunocytochemical analysis of normal skin as well as specific skin disorders was carried out. In normal skin, the expression of SULT2B1b was localized to the granular layer of the epidermis similar to that of filaggrin, an acknowledged late marker of differentiation and in contrast to that of involucrin, an early marker of terminal differentiation, which was expressed throughout the suprabasal region. The confinement of SULT2B1b to the granular layer coincides with this being the area with the highest cholesterol sulfate content suggesting that the physiologic action of cholesterol sulfate is likely carried out in this region of the living epidermis. Additionally, 88% of cholesterol sulfate in NHEK was membrane-associated further suggesting a cellular location for cholesterol sulfate action.

Glycogen storage disease type Ib (GSD-Ib) deficient in the glucose-6-phosphate transporter (G6PT or SLC37A4) is characterized impaired glucose homeostasis, myeloid dysfunction, and long-term complication of hepatocellular adenoma (HCA). We have shown that gene therapy mediated by a recombinant (r) AAV8 vector expressing G6PT directed by the chicken β-actin promoter/CMV enhancer enabled the G6pt-/- mice lived to over 51 weeks but all 5 transduced G6pt-/- mice expressed only low levels of hepatic G6PT activity and two developed multiple HCAs with one undergoing malignant transformation. We now examined the safety and efficacy of rAAV8-GPE-G6PT, a rAAV8 vector expressing G6PT directed by the gluconeogenic tissue-specific human G6PC promoter/enhancer (GPE). Of the fifteen rAAV8-GPE-G6PT-treated G6pt-/- mice that lived over age 60 weeks expressed 2-62% of wild-type hepatic G6PT activity with only one developed HCA. The treated mice, including the HCA-bearing mouse exhibit a leaner phenotype along with normal blood metabolite, display normal glucose tolerance profiles, maintain normoglycemia over a 24-hour fast, and retain insulin sensitivity. We further show that activation of hepatic ChREBP signaling that improves glucose tolerance and insulin sensitivity is one mechanism that protects the rAAV-GPE-G6PT-treated G6pt-/- mice against age-related obesity and insulin resistance.

Recently, immediate allergic reactions, including anaphylaxis, after Pachycondyla chinensis ant stings have been frequently reported in Korea. To estimate the prevalence of these reactions and the sensitization rate to P chinensis , we undertook a visit-questionnaire survey of the 327 adult residents living in a town in an ant-infested area in Korea. Skin prick tests with 6 common inhalant allergens, 3 bee venom allergens, and P chinensis whole body extract were performed on all ant-allergic subjects, on 86 asymptomatic residents, and on 37 controls outside the area. The serum-specific IgE to P chinensis extract was determined by ELISA. Seven subjects (2.1%) reported that they had experienced systemic allergic reactions to P chinensis stings; 4 of them had anaphylactic reactions. Large local reactions occurred in an additional 5 subjects (1.6%). All subjects with systemic allergic reactions had positive skin prick test results to P chinensis extract, whereas 23.3% of asymptomatic residents and 2.7% of the controls showed positive skin prick test results. The serum-specific IgE level was significantly higher in the subjects with systemic reactions than in the subjects with local reactions, the asymptomatic sensitizers, and the nonatopic controls. Sensitization to bee venom was found in 25% of the P chinensis -allergic subjects; this was significantly higher than the 3% rate seen in nonsensitized subjects. In conclusion, we report a 2.1% prevalence of systemic allergic reactions after P chinensis stings, based on self-reported symptoms, in an ant-infested area in Korea. (J Allergy Clin Immunol 2002;110:54-7.)

Allergic rhinitis (AR) is one of the most common chronic allergic respiratory diseases worldwide. Various practical guidelines for AR have been developed and updated to improve the care of AR patients; however, up to 40% patients remain symptomatic. The unmet need for AR care is one of the greatest public health problems in the world. The gaps between guideline and real-world practice, and differences according to the region, culture, and medical environments may be the causes of unmet needs for AR care. Because there is no evidence-based AR practical guideline reflecting the Korean particularity, various needs are increasing. The purpose of the study was to evaluate whether existing guidelines are sufficient for AR patient management in real practice and whether development of regional guidelines to reflect regional differences is needed in Korea. A total of 99 primary physicians comprising internists, pediatricians, and otolaryngologists (n=33 for each) were surveyed by a questionnaire relating to unmet needs for AR care between June 2 and June 16 of 2014. Among 39 question items, participants strongly agreed on 15 items that existing guidelines were highly insufficient and needed new guidelines. However, there was some disagreement according to specialties for another 24 items. In conclusion, the survey results demonstrated that many physicians did not agree with the current AR guideline, and a new guideline reflecting Korean particularity was needed.

Glycogen storage disease type Ib (GSD-Ib) is an autosomal-recessive syndrome characterized by neutropenia and impaired glucose homeostasis resulting from a deficiency in the glucose-6-phosphate (G6P) transporter (G6PT). The underlying cause of GSD-Ib neutropenia is an enhanced neutrophil apoptosis, but patients also manifest neutrophil dysfunction of unknown etiology. Previously, we showed G6PT interacts with the enzyme glucose-6-phosphatase-β (G6Pase-β) to regulate the availability of G6P/glucose in neutrophils. A deficiency in G6Pase-β activity in neutrophils impairs both their energy homeostasis and function. We now show that G6PT-deficient neutrophils from GSD-Ib patients are similarly impaired. Their energy impairment is characterized by decreased glucose uptake and reduced levels of intracellular G6P, lactate, adenosine triphosphate, and reduced NAD phosphate, whereas functional impairment is reflected in reduced neutrophil respiratory burst, chemotaxis, and calcium mobilization. We further show that the mechanism of neutrophil dysfunction in GSD-Ib arises from activation of the hypoxia-inducible factor-1α/peroxisome-proliferators–activated receptor-γ pathway.

Depression is an important comorbidity of asthma. However, little information is available about depression and its potential impact on asthma control in Korean adult asthma patients. We aimed to estimate the prevalence and risk factors for depression in Korean adults with persistent asthma. The 127 non-elderly (20-64 yr) and 75 elderly (≥65 yr) patients with asthma were recruited. Demographic and clinical data were extracted, and the patients completed the Asthma Specific Quality of Life (AQOL) questionnaire and asthma control test (ACT). Depression status was defined using the Korean version of the Patient Health Questionnaire-9 (PHQ-9). Depression was more prevalent in non-elderly (18.9%) than in elderly patients with asthma (13.3%). Patients with depression were significantly younger, had lower economic status, shorter disease duration, poorer asthma control, and worse AQOL scores (P

Summary Xylitol has many uses, such as in dentistry and as a sweetening agent, and is synthesized from fungus by fermentation. According to some publications, xylitol has no adverse effects on the lungs in either healthy people or patients with cystic fibrosis, but no studies have examined the effects of long-term exposure. Here, we report a patient who developed occupational asthma due to xylitol as confirmed with provocation tests.

Summary We report a case of bulla formation in usual interstitial pneumonia (UIP) after a 5-year follow-up, proven by open lung biopsy, in a 35-year-old Korean man with dermatomyositis. Chest computed tomography showed marked bulla formation and bilateral hydrothorax. To our knowledge, the end result of UIP is honeycombing, respiratory failure, and infection. We postulate that if patients show good compliance in dealing with their UIP, the next form of honeycombing is bulla formation. This is the first report of bulla formation in UIP with dermatomyositis after long-term follow-up.

Glucose-6-phosphatase-β (G6Pase-β or G6PC3) deficiency, also known as severe congenital neutropenia syndrome 4, is characterized not only by neutropenia but also by impaired neutrophil energy homeostasis and functionality. We now show the syndrome is also associated with macrophage dysfunction, with murine G6pc3−/− macrophages having impairments in their respiratory burst, chemotaxis, calcium flux, and phagocytic activities. Consistent with a glucose-6-phosphate (G6P) metabolism deficiency, G6pc3−/− macrophages also have a lower glucose uptake and lower levels of G6P, lactate, and ATP than wild-type macrophages. Furthermore, the expression of NADPH oxidase subunits and membrane translocation of p47phox are down-regulated, and G6pc3−/− macrophages exhibit repressed trafficking in vivo both during an inflammatory response and in pregnancy. During pregnancy, the absence of G6Pase-β activity also leads to impaired energy homeostasis in the uterus and reduced fertility of G6pc3−/− mothers. Together these results show that immune deficiencies in this congenital neutropenia syndrome extend beyond neutrophil dysfunction.

Background : An appreciable number of patients with toluene diisocyanate (TDI)-induced asthma have high serum levels of specific IgE (sIgE) antibody to TDI-human serum albumin conjugate (HSA). A recent investigation suggested a role of specific IgG (sIgG) in the development of TDI asthma. Methods : We observed the changes in the levels of specific IgE and IgG antibodies to TDIHSA conjugate in TDI-induced asthmatic patients during seven years avoidance. Results : Six subjects with high sIgE and five with high sIgG were enrolled. All of them had taken anti-asthmatic medications with complete avoidance. Serum levels of sIgE and sIgG to TDI-HSA conjugate were detected by ELISA. The level of sIgE continued to decline up to 7 years and the mean half-life was 3.9 years. The mean half-life of sIgG was 4.5 yrs. Conclusion : These findings suggest that both sIgE and sIgG to TDI-HSA conjugate may persist for several years after the last exposure to TDI.

Glycogen storage disease type Ia (GSD-Ia), which is characterized by impaired glucose homeostasis and chronic risk of hepatocellular adenoma (HCA), is caused by deficiencies in the endoplasmic reticulum (ER)-associated glucose-6-phosphatase-α (G6Pase-α or G6PC) that hydrolyzes glucose-6-phosphate (G6P) to glucose. G6Pase-α activity depends on the G6P transporter (G6PT) that translocates G6P from the cytoplasm into the ER lumen. The functional coupling of G6Pase-α and G6PT maintains interprandial glucose homeostasis. We have shown previously that gene therapy mediated by AAV-GPE, an adeno-associated virus (AAV) vector expressing G6Pase-α directed by the human G6PC promoter/enhancer (GPE), completely normalizes hepatic G6Pase-α deficiency in GSD-Ia (G6pc−/−) mice for at least 24 weeks. However, a recent study showed that within 78 weeks of gene deletion, all mice lacking G6Pase-α in the liver develop HCA. We now show that gene therapy mediated by AAV-GPE maintains efficacy for at least 70-90 weeks for mice expressing more than 3% of wild-type hepatic G6Pase-α activity. The treated mice displayed normal hepatic fat storage, had normal blood metabolite and glucose tolerance profiles, had reduced fasting blood insulin levels, maintained normoglycemia over a 24-hour fast, and had no evidence of hepatic abnormalities. After a 24-hour fast, hepatic G6PT messenger RNA levels in G6pc−/− mice receiving gene therapy were markedly increased. Because G6PT transport is the rate-limiting step in microsomal G6P metabolism, this may explain why the treated G6pc−/− mice could sustain prolonged fasts. The low fasting blood insulin levels and lack of hepatic steatosis may explain the absence of HCA. Conclusion: These results confirm that AAV-GPE–mediated gene transfer corrects hepatic G6Pase-α deficiency in murine GSD-Ia and prevents chronic HCA formation. (HEPATOLOGY 2012;56:1719–1729)

G6PC3 (or glucose-6-phosphatase-β) deficiency underlies a congenital neutropenia syndrome in which neutrophils exhibit enhanced endoplasmic reticulum (ER) stress, increased apoptosis, impaired energy homeostasis, and impaired functionality. Here we show that murine G6pc3−/− neutrophils undergoing ER stress activate protein kinase-like ER kinase and phosphatidylinositol 3,4,5-trisphosphate/Akt signaling pathways, and that neutrophil apoptosis is mediated in part by the intrinsic mitochondrial pathway. In G6PC3-deficient patients, granulocyte colony-stimulating factor (G-CSF) improves neutropenia, but its impact on neutrophil apoptosis and dysfunction is unknown. We now show that G-CSF delays neutrophil apoptosis in vitro by modulating apoptotic mediators. However, G6pc3−/− neutrophils in culture exhibit accelerated apoptosis compared with wild-type neutrophils both in the presence or absence of G-CSF. Limiting glucose (0.6mM) accelerates apoptosis but is more pronounced for wild-type neutrophils, leading to similar survival profiles for both neutrophil populations. In vivo G-CSF therapy completely corrects neutropenia and normalizes levels of p-Akt, phosphatidylinositol 3,4,5-trisphosphate, and active caspase-3. Neutrophils from in vivo G-CSF–treated G6pc3−/− mice exhibit increased glucose uptake and elevated intracellular levels of G6P, lactate, and adenosine-5′-triphosphate, leading to improved functionality. Together, the results strongly suggest that G-CSF improves G6pc3−/− neutrophil survival by modulating apoptotic mediators and rectifies function by enhancing energy homeostasis.

G6PC3 deficiency, characterized by neutropenia and neutrophil dysfunction, is caused by deficiencies in the endoplasmic reticulum (ER) enzyme glucose-6-phosphatase-β (G6Pase-β or G6PC3) that converts glucose-6-phosphate (G6P) into glucose, the primary energy source of neutrophils. Enhanced neutrophil ER stress and apoptosis underlie neutropenia in G6PC3 deficiency, but the exact functional role of G6Pase-β in neutrophils remains unknown. We hypothesized that the ER recycles G6Pase-β–generated glucose to the cytoplasm, thus regulating the amount of available cytoplasmic glucose/G6P in neutrophils. Accordingly, a G6Pase-β deficiency would impair glycolysis and hexose monophosphate shunt activities leading to reductions in lactate production, adenosine-5′-triphosphate (ATP) production, and reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity. Using annexin V–depleted neutrophils, we show that glucose transporter-1 translocation is impaired in neutrophils from G6pc3−/− mice and G6PC3-deficient patients along with impaired glucose uptake in G6pc3−/− neutrophils. Moreover, levels of G6P, lactate, and ATP are markedly lower in murine and human G6PC3-deficient neutrophils, compared with their respective controls. In parallel, the expression of NADPH oxidase subunits and membrane translocation of p47phox are down-regulated in murine and human G6PC3-deficient neutrophils. The results establish that in nonapoptotic neutrophils, G6Pase-β is essential for normal energy homeostasis. A G6Pase-β deficiency prevents recycling of ER glucose to the cytoplasm, leading to neutrophil dysfunction.

Glycogen storage disease type Ia (GSD-Ia) patients deficient in glucose-6-phosphatase-alpha (G6Pase-alpha or G6PC) manifest disturbed glucose homeostasis. We examined the efficacy of liver G6Pase-alpha delivery mediated by AAV-GPE, an adeno-associated virus (AAV) serotype 8 vector expressing human G6Pase-alpha directed by the human G6PC promoter/enhancer (GPE), and compared it to AAV-CBA, that directed murine G6Pase-alpha expression using a hybrid chicken beta-actin (CBA) promoter/cytomegalovirus (CMV) enhancer. The AAV-GPE directed hepatic G6Pase-alpha expression in the infused G6pc(-/-) mice declined 12-fold from age 2 to 6 weeks but stabilized at wild-type levels from age 6 to 24 weeks. In contrast, the expression directed by AAV-CBA declined 95-fold over 24 weeks, demonstrating that the GPE is more effective in directing persistent in vivo hepatic transgene expression. We further show that the rapid decline in transgene expression directed by AAV-CBA results from an inflammatory immune response elicited by the AAV-CBA vector. The AAV-GPE-treated G6pc(-/-) mice exhibit normal levels of blood glucose, blood metabolites, hepatic glycogen, and hepatic fat. Moreover, the mice maintained normal blood glucose levels even after 6 hours of fasting. The complete normalization of hepatic G6Pase-alpha deficiency by the G6PC promoter/enhancer holds promise for the future of gene therapy in human GSD-Ia patients.

This study was conducted to evaluate whether the sex of donor primordial germ cells (PGCs) influences production of chimeric semen from recipient hatchlings produced by interspecies transfer between pheasant (Phasianus colchicus) and chicken (Gallus gallus). Pheasant PGCs were retrieved from 7-d-old embryos and subsequently transferred into circulatory blood of 2.5-d-old (Stage 17) embryos. The sex of embryos was discerned 3 to 6 days after laying, and in preliminary study, overall rate of embryo survival after sexing was 74.6% with male-to-female ratio of 0.49 to 0.51. In Experiment 1, magnetic-activated cell sorting (MACS) using QCR1 antibody was effective for enriching the population of male and female PGCs in gonadal cells (9.2- to 12.5-fold and 10.8- to 19.5-fold increase, respectively). In Experiment 2, an increase in the number of hatchlings producing chimeric semen was detected after the homosexual transfer of male-to-male compared with that after the heterosexual transfer of female-to-male (68% to 88%). Significant increase was found in the frequency of chimeric semen production (0.96 to 1.68 times); production of pheasant progenies by artificial insemination using chimeric semen was also increased in the homosexual transfer (0 to 3 cases). In conclusion, the homosexual PGC transfer of male-to-male yielded better rate of generating pheasant progenies after test cross-reproduction than that of the heterosexual transfer of female-to-male, which could improve the efficiency of interspecies germ cell transfer system.

Object The authors propose that anterior callosotomy—thought to have some advantages over total callosotomy—is not superior to total callosotomy for prevention of seizure propagation or other complications. Methods The study comprised 34 patients in whom generalized epilepsy syndrome or frontal lobe seizures with a secondary generalization were diagnosed. Preoperatively, all patients suffered from disabling drop attacks or intense head-drop seizures, and some patients also experienced other types of seizures. The male/female ratio was 22:12, and patients ranged in age between 1 to 19 years (mean 8.7 years). The follow-up period ranged from 1.08 to 5.0 years (mean 2.58 years). Seizure outcome, parental assessment of daily function, and parental satisfaction with the outcome was assessed postoperatively. Results After undergoing 1-stage total callosotomy, drop attacks disappeared completely in 25 patients during the follow-up period, and in 6 patients the frequency of drop attacks decreased to < 10% of baseline. With regard to other types of seizures, seizures resolved completely in 12 patients, and in another 18 seizure frequency decreased. Two patients experienced a transient disconnection syndrome for 2 and 4 weeks. One patient experienced ataxic hemiparesis for 3 weeks before it completely abated. The overall daily function of the patients improved, and all parents were satisfied with the surgical outcome. Conclusions For pediatric generalized epilepsy syndrome, 1-stage total callosotomy will be the first choice in treatment for controlling generalized seizures.

Glycogen storage disease type Ib (GSD-Ib) is an autosomal recessive disorder caused by deficiencies in a glucose-6-phosphate (G6P) transporter (G6PT) that transloacates G6P from the cytoplasm into the lumen of the endoplasmic reticulum where it is hydrolyzed to glucose and phosphate by the glucose-6-phosphatase-α (G6Pase-α or G6PC) enzyme. The functional coupling of G6PT and G6Pase-α maintains interprandial blood glucose homeostasis. GSD-Ib patients manifest impaired glucose homeostasis characterized by fasting hypoglycemia, hepatomegaly, nephromegaly, hyperlipidemia, hyperuricemia, lactic acidemia, and growth retardation. The current dietary therapies enable patients to maintain normoglycemia but the underlying pathological processes remain uncorrected and long-term complications, including hepatocellular adenoma (HCA), are common. In this study, we examined the efficacy of liver G6PT delivery mediated by two human G6PT-expressing recombinant adeno-associated virus (AAV) pseudotype 2/8 (rAAV8) vectors. One is a single-stranded vector containing a 2864-bp of the human G6PC promoter/enhancer (rAAV8-GPE-G6PT) and the other is a double-stranded vector containing the minimal human G6PT promoter/enhancer (rAAV8-miGT-G6PT). GSD-Ib mice were infused with either vector neonatally, then at age 4 weeks, and metabolic profiles of the infused animals were monitored at age 12 weeks. We show that the rAAV8-GPE-G6PT vector directed significantly higher levels of hepatic G6PT expression, achieved greater reduction in hepatomegaly, and led to a better toleration of fasting in GSD-Ib mice than the rAAV8-miGT-G6PT vector. Our results suggest that the rAAV8-GPE-G6PT vector is an efficacious vector in correcting metabolic abnormalities in murine GSD-Ib.

Boda Nam, MD, Jung Hwa Hwang, MD*, Young Mok Lee, MD, Jai Soung Park, MD, Sung Shick Jou, MD, Youngbae Kim, MD 1Department of Radiology, Soonchunhyang University Hospital, Seoul, Korea 2Bangbae GF Allergy Clinic, Seoul, Korea 3Department of Radiology, Soonchunhyang University Bucheon Hospital, Bucheon, Korea 4Department of Radiology, Soonchunhyang University Cheonan Hospital, Cheonan, Korea 5Department of Preventive Medicine, Soonchunhyang University College of Medicine, Cheonan, Korea