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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.

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.

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.

The production of chimeric birds is an important tool for the investigation of vertebrate development, the conservation of endangered birds, and the development of various biotechnological applications. This study examined whether gamma (γ)-irradiation depletes endogenous primordial germ cells and enhances the efficiency of somatic chimerism in chickens. An optimal irradiation protocol for stage X embryos was determined after irradiation at various doses (0, 100, 300, 500, 600, 700, and 2,000 rad). Exposure to 500 rad of γ-irradiation for 73 s significantly decreased the number of primordial germ cells (P < 0.0001). Somatic chimera hatchlings were then produced by transferring blastodermal cells from a Korean Oge into either an irradiated (at 500 rad) or intact stage X White Leghorn embryo. An analysis of feather color pattern and polymerase chain reaction-based species-specific amplification of various tissues of the hatchlings confirmed chimerism in most organs of the chick produced from the irradiated recipient; a lesser degree of chimerism was observed in the non-irradiated control recipient. In conclusion, the exposure of chick embryos to an optimized dose of γ-irradiation effectively depleted germ cells and yielded greater somatic chimerism than non-irradiated control embryos. This technique can be applied to interspecies reproduction or the production of transgenic birds.

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

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.

We recently succeeded in inducing germline transmission by transferring chicken testicular cells into heterologous testes. This study was designed subsequently to identify pluripotent cells in the testicular cells, which would induce the germline transmission. Testicular cells retrieved from juvenile (4-wk-old) or adult (24-wk-old) White Leghorn (WL) chickens were stained with germ cell-specific markers anti-SSEA1, anti-SSEA3, anti-SSEA4, anti-EMA1, anti-ITGA6, and anti-ITGB1 antibodies; 2C9; and lectin-Solanum tuberosum agglutinin (STA). The percentages of the cells that were positive for each marker were within the ranges of 0.33% -0.44% and 0.029%-0.072% of the total testicular cell population in the juvenile and adult, respectively, and significant (P < 0.0002) differences were detected between the ages. When 1 x 10(6) testicular cells were cultured in Dulbecco minimum essential medium-based medium supplemented with leukemia inhibitory factor (LIF), basic fibroblast growth factor (FGF2), and/or insulinlike growth factor 1 (IGF1), colony formation was detected only in LIF++FGF2-containing or LIF+FGF2+IGF1-containing medium during primary culture, and the supplementation of LIF+FGF2+IGF1 was the most efficient for maintaining the colony-forming cells through subculture. The established cells retrieved at the end of the primary culture or the 20th subpassage were positive for chicken germ cell-specific periodic acid-Schiff (PAS), EMA1, 2C9, SSEA1, SSEA3, SSEA4, ITGA6, and ITGB1; and lectin-STA markers (evaluated after 11th subpassage). Double staining of lectin-STA with anti-SSEA1, anti-SSEA3, anti-SSEA4, anti-ITGA6, and anti-ITGB1 also was possible. They differentiated spontaneously into embryoid bodies after being cultured in LIF-free medium. We conclude that germline stem cell-like cells are present in chicken testicular cells retrieved from both juvenile and adult testes, which can be identified with the specific markers for primordial germ cells or embryonic germ cells.

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.

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.

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.)

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.

To evaluate the transcrystalline effects caused by various fibers, which were untreated, or treated with sodium hydroxide and cellulase, isothermal crystallization was performed. It was observed that the untreated and cellulase-treated cellulose fibers (cellulose I) had a nucleating ability to transcrystallize at PP matrix. Especially, cellulose fibers treated with Sodium hydroxide (cellulose II) transcystallized at PP matrix. This result was different from other's. Cellulose fibers also transcrystallized at PP/MAH-PP matrix irrespective of the type of cellulose crystalline structure. In PP/MAH-PP/CELL system, MAH-PP was located around the fiber surface at initial crystallization time, but was gradually expelled from that with the increase of crystallization time, and existed at outer boundaries of transcrstalline region at the final crystallization time. These phenomena were confirmed by IR-IRS spectra. The tensile strength of PP/CELL and PP/MAH-PP/CELL composites decreased with the increase of isothermal crystallization time. Therefore, it is thought that transcrystallinity gives rise to negative effect of tensile strength.

Low-density polyethylene (LDPE) composites containing untreated cellulose, cellulose treated with cellulase, and cellulose treated with cellulase after pretreating with NaOH were prepared. The purpose of the cellulase treatment of cellulose is to enhance the interfacial adhesion between the cellulose and the matrix. The effect of this treatment was studied by viscometry and X-ray diffraction. Maleic anhydrideg-polyethylene (MAH-PE) was also added to the composite to enhance interfacial interactions between the cellulose and the matrix and to increase the dispersibility and the wettability of cellulose. The increased adhesion in composites containing the treated cellulose and MAH-PE was studied by FTIR. In addition, the thermal, dynamic mechanical, and tensile properties of the composites were investigated. When MAH-PE was added to the composite, it was found that the reaction between MAH-PE and treated cellulose occurred more easily.

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.

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.

We report a case of hypersensitivity pneumonitis in a 30-yr-old female housewife caused by Penicillium species found in her home environment. The patient was diagnosed according to history, chest radiograph, spirometry, high-resolution chest CT, and transbronchial lung biopsy. To identify the causative agent, cultured aeromolds were collected by the open-plate method. From the main fungi cultured, fungal antigens were prepared, and immunoblot analysis with the patient's serum and each fungal antigen was performed. A fungal colonies were isolated from the patient's home. Immunoblotting analysis with the patient's sera demonstrated a IgG-binding fractions to Penicillium species extract, while binding was not noted with control subject. This study indicates that the patient had hypersensitivity pneumonitis on exposure to Penicillium species in her home environment.

Cytokines, having central functions in immunological and inflammatory process, are always expected to play important roles in the pathogenesis of various diseases, such as asthma. Genetic polymorphisms of those cytokine and cytokine receptor genes are the focus of genetic association studies. In an effort to identify gene(s) whose variant(s) are involved in the development of asthma, we examined the genetic effects of 19 single nucleotide polymorphisms in eight cytokine and cytokine receptor genes, including IL1A, IL1B, IL2, IL3, IL4, IL8, IL10, and IL5RA, on asthma and atopy. Nineteen single nucleotide polymorphisms in eight cytokine and cytokine receptor genes were genotyped using the single-base extension method in a Korean asthma cohort (n = 723). Logistic regression and multiple regressions were used for statistical analyses controlling for smoking, age, and gender as covariables. Genetic association analysis of polymorphisms revealed that one exonic (exon 1), IL3 + 79TC ( Ser27Pro), showed significant association with the risk of asthma and atopy. The Pro allele had shown dominant and protective effects on development of asthma in nonatopic subjects (P = 0.002) and also showed significant association with the risk of atopy in normal control subjects (P = 0.007). This information about the genetic association of important genes with asthma might provide valuable insights into strategies for the pathogenesis of asthma and atopy.

The Eotaxin gene family (Eotaxin1, Eotaxin2 and Eotaxin3) recruits and activates CCR3-bearing cells such as eosinophils, mast cells and Th2 lymphocytes that play a major role in allergic disorders. To date, the effect of polymorphisms of Eotaxin genes on asthma phenotypes has not been thoroughly examined. In our research, we sequenced whole regions of the Eotaxin gene family to identify polymorphisms, which may be involved in the development of asthma and total serum IgE. We have identified 37 SNPs in the Exotaxin gene family (Exotaxin1, 2 and 3), and 17 common polymorphic sites were selected for genotyping in our asthma cohort (n ¼ 721). Statistical analysis revealed that the EOT2þ1265A> GG * allele showed significantly lower frequency in asthmatics than in normal healthy controls (0.14 versus 0.23, P ¼ 0.002), and that distribution of the EOT2þ1265A> GG * allele-containing genotypes was also much lower in asthmatics (26.3 versus 40.8%, P ¼ 0.003). In addition, a non-synonymous SNP in Eotaxin1, EOT1þ123Ala>Thr showed significant association with total serum IgE levels (P ¼ 0.002–0.02). The effect of EOT1þ123Ala>Thr on total serum IgE appeared in a gene-dose-dependent manner. Our findings suggest that the development of asthma may be associated with EOT2þ1265A>G polymorphisms, and the susceptibility to high IgE production may be attributed to the EOT1þ123Ala>Thr polymorphism. Eotaxin variation/haplotype information identified in this study might provide valuable insights into strategies for the control of asthma.

Background: Airway eosinophilia is frequently observed during acute exacerbation of asthma. Interleukin-5 (IL-5) and eotaxin are directly involved in the airway eosinophilia found in persistent asthma. Interrelation between these cytokines is expected to occur in acute exacerbation of asthma. Thus, we evaluated the relevance of interaction between eotaxin and IL-5 in the airway inflammation of acute exacerbation. Methods: We measured the number of inflammatory cells and the amount of eotaxin and IL-5 in sputum from 22 healthy subjects, 21 asthmatics with acute exacerbation and 16 patients with mild persistent asthma, and reassessed these values in 7 subjects with acute exacerbation after 7 days’ treatment with systemic steroid (2 mg/kg/day). Sources of IL-5 and eotaxin were investigated by immunohistochemical staining of sputum cells of 4 cases from each group. Results: Both IL-5 and eotaxin levels were higher in patients with acute exacerbation of asthma than in patients with persistent asthma and normal subjects. IL-5 and eotaxin levels were significantly correlated with eosinophil percentages in mild persistent asthma. Eotaxin staining was found mainly on macrophages and occasionally on eosinophils. Steroid treatment markedly decreased eosinophil percentages and IL-5 levels within 7 days but did not alter eotaxin levels. Conclusions: Both IL-5 and eotaxin are associated with acute exacerbation of asthma. IL-5 rather than eotaxin is effectively decreased by the inhibitory effect of steroid in acute exacerbation.

Signaling by hepatocyte growth factor (HGF) via its cell surface receptor, Met, drives cell survival, growth and motility, as well as tumor growth and metastasis in several prevalent human cancers. HGF and Met have become highly sought targets for cancer drug development: 165 clinical trials of 21 experimental agents have been completed or are in progress. We reported previously that a selective HGF antagonist failed to block growth of the human prostate adenocarcinoma derived cell line PC3M as primary tumor xenografts in mice, but potently blocked spontaneous PC3M tumor metastasis, as well as cell motility and invasion in vitro. Thus partial activation of the Met pathway in PC3M cells by murine HGF (mHGF) is capable of driving invasion but not proliferation, whereas human HGF (hHGF) is capable of driving both activities. To identify the signaling and gene modulation events critical for Met-driven cell invasion and to distinguish them from Met-driven proliferation, mRNA expression array and pathway profiling analyses of hHGF and mHGF-treated PC3M cells were performed. Ingenuity Pathway Analysis (IPA) of 344 hHGF-specific statistically significant gene modulation events of 2-fold or greater showed significant overlap with cell cycle progression and proliferation pathways, cancer and upstream activation of the cyclin D1 and choriogonadotropin pathways. IPA analysis of 520 gene modulation events associated with motility and invasion showed significant overlap with cell migration and invasion pathways, upstream activation of the MEK/MAPK and mir-155 pathways and upstream inhibition of PI3K and pro-inflammatory cytokine pathways. HGF-specific blockade of spontaneous PC3M metastasis in our model prompted us to further compare significant gene modulation events related to cell invasion with those reported for prostate cancer tumor samples as part of The Cancer Genome Atlas Project (TCGA; MSKCC, 2010; 216 cases). Of 520 altered genes in PC3M, 345 were also interrogated in TCGA: 150 of these (47%) were similarly modulated in prostate cancer patient samples. Twenty-four gene modulation events (7%) were associated with significant change in disease-free survival (DFS) in 88% of cases: 1 gene with increased DFS (logrank p: 0.002747, 56% of cases affected) and 23 with decreased DFS (logrank p: 0.007542; 65% of cases affected). Together these findings identify critical differences in intracellular pathways downstream of HGF/Met interaction leading to cell proliferation vs invasion and reveal that a substantial fraction of prostate cancer patients who show gene modulation events associated with increased tumor cell invasion and metastasis in the PC3M model described here also experience significantly accelerated disease progression. Citation Format: Fabiola Cecchi, Jason Lih, Young Lee, William Walsh, Mickey Williams, Donald P. Bottaro. Gene expression array and pathway profiling analyses distinguish HGF/Met pathways driving cell proliferation from invasion and identify events correlated with prostate cancer progression. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5259. doi:10.1158/1538-7445.AM2014-5259

Background Respiratory syncytial virus (RSV)–specific IgE is a component of the host response to RSV infection, but its role in the subsequent enhancement of altered airway responsiveness is unknown. Objective To define the role of RSV-specific IgE in the enhancement of airway responsiveness on reinfection of newborn mice. Methods Mice were infected as newborns with RSV and were reinfected 5 weeks later. The role of IgE was determined by documenting RSV-specific IgE response after neonatal infection, and by assessing airway responsiveness on reinfection. Results After neonatal infection, wild-type (WT) mice developed an RSV-specific IgE response. On reinfection, these mice developed enhanced airway hyperresponsiveness (AHR), airway eosinophilia, and mucus hyperproduction, and their T-cell cytokine response was skewed toward a T H 2 phenotype. None of these altered responses developed on reinfection of IL-4 −/− /IL-13 −/− mice, and no RSV-specific IgE could be detected after neonatal infection of these mice. FcɛRI −/− mice did not develop the enhanced AHR on reinfection, and airway eosinophilia and mucus production were significantly attenuated. These responses could be restored in deficient mice reconstituted with WT mast cells. In RSV-infected newborn WT mice, administration of anti-IgE prevented the enhancement of AHR and attenuated eosinophilia and mucus hyperproduction on reinfection, an effect that was associated with diminished T H 2 cytokine production and increased IFN-γ production. Conclusion Respiratory syncytial virus–specific IgE enhances the development of T H 2-biased airway responsiveness on reinfection of mice initially infected as newborns.

결핵균은 공기를 매개로 전파되고 결핵균에 노출된 시간과 최초의 노출 정도가 전파의 위험을 증가 시키는 중요한 인자로 알려져 있다. 활동성 폐결핵 환자와 함께 생활하는 가족 내에서 감염이 현저히 증가하는데 이는 결핵균에 노출되는 기회 및 시간이 증가하기 때문이다. 하지만 고식적인 접촉자 조사만으로는 흔히 결핵 발생률을 과소평가할 수 있기 때문에 접촉자 조사와 결핵균의 유전형 조사를 병행하는 것이 감염의 위험이 높은 집단을 선별해내고 발생빈도 및 전염경로를 밝히는 데 보다 효과적인 것으로 보고되고 있다. 이에 접촉자 조사와 결핵균의 유전형 조사를 통해 가족 내 결핵균 전파를 증명한 1예를 보고하는 바이다. (Tuberc Respir Dis 2008;65:512-516)

Background : Diagnosing a pulmonary embolism is difficult because its presenting symptoms are nonspecific and there are limitations with all of the objective tests. The D-dimer is known to be a marker of the lysis of intravascular cross-linked fibrin as a result of the activation of the endogenous fibrinolytic pathways, and the D-dimer assay is these an objective method for diagnosing a pulmonary embolism. This study assessed the benefits of the D-dimer test for diagnosing a pulmonary embolism using semiquantitative latex agglutination. Methods : The latex agglutination results of 185 patients were retrospectively reviewed. The D-dimer test was performed at the time a pulmonary embolism was suspected. Ninety patients(group I) were diagnosis with PE through spiral chest CT or a chest CT angiogram, perfusion/ventilation scans, and/or pulmonary angiogram. Ninety-five patients (group II) were found not to have a pulmonary embolism through the above tests. Results : The male to female ratio and mean age in groups I and II was 37:55, and 57 years old to 50:45 and 52 years old, respectively. When the cut off value for a positive D-dimer assay was set to , the sensitivity, positive predictive value, negative predictive value and specificity was 86.7%, 61.4%, 79.3%, and 48.4%, respectively. Conclusion : The semiquantitative latex agglutination method in the D-dimer test has a lower sensitivity and negative predictive value than the well known ELISA test particularly for small emboli. Therefore, this test is not a suitable screening test for excluding a pulmonary embolism.

Aspergillus fumigatus causes a variety clinical syndrome in lung including aspergilloma, chronic necrotizing aspergillosis, invasive pulmonary aspergillosis, and allergic bronchopulmonary aspergillosis. Aspergilloma develops by a colonization and growing of Aspergillus inside lung cavities with underlying lung disease. There is a few report of endobronchial aspergilloma without lung parenchymal lesion. We experienced a case of endobronchial aspergilloma did not fit any category of Aspergillus-induced lesion, who show minimal fibrostreaky denstities on chest PA and chest CT. Massive hemoptysis was improved by a removal of the aspergilloma in this patient. Here, we report a rare case of endobronchial aspergilloma showing massive hemoptysis with review of literatures. (Tuberc Respir Dis 2004; 57:589-593)

Eosinophil and mast cell infiltrations are consistent findings in nasal polyp tissue. Previous studies have shown that matrix metalloproteinases (MMPs) may be involved in eosinophil infiltration in airway mucosa of asthmatic patients, and that transforming growth factor-beta1 (TGF-beta1) induces extracellular matrix deposition in nasal polyp tissue. The aim of this study was to evaluate the role of MMPs and tissue-inhibitor of metalloproteinase-1 (TIMP-1) in association with TGF-beta1, eosinophils and mast cell activation in nasal polyp tissue. Nasal polyp tissues from 20 patients who underwent polypectomies were collected and prepared into tissue homogenate. Eosinophil cationic protein (ECP) and tryptase levels were measured by CAP system (Pharmacia, Sweden). MMP-2, MMP-9, TIMP-1 and TGF-beta1 levels were measured by enzyme-liked immunosorbent assay. MMP-2 was the predominant form of MMPs, followed by MMP-9 and TIMP-1. There were significant correlations between ECP, and MMP-9, MMP-2, TGF-beta1 and tryptase, but not with TIMP-1. Significant correlations were noted between tryptase, and MMP-2, MMP-9, and TGF-beta1, but not with TIMP-1. Close correlations were noted between TGF-beta1, and MMP-9 and MMP-2, but not with TIMP-1. MMP-2, MMP-9, and TGF-beta1 may contribute to eosinophil and mast cell migrations into nasal polyp tissue.

Background: TNF-alpha is related to the generation of lung fibrosis in patients with UIP. The precise mechanism leading to lung fibrosis by TNF-alpha is unknown. However, the activation of a transcription factor like AP-1(down stream of c-jun N-terminal kinase, JNK) by TNF-alpha may be related to the induction of fibrogenic cytokines like PDGF or IGF-I. Furthermore, JNK was reported to be activated in the radiation-induced lung fibrosis model. This study examined JNK activity in patients with UIP. Methods : The expression of phosphorous JNK(p-JNK), macrophage/monocyte specific markers, CD68, and cytokeratin was evaluated by immunohistochemical(IHC) staining of lung tissues from patients with UIP and lung cancer. An in vitro kinase assay was performed with alveolar macrophages obtained by a bronchol-avleolar lavage from patients with UIP and healthy persons as the control. Results : The IHC stain showed that p-JNK is expressed in the almost all of the alveolar macrophages and smooth muscle cells in patients with UIP. In case of the normal areas of the lung from patients with lung cancer, the alveolar macrophages showed little p-JNK expression. Interestingly, increased JNK activity was not found in the in vitro kinase assay of the alveolar macrophages obtained from both patients with UIP and healthy persons as the control. Furthermore, 10 ng/mL of TNF-alpha failed to increase the JNK activity of the alveolar macrophages in both patients with UIP and healthy people. Conclusion : The JNK was activated constitutionally in patients with UIP. However, the role of JNK in the pathogenesis of lung fibrosis needs to be clarified.

Purpose : An anthracofibrosis(AF), dark multiple anthracotic pigmentations combined with narrowing and obstruction of bronchi, was reported to be strongly related with past and active pulmonary tuberculosis. This study was performed to determine whether anti-tuberculous regiemens would be helpful in patients with anthracofibrosis who failed to demonstrate the evidences of pulmonary tuberculosis. Methods : Twenty-two patients with multiple anthracotic pigmentations in bronchial mucosa with luminal narrowing were enrolled in this study. The bacteriological and histological findings for Mycobacterium tuberculosis was reviwed in each patients. They are composed of 8 males and 14 females ranging from 55 to 85 years old in age. Results: The most common symptoms were coughing(73%, 16/22), followed by sputum(41%, 9/22), dyspnea on exertion(32%, 7/22), and hemoptysis(27%, 6/22). The evidence of pulmonary tuberculosis, defined by positive AFB smear or culture of Mycobacterium tuberculosis from sputum or bronchial washing fluid or histological findings of granuloma with caseous necrosis, were found in eleven patients(50%) and the others has showed no evidences. Among 11 patients without pulmonary tuberculosis, only one patient showed the evidences of pulmonary tuberculosis after 16 months, and the 8 patients still showed no evidence of pulmonary tuberculosis during follow-up periods of ranging from 8 months to 60 months. Conclusions : Beause the anthracofibrosis is closely related to tuberculosis, it needs to find out extensively the evidences of tuberculosis in patients with anthracofibrosis. Chemotherapy for tuberculosis should be administrated only with confirmation of tuberculosis on bacteriologic study.

Background : The underlying pathogenesis of radiation-induced lung fibrosis (RTLF) has not been very well defined. However, the role of TGF- in the generation of RTLF has been a major focus because there is an increase in the expression of both the TGF--RNA and its protein preceding RTLF lesions. The down stream signal after a TGF- stimulated lung fibrosis includes the activation of many mediators such as Smad and c-Jun N-terminal kinase (JNK) through TAK1. It is we hypothesized that JNK activation may play a pivotal role in RTLF pathogenesis through increased transcription of the fibrogenic cytokines. The present study evaluates JNK activity in alveolar macrophages after irradiation and the relationship between JNK activity and the amount of collagen in the lung tissues. Methods : C57BL/6 mice(20-25 gr, males) received chlorotetracycline(2g/L) in their drinking water 1 week prior to irradiation and continuously there after. The mice were irradiated once with 1400 cGy of -ray over the whole chest. The cellular composition of the whole lung bronchoalveoalr lavage fluids(BALF), elastin expression in the lung tissues, the level of hydroxyproline in lung tissues, and an in vitro JNK assay was measured before irradiation and one, four, and eight weeks after irradiation (RT). Results : The volumes of BALF retrieved from instilled 4 mL of saline with 2% heparin were 3.7-3.8 mL for each group. The cell numbers were similar before() and 1 week() after RT. At four and eight weeks after RT, the cell number reached to and , respectively. There we no changes in the lymphocytes and neutrophils population observed in the BALF after RT. The H-E stain of the lung tissues did not show any structural and fibrotic change in the lung tissues at 4 and 8 weeks after RT. In addition, the amount of elastin and collagen were not different on Verhoeff staining of the lung tissues before RT to eight weeks after RT. The hydroxyproine content was measured with the left lung dissected from the left main bronchus. The lung were homogenized and hydrolyzed with 6 N Hel for 12 hours at then measured as previously described. The content of hydroxyproline, standardized with a lung protein concentration, reached a peak 4 weeks after RT, and thereafter showed a plateau. AnIn vitro JNK assay using c--GST sepharose beads were performed with the alveolar macrophages obtained from the BAL. JNK activity was not detected prior to RT, However, the JNK activity increased from one week after RT and reached a peak four weeks after RT. Conclusion : JNK may be involved in the pathogenesis because the JNK activity showed similar pattern observed with the hydroxyproine content. However, it is necessary to clarify that the JNK increases the transcription of fibrogenic cyiokines through the transcription factor.

To confirm local production of IgE, and evaluate role of immunoglobulins on eosinophil activation in nasal polyp (NP) tissue, we measured IgG, IgA, secretory IgA(sIgA), total (tIgE) and specific IgE (sIgE) to Dermatophagoides pteronyssinus(DP) by ELISA in NP tissue homogenates from 51 subjects. They were classified according to skin reactivity to DP: group I, 15 highly atopic subjects; group II, 18 weakly atopic subjects; and group III, 18 non-atopic subjects. Eosinophil cationic protein (ECP) level was measured by CAP system. Highest level of DP-sIgE was noted in group I, followed by group II and III (p