Nagoya City University

autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.

Follow-up data were obtained for 96 cases of thymoma. The one-year survival rate was 84.3%, the three-year 77.1%, the five-year 74.1%, and the ten-year 57.1%. The five-year survival rate of total resection group was 88.9%; that of non-radically treated group was 44.4%. Clinical stages were defined: Stage I—macroscopically encapsulated and microscopically no capsular invasion; Stage II—1. macroscopic invasion into surrounding fatty tissue or mediastinal pleura, or 2. microscopic invasion into capsule; Stage III—macroscopic invasion into neighboring organ; Stage IVa—pleural or pericardial dissemination; Stage IVb—lymphogenous or hematogenous metastasis. Five-year survival rates of each clinical stage were 92.6% in Stage I, 85.7% in Stage II, 69.6% in Stage III, and 50% in Stage IV. Recurrence after total resection was found in six of 69 cases. Seven of 13 patients treated by subtotal resection survived more than five years with postopertive radiotherapy.

BACKGROUND: IgG4-related disease (IgG4-RD) is a novel clinical disease entity characterized by elevated serum IgG4 concentration and tumefaction or tissue infiltration by IgG4+ plasma cells. Although IgG4-RD is not rare and is clinically important, its clinical diagnostic criteria have not been established. Comprehensive diagnostic criteria for IgG4-RD, including the involvement of various organs, are intended for the practical use of general physicians and nonspecialists. METHODS: Two IgG4-RD study groups, the Umehara and Okazaki teams, were organized by the Ministry of Health, Labor and Welfare Japan. As IgG4-RD comprises a wide variety of diseases, these groups consist of physicians and researchers in various disciplines, including rheumatology, hematology, gastroenterology, nephrology, pulmonology, ophthalmology, odontology, pathology, statistics, and basic and molecular immunology throughout Japan, with 66 and 56 members of the Umehara and Okazaki teams, respectively. Collaborations of the two study groups involved detailed analyses of clinical symptoms, laboratory results, and biopsy specimens of patients with IgG4-RD, resulting in the establishment of comprehensive diagnostic criteria for IgG4-RD. RESULTS: Although many patients with IgG4-RD have lesions in several organs, either synchronously or metachronously, and the pathological features of each organ differ, consensus has been reached on two diagnostic criteria for IgG4RD: (1) serum IgG4 concentration >135 mg/dl, and (2) >40% of IgG+ plasma cells being IgG4+ and >10 cells/high powered field of biopsy sample. Although the comprehensive diagnostic criteria are not sufficiently sensitive for the diagnosis of type 1 IgG4-related autoimmune pancreatitis (IgG4-related AIP), they are adequately sensitive for IgG4-related Mikulicz's disease (MD) and kidney disease (KD). In addition, the comprehensive diagnostic criteria, combined with organ-specific diagnostic criteria, have increased the sensitivity of diagnosis to 100% for IgG4-related MD, KD, and AIP. CONCLUSION: Our comprehensive diagnostic criteria for IgG4-RD are practically useful for general physicians and nonspecialists.

BACKGROUND: Two new screening scales for psychological distress, the K6 and K10, have been developed but their relative efficiency has not been evaluated in comparison with existing scales. METHOD: The Australian National Survey of Mental Health and Well-Being, a nationally representative household survey, administered the WHO Composite International Diagnostic Interview (CIDI) to assess 30-day DSM-IV disorders. The K6 and K10 were also administered along with the General Health Questionnaire (GHQ-12), the current de facto standard of mental health screening. Performance of the three screening scales in detecting CIDI/DSM-IV mood and anxiety disorders was assessed by calculating the areas under receiver operating characteristic curves (AUCs). Stratum-Specific Likelihood Ratios (SSLRs) were computed to help produce individual-level predicted probabilities of being a case from screening scale scores in other samples. RESULTS: The K10 was marginally better than the K6 in screening for CIDI/DSM-IV mood and anxiety disorders (K10 AUC: 0.90, 95%CI: 0.89-0.91 versus K6 AUC: 0.89, 95%CI: 0.88-0.90), while both were significantly better than the GHQ-12 (AUC: 0.80, 95%CI: 0.78-0.82). The SSLRs of the K10 and K6 were more informative in ruling in or out the target disorders than those of the GHQ-12 at both ends of the population spectrum. The K6 was more robust than the K10 to subsample variation. CONCLUSIONS: While the K10 might outperform the K6 in screening for severe disorders, the K6 is preferred in screening for any DSM-IV mood or anxiety disorder because of its brevity and consistency across subsamples. Precision of individual-level prediction is greatly improved by using polychotomous rather than dichotomous classification.

IgG4-related disease (IgG4-RD) is a novel clinical disease entity characterized by elevated serum IgG4 concentration and tumefaction or tissue infiltration by IgG4+ plasma cells. Although IgG4-RD is not rare and is clinically important, its clinical diagnostic criteria have not been established. Comprehensive diagnostic criteria for IgG4-RD, including the involvement of various organs, are intended for the practical use of general physicians and nonspecialists. Background: IgG4-related disease (IgG4-RD) is a novel clinical disease entity characterized by elevated serum IgG4 concentration and tumefaction or tissue infiltration by IgG4+ plasma cells. Although IgG4-RD is not rare and is clinically important, its clinical diagnostic criteria have not been established. Comprehensive diagnostic criteria for IgG4-RD, including the involvement of various organs, are intended for the practical use of general physicians and nonspecialists. Methods: Two IgG4-RD study groups, the Umehara and Okazaki teams, were organized by the Ministry of Health, Labor and Welfare Japan. As IgG4-RD comprises a wide variety of diseases, these groups consist of physicians and researchers in various disciplines, including rheumatology, hematology, gastroenterology, nephrology, pulmonology, ophthalmology, odontology, pathology, statistics, and basic and molecular immunology throughout Japan, with 66 and 56 members of the Umehara and Okazaki teams, respectively. Collaborations of the two study groups involved detailed analyses of clinical symptoms, laboratory results, and biopsy specimens of patients with IgG4-RD, resulting in the establishment of comprehensive diagnostic criteria for IgG4-RD. Results: Although many patients with IgG4-RD have lesions in several organs, either synchronously or metachronously, and the pathological features of each organ differ, consensus has been reached on two diagnostic criteria for IgG4RD: (1) serum IgG4 concentration >135 mg/dl, and (2) >40% of IgG+ plasma cells being IgG4+ and >10 cells/high powered field of biopsy sample. Although the comprehensive diagnostic criteria are not sufficiently sensitive for the diagnosis of type 1 IgG4-related autoimmune pancreatitis (IgG4-related AIP), they are adequately sensitive for IgG4-related Mikulicz’s disease (MD) and kidney disease (KD). In addition, the comprehensive diagnostic criteria, combined with organ-specific diagnostic criteria, have increased the sensitivity of diagnosis to 100% for IgG4-related MD, KD, and AIP. Conclusion: Our comprehensive diagnostic criteria for IgG4-RD are practically useful for general physicians and nonspecialists.

Two new screening scales for psychological distress, the K6 and K10, have been developed using the item response theory and shown to outperform existing screeners in English. We developed their Japanese versions using the standard back-translaton method and included them in the World Mental Health Survey Japan (WMH-J), which is a psychiatric epidemiologic study conducted in seven communities across Japan with 2436 participants. The WMH-J used the WMH Survey Initiative version of the Composite International Diagnostic Interview (CIDI) to assess the 30-day Diagnostic and Statistical Manual of Mental Disorders--Fourth Edition (DSM-IV). Performance of the two screening scales in detecting DSM-IV mood and anxiety disorders, as assessed by the areas under receiver operating characteristic curves (AUCs), was excellent, with values as high as 0.94 (95% confidence interval = 0.88 to 0.99) for K6 and 0.94 (0.88 to 0.995) for K10. Stratum-specific likelihood ratios (SSLRs), which express screening test characteristics and can be used to produce individual-level predicted probabilities of being a case from screening scale scores and pretest probabilities in other samples, were strikingly similar between the Japanese and the original versions. The Japanese versions of the K6 and K10 thus demonstrated screening performances essentially equivalent to those of the original English versions.

International standardization and coordination of the nomenclature of variants of hepatitis C virus (HCV) is increasingly needed as more is discovered about the scale of HCV-related liver disease and important biological and antigenic differences that exist between variants. A group of scientists expert in the field of HCV genetic variability, and those involved in development of HCV sequence databases, the Hepatitis Virus Database (Japan), euHCVdb (France), and Los Alamos (United States), met to re-examine the status of HCV genotype nomenclature, resolve conflicting genotype or subtype names among described variants of HCV, and draw up revised criteria for the assignment of new genotypes as they are discovered in the future. A comprehensive listing of all currently classified variants of HCV incorporates a number of agreed genotype and subtype name re-assignments to create consistency in nomenclature. The paper also contains consensus proposals for the classification of new variants into genotypes and subtypes, which recognizes and incorporates new knowledge of HCV genetic diversity and epidemiology. A proposal was made that HCV variants be classified into 6 genotypes (representing the 6 genetic groups defined by phylogenetic analysis). Subtype name assignment will be either confirmed or provisional, depending on the availability of complete or partial nucleotide sequence data, or remain unassigned where fewer than 3 examples of a new subtype have been described. In conclusion, these proposals provide the framework by which the HCV databases store and provide access to data on HCV, which will internationally coordinate the assignment of new genotypes and subtypes in the future.

Data are reported on the background and performance of the K6 screening scale for serious mental illness (SMI) in the World Health Organization (WHO) World Mental Health (WMH) surveys. The K6 is a six-item scale developed to provide a brief valid screen for Diagnostic and Statistical Manual of Mental Disorders 4th edition (DSM-IV) SMI based on the criteria in the US ADAMHA Reorganization Act. Although methodological studies have documented good K6 validity in a number of countries, optimal scoring rules have never been proposed. Such rules are presented here based on analysis of K6 data in nationally or regionally representative WMH surveys in 14 countries (combined N = 41,770 respondents). Twelve-month prevalence of DSM-IV SMI was assessed with the fully-structured WHO Composite International Diagnostic Interview. Nested logistic regression analysis was used to generate estimates of the predicted probability of SMI for each respondent from K6 scores, taking into consideration the possibility of variable concordance as a function of respondent age, gender, education, and country. Concordance, assessed by calculating the area under the receiver operating characteristic curve, was generally substantial (median 0.83; range 0.76-0.89; inter-quartile range 0.81-0.85). Based on this result, optimal scaling rules are presented for use by investigators working with the K6 scale in the countries studied.

In the 2016/17 winter season in Japan, HuNoV GII.P16-GII.2 strains (2016 strains) emerged and caused large outbreaks of acute gastroenteritis. To better understand the outbreaks, we examined the molecular evolution of the VP1 gene and RdRp region in 2016 strains from patients by studying their time-scale evolutionary phylogeny, positive/negative selection, conformational epitopes, and phylodynamics. The time-scale phylogeny suggested that the common ancestors of the 2016 strains VP1 gene and RdRp region diverged in 2006 and 1999, respectively, and that the 2016 strain was the progeny of a pre-2016 GII.2. The evolutionary rates of the VP1 gene and RdRp region were around 10−3 substitutions/site/year. Amino acid substitutions (position 341) in an epitope in the P2 domain of 2016 strains were not found in pre-2016 GII.2 strains. Bayesian skyline plot analyses showed that the effective population size of the VP1 gene in GII.2 strains was almost constant for those 50 years, although the number of patients with NoV GII.2 increased in 2016. The 2016 strain may be involved in future outbreaks in Japan and elsewhere.

Covalent modification of histone tails is crucial for transcriptional regulation, mitotic chromosomal condensation, and heterochromatin formation. Histone H3 lysine 9 (H3-K9) methylation catalyzed by the Suv39h family proteins is essential for establishing the architecture of pericentric heterochromatin. We recently identified a mammalian histone methyltransferase (HMTase), G9a, which has strong HMTase activity towards H3-K9 in vitro. To investigate the in vivo functions of G9a, we generated G9a-deficient mice and embryonic stem (ES) cells. We found that H3-K9 methylation was drastically decreased in G9a-deficient embryos, which displayed severe growth retardation and early lethality. G9a-deficient ES cells also exhibited reduced H3-K9 methylation compared to wild-type cells, indicating that G9a is a dominant H3-K9 HMTase in vivo. Importantly, the loss of G9a abolished methylated H3-K9 mostly in euchromatic regions. Finally, G9a exerted a transcriptionally suppressive function that depended on its HMTase activity. Our results indicate that euchromatic H3-K9 methylation regulated by G9a is essential for early embryogenesis and is involved in the transcriptional repression of developmental genes.

Antibody‐mediated rejection (AbAR) is increasingly recognized in the renal allograft population, and successful therapeutic regimens have been developed to prevent and treat AbAR, enabling excellent outcomes even in patients highly sensitized to the donor prior to transplant. It has become critical to develop standardized criteria for the pathological diagnosis of AbAR. This article presents international consensus criteria for and classification of AbAR developed based on discussions held at the Sixth Banff Conference on Allograft Pathology in 2001. This classification represents a working formulation, to be revisited as additional data accumulate in this important area of renal transplantation. Antibody‐mediated rejection (AbAR) is increasingly recognized in the renal allograft population, and successful therapeutic regimens have been developed to prevent and treat AbAR, enabling excellent outcomes even in patients highly sensitized to the donor prior to transplant. It has become critical to develop standardized criteria for the pathological diagnosis of AbAR. This article presents international consensus criteria for and classification of AbAR developed based on discussions held at the Sixth Banff Conference on Allograft Pathology in 2001. This classification represents a working formulation, to be revisited as additional data accumulate in this important area of renal transplantation.

A. Khosroshahi, Z. S. Wallace, J. L. Crowe, T. Akamizu, A. Azumi, M. N. Carruthers, S. T. Chari, E. Della-Torre, L. Frulloni, H. Goto, P. A. Hart, T. Kamisawa, S. Kawa, M. Kawano, M. H. Kim, Y. Kodama, K. Kubota, M. M. Lerch, M. L€ ohr, Y. Masaki, S. Matsui, T. Mimori, S. Nakamura, T. Nakazawa, H. Ohara, K. Okazaki, J. H. Ryu, T. Saeki, N. Schleinitz, A. Shimatsu, T. Shimosegawa, H. Takahashi, M. Takahira, A. Tanaka, M. Topazian, H. Umehara, G. J. Webster, T. E. Witzig, M. Yamamoto, W. Zhang, T. Chiba, and J. H. Stone

In this article, we investigate the long‐run relationships among disasters, capital accumulation, total factor productivity, and economic growth. The cross‐country empirical analysis demonstrates that higher frequencies of climatic disasters are correlated with higher rates of human capital accumulation, increases in total factor productivity, and economic growth. Though disaster risk reduces the expected rate of return to physical capital, risk also serves to increase the relative return to human capital. Thus, physical capital investment may fall, but there is also a substitution toward human capital investment. Disasters also provide the impetus to update the capital stock and adopt new technologies, leading to improvements in total factor productivity.

Microglia survey brain parenchyma, responding to injury and infections. Microglia also respond to systemic disease, but the role of blood-brain barrier (BBB) integrity in this process remains unclear. Using simultaneous in vivo imaging, we demonstrated that systemic inflammation induces CCR5-dependent migration of brain resident microglia to the cerebral vasculature. Vessel-associated microglia initially maintain BBB integrity via expression of the tight-junction protein Claudin-5 and make physical contact with endothelial cells. During sustained inflammation, microglia phagocytose astrocytic end-feet and impair BBB function. Our results show microglia play a dual role in maintaining BBB integrity with implications for elucidating how systemic immune-activation impacts neural functions.

PURPOSE: Two similarly designed phase 3 trials (HAWK and HARRIER) compared brolucizumab, a single-chain antibody fragment that inhibits vascular endothelial growth factor-A, with aflibercept to treat neovascular age-related macular degeneration (nAMD). DESIGN: Double-masked, multicenter, active-controlled, randomized trials. PARTICIPANTS: Patients (N = 1817) with untreated, active choroidal neovascularization due to age-related macular degeneration in the study eye. INTERVENTION: Patients were randomized to intravitreal brolucizumab 3 mg (HAWK only) or 6 mg or aflibercept 2 mg. After loading with 3 monthly injections, brolucizumab-treated eyes received an injection every 12 weeks (q12w) and were interval adjusted to every 8 weeks (q8w) if disease activity was present; aflibercept-treated eyes received q8w dosing. MAIN OUTCOME MEASURES: The primary hypothesis was noninferiority in mean best-corrected visual acuity (BCVA) change from baseline to Week 48 (margin: 4 letters). Other key end points included the percentage of patients who maintained q12w dosing through Week 48 and anatomic outcomes. RESULTS: At Week 48, each brolucizumab arm demonstrated noninferiority to aflibercept in BCVA change from baseline (least squares [LS] mean, +6.6 [6 mg] and +6.1 [3 mg] letters with brolucizumab vs. +6.8 letters with aflibercept [HAWK]; +6.9 [brolucizumab 6 mg] vs. +7.6 [aflibercept] letters [HARRIER]; P < 0.001 for each comparison). Greater than 50% of brolucizumab 6 mg-treated eyes were maintained on q12w dosing through Week 48 (56% [HAWK] and 51% [HARRIER]). At Week 16, after identical treatment exposure, fewer brolucizumab 6 mg-treated eyes had disease activity versus aflibercept in HAWK (24.0% vs. 34.5%; P = 0.001) and HARRIER (22.7% vs. 32.2%; P = 0.002). Greater central subfield thickness reductions from baseline to Week 48 were observed with brolucizumab 6 mg versus aflibercept in HAWK (LS mean -172.8 μm vs. -143.7 μm; P = 0.001) and HARRIER (LS mean -193.8 μm vs. -143.9 μm; P < 0.001). Anatomic retinal fluid outcomes favored brolucizumab over aflibercept. Overall, adverse event rates were generally similar with brolucizumab and aflibercept. CONCLUSIONS: Brolucizumab was noninferior to aflibercept in visual function at Week 48, and >50% of brolucizumab 6 mg-treated eyes were maintained on q12w dosing interval through Week 48. Anatomic outcomes favored brolucizumab over aflibercept. Overall safety with brolucizumab was similar to aflibercept (ClinicalTrials.gov; NCT02307682, NCT02434328).

BACKGROUND: Most studies that have evaluated the association between the body-mass index (BMI) and the risks of death from any cause and from specific causes have been conducted in populations of European origin. METHODS: We performed pooled analyses to evaluate the association between BMI and the risk of death among more than 1.1 million persons recruited in 19 cohorts in Asia. The analyses included approximately 120,700 deaths that occurred during a mean follow-up period of 9.2 years. Cox regression models were used to adjust for confounding factors. RESULTS: In the cohorts of East Asians, including Chinese, Japanese, and Koreans, the lowest risk of death was seen among persons with a BMI (the weight in kilograms divided by the square of the height in meters) in the range of 22.6 to 27.5. The risk was elevated among persons with BMI levels either higher or lower than that range--by a factor of up to 1.5 among those with a BMI of more than 35.0 and by a factor of 2.8 among those with a BMI of 15.0 or less. A similar U-shaped association was seen between BMI and the risks of death from cancer, from cardiovascular diseases, and from other causes. In the cohorts comprising Indians and Bangladeshis, the risks of death from any cause and from causes other than cancer or cardiovascular disease were increased among persons with a BMI of 20.0 or less, as compared with those with a BMI of 22.6 to 25.0, whereas there was no excess risk of either death from any cause or cause-specific death associated with a high BMI. CONCLUSIONS: Underweight was associated with a substantially increased risk of death in all Asian populations. The excess risk of death associated with a high BMI, however, was seen among East Asians but not among Indians and Bangladeshis.

Diabetic retinopathy is a leading cause of adult vision loss and blindness. Much of the retinal damage that characterizes the disease results from retinal vascular leakage and nonperfusion. This study shows that diabetic retinal vascular leakage and nonperfusion are temporally and spatially associated with retinal leukocyte stasis (leukostasis) in the rat model of streptozotocin-induced diabetes. Retinal leukostasis increases within days of developing diabetes and correlates with the increased expression of retinal intercellular adhesion molecule-1 (ICAM-1). ICAM-1 blockade with a mAb prevents diabetic retinal leukostasis and vascular leakage by 48.5% and 85.6%, respectively. These data identify the causal role of leukocytes in the pathogenesis of diabetic retinopathy and establish the potential utility of ICAM-1 inhibition as a therapeutic strategy for the prevention of diabetic retinopathy.

Cancer tissues are composed of cancer cells and the surrounding stromal cells (e.g., fibroblasts, vascular endothelial cells, and immune cells), in addition to the extracellular matrix. Most studies investigating carcinogenesis and the progression, invasion, metastasis, and angiogenesis of cancer have focused on alterations in cancer cells, including genetic and epigenetic changes. Recently, interactions between cancer cells and the stroma have attracted considerable attention, and increasing evidence has accumulated on this. Several researchers have gradually clarified the origins, features, and roles of cancer-associated fibroblasts (CAFs), a major component of the cancer stroma. CAFs function in a similar manner to myofibroblasts during wound healing. We previously reported the relationship between CAFs and angiogenesis. Interleukin-6 (IL-6), a multifunctional cytokine, plays a central role in regulating inflammatory and immune responses, and important roles in the progression, including proliferation, migration, and angiogenesis, of several cancers. We showed that CAFs are an important IL-6 source and that anti-IL-6 receptor antibody suppressed angiogenesis and inhibited tumor-stroma interactions. Furthermore, CAFs contribute to drug-resistance acquisition in cancer cells. The interaction between cancer cells and the stroma could be a potential target for anti-cancer therapy.

We found that caveolin-2 is targeted to the surface of lipid droplets (Fujimoto, T., Kogo, H., Ishiguro, K., Tauchi, K., and Nomura, R. (2001) J. Cell Biol. 152, 1079–1085) and hypothesized that the lipid droplet surface is a kind of membrane. To elucidate the characteristics of the lipid droplet surface, we isolated lipid droplets from HepG2 cells and analyzed them by cryoelectron microscopy and by mass spectrometry. By use of cryoelectron microscopy at the stage temperature of 4.2 K, the lipid droplet surface was observed as a single line without any fixation or staining, indicating the presence of a single layer of phospholipids. This result appeared consistent with the hypothesis that the lipid droplet surface is derived from the cytoplasmic leaflet of the endoplasmic reticulum membrane and may be continuous to it. However, mass spectrometry revealed that the fatty acid composition of phosphatidylcholine and lysophosphatidylcholine in lipid droplets is different from that of the rough endoplasmic reticulum. The ample presence of free cholesterol in lipid droplets also suggests that their surface is differentiated from the bulk endoplasmic reticulum membrane. On the other hand, although caveolin-2β and adipose differentiation-related protein, both localizing in lipid droplets, were enriched in the low density floating fraction, the fatty acid composition of the fraction was distinct from lipid droplets. Collectively, the result indicates that the lipid droplet surface is a hemi-membrane or a phospholipid monolayer containing cholesterol but is compositionally different from the endoplasmic reticulum membrane or the sphingolipid/cholesterol-rich microdomain. We found that caveolin-2 is targeted to the surface of lipid droplets (Fujimoto, T., Kogo, H., Ishiguro, K., Tauchi, K., and Nomura, R. (2001) J. Cell Biol. 152, 1079–1085) and hypothesized that the lipid droplet surface is a kind of membrane. To elucidate the characteristics of the lipid droplet surface, we isolated lipid droplets from HepG2 cells and analyzed them by cryoelectron microscopy and by mass spectrometry. By use of cryoelectron microscopy at the stage temperature of 4.2 K, the lipid droplet surface was observed as a single line without any fixation or staining, indicating the presence of a single layer of phospholipids. This result appeared consistent with the hypothesis that the lipid droplet surface is derived from the cytoplasmic leaflet of the endoplasmic reticulum membrane and may be continuous to it. However, mass spectrometry revealed that the fatty acid composition of phosphatidylcholine and lysophosphatidylcholine in lipid droplets is different from that of the rough endoplasmic reticulum. The ample presence of free cholesterol in lipid droplets also suggests that their surface is differentiated from the bulk endoplasmic reticulum membrane. On the other hand, although caveolin-2β and adipose differentiation-related protein, both localizing in lipid droplets, were enriched in the low density floating fraction, the fatty acid composition of the fraction was distinct from lipid droplets. Collectively, the result indicates that the lipid droplet surface is a hemi-membrane or a phospholipid monolayer containing cholesterol but is compositionally different from the endoplasmic reticulum membrane or the sphingolipid/cholesterol-rich microdomain. Lipid droplets have been regarded as a depot of neutral lipids. They exist most abundantly in adipose cells and steroid-producing cells but can be found in virtually any kind of cell. The core of lipid droplets is occupied by triacylglycerol and cholesterol ester in various ratios depending on the cell type (1Zweytick D. Athenstaedt K. Daum G. Biochim. Biophys. Acta. 2000; 1469: 101-120Crossref PubMed Scopus (278) Google Scholar), but information on the lipid droplet surface has been scarce. Recently we as well as others showed that caveolins can exist in the lipid droplet surface (2Fujimoto T. Kogo H. Ishiguro K. Tauchi K. Nomura R. J. Cell Biol. 2001; 152: 1079-1085Crossref PubMed Scopus (213) Google Scholar, 3Ostermeyer A.G. Paci J.M. Zeng Y. Lublin D.M. Munro S. Brown D.A. J. Cell Biol. 2001; 152: 1071-1078Crossref PubMed Scopus (213) Google Scholar, 4Pol A. Luetterforst R. Lindsay M. Heino S. Ikonen E. Parton R.G. J. Cell Biol. 2001; 152: 1057-1070Crossref PubMed Scopus (278) Google Scholar). Caveolins, i.e. caveolin-1, 2, 3, are membrane proteins that are incorporated to the sphingolipid/cholesterol-enriched membrane microdomain and form the framework of caveolae (5Parton R.G. Curr. Opin. Cell Biol. 1996; 8: 542-548Crossref PubMed Scopus (495) Google Scholar). Furthermore, lipid droplets were reported to contain other microdomain proteins,i.e. Lyn and mitogen-activated protein kinase, as well as abundant free cholesterol (6Yu W. Bozza P.T. Tzizik D.M. Gray J.P. Cassara J. Dvorak A.M. Weller P.F. Am. J. Pathol. 1998; 152: 759-769PubMed Google Scholar, 7Yu W. Cassara J. Weller P.F. Blood. 2000; 95: 1078-1085Crossref PubMed Google Scholar, 8Prattes S. Horl G. Hammer A. Blaschitz A. Graier W.F. Sattler W. Zechner R. Steyrer E. J. Cell Sci. 2000; 113: 2977-2989Crossref PubMed Google Scholar). These results suggest that the lipid droplet surface is a kind of membrane and that it might have some similarity to the microdomain. However, electron microscopy of conventional resin-embedded ultrathin sections cannot visualize any membranous structure around the lipid droplet. In the ultrathin section of specimens fixed by aldehydes and then by osmium tetroxide, the lipid droplet content appears vacant, and its periphery is usually seen as a thin intermittent line. In many diagrams, the lipid droplet surface has been depicted as a phospholipid monolayer with the hydrophilic headgroup facing the cytoplasm and the hydrophobic acyl chains extending into the lipid droplet content (9Murphy D.J. Vance J. Trends Biochem. Sci. 1999; 24: 109-115Abstract Full Text Full Text PDF PubMed Scopus (491) Google Scholar,10Brown D.A. Curr. Biol. 2001; 11: R446-R449Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar). It has also been assumed that lipid droplets form by accumulation of neutral lipids between the two leaflets of the endoplasmic reticulum (ER) 1The abbreviations used are: ER, endoplasmic reticulum; ADRP, adipose differentiation-related protein; Cap-LC/ESI, capillary liquid chromatography/electrospray ionization; PC, phosphatidylcholine; TIFF, Triton X-100-insoluble floating fraction membrane. However, evidence to support the above assumptions is scarce. Only freeze-fracture electron microscopy showed that the fracture plane along the lipid droplet surface is occasionally continuous with the cytoplasmic leaflet of the ER membrane (11Blanchette Mackie E.J. Dwyer N.K. Barber T. Coxey R.A. Takeda T. Rondinone C.M. Theodorakis J.L. Greenberg A.S. Londos C. J. Lipid Res. 1995; 36: 1211-1226PubMed Google Scholar, 12Peixoto de Menezes A. Pinto da Silva P. Lab. Invest. 1979; 40: 545-553PubMed Google Scholar). We wanted to examine whether the lipid droplet surface is really a phospholipid monolayer. Because it is difficult to retain lipid-rich structures by conventional morphological methods, we took advantage of cryoelectron microscopy that can observe biological specimens at the atomic resolution without fixation or staining (13Fujiyoshi Y. Mizusaki T. Morikawa K. Yamagishi H. Aoki Y. Kihara H. Harada Y. Ultramicroscopy. 1991; 38: 241-251Crossref Scopus (175) Google Scholar). Furthermore, to gain information about the lipid droplet phospholipids, we analyzed the fatty acid composition of phospholipids by mass spectrometry. The microscopy showed that the lipid droplet surface is indeed a hemi-membrane, or a phospholipid monolayer, and mass spectrometry revealed that the fatty acid composition of the lipid droplet phospholipids is distinct from that of rough ER and cholesterol/sphingolipid-rich microdomain. The result not only questions the current hypothesis on the mechanism of lipid droplet formation but also provides a firm basis for further studies on the physiological function of lipid droplets. Mouse anti-caveolin-2 antibody (BD Transduction Laboratories), mouse anti-adipose differentiation-related protein (ADRP) antibody (Progen), and colloidal gold-conjugated secondary antibody (Amersham Biosciences) were obtained from respective suppliers. HepG2 cells were grown in Dulbecco's modified Eagle's medium added with 10% fetal calf serum. A HepG2 cell line stably expressing human caveolin-2β (clone A-8) (2Fujimoto T. Kogo H. Ishiguro K. Tauchi K. Nomura R. J. Cell Biol. 2001; 152: 1079-1085Crossref PubMed Scopus (213) Google Scholar), kept in the presence of 200 μg/ml G418, was also used for some experiments. Cells were disrupted by nitrogen cavitation and subjected to sucrose density gradient ultracentrifugation as described (2Fujimoto T. Kogo H. Ishiguro K. Tauchi K. Nomura R. J. Cell Biol. 2001; 152: 1079-1085Crossref PubMed Scopus (213) Google Scholar). The lipid droplet layer floating on the surface was used in subsequent studies. Absence of contamination by other organelles was confirmed by Western blotting of marker molecules. Triton X-100-insoluble floating fraction (TIFF) was obtained by treating cells with 1% Triton X-100; a light scattering band at the interface of 5%/35% sucrose solutions after ultracentrifugation was collected by aspiration (14Brown D.A. Rose J.K. Cell. 1992; 68: 533-544Abstract Full Text PDF PubMed Scopus (2658) Google Scholar). In some experiments, 1% Triton X-100 was substituted with either 0.025% Triton X-100 (15Field K.A. Holowka D. Baird B. J. Biol. Chem. 1997; 272: 4276-4280Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar) or 500 mm sodium carbonate (pH 11) (16Song K.S., Li Shengwen Okamoto T. Quilliam L.A. Sargiacomo M. Lisanti M.P. J. Biol. Chem. 1996; 271: 9690-9697Abstract Full Text Full Text PDF PubMed Scopus (924) Google Scholar), and fractions were obtained from the top. In this report, only the fraction obtained by the 1% Triton X-100 procedure is called TIFF, and the others are referred to by separate names. Rough microsome, representing rough ER, was obtained from cycloheximide-treated cells as a fraction at the interface of 1.5 m/1.8 msucrose (17Morimoto T. Sabatini D.D. Spector. D.L. Goldman. R.D. Leinwand. L.A. Cells: A Laboratory Manual. 1. Cold Spring Harbor Press, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1998Google Scholar). Crude microsome was prepared by centrifuging a postmitochondrial supernatant for 60 min at 140,000 ×g. Lipid droplet suspension placed on microgrid was rapidly plunged into liquid ethane cooled by liquid nitrogen (18Adrian M. Dubochet J. Lepault J. McDowall A.W. Nature. 1984; 308: 32-36Crossref PubMed Scopus (1008) Google Scholar). The specimen was transferred into a JEOL 3000SSF cryoelectron microscope using a cryotransfer device and observed at a stage temperature of 4.2 K. Micrographs were taken using the minimum dose system to alleviate the radiation damage (19Fujiyoshi Y. Kobayashi T. Ishizuka T. Uyeda N. Ishida Y. Harada Y. Ultramicroscopy. 1980; 5: 459-468Crossref Scopus (94) Google Scholar). A defocus value was set at 1–2 μm to increase the image contrast. The electron radiation to the specimen was less than 5,000 electrons/nm2, which does not seriously damage the vitrified biological specimen at the temperature. Lipid droplet suspension prepared from clone A-8 was placed between thin copper plates and rapidly frozen by the metal sandwich method (20Fujimoto T. Fujimoto K. J. Histochem. Cytochem. 1997; 45: 595-598Crossref PubMed Scopus (29) Google Scholar) and then freeze-fractured in a Balzers BAF400D. Platinum/carbon replicas were treated with 1% SDS and labeled for caveolin-2 by the procedure described previously (21Fujimoto K. J. Cell Sci. 1995; 108: PubMed Google Scholar). The lipids were from lipid droplets and microsome Y. J. Biol. Chem. Full Text PDF Google Scholar). the from lipid droplets was subjected to to separate and lipids S. J. Lipid Res. 24: Full Text PDF PubMed Google Scholar). They were on plates by and then by acid and by cholesterol and phospholipids in lipid were and the was obtained G. S. PubMed Scopus Google Scholar, W. M. J. J. Lipid Res. Full Text PDF PubMed Google Scholar). The lipids from isolated lipid droplets were subjected to to the content of neutral which the of phospholipids by mass spectrometry. The mass spectrometry was as described previously R. J. Y. Ishida M. J. 2000; PubMed Scopus Google Scholar). after of phospholipids by Nomura the was analyzed by a mass with of of phospholipid was by the mass electron microscopy of resin-embedded sections not membrane structure in the of lipid droplets in HepG2 cells not To visualize the lipid droplet surface we cryoelectron microscopy that can visualize at or a phospholipid was observed as two by this method without any fixation or staining Y. Y. PubMed Scopus Google Scholar). By isolated lipid droplets were in thin in microgrid On the stage cooled to 4.2 K, the specimen was at a low with a minimum electron in a from the were taken by using the minimum dose system (19Fujiyoshi Y. Kobayashi T. Ishizuka T. Uyeda N. Ishida Y. Harada Y. Ultramicroscopy. 1980; 5: 459-468Crossref Scopus (94) Google Scholar). The of isolated lipid droplets observed by cryoelectron microscopy was It was than which was observed as the of lipid droplets in resin-embedded ultrathin sections of HepG2 cells by conventional electron The indicates that lipid droplets were disrupted into Lipid droplets were observed as structures the content was of low electron but the was observed as a single line of about in The result that a single of or a phospholipid monolayer, the lipid droplet A of content showed a electron density than lipid droplets, were with two of the are to be a membrane a membrane. lipid droplets were seen to have The line was the the and A structure that to to the was seen by freeze-fracture of isolated lipid lipid droplets with some showed fracture or Furthermore, anti-caveolin-2 antibody labeled not only the fracture but also the fracture we that the lipid droplets with the a we assumed that may be phospholipid and the of lipid D.J. R.G. J. Cell Biol. PubMed Scopus Google Scholar, G. J. Cell Biol. 2001; 152: PubMed Scopus Google Scholar). However, in to membrane observed by conventional electron the are with without any and in cells were rapidly frozen and the was found only not we that the be by temperature the However, further studies be to the of the in isolated lipid droplets. In HepG2 expressing caveolin-2β without caveolin-1, caveolin-2β was found around lipid droplets by The as or not as reported previously for other cell S. D. Sargiacomo M. K. Lisanti M.P. 1998; PubMed Scopus Google Scholar). the cell was in the presence of 1% Triton X-100 and subjected to sucrose density gradient caveolin-2β in fractions and was not from the fractions not However, by 1% Triton X-100 with 500 mm sodium carbonate or 0.025% Triton caveolin-2β was from the low density fractions By lipid droplets were to contain phosphatidylcholine and free cholesterol as well as abundant cholesterol ester and On the other hand, other phospholipids, and not be by the method we used not showed that the of phospholipids and free cholesterol is for lipid droplet and for postmitochondrial The microsome contain the membrane and the ER as well as other that most free cholesterol in the membrane Y. J. Biol. Chem. Full Text PDF PubMed Google Scholar), the result indicates that the content of free cholesterol in the lipid droplet surface is than that in the membrane but than that in the In to the result in S. Horl G. Hammer A. Blaschitz A. Graier W.F. Sattler W. Zechner R. Steyrer E. J. Cell Sci. 2000; 113: 2977-2989Crossref PubMed Google Scholar), not lipid droplets in HepG2 cells not The result suggests that the free cholesterol content of lipid droplets may be different between and Lipid droplets have been hypothesized to by accumulation of neutral lipids between the two leaflets of the ER membrane (9Murphy D.J. Vance J. Trends Biochem. Sci. 1999; 24: 109-115Abstract Full Text Full Text PDF PubMed Scopus (491) Google may either from the ER or to it. The surface of lipid droplet is assumed to be derived from the cytoplasmic leaflet of the ER membrane. the lipid droplet surface is continuous with the ER composition of phospholipids in the lipid droplet surface may be to that of the accumulation of caveolin-2β and its in low density might the lipid droplet surface is to the microdomain. To examine we the fatty acid composition of and in lipid droplets, rough microsome rough and the by mass spectrometry. The fatty acid of obtained by a low was between lipid droplet and rough of and were observed as In in was enriched with fatty with a of and were The obtained by this the mass of and not fatty On the other hand, by using a acyl chains are from by and can be as a By this lipid droplets and rough microsome showed distinct for the for lipid droplets, but the of and were seen for rough The result indicates that the of was of in lipid droplets, the in rough microsome a of By the the of that the of in lipid droplets may contain than in rough microsome, the may be enriched with Furthermore, analyzed by the also distinct for lipid droplets and rough microsome of and were in lipid droplets, were low or in rough microsome and TIFF, and were in the that the lipid droplet surface is distinct from the rough ER is it to the sphingolipid/cholesterol-rich microdomain. The surface of lipid droplets has been to be a phospholipid monolayer hydrophobic lipid may exist stably in the cytoplasm only with with hydrophilic facing most the only result that the has been a freeze-fracture the of a ER membrane leaflet and the lipid droplet surface (11Blanchette Mackie E.J. Dwyer N.K. Barber T. Coxey R.A. Takeda T. Rondinone C.M. Theodorakis J.L. Greenberg A.S. Londos C. J. Lipid Res. 1995; 36: 1211-1226PubMed Google Scholar, 12Peixoto de Menezes A. Pinto da Silva P. Lab. Invest. 1979; 40: 545-553PubMed Google Scholar). In the we a cryoelectron which is to visualize biological specimens with damage and has been used to structures at atomic resolution C. Y. K. K. M. A. Y. Nature. 2001; PubMed Scopus Google Scholar). The cryoelectron microscopy has previously that the of the A is a phospholipid monolayer with a protein layer Y. K. J. PubMed Scopus Google Scholar). By this a single line representing a of was observed in the surface of isolated lipid droplets. It with two seen in the membrane Y. Y. PubMed Scopus Google Scholar), which is a phospholipid and for the that the lipid droplet surface is a phospholipid monolayer. The presence of a phospholipid monolayer in the surface is consistent with the current that lipid droplets are by lipid ester between the two leaflets of the ER membrane and may to it (9Murphy D.J. Vance J. Trends Biochem. Sci. 1999; 24: 109-115Abstract Full Text Full Text PDF PubMed Scopus (491) Google Scholar, D.A. Curr. Biol. 2001; 11: R446-R449Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar). of a that cholesterol in the ER J. D. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google and not to that lipid droplets may along the membrane. However, mass spectrometry showed that fatty acid of and in lipid droplets are distinct from in rough microsome, which is to be to the rough The result does not the that the lipid droplet surface is from the ER membrane but indicates that the is a differentiated lipid droplets may exist as a structure of the the lipid droplet may be to the ER, but some mechanism may the lipid droplet surface from the bulk ER membrane as for other ER Vance J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). is the we that in of the ER not but are to to lipid droplets. or other lipid proteins may be in the A of lipid droplets revealed by mass spectrometry is the of fatty in and the was is not but reported to exist in lipid droplets of (6Yu W. Bozza P.T. Tzizik D.M. Gray J.P. Cassara J. Dvorak A.M. Weller P.F. Am. J. Pathol. 1998; 152: 759-769PubMed Google Scholar), may be in lipid droplets is derived from a of with acyl in 1. The of with two acyl chains and in lipid droplets is in line with the The of the is also They may be in the ER and to lipid with neutral lipids might be in the accumulation of acyl However, it is also that the were in lipid in this the continuous of lipid and the and of fatty are to be to the surface contamination in the lipid droplet fraction is by the of marker of other organelles (2Fujimoto T. Kogo H. Ishiguro K. Tauchi K. Nomura R. J. Cell Biol. 2001; 152: 1079-1085Crossref PubMed Scopus (213) Google Scholar), but a of contamination by phospholipids may to be However, using the cell as a with two acyl chains are found abundantly only in the lipid droplet and less in the microsome or Furthermore, using the mass obtained from cell and membrane not the characteristics found with the lipid droplet. These results it that the found in the lipid droplet in a and to any membrane In of caveolins to lipid droplets was found to be or cells were treated with A (2Fujimoto T. Kogo H. Ishiguro K. Tauchi K. Nomura R. J. Cell Biol. 2001; 152: 1079-1085Crossref PubMed Scopus (213) Google Scholar, 3Ostermeyer A.G. Paci J.M. Zeng Y. Lublin D.M. Munro S. Brown D.A. J. Cell Biol. 2001; 152: 1071-1078Crossref PubMed Scopus (213) Google Scholar). The result was to that caveolins in the ER membrane and then to the lipid droplet by the membrane However, two questions were this was caveolins be in lipid droplets and the other was caveolins to lipid droplets were not after A was The may be by that the lipid droplet surface is not continuous to the ER membrane. the can be only by a mechanism of caveolins appears to in lipid droplets. a showed that cholesterol are incorporated to droplets containing G. B. M.P. S. J. Lipid Res. 2001; Full Text Full Text PDF PubMed Google Scholar). It is that lipid along with phospholipids, may be from the ER to lipid droplets by and caveolins might the some mass spectrometry also showed that the lipid droplet surface is different from the sphingolipid/cholesterol-rich microdomain in the fatty acid a of fatty than rough microsome, lipid droplet The result indicates that not only between and cholesterol but also between phospholipid and cholesterol are in the as from membrane studies R. E. Brown D. Sci. S. A. PubMed Scopus Google Scholar). on the of we previously that the lipid droplet surface might have a to the microdomain (2Fujimoto T. Kogo H. Ishiguro K. Tauchi K. Nomura R. J. Cell Biol. 2001; 152: 1079-1085Crossref PubMed Scopus (213) Google Scholar). However, the result does not support the in of the fatty acid Furthermore, showed that the of and free cholesterol in with is less in lipid droplets than in not lipid composition does not to be the of to lipid droplets. as HepG2 cells are caveolin-2β was found around lipid droplets, but was found in the and only in lipid was not to lipid droplets treated with A (2Fujimoto T. Kogo H. Ishiguro K. Tauchi K. Nomura R. J. Cell Biol. 2001; 152: 1079-1085Crossref PubMed Scopus (213) Google Scholar). The of caveolins may a to elucidate the mechanism of to lipid droplets. obtained by the 1% Triton X-100 method has been as of the sphingolipid/cholesterol-rich microdomain D.A. E. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). We found that caveolin-2β in the lipid droplet was with 1% Triton X-100 in the but to low density fractions 500 mm sodium carbonate or 0.025% Triton X-100 was used in cell The low density fractions obtained by the sodium carbonate were reported to be enriched with the as (16Song K.S., Li Shengwen Okamoto T. Quilliam L.A. Sargiacomo M. Lisanti M.P. J. Biol. Chem. 1996; 271: 9690-9697Abstract Full Text Full Text PDF PubMed Scopus (924) Google Scholar). However, whether the two really the membrane has not been On the other hand, the fractions collected by the 0.025% Triton X-100 method were to contain with less to the sphingolipid/cholesterol-rich microdomain (15Field K.A. Holowka D. Baird B. J. Biol. Chem. 1997; 272: 4276-4280Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar). The result showed that although the lipid composition is in the lipid droplet surface are enriched in the fraction as the sphingolipid/cholesterol-rich microdomain. It further studies to whether the result indicates some similarity in the of the two or indicates that the low density fractions obtained by the sodium carbonate and 0.025% Triton X-100 contain In the showed that the lipid droplet surface is a phospholipid monolayer with a fatty acid Lipid droplets been regarded as a of neutral lipids and as However, in of indicating accumulation of proteins to various D.D. T. S. D. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google H. PubMed Scopus Google Scholar), it is to lipid droplets are and The revealed a firm basis for the studies. We are most to Y. for the use of cryoelectron and the We also M. and T. for

OBJECTIVE: This trial compared the efficacy and safety of transarterial chemoembolisation (TACE) plus sorafenib with TACE alone using a newly established TACE-specific endpoint and pre-treatment of sorafenib before initial TACE. DESIGN: Patients with unresectable hepatocellular carcinoma (HCC) were randomised to TACE plus sorafenib (n=80) or TACE alone (n=76). Patients in the combination group received sorafenib 400 mg once daily for 2-3 weeks before TACE, followed by 800 mg once daily during on-demand conventional TACE sessions until time to untreatable (unTACEable) progression (TTUP), defined as untreatable tumour progression, transient deterioration to Child-Pugh C or appearance of vascular invasion/extrahepatic spread. Co-primary endpoints were progression-free survival (PFS), which is not a conventional one but defined as TTUP, or time to any cause of death plus overall survival (OS). Multiplicity was adjusted by gatekeeping hierarchical testing. RESULTS: Median PFS was significantly longer in the TACE plus sorafenib than in the TACE alone group (25.2 vs 13.5 months; p=0.006). OS was not analysed because only 73.6% of OS events were reached. Median TTUP (26.7 vs 20.6 months; p=0.02) was also significantly longer in the TACE plus sorafenib group. OS at 1 year and 2 years in TACE plus sorafenib group and TACE alone group were 96.2% and 82.7% and 77.2% and 64.6%, respectively. There were no unexpected toxicities. CONCLUSION: TACE plus sorafenib significantly improved PFS over TACE alone in patients with unresectable HCC. Adverse events were consistent with those of previous TACE combination trials. TRIAL REGISTRATION NUMBER: NCT01217034.