Reduction in all-cause medical and caregiving costs through innovative health awareness projects in a rural area in Japan: a retrospective cohort study.

BACKGROUND: This study evaluates cost reduction in participants of a health awareness program (the Center of Healthy Aging Program, CHAP) in a Japanese rural area, characterized by an annual check-up and personalized interview on health issues and related risks immediately after the check-up. METHODS: This is a cross-sectional study using medical and caregiving costs and Japan-specific health check-up results in Hirosaki residents stored by the local government, which were individually-based linked to the CHAP information collected by Hirosaki University. This is the first study that used anonymized data with individually-based linkages to both a research institute and a local government in Japan under a strict limitation regarding linking to third-party data. We included residents who had been continuously enrolled for > 6 months as of 1 July 2015. We compared 5-year all-cause costs between three groups (with CHAP, with Japan-specific health check-up, and no check-up) using a multivariate negative binomial regression model considering risk factors including lifestyle habits and an inverse probability weight to adjust for baseline characteristics: age, sex, Charlson comorbidity index, baseline care level, and risk score of coronary heart diseases. RESULTS: A total of 384, 9805, and 32,630 residents aged 40-74 years were included for the CHAP, Japan-specific health check-up, and no check-up groups, respectively. The Japan-specific health check-up group showed older and higher Charlson comorbidity index than the others. After inverse probability weight adjusting, the amount of all-cause medical costs was significantly lower only in the CHAP group. Faster walking speed and exercise habits were independently associated with lower all-cause medical and caregiving costs. CONCLUSIONS: We demonstrated a 5-year all-cause cost reduction in residents who participated in the CHAP and also suggested the effect of exercise habits in Hirosaki, which indicated the significance of individually-based data linkages to external third-party data for all local governments to improve the health condition of residents.

Risk of hospitalized infections in older elderly patients with rheumatoid arthritis treated with tocilizumab or other biological/targeted synthetic disease-modifying antirheumatic drugs: Evaluation of data from a Japanese claims database.

OBJECTIVE: We compared the incidence rates of hospitalized infections (HIs) between tocilizumab (TCZ) and other biological/targeted synthetic disease-modifying antirheumatic drugs (b/tsDMARDs) in adults aged ≥75 years with rheumatoid arthritis (RA). METHODS: We used a Japanese claims database from Medical Data Vision Co., Ltd (Tokyo, Japan) to perform a retrospective longitudinal population-based study in patients with RA who were prescribed b/tsDMARDs between 2014 and 2019. We calculated adjusted risk ratios (aRRs) for HIs in three age groups (<65, ≥65 and <75, and ≥75 years). RESULTS: Of 5506 patients, 2265 (41.1%) were <65 years, 1709 (31.0%) were 65-74 years, and 1532 (27.8%) were ≥75 years. Crude incidence rates (/100 person-years) of HIs were 3.99, 7.27, and 10.77, respectively. In the oldest group, aRRs (95% confidence interval) for HIs (b/tsDMARDs versus TCZ) were as follows: etanercept, 2.40 (1.24-4.61); adalimumab, 1.90 (0.75-4.83); golimumab, 1.21 (0.66-2.23); and abatacept, 0.89 (0.49-1.62). In the other age groups, the noticeable difference was a lower aRR of etanercept versus TCZ in the youngest group (0.30, 0.11-0.85). CONCLUSION: In patients with RA aged ≥75 years, b/tsDMARDs have a similar risk of HIs to tocilizumab except for etanercept.

Patient-Physician Communication and Perception of Treatment Goals in Rheumatoid Arthritis: An Online Survey of Patients and Physicians.

INTRODUCTION: To evaluate patient-physician communication and patients' understanding of treatment goals in rheumatoid arthritis (RA). METHODS: A cross-sectional online survey of patients with RA and physicians treating RA was conducted between 16 and 30 June 2021. Participants were asked to rate the importance of 17 goals on a 6-point Likert scale, and mean scores were compared between patients and physicians by the Wilcoxon rank sum test. Patients' satisfaction with physician communication and their understanding of treatment goals were also assessed. RESULTS: The responses of 502 patients and 216 physicians were analyzed. The most common patient age group was 50-59 years (28.5%), and the mean disease duration was 10.3 years. Physicians had a mean of 19.2 years of treatment experience and were treating a mean of 44.3 patients. Among the 17 goals assessed, patients placed significantly more importance on drug tapering or discontinuation as short-term goals (3-6 months) and on performing basic activities of daily living, being able to engage in daily tasks, achieving and maintaining remission, maintaining better laboratory values, and drug tapering or discontinuation as long-term goals (5-10 years; all adjusted p < 0.05). Patient treatment satisfaction was significantly associated with disease activity, a feeling of treatment effectiveness, satisfaction with physician communication, and agreement with physician goals. CONCLUSION: Differences exist among patients with RA and physicians treating RA regarding the importance of short- and long-term treatment goals. Good patient-physician communication appears to be important for improving patient satisfaction. TRIAL REGISTRATION: University Hospital Medical Information Network identifier: UMIN000044463.

Cost-effectiveness analysis of cardiac implantable electronic devices with reactive atrial-based antitachycardia pacing.

AIMS: Reactive atrial-based anti-tachycardia pacing (rATP) in pacemakers (PMs) and cardiac resynchronization therapy defibrillators (CRT-Ds) has been reported to prevent progression of atrial fibrillation, and this reduced progression is expected to decrease the risk of complications such as stroke and heart failure (HF). This study aimed to assess the cost-effectiveness of rATP in PMs and CRT-Ds in the Japanese public health insurance system. METHODS AND RESULTS: We developed a Markov model comprising five states: bradycardia, post-stroke, mild HF, severe HF, and death. For devices with rATP and control devices without rATP, we compared the incremental cost-effectiveness ratio (ICER) from the payer's perspective. Costs were estimated from healthcare resource utilisation data in a Japanese claims database. We evaluated model uncertainty by analysing two scenarios for each device. The ICER was 763 729 JPY/QALY (5616 EUR/QALY) for PMs and 1,393 280 JPY/QALY (10 245 EUR/QALY) for CRT-Ds. In all scenarios, ICERs were below 5 million JPY/QALY (36 765 EUR/QALY), supporting robustness of the results. CONCLUSION: According to a willingness to pay threshold of 5 million JPY/QALY, the devices with rATP were cost-effective compared with control devices without rATP, showing that the higher reimbursement price of the functional categories with rATP is justified from a healthcare economic perspective.

Characteristics of subjects with type 2 diabetes enrolled in randomized controlled trials and non-randomized controlled trials in Japan: A systematic review.

AIMS/INTRODUCTION: This study aimed to understand the characteristics of type 2 diabetes subjects enrolled in randomized controlled trials (RCTs) and non-RCTs according to therapeutic regimens through systematic literature review. MATERIALS AND METHODS: PubMed and the database of the Japanese Medical Abstract Society (ICHUSHI) were searched for studies published from 2010 to 2019 reporting the efficacy and safety of glucose-lowering drugs in Japanese individuals with suboptimally controlled type 2 diabetes, and therapeutic regimens, demographics and clinical characteristics at the baseline were extracted. We evaluated the treatment arms, not the placebo arms. RESULTS: The literature searches identified 2,656 publications, 145 of which met all eligibility criteria and included 282 eligible arms. In the past 10 years, dipeptidyl peptidase-4 inhibitor was the most frequently studied in both RCTs and non-RCTs. Regarding the characteristics of enrolled subjects, sodium-glucose cotransporter 2 inhibitor and glucagon-like peptide-1 receptor agonist have been studied more in relatively obese subjects, and insulin has been studied in higher proportion of subjects with disease duration ≥10 years. Most of the RCTs included subjects aged 55-64 years, whereas a higher proportion of dipeptidyl peptidase-4 inhibitor and insulin arms in the non-RCTs included those aged ≥65 years. Dipeptidyl peptidase-4 inhibitor and sodium-glucose cotransporter 2 inhibitor were evaluated in subjects with no abnormalities in blood pressure or lipid parameters; however, only a few reports of those parameters have been assessed with glucagon-like peptide-1 receptor agonist and insulin. CONCLUSIONS: As RCTs and non-RCTs differ in the baseline characteristics of type 2 diabetes subjects, it is necessary to integrate and evaluate both to understand the actual treatment status of type 2 diabetes.

Cost-effectiveness analyses of biologic and targeted synthetic disease-modifying anti-rheumatic diseases in patients with rheumatoid arthritis: Three approaches with a cohort simulation and real-world data.

OBJECTIVE: To assess the cost-effectiveness of biologic and targeted synthetic disease-modifying anti-rheumatic drugs (b/tsDMARDs) in rheumatoid arthritis. METHODS: We conducted three analyses: a lifetime analysis with a cohort model (Study A) and two short-term analyses (Studies B and C). Study A evaluated the incremental cost-effectiveness ratio (ICER) per quality-adjusted life-year (QALY) gained from costs of standard treatments. Study B evaluated yearly costs per person achieving American College of Rheumatology (ACR) response (ACR20, ACR50, and ACR70), and Study C evaluated costs per person achieving previously defined claims-based effectiveness (equivalent to 28-joint Disease Activity Score ≤ 3.2). The proportion of ACR responders to the drugs of interest were determined by mixed treatment comparisons. Studies B and C estimated costs using a claims database. RESULTS: In Study A, ICERs of all b/tsDMARDs were lower than 5.0 million Japanese yen (JPY) per QALY. In Study B, yearly costs per person with ACR50 response were lower for subcutaneous tocilizumab (TCZ-SC; 1.9 million JPY) and SC abatacept (2.3 million JPY). In Study C, costs per person were lower for TCZ-SC (1.3 million JPY) and intravenous TCZ (1.6 million JPY) and effectiveness rates were higher for intravenous TCZ (45.3%) and infliximab (43.0%). CONCLUSION: The b/tsDMARDs with lower prices showed higher cost-effectiveness.

Reduction in medical costs for cardiovascular diseases through innovative health awareness projects in a rural area in Japan.

To promote health awareness and improve life expectancy in Hirosaki, a Japanese rural area, the Center of Healthy Aging Program (CHAP) was founded in 2013. The most important characteristic of CHAP is a personalized interview just after the checkup to discuss individual results. We evaluated the clinical and economic effects of CHAP by analyzing the cohort data of voluntary participants from annual health checkups since 2005 in the Iwaki district of Hirosaki. We calculated 10-year incidence risk scores for coronary heart diseases (CHDs) and stroke, and compared the risk-score trend before and after the start of CHAP by adjusting other risk factors using multivariate generalized linear regression analyses. We also predicted the 10-year incidences of CHDs and stroke based on the risk scores, for future scenarios of the two conditions, with and without CHAP, and compared them to their treatment costs between scenarios. The number of participants ranged between 808 and 1,167, from 2008 to 2016. The mean age (55 years) and proportion of women (60%) did not significantly change throughout the period. After adjusting for sex, age, outside temperature on the checkup date, the preparation effect of CHAP in 2012, and risk scores in the previous year, the annual increases in risk scores of CHDs and stroke were significant even after CHAP (+0.413, p <0.001; +0.169, p <0.001, respectively), but slightly less compared to before CHAP (+2.638, p <0.001; +1.155, p <0.001, respectively). Assuming the trend continued until 2021, the 10-year incidences of CHDs and stroke have decreases by 22,486 and 9,603, respectively, and treatment costs decreased by JPY 21,973 and 16,056 million, respectively. CHAP contributes to a significant decrease in the incidences of CHDs and stroke, and reduces economic burden on the local government.

Cost-consequence of abatacept as first-line therapy in Japanese rheumatoid arthritis patients using IORRA real-world data.

OBJECTIVES: To investigate the cost-effectiveness of abatacept (ABA) as first-line (1L) therapy in Japanese rheumatoid arthritis (RA) patients using data from the Institute of Rheumatology, Rheumatoid Arthritis database. METHODS: A decision-analytic model was used to estimate the cost per American College of Rheumatology response of at least 50% improvement (ACR50) responder and per patient in Clinical Disease Activity Index (CDAI) and Simplified Disease Activity Index (SDAI) remission from a Japanese healthcare payers' perspective over a 2-year time horizon. Clinical characteristics of patients on ABA-1L were matched with those of patients on ABA second or later line (2L+) or tumour necrosis factor inhibitor (TNFi)-1L directly or using propensity scores. Resource utilisation and medical costs were calculated from the Japan Medical Data Center claims database. Parameter uncertainty was addressed by sensitivity and subgroup analyses (age, treatment duration, Japanese version of Health Assessment Questionnaire [J-HAQ] score). RESULTS: Incremental costs per member per month (ΔPMPM) for ABA-1L versus TNFi-1L and ABA-2L+ were -1,571 Japanese Yen (JPY) and 81 JPY, respectively. For ABA-1L versus TNFi-1L, ΔPMPM by ACR50 response was -11,715 JPY and by CDAI and SDAI remission 11,602 JPY and 47,003 JPY, respectively. Corresponding costs for ABA-1L were lower for all outcome parameters versus those for ABA-2L+. Scenario analyses showed that ABA-1L was cost-effective over TNFi-1L in patients <65 years for any outcome. Furthermore, ABA-1L was cost-effective over ABA-2L+ for all outcomes in patients with age <65 years, disease duration <5 years and J-HAQ ≥1.5. CONCLUSIONS: ABA-1L demonstrated a favourable cost-effectiveness profile in RA patients, accruing savings for the Japanese healthcare payers.

Efficacy of Low-Dose Estrogen-Progestins and Progestins in Japanese Women with Dysmenorrhea: A Systematic Review and Network Meta-analysis.

INTRODUCTION: Although several studies suggest beneficial effects of low-dose estrogen-progestins (LEPs) and progestins on dysmenorrhea in Japanese women, the difference in efficacy between drugs remains unknown. METHODS: We identified studies by searching the MEDLINE, Cochrane Library, and ICHUSHI databases and included randomized controlled trials (RCTs) that used total dysmenorrhea score and visual analogue scale (VAS) as outcome measures to evaluate LEPs and progestins for primary and secondary dysmenorrhea. We analyzed results by meta-analysis and network meta-analysis (NMA). RESULTS: We identified 10 articles on eight RCTs and included seven drugs (six LEPs and one progestin, i.e., dienogest) and placebo in the analysis. Meta-analysis showed improvements in total dysmenorrhea score and VAS for almost all drugs compared with placebo. In NMA, VAS in secondary dysmenorrhea improved more with dienogest than with norethisterone/ethinylestradiol (mean difference - 25.84 [95% CrI - 44.46 to - 7.15]). In the comparison of administration regimens, VAS improved more with progestin-continuous than LEP-cyclic and the surface under the cumulative ranking (SUCRA) of LEP-extended and progestin-continuous appeared to be higher than that of LEP-cyclic. CONCLUSIONS: We confirmed that LEPs and dienogest are effective for primary and secondary dysmenorrhea and suggest that continuous regimens may be more effective than cyclic regimens in improving outcomes.

30-year trends of hip and vertebral fracture incidence in Japan: a systematic review and meta-analysis.

INTRODUCTION: A previous review study showed the trends of declining age- and sex-adjusted incidence of hip fractures in almost all the countries, except Austria and Japan, in the most recent of the periods studied. However, the expansion of devices for diagnosis and drugs for fracture prevention in Japan was roughly the same as in the other countries. This study aimed to conduct a comprehensive systematic review and meta-analyses of incidence rates (IRs) of osteoporotic fractures reported over 30-years in multiple communities in Japan and to evaluate secular trends. MATERIALS AND METHODS: We searched MEDLINE for observational studies reporting IRs of osteoporotic fractures in the general population. Additional studies were identified by hand searches of reference lists of published studies. Two reviewers and 1 expert independently assessed study eligibility. Pooled analyses of IRs were conducted by a random-effect model. Data from 3 periods (1985-1999, 2000-2009, and from 2010) were compared. RESULTS: Of 47 eligible studies, 21 with IRs or information enabling recalculation of IRs in the population aged 50 years and older were included. IRs of hip fractures per 100,000 person-years significantly increased over time in men: 1985-1999, 0.79 (95% CI 0.71-0.87); 2000-2009, 1.18 (95% CI 1.09-1.28); and 2010 onwards, 1.31 (95% CI 1.17-1.45). They also significantly increased in women: 2.23 (95% CI 1.89-2.58); 3.99 (95% CI 3.70-4.28); and 4.39 (95% CI 4.00-4.78), respectively. CONCLUSION: IRs of hip fractures continuously increased in the past 30-years in men and women despite improved the diagnosis, treatment, and care. Such data from Japan, a leading aging society, provide important information for other countries.

A Systematic Review and Network Meta-Analysis on the Efficacy of Medications in the Treatment of Chronic Idiopathic Constipation in Japan.

BACKGROUND: In the 2010s, medications with new mechanisms were introduced in Japan for the treatment of chronic idiopathic constipation (CIC). A few systematic reviews have compared medications' relative efficacy, but the reviews included studies on patients from various races, even though the mechanism of CIC is considered to differ between races. The aim of this study was to use a systematic review and network meta-analysis to compare the relative efficacy of these medications in Japanese patients. METHODS: We conducted a meta-analysis and report it here according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA). We identified studies by searching MEDLINE (via the PubMed interface) and the Cochrane Library and ICHUSHI databases and included randomized clinical trials that compared medications for CIC with placebo in Japanese adults. Two reviewers independently screened and assessed articles, abstracted data, and assessed the risk of bias. We pooled data by random-effects meta-analyses and also performed a Bayesian network meta-analysis to indirectly compare data. RESULTS: The present systematic review and meta-analyses included 1460 patients in 6 randomized clinical trials: 2 on linaclotide, 3 on elobixibat, 2 on lubiprostone, and 1 on lactulose. The results of direct comparisons showed that linaclotide, elobixibat, and lubiprostone were superior to placebo in the change of spontaneous bowel movements (SBMs) within 1 week: linaclotide, 1.95 (95% CI, 1.51-2.39); elobixibat, 5.69 (95% CI, 3.31-8.07); and lubiprostone, 2.41 (95% CI, 0.82-4.01). The Bayesian network meta-analysis showed consistent results. Elobixibat 10 mg was ranked first for the increase in SBMs and complete SBMs within 1 week and the time to first SBM. Lubiprostone 48 µg was ranked first for the proportion of patients with SBM within 24 hours. CONCLUSION: Our direct and indirect meta-analyses revealed that the new CIC medications available in Japan have equal efficacy but that elobixibat and lubiprostone are highly likely to be more efficacious.

Trends in Corticosteroid Prescriptions for Ulcerative Colitis and Factors Associated with Long-Term Corticosteroid Use: Analysis Using Japanese Claims Data from 2006 to 2016.

BACKGROUND AND AIMS: Recent treatment guidelines for ulcerative colitis [UC] do not recommend long-term corticosteroid [CS] use. The present study aimed to capture the changes in CS use from 2006 to 2016 and to identify factors associated with long-term CS use after 2014, when the first two anti-tumour necrosis factor antibodies [infliximab and adalimumab] became available. METHODS: A retrospective study using the JMDC Claims Database included UC patients who initiated UC medications in any year from January 2006 to December 2016, or after January 2014, who were under continuous observation from 6 months before to 12 months after initiation. Patients with Crohn's disease before initiation and those prescribed <8 days of CSs were excluded. RESULTS: Among 7907 UC patients who initiated UC medications within the study period, 1555 were prescribed CSs. The proportion of patients using CSs in each year decreased from 2011 as use of thiopurines and biologics increased. The proportion of patients with a starting dose ≥30 mg/day of CSs and patients continuing CSs for <90 days increased from 2011, reaching 49.1% and 41.0%, respectively, in 2016. However, even in 2016, 34.3% continued to use CSs for ≥180 days. Among 1230 patients with CS use after January 2014, low initial CS dose [<10 mg/day] was most strongly associated with long-term CS use [≥180 days]. CONCLUSIONS: CS use became more appropriate as use of thiopurine and biologics increased, although there were still many cases of inappropriate use. Long-term CS use was most strongly associated with low initial doses of CSs.

Cost-effectiveness analysis of treatment regimens with obinutuzumab plus chemotherapy in Japan for untreated follicular lymphoma patients.

AIMS: Obinutuzumab (GA101; G) is a new treatment for follicular lymphoma (FL) that is anticipated to have greater efficacy than the current treatment, rituximab (R). The aim of this study was to evaluate the cost-effectiveness of G plus chemotherapy (G + Chemo) against that of R plus chemotherapy (R + Chemo) for patients in Japan with previously untreated FL. MATERIALS AND METHODS: We localized a previously reported cost-effectiveness model using the Japanese cost data. For estimating costs adapted in the model, FL patients treated with R were identified from Japanese hospital-based claims database and classified into three treatment regimen groups according to chemotherapies used with R: CHOP, CVP, and bendamustine (B). Based on services per patient and cost items, parameters determining the cost amounts were derived using a multivariate generalized linear mixed model to estimate the direct medical costs for each treatment regimen group for G and R. For the utility, same values in the previous model were used. Lifetime cost and quality-adjusted life year (QALY) were estimated under the payer's perspective. RESULTS: The calculated incremental cost-effectiveness ratios (ICERs) in million JPY per QALY for G-CHOP vs. R-CHOP, G-CVP vs. R-CVP, and G-B vs. R-B were 5.4, 4.3, and 4.8, respectively. ICERs were lower than 7.5 million JPY per QALY, which is the cost-effectiveness threshold for cancer treatments, and showed that G + Chemo is a cost-effective treatment regimen for Japanese patients with previously untreated FL. Lifetime direct medical costs were lowest in the R-B group because hospitalization costs that accounted for most of the total cost were lowest in this group. Limitations and conclusions: The cost-effectiveness of G + Chemo is acceptable in Japan. Differences in the direct medical costs among treatment regimen groups were mostly due to hospitalization costs. This is probably because many Japanese hematologists choose inpatient treatments over outpatient treatments in CHOP-based induction therapy.

Serum d- and l-lactate, pyruvate and glucose levels in individuals with at-risk mental state and correlations with clinical symptoms.

AIM: Little information exists on the peripheral metabolite levels in individuals with at-risk mental state who meet the criteria for a high-risk state of psychosis. Here, we aimed to investigate serum levels of glucose, pyruvate and d- and l-lactate, which may act as a signalling molecule for learning and memory in neuronal cells. METHODS: High performance liquid chromatography or commercial kits were used to assess serum metabolites in individuals with attenuated psychosis symptoms of at-risk mental state (n = 24, men = 12) who were not receiving antipsychotics. The metabolite levels of these individuals were compared with those of age- and sex-matched healthy individuals (controls, n = 23, men = 11). Correlations between the metabolites and clinical symptoms of at-risk mental state were also examined. RESULTS: Individuals with at-risk mental state had higher serum glucose levels than did controls (P = 2.18 × 10-3 ), while no significant difference in pyruvate levels were observed between the groups. Individuals with at-risk mental state had significantly lower serum l-lactate levels than did controls (P = 6.31 × 10-5 ), while no differences in d-lactate levels were observed. Furthermore, a negative correlation was identified between serum l-lactate levels and Positive and Negative Syndrome Scale negative symptoms scores (r = -0.5651, P = 4.01 × 10-3 ) in individuals with at-risk mental state. CONCLUSIONS: We found that, compared with controls, individuals with at-risk mental state have reduced serum l-lactate levels, which may predate psychosis onset, and may be involved in the related negative symptoms.

Spectral sensitivity of guppy visual pigments reconstituted in vitro to resolve association of opsins with cone cell types.

The guppy (Poecilia reticulata) shows remarkable variation of photoreceptor cells in the retina, especially those sensitive to middle-to-long wavelengths of light. Microspectrophotometry (MSP) has revealed varying "green", "green-yellow" and "yellow" cone cells among guppies in Trinidad and Venezuela (Cumana). In the guppy genome, there are four "long-wave" opsin loci (LWS-1, -2, -3 and -4). Two LWS-1 alleles have potentially differing spectral sensitivity (LWS-1/180Ser and LWS-1/180Ala). In addition, two "middle-wave" loci (RH2-1 and -2), two "short-wave" loci (SWS2-A and -B), and a single "ultraviolet" locus (SWS1) as well as a single "rhodopsin" locus (RH1) are present. However, the absorption spectra of these photopigments have not been measured directly and the association of cell types with these opsins remains speculative. In the present study, we reconstituted these opsin photopigments in vitro. The wavelengths of maximal absorbance (λmax) were 571nm (LWS-1/180Ser), 562nm (LWS-1/180Ala), 519nm (LWS-3), 516nm (LWS-2), 516nm (RH2-1), 476nm (RH2-2), 438nm (SWS2-A), 408nm (SWS2-B), 353nm (SWS1) and 503nm (RH1). The λmax of LWS-3 is much shorter than the value expected (560nm) from the "five-sites" rule. The two LWS-1 alleles could explain difference of the reported MSP λmax values for the yellow cone class between Trinidad and Cumana guppies. Absence of the short-wave-shifted LWS-3 and the green-yellow cone in the green swordtail supports the hypothesis that this cell class of the guppy co-expresses the LWS-1 and LWS-3. These results reveal the basis of variability in the guppy visual system and provide insight into the behavior and ecology of these tropical fishes.

Reduced argininosuccinate synthetase is a predictive biomarker for the development of pulmonary metastasis in patients with osteosarcoma.

Pulmonary metastasis is the most significant prognostic determinant for osteosarcoma, but methods for its prediction and treatment have not been established. Using oligonucleotide microarrays, we compared the global gene expression of biopsy samples between seven osteosarcoma patients who developed pulmonary metastasis within 4 years after neoadjuvant chemotherapy and curative resection, and 12 patients who did not relapse. We identified argininosuccinate synthetase (ASS) as a gene differentially expressed with the highest statistical significance (Welch's t test, P = 2.2 x 10(-5)). Immunohistochemical analysis of an independent cohort of 62 osteosarcoma cases confirmed that reduced expression of ASS protein was significantly correlated with the development of pulmonary metastasis after surgery (log-rank test, P < 0.05). Cox regression analysis revealed that ASS was the sole significant predictive factor (P = 0.039; hazard ratio, 0.319; 95% confidence interval, 0.108-0.945). ASS is one of the enzymes required for the production of a nonessential amino acid, arginine. We showed that osteosarcoma cells lacking ASS expression were auxotrophic for arginine and underwent G(0)-G(1) arrest in arginine-free medium, suggesting that an arginine deprivation therapy could be effective in patients with osteosarcoma. Recently, phase I and II clinical trials in patients with melanoma and hepatocellular carcinoma have shown the safety and efficacy of plasma arginine depletion by stabilized arginine deiminase. Our data indicate that in patients with osteosarcoma, reduced expression of ASS is not only a novel predictive biomarker for the development of metastasis, but also a potential target for pharmacologic intervention.

Survival prediction for pancreatic cancer patients receiving gemcitabine treatment.

Although gemcitabine monotherapy is the standard treatment for advanced pancreatic cancer, patient outcome varies significantly, and a considerable number do not benefit adequately. We therefore searched for new biomarkers predictive of overall patient survival. Using LC-MS, we compared the base-line plasma proteome between 29 representative patients with advanced pancreatic cancer who died within 100 days and 31 patients who survived for more than 400 days after receiving at least two cycles of the same gemcitabine monotherapy. Identified biomarker candidates were then challenged in a larger cohort of 304 patients treated with the same protocol using reverse-phase protein microarray. Among a total of 45,277 peptide peaks, we identified 637 peaks whose intensities differed significantly between the two groups (p < 0.001, Welch's t test). Two MS peaks with the highest statistical significance (p = 2.6 x 10(-4) and p = 5.0 x 10(-4)) were revealed to be derived from alpha(1)-antitrypsin and alpha(1)-antichymotrypsin, respectively. The levels of alpha(1)-antitrypsin (p = 8.9 x 10(-8)) and alpha(1)-antichymotrypsin (p = 0.001) were significantly correlated with the overall survival of the 304 patients. We selected alpha(1)-antitrypsin (p = 0.0001), leukocyte count (p = 0.066), alkaline phosphatase (p = 8.3 x 10(-8)), and performance status (p = 0.003) using multivariate Cox regression analysis and constructed a scoring system (nomogram) that was able to identify a group of high risk patients having a short median survival time of 150 days (95% confidence interval, 123-187 days; p = 2.0 x 10(-15), log rank test). The accuracy of this model for prognostication was internally validated and showed good calibration and discrimination with a bootstrap-corrected concordance index of 0.672. In conclusion, an increased level of alpha(1)-antitrypsin is a biomarker that predicts short overall survival of patients with advanced pancreatic cancer receiving gemcitabine monotherapy. Although an external validation study will be necessary, the current model may be useful for identifying patients unsuitable for the standardized therapy.

Adiponectin does not bind to gelatin: a new and easy way to purify high-molecular-weight adiponectin from human plasma.

Human plasma contains three forms of adiponectin, a trimer, a hexamer, and a high-molecular-weight (HMW) multimer. We previously reported HMW adiponectin was a gelatin-binding protein of 28 kDa (GBP28), it having been purified due to its affinity to gelatin-Cellulofine (Nakano, Y., et al. Isolation and characterization of GBP28, a novel gelatin-binding protein purified from human plasma. J. Biochem. 1996. 120: 803-12). Although HMW adiponectin binds to gelatin-Cellulofine, it cannot bind to gelatin-Sepharose. Gelatin-Cellulofine was made of formyl-Cellulofine and gelatin, and we found that HMW adiponectin binds to reduced formyl-Cellulofine with similar affinity as to gelatin-Cellulofine. Through only two steps using reduced formyl-Cellulofine and DEAE-Sepharose, HMW adiponectin can be effectively purified from human plasma.

Functional genome screen for therapeutic targets of osteosarcoma.

Osteosarcoma (OS) is the most frequent primary malignant bone tumor of children and young adults. Although the introduction of combined neoadjuvant chemotherapy has markedly improved survival, the outcome of OS patients with distant metastasis and/or poor response to chemotherapy is still unsatisfactory. Therefore there is a need to develop new therapeutic agents that suppress OS cell proliferation with higher efficacy. The protein kinases are a family of genes that play critical roles in various signaling pathways. Some cancer cells show addiction to constitutive activation of certain signaling pathways for proliferation and survival. To identify new drug targets for OS, we screened a panel of small interfering RNAs (siRNAs) that target 691 genes encoding human protein kinases and related proteins. We found that different constructs of siRNA specifically targeting polo-like 1 kinase (PLK1) significantly caused mitotic cell cycle arrest and subsequent apoptotic cell death in a variety of OS cell lines. siRNA targeting PLK1 also suppressed the growth of OS xenografts established in immunodeficient mice. Recently, phase I clinical trials of PLK1 chemical inhibitors have been reported. Our results indicate that PLK1 is a promising molecular target for pharmacologic intervention in OS.

Prolyl 4-hydroxylation of alpha-fibrinogen: a novel protein modification revealed by plasma proteomics.

Plasma proteome analysis requires sufficient power to compare numerous samples and detect changes in protein modification, because the protein content of human samples varies significantly among individuals, and many plasma proteins undergo changes in the bloodstream. A label-free proteomics platform developed in our laboratory, termed "Two-Dimensional Image Converted Analysis of Liquid chromatography and mass spectrometry (2DICAL)," is capable of these tasks. Here, we describe successful detection of novel prolyl hydroxylation of alpha-fibrinogen using 2DICAL, based on comparison of plasma samples of 38 pancreatic cancer patients and 39 healthy subjects. Using a newly generated monoclonal antibody 11A5, we confirmed the increase in prolyl-hydroxylated alpha-fibrinogen plasma levels and identified prolyl 4-hydroxylase A1 as a key enzyme for the modification. Competitive enzyme-linked immunosorbent assay of 685 blood samples revealed dynamic changes in prolyl-hydroxylated alpha-fibrinogen plasma level depending on clinical status. Prolyl-hydroxylated alpha-fibrinogen is presumably controlled by multiple biological mechanisms, which remain to be clarified in future studies.