Association between circadian clock gene expressions and meal timing in young and older adults.

Ageing is associated with a decline in circadian clock systems, which correlates with the development of ageing-associated diseases. Chrononutrition is a field of chronobiology that examines the relationship between the timing of meal/nutrition and circadian clock systems. Although there is growing evidence regarding the role of chrononutrition in the prevention of lifestyle and ageing-related diseases, the optimal timing of meal intake to regulate the circadian clock in humans remains unknown. In this study, we investigated the relationship between clock gene expression and meal timing in young and older adults. In this cross-sectional study, we enrolled 51 healthy young men and 35 healthy older men (age, mean±standard deviation: 24 ± 4 and 70 ± 4 y, respectively). Under daily living conditions, beard follicle cells were collected at 4-h intervals over a 24-h period to evaluate clock gene expression. Participants were asked to record the timing of habitual sleep and wake-up, breakfast, lunch, and dinner. From these data, we calculated "From bedtime to breakfast time," "From wake up to first meal time," and "From dinner to bed time." NR1D1 and PER3 expressions in older adults at 06:00 h were significantly higher than those in young adults (P = 0.001). There were significant differences in the peak time for NR1D2 (P = 0.003) and PER3 (P = 0.049) expression between young and older adults. "From bedtime to breakfast time" was significantly longer in older adults than in young adults. In contrast, "From dinner to bed time" was significantly shorter in older adults than in young adults. Moreover, higher rhythmicity of NR1D1 correlated with longer "From bedtime to breakfast time" (r = -0.470, P = 0.002) and shorter "From wake up to first meal time" in young adults (r = 0.302, P = 0.032). Higher rhythmicity of PER3 correlated with longer "From bedtime to breakfast time" in older adults (r = -0.342, P = 0.045). These results suggest that the peak time of clock gene expression in older adults may be phase-advanced compared to that in young adults. In addition, a longer fasting duration from bedtime to breakfast in both young and older adults and earlier intake of meals after waking up in young adults may correlate with robust clock gene expression rhythms.

Effects of the timing of acute mulberry leaf extract intake on postprandial glucose metabolism in healthy adults: a randomised, placebo-controlled, double-blind study.

BACKGROUND/OBJECTIVES: Glucose tolerance is controlled by the internal clock and is worse in the evening. From a chrononutrition perspective, diabetes prevention requires evaluating the antidiabetic effects of the timing of functional ingredients and nutrient intake. The purpose of this study was to investigate the timing effects of acute mulberry leaf extract (MLE) intake on postprandial glucose levels in young adults. SUBJECTS/METHODS: Twelve young adults underwent four trials. Blood samples were collected in a fasting state and at 30, 60, 120, and 180 min after eating a mixed meal. The study had a randomised, placebo-controlled, double-blind trial design involving: (1) morning placebo trial (08:00 h; MP trial), (2) evening placebo trial (18:00 h; EP trial), (3) morning MLE trial (08:00 h; MM trial), and (4) evening MLE trial (18:00 h; EM trial). RESULTS: The incremental area under the blood glucose curve (iAUC) in the EM trials was significantly lower than that in the EP trials (P = 0.010). The postprandial glucose concentrations 120 min after the meal were significantly lower in the EM trials than those in the EP trials (P = 0.006). The postprandial insulin concentrations at 120 min were significantly lower in the MM trials than those in the MP trials (P = 0.034). Moreover, the postprandial insulin concentrations 180 min after the meal were significantly lower in the EM trials than those in the EP trials (P = 0.034). CONCLUSIONS: MLE intake in the evening, but not in the morning, was effective in improving glucose tolerance. TRIAL REGISTRATION: Clinical trial reference: UMIN 000045301; website of trial registry: https://center6.umin.ac.jp/cgi-open-bin/ctr/ctr_view.cgi?recptno=R000051340 .

Effect of lunch with different calorie and nutrient balances on dinner-induced postprandial glucose variability.

AIM: This study aimed to examine the effect of lunches with different caloric contents (Study 1) and nutrient balances (Study 2) on dinner-induced postprandial glucose fluctuation. METHODS: Energy trial (Study 1): Thirteen healthy young participants (n = 10 men, n = 3 women) were investigated to determine the effects of different caloric intakes at lunch on glucose level variability. The study was comprised of four trials (no lunch, low lunch, standard lunch, and high-energy lunch). Energy balance trial (Study 2): Fourteen healthy young adults (n = 8 men, n = 6 women) were investigated to determine the effect of different nutrient balances during lunch on glucose level variability. The study consisted of four trials (standard, protein-rich, fat-rich, and carbohydrate-rich). In studies 1 and 2, each trial was spaced at least 24 full hours apart, and breakfast and dinner were tested as meals. The mealtimes for each trial were then aligned. Continuous glucose monitoring was used to assess the blood glucose fluctuations. RESULTS: Study 1: The no-lunch (95% CI 95.5-149.7) and low-energy lunch (95% CI 90.8-143.1) trials had significantly higher values in the incremental area under the curve (iAUC) of postprandial blood glucose at dinner compared to the standard (95% CI 55.4-90.0) and high-energy lunch (95% CI 29.3-54.6) trials (P = 0.006, P = 0.001 vs. none), (P = 0.004, P = 0.001 vs. low-energy trial). Study 2: A significantly higher postprandial blood glucose iAUC for dinner was found in the fat-rich trial (95% CI 58.5-114.0) than that in the protein-rich (95% CI 25.6-63.9) and standard (95% CI 25.6-112.4) trials, (P = 0.006, P = 0.035 vs. fat-rich trial). CONCLUSIONS: Our findings indicate that skipping lunch and low-calorie or high-lipid intake increased postprandial blood glucose levels after dinner.

Supplementation of Protein at Breakfast Rather Than at Dinner and Lunch Is Effective on Skeletal Muscle Mass in Older Adults.

Background: The effects of different intake patterns of meal protein on muscle mass have not been clarified. We cross-sectionally and longitudinally examined the effect of different timing of protein intake on sarcopenia-related factors in older adults. Methods: This cross-sectional study 1 included 219 (male, n = 69, female, n = 150) elderly subjects aged ≥65 years. Subjects who consumed more protein at breakfast than at dinner were grouped into the morning group (MG, n = 76; male, n = 26; female, n = 50), and those who consumed more protein at dinner than at breakfast were grouped into the evening group (EG, n = 143; male, n = 43; female, n = 100). In cross-sectional study 2-1 (female, n = 125), the subjects were classified into four groups according to the number of meals with sufficient protein intake. In cross-sectional studies 2-2 (female, n = 125) and 2-3 (female, n = 27), the subjects were classified into eight groups and three groups according to whether they had consumed sufficient protein at three meals; sarcopenia-related factors were compared. The intervention study was a placebo-controlled, double-blind, randomized controlled trial that included 40 elderly women with low daily breakfast protein intake. The subjects were divided into four groups: morning protein and placebo intake groups and evening protein and placebo intake groups. Each group consumed the test food (containing 10 g milk protein) or placebo in the morning or evening for 12 weeks. Blood indices and physical function were assessed before and after the intervention. Results: Comparing all subjects, MG showed significantly higher handgrip strength than did EG (P < 0.05). The higher ratio of morning protein intake relative to the total protein intake, the better the muscle mass (r = 0.452, P < 0.05) and handgrip strength (r = 0.383, P < 0.05). The intervention study showed an increase in muscle mass with the intake of milk protein in the morning rather than in the evening (P < 0.05). Conclusions: Protein intake at breakfast might have relatively stronger effects on skeletal muscle mass than at lunch and dinner.

Effects of timing of acute catechin-rich green tea ingestion on postprandial glucose metabolism in healthy men.

Green tea polyphenols, particularly catechins, decrease fasting and postprandial glucose. However, no studies have compared the timing of green tea ingestion on glucose metabolism and changes in catechin concentrations. Here, we examined the effects of timing of acute catechin-rich green tea ingestion on postprandial glucose metabolism in young men. Seventeen healthy young men completed four trials involving blood collection in a fasting state and at 30, 60, 120, and 180 min after meal consumption in a random order: 1) morning placebo trial (09:00 h; MP trial), 2) evening placebo trial (17:00 h; EP trial), 3) morning catechin-rich green tea trial (09:00 h; MGT trial), and 4) evening catechin-rich green tea trial (17:00 h; EGT trial). The concentrations of glucose at 120 min (P=.031) and 180 min (P=.013) after meal intake were significantly higher in the MGT trials than in the MP trials. Additionally, the concentration of glucose was significantly lower in EGT trials than in the EP trials at 60 min (P=.014). Moreover, the concentrations of glucose-dependent insulinotropic polypeptide were significantly lower in the green tea trials than in the placebo trials at 30 min (morning: P=.010, evening: P=.006) and 60 min (morning: P=.001, evening: P=.006) after meal intake in both the morning and evening trials. Our study demonstrated that acute ingestion of catechin-rich green tea in the evening reduced postprandial plasma glucose concentrations.

The effect of night shift work on the expression of clock genes in beard hair follicle cells.

OBJECTIVE: Shift work encompasses a broad range of work time arrangements. However, how shift work affects the circadian expression of clock genes remains to be explored. The objective of this study was to evaluate the pattern of clock gene expression in shift workers in the field. METHODS: We examined clock gene expression in Japanese men who work: (1) one night shift followed by a day off (caregivers: nurses and doctors; the one-night group); (2) three or more consecutive night shifts (factory workers; the consecutive-night group); or (3) daytime only (the daytime group), using beard follicle samples. The expression of Period3, Nuclear Receptor Subfamily 1 Group D Member 1 (Nr1d1), and Nuclear Receptor Subfamily 1 Group D Member 2 (Nr1d2) was examined by real-time polymerase chain reaction. RESULTS: Period3 expression in the daytime and one-night groups together with Nr1d2 expression in the one-night group fitted a 24-h-period cosine curve better than in the consecutive-night group (p = 0.004, 0.012, and 0.001, respectively). The level of overall Period3 gene expression, calibrated with that of 18S-rRNA, was decreased in the consecutive-night group compared with that in the daytime group (p = 0.006). The patterns of Period3 and Nr1d2 expression in the daytime and one-night groups were more coherent than those in the consecutive-night group. CONCLUSIONS: These results suggest that night shift work affects the rhythms and levels of circadian Period3 and Nr1d2 expression dependent on the shift schedule or type of the shift; however, there is substantial variation between individuals.

Chronotype and social jetlag influence human circadian clock gene expression.

We examined the relationships between chronotype or social jetlag and clock gene expression. Twenty-four young men [Chronotype: morningness, n = 8; intermediate, n = 8, eveningness, n = 8], aged 27 ± 2 years old (mean ± SE), completed two trials in a randomized order: (1) a Friday trial and (2) a Monday trial. In both trials, hair follicle cells were collected to evaluate the expression of clock genes over a 24-hour period at 4-hour intervals. There was a significant main effect of time on the expression of NR1D1, NR1D2, and PER3 (P < 0.001) in the morningness group, but not in the eveningness group. Changes in the peak time of expression of NR1D1 (r = 0.434, P = 0.034), NR1D2 (r = 0.481, P = 0.017), and PER3 (r = 0.457, P = 0.025) from the Friday to Monday trials were positively correlated with social jetlag (SJL) time. Our findings indicate that there was no change in the patterns of clock gene expression between workdays and the day after the holiday in the morningness group, and that SJL time influences the peak time of clock gene expression, moving it from the early to late workday, after a holiday.

A randomized, double-blind and placebo-controlled crossover trial on the effect of l-ornithine ingestion on the human circadian clock.

In mammals, daily physiological events are regulated by the circadian rhythm, which comprises two types of internal clocks: the central clock and peripheral clocks. Circadian rhythm plays an important role in maintaining physiological functions including the sleep-wake cycle, body temperature, metabolism and organ functions. Circadian rhythm disorder, which is caused, for example, by an irregular lifestyle or long-haul travel, increases the risk of developing disease; therefore, it is important to properly maintain the rhythm of the circadian clock. Food and the circadian clock system are known to be closely linked. Studies on rodents suggest that ingesting specific food ingredients, such as the flavonoid nobiletin, fish oil, the polyphenol resveratrol and the amino acid L-ornithine affects the circadian clock. However, there are few reports on the foods that affect these circadian clocks in humans. In this study, therefore, we examined whether L-ornithine affects the human central clock in a crossover design placebo-controlled human trial. In total, 28 healthy adults (i.e. ≥20 years) were randomly divided into two groups and completed the study protocol. In the 1st intake period, participants were asked to take either L-ornithine (400 mg) capsules or placebo capsules for 7 days. After 7 days' interval, they then took the alternative test capsules for 7 days in the 2nd intake period. On the final day of each intake period, saliva was sampled at various time points in the dim light condition, and the concentration of melatonin was quantified to evaluate the phase of the central clock. The results revealed that dim light melatonin onset, a recognized marker of central circadian phase, was delayed by 15 min after ingestion of L-ornithine. Not only is this finding an indication that L-ornithine affects the human central clock, but it also demonstrates that the human central clock can be regulated by food ingredients.

Compositional Analysis of Coronary Artery Calcification in Dialysis Patients in vivo by Dual-Energy Computed Tomography Angiography.

While vascular calcification is an important factor regulating prognosis in dialysis patients, its components have not been adequately studied. We analyzed in vivo components of calcification in the coronary arteries of dialysis patients using the effective atomic number from dual-energy computed tomography. In dialysis patients (hemodialysis, N = 10; peritoneal dialysis, N = 12), average of median effective atomic number was 13.8 in the hemodialysis group, and 13.7 in the peritoneal dialysis group. No significant differences were seen between groups, with calcium oxalate monohydrate identified as the most common component in each. To confirm the accuracy of this method, we investigated the composition of surgically removed calcified tissues using already established methods. Comparison with the effective atomic number from dual-energy computed tomography showed that the results of calcification analysis were the same. We concluded that calcium oxalate monohydrate might be one of the major components of coronary artery calcification in dialysis patients.

Clinical application of effective atomic number for classifying non-calcified coronary plaques by dual-energy computed tomography.

BACKGROUND AND AIMS: Coronary computed tomography (CT) angiography allows non-invasive classification of non-calcified coronary plaques (NCCPs) based on Hounsfield unit (HU) values. This methodology, however, is somewhat limited for reliable classification of NCCPs. Therefore, we evaluated the effective atomic number (EAN) for classifying NCCPs by single-source dual-energy CT with fast tube voltage switching (SSDECT). METHODS: We prospectively enrolled 18 patients undergoing both SSDECT and intravascular ultrasonography (IVUS). Monochromatic images at 70 keV and EAN images were reconstructed from SSDECT data sets. Regions of interest (ROIs) within NCCPs were placed on IVUS-matched SSDECT images, and mean HU values and EANs for soft and fibrous plaques, classified using IVUS, were compared with an unpaired t-test. RESULTS: We placed 96 ROIs in 29 soft plaques and 37 ROIs in 15 fibrous plaques in 12 coronary arteries of 11 patients. The mean HU value in soft plaques (58.2 ± 32.8 HU) was significantly lower than that in fibrous plaques (103.9 ± 48.3 HU) (p < 0.001). The mean EAN in soft plaques (8.7 ± 0.5) was also significantly lower than that in fibrous plaques (9.6 ± 0.5) (p < 0.0001). Area under the curve for EAN (0.91) was significantly higher than that for HU value (0.79) in receiver operating characteristic curve analysis (p = 0.046). With a cutoff EAN of 9.3, sensitivity was 90% and specificity, 87%; whereas with a cutoff HU value of 55.0 HU, sensitivity was 62% and specificity, 93%. CONCLUSIONS: EAN measurement by SSDECT can be clinically useful for accurately classifying soft and fibrous coronary plaques.

Positive association between physical activity and PER3 expression in older adults.

The circadian clock regulates many physiological functions including physical activity and feeding patterns. In addition, scheduled exercise and feeding themselves can affect the circadian clock. The purpose of the present study was to investigate the relationship between physical/feeding activity and expression of clock genes in hair follicle cells in older adults. Twenty adult men (age, 68 ± 7 years, mean ± SE) were examined in this cross-sectional study. Prior to hair follicle cell collection, the participants were asked to wear a uniaxial accelerometer for one week. The timings of breakfast, lunch, and dinner were also recorded. Hair follicle cells were then collected over a 24 h period at 4 h intervals. The amplitude of PER3 expression was positively correlated with moderate and vigorous physical activity (r = 0.582, p = 0.007) and peak oxygen uptake (r = 0.481, p = 0.032), but these correlations were not observed for NR1D1 or NR1D2. No association was noted between meal times and the amplitude or the acrophase for any of these three clock genes. These findings suggest that rhythmic expression of the circadian clock gene PER3 is associated with the amount of daily physical activity and physical fitness in older adults.

Depression and Altitude: Cross-Sectional Community-Based Study Among Elderly High-Altitude Residents in the Himalayan Regions.

Suicide rates are higher at high altitudes, and some hypothesize that hypoxia is the cause. There may be a significant correlation between rates of depression and altitude, but little data exist outside the United States. The purpose of the present study is to conduct a survey of depression among the elderly highlanders in Asia. We enrolled 114 persons aged 60 years or older (mean, 69.2 ± 6.7 years; women, 58.8%) in Domkhar (altitude, 3800 m), Ladakh, India and 173 ethnic Tibetans (mean, 66.5 ± 6.1 years; women, 61.3%) in Yushu (altitude, 3700 m), Qinghai Province, China. The two-item Patient Health Questionnaire (PHQ-2) and the geriatric depression scale were administered. A psychiatrist interviewed the subjects who had a positive score on the PHQ-2. The results of the interview with the residents conducted by the specialist showed that two cases (1.8%) from Domkhar and four (2.3%) from Qinghai had depression. Despite the high altitude, the probability of depression was low in elderly highlander in Ladakh and Qinghai. Our finding seems to indicate that cultural factors such as religious outlook and social/family relationship inhibit the development of depression.

Vasodilatation of multiple cerebral arteries in early stage of stroke-like episode with MELAS.

We describe a patient with mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS), with multiple cerebral vasodilatations in a stroke-like episode visualised by using magnetic resonance angiography (MRA) and CT angiography (CTA). In the acute stroke-like episode stage, T2-weighted and fluid-attenuated inversion recovery MRI showed high-intensity areas in the left occipital area. In addition, MRA and CTA revealed prominent dilatation of the left posterior cerebral artery and temporal branches of the middle cerebral artery with focal hyperperfusions using CT perfusion (CTP) that corresponded to the MRI. After 10 days, with the development of aphasia, MRI indicated the lesions had spread to the temporal and parietal regions, and this distribution was not confined to major vascular territories. The patient's symptoms gradually improved, accompanied by the attenuation of MRI, CTA, and CTP findings. These characteristic features along with the MRI changes that spread beyond vascular boundaries and the multiple cerebral vasodilatations prior to the development of clinical symptoms are not fully explained by the mitochondrial angiopathy or cytopathy theories. These findings provide further evidence supporting neuronal hyperexcitability in stroke-like episodes of MELAS.

Analysis of coronary arterial calcification components with coronary CT angiography using single-source dual-energy CT with fast tube voltage switching.

Clinical cardiac applications of single-source dual-energy computed tomography (DECT) have recently been introduced. This study aimed to analyze the components of coronary arterial calcification (CAC) in vivo by material decomposition achieved with DECT. We reconstructed computed tomography (CT) angiography images for 51 consecutive patients with CACs who had undergone electrocardiography-gated coronary CT angiography by single-source DECT with fast tube voltage switching. We placed regions of interest (ROIs) within the CAC with margins of at least 0.5 mm to minimize partial volume averaging. We compared histograms for the effective atomic number (EAN) and the median, mean, and maximum EANs for each CAC with the theoretical EANs for possible CAC components, including hydroxyapatite (HA), calcium oxalate monohydrate (COM), and dicalcium phosphate dehydrate. We also investigated the in vivo EAN for COM and in vitro EAN for HA by our phantom experiment. Analysis of the CAC components was feasible in 177 ROIs from 28 patients. The median EAN was 13.8 ± 0.8 (95% confidence interval 13.7-13.9), which is similar to the theoretical EAN for COM (13.8). The EAN for HA in vitro was 16.5 ± 0.1, which was slightly higher than the theoretical EAN value for HA (16.1). Notably, the median EAN in 144 ROIs (81.4%) was between 11.2 and 14.4, which is the reported range of the in vivo EAN for COM. Our results suggest that COM might be a more frequent CAC component than previously reported.

[Association between high altitude and depression in the Himalayas and the Andes].

AIM: Suicide rates in the United States are higher in higher altitude areas, and hypoxia has been cited as a factor in these higher rates. There may be a significant correlation between rates of depression and altitude, but little data exist outside the United States. The purpose of the present study is to conduct a survey of depression among the elderly residing in the Himalayas and the Andes. METHOD: We visited Ladakh (altitude 3,800-4,800 m) in India, Qinghai (3,700 m) in China and Puyca (3,600 m) in Peru between July 2009 and July 2011. We recruited 114 farmers from Domkhar in Ladakh (mean age, 69.2 years; female-male ratio, 58.8%), 206 nomads from Changthang in Ladakh (55.1 years; 43.7%), 173 Tibetan subjects from Qinghai (66.5 years; 61.3%) and 103 indigenous Andean subjects from Puyca (69.0 years; 68.0%). The two-item Patient Health Questionnaire (PHQ-2) was administered to the subjects. A psychiatrist interviewed the residents with single or double positive scores on the PHQ-2. RESULT: The ratio of subjects with one or more positive score in PHQ-2 was significantly higher in Qinghai than in other regions. (Domkhar vs. Changthang vs. Qinghai vs. Puyca = 7.0% vs. 5.3% vs. 36.9% vs. 15.5%, P<0.001). However, prevalence of depression by interview did not change in these regions. (1.8% vs. 1.9% vs. 2.3% vs. 2.9%). CONCLUSION: Despite the high altitude, the prevalence of depression was low in elderly highlanders in the Himalayas and the Andes. These results may relate to being presumed to related to a deep devotion to a religion and tight interpersonal networks.

[Links between circadian system and human health].

There is now reason to speculate that disruption of circadian rhythms of physiology and behavior may have broader implications for human health. A long history of clinical epidemiology in humans demonstrates an increased incidence of obesity, cardiovascular disease and cancer among shift workers. Clues from studies on the molecular genetics of circadian clock genes may offer insight into the molecular mechanisms underlying the circadian variation of metabolic coordination. A better understanding of the impact of circadian gene networks on nutrient balance at the molecular, cellular, and system levels promises to shed light on the emerging association between disorders of diabetes, obesity, sleep, and circadian timing.

Profiling of immune-related microRNA expression in human cord blood and adult peripheral blood cells upon proinflammatory stimulation.

OBJECTIVES: Cord blood (CB) transplantation has advantages in terms of incidence and severity of acute graft-versus-host disease (GVHD), while it has disadvantages in terms of infection. Our aim is to elucidate the molecular mechanism underlying the immune response of CB-derived cells during acute GVHD and infection following CB transplantation. METHODS: We examined expression of 69 immune-relating microRNAs in CD4(+), CD8(+), and CD14(+) cells of CB and adult peripheral blood (APB) upon interferon-γ or lipopolysaccharide (LPS) stimulation. RESULTS: Under basal condition, 20 microRNAs showed differential expression between CB and APB. Compared to APB counterparts, six microRNAs (miR-21, miR-22, miR-29a, miR-29b, miR-29c, and let-7c) were underexpressed in at least two cell lineages of CB, while five microRNAs (miR-15b, miR-181a, miR-181c, miR-363, and miR-424) were overexpressed in CD4(+) and CD8(+) CB cells. Upon interferon-γ stimulation, seven microRNAs (miR-29a, miR-29b, miR-34c-5p, miR-132, miR-146a, miR-146b-5p, and miR-155) changed in expression mainly in CD14(+) CB cells, while only two microRNAs (miR-18a and miR-155) changed in expression in CD14(+) CB cells upon LPS stimulation. These results suggest that the mechanisms regulating the expression of such immune-relating microRNAs in CD14(+) CB cells are much more sensitive to proinflammatory stimuli than those in APB CD14(+) cells, which might be related to the poor immunoreactivity of CD14(+) CB cells. CONCLUSIONS: Our results suggest essential roles of specific microRNAs in regulating immune function of CB cells, providing insight into the underlying molecular mechanism.

Gastrointestinal bleeding during anti-angiogenic peptide vaccination in combination with gemcitabine for advanced pancreatic cancer.

Most pancreatic cancer patients are diagnosed at the advanced stages, and no therapy is superior to gemcitabine alone. To confirm the feasibility and efficacy of a novel clinical intervention using tumor vessel-specific anti-angiogenic peptide vaccination, we conducted a clinical phase I/II trial using HLA-A*2402/A*0201-restricted vascular endothelial growth factor receptor type 1 (VEGFR1)-derived peptide vaccination in combination with gemcitabine for advanced pancreatic cancer (http://www.clinical-trials.gov; NCT00683358 and NCT00683085). Four of the enrolled patients (n = 2 for HLA-A*2402 and n = 2 for HLA-A*0201 protocol, respectively), defined as having progressive disease according to the Response Evaluation Criteria in Solid Tumors version 1.0 (RECIST v.1.0), failed to respond to the therapy. Another two patients enrolled in HLA-A*2402 protocol dropped out of the study due to rapid disease progression. Grade 2-3 hematologic toxicities were observed in all cases, but the treatment was well tolerated with minimal systemic adverse events. One case in HLA-A*2402 protocol and another case in HLA-A*0201 protocol suffered complicated gastrointestinal (GI) bleeding during vaccination. The causal relationship between GI bleeding and VEGFR1-peptide vaccination is unclear according to the pathologic examination. These studies terminated prematurely because of the advanced stage of the disease in the enrolled patients on entry to the study. Despite GI bleeding, peptide vaccination provides a feasible treatment option for many advanced pancreatic cancer patients.

Inhibition of B16 melanoma growth and metastasis in C57BL mice by vaccination with a syngeneic endothelial cell line.

BACKGROUND: Key role of angiogenesis in tumor growth and metastasis based on accumulating evidence and recent progress of immunotherapy have led us to investigate vaccine therapy targeting tumor angiogenesis. METHODS: C57BL/6J mice were vaccinated with a syngeneic endothelial cell line Tpit/E by subcutaneous injection once a week. Prior to ninth vaccination, the mice were challenged with B16/F10 melanoma cells by subcutaneous inoculation on the back for the tumor growth model or by tail venous injection for the lung metastasis model. Development of subcutaneous tumor and lung metastasis was monitored by computed tomography scanning, which enabled accurate evaluation with the minimized sacrifice of mice. RESULTS: Vaccination with Tpit/E cells inhibited subcutaneous tumor growth and appearance of lung metastasis compared to control. Survival period was elongated in the Tpit/E vaccination in both of the two models. We also obtained hybridomas secreting specific antibodies to Tpit/E cells from a mouse vaccinated with the cells, indicating that specific immune response to the syngeneic endothelial cells was elicited. CONCLUSION: These results suggest that vaccination with an autologous endothelial cell line may be effective against melanoma.