Transcriptome analysis of cynomolgus macaques throughout their lifespan reveals age-related immune patterns.

Despite the different perspectives by diverse research sectors spanning several decades, aging research remains uncharted territory for human beings. Therefore, we investigated the transcriptomic characteristics of eight male healthy cynomolgus macaques, and the annual sampling was designed with two individuals in four age groups. As a laboratory animal, the macaques were meticulously shielded from all environmental factors except aging. The results showed recent findings of certain immune response and the age-associated network of primate immunity. Three important aging patterns were identified and each gene clusters represented a different immune response. The increased expression pattern was predominantly associated with innate immune cells, such as Neutrophils and NK cells, causing chronic inflammation with aging whereas the other two decreased patterns were associated with adaptive immunity, especially "B cell activation" affecting antibody diversity of aging. Furthermore, the hub gene network of the patterns reflected transcriptomic age and correlated with human illness status, aiding in future human disease prediction. Our macaque transcriptome profiling results offer systematic insights into the age-related immunological features of primates.

Heterologous vaccination utilizing viral vector and protein platforms confers complete protection against SFTSV.

Severe fever with thrombocytopenia syndrome virus was first discovered in 2009 as the causative agent of severe fever with thrombocytopenia syndrome. Despite its potential threat to public health, no prophylactic vaccine is yet available. This study developed a heterologous prime-boost strategy comprising priming with recombinant replication-deficient human adenovirus type 5 (rAd5) expressing the surface glycoprotein, Gn, and boosting with Gn protein. This vaccination regimen induced balanced Th1/Th2 immune responses and resulted in potent humoral and T cell-mediated responses in mice. It elicited high neutralizing antibody titers in both mice and non-human primates. Transcriptome analysis revealed that rAd5 and Gn proteins induced adaptive and innate immune pathways, respectively. This study provides immunological and mechanistic insight into this heterologous regimen and paves the way for future strategies against emerging infectious diseases.

Assessing the Utility of Mental Health First Aid Training for Physician Assistant Students.

INTRODUCTION: Physician assistant (PA) students are at increased risk for anxiety and depression compared with the general population. The demands of studying medicine can contribute to a decline in mental health, which may impede a student's ability to progress in his or her PA program. Mental Health First Aid (MHFA) is one strategy that can be used to increase mental health literacy, promote help-seeking behavior, reduce stigma, and improve confidence in providing help to individuals showing signs of a mental health disorder. The aim of this outcomes assessment was to assess PA students' satisfaction with the MHFA course and their posttraining confidence in using the skills learned. METHODS: Participants were PA students (N = 435) across 9 PA programs in their didactic year of training who had completed an MHFA course through the PA Foundation's Mental Health Outreach Fellowship. MHFA postcourse evaluations, completed by the PA students, were analyzed to determine PA students' satisfaction with the course and their posttraining confidence in using the skills learned to help both themselves as well as the general population. RESULTS: The mean of the MHFA course evaluation items corresponding to satisfaction with the course was 4.82 (maximum score of 5), and the mean of the items corresponding to posttraining confidence in using the skills learned was 4.74 (maximum score of 5). When asked, "Would you recommend this course to others?" 99.3% of the PA students trained in MHFA answered "yes," indicating a high level of satisfaction with the training they had received. DISCUSSION: PA students who received MHFA training from the PA Foundation's Mental Health Outreach fellows showed high levels of satisfaction with the MHFA course and posttraining confidence in using the skills learned.

Coronary Circulatory Indexes Before and After Percutaneous Coronary Intervention in a Porcine Tandem Stenoses Model.

Background In tandem stenoses, nonhyperemic pressure ratio pullback is the preferred method to fractional flow reserve (FFR), based on the assumption of stable resting coronary flow. This study aimed to evaluate temporal changes of coronary circulatory indexes in tandem stenoses before and after angioplasty for proximal stenosis. Methods and Results Coronary tandem stenoses were created by porcine restenosis model with 2 bare metal stents in the left anterior descending artery. Four weeks later, changes in distal coronary pressure (Pd), averaged peak velocity, microvascular resistance, transstenotic pressure gradient across distal stenosis, resting Pd/aortic pressure, and FFR were measured before and 1, 5, 10, 15, and 20 minutes after balloon angioplasty for proximal stenosis. After angioplasty, there were significant changes in both resting and hyperemic Pd, averaged peak velocity, microvascular resistance, and transstenotic pressure gradient across distal stenosis (all P values <0.01). After initial acute changes, hyperemic averaged peak velocity and microvascular resistance did not show significant difference from the baseline values (P=0.712 and 0.972, respectively). Conversely, resting averaged peak velocity remained increased (10.1±0.7 to 17.8±0.7; P<0.001) and resting microvascular resistance decreased (6.0±0.1 to 2.2±0.7; P<0.001). Transstenotic pressure gradient across distal stenosis was significantly increased in both resting (13.1±7.6 to 25.3±4.2; P=0.040) and hyperemic conditions (11.0±3.0 to 27.4±3.3 mm Hg; P<0.001). Actual post-percutaneous coronary intervention Pd/aortic pressure and FFR were significantly lower than predicted values (Pd/aortic pressure, 0.68±0.22 versus 0.85±0.14; P<0.001; FFR, 0.63±0.08 versus 0.81±0.08; P<0.001). Conclusions After angioplasty for proximal stenosis, transstenotic pressure gradient across distal stenosis showed similar changes between resting and hyperemic conditions. Both actual post-percutaneous coronary intervention resting Pd/aortic pressure and FFR were significantly lower than predicted values.

Impaired Hand Dexterity Function in a Non-human Primate Model with Chronic Parkinson's Disease.

Symptoms of Parkinson's disease (PD) caused by loss of dopaminergic neurons are accompanied by movement disorders, including tremors, rigidity, bradykinesia, and akinesia. Non-human primate (NHP) models with PD play an essential role in the analysis of PD pathophysiology and behavior symptoms. As impairments of hand dexterity function can affect activities of daily living in patients with PD, research on hand dexterity function in NHP models with chronic PD is essential. Traditional rating scales previously used in the evaluation of animal spontaneous behavior were insufficient due to factors related to subjectivity and passivity. Thus, experimentally designed applications for an appropriate apparatus are necessary. In this study, we aimed to longitudinally assess hand dexterity function using hand dexterity task (HDT) in NHP-PD models. To validate this assessment, we analyzed the alteration in Parkinsonian tremor signs and the functionality of presynaptic dopaminergic neuron using positron emission tomography imaging of dopamine transporters in these models. In addition, a significant inverse correlation between HDT and DAT level was identified, but no local bias was found. The correlation with intention tremor signs was lower than the resting tremor. In conclusion, the evaluation of HDT may reflect behavioral symptoms of NHP-PD models. Furthermore, HDT was effectively used to experimentally distinguish intention tremors from other tremors.

Establishment and Characterization of Immortalized Miniature Pig Pancreatic Cell Lines Expressing Oncogenic K-RasG12D.

In recent decades, many studies on the treatment and prevention of pancreatic cancer have been conducted. However, pancreatic cancer remains incurable, with a high mortality rate. Although mouse models have been widely used for preclinical pancreatic cancer research, these models have many differences from humans. Therefore, large animals may be more useful for the investigation of pancreatic cancer. Pigs have recently emerged as a new model of pancreatic cancer due to their similarities to humans, but no pig pancreatic cancer cell lines have been established for use in drug screening or analysis of tumor biology. Here, we established and characterized an immortalized miniature pig pancreatic cell line derived from primary pancreatic cells and pancreatic cancer-like cells expressing K-rasG12D regulated by the human PTF1A promoter. Using this immortalized cell line, we analyzed the gene expression and phenotypes associated with cancer cell characteristics. Notably, we found that acinar-to-ductal transition was caused by K-rasG12D in the cell line constructed from acinar cells. This may constitute a good research model for the analysis of acinar-to-ductal metaplasia in human pancreatic cancer.

Prevalence and characterization of Clostridium perfringens isolated from feces of captive cynomolgus monkeys (Macaca fascicularis).

Clostridium perfringens is ubiquitous in the environment and the gastrointestinal tract of warm-blooded animals. While part of the gut microbiome, abnormal growth of C. perfringens causes histotoxic, neurologic, and enteric diseases in a variety of animal species, including humans, due to the production of toxins. There is extremely limited information on C. perfringens infection in non-human primates. Presently, 10 strains were successfully isolated from 126 monkeys and confirmed by molecular and biochemical analyses. All isolates were genotype A based on molecular analysis. Alpha toxin was identified in all isolates. Beta 2 toxin was detected in only three isolates. No other toxins, including enterotoxin, beta, iota, epsilon, and net B toxin, were identified in any isolate. All isolates were highly susceptible to ß-lactam antibiotics. Double hemolysis and lecithinase activity were commonly observed in all strains. Biofilm formation, which can increase antibiotic resistance, was identified in 90% of the isolates. The data are the first report the prevalence and characteristics of C. perfringens isolated from captive cynomolgus monkeys.

Chronic Infiltration of T Lymphocytes into the Brain in a Non-human Primate Model of Parkinson's Disease.

Study of interactions between the nervous system and immunity offers insights into the pathogenesis of Parkinson's disease (PD) and potential therapeutic strategies for neurodegenerative diseases. Studies on rodents have revealed regulatory mechanisms of microglial activation and T lymphocyte recruitment in PD. However, the mechanisms underlying chronic T lymphocyte infiltration into the brain after 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) injection into a non-human primate (NHP) model of PD remain unknown. This study aimed to investigate changes in serum RANTES (regulated on activation, normal T cell expression and secretion) and analyze the chronic infiltration of T lymphocytes into the brain and microglia activation in NHPs at 48 weeks post-MPTP administration. We found selective and local chronic infiltration of CD4+ and CD8+ T lymphocytes, loss of dopaminergic neurons, dopamine transporter expression, chronic normalization of RANTES in the peripheral blood, and altered microglial morphology at 48 weeks after MPTP injection. This study confirms the involvement of CD4+ and CD8+ T lymphocyte infiltration in MPTP-induced NHP models of PD. Additionally, we corroborated previous findings regarding the mechanisms of T lymphocyte-induced neurodegeneration. The findings of chronic infiltration of T lymphocytes in our NHP model of PD provide novel insights into PD pathogenesis and the development of preventive and therapeutic agents.

Idiopathic chronic diarrhea associated with dysbiosis in a captive cynomolgus macaque (Macaca fascicularis).

Chronic inflammatory enteric diseases occur commonly in humans and animals, especially in captive bred macaques. However, information about the etiology of idiopathic chronic inflammatory diarrhea in cynomolgus monkeys is limited. In this paper, we reported the unusual case of idiopathic chronic diarrhea in a captive cynomolgus monkey based on microbial, imaging, and microbiome examinations.

A pilot study on assessment of locomotor behavior using a video tracking system in minipigs.

Pigs are often selected for large animal models including for neuroscience and behavioral research, because their anatomy and biochemistry are similar to those of humans. However, behavioral assessments, in combination with objective long-term monitoring, is difficult. In this study, we introduced an automated video tracking system which was previously used in rodent studies, for use with pig models. Locomotor behaviors (total distance, number of zone transitions, and velocity) were evaluated and their changes were validated by different 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) administration methods and dosing regimens. Three minipigs (23-29 kg) received subcutaneous or intravenous MPTP, either 1 or 3 times per week. Immediately after MPTP injection, the minipigs remained in a corner and exhibited reduced trajectory. In addition, the total distance travelled, number of zone transitions, and velocity were greatly reduced at every MPTP administration in all the minipigs, accompanying to increased resting time. However, the MPTP-induced symptoms were reversed when MPTP administration was terminated. In conclusion, this automated video-tracking system was able to monitor long-term locomotor activity and differentiate detailed alterations in large animals. It has the advantages of being easy to use, higher resolution, less effort, and more delicate tracking. Additionally, as our method can be applied to the animals' home pen, no habituation is needed.

Embryo aggregation regulates in vitro stress conditions to promote developmental competence in pigs.

Embryo aggregation is a useful method to produce blastocysts with high developmental competence to generate more offspring in various mammals, but the underlying mechanism(s) regarding the beneficial effects are largely unknown. In this study, we investigated the effects of embryo aggregation using 4-cell stage embryos in in vitro developmental competence and the relationship of stress conditions in porcine early embryogenesis. We conducted aggregation using the well of the well system and confirmed that aggregation using two or three embryos was useful for obtaining blastocysts. Aggregated embryos significantly improved developmental competence, including blastocyst formation rate, blastomere number, ICM/TE ratio, and cellular survival rate, compared to non-aggregated embryos. Investigation into the relationship between embryo aggregation and stress conditions revealed that mitochondrial function increased, and oxidative and endoplasmic reticulum (ER)-stress decreased compared to 1X (non-aggregated embryos) blastocysts. In addition, 3X (three-embryo aggregated) blastocysts increased the expression of pluripotency, anti-apoptosis, and implantation related genes, and decreased expression of pro-apoptosis related genes. Therefore, these findings indicate that embryo aggregation regulates in vitro stress conditions to increase developmental competence and contributes to the in vitro production of high-quality embryos and the large-scale production of transgenic and chimeric pigs.

Abnormal Mitochondria in a Non-human Primate Model of MPTP-induced Parkinson's Disease: Drp1 and CDK5/p25 Signaling.

Mitochondria continuously fuse and divide to maintain homeostasis. An impairment in the balance between the fusion and fission processes can trigger mitochondrial dysfunction. Accumulating evidence suggests that mitochondrial dysfunction is related to neurodegenerative diseases such as Parkinson's disease (PD), with excessive mitochondrial fission in dopaminergic neurons being one of the pathological mechanisms of PD. Here, we investigated the balance between mitochondrial fusion and fission in the substantia nigra of a non-human primate model of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD. We found that MPTP induced shorter and abnormally distributed mitochondria. This phenomenon was accompanied by the activation of dynamin-related protein 1 (Drp1), a mitochondrial fission protein, through increased phosphorylation at S616. Thereafter, we assessed for activation of the components of the cyclin-dependent kinase 5 (CDK5) and extracellular signal-regulated kinase (ERK) signaling cascades, which are known regulators of Drp1(S616) phosphorylation. MPTP induced an increase in p25 and p35, which are required for CDK5 activation. Together, these findings suggest that the phosphorylation of Drp1(S616) by CDK5 is involved in mitochondrial fission in the substantia nigra of a non-human primate model of MPTP-induced PD.

Evaluation of fecal microbiomes associated with obesity in captive cynomolgus monkeys (Macaca fascicularis).

Microorganisms play important roles in obesity; however, the role of the gut microbiomes in obesity is controversial because of the inconsistent findings. This study investigated the gut microbiome communities in obese and lean groups of captive healthy cynomolgus monkeys reared under strict identical environmental conditions, including their diet. No significant differences in the relative abundance of Firmicutes, Bacteroidetes and Prevotella were observed between the obese and lean groups, but a significant difference in Spirochetes (p < 0.05) was noted. Microbial diversity and richness were similar, but highly variable results in microbial composition, diversity, and richness were observed in individuals, irrespective of their state of obesity. Distinct clustering between the groups was not observed by principal coordinate analysis using an unweighted pair group method. Higher sharedness values (95.81% ± 2.28% at the genus level, and 79.54% ± 5.88% at the species level) were identified among individual monkeys. This paper reports the association between the gut microbiome and obesity in captive non-human primate models reared under controlled environments. The relative proportion of Firmicutes and Bacteroidetes as well as the microbial diversity known to affect obesity were similar in the obese and lean groups of monkeys reared under identical conditions. Therefore, obesity-associated microbial changes reported previously appear to be associated directly with environmental factors, particularly diet, rather than obesity.

The effects of kinase modulation on in vitro maturation according to different cumulus-oocyte complex morphologies.

Successful production of transgenic pigs requires oocytes with a high developmental competence. However, cumulus-oocyte complexes (COCs) obtained from antral follicles have a heterogeneous morphology. COCs can be classified into one of two classes: class I, with five or more layers of cumulus cells; and class II, with one or two layers of cumulus cells. Activator [e.g., epidermal growth factor (EGF)] or inhibitors (e.g., wortmannin and U0126) are added to modulate kinases in oocytes during meiosis. In the present study, we investigated the effects of kinase modulation on nuclear and cytoplasmic maturation in COCs. Class I COCs showed a significantly higher developmental competence than class II COCs. Moreover, the expression of two kinases, AKT and ERK, differed between class I and class II COCs during in vitro maturation (IVM). Initially, inhibition of the PI3K/AKT signaling pathway in class I COCs during early IVM (0-22 h) decreased developmental parameters, such as blastocyst formation rate, blastomere number, and cell survival. Conversely, EGF-mediated AKT activation in class II COCs enhanced developmental capacity. Regarding the MAPK signaling pathway, inhibition of ERK by U0126 in class II COCs during early IVM impaired developmental competence. However, transient treatment with U0126 in class II COCs increased oocyte maturation and AKT activity, improving embryonic development. Additionally, western blotting showed that inhibition of ERK activity negatively regulated the AKT signaling pathway, indicative of a relationship between AKT and MAPK signaling in the process underlying meiotic progression in pigs. These findings may help increase the developmental competence and utilization rate of pig COCs with regard to the production of transgenic pigs and improve our understanding of kinase-associated meiosis events.

Chronic Insulin Infusion Down-Regulates Circulating and Urinary Nitric Oxide (NO) Levels Despite Molecular Changes in the Kidney Predicting Greater Endothelial NO Synthase Activity in Mice.

Insulin therapy is often needed to overcome insulin receptor resistance in type 2 diabetes; however, the impact of providing additional insulin to already hyperinsulinemic subjects is not clear. We infused male TALLYHO/Jng (TH) mice (insulin resistant) with insulin (50 U/kg·bw/d) or vehicle (control) by osmotic minipump for 14 days. One group of insulin-infused mice was switched to 4% NaCl diet (high-sodium diet, HSD) in the second week. Blood chemistry revealed a significantly higher anion gap and blood sodium concentrations with insulin infusion, i.e., relative metabolic acidosis. Systolic BP and heart rate were slightly (~5 mm Hg) higher in insulin-infused versus control mice. HSD resulted in a modest and transient rise in mean arterial blood pressure (BP), relative to control or insulin-infused, normal-NaCl-fed mice. In kidney, insulin infusion: (1) increased total and phosphorylated (serine-1177) endothelial nitric oxide synthase (eNOS) band densities; (2) reduced band density of the uncoupled form of eNOS; and (3) increased renal homogenate nitric oxide synthase (NOS) activity. Despite this, plasma and urine levels of nitrates plus nitrites (NOx) fell with insulin infusion, by day 14 (40⁻50%) suggesting worsening of resistance. Overall, insulin infusion ramps up the cellular means in kidney to increase vasodilatory and natriuretic NO, but in the long term may be associated with worsening of insulin receptor resistance.

Absence of renal enlargement in fructose-fed proximal-tubule-select insulin receptor (IR), insulin-like-growth factor receptor (IGF1R) double knockout mice.

The major site of fructose metabolism in the kidney is the proximal tubule (PT). To test whether insulin and/or IGF1 signaling in the PT is involved in renal structural/functional responses to dietary fructose, we bred mice with dual knockout (KO) of the insulin receptor (IR) and the IGF1 receptor (IGF1R) in PT by Cre-lox recombination, using a γ-glutamyl transferase promoter. KO mice had slightly (~10%) reduced body and kidney weights, as well as, a reduction in mean protein-to-DNA ratio in kidney cortex suggesting smaller cell size. Under control diet, IR and IGF1R protein band densities were 30-50% (P < 0.05) lower than WT, and the relative difference was greater in male animals. Male, but not female KO, also had significantly reduced band densities for Akt (protein kinase B), phosphorylated AktT308 and IRY1162/1163 A high-fructose diet (1-month) led to a significant increase in kidney weight in WT males (12%), but not in KO males or in either genotype of female mice. Kidney enlargement in the WT males was accompanied by a small, insignificant fall in protein-to-DNA ratio, supporting hyperplasia rather than hypertrophy. Fructose feeding of male WT mice led to significantly higher sodium bicarbonate exchanger (NBCe1), sodium hydrogen exchanger (NHE3), sodium phosphate co-transporter (NaPi-2), and transforming growth factor-ß (TGF-ß) abundances, as compared to male KO, suggesting elevated transport capacity and an early feature of fibrosis may have accompanied the renal enlargement. Overall, IR and/or IGF1R appear to have a role in PT cell size and enlargement in response to high-fructose diet.

Endoscopic evaluation of gastric emptying and effect of mosapride citrate on gastric emptying.

PURPOSE: Gastric emptying has been evaluated by scintigraphy in spite of its limitations of time consumption, cost, and danger of radioisotope. Endoscopy is a simple technique, however, its validation for gastric emptying and quantification of food has not yet been investigated. The aim of our study was to assess endoscopic gastric emptying compared with scintigraphy and radiopaque markers (ROMs) studies. We also investigated the effect of a single dose of mosapride on gastric emptying. MATERIALS AND METHODS: Fifteen healthy volunteers underwent scintigraphy. Next day, subjects received a standard solid meal with ROMs and underwent endoscopy and simple abdomen X-ray after 3 hrs. After one week, the same procedure was repeated after ingestion of mosapride (5 mg for group 1, n = 8; 10 mg for group 2, n = 7) 15 min before the meal. Quantification of gastric residue by endoscopy was scored from 0 to 3, and the scores were added up. RESULTS: All subjects completed the study without any complication. The gastric emptying rate [T(1/2) (min)] was in normal range (65.6 +/- 12.6 min). Endoscopic gastric emptying was correlated significantly with gastric clearance of ROMs (r = 0.627, p = 0.012). Endoscopic gastric emptying and gastric clearance of ROMs after administration of mosapride showed significant differences in the 10 mg group (p < 0.05). CONCLUSION: Endoscopy can evaluate gastric emptying safely and simply on an outpatient basis. A 10 mg dose of mosapride enhanced gastric emptying, assessed by both endoscopy and ROMs.

[Extrapancreatic tumors in intraductal papillary mucinous neoplasm of the pancreas].

BACKGROUND/AIMS: Intraductal papillary mucinous neoplasm (IPMN) of the pancreas has a favorable prognosis, but seems to be associated with a high incidence of extrapancreatic tumors. The purpose of this study was to evaluate the incidence and clinicopathological features of extrapancreatic tumors associated with IPMN. METHODS: Thirty-seven patients with IPMN of the pancreas, confirmed by surgical resection and typical findings of endoscopic ultrasonography and CT imaging between October 1, 1998 and August 31, 2006 were included. Seventeen patients were diagnosed with surgical resection and biopsy, and others by typical imaging findings of IPMN. These patients were examined for the development of extrapancreatic tumors. RESULTS: Of 37 patients with IPMN, 14 (38%) had 18 extrapancreatic tumors, and 10 (27%) had 13 extrapancreatic malignancies. Five, six, and two extrapancreatic malignancies had been diagnosed before, during, and after the diagnosis of IPMN. Gastric adenocarcinoma (3 patients, 23%) and colorectal carcinoma (3 patients, 23%) were the most common neoplasms. Other extrapancreatic tumors included lung cancer (n=2), prostatic cancer (n=1), renal cell carcinoma (n=1), cholangiocellular carcinoma (n=1), urinary bladder cancer (n=1), and gallbladder cancer (n=1), respectively. As benign tumor, there were two gallbladder adenoma, one gastric adenoma, one colonic adenoma and one benign ovarian cystic neoplasm, respectively. CONCLUSIONS: IPMN is associated with high incidence of extrapancreatic tumors, particularly gastric and colorectal neoplasms. Upper gastrointestinal endoscopy and colonoscopy should be done, and systemic surveillance for the possible occurrence of other tumors may allow early detection of extrapancreatic tumor in patients with IPMN.

[A phase II study of combination chemotherapy with gemcitabine, 5-fluorouracil, and cisplatin for advanced pancreatic cancer].

BACKGROUND/AIMS: Gemcitabine has been the standard regimen for advanced pancreatic cancer, but the effect on the response rate and survival is still disappointing, leading to many trials of combination chemotherapy. 5-FU and cisplatin were combined with gemcitabine in this trial, as they are synergistic with gemcitabine and each other as well. This study was aimed to assess the effectiveness and safety of combination chemotherapy with gemcitabine, 5-FU, and cisplatin for advanced pancreatic cancer. METHODS: Patients with advanced pancreatic cancer were entered into this study. Gemcitabine at a dose of 800 mg/m2 on day 1 and 8, 5-FU 1,000 mg/m2/day from day 1 to 3 for 72 hours, and cisplatin 60 mg/m2 on day 2, 24 hours after the start of gemcitabine were administered every 3 weeks. RESULTS: From December 2001 to January 2004, twenty patients were enrolled in this study. Among 17 of these patients assessable, 3 patients had a partial remission with the response rate of 23.6% (95% confidence interval, 6.2-41.0%). The median time to disease progression was 230 days and median duration of survival was 322 days. Among total of 91 cycles, leukopenia, neutropenia, and thrombocytopenia of grade 3 or 4 occurred in 12 cycles (13.2%), 12 cycles (13.2%), and 23 cycles (24.4%), respectively. Grade 3 or 4 mucositis developed at 2 cycles (2.2%), and nausea and vomiting were encountered in 3 cycles (3.3%). CONCLUSIONS: Combination chemotherapy with gemcitabine, 5-FU, and cisplatin for advanced pancreatic cancer is active and well-tolerated, warranting a phase III study.