Fully Automated Radiosynthesis of [18F]mG4P027 for mGluR4 Imaging.

Fluorine-18 labeled N-(4-chloro-3-(((fluoro-18F)methyl-d2)thio)phenyl)picolinamide, [18F]mG4P027, is a potent positron emission tomography (PET) radiotracer for metabotropic glutamate receptor 4 (mGluR4). Our previous in vitro and in vivo evaluations have demonstrated that this tracer is promising for further translational studies. To automate the radiosynthesis of [18F]mG4P027, significant modifications were made to the manual process by carefully examining this process and addressing the root causes of the challenges associated with its automation. We successfully implemented its automated radiosynthesis using the TRACERlab FX2N module and consequently, obtained a high-purity radiolabeled [18F]mG4P027 in high yield, meeting the requirements for future human studies.

Detailed radiosynthesis of [18 F]mG4P027 as a positron emission tomography radiotracer for mGluR4.

We report here the detailed radiosynthesis of [18 F]mG4P027, a metabotropic glutamate receptor 4 (mGluR4) PET radiotracer, which showed superior properties to the currently reported mGluR4 radiotracers. The radiosynthesis in the automated system has been challenging, therefore we disclose here the major limiting factors for the synthesis via step-by-step examination. And we hope this thorough study will help its automation for human use in the future.

Organomediated cleavage of benzoyl group enables an efficient synthesis of 1-(6-nitropyridin-2-yl)thiourea and its application for developing 18F-labeled PET tracers.

A novel organomediated cleavage of benzoyl group using ethane-1,2-diamine and acetic acid under neutral condition enables an efficient synthesis of 1-(6-nitropyridin-2-yl)thiourea, which previously has been challenging to prepare by conventional methods. The successful synthesis of 1-(6-nitropyridin-2-yl)thiourea as a synthon permits development of a variety of 18F labeled heterocycles as PET imaging ligands such as N-(pyridin-2-yl)thiazol-2-amine derivatives. The utility of this synthon is demonstrated with the synthesis of a 18F-labeled PET tracer for studying prion disease. In vitro autoradiography using this PET tracer on sagittal rat brain slices showed highest accumulation of radioactivity in the hippocampus, cortex, and striatum, in accordance with reported immunostaining of PrPc in rat brain.

Synthesis and Characterization of [18F]JNJ-46356479 as the First 18F-Labeled PET Imaging Ligand for Metabotropic Glutamate Receptor 2.

PURPOSE: Metabotropic glutamate receptor 2 (mGluR2) has been implicated in various psychiatric and neurological disorders, such as schizophrenia and Alzheimer's disease. We have previously developed [11C]7 as a PET radioligand for imaging mGluR2. Herein, [18F]JNJ-46356479 ([18F]8) was synthesized and characterized as the first 18F-labeled mGluR2 imaging ligand to enhance diagnostic approaches for mGluR2-related disorders. PROCEDURES: JNJ-46356479 (8) was radiolabeled via the copper (I)-mediated radiofluorination of organoborane 9. In vivo PET imaging experiments with [18F]8 were conducted first in C57BL/6 J mice and Sprague-Dawley rats to obtain whole body biodistribution and brain uptake profile. Subsequent PET studies were done in a cynomolgus monkey (Macaca fascicularis) to investigate the uptake of [18F]8 in the brain, its metabolic stability, as well as pharmacokinetic properties. RESULTS: JNJ-46356479 (8) exhibited excellent selectivity against other mGluRs. In vivo PET imaging studies showed reversible and specific binding characteristic of [18F]8 in rodents. In the non-human primate, [18F]8 displayed good in vivo metabolic stability, excellent brain permeability, fast and reversible kinetics with moderate heterogeneity across brain regions. Pre-treatment studies with compound 7 revealed time-dependent decrease of [18F]8 accumulation in mGluR2 rich regions based on SUV values with the highest decrease in the nucleus accumbens (18.7 ± 5.9%) followed by the cerebellum (18.0 ± 7.9%), the parietal cortex (16.9 ± 7.8%), and the hippocampus (16.8 ± 6.9%), similar to results obtained in the rat studies. However, the volume of distribution (VT) results derived from 2T4k model showed enhanced VT from a blocking study with compound 7. This is probably because of the potentiating effect of compound 7 as an mGluR2 PAM as well as related non-specific binding in the tissue data. CONCLUSIONS: [18F]8 readily crosses the blood-brain barrier and demonstrates fast and reversible kinetics both in rodents and in a non-human primate. Further investigation of [18F]8 on its binding specificity would warrant translational study in human.

Design, Synthesis, and Characterization of Benzimidazole Derivatives as Positron Emission Tomography Imaging Ligands for Metabotropic Glutamate Receptor 2.

Three benzimidazole derivatives (13-15) have been synthetized as potential positron emission tomography (PET) imaging ligands for mGluR2 in the brain. Of these compounds, 13 exhibits potent binding affinity (IC50 = 7.6 ± 0.9 nM), positive allosteric modulator (PAM) activity (EC50 = 51.2 nM), and excellent selectivity against other mGluR subtypes (>100-fold). [11C]13 was synthesized via O-[11C]methylation of its phenol precursor 25 with [11C]methyl iodide. The achieved radiochemical yield was 20 ± 2% (n = 10, decay-corrected) based on [11C]CO2 with a radiochemical purity of >98% and molar activity of 98 ± 30 GBq/µmol EOS. Ex vivo biodistribution studies revealed reversible accumulation of [11C]13 and hepatobiliary and urinary excretions. PET imaging studies in rats demonstrated that [11C]13 accumulated in the mGluR2-rich brain regions. Pre-administration of mGluR2-selective PAM, 17 reduced the brain uptake of [11C]13, indicating a selective binding. Therefore, [11C]13 is a potential PET imaging ligand for mGluR2 in different central nervous system-related conditions.

Current Progress and Perspective: Clinical Imaging of Islet Transplantation.

Islet transplantation has great potential as a cure for type 1 diabetes. At present; the lack of a clinically validated non-invasive imaging method to track islet grafts limits the success of this treatment. Some major clinical imaging modalities and various molecular probes, which have been studied for non-invasive monitoring of transplanted islets, could potentially fulfill the goal of understanding pathophysiology of the functional status and viability of the islet grafts. In this current review, we summarize the recent clinical studies of a variety of imaging modalities and molecular probes for non-invasive imaging of transplanted beta cell mass. This review also includes discussions on in vivo detection of endogenous beta cell mass using clinical imaging modalities and various molecular probes, which will be useful for longitudinally detecting the status of islet transplantation in Type 1 diabetic patients. For the conclusion and perspectives, we highlight the applications of multimodality and novel imaging methods in islet transplantation.

Synthesis and Characterization of Fluorine-18-Labeled N-(4-Chloro-3-((fluoromethyl-d2)thio)phenyl)picolinamide for Imaging of mGluR4 in Brain.

We have synthesized and characterized [18F]-N-(4-chloro-3-((fluoromethyl-d2)thio)phenyl)-picolinamide ([18F]15) as a potential ligand for the positron emission tomography (PET) imaging of mGluR4 in the brain. Radioligand [18F]15 displays central nervous system drug-like properties, including mGluR4 affinity, potent mGluR4 PAM activity, and selectivity against other mGluRs, as well as sufficient metabolic stability. Radiosynthesis was carried out in two steps. The radiochemical yield of [18F]15 was 11.6 ± 2.9% (n = 7, decay corrected) with a purity of 99% and a molar activity of 84.1 ± 11.8 GBq/µmol. Ex vivo biodistribution studies showed reversible binding of [18F]15 in all investigated tissues including the brain, liver, heart, lungs, and kidneys. PET imaging studies in male Sprague Dawley rats showed that [18F]15 accumulates in the brain regions known to express mGluR4. Pretreatment with the unlabeled mGluR4 PAM compounds 13 (methylthio analogue) and 15 showed significant dose-dependent blocking effects. These results suggest that [18F]15 is a promising radioligand for PET imaging mGluR4 in the brain.

Efficient synthesis of NIR emitting bis[2-(2'-hydroxylphenyl)benzoxazole] derivative and its potential for imaging applications.

Unassymetric bis[2-(2'-hydroxyphenylbenzoxole)] bis(HBO) derivatives with a DPA functionality for zinc binding have been developed with an efficient synthetic route, using the retrosynthetic analysis. Comparison of bis(HBO) derivatives with different substitution patterns allows us to verify and optimize their unique fluorescence properties. Upon binding zinc cation, bis(HBO) derivatives give a large fluorescence turn-on in both visible (λem ≈ 536 nm) and near-infrared (NIR) window (λem ≈ 746 nm). The probes are readily excitable by a 488 nm laser, making this series of compounds a suitable imaging tool for in vitro and in vivo study on a confocal microscope. The application of zinc binding-induced fluorescence turn-on is successfully demonstrated in cellular environments and thrombus imaging.

Improved Synthesis of the Thiophenol Precursor N-(4-Chloro-3-mercaptophenyl)picolinamide for Making the mGluR4 PET Ligand.

Recently [11C]mG4P012 (previously [11C]KALB012 and presently named as [11C]PXT012253 by Prexton Therapeutics) had been used as a biomarker during the preclinical development of a potential therapeutic drug, PXT0002331 (an mGluR4 PAM), for PD and L-dopa-induced dyskinesia. [11C]mG4P012 was shown to be a promising PET radioligand for mGluR4 in the monkey brain and for further development in human subjects. However, the previously reported multi-step synthesis of the thiophenol precursor suffered from low yields and difficult workup procedures. To support the translational research of [11C]mG4P012 and the other potential applications, we have developed a new route for synthesis of the thiophenol precursor and optimized the reaction conditions. The synthesis of N-(4-chloro-3-mercaptophenyl)picolinamide from 1-chloro-4-nitrobenzene has been greatly improved from 8% to 52% total yield with easy handling and in gram scales.

[Effects of RAGE on Cell Proliferation and Tumor Growth in Pancreatic Cancer].

OBJECTIVES: To investigate the effect of receptor for advanced glycation end products (RAGE) on cell proliferation and tumor growth in nude mice with pancreatic cancer. METHODS: PANC-1 cells were transfected with shRNA RAGE -1, -2, -3 to down-regulate the expression of RAGE. Cholecystokinin octopeptide-8 (CCK-8), real-time PCR and Western blot were performed to test the impact of shRNA RAGE on the expressions of mRNAs and proteins of RAGE, matrix metalloproteinase-2 (MMP-2), MMP-9, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), and vascular endothelial growth factor (VEGF). Tumor growth and microvessel density in the nude mice implanted with shRNA RAGE transfected PANC-1 cells were observed using immunohistochemistry. RESULTS: The shRNA RAGE -1, -2, -3 transfected cells had lower absorbance values than the controls 24 h after transfection, and the absorbance value reached the lowest at 48 h. The specific shRNA sequences significantly inhibited the expressions of mRNA and protein of RAGE. The mice implanted with shRNA RAGE -2 had lower tumor volume and microvessel density than shRNA RAGE -1, -3. The expressions of mRNAs and proteins of RAGE, MMP-2, NF-κB, MMP-9 and VEGF were lower in the cells transfected with shRNA RAGE -2 compared with shRNA RAGE -1, -3. CONCLUSIONS: RAGE is involved in the progression of pancreatic cancerin vitro and in vivo . The RAGE expression could influence the process of tumor angiogenesis.

Transjugular intrahepatic portosystemic shunt versus open splenectomy and esophagogastric devascularization for portal hypertension with recurrent variceal bleeding.

BACKGROUND: Transjugular intrahepatic portosystemic shunt (TIPS) and open splenectomy and esophagogastric devascularization (OSED) are widely used to treat patients with portal hypertension and recurrent variceal bleeding (PHRVB). This study aimed to compare the effectiveness between TIPS and OSED for the treatment of PHRVB. METHODS: The data were retrospectively retrieved from 479 cirrhotic patients (Child-Pugh A or B class) with PHRVB, who had undergone TIPS (TIPS group) or OSED (OSED group) between January 1, 2010 and October 31, 2014. RESULTS: A total of 196 patients received TIPS, whereas 283 underwent OSED. Within one month after TIPS and OSED, the rebleeding rates were 6.1% and 3.2%, respectively (P=0.122). Significantly lower incidence of pleural effusion, splenic vein thrombosis, and pulmonary infection, as well as higher hepatic encephalopathy rate, shorter postoperative length of hospital stay, and higher hospital costs were observed in the TIPS group than those in the OSED group. During the follow-up periods (29 months), significantly higher incidences of rebleeding (15.3% vs 4.6%, P=0.001) and hepatic encephalopathy (17.3% vs 3.9%, P=0.001) were observed in the TIPS group than in the OSED group. The incidence of in-stent stenosis was 18.9%. The survival rates were 91.3% in the TIPS group and 95.1% in the OSED group. The long-term liver function did not worsen after either TIPS or OSED. CONCLUSION: For the patients with liver function in the Child-Pugh A or B class, TIPS is not superior over OSED in terms of PHRVB treatment and rebleeding prevention.

Early complications after interventions in patients with acute pancreatitis.

AIM: To identify the possible predictors of early complications after the initial intervention in acute necrotizing pancreatitis. METHODS: We collected the medical records of 334 patients with acute necrotizing pancreatitis who received initial intervention in our center. Complications associated with predictors were analyzed. RESULTS: The postoperative mortality rate was 16% (53/334). Up to 31% of patients were successfully treated with percutaneous catheter drainage alone. The rates of intra-abdominal bleeding, colonic fistula, and progressive infection were 15% (50/334), 20% (68/334), and 26% (87/334), respectively. Multivariate analysis indicated that Marshall score upon admission, multiple organ failure, preoperative respiratory infection, and sepsis were the predictors of postoperative progressive infection (P < 0.05). Single organ failure, systemic inflammatory response syndrome upon admission, and C-reactive protein level upon admission were the risk factors of postoperative colonic fistula (P < 0.05). Moreover, preoperative Marshall score, organ failure, sepsis, and preoperative systemic inflammatory response syndrome were the risk factors of postoperative intra-abdominal bleeding (P < 0.05). CONCLUSION: Marshall score, organ failures, preoperative respiratory infection, sepsis, preoperative systemic inflammatory response syndrome, and C-reactive protein level upon admission are associated with postoperative complications.

Laparoscopic Splenectomy for the Elderly Liver Cirrhotic Patients With Hypersplenism: A Retrospective Comparable Study.

A growing body of evidence has supported the benefits of laparoscopic splenectomy (LS) for hypersplenism due to liver cirrhosis. With the increased proportion of elderly persons worldwide, it is necessary to investigate the risks and benefits of LS in elderly liver cirrhotic patients.From September 2003 to March 2012, LS and open splenectomy (OS) were performed for 21 (Group 1) and 19 (Group 3) patients, respectively, all of whom were 65 years of age and older; in addition, 39 patients who were <65 years old were treated with LS and referred to as Group 2. Data (i.e., demographic characteristics and preoperative, intraoperative, and postoperative information) were retrospectively collected. Between-group comparisons were performed for the above-mentioned data.Compared with the patients in Group 3, the patients in Group 1 required longer operative times, fewer transfusions, less intensive care, a shorter postoperative course, and a shorter time to the first oral intake, and they had less blood loss and fewer postoperative short-term complications. During the follow-up period, compared with the preoperative status, significant changes in hemoglobin, leukocyte, platelet, and albumin levels were observed in all groups, whereas changes in the total BILirubin (BIL), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) levels were inconspicuous.Patients >65 years of age with hypersplenism caused by liver cirrhosis can safely undergo LS.

Learning Curve for Laparoscopic Pancreaticoduodenectomy: a CUSUM Analysis.

BACKGROUND: Laparoscopic pancreaticoduodenectomy (LPD), an advanced minimally invasive technique, has demonstrated advantages to open pancreaticoduodenectomy (OPD). However, this complex procedure requires a relatively long training period to ensure technical proficiency. This study was therefore designed to analyze the learning curve for LPD. METHODS: From October 2010 to September 2015, 63 standard pancreaticoduodenectomy procedures were to be performed laparoscopically by a single surgeon at the Department of Pancreatic Surgery, West China Hospital, Sichuan University, China. After applying the inclusion and exclusion criteria, a total of 57 patients were included in the study. Data for all the patients, including preoperative, intraoperative, and postoperative variables, were prospectively collected and analyzed. The learning curve for LPD was evaluated using both cumulative sum (CUSUM) and risk-adjusted CUSUM (RA-CUSUM) methods. All of the variables among the learning curve phases were compared. RESULTS: Based on the CUSUM and the RA-CUSUM analyses, the learning curve for LPD was grouped into three phases: phase I was the initial learning period (cases 1-11), phase II represented the technical competence period (cases 12-38), and phase III was regarded as the challenging period (cases 39-57). The operative time, intraoperative blood loss, and postoperative ICU demand significantly decreased with the learning curve. More lymph nodes were collected after the initial learning period. There were no significant differences in terms of postoperative complications or the 30-day mortality among the three phases. More challenging cases were encountered in phase III. CONCLUSIONS: According to this study, the learning curve for LPD consisted of three phases. Conservatively, to attain technical competence for performing LPD, a minimum of 40 cases are required for laparoscopic surgeons with a degree of laparoscopic experience.

Regulating the expression of CD80/CD86 on dendritic cells to induce immune tolerance after xeno-islet transplantation.

BACKGROUND: Antigen present cells (APCs) have been demonstrated to play dual roles in immune tolerance. Recently, compelling evidence indicates that APCs that express CD80, but not CD86 can protect allograft. We investigated whether modulation of CD80 in dendritic cells (DCs) offer protection for xeno-islets. METHODS: In vitro, isolated mature murine DCs received untransfection, transfection with CD86 siRNA or negative control siRNA. The DCs were used in mixed lymphocyte reaction in which rat islets and murine splenocytes were further added. On day 3 of co-culturing, the proliferation of lymphocytes was measured and interleukin (IL)-2, IL-4, IL-10, transforming growth factor ß (TGF-ß), interferon γ (INF-γ) and indoleamine 2,3-dioxygenase (IDO) from the supernatants were determined. Islets viability and function were also assessed. In vivo, streptozotocin-induced diabetic mice underwent rat islets transplantation were pre-treated with above DCs. At designated time, xeno-islets were subjected to histopathology, immunohistochemistry, survival time and functional tests. Peripheral blood T lymphocyte profiles were also examined. RESULTS: CD86-silenced-DCs had unchanged expression of CD80 and significantly suppressed the proliferation of lymphocytes. CD86-silenced-DCs simultaneously reduced IL-2 and INF-γ and increased IL-10, TGF-ß and IDO, while had minimal effect on IL-4. The CD86-silenced-DCs also improved cell viability and function of xeno-islets when compared to untransfection and transfection control groups. In xeno-islets transplanted diabetic mice, transfer of CD86-silenced-DCs resulted in improved histopathology and dramatically prolonged survival time of the islets. These effects were also mirrored by the functional tests. Further analysis revealed that CD86-silenced-DCs had up-regulated levels of CD4(+)CD25(+)T cells in the peripheral blood compared to the other groups. CONCLUSIONS: CD86-silenced-DCs induced immune tolerance of rat xeno-islets in recipient diabetic mice with up-regulated peripheral blood CD4(+)CD25(+)T cells.

Synthesis and evaluation of N-(methylthiophenyl)picolinamide derivatives as PET radioligands for metabotropic glutamate receptor subtype 4.

In recent years, mGlu4 has received great research attention because of the potential benefits of mGlu4 activation in treating numerous brain disorders, such as Parkinson's disease (PD). A specific mGlu4 PET radioligand could be an important tool in understanding the role of mGlu4 in both healthy and disease conditions, and also for the development of new drugs. In this study, we synthesized four new N-(methylthiophenyl)picolinamide derivatives 11-14. Of these ligands, 11 and 14 showed high in vitro binding affinity for mGlu4 with IC50 values of 3.4nM and 3.1nM, respectively, and suitable physicochemical parameters. Compound 11 also showed enhanced metabolic stability and good selectivity to other mGluRs. [(11)C]11 and [(11)C]14 were radiolabeled using the [(11)C]methylation of the thiophenol precursors 20a and 20c with [(11)C]CH3I in 19.0% and 34.8% radiochemical yields (RCY), and their specific activities at the end of synthesis (EOS) were 496±138GBq/µmol (n=6) and 463±263GBq/µmol (n=4), respectively. The PET studies showed that [(11)C]11 accumulated fast into the brain and had higher uptake, slower washout and 25% better contrast than [(11)C]2, indicating improved imaging characteristics as PET radiotracer for mGlu4 compared to [(11)C]2. Therefore, [(11)C]11 will be a useful radioligand to investigate mGlu4 in different biological applications.

Functional modulation of G-protein coupled receptors during Parkinson disease-like neurodegeneration.

G-protein coupled dopamine and metabotropic glutamate receptors (mGlu) can modulate neurotransmission during Parkinson's disease (PD)-like neurodegeneration. PET imaging studies in a unilateral dopamine denervation model (6-OHDA) showed a significant inverse correlation of presynaptic mGlu4 and postsynaptic mGlu5 expression in the striatum and rapidly declining mGlu4 and enhanced mGlu5 expression in the hippocampus during progressive degeneration over time. Immunohistochemical studies verified the decreased mGlu4 expression in the hippocampus on the lesion side but did not show difference in mGlu5 expression between lesion and control side. Pharmacological MRI studies showed enhanced hemodynamic response in several brain areas on the lesion side compared to the control side after challenge with mGlu4 positive allosteric modulator or mGlu5 negative allosteric modulator. However, mGlu4 response was biphasic having short enhancement followed by negative response on both sides of brain. Studies in mGlu4 expressing cells demonstrated that glutamate induces cooperative increase in binding of mGlu4 ligands - especially at high glutamate levels consistent with in vivo concentration. This suggests that mGlu allosteric modulators as drug candidates will be highly sensitive to changes in glutamate concentration and hence metabolic state. These experiments demonstrate the importance of the longitudinal imaging studies to investigate temporal changes in receptor functions to obtain individual response for experimental drugs.

Clinical outcomes of combined necrotizing pancreatitis versus extrapancreatic necrosis alone.

INTRODUCTION: Extrapancreatic necrosis (EPN) alone, i.e., in the absence of pancreatic parenchyma necrosis has gradually come to be regarded as a separate entity of acute necrotizing pancreatitis (ANP). However, data regarding the prognostic significance of EPN are quite limited, and the outcomes of interventions for patients with EPN alone are not well elucidated. The aim of this study was to explore the differences in the outcomes of patients with EPN alone and patients with both the pancreatic parenchyma and extrapancreatic necrosis (combined necrosis). METHODS: From January 2009 to December 2013, a total of 334 patients with ANP who had received interventions in the West China Hospital in China were included. Based on the extent of necrosis as assessed with contrast-enhanced CT, the patients were divided into Group 1 (n = 285) in which the necrosis involved both the pancreatic parenchyma and extrapancreatic tissues (combined necrosis) and Group 2 (n = 49) in which the necrosis involved only the extrapancreatic tissues. Additionally, Group 3 included 443 patients with interstitial pancreatitis who were also included in the analyses. The demographic characteristics, support treatment information, organ failure information, infection necrosis, persistent systemic inflammatory response syndrome (SIRS) in the first week of onset, CT severity index, and intervention types, as well as the postoperative stay lengths, ICU utility, and complications were collected and compared. RESULTS: Compared with the patients in Group 1, the patients in Group 2 suffered less persistent SIRS in the first week of onset (12/24.5% vs. 145/50.9%; P < 0.05), less persistent organ failure (6/12.2% vs. 95/33.3%; P < 0.05), less persistent multiple organ failure (3/6.1% vs. 67/23.5%; P < 0.05), and less bacteremia (5/10.2% vs. 107/37.5%; P < 0.001). The intervention types were significantly different between the two groups (P < 0.001); initial open necrosectomy was performed in 174/61.6% and 8/16.3% of the patients in Groups 1 and 2, respectively, and initial percutaneous catheter drainage (PCD) was performed in 73/25.6% and 29/59.2% of the patients in the two respective groups. Second open necrosectomies following PCD were required in 61/83.5% and 9/31.0% of the patients in Groups 1 and 2, respectively (P < 0.001). A greater number of patients in Group 1 were diagnosed with infected necrosis (204/71.6% vs. 10/20.4%; P < 0.001) and had to be sent to the ICU for further postoperative care (221/77.5% vs. 23/46.9%; P < 0.001). The postoperative stay was longer for Group 1 (median: 43.0 vs. 26.5 days; P < 0.001). Residual necrotic tissue or abscess was the most common postoperative complication in both groups. The mortality was higher in Group 1 (52/18.2% vs. 1/2.1%; P < 0.05). Compared with the patients in Group 2, the patients with interstitial pancreatitis exhibited milder courses and better outcomes. Subgroup comparisons with Group 1 indicated that early multiple organ failure was significantly associated with higher mortality. CONCLUSION: The patients with EPN alone exhibited significantly better prognoses than those with combined necrosis, and EPN alone should be regarded as a separate group of acute necrotizing pancreatitis. Open necrosectomy can be avoided in the majority of patients with EPN alone, who receive PCD as the initial first intervention.

[Effect of CD86 Gene Modified Recipient Dendritic Cell on Mix Cultured Donor-derived Islet with Recipient-derived Lymphocyte in vitro].

OBJECTIVE: To investigate the effect of CD86 gene modified recipient dendritic cell (DC) on mix cultured donor-derived islet with recipient-derived lymphocyte in vitro. METHODS: DCs were separated from bone marrow of BALB/c mice and identified by flow cytometry. Chemically synthesized CD86 siRNA was transferred into DC. Donor islets were separated from the pancreas of SD rats. Acridine orange (AO)/Propidium iodide (PI) staining was conducted to assess the viability of islets. Lymphocytes were collected from the spleen of SD rats and then co-cultured with CD86 gene modified recipient DCs. CD86 gene modified recipient DC, donor-derived islet (400 IEQ) and recipient-derived lymphocyte (1 x 10(6)) were mix cultured in vitro. Four groups were set: blank group (islets of SD rat only), control 1 group (islets of SD rat with splenic lymphocyte of BALB/c mice) , control 2 group (islets of SD rat, splenic lymphocyte of BALB/c mice with normal recipient DC) and experimental group (islets of rat, splenic lymphocyte of BALB/c mice with CD86 gene modified recipient DC). After 3 days culture, the cellular morphology of culture was observed with light inverted microscope. The levels of IL-2, IL-4, IL-10 and IFN-γ in the culture supernatant were tested, and islets viability was assessed by AO/PI staining. GSIS was conducted and stimulation index (SD was calculated. RESULTS: Typical DC morphology was found from the collected cells. The positive rates of CD1lc, CD80 and CD86 protein expression on DCs were 86.26% ± 9.73%, 72.64% ± 8.55% and 77.18% ± 10.23%, respectively. The positive rate of CD86 protein expression on DCs after transfection was 23.64% ± 5.25%. The viability of islets was over 95%. After 3 days culture, the level of IL-10 increased significantly and the levels of IL-2 and INF-γ decreased significantly in experimental group (vs. control 1 and control 2 groups, P < 0.05). The level of IL-4 was similar in control 1, control 2 and experimental groups, but the proliferation rate of lymphocyte in the experimental group was the lowest one, the viability of islets in the experimental group was the best and the SI was the highest. The levels of IL-2, IL-4, IL-10 and IFN-γ in the experimental group were higher than those in the blank group. CONCLUSION: CD86 gene modified recipient DC loaded with donor-derived antigen could protect the islet function in vitro to some extent.

Re-exploring the N-phenylpicolinamide derivatives to develop mGlu4 ligands with improved affinity and in vitro microsomal stability.

In recent years, mGlu4 has received great attention and research effort because of the potential benefits of mGlu4 activation in treating numerous brain disorders, such as Parkinson's disease (PD). Many positive allosteric modulators of mGlu4 have been developed. To better understand the role of mGlu4 in healthy and disease conditions, we are interested in developing an mGlu4 selective radioligand for in vivo studies. Thus, we had synthesized and studied [(11)C]2 as a PET tracer for mGlu4, which demonstrated some promising features as a PET radioligand as well as the limitation need to be improved. In order to develop an mGlu4 ligand with enhanced affinity and improved metabolic stability, we have modified, synthesized and evaluated a series of new N-phenylpicolinamide derivatives. The SAR study has discovered a number of compounds with low nM affinity to mGlu4. The dideuteriumfluoromethoxy modified compound 24 is identified as a very promising mGlu4 ligand, which has demonstrated enhanced affinity, improved in vitro microsomal stability, good selectivity and good permeability.