Sitagliptin improved glucose assimilation in detriment of fatty-acid utilization in experimental type-II diabetes: role of GLP-1 isoforms in Glut4 receptor trafficking.

BACKGROUND: The distribution of glucose and fatty-acid transporters in the heart is crucial for energy consecution and myocardial function. In this sense, the glucagon-like peptide-1 (GLP-1) enhancer, sitagliptin, improves glucose homeostasis but it could also trigger direct cardioprotective actions, including regulation of energy substrate utilization. METHODS: Type-II diabetic GK (Goto-Kakizaki), sitagliptin-treated GK (10 mg/kg/day) and wistar rats (n = 10, each) underwent echocardiographic evaluation, and positron emission tomography scanning for [18F]-2-fluoro-2-deoxy-D-glucose (18FDG). Hearts and plasma were isolated for biochemical approaches. Cultured cardiomyocytes were examined for receptor distribution after incretin stimulation in high fatty acid or high glucose media. RESULTS: Untreated GK rats exhibited hyperglycemia, hyperlipidemia, insulin resistance, and plasma GLP-1 reduction. Moreover, GK myocardium decreased 18FDG assimilation and diastolic dysfunction. However, sitagliptin improved hyperglycemia, insulin resistance, and GLP-1 levels, and additionally, enhanced 18FDG uptake and diastolic function. Sitagliptin also stimulated the sarcolemmal translocation of the glucose transporter-4 (Glut4), in detriment of the fatty acyl translocase (FAT)/CD36. In fact, Glut4 mRNA expression and sarcolemmal translocation were also increased after GLP-1 stimulation in high-fatty acid incubated cardiomyocytes. PI3K/Akt and AMPKα were involved in this response. Intriguingly, the GLP-1 degradation metabolite, GLP-1(9-36), showed similar effects. CONCLUSIONS: Besides of its anti-hyperglycemic effect, sitagliptin-enhanced GLP-1 may ameliorate diastolic dysfunction in type-II diabetes by shifting fatty acid to glucose utilization in the cardiomyocyte, and thus, improving cardiac efficiency and reducing lipolysis.

Inorganic oxides with potential application in the preparation of a 68Ge/68Ga generator system.

The ion exchange properties of some tin and titanium oxides with potential application in the development of a 68Ge/68Ga generator were determined. The best potential candidates, SnO2 and calcined SnO2, were further characterized by powder X-Ray Diffraction (XRD), Scanning Electron Microscopy (SEM) and Brunauer-Emmett-Teller (BET) surface area analysis and its radiation stability was also determined. Two 68Ge/68Ga pilot generators (1.85MBq) based on SnO2 and calcined SnO2 were developed and evaluated over 100 and 200 elution cycles respectively, using as eluent different concentrations of HCl. The generator based on calcined SnO2 showed higher 68Ga elution yield and lower 68Ge content in the eluate (75-80% and <3×10-3% respectively, 1-2M HCl) than the generator based on unheated SnO2 (60-65% and <1×10-1% respectively, 1-2M HCl). Nano-crystalline calcined SnO2 proved to be a promising sorbent; therefore it should be considered as an attractive candidate to develop 68Ge/68Ga generators to produce gallium-68 for biomedical purposes.

Targeting MT1-MMP as an ImmunoPET-Based Strategy for Imaging Gliomas.

BACKGROUND: A critical challenge in the management of Glioblastoma Multiforme (GBM) tumors is the accurate diagnosis and assessment of tumor progression in a noninvasive manner. We have identified Membrane-type 1 matrix metalloproteinase (MT1-MMP) as an attractive biomarker for GBM imaging since this protein is actively involved in tumor growth and progression, correlates with tumor grade and is closely associated with poor prognosis in GBM patients. Here, we report the development of an immunoPET tracer for effective detection of MT1-MMP in GBM models. METHODS: An anti-human MT1-MMP monoclonal antibody (mAb), LEM2/15, was conjugated to p-isothiocyanatobenzyl-desferrioxamine (DFO-NCS) for 89Zr labeling. Biodistribution and PET imaging studies were performed in xenograft mice bearing human GBM cells (U251) expressing MT1-MMP and non-expressing breast carcinoma cells (MCF-7) as negative control. Two orthotopic brain GBM models, patient-derived neurospheres (TS543) and U251 cells, with different degrees of blood-brain barrier (BBB) disruption were also used for PET imaging experiments. RESULTS: 89Zr labeling of DFO-LEM2/15 was achieved with high yield (>90%) and specific activity (78.5 MBq/mg). Biodistribution experiments indicated that 89Zr-DFO-LEM2/15 showed excellent potential as a radiotracer for detection of MT1-MMP positive GBM tumors. PET imaging also indicated a specific and prominent 89Zr-DFO-LEM2/15 uptake in MT1-MMP+ U251 GBM tumors compared to MT1-MMP- MCF-7 breast tumors. Results obtained in orthotopic brain GBM models revealed a high dependence of a disrupted BBB for tracer penetrance into tumors. 89Zr-DFO-LEM2/15 showed much higher accumulation in TS543 tumors with a highly disrupted BBB than in U251 orthotopic model in which the BBB permeability was only partially increased. Histological analysis confirmed the specificity of the immunoconjugate in all GBM models. CONCLUSION: A new anti MT1-MMP-mAb tracer, 89Zr-DFO-LEM2/15, was synthesized efficiently. In vivo validation showed high-specific-contrast imaging of MT1-MMP positive GBM tumors and provided strong evidence for utility of MT1-MMP-targeted immunoPET as an alternate to nonspecific imaging of GBM.

P38MAPK is a major determinant of the balance between apoptosis and autophagy triggered by 5-fluorouracil: implication in resistance.

5-Fluorouracil (5-FU), together with other drugs such as oxaliplatin, is one of the most important pharmacological agents in the treatment of colorectal cancer. Although mitogen-activated protein kinases (MAPKs) have been extensively connected with resistance to platinum compounds, no role has been established in 5-FU resistance. Here we demonstrate that p38MAPK activation is a key determinant in the cellular response to 5-FU. Thus, inhibition of p38MAPKα by SB203580 compound or by short-hairpin RNA interference-specific knockdown correlates with a decrease in the 5-FU-associated apoptosis and chemical resistance in both HaCaT and HCT116 cells. Activation of p38MAPK by 5-FU was dependent on canonical MAP2K, MAPK kinase (MKK)-3 and MKK6. In addition, ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3 related (ATR) showed a redundancy of function for the final activation of p38MAPK. Resistance associated with p38MAPK inhibition correlates with an autophagic response that was mediated by a decrease in p53-driven apoptosis, without effect onto p53-dependent autophagy. Moreover, the results with colorectal cancer-derived cell lines with different p53 status and patterns of resistance to 5-FU suggest that de novo and acquired resistance was controlled by similar mechanisms. In summary, our data demonstrate a critical role for the p38MAPK signaling pathway in the cellular response to 5-FU by controlling the balance between apoptosis and autophagy.

Feasibility of adequate resectable rectal cancer treatment in a third-level hospital.

PURPOSE: The aim of this study was to determine the feasibility, concerning compliance to protocol and recommended clinical practice guidelines, as well as efficacy results of multidisciplinary treatment (surgery, radiotherapy and chemotherapy) of resectable rectal cancer in a third-level hospital devoid of radiotherapy and clinical oncology units. PATIENTS AND METHODS: A retrospective, single-institution analysis was completed for 45 consecutive patients diagnosed with resectable rectal cancer who entered an officially proposed multidisciplinary treatment protocol from October 1998 to September 2003. Adequacy of patient inclusion, according to clinical stage, was reviewed. Neoadjuvant radiotherapy schedule, surgery procedures and adjuvant chemotherapy indication were assessed. All treatment time intervals were analysed. Finally, efficacy results are discussed and contextualised by comparison with results of clinical trials which support this treatment strategy. RESULTS: According to an independent board review, 3 patients (6.7%) with stage I rectal cancer, 31 patients (68.9%) with stage II and 11 patients (24.4%) with stage III rectal cancer were included. Radiotherapy dosage, volume and schedule were as planned. Median time from diagnosis to start of radiotherapy was 26.36 days (24.26- 28.57; CI 95%). Median duration of radiotherapy was 6.00 days (5.56-6.44; CI 95%). Median time from start of radiotherapy to surgery was 15.67 days (14.47-16.87; CI 95%). Median time from completion of radiotherapy to surgery was 10.67 days (9.53-11.81; CI 95%). Most of the patients underwent low anterior resection [23 patients (51.2%)] and abdominoperineal resection [16 patients (35.6%)]. Correlation between clinical and pathologic staging was as expected. Twenty-nine patients (64.4%) of the 45 that were initially included started adjuvant chemotherapy. A statistically significant relationship between pathologic stage (grouped I-II vs. III) and the use of adjuvant chemotherapy was found (p=0.033; chi-square test). Radiotherapy- and chemotherapy-induced toxicity did not differ from that previously reported. With a median follow-up of 65.46 months, a total of 10 recurrences have been diagnosed, all of them in stage III patients. Overall survival rate at five years was 76% for the complete population included. CONCLUSION: Multidisciplinary treatment of resectable rectal cancer in a third-level hospital is feasible. Although efficacy results are comparable to those previously reported in the literature, further improvements in clinical staging as well as in adjuvant chemotherapy indication are desirable.

Feasibility of adequate resectable rectal cancer treatment in a third-level hospital

PURPOSE: The aim of this study was to determine the feasibility, concerning compliance to protocol and recommended clinical practice guidelines, as well as efficacy results of multidisciplinary treatment (surgery, radiotherapy and chemotherapy) of resectable rectal cancer in a third-level hospital devoid of radiotherapy and clinical oncology units. PATIENTS AND METHODS: A retrospective, single-institution analysis was completed for 45 consecutive patients diagnosed with resectable rectal cancer who entered an officially proposed multidisciplinary treatment protocol from October 1998 to September 2003. Adequacy of patient inclusion, according to clinical stage, was reviewed. Neoadjuvant radiotherapy schedule, surgery procedures and adjuvant chemotherapy indication were assessed. All treatment time intervals were analysed. Finally, efficacy results are discussed and contextualised by comparison with results of clinical trials which support this treatment strategy. RESULTS: According to an independent board review, 3 patients (6.7%) with stage I rectal cancer, 31 patients (68.9%) with stage II and 11 patients (24.4%) with stage III rectal cancer were included. Radiotherapy dosage, volume and schedule were as planned. Median time from diagnosis to start of radiotherapy was 26.36 days (24.26- 28.57; CI 95%). Median duration of radiotherapy was 6.00 days (5.56-6.44; CI 95%). Median time from start of radiotherapy to surgery was 15.67 days (14.47-16.87; CI 95%). Median time from completion of radiotherapy to surgery was 10.67 days (9.53-11.81; CI 95%). Most of the patients underwent low anterior resection [23 patients (51.2%)] and abdominoperineal resection [16 patients (35.6%)]. Correlation between clinical and pathologic staging was as expected. Twenty-nine patients (64.4%) of the 45 that were initially included started adjuvant chemotherapy. A statistically significant relationship between pathologic stage (grouped I-II vs. III) and the use of adjuvant chemotherapy was found (p=0.033; chi-square test). Radiotherapy- and chemotherapy-induced toxicity did not differ from that previously reported. With a median follow-up of 65.46 months, a total of 10 recurrences have been diagnosed, all of them in stage III patients. Overall survival rate at five years was 76% for the complete population included. CONCLUSION: Multidisciplinary treatment of resectable rectal cancer in a third-level hospital is feasible. Although efficacy results are comparable to those previously reported in the literature, further improvements in clinical staging as well as in adjuvant chemotherapy indication are desirable (AU)

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Inositol hexaphosphate: a potential chelating agent for uranium.

Chelation therapy is an optimal method to reduce the radionuclide-related risks. In the case of uranium incorporation, the treatment of choice is so far i.v infusion of a 1.4% sodium bicarbonate solution, but the efficacy has been proved to be not very high. In this study, we examine the efficacy of some substances: bicarbonate, citrate, diethylenetriamine pentaacetic acid (DTPA), ethidronate (EHBP) and inositol hexaphosphate (phytic acid) to chelate uranium using a test developed by Braun et al. Different concentrations of phytic acid, an abundant component of plant seeds that is widely distributed in animal cells and tissues in substantial levels, were tested and compared to the same concentrations of sodium citrate, bicarbonate, EHBP and DTPA. The results showed a strong affinity of inositol hexaphosphate for uranium, suggesting that it could be an effective chelating agent for uranium in vivo.

TIARA: treatment initiatives after radiological accidents.

This paper describes the objectives, and reviews the progress, of the European project 'Treatment Initiatives After Radiological Accidents' (TIARA). TIARA forms part of the 'Preparatory Action for Security Research' (PASR) launched by the European Commission in 2004. The Preparatory Action is intended to reach preliminary conclusions on the needs for the security of EU citizens. It prepared a comprehensive Security Research Programme as part of the Commission's Seventh Framework Programme proposal, which was adopted in 2006 and launched in 2007. The principal purpose of TIARA is to constitute a European network that will participate in facilitating the management of a crisis in the event of the malevolent dispersal of radionuclides into the public environment.

Treatment of accidental intakes of plutonium and americium: guidance notes.

The scientific basis for the treatment of the contamination of the human body by plutonium, americium and other actinides is reviewed. Guidance Notes are presented for the assistance of physicians and others who may be called upon to treat workers or members of the public who may become contaminated internally with inhaled plutonium nitrate, plutonium tributyl phosphate, americium nitrate or americium oxide.

Astrocyte-targeted expression of interleukin-3 and interferon-alpha causes region-specific changes in metallothionein expression in the brain.

Transgenic mice expressing IL-3 and IFN-alpha under the regulatory control of the GFAP gene promoter (GFAP-IL3 and GFAP-IFNalpha mice) exhibit a cytokine-specific, late-onset chronic-progressive neurological disorder which resemble many of the features of human diseases such as multiple sclerosis, Aicardi-Goutières syndrome, and some viral encephalopathies including HIV leukoencephalopathy. In this report we show that the metallothionein-I+II (MT-I+II) isoforms were upregulated in the brain of both GFAP-IL3 and GFAP-IFNalpha mice in accordance with the site and amount of expression of the cytokines. In the GFAP-IL3 mice, in situ hybridization analysis for MT-I RNA and radioimmunoassay results for MT-I+II protein revealed that a significant upregulation was observed in the cerebellum and medulla plus pons at the two ages studied, 1-3 and 6-10 months. Increased MT-I RNA levels occurred in the Purkinje and granular layers of the cerebellum, as well as in its white matter tracts. In contrast to the cerebellum and brain stem, MT-I+II were downregulated by IL-3 in the hippocampus and the remaining brain in the older mice. In situ hybridization for MT-III RNA revealed a modest increase in the cerebellum, which was confirmed by immunohistochemistry. MT-III immunoreactivity was present in cells that were mainly round or amoeboid monocytes/macrophages and in astrocytes. MT-I+II induction was more generalized in the GFAP-IFNalpha (GIFN12 and GIFN39 lines) mice, with significant increases in the cerebellum, thalamus, hippocampus, and cortex. In the high expressor line GIFN39, MT-III RNA levels were significantly increased in the cerebellum (Purkinje, granular, and molecular layers), thalamus, and hippocampus (CA2/CA3 and especially lacunosum molecular layers). Reactive astrocytes, activated rod-like microglia, and macrophages, but not the perivenular infiltrating cells, were identified as the cellular sources of the MT-I+II and MT-III proteins. The pattern of expression of the different MT isoforms in these transgenic mice differed substantially, demonstrating unique effects associated with the expression of each cytokine. The results indicate that the MT expression in the CNS is significantly affected by the cytokine-induced inflammatory response and support a major role of these proteins during CNS injury.

Effect of gamma irradiation on liver metallothionein synthesis and lipid peroxidation in rats.

Several studies have recently shown that metallothionein (MT), a protein characterized by a high thiol content and that binds Zn2+ and Cu+, might be involved in the protection against oxidative stress and can act as a free radical scavenger. Oxidative stresses, such as irradiation, increase lipid peroxidation (LP) and subsequent tissue damage through free radical production. The induction of hepatic MT synthesis by gamma-irradiation (20 Gy) at 8, 24, 30 and 48 hrs. post-irradiation in two different age groups of Sprague-Dawley rats (39-40 and 48-49 days old) was studied. LP measured by the thiobarbituric acid reactive substances (TBARS) assay and Cu and Zn levels in liver have also been determined. In the younger group, the gamma-irradiation induced hepatic MT synthesis and increased LP that peaked 24 hrs. after irradiation. During the first 30 hrs. post-irradiation, a positive and statistically significant correlation between hepatic MT content and LP level in liver was found. In the older group, liver MT synthesis was only increased 1.7-fold and LP levels were not altered at 24 hrs. post-irradiation compared with sham-irradiated rats. Therefore it appears that LP is not necessary for induction of MT synthesis by gamma-irradiation.

Mercury distribution and renal metallothionein induction after subchronic oral exposure in rats.

The effects of long-term daily intake of low and high levels of mercury on its organ distribution and binding to renal metallothionein (MT) in male rats were studied. The animals were exposed to mercuric chloride labelled with 203Hg via drinking water for 8 weeks (5, 50 and 500 microM Hg). The greatest concentration of mercury was found in the kidneys. Similar levels of radioactivity in the buccal cavity and oesophagus were also observed by whole-body autoradiography. In the kidneys, the mercury was accumulated in the outer stripe of the outer zone of the medulla and, to a minor degree, in the renal cortex. Almost 50% the total renal mercury was associated to MT. The binding capacity of the renal MT for mercury tends to saturate with increasing doses, thus this means that the capacity of the kidneys to accumulate mercury is limited.

Whole-body retention, and urinary and fecal excretion of mercury after subchronic oral exposure to mercuric chloride in rats.

The effects of long-term daily intake of mercury on its urinary and fecal excretion, whole-body retention, and blood concentration in male rats were observed. The animals were exposed to mercuric, chloride labeled with 203Hg via drinking water for 8 weeks (5, 50 and 500 microM Hg). 203Hg in urine, feces and blood was quantified. The blood mercury concentration did not keep a linear relationship with the increasing dose. The percentage of the total amount of mercury intake which is excreted by the fecal route in rats exposed to 500 microM Hg was significantly lower than in those exposed to 5 and 50 microM. The daily dose percentage of mercury excreted in urine increased with dose size. The results show that the absorption fraction of mercury through the gastrointestinal tract (30-40%) was higher than values previously reported.

Separation and characterization of rat kidney isometallothioneins induced by exposure to inorganic mercury.

High-performance liquid chromatography (HPLC) was applied to the separation of metallothionein (MT) isoforms from different tissues from a variety of eukaryotic species. Recently we reported an analytical method for 203Hg-metallothionein, which detects the radioisotope bound to each iso-MT after separation by HPLC on a size-exclusion column coupled with on-line radioactivity flow detection. The MTs can be separated as distinct isoprotein peaks by elution with alkaline buffer solution owing to cation-exchange chromatographic action. In the present work, renal MT from rats exposed to inorganic mercury was separated into four peaks by UV and 203Hg detection. Moreover, it was resolved into four components by non-denaturing polyacrylamide gel electrophoresis. The two major components correspond to MT-1 and MT-2, which were characterized by amino acid analysis. Finally, Hg induces and binds to both iso-MTs.

Isolation of 203mercury-induced metallothionein in rat kidney by direct connection of HPLC to a beta radioactivity detector.

Rat kidney 203Hg-induced metallothionein (HgMT) was separated on a high performance liquid chromatograph equipped with a gel permeation column and an on-line beta radioactivity detector, in order to obtain the simultaneous measurements of renal MT by UV detection and MT-associated 203Hg by a beta radioactivity detector. Metallothionein was separated in three major species by both UV detection at 254 nm and 203Hg detection, probably due to the presence of mercury and copper. A standard curve was prepared which demonstrated excellent linear correlation between the integrated HgMT peaks area and the quantity of HgMT injected into the column. In contrast to the results with the gel permeation column above mentioned, rat kidney HgMT was separated in four peaks by reversed-phase height performance liquid chromatography.