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AIMS: Perfusion of the pancreas and the islets of Langerhans is sensitive to physiological stimuli and is dysregulated in metabolic disease. Pancreatic perfusion can be assessed by both positron emission tomography (PET) and magnetic resonance imaging (MRI), but the methods have not been directly compared or benchmarked against the gold-standard microsphere technique. METHODS: Pigs (n = 4) were examined by [15O]H2O PET and intravoxel incoherent motion (IVIM) MRI technique simultaneously using a hybrid PET/MRI scanner. The pancreatic perfusion was measured both at basal conditions and after intravenous (IV) administration of up to 0.5 g/kg glucose. RESULTS: Pancreatic perfusion increased by 35%, 157%, and 29% after IV 0.5 g/kg glucose compared to during basal conditions, as assessed by [15O]H2O PET, IVIM MRI, and microspheres, respectively. There was a correlation between pancreatic perfusion as assessed by [15O]H2O PET and IVIM MRI (r = 0.81, R2 = 0.65, p

AIM: The zinc transporter 8 (ZnT8) has been suggested as a suitable target for non-invasive visualization of the functional pancreatic beta cell mass, due to both its pancreatic beta cell restricted expression and tight involvement in insulin secretion. METHODS: In order to examine the potential of ZnT8 as a surrogate target for beta cell mass, we performed mRNA transcription analysis in pancreatic compartments. A novel ZnT8 targeting antibody fragment Ab31 was radiolabeled with iodine-125, and evaluated by in vitro autoradiography in insulinoma and pancreas as well as by in vivo biodistribution. The evaluation was performed in a direct comparison with radio-iodinated Exendin-4. RESULTS: Transcription of the ZnT8 mRNA was higher in islets of Langerhans compared to exocrine tissue. Ab31 targeted ZnT8 in the cytosol and on the plasma membrane with 108 nM affinity. Ab31 was successfully radiolabeled with iodine-125 with high yield and > 95% purity. [(125)I]Ab31 binding to insulinoma and pancreas was higher than for [(125)I]Exendin-4, but could only by partially competed away by 200 nM Ab31 in excess. The in vivo uptake of [(125)I]Ab31 was higher than [(125)I]Exendin-4 in most tissues, mainly due to slower clearance from blood. CONCLUSIONS: We report a first-in-class ZnT8 imaging ligand for pancreatic imaging. Development with respect to ligand miniaturization and radionuclide selection is required for further progress. Transcription analysis indicates ZnT8 as a suitable target for visualization of the human endocrine pancreas.

Purpose Heat shock protein 90 (HSP90) is essential for the activation and stabilization of numerous oncogenic client proteins. AT13387 is a novel HSP90 inhibitor promoting degradation of oncogenic proteins upon binding, and may also act as a radiosensitizer. For optimal treatment there is, however, the need for identification of biomarkers for patient stratification and therapeutic response monitoring, and to find suitable targets for combination treatments. The aim of this study was to assess the response of surface antigens commonly expressed in squamous cell carcinoma to AT13387 treatment, and to find suitable biomarkers for molecular imaging and radioimmunotherapy in combination with HSP90 inhibition. Methods Cancer cell proliferation and radioimmunoassays were used to evaluate the effect of AT13387 on target antigen expression in vitro. Inhibitor effects were then assessed in vivo in mice-xenografts. Animals were treated with AT13387 (5 × 50 mg/kg), and were imaged with PET using either 18F-FDG or 124I-labelled tracers for EGFR and CD44v6, and this was followed by ex-vivo biodistribution analysis and immunohistochemical staining. Results AT13387 exposure resulted in high cytotoxicity and possible radiosensitization with IC50 values below 4 nM. Both in vitro and in vivo AT13387 effectively downregulated HSP90 client proteins. PET imaging with 124I-cetuximab showed a significant decrease of EGFR in AT13387-treated animals compared with untreated animals. In contrast, the squamous cell carcinoma-associated biomarker CD44v6, visualized with 124I-AbD19384 as well as 18F-FDG uptake, were not significantly altered by AT13387 treatment. Conclusion We conclude that AT13387 downregulates HSP90 client proteins, and that molecular imaging of these proteins may be a suitable approach for assessing treatment response. Furthermore, radioimmunotherapy targeting CD44v6 in combination with AT13387 may potentiate the radioimmunotherapy outcome due to radiosensitizing effects of the drug, and could potentially lead to a lower dose to normal tissues. Electronic supplementary material The online version of this article (doi:10.1007/s00259-015-3260-x) contains supplementary material, which is available to authorized users.

To address questions regarding onset and progression of types 1 and 2 diabetes (T1D/T2D), surrogate imaging biomarkers for beta cell function and mass are needed. Here, we assess the potential of GPR44 as a surrogate marker for beta cells, in a direct comparison with clinically used biomarker VMAT2.GPR44 surface availability was assessed by flow cytometry of human beta cells. RNA transcription levels in different pancreas compartments were evaluated. The density of GPR44 receptor in endocrine and exocrine tissues was assessed by the radiolabeled GPR44 ligand [(3)H]AZD 3825. A direct comparison with the established beta cell marker VMAT2 was performed by radiolabeled [(3)H]DTBZ.GPR44 was available on the cell surface, and pancreatic RNA levels were restricted to the islets of Langerhans. [(3)H]AZD 3825 had nanomolar affinity for GPR44 in human islets and EndoC-βH1 beta cells, and the specific binding to human beta cells was close to 50 times higher than in exocrine preparations. The endocrine-to-exocrine binding ratio was approximately 10 times higher for [(3)H]AZD 3825 than for [(3)H]DTBZ.GPR44 is a highly beta cell-specific target, which potentially offers improved imaging contrast between the human beta cell and the exocrine pancreas.

The glucagon-like peptide-1 receptor (GLP-1R) has been proposed as a target for molecular imaging of beta cells. The feasibility of non-invasive imaging and quantification of GLP-1R in pancreas using the positron emission tomography (PET) tracer [(68)Ga]Ga-DO3A-VS-Cys(40)-Exendin-4 in non-diabetic and streptozotocin (STZ)-induced diabetic pigs treated with insulin was investigated.Non-diabetic (n = 4) and STZ-induced diabetic pigs (n = 3) from the same litter were examined. Development of diabetes was confirmed by blood glucose values, clinical examinations and insulin staining of pancreatic sections post mortem. Tissue perfusion in the pancreas and kidneys was evaluated by [(15)O]water PET/computed tomography (CT) scans. The in vivo receptor specificity of [(68)Ga]Ga-DO3A-VS-Cys(40)-Exendin-4 was assessed by administration of either tracer alone or by competition with 3-6.5 μg/kg of Exendin-4. Volume of distribution and occupancy in the pancreas were quantified with a single tissue compartment model.[(15)O]water PET/CT examinations showed reduced perfusion in the pancreas and kidneys in diabetic pigs. [(68)Ga]Ga-DO3A-VS-Cys(40)-Exendin-4 uptake in the pancreas of both non-diabetic and diabetic pigs was almost completely abolished by co-injection of unlabeled Exendin-4 peptide. [(68)Ga]Ga-DO3A-VS-Cys(40)-Exendin-4 uptake did not differ between non-diabetic and diabetic pigs. In all animals, administration of the tracer resulted in an immediate increase in the heart rate (HR).Pancreatic uptake of [(68)Ga]Ga-DO3A-VS-Cys(40)-Exendin-4 was not reduced by destruction of beta cells in STZ-induced diabetic pigs.

Muscle spindles are skeletal muscle mechanoreceptors that provide proprioceptive information to the central nervous system. The human adult masseter muscle has greater number, larger and more complex muscle spindles than the adult biceps. For a better knowledge of muscle diversity and physiological properties, this study examined the myosin heavy chain (MyHC) expression of muscle spindle intrafusal fibres in the human young masseter and young biceps muscles by using a panel of monoclonal antibodies (mAbs) against different MyHC isoforms. Eight MyHC isoforms were detected in both muscles-slow-tonic, I, IIa, IIx, foetal, embryonic, α-cardiac and an isoform not previously reported in intrafusal fibres, termed IIx'. Individual fibres co-expressed 2-6 isoforms. MyHC-slow tonic separated bag1, AS-bag1 and bag2 fibres from chain fibres. Typically, bag fibres also expressed MyHC-I and α-cardiac, whereas chain fibres expressed IIa and foetal. In the young masseter 98 % of bag1 showed MyHC-α cardiac versus 30 % in the young biceps, 35 % of bag2 showed MyHC-IIx' versus none in biceps, 17 % of the chain fibres showed MyHC-I versus 61 % in the biceps. In conclusion, the result showed fundamental similarities in intrafusal MyHC expression between young masseter and biceps, but also marked differences implying muscle-specific proprioceptive control, probably related to diverse evolutionary and developmental origins. Finding of similarities in MyHC expression between young and adult masseter and biceps muscle spindles, respectively, in accordance with previously reported similarities in mATPase fibre type composition suggest early maturation of muscle spindles, preceding extrafusal fibres in growth and maturation.

The myosin heavy chain (MyHC) content in different parts of, two jaw opening muscle, the human lateral pterygoid and the digastric muscles of five young adult and five elderly subjects (mean age 22 and 73 years, respectively) was determined, using gel electrophoresis and immunohistochemical methods. The lateral pterygoid of both young and elderly contained predominantly slow MyHC, and fast A MyHC was the major fast isoform. In contrast, the digastric was composed of slow, fast A and fast X MyHCs in about equal proportions in both age groups. About half of the lateral pterygoid fibres contained mixtures of slow and fast MyHCs, often together with alpha-cardiac MyHC. In the digastric, co-existence of slow and fast MyHCs was rare, and alpha-cardiac MyHC was lacking. On the other hand, co-expression of fast A and fast X MyHCs was found more often in the digastric than in the lateral pterygoid. In both age groups about half of the digastric IIB fibres contained solely fast X MyHC. In the lateral pterygoid, type IIB fibres with pure fast X MyHC was found in only one subject. The lateral pterygoid in elderly showed a significant amount of fibres with solely fast A MyHC, which were occasionally found in young adults. In the digastric, no significant differences were found between young and elderly, although the muscles of elderly contained lower mean value of slow MyHC, as compared to that of young muscles. It is concluded that the lateral pterygoid and the digastric muscles differ not only in the MyHC composition but also in modifications of the MyHC phenotypes during aging, suggesting that they have separate roles in jaw opening function.

The myosin heavy chain (MyHC) content in functionally different parts of the human masseter muscle of six elderly and five young adult subjects (mean age 74 and 22 years, respectively) was determined, using gel electrophoresis. The MyHC composition of the old masseter was also studied by enzyme- and immunohistochemical methods and compared with previous data for young adults. For comparison, the biceps brachii muscle of the same subjects was also analysed. The old masseter contained smaller amounts of slow and larger amounts of fast and fetal MyHCs. These differences were region-dependent and were more pronounced in the superficial portion. There was also a larger proportion of “hybrid” fibres, containing two to four MyHC isoforms (42%), compared with the young adult masseter (23%). No such differences were observed between old and young biceps. In contrast to the masseter, the old biceps contained more slow MyHC and less fast MyHC. This investigation demonstrates that the aging process in human skeletal muscle is accompanied by a modification in the muscle phenotype which is both muscle and region specific; a transformation towards a fast and fetal phenotype concomitant with an increased number of fibres with a mixture of different MyHC isoforms in the masseter; and an opposite shift towards a slower phenotype in the biceps brachii. The results might reflect differences between jaw and limb muscles in genetic programs and adaptive responses to changed functional demands following aging.

In this report we show that immunostaining of the capillary basement membrane with an antibody directed against laminin is a useful alternative in detecting the capillaries in human muscle. Using this method, the capillary supply of three embryologically, morphologically and functionally different muscle groups, oro-facial, masticatory and limb muscles, were analysed. Significant differences in capillarization between muscles and muscle groups were found. The oro-facial and masseter muscles showed significantly higher density of capillaries than the limb muscles, taken in consideration the muscle cross-sectional area, with the masseter having the highest capillary density ever reported for human sedentary skeletal muscles. Judged from the number of capillaries per muscle fibre, the limb muscles showed the best capillarization. However, since the three muscle groups investigated differed with respect to fibre diameter, our evaluation of number of capillaries per muscle fibre was related also to fibre diameter. Thus, capillary constants were created which take fibre diameter into account. Based on these new parameters, the oro-facial and masseter muscles were found to be relatively better supplied by capillaries than the limb muscles. These results suggest differences in tasks and functional activity between muscles, and reflect a relatively higher need and demand for blood supply in the masticatory and oro-facial muscles than in limb muscles.

Enzyme-histochemical methods were used to analyse the activities of alkaline phosphatase (AP), dipeptidylpeptidase IV (DPP IV) and adenosine triphosphatase (ATPase) in capillaries of four different human oro-facial muscles, the major and minor zygomatic, the orbicularis oris and buccinator, one masticatory, the masseter and two limb muscles, the biceps brachii and first dorsal interosseus muscles. In all muscles, except for the orbicularis oris, the majority of the capillaries lacked enzyme activity. Therefore, none of these enzymes seems to be reliable as a general marker for human muscle capillaries. In general, the capillaries of the limb muscles and the major and minor zygomatic and the buccinator, were similar in their staining pattern for AP and ATPase, but differed in DPP IV staining. The orbicularis oris muscle differed from the other muscles by showing the largest proportion of capillaries with AP and ATPase activity. The masseter muscle had the largest proportion of capillaries stained for DPP IV. The muscle specific differences in enzyme activity of the capillaries are in agreement with our previous findings of specific differences between limb, oro-facial and masticatory muscles with respect to capillary supply and composition of fibre types and myosins. The results reflect functional specialization of the capillary bed of human muscles.

We have investigated the reactivity of different human, rat and cat muscles to a monoclonal antibody directed against human alpha-cardiac myosin heavy chain. We have found that special fiber subpopulations of human masseter and extraocular muscles, as well as the bag fibers of human, rat and cat muscle spindles, were reactive to this antibody, indicating that these fibers expressed alpha-cardiac myosin heavy chain or a closely related isoform. This isomyosin was present in the spindle bag fibers at early fetal stages, whereas its expression in masseter and extraocular muscle fibers was not detected during the first 22 weeks of gestation. Our results add to the list of muscle proteins which are expressed in locations or at developmental stages other than those initially described, suggesting that a revision of the present nomenclature of the subgroups of myosin heavy chains might be considered in the future.

Renal function and excretion of water, salt, and the prostacyclin hydration product (6-keto-PGF1α) were evaluated in 10 furosemide-treated patients with well-controlled congestive heart failure. Four doses of sulindac (200 mg b.i.d.) and naproxen (500 mg b.i.d.) were given every 12 hours in a double-blind crossover design. Naproxen significantly decreased the urinary excretion of water (19%), sodium (26%), chloride (26%), and 6-keto PGF1α (76%) and decreased osmolal clearance (18%). No significant changes in these functions were observed in the patients receiving sulindac. Plasma renin activity, plasma aldosterone, freewater clearance, or clearance of furosemide did not change significantly with either treatment. Although the basal glomerular filtration rate (GFR) and renal plasma flow (RPF) were reduced, these patients with cardiac disease, with normal serum sodium concentration, did not have any further reduction of GFR or RPF despite naproxen-induced inhibition of renal prostacyclin synthesis. It is concluded that renal prostaglandins contribute to the natriuretic effect of oral furosemide in patients with compensated congestive heart failure. In this clinical setting, GFR and RPF are not critically dependent on intact renal PGI2 synthesis. The lack of effect on renal prostaglandin synthesis and the renal response to oral furosemide supports the concept of a renal sparing effect of sulindac. Clinical Pharmacology and Therapeutics (1987) 42, 646–654; doi:10.1038/clpt.1987.213

The reliability of muscle fibre typing of post mortem specimens was investigated with special reference to the influence of time and temperature. In specimens stored at +4 degrees C, muscle fibre typing could be reliably performed up to at least ten and fifteen days post mortem for the masseter and biceps brachii muscles respectively. The corresponding figures for storage at room temperature were three and six days. The difference in the preservation of enzyme activity between masticatory and limb muscles might be related to the demonstrated difference in the fibre type composition and thus the enzyme content and energy sources.