Surrogate markers for cerebral blood flow correlate with [18F]‐fallypride binding potential at dopamine D2/3 receptors in human striatum

Positron emission tomography (PET) with the high affinity dopamine D_2/3 receptor ligand [¹⁸F]‐fallypride affords estimates of the binding potential (BP_ND) in extra‐striatal regions of low receptor abundance, but the sufficient recording time for accurate measurements in striatum has been called into question. We have earlier argued that transient equilibrium measurements are obtained in striatum with [¹⁸F]‐fallypride PET recordings of 3 h duration, which may be the practical limit for clinical investigations without interrupted scanning. However, the high extraction fraction of [¹⁸F]‐fallypride predicts flow‐dependence of tracer delivery to brain, which may be a source of variance of the apparent BP_ND in regions of high binding. To test this prediction, we conducted a retrospective analysis of [¹⁸F]‐fallypride PET data from a group of 50 healthy volunteers (age 18–58 years [mean ± SD: 32.6 ± 10.6), who had participated in clinical studies without arterial input measurements. We used the initial 120‐s integral (AUC) of the venous confluence (VC) as a surrogate marker for cerebral blood flow (CBF) and tested for correlations between regional estimates of BP_ND calculated by the simplified reference tissue model (SRTM) and the individual VC‐AUC. The magnitude of BP_ND in a high binding region (putamen), but not in a low binding region (thalamus) correlated positively with VC‐AUC, suggesting that approximately 9% of the variance in the [¹⁸F]‐fallypride BP_ND in putamen can be attributed to individual differences in this surrogate marker for CBF, a contribution equal in magnitude to the effects of age on BP_ND in putamen of the present healthy control group. Synapse, 2013. © 2012 Wiley Periodicals, Inc.     

18F]Fluoroethylflumazenil: a novel tracer for PET imaging of human benzodiazepine receptors.

5-(2'-[18F]Fluoroethyl)flumazenil ([18F]FEF) is a fluorine-18 labelled positron emission tomography (PET) tracer for central benzodiazepine receptors. Compared with the established [11C]flumazenil, it has the advantage of the longer half-life of the fluorine-18 label. After optimisation of its synthesis and determination of its in vitro receptor affinities, we performed first PET studies in humans. PET studies in seven healthy human volunteers were performed on a Siemens ECAT EXACT whole-body scanner after injection of 100-280 MBq [L8F]FEF. In two subjects, a second PET scan was conducted after pretreatment with unlabelled flumazenil (1 mg or 2.5 mg i.v., 3 min before tracer injection). A third subject was studied both with [18F]FEF and with [11C]flumazenil. Brain radioactivity was measured for 60-90 min p.i. and analysed with a region of interest-oriented approach and on a voxelwise basis with spectral analysis. Plasma radioactivity was determined from arterial blood samples and metabolites were determined by high-performance liquid chromatography. In human brain, maximum radioactivity accumulation was observed 4 +/- 2 min p.i., with a fast clearance kinetics resulting in 50% and 20% of maximal activities at about 10 and 30 min, respectively. [18F]FEF uptake followed the known central benzodiazepine receptor distribution in the human brain (occipital cortex >temporal cortex >cerebellum >thalamus >pons). Pretreatment with unlabelled flumazenil resulted in reduced tracer uptake in all brain areas except for receptor-free reference regions like the pons. Parametric images of distribution volume and binding potential generated on a voxelwise basis revealed two- to three-fold lower in vivo receptor binding of [18F]FEF compared with [11C]flumazenil, while relative uptake of [18F]FEF was higher in the cerebellum, most likely owing to its relatively higher affinity for benzodiazepine receptors containing the alpha6 subunit. Metabolism of [18F]FEF was very rapid. Polar metabolites represented about 50%-60% of total plasma radioactivity at 5 min and 80%-90% at 20 min p.i. Although [11C]flumazenil has some advantages over [18F]FEF (higher affinity, slower metabolism, slower kinetics), our results indicate that [18F]FEF is a suitable PET ligand for quantitative assessment of central benzodiazepine receptors, which can be used independently of an on-site cyclotron.     

Isolation and characterization of a trisulfide variant of recombinant human growth hormone formed during expression in Escherichia coli

A new variant of human growth hormone was recently found [Pavlu, B. & Gellerfors, P. (1993) Bioseparation 3, 257-265]. We report here the identification and the structural determination of this variant. The variant, which is formed during the expression of human growth hormone in Escherichia coli, was found to be more hydrophobic than rhGH as judged by its prolonged elution time by hydrophobic interaction chromatography. The rhGH hydrophobic variant (rhGH-HV) was isolated and subjected to trypsin digestion and RP-HPLC analysis, resulting in an altered retention time of one single tryptic peptide as compared to the corresponding fragment of rhGH. This tryptic peptide constitutes the C-terminus (aa 179-191) of hGH and contains one of the two disulfide bridges in hGH, viz. CySl82-Cys189. Amino acid sequences and composition analyses of the tryptic peptide from rhGH-HV (T^v_{18 + 19}) and the corresponding tryptic peptide from rhGH (T_{18 + 19}) were identical. Electrospray mass spectrometry (ES/MS) of T^v_{18 + 19} isolated from rhGH-HV revealed a monoisotopic mass increase of 32.7, as compared to T_{18 + 19} from rhGH. A synthetic T_{18 + 19} peptide having a trisulfide bridge between Cys182 and Cys189 showed identical fragments in ES/MS compared to T^v_{18 + 19} isolated from rhGH-HV, i.e. m/z 617.7 and 682.9. These fragments are formed through a unique cleavage in the trisulfide (Cysl82-SSS-Cys189) bridge not found in the corresponding T_{18 + 19} disulfide peptide. Furthermore, the synthetic T^v_{18 + 19} co-eluted in RP-HPLC with T_{18 + 19} isolated from rhGH-HV. Two-dimensional NMR spectroscopy of the synthetic T_{18 + 19} and T^v_{18 + 19} peptides were performed. Using these data all protons were assigned. The major chemical shift changes (δ§>0.05 ppm) observed were for the β-protons of Cys182 and Cys189 in T^v_{18 + 19} as compared to T_{18 + 19}. CD spectroscopy data were also in agreement with the above results. Based on these physico-chemical data, rhGH-HV has been structurally defined as a trisulfide variant of rhGH. The receptor binding properties of rhGH-HV was studied by a biosensor device, BIAcore^TM. The binding capacity of rhGH-HV was similar to rhGH with a binding stoichiometry to the rhGHBP of 1:1.6 and 1:1.5, respectively, indicating that the trisulfide modification did not affect its receptor binding properties. © Munksgaard 1996.     

Respiratory function in the prune-belly syndrome

Respiratory function was evaluated in 11 patients with prune-belly syndrome. Nine had evidence of gas trapping and six of restrictive lung disease. These abnormalities of lung function appear to be secondary to the musculoskeletal disorder associated with prune-belly syndrome rather than parenchymal lung disease.     

Dummies and the sudden infant death syndrome

The association between dummy use and sudden infant death syndrome (SIDS) was investigated in 485 deaths due to SIDS in the postneonatal age group and compared with 1800 control infants. Parental interviews were completed in 87% of subjects. The prevalence of dummy use in New Zealand is low and varies within New Zealand. Dummy use in the two week period before death was less in cases of SIDS than in the last two weeks for controls (odds ratio (OR) 0.76, 95% confidence interval (CI) 0.57 to 1.02). Use of a dummy in the last sleep for cases of SIDS or in the nominated sleep for controls was significantly less in cases than controls (OR 0.44, 95% CI 0.26 to 0.73). The OR changed very little after controlling for a wide range of potential confounders. It is concluded that dummy use may protect against SIDS, but this observation needs to be repeated before dummies can be recommended for this purpose. If dummy sucking is protective then it is one of several factors that may explain the higher mortality from SIDS in New Zealand than in other countries, and may also explain in part the regional variation within New Zealand.     

Prognostic value of tissue protein expression levels of MIB‐1 (Ki‐67) in Danish ovarian cancer patients. From the ‘MALOVA’ ovarian cancer study

The primary objective of this study was to assess the expression of MIB‐1 (Ki‐67) in tumour tissues from 808 patients with epithelial ovarian tumours. The second was to evaluate, whether MIB‐1 (Ki‐67) tissue expression levels correlate with clinicopathological parameters and prognosis of the disease. Using tissue arrays (TA), we analysed the MIB‐1 (Ki‐67) expression levels in tissues from 202 women with borderline ovarian tumours (BOT) (177 stage I, 5 stage II, 19 stage III, 1 stage IV) and 606 ovarian cancer (OC) patients (177 stage I, 64 stage II, 311 stage III, 54 stage IV). Using a 10% cut‐off level for MIB‐1 (Ki‐67) overexpression, 12% of the BOTs and 51% of the OCs were positive for MIB‐1 (Ki‐67) expression. The frequency of MIB‐1 (Ki‐67) expression‐positive OC increased with increasing FIGO stage (p = 0.003), increasing histological grade (p ≤ 0.0001), and a significantly different distribution of MIB‐1 (Ki‐67) positive and negative tumours were found in adenocarcinoma NOS, serous adenocarcinomas, mucinous adenocarcinomas, endometrioid adenocarcinomas, non‐epithelial and clear‐cell carcinomas (p = 0.016). Univariate Kaplan–Meier survival analysis performed on all OC cases showed a significant shorter disease specific survival in patients with positive MIB‐1 (Ki‐67) expression in the tumour tissue (p ≤ 0.0001). In a Cox survival analysis including 606 FIGO stages I to IV OC cases, FIGO stage (II vs I: HR = 3.00, 95% CI: 1.81–4.99, III–I: HR = 6.41, 95% CI: 3.90–10.50, IV vs I: HR = 12.69, 95% CI: 7.21–22); age at diagnosis pr.10 years (HR = 1.27, 95% CI: 1.15–1.40), residual tumour after surgery (HR = 1.95, 95% CI: 1.40–2.73) and MIB‐1 (Ki‐67) expression (HR = 1.31, 95% CI: 1.08–1.60) had a significant independent impact on survival. Histological grade (p = 0.14) and histological tumour type (p = 0.35) had no significant independent impact on survival. In conclusion, our results predict that an increased level of MIB‐1 (Ki‐67) expression in tumour tissue, points to a less favourable outcome for OC patients.