Imaging expression of cytosine deaminase-herpes virus thymidine kinase fusion gene (CD/TK) expression with [124I]FIAU and PET

Double prodrug activation gene therapy using the Escherichia coli cytosine deaminase (CD)-herpes simplex virus type 1 thymidine kinase (HSV1-tk) fusion gene (CD/TK) with 5-fluorocytosine (5FC), ganciclovir (GCV), and radiotherapy is currently under evaluation for treatment of different tumors. We assessed the efficacy of noninvasive imaging with [124I]FIAU (2'-fluoro-2'-deoxy-1-beta-D-arabinofuranosyl-5-iodo-uracil) and positron emission tomography (PET) for monitoring expression of the CD/TK fusion gene. Walker-256 tumor cells were transduced with a retroviral vector bearing the CD/TK gene (W256CD/TK cells). The activity of HSV1-TK and CD subunits of the CD/TK gene product was assessed in different single cell-derived clones of W256CD/TK cells using the FIAU radiotracer accumulation assay in cells and a CD enzyme assay in cell homogenates, respectively. A linear relationship was observed between the levels of CD and HSV1-tk subunit expression in corresponding clones in vitro over a wide range of CD/TK expression levels. Several clones of W256CD/TK cells with significantly different levels of CD/TK expression were selected and used to produce multiple subcutaneous tumors in rats. PET imaging of HSV1-TK subunit activity with [124I]FIAU was performed on these animals and demonstrated that different levels of CD/TK expression in subcutaneous W256CD/TK tumors can be imaged quantitatively. CD expression in subcutaneous tumor sample homogenates was measured using a CD enzyme assay. A comparison of CD and HSV1-TK subunit enzymatic activity of the CD/TK fusion protein in vivo showed a significant correlation. Knowing this relationship, the parametric images of CD subunit activity were generated. Imaging with [124I]FIAU and PET could provide pre- and posttreatment assessments of CD/TK-based double prodrug activation in clinical gene therapy trials.     

Detection of Pancreatic Carcinomas by Imaging Lactose-Binding Protein Expression in Peritumoral Pancreas Using [18F]Fluoroethyl-Deoxylactose PET/CT

Background

Early diagnosis of pancreatic carcinoma with highly sensitive diagnostic imaging methods could save lives of many thousands of patients, because early detection increases resectability and survival rates. Current non-invasive diagnostic imaging techniques have inadequate resolution and sensitivity for detection of small size (∼2–3 mm) early pancreatic carcinoma lesions. Therefore, we have assessed the efficacy of positron emission tomography and computer tomography (PET/CT) imaging with β-O-D-galactopyranosyl-(1,4′)-2′-deoxy-2′-[¹⁸F]fluoroethyl-D-glucopyranose ([¹⁸F]FEDL) for detection of less than 3 mm orthotopic xenografts of L3.6pl pancreatic carcinomas in mice. [¹⁸F]FEDL is a novel radioligand of hepatocarcinoma-intestine-pancreas/pancreatitis-associated protein (HIP/PAP), which is overexpressed in peritumoral pancreatic acinar cells.

Methodology/Principal Findings

Dynamic PET/CT imaging demonstrated rapid accumulation of [¹⁸F]FEDL in peritumoral pancreatic tissue (4.04±2.06%ID/g), bi-exponential blood clearance with half-lives of 1.65±0.50 min and 14.14±3.60 min, and rapid elimination from other organs and tissues, predominantly by renal clearance. Using model-independent graphical analysis of dynamic PET data, the average distribution volume ratio (DVR) for [¹⁸F]FEDL in peritumoral pancreatic tissue was estimated as 3.57±0.60 and 0.94±0.72 in sham-operated control pancreas. Comparative analysis of quantitative autoradiographic images and densitometry of immunohistochemically stained and co-registered adjacent tissue sections demonstrated a strong linear correlation between the magnitude of [¹⁸F]FEDL binding and HIP/PAP expression in corresponding regions (r = 0.88). The in situ analysis demonstrated that at least a 2–4 fold apparent lesion size amplification was achieved for submillimeter tumors and to nearly half a murine pancreas for tumors larger than 3 mm.

Conclusion/Significance

We have demonstrated the feasibility of detection of early pancreatic tumors by non-invasive imaging with [¹⁸F]FEDL PET/CT of tumor biomarker HIP/PAP over-expressed in peritumoral pancreatic tissue. Non-invasive non-invasive detection of early pancreatic carcinomas with [¹⁸F]FEDL PET/CT imaging should aid the guidance of biopsies and additional imaging procedures, facilitate the resectability and improve the overall prognosis.     

Combination of [68Ga]Ga-PSMA PET/CT and [18F]FDG PET/CT in demonstrating dedifferentiation in castration-resistant prostate cancer

Aim of the studyIn this study, we aimed to determine the factors affecting increased glucose metabolism, which is one of the dedifferentiation mechanisms, by using [¹⁸F]FDG and [⁶⁸Ga]Ga-PSMA PET/CT in patients with castration-resistant prostate cancer (CRPC).Materials and methodNinety-three patients with CRPC were included in the study. Gleason score (GS), and total PSA and free PSA levels of the patients were recorded. Patient- and organ-based evaluations were performed according to the lesion uptakes as follows: score 0: PSMA (-) FDG (-), score 1: PSMA (+) FDG (-), score 2: PSMA (+) FDG (+) (FDG < PSMA), score 3: PSMA (+) FDG (+) (FDG = PSMA), score 4: PSMA (+) FDG (+) (FDG > PSMA), and score 5: PSMA (-) FDG (+). scores 1 and 2 were classified as group 1, and scores 3 to 5 were classified as group 2.ResultsThe median age of our patients was 70 (51–88) years. Eighty-eight patients (94.6%) were PSMA-positive, 78 patients (83.8%) were FDG-positive, and 89 patients (95.6%) were or PSMA or FDG positive. When the two groups were compared in terms of patient-based parameters, the median age and GS were found to be significantly higher in group 2. ROC analyses revealed that age and GS were significant in predicting group 2.ConclusionSince glucose metabolism can increase in CRPC patients with advanced age and high GS, we recommend combining [¹⁸F]FDG PET/CT with [⁶⁸Ga]Ga-PSMA PET/CT in routine clinical practice in order to identify this patient subset and refer them to additional therapies.     

Post-traumatic infections of the musculoskeletal locomotor system; prophylaxis and treatment]

An analysis of epidemiology of the post-traumatic infections of the locomotive system, prophylaxis and treatment in presented. Infections are the most severe complications of the open bone fractures and orthopedic surgery. An incident of inflammatory complications following the open bone fractures is very high (20% according to Burrie). An incidence of complications following orthopedic surgeries is somewhat lower--0.2-9.0%. Within 1976-1986, as much as 585 patients with chronic osteitis, including 222 patients with post-traumatic infections, were treated at the Department of Orthopedic Surgery, Medical Academy in Warsaw. Therapy depended on the general and local health status, and the types of surgery were diversified. The treatment included, first of all, radical removal of crushed and necrotic tissues, stabilization of the fractured bone fragments with the external stabilizer, AO plate or the Polish original method of the stable internal osteosynthesis--ZESPOL, decompression of the crack with the aid of permanent flow or suction drainage, systemic or local antibiotic therapy, administration of vasodilating agents, and the primary skin plasty in some cases.     

Purification of [I]-vasoactive intestinal peptide by reverse-phase HPLC

VIP was labeled with sodium ¹²⁵iodide, and ¹²⁵I-VIP was purified by reverse-phase high performance liquid chromatography. Optimal separations of ¹²⁵I-VIP and unlabeled VIP were obtained using two C₁₈-Novapak columns in series and a gradient of acetonitrile in triethylamine phosphate for elution. The specific activity of the ¹²⁵I-VIP was 1.99±0.21 Ci/μmole, approaching the maximum specific activity of monoiodinated VIP (2.26 Ci/μmole). Radioimmunoassay and radioreceptorassay for VIP were more sensitive (2.6-fold, and 2.5-fold, respectively) using ¹²⁵I-VIP purified by HPLC compared to ¹²⁵I-VIP obtained from an open-end cellulose column. These results demonstrate the advantage of preparing purified ¹²⁵I-VIP by HPLC for the accurate assay of VIP and VIP-receptors in tissues and biological fluids.     

Autoradiographic localizatio of [125I]-C-ANP compared to [125I]-ANP in rat tissue

Summary Whole-body autoradiography demonstrated the different distribution of [¹²⁵I]-C-ANP and [¹²⁵I]-ANP to rat tissues. Highest enrichment of radioactivity of both labelled peptides was found in the kidney. In some organs we found remarkable differences between [¹²⁵I]-ANP and [¹²⁵I]-C-ANP. In the kidney cortex, especially in the glomeruli, as well as in the endocardium, the zona glomerulosa and the medulla of the adrenal gland, where high levels of radioactivity after [¹²⁵I]-ANP administration were detected, no or just few radioactivity was found after administration of [¹²⁵I]-C-ANP. On the other hand in the kidney papilla and the outer subcortical medulla, characteristic blackening was found after [¹²⁵I]-C-ANP administration. Those differences might be important for the understanding of pharmacological actions of ANP analogues.This work is part of the doctoral thesis of Frank Heidemann to be presented at the Ludwig-Maximilians-Universität München, FRG     

Basis for changes in DNA recognition by the EcoR124 and EcoR124/3 type I DNA restriction and modification enzymes

EcoR124 and EcoR124/3 are type I DNA restriction and modification systems. The EcoR124/3 system arose from the EcoR124 system some 15 years ago and at the electron microscopic DNA heteroduplex level the genes for both systems are still apparently identical. We have shown that the DNA sequences recognized by the two systems are GAA(N6)RTCG for EcoR124 and GAA(N7)RTCG for EcoR124/3. The sequences thus differ only in the length of the non-specific spacer. This difference nevertheless places the two specific domains of the EcoR124/3 recognition sequence 0.34 nm further apart and rotates them 36 degrees with respect to those of EcoR124, which implies major structural differences in the proteins recognizing these sequences. We have now determined the nucleotide sequences of the hsdS and hsdM genes of both systems and of the hsdR gene of EcoR124/3. The hsdS gene products provide DNA sequence specificity in both restriction and modification, the hsdM gene products are necessary for modification and all three hsd gene products are required for restriction. The only difference that we have detected between the two systems is that a 12 base-pair sequence towards the middle of the hsdS gene is repeated twice in the EcoR124 gene and three times in the EcoR124/3 gene. We have deleted one of the repeats in the EcoR124/3 gene and shown that this changes the specificity to that of EcoR124. Thus, the extra four amino acids in the middle of the EcoR124/3 hsdS gene product, which in an alpha-helical configuration would extend 0.6 nm, are sufficient to explain the differences in sequence recognition. We suggest that the EcoR124/3 system was generated by an unequal crossing over and argue that this kind of specificity change should not be rare in Nature.     

Comparison of cell inactivation by Auger electrons using the two reagents 4-[123I]iodoantipyrine and [123I]NaI

The Auger electron emitter ¹²³I was examined in the form of 4-[¹²³I]iodoantipyrine and as [¹²³I]NaI for its effectiveness in killing cells of different sensitivity to photon irradiation. Micronucleus assays showed that 4-[¹²³I]iodoantipyrine is 2–3 times more effective in cell inactivation than [¹²³I]NaI. This can be attributed to the fact that antipyrine, for reason of its lipid solubility, can enter cells and can reach the nucleus, whereas [¹²³I]NaI is excluded from the cytoplasm. In the nucleus Auger decay is conceivably located on the DNA where it may invoke high-LET irradiation damage. Irradiation damage by [¹²³I]NaI is by long range Auger and internal conversion electrons and hence less densely ionising. Results of the present study demonstrate, however, that the enhancement of micronuclei frequency (MNF) seen with 4-[¹²³I]iodoantipyrine as compared to [¹²³I]NaI is similar for all cell lines and that the ratio of 4-[¹²³I]iodoantipyrine/[¹²³I]NaI MN response remains the same. Experiments with the free radical scavenger DMSO, indicated nearly identical dose reduction factors for both ¹²³I carriers. These two observations strongly suggest that the cell inactivation by 4-[¹²³I]iodoantipyrine is not by direct high-LET ionisation of DNA, but is due to an indirect effect. The indirect radiation effect of Auger decay in the nucleus could arise because 4-[¹²³I]iodoantipyrine is not incorporated into the DNA, but is only associated with chromatin where the DNA is shielded by histones. Received: 24 May 2000 / Accepted: 1 November 2000     

Autoradiographic localization of [125I]-C-ANP compared to [125I]-ANP in rat tissue.

Whole-body autoradiography demonstrated the different distribution of [125I]-C-ANP and [125I]-ANP to rat tissues. Highest enrichment of radioactivity of both labelled peptides was found in the kidney. In some organs we found remarkable differences between [125I]-ANP and [125I]-C-ANP. In the kidney cortex, especially in the glomeruli, as well as in the endocardium, the zona glomerulosa and the medulla of the adrenal gland, where high levels of radioactivity after [125I]-ANP administration were detected, no or just few radioactivity was found after administration of [125I]-C-ANP. On the other hand in the kidney papilla and the outer subcortical medulla, characteristic blackening was found after [125I]-C-ANP administration. Those differences might be important for the understanding of pharmacological actions of ANP analogues.     

Purification of [125I]-vasoactive intestinal peptide by reverse-phase HPLC

VIP was labeled with sodium ¹²⁵iodide, and ¹²⁵I-VIP was purified by reverse-phase high performance liquid chromatography. Optimal separations of ¹²⁵I-VIP and unlabeled VIP were obtained using two C₁₈-Novapak columns in series and a gradient of acetonitrile in triethylamine phosphate for elution. The specific activity of the ¹²⁵I-VIP was 1.99±0.21 Ci/μmole, approaching the maximum specific activity of monoiodinated VIP (2.26 Ci/μmole). Radioimmunoassay and radioreceptorassay for VIP were more sensitive (2.6-fold, and 2.5-fold, respectively) using ¹²⁵I-VIP purified by HPLC compared to ¹²⁵I-VIP obtained from an open-end cellulose column. These results demonstrate the advantage of preparing purified ¹²⁵I-VIP by HPLC for the accurate assay of VIP and VIP-receptors in tissues and biological fluids.     

Ssa1 Overexpression and [PIN] Variants Cure [PSI] by Dilution of Aggregates

Several cellular chaperones have been shown to affect the propagation of the yeast prions [PSI⁺], [PIN⁺] and [URE3]. Ssa1 and Ssa2 are Hsp70 family chaperones that generally cause pro-[PSI⁺] effects, since dominant-negative mutants of Ssa1 or Ssa2 cure [PSI⁺], and overexpression of Ssa1 enhances de novo [PSI⁺] appearance and prevents curing by excess Hsp104. In contrast, Ssa1 was shown to have anti-[URE3] effects, since overexpression of Ssa1 cures [URE3]. Here we show that excess Ssa1 or Ssa2 can also cure [PSI⁺]. This curing is enhanced in the presence of [PIN⁺]. During curing, Sup35-GFP fluorescent aggregates get bigger and fewer in number, which leads to their being diluted out during cell division, a phenotype that was also observed during the curing of [PSI⁺] by certain variants of [PIN⁺]. The sizes of the detergent-resistant [PSI⁺] prion oligomers increase during [PSI⁺] curing by excess Ssa1. Excess Ssa1 likewise leads to an increase in oligomer sizes of low, medium and very high [PIN⁺] variants. While these phenotypes are also caused by inhibition of Hsp104 or Sis1, the overexpression of Ssa1 did not cause any change in Hsp104 or Sis1 levels.