Application of 89Zr-DFO*-immuno-PET to assess improved target engagement of a bispecific anti-amyloid-ß monoclonal antibody.

PURPOSE: The recent conditional FDA approval of Aducanumab (Adu) for treating Alzheimer's disease (AD) and the continued discussions around that decision have increased interest in immunotherapy for AD and other brain diseases. Reliable techniques for brain imaging of antibodies may guide decision-making in the future but needs further development. In this study, we used 89Zr-immuno-PET to evaluate the targeting and distribution of a bispecific brain-shuttle IgG based on Adu with transferrin receptor protein-1 (TfR1) shuttling mechanism, mAbAdu-scFab8D3, designated Adu-8D3, as a candidate theranostic for AD. We also validated the 89Zr-immuno-PET platform as an enabling technology for developing new antibody-based theranostics for brain disorders. METHODS: Adu, Adu-8D3, and the non-binding control construct B12-8D3 were modified with DFO*-NCS and radiolabeled with 89Zr. APP/PS1 mice were injected with 89Zr-labeled mAbs and imaged on days 3 and 7 by positron emission tomography (PET). Ex vivo biodistribution was performed on day 7, and ex vivo autoradiography and immunofluorescence staining were done on brain tissue to validate the PET imaging results and target engagement with amyloid-ß plaques. Additionally, [89Zr]Zr-DFO*-Adu-8D3 was evaluated in 3, 7, and 10-month-old APP/PS1 mice to test its potential in early stage disease. RESULTS: A 7-fold higher brain uptake was observed for [89Zr]Zr-DFO*-Adu-8D3 compared to [89Zr]Zr-DFO*-Adu and a 2.7-fold higher uptake compared to [89Zr]Zr-DFO*-B12-8D3 on day 7. Autoradiography and immunofluorescence of [89Zr]Zr-DFO*-Adu-8D3 showed co-localization with amyloid plaques, which was not the case with the Adu and B12-8D3 conjugates. [89Zr]Zr-DFO*-Adu-8D3 was able to detect low plaque load in 3-month-old APP/PS1 mice. CONCLUSION: 89Zr-DFO*-immuno-PET revealed high and specific uptake of the bispecific Adu-8D3 in the brain and can be used for the early detection of Aß plaque pathology. Here, we demonstrate that 89Zr-DFO*-immuno-PET can be used to visualize and quantify brain uptake of mAbs and contribute to the evaluation of biological therapeutics for brain diseases.

Height system connection between island and mainland using a hydrodynamic model: a case study connecting the Dutch Wadden islands to the Amsterdam ordnance datum (NAP).

We present an efficient and flexible alternative method to connect islands and offshore tide gauges with the height system on land. The method uses a regional, high-resolution hydrodynamic model that provides total water levels. From the model, we obtain the differences in mean water level (MWL) between tide gauges at the mainland and at the islands or offshore platforms. Adding them to the MWL relative to the national height system at the mainland's tide gauges realizes a connection of the island and offshore platforms with the height system on the mainland. Numerical results are presented for the connection of the Dutch Wadden islands with the national height system (Normaal Amsterdams Peil, NAP). Several choices of the period over which the MWLs are computed are tested and validated. The best results were obtained when we computed the MWL only over the summer months of our 19-year simulation period. Based on this strategy, the percentage of connections for which the absolute differences between the observation- and model-derived MWL differences are ≤ 1  cm is about 34% (46 out of 135 possible leveling connections). In this case, for each Wadden island we can find several connections that allow the transfer of NAP with (sub-)centimeter accuracy.

UGT1A7 polymorphisms in chronic pancreatitis: an example of genotyping pitfalls.

UDP-glucuronosyltransferases (UGT) catalyze the glucuronidation of various compounds and thus inactivate toxic substrates. Genetic variations reducing the activity of UGT1A7 have been associated with various gastrointestinal cancers. Most recently, the UGT1A7*3 allele has been reported as a significant risk factor for pancreatic disorders, but we could not confirm these data. This study focused on the possible causes for the noted discrepancy. UGT1A7 genotypes were assessed in 37 samples, which were previously analyzed for UGT1A7 polymorphisms by others. We determined genotypes by melting curve analysis and by DNA sequencing. Additionally, we produced UGT1A7*1 and *3 constructs with or without a mutation at position - 57 of UGT1A7 and analyzed various combinations of these constructs. In 14/37 samples UGT1A7 genotyping results differed. The discrepancy could be explained by polymerase chain reaction bias owing to an unbalanced allelic amplification which was caused by a -57T>G variant located within the sequence of the chosen primer template in previous studies. Our findings indicate that most of the previously reported genetic associations between UGT1A7 and gastrointestinal cancers are based on primer-dependent genotyping errors.

Hepatic artery infusion of high-dose melphalan at reduced flow during isolated hepatic perfusion for the treatment of colorectal metastases confined to the liver: a clinical and pharmacologic evaluation.

Isolated hepatic perfusion (IHP) offers the advantage of high local drug exposure with limited systemic toxicity. To increase local drug exposure, we administered melphalan at a reduced flow in the hepatic artery during IHP (hepatic artery infusion, hepatic artery-portal vein perfusion, HI-HPP). Between December 2001 and December 2004, 30 patients with colorectal cancer liver metastases underwent HI-HPP with 200mg melphalan. Samples of the perfusate were taken for pharmacokinetic analysis. Patients were monitored for response, toxicity and survival. Perfusion was aborted prematurely in 2 patients due to leakage. During melphalan administration in the hepatic inflow cannula a mean flow rate of 121.3 mL/min and mean pressure of 62.5mm Hg were achieved. One patient died within 30 days after HI-HPP. Four patients developed veno-occlusive disease (VOD), while 2 patients showed signs of VOD. Twelve patients showed hepatic response, with a median duration of response of 11.5 months, according to WHO criteria. Although HI-HPP results in high perfusate melphalan concentration levels, it is associated with a relatively high level of hepatotoxicity and a limited response rate. We believe that the low flow and pressure rates found in this study can result in reduced drug penetration of the tumour and thus limited tumour response.

Sustainability of the prevention of passive infant smoking within well-baby clinics.

This study assessed the antecedents of continued use of an education program to prevent passive smoking in infants. It consists of a booklet for parents and a manual for health professionals describing a five-step procedure for discussing passive smoking. A questionnaire was sent to 67 managers, 670 nurses, and 335 physicians working in well-baby clinics (response rate: 70%, 53%, 47% respectively). Questions concerned the completeness of use, level of institutionalization, and characteristics of the organization, the user, and the dissemination strategy. Seventy-one percent of nurses and 42% of physicians worked with the program. They foremost provided the first three steps of the five-step procedure. Physicians' completeness of use was related to their perceived responsibility in providing this education, and nurses' use was related to their perceived self-efficacy, responsibility, training attendance, participation in the adoption decision, and level of institutionalization. Diffusion efforts should focus on improving the completeness of use and level of institutionalization.

Polymorphisms of UDP-glucuronosyltransferase 1A7 are not involved in pancreatic diseases.

BACKGROUND: Xenobiotic mediated cellular injury is thought to play a major role in the pathogenesis of pancreatic diseases. Genetic variations that reduce the expression or activity of detoxifying phase II biotransformation enzymes such as the UDP-glucuronosyltransferases might be important in this respect. Recently, a UGT1A7 low detoxification activity allele, UGT1A7*3, has been linked to pancreatic cancer and alcoholic chronic pancreatitis. OBJECTIVE: To investigate whether UGT1A7 polymorphisms contribute to the risk of pancreatitis and pancreatic cancer. METHODS: Genetic polymorphisms in the UGT1A7 gene were assessed in a large cohort of patients with different types of pancreatitis and pancreatic cancer originating from the Czech Republic (n = 93), Germany (n = 638), Netherlands (n = 136), and Switzerland (n = 106), and in healthy (n = 1409) and alcoholic (n = 123) controls from the same populations. Polymorphisms were determined by melting curve analysis using fluorescence resonance energy transfer probes. In addition, 229 Dutch subjects were analysed by restriction fragment length polymorphism. RESULTS: The frequencies of UGT1A7 genotypes did not differ between patients with acute or chronic pancreatitis or pancreatic adenocarcinoma and alcoholic and healthy controls. CONCLUSIONS: The data suggest that, in contrast to earlier studies, UGT1A7 polymorphisms do not predispose patients to the development of pancreatic cancer and pancreatitis.

Upscaling and reversibility of Taylor dispersion in heterogeneous porous media.

Tracer flow in stratified porous media is dominated by the interaction between convective transport and transverse diffusive mixing. By averaging the tracer concentration in the transverse direction, a one-dimensional non-Fickian dispersion model is derived. The model accounts for the relaxation process that reduces the convective transport to dispersive mixing. This process is (short-) time correlated and partially reversible upon reversal of flow direction. For multiscale velocity fields, the relaxation is a multiscale process. To date only single scale processes have been successfully upscaled. Our procedure extends this to multiscale processes, using scale separation. The model parameters can be calculated a priori based on the velocity profile. For periodic flow reversal, the results are essentially the same. Despite the non-Fickian behavior during a cycle, the net contribution of each cycle to the spreading relaxes to a Fickian process in a similar way as for unidirectional flow. The cycle time averaged dispersion coefficient is a monotonically increasing function of the reversal time. It asymptotically converges towards the effective dispersion coefficient in the absence of flow reversal.

Gastrointestinal symptoms and ethanol metabolism in alcoholics.

Excessive alcohol intake frequently results in gastrointestinal discomfort. It is an empirical fact that the severity of gastrointestinal discomfort induced by alcohol abuse is subject to interindividual variation. The aim of this study was to determine whether genetic polymorphism in alcohol dehydrogenase 3 (ADH3) and cytochrome P450 2E1 (CYP2E1), important first-pass enzymes in the metabolism of ethanol, predispose to the development of gastrointestinal symptoms in alcoholics, Blood samples were obtained from 92 adult alcoholics admitted for detoxification. The samples were analyzed for genetic polymorphism in ADH3 and CYP2E1 by polymerase chain reaction followed by restriction fragment length polymorphism analyses. During an interview on the first day of hospital admission, patient characteristics and gastrointestinal symptoms in the week before admission were assessed. A total of 75 of 92 alcoholics (83%) reported symptoms: 66 patients had upper gastrointestinal symptoms (72%), 70 patients had lower gastrointestinal symptoms (76%), and 59 patients reported alarming symptoms (64%). Patients with gastrointestinal symptoms less often abused beer in comparison to those without gastrointestinal symptoms (P = 0.05). The numbers of patients with the homozygous y1y1 genotype, the heterozygous y1y2 genotype, and the homozygous y2y2 genotype in ADH3 who reported gastrointestinal symptoms were 20 (83%), 34 (76%), and 15 (88%), respectively. The number of patients with the heterozygous c1c2 CYP2E1 genotype (5%) and the heterozygous DC CYP2E1 genotype (14%) was low and also unrelated to gastrointestinal symptoms. Our data suggest that the ethanol concentrations of the consumed beverages, and not interindividual variations in the activities of first-pass alcohol-metabolizing enzymes, are associated with gastrointestinal symptoms in alcoholics.

A specific lysine in c-Jun is required for transcriptional repression by E1A and is acetylated by p300.

The adenovirus E1A protein regulates transcription of cellular genes via its interaction with the transcriptional coactivators p300/CBP. The collagenase promoter activated by the c-Jun protein is repressed by E1A. Here we show that E1A repression is specific for c-Jun, as E1A does not repress the collagenase promoter activated by the homologous transcription factor EB1. Using chimeras of c-Jun and EB1, we demonstrate that a 12 amino acid region in the basic region of the c-Jun DNA-binding domain is essential for repression by E1A. Since repression requires the binding of p300 to E1A, we studied the involvement of p300 acetyltransferase activity in the repression mechanism. We demonstrate that c-Jun is acetylated in vivo, and mutational analysis identified Lys271 in the c-Jun basic region to be essential for repression of the collagenase promoter by E1A. In addition, Lys271 is acetylated both in vitro and in vivo. These results suggest that the specific repression of the collagenase promoter by E1A involves acetylation of c-Jun.

The N-terminal transactivation domain of ATF2 is a target for the co-operative activation of the c-jun promoter by p300 and 12S E1A.

The adenovirus E1A proteins activate the c-jun promoter through two Jun/ATF-binding sites, jun1 and jun2. P300, a transcriptional coactivator of several AP1 and ATF transcription factors has been postulated to play a role in this activation. Here, we present evidence that p300 can control c-jun transcription by acting as a cofactor for ATF2: (1) Over-expression of p300 was found to stimulate c-jun transcription both in the presence and absence of E1A. (2) Like E1A, p300 activates the c-jun promoter through the junl and jun2 elements and preferentially activates the N-terminal domain of ATF2. (3) Co-immunoprecipitation assays of crude cell extracts indicate that endogenous p300/CBP(-like) proteins and ATF2 proteins are present in a multiprotein complex that can bind specifically to the jun2 element. We further demonstrate that the Stress-Activated-Protein-Kinase (SAPK) target sites of ATF2, Thr69 and Thr71 are not required for the formation of the p300/CBP-ATF2 multiprotein complex. These data indicate that E1A does not inhibit all transcription activation functions of p300, and, in fact, cooperates with p300 in the activation of the ATF2 N-terminus.

Comparison of the internal transcribed spacer, ITS 1, from Toxoplasma gondii isolates and Neospora caninum.

The internal transcribed spacer (ITS1) region and the 5' part of the 5.8S ribosomal RNA gene of the ribosomal DNA repeat from 20 Toxoplasma gondil isolates was sequenced and found to be identical in all isolates, independent of host origin or virulence to mice. The ITS1 region from the closely related coccidian parasite Neospora caninum differed in 22% of its nucleotides. Hence, the ITS1 region provides a good marker for the distinction of T. gondii and N. caninum but is not useful for epidemiology studies of T. gondii.

Classification of Enterobacteriaceae by minimization of stochastic complexity.

A new method for classifying bacteria is presented and applied to a large set of biochemical data for the Enterobacteriaceae. The method minimizes the bits needed to encode the classes and the items or, equivalently, maximizes the information content of the classification. The resulting taxonomy of Enterobacteriaceae corresponds well to the general structure of earlier classifications. Minimization of stochastic complexity can be considered as a useful tool to create bacterial classifications that are optimal from the point of view of information theory.

Altered AP-1/ATF complexes in adenovirus-E1-transformed cells due to EIA-dependent induction of ATF3.

The adenovirus (Ad) E1A proteins alter the expression level and activity of AP-1/ATF transcription factors. Previously we have shown that in AdE1-transformed cells cJun is hyperphosphorylated in its N-terminal transactivation domain, which parallels enhanced transactivation function. To find out whether the interaction between cJun and other cellular proteins is altered, we have searched for proteins which would physically associate with cJun. In this report we show that in AdE1-transformed cells cJun specifically associates with two proteins of 21 and 23 kD. These proteins are not expressed at detectable levels in the parental cells or in cells transformed by oncogenes other than AdE1. The cJun-associated proteins represent different forms of the bZIP transcription factor ATF3, the human homolog of rat LRF1. The expression of ATF3 is induced in AdE1-transformed cells and is a direct effect of the expression of E1A. Through induction of ATF3 expression and the subsequent formation of cJun/ATF3 heterodimers, E1A alters the repertoire of AP-1/ATF factors and may thereby redirect the corresponding gene-expression program. Since the induction of ATF3 is a function of sequences within the transforming 12S-ElA protein, cJun/ATF3 complexes might be involved in establishing cellular transformation by AdE1A.