Evaluation of Novel Benzo-annelated 1,4-dihydropyridines as MDR Modulators in Cancer Cells.

BACKGROUND: Multidrug resistance (MDR) is the main problem in anticancer therapy today. Causative transmembrane efflux pumps in cancer cells have been reconsidered as promising anticancer target structures to restore anticancer drug sensitivity by various strategies, including MDR modulators. MDR modulators interfere with the efflux pumps and improve the cellular efficiency of chemotherapeutics. So far, only a few candidates have gone through clinical trials with disappointing results because of low specificity and toxic properties. AIM: This study aimed to find Novel MDR modulators to effectively combat multidrug resistance in cancer cells. OBJECTIVE: We synthesized various novel benzo-annelated 1,4-dihydropyridines to evaluate them as MDR modulators towards ABCB1 in cancer cells. METHODS: Synthesized compounds were purified by column chromatography. The MDR modulation of ABCB1 was determined in cellular efflux assays using the flow cytometry technique and cellular fluorescent measurements by the use of each fluorescent substrate. RESULTS: Compounds were yielded in a two-step reaction with structurally varied components. Further, substituent- dependent effects on the determined MDR inhibiting properties towards ABCB1 were discussed. Cellular studies prove that there is no toxicity or restoration of cancer cell sensitivity towards the used anticancer drug. CONCLUSION: Novel MDR modulators could be identified with favorable methoxy and ester group functions. Their use in both ABCB1 non-expressing and overexpressing cells proves a selective toxicity-increasing effect of the applied anticancer agent in the ABCB1 overexpressing cells, whereas the toxicity effect of the anticancer drug was almost unchanged in the non-expressing cells. These results qualify our novel compounds as perspective anticancer drugs compared to MDR modulators with nonselective toxicity properties.

Ceruloplasmin expression in renal cell carcinoma correlates with higher-grade and shortened survival.

Aim: We aimed to explore ceruloplasmin (CP) expression in clear cell renal cell carcinoma (ccRCC). Materials & methods: CP was analyzed in biofluid samples of 63 ccRCC patients, divided into three grading groups, and immunohistochemically, in 308 ccRCC. Results: Significant differences of mean plasma and urine CP levels in different grading groups were found. CP immunoreactivity was significantly linked to high-grade disease. Log rank tests showed a significant shorter overall survival rate in CP-positive cases (all p < 0.05). Conclusion: CP protein levels in biofluid samples confirmed differential CP expressions, depending on nuclear grade in ccRCC as previously seen in RNA expression analysis. CP expression was linked to high-grade disease and reduced survival rate in RCC.

Novel Symmetrical Cage Compounds as Inhibitors of the Symmetrical MRP4-Efflux Pump for Anticancer Therapy.

Within the last decades cancer treatment improved by the availability of more specifically acting drugs that address molecular target structures in cancer cells. However, those target-sensitive drugs suffer from ongoing resistances resulting from mutations and moreover they are affected by the cancer phenomenon of multidrug resistance. A multidrug resistant cancer can hardly be treated with the common drugs, so that there have been long efforts to develop drugs to combat that resistance. Transmembrane efflux pumps are the main cause of the multidrug resistance in cancer. Early inhibitors disappointed in cancer treatment without a proof of expression of a respective efflux pump. Recent studies in efflux pump expressing cancer show convincing effects of those inhibitors. Based on the molecular symmetry of the efflux pump multidrug resistant protein (MRP) 4 we synthesized symmetric inhibitors with varied substitution patterns. They were evaluated in a MRP4-overexpressing cancer cell line model to prove structure-dependent effects on the inhibition of the efflux pump activity in an uptake assay of a fluorescent MRP4 substrate. The most active compound was tested to resentisize the MRP4-overexpressing cell line towards a clinically relevant anticancer drug as proof-of-principle to encourage for further preclinical studies.

Transient Howell-Jolly-body-like cytoplasmic inclusions in neutrophils after severe intoxication with alpha-lipoic acid.

After intravenous supplementation of an unintentionally high dose of the antioxidant alpha-lipoic acid (ALA), a 53-year-old female complained of myalgia, chills and nausea, and showed signs of haemorrhagic diathesis. The laboratory findings were excessive hyperferritinemia, leukoerythroblastosis, severe thrombocytopenia, elevated liver enzymes and impaired coagulation. The toxicological tests resulted in an ALA serum concentration of 10 280 µg/L. The peripheral blood film of the patient showed some neutrophil dysplasia with unusual small dark-blue stained round cytoplasmic inclusions resembling 'Howell-Jolly-body-like' (HJBL) cytoplasmic inclusions, aptly named due to the morphologic similarity to their erythrocytic counterparts. Such HJBL inclusions are occasionally associated with acquired immunodeficiency, or immunosuppressive or cytostatic treatment. An association with ALA intoxication has not been described before. There are only a few reports on unintentional, harmful and lethal intoxications with ALA. The underlying molecular background of its toxicity on liver function or haematopoiesis is not yet known in detail, but ALA seems to interact with enzyme functions, e.g. with mitochondrial enzyme-complexes, possibly due to its pro-oxidant potential at high doses.

Performance of the XN-2000 WPC channel-flagging to differentiate reactive and neoplastic leukocytosis.

BACKGROUND: The distinction between reactive and neoplastic leukocytes, especially atypical lymphocytes suspected to be reactive or neoplastic, is a particular challenge in automated hematological cell differentiation. The aim of the study was to evaluate the performance of the XN analyzer supplemented with the WPC channel for differentiating between reactive and neoplastic leukocytosis. METHODS: Blood samples of 253 patients with viral infections, lymphoma or leukemia were analyzed by the Sysmex XN-2000 analyzer equipped with the WPC channel. The results were compared to routine leukocyte differentiation using the routine Sysmex XE-2100 analyzer and automated digital microscopy (DM96). The combined information from standard morphology, immune phenotyping and clinical diagnosis served as a reference. RESULTS: The XN WPC channel demonstrated an excellent performance for differentiating neoplastic (AUC=0.933) and reactive leukocytosis (AUC=0.900) as compared to morphological smear examination (AUC=0.949 and AUC=0.968, respectively) or to the differentiation results of our routine hematology analyzer (AUC=0.630 and AUC=0.635, respectively). CONCLUSIONS: Our data show that the combined WDF/WPC of the Sysmex XN-Series analyzer is advantageous in the automated differentiation of neoplastic and reactive leukocytosis, thus supporting the correct diagnostic decision in the daily laboratory routine.

Exclusion of thrombocytopenia as a contraindication for invasive radiofrequency ablation in a patient with paroxysmal atrial fibrillation by using magnesium anticoagulation instead of EDTA: another case of anticoagulant-induced pseudo-thrombocytopenia.

Thrombocytopenia might be an exclusion criterion for invasive radiofrequency catheter ablation; therefore it is necessary to differentiate between pseudo-thrombocytopenia and a low platelet count due to other etiologies.A 69-year-old female presented to the cardiology department with recurrent atrial fibrillation that was resistant to conventional drug treatment. The initial laboratory findings were within the normal ranges, except for low platelet counts that occurred without a specific bleeding history. The reason for thrombocytopenia was anticoagulant-induced in vitro aggregation of platelets in the presence of EDTA as well as in citrated blood samples. As recently communicated, magnesium anticoagulated blood samples prevent platelet aggregation in individuals with anticoagulant-associated pseudo-thrombocytopenia. Although its aggregation-inhibiting effect is known from previous clinical observations, magnesium sulphate has not been introduced as an anticoagulant in analytical medicine.Based on our observations, blood anticoagulated with magnesium sulphate is recommended to verify low routine platelet counts before final clinical decisions are made.

Lean body mass correction of standardized uptake value in simultaneous whole-body positron emission tomography and magnetic resonance imaging.

This study explores the possibility of using simultaneous positron emission tomography--magnetic resonance imaging (PET-MRI) to estimate the lean body mass (LBM) in order to obtain a standardized uptake value (SUV) which is less dependent on the patients' adiposity. This approach is compared to (1) the commonly-used method based on a predictive equation for LBM, and (2) to using an LBM derived from PET-CT data. It is hypothesized that an MRI-based correction of SUV provides a robust method due to the high soft-tissue contrast of MRI. A straightforward approach to calculate an MRI-derived LBM is presented. It is based on the fat and water images computed from the two-point Dixon MRI primarily used for attenuation correction in PET-MRI. From these images, a water fraction was obtained for each voxel. Averaging over the whole body yielded the weight-normalized LBM. Performance of the new approach in terms of reducing variations of (18)F-Fludeoxyglucose SUVs in brain and liver across 19 subjects was compared with results using predictive methods and PET-CT data to estimate the LBM. The MRI-based method reduced the coefficient of variation of SUVs in the brain by 41 ± 10% which is comparable to the reduction by the PET-CT method (35 ± 10%). The reduction of the predictive LBM method was 29 ± 8%. In the liver, the reduction was less clear, presumably due to other sources of variation. In conclusion, employing the Dixon data in simultaneous PET-MRI for calculation of lean body mass provides a brain SUV which is less dependent on patient adiposity. The reduced dependency is comparable to that obtained by CT and predictive equations. Therefore, it is more comparable across patients. The technique does not impose an overhead in measurement time and is straightforward to implement.

Integrated PET/MRI for planning navigated biopsies in pediatric brain tumors.

INTRODUCTION: An integrated PET/MRI scanner has been used in selected cases of pediatric brain tumor patients to obtain additional metabolic information about lesions for preoperative biopsy planning and navigation. PATIENTS AND METHODS: Four patients, age 9-16 years, received PET/MRI scans employing [(11)C]methionine positron emission tomography (PET) and contrast-enhanced 3D-MR sequences for neuronavigation. PET and MR sequences have been matched for neurosurgical guidance. An infrared camera-based neuronavigation system was employed with co-registered MR and PET images fused to hybrid images for preoperative planning, stereotactic biopsy planning, and/or intraoperative guidance. RESULTS: All patients showed hot spots of increased amino acid transport in PET and contrast-enhancing lesions in MRI. In three of the four patients, PET hot spots were congruent with contrast-enhancing areas in MRI. In two patients, frame-based stereotactic biopsies were taken from thalamo-mesencephalic lesions. One patient underwent second-look surgery for the suspicion of recurrent malignant glioma of the posterior fossa. One incidental frontal mass lesion was subtotally resected. No complications occurred. Hybrid imaging was helpful during the procedures to obtain representative histopathologic specimens and for surgical guidance during resection. Co-registered images did match with intraoperative landmarks, tumor borders, and histopathologic specimens. CONCLUSION: The integrated PET/MRI scanner offers co-registered multimodal, high-resolution data for neuronavigation with reduced radiation exposure compared to PET/CT scans. One examination session provides all necessary data for neuronavigation and preoperative planning, avoiding additional anesthesia in the small patients. Hybrid multimodality imaging may improve safety and yield additional information when obtaining representative histopathologic specimens of brain tumors.

The control of seed oil polyunsaturate content in the polyploid crop species Brassica napus.

Many important plant species have polyploidy in their recent ancestry, complicating inferences about the genetic basis of trait variation. Although the principal locus controlling the proportion of polyunsaturated fatty acids (PUFAs) in seeds of Arabidopsis thaliana is known (fatty acid desaturase 2; FAD2), commercial cultivars of a related crop, oilseed rape (Brassica napus), with very low PUFA content have yet to be developed. We showed that a cultivar of oilseed rape with lower than usual PUFA content has non-functional alleles at three of the four orthologous FAD2 loci. To explore the genetic basis further, we developed an ethyl methanesulphonate mutagenised population, JBnaCAB_E, and used it to identify lines that also carried mutations in the remaining functional copy. This confirmed the hypothesised basis of variation, resulting in an allelic series of mutant lines showing a spectrum of PUFA contents of seed oil. Several lines had PUFA content of ~6 % and oleic acid content of ~84 %, achieving a long-standing industry objective: very high oleic, very low PUFA rapeseed without the use of genetic modification technology. The population contains a high rate of mutations and represents an important resource for research in B. napus.

Biomimetic magnetite formation: from biocombinatorial approaches to mineralization effects.

Biological materials typically display complex morphologies and hierarchical architectures, properties that are hardly matched by synthetic materials. Understanding the biological control of mineral properties will enable the development of new synthetic approaches toward biomimetic functional materials. Here, we combine biocombinatorial approaches with a proteome homology search and in vitro mineralization assays to assess the role of biological determinants in biomimetic magnetite mineralization. Our results suggest that the identified proteins and biomimetic polypeptides influence nucleation in vitro. Even though the in vivo role cannot be directly determined from our experiments, we can rationalize the following design principles: proteins, larger complexes, or membrane components that promote nucleation in vivo are likely to expose positively charged residues to a negatively charged crystal surface. In turn, components with acidic (negatively charged) functionality are nucleation inhibitors, which stabilize an amorphous structure through the coordination of iron.

PET/MR in children. Initial clinical experience in paediatric oncology using an integrated PET/MR scanner.

Use of PET/MR in children has not previously been reported, to the best of our knowledge. Children with systemic malignancies may benefit from the reduced radiation exposure offered by PET/MR. We report our initial experience with PET/MR hybrid imaging and our current established sequence protocol after 21 PET/MR studies in 15 children with multifocal malignant diseases. The effective dose of a PET/MR scan was only about 20% that of the equivalent PET/CT examination. Simultaneous acquisition of PET and MR data combines the advantages of the two previously separate modalities. Furthermore, the technique also enables whole-body diffusion-weighted imaging (DWI) and statements to be made about the biological cellularity and nuclear/cytoplasmic ratio of tumours. Combined PET/MR saves time and resources. One disadvantage of PET/MR is that in order to have an effect, a significantly longer examination time is needed than with PET/CT. In our initial experience, PET/MR has turned out to be an unexpectedly stable and reliable hybrid imaging modality, which generates a complementary diagnostic study of great additional value.

Discovery and development of exome-based, co-dominant single nucleotide polymorphism markers in hexaploid wheat (Triticum aestivum L.).

Globally, wheat is the most widely grown crop and one of the three most important crops for human and livestock feed. However, the complex nature of the wheat genome has, until recently, resulted in a lack of single nucleotide polymorphism (SNP)-based molecular markers of practical use to wheat breeders. Recently, large numbers of SNP-based wheat markers have been made available via the use of next-generation sequencing combined with a variety of genotyping platforms. However, many of these markers and platforms have difficulty distinguishing between heterozygote and homozygote individuals and are therefore of limited use to wheat breeders carrying out commercial-scale breeding programmes. To identify exome-based co-dominant SNP-based assays, which are capable of distinguishing between heterozygotes and homozygotes, we have used targeted re-sequencing of the wheat exome to generate large amounts of genomic sequences from eight varieties. Using a bioinformatics approach, these sequences have been used to identify 95 266 putative single nucleotide polymorphisms, of which 10 251 were classified as being putatively co-dominant. Validation of a subset of these putative co-dominant markers confirmed that 96% were true polymorphisms and 65% were co-dominant SNP assays. The new co-dominant markers described here are capable of genotypic classification of a segregating locus in polyploid wheat and can be used on a variety of genotyping platforms; as such, they represent a powerful tool for wheat breeders. These markers and related information have been made publically available on an interactive web-based database to facilitate their use on genotyping programmes worldwide.

Role of lipid apheresis in changing times.

During the last decades, LDL-apheresis was established as an extracorporeal treatment option for patients with severe heterozygous or homozygous familial hypercholesterolemia (FH) that is resistant to conventional treatment strategies such as diet, drugs, and changes in lifestyle. Nearly half a century ago, the first LDL-apheresis treatment was performed by plasma exchange in a child with homozygous FH. At the beginning of the 1970s, the clinical advantage of regular extracorporeal LDL-elimination was demonstrated in siblings suffering from homozygous FH. These findings encouraged researchers especially from Germany and Japan to develop extracorporeal devices to selectively eliminate LDL-cholesterol in the 1980s. Although the selectivity of the currently available LDL-apheresis devices is different, the efficacy of LDL-elimination during a single treatment is rather similar and ranges between 55 and 65 % of the pretreatment LDL plasma concentration.In the 1990s, the patients regularly treated by extracorporeal LDL-elimination, diet, and drugs were included in regression studies assessed by angiography. It was shown that the combined treatment with LDL-apheresis, diet, and drugs resulted in less progression of coronary lesions than drugs and/or diet alone. However, although a tendency was evident, results did not reach criteria for significance. During the last decade, apheresis registries were established to collect data on efficiency, safety, and clinical outcome of regular long-term LDL-apheresis. The evaluation of registry data will certainly permit further insights in the therapeutic benefit of this expensive and time-consuming therapeutic approach. Furthermore, the future of LDL-apheresis will depend upon the availability of highly efficient new drugs and molecular genetic approaches such as RNA silencing of the apoB gene, whereas the liver transplantation and gene therapy of the LDL-receptor deficiency will not replace LDL-apheresis in severe familial hypercholesterolemia in the near future.

Health care consequences of demographic changes in Mecklenburg-West Pomerania: projected case numbers for age-related diseases up to the year 2020, based on the Study of Health in Pomerania (SHIP).

BACKGROUND: The population in the German federal state of Mecklenburg-West Pomerania is growing older. A resulting rise in age-related diseases will likely lead to a greater need for medical care, even though the population as a whole is declining. The predicted number of patients affected by these diseases varies from one district to another because of local differences in demographic trends. METHODS: Case numbers were forecasted on the basis of representative data on the morbidity from chronic diseases, which were derived from the Study of Health in Pomerania (SHIP), the conjoint cancer registry of the East German federal states (GKR), and a study on dementia morbidity. These data were combined with demographic prognoses for Mecklenburg-West Pomerania and its rural and urban districts up to the year 2020. RESULTS: The largest increases in case numbers are predicted for dementia (+91.1%), myocardial infarction (+28.3%), diabetes mellitus (+21.4%), and incident colon carcinoma (+31.0%; all figures are expressed in relation to the year 2005 as a baseline). The predicted changes in case numbers vary widely from one district to another. CONCLUSION: All of the German federal states located in the former East Germany are likely to experience similar developments to those predicted for Mecklenburg-West Pomerania, as will many rural areas of the former West Germany, in which a demographic transition is already evident. Because of the predicted rise in the number of patients, new health care concepts will have to be rapidly developed, implemented, and evaluated in order to ensure that comprehensive medical care will be delivered where it is needed.

Renal Insufficiency Following Radiocontrast Exposure Trial (REINFORCE): a randomized comparison of sodium bicarbonate versus sodium chloride hydration for the prevention of contrast-induced nephropathy.

INTRODUCTION: For the prevention of contrast-induced nephropathy (CIN) after coronary angiography, hydration by 0.9% sodium chloride solution and N-acetylcysteine is currently recommended. However, it is unclear whether volume supplementation with sodium bicarbonate is better than with sodium chloride when used in conjunction with nonionic, low-osmolar iopamidol. The aim of this study was to analyze and compare the effects of sodium bicarbonate and sodium chloride on renal function in 145 patients exposed to nonionic iso-osmolar contrast medium iodixanol in a randomized study. PATIENTS AND METHODS: Renal Insufficiency Following Radiocontrast Exposure is a prospective, randomized, single-center, double-blinded trial of 145 patients (age 72.6+/-6.7 years) with elevated baseline serum creatinine levels (mean 132.6+/-29.3 micromol/l). Eligible patients were randomized to either a 154 mEq/l infusion of sodium bicarbonate (n=71, group I) or sodium chloride 0.9% solution (n=74, group II). The primary endpoint was serum creatinine elevation beyond 25% or 44 micromol/l on the first or second day following exposure to the contrast medium. Serum creatinine, serum cystatin C, plasma viscosity, urinary enzymes alanine aminopeptidase and N-acetyl-beta-D-glucosaminidase, and alpha1-microglobulin were measured at baseline and on days 1 and 2 after contrast medium administration. RESULTS: An overall proportion of five CIN (3.4%) was observed with equal distribution among the groups (4.2% in sodium bicarbonate group vs. 2.7% in sodium chloride group; P=0.614). Parameters of renal function demonstrated no differences between the two hydration regimens on day 1 after angiography; even on day 2 most parameters were similar in groups I and II. CONCLUSION: Renal Insufficiency Following Radiocontrast Exposure demonstrates a homogeneously low rate of CIN after exposure to nonionic, iso-osmolar iodixanol regardless of the use of either bicarbonate sodium or sodium chloride solution for volume supplementation. Low-toxicity contrast media and any hydration may offset potential antioxidant effects of sodium bicarbonate.

Thrombin/thrombin receptor (PAR-1)-mediated induction of IL-8 and VEGF expression in prostate cancer cells.

Interleukin 8 (IL-8) and vascular endothelial growth factor (VEGF) are two cytokines promoting prostate tumor growth and angiogenesis. The main coagulation protease thrombin may modulate the malignant phenotype of prostate cancer cells via its cellular receptor(s). We aimed to investigate the effects of thrombin on IL-8 and VEGF expression in DU 145 prostate cancer cells. Thrombin induced the expression and secretion of IL-8 and VEGF, with more pronounced effects on IL-8. Target-specific siRNA-induced protease-activated receptor 1 (PAR-1) knockdown completely neutralized thrombin-enhanced cytokine secretion, demonstrating the essential role of PAR-1. Inhibitors of either extracellular signal-regulating kinase (ERK) or phosphatidylinositol 3-kinase (PI3K) partly reversed the thrombin-induced cytokine expression, suggesting that both ERK and PI3K kinase pathways may be involved in IL-8 and VEGF expression. The results suggest that the thrombin/PAR-1 system upregulates cytokines in prostate cancer cells which in turn may contribute to the progression of prostate cancer.