Preparation, characterization and evaluation of HPßCD-PTX/PHB nanoparticles for pH-responsive, cytotoxic and apoptotic properties.

Paclitaxel (PTX) is a potent anticancer drug. However, PTX exhibits extremely poor solubility in aqueous solution along with severe side effects. Therefore, in this study, an inclusion complex was prepared between PTX and hydroxypropyl-ß-cyclodextrin (HPßCD) by solvent evaporation to enhance the drug's solubility. The HPßCD-PTX inclusion complex was then encapsulated in poly-3-hydroxybutyrate (PHB) to fabricate drug-loaded nanoparticles (HPßCD-PTX/PHB NPs) by nanoprecipitation. The HPßCD-PTX/PHB NPs depicted a higher release of PTX at pH 5.5 thus demonstrating a pH-dependent release profile. The cytotoxic properties of HPßCD-PTX/PHB NPs were tested against MCF-7, MDA-MB-231 and SW-620 cell lines. The cytotoxic potential of HPßCD-PTX/PHB NPs was 2.59-fold improved in MCF-7 cells in comparison to free PTX. Additionally, the HPßCD-PTX/PHB NPs improved the antimitotic (1.68-fold) and apoptotic (8.45-fold) effects of PTX in MCF-7 cells in comparison to PTX alone. In summary, these pH-responsive nanoparticles could be prospective carriers for enhancing the cytotoxic properties of PTX for the treatment of breast cancer.

Altered serum metabolome as an indicator of paraneoplasia or concomitant cancer in patients with rheumatic disease.

OBJECTIVES: A timely diagnosis is imperative for curing cancer. However, in patients with rheumatic musculoskeletal diseases (RMDs) or paraneoplastic syndromes, misleading symptoms frequently delay cancer diagnosis. As metabolic remodelling characterises both cancer and RMD, we analysed if a metabolic signature can indicate paraneoplasia (PN) or reveal concomitant cancer in patients with RMD. METHODS: Metabolic alterations in the sera of rheumatoid arthritis (RA) patients with (n=56) or without (n=52) a history of invasive cancer were quantified by nuclear magnetic resonance analysis. Metabolites indicative of cancer were determined by multivariable regression analyses. Two independent RA and spondyloarthritis (SpA) cohorts with or without a history of invasive cancer were used for blinded validation. Samples from patients with active cancer or cancer treatment, pulmonary and lymphoid type cancers, paraneoplastic syndromes, non-invasive (NI) precancerous lesions and non-melanoma skin cancer and systemic lupus erythematosus and samples prior to the development of malignancy were used to test the model performance. RESULTS: Based on the concentrations of acetate, creatine, glycine, formate and the lipid ratio L1/L6, a diagnostic model yielded a high sensitivity and specificity for cancer diagnosis with AUC=0.995 in the model cohort, AUC=0.940 in the blinded RA validation cohort and AUC=0.928 in the mixed RA/SpA cohort. It was equally capable of identifying cancer in patients with PN. The model was insensitive to common demographic or clinical confounders or the presence of NI malignancy like non-melanoma skin cancer. CONCLUSIONS: This new set of metabolic markers reliably predicts the presence of cancer in arthritis or PN patients with high sensitivity and specificity and has the potential to facilitate a rapid and correct diagnosis of malignancy.

Khasianine Affects the Expression of Sugar-Sensitive Proteins in Pancreatic Cancer Cells, Which Are Altered in Data from the Rat Model and Patients.

Pancreatic ductal adenocarcinoma (PDAC) is a deadly malignancy with no effective treatment, particularly in the advanced stage. This study explored the antiproliferative activity of khasianine against pancreatic cancer cell lines of human (Suit2-007) and rat (ASML) origin. Khasianine was purified from Solanum incanum fruits by silica gel column chromatography and analyzed by LC-MS and NMR spectroscopy. Its effect in pancreatic cancer cells was evaluated by cell proliferation assay, chip array and mass spectrometry. Proteins showing sensitivity to sugars, i.e. sugar-sensitive lactosyl-Sepharose binding proteins (LSBPs), were isolated from Suit2-007 cells by competitive affinity chromatography. The eluted fractions included galactose-, glucose-, rhamnose- and lactose-sensitive LSBPs. The resulting data were analyzed by Chipster, Ingenuity Pathway Analysis (IPA) and GraphPad Prism. Khasianine inhibited proliferation of Suit2-007 and ASML cells with IC50 values of 50 and 54 µg/mL, respectively. By comparative analysis, khasianine downregulated lactose-sensitive LSBPs the most (126%) and glucose-sensitive LSBPs the least (85%). Rhamnose-sensitive LSBPs overlapped significantly with lactose-sensitive LSBPs and were the most upregulated in data from patients (23%) and a pancreatic cancer rat model (11.5%). From IPA, the Ras homolog family member A (RhoA) emerged as one of the most activated signaling pathways involving rhamnose-sensitive LSBPs. Khasianine altered the mRNA expression of sugar-sensitive LSBPs, some of which were modulated in data from patients and the rat model. The antiproliferative effect of khasianine in pancreatic cancer cells and the downregulation of rhamnose-sensitive proteins underscore the potential of khasianine in treating pancreatic cancer.

Reduction of Proinflammatory Effector Functions Through Remodeling of Fatty Acid Metabolism in CD8+ T Cells From Rheumatoid Arthritis Patients.

OBJECTIVE: Rheumatoid arthritis (RA) CD8+ T cells maintain their effector proinflammatory phenotype by changing their metabolism toward aerobic glycolysis. However, their massive energy and biosynthesis needs may require additional substrates other than glucose. Since systemic alterations in lipid metabolism have been reported in RA patients, we explored the role of fatty acid (FA) metabolism in CD8+ T cells to identify potential targets to curb their proinflammatory potential. METHODS: The expression of FA metabolism-related genes was analyzed for total CD8+ T cells and CD8+ T cell subsets in the data of RA patients and healthy controls retrieved from the GEO database. Functional assays were performed using peripheral blood CD8+ T cells isolated from RA (n = 31), psoriatic arthritis (n = 26), and spondyloarthritis (n = 21) patients receiving different therapies (disease-modifying antirheumatic drugs, biologics, and JAK inhibitors) and from healthy controls (n = 14). We quantified the expression of FA transporters, lipid uptake, intracellular FA content, cytokine production, activation, proliferation, and capacity to inhibit tumor cell growth, either with or without FA metabolism inhibitors. RESULTS: The CD8+ T cell gene expression profile of FA metabolism-related genes was significantly different between untreated RA patients and healthy controls. RA patients who had a good clinical response after 6 months of methotrexate therapy had significantly increased expression of FA metabolism-related genes. Cell surface expression of the FA transporters FA binding protein 4 (FABP4) and G protein-coupled receptor 84 (GPR84) and FA uptake were higher in effector and memory CD8+ T cells from RA patients compared to those from healthy controls. In vitro blockade of FA metabolism significantly impaired CD8+ T cell effector functions. CONCLUSION: RA CD8+ T cells present an altered FA metabolism, which could provide potential therapeutic targets to control their proinflammatory profile, particularly therapies directed against the transport and oxidation of free FA.

Distinct immune-effector and metabolic profile of CD8+ T cells in patients with autoimmune polyarthritis induced by therapy with immune checkpoint inhibitors.

OBJECTIVES: Rheumatic immune-related adverse events (irAE) such as (poly)arthritis in patients undergoing immune checkpoint inhibitor (ICI) treatment pose a major clinical challenge. ICI therapy improves CD8+ T cell (CD8) function, but CD8 contributes to chronic inflammation in autoimmune arthritis (AA). Thus, we investigated whether immune functional and metabolic changes in CD8 explain the development of musculoskeletal irAE in ICI-treated patients. METHODS: Peripheral CD8 obtained from ICI-treated patients with and without arthritis irAEs and from AA patients with and without a history of malignancy were stimulated in media containing 13C-labelled glucose with and without tofacitinib or infliximab. Changes in metabolism, immune-mediator release, expression of effector cell-surface molecules and inhibition of tumour cell growth were quantified. RESULTS: CD8 from patients with irAE showed significantly lower frequency and expression of cell-surface molecule characteristic for activation, effector-functions, homing, exhaustion and apoptosis and reduced release of cytotoxic and proinflammatory immune mediators compared with CD8 from ICI patients who did not develop irAE. This was accompanied by a higher glycolytic rate and ATP production. Gene-expression analysis of pre-ICI-treated CD8 revealed several differentially expressed transcripts in patients who later developed arthritis irAEs. In vitro tofacitinib or infliximab treatment did not significantly change the immune-metabolic profile nor the capacity to release cytolytic mediators that inhibit the growth of the human lung cancer cell line H838. CONCLUSIONS: Our study shows that CD8 from ICI-treated patients who develop a musculoskeletal irAE has a distinct immune-effector and metabolic profile from those that remain irAE free. This specific irAE profile overlaps with the one observed in CD8 from AA patients and may prove useful for novel therapeutic strategies to manage ICI-induced irAEs.

Synthesis of a polyaminocarboxylate-based aluminum complex and its structural studies using 1H{13C}-HMBC NMR and a Karplus-type function.

The HBED chelator is used to stabilize small and hard metal ions such as Fe3+, Ti4+, Ga3+ and Al3+ in both medicine and industry. While the coordination of hexadentate HBED4- is known in the case of Fe3+, Ti4+ and Ga3+, it is unknown in the case of the small Al3+ ion since its corresponding complex has never been fully characterized. Thus, in this work the coordination pattern in a newly synthesized aluminum HBED-based complex ([Al-HBED-NN]-Na+) was determined using 2D NMR in conjunction with DFT calculations.

Synthesis, Characterization and Biological Investigations of Half-Sandwich Ruthenium(II) Complexes Containing Benzimidazole Moiety.

Half-sandwich Ru(II) complexes belong to group of biologically active metallo-compounds with promising antimicrobial and anticancer activity. Herein, we report the synthesis and characterization of arene ruthenium complexes containing benzimidazole moiety, namely, [(η6-p-cymene)RuCl(bimCOO)] (1) and [(η6-p-cymene)RuCl2(bim)] (2) (where bimCOO = benzimidazole-2-carboxylate and bim = 1-H-benzimidazole). The compounds were characterized by 1H NMR, 13C NMR, IR, UV-vis and CV. Molecular structures of the complexes were determined by SC-XRD analysis, and the results indicated the presence of a pseudo-tetrahedral (piano stool) geometry. Interactions in the crystals of the Ru complexes using the Hirshfeld surface analysis were also examined. In addition, the biological studies of the complexes, such as antimicrobial assays (against planktonic and adherent microbes), cytotoxicity and lipophilicity, were performed. Antibacterial activity of the complexes was evaluated against S. aureus, E. coli, P. aeruginosa PAO1 and LES B58. Cytotoxic activity was tested against primary human fibroblasts and adenocarcinoma human alveolar basal epithelial cells. Obtained biological results show that the ruthenium compounds have bacteriostatic activity toward Pseudomonas aeruginosa PAO1 strain and are not toxic to normal cells. A molecular docking study was applied as a predictive source of information about the plausibility of examined structures binding with HSA as a transporting system.

Synthesis and application of a thiol-reactive HBED-type chelator for development of easy-to-produce Ga-radiopharmaceutical kits and imaging probes.

In radiopharmaceutical syntheses, maleimide is commonly used for linking thiol-bearing bioactive molecules to metal-complexing ligands (chelators). However, due to instability of the resulting linkage, phenyloxadiazolyl methylsulfone (PODS) was developed as an alternative to maleimide. This coupling strategy has never been attempted with HBED which is a powerful chelator for gallium-radiolabeling especially at ambient temperature. Here we present HBED-CC-PODS as a bifunctional chelator scaffold for the site-selective conjugation of thiol-bearing vectors and [68Ga]Ga-radiolabeling.

Anticancer potential of rhizome extract and a labdane diterpenoid from Curcuma mutabilis plant endemic to Western Ghats of India.

Zingiberaceae plants are well known for their use in ethnomedicine. Curcuma mutabilis Skornick., M. Sabu & Prasanthk., is an endemic Zingiberaceae species from Western Ghats of Kerala, India. Here, we report for the first time, the anticancer potential of petroleum ether extract from C. mutabilis rhizome (CMRP) and a novel labdane diterpenoid, (E)-14, 15-epoxylabda-8(17), 12-dien-16-al (Cm epoxide) isolated from it. CMRP was found to be a mixture of potent bioactive compounds including Cm epoxide. Both the extract and the compound displayed superior antiproliferative activity against several human cancer cell lines, without any display of cytotoxicity towards normal human cells such as peripheral blood derived lymphocytes and erythrocytes. CMRP treatment resulted in phosphatidylserine externalization, increase in the levels of intracellular ROS, Ca2+, loss of mitochondrial membrane potential as well as fragmentation of genomic DNA. Analyses of transcript profiling and immunostained western blots of extract-treated cancer cells confirmed induction of apoptosis by both intrinsic and extrinsic pathways. The purified compound, Cm epoxide, was also found to induce apoptosis in many human cancer cell types tested. Both CMRP and the Cm epoxide were found to be pharmacologically safe in terms of acute toxicity assessment using Swiss albino mice model. Further, molecular docking interactions of Cm epoxide with selected proteins involved in cell survival and death were also indicative of its druggability. Overall, our findings reveal that the endemic C. mutabilis rhizome extract and the compound Cm epoxide isolated from it are potential candidates for development of future cancer chemotherapeutics.

Riproximin Exhibits Diversity in Sugar Binding, and Modulates some Metastasis-Related Proteins with Lectin like Properties in Pancreatic Ductal Adenocarcinoma.

Riproximin (Rpx) is a type II ribosome-inactivating protein with specific anti-proliferative activity. It was purified from Ximenia americana by affinity chromatography using a resin coupled with lactosyl residues. The same technique facilitated isolation of proteins with lectin-like properties from human Suit2-007 and rat ASML pancreatic cancer cells, which were termed lactosyl-sepharose binding proteins (LSBPs). The role of these proteins in cancer progression was investigated at mRNA level using chip array data of Suit2-007 and ASML cells re-isolated from nude rats. These data compared significant mRNA expression changes when relating primary (pancreas) and metastatic (liver) sites following orthotopic and intraportal implantation of Pancreatic Ductal Adenocarcinoma (PDAC) cells, respectively. The affinity of Rpx to 13 simple sugar structures was modeled by docking experiments, the ranking of which was principally confirmed by NMR-spectroscopy. In addition, Rpx and LSBPs were evaluated for anti-proliferative activity and their cellular uptake was assessed by fluorescence microscopy. From 13 monosaccharides evaluated, open-chain rhamnose, ß-d-galactose, and α-l-galactopyranose showed the highest affinities for site 1 of Rpx's B-chain. NMR evaluation yielded a similar ranking, as galactose was among the best binders. Both, Rpx and LSBPs reduced cell proliferation in vitro, but their anti-proliferative effects were decreased by 15-20% in the presence of galactose. The program "Ingenuity Pathway Analysis" identified 2,415 genes showing significantly modulated mRNA expression following exposure of Suit2-007 cells to Rpx in vitro. These genes were then matched to those 1,639 genes, which were significantly modulated in the rat model when comparing primary and metastatic growth of Suit2-007 cells. In this overlap analysis, LSBP genes were considered separately. The potential suitability of Rpx for treating metastatic Suit2-007 PDAC cells was reflected by those genes, which were modulated by Rpx in a way opposite to that observed in cancer progression. Remarkably, these were 14% of all genes modulated during cancer progression, but 71% of the respective LSBP gene subgroup. Based on these findings, we predict that Rpx has the potential to treat PDAC metastasis by modulating genes involved in metastatic progression, especially by targeting LSBPs.

Effect of Increased Lactate Dehydrogenase A Activity and Aerobic Glycolysis on the Proinflammatory Profile of Autoimmune CD8+ T Cells in Rheumatoid Arthritis.

OBJECTIVE: CD8+ T cells contribute to rheumatoid arthritis (RA) by releasing proinflammatory and cytolytic mediators, even in a challenging hypoxic and nutrient-poor microenvironment such as the synovial membrane. This study was undertaken to explore the mechanisms through which CD8+ T cells meet their metabolic demands in the blood and synovial membrane of patients with RA. METHODS: Purified blood CD8+ T cells from patients with RA, patients with psoriatic arthritis (PsA), and patients with spondyloarthritis (SpA), as well as healthy control subjects, and CD8+ T cells from RA synovial membrane were stimulated in medium containing 13 C-labeled metabolic substrates in the presence or absence of metabolic inhibitors, under conditions of normoxia or hypoxia. The production of metabolic intermediates was quantified by 1 H-nuclear magnetic resonance. The expression of metabolic enzymes, transcription factors, and immune effector molecules was assessed at both the messenger RNA (mRNA) and protein levels. CD8+ T cell functional studies were performed. RESULTS: RA blood CD8+ T cells met their metabolic demands through aerobic glycolysis, production of uniformly 13 C-enriched lactate in the RA blood (2.6 to 3.7-fold higher than in patients with SpA, patients with PsA, and healthy controls; P < 0.01), and induction of glutaminolysis. Overexpression of Warburg effect-linked enzymes in all RA CD8+ T cell subsets maintained this metabolic profile, conferring to the cells the capacity to proliferate under hypoxia and low-glucose conditions. In all RA CD8+ T cell subsets, lactate dehydrogenase A (LDHA) was overexpressed at the mRNA level (P < 0.03 versus controls; n = 6 per group) and protein level (P < 0.05 versus controls; n = 17 RA patients, n = 9 controls). In RA blood, inhibition of LDHA with FX11 led to reductions in lipogenesis, migration and proliferation of CD8+ T cells, and CD8+ T cell effector functions, while production of reactive oxygen species was increased by 1.5-fold (P < 0.03 versus controls). Following inhibition of LDHA with FX11, RA CD8+ T cells lost their capacity to induce healthy B cells to develop a proinflammatory phenotype. Similar metabolic alterations were observed in RA CD8+ T cells from the synovial membrane. CONCLUSION: Remodeling glucose and glutamine metabolism in RA CD8+ T cells by targeting LDHA activity can reduce the deleterious inflammatory and cytolytic contributions of these cells to the development of autoimmunity.

Vancomycin Resistance Is Overcome by Conjugation of Polycationic Peptides.

Multidrug-resistant bacteria represent one of the biggest challenges facing modern medicine. The increasing prevalence of glycopeptide resistance compromises the efficacy of vancomycin, for a long time considered as the last resort for the treatment of resistant bacteria. To reestablish its activity, polycationic peptides were conjugated to vancomycin. By site-specific conjugation, derivatives that bear the peptide moiety at four different sites of the antibiotic were synthesized. The most potent compounds exhibited an approximately 1000-fold increased antimicrobial activity and were able to overcome the most important types of vancomycin resistance. Additional blocking experiments using d-Ala-d-Ala revealed a mode of action beyond inhibition of cell-wall formation. The antimicrobial potential of the lead candidate FU002 for bacterial infection treatments could be demonstrated in an in vivo study. Molecular imaging and biodistribution studies revealed that conjugation engenders superior pharmacokinetics.

Differences in the serum metabolome and lipidome identify potential biomarkers for seronegative rheumatoid arthritis versus psoriatic arthritis.

OBJECTIVES: The differential diagnosis of seronegative rheumatoid arthritis (negRA) and psoriasis arthritis (PsA) is often difficult due to the similarity of symptoms and the unavailability of reliable clinical markers. Since chronic inflammation induces major changes in the serum metabolome and lipidome, we tested whether differences in serum metabolites and lipids could aid in improving the differential diagnosis of these diseases. METHODS: Sera from negRA and PsA patients with established diagnosis were collected to build a biomarker-discovery cohort and a blinded validation cohort. Samples were analysed by proton nuclear magnetic resonance. Metabolite concentrations were calculated from the spectra and used to select the variables to build a multivariate diagnostic model. RESULTS: Univariate analysis demonstrated differences in serological concentrations of amino acids: alanine, threonine, leucine, phenylalanine and valine; organic compounds: acetate, creatine, lactate and choline; and lipid ratios L3/L1, L5/L1 and L6/L1, but yielded area under the curve (AUC) values lower than 70%, indicating poor specificity and sensitivity. A multivariate diagnostic model that included age, gender, the concentrations of alanine, succinate and creatine phosphate and the lipid ratios L2/L1, L5/L1 and L6/L1 improved the sensitivity and specificity of the diagnosis with an AUC of 84.5%. Using this biomarker model, 71% of patients from a blinded validation cohort were correctly classified. CONCLUSIONS: PsA and negRA have distinct serum metabolomic and lipidomic signatures that can be used as biomarkers to discriminate between them. After validation in larger multiethnic cohorts this diagnostic model may become a valuable tool for a definite diagnosis of negRA or PsA patients.

HBED-NN: A Bifunctional Chelator for Constructing Radiopharmaceuticals.

Radiometal-based radiopharmaceuticals bearing bifunctional HBED chelators are powerful radiotracers for cancer diagnosis and therapy. Bifunctional HBED chelators make strong complexes with trivalent gallium and are able to bind to bioactive molecules through covalent bonds. However, thus far, no bifunctional HBED chelator capable of direct conjugation via click chemistry has been reported. We hereby introduce HBED-NN as a structurally new bifunctional HBED chelator for direct click coupling. We also investigated the complex chemistry of [Ga-(HBED-NN)] for potential use in gallium-based radiopharmaceuticals.

Relaxation-compensated APT and rNOE CEST-MRI of human brain tumors at 3 T.

PURPOSE: Relaxation-compensated CEST-MRI (i.e., the inverse metrics magnetization transfer ratio and apparent exchange-dependent relaxation) has already been shown to provide valuable information for brain tumor diagnosis at ultrahigh magnetic field strengths. This study aims at translating the established acquisition protocol at 7 T to a clinically relevant magnetic field strength of 3 T. METHODS: Protein model solutions were analyzed at multiple magnetic field strengths to assess the spectral widths of the amide proton transfer and relayed nuclear Overhauser effect (rNOE) signals at 3 T. This prior knowledge of the spectral range of CEST signals enabled a reliable and stable Lorentzian-fitting also at 3 T where distinct peaks are no longer resolved in the Z-spectrum. In comparison to the established acquisition protocol at 7 T, also the image readout was extended to three dimensions. RESULTS: The observed spectral range of CEST signals at 3 T was approximately ±15 ppm. Final relaxation-compensated amide proton transfer and relayed nuclear Overhauser effect contrasts were in line with previous results at 7 T. Examination of a patient with glioblastoma demonstrated the applicability of this acquisition protocol in a clinical setting. CONCLUSION: The presented acquisition protocol allows relaxation-compensated CEST-MRI at 3 T with a 3D coverage of the human brain. Translation to a clinically relevant magnetic field strength of 3 T opens the door to trials with a large number of participants, thus enabling a comprehensive assessment of the clinical relevance of relaxation compensation in CEST-MRI.

Nutraceutical compounds: Echinoids, flavonoids, xanthones and caffeine identified and quantitated in the leaves of Coffea arabica trees from three regions of Brazil.

There are relatively few studies concerning the use of coffee leaves for medicinal purposes and the composition of secondary plant substances. Therefore, we identified and quantitated polyphenolic compounds along with caffeine present in methanol extracts of Coffea arabica leaves from three different regions of Brazil (Ceará, Minas Gerais and São Paulo) by HPLC-ESI-MS. In addition, correlations between polyphenolic content of the coffee leaves and antioxidant assays DPPH, FRAP and ORAC were evaluated. Fifteen compounds belonging to three classes of polyphenols (xanthones, chlorogenic acids and flavonoids) along with the alkaloid caffeine were detected. The mean concentration of total polyphenolic compounds in the leaves of C. arabica, harvested from three different regions of Brazil was quite variable. The highest values were detected in the coffee leaves harvested in Minas Gerais (n = 4) at 40.80(13.00) g/kg (SD), followed by coffee leaves harvested in São Paulo (n = 20) at 24.79(20.19) g/kg, and the lowest in coffee leaves harvested in Ceará (n = 11) in the Northeast of Brazil at 10.30(5.61) g/kg. The three classes of polyphenols, all showed excellent correlations in the antioxidant assays. Coffee leaf tea, appears to be an excellent functional beverage, with its high content of polyphenolic compounds, which may render positive biologic effects, when inbibed as part of the normal human diet.

Reactive Oxygen Species Deficiency Due to Ncf1-Mutation Leads to Development of Adenocarcinoma and Metabolomic and Lipidomic Remodeling in a New Mouse Model of Dextran Sulfate Sodium-Induced Colitis.

Inflammatory bowel disease is characterized by chronic relapsing idiopathic inflammation of the gastrointestinal tract and persistent inflammation. Studies focusing on the immune-regulatory function of reactive oxygen species (ROS) are still largely missing. In this study, we analyzed an ROS-deficient mouse model leading to colon adenocarcinoma. Colitis was induced with dextran sulfate sodium (DSS) supplied via the drinking water in wild-type (WT) and Ncf1-mutant (Ncf1) B10.Q mice using two different protocols, one mimicking recovery after acute colitis and another simulating chronic colitis. Disease progression was monitored by evaluation of clinical parameters, histopathological analysis, and the blood serum metabolome using 1H nuclear magnetic resonance spectroscopy. At each experimental time point, colons and spleens from some mice were removed for histopathological analysis and internal clinical parameters. Clinical scores for weight variation, stool consistency, colorectal bleeding, colon length, and spleen weight were significantly worse for Ncf1 than for WT mice. Ncf1 mice with only a 7-day exposure to DSS followed by a 14-day resting period developed colonic distal high-grade dysplasia in contrast to the low-grade dysplasia found in the colon of WT mice. After a 21-day resting period, there was still ß-catenin-rich inflammatory infiltration in the Ncf1 mice together with high-grade dysplasia and invasive well-differentiated adenocarcinoma, while in the WT mice, high-grade dysplasia was prominent without malignant invasion and only low inflammation. Although exposure to DSS generated less severe histopathological changes in the WT group, the blood serum metabolome revealed an increased fatty acid content with moderate-to-strong correlations to inflammation score, weight variation, colon length, and spleen weight. Ncf1 mice also displayed a similar pattern but with lower coefficients and showed consistently lower glucose and/or higher lactate levels which correlated with inflammation score, weight variation, and spleen weight. In our novel, DSS-induced colitis animal model, the lack of an oxidative burst ROS was sufficient to develop adenocarcinoma, and display altered blood plasma metabolic and lipid profiles. Thus, oxidative burst seems to be necessary to prevent evolution toward cancer and may confer a protective role in a ROS-mediated self-control mechanism.

Dual-frequency irradiation CEST-MRI of endogenous bulk mobile proteins.

A novel MRI contrast is proposed which enables the selective detection of endogenous bulk mobile proteins in vivo. Such a non-invasive imaging technique may be of particular interest for many diseases associated with pathological alterations of protein expression, such as cancer and neurodegenerative disorders. Specificity to mobile proteins was achieved by the selective measurement of intramolecular spin diffusion and the removal of semi-solid macromolecular signal components by a correction procedure. For this purpose, the approach of chemical exchange saturation transfer (CEST) was extended to a radiofrequency (RF) irradiation scheme at two different frequency offsets (dualCEST). Using protein model solutions, it was demonstrated that the dualCEST technique allows the calculation of an image contrast which is exclusively sensitive to changes in concentration, molecular size and the folding state of mobile proteins. With respect to application in humans, dualCEST overcomes the selectivity limitations at relatively low magnetic field strengths, and thus enables examinations on clinical MR scanners. The feasibility of dualCEST examinations in humans was verified by a proof-of-principle examination of a brain tumor patient at 3 T. With its specificity for the mobile fraction of the proteome, its comparable sensitivity to conventional water proton MRI and its applicability to clinical MR scanners, this technique represents a further step towards the non-invasive imaging of proteomic changes in humans.

Carnosine Catalyzes the Formation of the Oligo/Polymeric Products of Methylglyoxal.

BACKGROUND/AIMS: Reactive dicarbonyl compounds, such as methylglyoxal (MG), contribute to diabetic complications. MG-scavenging capacities of carnosine and anserine, which have been shown to mitigate diabetic nephropathy, were evaluated in vitro and in vivo. METHODS: MG-induced cell toxicity was characterized by MTT and MG-H1-formation, scavenging abilities by Western Blot and NMR spectroscopies, cellular carnosine transport by qPCR and microplate luminescence and carnosine concentration by HPLC. RESULTS: In vitro, carnosine and anserine dose-dependently reduced N-carboxyethyl lysine (CEL) and advanced glycation end products (AGEs) formation. NMR studies revealed the formation of oligo/polymeric products of MG catalyzed by carnosine or anserine. MG toxicity (0.3-1 mM) was dose-dependent for podocytes, tubular and mesangial cells whereas low MG levels (0.2 mM) resulted in increased cell viability in podocytes (143±13%, p<0.001) and tubular cells (129±3%, p<0.001). Incubation with carnosine/anserine did not reduce MG-induced toxicity, independent of incubation times and across large ranges of MG to carnosine/anserine ratios. Cellular carnosine uptake was low (<0.1% in 20 hours) and cellular carnosine concentrations remained unaffected. The putative carnosine transporter PHT1 along with the taurine transporter (TauT) was expressed in all cell types while PEPT1, PEPT2 and PHT2, also belonging to the proton-coupled oligopeptide transporter (POT) family, were only expressed in tubular cells. CONCLUSION: While carnosine and anserine catalyze the formation of MG oligo/polymers, the molar ratios required for protection from MG-induced cellular toxicity are not achievable in renal cells. The effect of carnosine in vivo, to mitigate diabetic nephropathy may therefore be independent upon its ability to scavenge MG and/or carnosine is mainly acting extracellularly.