Liver Transplantation for Primary Sclerosing Cholangitis (PSC) With or Without Inflammatory Bowel Disease (IBD)-A European Society of Organ Transplantation (ESOT) Consensus Statement.

Primary sclerosing cholangitis (PSC) is the classical hepatobiliary manifestation of inflammatory bowel disease (IBD) and a lead indication for liver transplantation (LT) in the western world. In this article, we present a Consensus Statement on LT practice, developed by a dedicated Guidelines' Taskforce of the European Society of Organ Transplantation (ESOT). The overarching goal is to provide practical guidance on commonly debated topics, including indications and timing of LT, management of bile duct stenosis in patients on the transplant waiting list, technical aspects of transplantation, immunosuppressive strategies post-transplant, timing and extension of intestinal resection and futility criteria for re-transplantation.

Prognostic impact of inter-metastatic heterogeneity of viable tumour cells in colorectal liver metastases.

BACKGROUND: Heterogenous response to neoadjuvant chemotherapy in patients with multiple colorectal liver metastases (CRLM) has been associated with an acquired resistance to systemic therapy. This study evaluated the occurrence of a heterogenous inter-metastatic tumour response with regards to the proportion of viable tumour cells, and its prognostic impact. METHODS: A retrospective cohort study was conducted, including all patients with CRLM surgically treated at Karolinska University Hospital, Stockholm, Sweden, from 2013 to 2018. Factors associated with the proportion of viable tumour cells and inter-metastatic heterogeneity were analysed with regression and survival analyses. RESULTS: Out of 640 surgically treated patients, 405 patients (1357 CRLM), received neoadjuvant chemotherapy. Multiple CRLM were present in 314 patients (78%), out of whom 72 patients (23%) presented with a heterogenous tumour response. The median overall survival (OS) for patients with a heterogenous inter-metastatic tumour response was 36 months, compared to 57 months for patients with a homogenous inter-metastatic tumour response (p < .001). Poor OS in patients receiving preoperative chemotherapy was significantly associated with a heterogenous inter-metastatic tumour response (hazard ratio (HR) 1.68 (1.02-2.78)), right-sided primary tumour (HR 2.01 (1.29-3.43)) and CRLM diameter >5 cm (HR 1.83 (1.06-3.17)). CONCLUSION: Outcome in patients with a heterogenous inter-metastatic tumour response, illustrated by the proportion of viable tumour cells, is inferior to that of patients with a homogenous response. These results suggest that heterogeneity in treatment response is an important marker of aggressive disease and could be of clinical value for decisions on post-operative therapy.

Conversion therapy in patients with colorectal liver metastases.

BACKGROUND: The occurrence of colorectal liver metastases (CRLM) impairs prognosis, yet long-term survival can be achieved by enabling liver resection. This study aims to describe factors associated with conversion therapy leading to liver surgery and treatment outcome. METHODS: A retrospective cohort study was conducted including all patients with CRLM discussed at multidisciplinary team conference at Karolinska University Hospital, Stockholm, Sweden, from 2013 to 2018. Factors associated with conversion therapy and outcome following conversion therapy were analysed with logistic regression and survival analyses. RESULTS: Out of 1023 patients with CRLM, 100 patients (10%) received conversion chemotherapy, out of whom 31 patients (31%) subsequently underwent liver resection. Patients in whom conversion chemotherapy resulted in liver resection were younger (median age 61 vs. 66 years, p = .024), less likely to have a KRAS/NRAS-mutated primary tumours (25% vs. 53%, p = .039) and more likely to have received anti-EGFR agents (32% vs. 4%, p = .001) than patients progressing during conversion chemotherapy. The median OS for patients treated with conversion chemotherapy leading to liver resection was 24 months, compared to 14 months for patients progressing during conversion chemotherapy, p < .001. The OS for patients progressing during conversion chemotherapy was similar to patients given palliative chemotherapy, approximately 13 months. CONCLUSION: Conversion therapy offers a survival benefit in selected patients. Despite treatment advances, the majority of patients undergoing conversion chemotherapy never become eligible for curative treatment.

Inhibitor of apoptosis protein expression in glioblastomas and their in vitro and in vivo targeting by SMAC mimetic GDC-0152.

Glioblastomas (GBMs) are the most aggressive primary brain tumors in adult and remain a therapeutic challenge. Targeting key apoptosis regulators with the ultimate aim to restore apoptosis in tumor cells could be an interesting therapeutic strategy. The inhibitors of apoptosis proteins (IAPs) are regulators of cell death and represent attractive targets, especially because they can be antagonized by SMAC mimetics. In this study, we first investigated the expression of cIAP1, cIAP2, XIAP and ML-IAP in human GBM samples and in four different cell lines. We showed that all GBM samples and GBM cell lines expressed all these IAPs, although the expression of each IAP varied from one case to another. We then showed that high level of ML-IAP predicted worse progression-free survival and overall survival in both univariate and multivariate analyses in two independent cohorts of 58 and 43 primary human GBMs. We then used GDC-0152, a SMAC mimetic that antagonizes these IAPs and confirmed that GDC-0152 treatment in vitro decreased IAPs in all the cell lines studied. It affected cell line viability and triggered apoptosis, although the effect was higher in U87MG and GL261 than in GBM6 and GBM9 cell lines. In vivo, GDC-0152 effect on U87MG orthotopic xenografts was dose dependent; it postponed tumor formation and slowed down tumor growth, significantly improving survival of GBM-bearing mice. This study revealed for the first time that ML-IAP protein expression correlates with GBM patient survival and that its antagonist GDC-0152 improves outcome in xenografted mouse.

Fluorimetric screening assay for protein carbonyl evaluation in biological samples.

Many assays are available for the detection of protein carbonyls (PCs). Currently, the measurement of PC groups after their derivatization with 2,4-dinitrophenol hydrazine (DNPH) is widely used for measuring protein oxidation in biological samples. However, this method includes several washing steps. In this context, we have developed a rapid, sensitive, and accurate fluorimetric method adapted to 96-well microplates for the convenient assessment of protein carbonyl level in biological samples. The method reported here is based on the reaction of carbonyl content in proteins with 7-hydrazino-4-nitrobenzo-2,1,3-oxadiazole (NBDH) to form highly fluorescent derivatives via hydrazone formation. PCs were determined using the DNPH and NBDH assays in fully reduced bovine serum albumin (BSA) and plasma and liver homogenates obtained from healthy control rats up the addition of various amounts of HOCl-oxidized BSA (OxBSA). Using the NBDH assay, PC concentrations as low as 0.2 nmol/mg were detected with precision as low as 5%. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectroscopy was used to successfully identify the formation of the NBDH adducts after derivatization with standard oxidized peptides. Finally, the two methods were further used for PC determination in plasma and liver samples from diabetic and normal rats, showing that the NBDH assay can be reliably used in biological experiments.

Small RNA Deep-Sequencing Analyses Reveal a New Regulator of Virulence in Agrobacterium fabrum C58.

Novel ways of regulating Ti plasmid functions were investigated by studying small RNAs (sRNAs) that are known to act as posttranscriptional regulators in plant pathogenic bacteria. sRNA-seq analyses of Agrobacterium fabrum C58 allowed us to identify 1,108 small transcripts expressed in several growth conditions that could be sRNAs. A quarter of them were confirmed by bioinformatics or by biological experiments. Antisense RNAs represent 24% of the candidates and they are over-represented on the pTi (with 62% of pTi sRNAs), suggesting differences in the regulatory mechanisms between the essential and accessory replicons. Moreover, a large number of these pTi antisense RNAs are transcribed opposite to those genes involved in virulence. Others are 5'- and 3'-untranslated region RNAs and trans-encoded RNAs. We have validated, by rapid amplification of cDNA ends polymerase chain reaction, the transcription of 14 trans-encoded RNAs, among which RNA1111 is expressed from the pTiC58. Its deletion decreased the aggressiveness of A. fabrum C58 on tomatoes, tobaccos, and kalanchoe, suggesting that this sRNA activates virulence. The identification of its putative target mRNAs (6b gene, virC2, virD3, and traA) suggests that this sRNA may coordinate two of the major pTi functions, the infection of plants and its dissemination among bacteria.

Ruminococcin C, a new anti-Clostridium perfringens bacteriocin produced in the gut by the commensal bacterium Ruminococcus gnavus E1.

When colonizing the digestive tract of mono-associated rats, Ruminococcus gnavus E1 - a bacterium isolated from human faeces - produced a trypsin-dependent anti-Clostridium perfringens substance collectively named Ruminococcin C (RumC). RumC was isolated from the caecal contents of E1-monocontaminated rats and found to consist of two antimicrobial fractions: a single peptide (RumCsp) of 4235 Da, and a mixture of two other peptides (RumCdp) with distinct molecular masses of 4324 Da and 4456 Da. Both RumCsp and RumCdp were as effective as metronidazole in combating C. perfringens and their activity spectra against different pathogens were established. Even if devoid of synergistic activity, the combination of RumCsp and RumCdp was observed to be much more resistant to acidic pH and high temperature than each fraction tested individually. N-terminal sequence analysis showed that the primary structures of these three peptides shared a high degree of homology, but were clearly distinct from previously reported amino acid sequences. Amino acid composition of the three RumC peptides did not highlight the presence of any Lanthionine residue. However, Edman degradation could not run beyond the 11th amino acid residue. Five genes encoding putative pre-RumC-like peptides were identified in the genome of strain E1, confirming that RumC was a bacteriocin. This is the first time that a bacteriocin produced in vivo by a human commensal bacterium was purified and characterized.

Microtubule targeting agents: from biophysics to proteomics.

This review explores various aspects of the interaction between microtubule targeting agents and tubulin, including binding site, affinity, and drug resistance. Starting with the basics of tubulin polymerization and microtubule targeting agent binding, we then highlight how the three-dimensional structures of drug-tubulin complexes obtained on stabilized tubulin are seeded by precise biological and biophysical data. New avenues opened by thermodynamics analysis, high throughput screening, and proteomics for the molecular pharmacology of these drugs are presented. The amount of data generated by biophysical, proteomic and cellular techniques shed more light onto the microtubule-tubulin equilibrium and tubulin-drug interaction. Combining these approaches provides new insight into the mechanism of action of known microtubule interacting agents and rapid in-depth characterization of next generation molecules targeting the interaction between microtubules and associated modulators of their dynamics. This will facilitate the design of improved and/or alternative chemotherapies targeting the microtubule cytoskeleton.

Sialome individuality between Aedes aegypti colonies.

Aedes aegypti is responsible for the transmission of arboviruses. The Yellow Fever, Dengue and Chikungunya viruses are transmitted to the vertebrate host by injection of infected saliva during the blood meal of its vectors. Saliva contains different components with various biochemical activities; anti-hemostatic, angiogenic, inflammatory, and immunomodulatory. This work compares the sialomes of three Ae. aegypti colonies (Rockefeller, PAEA, and Formosus), where the repertoire of salivary proteins from these colonies was analyzed by a proteomic approach. This study indicated that major proteins were detectable in the three colonies. However, differences in the abundance of some saliva proteins have been observed between the three Ae. aegypti colonies.

Magneto-optical setup for in situ strain and transport measurements on superconductors.

A recently developed magneto-optical (MO) imaging setup for investigations on superconductors is reported. The main originality of our setup is its ability to combine both strain and transport measurements in the temperature range of 6-300 K with magneto-optical observations. We give here some theoretical considerations on the cryostat conception, which is a key point of our setup. In particular, the thermal and mechanical aspects are discussed. A detailed description of the MO setup and of the associated strain apparatus is given. Additionally, an example of MO strain and transport study on DyBCO coated conductors is given. Evidence of Luders Bands formation under strain in the Hastelloy is revealed by the field penetration inside cracks in the DyBCO and MgO layers. A correlation between the damaging morphology and the critical current at 70 K versus strain has been established.

[Evaluation of the analytical performances of CRP Diasys reagent on Roche Hitachi 917]. / Evaluation des performances analytiques du réactif CRP Diasys sur Roche Hitachi 917.

C reactive protein, the most sensible acute phase protein of inflammation and the labororatory should perform CRP testing on a continous 24 hour basis. The measurement is mainly performed by immunoturbimetry and immunonephelemetry methods available on multiparametric biochemical analyzer. In this study, we evaluated the analytical performances, precision and exactitude, of the CRP Diasys reagent on Roche Hitachi 917. The results were compared to those obtained with a CRP latex immunoassay (Roche). The reagent showed high analytical characteristics and especially a significant precision in a large range of CRP levels including low levels between 1 and 3 mg/L. Although this reagent is not considered as a high-sensitive CRP reagent, the measurement quality obtained in the 1-3 mg/L range allows an utilization as a cardiovascular risk predictor.

Structure-function relationships in the carboxylic-ester-hydrolase superfamily. Disulfide bridge arrangement in porcine intestinal glycerol-ester hydrolase.

CNBr fragments from porcine intestinal glycerol-ester hydrolase were separated by SDS/PAGE under reducing and nonreducing conditions, and their amino-acid sequences were analysed. Two intra-chain disulfide bridges were identified, namely Cys70-Cys99 (loop A) and Cys256-Cys267 (loop B). As the Cys71 sulfhydryl group could not be alkylated with iodoacetamide, it is suggested that the residue is blocked rather than being present in the free form. The two disulfide bridges of intestinal glycerol-ester hydrolase are present in the cholinesterase family, although the enzyme showed only about 35% identity with these proteins. Furthermore, the finding that glycerol-ester hydrolase was partly inactivated under reducing conditions suggests that one or both disulfide bridges are important for the enzyme conformation. Lastly, glycerol-ester hydrolase was also found to hydrolyse cholinergic substrates, although residues Trp86 and Asp74 which are considered to be the main constituents of the 'anionic' subsite responsible for substrate binding in cholinesterases were absent from loop A. Other amino-acid residues in the glycerol-ester hydrolase may therefore be responsible for the binding of cholinergic substrates to the enzyme.

Purification and molecular cloning of porcine intestinal glycerol-ester hydrolase--evidence for its identity with carboxylesterase.

A glycerol-ester hydrolase was purified to homogeneity from porcine intestinal mucosa using a partial delipidation method and an eight-step purification procedure. The isolation scheme used gave a 483-fold purification, resulting in a pure enzyme with a specific activity on tributyrin of 290 micromol x min(-1) x mg(-1). The molecular mass of the enzyme was estimated at 240 kDa, based on the results of size-exclusion chromatography, and at 60 kDa, as determined by SDS/PAGE analysis. The isoelectric focusing data obtained indicated that only one isoform with a pI of 5.1 was present. Complete identity was found to exist between the N-terminal sequence of the first 25 amino acid residues and that of a porcine liver carboxylesterase. A full-length cDNA coding for the enzyme was isolated from pig small intestine. We observed that the corresponding protein originally named intestinal glycerol-ester hydrolase definitely belongs to the carboxylesterase family. The deduced amino acid sequence consisted of 565 residues and showed 97% identity with that of porcine liver carboxylesterase and more than 50% identity with those of other carboxylesterases from different mammalian species.

Comparative quantitative analysis of sucrose and related compounds using ion exchange and reverse phase chromatographic methods.

Two analytical methods of sugar determination, namely ion exchange chromatography on an anionic resin coupled with electrochemical detection, and reverse phase chromatography on Nucleosil-NH2 resin equipped with a light scattering detector were tested and compared as regards their rapidity, sensitivity and accuracy with sucrose, fructose, glucose, raffinose, maltose, arabinose, fucose, rhamnose and xylose. Excellent resolution and highly reproducible results were obtained in both cases. Greater sensitivity up to the picomolar range was possible however only with ion exchange chromatography. Reverse phase chromatography was successfully applied to the time course of sucrose hydrolysis under chemical (acid) and enzymatic (invertase) conditions. The hydrolysis was monitored by determining sucrose degradation and the corresponding formation of glucose and fructose.

Evaluation of a new automated ELISA test for von Willebrand factor using two monoclonal antibodies.

A new automated quantitative enzyme linked immunosorbent assay (ELISA) for the detection of human von Willebrand factor (vWF), VIDAS vWF, has been developed for use on the VIDAS analyser (bioMérieux). The two-step capture/tag test relies on two monoclonal antibodies (mAbs), the second one being labelled with alkaline phosphatase. The lower limit of detection of the assay is < 1 IU/dl, and the upper limit of detection is 120 IU/dl. There is no hook effect for concentrations up to 1100 IU/dl. Intra- and inter-assay precision ranges from 3% and 5%. Assays are performed preferentially using citrated plasma and in these conditions the 95% confidence intervals for normal values are 52-154 IU/dl and 60-200 IU/dl for O blood group and non-O blood group subjects, respectively. Using the lower limits of the normal range as the cut-off level, all patients tested with type 1 (n = 29) or 3 (n = 2) von Willebrand disease (vWD) would be accurately classified with the new ELISA. Comparing the VIDAS test with a conventional ELISA gave a good correlation and comparable results with type 1 and 3 vWD patients (n = 31; r = 0.99; y = 0.99x + 0.24), type 2A and 2B vWD patients (n = 20; r = 0.99; y = 1.05x-1.65) and normal subjects (n = 204; r = 0.94; y = 1.06x-2.6).

VIDAS D-dimer: fast quantitative ELISA for measuring D-dimer in plasma.

VIDAS D-dimer (bioMérieux) is a new quantitative ELISA for D-dimer determination designed for the VIDAS automated system. The test contains single-dose, ready-to-use reagents and is completed within 35 min. Quantitative results are obtained from a calibration curve stored in the software of the system and expressed as fibrinogen equivalent units. The two-step capture/tag test relies on two complementary monoclonal anti-D-dimer antibodies, the second one being labeled with alkaline phosphatase. The upper limit of the measuring range is 1000 micrograms/L and the lower detection limit is <50 micrograms/L, which is below the lower limit of the reference interval (68-494 micrograms/L). Reproducibility (CV) within and between runs ranges from 5% to 7%. There is no interference from heparin, bilirubin, hemoglobin, fibrinogen degradation products, or plasma turbidity. Comparison with a conventional ELISA (y) gave good correlation (r= 0.91, n= 579) and comparable results (y= 1.35x - 148, S(y/x)= 750), especially for D-dimer concentrations ranging from 0 to 1000 micrograms/L (y= 1.09x - 10.6, r= 0.88, S(y/x)= 170).

Comparative clinical trial of AMO Vitrax and Healon use in extracapsular cataract extraction.

This randomized, single-masked, multicenter clinical trial, comprising 95 patients enrolled at five sites, evaluated the performance of AMO Vitrax and Healon viscoelastic materials during cataract surgery. Patients were examined preoperatively and at one day, four days, one month, and three months postoperatively. The following measurements were recorded and analyzed: percentage of endothelial cell loss from preoperative to three months postoperative; change in intraocular pressure (IOP) from preoperative to 24 hours postoperatively; postoperative corrected visual acuity; subjective assessment of ability of viscoelastic to create and maintain tissue space; intraocular transparency; ease of evacuation. Three months postoperatively, endothelial cell loss was 4.9% (+/- 8.3%) for the AMO Vitrax group and 6.3% (+/- 10.5%) for the Healon group. One day postoperatively, IOP decreased by 1.6 mm Hg and increased by +1.1 mm Hg, respectively. Postoperative visual acuities were similar between the two groups at three months. Subjective assessment of transparency was higher for Healon. Assessment of tissue space maintenance was similar between the two materials. Healon was rated as slightly easier to evacuate.