Optimising the clinical strategy for autoimmune liver diseases: Principles of value-based medicine

Background

Autoimmune hepatitis, primary biliary cholangitis, and primary sclerosing cholangitis represent the three major autoimmune liver diseases (AILDs). Their management is highly specialized, requires a multidisciplinary approach and often relies on expensive, orphan drugs. Unfortunately, their treatment is often unsatisfactory, and the care pathway heterogeneous across different centers. Disease-specific clinical outcome indicators (COIs) able to evaluate the whole cycle of care are needed to assist both clinicians and administrators in improving quality and value of care. Aim of our study was to generate a set of COIs for the three AILDs. We then prospectively validated these indicators based on a series of consecutive patients recruited at three tertiary clinical centers in Lombardy, Italy.

Cofacial and antarafacial indenyl bimetallic isomers: a descriptive MO picture and implications for the indenyl effect on ligand substitution reactions

The hetero-bimetallic Ind complexes (Ind=indenyl anion) with the formula (CO)₃Cr(μ-Ind)RhL₂ (L=CO, ethylene; L₂=COD, COT, NBD) have two possible conformers, one with the metals at antipodal sides of the condensed bicyclic polyene (antarafacial, anti) and the other with both metals on the same side (cofacial, syn). These systems can be considered to contain 34 valence electrons by assuming that Ind is capable of donating all of its ten π electrons to the metals. A qualitative MO analysis, based on EHMO calculations, is presented. The anti conformer is compared to the homometallic 34e species, namely [CpRu(μ-Ind)RuCp]⁺. Here, the pseudo-symmetrical relationship between the two identical metal fragments strongly suggests that one Ind σ-bonding MO donates part of its electron density to each of the two metals, which thus become formally saturated (36e). This argument is extended to the anti Cr---Rh derivative in which a critical role is played by an empty FMO of the (CO)₂Rh(I) fragment with π symmetry (i.e. lying in the molecular mirror plane). The latter, in spit the large π_CO^* contributions, has good acceptor capabilities at the metal.The calculations suggest that the so-called indenyl effect, namely the increased rate of substitution of one CO ligand by another σ donor (e.g. a phosphine), although a structurally dynamic process, also depends on the electron density which accumulates on the aforementioned π FMO. In the series (η⁵-C₅H₅)Rh(CO)₂, (η⁵-C₇H₉Rh(CO)₂ and {η⁵-[(CO)₃CrC₇H₉]}-Rh(CO)₂ the progressively reduced donor capabilities of the cyclic polyene toward the (CO)₂Rh(I) fragment induce a larger electrophilicity in rhodium, in good agreement with the experimental data on substitution reactivity. Finally, the electronic features of the cofacial conformer (CO)₃Cr(μ-Ind)Rh(CO)₂ are examined. In spite of the long Cr---Rh separation (> 3 Å) the graphically illustrated MO analysis indicates a direct, heterodox, intermetallic linkage, which helps the metals to achieve a formal electronic saturation. In this case, the limited propensity of the complex toward any CO substitution reactions is likely attributable to the hindered mobility of the (CO)₂Rh(I) fragment.     

The origin of the genetic code and protein synthesis

A model for a parallel evolution of the genetic code and protein synthesis is presented. The main tenet of this model is that the genetic code, that is, a correspondence between nucleotide and aminoacid coding units, originated from sequence-specific interaction between abiotically synthesized polynucleotides and polypeptides. A sequence-specific binding between oligonucleotides and oligopeptides is supported by experimental findings. Moreover, it is parsimonious enough to be consistent with the relatively simple chemistry of a primordial environment. Proximity between peptides and RNA increased the rate of formation of ester bonds between them. This lead to the accumulation of sequence-specific polypeptide-polynucleotide pairs, that is, of primordial-loaded tRNA. Condensation of short polypeptides into longer products could be catalyzed by a sequence-specific juxtaposition of loaded tRNA over complementary RNA, originating the core of protein synthesis. The accumulation of useful encoded products, for example, catalysts for tRNA loading (primordial aminoacyl-tRNA synthetases) or stabilizers of tRNA-mRNA interactions (primordial ribosomes), permitted the subsequent evolution of protein synthesis and of the genetic code to their mature form. This occurred via a parallel reduction in length of the interacting polynucleotides and polypeptides. Thus, it maintained the correct reading frame of mRNA from the preceding stages of evolution. Received: 27 September 1996 / Accepted: 17 May 1997     

Enhanced resolution of specific chromosome and nuclear regions by reflectance laser scanning confocal microscopy

 DNA sequences digested by HaeIII and reconstructed by in situ nick translation employing digoxigenin-labelled nucleotides are usually revealed either by horseradish peroxidase or FITC fluorescence. To obtain a significant improvement in terms of resolution, sensitivity and specificity, colloidal gold has been used instead of FITC (as the reporter molecule) to reveal the labelled DNA. Colloidal gold and propidium iodide were visualised by employing the reflectance mode and the 488-nm laser line of a confocal laser scanning microscope. In chromosomes, the fluorescent reaction pattern showed diffuse areas of labelling in which it was impossible to identify any specific kind of banding along the arms. In some chromosomes and, in particular, 1 and 9, a C-negative banding due to the negativity of the centromeric areas was seen. A more accurate localisation on chromosomes, including telomeric regions, often organised in spot pairs that resembled an R-like banding, was detected using 1-nm colloidal gold. A fine labelling was also demonstrated in nuclei, especially at their peripheral heterochromatin. The non-fading properties of colloidal gold combined with visualisation by reflectance confocal laser scanning microscopy demonstrated the possibility of obtaining a higher spatial resolution than when using conventional fluorophores or higher laser wavelength. This improved way to study the localization of HaeIII digestion sites in single chromosomes and in interphase nuclei made the reaction a valuable tool for the detection of antigens or of specific DNA sequences in biological preparations. Accepted: 5 September 1996     

Chemical and pathological oxidative influences on band 3 protein anion-exchanger.

BACKGROUND/AIMS: The erythrocyte is a cell exposed to a high level of oxygen pressure and to oxidative chemical agents. This stress involves SH-groups oxidation, cell shrinkage by activation of K-Cl co-transport (KCC) and elevation of the band 3 tyrosine phosphorylation level. The aim of our study was to test whether oxidative stress could influence band 3-mediated anion transport in human red blood cells. METHODS: To evaluate this hypothesis, normal and pathological (glucose 6 phosphate dehydrogenase (G6PDH) defficient) erythrocytes were treated with known sulphydryl-blocking or thiol-oxidizing agents, such as N-ethylmaleimide (NEM), azodicarboxylic acid bis[dimethylamide] (diamide), orthovanadate, Mg2+ and tested for sulphate (SO4-) uptake, K+ efflux, G6PDH activity and glutathione (GSH) concentration. RESULTS: In normal red blood cells, the rate constants of SO4- uptake decreased by about 28 % when cells were incubated with NEM, diamide and orthovanadate. In G6PDH-deficient red blood cells, in which oxidative stress occurs naturally, the rate constant of sulphate uptake was decreased by about 40% that of normal red cells. Addition of oxidizing and phosphatase inhibitor agents to pathological erythrocytes further decreased anion transport. In contrast, G6PDH activity was increased under oxidative stress in normal as well as in pathological cells and was lower in the presence of exogenous Mg2+ in parallel to a significant increase in sulphate transport. In both cells, the oxidizing agents increased K+ efflux with depletion of GSH. CONCLUSION: The data are discussed in light of the possible opposite effects exerted by oxidative agents and Mg2+ on KCC and on the protein tyrosine kinase (PTK)-protein tyrosine phosphatase (PTP) equilibrium. The decreased sulphate uptake observed in the experimental and pathological conditions could be due to band 3 SH-groups oxidation or to oxidative stress-induced K-Cl symport-mediated cell shrinkage with concomitant band 3 tyrosine phosphorylation.     

Regional-dependent increase of sympathetic innervation in rat white adipose tissue during prolonged fasting.

White adipose tissue (WAT) is innervated by the sympathetic nervous system. A role for WAT sympathetic noradrenergic nerves in lipid mobilization has been suggested. To gain insight into the involvement of nerve activity in the delipidation process, WAT nerves were investigated in rat retroperitoneal and epididymal depots after prolonged fasting. A significant increase in tyrosine hydroxylase (TH) content was found in epididymal and, especially, retroperitoneal WAT by Western blotting. Accordingly, an increased immunoreactivity for TH was detected by immunohistochemistry in epididymal and, especially, retroperitoneal vascular and parenchymal noradrenergic nerves. Neuropeptide Y (NPY)-containing nerves were found around arteries and in the parenchyma. Double-staining experiments and confocal microscopy showed that most perivascular and some parenchymal noradrenergic nerves also contained NPY. Detection of protein gene product (PGP) 9.5, a general marker of peripheral nerves, by Western blotting and PGP 9.5-TH by double-staining experiments showed significantly increased noradrenergic nerve density in fasted retroperitoneal, but not epididymal depots, suggesting that formation of new nerves takes place in retroperitoneal WAT in fasting conditions. On the whole, these data confirm the important role of sympathetic noradrenergic nerves in WAT lipid mobilization during fasting but also raise questions about the physiological role of regional-dependent nerve adjustments and their functional significance in relation to white adipocyte secretory products.     

Bovine Doppel (Dpl) and prion protein (PrP) expression on lymphoid tissue and circulating leukocytes.

Doppel (Dpl) protein shares some structural features with prion protein (PrP), whose pathologic isoform (PrPsc) is considered to be the causative agent of transmissible spongiform encephalopathies. Dpl is mainly expressed in testes but, when ectopically expressed in the central nervous system, is neurotoxic. We have examined the expression pattern of Dpl and PrP on bovine lymphoid tissues and circulating leukocytes. A polyclonal anti-Dpl antibody along with a panel of monoclonal antibodies specific for leukocyte membrane antigens or PrP were used to examine frozen sections from spleen, lymph nodes, and bone marrow by immunohistochemistry. Blood was analyzed by flow cytometry. Double staining was used to study the possible coexpression of the two proteins and to characterize cells expressing Dpl and/or PrP. Dpl was expressed in B-cells, in dendritic cells within lymphoid follicles, bone marrow, circulating myeloid cells, and circulating B-cells. The distribution of Dpl was quite similar to that of PrP. The only differences in expression observed concerned the low number of Dpl+ cells in lymph nodes and the strong Dpl positivity of circulating granulocytes. The two proteins were rarely co-expressed, suggesting an independent expression mechanism in resting cells. The role of Dpl+ leukocytes in the pathogenesis of Dpl- or PrP-induced diseases merits further investigation.     

Pathology Tissue-quantitative Mass Spectrometry Analysis to Profile Histone Post-translational Modification Patterns in Patient Samples

Histone post-translational modifications (hPTMs) generate a complex combinatorial code that has been implicated with various pathologies, including cancer. Dissecting such a code in physiological and diseased states may be exploited for epigenetic biomarker discovery, but hPTM analysis in clinical samples has been hindered by technical limitations. Here, we developed a method (PAThology tissue analysis of Histones by Mass Spectrometry - PAT-H-MS) that allows to perform a comprehensive, unbiased and quantitative MS-analysis of hPTM patterns on formalin-fixed paraffin-embedded (FFPE) samples. In pairwise comparisons, histone extracted from formalin-fixed paraffin-embedded tissues showed patterns similar to fresh frozen samples for 24 differentially modified peptides from histone H3. In addition, when coupled with a histone-focused version of the super-SILAC approach, this method allows the accurate quantification of modification changes among breast cancer patient samples. As an initial application of the PAThology tissue analysis of Histones by Mass Spectrometry method, we analyzed breast cancer samples, revealing significant changes in histone H3 methylation patterns among Luminal A-like and Triple Negative disease subtypes. These results pave the way for retrospective epigenetic studies that combine the power of MS-based hPTM analysis with the extensive clinical information associated with formalin-fixed paraffin-embedded archives.Histone post-translational modifications (hPTMs)¹ generate a complex combinatorial code that plays a critical role during the physiological and pathological regulation of gene expression (1). Alterations in histone modification patterns have been linked with various diseases, including cancer, often as a result of the aberrant expression or localization of histone modifying enzymes (2). Therefore, accurately dissecting hPTM patterns in normal and diseased tissues could yield epigenetic biomarkers useful for prognostic, diagnostic, and therapeutic purposes. Immunohistochemistry studies have shown the potential of this strategy (3, 4), but they were limited to the analysis of only a few hPTMs. In addition, despite their sensitivity and ease of use, antibody-based assays are hindered by issues such as the difficulty in detecting adjacent modifications and the limited linearity of the signal. As an alternative to traditional antibody-based methods, in recent years MS has become the elective method to analyze hPTMs, thanks to its unbiased nature, accuracy and its ability to quantitate modifications and detect their combinations. Various MS-based workflows optimized for hPTM analysis have been developed (5), but most of the studies focused on cell lines and animal tissue, whereas the potential offered by the analysis of clinical samples has been left largely unexploited. In particular, the MS-based analysis of hPTMs from formalin-fixed paraffin-embedded (FFPE) samples has never been addressed.Paraffin embedding following fixation in buffered formalin is the storage method of choice for clinical specimens, thus representing an invaluable source of clinical samples linked to retrospective patient information. Large formalin-fixed paraffin embedded (FFPE) archives, which are available in many hospitals, have been successfully exploited for DNA and RNA analyses, including chromatin immunoprecipitation (6, 7). However, the extensive protein cross-linking generated by formaldehyde fixation has hindered the proteomic study of this type of tissue. This problem has been addressed and overcome only recently in global proteomic studies by taking advantage of extraction protocols based on heat-induced antigen retrieval techniques derived from immunohistochemistry (8, 9). Moreover, a few studies showed the possibility to globally analyze protein post-translational modifications, such as glycosylation and phosphorylation, from fixed and embedded tissues (10–12).Here, we report for the first time the successful application of MS-based analysis of hPTMs to human clinical samples, focusing in particular on the development and validation of a method (PAT-H-MS) to extract histones from FFPE tissues in yield and purity sufficient to enable the subsequent use of a proteomic workflow optimized for hPTM analysis (13). By using this method we were able to profile in a quantitative manner 24 distinct modified histone peptides from human FFPE breast cancer samples belonging to different subtypes, identifying differences in histone methylation patterns of potential clinical relevance. Thus, PAT-H-MS represents a valid approach for hPTM analysis of clinical samples.     

Computational Modeling of PI3K/AKT and MAPK Signaling Pathways in Melanoma Cancer

Background

Malignant melanoma is an aggressive tumor of the skin and seems to be resistant to current therapeutic approaches. Melanocytic transformation is thought to occur by sequential accumulation of genetic and molecular alterations able to activate the Ras/Raf/MEK/ERK (MAPK) and/or the PI3K/AKT (AKT) signalling pathways. Specifically, mutations of B-RAF activate MAPK pathway resulting in cell cycle progression and apoptosis prevention. According to these findings, MAPK and AKT pathways may represent promising therapeutic targets for an otherwise devastating disease.

Result

Here we show a computational model able to simulate the main biochemical and metabolic interactions in the PI3K/AKT and MAPK pathways potentially involved in melanoma development. Overall, this computational approach may accelerate the drug discovery process and encourages the identification of novel pathway activators with consequent development of novel antioncogenic compounds to overcome tumor cell resistance to conventional therapeutic agents. The source code of the various versions of the model are available as S1 Archive.     

Site of participation of cytochrome b5 in hepatic microsomal fatty acid chain elongation. Electron input in the first reduction step

The present study provides strong evidence for the involvement of rat liver microsomal cytochrome b5 in the first reduction step of fatty acid chain elongation. The rate of reoxidation of NADH-reduced microsomal cytochrome b5 was markedly stimulated (up to 3-fold) by the addition of increasing concentrations of beta-ketohexadecanoyl-CoA (1-8 microM). A quantitative analysis of product formation, the effect of cyanide, and anaerobiosis completely exclude the possibility that desaturase activity accounted for the beta-ketohexadecanoyl-CoA-induced stimulation of the cytochrome b5 reoxidation rate. Using liver microsomes from untreated rats, the beta-keto substrate was found to stimulate the rate of reoxidation of cytochrome b5 by 30%. However, when liver microsomes from fat-free diet rats were employed the stimulation was more than 3-fold, suggesting that the beta-ketoacyl-CoA reductase is inducible by a high carbohydrate, fat-free diet. This study also provides evidence for the noninvolvement of cytochrome b5 in the terminal reaction step (second reduction step of chain elongation), which is catalyzed by the trans-2-enoyl-CoA reductase. Although trans-2-hexadecenoyl-CoA significantly stimulated the NADH-reduced cytochrome b5 reoxidation rate under aerobic conditions, it did not have any stimulatory effect under anaerobic conditions. One interpretation of these results is that the trans-2-hexadecenoyl-CoA is substrate for the microsomal delta 9 desaturase system. Consistent with this conclusion was the fact that the trans-2-hexadecenoyl-CoA inhibited the liver microsomal delta 9 desaturation of stearoyl-CoA to oleoyl-CoA.