Canakinumab in adults with steroid-refractory pyoderma gangrenosum.

BACKGROUND: Pyoderma gangrenosum (PG) is a rare, neutrophilic, ulcerative skin disease that is difficult to treat, especially when unresponsive to steroids. OBJECTIVES: To determine whether canakinumab is an effective and safe treatment in PG. METHODS: Five adult patients with clinically and histologically confirmed steroid-refractory PG were enrolled in this prospective open-label study. They received canakinumab 150 mg subcutaneously at week 0 with an optional 150 mg at week 2 in case of an inadequate response [Physician's Global Assessment (PGA) ≥ 2], and an optional 150-300 mg at week 8 depending on PGA. The primary clinical end point was clinical improvement (PGA at least -1 from baseline) and/or complete remission (PGA 0 or 1) at week 16. Real-time quantitative polymerase chain reaction was performed on skin samples to quantify cytokine mRNA levels. RESULTS: Interleukin (IL)-1ß and its known target genes IL6, CXCL8 and IL36A were significantly increased in lesional skin of PG. Under canakinumab therapy, four of five patients showed a decrease in target-lesion size, PGA and Dermatology Life Quality Index (DLQI), and three of five achieved complete remission. The mean diameter of target lesions decreased from 4·32 ± 2·6 cm at visit 1 to 0·78 ± 1·3 cm at visit 7 (P = 0·03). Mean DLQI decreased from 15 ± 5 at visit 1 to 8 ± 4 by visit 7 (P = 0·01). Adverse effects were reported in two patients: fatigue in one and worsening of disease at a nontarget lesion in the other. CONCLUSIONS: Our data indicate that IL-1ß plays a key pathogenic role in PG and canakinumab may represent a therapeutic option for steroid-refractory PG.

Efficacy of ustekinumab in the treatment of patients with Crohn's disease with failure to previous conventional or biologic therapy: a prospective observational real-life study.

Ustekinumab (UST), a human anti-IL12/23p40 monoclonal antibody, was approved by FDA and EMA for the treatment of moderate to severe Crohn's disease (CD). Whether UST is effective in inducing deep remission, including mucosal healing and transmural healing, in patients with CD in a real life setting is not completely clear. This study was performed on 92 subjects with confirmed diagnosis of moderate to severe Crohn's disease and no neoplasia. Before inclusion, all patients had been exposed and had failed to respond to conventional and/or at least one biological therapy. All patients underwent endoscopic examination and bowel MRI and ultrasonography at baseline (T0). At week 52 (T52), patients underwent colonoscopy for assessment of mucosal healing and MRI or ultrasonography for assessment of transmural healing. CDAI was used for the assessment of clinical response and clinical remission. SES-CD was used to assess endoscopic response and remission. Incidence of treatment-related adverse events (TRAEs) was recorded during the study period. Clinical response at week 52 was achieved in 38 (50.5%) patients and clinical remission in 29 (39%). Twenty-six (34%) patients showed mucosal healing, 34 (45%) showed partial endoscopic response. We observed a reduction in SES-CD of at least 50% in 34 (45%) patients as well as an SES-CD ≤ 2 in 26 (35%) patients. All patients with mucosal healing also showed transmural healing. No major TRAEs were observed during treatment. In this multicenter, real life study, we show that UST was well tolerated and effective in inducing clinical response and clinical remission in patients with moderate to severe CD who had previously failed to respond to conventional or biologic therapy. UST showed limited efficacy in inducing deep remission (i.e. mucosal+transmural healing).

Comparative study of cytokine content in the plasma and wound exudate from children with severe burns.

The content of 27 cytokines was measured in blood plasma from 19 children with severe uncomplicated burns (group 1) and complicated burns (septic toxemia, toxemia, and pneumonia; group 2). Before surgical treatment (day 4 (+/-2) after burn), significant differences were found in the concentrations of interleukin-1 receptor antagonist, interleukin-6, interleukin-8, interleukin-10, tumor necrosis factor-alpha, interferon-gamma, MCP-1, and granulocyte colony-stimulating factor. Cytokine concentration in group 2 patients was much higher than in group 1 patients and healthy children. The concentrations of interleukin-6, interleukin-8, and MCP-1 in group 1 patients significantly surpassed the normal level. Cytokine concentration in the plasma and wound exudates and myeloperoxidase activity in wound exudates from 4 patients of group 2 were measured over 18 days after burn. The inflammatory response was characterized by an increase in the content of interleukin-1beta, interleukin-8, MCP-1, tumor necrosis factor-alpha, MIP-1alpha, and granulocyte-macrophage colony-stimulating factor in the wound (as compared to that in the plasma). Activity of myeloperoxidase in all patients was shown to correlate with the amount of MIP-1alpha (r=0.47), tumor necrosis factor-alpha (r=0.47), and granulocyte-macrophage colony-stimulating factor (r=0.55, p<0.05). Interleukin-8 concentration was beyond the limits of calibration. No correlation was found between the concentration of any of 27 cytokines in blood plasma and exudate. Our results indicate that during active surgical treatment, the wound serves as the source of inflammatory cytokines. Cytokines play a role in the systemic response and increase the degree of local inflammation, which modulates the number and activity of wound neutrophils.

Pre-Processing MALDI/TOF Mass Spectra by Using Geena 2.

Geena 2 is a tool for filtering, averaging, and aligning MALDI/TOF mass spectra, designed to assist scientists in the analysis of high volumes of data and support them for comparative studies. Three web interfaces are available with different levels of complexity. In this manuscript, we explain how to use Geena 2 with these three interfaces to perform analyses of one's own data. Two support protocols showing how to check the example input file and how to create an input file with own data are also presented. © 2018 by John Wiley & Sons, Inc.

Simulation of conformational changes occurring when a protein interacts with its receptor.

In order to simulate the conformational changes occurring when a protein interacts with its receptor, we firstly evaluated the structural differences between the experimental unbound and bound conformations for selected proteins and created theoretical complexes by replacing, in each experimental complex, the protein-bound with the protein-unbound chain. The theoretical models were then subjected to additional modeling refinements to improve the side chain geometry. Comparing the theoretical and experimental complexes in term of structural and energetic factors is resulted that the refined theoretical complexes became more similar to the experimental ones. We applied the same procedure within an homology modeling experiment, using as templates the experimental structures of human interleukin-1beta (IL-1beta) unbound and bound with its receptor, to build models of the homologous proteins from mouse and trout in unbound and bound conformations and to simulate the interaction with the related receptors. Our results suggest that homology modeling techniques are sensitive to differences between bound and unbound conformations, and that modeling with accuracy the side chains in the complex improves the interaction and molecular recognition. Moreover, our refinement procedure could be used in protein-protein interaction studies and, also, applied in conjunction with rigid-body docking when is not available the protein-bound conformation.

Characterization and localization of the TCL-1 oncogene product.

The TCL-1 gene maps at chromosome 14q32.1 and is activated in T cell leukemias and lymphomas by either chromosome translocations or inversions that juxtapose the TCL-1 gene to the alpha/delta or the beta locus of the T cell receptor. The open reading frame of the TCL-1 gene, coding for a protein of 114 amino acids, was expressed in bacteria and antisera were raised against it. The antibodies recognized the predicted TCL-1 M(r) 14,000 protein product in cells expressing TCL-1 mRNA. Cell fractionation experiments indicated that the TCL-1 protein is present in the microsomal fraction. These results were confirmed by confocal microscopy. The TCL-1 protein has considerable sequence similarities to the product of the MTCP-1 gene on chromosome Xq28, which is involved in T cell lympho-proliferative diseases. Thus, TCL-1 may represent a member of a novel family of genes involved in lymphoid proliferation and/or survival and in T cell malignancies.

The fluorescent monomeric protein Kusabira Orange. Pressure effect on its structure and stability.

The structure and stability of the fluorescent protein monomeric Kusabira Orange (mKO), a GFP-like protein, was studied under different pressure levels and in different chemical environments. At different pH values (between pH 7.4 and pH 4.0) and under a pressure up to 600 MPa (at 25 °C), mKO did not show significant fluorescence spectral changes, indicating a structural stability of the protein. In more extreme chemical conditions (at pH 4.0 in the presence of 0.8 M guanidine hydrochloride), a marked reduction of mKO fluorescence intensity emission was observed at pressures above 300 MPa. This fluorescence emission quenching may be due to the loss of the intermolecular bonds and, consequently, to the destructuration of the mKO chromophore structure. Since the electrostatic and hydrophobic interactions as well as the salt bridges present in proteins are usually perturbed under high pressure, the reduction of mKO fluorescence intensity emission is associated to the perturbation of the protein salt bridges network.

The murine Tcl1 oncogene: embryonic and lymphoid cell expression.

In human leukemias and lymphomas nonrandom chromosomal rearrangements cause changes in cell growth and/or survival in such a way as to promote malignancy. The detailed study of the biochemical and genetic pathways altered in human cancer requires the identification or development of models to allow the study and manipulation of cancer gene function. Recently, the breakpoint gene TCL1, involved in chromosome translocations observed mostly in mature T-cell proliferations and chronic lymphocytic leukemias (CLL), was isolated and characterized, and showed to be part of a new gene family of proteins involved in these tumors. The murine Tcl1 gene, is similar in sequence to the murine and human MTCP1 gene also involved in T cell leukemias. The murine Tcl1 gene was shown to reside on mouse chromosome 12 in a region syntenic to human chromosome 14. Furthermore, we show that the murine Tcl1 gene is expressed early in mouse embryonic development and demonstrates expression in fetal hematopoietic organs as well as in immature T and B cells. Characterization of the murine Tcl1 gene will help in developing a mouse model of CLL and would provide the best opportunity to study and decipher the role of TCL1 in malignant transformation.

Molecular properties of glutamate dehydrogenase from the extreme thermophilic archaebacterium Sulfolobus solfataricus.

This study is concerned with the structural characterization in solution of the glutamate dehydrogenase from the Archaeon Sulfolobus solfataricus. At neutral pH both alpha-helix and beta-sheet constitute the secondary structure of this enzyme, on the basis of circular dichroism. A complex, temperature dependent self-association equilibrium regulates the formation of the enzyme quaternary structure, which seems to be accompanied by a reversible structural change. At 25 degrees C the enzyme is mostly represented by monomeric subunits at concentrations lower than 0.02 mg/ml, while oligomers are predominant at concentrations higher than 0.12 mg/ml. The mid-point of the association curve shifts from 0.05 mg/ml at 25 degrees C to about 0.1 mg/ml at 45 degrees C. Only the oligomeric form appears to be temperature resistant. Monomeric and oligomeric enzyme show distinct behaviour on guanidine hydrochloride perturbation at neutral pH. The monomer denaturation, although complex, is reversible. Two fluorescent tryptophan classes are detectable in the monomer, monitoring the independent unfolding of two regions through a multistate transition. Instead, the oligomeric protein shows a complex denaturation pattern with the tendency to aggregate irreversibly at high denaturant concentration.

Identification of a beta-lactoglobulin lactosylation site.

Thermal treatment of milk leads to non-enzymatic glycosylation of proteins through Maillard reaction. Free NH2 groups of basic amino acids react with the reducing carbonyl group of lactose forming the so-called Amadori products. Electrospray mass spectrometry analysis shows that beta-lactoglobulin (beta-LG), the major whey protein, undergoes lactosylation under industrial thermal treatment. In order to investigate the specificity of reactive sites for lactose binding the analysis of trypsin hydrolysates of beta-LG isolated from different industrial milks was performed. Results demonstrate that Lys-100 is a preferential lactosylation site of beta-LG during industrial milk treatment. These results were confirmed by an analysis of the three-dimensional model of the protein which showed that Lys-100 had the highest solvent accessibility and proximity to another amino group making Lys-100 the best candidate to lactosylation. Lys-47, previously identified by other authors, showed a good proximity to another Lys residue, but an intermediate level of exposition to solvent.

Inhibition of interleukin-1 release and activity by the rat seminal vesicle protein SV-IV.

SV-IV, a 9.8-kd protein isolated from rat seminal vesicle secretion, has been shown to have strong non-species-specific immunosuppressive, anti-inflammatory, antithrombotic, and antiphospholipase A2 properties. The present investigation was undertaken to determine the mechanism of action of its immunosuppressive effects. It was found that SV-IV is a potent inhibitor of interleukin-1 (IL-1) release from lipopolysaccharide (LPS)-activated human adherent monocytes and an effective inhibitor of IL-1-induced thymocyte proliferation. The ability of SV-IV to form a noncovalent dimeric association with IL-1 alpha but not with IL-1 beta, its ability to induce a marked decrease of IL-1 binding to its own receptors on the thymocyte surface, and its capacity to bind specifically to the macrophage plasma membrane might play an important role in the molecular mechanism of these inhibitory effects.

Clinical and molecular spectra in galactosemic patients from neonatal screening in northeastern Italy: structural and functional characterization of new variations in the galactose-1-phosphate uridyltransferase (GALT) gene.

Classical galactosemia is an autosomal recessive inborn error of metabolism due to mutations of the GALT gene leading to toxic accumulation of galactose and derived metabolites. With the benefit of early diagnosis by neonatal screening and early therapy, the acute presentation of classical galactosemia can be prevented. However, despite early diagnosis and treatment, the long term outcome for these patients is still unpredictable because they may go on to develop cognitive disability, speech problems, neurological and/or movement disorders and, in females, ovarian dysfunction. The objectives of the current study were to report our experience with a group of galactosemic patients identified through the neonatal screening programs in northeastern Italy during the last 30years. No neonatal deaths due to galactosemia complications occurred after the introduction of the neonatal screening program. However, despite the early diagnosis and dietary treatment, the patients with classical galactosemia showed one or more long-term complications. A total of 18 different variations in the GALT gene were found in the patient cohort: 12 missense, 2 frameshift, 1 nonsense, 1 deletion, 1 silent variation, and 1 intronic. Six (p.R33P, p.G83V, p.P244S, p.L267R, p.L267V, p.E271D) were new variations. The most common variation was p.Q188R (12 alleles, 31.5%), followed by p.K285N (6 alleles, 15.7%) and p.N314D (6 alleles, 15.7%). The other variations comprised 1 or 2 alleles. In the patients carrying a new mutation, the biochemical analysis of GALT activity in erythrocytes showed an activity of <1%. In silico analysis (SIFT, PolyPhen-2 and the computational analysis on the static protein structure) showed potentially damaging effects of the six new variations on the GALT protein, thus expanding the genetic spectrum of GALT variations in Italy. The study emphasizes the difficulty in establishing a genotype-phenotype correlation in classical galactosemia and underlines the importance of molecular diagnostic testing prior to making any treatment.

Mutant p53 gains new function in promoting inflammatory signals by repression of the secreted interleukin-1 receptor antagonist.

The TP53 tumor-suppressor gene is frequently mutated in human cancer. Missense mutations can add novel functions (gain-of-function, GOF) that promote tumor malignancy. Here we report that mutant (mut) p53 promotes tumor malignancy by suppressing the expression of a natural occurring anti-inflammatory cytokine, the secreted interleukin-1 receptor antagonist (sIL-1Ra, IL1RN). We show that mutp53 but not wild-type (wt) p53 suppresses the sIL-1Ra production in conditioned media of cancer cells. Moreover, mutp53, but not wtp53, binds physically the sIL-1Ra promoter and the protein-protein interaction with the transcriptional co-repressor MAFF (v-MAF musculoaponeurotic fibrosarcoma oncogene family, protein F) is required for mutp53-induced sIL-1Ra suppression. Remarkably, when exposed to IL-1 beta (IL-1ß) inflammatory stimuli, mutp53 sustains a ready-to-be-activated in vitro and in vivo cancer cells' response through the sIL-1Ra repression. Taken together, these results identify sIL-1Ra as a novel mutp53 target gene, whose suppression might be required to generate a chronic pro-inflammatory tumor microenvironment through which mutp53 promotes tumor malignancy.

Alteration in the ubiquitin structure and function in the human lens: a possible mechanism of senile cataractogenesis.

High-performance liquid chromatography purification followed by mass spectrometry analyses highlighted that human senile cataractous lens includes a 8182 Da species which is absent in the normal lens, whereas a 8566/8583 Da species is present in both lenses. Western blot analysis identified both species as ubiquitin. The species at lower molecular weight is a shorter form due to the cleavage of the C-terminal residues 73-76. As it is the last amino acid of ubiquitin which is involved in the protein degradation mechanism, we suggest that this structure modification compromises the function of ubiquitin and consequently the physiologically occurring degradation of the lens proteins.

Internal repeats of prion protein and A beta PP, and reciprocal similarity with the amyloid-related proteins.

We are currently interested in a group of proteins associated with the dementias characterized by amyloid deposition in the brain: amyloid beta protein precursor (A beta PP) of Alzheimer's disease (AD) and the abnormal isoform of prion protein (PrP) of spongiform encephalopathies such as kuru, Creutzfeldt-Jacob disease (CJD) and Gerstmann-Straussler-Scheinker disease (GSSD). Here we show that both A beta PP and prion protein (PrP) consist of peculiar internal repeats and share regions of sequence similarity. Further analysis revealed that local homology is also shared with the other proteins involved in specific forms of amyloidosis--i.e., the amyloid related proteins (ARP).

Thyroid hormone binding to isolated human apolipoproteins A-II, C-I, C-II, and C-III: homology in thyroxine binding sites.

Thyroid hormone binding to lipid-free apolipoprotein (apo) A-II, C-I, C-II, and C-III isolated from human plasma was investigated by photoaffinity labeling with [125I]T4 and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Both the monomeric and polymeric forms were specifically labeled. Inhibition by 10 microM unlabeled L-T4 was > or = 50%, suggesting affinity constants in the nM to microM range; the least inhibition was seen with apoA-II. Unlabeled D-T4 and reverse T3 (rT3) gave the same inhibition as unlabeled L-T4. Inhibitors of thyroid hormone binding to plasma proteins showed a different inhibitor potency with each apolipoprotein and a pattern different from that seen with T4 binding globulin (TBG) and transthyretin (TTR). Also in contrast to TBG, where only unsaturated nonesterified fatty acids (NEFA) are effective inhibitors, both unsaturated and saturated NEFA as well as other lipids inhibited T4 labeling. The flavonoid EMD 21388 was ineffective, confirming that it is a selective inhibitor of T4 binding to TTR. T4 binding to the apoCs was confirmed by the quenching of tryptophan fluorescence by unlabeled L-T4. (ApoA-II was not studied since it lacks tryptophan). Since the self-association of apolipoproteins involves interaction between amphipathic alpha-helices, and since the polymeric forms show specific T4 binding properties as in the parent monomer, the T4-binding domain appears to be outside the alpha-helical domain, as previously seen with apoA-I.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of pancreatic phospholipase A2 activity by uteroglobin and antiflammin peptides: possible mechanism of action.

We investigated the possible mechanism of inhibition of porcine pancreatic phospholipase A2 in vitro by rabbit uteroglobin and by the antiflammin peptides. We optimized the conditions of phospholipase A2 assay using a deoxycholate-phosphatidylcholine mixed micellar substrate and established the activity of these inhibitors under optimized conditions. The results of fluorescence studies and crosslinking experiments indicate that the inhibitors interact with the enzyme in solution and affect the increase in intrinsic fluorescence of phospholipase A2 observed upon interaction with a mixed micellar substrate. In addition, we identified a sequence similarity between the antiflammin peptides, the putative active region of uteroglobin and a region in pancreatic phospholipase A2. This region of phospholipase A2 has been previously identified as being involved in the regulation of dimerization of this enzyme, and is conserved in the pancreatic-type enzymes. Taken together, these observations suggest that uteroglobin and antiflammins interact with porcine pancreatic phospholipase A2 and this may, at least in part, explain the enzyme inhibitory effect of these molecules observed in vitro. One possible mechanism of this effect may be an interference with the dimerization process of phospholipase A2 which is associated with interfacial activation.

Amino acid composition and N-terminal sequence of purified cystine binding protein of Escherichia coli.

Cystine Binding Protein (CBP), a commercially available crude protein extract obtained by osmotic shock of Escherichia coli (E. coli), was studied to characterize further its cystine binding properties and to elucidate its cystine transport activity. We report here the amino acid composition, the N-terminal amino acid sequence analysis and some binding characteristics of the purified cystine binding component of CBP. A search of the Swiss-Prot version 20 data base revealed that this sequence is unique.

Protein structure prediction and biomolecular recognition: from protein sequence to peptidomimetic design with the human beta3 integrin.

Computational tools can bridge the gap between sequence and protein 3D structure based on the notion that information is to be retrieved from the databases and that knowledge-based methods can help in approaching a solution of the protein-folding problem. To this aim our group has implemented neural network-based predictors capable of performing with some success in different tasks, including predictions of the secondary structure of globular and membrane proteins, the topology of membrane proteins and porins and stable alpha-helical segments suited for protein design. Moreover we have developed methods for predicting contact maps in proteins and the probability of finding a cysteine in a disulfide bridge, tools which can contribute to the goal of predicting the 3D structure starting from the sequence (the so called ab initio prediction). All our predictors take advantage of evolution information derived from the structural alignments of homologous (evolutionary related) proteins and taken from the sequence and structure databases. When it is necessary to build models for proteins of unknown spatial structure, which have very little homology with other proteins of known structure, non-standard techniques need to be developed and the tools for protein structure predictions may help in protein modeling. The results of a recent simulation performed in our lab highlights the role of high performing computing technology and of tools of computational biology in protein modeling and peptidomimetic design.