Specific processing of tenascin-C by the metalloprotease meprin beta neutralizes its inhibition of cell spreading

The metalloprotease meprin has been implicated in tissue remodelling due to its capability to degrade extracellular matrix components. Here, we investigated the susceptibility of tenascin-C to cleavage by meprin beta and the functional properties of its proteolytic fragments. A set of monoclonal antibodies against chicken and human tenascin-C allowed the mapping of proteolytic fragments generated by meprin beta. In chicken tenascin-C, meprin beta processed all three major splicing variants by removal of 10 kDa N-terminal and 38 kDa C-terminal peptides, leaving a large central part of subunits intact. IN similar cleavage pattern was found for large human tenascin-C variant where two N-terminal peptides (10 or 15 kDa) and two C-terminal fragments (40 and 55 kDa) were removed from the intact subunit. N-terminal sequencing revealed the exact amino acid positions of cleavage sites. In both chicken and human tenascin-C N-terminal cleavages occurred just before and/or after the heptad repeats involved in subunit oligomerization. In the human protein, an additional cleavage site was identified in the alternative fibronectin type III repeat D. Whereas all these sites are known to be attacked by several other proteases, a unique cleavage by meprin beta was located to the 7th constant fibronectin type III repeat in both chicken and human tenascin-C, thereby removing the C-terminal domain involved in its anti-adhesive activity. In cell adhesion assays meprin beta-digested human tenascin-C was not able to interfere with fibronectin-mediated cell spreading, confirming cleavage in the anti-adhesive domain. Whereas the expression of meprin beta and tenascin-C does not overlap in normal colon tissue, inflamed lesions of the mucosa from patients with Crohn's disease exhibited many meprin beta-positive leukocytes in regions where tenascin-C was strongly induced. Our data indicate that, at least under pathological conditions, meprin beta might attack specific functional sites in tenascin-C that are important for its oligomerization and anti-adhesive activity. (C) 2009 Elsevier B.V. All rights reserved.

Effect of mouse antisera targeting the Phlebotomus papatasi midgut chitinase PpChit1 on sandfly physiology and fitness

In sandflies, the absence of the peritrophic matrix (PM) affects the rate of blood digestion. Also, the kinetics of PM secretion varies according to species. We previously characterised PpChit1, a midgut-specific chitinase secreted in Phlebotomus papatasi (PPIS) that is involved in the maturation of the PM and showed that antibodies against PpChit1 reduce the chitinolytic activity in the midgut of several sandfly species. Here, sandflies were fed on red blood cells reconstituted with naïve or anti-PpChit1 sera and assessed for fitness parameters that included blood digestion, oviposition onset, number of eggs laid, egg bouts, average number of eggs per bout and survival. In PPIS, anti-PpChit1 led to a one-day delay in the onset of egg laying, with flies surviving three days longer compared to the control group. Anti-PpChit1 also had a negative effect on overall ability of flies to lay eggs, as several gravid females from all three species were unable to lay any eggs despite having lived longer than control flies. Whereas the longer survival might be associated with improved haeme scavenging ability by the PM, the inability of females to lay eggs is possibly linked to changes in PM permeability affecting nutrient absorption.

The rise of soluble TWEAK levels in severely obese subjects after bariatric surgery may affect adipocyte-cytokine production induced by TNFα.

CONTEXT Soluble TNF-like weak inducer of apoptosis (sTWEAK) is generated by the intracellular proteolytic cleavage of full-length membrane-bound TNF-like weak inducer of apoptosis (mTWEAK). sTWEAK levels are reduced in diseases with an inflammatory component. Additionally, sTWEAK hampers TNFα activity in human cells. OBJECTIVES The objectives of the study were as follows: 1) to determine circulating sTWEAK in severe obesity and after bariatric surgery; 2) to study m/sTWEAK and its receptor fibroblast growth factor-inducible 14 (Fn14) protein expression in sc adipose tissue (SAT) of severely obese subjects, in SAT stromal vascular fraction (SVF), and isolated adipocytes and in human monocyte-derived macrophages; and 3) to explore, on human adipocytes, the sTWEAK effect on TNFα proinflammatory activity. DESIGN sTWEAK levels were measured in cohort 1: severely obese subjects (n = 23) and a control group (n = 35); and in cohort 2: (n = 23) severely obese subjects before and after surgery. The m/sTWEAK and Fn14 expressions were determined in SAT biopsies, SVF, and isolated adipocytes from severely obese and control subjects and in human monocyte-derived macrophages. In human primary cultured adipocytes, sTWEAK pretreated and TNFα challenged, IL-6, IL-8, and adiponectin protein and gene expressions were determined and nuclear factor-κ B and MAPK signaling analyzed. RESULTS sTWEAK levels were reduced in severely obese subjects. After surgery, sTWEAK levels rose in 69% of patients. mTWEAK protein expression was increased in SAT and SVF of severely obese subjects, whereas Fn14 was up-regulated in isolated adipocytes. M2 human monocyte-derived macrophages overexpress mTWEAK. In human adipocytes, sTWEAK down-regulates TNFα cytokine production by hampering TNFα intracellular signaling events. CONCLUSION The decrease of sTWEAK in severely obese patients may favor the proinflammatory activity elicited by TNFα.

Influencia de un programa de actividad física en niños y adolescentes obesos; evaluación del estrés fisiológico mediante compuestos en la saliva; protocolo de estudio.

Different studies4, 5 relate that stress increases in children and adolescents with overweight and obesity, and consequently their salivary physiological responses (AEA salivary alpha-amylase, cortisol, cytokines, leptin), so in this study we relate these two parameters to see their progress through a program of physical activity. If we manage to reduce overweight or obesity, these physiological responses and stress should also be reduced, thus improving the overall health status of these children and adolescents. The overall objective of the study was to determine the influence of physical activity in obese children and adolescents in perceived stress. An observational, descriptive, prospective and longitudinal study will be carried out. The universe is made up of 60 overweight / obese children and adolescents aged between 10 and 18 years. The assessment will take place from September 2012 to September 2013. To collect saliva samples, the ELISA8 method will be used. Variables such as BMI, lifestyle and diet will also be collected. Among the expected results are to lower overweight and obesity in children through physical activity program. To reduce stress and to normalize physiological salivary parameters.

Structural determinants for membrane association and dynamic organization of the hepatitis C virus NS3-4A complex.

Hepatitis C virus (HCV) NS3-4A is a membrane-associated multifunctional protein harboring serine protease and RNA helicase activities. It is an essential component of the HCV replication complex and a prime target for antiviral intervention. Here, we show that membrane association and structural organization of HCV NS3-4A are ensured in a cooperative manner by two membrane-binding determinants. We demonstrate that the N-terminal 21 amino acids of NS4A form a transmembrane alpha-helix that may be involved in intramembrane protein-protein interactions important for the assembly of a functional replication complex. In addition, we demonstrate that amphipathic helix alpha(0), formed by NS3 residues 12-23, serves as a second essential determinant for membrane association of NS3-4A, allowing proper positioning of the serine protease active site on the membrane. These results allowed us to propose a dynamic model for the membrane association, processing, and structural organization of NS3-4A on the membrane. This model has implications for the functional architecture of the HCV replication complex, proteolytic targeting of host factors, and drug design.

HCF-1 self-association via an interdigitated Fn3 structure facilitates transcriptional regulatory complex formation.

Host-cell factor 1 (HCF-1) is an unusual transcriptional regulator that undergoes a process of proteolytic maturation to generate N- (HCF-1(N)) and C- (HCF-1(C)) terminal subunits noncovalently associated via self-association sequence elements. Here, we present the crystal structure of the self-association sequence 1 (SAS1) including the adjacent C-terminal HCF-1 nuclear localization signal (NLS). SAS1 elements from each of the HCF-1(N) and HCF-1(C) subunits form an interdigitated fibronectin type 3 (Fn3) tandem repeat structure. We show that the C-terminal NLS recruited by the interdigitated SAS1 structure is required for effective formation of a transcriptional regulatory complex: the herpes simplex virus VP16-induced complex. Thus, HCF-1(N)-HCF-1(C) association via an integrated Fn3 structure permits an NLS to facilitate formation of a transcriptional regulatory complex.

X-ray structures of Sap1 and Sap5: structural comparison of the secreted aspartic proteinases from Candida albicans.

Proteolytic activity is an important virulence factor for Candida albicans (C. albicans). It is attributed to the family of the secreted aspartic proteinases (Saps) from C. albicans with a minimum of 10 members. Saps show controlled expression and regulation for the individual stages of the infection process. Distinct isoenzymes can be responsible for adherence and tissue damage of local infections, while others cause systemic diseases. Earlier, only the structures of Sap2 and Sap3 were known. In our research, we have now succeeded in solving the X-ray crystal structures of the apoenzyme of Sap1 and Sap5 in complex with pepstatin A at 2.05 and 2.5 A resolution, respectively. With the structure of Sap1, we have completed the set of structures of isoenzyme subgroup Sap1-3. Of subgroup Sap4-6, the structure of the enzyme Sap5 is the first structure that has been described up to now. This facilitates comparison of structural details as well as inhibitor binding modes among the different subgroup members. Structural analysis reveals a highly conserved overall secondary structure of Sap1-3 and Sap5. However, Sap5 clearly differs from Sap1-3 by its electrostatic overall charge as well as through structural conformation of its entrance to the active site cleft. Design of inhibitors specific for Sap5 should concentrate on the S4 and S3 pockets, which significantly differ from Sap1-3 in size and electrostatic charge. Both Sap1 and Sap5 seem to play a major part in superficial Candida infections. Determination of the isoenzymes' structures can contribute to the development of new Sap-specific inhibitors for the treatment of superficial infections with a structure-based drug design program.

Análisis de anticuerpos antifosfolipídicos y cistatina C en pacientes con esclerosis múltiple

Cystatin C is considered the most important physiological inhibitor of endogenous cysteine proteases; the role of cystatin C is believed to be to modulate the activity of proteases secreted or released from damaged cells or in the process of necrosis, therefore cystatins being fundamental regulatory processes and a potential prevention of local proteolytic damage. Antiphospholipid antibodies are used to clarify the diagnosis of diseases like multiple sclerosis (MS) and other pathologies could present similar symptoms or paraclinical findings. The objective of the present work is to analyze the concentration of cystatin C and the presence or absence of antiphospholipid antibodies in patients diagnosed with relapsing remitting multiple sclerosis (RRMS) as markers of demyelization. This work was carried out jointly by the Vascular Risk Laboratory, the Laboratory of Autoimmunity and Multiple Sclerosis Unit, Hospital Universitario Virgen Macarena in Seville in one year. Two types of people were selected: Group 1 (n = 30) RRMS group and a control group, n = 30. Cystatin C and antiphospholipid antibodies IgG and IgM, IgG and IgM anticardiolipin, β2 glycoprotein IgG and IgM were determined. Patients showed negative titers of antiphospholipid antibodies IgG and IgM, IgG and IgM anticardiolipin, β2 glycoprotein IgG and IgM. Cystatin C concentration is lower in the group of patients diagnosed with MS, which could give rise to a decrease in the modulation of endogenous cysteine proteases. This would exacerbate the progress of demyelization in MS.

First-line therapies in inflammatory bowel disease.

Background and Aims: Medical therapy of inflammatory bowel disease (IBD) is becoming more complex, given the increasing choice of drugs to treat Crohn's disease (CD) and ulcerative colitis (UC). We aimed to summarize the current guidelines for first-line treatments in IBD. Methods: An extensive literature search with focus on the guidelines of the European Crohn's and Colitis Organisation for the diagnosis and treatment of CD and UC was performed. First-line treatments were defined as the following drug categories: 5-aminosalicylates, budesonide, systemic steroids, azathioprine, 6-mercaptopurine, methotrexate, infliximab, adalimumab and certolizumab pegol. The following drug categories were not included: cyclosporine and tacrolimus (not yet approved by Swissmedic for IBD treatment). Results: Treatment recommendations for the following clinically frequent situations are presented according to disease severity: ileocecal CD, colonic CD, proximal small bowel CD and perianal CD. For UC the following situations are presented: ulcerative proctitis, left-sided colitis and pancolitis. Conclusions: We provide a summary on the use of first-line therapies for clinically frequent situations in patients with CD and UC.

Plasmodium falciparum metacaspase PfMCA-1 triggers a z-VAD-fmk inhibitable protease to promote cell death.

Activation of proteolytic cell death pathways may circumvent drug resistance in deadly protozoan parasites such as Plasmodium falciparum and Leishmania. To this end, it is important to define the cell death pathway(s) in parasites and thus characterize proteases such as metacaspases (MCA), which have been reported to induce cell death in plants and Leishmania parasites. We, therefore, investigated whether the cell death function of MCA is conserved in different protozoan parasite species such as Plasmodium falciparum and Leishmania major, focusing on the substrate specificity and functional role in cell survival as compared to Saccharomyces cerevisae. Our results show that, similarly to Leishmania, Plasmodium MCA exhibits a calcium-dependent, arginine-specific protease activity and its expression in yeast induced growth inhibition as well as an 82% increase in cell death under oxidative stress, a situation encountered by parasites during the host or when exposed to drugs such as artemisins. Furthermore, we show that MCA cell death pathways in both Plasmodium and Leishmania, involve a z-VAD-fmk inhibitable protease. Our data provide evidence that MCA from both Leishmania and Plasmodium falciparum is able to induce cell death in stress conditions, where it specifically activates a downstream enzyme as part of a cell death pathway. This enzymatic activity is also induced by the antimalarial drug chloroquine in erythrocytic stages of Plasmodium falciparum. Interestingly, we found that blocking parasite cell death influences their drug sensitivity, a result which could be used to create therapeutic strategies that by-pass drug resistance mechanisms by acting directly on the innate pathways of protozoan cell death.

Penetrating or Stricturing Diseases are the Major Determinants of Time to First and Repeat Resection Surgery in Crohn's Disease.

Background: About 80% of patients with Crohn's disease (CD) require bowel resection and up to 65% will undergo a second resection within 10 years. This study reports clinical risk factors for resection surgery (RS) and repeat RS. Methods: Retrospective cohort study, using data from patients included in the Swiss Inflammatory Bowel Disease Cohort. Cox regression analyses were performed to estimate rates of initial and repeated RS. Results: Out of 1,138 CD cohort patients, 417 (36.6%) had already undergone RS at the time of inclusion. Kaplan-Meier curves showed that the probability of being free of RS was 65% after 10 years, 42% after 20 years, and 23% after 40 years. Perianal involvement (PA) did not modify this probability to a significant extent. The main adjusted risk factors for RS were smoking at diagnosis (hazard ratio (HR) = 1.33; p = 0.006), stricturing with vs. without PA (HR = 4.91 vs. 4.11; p < 0.001) or penetrating disease with vs. without PA (HR = 3.53 vs. 4.58; p < 0.001). The risk factor for repeat RS was penetrating disease with vs. without PA (HR = 3.17 vs. 2.24; p < 0.05). Conclusion: The risk of RS was confirmed to be very high for CD in our cohort. Smoking status at diagnosis, but mostly penetrating and stricturing diseases increase the risk of RS.

Lack of tumor recognition by hTERT peptide 540-548-specific CD8(+) T cells from melanoma patients reveals inefficient antigen processing.

Telomerase is a ribonucleoprotein complex responsible for the maintenance of the length of the telomeres during cell division, which is active in germ-line cells as well as in the vast majority of tumors but not in most normal tissues. The wide expression of the human telomerase catalytic subunit (hTERT) in tumors makes it an interesting candidate vaccine for cancer. hTERT-derived peptide 540-548 (hTERT(540)) has been recently shown to be recognized in an HLA-A*0201-restricted fashion by T cell lines derived from peptide-stimulated peripheral blood mononuclear cells (PBMC) from healthy donors. As a first step to the inclusion of this peptide in immunotherapy clinical trials, it is crucial to assess hTERT(540)-specific T cell reactivity in cancer patients as well as the ability of hTERT-specific CD8(+) T lymphocytes to recognize and lyse hTERT-expressing target cells. Here, we have analyzed the CD8(+) T cell response to peptide hTERT(540) in HLA-A*0201 melanoma patients by using fluorescent HLA-A*0201/hTERT(540) peptide tetramers. HLA-A*0201/hTERT(540) tetramer(+) CD8(+) T cells were readily detected in peptide-stimulated PBMC from a significant proportion of patients and could be isolated by tetramer-guided cell sorting. hTERT(540)-specific CD8(+) T cells were able to specifically recognize HLA-A*0201 cells either pulsed with peptide or transiently transfected with a minigene encoding the minimal epitope. In contrast, they failed to recognize hTERT-expressing HLA-A*0201(+) target cells. Furthermore, in vitro proteasome digestion studies revealed inadequate hTERT processing. Altogether, these results raise questions on the use of hTERT(540) peptide for cancer immunotherapy.

Production of human secretory component with dimeric IgA binding capacity using viral expression systems.

The cDNA encoding the NH2-terminal 589 amino acids of the extracellular domain of the human polymeric immunoglobulin receptor was inserted into transfer vectors to generate recombinant baculo- and vaccinia viruses. Following infection of insect and mammalian cells, respectively, the resulting truncated protein corresponding to human secretory component (hSC) was secreted with high efficiency into serum-free culture medium. The Sf9 insect cell/baculovirus system yielded as much as 50 mg of hSC/liter of culture, while the mammalian cells/vaccinia virus system produced up to 10 mg of protein/liter. The M(r) of recombinant hSC varied depending on the cell line in which it was expressed (70,000 in Sf9 cells and 85-95,000 in CV-1, TK- 143B and HeLa). These variations in M(r) resulted from different glycosylation patterns, as evidenced by endoglycosidase digestion. Efficient single-step purification of the recombinant protein was achieved either by concanavalin A affinity chromatography or by Ni(2+)-chelate affinity chromatography, when a 6xHis tag was engineered to the carboxyl terminus of hSC. Recombinant hSC retained the capacity to specifically reassociate with dimeric IgA purified from hybridoma cells.

Identification of Leishmania major cysteine proteinases as targets of the immune response in humans.

In this study, we report the identification of two parasite polypeptides recognized by human sera of patients infected with Leishmania major. Isolation and sequencing of the two genes encoding these polypeptides revealed that one of the genes is similar to the L. major cathepsin L-like gene family CPB, whereas the other gene codes for the L. major homologue of the cysteine proteinase a (CPA) of L. mexicana. By restriction enzyme digestion of genomic DNA, we show that the CPB gene is present in multiple copies in contrast to the cysteine proteinase CPA gene which could be unique. Specific antibodies directed against the mature regions of both types expressed in Escherichia coli were used to analyze the expression of these polypeptides in different stages of the parasite's life cycle. Polypeptides of 27 and 40 kDa in size, corresponding to CPA and CPB respectively, were detected at higher level in amastigotes than in stationary phase promastigotes. Purified recombinant CPs were also used to examine the presence of specific antibodies in sera from either recovered or active cases of cutaneous leishmaniasis patients. Unlike sera from healthy uninfected controls, all the sera reacted with recombinant CPA and CPB. This finding indicates that individuals having recovered from cutaneous leishmaniasis or with clinically apparent disease have humoral responses to cysteine proteinases demonstrating the importance of these proteinases as targets of the immune response and also their potential use for serodiagnosis.

Review article: Intestinal epithelia and barrier functions.

The mucosal epithelia of the digestive tract acts as a selective barrier, permeable to ions, small molecules and macromolecules. These epithelial cells aid the digestion of food and absorption of nutrients. They contribute to the protection against pathogens and undergo continuous cell renewal which facilitates the elimination of damaged cells. Both innate and adaptive defence mechanisms protect the gastrointestinal-mucosal surfaces against pathogens. Interaction of microorganisms with epithelial cells triggers a host response by activating specific transcription factors which control the expression of chemokines and cytokines. This host response is characterized by the recruitment of macrophages and neutrophils at the site of infection. Disruption of epithelial signalling pathways that recruit migratory immune cells results in a chronic inflammatory response. The adaptive defence mechanism relies on the collaboration of epithelial cells (resident sampling system) with antigen-presenting and lymphoid cells (migratory sampling system); in order to obtain samples of foreign antigen, these samples must be transported across the barriers without affecting the integrity of the barrier. These sampling systems are regulated by both environmental and host factors. Fates of the antigen may differ depending on the way in which they cross the epithelial barrier, i.e. via interaction with motile dendritic cells or epithelial M cells in the follicle-associated epithelium.