Enhanced activity of meprin-?, a pro-migratory and pro-angiogenic protease, in colorectal cancer

Meprin-α is a metalloprotease overexpressed in cancer cells, leading to the accumulation of this protease in a subset of colorectal tumors. The impact of increased meprin-α levels on tumor progression is not known. We investigated the effect of this protease on cell migration and angiogenesis in vitro and studied the expression of meprin-α mRNA, protein and proteolytic activity in primary tumors at progressive stages and in liver metastases of patients with colorectal cancer, as well as inhibitory activity towards meprin-α in sera of cancer patient as compared to healthy controls. We found that the hepatocyte growth factor (HGF)-induced migratory response of meprin-transfected epithelial cells was increased compared to wild-type cells in the presence of plasminogen, and that the angiogenic response in organ-cultured rat aortic explants was enhanced in the presence of exogenous human meprin-α. In patients, meprin-α mRNA was expressed in colonic adenomas, primary tumors UICC (International Union Against Cancer) stage I, II, III and IV, as well as in liver metastases. In contrast, the corresponding protein accumulated only in primary tumors and liver metastases, but not in adenomas. However, liver metastases lacked meprin-α activity despite increased expression of the corresponding protein, which correlated with inefficient zymogen activation. Sera from cancer patients exhibited reduced meprin-α inhibition compared to healthy controls. In conclusion, meprin-α activity is regulated differently in primary tumors and metastases, leading to high proteolytic activity in primary tumors and low activity in liver metastases. By virtue of its pro-migratory and pro-angiogenic activity, meprin-α may promote tumor progression in colorectal cancer.

Evaluation of chronic HCV infection in transplanted livers using a modified histological activity index

The majority of histopathological classifications of primary chronic viral hepatitis and recurrence of HCV infection in liver transplants is based on the histological activity index (HAI) introduced by Knodell et al in 1981; however, correlation between HAI and clinical/laboratory data is poor. Therefore, the aim of this study was to present a modification of HAI (mHAI) adapted to distinct features of graft infection, and to evaluate its usefulness in the description of disease activity.

Genetic evidence supporting the association of protease and protease inhibitor genes with inflammatory bowel disease: a systematic review

As part of the European research consortium IBDase, we addressed the role of proteases and protease inhibitors (P/PIs) in inflammatory bowel disease (IBD), characterized by chronic mucosal inflammation of the gastrointestinal tract, which affects 2.2 million people in Europe and 1.4 million people in North America. We systematically reviewed all published genetic studies on populations of European ancestry (67 studies on Crohn's disease [CD] and 37 studies on ulcerative colitis [UC]) to identify critical genomic regions associated with IBD. We developed a computer algorithm to map the 807 P/PI genes with exact genomic locations listed in the MEROPS database of peptidases onto these critical regions and to rank P/PI genes according to the accumulated evidence for their association with CD and UC. 82 P/PI genes (75 coding for proteases and 7 coding for protease inhibitors) were retained for CD based on the accumulated evidence. The cylindromatosis/turban tumor syndrome gene (CYLD) on chromosome 16 ranked highest, followed by acylaminoacyl-peptidase (APEH), dystroglycan (DAG1), macrophage-stimulating protein (MST1) and ubiquitin-specific peptidase 4 (USP4), all located on chromosome 3. For UC, 18 P/PI genes were retained (14 proteases and 4 protease inhibitors), with a considerably lower amount of accumulated evidence. The ranking of P/PI genes as established in this systematic review is currently used to guide validation studies of candidate P/PI genes, and their functional characterization in interdisciplinary mechanistic studies in vitro and in vivo as part of IBDase. The approach used here overcomes some of the problems encountered when subjectively selecting genes for further evaluation and could be applied to any complex disease and gene family.

IL28B alleles associated with poor hepatitis C virus (HCV) clearance protect against inflammation and fibrosis in patients infected with non-1 HCV genotypes

Genetic polymorphisms near IL28B are associated with spontaneous and treatment-induced clearance of hepatitis C virus (HCV), two processes that require the appropriate activation of the host immune responses. Intrahepatic inflammation is believed to mirror such activation, but its relationship with IL28B polymorphisms has yet to be fully appreciated. We analyzed the association of IL28B polymorphisms with histological and follow-up features in 2335 chronically HCV-infected Caucasian patients. Assessable phenotypes before any antiviral treatment included necroinflammatory activity (n = 1,098), fibrosis (n = 1,527), fibrosis progression rate (n = 1,312), and hepatocellular carcinoma development (n = 1,915). Associations of alleles with the phenotypes were evaluated by univariate analysis and multivariate logistic regression, accounting for all relevant covariates. The rare G allele at IL28B marker rs8099917-previously shown to be at risk of treatment failure-was associated with lower activity (P = 0.04), lower fibrosis (P = 0.02) with a trend toward lower fibrosis progression rate (P = 0.06). When stratified according to HCV genotype, most significant associations were observed in patients infected with non-1 genotypes (P = 0.003 for activity, P = 0.001 for fibrosis, and P = 0.02 for fibrosis progression rate), where the odds ratio of having necroinflammation or rapid fibrosis progression for patients with IL28B genotypes TG or GG versus TT were 0.48 (95% confidence intervals 0.30-0.78) and 0.56 (0.35-0.92), respectively. IL28B polymorphisms were not predictive of the development of hepatocellular carcinoma.

The SerpinB1 knockout mouse a model for studying neutrophil protease regulation in homeostasis and inflammation

SerpinB1 is a clade B serpin, or ov-serpin, found at high levels in the cytoplasm of neutrophils. SerpinB1 inhibits neutrophil serine proteases, which are important in killing microbes. When released from granules, these potent enzymes also destroy host proteins and contribute to morbidity and mortality in inflammatory diseases including emphysema, chronic obstructive pulmonary disease, cystic fibrosis, arthritis, and sepsis. Studies of serpinB1-deficient mice have established a crucial role for this serpin in Pseudomonas aeruginosa infection by preserving lung antimicrobial proteins from proteolysis and by protecting lung-recruited neutrophils from a premature death. SerpinB1⁻/⁻ mice also have a severe defect in the bone marrow reserve of mature neutrophils demonstrating a key role for serpinB1 in cellular homeostasis. Here, key methods used to generate and characterize serpinB1⁻/⁻ mice are described including intranasal inoculation, myeloperoxidase activity, flow cytometry analysis of bone marrow myeloid cells, and elastase activity. SerpinB1-knockout mice provide a model to dissect the pathogenesis of inflammatory disease characterized by protease:antiprotease imbalance and may be used to assess the efficacy of therapeutic compounds.

Constrained pattern of viral evolution in acute and early HCV infection limits viral plasticity

Cellular immune responses during acute Hepatitis C virus (HCV) and HIV infection are a known correlate of infection outcome. Viral adaptation to these responses via mutation(s) within CD8+ T-cell epitopes allows these viruses to subvert host immune control. This study examined HCV evolution in 21 HCV genotype 1-infected subjects to characterise the level of viral adaptation during acute and early HCV infection. Of the total mutations observed 25% were within described CD8+ T-cell epitopes or at viral adaptation sites. Most mutations were maintained into the chronic phase of HCV infection (75%). The lack of reversion of adaptations and high proportion of silent substitutions suggests that HCV has structural and functional limitations that constrain evolution. These results were compared to the pattern of viral evolution observed in 98 subjects during a similar phase in HIV infection from a previous study. In contrast to HCV, evolution during acute HIV infection is marked by high levels of amino acid change relative to silent substitutions, including a higher proportion of adaptations, likely reflecting strong and continued CD8+ T-cell pressure combined with greater plasticity of the virus. Understanding viral escape dynamics for these two viruses is important for effective T cell vaccine design.

Management of hepatitis C virus (HCV) infection in drug substitution programs

In Switzerland, intravenous drug use (IDU) accounts for 80% of newly acquired hepatitis C virus (HCV) infections. Early HCV treatment has the potential to interrupt the transmission chain and reduce morbidity/mortality due to decompensated liver cirrhosis and hepatocellular carcinoma. Nevertheless, patients in drug substitution programs are often insufficiently screened and treated.

Mannan-binding lectin (MBL) and MBL-associated serine protease-2 in children with cancer

Mannan-binding lectin (MBL) and MBL-associated serine protease-2 (MASP-2) are two key components of the lectin-pathway of complement-activation. Information on the potential role of lectin-pathway components in carcinogenesis versus immune surveillance of cancer is scarce. This study aimed to determine if serum concentrations of MBL and MASP-2 differ between children with cancer and healthy age-matched controls.

Serum concentrations of lectin-pathway components in healthy neonates, children and adults: mannan-binding lectin (MBL), M-, L-, and H-ficolin, and MBL-associated serine protease-2 (MASP-2)

This study aimed to measure serum concentrations of five lectin-pathway components, mannan-binding lectin (MBL), M-ficolin, L-ficolin, H-ficolin, and MBL-associated serine protease-2 (MASP-2), in healthy neonates and children, to determine if they change with age and to compare them with serum concentrations in healthy adults. Concentrations were measured in 141 preterm and 30 term neonates, in 120 children including infants and adolescents, and in 350 adults (97 for L-ficolin) by inhouse time-resolved immunofluorometric assays or commercially available enzyme-linked immunosorbent assays. The adjacent categories method applying Wilcoxon-Mann-Whitney tests was used to determine age categories where concentrations differed significantly. Displaying serum concentration vs. age, an inverted-U shape (higher concentrations in children than in neonates and adults) was found for MBL and the ficolins, and an S-shape for MASP-2. Serum concentrations of all five lectin-pathway components were significantly lower in preterm neonates <32-wk gestational age compared to older neonates, infants, and children. Only M-ficolin in children >1 yr and H-ficolin in term neonates and in children were found to be comparable with adult values. MBL, M-, L-, and H-ficolin, and MASP-2 serum concentrations show important changes with age. The respective normal ranges for adults should not be used in the pediatric population. The age-specific pediatric ranges established here may be used instead.

Structural basis for the regulation of cysteine-protease activity by a new class of protease inhibitors in Plasmodium

Plasmodium cysteine proteases are essential for host-cell invasion and egress, hemoglobin degradation, and intracellular development of the parasite. The temporal, site-specific regulation of cysteine-protease activity is a prerequisite for survival and propagation of Plasmodium. Recently, a new family of inhibitors of cysteine proteases (ICPs) with homologs in at least eight Plasmodium species has been identified. Here, we report the 2.6 A X-ray crystal structure of the C-terminal, inhibitory domain of ICP from P. berghei (PbICP-C) in a 1:1 complex with falcipain-2, an important hemoglobinase of Plasmodium. The structure establishes Plasmodium ICP as a member of the I42 class of chagasin-like protease inhibitors but with large insertions and differences in the binding mode relative to other family members. Furthermore, the PbICP-C structure explains why host-cell cathepsin B-like proteases and, most likely, also the protease-like domain of Plasmodium SERA5 (serine-repeat antigen 5) are no targets for ICP.

Acquisition of complement inhibitor serine protease factor I and its cofactors C4b-binding protein and factor H by Prevotella intermedia

Infection with the Gram-negative pathogen Prevotella intermedia gives rise to periodontitis and a growing number of studies implies an association of P. intermedia with rheumatoid arthritis. The serine protease Factor I (FI) is the central inhibitor of complement degrading complement components C3b and C4b in the presence of cofactors such as C4b-binding protein (C4BP) and Factor H (FH). Yet, the significance of complement inhibitor acquisition in P. intermedia infection and FI binding by Gram-negative pathogens has not been addressed. Here we show that P. intermedia isolates bound purified FI as well as FI directly from heat-inactivated human serum. FI bound to bacteria retained its serine protease activity as shown in degradation experiments with (125)I-labeled C4b. Since FI requires cofactors for its activity we also investigated the binding of purified cofactors C4BP and FH and found acquisition of both proteins, which retained their activity in FI mediated degradation of C3b and C4b. We propose that FI binding by P. intermedia represents a new mechanism contributing to complement evasion by a Gram-negative bacterial pathogen associated with chronic diseases.

Periodontal pathogens affect the level of protease inhibitors in gingival crevicular fluid

In periodontitis, an effective host-response is primarily related to neutrophils loaded with serine proteases, including elastase (NE) and protease 3 (PR3), the extracellular activity of which is tightly controlled by endogenous inhibitors. In vitro these inhibitors are degraded by gingipains, cysteine proteases produced by Porphyromonas gingivalis. The purpose of this study was to determine the level of selected protease inhibitors in gingival crevicular fluid (GCF) in relation to periodontal infection. The GCF collected from 31 subjects (nine healthy controls, seven with gingivitis, five with aggressive periodontitis and 10 with chronic periodontitis) was analyzed for the levels of elafin and secretory leukocyte protease inhibitor (SLPI), two main tissue-derived inhibitors of neutrophil serine proteases. In parallel, activity of NE, PR3 and arginine-specific gingipains (Rgps) in GCF was measured. Finally loads of P. gingivalis, Aggregatibacter actinomycetemcomitans, Tannerella forsythia and Treponema denticola were determined. The highest values of elafin were found in aggressive periodontitis and the lowest in controls. The quantity of elafin correlated positively with the load of P. gingivalis, Ta. forsythia and Tr. denticola, as well as with Rgps activity. In addition, NE activity was positively associated with the counts of those bacterial species, but not with the amount of elafin. In contrast, the highest concentrations of SLPI were found in periodontally healthy subjects whereas amounts of this inhibitor were significantly decreased in patients infected with P. gingivalis. Periodontopathogenic bacteria stimulate the release of NE and PR3, which activities escape the control through degradation of locally produced inhibitors (SLPI and elafin) by host-derived and bacteria-derived proteases.