Glucocorticoids induce the production of the chemoattractant CCL20 in airway epithelium.

Th17-mediated neutrophilic airway inflammation has been implicated in decreased response to glucocorticoids in asthma. We aimed to investigate the effect of glucocorticoids on the airway epithelial release of the neutrophilic and Th17-cell chemoattractant CCL20. We studied CCL20 and CXCL8 sputum levels in asthmatic subjects using inhaled glucocorticoids or not, and the effect of budesonide on CCL20 and CXCL8 production in primary bronchial epithelial cells. The mechanism behind the effect of budesonide-induced CCL20 production was studied in 16HBE14o- cells using inhibitors for the glucocorticoid receptor, intracellular pathways and metalloproteases. We observed higher levels of CCL20, but not CXCL8, in the sputum of asthmatics who used inhaled glucocorticoids. CCL20 levels correlated with inhaled glucocorticoid dose and sputum neutrophils. Budesonide increased tumour necrosis factor (TNF)-α-induced CCL20 by primary bronchial epithelium, while CXCL8 was suppressed. In 16HBE14o- cells, similar effects were observed at the CCL20 protein and mRNA levels, indicating transcriptional regulation. Although TNF-α-induced CCL20 release was dependent on the ERK, p38 and STAT3 pathways, the increase by budesonide was not. Inhibition of glucocorticoid receptor or ADAM17 abrogated the budesonide-induced increase in CCL20 levels. We show that glucocorticoids enhance CCL20 production by bronchial epithelium, which may constitute a novel mechanism in Th17-mediated glucocorticoid-insensitive inflammation in asthma.

PMS2 involvement in patients suspected of Lynch syndrome.

It is well-established that germline mutations in the mismatch repair genes MLH1, MSH2, and MSH6 cause Lynch syndrome. However, mutations in these three genes do not account for all Lynch syndrome (suspected) families. Recently, it was shown that germline mutations in another mismatch repair gene, PMS2, play a far more important role in Lynch syndrome than initially thought. To explore this further, we determined the prevalence of pathogenic germline PMS2 mutations in a series of Lynch syndrome-suspected patients. Ninety-seven patients who had early-onset microsatellite instable colorectal or endometrial cancer, or multiple Lynch syndrome-associated tumors and/or were from an Amsterdam Criteria II-positive family were selected for this study. These patients carried no pathogenic germline mutation in MLH1, MSH2, or MSH6. When available, tumors were investigated for immunohistochemical staining (IHC) for PMS2. PMS2 was screened in all patients by exon-by-exon sequencing. We identified four patients with a pathogenic PMS2 mutation (4%) among the 97 patients we selected. IHC of PMS2 was informative in one of the mutation carriers, and in this case, the tumor showed loss of PMS2 expression. In conclusion, our study confirms the finding of previous studies that PMS2 is more frequently involved in Lynch syndrome than originally expected.

A novel MSH2 germline mutation in a Druze HNPCC family.

Germline mutations in DNA mismatch repair (DNA-MMR) genes, mainly MLH1, MSH2, and MSH6, underlie Hereditary non-polyposis colorectal cancer (HNPCC) and are mostly family-specific, with few reported founder mutations in MSH2 (Ashkenazim) MLH1 (Finnish). No mutations in colon cancer susceptibility genes have ever been reported in Druze individuals, a Moslem related faith encompassing approximately 1,000,000 individuals worldwide. A novel MSH2 mutation is described in a Druze HNPCC family: a multigenerational family with 10 members in 4 generations affected with colorectal cancer (mean age of diagnosis 46.5 years), two with gastric cancer and one--endometrial cancer. Mutational analysis of the MSH2 gene using denaturing gradient gel electrophoresis (DGGE) and direct sequencing revealed the c.702delA mutation in codon 234 of exon 4 of the MSH2 gene leading to a premature early stop in codon 245, p.Thr234fsX245. Analysis of mutation-carrying or presumed carriers individuals' offspring, revealed 11/42 asymptomatic mutation carriers, age range 17-50 years. The mutation was not present in two additional Druze HNPCC families and 20 Druze sporadic colon cancer patients. This is the first mutation ever reported in a colon cancer susceptibility gene in a Druze family and it appears not to be a founder mutation in Druze individuals with HNPCC.

Metalloproteinase Profiling in Lung Transplant Recipients With Good Outcome and Bronchiolitis Obliterans Syndrome.

BACKGROUND: Bronchiolitis obliterans syndrome (BOS), the major cause of death on lung transplantation, is characterized by bronchiolar inflammation and tissue remodeling. Matrix metalloproteinases (MMPs) have been implicated in these processes, although it is still unclear whether MMP activity and binding to their endogenous inhibitors, tissue inhibitors of metalloproteinases (TIMPs), is abnormal in BOS. METHODS: We studied total MMP-1,-2,-3,-7,-8,-9,-12,-13 levels, their activity state using activity-based extraction and their binding to TIMP-1, -2, -3, and -4 in bronchoalveolar lavage (BAL) of lung transplant recipients with good outcome and BOS using a multiplex immunoassay. RESULTS: The BAL levels of TIMP-1 and -2 and MMP-2, -3, -7, -8, and -9 were significantly increased in BOS compared to good outcome recipients. Interestingly, activity of MMP-7, but none of the other MMPs, was detected in good outcome recipients, whereas no active MMPs were observed in BOS recipients. However, BAL levels of TIMP-bound MMP-8 and -9 were higher in BOS than in good outcome recipients, suggesting activity of these MMPs in an earlier stage. CONCLUSIONS: We demonstrate that development of BOS is associated with increased levels of TIMP-1 and -2 and total MMP-2, -3, -7, -8, and -9. Although active MMP-7 was only observed in good outcome recipients, levels of TIMP-bound MMP-8 and -9 were higher in BOS. By enabling profiling of active and TIMP-bound MMPs, our novel method may open opportunities for the screening of early predictors for BOS.

MicroPET Evaluation of a Hydroxamate-Based MMP Inhibitor, [(18)F]FB-ML5, in a Mouse Model of Cigarette Smoke-Induced Acute Airway Inflammation.

Matrix metalloproteinases (MMPs) are the main proteolytic enzymes involved in the pathogenesis of chronic obstructive pulmonary disease (COPD). A radiolabeled MMP inhibitor, [(18)F]FB-ML5, was prepared, and its in vivo kinetics were tested in a mouse model of pulmonary inflammation. BALB/c mice were exposed for 4 days to cigarette smoke (CS) or air. On the fifth day, a dynamic microPET scan was made with [(18)F]FB-ML5. Standardized uptake values (PET-SUVmean) were 0.19 ± 0.06 in the lungs of CS-exposed mice (n = 6) compared to 0.11 ± 0.03 (n = 5) in air-exposed controls (p < 0.05), 90 min post-injection MMP-9 levels in bronchoalveolar lavage fluid (BALF) were increased from undetectable level to 4615 ± 1963 pg/ml by CS exposure. Increased MMP expression in a COPD mouse model was shown to lead to increased retention of [(18)F]FB-ML5.

Staphylococcal superantigen-like 10 inhibits CXCL12-induced human tumor cell migration.

PURPOSE: Tumor cell migration and metastasis share many similarities with leukocyte trafficking, which is critically regulated by chemokines and their receptors. CXCR4 is the most widely expressed chemokine receptor in many different types of cancer and has been linked to tumor dissemination and poor prognosis. Several CXCR4 antagonists have been synthesized. A totally novel approach to discover chemokine receptor antagonists is the use of bacteria. Bacteria produce chemokine receptor inhibitors to prevent neutrophil extravasation and migration toward the infection site to escape clearance by innate immune cells. The aim of the current study was to find and identify the mechanism of a bacterial protein that specifically targets CXCR4, a chemokine receptor shared by neutrophils and cancer cells. EXPERIMENTAL DESIGN: Several staphylococcal proteins were screened for their capacity to prevent binding of a function-blocking antibody against CXCR4. RESULTS: Staphylococcal superantigen-like 10 was found to bind CXCR4 expressed on human T acute lymphoblastic leukemia, lymphoma, and cervical carcinoma cell lines. It potently inhibited CXCL12-induced calcium mobilization and cell migration. CONCLUSIONS: Staphylococcal superantigen-like 10 is a potential lead in the development of new anticancer compounds preventing metastasis by targeting CXCR4.