Dose-dependent effects of the once-daily GLP-1 receptor agonist lixisenatide in patients with Type 2 diabetes inadequately controlled with metformin:a randomized, double-blind, placebo-controlled trial

To evaluate the dose-response relationship of lixisenatide (AVE0010), a glucagon-like peptide-1 (GLP-1) receptor agonist, in metformin-treated patients with Type 2 diabetes.

Rationale and Means to Target Pro-Inflammatory Interleukin-8 (CXCL8) Signaling in Cancer

It is well established that chronic inflammation underpins the development of a number of human cancers, with pro-inflammatory signaling within the tumor microenvironment contributing to tumor progression and metastasis. CXCL8 is an ELR+ pro-inflammatory CXC-chemokine which mediates its effects via signaling through two G protein-coupled receptors, CXCR1 and CXCR2. Elevated CXCL8-CXCR1/2 signaling within the tumor microenvironment of numerous cancers is known to enhance tumor progression via activation of signaling pathways promoting proliferation, angiogenesis, migration, invasion and cell survival. This review provides an overview of established roles of CXCL8-CXCR1/2 signaling in cancer and subsequently, discusses the possible strategies of targeting CXCL8-CXCR1/2 signaling in cancer, covering indirect strategies (e.g., anti-inflammatories, NFκB inhibitors) and direct CXCL8 or CXCR1/2 inhibition (e.g., neutralizing antibodies, small molecule receptor antagonists, pepducin inhibitors and siRNA strategies). Reports of pre-clinical cancer studies and clinical trials using CXCL8-CXCR1/2-targeting strategies for the treatment of inflammatory diseases will be discussed. The future translational opportunities for use of such agents in oncology will be discussed, with emphasis on exploitation in stratified populations.

Characterization of protein C receptor expression in monocytes

Many sequelae associated with endotoxaemic-induced shock result from excessive production of the cytokine mediators, tumour necrosis factor alpha (TNF-alpha), interleukin 1 (IL-1) and IL-6 from lipopolysaccharide (LPS)-activated monocytes. Protein C (PC)/activated protein C (APC) has potent cytokine-modifying properties and is protective in animal models and human clinical trials of sepsis. The precise mechanism by which this anti-inflammatory response is achieved remains unknown; however, the recently described endothelial protein C receptor (EPCR) appears to be essential for this function. The pivotal role that monocytes play in the pathophysiology of septic shock led us to investigate the possible expression of a protein C receptor on the monocyte membrane. We used similarity algorithms to screen human sequence databases for paralogues of the EPCR but found none. However, using reverse transcription-polymerase chain reaction (RT-PCR), we detected an mRNA transcribed in primary human monocytes and THP1 cells that was identical to human EPCR mRNA. We also used immunocytochemical analysis to demonstrate the expression of a protein C receptor on the surface of monocytes encoded by the same gene as EPCR. These results confirm a new member of the protein C pathway involving primary monocytes. Further characterization will be necessary to compare and contrast its biological properties with those of EPCR.

Interleukin-8 and its receptor CXCR2 in the tumour microenvironment promote colon cancer growth, progression and metastasis

BACKGROUND: Colorectal cancer (CRC) is a leading cause of death in the United States. Increased level of interleukin-8 (IL-8) and CXCR2 on tumours and in the tumour microenvironment has been associated with CRC growth, progression and recurrence in patients. Here, we aimed to evaluate the effects of tissue microenvironment-encoded IL-8 and CXCR2 on colon cancer progression and metastasis.

METHODS: A novel immunodeficient, skin-specific IL-8-expressing transgenic model was generated to evaluate colon cancer growth and metastasis. Syngeneic mouse colon cancer cells were grafted in CXCR2 knockout (KO) mice to study the contribution of CXCR2 in the microenvironment to cancer growth.

RESULTS: Elevated levels of IL-8 in the serum and tumour microenvironment profoundly enhanced the growth of human and mouse colon cancer cells with increased peri-tumoural angiogenesis, and also promoted the extravasation of the cancer cells into the lung and liver. The tumour growth was inhibited in CXCR2 KO mice with significantly reduced tumour angiogenesis and increased tumour necrosis.

CONCLUSION: Increased expression of IL-8 in the tumour microenvironment enhanced colon cancer growth and metastasis. Moreover, the absence of its receptor CXCR2 in the tumour microenvironment prevented colon cancer cell growth. Together, our study demonstrates the critical roles of the tumour microenvironment-encoded IL-8/CXCR2 in colon cancer pathogenesis, validating the pathway as an important therapeutic target.

Cathepsin S Cleavage of Protease-Activated Receptor-2 on Endothelial Cells Promotes Microvascular Diabetes Complications

Endothelial dysfunction is a central pathomechanism in diabetes-associated complications. We hypothesized a pathogenic role in this dysfunction of cathepsin S (Cat-S), a cysteine protease that degrades elastic fibers and activates the protease-activated receptor-2 (PAR2) on endothelial cells. We found that injection of mice with recombinant Cat-S induced albuminuria and glomerular endothelial cell injury in a PAR2-dependent manner. In vivo microscopy confirmed a role for intrinsic Cat-S/PAR2 in ischemia-induced microvascular permeability. In vitro transcriptome analysis and experiments using siRNA or specific Cat-S and PAR2 antagonists revealed that Cat-S specifically impaired the integrity and barrier function of glomerular endothelial cells selectively through PAR2. In human and mouse type 2 diabetic nephropathy, only CD68(+) intrarenal monocytes expressed Cat-S mRNA, whereas Cat-S protein was present along endothelial cells and inside proximal tubular epithelial cells also. In contrast, the cysteine protease inhibitor cystatin C was expressed only in tubules. Delayed treatment of type 2 diabetic db/db mice with Cat-S or PAR2 inhibitors attenuated albuminuria and glomerulosclerosis (indicators of diabetic nephropathy) and attenuated albumin leakage into the retina and other structural markers of diabetic retinopathy. These data identify Cat-S as a monocyte/macrophage-derived circulating PAR2 agonist and mediator of endothelial dysfunction-related microvascular diabetes complications. Thus, Cat-S or PAR2 inhibition might be a novel strategy to prevent microvascular disease in diabetes and other diseases.

Endothelium-derived intermedin/adrenomedullin-2 protects human ventricular cardiomyocytes from ischaemia-reoxygenation injury predominantly via the AM1 receptor

Application of intermedin/adrenomedullin-2 (IMD/AM-2) protects cultured human cardiac vascular cells and fibroblasts from oxidative stress and simulated ischaemia-reoxygenation injury (I-R), predominantly via adrenomedullin AM1 receptor involvement; similar protection had not been investigated previously in human cardiomyocytes (HCM). Expression of IMD, AM and their receptor components was studied in HCM. Receptor subtype involvement in protection by exogenous IMD against injury by simulated I-R was investigated using receptor component-specific siRNAs. Direct protection by endogenous IMD against HCM injury, both as an autocrine factor produced in HCM themselves and as a paracrine factor released from HCMEC co-cultured with HCM, was investigated using peptide-specific siRNA for IMD. IMD, AM and their receptor components (CLR, RAMPs1-3) were expressed in HCM. IMD 1 nmol L−1, applied either throughout ischaemia (3 h) and re-oxygenation (1 h) or during re-oxygenation (1 h) alone, attenuated HCM injury (P < 0.05); cell viabilities were 59% and 61% respectively vs. 39% in absence of IMD. Cytoskeletal disruption, protein carbonyl formation and caspase activity followed similar patterns. Pre-treatment (4 days) of HCM with CLR and RAMP2 siRNAs attenuated (P < 0.05) protection by exogenous IMD. Pre-treatment of HCMEC with IMD (and AM) siRNA augmented (P < 0.05) I-R injury: cell viabilities were 22% (and 32%) vs. 39% untreated HCMEC. Pre-treatment of HCM with IMD (and AM) siRNA did not augment HCM injury: cell viabilities were 37% (and 39%) vs. 39% untreated HCM. Co-culture with HCMEC conferred protection from injury on HCM; such protection was attenuated when HCMEC were pre-treated with IMD (but not AM) siRNA before co-culture. Although IMD is present in HCM, IMD derived from HCMEC and acting in a paracrine manner, predominantly via AM1 receptors, makes a marked contribution to cardiomyocyte protection by the endogenous peptide against acute I-R injury.

Protease activated receptor 2 expression and functionality in caries

Introduction: Protease activated receptors (PARs) are G-protein-coupled transmembrane receptors that are expressed on many cell types and implicated in various inflammatory processes in vivo. The induction of PAR2 as a result of the inflammatory response associated with dental caries remains to be determined. Objectives: The aim was to localise the expression of PAR2 in human dental pulp from carious teeth and to confirm receptor functionality using an in vitro assay. Methods: Dental pulp sections from decalcified carious teeth were examined by immunocytochemsitry. Membrane preparations from cultured pulp fibroblasts were subject to SDS-PAGE and immunoblotting to confirm fibroblast-associated immunoreactivity. The functionality of PAR2 on dental pulp fibroblasts was studied using calcium imaging in the presence of several potential activators including a PAR2 agonist (PAR2-AP), trypsin and pulpal enzymes from a carious tooth. Results: Immunocytochemistry revealed intense PAR2 immunoreactivity on pulpal fibroblasts subjacent to carious lesions but not in surrounding regions of the dental pulp. Pulp specimens from a dental injury model showed no expression of PAR2, suggesting its expression was related to cellular changes associated with ongoing caries. The localisation of PAR2 staining to pulpal fibroblasts in carious teeth was confirmed by Western blotting which revealed PAR2 immunoreactive bands in membrane fractions prepared from pulp fibroblasts. In functional studies, challenge of cultured pupal fibroblasts with PAR2-AP, trypsin and an extract of proteolytic enzymes from a carious dental pulp, showed specific activation of PAR2. Conclusions: This work demonstrates that PAR2 is functional and inducible in human dental pulp fibroblasts in response to caries and that endogenous pulpal enzymes can activate PAR2.

Novel free fatty acid receptor 1 (GPR40) agonists based on 1,3,4-thiadiazole-2-carboxamide scaffold

Free fatty acid receptor 1 (FFA1), previously known as GPR40 is a G protein-coupled receptor and a new target for treatment of type 2 diabetes. Two series of FFA1 agonists utilizing a 1,3,4-thiadiazole-2-caboxamide scaffold were synthetized. Both series offered significant improvement of the potency compared to the previously described 1,3,4-thiadiazole-based FFA1 agonists and high selectivity for FFA1. Molecular docking predicts new aromatic interactions with the receptor that improve agonist potency. The most potent compounds from both series were profiled for in vitro ADME properties (plasma and metabolic stability, LogD, plasma protein binding, hERG binding and CYP inhibition). One series suffered very rapid degradation in plasma and in presence of mouse liver microsomes. However, the other series delivered a lead compound that displayed a reasonable ADME profile together with the improved FFA1 potency.

Design and standardization of PCR primers and protocols for detection of clonal immunoglobulin and T-cell receptor gene recombinations in suspect lymphoproliferations: report of the BIOMED-2 Concerted Action BMH4-CT98-3936.

In a European BIOMED-2 collaborative study, multiplex PCR assays have successfully been developed and standardized for the detection of clonally rearranged immunoglobulin (Ig) and T-cell receptor (TCR) genes and the chromosome aberrations t(11;14) and t(14;18). This has resulted in 107 different primers in only 18 multiplex PCR tubes: three VH-JH, two DH-JH, two Ig kappa (IGK), one Ig lambda (IGL), three TCR beta (TCRB), two TCR gamma (TCRG), one TCR delta (TCRD), three BCL1-Ig heavy chain (IGH), and one BCL2-IGH. The PCR products of Ig/TCR genes can be analyzed for clonality assessment by heteroduplex analysis or GeneScanning. The detection rate of clonal rearrangements using the BIOMED-2 primer sets is unprecedentedly high. This is mainly based on the complementarity of the various BIOMED-2 tubes. In particular, combined application of IGH (VH-JH and DH-JH) and IGK tubes can detect virtually all clonal B-cell proliferations, even in B-cell malignancies with high levels of somatic mutations. The contribution of IGL gene rearrangements seems limited. Combined usage of the TCRB and TCRG tubes detects virtually all clonal T-cell populations, whereas the TCRD tube has added value in case of TCRgammadelta(+) T-cell proliferations. The BIOMED-2 multiplex tubes can now be used for diagnostic clonality studies as well as for the identification of PCR targets suitable for the detection of minimal residual disease.