Elevated expression of the adhesion GPCR ADGRL4/ELTD1 promotes endothelial sprouting angiogenesis without activating canonical GPCR signalling.

ADGRL4/ELTD1 is an orphan adhesion GPCR (aGPCR) expressed in endothelial cells that regulates tumour angiogenesis. The majority of aGPCRs are orphan receptors. The Stachel Hypothesis proposes a mechanism for aGPCR activation, in which aGPCRs contain a tethered agonist (termed Stachel) C-terminal to the GPCR-proteolytic site (GPS) cleavage point which, when exposed, initiates canonical GPCR signalling. This has been shown in a growing number of aGPCRs. We tested this hypothesis on ADGRL4/ELTD1 by designing full length (FL) and C-terminal fragment (CTF) ADGRL4/ELTD1 constructs, and a range of potential Stachel peptides. Constructs were transfected into HEK293T cells and HTRF FRET, luciferase-reporter and Alphascreen GPCR signalling assays were performed. A stable ADGRL4/ELTD1 overexpressing HUVEC line was additionally generated and angiogenesis assays, signalling assays and transcriptional profiling were performed. ADGRL4/ELTD1 has the lowest GC content in the aGPCR family and codon optimisation significantly increased its expression. FL and CTF ADGRL4/ELTD1 constructs, as well as Stachel peptides, did not activate canonical GPCR signalling. Furthermore, stable overexpression of ADGRL4/ELTD1 in HUVECs induced sprouting angiogenesis, lowered in vitro anastomoses, and decreased proliferation, without activating canonical GPCR signalling or MAPK/ERK, PI3K/AKT, JNK, JAK/HIF-1α, beta catenin or STAT3 pathways. Overexpression upregulated ANTXR1, SLC39A6, HBB, CHRNA, ELMOD1, JAG1 and downregulated DLL4, KIT, CCL15, CYP26B1. ADGRL4/ELTD1 specifically regulates the endothelial tip-cell phenotype through yet undefined signalling pathways.

Immunoblot analysis of sera in uncomplicated typhoid fever & with typhoid ileal perforation.

BACKGROUND & OBJECTIVE: Ileal perforation is a serious complication of typhoid fever. The exact reasons for the development of perforation in only a few of those infected with Salmonella Typhi is unknown, and it is likely that immunological factors are involved. Therefore we undertook this study to compare the antibody profile in patients with uncomplicated typhoid fever with those having ileal perforation by immunoblotting. METHODS: Two groups of patients were included in the study. Group II comprised patients with uncomplicated typhoid fever (n=47), and group I with typhoid ileal perforation (n=33). The flagellar (H), lipopolysaccharide (LPS) and outer membrane protein (OMP) antigens of Salmonella Typhi were extracted and used to test patient sera for antibodies by immunoblotting RESULTS: Immunoblotting using S. Typhi antigens enabled the detection of S. Typhi antibodies in the two groups of patients. A significant difference was seen in the response of these two groups of patients with respect to antibodies to flagella, lipopolysaccharide and outer membrane proteins. Antibodies to flagella were more pronounced among patients with uncomplicated typhoid fever, while anti-OMP antibodies were significantly associated with typhoid ileal perforation. INTERPRETATION & CONCLUSION: A comparison of antibodies in patients with uncomplicated typhoid fever and with ileal perforation revealed the differences in the antibody profiles of the two groups. Our study suggests that the difference in antibody response may in some way play a role in the pathogenesis of typhoid ileal perforation which can also potentially be exploited to develop suitable diagnostic tests.