Development and characterisation of a novel glucagon like peptide-1 receptor antibody.

AIMS/HYPOTHESIS: Glucagon like peptide-1 (GLP-1) enhances glucose-dependent insulin secretion by binding to GLP-1 receptors (GLP1Rs) on pancreatic beta cells. GLP-1 mimetics are used in the clinic for the treatment of type 2 diabetes, but despite their therapeutic success, several clinical effects of GLP-1 remain unexplained at a mechanistic level, particularly in extrapancreatic tissues. The aim of this study was to generate and characterise a monoclonal antagonistic antibody for the GLP1R for use in vivo. METHODS: A naive phage display selection strategy was used to isolate single-chain variable fragments (ScFvs) that bound to GLP1R. The ScFv with the highest affinity, Glp1R0017, was converted into a human IgG1 and characterised further. In vitro antagonistic activity was assessed in a number of assays: a cAMP-based homogenous time-resolved fluorescence assay in GLP1R-overexpressing cell lines, a live cell cAMP imaging assay and an insulin secretion assay in INS-1 832/3 cells. Glp1R0017 was further tested in immunostaining of mouse pancreas, and the ability of Glp1R0017 to block GLP1R in vivo was assessed by both IPGTT and OGTT in C57/Bl6 mice. RESULTS: Antibodies to GLP1R were selected from naive antibody phage display libraries. The monoclonal antibody Glp1R0017 antagonised mouse, human, rat, cynomolgus monkey and dog GLP1R. This antagonistic activity was specific to GLP1R; no antagonistic activity was found in cells overexpressing the glucose-dependent insulinotropic peptide receptor (GIPR), glucagon like peptide-2 receptor or glucagon receptor. GLP-1-stimulated cAMP and insulin secretion was attenuated in INS-1 832/3 cells by Glp1R0017 incubation. Immunostaining of mouse pancreas tissue with Glp1R0017 showed specific staining in the islets of Langerhans, which was absent in Glp1r knockout tissue. In vivo, Glp1R0017 reversed the glucose-lowering effect of liraglutide during IPGTTs, and reduced glucose tolerance by blocking endogenous GLP-1 action in OGTTs. CONCLUSIONS/INTERPRETATION: Glp1R0017 is a monoclonal antagonistic antibody to the GLP1R that binds to GLP1R on pancreatic beta cells and blocks the actions of GLP-1 in vivo. This antibody holds the potential to be used in investigating the physiological importance of GLP1R signalling in extrapancreatic tissues where cellular targets and signalling pathways activated by GLP-1 are poorly understood

An investigation into sex-differences into sex-differences in leukocyte mobilisation and recruitment in response to acute inflammation

Females are relatively protected from inflammatory diseases, particularly conditions that are characterised by excessive tissue infiltration of neutrophils (PMNs), such as ischaemia/reperfusion (I/R) injury. Therefore, understanding sex-differences is very important particularly for appropriate treatment of inflammatory disorders in men and women. Unfortunately, efforts to exploit sex-differences therapeutically have been unsuccessful since the precise mechanisms that confer the protective advantage in females over males are unclear. Many fundamental aspects ofthe nature of sex-differences have not been investigated, particularly in the regulation of PMN mobilisation from the bone marrow during acute inflammation. The aim ofthis thesis is to determine the nature and mechanism of leukocyte activation and recruitment in males and females, particularly in I/R. This thesis demonstrates for the first time that regulation of PMN mobilisation during acute inflammation is distinct in females. In comparison to males, females demonstrate reduced expression of mediators that cause the release of PMNs, including GCSF, CXCR2 and CXCLS, and increased expression of CXCR4 and CXCl12, which mediate PMN retention in the bone marrow. Reduced granulopoiesis, PMN mobilisation and recruitment into tissues in response to inflammogens protect females from collateral damage incurred by PMN-derived mediators that contribute to tissue injury and loss of function. This thesis has also revealed a novel, and possibly predominant, role for the ElR+ CXC chemokine CXClS in mobilisation of tissue-damaging PMNs from the bone marrow, whereby CXCLS production, PMN mobilisation and tissue infiltration was profoundly greater in males during acute inflammation. CXClS appears to stimulate PMN mobilisation by 1) upregulating CXCR2 iv / expression on bone marrow cells and simultaneously downregulating CXCR4 expression; 2) inducing its own expression; 3) stimulating GCSF expression; and 4) increasing PMN expression of J32 integrin. Thus, the thesis proposes that reduced CXCLS expression, and increased CXCR4/CXCL12 expression, in females during acute inflammation may be a novel mechanism underlying the protection against tissue injury. The fact that these sex- differences were apparent in different species (rat, mouse), tissues (mesenteric, lung, kidney, heart), in response to different stimuli (mesenteric, renal, myocardial I/R and carrageenan-induced pleurisy) and shown both in vivo and in vitro, suggests that this is a fundamental, and more generalised, phenomenon in males and females following acute inflammation. The inherent differences between the sexes have important clinical implications in that they demonstrate the need of considering sex-differences in research. This thesis demonstrates that sex-differences must be taken into account when developing such therapies for specific inflammatory diseases such as I/R injury as there are major differences in the temporal profile of chemokines and the extent of PMN infiltration.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Injectable Biodegradable Silica Depot: Two Months of Sustained Release of the Blood Glucose Lowering Peptide, Pramlintide

Diabetes mellitus is a major healthcare challenge. Pramlintide, a peptide analogue of the hormone amylin, is currently used as an adjunct with insulin for patients who fail to achieve glycemic control with only insulin therapy. However, hypoglycemia is the dominant risk factor associated with such approaches and careful dosing of both drugs is needed. To mitigate this risk factor and compliance issues related to multiple dosing of different drugs, sustained delivery of Pramlintide from silica depot administered subcutaneously (SC) was investigated in a rat model. The pramlintide-silica microparticle hydrogel depot was formulated by spray drying of silica sol-gels. In vitro dissolution tests revealed an initial burst of pramlintide followed by controlled release due to the dissolution of the silica matrix. At higher dosing, pramlintide released from subcutaneously administered silica depot in rats showed a steady concentration of 500 pM in serum for 60 days. Released pramlintide retained its pharmacological activity in vivo, as evidenced by loss of weight. The biodegradable silica matrix offers a sustained release of pramlintide for at least two months in the rat model and shows potential for clinical applications

Additional file 3: Figure S2. of Sex-specific regulation of chemokine Cxcl5/6 controls neutrophil recruitment and tissue injury in acute inflammatory states

No effect of sex or ischemia on hemodynamics during reperfusion. Male and female rats were subjected to 30-min mesenteric ischemia followed by up to 2-h reperfusion. (A) Mean arterial blood pressure (MABP) measured by cannulation of carotid artery. (B) Red blood cell (RBC) velocity and (C) wall shear rate, calculated from RBC velocity and venule diameter, as described in Supplementary Methods. Sham, n = 4 rats/group; I/R, n = 4 rats/group. Data are presented as mean ± sem. All comparisons P > 0.05 by two-way ANOVA. (TIFF 1174 kb

Additional file 2: Figure S1. of Sex-specific regulation of chemokine Cxcl5/6 controls neutrophil recruitment and tissue injury in acute inflammatory states

Distinct temporal regulation of neutrophil recruitment increases tissue I/R injury in males. Male and female rats were subjected to 30-min mesenteric ischemia followed by up to 2-h reperfusion. (A) Representative images of portions of male and female small intestine at the end of reperfusion, demonstrating redness and edema in males following I/R but no observable change in females. (B-C) Leukocyte/vessel wall interactions throughout reperfusion in mesenteric venules, measured by intravital microscopy: (B) Leukocyte adhesion and (C) number of tissue leukocytes at different distances away from venule (sham, n = 5 rats/group; I/R, n = 8 rats/group). (D) Circulating CD68+ monocytes in males and females during ischemia and reperfusion. Data are presented as mean ± sem. §P < 0.001 by two-way ANOVA and ‡P < 0.05 or #P < 0.001 by Bonferroni’s post-test. ns denotes P > 0.05 by one-way ANOVA. (PDF 811 kb

Additional file 1:Tables S1 and S2. of Sex-specific regulation of chemokine Cxcl5/6 controls neutrophil recruitment and tissue injury in acute inflammatory states

Table S1. Details of antibodies used for flow cytometry. Table S2. Sequence of primers used for real-time quantitative PCR with SYBR green. (PDF 96 kb

Additional file 4: Figure S3. of Sex-specific regulation of chemokine Cxcl5/6 controls neutrophil recruitment and tissue injury in acute inflammatory states

Increased induction of neutrophil integrins in males. Male and female rats were subjected to 30-min mesenteric ischemia followed by 2-h reperfusion. Surface expression of integrins β1, α4, αL, and αM on RP1+ neutrophils in BM and circulation of male and female rats, measured at 2-h reperfusion. Sham, n = 3 rats/group; I/R, n = 5 rats/group. Data are presented as mean ± sem. *P < 0.05, **P < 0.01 by one-way ANOVA followed by Bonferroni’s post-test. (TIFF 540 kb

Additional file 6: Figure S5. of Sex-specific regulation of chemokine Cxcl5/6 controls neutrophil recruitment and tissue injury in acute inflammatory states

Cxcl5-induced leukocyte/vessel wall interactions. Male rats were treated with 3 μg/kg Cxcl5 (ip, n = 5 rats) or PBS (n = 3 rats) for 2 h. (A) Leukocyte flux, (B) adherent leukocytes, (C) emigrated leukocytes in mesenteric tissues at 2 h. Data are presented as mean ± sem. *P < 0.05,**P < 0.01, ***P < 0.001 compared to PBS by Student’s t test. (TIFF 448 kb