Quantitation of fibroblast activation protein (FAP)-specific protease activity in mouse, baboon and human fluids and organs.

The protease fibroblast activation protein (FAP) is a specific marker of activated mesenchymal cells in tumour stroma and fibrotic liver. A specific, reliable FAP enzyme assay has been lacking. FAP's unique and restricted cleavage of the post proline bond was exploited to generate a new specific substrate to quantify FAP enzyme activity. This sensitive assay detected no FAP activity in any tissue or fluid of FAP gene knockout mice, thus confirming assay specificity. Circulating FAP activity was ∼20- and 1.3-fold less in baboon than in mouse and human plasma, respectively. Serum and plasma contained comparable FAP activity. In mice, the highest levels of FAP activity were in uterus, pancreas, submaxillary gland and skin, whereas the lowest levels were in brain, prostate, leukocytes and testis. Baboon organs high in FAP activity included skin, epididymis, bladder, colon, adipose tissue, nerve and tongue. FAP activity was greatly elevated in tumours and associated lymph nodes and in fungal-infected skin of unhealthy baboons. FAP activity was 14- to 18-fold greater in cirrhotic than in non-diseased human liver, and circulating FAP activity was almost doubled in alcoholic cirrhosis. Parallel DPP4 measurements concorded with the literature, except for the novel finding of high DPP4 activity in bile. The new FAP enzyme assay is the first to be thoroughly characterised and shows that FAP activity is measurable in most organs and at high levels in some. This new assay is a robust tool for specific quantitation of FAP enzyme activity in both preclinical and clinical samples, particularly liver fibrosis.

Characterization of selective Calcium-Release Activated Calcium channel blockers in mast cells and T-cells from human, rat, mouse and guinea-pig preparations.

Loss of function mutations in the two key proteins which constitute Calcium-Release Activated Calcium (CRAC) channels demonstrate the critical role of this ion channel in immune cell function. The aim of this study was to demonstrate that inhibition of immune cell activation could be achieved with highly selective inhibitors of CRAC channels in vitro using cell preparations from human, rat, mouse and guinea-pig. Two selective small molecule blockers of CRAC channels; GSK-5498A and GSK-7975A were tested to demonstrate their ability to inhibit mediator release from mast cells, and pro-inflammatory cytokine release from T-cells in a variety of species. Both GSK-5498A and GSK-7975A completely inhibited calcium influx through CRAC channels. This led to inhibition of the release of mast cell mediators and T-cell cytokines from multiple human and rat preparations. Mast cells from guinea-pig and mouse preparations were not inhibited by GSK-5498A or GSK-7975A; however cytokine release was fully blocked from T-cells in a mouse preparation. GSK-5498A and GSK-7975A confirm the critical role of CRAC channels in human mast cell and T-cell function, and that inhibition can be achieved in vitro. The rat displays a similar pharmacology to human, promoting this species for future in vivo research with this series of molecules. Together these observations provide a critical forward step in the identification of CRAC blockers suitable for clinical development in the treatment of inflammatory disorders.

Radiation dosimetry of the translocator protein ligands [18F]PBR111 and [18F]PBR102.

INTRODUCTION: The translocator protein (TSPO) ligands [18F]PBR111 and [18F]PBR102 show promise for imaging neuroinflammation. Our aim was to estimate the radiation dose to humans from primate positron emission tomography (PET) studies using these ligands and compare the results with those obtained from studies in rodents. METHODS: [18F]PBR111 and [18F]PBR102 PET-computed tomography studies were carried out in baboons. The cumulated activity in the selected source organs was obtained from the volume of interest time-activity curves drawn on coronal PET slices and adjusted for organ mass relative to humans. Radiation dose estimates were calculated in OLINDA/EXM Version 1.1 from baboon studies and compared with those calculated from Sprague-Dawley rat tissue concentration studies, also adjusted for relative organ mass. RESULTS: In baboons, both ligands cleared rapidly from brain, lung, kidney and spleen and more slowly from liver and heart. For [18F]PBR111, the renal excretion fraction was 6.5% and 17% for hepatobiliary excretion; for [18F]PBR102, the renal excretion was 3.0% and 15% for hepatobiliary excretion. The estimated effective dose in humans from baboon data was 0.021 mSv/MBq for each ligand, whilst from rat data, the estimates were 0.029 for [18F]PBR111 and 0.041 mSv/MBq for [18F]PBR102. CONCLUSION: Biodistribution in a nonhuman primate model is better suited than the rat model for the calculation of dosimetry parameters when translating these ligands from preclinical to human clinical studies. Effective dose calculated from rat data was overestimated compared to nonhuman primate data. The effective dose coefficient for both these TSPO ligands determined from PET studies in baboons is similar to that for [18F]FDG.

Tumor necrosis factor α induces a model of preeclampsia in pregnant baboons (Papio hamadryas).

Preeclampsia is a common disease of pregnancy characterised by maternal hypertension and proteinuria. Abnormal placentation in early pregnancy and abnormal cytokine and anti-angiogenic factor expression are thought to contribute to the clinical syndrome of endothelial dysfunction evident in the second half of gestation. The mechanisms underlying both the placental pathology and its translation to the maternal clinical syndrome are not fully understood. A model of preeclampsia manifest by clinically evident endothelial dysfunction (increased blood pressure and proteinuria) was induced by administration of low-dose TNF-α for 2weeks at mid-gestation in pregnant baboons (Papio hamadryas). Blood pressure was monitored continuously and remotely by intra-arterial radiotelemetry. Following TNF-α infusion, there was an increase in systolic and diastolic blood pressure and development of proteinuria in pregnant treated animals, but not in pregnant saline controls nor in non-pregnant TNF-α treated animals. The treated pregnant animals also developed elevated plasma soluble FMS-like tyrosine kinase-1 (sFLT-1) and increased placental mRNA expression of sFLT-1 and soluble endoglin (sEng). These results clearly demonstrate that the cytokine TNF-α can induce the clinical and biochemical features of human preeclampsia. The results identify a link between cytokines, placental dysfunction and endothelial dysfunction resulting in a loss of maternal blood pressure control.

The effects of diabetes and aminoguanidine treatment on endothelial function in a primate model of type 1 diabetes.

Abnormalities of endothelial function have been demonstrated in diabetes and are thought to play a role in the pathogenesis of diabetic complications. The aims of this study were to determine whether aminoguanidine, an inhibitor of glycation, can prevent endothelial and microcirculation abnormalities in a primate model of type 1 diabetes. Male baboons (Papio hamadryas) were assigned to one of the four groups: control, diabetes, control treated with aminoguanidine or diabetes treated with aminoguanidine. Diabetes was induced by streptozocin (60 mg/kg) and treated with once daily injection of insulin. Aminoguanidine was given subcutaneously (10 mg/kg), once a day. Diabetic animals had a mean duration of diabetes of 8.9 +/- 3.4 years and HbA1c of 8.9 +/- 1.1%. Microvascular function was measured by laser Doppler velocimetry, with examination of endothelium-dependent increase in skin blood flow (SkBF) following iontophoresis of acetylcholine (ACh) and endothelium-independent increase in SkBF in response to the nitric oxide (NO) donor sodium nitroprusside (SNP). Multiple regression analysis identified diabetes (P = 0.049) and aminioguanidine treatment (P = 0.026) as significant determinants of ACh response. The diabetic baboons treated with aminoguanidine had less Ach-mediated SkBF response compared with controls (1.39 +/- 0.32 vs. 2.26 +/- 0.61, F = 3.3, P = 0.04), but there was no difference between groups in SkBF response to SNP. We conclude that endothelial dysfunction can be demonstrated in this primate model of type 1 diabetes at a stage when overt diabetic complications are not present. This occurred in the absence of insulin resistance or significant hypercholesterolemia. Administration of aminoguanidine from the onset of diabetes was not able to prevent this abnormality and in fact aggravated the endothelial response. Effects of aminoguanidine on NO synthase may contribute to this phenomenon.

Renal connective tissue growth factor correlates with glomerular basement membrane thickness and prospective albuminuria in a non-human primate model of diabetes: possible predictive marker for incipient diabetic nephropathy.

UNLABELLED: Diabetic renal disease is characterized by accumulation of extracellular matrix, glomerulosclerosis, and tubulointerstitial fibrosis. Connective tissue growth factor (CTGF) is implicated in these changes, as it contributes to new matrix synthesis and is increased in the diabetic kidney. CTGF also inhibits mesangial matrix degradation through up-regulation of the tissue inhibitor of matrix metalloproteinase 1 (TIMP-1). In a non-human primate model of diabetes, we determined whether the level of renal CTGF protein before development of albuminuria correlated with renal matrix and TIMP-1 changes and whether renal CTGF predicts progression to albuminuria. METHODS: In a group of diabetic (n=9) and control (n=6) baboons after a 5-year duration of diabetes, renal tissue CTGF and TIMP-1 were detected by immunohistochemistry and compared with glomerular basement membrane (GBM) thickness and mesangial volume measurements from electron photomicrographs of renal biopsies. Urinary albumin levels were measured at 5 and 10 years of diabetes. RESULTS: GBM thickness, CTGF protein, and TIMP-1 protein were increased after 5 years of diabetes (each P<.05). Tubular fibronectin scores correlated with tubular CTGF scores (r=0.72, P=.002). In diabetic animals, GBM thickness correlated with tubular and total CTGF levels (P=.002 and P=.04, respectively), whereas mesangial cell and total matrix volume correlated with glomerular TIMP-1 (P=.02 and P=.01, respectively). Tubular CTGF scores (P=.008) and GBM thickness (P=.03) at 5 years in diabetes each predicted the degree of albuminuria at 10 years. CONCLUSIONS: These results suggest that early increases in renal CTGF protein contribute to incipient diabetic nephropathy and that renal CTGF may have utility as an early marker for progression to dysfunction in the diabetic kidney.

Maternal parity affects neonatal survival rate in a colony of captive bred baboons (Papio hamadryas).

BACKGROUND: Non-human primates, particularly baboons, are valuable as animal models for reproductive research, because of their similarity to humans. Knowledge of colony-specific pregnancy and neonatal outcomes is essential for interpretation of such research. METHODS: A retrospective review of the reproductive records of the Australian National Baboon Colony (ANBC) from 1994 to 2006 was performed. RESULTS: The overall live birth rate was over 70% of recognized pregnancies. Pregnancy loss was due to equal proportions of spontaneous abortion and stillbirth, and was not affected by maternal age or parity. Stillbirth rates were increased by the use of animals in novel late pregnancy experimental protocols. Neonatal mortality rates were low overall, but significantly higher in primiparous compared with multiparous mothers (P < 0.05). There were no cases of maternal mortality. CONCLUSIONS: The success of the ANBC breeding programme is demonstrated by the low rate of pregnancy loss, high neonatal survival rate and lack of maternal mortality.

Growth factors in diabetic complications.

Diabetes mellitus is characterized by a lack of insulin, causing elevated blood glucose, often with associated insulin resistance. Over time, especially in genetically susceptible individuals, such chronic hyperglycemia can cause tissue injury. Dysregulation of growth factors in diabetes occurs through biochemical and hemodynamic pathways. In some tissues affected by diabetes, growth factors are induced to an excess, while in other sites a relative deficit of growth factors occurs. There is evidence that these growth factor changes contribute to the tissue pathology in diabetes, whether it be fibrosis, persistent inflammation or a combination of the two. This review focuses on the role of growth factors in diabetic nephropathy and wound healing in diabetes. Growth factor therapy in diabetic foot ulcers is already a clinical reality. As methods to finely regulate growth factors in a tissue- and time-specific manner are further developed and tested, downregulation of growth factors in vivo may well become a therapy to prevent and treat diabetic nephropathy.

Synthesis and in vivo evaluation of a novel peripheral benzodiazepine receptor PET radioligand.

The novel pyrazolopyrimidine ligand, N,N-diethyl-2-[2-(4-methoxyphenyl)-5,7-dimethyl-pyrazolo[1,5-a]pyrimidin-3-yl]-acetamide 1 (DPA-713), has been reported as a potent ligand for the peripheral benzodiazepine receptor (PBR) displaying an affinity of K(i)=4.7 nM. In this study, 1 was successfully synthesised and demethylated to form the phenolic derivative 6 as precursor for labelling with carbon-11 (t(1/2) = 20.4 min). [11C]1 was prepared by O-alkylation of 6 with [11C]methyl iodide. The radiochemical yield of [(11)C]1 was 9% (non-decay corrected) with a specific activity of 36 GBq/micromol at the end of synthesis. The average time of synthesis including formulation was 13.2 min with a radiochemical purity >98%. In vivo assessment of [11C]1 was performed in a healthy Papio hamadryas baboon using positron emission tomography (PET). Following iv administration of [11C]1, significant accumulation was observed in the baboon brain and peripheral organs. In the brain, the radioactivity peaked at 20 min and remained constant for the duration of the imaging experiment. Pre-treatment with the PBR-specific ligand, PK 11195 (5 mg/kg), effectively reduced the binding of [11C]1 at 60 min by 70% in the whole brain, whereas pre-treatment with the central benzodiazepine receptor ligand, flumazenil (1mg/kg), had no inhibitory effect on [11C]1 uptake. These results indicate that accumulation of [11C]1 in the baboon represents selective binding to the PBR. These exceptional in vivo binding properties suggest that [11C]1 may be useful for imaging the PBR in disease states. Furthermore, [11C]1 represents the first ligand of its pharmacological class to be labelled for PET studies and therefore has the potential to generate new information on the pathological role of the PBR in vivo.