Isolation of human capillary endothelial cells from abdominal adipose tissue.

INTRODUCTION: This protocol outlines a technique to isolate human capillary endothelial cells from human abdominal wall adipose tissue. The relative lack of cell-type complexity within fat tissue makes it an attractive source of endothelial cells. In addition, collaboration with a local plastic surgeon can yield significant quantities of subcutaneous adipose tissue from liposuction without the need to be on call for cadaveric or post-surgical samples.

Isolation of human capillary endothelial cells using paramagnetic beads conjugated to anti-PECAM antibodies.

INTRODUCTION: The protocol described below outlines a technique to isolate and maintain pure populations of human capillary endothelial cells using an active selection technique that targets the platelet endothelial cell adhesion molecule (PECAM or CD31). Human abdominal wall adipose tissue is used as the source for capillary endothelial cells, which are partially purified before selection.

A TPO receptor agonist, ALXN4100TPO, mitigates radiation-induced lethality and stimulates hematopoiesis in CD2F1 mice.

Thrombopoietin (TPO) receptor agonists lacking sequence homology to TPO were designed by grafting a known peptide sequence into the hinge and/or kappa constant regions of a human anti-anthrax antibody. Some of these proteins were equipotent to TPO in stimulating cMpl-r activity in vitro and in increasing platelet levels in vivo. ALXN4100TPO (4100TPO), the best agonist in this series with a K(d) of 30 nM for cMpl-r, exhibited potent activity as a radiation countermeasure in CD2F1 mice exposed to lethal total-body radiation from a cobalt-60 γ-ray source. 4100TPO (2 mg/kg, s.c.) administered once either 24 h before or 6 h after TBI showed superior protection to five daily doses given before or after TBI. Prophylactic administration (69 to 94% survival) was superior to therapeutic schedules (60% survival). 4100TPO conferred a significant survival benefit (P < 0.01) when administered 4 days before or even 12 h after exposure and across a dose range of 0.1 to 8 mg/kg. The dose reduction factors (DRFs) with a single dose of 1 mg/kg 4100TPO 24 h before or 12 h after TBI were 1.32 and 1.11, respectively (P < 0.0001). Furthermore, 4100TPO increased bone marrow cellularity and megakaryocytic development and accelerated multi-lineage hematopoietic recovery in irradiated mice, demonstrating the potential of 4100TPO as both a protector and a mitigator in the event of a radiological incident.

A rationally designed agonist antibody fragment that functionally mimics thrombopoietin.

By using rational design, antibody fragments (Fabs) that mimic thrombopoietin (TPO) were created. A peptide with cMpl receptor-binding capability was grafted into different complementarity-determining regions of a fully human Fab scaffold. Functional presentation of the peptide was optimized by using phage display and cell-based panning. Select antibodies and fragments containing two grafted peptides were assayed for their ability to stimulate the cMpl receptor in vitro. Several candidates demonstrated agonist activity in an in vitro cMpl receptor signaling reporter assay, including Fab59, which was estimated to be equipotent to TPO. Fab59 additionally was able to effectively stimulate platelet production in normal mice. These rationally designed mimetic Fabs may provide a therapeutic intervention for thrombocytopenia while avoiding the potential generation of neutralizing antibodies to endogenous TPO. Furthermore, this study demonstrates a method by which short-lived linear peptides with binding activity may be converted to more stable and potent agonists capable of activating cell surface receptors.