Antibody combination therapy targeting CD25, CD70 and CD8 reduces islet inflammation and improves glycaemia in diabetic mice.

Destruction of pancreatic islets in type 1 diabetes is caused by infiltrating, primed and activated T cells. In a clinical setting this autoimmune process is already in an advanced stage before intervention therapy can be administered. Therefore, an effective intervention needs to reduce islet inflammation and preserve any remaining islet function. In this study we have investigated the role of targeting activated T cells in reversing autoimmune diabetes. A combination therapy consisting of CD25-, CD70- and CD8-specific monoclonal antibodies was administered to non-obese diabetic (NOD) mice with either new-onset diabetes or with advanced diabetes. In NOD mice with new-onset diabetes antibody combination treatment reversed hyperglycaemia and achieved long-term protection from diabetes (blood glucose <13·9 mmol/l) in >50% of mice. In contrast, in the control, untreated group blood glucose levels continued to increase and none of the mice were protected from diabetes (P < 0·0001). Starting therapy early when hyperglycaemia was relatively mild proved critical, as the mice with advanced diabetes showed less efficient control of blood glucose and shorter life span. Histological analysis (insulitis score) showed islet preservation and reduced immune infiltration in all treated groups, compared to their controls. In conclusion, antibody combination therapy that targets CD25, CD70 and CD8 results in decreased islet infiltration and improved blood glucose levels in NOD mice with established diabetes.

Surface and bulk effects on platelet adhesion and aggregation during simple (laminar) shear flow of whole blood.

This study attempts to clarify the role of the artificial surface and the fluid bulk on platelet adhesion and aggregation events during simple shear flow of whole blood. The experimental approach involved the shearing of fresh whole blood samples over the shear rate range of 720-5680 s-1, which corresponded to a shear stress maximum of about 150 dyn cm-2. Results on platelet adhesion, measured as surface coverage by platelets, and platelet aggregation, measured in terms of reduction in platelet count and adenosine diphosphate (ADP) release, were determined as a function of the surface to volume ratio (S/V); and artificial surface used. Both shear-induced platelet adhesion and platelet count reduction showed significant variation over the range of S/V employed. The ratios between the three different S/V values used in this system (10:6:4) were about the same as the ratio of the shear rate-averaged results obtained. Also, for shear-induced hemolysis, an increase in the release of hemoglobin from red blood cells was found as S/V was increased, again with ratios between the shear rate-averaged values similar to the ratio of S/V values employed. The shear-induced release of ADP, presumably from platelets and from red blood cells indicated a different dependence of ADP release on S/V than was observed for the other parameters reported. Irreversible platelet aggregation was expected to occur because the amount of ADP that was released as a result of the shear was substantial. Models proposed to explain the experimental results were found to support a surface-controlled mechanism.

Effect of hirudin on platelet deposition to an artificial surface during low-stress shear flow of whole blood.

The effect of hirudin, a known inhibitor of thrombin, was evaluated for whole blood samples in terms of platelet deposition/adhesion to a non-biological test surface (tetrafluoroethylene-propylene copolymer), adenosine diphosphate (ADP) release and reduction in platelet count during laminar shear flow for a shear rate to 5680 s-1 (corresponding to a shear stress of about 150 dynes/cm2). Experiments were done in a cone-and-plate viscometer for samples of whole blood with and without the addition of hirudin. Whole blood samples containing hirudin showed about a 50% reduction in platelet surface coverage compared with blood samples not containing hirudin. Results on low-stress, shear-induced release of ADP showed that for shear rates of 2860 s-1 and above there was an increase in ADP release for the blood samples not containing hirudin compared with the hirudin-treated samples. However, no differences in haemoglobin leakage from red blood cells as well as residual platelet count following shear were observed between both types of blood samples.

The effects of shear stress on endothelial cell retention and function on expanded polytetrafluoroethylene.

We evaluated the adherence of indium 111-radiolabeled endothelial cells to fibronectin-treated expanded polytetrafluoroethylene surfaces exposed to high (437 s-1) vs low (218 s-1) shear and the influence of shear on prostacyclin production. Canine jugular vein factor VIII-positive endothelial cells in passages 3 through 6 were incubated with 111Indium-oxine, and labeled cells were seeded onto fibronectin-treated expanded polytetrafluoroethylene patches. Patches with confluent cells were exposed to shear in a Weissenberg rheogoniometer for intervals ranging up to 60 minutes. Percent endothelial cell retention was determined by gamma counting of patches and media and by histologic evaluation. Prostacyclin production (tritiated radioimmunoassay of 6-keto-prostaglandin F1 alpha) was assayed on perfusing media. Results showed no differences in 6-keto-prostaglandin F1 alpha production between shear rates or time periods. Endothelial cell retention did not differ between the shear rates. Rotational shear caused persistent cell loss over time in either high- or low-shear conditions. This persistent cell loss in response to steady rotational shear differs from that in response to identical rates of pulsatile linear shear in our laboratory where cell loss approached zero after 15 minutes.

Artificial surface effect on red blood cells and platelets in laminar shear flow.

Red blood cell (RBC) effects on platelet adhesion to a nonbiologic test surface (tetrafluoroethylene propylene copolymer) and platelet aggregation during laminar shear flow for shear rates to 5,680 s-1 (corresponding to shear stress to 200 dyne/cm2) were investigated. Results on hemoglobin (Hb) and adenosine diphosphate (ADP) release from RBCs, percent decrease of single platelets in the bulk, and percent of test surface covered with platelets were obtained in a cone-and-plate (CP) viscometer for samples of whole blood, suspensions of RBC ghosts in platelet-rich plasma (PRP), and suspensions of RBCs in either PRP or platelet-poor plasma. Results obtained over the shear rate range studied for samples of normal hematocrit indicated that low-stress shearing led to ADP and Hb release from intact RBCs; shear-induced release of ADP from RBCs was about twice that of platelets, and of the total ADP released, the ADP released from RBCs contributed about six times that of the platelets to single platelet reduction in the bulk and about twice that of the platelets to platelet adhesion, ie, coverage of the test surface with platelets. Results obtained for various hematocrits showed that above a threshold hematocrit of about 25% to 35% the RBCs (suspended in PRP) had a greater contribution to ADP release, platelet adhesion, and platelet aggregation than the platelets themselves. Single platelet reduction for samples of RBC ghosts suspended in PRP correlated with shear rate level and not with shear stress.

Red blood cell effect on platelet adhesion and aggregation in low-stress shear flow. Myth or fact?

Evidence has been accumulating which suggests that red blood cells affect platelet adhesion on nonbiological surfaces and platelet aggregation in the bulk; this in vitro study investigated these red blood cell effects. For samples of either whole blood or suspensions of either ghosts or red blood cells in platelet rich plasma undergoing low-stress simple shear flow, data on hemoglobin and adenosine diphosphate (ADP) released from red blood cells, single platelet reduction (which is a measure of platelet adhesion to nonbiological surfaces and platelet aggregation in the bulk) and percent of surface covered with platelets and platelet aggregates were obtained in a cone-and-plate viscometer for shear rates up to 5680 s-1. The results obtained suggest that red blood cells release a significant fraction of their ADP (2% at 5680 s-1), which is enough to induce platelet aggregation, and contribute about 65% to the total ADP release for a sheared blood sample; ADP released from red blood cells contributes about 60% to single platelet reduction and about 28% to platelet adhesion, whereas ADP from platelets contributes about 8% and 14%, respectively; and the physical effect of red blood cells, which is coupled to the chemical effect, acts to catalyze (enhance) the chemical effects. Based on the results obtained a mechanism was developed to describe both the chemical and physical nature of the red blood cell effect.