|CINCINNATI MAY, 2000
We made our second trip to Cincinnati the week of May 7. Randy went early to participate in an explant of a sheet from the autograft (pre clinical animal experiments #2). We planned to do another "pilot" canine allograft -- this one being a bit more careful that in February -- then go home. But things don't always work out as planned....
We got to meet Cindy Steffen -- Horacio's second tech. We had already been in frequent communication on experimental planning and data transfer. She is a delight to have around. She and Lisa do most of the technical work in Horacio Rilo's laboratory.
Randy arrived on Monday planning to explant one of the sheets in Cincinnati pre clinical animal experiment #2, the autograft. We had not really expected the allograft (Cincinnati pre clinical animal experiment #1) to do so well. Euglycemia without injected insulin seemed over-the-moon! So the autograft was sort of a fifth wheel of an experiment. We thought having a look at a sheet would tell us something about how the sheet and the omentum were "getting along."
Horacio and Randy were shocked to see a golf ball size mass on the omentum. And then they opened it up -- the sheet had folded on itself and crumpled. There was fat, fibrosis -- and bacteria! We knew we had bacterial contamination in February because of a break in sterile technique, but we figured that the metabolic results meant that the antibiotics had killed all the bacteria. They hadn't. We had an obvious bacterial contamination, which had partially dissolved the sheet. (The sheet was designed to withstand mammalian enzymes, not bacterial enzymes.) There were even some exposed islets. They weren't rejected because it was an autograft.
Rick and I (Scott) arrived Monday night and we all puzzled about it. We were stumped. If the islet preparations were contaminated with bacteria that dissolved the sheets, how could islets have survived for 10 weeks in the allograft dog? Was the pancreatectomy incomplete? A little pancreas nub might explain the data. Was there less or different contamination? Had tolerance developed? We decided we had to find out. I continued on my itinerary and Rick and Randy stayed to remove the sheets from dog #1 on Thursday the 11th.
The picture above is what they found. The omentum had folded back on itself, making a mass filled with crumpled sheets and some bacteria. The surrounding tissue was clearly reacting to bacteria, but at the same time seemed to be producing additional vasculature and fat cells -- what would be expected as a reaction to viable islets. It was clear we were fortunate -- whatever the contaminant, it did not dissolve the sheets. The islets were still inside. We were unlucky because the bacteria made our results unable to address bio-inertness of the sheets.
Dithizone stained islets immediately after explantation.
And the islets were viable. The explant above was frozen for sectioning and histology. But first Rick took a bit of sheet and stained it with dithizone, a stain that only works with viable islets containing insulin. You can see the orange islet cells in the micrograph above. So we really were curing diabetes with islets in our sheets!
The other question was, did any pancreatic islets remain? The proof would be subsequent development of diabetes. We waited anxiously for the blood sugars when we returned to San Francisco. And sure enough, the dog became diabetic with consecutive blood sugars over 200 mg/dL (click here). When the dog was sacrificed the duodenum was removed and will be examined for residual pancreas and islet tissue. But we can already say, without doubt, that the dog did not have enough islets of its own to control its diabetes. The sheets made the difference.
While we were in Cincinnati we did another allograft, this time making sure that the dog really did have diabetes before the Islet Sheets were implanted. (Some people were skeptical about dog #1.) Dog #3's blood sugars were certainly diabetic a week after pancreatectomy.
The isolation of islets on Tuesday the 9th went according to plan. Following the usual islet isolation and purification, we had nice looking islets:
Another dithizone stain of freshly isolated islets:
Sheet fabrication went well. Because the sheets are small, Horacio was able to make a small incision and pull the omentum out. The two sheets were sutured to opposite sides of the omentum (perhaps this will minimize the folding and sticking problem) then stuffed back into the dog.
One of the two Islet Sheets being sutured to the dog's omentum.
So we were happy, having learned a lot. To our astonishment, the Islet Sheet works even when contaminated with bacteria! So our own confidence when we have clean islets to put in the sheets has gone up. On the other hand, the omentum might be too reactive. We don't really know because the bacteria "contaminate" the results. Next time we will probably try some other sites.
Steve Santen (a diabetes activist in Cincinnati and a regular at The Islet Forum) dropped by the laboratory on the 9th, and we had a good chat. And my sister, Debby Mustard, lives near Cincinnati. She came by too: