Procedure cures some diabetic mice, but not in the way previously reported

Mar 28 2006

Researchers attempting to reproduce a controversial 2003 mouse experiment suggestive of a cure for type 1 diabetes have found evidence that the experimental procedure does eliminate diabetic symptoms in a small fraction of the mice exposed to it.

However, scientists from Washington University School of Medicine in St. Louis found no signs that the procedure was working in the manner reported by the group of scientists at Harvard University who originated it.

The Washington University group is one of three labs reporting in the March 24 issue of Science on attempts to reproduce the earlier experiment. All three groups independently found no evidence of a key claim of the earlier study: that cells injected from the spleens of healthy mice had formed new insulin-producing beta cells in the diabetic mice, a finding that created hope that the approach might be used to cure diabetes in humans.

"We showed that various immunological processes had rejected the injected cells," says senior author Emil R. Unanue, M.D., Mallinckrodt Professor of Pathology and Immunology. "In the mice who were cured, we found no evidence linking restoration of beta cell function to the spleen cell injections."

Researchers are following up on the study with new experiments designed to determine how the mice were cured.

"It's a positive thing that 4 of 22 mice recovered beta cell function, and we're investigating where that recovery of beta cell function came from," says lead author Anish Suri, Ph.D., a research assistant professor of pathology and immunology in Unanue's lab. "Conceivably, controlling the autoimmune response in patients with early diabetes may allow for recovery of some beta cell function and a degree of reversion of the diabetic process."

As in the 2003 experiment, researchers performed their studies in female mice from the NOD mouse strain, which develops diabetes in a manner very similar to human type 1 diabetes mellitus. Between the age of 20 and 30 weeks, immune system cells in the mice begin attacking beta cells in the pancreas, leading to death of the cells and onset of diabetic symptoms such as hyperglycemia, or abnormally high blood sugar levels.

Following the procedures developed for the prior study by Harvard researcher Denise L. Faustman, Washington University scientists gave the mice injections of a solution called complete Freunds adjuvant (CFA) that contains water, oil and portions of dead bacteria. Scientists had previously established that such injections stop immune attacks on beta cells.

Researchers also gave the mice repeated large injections of spleen cells from healthy male mice. Faustman's group has hypothesized that the spleen is erroneously promoting the survival and reproduction of immune cells that attack beta cells; they theorize that injections of spleen cells from healthy mice will help reset this dysfunctional selection mechanism.

Finally, scientists took islets, structures in the pancreas containing insulin-producing beta cells, from healthy mice and transplanted them into one of the diabetic mice's kidneys.

The experimental procedures began when the mice first became hyperglycemic, indicating that a substantial portion of their beta cells had died off as a result of immune assault. In 22 of 53 mice, the injections and the transplant restored normal blood sugar levels. Scientists followed these mice for at least 120 days, continuing to give them spleen cell injections. Then they removed the kidney with the transplanted islets.

This caused 18 of the 22 mice to revert to hyperglycemia and diabetes. Studies of the four mice that continued to have normal blood-sugar levels revealed signs that the pancreas was regaining beta cell mass and function.

In the 2003 study, scientists looked in the pancreas of cured mice for indications of the presence of the Y chromosome, the male sex chromosome. When they found it, they interpreted this as proof that stem cells from the spleen cell injections, which all came from male mice, had developed into new beta cells in the diabetic mice, which were all female.

Washington University scientists did not see this same indicator or any other signs that the male spleen cells had survived in cured mice.

"This idea that spleen cell injections not matched for immune compatibility could lead to new beta cells was very controversial," Unanue says. "Everything we know in immunology suggests that such injections should be rejected by the immune system."

In a second test performed after they repeated the earlier experiment, researchers gave the mice the CFA injection and the islet transplants but did not give spleen cell injections. For periods of more than 100 days, this temporarily cured diabetes in 69 percent of the mice that received it (20 of 29). The procedure that included the spleen injection had a temporary cure rate of 42 percent.