J Korean Diabetes Assoc.  2006 Jan;30(1):54-63.

The effects of mixed chimerism conducted by natural killer cell depletion with non myeloablation on islet allograft rejection

• Park HS
• Cho SG
• Park CG
• Hong OK
• Kim JW
• Kim BR
• Yoon KH

Abstract

BACKGROUND: Because of the shortage of human pancreas and immunorejection, very small fraction of patients with type 1 diabetes can be treated with islet transplantation. The immune tolerance induction for overcoming the immume rejectin of trausplamted islets could be conducted by hematopoietic mixed chimerims with various invasive methods. The purpose of this study is to investigate the effect of mixed chimerims conducted by newly developed minimally invasive methods on islet allografts rejection in streptozotocin induced diabetic mice.
METHODS
Recipient, Balb/c(H-2Kd) mice were injected intraperitoneally with anti- asialoGM1 antibody at one day before bone marrow transplantation. There were received total body irradiation at a dose of 500 cGy and followed by tail vein injection of the 2 x 10(7) T-cell depleted bone marrow cells from C57BL/6(H-2Kb). Mixed chimerism mice were determined by gDNA PCR of lymphocyte MHC class I gene (H-2K) on 21st day. Streptozotocin induced diabetic mixed chimera mice were received islet transplantation from bone marrow donors. Grafts, spleen and peripheral blood were obtained from the mixed chimera mice, and there were used by Immunohistochimeical staining, flow cytometric analysis and gDNA PCR on 21st day.
RESULTS
The blood glucose levels of streptozotocin induced diabetic mice were normalized by transplantation of bone marrow donor islets and maintained during 30 days. After removal of first islet allografts, hyperglycemia was re-established. We could re-confirmed donor specific tolerance of transplanted islets by second transplantation of bone marrow donor islets. Normoglycemia was maintained during 21 days after second islet transplantation. Furthermore islet grafts from MHC-mismatched third party mice were immediately rejected. Flow cytometric analysis results suggest that the mixed chimerism mice were maintain during the whole study period.
CONCLUSION
The mixed chimerism model conducted by newly developed and minimally invasive method effectively prevents the islet allo grafts rejection in STZ-induced mixed chimerism mice.

Keyword

Insulin; Islet; Transplantation; Mixed chimera; Type 1 diabete

MeSH Terms

Allografts*
Animals
Blood Glucose
Bone Marrow
Bone Marrow Cells
Bone Marrow Transplantation
Chimera
Chimerism*
Genes, MHC Class I
Humans
Hyperglycemia
Immune Tolerance
Insulin
Islets of Langerhans Transplantation
Killer Cells, Natural*
Lymphocytes
Mice
Pancreas
Polymerase Chain Reaction
Spleen
Streptozocin
T-Lymphocytes
Tissue Donors
Transplantation
Transplants
Veins
Whole-Body Irradiation
Blood Glucose
Insulin
Streptozocin

Figure

• 

  Fig. 1 Schematic of experimental design. (A) Method of mixed chimerism mice induction. -1 day. Anti-asialoGM1 was injected to Balb/c. Total body irradiated and 2×107 Bone marrow cells were transplanted to the Balb/c on 0 day. (B) Islet transplantation schedule in STZ induced type 1 DM mixed chimerism mice. On 21 days before islets transplantation to induced mixed chimerism in Balb/c. On 6day before transplantation, type 1 diabetes was induced by STZ injection. On day 0. 1st B6 islets transplantation to the mixed chimerism mice. Graft removal or sham op was done on 30th day. On the 35th day, the second islet transplantation was performed, at the same time third party islets were co-transplanted on the other side of the same kidney. Finally, on the 56th day and the 65th day, all grafts, spleen, peripheral blood and pancreas were harvested. TBI: Total body irradiation BMCs: Bone marrow cells BMT: Bone marrow transplantation STZ: Streptozotocin DM: Diabetes mellitus

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  Fig. 2 Pancreatic damage after streptozotocin treatment. Insulin immunohistochemical staining(Arrow) in wild Balb/c(B) and mixed chimerism Balb/c(A) pancreatic islet on 1 day before islet transplantation. BrdU immunohistochemical staining in wild Balb/c(D) and mixed chimerism Balb/c(C). pancreatic islet on 6th day after STZ treatment. Insulin immunohistochemical staining in Mixed chimerism Balb/c(E). on 65th day after islet transplanted of bone marrow cell donor. Beta cell percentage in islets of wild Balb/c and mixed chimerism Balb/c on -1 day(F). (Original magnification ×400, * P≤0.001)

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  Fig. 3 Induced mixed chimerims Balb/c confirmed by genomic DNA PCR in peripheral blood.

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  Fig. 4 Blood glucose levels were measured after islet transplantation. Islets of bone marrow cell donor were transplanted to STZ-induced diabetic mixed chimerims Balb/c. Islet of bone marrow cell donor and third party were re-transplanted after depleted(30th day) of the first graft(-●- 1st graft depleted group n=4, -○- 1st graft did not deplete group n=2). Islets of bone marrow cell donor were transplanted to wild Balb/c mice(-▼- n=14) *P≤0.001 (-●- vs -▼- & -○- vs -▼-) †Streptozotocin injection (300mg/Kg) ‡Islet transplantation §IP-GTT(-●- & -○-) ∥End point of death(-▼-)

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  Fig. 5 Insulin immunohistochemical staining in islet graft. (A) Islet of bone marrow cell donor in the graft was stained with insulin immunohistochemical staining at 56th day. (B) Second islet 1st graft depleted group were stained with insulin immunohistochemical staining at 26 days after 2nd islet transplantation. (Original magnification ×400)

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  Fig. 6 The diagram of flowcytometric analysis in mixed chimerism Balb/c. Splenocytes were stained with C57BL/6 marker CD45.2 on mixed chimerism mice. Histogram of C57BL/6 origin lymphocyte (CD4, CD8a, CD45R/B220;B-cell) has been shown from the CD45.2 positive region.

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