Endoscopic biopsy of islet transplants in the gastric submucosal space provides evidence of islet graft rejection in diabetic pigs

Figures & data

Table 1. Summary of individual experiments in STZ-induced diabetic pigs.

Group	IS	BMX-001	Pig #	Body weight (kg)	Islets (kIEq /kg)	Islets/ site (kIEq /site)	Insulin before Tx	Insulin after Tx	BG before Tx	BG after Tx	Biopsy (day)	Day of euthanasia /necropsy	Reason for euthanasia
1A	TAC, MP	No	3780	13.6	10.3	35.0	0.86	0.98	317	224	14	25	Pulmonary infection
1A	TAC, MP	No	3786	14.0	10.0	35.0	1.04	0.96	308	312	7	28	Elective
1B	TAC, MP, MMF	No	3779	13.6	10.3	35.0	1.07	0.98	384	217	14	28	Elective
1B	TAC, MP, MMF	No	3777	14.0	10.0	35.0	0.67	0.57	182	215	7	23	Pulmonary infection
2	TAC, MP, MMF	Yes	5401	18.4	14.7	38.6	1.10	0.69	357	161	14	28	Elective
2	TAC, MP, MMF	Yes	5429	17.7	15.3	38.6	1.15	0.76	344	166	14	28	Elective
3	None	No	3781	19.5	ND	ND	0.96	ND	328	ND	ND	46	Elective
3	None	No	3802	20.0	ND	ND	1.02	ND	330	ND	ND	46	Elective

IS = immunosuppressive therapy. MMF = mycophenolate mofetil (Roche, Indianapolis, IN; 100–200mg/kg p.o. twice daily to maintain trough levels at 2–6µg/mL). MP = methylprednisolone (Pfizer, New York, NY; 2 mg/kg i.v. daily, tapering to 0.25mg/kg/day by day 7). ND = not done. TAC = tacrolimus (Astellas, Northbrook, IL; 0.05mg/kg i.m. twice daily to maintain trough levels at 10–15 ng/mL). Tx = transplantation

^† Insulin requirement is shown as daily dose per body weight (IU/kg/day)

^‡ Average of daily blood glucose (BG) level is shown as mg/dL. In Pig 3780 (Group1A) blood glucose level after transplantation was significantly lower than that during the period between STZ and islet transplantation

* (p<0.05). In Pig 3779 (Group1B) blood glucose level after transplantation was significantly lower than that during the period between STZ and islet transplantation

** (p<0.01). In Pig 5401 and Pig 5429 (Group2) both blood glucose level and insulin requirement were significantly lower than those during the period between STZ and islet transplantation

** (p<0.01). Survival of Group3 pigs is indicated as the number days after the administration of STZ (as no islet transplant was performed).

Table 2. In vitro insulin secretion of donor islets.

Donor ^#	Group	Recipient pig^#	SI (high glucose/basal)	SI (theophylline+high glucose/basal)
8004	1A / 1B	3786 / 3777	4.6	12.4
9782	1A / 1B	3780 / 3779	3.5	14.4
0906	2	5401	5.7	19.0
1098	2	5429	8.5	15.0

SI = stimulation index of insulin secretion.

^† Islets obtained from 8004 and 9782 were each transplanted into 2 recipients

^‡ Insulin secretion was higher when the islets had been cultured in the presence of BMX-001 (Group2), but no statistical analyses were performed because of the limited number of pigs in each group (n = 2).

Figure 1. Suppression of T cell proliferation and activation by BMX-001 PBMCs from pigs (n = 4) and humans (n = 3) were stimulated with PHA for 72h with/without 10µM BMX-001. BMX-001 significantly reduced proliferation of both pig (A) and human (D) PBMCs stimulated with PHA (*p<0.01). Results are expressed as ³H-thymidine incorporation (counts per minute). The percentages of T cells in pigs (B,C) and humans (E,F) secreting the inflammatory cytokines, IFN-γ or TNF-α, were measured after intracellular staining of whole blood. In the presence of 10µM BMX-001, the percentages of both IFN-γ- or TNF-α-secreting CD4⁺ and CD8⁺ T cells were significantly decreased in humans (*p<0.01) whereas only the percentage of IFN-γ secreting CD8⁺ T cells was significantly decreased in pigs (*p<0.05). Data are expressed as mean ± SEM.

Figure 2. Control data of diabetic pigs (A) Average daily blood glucose levels (left y-axis) and exogenous insulin requirements (right y-axis) in Group3 pigs after STZ. (B) Blood glucose levels (left panel) and exogenous insulin requirements (right panel) during the first 2 weeks after STZ (0–2 weeks, white bar), the next 2 weeks (2–4 weeks, striped bar), and thereafter (to 6 weeks, black bar). The daily values (as documented in A above) were divided by the average of the first 2 weeks, and the ratio of each period is shown. Data are mean ± SEM. Neither glucose level nor insulin requirement showed significant changes in Group3. (C) C-peptide levels in response to arginine stimulation before STZ, and 2 weeks or 4 weeks after STZ. C-peptide secretion was abrogated by STZ.

Figure 3. An increased number of transplanted islets with BMX-001 treatment of islets lead to improved glyco-metabolic control. (A) Daily blood glucose levels (left y-axis) and exogenous insulin requirements (right y-axis) in each pig in Groups 1 and 2. Blood glucose levels (but not insulin requirements) decreased in one Group1A pig (3780; p<0.05) and in one Group1B pig (3779; p<0.01). Blood glucose levels and insulin requirements decreased significantly in both Group2 pigs (p<0.01). (B) Mean blood glucose levels (left panel) and mean exogenous insulin requirements (right panel) before islet transplantation (pre-transplant, white bar), during the first 2 weeks after transplantation (striped bar), and thereafter (2 to 4 weeks, black bar) in each group were calculated to compare the changes after transplantation without the influence of daily fluctuation. The mean daily values in each group were divided by the mean of the pre-transplant period, and the ratio of this value for each group is shown (n = 2). Both glucose levels and insulin requirements decreased after transplantation in Group2. Data are mean ± SEM. (C) C-peptide levels in response to arginine stimulation before (left panel), after STZ (middle panel), and 2 weeks after transplantation (right panel). Following baseline blood sampling at 0 min, arginine was injected over a 1-min period. Additional blood samples were collected at 2, 3, 4, and 5 min following injection. C-peptide secretion was abrogated by STZ, and partially recovered following islet transplantation (n = 2).

Figure 4. Body weight changes in each recipient pig. The weights of all recipient pigs remained stable after transplantation, except Pig 3777, which lost weight and underwent euthanasia on day 23 due to pulmonary infection.

Figure 5. Anti-donor IgM or IgG antibody levels in recipients after islet Tx into the GSMS. To evaluate the sensitization to donor antigens, anti-donor IgM (left panels) and IgG (right panels) antibody levels were determined before, 2 weeks and 4 weeks after transplantation, except when recipient animals (Pig 3780, Pig 3777) were euthanized prior to 4 weeks due to pulmonary infection. Serum was tested at 3 weeks for these animals. IgM antibody levels were slightly elevated, but the level returned to the pre-transplant level in Pig 3780 (Group1A) (A). In contrast, there was no increase in IgM or IgG antibodies in Group1B (B) or Group2 pigs (C).

Figure 6. Comparison of histopathological examination in Groups 1 and 2 (A). Histopathological examination of islet allografts at biopsy (2 weeks after transplantation) of sections stained with hematoxylin and eosin (H&E, left columns), immunostained for insulin (second columns panels), CD3 cells (third column), and CD68 cells (right column). While Group1A or Group1B insulin-positive islets were fragmented, Group2 grafts showed preserved islet morphology. Cell infiltration was observed in H&E staining in biopsies from all groups, and most of the infiltrating cells were positive for CD3. CD68-positive cell infiltration was possibly more intensive in the Group1A pigs. (B) Histopathological examination of islet allografts at necropsy was performed 4 weeks after transplantation. While Group1A or Group1B insulin-positive islets were fragmented, Group2 grafts showed preserved islet morphology. Scale bars: 50μm.