Long-Term Metabolic and Immunological Follow-Up of Nonimmunosuppressed Patients With Type 1 Diabetes Treated With Microencapsulated Islet Allografts: Four cases

HIs were isolated from single donor pancreases, according to the Edmonton protocol (3). Islet preparations from our laboratory were grafted in patients 1 and 2. We also used HIs procured at the University of Illinois at Chicago (UIC) (patients 3 and 4). The “UIC HIs” were isolated using a modified Ricordi’s method and passed the product release criteria including viability, purity, and endotoxin levels <5 endotoxin units/g (EU/g), as required by the U.S. Food and Drug Administration. The UIC HIs could be used in our Center because there was no suitable U.S. recipient available for such a given islet preparation. This scenario happened because the HI yield was insufficient to achieve the required 5,000 islet equivalents (IEQ)/kg body wt of listed U.S. recipients. Islet morphology, viability, and functionality assessments were performed before and after microencapsulation, showing 1) purity >80%; 2) viability >90%; and 3) stimulation index upon static incubation with glucose >5 above baseline.

The selected islet batches were encapsulated in ultra-purified, endotoxin-free sodium alginate prepared in-house (patent number WO 2009/093184 A1) by our method (4).

Four patients with long-standing type 1 diabetes (T1DM) were selected, as previously reported (1).

All clinical and metabolic parameters were carefully acquired before and strictly monitored after TX.

Complete blood chemistry, including all metabolic parameters (HbA_1c; daily glucose profiles after and 3 consecutive months before entering the trial), was performed.

All grafted patients underwent hourly BG and exogenous insulin supplement monitoring to keep BG within the prefixed range (120–150 mg/dL).

All patients, upon TX, underwent either an oral glucose tolerance test (75 g; patient 1 only) or a glucagon (1 mg i.v.) or arginine test (10 g in 250 mL saline i.v.; patient 1 only) to determine basal and poststimulation serum C-peptide response by radioimmunoassay (Myria, Milan, Italy). Islet cell antibodies, anti-GAD65 antibodies, and anti-major histocompatibility complex (MHC) class I–II antibodies were assessed before and after transplantation (Table 1) on a long-term follow-up basis. Anti-MHC class I–II antibodies were assessed by ELISA (Biotest, Waukesha, WI).

Abdominal MRI was scheduled only if necessary, to exclude the occurrence of post-TX peritoneal lesions.

All patients received a transplant of microencapsulated HIs intraperitoneally, under ecography guidance and local anesthesia. The encapsulated islet suspension in saline was placed in a 60-mL syringe barrel and slowly delivered through a polypropylene catheter into the peritoneal cavity through a small incision of the abdominal wall. The injected total graft volume did not exceed 100 mL (capsules + saline). Patient 4, on his third TX, underwent abdominal laparoscopy, under general anesthesia, to visually select and optimize the TX site and possibly avoid capsule injection errors (see patient 1). In this instance, the microcapsules were evenly distributed beneath the liver and the spleen, where blood supply is high. In all TX procedures, care was taken to dispense the capsular suspension as thoroughly as possible, to prevent formation of capsules clusters. Human islet dosing varied between recipients, ranging from 5,000 to 15,000 IEQ/kg/TX (range 540,000–1,600,000 IEQ/patient). This result strictly depended on organ availability and the islet isolation rate per pancreas.

None of the TX recipients showed any acute, significant postoperatory side effects. BG levels were stable, both short and long term after TX. In particular, throughout 24 months of TX, daily mean BG was stable in all patients, whereas after such time, the values tended to slightly but progressively raise. Interestingly, patient 1, who had suffered for severe nocturnal hypoglycemic episodes, showed evident recovery, in conjunction with stabilization of BG profiles. The natural history of this pilot study is summarized in Table 1, throughout 3 consecutive years of post-TX follow-up. At this time, the study was terminated and the patients were seen once a year. So far, at 7 years post-TX, all patients are in good health and are fully back to their original exogenous insulin therapy regimens.

As a unique finding, no anti-MHC class I–II or anti-GAD65 antibodies or islet cell antibodies were detected in any of the transplanted patients throughout 5 years of post-TX follow-up (Table 1).

Patient 1, 5 years after TX, back to his original insulin schedule, started complaining of abdominal discomfort. In our Center, upon palpation of the abdomen, we found a small mass that ultrasound scan identified as a hyperechoic cyst-like formation. The cyst, situated in the rectus anterior muscle, was surgically removed and consisted of a 3- to 4-cm fibrotic lump that contained mostly intact capsules with no more viable islet cells inside. Obviously, the original capsules, in this instance, had been mistakenly injected beneath the muscle fascia rather than intraperitoneally, thus resulting in the cyst formation.

Our alginate/polyaminoacidic encapsulation system has been confirmed to represent a powerful tool for immunoprotection of HI grafts (5), as proven by the absence of a wide array of islet cell-directed as well as anti-MHC class I–II antibodies (6). Hence, microcapsules provided the islet grafts with bioinvisibility, according to U.S. Food and Drug Administration criteria. In our opinion, this was the most important finding of the study. Obviously, the partial and transient metabolic benefits obtained by the treatment reflect limitations of this microcapsules generation, with special regard to their size in relation to TX site. Moreover, HIs can be moved through long distances with no loss of their viability and function (7). We maintain that smaller-size microcapsules could permit access to TX sites possibly associated with better functional exchange, thereby complying, more efficiently, with metabolic requirements of patients with T1DM.

No potential conflicts of interest relevant to this article were reported.

G.B. and P.M. researched data, contributed to discussion, wrote the manuscript, and reviewed and edited the manuscript. G.L. contributed to discussion. C.B. and G.N. researched data. B.B. and M.Q. researched data and reviewed and edited the manuscript. K.P.K. reviewed and edited the manuscript. J.O. contributed to discussion and reviewed and edited the manuscript. R.C. contributed to discussion and wrote, reviewed, and edited the manuscript.

The authors gratefully acknowledge the technical support of the “Consorzio Interuniversitario per i Trapianti d’Organo,” Rome, Italy. They also acknowledge the technical assistance of Rita Tognellini, PhD (“Centro Regionale di Tipizzazione Tissutale ed Immunologia dei Trapianti,” Perugia University Hospital, Perugia, Italy), who assisted with the anti-MCH antibody assays.