his were isolated from single donor pancreases , according to the edmonton protocol ( 3 ) . 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 . 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 . 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 ( t1 dm ) were selected , as previously reported ( 1 ) . complete blood chemistry , including all metabolic parameters ( hba1c ; 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 ( 120150 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 ) . summary of clinical , metabolic , and immunological data of the transplanted patients throughout long - term follow - up ( patients 1 and 4 received more than one graft ) scpr , serum c - peptide response . patient 4 got a second graft 7 days after the first one and a third graft 6 months after the first . 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 540,0001,600,000 ieq / patient ) . his were isolated from single donor pancreases , according to the edmonton protocol ( 3 ) . 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 . 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 . 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 ( t1 dm ) were selected , as previously reported ( 1 ) . complete blood chemistry , including all metabolic parameters ( hba1c ; 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 ( 120150 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 ) . summary of clinical , metabolic , and immunological data of the transplanted patients throughout long - term follow - up ( patients 1 and 4 received more than one graft ) scpr , serum c - peptide response . patient 4 got a second graft 7 days after the first one and a third graft 6 months after the first . abdominal mri was scheduled only if necessary , to exclude the occurrence of post - tx peritoneal lesions . complete blood chemistry , including all metabolic parameters ( hba1c ; 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 ( 120150 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 ) . summary of clinical , metabolic , and immunological data of the transplanted patients throughout long - term follow - up ( patients 1 and 4 received more than one graft ) scpr , serum c - peptide response . patient 4 got a second graft 7 days after the first one and a third graft 6 months after the first . 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 540,0001,600,000 ieq / patient ) . 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 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 . 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 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 t1 dm .
objectiveto assess long - term metabolic and immunological follow - up of microencapsulated human islet allografts in nonimmunosuppressed patients with type 1 diabetes ( t1dm).research design and methodsfour nonimmunosuppressed patients , with long - standing t1 dm , received intraperitoneal transplant ( tx ) of microencapsulated human islets . anti - major histocompatibility complex ( mhc ) class i  ii , gad65 , and islet cell antibodies were measured before and long term after tx.resultsall patients turned positive for serum c - peptide response , both in basal and after stimulation , throughout 3 years of posttransplant follow - up . 
 daily mean blood glucose , as well as hba1c levels , significantly improved after tx , with daily exogenous insulin consumption declining in all cases and being discontinued , just transiently , only in patient 4 . 
 anti - mhc class i  ii and gad65 antibodies all tested negative at 3 years after tx.conclusionsthe grafts did not elicit any immune response , even in the cases where more than one preparation was transplanted , as a unique finding , compatible with encapsulation - driven 
 bioinvisibility  of the grafted islets . 
 this result had never been achieved with the recipient s general immunosuppression .