Male C57BL/6 and BALB-c mice were purchased from Charles River Laboratories. Cxcr2–/–
mice on BALB-c background [strain C129S2 (B6)-IL8RB] were provided by Dompè spa. Pancreatic islets were isolated and transplanted via the portal vein as previously described (11
). Reparixin was provided by Dompè spa and administered by s.c. continuous infusion (Alzet Osmotic pump; Alza Corp.) starting from day –1 up to day 6 after islet transplantation at a dose of 5.4 mg/kg/h. Recipient mice were rendered diabetic with i.v. alloxan injection (60–75 mg/kg; Sigma-Aldrich). Blood glucose measurements were performed using a Glucometer Elite (Bayer Canada). Euglycemia was defined as nonfasting blood glucose concentrations of less than 200 mg/dl in 2 consecutive measurements. For evaluation of intrahepatic leukocytes, single-cell suspensions were prepared from 2 liver lobes of known weight, and analysis of IHL population was performed by flow cytometry. Cells were stained with FITC-, PE-, or allophycocyanin-labeled (APC-labeled) anti-CD4, anti-CD8, anti-CD3, anti-CD19, anti-TCRb, anti-NK1.1, anti-CD11, anti–Gr-1, anti-CD11b, anti-Ly6C, or anti-CD11c Abs (BD Biosciences — Pharmingen).
Human islet transplantation.
Circulating cytokine/chemokine concentrations after islet infusion were measured in 11 patients with type 1 diabetes receiving islet transplantation alone at San Raffaele Scientific Institute. 8 recipients (NCT01346085) were treated with: (a) pretransplant rapamycin (0.1 mg/kg/d, targeting serum trough levels of 8–10 ng/ml) for at least 30 days prior to first islet infusion; (b) ATG induction therapy (1.5 mg/kg/d for 4 days) and a steroid bolus (methyl-prednisolone, 500 mg bolus) plus low-dose steroids (prednisone, 10 mg/d) and Anakirna (100 mg/d) for 2 weeks; (c) maintenance immunosuppressive therapy with rapamycin (0.1 mg/kg/d, targeting serum trough levels of 12–15 ng/ml) plus MMF (2 g/d). 3 recipients (NCT01220856) were treated with: (a) ATG induction therapy (1.5 mg/kg/d for 4 days) and a steroid bolus (methyl-prednisolone, 500 mg bolus); (b) maintenance immunosuppressive therapy with FK506 (targeting serum trough levels of 8–10 ng/ml) plus MMF (2 g/d). The characteristics of islet preparations, donors, and recipients are reported in Supplemental Table 3.
Reparixin in pancreatic islet transplantation trial.
A phase 2 randomized, open-label, pilot study to assess the efficacy and safety of reparixin after single-infusion islet transplantation in patients with type 1 diabetes mellitus was initiated in July 2010 (NCT01220856). Inclusion criteria restricted enrolment to patients expected to receive an islet mass in the lower range of the currently accepted transplantable islet amount (3,000–7,000 IEQ/kg BW). Patients were on an immunosuppression regimen consisting of: (a) ATG induction therapy (1.5 mg/kg/d for 4 days starting at day –1) and a steroid bolus (methyl-prednisolone, 500 mg bolus, day –1), (b) maintenance immunosuppressive therapy with mycophenolate mofetil (2 g/d starting on day –1 of islet infusion) plus tacrolimus (0.087 mg/kg twice daily, targeting serum trough levels of 8–10 ng/ml) replaced by rapamycin from month 3 after transplant (0.1 mg/kg/d, targeting serum trough levels of 10–12 ng/ml). Patients were randomly (1:1) assigned to receive either no additional experimental intervention (control group) or reparixin treatment (2.772 mg/kg BW/h i.v. continuous infusion for 7 days from day –1).
Basal release of chemokines and cytokines in freshly isolated mouse and human islets.
Human and mouse cytokines/chemokines were detected using multiplex bead-based assays (Bio-Plex Human Cytokine 27-Plex Panel; Bio-Plex Human Group II Cytokine 23-Plex Panel; Bio-Plex Mouse Cytokine 23-Plex Panel; Biorad Laboratories).
Analysis of data was performed using the SPSS statistical package for Windows (SPSS Inc.). A 2-tailed P value less than 0.05 was considered significant.
All patients gave informed consent for the investigations. The ethical committee of the Istituto Scientifico Ospedale San Raffaele approved the islet transplant protocols and investigations. Investigations were carried out in accordance with the principles of the Declaration of Helsinki, as revised in 2000. All mouse experiments were in accordance with protocols approved by the Animal Care and Use Committee of San Raffaele Scientific Institute.