Human small intestine contains 2 functionally distinct regulatory T-cell subsets.

BACKGROUND: Regulatory T (Treg) CD4 cells in mouse gut are mainly specific for intestinal antigens and play an important role in the suppression of immune responses against harmless dietary antigens and members of the microbiota. However, information about the phenotype and function of Treg cells in the human gut is limited. OBJECTIVE: We performed a detailed characterization of Foxp3+ CD4 Treg cells in human normal small intestine (SI) as well as from transplanted duodenum and celiac disease lesions. METHODS: Treg cells and conventional CD4 T cells derived from SI were subjected to extensive immunophenotyping and their suppressive activity and ability to produce cytokines assessed. RESULTS: SI Foxp3+ CD4 T cells were CD45RA-CD127-CTLA-4+ and suppressed proliferation of autologous T cells. Approximately 60% of Treg cells expressed the transcription factor Helios. When stimulated, Helios-negative Treg cells produced IL-17, IFN-γ, and IL-10, whereas Helios-positive Treg cells produced very low levels of these cytokines. By sampling mucosal tissue from transplanted human duodenum, we demonstrated that donor Helios-negative Treg cells persisted for at least 1 year after transplantation. In normal SI, Foxp3+ Treg cells constituted only 2% of all CD4 T cells, while in active celiac disease, both Helios-negative and Helios-positive subsets expanded 5- to 10-fold. CONCLUSION: The SI contains 2 subsets of Treg cells with different phenotypes and functional capacities. Both subsets are scarce in healthy gut but increase dramatically in active celiac disease.

The Presence of Activated T Cell Subsets prior to Transplantation Is Associated with Increased Rejection Risk in Pancreas Transplant Recipients.

Pancreas and islet transplantation (PTx) are currently the only curative treatment options for type 1 diabetes. CD4+ and CD8+ T cells play a pivotal role in graft function, rejection, and survival. However, characterization of immune cell status from patients with and without rejection of the pancreas graft is lacking. We performed multiparameter immune phenotyping of T cells from PTx patients prior to and 1 y post-PTx in nonrejectors and histologically confirmed rejectors. Our results suggest that rejection is associated with presence of elevated levels of activated CD4+ and CD8+ T cells with a gut-homing phenotype both prior to and 1 y post-PTx. The CD4+ and CD8+ T cells were highly differentiated, with elevated levels of type 1 inflammatory markers (T-bet and INF-γ) and cytotoxic components (granzyme B and perforin). Furthermore, we observed increased levels of activated FOXP3+ regulatory T cells in rejectors, which was associated with a hyporesponsive phenotype of activated effector T cells. Finally, activated T and B cell status was correlated in PTx patients, indicating a potential interplay between these cell types. In vitro treatment of healthy CD4+ and CD8+ T cells with tacrolimus abrogated the proliferation and cytokine (INF-γ, IL-2, and TNF-α) secretion associated with the type 1 inflammatory phenotype observed in pre- and post-PTx rejectors. Together, our results suggest the presence of activated CD4+ and CD8+ T cells prior to PTx confer increased risk for rejection. These findings may be used to identify patients that may benefit from more intense immunosuppressive treatment that should be monitored more closely after transplantation.

Validating the US pancreas donor risk index in a Norwegian population, a retrospective observational study.

OBJECTIVES: Despite advances in immunosuppression and surgical technique, pancreas transplantation is still associated with a significant graft loss rate. The Pancreas Donor Risk Index (PDRI) is a pre-transplant scoring tool derived from a US population. We sought to validate the PDRI in a Norwegian population. METHODS: We retrospectively retrieved donor data for 344 pancreas transplants undertaken in Norway between 2000 and 2019, utilising the Scandiatransplant database, and matched these to the respective recipients. The PDRI score was calculated for each transplanted pancreas, these were then stratified into quintiles. The association between the PDRI quintiles and 1-year graft survival was calculated, and this was repeated for the different types of pancreas transplantation. The association between PDRI as a continuous variable, and graft survival was determined. Donor and recipient data were compared to the original US population. RESULTS: The overall 1-year graft survival was 82.7%. There were no significant differences in survival between the different PDRI quintiles. When viewed as a continuous variable, increased PDRI score was not associated with decreased graft survival. Significant differences between the Norwegian and US populations were found. CONCLUSIONS: When applied to a Norwegian population, the PDRI score was unable to predict 1-year graft survival.

Insulin secretion and action after pancreas transplantation. A retrospective single-center study.

We explored glucometabolic and renal function after engraftment in all 159 consecutive patients with type 1 diabetes who received pancreas transplantation alone (PTA, n = 80) or simultaneous pancreas and kidney transplantation (SPK, n = 79) in Norway from 2012 until 2017. We report fasting levels of plasma glucose (FPG), C-peptide, eGFR and the homeostasis model assessment of insulin sensitivity (HOMA2(%S)) and beta-cell function (HOMA2(%B)) measured one to three times weekly during the first 8 and at 52 weeks after transplantation. One year after engraftment, in the PTA and SPK groups 52 and 64 were normoglycaemic without exogenous insulin, and two and zero patients were dead. Data at the 52-week visit were missing for 5 and 6 patients in the respective groups. During the first 8 weeks, FPG was lower, C-peptide and HOMA2(%S) were higher and eGFR was lower in the SPK group as compared with the PTA group (all p < .05). 30 out of 157 living patients needed insulin treatment 52 weeks after transplantation, 9/79 in the SPK group and 21/78 in the PTA group (p = .02). In conclusion, patients who underwent SPK showed lower insulin sensitivity, but higher insulin secretory capacity and lower mean blood glucose levels the first 8 weeks after transplantation. Also, a higher proportion of patients in the SPK group were insulin-free after 1 year, compared with the PTA group.

Endothelial function after pancreas transplantation-A single-center observational study.

BACKGROUND: Patients with diabetes mellitus treated with successful pancreas transplantation (PTX) normalize hyperglycemia, but are exposed to immunosuppressive drugs that may impair endothelial function. This study aimed to evaluate endothelial function in single PTX recipients. METHODS: Flow-mediated dilatation (FMD) in the brachial artery was measured by ultrasound 8 weeks after transplantation in single PTX (n = 27) and compared with healthy controls (n = 58), simultaneous pancreas and kidney recipients (n = 9), and kidney transplant recipients with (n = 41) and without (n = 95) diabetes mellitus. Adjustments for age, gender, blood pressure, and body mass index were included in a linear regression model. Changes in FMD from before to 1 year after transplantation were assessed in a subgroup of PTX recipients (n = 9). RESULTS: Flow-mediated dilatation% in PTX recipients was not inferior to healthy controls (8.7 ± 3.6 vs 7.7 ± 3.3, P = .06) and simultaneous pancreas and kidney recipients (6.7 ± 4.5, P = .24) in an adjusted model, and superior to kidney recipients with and without diabetes (3.0 ± 3.0 and 4.8 ± 3.3, respectively, both P < .005). FMD% improved significantly from eight weeks to one year after PTX, mean 7.9 ± 4.2% vs 11.8 ± 4.8% (N = 9; P = .03). CONCLUSION: Flow-mediated dilatation is well preserved in patients undergoing pancreas transplantation and is not impaired when immunosuppressive drugs are introduced.

Long-term cardiovascular outcomes in type 1 diabetic patients after simultaneous pancreas and kidney transplantation compared with living donor kidney transplantation.

AIMS/HYPOTHESIS: Mortality due to cardiovascular disease (CVD), particularly coronary artery disease (CAD), is high in type 1 diabetic patients with end-stage renal disease (ESRD). We aimed to determine whether normoglycaemia, as achieved by successful simultaneous pancreas and kidney (SPK) transplantation, could improve long-term outcomes compared with living donor kidney-alone (LDK) transplantation. METHODS: We studied 486 type 1 diabetic patients with ESRD who underwent a first SPK (n = 256) or LDK (n = 230) transplant between 1983 and 2012 and were followed to the end of 2014. Data were retrieved from the Norwegian Renal Registry and hospital records. Kaplan-Meier plots and multivariate Cox regression, with correction for recipient, donor and transplant factors, were used to examine potential associations between transplant type and all-cause and CVD- and CAD-related mortality. RESULTS: Median follow-up time was 7.9 years (interquartile range 4.3, 12.9). The adjusted HR for CVD-related deaths in SPK recipients compared with LDK recipients was 0.63 (95% CI 0.40, 0.99; p = 0.047), while the HRs for all-cause and CAD-related mortality were 0.81 (95% CI 0.57, 1.16; p = 0.25) and 0.63 (95% CI 0.36, 1.12; p = 0.12), respectively. Compared with the LDK group, SPK recipients were younger and received grafts from younger donors. Cardiovascular mortality was higher in patients transplanted between 1983 and 1999 compared with those who received their grafts in subsequent years. CONCLUSIONS/INTERPRETATION: In patients with type 1 diabetes and ESRD, SPK transplantation was associated with reduced long-term cardiovascular mortality compared with LDK transplantation.

In patients with type 1 diabetes simultaneous pancreas and kidney transplantation preserves long-term kidney graft ultrastructure and function better than transplantation of kidney alone.

AIMS/HYPOTHESIS: In patients with type 1 diabetes and end-stage renal disease (ESRD) we aimed to determine whether long-term normoglycaemia, as achieved by successful simultaneous pancreas and kidney (SPK) transplantation, would preserve kidney graft structure and function better than live donor kidney (LDK) transplantation alone. METHODS: Estimated GFR (eGFR) was calculated in SPK (n = 25) and LDK (n = 17) recipients in a stable phase 3 months after transplantation and annually during follow-up. Kidney graft biopsies were obtained at follow-up for measurement of glomerular volume (light microscopy), glomerular basement membrane (GBM) and podocyte foot process widths and mesangial volume fraction (electron microscopy). RESULTS: SPK and LDK recipients were similar in age and diabetes duration at engraftment. Donor age was higher in the LDK group. Median follow-up time was 10.1 years. Mean HbA1c levels during follow-up were 5.5 ± 0.4% (37 ± 5 mmol/mol) and 8.3 ± 1.5% (68 ± 16 mmol/mol) in the SPK and LDK group, respectively (p < 0.001). Compared with SPK recipients, LDK recipients had wider GBM (369 ± 109 nm vs 281 ± 57 nm; p = 0.008) and increased mesangial volume fraction (median 0.23 [range 0.13-0.59] vs 0.16 [0.10-0.41]; p = 0.007) at follow-up. Absolute eGFR change from baseline was -11 ± 21 and -23 ± 15 ml min(-1) 1.73 m(-2) (p = 0.060), whereas eGFR slope was -1.1 (95% CI -1.7, -0.5) and -2.6 (95% CI -3.1, -2.1) ml min(-1) 1.73 m(-2) per year in the SPK and LDK group, respectively (p = 0.001). CONCLUSIONS/INTERPRETATION: In patients with type 1 diabetes and long-term normoglycaemia after successful SPK transplantation, kidney graft ultrastructure and function were better preserved compared with LDK transplantation alone.

Local Postoperative Graft Inflammation in Pancreas Transplant Patients With Early Graft Thrombosis.

Background: Graft thrombosis is the main cause of early graft loss following pancreas transplantation, and is more frequent in pancreas transplant alone (PTA) compared with simultaneous pancreas-kidney (SPK) recipients. Ischemia-reperfusion injury during transplantation triggers a local thromboinflammatory response. We aimed to evaluate local graft inflammation and its potential association with early graft thrombosis. Methods: In this observational study, we monitored 67 pancreas-transplanted patients using microdialysis catheters placed on the pancreatic surface during the first postoperative week. We analyzed 6 cytokines, interleukin-1 receptor antagonist (IL-1ra), IL-6, IL-8, interferon gamma-induced protein 10 (IP-10), macrophage inflammatory protein 1ß (MIP-1ß), IL-10, and the complement activation product complement activation product 5a (C5a) in microdialysis fluid. We compared the dynamic courses between patients with pancreas graft thrombosis and patients without early complications (event-free) and between PTA and SPK recipients. Levels of the local inflammatory markers, and plasma markers C-reactive protein, pancreas amylase, and lipase were evaluated on the day of thrombosis diagnosis compared with the first week in event-free patients. Results: IL-10 and C5a were not detectable. Patients with no early complications (n = 34) demonstrated high IL-1ra, IL-6, IL-8, IP-10, and MIP-1ß concentrations immediately after surgery, which decreased to steady low levels during the first 2 postoperative days (PODs). Patients with early graft thrombosis (n = 17) demonstrated elevated IL-6 (P = 0.003) concentrations from POD 1 and elevated IL-8 (P = 0.027) concentrations from POD 2 and throughout the first postoperative week compared with patients without complications. IL-6 (P < 0.001) and IL-8 (P = 0.003) were higher on the day of thrombosis diagnosis compared with patients without early complications. No differences between PTA (n = 35) and SPK (n = 32) recipients were detected. Conclusions: Local pancreas graft inflammation was increased in patients experiencing graft thrombosis, with elevated postoperative IL-6 and IL-8 concentrations, but did not differ between PTA and SPK recipients. Investigating the relationship between the local cytokine response and the formation of graft thrombosis warrants further research.

Thromboinflammatory response is increased in pancreas transplant alone versus simultaneous pancreas-kidney transplantation and early pancreas graft thrombosis is associated with complement activation.

Background: Pancreas transplant alone (PTA) recipients are more affected by pancreas graft thrombosis, and graft loss compared to simultaneous pancreas-kidney (SPK) recipients. The pathophysiology is unknown, but an increased immune response has been suggested in the PTA recipients. In this observational study, we compared perioperative thromboinflammation between PTA (n=32) and SPK (n=35) recipients, and between PTA recipients with (n=14) versus without (n=18) early graft thrombosis. Methods: We measured C-reactive protein (CRP), plasma markers of activated coagulation and complement, and cytokines preoperatively and daily during the first postoperative week. Results: Preoperatively, coagulation and complement activation markers were comparable between PTA and SPK recipients, while cytokine concentrations were higher in SPK recipients (TNF, IL-8, IP-10, MCP-1, MIP-1α; all p<0.05). On the first postoperative day, PTA recipients had higher coagulation activation, measured as thrombin-antithrombin complex (TAT), than SPK recipients (p=0.008). In the first postoperative week, PTA recipients showed higher relative cytokine release (IL-6, IL-8, G-CSF, IP-10, MCP-1, and MIP-1α; all p<0.05) while SPK recipients showed higher absolute cytokine concentrations (TNF, IL-1ra, IL-8, MIP-1α, and IL-4; all p<0.05). PTA and SPK recipients showed similar terminal complement complex (TCC, sC5b-9) activation. On the first postoperative day, TCC (OR 1.2 [95% CI 1.0-1.5] for 0.1 CAU/ml increase, p=0.02) and CRP (OR 1.2 [95% CI 1.0-1.3] for 10 mg/L increase, p=0.04) were associated with an increased risk of early graft thrombosis. TCC was specific for graft thrombosis, while CRP increased with several complications. PTA recipients with compared to those without graft thrombosis had higher TCC pre- (p=0.04) and postoperatively (p=0.03). Conclusion: The relative increase in postoperative thromboinflammatory response was more pronounced in PTA recipients. Complement activation was associated with an increased risk of graft thrombosis. This study indicates that innate immune activation rather than elevated levels may affect early postoperative pancreas graft thrombosis. Clinical trial registration: https://clinicaltrials.gov/ct2/show/NCT01957696, identifier NCT01957696.

Microdialysis and CO2 sensors detect pancreatic ischemia in a porcine model.

BACKGROUND: Pancreatic transplantation is associated with a high rate of early postoperative graft thrombosis. If a thrombosis is detected in time, a potentially graft-saving intervention can be initiated. Current postoperative monitoring lacks tools for early detection of ischemia. The aim of this study was to investigate if microdialysis and tissue pCO2 sensors detect pancreatic ischemia and whether intraparenchymal and organ surface measurements are comparable. METHODS: In 8 anaesthetized pigs, pairs of lactate monitoring microdialysis catheters and tissue pCO2 sensors were simultaneously inserted into the parenchyma and attached to the surface of the pancreas. Ischemia was induced by sequential arterial and venous occlusions of 45-minute duration, with two-hour reperfusion after each occlusion. Microdialysate was analyzed every 15 minutes. Tissue pCO2 was measured continuously. We investigated how surface and parenchymal measurements correlated and the capability of lactate and pCO2 to discriminate ischemic from non-ischemic periods. RESULTS: Ischemia was successfully induced by arterial occlusion in 8 animals and by venous occlusion in 5. During all ischemic episodes, lactate increased with a fold change of 3.2-9.5 (range) in the parenchyma and 1.7-7.6 on the surface. Tissue pCO2 increased with a fold change of 1.6-3.5 in the parenchyma and 1.3-3.0 on the surface. Systemic lactate and pCO2 remained unchanged. The area under curve (AUC) for lactate was 0.97 (95% confidence interval (CI) 0.93-1.00) for parenchymal and 0.90 (0.83-0.97) for surface (p<0.001 for both). For pCO2 the AUC was 0.93 (0.89-0.96) for parenchymal and 0.85 (0.81-0.90) for surface (p<0.001 for both). The median correlation coefficients between parenchyma and surface were 0.90 (interquartile range (IQR) 0.77-0.95) for lactate and 0.93 (0.89-0.97) for pCO2. CONCLUSIONS: Local organ monitoring with microdialysis and tissue pCO2 sensors detect pancreatic ischemia with adequate correlation between surface and parenchymal measurements. Both techniques and locations seem feasible for further development of clinical pancreas monitoring.

Patient selection for islet or solid organ pancreas transplantation: experiences from a multidisciplinary outpatient-clinic approach.

OBJECTIVE: ß-cell replacement therapy (ßCRT), including pancreas transplantation alone (PTA) and islet transplantation (ITX), is a treatment option for selected type 1 diabetes patients. All potential candidates for ßCRT in Norway are referred to one national transplant centre for evaluation before any pre-transplant workup is started. This evaluation was performed by a transplant nephrologist alone prior to 2015 and by a multidisciplinary team (MDT) from 2015. We have reviewed the allocation of patients to treatment modality and the 1-year clinical outcome for the patients after transplantation. RESEARCH DESIGN AND METHODS: Medical charts of all patients evaluated for ßCRT between 2010 and 2020 in Norway were retrospectively analysed and the outcome of patients receiving ßCRT were studied. RESULTS: One hundred and forty-four patients were assessed for ßCRT eligibility between 2010 and 2020. After MDT evaluation was introduced for ßCRT eligibility in 2015, the percentage of referred patients accepted for the transplant waiting list fell from 84% to 40% (P < 0.005). One year after transplantation, 73% of the PTA and none of the ITX patients were independent of exogenous insulin, 8% of the PTA and 90% of the ITX patients had partial graft function while 19% of the PTA and 10% of the ITX patients suffered from graft loss. CONCLUSION: The acceptance rate for ßCRT was significantly reduced during a 10-year observation period and 81% of the PTA and 90% of the ITX patients had partial or normal graft function 1 year post-transplant.

CD4+ T cells persist for years in the human small intestine and display a TH1 cytokine profile.

Studies in mice and humans have shown that CD8+ T cell immunosurveillance in non-lymphoid tissues is dominated by resident populations. Whether CD4+ T cells use the same strategies to survey peripheral tissues is less clear. Here, examining the turnover of CD4+ T cells in transplanted duodenum in humans, we demonstrate that the majority of CD4+ T cells were still donor-derived one year after transplantation. In contrast to memory CD4+ T cells in peripheral blood, intestinal CD4+ TRM cells expressed CD69 and CD161, but only a minor fraction expressed CD103. Functionally, intestinal CD4+ TRM cells were very potent cytokine producers; the vast majority being polyfunctional TH1 cells, whereas a minor fraction produced IL-17. Interestingly, a fraction of intestinal CD4+ T cells produced granzyme-B and perforin after activation. Together, we show that the intestinal CD4+ T-cell compartment is dominated by resident populations that survive for more than 1 year. This finding is of high relevance for the development of oral vaccines and therapies for diseases in the gut.

Early detection of complications in pancreas transplants by microdialysis catheters, an observational feasibility study.

BACKGROUND: Despite advances in immunosuppression and surgical technique, pancreas transplantation is encumbered with a high rate of complication and graft losses. Particularly, venous graft thrombi occur relatively frequently and are rarely detected before the transplant is irreversibly damaged. METHODS: To detect complications early, when the grafts are potentially salvageable, we placed microdialysis catheters anteriorly and posteriorly to the graft in a cohort of 34 consecutive patients. Glucose, lactate, pyruvate, and glycerol were measured at the bedside every 1-2 hours. RESULTS: Nine patients with graft venous thrombosis had significant lactate and lactate-to-pyruvate-ratio increases without concomitant rise in blood glucose or clinical symptoms. The median lactate in these patients was significantly higher in both catheters compared to non-events (n = 15). Out of the nine thrombi, four grafts underwent successful angiographic extraction, one did not require intervention and four grafts were irreversibly damaged and explanted. Four patients with enteric anastomosis leakages had significantly higher glycerol measurements compared to non-events. As with the venous thrombi, lactate and lactate-to-pyruvate ratio were also increased in six patients with graft surrounding hematomas. CONCLUSIONS: Bedside monitoring with microdialysis catheters is a promising surveillance modality of pancreatic grafts, but differentiating between the various pathologies proves challenging.

Resident memory CD8 T cells persist for years in human small intestine.

Resident memory CD8 T (Trm) cells have been shown to provide effective protective responses in the small intestine (SI) in mice. A better understanding of the generation and persistence of SI CD8 Trm cells in humans may have implications for intestinal immune-mediated diseases and vaccine development. Analyzing normal and transplanted human SI, we demonstrated that the majority of SI CD8 T cells were bona fide CD8 Trm cells that survived for >1 yr in the graft. Intraepithelial and lamina propria CD8 Trm cells showed a high clonal overlap and a repertoire dominated by expanded clones, conserved both spatially in the intestine and over time. Functionally, lamina propria CD8 Trm cells were potent cytokine producers, exhibiting a polyfunctional (IFN-γ+ IL-2+ TNF-α+) profile, and efficiently expressed cytotoxic mediators after stimulation. These results suggest that SI CD8 Trm cells could be relevant targets for future oral vaccines and therapeutic strategies for gut disorders.

Preserved insulin secretion and kidney function in recipients with functional pancreas grafts 1 year after transplantation: a single-center prospective observational study

Objective: Successful simultaneous pancreas and kidney transplantation (SPK) or pancreas transplantation alone (PTA) restores glycemic control. Diabetes and impaired kidney function are common side effects of immunosuppressive therapy. This study addresses glucometabolic parameters and kidney function during the first year. Methods: We examined 67 patients with functioning grafts (SPK n = 30, PTA n = 37) transplanted between September 2011 and November 2016 who underwent repeated oral glucose tolerance tests (OGTTs) 8 and 52 weeks after transplantation. Another 19 patients lost their graft the first year post-transplant and 28 patients did not undergo repeated OGTTs and could not be studied. All patients received ATG induction therapy plus tacrolimus, mycophenolate and prednisolone. Glomerular filtration rate was measured before and 8 and 52 weeks after transplantation by serum clearance methods. Results: From week 8 to 52 after transplantation, mean fasting glucose decreased (SPK: 5.4 ± 0.7 to 5.1 ± 0.8 mmol/L, PTA: 5.4 ± 0.6 to 5.2 ± 0.7 mmol/L; both P < 0.05), and also 120-min post-OGTT glucose (SPK: 6.9 ± 2.9 to 5.7 ± 2.2 mmol/L; P = 0.07, PTA: 6.5 ± 1.7 to 5.7 ± 1.2 mmol/L; P < 0.05). Fasting C-peptide levels also decreased (SPK: 1500 ± 573 to 1078 ± 357 pmol/L, PTA: 1210 ± 487 to 1021 ± 434 pmol/L, both P < 0.005). Measured GFR decreased from enlistment to 8 weeks post transplant in PTA patients (94 ± 22 to 78 ± 19 mL/min/1.73 m2; P < 0.005), but did not deteriorate from week 8 to week 52 (SPK: 55.0 ± 15.1 vs 59.7 ± 11.3 ml/min/1.73 m²; P = 0.19, PTA: 76 ± 19 vs 77 ± 19 mL/min/1.73 m²; P = 0.74). Conclusion: Glycemic control and kidney function remain preserved in recipients with functioning SPK and PTA grafts 1 year after transplantation.

Calcium: A Crucial Potentiator for Efficient Enzyme Digestion of the Human Pancreas.

BACKGROUND: Effective digestive enzymes are crucial for successful islet isolation. Supplemental proteases are essential because they synergize with collagenase for effective pancreatic digestion. The activity of these enzymes is critically dependent on the presence of Ca2+ ions at a concentration of 5-10 mM. The present study aimed to determine the Ca2+ concentration during human islet isolation and to ascertain whether the addition of supplementary Ca2+ is required to maintain an optimal Ca2+ concentration during the various phases of the islet isolation process. METHODS: Human islets were isolated according to standard methods and isolation parameters. Islet quality control and the number of isolations fulfilling standard transplantation criteria were evaluated. Ca2+ was determined by using standard clinical chemistry routines. Islet isolation was performed with or without addition of supplementary Ca2+ to reach a Ca2+ of 5 mM. RESULTS: Ca2+ concentration was markedly reduced in bicarbonate-based buffers, especially if additional bicarbonate was used to adjust the pH as recommended by the Clinical Islet Transplantation Consortium. A major reduction in Ca2+ concentration was also observed during pancreatic enzyme perfusion, digestion, and harvest. Additional Ca2+ supplementation of media used for dissolving the enzymes and during digestion, perfusion, and harvest was necessary in order to obtain the concentration recommended for optimal enzyme activity and efficient liberation of a large number of islets from the human pancreas. CONCLUSIONS: Ca2+ is to a large extent consumed during clinical islet isolation, and in the absence of supplementation, the concentration fell below that recommended for optimal enzyme activity. Ca2+ supplementation of the media used during human pancreas digestion is necessary to maintain the concentration recommended for optimal enzyme activity. Addition of Ca2+ to the enzyme blend has been implemented in the standard isolation protocols in the Nordic Network for Clinical Islet Transplantation.

Transcriptional and functional profiling defines human small intestinal macrophage subsets.

Macrophages (Mfs) are instrumental in maintaining immune homeostasis in the intestine, yet studies on the origin and heterogeneity of human intestinal Mfs are scarce. Here, we identified four distinct Mf subpopulations in human small intestine (SI). Assessment of their turnover in duodenal transplants revealed that all Mf subsets were completely replaced over time; Mf1 and Mf2, phenotypically similar to peripheral blood monocytes (PBMos), were largely replaced within 3 wk, whereas two subsets with features of mature Mfs, Mf3 and Mf4, exhibited significantly slower replacement. Mf3 and Mf4 localized differently in SI; Mf3 formed a dense network in mucosal lamina propria, whereas Mf4 was enriched in submucosa. Transcriptional analysis showed that all Mf subsets were markedly distinct from PBMos and dendritic cells. Compared with PBMos, Mf subpopulations showed reduced responsiveness to proinflammatory stimuli but were proficient at endocytosis of particulate and soluble material. These data provide a comprehensive analysis of human SI Mf population and suggest a precursor-progeny relationship with PBMos.

Cardiac Assessment of Patients With Type 1 Diabetes Median 10 Years After Successful Simultaneous Pancreas and Kidney Transplantation Compared With Living Donor Kidney Transplantation.

BACKGROUND: In recipients with type 1 diabetes, we aimed to determine whether long-term normoglycemia achieved by successful simultaneous pancreas and kidney (SPK) transplantation could beneficially affect progression of coronary artery disease (CAD) when compared with transplantation of a kidney-alone from a living donor (LDK). METHODS: In 42 kidney transplant recipients with functioning grafts who had received either SPK (n = 25) or LDK (n = 17), we studied angiographic progression of CAD between baseline (pretransplant) and follow-up at 7 years or older. In addition, computed tomography scans for measures of coronary artery calcification and echocardiographic assessment of left ventricular systolic function were addressed at follow-up. RESULTS: During a median follow-up time of 10.1 years (interquartile range [IQR], 9.1-11.5) progression of CAD occurred at similar rates (10 of 21 cases in the SPK and 5 of 14 cases in the LDK group; P = 0.49). Median coronary artery calcification scores were high in both groups (1767 [IQR, 321-4035] for SPK and 1045 [IQR, 807-2643] for LDK patients; P = 0.59). Left ventricular systolic function did not differ between the 2 groups. The SPK and LDK recipients were similar in age (41.2 ± 6.9 years vs 40.5 ± 10.3 years; P = 0.80) and diabetes duration at engraftment but with significant different mean HbA1c levels of 5.5 ± 0.4% for SPK and 8.3 ± 1.5% for LDK patients (P < 0.001) during follow-up. CONCLUSIONS: In patients with both type 1 diabetes and end-stage renal disease, SPK recipients had similar progression of CAD long-term compared with LDK recipients. Calcification of coronary arteries is a prominent feature in both groups long-term posttransplant.

Antibody-secreting plasma cells persist for decades in human intestine.

Plasma cells (PCs) produce antibodies that mediate immunity after infection or vaccination. In contrast to PCs in the bone marrow, PCs in the gut have been considered short lived. In this study, we studied PC dynamics in the human small intestine by cell-turnover analysis in organ transplants and by retrospective cell birth dating measuring carbon-14 in genomic DNA. We identified three distinct PC subsets: a CD19+ PC subset was dynamically exchanged, whereas of two CD19- PC subsets, CD45+ PCs exhibited little and CD45- PCs no replacement and had a median age of 11 and 22 yr, respectively. Accumulation of CD45- PCs during ageing and the presence of rotavirus-specific clones entirely within the CD19- PC subsets support selection and maintenance of protective PCs for life in human intestine.