The long lifespan and low turnover of human islet beta cells estimated by mathematical modelling of lipofuscin accumulation.

AIMS/HYPOTHESIS: Defects in pancreatic beta cell turnover are implicated in the pathogenesis of type 2 diabetes by genetic markers for diabetes. Decreased beta cell neogenesis could contribute to diabetes. The longevity and turnover of human beta cells is unknown; in rodents <1 year old, a half-life of 30 days is estimated. Intracellular lipofuscin body (LB) accumulation is a hallmark of ageing in neurons. To estimate the lifespan of human beta cells, we measured beta cell LB accumulation in individuals aged 1-81 years. METHODS: LB content was determined by electron microscopical morphometry in sections of beta cells from human (non-diabetic, n = 45; type 2 diabetic, n = 10) and non-human primates (n = 10; 5-30 years) and from 15 mice aged 10-99 weeks. Total cellular LB content was estimated by three-dimensional (3D) mathematical modelling. RESULTS: LB area proportion was significantly correlated with age in human and non-human primates. The proportion of human LB-positive beta cells was significantly related to age, with no apparent differences in type 2 diabetes or obesity. LB content was low in human insulinomas (n = 5) and alpha cells and in mouse beta cells (LB content in mouse <10% human). Using 3D electron microscopy and 3D mathematical modelling, the LB-positive human beta cells (representing aged cells) increased from >or=90% (<10 years) to >or=97% (>20 years) and remained constant thereafter. CONCLUSIONS/INTERPRETATION: Human beta cells, unlike those of young rodents, are long-lived. LB proportions in type 2 diabetes and obesity suggest that little adaptive change occurs in the adult human beta cell population, which is largely established by age 20 years.

Administration of 75 mg of aspirin daily for 28 days is sufficient prophylaxis against renal transplant vein thrombosis.

INTRODUCTION: Early postoperative renal transplant vein thrombosis results in graft loss. We evaluate the effect of administering aspirin 75 mg daily for 28 days following transplantation. METHODS: Prospectively collected data on the outcome of all transplants undertaken in our unit in the five-year period from January 1997 to January 2002 were reviewed, and in cases of graft failure before three months the cause was defined. RESULTS: In the study period, a total of 401 transplants were undertaken (311 cadaveric and 90 living related). There was one case of renal transplant vein thrombosis (0.25%). This represents a significant reduction on the unit's historical incidence of 5.8%, P < 0.001. CONCLUSION: Aspirin 75 mg daily is adequate to virtually abolish renal transplant vein thrombosis and has a role in thromboprophylaxis in other situations where heparin is contraindicated.

Comparison of the collagen VI content within the islet-exocrine interface of the head, body, and tail regions of the human pancreas.

Efficient islet isolation depends on the use of collagenase to digest the extracellular matrix within the islet-exocrine interface, the molecular structure of which is poorly understood. Recently it has been reported that transplantable yields of islets can be isolated from the tail segment of the pancreas alone. This study aimed to quantify and compare the amount of collagenase-resistant collagen VI within the islet-exocrine interface of the head, body, and tail of the human pancreas. Human adult pancreata (n = 5) were retrieved from heart-beating donors (age range, 40-62 years; cold ischemia times <10 hours). Tissue blocks from the head, body, and tail region of each pancreas were fixed in formalin and processed for immuno-labelling of collagen VI, which was quantified in the islet-exocrine interface using a Zeiss KS-400 image analysis system. Data were expressed as area of collagen at the interface relative to the islet area. Statistical analysis was done using paired t test. The mean islet areas in the head, body, and tail regions were not significantly different. Collagen VI was uniformly present within the islet-exocrine interface of all regions of the pancreas and was 0.326 +/- 0.064, 0.324 +/- 0.060, and 0.334 +/- 0.052 microm(2)/islet area (P = .441) in the head, body, and tail, respectively. The content of collagen VI within the islet-exocrine interface was uniform throughout all parts of the adult pancreas. Targeting this collagen subtype with novel collagenase blends may result in consistently improved islet yields and enable a wider number of available donor pancreata to be used.

Development of a novel digestion chamber for human and porcine islet isolation.

BACKGROUND: The current technique of human pancreas digestion for islet isolation relies on selective distribution of collagenase delivered via the pancreatic duct to produce digestion and removal of peri-acinar fibrous tissue. However, the collagenase has relatively little effect on the interlobular fibrous tissue, which must therefore be broken down by mechanical means within the digestion chamber so as to release the contained acini and islets. The current way of achieving this in the Ricordi chamber is to place five or six stainless steel balls within the chamber and shake vigorously. The shaking presumably breaks down the interlobular fibrous tissue by a combination of shear force induced by the movement of tissue through the shaking process, assisted by numerous blows from the steel balls. Intuitively, one would expect some islets would be destroyed rather than released by such a battering. METHODS: In an attempt to improve the efficiency of islet isolation we have designed a new digestion/filtration chamber that consists of a glass cylinder, sealed with Teflon plates holding in mesh filters at each end, secured in place by a central threaded tie-rod and external knurled nuts. A ring-shaped piston within the cylinder can be pushed up and down the travel by two rods passing out through sealed ports in the Teflon disk at one end and connected to an external handle. The handle is used to gently push the piston up and down the travel of the cylinder, which pushes the fluid and tissue through the central lumen of the ring-piston. A series of hooks attached to the central tie-rod catch the fibrous strands of the passing tissue; the shearing forces produced cause disruption by a process thought to be similar to teasing the tissue apart with fine forceps. RESULTS: A series of initial experiments with human pancreas showed the prototype to be too large, causing temperature control problems, and a redesigned smaller chamber was produced, maintaining the crucial design features. Experience processing five human pancreata has now demonstrated that in three of five pancreata the new chamber produced a good yield (>200,000 I.E.) of remarkably well separated and intact islets, the entire dispersion process being under 1 hour. However, in two isolations the collagenase digestion was poor, with few free islets. A copy of the new chamber (reserved for porcine work only) has been produced, as well as a copy of the Ricordi chamber. We have confirmed that the new chamber can isolate porcine islets in large numbers (>5000 islets/g pancreas [n = 2], but note that pig islets are small). CONCLUSION: These preliminary studies are sufficiently encouraging to justify further direct comparison with the Ricordi chamber for the purpose of animal and human islet isolation.

Lymphoproliferative disorders in Oxford renal transplant recipients.

BACKGROUND: Increased cancer incidence, particularly lymphoproliferative disease, is a complication of immunosuppression in organ transplantation. Non-Hodgkin's lymphomas (NHLs) occur frequently during the first year after transplantation, more so in North America than in Europe. METHODS: This study audited and correlated the demographic, clinical, pathological, and outcome features of post-transplant lymphoproliferative disorders (PTLDs) in a large centre in Oxford, and assessed whether the time of onset fitted more with the European or North American pattern. RESULTS: There were 1383 renal transplants in the study period and 27 patients developed lymphoma: 26 NHLs and one Hodgkin's disease (1.95%). Four of the patients never received cyclosporin. The mean time of diagnosis after transplant was 46 months. Most tumours (21/27) presented extranodally. Management included reduction of immunosuppression, surgical excision, antiviral treatment, radiotherapy, and chemotherapy. Three patients presented in the first post-transplant year-0.34% of cyclosporin managed patients-similar to the North American incidence, although the incidence of extranodal late PTLDs was also high (mean onset, 36 months v 15 months international mean). Post-transplant lymphomas were the most common malignancy associated with death in transplant patients. CONCLUSIONS: PTLDs occurred in 2% of renal transplant patients, presenting both in the first year in association with cyclosporin use, as in North America, but also in subsequent years, giving an overall presentation time later than the international mean. The disease usually presented extranodally, accounting for the wide range of symptoms and signs. Despite awareness and active management, the disease contributed to death in more that 50% of patients with PTLDs.

Rising incidence of post-transplant lymphoproliferative disease in kidney transplant recipients.

BACKGROUND: The purpose of this study was to determine whether the incidence of post-transplant lymphoproliferative disease (PTLD) has been increasing in renal transplant recipients in this centre. METHODS: Prospectively gathered data were analysed to establish trends in the epidemiology of PTLD in 1537 patients. RESULTS: Overall, PTLD occurred in 2.3 per cent of renal transplant recipients. An increase in its incidence coincided with the introduction of cyclosporin in the 1980s. However, there was a further increase in the incidence of PTLD in the 1990s when the only change in immunosuppressive policy was the abandonment of pretransplantation blood transfusion. The latter increase was particularly pronounced in patients with early-onset PTLD in whom it presented within 600 days after transplantation. CONCLUSION: The incidence of PTLD has been increasing in renal transplant recipients. The recent increase appears to be independent of cyclosporin and may reflect the reduction in pretransplant blood transfusion. Changes in the incidence of PTLD may also mirror changes in the epidemiology of non-Hodgkin lymphoma in the general population.

Islet transplantation and glucose regulation.

Transplantation of isolated islets of Langerhans for treatment of diabetes has been developed through experimental research in several species and is now being applied to humans with some success albeit limited. A significant problem for human islet allotransplantation or autotransplantation (following pancreatectomy) is the relatively poor yield of islets available for transplantation. The metabolic function of islet transplant recipients that have achieved insulin independence reflects the relatively small mass of insulin-secreting tissue implanted and the fact that only the intraportal site of transplantation appears to allow sufficient graft function to achieve insulin independence. The long-term function of such grafts has been poor, with most grafts showing deterioration in function within 5 years. Studies of islet transplantation in other species showed a similar result, although other sites for islet graft implantation, such as the spleen or kidney capsule, may be associated with a better outcome. These studies, however, also suffer from problems of relatively limited islet mass. Only in the rodent model where isogeneic strains are available is it possible to transplant sufficient numbers of islets to obtain an equivalent functional islet mass similar to that found in the normal pancreas; and in this case near-normal glucose metabolism is obtained and is maintained for the life-span of the animal.

Transplantation of mouse pancreatic islets into primates--in vivo and in vitro evaluation.

BACKGROUND: Islets transplanted from other species to man has the potential to cure diabetes but whether islets are subject to hyperacute rejection after xenotransplantation is contentious. We transplanted mouse pancreatic islets of mouse beneath the primate renal capsule and assessed natural xenoantibody binding, complement activation and cell lysis in vitro. METHODS: Freshly isolated mouse islets were transplanted in a blood clot under the renal capsule of cynolmogus monkeys. The graft was removed after 24 hr for histological and ultrastructural analysis. Freshly isolated mouse pancreatic islets were analyzed in vitro by immunohistochemistry for Gal(alpha1,3)Gal and Von Willebrand factor expression and for IgG, IgM, C3, C4, and C5b-9 binding after incubation in 100% human serum. Complement mediated cell lysis was evaluated by 51Cr release assays after incubation of islets for 4 hr in human serum, plasma, and lymph with and without added neutrophils. RESULTS: Mouse islets transplanted under the renal capsule of cynomolgus monkeys were destroyed within 24 hr by a process involving necrosis with neutrophil and mononuclear cell infiltration. Gal(alpha1,3)Gal was strongly positive on only 10% of islet cells. After islet incubation in 100% human serum before frozen section, human IgG and IgM, C3, C4, and C5b-9 was deposited on islets with increased intensity in the periphery. Measurement of 51Cr release from labeled fresh islets after four hours incubation in 100% human serum showed 17% lysis and was not changed by addition of neutrophils. CONCLUSION: These results indicate that mouse islets in a primate recipient undergo rapid destruction by a process that has features similar to hyperacute rejection in vascularized organs and we propose the same term be used.

An overview of the immune system with specific reference to membrane encapsulation and islet transplantation.

The concept of immunoisolation by use of a bioartificial membrane is discussed, concentrating on the immunological mechanisms that are likely to be operative in the light of recent information on the workings of the immune system. Special attention is given to the use of encapsulation for the purpose of treating autoimmune diabetes by implantation of xenogeneic islet tissue. It is argued that the term immunoisolation is misleading because the immune system is always activated by the indirect pathway of antigen presentation and that the term immunomodulation would be more appropriate.

Low dose aspirin as prophylaxis against renal-vein thrombosis in renal-transplant recipients.

BACKGROUND: Renal-vein thrombosis (RVT) is an infrequent event that accounts for a high proportion of early renal allograft losses, since graft failure secondary to acute irreversible rejection is now relatively rare. The cause of RVT may be related to technical problems, clotting disorders, diabetes, or cyclosporin, but is often difficult to define. METHODS: This retrospective study was performed to examine the influence of aspirin on the incidence of RVT in cadaveric and living-related renal transplant recipients receiving cyclosporin-based triple immunosuppression. The Oxford Transplant Centre database was used to identify all early (<30 day) non-immunological graft failures and case histories were examined for clinical and pathological evidence of RVT. In July 1991, aspirin (75 mg o.d. starting immediately before and continuing for 1 month post-transplant) was introduced as routine prophylaxis against RVT. Prior to this, aspirin prophylaxis was not used. RESULTS: In the 6-year period from July 1985 to June 1991, there were 27 cases of RVT in 475 transplants (5.6%). In the subsequent 6-year period, there were six cases of RVT in 480 transplants (1.2%) (P:<0.01). CONCLUSION: Although not abolished, this indicates a significant reduction in the incidence of RVT with the addition of low-dose aspirin.

Chronic allograft failure in human renal transplantation: a multivariate risk factor analysis.

OBJECTIVE: To identify potential risk factors for the development of chronic renal allograft failure. SUMMARY BACKGROUND DATA: Chronic allograft failure (CAF) is the leading cause of late graft loss in renal transplantation. The authors studied the risk factors for the development of CAF in a single center during a period in which a consistent baseline immunosuppression regimen (cyclosporine, azathioprine, and prednisolone) was used. METHODS: Data from the Oxford Transplant Center Database were assessed on 862 renal allografts during a 10-year period. Risk factors were identified using multivariate logistic regression analysis. RESULTS: Biopsy-proven CAF occurred in 77 patients (9.2%) in the entire group. Multivariate risk factor analysis revealed that early and late acute rejection episodes, proteinuria, and serum triglycerides were significant factors. Acute rejection after 3 months was more important than early acute rejection. Serum triglyceride level and proteinuria at 1 year were both elevated in the CAF group. Male sex provided a protective effect. Serum creatinine levels at 6 months after the transplant were not predictive of the risk of developing CAF. CONCLUSIONS: These results from the largest single-center review to date suggest that both antigen-dependent and -independent factors are involved in the pathogenesis of CAF. Acute rejection at all time points has a significant impact on the development of CAF.

Islet cell transplantation for insulin-dependant diabetes mellitus: perspectives from the present and prospects for the future.

The long-term complications of insulin-dependant diabetes mellitus have become a major health care problem, and it is now clear that they arise from inadequate homeostatic control of blood glucose by injected replacement insulin. Transplantation of pancreatic islets is arguably the most logical approach to restoring metabolic homeostasis in people with diabetes. This review looks at the current status of human islet transplantation and the problems that remain. These include: (1) the limited supply of human islet tissue available for transplantation; (2) the adverse effects of current immunosuppressive protocols on diabetic patients; (3) the problems of primary nonfunction of the transplanted islets; (4) the rejection of islets; and (5) the recurrence of autoimmune diabetic disease. Some of the approaches that might solve these problems are then examined: (1) immune modulation to reduce or prevent immune attack by the recipient's immune system; (2) immunoisolation to prevent recognition of the islet graft; (3) induction of tolerance; (4) xenotransplantation using islets derived from animals; and (5) gene therapy.

An evaluation of the Banff classification of early renal allograft biopsies and correlation with outcome.

BACKGROUND: The Banff classification for assessment of renal allograft biopsies was introduced as a standardized international classification of renal allograft pathology and acute rejection. Subsequent debate and evaluation studies have attempted to develop and refine the classification. A recent alternative classification, known as the National Institutes of Health Collaborative Clinical Trials in Transplantation (NIH-CCTT) classification, proposed three distinct types of acute rejection. The 1997 Fourth Banff meeting appeared to move towards a consensus for describing transplant biopsies, which incorporated both approaches. Patients who received a renal allograft at the Oxford Transplant Centre were managed by a combination of protocol and clinically indicated biopsies. We have undertaken a retrospective analysis of the biopsies correlated with the clinical outcome to test the prognostic value of the original Banff (Banff 93-95) and NIH-CCTT classifications. METHODS: Three hundred and eighty-two patients received renal allografts between May 1985 and December 1989, and were immunosuppressed using a standard protocol of cyclosporine, azathioprine and steroid. Adequate 5-year follow-up data were available on 351 patients, and of these, 293 had at least one satisfactory biopsy taken between days 2 and 35 after transplantation, the latter patients forming the study group. The D2-35 biopsies taken from these patients, which were not originally reported according to the Banff classification, were re-examined and classified according to the Banff 93-95 protocols. For each patient the biopsy found to be the most severely abnormal was selected, and the Banff and NIH-CCTT grading compared with the clinical outcome. RESULTS: Seven hundred and forty-three biopsies taken from 293 patients between days 2 and 35 after transplantation were examined and the patients categorized on the basis of the 'worst' Banff grading as follows. Normal or non-rejection, 20%; borderline, 34%; acute rejection grade I (AR I), 18%; AR IIA, 6%; AR IIB, 14%; AR III, 1%; AR IIIC, 3%; widespread necrosis 3%. The clinical outcome for the last two groups combined was very poor with 18% of grafts functioning at 3 months and 6% at 5 years. The other groups with vascular rejection (AR IIB and AR III) had an intermediate outcome, graft survival being 78% at 3 months and 61% at 5 years. The remaining four groups (normal, borderline, cellular AR I and AR IIA) had the best outcome: graft survival 95% at 3 months and 78% at 5 years with virtually no difference between the four groups. Three forms of acute rejection, namely tubulo-interstitial, vascular and transmural vascular, were identified, but only the latter two categories were associated with a poor outcome. CONCLUSIONS: The eight sub-categories of the Banff classification of renal allograft biopsies are associated with three different prognoses with respect to graft survival in the medium term. These three prognostic groups correspond to the three NIH-CCTT types. The data provide support for the consensus developed at Banff 97 separating tubulo-interstitial, vascular and transmural vascular rejection (types I, II and III acute rejection).

Receptors mediating CGRP-induced relaxation in the rat isolated thoracic aorta and porcine isolated coronary artery differentiated by h(alpha) CGRP(8-37).

1 Receptors mediating CGRP-induced vasorelaxation were investigated in rat thoracic aorta and porcine left anterior descending (LAD) coronary artery and anterior interventricular artery (AIA), using CGRP agonists, homologues and the antagonist h(alpha) CGRP(8-37). 2 In the endothelium-intact rat aorta, h(alpha) CGRP, h(beta) CGRP, rat beta CGRP and human adrenomedullin caused relaxation with similar potencies. Compared with h(alpha) CGRP, rat amylin was about 25 fold less potent, while [Cys(ACM2,7)] h(alpha) CGRP and salmon calcitonin were at least 1000 fold weaker. 3 H(alpha) CGRP(8-37) (up to 10(-5) M) did not antagonize responses to h(alpha) CGRP, h(beta) CGRP or rat beta CGRP (apparent pKB <5). Peptidase inhibitors did not increase either the effect of h(alpha) CGRP or [Cys(ACM,2,7)] h(alpha) CGRP, while h(alpha) CGRP(8-37) remained inactive. Endothelium-dependent relaxation produced by h(alpha) CGRP was accompanied by increases in cyclic AMP and cyclic GMP, that were not inhibited by h(alpha) CGRP(8-37) (10(-5) M). 4 In porcine LAD and AIA, h(alpha) CGRP produced relaxation in an endothelium-independent manner. H(alpha) CGRP(8-37) competitively antagonized h(alpha) CGRP responses (pA2 6.3 and 6.7 (Schild slope 0.9+/-0.1, each), in LAD and AIA, respectively). In LAD artery, h(alpha) CGRP-induced relaxation was accompanied by increases in cyclic AMP that were inhibited by h(alpha) CGRP(8-37) (10(-7)-10(5 )). 5 In conclusion, the antagonist affinity for h(alpha) CGRP(8-37) in porcine coronary artery is consistent with a CGRP1 receptor, while the lack of h(alpha) CGRP(8-37) antagonism in rat aorta could suggest either a CGRP receptor different from CGRP1 and CGRP2 type, or a non-CGRP receptor.

Type 1 diabetes: the facts fit a deficient inhibitory signal given by MHC class II.

This paper presents a hypothesis regarding the aetiology of Type 1 (autoimmune) diabetes, which suggests that autoimmunity is normally prevented by an inhibitory or negative signal delivered by MHC molecules, and that in Type 1 diabetes it is the inability of beta cells to deliver sufficient negative signal from MHC Class II that drives the underlying autoimmune process. Based on a broad survey of the diabetes literature, a list of clinical, pathological, experimental and epidemiological 'facts' about Type 1 diabetes is presented which are considered to be widely accepted as proven. The new theory is then compared to other recent theories on the aetiology of diabetes with regard to its ability to explain these accepted 'facts'.