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Biomedical subjects

C Ricordi

Publications and source records attributed to C Ricordi.

At least 145 records · Page 8Linked to original sources
PubMed1997

The insulin gene is transcribed in the human thymus and transcription levels correlated with allelic variation at the INS VNTR-IDDM2 susceptibility locus for type 1 diabetes.

Type 1, or insulin-dependent diabetes mellitus (IDDM) is an autoimmune disease associated with loss of tolerance to several pancreatic islet cell molecules, including insulin, glutamic acid decarboxylase (GAD), ICA69 and the tyrosine phosphatase IA-2 (refs 1-3). Among several predisposing loci, IDDM2 maps to the insulin gene (INS) VNTR (variable number of tandem repeats) minisatellite on chromosome 11p15 (refs 4-9). Allelic variation at this VNTR locus correlates with steady-state levels of INS mRNA in pancreas and transfected rodent cell lines, but it is difficult to reconcile the association of lower INS mRNA levels in the pancreas with class III VNTRs that are dominantly protective from IDDM. We show that during fetal development and childhood, mRNAs for insulin and other islet cell autoantigens (GAD, ICA69, IA-2) are expressed at low levels in the human thymus. Critically, we also detect proinsulin and insulin protein. VNTR alleles correlate with differential INS mRNA expression in the thymus where, in contrast to the pancreas, protective class III VNTRs are associated with higher steady-state levels of INS mRNA expression. This finding provides a plausible explanation for the dominant protective effect of class III VNTRs, and suggests that diabetes susceptibility and resistance associated with IDDM2 may derive from the VNTR influence on INS transcription in the thymus. Higher levels of (pro)insulin in the thymus may promote negative selection (deletion) of insulin-specific T-lymphocytes which play a critical role in the pathogenesis of type-1 diabetes.

Aging↗
PubMed1997

Effects of ketamine sedation on glucose clearance, insulin secretion and counterregulatory hormone production in baboons (Papio hamadryas).

Since the effects of ketamine sedation seem to differ between subspecies of baboons, we assessed the endocrine response to an intravenous glucose tolerance test (IVGTT) in 12 hamadryas baboons. The first phase insulin secretion, basal insulin, and glucose levels, as well as the glucose clearance, were significantly lower in sedated baboons as compared to fully awake animals. Glucagon and cortisol were significantly higher, while growth hormone was lower during ketamine sedation. Papio hamadryas appears to be a promising pre-clinical model for the study of endocrine replacement therapy in insulin-dependent diabetes. However, the data obtained must be interpreted with the knowledge that the anesthetic employed to allow for testing of the animals does have an effect on the parameters described in this report.

Anesthetics, Dissociative↗
PubMed1997

Long-term function (6 years) of islet allografts in type 1 diabetes.

Eight type 1 diabetic patients, ages 29-41 years, with mean diabetes duration of 23 years (range 18-29 years) received islet transplants from 1 to 5 donors. Seven patients had stable kidney allografts 1-11 years before the islet transplant, and one patient had a simultaneous islet-kidney allograft. Patients' blood glucose control was poor as reflected by the mean +/- SD HbA1c of 9.1 +/- 1.7% before transplant. Of the first three patients, two (1 and 3) achieved insulin independence for 36 and 38 days, respectively. Two recipients rejected their islet grafts within 1 month (2 and 8) and therefore were excluded from analysis. The HbA1c and insulin requirement of the six remaining patients who had persistent islet function for more than 60 days was significantly reduced from 9.3 +/- 1.9 to 6.4 +/- 1.0% (P = 0.002) and from 0.75 +/- 0.15 to 0.35 +/- 0.12 U x kg(-1) x day(-1) (P < 0.001), respectively. The two patients with the longest graft survival (4 and 6) achieved a normalization or near-normalization of their HbA1c levels during 6 years in the absence of severe episodes of hypoglycemia. As demonstrated by a decline in C-peptide response during Sustacal challenge tests over a 6-year period, there was a diminution of islet allograft function over time, despite persistence of normal or near normal HbA1c. We concluded that transplantation of allogeneic islets with an islet mass comparable with whole or segmental pancreas transplants in type 1 diabetic patients can result in long-term islet allograft function; further, we concluded that, in conjunction with small dosages of exogenous insulin, a functioning islet allograft can result in near-normalization of blood glucose levels and significant improvement in HbA1c. The occurrence of severe hypoglycemic episodes observed for patients in the Diabetes Control and Complications Trial was not observed in recipients with functioning islet transplants, despite the continuous need for exogenous insulin therapy to sustain normal HbA1c over the 6-year follow-up. The significant improvement in metabolic control observed for the patients described in this study, and the potential to significantly decrease or halt the progression of diabetic complications, support the continued application of islet allotransplantation as a treatment modality for type 1 diabetic patients.

Adult↗
PubMed1997

Trehalose: a cryoprotectant that enhances recovery and preserves function of human pancreatic islets after long-term storage.

The scarcity of available tissue for transplantation in diabetes and the need for multiple donors make it mandatory to use an optimal cryopreservation method that allows maximal recovery and preservation of beta-cell function. We have developed a method to cryopreserve islets with excellent survival of endocrine cells. Current methods use DMSO as cryoprotectant. Our method involves introducing both DMSO and the disaccharide trehalose into the cells during cooling. Uptake and release of trehalose occurred during the thermotropic lipid-phase transition measured in pancreatic endocrine cells between 5 degrees and 9 degrees C, using [14C]trehalose. Recovery of adult islets after cryopreservation with 300 mmol/l trehalose was 92 vs. 58% using DMSO alone. In vitro function, in terms of insulin content and release in response to secretagogues, was indistinguishable from fresh islets. Grafts from islets cryopreserved with trehalose contained 14-fold more insulin than grafts from islets cryopreserved without trehalose. Results with human fetal islet-like cell clusters (ICCs) were more pronounced: recovery from cryopreservation was 94%, compared with 42% without trehalose. Complete functionality of fetal cells was also restored; tritiated thymidine incorporation and insulin content and release were similar to fresh tissue. After transplantation in nude mice, there was a 15-fold increase in insulin content of grafts from ICCs cryopreserved with trehalose compared with ICCs cryopreserved without trehalose. Thus, the addition of trehalose to cryopreservation protocols leads to previously unobtainable survival rates of human pancreatic endocrine tissue.

Adult↗
PubMed1997

Improved human islet isolation using a new enzyme blend, liberase.

Enzymatic digestion of donor pancreases is a vital step in human and large mammalian islet isolation. The variable enzymatic activities of different batches of commercially available collagenase is a major obstacle in achieving reproducibility in islet isolation procedures. In the present work, the effectiveness of Liberase, a standardized mixture of highly purified enzymes recently developed for the separation of human islets, was compared with that of a traditional collagenase preparation (type P). The results of 50 islet isolations using Liberase enzyme were compared with those of 36 isolations with collagenase, type P. No significant differences in donor age, cold ischemia time, digestion time, or weight of the pancreases were observed between the two groups. Islet yield was significantly higher in the group where the Liberase enzyme was used. All parameters examined (islet number, islet number per gram of tissue, islet equivalent number, and islet equivalent number per gram of tissue) were significantly improved when Liberase enzyme was used. Different lots of Liberase enzyme were tested, and no difference was observed. Islets isolated with Liberase enzyme were also of larger size and were much less fragmented, suggesting a gentler enzymatic action and better preservation of anatomical integrity. Islets isolated with Liberase enzyme, assessed both in vitro and in vivo, revealed a functional profile similar to that of islets separated with collagenase. Liberase enzyme appears, therefore, to represent a new powerful tool for improving the quality of human islet isolation.

Adult↗
PubMed1996

The role of the perforin and Fas pathways of cytotoxicity in skin graft rejection.

Cell-mediated cytotoxicity is believed to play an important role in the mechanisms of rejection following organ and tissue transplantation. We have investigated the role of the perforin and Fas pathways of cell-mediated cytotoxicity on the survival of xenogeneic, allogeneic and hemiallogeneic skin grafts. Wild type C57BL/6 (B6), perforin deficient C57BL/6 (B6 PKO) and Fas ligand deficient (B6 gld/gld) mice were grafted with xenogeneic Lewis rat skin grafts, or allogeneic B10.BR, class I disparate B6 bml, class II disparate B6 bm12, and Fas deficient allogeneic C3H lpr/lpr grafts. No significant difference was observed in the timing of rejection with B6 PKO mice as recipients when compared with normal B6 controls. All grafts were also rejected by B6 gld/gld recipients, but with a significantly delayed pattern of rejection, showing, therefore, that Fas, although not critical, may participate to the mechanisms of rejection. Neither perforin nor Fas, however, seem to be essential for rejection of a skin graft, and it is likely that alternative mechanisms assume an important role in the effector phase of rejection once these cell-cytotoxic pathways are abrogated.

Animals↗
PubMed1996

An assessment of the effects of cadaver donor bone marrow on kidney allograft recipient blood cell chimerism by a novel technique combining PCR and flow cytometry.

A new technique, the PCR-flow assay is described that has allowed for the serial identification and quantitation of discrete mononuclear cell subsets of donor (or recipient) bone marrow derived cells in cadaver kidney transplant recipients infused postoperatively with donor vertebral body bone marrow cells. With fixed permeabilized cells in flow cytometry the amplification power of the polymerase chain reaction (PCR), using fluorescent-labeled primers to identify single copy HLA class II DRbeta1 genes of either donor or recipient origin, is combined with multi-color fluorochrome-labeled CD epitope-specific monoclonal antibodies. The details of the methodology are described; these support the utility of the assay. Initial observations were made on the chimeric makeup of the peripheral blood as well as iliac crest bone marrow between six months and one year posttransplantation in recipients serially followed weekly and then monthly, concomitantly compared with a control group of stable kidney transplant recipients using similar therapeutic protocols, who did not receive cadaver bone marrow. Several findings are of note. In 14 recipients of two bone marrow infusions totalling a mean of 6.29+/-2.18x10(10) cells, donor CD34 positive (+) (immature) cells were fourteen times as numerous in peripheral blood six months postoperatively as in six recipients given half as many bone marrow cells in one infusion (averaging 3.02+/-0.5x10(10)). These donor CD34+ cells unexpectedly averaged 36+/-7% of the total (donor plus recipient) CD34+ subset counted. Moreover, iliac crest bone marrow aspirates contained an average of thirteen times this number of CD34+ cells than in the peripheral blood, supporting the notion of engraftment. Of additional interest, between six months and one year posttransplant although no donor cells could be detected in peripheral blood of the controls there was an identifiable presence of donor CD34+ cells in their iliac crest bone marrow, albeit 10-fold less than the marrow-infused patients. In the clinical follow-up, although there were three unrelated mortalities, there were no additional kidney losses with current serum creatinine concentrations averaging 1.3+/-0.06 mg/dl. In conclusion, the PCR-flow assay presents the possibility of identifying discrete subsets of donor or recipient cells that may have an immunoregulatory function.

Adoptive Transfer↗
PubMed1996

A hammerhead ribozyme that cleaves perforin and fas-ligand RNAs in vitro.

Polyfunctional ribozymes targeting more than one RNA might be ideal for treatment of diseases caused by the expression of more than one mRNA. This is the case of Graft versus Host Disease, which in the mouse model is due to the action of two proteins responsible for different lytic pathways: perforin and fasligand. We have created a bifunctional ribozyme fusing two antiperforin and antifas-ligand hammerhead ribozymes with a stretch of CA dinucleotides. This bifunctional ribozyme is able to recognize and cleave in vitro perforin and fas-ligand mRNA in a specific and selective fashion, with a catalytic efficiency similar to that of its individual components.

Animals↗
PubMed1996

Liver transplantation without immunosuppression: future perspectives.

It has been hypothesized that a state of microchimerism in recipients of organ transplants may result in donor-specific tolerance to the graft. Numerous studies show that infusion of donor-derived bone marrow cells can, indeed, achieve systemic chimerism in the recipient and effectively prolong allograft survival. We have compared organ and patient survival in recipients of liver allografts alone (controls) or in combination with single or multiple infusions of donor bone marrow cells; recipients were infused either at day 0 (perioperatively) or at day 0 and 11 post-transplant. The incidence of rejection episodes and survival of the liver allograft were significantly reduced in recipients of two bone marrow infusions compared to controls; recipients of one infusion, conversely, experienced a higher number of rejection episodes when compared to controls, pointing to a possible sensitizing role of a single bone marrow infusion if administered perioperatively. Variables such as timing, number and composition of the bone marrow inocula still remain to be elucidated but may be of critical importance for the attainment of a state of donor-specific tolerance without the need for immunosuppressive therapy in recipients of organ allografts.

Animals↗
PubMed1996

Status of liver and gastrointestinal transplantation at the University of Miami.

The liver transplant program at the University of Miami, established in 1987, was rejuvenated in June 1994 with the addition of new staff and expanded to include all organs of the gastrointestinal tract. Since its inception, 630 patients have been transplanted in the program. During the past 2 years we performed 349 transplants in 318 patients (livers n = 323 in 298 patients, liver + kidneys n = 13, liver + islet n = 10, liver + kidney + islets n = 1, liver + heart n = 10, liver + lung n = 1). These included 4 split-liver, 3 living-related, multiple reduced-sized and one "Domino" liver transplant. We have an active pediatric program and 10% of our transplanted patients are pediatric. Our overall patient and graft survival rates were 81% and 78%, respectively. The intestinal transplant program was launched in August 1994. To date we have performed 22 intestinal transplants, in 9 adults and 13 children. These transplants included 4 isolated intestinal, 11 combined liver-intestinal and 7 multivisceral transplants. Overall patient and graft survival rates were 55% and 50%, respectively. During the past 2 years several studies involving immunosuppressive agents were carried out: 1)Mycophenolate Mofetil (MMF) was used as induction therapy and as rescue therapy in patients with steroid-resistant rejection. Tacrolimus toxicity, and chronic rejection; 2) Neoral was compared with Tacrolimus in patients with Hepatitis C; and 3) MMF was added as triple therapy for the intestinal transplants. We used alpha interferon-2b (alpha-IFN) in hepatitis C positive patients in the early posttransplant period and found that it appears to be a safe drug. There was no increase in rejection in patients receiving alpha-IFN, and patient and graft survival were the same as in our overall patient population. A combination a-IFN with Ribavirin will be undertaken in the near future. The use of Lamivudine in hepatitis B patients was shown to be effective in preventing and treating recurrence of hepatitis B posttransplant. Unmodified donor bone marrow cells (DBMC) were isolated from the vertebral bodies of the same cadaveric liver donors. Donor bone marrow dose, number of cells and/or number (or timing) of infusions were investigated to determine which variables affected the ability of DBMC to engraft in the liver recipient. The long-term benefit of DBMC needs further follow-up. Although, our patient and graft survival for liver transplant recipients is comparable to other large centers nationally and internationally, we still have some challenges to overcome. These include: 1) control and prevention of recurrent HCV, 2) improved treatment for hepatocellular cancer pre- and posttransplant, and 3) treatment and prevention of chronic rejection. Intestinal transplantation remains an even greater challenge. Diagnostic tests to determine intestinal function need further development and although MMF has shown some promise in this field, newer immunosuppressive medications need to be investigated to prevent rejection and avoid over immunosuppression.

Adult↗