Donor bone marrow cells play a role in the prevention of accelerated graft rejection induced by semi-allogeneic spleen cells in transplantation

Spleen or spleen plus bone marrow cells from (BALB/c x C57Bl/6)F1 donors were transferred into BALB/c recipients 21 days before skin or cardiac transplantation. Prolonged graft survival was observed on recipients treated with the mixture of donor-derived cells as compared to those treated with spleen cells alone. We evaluated the expression of CD45RB and CD44 by splenic CD4(+) and CD8(+) T cells 7 and 21 days after donor cell transfer. The populations of CD8(+)CD45RB(low) and CD8(+)CD44(high) cells were significantly decreased in mice pre-treated with donor spleen and bone marrow cells as compared to animals treated with spleen cells only, although these cells expanded in both groups when compared to an earlier time-point. No differences were observed regarding CD4+ T cell population when recipients of donor-derived cells were compared. An enhanced production of IL-10 was observed seven days after transplantation in the supernatants of spleen cell cultures of mice treated with spleen and bone marrow cells. Taken together these data suggest that donor-derived bone marrow cells modulate the sensitization of the recipient by semi-allogeneic spleen cells in part by delaying the generation of activated/memory CD8(+) T cells leading to enhanced graft survival. (c) 2007 Elsevier B.V. All rights reserved.

Vimentin Expression and Myofibroblast Infiltration Are Early Markers of Renal Dysfunction in Kidney Transplantation: An Early Stage of Chronic Allograft Dysfunction?

Introduction. The objective of this study was to show the morphologic characteristics of allograft renal biopsies in renal transplant patients with stable renal function, which can potentially be early markers of allograft dysfunction, after 5 years of follow-up. Methods. Forty-nine renal transplant patients with stable renal function were submitted to renal biopsies and simultaneous measurement of serum creatinine (Cr). Histology was evaluated using Banff scores, determination of interstitial fibrosis by Sirius red staining and immunohistochemical study of proximal tubule and interstitial compartment (using cytokeratin, vimentin, and myofibroblasts as markers). Biopsies were evaluated according to the presence or absence of the epitheliomesenchymal transition (EMT). The interstitial presence of myofibroblasts and tubular presence of vimentin was also analyzed simultaneously. Renal function was measured over the follow-up period to estimate the reduction of graft function. Results. Median posttransplant time at enrollment was 105 days. Patients were followed for 64.3 +/- 8.5 months. The mean Cr at biopsy time was 1.44 +/- 0.33 mg/dL, and after the follow-up it was 1.29 +/- 0.27 mg/dL. Nine patients (19%) had a reduction of their graft function. Eleven biopsies (22%) had tubulointerstitial alterations according to Banff score. Seventeen biopsies (34%) presented EMT. Fifteen biopsies (32%) had high interstitial expression of myofibroblasts and tubular vimentin. Using Cox multivariate analysis, HLA and high expression of interstitial myofibroblasts and tubular vimentin were associated with reduction of graft function, yielding a risk of 3.3 (P = .033) and 9.8 (P = .015), respectively. Conclusion. Fibrogenesis mechanisms occur very early after transplantation and are risk factors for long-term renal function deterioration.

Islet neogenesis-associated protein signaling in neonatal pancreatic rat islets: involvement of the cholinergic pathway

Islet neogenesis associated protein (INGAP) increases islet mass and insulin secretion in neonatal and adult rat islets. lit the Present Study, we measured the short- and long-term effects of INGAP-PP (a pentadecapeptide having the 104-118 amino acid sequence of INGAP) upon islet protein expression and phosphorylation of components of the PI3K, MAPK and cholinergic pathways, and on insulin secretion. Short-term exposure of neonatal islets to INGAP-PP (90 s, 5, 15, and 30 min) significantly increased Akt1(-Ser473) and MAPK3/1(-Thr202/Tyr204) phosphorylation and INGAP-PP also acutely increased insulin secretion from islets perifused with 2 and 20 mM glucose. Islets cultured for 4 days in the presence of INGAP-PP showed an increased expression of Akt1, Frap1, and Mapk1 mRNAs as well as of the muscarinic M3 receptor subtype, and phospholipase C (PLC)-beta 2 proteins. These islets also showed increased Akt1 and MAPK3/1 protein phosphorylation. Brief exposure of INGAP-P-treated islets to carbachol (Cch) significantly increased P70S6K(-Thr389) and MAPK3/1 phosphorylation and these islets released more insulin when challenged with Cch that was prevented by the M3 receptor antagonist 4-DAMP in a concentration-dependent manner. In conclusion, these data indicate that short- and long-term exposure to INGAP-PP significantly affects the expression and the phosphorylation of proteins involved in islet PI3K and MAPK signaling pathways. The observations of INGAPP-PP-stimulated up-regulation of cholinergic M3 receptors and PLC-beta 2 proteins, enhanced P70S6K and MAIIK3/1 phosphorylation and Cch-induced insulin secretion suggest a participation of the cholinergic pathway in INGAP-PP-mediated effects.

Endurance training activates AMP-activated protein kinase, increases expression of uncoupling protein 2 and reduces insulin secretion from rat pancreatic islets

Endurance exercise is known to enhance peripheral insulin sensitivity and reduce insulin secretion. However, it is unknown whether the latter effect is due to the reduction in plasma substrate availability or alterations in beta-cell secretory machinery. Here, we tested the hypothesis that endurance exercise reduces insulin secretion by altering the intracellular energy-sensitive AMP-activated kinase (AMPK) signaling pathway. Male Wistar rats were submitted to endurance protocol training one, three, or five times per week, over 8 weeks. After that, pancreatic islets were isolated, and glucose-induced insulin secretion (GIIS), glucose transporter 2 (GLUT2) protein content, total and phosphorylated calmodulin kinase kinase (CaMKII), and AMPK levels as well as peroxisome proliferator-activated receptor-gamma coactivator-1-alpha (PGC-1 alpha) and uncoupling protein 2 (UCP2) content were measured. After 8 weeks, chronic endurance exercise reduced GIIS in a dose-response manner proportionally to weekly exercise frequency. Contrariwise, increases in GLUT2 protein content, CaMKII and AMPK phosphorylation levels were observed. These alterations were accompanied by an increase in UCP2 content, probably mediated by an enhancement in PGC-1 alpha protein expression. In conclusion, chronic endurance exercise induces adaptations in beta-cells leading to a reduction in GIIS, probably by activating the AMPK signaling pathway. Journal of Endocrinology (2011) 208, 257-264

Effect of Age on Outcome of Reduced-Intensity Hematopoietic Cell Transplantation for Older Patients With Acute Myeloid Leukemia in First Complete Remission or With Myelodysplastic Syndrome

Purpose Acute myelogenous leukemia (AML) and myelodysplastic syndrome (MDS) primarily afflict older individuals. Hematopoietic cell transplantation (HCT) is generally not offered because of concerns of excess morbidity and mortality. Reduced-intensity conditioning (RIC) regimens allow increased use of allogeneic HCT for older patients. To define prognostic factors impacting long-term outcomes of RIC regimens in patients older than age 40 years with AML in first complete remission or MDS and to determine the impact of age, we analyzed data from the Center for International Blood and Marrow Transplant Research (CIBMTR). Patients and Methods We reviewed data reported to the CIBMTR (1995 to 2005) on 1,080 patients undergoing RIC HCT. Outcomes analyzed included neutrophil recovery, incidence of acute or chronic graft-versus-host disease (GVHD), nonrelapse mortality (NRM), relapse, disease-free survival (DFS), and overall survival (OS). Results Univariate analyses demonstrated no age group differences in NRM, grade 2 to 4 acute GVHD, chronic GVHD, or relapse. Patients age 40 to 54, 55 to 59, 60 to 64, and >= 65 years had 2-year survival rates as follows: 44% (95% Cl, 37% to 52%), 50% (95% Cl, 41% to 59%), 34% (95% Cl, 25% to 43%), and 36% (95% Cl, 24% to 49%), respectively, for patients with AML (P = .06); and 42% (95% Cl, 35% to 49%), 35% (95% Cl, 27% to 43%), 45% (95% Cl, 36% to 54%), and 38% (95% Cl, 25% to 51%), respectively, for patients with MDS (P = .37). Multivariate analysis revealed no significant impact of age on NRM, relapse, DFS, or OS (all P>.3). Greater HLA disparity adversely affected 2-year NRM, DFS, and OS. Unfavorable cytogenetics adversely impacted relapse, DFS, and OS. Better pre-HCT performance status predicted improved 2-year OS. Conclusion With these similar outcomes observed in older patients, we conclude that older age alone should not be considered a contraindication to HCT.

Immobilization of primary cultures of insulin-releasing human pancreatic cells

Transplantation of pancreatic islets isolated from organ donors constitutes a promising alternative treatment for type 1 diabetes, however, it is severely limited by the shortage of organ donors. Ex vivo islet cell cultures appear as an attractive but still elusive approach for curing type 1 diabetes. It has recently been shown that, even in the absence of fibrotic over-growth, several factors, such as insufficient nutrition of the islet core, represent a major barrier for long-term survival of islets grafts. The use of immobilized dispersed cells may contribute to solve this problem due to conceivably easier nutritional and oxygen support to the cells. Therefore, we set out to establish an immobilization method for primary cultures of human pancreatic cells by adsorption onto microcarriers (MCs). Dispersed human islets cells were seeded onto Cytodex1 microcarriers and cultured in bioreactors for up to eight days. The cell number increased and islet cells maintained their insulin secretion levels throughout the time period studied. Moreover, the cells also presented a tendency to cluster upon five days culturing. Therefore, this procedure represents a useful tool for controlled studies on islet cells physiology and, also, for biotechnological applications.

Low protein diet confers resistance to the inhibitory effects of interleukin1 beta on insulin secretion in pancreatic islets

High protein content in the diet during childhood and adolescence has been associated to the onset insulin-dependent diabetes mellitus. We investigated the effect of interleukin-1 beta (IL-I beta) on insulin secretion, glucose metabolism, and nitrite formation by islets isolated from rats fed with normal protein (NP, 17%) or low protein (LP, 6%) after weaning. Pretreatment of islets with IL-1 beta for 1 h or 34 h inhibited the insulin secretion induced by glucose in both groups, but it was less marked in LP than in NP group. Islets from LP rats exhibited a decreased IL-1 beta -induced nitric oxide (NO) production, lower inhibition of D-[(UC)-C-14]-glucose oxidation to (CO2)-C-14, and less pronounced effect of IL-1 beta on alpha -ketoisocaproic acid-induced insulin secretion than NP islets. However, when the islets were stimulated by high concentrations of K+ the inhibitory effect of IL-1 beta on insulin secretion was not different between groups. In conclusion, protein restriction protects beta -cells of the deleterious effect of IL-1 beta, apparently, by decreasing NO production. The lower NO generation in islets from protein deprived rats may be due to increased free fatty acids oxidation and consequent alteration in Ca2+ homeostasis. (C) 2001 Elsevier B.V. All rights reserved.

Pancreas Transplantation Prevents Morphologic and Ultrastructural Changes in Pulmonary Parenchyma of Alloxan-Induced Diabetic Rats

Purpose. The aim of this study was to evaluate whether pancreas transplantation (PT) is a suitable method for controlling histopathologic changes in lungs of alloxan-induced diabetic rats.Methods. Sixty inbred male Lewis rats were randomly assigned to 3 experimental groups: NC, 20 nondiabetic control rats; DC, 20 untreated diabetic control rats; and PT, 20 diabetic rats that received syngeneic PT from normal donor Lewis rats. Each group was further divided into 2 subgroups of 10 rats each, which were killed after 4 and 12 weeks of follow-up. Clinical and laboratory parameters, fresh and fixed lung weights, and fixed lung volumes were recorded for all rats. Total number of alveoli, alveolar perimeter, alveolar surface area, and alveolar epithelial (AE) and endothelial capillary (EC) basal laminae thickening were randomly measured in 5 rats from each subgroup by using an image analyzer. For light microscopy, 250 alveoli were analyzed in each subgroup. For electron microscopy, 50 electron micrographs were examined for each subgroup.Results. The DC rats showed elevated blood glucose and glycosylated hemoglobin levels, with insulin blood levels significantly lower than the NC rats (P < .001). Fresh and fixed lung weights and fixed volumes were significantly reduced in these rats, although their proportions to body weight were increased at 12 weeks (P < .01). The total number of alveoli in diabetic rats was higher than in control rats, whereas alveolar perimeter and surface area were significantly diminished (P < .01). AE and EC basal laminae were significantly thicker in DC than in NC (P < .01). Successful PT corrected all clinical and metabolic changes in diabetic rats, with sustained normoglycemia throughout the study. Morphologic and morphometric changes observed in diabetic lungs were completely prevented in PT rats from 4 weeks after transplant.Conclusion. We conclude that PT can control morphologic and ultrastructural changes in pulmonary parenchyma, suggesting a promising perspective for preventing other chronic diabetic lesions.

Opinion of hepatic transplant waiting list patients regarding split liver transplantation

Background. Split liver transplantation (SLT) increases organ supply for hepatic transplantation. Long-term patient survival and complication rates seem to be equivalent between orthotopic liver transplantation (OLT) and SLT. There are controversies among transplant physicians due to an ethical dilemma between benefiting individual needs or those of society. Barshes and Goss (Am J Transplant 5:2047, 2005) demonstrated that the majority of adult liver transplant candidates are favorable to SLT. The aim of our study was to evaluate the opinions of patients at a Brazilian university hospital regarding SLT.Materials and Methods. A questionnaire with 14 questions was applied to 50 patients included in a hepatic transplant waiting list regarding SLT.Results. The overall attitudes of 66% of the participants were classified as utilitarian, 31% were classified as self-preserving, and 3% were undecided. Ninety-one percent of patients would be willing to share even if their expected survival after SLT was shorter than that with OLT. For 77% of patients, children must have priority over adults. However, 83% were unaware of the donors for pediatric transplantations.Conclusions. SLT is a consistent solution for organ demand despite controversies among transplant physicians. The present study demonstrated that most patients were favorable to SLT. In conclusion, attitudes toward graft sharing are not barriers to SLT.

Experimental pancreas transplantation: the consequences of the portocaval shunt on the blood glucose, plasma insulin, and glucagon

A proposição foi realizada, no período pós-operatório imediato, em 40 ratos Wistar, distribuídos por sorteio em doi grupos: grupo NC, vinte ratos correspondentes ao grupo controle, não diabético, submetidos a operação simulada e o grupo PT, 20 ratos correspondentes ao grupo diabético que recebeu transplante de pâncreas heterotópico de ratos Wistar normais. Durante sete dias, antes do transplante, e 1, 3, 6, 12, 24, 48, 72, 96 horas após, determinava-se a glicose sanguínea, a insulina plasnática e o glucagon. Estes parâmetros eram obtidos também do grupo NC. Diabetes mellitus experimental era induzida pela administração intravenosa de aloxana. O grupo PT era imunosuprimido com ciclosporina A. O grupo NC apresentou níveis normais de glicose sanguínea, de insulina plásmica e de glucagon, durante todo o experimento. Foi encontrada nítida hiperinsulinemia no sangue venoso periférico do grupo PT. A insulina plasmática era significantemente maior no grupo PT comparada ao grupo NC começando 72 horas após o transplante. O glucagon plasmático, elevado no período pré-transplante, não se alterou após o transplante. Apesar de hiperinsulinemia e hiperglucagonemia, os níveis de glicose sanguínea eram elevados 6 horas após o transplante e mantiveram-se normais após este período. Considerando-se os níveis de glicose sanguínea 12 horas pós-transplante, não houve diferença estatisticamente significante entre os grupos PT e NC, até o sacrifício.

Autologous transplantation of adult adipose derived stem cells into rabbit urethral wall

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Effect of amniotic membrane transplantation on corneal healing and proteoglycan expression in an experimental model of limbal deficiency in rabbits

PURPOSE. Amniotic membrane transplantation (AMT) has been used as a graft or as a dressing in ocular surface reconstruction, facilitating epithelization, maintaining normal epithelial phenotype, and reducing inflammation, vascularization, and scarring. The corneal transparency is due, at least in part, to the arrangement in orthogonal lamellae of collagen fibrils, surrounded by proteoglycans (PGs). These PGs regulate fibrilogenesis, the matrix assembly, and ultimately the corneal transparency. The purpose of the present study was to investigate the effects of AMT upon the corneal PGs after severe limbal injury.METHODS. Experiments were performed on the right corneas of 22 New Zealand female albino rabbits, and their left corneas were used as matched controls. These animals were divided into 3 groups: G1 (n = 10): total peritomy and keratolimbectomy, followed by application of 0.5 M NaOH; G2 (n = 10): submitted to the same trauma as G1, and treated by AMT; G3: no trauma, only AMT (n = 2). The right corneas of G2 and G3 were covered by DMSO 4 cryopreserved human amniotic membrane, fixed by interrupted 9-0 mononylon sutures, with its stromal face toward the ocular surface. After 7 or 30 days, the corneas were removed and PGs were extracted.RESULTS. Normal corneas contained approximately 9 mg of PGs per gram of dry tissue. AMT on intact cornea (G3) did not cause any changes in the concentration of PGs. In contrast, injured corneas contained much less PGs, both on the seventh and on the 30th day posttrauma. The PG concentration was even lower in injured corneas treated by AMT. This decrease was due almost exclusively to dermatan sulfate PGs, and the structure of dermatan sulfate was also modified, indicating changes in the biosynthesis patterns.CONCLUSIONS. Although beneficial effects have been observed on clinical observation and concentration of soluble proteins after AMT, the normal PG composition of cornea was not attained, even 30 days postinjury, indicating that the normal ocular surface reconstruction, if possible, is a long-term process. (Eur J Ophthalmol 2010; 20: 290-9)