Leda Racanicchi

Grafts of microencapsulated pancreatic islet cells for the therapy of diabetes mellitus in non-immunosuppressed animals.

Pancreatic‐islet‐cell transplantation may reverse hyperglycaemia in diabetic recipients that undertake general pharmacological immunosuppression. A major challenge that remains is the need to avoid immunosuppression associated with the use of allogeneic or heterologous islet cells. In the present study we demonstrate the use of microencapsulation of cells using artificial biocompatible and permselective membranes prepared with alginic acid derivatives and polyamino acids. While characterization of the microcapsule constituent polymers continues to progress, other technical issues such as definition of the immunobarrier capacity, biocompatibility, size, shape and graft site have come into sharper focus. Assessment of microcapsules properties, in order to establish possible guidelines for fabrication of reproducible membranes, and results from both in vitro functional testing, and in vivo encapsulated‐islet‐transplant outcome in several animal models of diabetes are reported.

Up-regulation of Glycohydrolases in Alzheimer's Disease Fibroblasts Correlates with Ras Activation

The lysosomal system is up-regulated in the brain of patients with Alzheimers Disease (AD), as demonstrated by previous experiments carried out in postmortem samples of brain patients. In this paper we provide evidence that an up-regulation of lysosomal glycohydrolases (α-d-mannosidase, β-d-hexosaminidase, and β-d-galactosidase) takes place in skin fibroblasts from AD patients affected either by sporadic or familial forms and is detectable also in presymptomatic subjects carrying the above mutations but healthy at the time of skin biopsy. This increase of enzyme activity is consequent to a transcriptional up-regulation. The oncogene Ras appears to be involved in the regulation of enzymatic activity. A parallel increase of Ras transcript and Ras protein, without an increase of p44/p42 MAPK activation was revealed in the same AD fibroblasts. An activation of p38 MAPK already described to occur in neurodegenerative diseases such as Alzheimers, was also found in fibroblasts derived from AD patients. High levels of expression of the constitutively active form of Ras in normal or AD fibroblasts induced glycohydrolases up-regulation. Overall results demonstrated that glycohydrolases up-regulation, as well as Ras up-regulation, are early markers of AD, detectable at peripheral level, and good candidates to be exploited for diagnostic purposes. These data also provide the first proof for a role of Ras in regulating lysosomal glycohydrolases expression.

Calcium and Fos Involvement in Brain‐Derived Ca2+‐Binding Protein (S100)‐Dependent Apoptosis in Rat Phaeochromocytoma Cells

Brain‐derived calcium‐binding protein S100 induces apoptosis in a significant fraction of rat phaeochromocytoma (PC12) cells. We used single cell techniques (patch clamp, videomicroscopy and immunocytochemistry) to clarify some of the specific aspects of S100‐induced apoptosis, the modality(ies) of early intracellular Ca2+ concentration increase and the expression of some classes of genes (c‐fos, c‐jun, bax, bcl‐x, p‐15, p‐21) known to be implicated in apoptosis of different cells. The results show that S100: (1) causes an increase of [Ca2+]i due to an increased conductance of L‐type Ca2+ channels; (2) induces a sustained increase of the Fos levels which is evident since the first time point tested (3 h) and remains elevated until to the last time point (72 h). All these data suggest that S100‐derived apoptosis in PC12 cells may be the consequence of a system involving an increase in L‐type Ca2+ channel conductance with consequent [Ca2+]i increase which up‐regulates, directly or indirectly, the expression of Fos.

Long-term cultured neonatal porcine islet cell monolayers: a potential tissue source for transplant in diabetes.

Abstract:  Background:  The restricted availability of cadaveric human donor pancreases mandates validation of possibly inexhaustible, alternative sources of insulin secretory cells in order to expand islet transplant for the therapy of insulin dependent diabetes mellitus (T1DM).

Effects of Streptozotocin on In Vitro Long-Term Cultured Human Islet Cell Monolayers

Replacement beta cells may be generated from stem/progenitor cells, possibly residing within the pancreatic islet cells. We sought to investigate the possible use of human islet (HI)-derived cell monolayers as a possible source for beta cells. These cells could be propagated in vitro and potentially induced to acquire glucose-stimulated insulin release (GSIR) ability. Loss of three-dimensional architecture, following monolayer establishment, resulted in either rearrangement of both pancreatic hormone expression and key transcription factors or decrease in insulin production or GSIR disappearance. We showed that cell deregulation/dedifferentiation was reversible by treating the cell monolayers, after five passages with streptozotocin (STZ), a well-known beta-cell toxin. When used at subtoxic concentrations, STZ promoted differentiation of HI-derived cell monolayers and GSIR recovery. This effect allowed production of insulin-secreting cells starting from cell monolayers doubled five times in vitro, meaning a 400-fold increase in the cellular starting material. Such islet cell expansion capability in vitro might elucidate a new source of insulin-secreting cells thereby possibly overcoming the problem posed by searching for human organ donation.

Effects of poly-L-lysine and collagen on FH-B-TPN cell differentiation into endocrine cell phenotype.

Pdx-1 genetically engineered FH-B-TPN cells might represent a source for insulin-secreting cells. We then have tested whether poly-L-lysine (PLL) and collagen (C) exposure in vitro promote three-dimensional particle formation and differentiation toward an endocrine cell phenotype. On these matrices, we observed that FH-B-TPN cells showed a tendency to either aggregate when seeded on PLL or to form uniform cell monolayers, but not to aggregate on C. While insulin was released in any condition, GSIR was only associated with PLL mainly at 24 and 72 hours of culture. Various culture matrices influenced the expression of glucose transporter type 2 and gluco kinase, being they expressed more intensively on PLL rather than C or in controls. mRNA expression for NeuroD/Beta2, Isl-1, Ras, Metalloproteinase-2 (MMP-2), -9 and -7 also were affected, with PLL inducing increased expression of NeuroD/Beta2 of Isl-1, and no difference between C and control. PLL, unlike C, strongly increased Ras through observation times. MPP-2 and -9 were decreased by both PLL and C, whereas MMP-7 was increased by PLL. PLL, usually employed to promote culture cell adhesion, has been proven capable to stimulate pancreatic endocrine function and cell aggregation and to stimulate gene expression of key markers for either insulin transcription or MMP-7.