Stefano Olivieri

Activation of the aryl hydrocarbon receptor promotes allograft-specific tolerance through direct and dendritic cell–mediated effects on regulatory T cells

VAF347 is a low-molecular-weight compound, which activates the aryl hydrocarbon receptor (AhR). Herein, we report that oral administration of a water-soluble derivative of VAF347 (VAG539) promotes long-term graft acceptance and active tolerance in Balb/c mice that receive a transplant of MHC-mismatched pancreatic islet allografts. In vivo VAG539 treatment results in increased frequency of splenic CD4(+) T cells expressing CD25 and Foxp3, markers associated with regulatory T (Tr) cells, and in vitro VAF347 treatment of splenic CD4(+) T cells improved CD4(+)CD25(+)Foxp3(+) T-cell survival. Interestingly, transfer of CD11c(+) dendritic cells (DCs), but not of CD4(+) T or CD19(+) B cells, from VAG539-treated long-term tolerant hosts into mice that recently underwent transplantation resulted in donor (C57Bl/6)-specific graft acceptance and in a significantly higher frequency of splenic CD4(+)CD25(+)Foxp3(+) Tr cells. Furthermore, the transfer of CD4(+)CD25(+) T cells from these mice into mice that recently underwent transplantation promoted graft acceptance. Similarly, cell therapy with in vitro VAF347-treated bone marrow-derived mature DCs prevented islet graft rejection, and reduced OVA-specific T-cell responses in OVA-immunized mice. Collectively, our data indicate that AhR activation induces islet allograft-specific tolerance through direct as well as DC-mediated effects on Tr-cell survival and function.

Mitochondrial ferritin expression in adult mouse tissues

Mitochondrial ferritin (FtMt) is a novel ferritin type specifically targeted to mitochondria. It is highly expressed in the human testis and in sideroblasts from patients with sideroblastic anemia, but other organs have not been studied. To study its expression in the main organs of the mouse, we first used RT-PCR and then produced recombinant mouse FtMt and specific antibodies. Immunohistochemistry analyses confirmed that FtMt is highly expressed in mouse testis, particularly in spermatocytes and interstitial Leydig cells. The protein was also identified in other organs including heart, brain, spinal cord, kidney, and pancreatic islet of Langerhans but not in liver and splenocytes, which have iron storage function and express high levels of cytosolic ferritins. Results indicate that the primary function of ferritin FtMt is not involved in storing cellular or body iron, but its association with cell types characterized by high metabolic activity and oxygen consumption suggests a role in protecting mitochondria from iron-dependent oxidative damage.

Up-regulation of CD1d expression restores the immunoregulatory function of NKT cells and prevents autoimmune diabetes in nonobese diabetic mice.

The immunoregulatory function of NKT cells is crucial for prevention of autoimmunity. The prototypical NKT cell Ag α-galactosylceramide is not present in mammalian cells, and little is known about the mechanism responsible for NKT cell recruitment and activation. Up-regulation of CD1d, the NKT cell restriction molecule, expressed on mononuclear cells infiltrating the target organ, could represent the physiological trigger for NKT cells to self-contain T cell immunity and to prevent autoimmune disease. Recognition of CD1d, either by itself or bound to self-ligands (selfCD1d), could drive NKT cells toward an immunoregulatory phenotype. Hence, ineffective NKT cell-mediated immunoregulation in autoimmune-prone individuals including nonobese diabetic (NOD) mice could be related to defective signals that regulate CD1d expression at time and site of autoimmunity. To test this hypothesis, we transgenically overexpressed CD1d molecules under the control of the insulin promoter within the pancreatic islets of NOD mice (insCD1d). Recognition of overexpressed CD1d molecules rescued NKT cell immunoregulatory function and prevented autoimmune diabetes in insCD1d transgenic NOD mice. Protection from diabetes was associated with a biased IL-4-secreting cytokine phenotype of NKT cells and alteration of the cytokine microenvironment in the pancreatic lymph nodes of transgenic mice. The net effect was a reduced development of the autoimmune T cell repertoire. Our findings suggest that up-regulation of CD1d expression during inflammation is critical to maintain T cell homeostasis and to prevent autoimmunity.

Amplification and overexpression of PRUNE in human sarcomas and breast carcinomas - a possible mechanism for altering the nm23-H1 activity

PRUNE, the human homologue of the Drosophila gene, is located in 1q21.3, a region highly amplified in human sarcomas, malignant tumours of mesenchymal origin. Prune protein interacts with the metastasis suppressor nm23-H1, but shows impaired affinity towards the nm23-H1 S120G mutant associated with advanced neuroblastoma. Based on these observations, we previously suggested that prune may act as a negative regulator of nm23-H1 activity. We found amplification of PRUNE in aggressive sarcoma subtypes, such as leiomyosarcomas and malignant fibrous histiocytomas (MFH) as well as in the less malignant liposarcomas. PRUNE amplification was generally accompanied by high mRNA and moderate to high protein levels. The sarcoma samples expressed nm23-H1 mostly at low or moderate levels, whereas mRNA and protein levels were moderate to high in breast carcinomas. For the more aggressive sarcoma subtypes, 9/13 patients with PRUNE amplification developed metastases. A similar situation was observed in all breast carcinomas with amplification of PRUNE. Infection of NIH3T3 cells with a PRUNE recombinant retrovirus increased cell proliferation. Possibly, amplification and overexpression of PRUNE has the same effect in the tumours. We suggest that amplification and overexpression of PRUNE could be a mechanism for inhibition of nm23-H1 activity that affect the development or progression of these tumours.

Differential expression of ceruloplasmin isoforms in the cerebrospinal fluid of amyotrophic lateral sclerosis patients

Amyotrophic lateral sclerosis (ALS) a fatal degenerative disease that selectively affects motor neurons, likely results from a complex interplay among oxidative injury, excitotoxic stimulation, protein aggregation and genetic factors. Ceruloplasmin (Cp) protein is a ferroxidase that oxidizes toxic ferrous iron to its nontoxic ferric form, protecting the central nervous system (CNS) from iron deposition. Cp is thus considered as one of the main systems dedicated to the protection of the CNS from oxidative stress damage. We investigated Cp protein behaviour in the cerebrospinal fluid (CSF) of ALS patients of recent onset. An increased expression of Cp was observed in ALS (n = 16) compared to two control groups (healthy subjects, n = 11 and peripheral neuropathy patients, n = 10). 2‐DE analysis revealed a differential expression of Cp isoforms in ALS patients compared to controls. ALS samples showed an increase in the relative abundance of more basic Cp forms, corresponding to the nonsialylated proteins. Despite the increase in protein expression, ferroxidase activity evaluated in the CSF of ALS patients was comparable to that of the controls, indicating a Cp functional impairment. Ceruloplasmin isoforms profile may be proposed as disease feature that could provide insight into the molecular mechanisms of ALS pathogenesis.

Serological immunoreactivity against colon cancer proteome varies upon disease progression.

Sera from colon carcinoma patients were used to identify tumor-associated antigens (TAAs) by screening tumor proteome resolved by 2D electrophoresis. A panel of six TAAs eliciting a serological immune response in colorectal cancer was identified, showing a modification in antigen recognition by B cells in patients as a function of colon cancer progression. The expression of these proteins was either confined or increased in tumor as compared to normal mucosa.

Prolonged islet allograft survival in diabetic mice upon macrophage depletion by clodronate-loaded erythrocytes

Early impairment of islet function and graft loss strongly limit the success of allogenic islet transplantation in insulin-dependent diabetes. Macrophages play a key role in this process thus the depletion of these cells may strongly affect islet survival. In this study, we have evaluated the effect of the depletion of macrophages in mouse allograft rejection using a new approach based on a single infusion of red blood cells loaded with the synthetic analogue of pyrophosphate clodronate. Graft survival was 19.4+/-0.89 and 20+/-2 days in the two control groups treated with physiological solution and unloaded erythrocytes, respectively; 25+/-1.9 days in the group treated with free-clodronate and 35+/-6 days in the erythrocytes-loaded group. Our results indicate clodronate selectively targeted to the macrophagic cells by a single administration of engineered erythrocytes can significantly prolong islet graft survival and open new therapeutic strategies in islet transplantation.

Acclimation increases freezing stress response of Arabidopsis thaliana at proteome level.

This study used 2DE to investigate how Arabidopsis thaliana modulates protein levels in response to freezing stress after sub-lethal exposure at -10°C, both in cold-acclimated and in non-acclimated plants. A map was implemented in which 62 spots, corresponding to 44 proteins, were identified. Twenty-two spots were modulated upon treatments, and the corresponding proteins proved to be related to photosynthesis, energy metabolism, and stress response. Proteins demonstrated differences between control and acclimation conditions. Most of the acclimation-responsive proteins were either not further modulated or they were down-modulated by freezing treatment, indicating that the levels reached during acclimation were sufficient to deal with freezing. Anabolic metabolism appeared to be down-regulated in favor of catabolic metabolism. Acclimated plants and plants submitted to freezing after acclimation showed greater reciprocal similarity in protein profiles than either showed when compared both to control plants and to plants frozen without acclimation. The response of non-acclimated plants was aimed at re-modulating photosynthetic apparatus activity, and at increasing the levels of proteins with antioxidant-, molecular chaperone-, or post-transcriptional regulative functions. These changes, even less effective than the acclimation strategy, might allow the injured plastids to minimize the production of non-useful metabolites and might counteract photosynthetic apparatus injuries.

Evaluation of two-dimensional gel electrophoresis maps by local tangent space alignment: An application to neuroproteomics

Proteomic analysis may be useful to investigate disorders of the central nervous system, in order to explore the protein content of cells and of biological fluids in respect of the onset and evolution of diseases. Today, one of the most used proteomic approach includes the separation and visualization of proteins by means of two-dimensional gel electrophoresis (2DE). However the development of fully automatic strategies in extracting information from gel images is still a challenging task. In this paper we applied a computational strategy to the aim of obtaining a compact representation of the original data. This method was applied to an experimental protocol including two different clinical groups of amyotrophic lateral sclerosis (ALS) and peripheral neuropathy patients : 32 2DE maps generated from cerebrospinal fluid (24 pathologic and 8 control subjects) were processed. Quantitative features were extracted to describe each image and dealt with the dimension reduction technique of local tangent space alignment (LTSA). The discovered low-dimensional structure allows to see the gel of the dataset as separable, according to their clinical conditions, showing the informativeness of the adopted descriptors and providing the bases for classification of this kind of samples.