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Objectives GDI1 gene encodes for αGDI, a protein controlling the cycling of small GTPases, reputed to orchestrate vesicle trafficking. Mutations in human GDI1 are responsible for intellectual disability (ID). In mice with ablated Gdi1, a model of ID, impaired working and associative short-term memory was recorded. This cognitive phenotype worsens if the deletion of αGDI expression is restricted to neurons. However, whether astrocytes, key homeostasis providing neuroglial cells, supporting neurons via aerobic glycolysis, contribute to this cognitive impairment is unclear. Methods We carried out proteomic analysis and monitored [18F]-fluoro-2-deoxy-d-glucose uptake into brain slices of Gdi1 knockout and wild type control mice. d-Glucose utilization at single astrocyte level was measured by the Förster Resonance Energy Transfer (FRET)-based measurements of cytosolic cyclic AMP, d-glucose and L-lactate, evoked by agonists selective for noradrenaline and L-lactate receptors. To test the role of astrocyte-resident processes in disease phenotype, we generated an inducible Gdi1 knockout mouse carrying the Gdi1 deletion only in adult astrocytes and conducted behavioural tests. Results Proteomic analysis revealed significant changes in astrocyte-resident glycolytic enzymes. Imaging [18F]-fluoro-2-deoxy-d-glucose revealed an increased d-glucose uptake in Gdi1 knockout tissue versus wild type control mice, consistent with the facilitated d-glucose uptake determined by FRET measurements. In mice with Gdi1 deletion restricted to astrocytes, a selective and significant impairment in working memory was recorded, which was rescued by inhibiting glycolysis by 2-deoxy-d-glucose injection. Conclusions These results reveal a new astrocyte-based mechanism in neurodevelopmental disorders and open a novel therapeutic opportunity of targeting aerobic glycolysis, advocating a change in clinical practice., Highlights • Mutations in human Gdi1, encoding αGDI, a protein controlling vesicle traffic, are responsible for Intellectual Disability. • Gdi1 knockout revealed significant changes in astrocyte-resident glycolytic enzymes and facilitated D-glucose utilization. • Astrocyte-selective Gdi1 deletion impairs working memory, which can be rescued by administration of 2-deoxy-D-glucose. • Astrocyte-based glycolysis is a new target to treat Intellectual Disability.

Aim. The aim of the current study was to compare clinical characteristics, laboratory findings, and major outcomes of patients hospitalized for COVID-19 pneumonia with COVID-associated hyperglycaemia or pre-existing diabetes. Methods. A cohort of 176 adult patients with a diagnosis of pre-existing diabetes (n = 112) or COVID-associated hyperglycaemia (n = 55) was studied. Results. Patients with COVID-associated hyperglycaemia had lower BMI, significantly less comorbidities, and higher levels of inflammatory markers and indicators of multi-organ injury than those with pre-existing diabetes. No differences between pre-existing diabetes and COVID-associated hyperglycaemia were evident for symptoms at admission, the humoral response against SARS-CoV-2, or autoantibodies to glutamic acid decarboxylase or interferon alpha-4. COVID-associated hyperglycaemia was independently associated with the risk of adverse clinical outcome, which was defined as ICU admission or death (HR 2.11, 95% CI 1.34–3.31, p = 0.001), even after adjustment for age, sex, and other selected variables associated with COVID-19 severity. Furthermore, at the same time, we documented a negative association (HR 0.661, 95% CI 0.43–1.02, p = 0.063) between COVID-associated hyperglycaemia to swab negativization. Conclusions. Recognizing hyperglycaemia as a specific clinical entity associated with COVID-19 pneumonia is relevant for early and appropriate patient management and close monitoring for the progression of disease severity.

Aims/hypothesis The aim of the study was to characterise the humoral response against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in patients with diabetes. Demonstrating the ability to mount an appropriate antibody response in the presence of hyperglycaemia is relevant for the comprehension of mechanisms related to the observed worse clinical outcome of coronavirus disease 2019 (COVID-19) pneumonia in patients with diabetes and for the development of any future vaccination campaign to prevent SARS-CoV-2 infection. Methods Using a highly specific and sensitive measurement of antibodies by fluid-phase luciferase immunoprecipitation assays, we characterised the IgG, IgM and IgA response against multiple antigens of SARS-CoV-2 in a cohort of 509 patients with documented diagnosis of COVID-19, prospectively followed at our institution. We analysed clinical outcomes and antibody titres according to the presence of hyperglycaemia, i.e., either diagnosed or undiagnosed diabetes, at the time of, or during, hospitalisation. Results Among patients with confirmed COVID-19, 139 (27.3%) had diabetes: 90 (17.7%) had diabetes diagnosed prior to the hospital admission (comorbid diabetes) while 49 (9.6%) had diabetes diagnosed at the time of admission (newly diagnosed). Diabetes was associated with increased levels of inflammatory biomarkers and hypercoagulopathy, as well as leucocytosis and neutrophilia. Diabetes was independently associated with risk of death (HR 2.32 [95% CI 1.44, 3.75], p = 0.001), even after adjustment for age, sex and other relevant comorbidities. Moreover, a strong association between higher glucose levels and risk of death was documented irrespective of diabetes diagnosis (HR 1.14 × 1.1 mmol/l [95% CI 1.08, 1.21], p

Islet volume and endocrine pancreas architecture (islet size distribution) may be independent determinants of β-cell function. Furthermore, the accuracy of homeostatic model assessment (HOMA) indexes in predicting β-cell mass has never been assessed. Here we investigated the relationships between islet volume, islet density, and islet size distribution, estimated after pancreatic tissue digestion, with established indexes of β-cell function in humans. We included in this study 42 patients who were candidates for islet autotransplantation and had well-characterized glucose metabolism. Indexes of insulin secretion were calculated and compared with the islet volume, as a surrogate of β-cell mass, obtained after digestion of pancreas. Islet counting analysis showed considerable interindividual variation in islet density and size. Islet volume, but not density nor size, was the only independent determinant of β-cell function assessed by insulin HOMA β-cell. Islet volume was significantly reduced in the patients with overt hyperglycemia, but not in patients with impaired fasting glucose. Insulin HOMA β-cell predicted islet volume better than other measures of fasting insulin secretion. In conclusion, the present study documented a close direct relationship between indexes of β-cell function and islet volume in humans. The insulin HOMA β-cell provides a more reliable estimate of pancreatic islet volume than fasting glucose before islet isolation.

Activation of the phosphoinositide 3‐kinase (PI3K) pathway is a key signaling event in cancer, inflammation, and other proliferative diseases. PI3K inhibitors are already approved for some specific clinical indications, but their systemic on‐target toxicity limits their larger use. In particular, whereas toxicity is tolerable in acute treatment of life‐threatening diseases, this is less acceptable in chronic conditions. In the past, the strategy to overcome this drawback was to block selected isoforms mainly expressed in leukocytes, but redundancy within the PI3K family members challenges the effectiveness of this approach. On the other hand, decreasing exposure to selected target cells represents a so‐far unexplored alternative to circumvent systemic toxicity. In this manuscript, we describe the generation of a library of triazolylquinolones and the development of the first prodrug pan‐PI3K inhibitor.

miR-204 has been proposed to modulate insulin expression in human pancreatic islets by regulating the expression of the MAFA transcript, and in turn insulin transcription. We investigated miR-204 expression in pancreatic endocrine tumors (PET), a panel of human tissues, tissues derived from pancreatic islet purification, and in induced pluripotent stem cells (iPSCs) differentiated towards a pancreatic endocrine phenotype by quantitative real time RT-PCR or droplet digital PCR (ddPCR). In addition, we evaluated the effect of miR-204 up- or down-regulation in purified human islets and in the EndoC-βH1 cell line, as an experimental model of human pancreatic β cells. Our results confirm that miR-204 was enriched in insulin producing PET, in β cells within healthy pancreatic islets, and highly expressed in EndoC-βH1 cells. Moreover, in iPSCs miR-204 increased stepwise upon stimulated differentiation to insulin producing cells. However, up- or down-regulation of miR-204 in human islets and in EndoC-βH1 cells resulted in modest and not significant changes of the MAFA and INS mRNAs measured by ddPCR or c-peptide release. Our data confirm the association of miR-204 with a β cell endocrine phenotype in human pancreatic islets, but do not support its direct role in regulating the levels of insulin mRNA through MAFA.

High levels of donor-derived high-mobility group box 1 (HMGB1) protein have been associated with poor islet graft outcome in mouse models. The aim of our work was to determine whether HMGB1 released by human islets had independent proinflammatory effects that influence engraftment in humans. Human islet preparations contained and released HMGB1 in different amounts, as determined by Western blot and ELISA (median 17 pg/ml/IEQ/24 h; min–max 0–211, n = 74). HMGB1 release directly correlated with brain death, donor hyperamilasemia, and factors related to the pancreas digestion procedure (collagenase and digestion time). HMGB1 release was significantly positively associated with the release of other cytokines/chemokines, particularly with the highly released “proinflammatory” CXCL8/IL-8, CXCL1/GRO-α, and the IFN-γ-inducible chemokines CXCL10/IP-10 and CXCL9/MIG. HMGB1 release was not modulated by Toll-like receptor 2, 3, 4, 5, and 9 agonists or by exposure to IL-1β. When evaluated after islet transplantation, pretransplant HMGB1 release was weakly associated with the activation of the coagulation cascade (evaluated as serum cross-linked fibrin products), but not with the immediate posttransplant inflammatory response. Concordantly, HMGB1 did not affect short-term human islet function. Our data show that human islet HMGB1 release is a sign of “damaged” islets, although without any independent direct role in graft failure.

Long-term clinical outcome of islet transplantation is hampered by the rejection and recurrence of autoimmunity. Accurate monitoring may allow for early detection and treatment of these potentially compromising immune events. Islet transplant outcome was analyzed in 59 consecutive pancreatic islet recipients in whom baseline and de novo posttransplant autoantibodies (GAD antibody, insulinoma-associated protein 2 antigen, zinc transporter type 8 antigen) and donor-specific alloantibodies (DSA) were quantified. Thirty-nine recipients (66%) showed DSA or autoantibody increases (de novo expression or titer increase) after islet transplantation. Recipients who had a posttransplant antibody increase showed similar initial performance but significantly lower graft survival than patients without an increase (islet autoantibodies P < 0.001, DSA P < 0.001). Posttransplant DSA or autoantibody increases were associated with HLA-DR mismatches (P = 0.008), induction with antithymocyte globulin (P = 0.0001), and pretransplant panel reactive alloantibody >15% in either class I or class II (P = 0.024) as independent risk factors and with rapamycin as protective (P = 0.006) against antibody increases. DSA or autoantibody increases after islet transplantation are important prognostic markers, and their identification could potentially lead to improved islet cell transplant outcomes.

Chemokines and their receptors have been associated with or implicated in the pathogenesis of type 1 diabetes (T1D), but the identification of a single specific chemokine/receptor pathway that may constitute a suitable target for the development of therapeutic interventions is still lacking. Here, we used multiple low-dose (MLD) streptozotocin (STZ) injections and the NOD mouse model to investigate the potency of CXCR1/2 inhibition to prevent inflammation- and autoimmunity-mediated damage of pancreatic islets. Reparixin and ladarixin, noncompetitive allosteric inhibitors, were used to pharmacologically blockade CXCR1/2. Transient blockade of said receptors was effective in preventing inflammation-mediated damage in MLD-STZ and in preventing and reversing diabetes in NOD mice. Blockade of CXCR1/2 was associated with inhibition of insulitis and modification of leukocytes distribution in blood, spleen, bone marrow, and lymph nodes. Among leukocytes, CXCR2+ myeloid cells were the most decreased subpopulations. Together these results identify CXCR1/2 chemokine receptors as “master regulators” of diabetes pathogenesis. The demonstration that this strategy may be successful in preserving residual β-cells holds the potential to make a significant change in the approach to management of human T1D.

Aerobiological data can be used as indirect but reliable measures of flowering phenology to analyze the response of plant species to ongoing climate changes. The aims of this study are to evaluate the performance of several phenological models for predicting the pollen start of season (PSS) in seven spring-flowering trees (Alnus glutinosa, Acer negundo, Carpinus betulus, Platanus occidentalis, Juglans nigra, Alnus viridis, and Castanea sativa) and in two summer-flowering herbaceous species (Artemisia vulgaris and Ambrosia artemisiifolia) by using a 26-year aerobiological data set collected in Turin (Northern Italy). Data showed a reduced interannual variability of the PSS in the summer-flowering species compared to the spring-flowering ones. Spring warming models with photoperiod limitation performed best for the greater majority of the studied species, while chilling class models were selected only for the early spring flowering species. For Ambrosia and Artemisia, spring warming models were also selected as the best models, indicating that temperature sums are positively related to flowering. However, the poor variance explained by the models suggests that further analyses have to be carried out in order to develop better models for predicting the PSS in these two species. Modeling the pollen season start on a very wide data set provided a new opportunity to highlight the limits of models in elucidating the environmental factors driving the pollen season start when some factors are always fulfilled, as chilling or photoperiod or when the variance is very poor and is not explained by the models.

Aims: New sources of insulin-secreting cells are strongly required for the cure of diabetes. Recent successes in differentiating embryonic stem cells, in combination with the discovery that it is possible to derive human induced pluripotent stem cells (iPSCs) from somatic cells, have raised the possibility that patient-specific beta cells might be derived from patients through cell reprogramming and differentiation. In this study, we aimed to obtain insulin-producing cells from human iPSCs and test their ability to secrete insulin in vivo. Methods: Human iPSCs, derived from both fetal and adult fibroblasts, were differentiated in vitro into pancreas-committed cells and then transplanted into immunodeficient mice at two different stages of differentiation (posterior foregut and endocrine cells). Results: IPSCs were shown to differentiate in insulin-producing cells in vitro, following the stages of pancreatic organogenesis. At the end of the differentiation, the production of INSULIN mRNA was highly increased and 5 ± 2.9 % of the cell population became insulin-positive. Terminally differentiated cells also produced C-peptide in vitro in both basal and stimulated conditions. In vivo, mice transplanted with pancreatic cells secreted human C-peptide in response to glucose stimulus, but transplanted cells were observed to lose insulin secretion capacity during the time. At histological evaluation, the grafts resulted to be composed of a mixed population of cells containing mature pancreatic cells, but also pluripotent and some neuronal cells. Conclusion: These data overall suggest that human iPSCs have the potential to generate insulin-producing cells and that these differentiated cells can engraft and secrete insulin in vivo.

BACKGROUND. Appropriate recruitment of dendritic cells (DC) at sites of inflammation and migration to secondary lymphoid organs is of critical importance for the initiation of Ag-specific immune responses. The proper localization of DC in selected tissues is guided primarily by the coordinated expression of chemokine receptors (CKR). Here we show that immunosuppressive drugs have divergent effects on the modulation of CKR in maturing DC. METHODS AND RESULTS. Dexamethazone (DEX) and IL-10 inhibited human DC migration to CCL19 in vitro and mouse DC migration to lymph nodes (LN) in vivo, by impairing CCR7 expression. The calcineurin inhibitors cyclosporine A (CsA) and tacrolimus (FK506) were characterized by the inability to modulate CKR expression and migratory activity. Rapamycin (RAPA) increased DC migration to CCL19 in vitro and to LN in vivo by enhancing CCR7 expression. This effect could be mediated, in LPS-maturing DC, by the inhibition of autocrine IL-10 production. The in vivo data obtained with ex vivo RAPA treated DC were confirmed in a model of in vivo drug administration in mice, suggesting a potential clinical relevance. CONCLUSIONS. These findings demonstrate that immunosuppressive agents differently modulate the CKR switch associated with maturing DC; in particular, RAPA selectively up-regulates CCR7 and enhances the migration of differentiated DC to regional LN. This study contributes to a better understanding of the role of immunosuppressive therapy on DC migration, a potentially relevant check point of immunosuppressive treatment.

The immunosuppressive activity of rapamycin (RAPA) and its efficacy as an anti-rejection agent in organ transplantation have been ascribed principally to its anti-proliferative effects on T cells, while the activity on monocytes is partially unknown. In vitro, RAPA reduced monocyte survival by inducing a caspase-independent cell death. RAPA-induced monocyte cell death (RAPA-CD) was impeded by activation of granulocyte macrophage-colony stimulating factor family receptors or toll-like receptor 4, and by exposure to inflammatory cytokines. In vivo, in patients who received RAPA monotherapy as part of pre-conditioning for islet transplantation, RAPA affected survival of myeloid lineage cells. In the peripheral blood, CD33(+) and CD14(+) cells decreased, whereas lymphocytes appeared unaffected. In the bone marrow, myeloid precursors such as CD15(+) and CD15(+)/CD16(+) were selectively and significantly decreased, but no major cytotoxic effects were observed. The RAPA-CD suggests a dependence of monocytes on mammalian target of RAPA pathways for nutrient usage, and this feature implies that RAPA could be selectively useful as a treatment to reduce monocytes or myeloid cells in conditions where these cells negatively affect patient, suggesting a potential anti-inflammatory action of this drug.

Plasticity is a hallmark of macrophages, and in response to environmental signals these cells undergo different forms of polarized activation, the extremes of which are called classic (M1) and alternative (M2). Rapamycin (RAPA) is crucial for survival and functions of myeloid phagocytes, but its effects on macrophage polarization are not yet studied. To address this issue, human macrophages obtained from six normal blood donors were polarized to M1 or M2 in vitro by lipopolysaccharide plus interferon-γ or interleukin-4 (IL-4), respectively. The presence of RAPA (10 ng/ml) induced macrophage apoptosis in M2 but not in M1. Beyond the impact on survival in M2, RAPA reduced CXCR4, CD206 and CD209 expression and stem cell growth factor-β, CCL18 and CCL13 release. In contrast, in M1 RAPA increased CD86 and CCR7 expression and IL-6, tumour necrosis factor-α and IL-1β release but reduced CD206 and CD209 expression and IL-10, vascular endothelial growth factor and CCL18 release. In view of the in vitro data, we examined the in vivo effect of RAPA monotherapy (0·1 mg/kg/day) in 12 patients who were treated for at least 1 month before islet transplant. Cytokine release by Toll-like receptor 4-stimulated peripheral blood mononuclear cells showed a clear shift to an M1-like profile. Moreover, macrophage polarization 21 days after treatment showed a significant quantitative shift to M1. These results suggest a role of mammalian target of rapamycin (mTOR) into the molecular mechanisms of macrophage polarization and propose new therapeutic strategies for human M2-related diseases through mTOR inhibitor treatment.

The standard of care for malignant pleural mesothelioma (MPM) is a combination of platinum compounds and Pemetrexed. Still, the median survival of MPM patients is low (about 12 months). Excision Repair Cross Complementing Group 1 (ERCC1) acts by removing DNA adducts formed by platinum compounds, whereas Pemetrexed inhibits Thymidylate Synthase (TS), which is involved in DNA synthesis. Pleural biopsies were collected from 140 MPM patients (98 males and 42 females, mean age 68 years) before treatment initiation. Average disease specific survival was 13 months (range 1-60). mRNA was extracted from formalin fixed paraffin blocks, reverse transcribed and assayed by qRT-PCR. Immunohistochemistry with anti-ERCC1 and anti-TS mAbs was scored semi-quantitatively. Of 140 patients, 102 were treated, the remaining were not because of bad performance status or advanced age. Patients with low ERCC1 protein expression survived longer (24.3 vs 12.5 months, P=.03). Likewise, patients with low levels of TS mRNA had better s...

Although long considered a promising treatment option for type 1 diabetes, pancreatic islet cell transformation has been hindered by immune system rejection of engrafted tissue. The identification of pathways that regulate post-transplant detrimental inflammatory events would improve management and outcome of transplanted patients. Here, we found that CXCR1/2 chemokine receptors and their ligands are crucial negative determinants for islet survival after transplantation. Pancreatic islets released abundant CXCR1/2 ligands (CXCL1 and CXCL8). Accordingly, intrahepatic CXCL1 and circulating CXCL1 and CXCL8 were strongly induced shortly after islet infusion. Genetic and pharmacological blockade of the CXCL1-CXCR1/2 axis in mice improved intrahepatic islet engraftrnent and reduced intrahepatic recruitment of polymorphonuclear leukocytes and NKT cells after islet infusion. In humans, the CXCR1/2 allosteric inhibitor reparixin improved outcome in a phase 2 randomized, open-label pilot study with a single infusion of allogeneic islets. These findings indicate that the CXCR1/2-mediated pathway is a regulator of islet damage and should be a target for intervention to improve the efficacy of transplantation. "Although long considered a promising treatment option for type 1 diabetes, pancreatic islet cell transformation has been hindered by immune system rejection of engrafted tissue. The identification of pathways that regulate post-transplant detrimental inflammatory events would improve management and outcome of transplanted patients. Here, we found that CXCR1\/2 chemokine receptors and their ligands are crucial negative determinants for islet survival after transplantation. Pancreatic islets released abundant CXCR1\/2 ligands (CXCL1 and CXCL8). Accordingly, intrahepatic CXCL1 and circulating CXCL1 and CXCL8 were strongly induced shortly after islet infusion. Genetic and pharmacological blockade of the CXCL1-CXCR1\/2 axis in mice improved intrahepatic islet engraftment and reduced intrahepatic recruitment of polymorphonuclear leukocytes and NKT cells after islet infusion. In humans, the CXCR1\/2 allosteric inhibitor reparixin improved outcome in a phase 2 randomized, open-label pilot study with a single infusion of allogeneic islets. These findings indicate that the CXCR1\/2-mediated pathway is a regulator of islet damage and should be a target for intervention to improve the efficacy of transplantation."

AIMS/HYPOTHESIS: The purpose of this study was to investigate whether the gut mucosa is a reservoir for enterovirus persistence in patients with type 1 diabetes.METHODS: Small intestine biopsy samples from 25 individuals at different stages of type 1 diabetes, 21 control individuals and 27 individuals with coeliac disease were analysed for the presence of enterovirus RNA by using both radioactive in-situ hybridisation and real-time RT-PCR and for the presence of enterovirus proteins by immunostaining with antibodies against VP1 and VP4-2-3 capsid proteins and virus polymerase. Lymphocytic enteropathy and serum anti-VP1 antibodies were also evaluated at the time of biopsy. Moreover, high-throughput sequencing was performed to identify viral transcripts or genomes.RESULTS: Enterovirus was not detected by in-situ hybridisation or RT-PCR in any of the individuals tested. Immunohistology revealed a few stained cells in the intestinal epithelium in a low number of individuals, with no difference between diabetic and non-diabetic individuals. Levels of serum IgG against VP1 did not differ between control individuals and those with diabetes or coeliac disease and no evidence of diabetes-related lymphocytic enteropathy was detected. High-throughput sequencing did not reveal specific enterovirus sequences in the gut mucosa of individuals with type 1 diabetes.CONCLUSIONS/INTERPRETATION: Prolonged/persistent enterovirus infections in gut mucosa are not common in patients with type 1 diabetes

Influenza A viruses commonly cause pancreatitis in naturally and experimentally infected animals. In this study, we report the results of in vivo investigations carried out to establish whether influenza virus infection could cause metabolic disorders linked to pancreatic infection. In addition, in vitro tests in human pancreatic islets and in human pancreatic cell lines were performed to evaluate viral growth and cell damage. Infection of an avian model with two low-pathogenicity avian influenza isolates caused pancreatic damage resulting in hyperlipasemia in over 50% of subjects, which evolved into hyperglycemia and subsequently diabetes. Histopathology of the pancreas showed signs of an acute infection resulting in severe fibrosis and disruption of the structure of the organ. Influenza virus nucleoprotein was detected by immunohistochemistry (IHC) in the acinar tissue. Human seasonal H1N1 and H3N2 viruses and avian H7N1 and H7N3 influenza virus isolates were able to infect a selection of human pancreatic cell lines. Human viruses were also shown to be able to infect human pancreatic islets. In situ hybridization assays indicated that viral nucleoprotein could be detected in beta cells. The cytokine activation profile indicated a significant increase of MIG/CXCL9, IP-10/CXCL10, RANTES/CCL5, MIP1b/CCL4, Groa/CXCL1, interleukin 8 (IL-8)/CXCL8, tumor necrosis factor alpha (TNF-alpha), and IL-6. Our findings indicate that influenza virus infection may play a role as a causative agent of pancreatitis and diabetes in humans and other mammals. Influenza A viruses commonly cause pancreatitis in naturally and experimentally infected animals. In this study, we report the results of in vivo investigations carried out to establish whether influenza virus infection could cause metabolic disorders linked to pancreatic infection. In addition, in vitro tests in human pancreatic islets and in human pancreatic cell lines were performed to evaluate viral growth and cell damage. Infection of an avian model with two low-pathogenicity avian influenza isolates caused pancreatic damage resulting in hyperlipasemia in over 50% of subjects, which evolved into hyperglycemia and subsequently diabetes. Histopathology of the pancreas showed signs of an acute infection resulting in severe fibrosis and disruption of the structure of the organ. Influenza virus nucleoprotein was detected by immunohistochemistry (IHC) in the acinar tissue. Human seasonal H1N1 and H3N2 viruses and avian H7N1 and H7N3 influenza virus isolates were able to infect a selection of human pancreatic cell lines. Human viruses were also shown to be able to infect human pancreatic islets. In situ hybridization assays indicated that viral nucleoprotein could be detected in beta cells. The cytokine activation profile indicated a significant increase of MIG/CXCL9, IP-10/CXCL10, RANTES/CCL5, MIP1b/CCL4, Groa/CXCL1, interleukin 8 (IL-8)/CXCL8, tumor necrosis factor alpha (TNF-alpha), and IL-6. Our findings indicate that influenza virus infection may play a role as a causative agent of pancreatitis and diabetes in humans and other mammals. OI Piemonti, Lorenzo/0000-0002-2172-2198; Lampasona, Vito/0000-0001-5162-8445; Patrono, Livia Victoria/0000-0003-3978-3607; bonfante, francesco/0000-0002-1276-2710 ZR 0 Z8 1 ZS 1

A simple HPLC method was developed to distinguish between 'poisonous' and 'non-poisonous' chemotypes of Ferula communis. The method was performed on a C8 reverse phase analytical column using a binary eluent (aqueous TFA 0.01%-TFA 0.01% in acetonitrile) under gradient condition. The two chemotypes showed different fingerprints. The identification of five coumarins and eleven daucane derivatives by HPLC-diode array detection (HPLC-DAD) and HPLC-MS is described. A coumarin, not yet described, was detected.

An HPLC-ELSD-ESI-MS method has been developed for the analysis of the steroidal saponins in the aerial parts of Tribulus terrestris. Protodioscin, a new saponin (5,6-dihydroprotodioscin, neoprotodioscin) and their respective sulfates were detected. The structure of the new compound was elucidated on the basis of NMR and ESI-MS spectral analysis.

Background: Data available on the immunomodulatory properties of neural stem/precursor cells (NPC) support their possible use as modulators for immune-mediated process. The aim of this study was to define whether NPC administered in combination with pancreatic islets prevents rejection in a fully mismatched allograft model. Methodology/Principal Finding: Diabetic Balb/c mice were co-transplanted under the kidney capsule with pancreatic islets and GFP(+) NPC from fully mismatched C57BL/6 mice. The following 4 groups of recipients were used: mice receiving islets alone; mice receiving islets alone and treated with standard immunosuppression (IL-2R alpha chain mAbs + FK506 + Rapamycin); mice receiving a mixed islet/NPC graft under the same kidney capsule (Co-NPC-Tx); mice receiving the islet graft under the left kidney capsule and the NPC graft under the right kidney capsule (NPC-Tx). Our results demonstrate that only the co-transplantation and co-localization of NPC and islets (Co-NPC-Tx) induce stable long-term graft function in the absence of immunosuppression. This condition is associated with an expansion of CD4(+)CD25(+)FoxP3(+) T regulatory cells in the spleen. Unfortunately, stable graft function was accompanied by constant and reproducible development of NPC-derived cancer mainly sustained by insulin secretion. Conclusion: These data demonstrate that the use of NPC in combination with islets prevents graft rejection in a fully mismatched model. However, the development of NPC-derived cancer raises serious doubts about the safety of using adult stem cells in combination with insulin-producing cells outside the original microenvironment. Background: Data available on the immunomodulatory properties of neural stem/precursor cells (NPC) support their possible use as modulators for immune-mediated process. The aim of this study was to define whether NPC administered in combination with pancreatic islets prevents rejection in a fully mismatched allograft model. Methodology/Principal Finding: Diabetic Balb/c mice were co-transplanted under the kidney capsule with pancreatic islets and GFP(+) NPC from fully mismatched C57BL/6 mice. The following 4 groups of recipients were used: mice receiving islets alone; mice receiving islets alone and treated with standard immunosuppression (IL-2R alpha chain mAbs + FK506 + Rapamycin); mice receiving a mixed islet/NPC graft under the same kidney capsule (Co-NPC-Tx); mice receiving the islet graft under the left kidney capsule and the NPC graft under the right kidney capsule (NPC-Tx). Our results demonstrate that only the co-transplantation and co-localization of NPC and islets (Co-NPC-Tx) induce stable long-term graft function in the absence of immunosuppression. This condition is associated with an expansion of CD4(+)CD25(+)FoxP3(+) T regulatory cells in the spleen. Unfortunately, stable graft function was accompanied by constant and reproducible development of NPC-derived cancer mainly sustained by insulin secretion. Conclusion: These data demonstrate that the use of NPC in combination with islets prevents graft rejection in a fully mismatched model. However, the development of NPC-derived cancer raises serious doubts about the safety of using adult stem cells in combination with insulin-producing cells outside the original microenvironment. BACKGROUND: Data available on the immunomodulatory properties of neural stem/precursor cells (NPC) support their possible use as modulators for immune-mediated process. The aim of this study was to define whether NPC administered in combination with pancreatic islets prevents rejection in a fully mismatched allograft model. METHODOLOGY/PRINCIPAL FINDING: Diabetic Balb/c mice were co-transplanted under the kidney capsule with pancreatic islets and GFP(+) NPC from fully mismatched C57BL/6 mice. The following 4 groups of recipients were used: mice receiving islets alone; mice receiving islets alone and treated with standard immunosuppression (IL-2Ralpha chain mAbs + FK506 + Rapamycin); mice receiving a mixed islet/NPC graft under the same kidney capsule (Co-NPC-Tx); mice receiving the islet graft under the left kidney capsule and the NPC graft under the right kidney capsule (NPC-Tx). Our results demonstrate that only the co-transplantation and co-localization of NPC and islets (Co-NPC-Tx) induce stable long-term graft function in the absence of immunosuppression. This condition is associated with an expansion of CD4(+)CD25(+)FoxP3(+) T regulatory cells in the spleen. Unfortunately, stable graft function was accompanied by constant and reproducible development of NPC-derived cancer mainly sustained by insulin secretion. CONCLUSION: These data demonstrate that the use of NPC in combination with islets prevents graft rejection in a fully mismatched model. However, the development of NPC-derived cancer raises serious doubts about the safety of using adult stem cells in combination with insulin-producing cells outside the original microenvironment

Adherent fibroblast-like cells have been reported to appear in cultures of human endocrine or exocrine pancreatic tissue during attempts to differentiate human beta cells from pancreatic precursors. A thorough characterization of these mesenchymal cells has not yet been completed, and there are no conclusive data about their origin. We demonstrated that the human mesenchymal cells outgrowing from cultured human pancreatic endocrine or exocrine tissue are pancreatic mesenchymal stem cells (pMSC) that propagate from contaminating pMSC. The origin of pMSC is partly extrapancreatic both in humans and mice, and by using green fluorescent protein (GFP(+)) bone marrow transplantation Adherent fibroblast-like cells have been reported to appear in cultures of human endocrine or exocrine pancreatic tissue during attempts to differentiate human beta cells from pancreatic precursors. A thorough characterization of these mesenchymal cells has not yet been completed, and there are no conclusive data about their origin.We demonstrated that the human mesenchymal cells outgrowing from cultured human pancreatic endocrine or exocrine tissue are pancreatic mesenchymal stem cells (pMSC) that propagate from contaminating pMSC. The origin of pMSC is partly extrapancreatic both in humans and mice, and by using green fluorescent protein (GFP(+)) bone marrow transplantation in the mouse model, we were able to demonstrate that these cells derive from the CD45(+) component of bone marrow. The pMSC express negligible levels of islet-specific genes both in basal conditions and after serum deprivation or exogenous growth factor exposure, and might not represent optimal candidates for generation of physiologically competent beta-cells. On the other hand, when cotransplanted with a minimal pancreatic islet mass, pMSC facilitate the restoration of normoglycemia and the neovascularization of the graft. These results suggest that pMSCs could exert an indirect role of "helper" cells in tissue repair processes

OBJECTIVE - Monocyte chemoattractant protein-1 (MCP-1/CCL2) is a chemokine involved into the pathogenesis of atherosclerosis and has prognostic value in the acute and chronic phases in patients with acute coronary syndromes. RESEARCH DESIGN AND METHODS - MCP-1/CCL2 concentration was measured in plasma fractions of 363 middle-aged overweight/obese individuals (aged 61 12 years BMI 30.1 +/- 6.6 kg/m(2), 15% with type 2 diabetes, and 12% with impaired glucose tolerance) of a population survey carried out in 1990-1991 in Lombardy, Italy (Cremona Study), and cardiovascular disease (CVD) mortality was assessed in 2006 through Regional Health Registry files. RESULTS - At baseline MCP-1/CCL2 was increased in individuals with type 2 diabetes (p < 0.05) and showed significant correlations with biochemical risk markers of atherosclerosis. After 15 years, among the 363 subjects, there were 82 deaths due to CVD. In univariate analysis age, sex, fasting glucose and insulin, fibrinogen, glucose tolerance status, smoking habit, and MCP-1/CCL2 were associated with CVD mortality. Age, sex, fasting serum glucose, MCP-1/CCL2, and smoking habit maintained an independent association with CVD mortality in multiple regression analysis. In a subgroup of 113 subjects in whom data for C-reactive protein (CRP) were available, its level was not predictive of CVD mortality. CONCLUSIONS - In middle-aged overweight/obese individuals MCP-1/CCL2 was independently associated with CVD mortality. Further studies will be necessary to establish its role as a surrogate biomarker and as a potential therapeutic target. OBJECTIVE Monocyte chemoattractant protein-1 (MCP-1/CCL2) is a chemokine involved into the pathogenesis of atherosclerosis and has prognostic value in the acute and chronic phases in patients with acute coronary syndromes. RESEARCH DESIGN AND METHODS MCP-1/CCL2 concentration was measured in plasma fractions of 363 middle-aged overweight/obese individuals (aged 61 ± 12 years, BMI 30.1 ± 6.6 kg/m2, 15% with type 2 diabetes, and 12% with impaired glucose tolerance) of a population survey carried out in 1990–1991 in Lombardy, Italy (Cremona Study), and cardiovascular disease (CVD) mortality was assessed in 2006 through Regional Health Registry files. RESULTS At baseline MCP-1/CCL2 was increased in individuals with type 2 diabetes (P < 0.05) and showed significant correlations with biochemical risk markers of atherosclerosis. After 15 years, among the 363 subjects, there were 82 deaths due to CVD. In univariate analysis age, sex, fasting glucose and insulin, fibrinogen, glucose tolerance status, smoking habit, and MCP-1/CCL2 were associated with CVD mortality. Age, sex, fasting serum glucose, MCP-1/CCL2, and smoking habit maintained an independent association with CVD mortality in multiple regression analysis. In a subgroup of 113 subjects in whom data for C-reactive protein (CRP) were available, its level was not predictive of CVD mortality. CONCLUSIONS In middle-aged overweight/obese individuals MCP-1/CCL2 was independently associated with CVD mortality. Further studies will be necessary to establish its role as a surrogate biomarker and as a potential therapeutic target.

While the mechanisms of specification and the reciprocal relationships of the four types of endocrine cell (alpha, beta, delta and pancreatic polypeptide cells) within the human endocrine pancreas are well described in adults and during fetal development, ghrelin-immunoreactive cells (epsilon cells) remain poorly understood. We studied epsilon cells in 24 human fetal pancreases between 11 and 39 weeks of development and in 32 pancreases from adult organ donors. We observed single epsilon cells scattered in primitive exocrine tissue from gestational week 13 in developing pancreas. Later in the developmental process, epsilon cells started to aggregate into clusters. From gestational week 21, epsilon cells were observed located around developing islets, forming an almost continuous layer at the peripheral rim of the islets. They remain localised on the mantle of the islets, although at different amounts, in the adult pancreas. Co-production of ghrelin with insulin, glucagon or somatostatin was not detected during fetal development. Co-production with pancreatic polypeptide was evident sporadically. Epsilon cells co-produced NK2 homeobox 2 and ISL LIM homeobox 1, but not NK6 homeobox 1 and paired box 6. A quantitative analysis was performed in the adult pancreas: there was an average of 1.17 + 1.17 epsilon cells per islet, the relative epsilon cell volume was 0.14 + 0.16% and the epsilon cell mass was 0.13 + 0.15 g. Neither sex nor age affected the epsilon cell mass, although there was a significant inverse correlation with BMI. During fetal development epsilon cells show an ontogenetic and morphogenetic pattern that is distinct from that of alpha and beta cells. AIMS/HYPOTHESIS: While the mechanisms of specification and the reciprocal relationships of the four types of endocrine cell (alpha, beta, delta and pancreatic polypeptide cells) within the human endocrine pancreas are well described in adults and during fetal development, ghrelin-immunoreactive cells (epsilon cells) remain poorly understood. METHODS: We studied epsilon cells in 24 human fetal pancreases between 11 and 39 weeks of development and in 32 pancreases from adult organ donors. RESULTS: We observed single epsilon cells scattered in primitive exocrine tissue from gestational week 13 in developing pancreas. Later in the developmental process, epsilon cells started to aggregate into clusters. From gestational week 21, epsilon cells were observed located around developing islets, forming an almost continuous layer at the peripheral rim of the islets. They remain localised on the mantle of the islets, although at different amounts, in the adult pancreas. Co-production of ghrelin with insulin, glucagon or somatostatin was not detected during fetal development. Co-production with pancreatic polypeptide was evident sporadically. Epsilon cells co-produced NK2 homeobox 2 and ISL LIM homeobox 1, but not NK6 homeobox 1 and paired box 6. A quantitative analysis was performed in the adult pancreas: there was an average of 1.17 + 1.17 epsilon cells per islet, the relative epsilon cell volume was 0.14 + 0.16% and the epsilon cell mass was 0.13 + 0.15 g. Neither sex nor age affected the epsilon cell mass, although there was a significant inverse correlation with BMI. CONCLUSIONS/INTERPRETATION: During fetal development epsilon cells show an ontogenetic and morphogenetic pattern that is distinct from that of alpha and beta cells.

Bone marrow–derived mesenchymal stem cells (BM-MSCs) are stromal cells with the ability to proliferate and differentiate into many tissues. Although they represent powerful tools for several therapeutic settings, mechanisms regulating their migration to peripheral tissues are still unknown. Here, we report chemokine receptor expression on human BM-MSCs and their role in mediating migration to tissues. A minority of BM-MSCs (2% to 25%) expressed a restricted set of chemokine receptors (CXC receptor 4 [CXCR4], CX3C receptor 1 [CX3CR1], CXCR6, CC chemokine receptor 1 [CCR1], CCR7) and, accordingly, showed appreciable chemotactic migration in response to the chemokines CXC ligand 12 (CXCL12), CX3CL1, CXCL16, CC chemokine ligand 3 (CCL3), and CCL19. Using human pancreatic islets as an in vitro model of peripheral tissue, we showed that islet supernatants released factors able to attract BM-MSCs in vitro, and this attraction was principally mediated by CX3CL1 and CXCL12. Moreover, cells with features of BM-MSCs were detected within the pancreatic islets of mice injected with green fluorescent protein (GFP)–positive BM. A population of bona fide MSCs that also expressed CXCR4, CXCR6, CCR1, and CCR7 could be isolated from normal adult human pancreas. This study defines the chemokine receptor repertoire of human BM-MSCs that determines their migratory activity. Modulation of homing capacity may be instrumental for harnessing the therapeutic potential of BM-MSCs.

Dendritic cells (DC) initiate immunity by the activation of naive T cells and control immunity through their ability to induce unresponsiveness of lymphocytes by mechanisms that include deletion and induction of regulatory cells. An inadequate presentation to T cells by tumor-induced “regulatory” DC, among several mechanisms, can explain tolerance to tumor-associated Ags. In this study, we show that tumor-derived mucin profoundly affects the cytokine repertoire of monocyte-derived DC and switch them into IL-10highIL-12low regulatory APCs with a limited capacity to trigger protective Th1 responses. In fact, DC cocultured with pancreatic tumor cell lines in a Transwell system did not reach full maturation, had low immunostimulatory functions, did not produce IL-12, and released high levels of IL-10. The involvement of known tumor-derived immune-suppressive factors (e.g., vascular endothelial growth factor, TGF-β, IL-6, and IL-10) was considered and excluded. We provide evidence that tumor-derived MUC1 mucins are responsible for the impaired DC maturation and function. DC obtained in the presence of tumor microenvironment preferentially polarized IL-4+ response. Moreover, T cells primed by these regulatory DC became anergic and behaved as suppressor/regulatory cells. These findings identify mucin secretion as a novel mechanism of tumor escape from immune surveillance and provide the basis for the generation of potentially tolerogenic DC.

There are a large number of stable pancreatic ductal carcinoma cell lines (PDCL) that are used by researchers worldwide. Detailed data about their differentiation status and genetic alterations are present in the literature, but a systematic correlation with cell biological behavior is often lacking. PDCL ( n=12) were clustered by source of tumor cell (ascites, primary tumor, metastasis), and the data of functional cell biology were correlated with the reported structural and genetic profiles. Major histocompatibility complex expression, chemosensitivity and aneuploidia appeared to be related to the source of PDCL, and proliferative capacity appeared to be related to the grade of differentiation. No correlation between genetic/structural features of PDCL and biological behavior was found. All the cell lines appeared generally insensitive to in vitro treatment with 5-fluorouracil and showed variable degrees of susceptibility to gemcitabine, raltitrexed and oxaliplatin. All the PDCL showed resistance to Fas-mediated apoptosis but were significantly sensitive to the pro-apoptotic effect of inflammatory cytokines [interleukin (IL)-1beta, tumor necrosis factor (TNF)alpha and interferon gamma]. PDCL were characterized for the secretion of several factors relevant to the tumor-immune cross talk. Vascular endothelial growth factor, CCL2, CCL5 and transforming growth factor beta were the factors most frequently released; less frequent was the secretion of CXCL8, CCL22, IL-6 and sporadically CXCL12, IL-10 and hepatocyte growth factor. The cytokines IL-1beta and TNFalpha were always undetectable. In conclusion, a clear correlation between structural/genetic features and function could not be detected, suggesting the weakness of a "morphological" classification for the in vitro studies of pancreatic cancer.

The macrocyclic lathyrane polyester Euphorbia factor L10 (1a) has been obtained from the seeds of the caper spurge (E. lathyris). The interaction of L10 (1a) and its acetyl derivative (1b) with P-glycoprotein, a multidrug transporter overexpressed in cancer cells and responsible for resistance to chemotherapy, was investigated. The results established lathyrane diterpenoids as a novel chemotype for P-glycoprotein inhibitors.

The expression of beclin-1, an oncosuppressor monoallelically deleted in >60% epithelial cancers, has been shown to be developmentally regulated in T and B lymphocytes. By interacting with either bcl-2 or class III phosphatidyl-inositol-3-phosphate kinase, beclin-1 regulates apoptosis and autophagy, two processes crucial for lymphatic tissue homeostasis. We analyzed the potential link between beclin-1-mediated autophagy and the malignant behaviour of lymphomas. The tissue expression of beclin-1 was analyzed in a large series of non-Hodgkin lymphomas and correlated with patient's clinical outcome. By immunofluorescence, beclin-1 staining showed faintly detectable and diffusely distributed in the cytoplasm (regarded as negative) or confined to the perinuclear region as large and brilliant puncta suggestive of macro-aggregate reactivity (regarded as positive). The positive expression of beclin-1 well correlated with the presence of LC3-positive autophagic vacuoles and was inversely correlated with the expression of bcl-2. Non-Hodgkin lymphomas in which > or =20% of tumour cells expressed high level of beclin-1 aggregates were associated with a complete (57%) or partial (35%) remission. The 5-year overall survival probability, calculated by the Kaplan-Meier method, was 92% and 42% in beclin-1-expressing non-Hodgkin lymphomas with > or =20% and

There is much evidence that climate is rapidly changing at a global scale, especially regarding mean annual temperatures, precipitations and evaporation. The consequences of this rapid environmental change on freshwater biota are still not clear, but undoubtedly they could be severe. Among the main effects of climate change, we can individuate the enhancement of water temperatures, particularly important for poikilothermic organisms, with the consequent diminution of dissolved oxygen, and the reduction of available habitats for most stenothermal organisms. Another consequence of climate change is the alteration of hydrologic cycles, with increasing intensity and frequency of extreme events such as droughts, especially in Southern Europe. This scenario could severely affect freshwater biota, especially in mid-latitude regions, such as the Italian peninsula: shifts in phenology, life cycles and distribution ranges are likely to be expected for many organisms, with the extinction of many sensitive s...

The structure of a new prenylated coumarin (E-omega-benzoyloxyferulenol, 1b) from the Sardinian giant fennel (Ferula communis) has been confirmed by synthesis. The parent compound ferulenol (1a) showed sub-micromolar antimycobacterial activity, which was partly retained in 1b and in the simplified synthetic analogue 3, but diminished in its omega-hydroxy and omega-acetoxy derivatives (1c and 1d, respectively). The outstanding activity of 1a, its low toxicity, and the evidence for definite structure-activity relationships make this prenylated 4-hydroxycoumarin an interesting antibacterial chemotype worth further investigation.

The receptor tyrosine kinase RET, with a known role in embryonic development and in human pathologies, is alternatively spliced to yield at least two functional isoforms, which differ only in their carboxyl terminal. Enigma protein is a member of the PDZ-LIM family and is known to interact with the short isoform of RET/PTC2, a chimeric oncoprotein isolated from papillary thyroid carcinoma. Here, we show that Enigma also interacts in intact cells with the short isoform of RET-wt and of its pathologic mutants associated to MEN2 syndromes, RET-C634R and RET-M918T. In contrast, Enigma binds all the corresponding RET long isoforms very poorly and colocalizes with short but not long RET/PTC2 isoforms. The RET docking tyrosine for Enigma is the last but one before the divergence between the two isoforms and we demonstrated that short-isoform-specific amino acid residues +2 to +4 to this tyrosine are required for the interaction of RET/PTC2 with Enigma.

Aim: The standard of care for malignant pleural mesothelioma (MPM) includes combination of platinum compounds and pemetrexed. Despite this, the median survival of MPM patients is still bad (12 months). Excision Repair Cross Complementing Group 1 (ERCC1) acts removing DNA adducts formed by platinum compounds, whereas Pemetrexed inhibits Thymidylate Synthase (TS), a folic enzyme also involved in ex novo synthesis of DNA. This study aims to analyse retrospectively, in a large series of MPM specimens, the ERCC1 and TS gene and protein expression to investigate their prognostic and predictive role. Methods: Pleural biopsies were collected from140 consecutive MPM patients (98 males and 42 females, mean age 68 years, range 27-90). Histologic subtypes were: epithelioid (#103, 73%), sarcomatoid (#19, 14%) and biphasic (#18, 13%). At the end of the study 113 patients were died (average disease specific survival (DSS) 13 months; range 1-60); mRNA was extracted from formalin fixed paraffin embedded blocks and reverse transcribed in cDNA to perform gene expression assay by qRT-PCR. Immunohistochemistry was performed by anti-ERCC1 and anti-TS mAbs and the results were scored from 0 to 3 depending on the percentage of positive tumour cells and staining intensity. The results were statistically correlated with the clinical features and with the DSS of the patients respectively by Fisher's exact test and Logrank test. Results: Of 140 patients 102 received platinum and pemetrexed (in single or in combination), the remaining were untreated due to bad performance status, advanced age or refusing therapy. Statistically significant correlation was found between the lack of protein expression of ERCC1 and DSS in the whole cohort (P = .03). Low levels of TS mRNA were significantly correlated with the DSS in the whole cohort (P = .01) and in the untreated subgroup (including patients not receiving pemetrexed) (P = .04), whereas did not in the subgroup of pemetrexed treated patients Conclusions: ERCC1 protein expression, scored by IHC, and TS gene expression, evaluated by mRNA analysis, seem both important prognostic markers; we suggest their evaluation in routine biopsies from patients with MPM. Disclosure: All authors have declared no conflicts of interest.

The RET/PTC oncogenes, generated by chromosomal rearrangements in papillary thyroid carcinomas, are constitutively activated versions of protoRET, a gene encoding two protein isoforms of a transmembrane tyrosine kinase receptor. By using Ret/ptc2 short isoform (iso9), we have previously demonstrated that Tyr586 (Tyr1062 of protoRet) is the docking site for both the PTB and the SH2 domains of Shc. To determine the relevance of this interaction for the transforming activity of Ret/ptc oncogenes, we have generated and characterized novel Ret/ptc mutants unable to activate Shc: Ret/ptc2 long isoform (iso51)-Y586F and both isoforms of Ret/ptc2-N583A. These mutants neither activate Shc nor transform NIH3T3 cells. Since Tyr1062 shows features of a multifunctional docking site, we have used a Shc mutant (Shc Y317F) to directly assess Shc role. We have demonstrated that in our cell system Shc Y317F behaves like a dominant interfering mutant on the activation of the Grb2-Sos pathway by endogenous Shc triggered by Ret/ptc2. A strong reduction of the transforming activity of Ret/ptc2 in presence of this mutant was also demonstrated. Our data suggest that Shc activation play a key role in the transforming pathways triggered by Ret/ptc oncoproteins. Moreover, we have shown that coexpression of the Shc-Y317F mutant with Ret/ptc2 specifically causes apoptosis, and that the surviving cells lose the long-term expression of one of the two genes.

The ultrastructure of the thymus was examined in young zincdeficient rats in whom alterations of immune functions were present. There was an increased number of macrophages, which contained large amounts of phagocytized material per cell. Signs of hyperactivity of epithelio-reticular cells, associated with a sharp reduction of thymocytes was observed. Thymic hormone activity was reduced. The possible correlation between zinc deficiency and changes of thymus function and structure in rats is discussed.

Objective: In this study we examined the effect of rapamycin (RAPA), a key component of the immunosuppressive regimen in clinical islet transplantation, on islet engraftment and function in vivo. Methods and results: Diabetic C57BL/6 or BALB/C recipient mice were transplanted with 350 syngeneic islets through the portal vein (PV-Tx; C57BL/6 n = 60; BALB/C n = 22) and treated with once-daily oral RAPA (1 mg/kg) or vehicle. No differences in post-transplant blood glucose concentrations and glucose tolerance were observed between RAPA-and vehicle-treated mice. The impact of RAPA on human islet engraftment was assessed in 10 patients with type 1 diabetes treated with 0.1 mg/kg/day rapamycin before islet transplantation. Compared to non pre-treated islet transplant recipents (n = 12), RAPA pre-treated patients had increased blood RAPA concentrations (p = 0.006) and fasting C-peptide concentrations (p = 0.005) in the two weeks post-transplant. RAPA pre-treatment was associated with a reduction in chemokines CCL2 and CCL3 concentrations pre-transplant (p < 0.01), and a dampened chemokine response (p = 0.005) post-transplant. Concordantly, in vitro RAPA inhibited the secretion of CCL2 and CCL3 by monocytes. Conclusion: Rapamycin does not adversely affect intrahepatic islet engraftment in the mouse, and potentially improves islet engraftment in humans by an anti-inflammatory mechanism.

High levels of donor-derived CCL2 have been associated with poor islet allograft outcome in patients with type 1 diabetes. The aim of our work was to determine whether CCL2 secreted by the islet has independent proinflammatory effects that influence engraftment and graft acceptance. Both in mice and humans CCL2 is significantly positively associated with other cytokines/chemokines, in particular with the highly released “proinflammatory” IL-6 and CXCL8 or CXCL1. Transplantation of CCL2-/- islets into syngenic recipients did not improve the transplant function. Transplantation of islets into CCL2-/- syngenic recipients led to a significant improvement of transplant function and partial abrogation of local hepatic inflammation. When evaluated in human islets CCL2 release was strongly related to the immediate local inflammatory response in the liver and impacted short-term human islet function dependently by the induced inflammatory response and independently by the immunosuppressive therapy. The data showed that islet CCL2 release is a sign of “inflamed” islets without having a direct role in graft failure. On the other hand, a causal effect for developing detrimental proinflammatory conditions after transplant was proved for recipient CCL2. Strategies to selectively decrease recipient, but not donor, CCL2 release may increase the success of islet transplantation.

The ultrastructural comparison between E and EA rosettes showed that, in the former, the rosetting lymphocytes are mostly round in shape and their interaction with sheep erythrocytes only consists of limited areas of membrane contact, in the latter, rosetting lymphocytes are mostly in the shape of uropods and surrounding ox red cells show pseudopods protruding towards the lymphocyte and coming into contact with it.

Severe pneumonia is a major challenge in the Intensive Care Unit, as it is characterized by high morbidity and mortality. A high rate of patients with pneumonia, in particular ventilator-associated poneumonia, develops septic shock. Although some interesting results have been reported in uncontrolled studies where IgM-enriched human intravenous immunoglobulins were added to the standard treatment of septic shock, a well-conducted clinical trial is missing[1]. Also, physiopathological data supporting such a trial are presently insufficient.