Extracellular vesicles as potential biomarkers of acute graft-vs-host disease.

Acute graft-vs-host disease (GVHD) is a serious complication after allografting. We carried out an exploratory study to investigate a potential correlation of surface antigens on extracellular vesicles (EVs) and acute GVHD. EVs were extracted from serum samples from 41 multiple myeloma patients who underwent allografting. EVs were characterized by flow cytometry using a panel of 13 antibodies against specific membrane proteins that were reported to be predictive of acute GVHD. We observed a correlation between three potential biomarkers expressed on EV surface and acute GVHD onset by both logistic regression analysis and Cox proportional hazard model. In our study, CD146 (MCAM-1) was correlated with an increased risk-by almost 60%-of developing GVHD, whereas CD31 and CD140-α (PECAM-1 and PDGFR-α) with a decreased risk-by almost 40 and 60%, respectively. These biomarkers also showed a significant change in signal level from baseline to the onset of acute GVHD. Our novel study encourages future investigations into the potential correlation between EVs and acute GVHD. Larger prospective multicenter studies are currently in progress.

Mesenchymal stromal cell-derived extracellular vesicles rescue radiation damage to murine marrow hematopoietic cells.

Mesenchymal stromal cells (MSCs) have been shown to reverse radiation damage to marrow stem cells. We have evaluated the capacity of MSC-derived extracellular vesicles (MSC-EVs) to mitigate radiation injury to marrow stem cells at 4 h to 7 days after irradiation. Significant restoration of marrow stem cell engraftment at 4, 24 and 168 h post irradiation by exposure to MSC-EVs was observed at 3 weeks to 9 months after transplant and further confirmed by secondary engraftment. Intravenous injection of MSC-EVs to 500cGy exposed mice led to partial recovery of peripheral blood counts and restoration of the engraftment of marrow. The murine hematopoietic cell line, FDC-P1 exposed to 500cGy, showed reversal of growth inhibition, DNA damage and apoptosis on exposure to murine or human MSC-EVs. Both murine and human MSC-EVs reverse radiation damage to murine marrow cells and stimulate normal murine marrow stem cell/progenitors to proliferate. A preparation with both exosomes and microvesicles was found to be superior to either microvesicles or exosomes alone. Biologic activity was seen in freshly isolated vesicles and in vesicles stored for up to 6 months in 10% dimethyl sulfoxide at -80 °C. These studies indicate that MSC-EVs can reverse radiation damage to bone marrow stem cells.

Obesity reduces the pro-angiogenic potential of adipose tissue stem cell-derived extracellular vesicles (EVs) by impairing miR-126 content: impact on clinical applications.

BACKGROUND/OBJECTIVES: Soluble factors and cell-derived extracellular vesicles (EVs) are crucial tissue repair mediators in cell-based therapy. In the present study, we investigate the therapeutic impact of EVs released by adipose tissue-derived stem cells (ASCs) recovered from obese subjects' visceral and subcutaneous tissues. METHODS: ASCs were recovered from 10 obese (oASCs) and 6 non-obese (nASCs) participants and characterized. In selected experiments, nASCs and oASCs were cultured with palmitic acid (PA) or high glucose (HG), respectively. EVs from obese (oEVs) and non-obese (nEVs) subjects' visceral and subcutaneous ASCs were collected after ultracentrifugation and analyzed for their cargo: microRNA-126 (miR-126), vascular endothelial growth factor (VEGF), and matrix metalloproteinase 2 (MMP-2), and for their biological effects on endothelial cells (ECs). Western blotting analysis and loss- and gain-of function experiments were performed. RESULTS: oEVs show impaired angiogenic potential compared with nEVs. This effect depends on EV cargo: reduced content of VEGF, MMP-2 and, more importantly, miR-126. We demonstrate, using gain- and loss-of-function experiments, that this reduced miR-126 content leads to Spred1 upregulation and the inhibition of the extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase pathway in ECs. We also show that PA treatment of nASCs translates into the release of EVs that recapitulate oEV cargo. Moreover, HG treatment of oASCs further reduces miR-126 EV content and EV-mediated in vitro angiogenesis. Finally, impaired pro-angiogenic potential is also detected in EVs released from obese subcutaneous adipose tissue-derived ASCs. CONCLUSIONS: These results indicate that obesity impacts on EV pro-angiogenic potential and may raise concerns about the use of adipose tissue-derived EVs in cell-based therapy in the obese setting.

Role of Lefty in the anti tumor activity of human adult liver stem cells.

Recent studies demonstrated that factors derived from embryonic stem cells inhibit the tumorigenicity of a variety of cancer cell lines. Embryonic stem cell-secreted Lefty, an inhibitor of Nodal-signalling pathway, was implicated in reprogramming cancer cells. Whether adult stem cells exhibited similar properties has not been explored. The aim of the present study was to investigate whether the conditioned medium (CM) derived from adult stem cells influence in vitro and in vivo tumor growth by a Nodal-dependent pathway. In particular we compared the anti-tumor effect of CM from human liver stem cells (HLSC) with that of bone marrow-derived mesenchymal stem cells (MSC). We found that HLSC-CM inhibited the in vitro growth and promoted apoptosis in HepG2 cells that expressed a deregulated Nodal pathway. The effect of HLSC-CM was related to the presence of Lefty A in the CM of HLSC. Silencing Lefty A in HLSC or Lefty A blockade with a blocking peptide abrogated the anti-proliferative and pro-apoptotic effect of HLSC-CM. Moreover, the administration of human recombinant Lefty A protein mimicked the effect of HLSC-CM indicating that Nodal pathway is critical for the growth of HepG2. At variance of HLSC, bone marrow-derived MSC did not express and release Lefty A and the MSC-CM did not exhibited an anti-tumor activity in vitro, but rather stimulated proliferation of HepG2. In addition, the intra-tumor administration of HLSC-CM was able to inhibit the in vivo growth of HepG2 hepatoma cells implanted subcutaneously in SCID mice. At variance, HLSC-CM derived from Lefty A silenced HLSC was unable to inhibit tumor growth. In conclusion, the results of present study suggest that Lefty A may account for the tumor suppressive activity of HLSC as a result of an inhibition of the Nodal-signalling pathway by a mechanism similar to that described for embryonic stem cells.

Tumor-derived microvesicles and the cancer microenvironment.

Tumor cells release microvesicles (MVs) that may remain in the extracellular space in proximity to the cell of origin, or that may migrate to distant sites by entering biological fluids. Increasing evidence indicates that MVs are mediators of cell-to-cell communication which are able to deliver specific signals, both within the tumor microenvironment and in the long-range. MVs are able to transfer bioactive lipids and proteins, including oncogene products and receptors, from the cell of origin to recipient cell. In addition, MVs may induce epigenetic changes in recipient cells by transferring genetic information in the form of mRNA, microRNA and oncogenes. Several changes in the phenotype and function that occur in stromal cells within the cancer microenvironment have been ascribed to tumor cell-derived MVs. In this review we discuss the various biological actions of tumor-derived MVs and their potential role in tumor biology.

The ghrelin gene products and exendin-4 promote survival of human pancreatic islet endothelial cells in hyperglycaemic conditions, through phosphoinositide 3-kinase/Akt, extracellular signal-related kinase (ERK)1/2 and cAMP/protein kinase A (PKA) signalling pathways.

AIMS/HYPOTHESIS: Pancreatic islet microendothelium exhibits unique features in interdependent relationship with beta cells. Gastrointestinal products of the ghrelin gene, acylated ghrelin (AG), unacylated ghrelin (UAG) and obestatin (Ob), and the incretin, glucagon-like peptide-1 (GLP-1), prevent apoptosis of pancreatic beta cells. We investigated whether the ghrelin gene products and the GLP-1 receptor agonist exendin-4 (Ex-4) display survival effects in human pancreatic islet microendothelial cells (MECs) exposed to chronic hyperglycaemia. METHODS: Islet MECs were cultured in high glucose concentration and treated with AG, UAG, Ob or Ex-4. Apoptosis was assessed by DNA fragmentation, Hoechst staining of the nuclei and caspase-3 activity. Western blot analyses and pharmacological inhibition of protein kinase B (Akt) and extracellular signal-related kinase (ERK)1/2 pathways, detection of intracellular cAMP levels and blockade of adenylyl cyclase (AC)/cAMP/protein kinase A (PKA) signalling were performed. Levels of NO, IL-1ß and vascular endothelial growth factor (VEGF)-A in cell culture supernatant fractions were measured. RESULTS: Islet MECs express the ghrelin receptor GHS-R1A as well as GLP-1R. Treatment with AG, UAG, Ob and Ex-4 promoted cell survival and significantly inhibited glucose-induced apoptosis, through activation of PI3K/Akt, ERK1/2 phosphorylation and intracellular cAMP increase. Moreover, peptides upregulated B cell lymphoma 2 (BCL-2) and downregulated BCL-2-associated X protein (BAX) and CD40 ligand (CD40L) production, and significantly reduced the secretion of NO, IL-1ß and VEGF-A. CONCLUSIONS/INTERPRETATION: The ghrelin gene-derived peptides and Ex-4 exert cytoprotective effects in islet MECs. The anti-apoptotic effects involve phosphoinositide 3-kinase (PI3K)/Akt, ERK1/2 and cAMP/PKA pathways. These peptides could therefore represent a potential tool to improve islet vascularisation and, indirectly, islet cell function.

Syndecan-1 promotes the angiogenic phenotype of multiple myeloma endothelial cells.

Angiogenesis is considered a hallmark of multiple myeloma (MM) progression. In the present study, we evaluated the morphological and functional features of endothelial cells (ECs) derived from bone marrow (BM) of patients affected by MM (MMECs). We found that MMECs compared with normal BM ECs (BMECs) showed increased expression of syndecan-1. Silencing of syndecan-1 expression by RNA interference technique decreased in vitro EC survival, proliferation and organization in capillary-like structures. In vivo, in severe combined immunodeficient mice, syndecan-1 silencing inhibited MMEC organization into patent vessels. When overexpressed in human umbilical vein ECs and BMECs, syndecan-1 induced in vitro and in vivo angiogenic effects. Flow-cytometric analysis of MMECs silenced for syndecan-1 expression indicated a decreased membrane expression of vascular endothelial growth factor (VEGF) receptor-2 (VEGFR-2). Immunoprecipitation and confocal analysis showed colocalization of VEGFR-2 with syndecan-1. Absence of nuclear translocation of VEGFR-2 in syndecan-1-knockdown cells together with the shift from perinuclear localization to recycling compartments suggest a role of syndecan-1 in modulation of VEGFR-2 localization. This correlated with an in vitro decreased VEGF-induced invasion and motility. These results suggest that syndecan-1 may contribute to the highly angiogenic phenotype of MMECs by promoting EC proliferation, survival and modulating VEGF-VEGFR-2 signalling.

IL-3 is a novel target to interfere with tumor vasculature.

Angiogenesis inhibiting agents are currently integral component of anticancer therapy. However, tumors, initially responsive to anti-angiogenic drugs or vascular targeting agents, can acquire resistance. The limited clinical efficacy might result from the heterogeneous nature of tumors or alternatively from the unique phenotype of tumor vascular cells, widely diverse from so-called 'normal' endothelium. Hence, defining the molecular mechanisms driving this diversity might provide a rational basis to design combinatory therapies that should be more effective in avoiding resistance. Herein, we demonstrated that tumor-derived endothelial cells (TECs) isolated from breast and kidney carcinomas retained an endothelial phenotype, but outspread independently of growth factors. Applying small interfering RNA approach, we demonstrated that interleukin (IL)-3, but not vascular endothelial growth factor, released by TECs, supports their autocrine growth and promotes in vivo vessel formation and tumor angiogenesis. Meanwhile, we found that the expression of the membrane-bound kit ligand (mbKitL) depends on IL-3, and it is crucial for adhesion of endothelial progenitor cells (EPCs) and inflammatory cells to TECs. These events required Akt activation. Finally, the finding that depletion of the mbKitL prevented EPC and inflammatory cell trafficking into vascular microenvironment, indicates that, as in bone marrow, the mbKitL can act as a membrane/adhesion molecule for c-Kit-expressing cells. These data provide evidences that an IL-3 autocrine loop can drive a tumor endothelial switch and that targeting IL-3 might confer a significant therapeutic advantage to hamper tumor angiogenesis.

Loss of nephrin expression in glomeruli of kidney-transplanted patients under m-TOR inhibitor therapy.

The development of proteinuria has been observed in kidney-transplanted patients on m-TOR inhibitor (m-TORi) treatment. Recent studies suggest that m-TORi(s) may alter the behavior and integrity of glomerular podocytes. We analyzed renal biopsies from kidney-transplanted patients and evaluated the expression of nephrin, a critical component of the glomerular slit-diaphragm. In a group of patients on 'de novo' m-TORi-treatment, the expression of nephrin within glomeruli was significantly reduced in all cases compared to pretransplant donor biopsies. Biopsies from control transplant patients not treated with m-TORi(s) failed to present a loss of nephrin. In a group of patients subsequently converted to m-TORi-treatment, a protocol biopsy performed before introduction of m-TORi was also available. The expression of nephrin in the pre-m-TORi biopsies was similar to that observed in the pretransplant donor biopsies but was significantly reduced after introduction of m-TORi(s). Proteinuria increased after the m-TORi inititiation in this group. However, in some cases proteinuria remained normal despite reduction of nephrin. In vitro, sirolimus downregulated nephrin expression by human podocytes. Our results suggest that m-TORi(s) may affect nephrin expression in kidney-transplanted patients, consistently with the observation in vitro on cultured podocytes.

[Mechanisms causing chronic renal injury in kidney disease and their possible reversibility]. / I meccanismi del danno cronico renale nelle nefropatie e la loro possibile reversibilita.

Much study has been dedicated to the understanding of the mechanisms leading to the progression of renal injury and to the development of strategies to limit this progression or possibly induce tissue regeneration. Among several identified mechanisms, the role of angiotensin II is widely recognized. Moreover, the progression of glomerular damage is characterized by capillary loss, reduction of the proliferative response, and production of antiangiogenic factors. Several lines of evidence support the potential effect of therapeutic startegies aimed at interfering with angiotensin II or stimulating angiogenesis in order to reduce the progression of renal injury. Recent work has underlined the potential of strategies involving the use of stem cells. Different populations of stem cells have been identified in the adult kidney. During renal injury, stem cells derived from the bone marrow that migrate through the circulation to the kidney may contribute to tissue repair. The regenerative potential of stem cells could be exploited by administration of ex vivo expanded stem cell populations or by the development of techniques to expand and differentiate local stem cells.

[The role of angiogenesis in renal carcinoma]. / Ruolo dell'angiogenesi nel carcinoma renale.

Renal cell carcinoma is characterized by intense angiogenesis associated with the inactivation of the von Hippel-Lindau oncosuppressor gene with consequent hyperexpression of proangiogenic factors. Functional and molecular characterization of renal tumor endothelial cells has demonstrated an increase in angiogenesis and cell survival. The proangiogenic phenotype was due to hyperactivation of the PI3K/Akt/mTor pathway, which downregulates the synthesis of the antiangiogenic factor thrombospondin-1. Moreover, renal tumor endothelial cells presented an immature and embryonic phenotype with expression of the embryonic kidney-specific gene PAX-2. It is conceivable that the endothelium present in renal carcinoma is heterogeneous, with a possible origin from adjacent vessels, resident or circulating stem cells, or from the tumor cells themselves. The relevance of the angiogenic process in renal carcinoma is underlined by the therapeutic effect of antiangiogenic drugs. Different drugs against VEGF, such as the anti-VEGF monoclonal antibody bevacizumab, and small molecule tyrosine-kinase inhibitors, such as sunitinib and sorafenib, showed a clinical effect in patients with metastatic carcinoma. However, antiangiogenic therapy, although beneficial, is not sufficient per se. These studies suggest a role for the angiogenic program in the growth and dissemination of renal carcinoma and indicate the need for new therapeutic strategies.

[Stem cells and repair of kidney damage]. / Cellule staminali e riparazione del danno renale.

In the adult kidney, different populations of progenitor cells (or stem cells) have been identified. These cells may represent a remnant of embryonic stem cells in the adult tissue, or populations of bone-marrow-derived stem cells homed within the kidney and modified by the local microenvironment. This modification may be the expression of a partial commitment or of different degrees of maturation. Resident stem cells may account for the growth of the organ during development, for the physiological cell turnover, and for the repair of kidney damage. In addition, stem cells derived from the bone marrow and migrated through the circulation to the site of the damage may contribute to tissue repair. Preliminary studies suggest that this regenerative potential of stem cells could be exploited for therapeutic purposes by administration of ex vivo expanded stem cell populations or by development of strategies aimed to expand and differentiate local stem cells.

Exogenous mesenchymal stem cells localize to the kidney by means of CD44 following acute tubular injury.

Mesenchymal stem cells (MSC) were recently shown to migrate to injured tissues when transplanted systemically. The mechanisms underlying the migration and homing of these cells is, however, unclear. In this study, we examine the role of CD44 and its major ligand, hyaluronic acid, in the trafficking of intravenously injected MSC in the glycerol-induced mouse model of acute renal failure (ARF). In vitro, hyaluronic acid promoted a dose-dependent migration of the stem cells that was inhibited by an anti-CD44 blocking monoclonal antibody. In vivo, stem cells injected into mice with ARF migrated to the injured kidney where hyaluronic acid expression was increased. Their presence correlated with morphological and functional recovery. Renal localization of the MSC was blocked by pre-incubation with the CD44 blocking antibody or by soluble hyaluronic acid. Stem cells derived from CD44 knockout mice did not localize to the injured kidney and did not accelerate morphological or functional recovery. Reconstitution by transfection of CD44 knockout stem cells with cDNA encoding wild-type CD44, but not a loss of function CD44 unable to bind hyaluronic acid, restored in vitro migration and in vivo localization of the cells to injured kidneys. We suggest that CD44 and hyaluronic acid interactions recruit exogenous MSC to injured renal tissue and enhance renal regeneration.

Insulin-like growth factor binding protein-3 induces angiogenesis through IGF-I- and SphK1-dependent mechanisms.

Angiogenesis is critical for development and repair, and is a prominent feature of many pathological conditions. Based on evidence that insulin-like growth factor binding protein (IGFBP)-3 enhances cell motility and activates sphingosine kinase (SphK) in human endothelial cells, we have investigated whether IGFBP-3 plays a role in promoting angiogenesis. IGFBP-3 potently induced network formation by human endothelial cells on Matrigel. Moreover, it up-regulated proangiogenic genes, such as vascular endothelial growth factor (VEGF) and matrix metalloproteinases (MMP)-2 and -9. IGFBP-3 even induced membrane-type 1 MMP (MT1-MMP), which regulates MMP-2 activation. Decreasing SphK1 expression by small interfering RNA (siRNA), blocked IGFBP-3-induced network formation and inhibited VEGF, MT1-MMP but not IGF-I up-regulation. IGF-I activated SphK, leading to sphingosine-1-phosphate (S1P) formation. The IGF-I effect on SphK activity was blocked by specific inhibitors of IGF-IR, PI3K/Akt and ERK1/2 phosphorylation. The disruption of IGF-I signaling prevented the IGFBP-3 effect on tube formation, SphK activity and VEGF release. Blocking ERK1/2 signaling caused the loss of SphK activation and VEGF and IGF-I up-regulation. Finally, IGFBP-3 dose-dependently stimulated neovessel formation into subcutaneous implants of Matrigel in vivo. Thus, IGFBP-3 positively regulates angiogenesis through involvement of IGF-IR signaling and subsequent SphK/S1P activation.

Apoptic renal carcinoma cells are better inducers of cross-presenting activity than their primary necrotic counterpart.

Vaccination with tumor-loaded dendritic cells (DC) is a promising treatment strategy for patients with renal cell carcinoma (RCC). Cells undergoing cell death proved useful as a source of tumor antigen for DC loading. Both apoptotic and necrotic tumor cells have been shown to efficiently load RCC-tumor antigens on DC. However, no direct comparison of these two kinds of death has been attempted in the same RCC. We compared DC pulsed with apoptotic cells, whole cell lysates or their supernatants of the cell line K1, derived from a patient with clear cell RCC, to determine their ability to activate T cells. Monocyte-derived DCs were pulsed with the different sources of tumor antigen, matured and co-cultured with autologouos peripheral blood lymphocytes. After three weekly re-stimulations with DCs, generation of cytotoxic T lymphocytes CTL was assessed by IFN-gamma release in an ELISpot assay in the presence of the sensitizing target. By comparison with lysate, apoptotic tumor cells induced a higher frequency of MHC class I-restricted IFN-gamma releasing lymphocytes. A higher CTL response was induced by pulsing DCs with cell lysate supernatant compared with whole cell lysate. These results indicate that, although necrotic death has been regarded as highly permissive when compared to apoptotic death, the immunogenicity of the death treatment may vary from one tumor to another.

Antiangiogenic and immunomodulatory effects of rapamycin on islet endothelium: relevance for islet transplantation.

Donor intra-islet endothelial cells contribute to neovascularization after transplantation. Several factors may interfere with this process and ultimately influence islet engraftment. Rapamycin, a central immunosuppressant in islet transplantation, is an mTOR inhibitor that has been shown to inhibit cancer angiogenesis. The aim of this study was to evaluate the effects of rapamycin on islet endothelium. Rapamycin inhibited the outgrowth of endothelial cells from freshly purified human islets and the formation of capillary-like structures in vitro and in vivo after subcutaneous injection within Matrigel plugs into SCID mice. Rapamycin decreased migration, proliferation and angiogenic properties of human and mouse islet-derived endothelial cell lines with appearance of apoptosis. The expression of angiogenesis-related factors VEGF, alphaVbeta3 integrin and thrombospondin-1 on islet endothelium was altered in the presence of rapamycin. On the other hand, rapamycin decreased the surface expression of molecules involved in immune processes such as ICAM-1 and CD40 and reduced the adhesion of T cells to islet endothelium. Our results suggest that rapamycin exerts dual effects on islet endothelium inducing a simultaneous inhibition of angiogenesis and a down-regulation of receptors involved in lymphocyte adhesion and activation.

Glomerular clusterin is associated with PKC-alpha/beta regulation and good outcome of membranous glomerulonephritis in humans.

Mechanisms for human membranous glomerulonephritis (MGN) remain elusive. Most up-to-date concepts still rely on the rat model of Passive Heymann Nephritis that derives from an autoimmune response to glomerular megalin, with complement activation and membrane attack complex assembly. Clusterin has been reported as a megalin ligand in immunodeposits, although its role has not been clarified. We studied renal biopsies of 60 MGN patients by immunohistochemistry utilizing antibodies against clusterin, C5b-9, and phosphorylated-protien kinase C (PKC) isoforms (pPKC). In vitro experiments were performed to investigate the role of clusterin during podocyte damage by MGN serum and define clusterin binding to human podocytes, where megalin is known to be absent. Clusterin, C5b-9, and pPKC-alpha/beta showed highly variable glomerular staining, where high clusterin profiles were inversely correlated to C5b-9 and PKC-alpha/beta expression (P=0.029), and co-localized with the low-density lipoprotein receptor (LDL-R). Glomerular clusterin emerged as the single factor influencing proteinuria at multivariate analysis and was associated with a reduction of proteinuria after a follow-up of 1.5 years (-88.1%, P=0.027). Incubation of podocytes with MGN sera determined strong upregulation of pPKC-alpha/beta that was reverted by pre-incubation with clusterin, serum de-complementation, or protein-A treatment. Preliminary in vitro experiments showed podocyte binding of biotinilated clusterin, co-localization with LDL-R and specific binding inhibition with anti-LDL-R antibodies and with specific ligands. These data suggest a central role for glomerular clusterin in MGN as a modulator of inflammation that potentially influences the clinical outcome. Binding of clusterin to the LDL-R might offer an interpretative key for the pathogenesis of MGN in humans.

The expression of CD154 by Kaposi's sarcoma cells mediates the anti-apoptotic and migratory effects of HIV-1-TAT protein.

Kaposi's sarcoma (KS) is a malignancy associated to conditions of immune system impairment such as HIV-1 infection and post-transplantation therapy. Here we report that HIV-1-Tat protein, at concentrations well below those detected in AIDS patients, up-regulates the expression of both CD40 and CD154 on KS cells. This occurred also in the presence of vincristine, that at doses shown to induce apoptosis decreased the expression of both CD40 and CD154 on KS cells. The treatment with a soluble CD40-muIg fusion protein (CD40 fp) that prevents the binding of CD154 with cell surface CD40, as well as the transfection with a vector for soluble CD40 (KS sCD40), decreased the anti-apoptotic effect of Tat. Moreover, Tat-induced motility of KS cells was inhibited by soluble CD40 fp. Tat also enhanced the expression of intracellular proteins known to transduce signals triggered by CD40 engagement, in particular TRAF-3. Tat as well as soluble CD154 (sCD154) prevented vincristine-induced reduction of TRAF-3 in KS cells transfected with a vector for neomycin resistance (KS psv-neo), but not in KS sCD40. Immunoprecipitation studies showed that Tat induced CD40 / TRAF-3 association and that this binding was abrogated upon the incubation with the soluble CD40 fp. These data suggest that Tat activates the CD40-CD154 pathway by enhancing the membrane expression of CD40 and in particular of CD154, and by activating the TRAF-3-dependent signaling pathway of CD40. These findings indicate that the CD40-CD154 pathway mediates the anti-apoptotic and migratory effects of HIV-1- Tat, suggesting the potential therapeutic benefits of blocking CD40 activation in HIV-1-associated KS.

Primary and immortalised human pancreatic islet endothelial cells: phenotypic and immunological characterisation.

AIMS/HYPOTHESIS: Studies on the biology of the microvascular endothelial cells (MECs) that surround and penetrate the pancreatic islets are hampered by difficulties in isolating and culturing large numbers of pure cells. We aimed to morphologically and functionally characterise primary MECs purified and cultured from human islets, and to establish a simian virus 40 (SV40)-immortalised cell line from these primary cultures. MATERIALS AND METHODS: Human islet MECs were extracted and purified using anti-CD105 coated immunomagnetic beads, and endothelial markers and surface molecules analysed by flow cytometric analysis. An immortalised cell line was then established by using a chimeric adeno5/SV40 virus. RESULTS: Islet MECs expressed classic and specific endothelial markers, a high basal level of intercellular adhesion molecule-1, and low levels of E-selectin and TNF (previously known as TNF-alpha) inducible vascular cell adhesion molecule-1. IFNG (previously known as IFN-gamma) induced expression of HLA class II molecules. The immortalised islet MECs expanded rapidly, exhibited increased DNA synthesis, and were passaged approximately 30 times, without signs of senescence. They retained the endothelial characteristics of the parental cells, and behaved as the primary cells in terms of TNF stimulation of expression of adhesion molecules and support of leucocyte adhesion and transmigration. CONCLUSIONS/INTERPRETATION: The immortalised islet MECs that we have established could effectively represent a substitute for primary counterparts for in vitro studies on the role of the microvasculature in pathophysiological processes involved in type 1 and type 2 diabetes.