Cinzia Scavullo

Anti-L-NGFR and -CD34 Monoclonal Antibodies Identify Multipotent Mesenchymal Stem Cells in Human Adipose Tissue

Stem cells hold great promise in tissue engineering for repairing tissues damaged by disease or injury. Mesenchymal stem cells (MSCs) are multipotent cells able to proliferate and differentiate into multiple mesodermal tissues such as bone, cartilage, muscle, tendon, and fat. We have previously reported that the low-affinity nerve growth factor receptor (L-NGFR or CD271) defines a subset of cells with high proliferative, clonogenic, and multipotential differentiation ability in adult bone marrow (BM). It has been recently shown that adipose tissue is an alternative source of adult multipotent stem cells and human adipose-derived stem cells, selected by plastic adherence (PA hASCs), have been extensively characterized for their functional potentials in vitro. In this study, immunoselected L-NGFR(+) and CD34(+) subpopulations have been analyzed and compared with the PA hASCs. Phenotypic profile of freshly purified subpopulations showed an enrichment in the expression of some stem cell markers; indeed, a great percentage of L-NGFR(+) cells co-expressed CD34 and CD117 antigens, whereas the endothelial-committed progenitor markers KDR and P1H12 were mainly expressed on CD34(+) cells. Differently from PA hASCs, the immunoseparated fractions showed high increments in cell proliferation, and the fibroblast colony-forming activity (CFU-F) was maintained throughout the time of culture. Furthermore, the immunoselected populations showed a greater differentiative potential toward adipocytes, osteoblasts, and chondrocyte-like cells, compared to PA hASCs. Our data suggest that both CD34(+) and L-NGFR(+) hASCs can be considered alternative candidates for tissue engineering and regenerative medicine applications.

Mu opioid receptor activation modulates Toll like receptor 4 in murine macrophages.

Opioids have been shown to affect both innate and adaptive immunity. We previously showed that morphine affects the macrophage production of pro-inflammatory cytokines after LPS in a NFkB dependent manner. Toll like receptors (TLRs) play a crucial role in the signaling pathways which lead to NFkB activation. TLR4 is considered the Lipopolysaccaride (LPS) receptor. The data here presented show that, in murine macrophages, morphine impacts on the immune function acting on the early step of pathogen recognition. Morphine, when added to RAW 264.7 cells and when injected into mice (s.c. 20mg/kg) is in fact able to decrease TLR4 both at mRNA and protein level in RAW cells and peritoneal macrophages. In the same cells, the mu opioid receptor (MOR) antagonist Naltrexone increases TLR4 levels, thus suggesting a role of the endogenous opioid system in TLR4 regulation. The effect of the two drugs is moreover lost in case of co-administration. Experiments with MOR KO mice and with DAMGO (MOR specific agonist) confirm that the effect of morphine on TLR4 mRNA in peritoneal macrophages is due to the MOR activation. Moreover the effect on TLR4 is blocked by PTX thus indicating the involvement of a G(i) protein after MOR binding. This work unveils a clear link between MOR activation and TLR4, suggesting a new possible mechanism at the basis of the peripheral immunosuppressive effect of opioids.

Antiendothelial cell antibodies induce apoptosis of bone marrow endothelial progenitors in systemic sclerosis.

Objective. Patients with systemic sclerosis (SSc) have significantly fewer and functionally impaired endothelial progenitor cells (EPC) in peripheral blood and bone marrow; further, endothelial apoptosis seems to play a primary role in the pathogenesis of vascular damage. We investigated whether the failure of bone marrow EPC is related to their apoptotic phenotype and analyzed the possible mechanisms inducing apoptosis. Methods. The presence of apoptotic cells was investigated in bone marrow aspirates taken from patients with SSc; microvessel density (MVD) and the immunohistochemical expression of vascular endothelial growth factor (VEGF) were also measured in bone marrow biopsies. A correlation between EPC apoptosis and the presence of antiendothelial cell antibodies (AECA) was also investigated. Results. We confirmed the presence of bone marrow EPC dysfunction in SSc, while hematopoiesis was not impaired. Bone marrow studies showed a high percentage of apoptotic progenitors, no signs of fibrosis or an altered MVD, and an increased VEGF index. The patients’ bone marrow plasma showed significant titers of AECA, and their presence correlated with that of apoptotic progenitors. These findings were further confirmed by an in vitro assay in which the apoptosis of normal progenitors was induced by the addition of AECA+ purified IgG. Conclusion. Our results showed that apoptosis in patients with SSc involves the source compartment of endothelial progenitors and correlates with AECA activity. These findings support the hypothesis that AECA may play a pathogenetic role by affecting the bone marrow EPC machinery that should repair the peripheral vascular lesions.

Novel second mitochondria-derived activator of caspases (Smac) mimetic compounds sensitize human leukemic cell lines to conventional chemotherapeutic drug-induced and death receptor-mediated apoptosis

SummaryThe Inhibitor of Apoptosis Proteins (IAPs) are important regulators of programmed cell death. XIAP is the most potent among them and is over-expressed in several hematological malignancies. Its activity is endogenously antagonized by SMAC/DIABLO, and also by small molecules mimicking Smac that can induce apoptosis in tumor cells. Here we describe the activity of 56 newly synthesized Smac-mimetics in human leukemic cell lines and normal CD34+ progenitor cells. Our compounds bind to XIAP with high affinity, reduce the levels of cIAP1 and are cytotoxic at nanomolar or low micromolar concentrations. Furthermore, the Smac-mimetics synergize with Cytarabine, Etoposide and especially with TRAIL in combination treatments. Apoptosis activation was clearly detectable by the occurrence of sub G1 apoptotic peak and the accumulation of cleaved PARP, caspase 8 and caspase 3. Interestingly, the down-regulation of XIAP sensitized Jurkat cells to drugs too, confirming the role of this protein in drug-resistance. In conclusion, while being very active in leukemic cells, our Smac-mimetics have modest effects on normal hematopoietic progenitors, suggesting their promising therapeutic potential as a new class of anticancer drugs in onco-hematology, particularly when combined with TRAIL, to overcome the resistance of cancer cells.

Dimeric Smac mimetics/IAP inhibitors as in vivo-active pro-apoptotic agents. Part II: Structural and biological characterization.

Novel pro-apoptotic, homodimeric and heterodimeric Smac mimetics/IAPs inhibitors connected through head-head (8), tail-tail (9) or head-tail linkers (10), were biologically and structurally characterized. In vitro characterization (binding to BIR3 and linker-BIR2-BIR3 domains from XIAP and cIAP1, cytotoxicity assays) identified early leads from each dimer family. Computational models and structural studies (crystallography, NMR, gel filtration) partially rationalized the observed properties for each dimer class. Tail-tail dimer 9a was shown to be active in a breast and in an ovary tumor model, highlighting the potential of dimeric Smac mimetics/IAP inhibitors based on the N-AVPI-like 4-substituted 1-aza-2-oxobicyclo[5.3.0]decane scaffold as potential antineoplastic agents.

Recombinant human erythropoietin stimulates vasculogenesis and wound healing in a patient with systemic sclerosis complicated by severe skin ulcers

Systemic sclerosis (SSc) is often complicated by severe skin ulcers that are unresponsive to traditional treatments. Vascular alterations are responsible for the ischaemic features of the disease in both the skin and visceral organs. Defective neoangiogenesis correlates with an abnormally reduced quantity of circulating endothelial progenitor cells (EPCs) caused by impaired maturation potential and proliferative capacity of bone‐marrow endothelial stem cells. We report a patient with nonhealing cutaneous ulcers successfully treated with recombinant human erythropoietin (rHuEPO). The possible biological effects of this drug were also investigated. Before rHuEPO treatment, the bone‐marrow sample contained reduced numbers of EPCs, which were functionally impaired. After a 6‐month rHuEPO cycle, a marked increase in endothelial progenitor markers was seen, along with a significant reduction in their apoptotic rates. The clinical and laboratory data variations before and after rHuEPO treatment give new insights into the pathogenetic role of impaired endothelial stem‐cell maturation and defective neoangiogenesis in patients with SSc.

Single-agent Smac-mimetic compounds induce apoptosis in B chronic lymphocytic leukaemia (B-CLL)

Defective apoptosis is a hallmark of the progression of B chronic lymphocytic leukaemia (B-CLL). Smac-mimetics have been shown to induce apoptosis in several tumours. We describe the in vitro pro-apoptotic activity and regulation of the molecular pathway induced by new Smac-mimetics in B-CLL. The cytotoxic effect was significantly higher in B-CLL samples than in healthy controls. No significant synergistic effect was observed in combined treatment. In conclusion one of our compounds (Smac66), used as monotherapy and not in combination, is highly active against B-CLL cells thus suggesting a promising therapeutic potential as a new class of antileukemic drugs in haematology.

Rational design, synthesis and characterization of potent, drug-like monomeric Smac mimetics as pro-apoptotic anticancer agents

A set of phenyl-substituted Smac mimetics/IAP inhibitor analogues of lead compound 2a was synthesized, aiming to retain its strong cell-free potency while increasing its bioavailability. Seventeen compounds 2b-r were prepared and characterized in vitro, using cell-free and cellular assays. Among them, the p-CF(3) substituted analogue 2m showed the best permeability through cell membranes, and was selected for further in vitro and in vivo studies due to its strong, sub-micromolar cellular potency.

SAT0029 Mesenchymal stem cells modulate in vitro fibroblast activity in systemic sclerosis

Background Bone marrow (BM)-derived mesenchymal stem cells (MSC) are currently under investigation for their potential role in the management of some autoimmune diseases. Besides their regenerative effects and ability to induce tissue repair, MSC seem to possess potent immunomodulatory and anti-inflammatory effects, either by direct cell-cell interaction or by secreting different molecules able to exert a paracrine effects on local environment. Objectives Aim of the study was to evaluate the paracrine effects of BM-MSCs mesenchymal stem cells (MSC) isolated from normal individuals and patients with systemic sclerosis (SSc), on proliferation and cytokine production of cutaneous fibroblasts. Methods Fibroblasts obtained from skin punch biopsies (10 dcSSc patients and 5 healthy donors -HD) were cultured in vitro. MSC were isolated by plastic adherence from SSc and HD bone marrow (BM). Co-cultures were assessed with HD or SSc fibroblasts with HD or SSc MSC. Fibroblast proliferation was assessed by WST-1 assay and data were expressed as median percentage of 3 replicates. Levels of TGF-β, Stem Cell Factor (SCF), and metalloproteinase MMP-9 were detected in the co-culture supernatants by commercial ELISA Kits. Results Co-culture assays confirmed our previous results: an inhibition of SSc fibroblast proliferation, higher when fibroblasts were cultured with nBM MSCs (% of fibroblast proliferation vs fibroblasts without MSCs (100%): 75.8±9.9% with SSc MSCs vs 64.7±8.3% with nBM MSCs; P=0.038). In the presence of fibroblasts from healthy subjects, MSCs from normal and SSc BM equally inhibited proliferation. In comparison with basal values, the levels of the cytokines showed only in the presence of SSc fibroblasts and HD MSC a significant reduction in the content of SCF (mean±SD: 14.68±3.4 pg/ml vs 11.48±4.4; P=0.01) and TGF-β (3169.8±810 pg/ml vs 2450±900; P=0.0098), while no significant changes were observed in the presence of SSc MSC. As regards MMP-9, the basal value (mean±SD: 0.008±0.01 ng/ml) significantly increased in the presence of both HD (0.035±0.02; P=0.003) and SSc (0.094±0.072 ng/ml; P=0.01) MSC. Conclusions Our findings demonstrate that normal MSC (more than SSc-MSC) may provide an important signal for skin fibroblasts, and particularly for SSc fibroblasts, by both exerting anti-proliferative effects and down-regulating their activation state. The present results may reinforce the perspective for a future use of BM-MSC as a therapeutic option in SSc, namely for their anti-fibrotic potential. Disclosure of Interest None Declared

Toll like receptor 4: A role in morphine immunomodulatory effect

253 The effects of corticotropin releasing factor (CRF) and CRF receptors on antibody production E.M. Smith , J. Tsang b a University of Texas Medical Branch, Microbiology & Immunology, Galveston, TX 77555, United States b University of Georgia, United States Corticotropin releasing factor (CRF) is a neuroendocrine hormone that is also synthesized and released by leukocytes. It enhances the antigen-specific, in vitro antibody response and also acts in a proinflammatory fashion. The goal was to determine if CRF enhances antibody production through specific CRF receptors; CRF-R1 and CRF-R2. Using the enzyme-linked immunospot (ELISPOT) assay, the effects of CRF, CRF-R1 and CRF-R2 on antibody production was determined. Treatments of CRF and/or CRF receptor antagonists (alpha-helical CRF (a-h-CRF) and antalarmin) were added to mouse splenocyte cultures along with an antigen, trinitrophenyl-lipopolysaccharide (TNP-LPS), to induce antibody production. The antagonist, a-h-CRF blocks both receptor subtypes while another antagonist, antalarmin only blocks CRF-R1. The preliminary data show that the ELISPOT which is a more sensitive assay reported an enhanced antibody response similar to that seen with the plaque-forming assay used previously. Further, the ELISPOT showed that CRF enhanced both the number of antibody forming cells and the amount of antibody produced. The results further showed a-h-CRF and antalarmin have similar effects in antibody production, both inhibiting the CRF enhancement of antibody production up to 100%. Since the antalarmin was as effective as the a-h-CRF it suggests that the CRF-R1 is the major mediator of the antibody enhancement. New CRF-R antagonists are being developed and these studies imply that they might be useful as immunomodulators. (Supported by NIAIDAI066273 and UTMB-SURP.) doi:10.1016/j.bbi.2010.07.047 Abstract # 254 Pro-inflammatory cytokine regulation of the serotonin transporter and serotonin autoreceptor 1b in rat serotonergic neurons N. Hengen , E.M. Smith b a Shenandoah University, Bernard J. Dunn School of Pharmacy, Winchester, VA, United States b University of Texas Medical Branch, United States The serotonin system in the brain, with the serotonin transporter (SERT) and serotonin autoreceptors (5-HT1A and 5-HT1B) in particular, play an important role in the pathogenesis of major depressive disorder (MDD) and therapeutic effects of antidepressants. Proinflammatory cytokines influence numerous brain functions and play a role in the pathogenesis of MDD. We used RN46A cells, derived from embryonic rat serotonergic neurons, to determine the effects of pro-inflammatory cytokines, tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) on SERT and 5-HT1B gene expression. Cells were grown in serum-free conditions (DMEM/F12 with B27 supplement), then treated with either TNFalpha (50 ng/ml) or IL-1beta (50 ng/ml) for 24 h. Quantitative realtime PCR with 18S RNA as a normalizer gene was used to compare SERT and 5-HT1B gene expression between cytokine-treated and control groups. Both cytokines increased SERT gene expression (225% control with TNF-alpha and 250% control with IL-1beta treatment, p < 0.001). At the same time, 5-HT1B gene expression was downregulated by both cytokines (24% control with TNF-alpha and 13% control with IL-1beta treatment, p < 0.001). These results show significant effects of pro-inflammatory cytokines on key components of serotonergic neurotransmission. In addition, RN46A cells provide a unique cell model system for examining the mechanisms by which cytokines may play a role in the pathogenesis of depression. Supported by the West Virginia IDeA Network of Biomedical Research Excellence Pilot Grant (NCRR P20RR016477). doi:10.1016/j.bbi.2010.07.048