Metformin shows anti-neoplastic properties by inhibition of oxidative phosphorylation and glycolysis in epidermolysis bullosa-associated aggressive cutaneous squamous cell carcinoma.

BACKGROUND: While most cutaneous squamous cell carcinomas (cSCCs) are treatable, certain high-risk cSCCs, such as those in recessive dystrophic epidermolysis bullosa (RDEB) patients, are particularly aggressive. Owing to repeated wounding, inflammation and unproductive healing, RDEB patients have a 68% cumulative risk of developing life-threatening cSCCs by the age of 35, and a 70% risk of death by the age of 45. Despite aggressive treatment, cSCC represents the leading cause of premature mortality in these patients, highlighting an unmet clinical need. Increasing evidence points to a role of altered metabolism in the initiation and maintenance of cSCC, making metabolism a potential therapeutic target. OBJECTIVES: We sought to determine the feasibility of targeting tumour cell energetics as a strategy to selectively hinder the growth advantage of aggressive cSCC. METHODS: We evaluated the cell energetics profiles of RDEB-SCC cells by analysing available gene expression data against multiple gene signatures and single-gene targets linked to metabolic reprogramming. Additionally, we employed real-time metabolic profiling to measure glycolysis and respiration in these cells. Furthermore, we investigated the anti-neoplastic properties of the metformin against human and murine high-risk cSCCs in vitro and in vivo. RESULTS: Gene expression analyses highlighted a divergence in cell energetics profiles between RDEB-SCC and non-malignant RDEB keratinocytes, with tumour cells demonstrating enhanced respiration and glycolysis scores. Real-time metabolic profiling supported these data and additionally highlighted a metabolic plasticity of RDEB-SCC cells. Against this background, metformin exerted an anti-neoplastic potential by hampering both respiration and glycolysis, and by inhibiting proliferation in vitro. Metformin treatment in an analogous model of fast-growing murine cSCC resulted in delayed tumour onset and slower tumour growth, translating to a 29% increase in median overall survival. CONCLUSIONS: Our data indicate that metformin exerts anti-neoplastic properties in aggressive cSCCs that exhibit high-risk features by interfering with respiration and glycolytic processes.

Optimizing antibiotic use for early onset sepsis: A tertiary NICU experience.

BACKGROUND: Neonatal antibiotic use is associated with a greater risk of nosocomial infection, necrotizing enterocolitis, and mortality. It can induce drug-resistant pathogens that contribute to increased neonatal morbidity/mortality, healthcare costs, and length of stay. Prior to the antibiotic stewardship program, decisions to obtain blood cultures and empiric antibiotics for possible Early-onset Sepsis (EOS) in late preterm and term infants upon NICU admission were provider-dependent rather than algorithm-based. We aimed to decrease empiric antibiotic prescription from 70% to 56% (20% decrease) in infants ≥34 weeks gestation admitted to the NICU. METHODS: The stewardship initiative comprised the following practice changes: (1) use of the Neonatal Sepsis Risk Calculator (SRC); and (2) a 36-hour time-out for prescribed empiric antibiotics. Data was retrospectively collected and analyzed for inborn infants pre-intervention (January 2015-December 2015; n = 263) and post-intervention (August 2016-September 2017; n = 279). Data regarding compliance with the new antibiotic guideline were collected and disseminated to the team every week. Overlap between CDC guidelines and calculator recommendations were studied. RESULTS: Pre-and post-intervention outcomes were analyzed using chi-square tests. There was a significant post-intervention reduction in the rate of both antibiotic prescriptions (29.4% decline; 70.3% vs. 49.6%; p < 0.001) and sepsis evaluations (24.3% decline; 90.9% vs. 68.8%; p < 0.001). No difference (p = 0.271) in culture-positive EOS cases was observed. There was 92% overlap in blood culture recommendations and 95% overlap between antibiotic recommendations when current CDC guidelines were compared to the SRC. CONCLUSION: A significant reduction in antibiotic use and sepsis evaluations was achieved for late preterm and term infants upon NICU admission. No clinical deterioration occurred in post-intervention infants who did not receive antibiotics. There is significant overlap between CDC guidelines and SRC recommendations.

Mesenchymal stromal cells from the human placenta promote neovascularization in a mouse model in vivo.

Cell transplantation is a promising strategy in regenerative medicine for revascularization of ischemic tissues. Based on our observation that placental mesenchymal stromal cells (PMSC) enhance endothelial cell viability in vitro via secretion of angiogenic factors, we asked whether PMSC support vascular growth in vivo. PMSC were isolated from amnion and placental endothelial cells (PLEC) from chorion and either separately or co-transplanted subcutaneously into immune-deficient mice. Co-transplantation resulted in a higher number of perfused human vessels (CD31+/vimentin+) containing mouse glycophorin A+ erythrocytes. Results indicate positive effects of PMSC on neovascularization in vivo, making them attractive candidates to create autologous PMSC/PLEC pairs for research and transplantation.

Hsa-mir-145 is the top EWS-FLI1-repressed microRNA involved in a positive feedback loop in Ewing's sarcoma.

EWS-FLI1 is a chromosome translocation-derived chimeric transcription factor that has a central and rate-limiting role in the pathogenesis of Ewing's sarcoma. Although the EWS-FLI1 transcriptomic signature has been extensively characterized on the mRNA level, information on its impact on non-coding RNA expression is lacking. We have performed a genome-wide analysis of microRNAs affected by RNAi-mediated silencing of EWS-FLI1 in Ewing's sarcoma cell lines, and differentially expressed between primary Ewing's sarcoma and mesenchymal progenitor cells. Here, we report on the identification of hsa-mir-145 as the top EWS-FLI1-repressed microRNA. Upon knockdown of EWS-FLI1, hsa-mir-145 expression dramatically increases in all Ewing's sarcoma cell lines tested. Vice versa, ectopic expression of the microRNA in Ewing's sarcoma cell lines strongly reduced EWS-FLI1 protein, whereas transfection of an anti-mir to hsa-mir-145 increased the EWS-FLI1 levels. Reporter gene assays revealed that this modulation of EWS-FLI1 protein was mediated by the microRNA targeting the FLI1 3'-untranslated region. Mutual regulations of EWS-FLI1 and hsa-mir-145 were mirrored by an inverse correlation between their expression levels in four of the Ewing's sarcoma cell lines tested. Consistent with the role of EWS-FLI1 in Ewing's sarcoma growth regulation, forced hsa-mir-145 expression halted Ewing's sarcoma cell line growth. These results identify feedback regulation between EWS-FLI1 and hsa-mir-145 as an important component of the EWS-FLI1-mediated Ewing's sarcomagenesis that may open a new avenue to future microRNA-mediated therapy of this devastating malignant disease.

Thiolated polyacrylic acid-modified iron oxide nanoparticles for in vitro labeling and MRI of stem cells.

PURPOSE: The purpose of this study was to develop and characterize new surface-modified iron oxide nanoparticles demonstrating the efficiency to be internalized by human endothelial progenitor cells (EPCs) from umbilical cord blood. METHODS: Iron oxide nanoparticles were coated with polyacrylic acid-cysteine (PAA-Cys) by either in situ precipitation or postsynthesis. The nanoparticles were characterized by X-ray powder diffraction. EPCs were labeled with PAA-Cys-modified iron oxide nanoparticles or with uncoated nanoparticles. The relaxivity of uncoated and coated iron oxide nanoparticles as well as EPCs labeled with PAA-Cys-modified iron oxide were determined. RESULTS: Addition of PAA-Cys increased the particle size from 10.4 to 144 and 197 nm, respectively. The X-ray powder diffraction pattern revealed that the particles consist of Fe(3)O(4) with a spinal structure. Postsynthesis coated particles showed a cellular uptake of 85% and 15.26 pg iron/cell. For both types of particles the relaxivity ratio was at least 2-fold higher than that of the gold standard Resovist(®). CONCLUSION: The PAA-Cys coated iron oxide nanoparticles are a promising tool for labeling living cells such as stem cells for diagnostic and therapeutic application in cell-based therapies due to their high relaxivities and their easy uptake by cells.

Basic research and clinical applications of non-hematopoietic stem cells, 4-5 April 2008, Tubingen, Germany.

From 4 to 5 April 2008, international experts met for the second time in Tubingen, Germany, to present and discuss the latest proceedings in research on non-hematopoietic stem cells (NHSC). This report presents issues of basic research including characterization, isolation, good manufacturing practice (GMP)-like production and imaging as well as clinical applications focusing on the regenerative and immunomodulatory capacities of NHSC.

IFPA meeting 2008 workshops report.

Workshops are an important part of the IFPA annual meeting. At the IFPA meeting 2008 diverse topics were discussed in 12 themed workshops. Topics covered included: immunology of placentation; galectins and trophoblast invasion; signaling in implantation and invasion; markers to identify trophoblast subpopulations; placental pathology; placental toxicology; stereology; placental transport of fatty acids; placental mesenchymal stem cells; comparative placentation; trophoblast and neoplasia; trophoblast differentiation. This report is a summary of the various topics covered.

RT-PCR and FISH analysis of acute myeloid leukemia with t(8;16)(p11;p13) and chimeric MOZ and CBP transcripts: breakpoint cluster region and clinical implications.

The translocation t(8;16)(p11;p13) is associated with acute myeloid leukemia displaying monocytic differentiation (AML FAB M4/5) and fuses the MOZ (also named MYST3) gene (8p11) with the CBP (also named CREBBP) gene (16p13). Detection of the chimeric RNA fusions has proven difficult; only three studies have described successful amplification of the chimeric MOZ-CBP and CBP-MOZ fusions by reverse transcriptase-polymerase chain reaction (RT-PCR). We analyzed four cases of AML M4/5 with t(8;16)(p11;p13) by RT-PCR and fluorescence in situ hybridization (FISH) and characterized the reciprocal RNA fusions from three cases. We cloned both genomic translocation breakpoints from one case by long-range PCR and successfully applied RT-PCR to monitor minimal residual disease (MRD) between clinical complete remission and relapse. In three cases, the genomic breakpoints occurred in MOZ intron 16 and CBP intron 2. In one case, no fusion transcript was detected. The available data suggest clustering of t(8;16)(p11;p13) breakpoints in these introns leading to reciprocal in-frame MOZ exon 16/CBP exon 3 and in-frame CBP exon 2/MOZ exon 17 chimeric transcripts in the majority of cases. The described RT-PCR strategy may be valuable both for the routine detection of the t(8;16)(p11;p13) as well as for monitoring of MRD in this prognostically unfavorable patient group.

Adoptive transfer of vitiligo after allogeneic bone marrow transplantation for non-Hodgkin's lymphoma.

Vitiligo developed in a 50-year-old man 9 months after allogeneic transplantation from his HLA-identical sister who had had this disease for several years. Our findings suggest adoptive transfer of vitiligo by haematopoietic stem cell transplantation, and lend support to the autoimmune nature of this disease.

Separation of sperm and vaginal cells based on ploidy, MHC class I-, CD45-, and cytokeratin expression for enhancement of DNA typing after sexual assault.

BACKGROUND: Successful DNA typing after rape is limited when only a few sperm and numerous vaginal cells are recovered from a swab, resulting in an extremely unfavorable ratio of male to female DNA. The goal of this study was to develop a protocol involving sperm cell sorting with flow cytometry based on differences in ploidy, major histocompatibility (MHC) class I, CD45 and cytokeratin expression. METHODS: Vaginal lavages were mixed with serially diluted ejaculate. After immunostaining and stoichiometric nuclear staining, spermatocytes were isolated by fluorescence-activated cell sorting. All sorted cells were used for DNA extraction and subsequent quantitative fluorescent multiplex polymerase chain reaction. The preferential lysis was performed for comparison. RESULTS: The sorting procedure was superior to the preferential lysis method within all tested dilutions. One documented case of rape was examined with both procedures and only after cell sorting with flow cytometry was the male DNA identified. CONCLUSIONS: We were able to show that separation of sperm and vaginal cells using cell sorting with flow cytometry may be crucial when there is only a few sperm detectable after rape.

A skin homing molecule defines the langerhans cell progenitor in human peripheral blood.

We have recently described a system for the generation of dendritic cells (DC) and Langerhans cells (LC) from defined CD34+ precursors purified from peripheral blood of healthy adult volunteers. This study has now been extended by the characterization of two distinct subpopulations of CD34+ cells in normal human peripheral blood as defined by the expression of the skin homing receptor cutaneous lymphocyte-associated antigen (CLA). CD34+/CLA+ cells from normal peripheral blood were found to be CD71LOW/CD11a+/CD11b+/CD49d+/CD45RA+ whereas CD34+/CLA- cells displayed the CD71+/CD11aLOW/CD11bLOW/CD49d(+)/ CD45RA(LOW) phenotype. To determine the differentiation pathways of these two cell populations, CD34+ cells were sorted into CLA+ and CLA- fractions, stimulated with GM-CSF and TNF-alpha in vitro, and then were cultured for 10 to 18 d. Similar to unfractionated CD34+ cells, the progeny of both cell populations contained sizable numbers (12-22%) of dendritically shaped, CD1a+/HLA-DR cells. In addition to differences in their motility, the two dendritic cell populations generated differed from each other by the expression of LC-specific structures. Only the precursors expressing the skin homing receptor were found to differentiate into LC as evidenced by the presence of Birbeck granules. In contrast, CLA precursor cells generated a CD1a+ DC population devoid of Birbeck granule-containing LC. Provided that comparable mechanisms as found in this study are also operative in vivo, we postulate that the topographic organization of the DC system is already determined, at least in part, at the progenitor level.

Single human T cells stimulated in the absence of feeder cells transcribe interleukin-2 and undergo long-term clonal growth in response to defined monoclonal antibodies and cytokine stimulation.

The two-signal model of T-cell activation postulates that T lymphocytes require at least two distinct signals for activation. This model has been established with bulk cultures of T cells in which T-cell-T-cell interaction can occur, possibly delivering further unrecognized costimulatory signals. The signal requirements of single T cells for the induction of clonal cell growth or the transcription of cytokines would best be studied in a cell cloning system in the absence of feeder cells; however, such an experimental system has not been reported so far. In this study, we report the long-term cloning of human resting peripheral blood CD4+CD45RO- T cells under feeder cell-free conditions in response to CD3 and CD28 stimulation in the presence of exogenous interleukin-2 (IL-2). Cloning efficiency ranged from 40% to 60% depending on the presence of additional cytokines IL-1 and IL-6. Single-call polymerase chain reaction showed that transcription of IL-2 occurred in cells stimulated through CD3 plus CD28 alone. T cells grown in response to CD3 plus CD28 plus IL-2 stimulation produced both IL-4 and interferon-gamma (IFN-gamma) on restimulation (Th0 cells) and could be functionally differentiated into Th1- or Th2-type cells by the addition of IFN-gamma or IL-4, respectively, during cell cloning. These data show on the single-cell level a two-signal model of T- cell activation for the transcription of IL-2. In addition, these experiments show that IFN-gamma and IL-4 exert their T-cell-differentiating effects directly on the T cell without any further need for antigen-presenting cells. Together, our experiments show the feasability of a defined long-term clonal cell culture system to study the growth and differentiation of human T lymphocytes.

Generation of human dendritic cells/Langerhans cells from circulating CD34+ hematopoietic progenitor cells.

Human Langerhans cells (LC) are CD1a+ dendritic cells (DC) that function as potent antigen-presenting cells for primary and secondary immune responses. Limitations in DC/LC numbers, imposed by difficult and tedious isolation procedures, have so far precluded their use as immunogens in the generation and/or augmentation of host responses against various pathogens. Therefore, we have developed a procedure for the generation of human DC/LC from CD34+ hematopoietic progenitor cells (HPC) isolated (mean: 0.7 x 10(6)/ buffy coat and 2.6 x 10(6)/leukapheresis product) and purified ( > 95%) from the peripheral blood of healthy adults. In vitro stimulation of these cells with granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor (TNF)-alpha led to their vigorous proliferation and differentiation resulting in the emergence of CD45+/CD68+/CD3-/CD19-/CD56- leukocytes some of which (mean: 12%) express CD1a and exhibit anti-CD4 and anti-major histocompatibility complex (MHC) class II reactivity. These CD1a- leukocytes include (1) LC as evidenced by the presence of Birbeck granules (BG), (2) CD14+ monocytes, and (3) Birbeck granule-negative cells with a dendritic morphology. Addition of interleukin (IL)-4 to the cytokine cocktail interfered with the development of monocytes and led to a reduction in the overall yield but, on the other hand, resulted in an increased percentage of CD1a+ cells (mean: 24%) among all cells generated. In vitro generated CD1a+, but not CD1a- HPC-derived cells are potent stimulators of the primary mixed leukocyte reaction and, as such, promising candidates for vaccination purposes.

Human melanocytes and melanoma cells constitutively express the Bcl-2 proto-oncogene in situ and in cell culture.

The Bcl-2 proto-oncogene regulates cell survival by antagonizing events that lead to apoptotic cell death and has been reported to be expressed in situ in lymphoid tissues, glandular epithelium, neurons, and basal epidermal cells. When we performed immunostaining on cryostat sections of normal skin, anti-Bcl-2 reactivity was confined to scattered dendritic cells in the basal epidermal layer. Double-staining experiments showed that the Bcl-2+ cells were positive for vimentin but negative for cytokeratins, CD1a, and CD45 antigens, excluding keratinocytes and Langerhans cells as possible candidates for constitutive Bcl-2 expression. Bcl-2+ epidermal cells also reacted with the monoclonal anti-melanocyte antibody NKI/beteb, and were absent from lesional skin in vitiligo, confirming that they represented epidermal melanocytes. Western blot analysis of cultured melanocytes and melanoma cell lines revealed a 26-kd protein specifically reacting with the anti-Bcl-2 monoclonal antibody. Immunostaining of pigmented lesions revealed strong expression of Bcl-2 by five of five nevocellular nevi and seven of seven melanomas. Our observations demonstrate that, within normal human epidermis, melanocytes are the only cells that express Bcl-2 constitutively and that Bcl-2 is expressed in benign and malignant pigmented tumors of the skin in situ.

Expression of monoclonal antibody HECA-452-defined E-selectin ligands on Langerhans cells in normal and diseased skin.

The cutaneous lymphocyte-associated antigen recognized by the monoclonal antibody HECA-452 has been thought to play a major role in the homing of memory T-cell subsets to the skin by virtue of its ability to bind to E-selectin of dermal microvascular endothelial cells. Considering that the homing of different leukocyte populations to the skin may involve similar mechanisms, we studied the expression of HECA-452-reactive molecules on CD1a+ epidermal Langerhans cells. Immunofluorescence double-labeling of cryostat sections and epidermal sheets of normal skin revealed HECA-452 immunoreactivity on a subpopulation of dermal and epidermal CD1a+ cells, whereas upon flow-cytometric analysis of epidermal single cell suspensions virtually all CD1a+ cells bound HECA-452 antibodies. We observed a marked upregulation of HECA-452-antigen expression on CD1a+ epidermal cells and a pronounced increase in the number of HECA-452+/CD1a+ dermal cells in lesional skin from inflammatory and neoplastic lymphocytic skin diseases, compared to normal skin. The molecule detected by the HECA-452 antibody on Langerhans cells is neuraminidase sensitive and contains a CD15 (LewisX) carbohydrate backbone. Because Langerhans cells react with the sialyl-LewisX-specific antibody CSLEX1, it is very likely that the HECA-452-reactive structure is or contains sialyl-LewisX. Our data are compatible with the view that i) resident epidermal Langerhans cells upregulate HECA-452-antigen expression due to the cytokine profile generated in the disease process or ii) that Langerhans cell precursors express HECA-452-antigens and show an enhanced immigration into lesional skin. The characterization of HECA-452+ cells in peripheral blood may not only clarify this issue but may also help to identify the still elusive Langerhans cell-precursor.

The influence of topical dermatological treatment modalities on epidermal Langerhans cells and contact sensitization in mice.

The influence of the widely used topical dermatological treatment modalities anthralin, coal tar and pyrogallol on surface markers of epidermal Langerhans cells and contact sensitization was studied and compared with that of a PUVA treatment. A common effect of all dermatological therapies tested was inhibition of Langerhans cell ATPase, whereas an effect on MHC class II antigens was found only after PUVA or tar treatment. The induction of contact hypersensitivity was inhibited only by PUVA, and not by the other treatments. These results show that various forms of topical therapy influence surface markers and immunological function of epidermal Langerhans cells differently.

Inhibition of Langerhans cell ATPase and contact sensitization by lanthanides--role of T-suppressor cells.

Lanthanides are rare earths, elements 55-71 in the periodic table, that are of interest in biologic systems as isomorphic competitors for calcium binding sites. Lanthanides were tested for their inhibitory influence on the Ca++/Mg(++)-dependent ATPase of epidermal langerhans cells in vitro, and on the immunologic function of Langerhans cells in vivo. The trivalent ions of lanthanides, lanthanum, and cerium completely inhibited the ATPase staining of Langerhans cells in vitro. When mice were sensitized with dinitrofluorobenzene on skin sites pretreated with topical lanthanum chloride, and challenged on untreated ear skin, a markedly reduced contact hypersensitivity response was observed. This hyporesponsiveness was found to be antigen specific, and could be passively transferred to naive syngeneic animals recipients by CD4-CD8+ spleen cells. These results suggest that inhibition of the epidermal Langerhans cell surface ATPase by application of topical lanthanum and the induction of antigen-specific immunologic tolerance may be related events.