Recognition and blocking of innate immunity cells by Candida albicans chitin.

Chitin is a skeletal cell wall polysaccharide of the inner cell wall of fungal pathogens. As yet, little about its role during fungus-host immune cell interactions is known. We show here that ultrapurified chitin from Candida albicans cell walls did not stimulate cytokine production directly but blocked the recognition of C. albicans by human peripheral blood mononuclear cells (PBMCs) and murine macrophages, leading to significant reductions in cytokine production. Chitin did not affect the induction of cytokines stimulated by bacterial cells or lipopolysaccharide (LPS), indicating that blocking was not due to steric masking of specific receptors. Toll-like receptor 2 (TLR2), TLR4, and Mincle (the macrophage-inducible C-type lectin) were not required for interactions with chitin. Dectin-1 was required for immune blocking but did not bind chitin directly. Cytokine stimulation was significantly reduced upon stimulation of PBMCs with heat-killed chitin-deficient C. albicans cells but not with live cells. Therefore, chitin is normally not exposed to cells of the innate immune system but is capable of influencing immune recognition by blocking dectin-1-mediated engagement with fungal cell walls.

Acute promyelocytic leukemia: a paradigm for differentiation therapy.

Acute promyelocytic leukemia(APL) is characterized by the t(15;17) chromosomal translocation leading to the formation of the PML-RARalpha oncoprotein. This leukemia has attracted considerable interest in recent years, being the first in which therapies that specifically target the underlying molecular lesion, i.e., all-trans retinoic acid (ATRA) and arsenic trioxide (ATO), leading to induction of differentiation and apoptosis have been successfully used in clinical practice. The advent of ATRA therapy has transformed APL from being a disease with a poor outlook to one of the most prognostically favorable subsets of acute myeloid leukemia. Further improvements in outcome may be achieved with the use of ATO, which achieves high rates of remission in the relatively small proportion of patients now relapsing following standard first-line therapy with ATRA and anthracycline-based chemotherapy. Moreover, recent studies have suggested that ATO and ATRA, or even ATO alone, used as front-line treatment of PML-RARA- associated APL can induce long-term remissions. This raises the possibility that some patients can be cured using differentiation therapies alone, without the need for chemotherapy, thereby potentially reducing treatment-related toxicity. It is clear that the success of such an approach is critically dependent upon molecular diagnostics and monitoring for minimal residual disease (MRD) to distinguish those patients who can potentially be cured with differentiation therapy from those requiring additional myelosuppressive agents. This represents an exciting new phase in the treatment of acute leukemia, highlighting the potential of molecularly targeted and MRD-directed therapies to achieve an individualized approach to patient management.

The platelet receptor CLEC-2 is active as a dimer.

The platelet receptor CLEC-2 binds to the snake venom toxin rhodocytin and the tumor cell surface protein podoplanin. Binding of either of these ligands promotes phosphorylation of a single tyrosine residue in the YXXL motif in the intracellular domain of CLEC-2. Phosphorylation of this tyrosine initiates binding of spleen tyrosine kinase (Syk) and triggers further downstream signaling events and ultimately potent platelet activation and aggregation. However, it is unclear how a single YXXL motif can interact efficiently with Syk, which usually recognizes two tandem YXXL repeats presented as an immunoreceptor tyrosine-based activation motif (ITAM). Using bioluminescence resonance energy transfer, coimmunopreciptitation, recombinant protein expression and analytical gel filtration chromatography, surface plasmon resonance, Western blotting, multiangle light scattering (MALS), and analytical ultracentrifugation, we show that CLEC-2 exists as a non-disulfide-linked homodimer which could allow each Syk molecule to interact with two YXXL motifs, one from each CLEC-2 monomer.

Renal cells activate the platelet receptor CLEC-2 through podoplanin.

We have recently shown that the C-type lectin-like receptor, CLEC-2, is expressed on platelets and that it mediates powerful platelet aggregation by the snake venom toxin rhodocytin. In addition, we have provided indirect evidence for an endogenous ligand for CLEC-2 in renal cells expressing HIV-1. This putative ligand facilitates transmission of HIV through its incorporation into the viral envelope and binding to CLEC-2 on platelets. The aim of the present study was to identify the ligand on these cells which binds to CLEC-2 on platelets. Recombinant CLEC-2 exhibits specific binding to HEK-293T (human embryonic kidney) cells in which the HIV can be grown. Furthermore, HEK-293T cells activate both platelets and CLEC-2-transfected DT-40 B-cells. The transmembrane protein podoplanin was identified on HEK-293T cells and was demonstrated to mediate both binding of HEK-293T cells to CLEC-2 and HEK-293T cell activation of CLEC-2-transfected DT-40 B-cells. Podoplanin is expressed on renal cells (podocytes). Furthermore, a direct interaction between CLEC-2 and podoplanin was confirmed using surface plasmon resonance and was shown to be independent of glycosylation of CLEC-2. The interaction has an affinity of 24.5+/-3.7 microM. The present study identifies podoplanin as a ligand for CLEC-2 on renal cells.

DNA topoisomerase II in therapy-related acute promyelocytic leukemia.

BACKGROUND: Chromosomal translocations leading to chimeric oncoproteins are important in leukemogenesis, but how they form is unclear. We studied acute promyelocytic leukemia (APL) with the t(15;17) translocation that developed after treatment of breast or laryngeal cancer with chemotherapeutic agents that poison topoisomerase II. METHODS: We used long-range polymerase chain reaction and sequence analysis to characterize t(15;17) genomic breakpoints in therapy-related APL. To determine whether topoisomerase II was directly involved in mediating breaks of double-stranded DNA at the observed translocation breakpoints, we used a functional in vitro assay to examine topoisomerase II-mediated cleavage in the normal homologues of the PML and RARA breakpoints. RESULTS: Translocation breakpoints in APL that developed after exposure to mitoxantrone, a topoisomerase II poison, were tightly clustered in an 8-bp region within PML intron 6. In functional assays, this "hot spot" and the corresponding RARA breakpoints were common sites of mitoxantrone-induced cleavage by topoisomerase II. Etoposide and doxorubicin also induced cleavage by topoisomerase II at the translocation breakpoints in APL arising after exposure to these agents. Short, homologous sequences in PML and RARA suggested the occurrence of DNA repair by means of the nonhomologous end-joining pathway. CONCLUSIONS: Drug-induced cleavage of DNA by topoisomerase II mediates the formation of chromosomal translocation breakpoints in mitoxantrone-related APL and in APL that occurs after therapy with other topoisomerase II poisons.

The molecular pathogenesis of acute promyelocytic leukaemia: implications for the clinical management of the disease.

Acute promyelocytic leukaemia (APL) is characterised by chromosomal rearrangements of 17q21, leading to fusion of the gene encoding retinoic acid receptor alpha (RARalpha) to a number of alternative partner genes (X), the most frequent of which are PML (>95%), PLZF (0.8%) and NPM (0.5%). Over the last few years, it has been established that the X-RARalpha fusion proteins play a key role in the pathogenesis of APL through recruitment of co-repressors and the histone deacetylase (HDAC)-complex to repress genes implicated in myeloid differentiation. Paradoxically, the X-RARalpha fusion protein has the potential to mediate myeloid differentiation at pharmacological doses of its ligand (all trans-retinoic acid (ATRA)), which is dependent on the dissociation of the HDAC/co-repressor complex. Arsenic compounds have also been shown to be promising therapeutic agents, leading to differentiation and apoptosis of APL blasts. It is now apparent that the nature of the RARalpha-fusion partner is a critical determinant of response to ATRA and arsenic, underlining the importance of cytogenetic and molecular characterisation of patients with suspected APL to determine the most appropriate treatment approach. Standard protocols involving ATRA combined with anthracycline-based chemotherapy, lead to cure of approximately 70% patients with PML-RARalpha-associated APL. Patients at high risk of relapse can be identified by minimal residual disease monitoring. The challenge for future studies is to improve complete remission rates through reduction of induction deaths, particularly due to haemorrhage, identification of patients at high risk of relapse who would benefit from additional therapy, and identification of a favourable-risk group, for which treatment intensity could be reduced, thereby reducing risks of treatment toxicity and development of secondary leukaemia/myelodysplasia. With the advent of ATRA and arsenic, APL has already provided the first example of successful molecularly targeted therapy; it is hoped that with further understanding of the pathogenesis of the disease, the next decade will yield further improvements in the outlook for these patients.

A chemically defined carrier for the delivery of human mesenchymal stem/stromal cells to skin wounds.

Skin has a remarkable capacity for regeneration, but age- and diabetes-related vascular problems lead to chronic non-healing wounds for many thousands of U.K. patients. There is a need for new therapeutic approaches to treat these resistant wounds. Donor mesenchymal stem/stromal cells (MSCs) have been shown to assist cutaneous wound healing by accelerating re-epithelialization. The aim of this work was to devise a low risk and convenient delivery method for transferring these cells to wound beds. Plasma polymerization was used to functionalize the surface of medical-grade silicone with acrylic acid. Cells attached well to these carriers, and culture for up to 3 days on the carriers did not significantly affect their phenotype or ability to support vascular tubule formation. These carriers were then used to transfer MSCs onto human dermis. Cell transfer was confirmed using an MTT assay to assess viable cell numbers and enhanced green fluorescent protein-labeled MSCs to demonstrate that the cells post-transfer attached to the dermis. We conclude that this synthetic carrier membrane is a promising approach for delivery of therapeutic MSCs and opens the way for future studies to evaluate its impact on repairing difficult skin wounds.

Regulation of ligands for the activating receptor NKG2D.

The outcome of an encounter between a cytotoxic cell and a potential target cell depends on the balance of signals from inhibitory and activating receptors. Natural Killer group 2D (NKG2D) has recently emerged as a major activating receptor on T lymphocytes and natural killer cells. In both humans and mice, multiple different genes encode ligands for NKG2D, and these ligands are non-classical major histocompatibility complex class I molecules. The NKG2D-ligand interaction triggers an activating signal in the cell expressing NKG2D and this promotes cytotoxic lysis of the cell expressing the ligand. Most normal tissues do not express ligands for NKG2D, but ligand expression has been documented in tumour and virus-infected cells, leading to lysis of these cells. Tight regulation of ligand expression is important. If there is inappropriate expression in normal tissues, this will favour autoimmune processes, whilst failure to up-regulate the ligands in pathological conditions would favour cancer development or dissemination of intracellular infection.