Quantitative mass spectrometry of human reticulocytes reveal proteome-wide modifications during maturation.

Erythropoiesis is marked by progressive changes in morphological, biochemical and mechanical properties of erythroid precursors to generate red blood cells (RBC). The earliest enucleated forms derived in this process, known as reticulocytes, are multi-lobular and spherical. As reticulocytes mature, they undergo a series of dynamic cytoskeletal re-arrangements and the expulsion of residual organelles, resulting in highly deformable biconcave RBCs (normocytes). To understand the significant, yet neglected proteome-wide changes associated with reticulocyte maturation, we undertook a quantitative proteomics approach. Immature reticulocytes (marked by the presence of surface transferrin receptor, CD71) and mature RBCs (devoid of CD71) were isolated from human cord blood using a magnetic separation procedure. After sub-fractionation into triton-extracted membrane proteins and luminal samples (isobaric tags for relative and absolute quantitation), quantitative mass spectrometry was conducted to identify more than 1800 proteins with good confidence and coverage. While most structural proteins (such as Spectrins, Ankyrin and Band 3) as well as surface glycoproteins were conserved, proteins associated with microtubule structures, such as Talin-1/2 and ß-Tubulin, were detected only in immature reticulocytes. Atomic force microscopy (AFM)-based imaging revealed an extended network of spectrin filaments in reticulocytes (with an average length of 48 nm), which shortened during reticulocyte maturation (average spectrin length of 41 nm in normocytes). The extended nature of cytoskeletal network may partly account for increased deformability and shape changes, as reticulocytes transform to normocytes.

LFA-1/ICAM-1 Ligation in Human T Cells Promotes Th1 Polarization through a GSK3ß Signaling-Dependent Notch Pathway.

In this study, we report that the integrin LFA-1 cross-linking with its ligand ICAM-1 in human PBMCs or CD4(+) T cells promotes Th1 polarization by upregulating IFN-γ secretion and T-bet expression. LFA-1 stimulation in PBMCs, CD4(+) T cells, or the T cell line HuT78 activates the Notch pathway by nuclear translocation of cleaved Notch1 intracellular domain (NICD) and upregulation of target molecules Hey1 and Hes1. Blocking LFA-1 by a neutralizing Ab or specific inhibition of Notch1 by a γ-secretase inhibitor substantially inhibits LFA-1/ICAM-1-mediated activation of Notch signaling. We further demonstrate that the Notch pathway activation is dependent on LFA-1/ICAM-1-induced inactivation of glycogen synthase kinase 3ß (GSK3ß), which is mediated via Akt and ERK. Furthermore, in silico analysis in combination with coimmunoprecipitation assays show an interaction between NICD and GSK3ß. Thus, there exists a molecular cross-talk between LFA-1 and Notch1 through the Akt/ERK-GSK3ß signaling axis that ultimately enhances T cell differentiation toward Th1. Although clinical use of LFA-1 antagonists is limited by toxicity related to immunosuppression, these findings support the concept that Notch inhibitors could be attractive for prevention or treatment of Th1-related immunologic disorders and have implications at the level of local inflammatory responses.

Long-Term Real-Time In Vivo Drug Release Monitoring with AIE Thermogelling Polymer.

A new drug concentration meter is developed. In vivo drug release can be monitored precisely via a self-indicating drug delivery system consisting of a new aggregation-induced emission thermoresponsive hydrogel. By taking the advantage of a self-indicating system, one can easily detect the depletion of drugs, and reinject to maintain a dosage in the optimal therapeutic window.

Adaptor regulation of LFA-1 signaling in T lymphocyte migration: Potential druggable targets for immunotherapies?

The integrin lymphocyte function associated antigen-1 (LFA-1) plays a key role in leukocyte trafficking and in adaptive immune responses through interactions with adhesive ligands, such as ICAM-1. Specific blockade of these interactions has validated LFA-1 as a therapeutic target in many chronic inflammatory diseases, however LFA-1 antagonists have not been clinically successful due to the development of a general immunosuppression, causing fatal side effects. Growing evidence has now established that LFA-1 mediates an array of intracellular signaling pathways by triggering a number of downstream molecules. In this context, a class of multimodular domain-containing proteins capable of recruiting two or more effector molecules, collectively known as "adaptor proteins," has emerged as important mediators in LFA-1 signal transduction. Here, we provide an overview of the adaptor proteins involved in the intracellular signaling cascades by which LFA-1 regulates T-cell motility and immune responses. The complexity of the LFA-1-associated signaling delineated in this review suggests that it may be an important and challenging focus for future research, enabling the identification of "tunable" targets for the development of immunotherapies.

Leukocyte function-associated antigen-1/intercellular adhesion molecule-1 interaction induces a novel genetic signature resulting in T-cells refractory to transforming growth factor-ß signaling.

The immunesuppressive cytokine TGF-ß plays crucial regulatory roles in the induction and maintenance of immunologic tolerance and prevention of immunopathologies. However, it remains unclear how circulating T-cells can escape from the quiescent state maintained by TGF-ß. Here, we report that the T-cell integrin leukocyte function-associated antigen-1 (LFA-1) interaction with its ligand intercellular adhesion molecule-1 (ICAM-1) induces a genetic signature associated with reduced TGF-ß responsiveness via up-regulation of SKI, E3 ubiquitin-protein ligase SMURF2, and SMAD7 (mothers against decapentaplegic homolog 7) genes and proteins. We confirmed that the expression of these TGF-ß inhibitory molecules was dependent on STAT3 and/or JNK activation. Increased expression of SMAD7 and SMURF2 in LFA-1/ICAM-1 cross-linked T-cells resulted in impaired TGF-ß-mediated phosphorylation of SMAD2 and suppression of IL-2 secretion. Expression of SKI caused resistance to TGF-ß-mediated suppression of IL-2, but SMAD2 phosphorylation was unaffected. Blocking LFA-1 by neutralizing antibody or specific knockdown of TGF-ß inhibitory molecules by siRNA substantially restored LFA-1/ICAM-1-mediated alteration in TGF-ß signaling. LFA-1/ICAM-1-stimulated human and mouse T-cells were refractory to TGF-ß-mediated induction of FOXP3(+) (forkhead box P3) and RORγt(+) (retinoic acid-related orphan nuclear receptor γt) Th17 differentiation. These mechanistic data suggest an important role for LFA-1/ICAM-1 interactions in immunoregulation concurrent with lymphocyte migration that may have implications at the level of local inflammatory response and for anti-LFA-1-based therapies.

Autophagy induction by silver nanowires: a new aspect in the biocompatibility assessment of nanocomposite thin films.

Nanomaterials and their enabled products have increasingly been attracting global attention due to their unique physicochemical properties. Among these emerging products, silver nanowire (AgNW)-based thin films are being developed for their promising applications in next generation nanoelectronics and nanodevices. However, serious concerns remain about possible health and safety risks they may pose. Here, we employed a multi-modal systematic biocompatibility assessment of thin films incorporating AgNW. To represent the possible routes of nanomaterial entry during occupational or environmental exposure, we employed four different cell lines of epithelial, endothelial, gastric, and phagocytic origin. Utilizing a cell-based automated image acquisition and analysis procedure in combination with real-time impedance sensing, we observed a low level of cytotoxicity of AgNW, which was dependent on cell type, nanowire lengths, doses and incubation times. Similarly, no major cytotoxic effects were induced by AgNW-containing thin films, as detected by conventional cell viability and imaging assays. However, transmission electron microscopy and Western immunoblotting analysis revealed AgNW-induced autophasosome accumulation together with an upregulation of the autophagy marker protein LC3. Autophagy represents a crucial mechanism in maintaining cellular homeostasis, and our data for the first time demonstrate triggering of such mechanism by AgNW in human phagocytic cells. Finally, atomic force microscopy revealed significant changes in the topology of cells attaching and growing on these films as substrates. Our findings thus emphasize the necessity of comprehensive biohazard assessment of nanomaterials in modern applications and devices and a thorough analysis of risks associated with their possible contact with humans through occupational or environmental exposure.

Analysis of dynamic tyrosine phosphoproteome in LFA-1 triggered migrating T-cells.

The ordered, directional migration of T-lymphocytes is a key process during immune surveillance and response. This requires cell adhesion to the high endothelial venules or to the extracellular matrix by a series of surface receptor/ligand interactions involving adhesion molecules of the integrin family including lymphocyte function associated molecule-1 (LFA-1) and intercellular adhesion molecules (ICAMs). Reversible protein phosphorylation is emerging as a key player in the regulation of biological functions with tyrosine phosphorylation playing a crucial role in signal transduction. Thus, the study of this type of post-translational modification at the proteomic level has great biological significance. In this work, phospho-enriched cell lysates from LFA-1-triggered migrating human T-cells were subjected to immunoaffinity purification of tyrosine phosphorylated proteins, mass spectrometric, and bioinformatic analysis. In addition to the identification of several well-documented proteins, the analysis suggested involvement of a number of new and novel proteins in LFA-1 induced T-cell migration. This dataset expands the list of the signaling components of the LFA-1 induced phosphotyrosine protein complexes in migrating T-cells that will be extremely useful in the study of their specific roles within LFA-1 associated signaling pathways. Identification of proteins previously not reported in the context of LFA-1 stimulated signal transduction might provide new insights into understanding the LFA-1 signaling networks and aid in the search for new potential therapeutic targets.

STAT3 knockdown by siRNA induces apoptosis in human cutaneous T-cell lymphoma line Hut78 via downregulation of Bcl-xL.

Cutaneous T-cell lymphomas (CTCLs) are non-Hodgkin's lymphomas resulting from clonal expansion and localization of malignant T-lymphocytes to the skin. CTCL cells have defective apoptosis. Signal transducers and activators of transcription (STAT) are a family of transcription factors known to play important roles in the development and progression of several human cancers by promoting cell proliferation and protecting against apoptosis. In this study, we investigated the specific role of STAT3, a major component of the STAT family, in growth and survival of human CTCL cell line Hut78. Western immunoblot analysis showed elevated expression of STAT3 and phospho-STAT3(Y705) in human CTCL cells as compared to freshly isolated peripheral blood lymphocytes (PBLs). Specific knockdown of STAT3 expression in Hut78 cells by RNA interference induced morphological and biochemical changes indicating apoptotic cell death. Moreover, STAT3 inhibition downregulated the expression of Bcl2 family of anti-apoptotic gene Bcl-xL. These observations suggest that STAT3 is required for the survival of CTCL cells and strongly indicate that targeting STAT3 using siRNA techniques may serve a novel therapeutic strategy for the treatment of CTCL.

The Citrus Flavanone Hesperetin Induces Apoptosis in CTCL Cells via STAT3/Notch1/NFκB-Mediated Signaling Axis.

BACKGROUND: Hesperetin is a natural compound known for its cholesterol-lowering effect and a wide range of pharmacological activities. OBJECTIVES: Investigating the potential anticancer activities of Hesperetin in malignant hematolymphoid cell lines HuT78 and MJ, derived from patients with Cutaneous T-Cell Lymphomas (CTCL). METHODS: The cytotoxic effect of Hesperetin on two different CTCL cell lines, HuT78 and MJ, was assessed by MTS-based colorimetric assay. Apoptosis, cell cycle, ROS (Reactive Oxygen Species) and molecular analysis were performed using flow-cytometry and immunoblotting. RESULTS: Hesperetin-treated CTCL cells were arrested at the sub-G1 phase of cell cycle with the concomitant decrease in the expression of the cell cycle regulator protein cyclin B. In addition, the study found that the cellular treatment with Hesperetin caused an induction of apoptosis, which was independent of ROS generation. Hesperetin caused a significant decrease in the expression level of anti-apoptotic protein Bcl-xL and an increase in cleaved caspase-3 and PARP proteins in CTCL cells. Furthermore, Hesperetin treatment in CTCL cells down-regulated the expression of Notch1 and phosphorylation of STAT3 (Tyr705) and inhibited NFκBp65. CONCLUSION: This study highlights the anticancer properties of Hesperetin. Which induces apoptosis in CTCL cells via STAT3/Notch1/NFκB mediated signaling pathway, suggesting that further development of this novel class of flavonoid may contribute to new drug discovery for certain hematolymphoid malignancies.

A new microtubule-targeting compound PBOX-15 inhibits T-cell migration via post-translational modifications of tubulin.

The ordered, directional migration of T-lymphocytes is a key process during immune surveillance, immune response, and development. A novel series of pyrrolo-1,5-benzoxazepines have been shown to potently induce apoptosis in variety of human chemotherapy resistant cancer cell lines, indicating their potential in the treatment of both solid tumors and tumors derived from the hemopoietic system. Pyrrolobenzoxazepine 4-acetoxy-5-(1-naphtyl)naphtho[2,3-b]pyrrolo[1,2-d][1,4]-oxazepine (PBOX-15) has been shown to depolymerize tubulin in vitro and in the MCF7 breast cancer cell line. We hypothesized that this may suggest a role for this compound in modulating integrin-induced T-cell migration, which is largely dependent on the microtubule dynamics. Experiments were performed using human T lymphoma cell line Hut78 and peripheral blood T-lymphocytes isolated from healthy donors. We observed that human T-lymphocytes exposed to PBOX-15 have severely impaired ability to polarize and migrate in response to the triggering stimulus generated via cross-linking of integrin lymphocyte function associated antigen-1 receptor. Here, we show that PBOX-15 can dramatically impair microtubule network via destabilization of tubulin resulting in complete loss of the motile phenotype of T-cells. We demonstrate that PBOX-15 inhibitory mechanisms involve decreased tubulin polymerization and its post-translational modifications. Novel microtubule-targeting effects of PBOX-15 can possibly open new horizons in the treatment of overactive inflammatory conditions as well as cancer and cancer metastatic spreading.

Design and Syntheses of Highly Potent Teixobactin Analogues against Staphylococcus aureus, Methicillin-Resistant Staphylococcus aureus (MRSA), and Vancomycin-Resistant Enterococci (VRE) in Vitro and in Vivo.

The cyclic depsipeptide, teixobactin, kills a number of Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA), and Mycobacterium tuberculosis without detectable resistance. To date, teixobactin is the only molecule in its class that has shown in vivo antibacterial efficacy. In this work, we designed and synthesized 10 new in vivo ready teixobactin analogues. These analogues showed highly potent antibacterial activities against Staphylococcus aureus, MRSA, and vancomycin-resistant enterococci (VRE) in vitro. One analogue, d-Arg4-Leu10-teixobactin, 2, was found to be noncytotoxic in vitro and in vivo. Moreover, topical instillation of peptide 2 in a mouse model of S. aureus keratitis decreased the bacterial bioburden (>99.0% reduction) and corneal edema significantly as compared to untreated mouse corneas. Collectively, our results have established the high therapeutic potential of a teixobactin analogue in attenuating bacterial infections and associated severities in vivo.

Bio-inspired in situ crosslinking and mineralization of electrospun collagen scaffolds for bone tissue engineering.

Bone disorders are the most common cause of severe long term pain and physical disability, and affect millions of people around the world. In the present study, we report bio-inspired preparation of bone-like composite structures by electrospinning of collagen containing catecholamines and Ca(2+). The presence of divalent cation induces simultaneous partial oxidative polymerization of catecholamines and crosslinking of collagen nanofibers, thus producing mats that are mechanically robust and confer photoluminescence properties. Subsequent mineralization of the mats by ammonium carbonate leads to complete oxidative polymerization of catecholamines and precipitation of amorphous CaCO3. The collagen composite scaffolds display outstanding mechanical properties with Young's modulus approaching the limits of cancellous bone. Biological studies demonstrate that human fetal osteoblasts seeded on to the composite scaffolds display enhanced cell adhesion, penetration, proliferation, differentiation and osteogenic expression of osteocalcin, osteopontin and bone matrix protein when compared to pristine collagen or tissue culture plates. Among the two catecholamines, mats containing norepinephrine displayed superior mechanical, photoluminescence and biological properties than mats loaded with dopamine. These smart multifunctional scaffolds could potentially be utilized to repair and regenerate bone defects and injuries.

PPAR-gamma expression modulates insulin sensitivity in C2C12 skeletal muscle cells.

Peroxisome proliferator-activated receptor-gamma (PPAR-gamma) expression is very low in skeletal muscle cells, which is one of the most important target tissues for insulin and plays a predominant role in glucose homeostasis. It has recently been shown that muscle-specific PPAR-gamma deletion in mouse causes insulin resistance. However, it is likely that the observed effects might be due to secondary interaction in whole animal. The aim of the study was to explore the role of muscle PPAR-gamma in insulin sensitivity. We stably transfected C2C12 skeletal muscle cells with plasmids containing sense or antisense constructs of PPAR-gamma and examined the effect of modulation of PPAR-gamma expression in terms of glucose uptake. Effect was also examined in insulin-resistant C2C12 skeletal muscle cells. In transfected C2C12 cell line, the inhibition of PPAR-gamma expression (23.0 +/-0.005%) was observed to induce insulin resistance as determined by functional assessment of 2-deoxyglucose incorporation. Overexpression of PPAR-gamma (28.5 +/- 0.008%) produced an additional effect on insulin (100 nM) and Pioglitazone (50 microM), resulting in 42.7 +/- 3.5% increase in glucose uptake as against 29.2+/-2.8% in wild-type C2C12 skeletal muscle cells differentiated under normal (2% horse serum) condition. Under similar treatment, PPAR-gamma overexpressing cells resistant to insulin exhibited enhanced glucose uptake upto 60.7 +/- 4.08%, as compared to 23.8 +/- 5.1% observed in wild-type C2C12 skeletal muscle cells. These data demonstrate a direct involvement of PPAR-gamma in insulin sensitization of TZD action on skeletal muscle cells, and suggest that pharmacological overexpression of muscle PPAR-gamma gene in skeletal muscle might be a useful strategy for the treatment of insulin resistance.

Combretastatin (CA)-4 and its novel analogue CA-432 impair T-cell migration through the Rho/ROCK signalling pathway.

The capacity of T-lymphocytes to migrate and localise in tissues is important in their protective function against infectious agents, however, the ability of these cells to infiltrate the tumour microenvironment is a major contributing factor in the development of cancer. T-cell migration requires ligand (ICAM-1)/integrin (LFA-1) interaction, activating intracellular signalling pathways which result in a distinct polarised morphology, with an actin-rich lamellipodium and microtubule (MT)-rich uropod. Combretastatin (CA)-4 is a MT-destabilising agent that possesses potent anti-tumour properties. In this study, the effect of CA-4 and its novel analogue CA-432 on human T-cell migration was assessed. Cellular pretreatment with either of CA compounds inhibited the migration and chemotaxis of the T-cell line HuT-78 and primary peripheral blood lymphocyte (PBL) T-cells. This migration-inhibitory effect of CA compounds was due to the disruption of the MT network of T-cells through tubulin depolymerisation, reduced tubulin acetylation and decreased MT stability. In addition, both CA compounds induced the RhoA/RhoA associated kinase (ROCK) signalling pathway, leading to the phosphorylation of myosin light chain (MLC). Furthermore, the siRNA-mediated depletion of GEF-H1, a MT-associated nucleotide exchange factor that activates RhoA upon release from MTs, in T-cells prevented CA-induced phosphorylation of MLC and attenuated the formation of actin-rich membrane protrusions and cell contractility. These results suggest an important role for a GEF-H1/RhoA/ROCK/MLC signalling axis in mediating CA-induced contractility of T-cells. Therapeutic agents that target cytoskeletal proteins and are effective in inhibiting cell migration may open new avenues in the treatment of cancer and metastasis.

The microtubule targeting agent PBOX-15 inhibits integrin-mediated cell adhesion and induces apoptosis in acute lymphoblastic leukaemia cells.

Although recent decades have seen an improved cure rate for newly diagnosed paediatric acute lymphoplastic leukaemia (ALL), the treatment options for adult ALL, T-cell ALL (T-ALL) and relapsed disease remain poor. We have developed a novel series of pyrrolo-1,5-benzoxazepine (PBOX) compounds and established their anticancer efficacy in a variety of human tumour cell types. Here, we demonstrate that PBOX-15 inhibits cell growth, and induces G2/M cell cycle arrest and apoptosis in both T-ALL and B-cell ALL (B-ALL) cells. In addition, prior to PBOX-15-induced apoptosis, PBOX-15 decreases ALL cell adhesion, spreading and migration. Concurrently, PBOX-15 differentially down-regulates ß1-, ß2- and α4-integrin expression in ALL cells and significantly decreases integrin-mediated cell attachment. PBOX-15 interferes with the lateral mobility and clustering of integrins in both B-ALL and T-ALL cells. These data suggest that PBOX-15 is not only effective in inducing apoptosis in ALL cells, but also has the potential to disrupt integrin-mediated adhesion of malignant lymphocytes, which represents a novel avenue for regulating leukaemic cell homing and migration.

Citrullination of proteins: a common post-translational modification pathway induced by different nanoparticles in vitro and in vivo.

AIM: Rapidly expanding manufacture and use of nanomaterials emphasize the requirements for thorough assessment of health outcomes associated with novel applications. Post-translational protein modifications catalyzed by Ca(2+)-dependent peptidylargininedeiminases have been shown to trigger immune responses including autoantibody generation, a hallmark of immune complexes deposition in rheumatoid arthritis. Therefore, the aim of the study was to assess if nanoparticles are able to promote protein citrullination. MATERIALS & METHODS: Human A549 and THP-1 cells were exposed to silicon dioxide, carbon black or single-walled carbon nanotubes. C57BL/6 mice were exposed to respirable single-walled carbon nanotubes. Protein citrullination, peptidylargininedeiminases activity and target proteins were evaluated. RESULTS: The studied nanoparticles induced protein citrullination both in cultured human cells and mouse lung tissues. Citrullination occurred via the peptidylargininedeiminase-dependent mechanism. Cytokeratines 7, 8, 18 and plectins were identified as intracellular citrullination targets. CONCLUSION: Nanoparticle exposure facilitated post-translational citrullination of proteins.

STAT3-stathmin interactions control microtubule dynamics in migrating T-cells.

T-cell migration is a complex highly coordinated process that involves cell adhesion to the high endothelial venules or to the extracellular matrix by surface receptor/ligand interactions, cytoskeletal rearrangements, and phosphorylation-dependent signaling cascades. The mechanism(s) that regulates T-cell migration is of considerable relevance for understanding the pathogenesis of various diseases, such as chronic inflammatory diseases and cancer metastasis. This study was designed to identify potential involvement of STAT3, a latent transcription factor, in mediating integrin-induced T-cell migration. Using our previously characterized in vitro model for lymphocyte migration, we demonstrate that STAT3 is activated and translocated to the nucleus during the process of active motility of Hut78 T-lymphoma cells triggered via LFA-1. Blocking STAT3 signaling by multiple approaches inhibited LFA-1-induced T-cell locomotion via destabilization of microtubules and post-translational modification of tubulin. Here, we show that STAT3 physically interacts with stathmin to regulate microtubule dynamics in migrating T-cells. These observations strongly indicate that STAT3 is critically important for T-cell migration and associated signaling events.

Miltefosine induces apoptosis in arsenite-resistant Leishmania donovani promastigotes through mitochondrial dysfunction.

The control of leishmaniasis in absence of vaccine solely depends on the choice of chemotherapy. The major hurdle in successful leishmanial chemotherapy is emergence of drug resistance. Miltefosine, the first orally administrable anti-leishmanial drug, has shown the potential against drug-resistant strains of Leishmania. However, there are discrepancies regarding the involvement of P-glycoprotein (Pgp) and sensitivity of miltefosine in multiple drug-resistant (MDR) cell lines that overexpress Pgp in Leishmania. To address this, the effect of miltefosine in arsenite-resistant Leishmania donovani (Ld-As20) promastigotes displaying an MDR phenotype and overexpressing Pgp-like protein was investigated in the current study. Results indicate that Ld-As20 is sensitive to miltefosine. Miltefosine induces process of programmed cell death in Ld-As20 in a time-dependent manner as determined by cell shrinkage, externalization of phosphatidylserine and DNA fragmentation. Miltefosine treatment leads to loss of mitochondrial membrane potential and the release of cytochrome C with consequent activation of cellular proteases. Activation of cellular proteases resulted in activation of DNase that damaged kinetoplast DNA and induced dyskinetoplasty. These data indicate that miltefosine causes apoptosis-like death in arsenite-resistant L. donovani.

Possible mechanism of miltefosine-mediated death of Leishmania donovani.

Miltefosine causes leishmanial death, but the possible mechanism(s) of action is not known. The mode of action of miltefosine was investigated in vitro in Leishmania donovani promastigotes as well as in extra- and intracellular amastigotes. Here, we demonstrate that miltefosine induces apoptosis-like death in L. donovani based on observed phenomena such as nuclear DNA condensation, DNA fragmentation with accompanying ladder formation, and in situ labeling of DNA fragments by the terminal deoxyribonucleotidyltransferase-mediated dUTP-biotin nick end labeling method. Understanding of miltefosine-mediated death will facilitate the design of new therapeutic strategies against Leishmania parasites.