9 research outputs found
Dendritic Cell-Based Immunotherapy Combined with Antimony-Based Chemotherapy Cures Established Murine Visceral Leishmaniasis
Dendritic cells (DCs) have been proposed to play a critical role as adjuvants in vaccination and immunotherapy. In this study we
evaluated the combined effect of soluble Leishmania donovani Ag (SLDA)-pulsed syngeneic bone marrow-derived DC-based
immunotherapy and antimony-based chemotherapy for the treatment of established murine visceral leishmaniasis. Three weekly
injections of SLDA-pulsed DCs into L. donovani-infected mice reduced liver and splenic parasite burden significantly, but could
not clear parasite load from these organs completely. Strikingly, the conventional antileishmanial chemotherapy (sodium antimony
gluconate) along with injections of SLDA-pulsed DCs resulted in complete clearance of parasites from both these organs. Repetitive
in vitro stimulation of splenocytes from uninfected or L. donovani-infected mice with SLDA-pulsed DCs led to the emergence
of CD4� T cells with characteristics of Th1 cells. Our data indicate that DC-based immunotherapy enhances the in vivo antileishmanial
potential of antimony or vice versa
Dihydrobetulinic Acid Induces Apoptosis in Leishmania donovani by Targeting DNA Topoisomerase I and II: Implications in Antileishmanial Therapy
Leishmaniasis is the second-most dreaded parasitic disease in the modern world, behind malaria. The lack of effective vaccines demand improved chemotherapy along with the development of lead compounds and newer targets. We report here that the pentacyclic triterpenoid, dihydrobetulinic acid (DHBA), is a novel lead compound for antileishmanial therapy. It acts by targeting DNA topoisomerases. DNA topoisomerase I and II activity was studied using relaxation and decatenation assays. Mechanistic studies were based on the decreased mobility of enzyme-bound DNA compared with free DNA and the differential mobility of nicked and supercoiled monomers in 1% agarose gel. Pulsed field gradient gel electrophoresis, confocal microscopy, and transmission electron microscopy were performed to assess cytotoxicity of the compound and ultrastructural damage of the parasite. Apoptosis was studied by the isolation of DNA from DHBA-treated parasites and subsequent electrophoresis in 1% agarose gel. DHBA inhibits growth of Leishmania donovani promastigotes and amastigotes with an IC(50) of 2.6 and 4.1 μM respectively. The compound is a dual inhibitor of DNA topoisomerases that fails to induce DNA cleavage and acts by preventing the formation of enzyme-DNA binary complex, ultimately inducing apoptosis. Treatment of infected golden hamsters with the compound markedly reduces (> 92%) parasitic burden, both in spleen and liver. Interestingly, the 17-decarboxylated analogue, dihydrolupeol, does not inhibit DNA topoisomerase I and II, has no effect on parasitic growth, and also fails to induce apoptosis. DHBA is a potent antileishmanial agent that induces apoptosis by primarily targeting DNA topoisomerases. Therefore it is a strong candidate for use in designing new antileishmanial drugs
Leishmania donovani Infection of Human Myeloid Dendritic Cells Leads to a Th1 Response in CD4+ T Cells from Healthy Donors and Patients with Kala-Azar.
The role played by dendritic cells (DCs) in Leishmania donovani infection is poorly understood. Here, we
report that L. donovani amastigotes efficiently infect human peripheral-blood monocyte–derived DCs. Opsonization
with normal human serum enhanced the infectivity of amastigotes and promastigotes only marginally.
Surface attachment versus internalization was distinguished by incubation of DCs with live, fluorescein isothiocyanate–labeled parasites, followed by quenching with crystal violet. Infection with amastigotes was accompanied by DC maturation, as was evident from the up-regulation of maturation-associated cell-surface markers, the nuclear translocation of RelB, and the release of cytokines. Amastigote-primed DCs produced inflammatory cytokines in response to subsequent treatment with interferon-g or anti-CD40 monoclonal antibody. When cocultured, amastigote-infected DCs induced T helper cell type 1 (Th1) responses both in naive allogeneic CD4+ T cells and in autologous CD4+ T cells from patients with kala-azar and up-regulated the expression of T-bet. Our data reveal that infection with L. donovani amastigotes induces a Th1 cytokine milieu in both DCs and T cells
N-acetyl cysteine enhances imatinib-induced apoptosis of Bcr-Abl+ cells by endothelial nitric oxide synthase-mediated production of nitric oxide
Introduction Imatinib, a small-molecule inhibitor of the
Bcr-Abl kinase, is a successful drug for treating chronic
myeloid leukemia (CML). Bcr-Abl kinase stimulates the
production of H2O2, which in turn activates Abl kinase. We
therefore evaluated whether N-acetyl cysteine (NAC), a
ROS scavenger improves imatinib efficacy.
Materials and methods Effects of imatinib and NAC
either alone or in combination were assessed on Bcr-Abl?
cells to measure apoptosis. Role of nitric oxide (NO) in
NAC-induced enhanced cytotoxicity was assessed using
pharmacological inhibitors and siRNAs of nitric oxide
synthase isoforms. We report that imatinib-induced apoptosis
of imatinib-resistant and imatinib-sensitive Bcr-Abl?
CML cell lines and primary cells from CML patients significantly enhanced by co-treatment with NAC compared
to imatinib treatment alone. In contrast, another ROS
scavenger glutathione reversed imatinib-mediated killing.
NAC-mediated enhanced killing correlated with cleavage
of caspases, PARP and up-regulation and down regulation
of pro- and anti-apoptotic family of proteins, respectively.
Co-treatment with NAC leads to enhanced production
of nitric oxide (NO) by endothelial nitric oxide synthase
(eNOS). Involvement of eNOS dependent NO in NACmediated
enhancement of imatinib-induced cell death was
confirmed by nitric oxide synthase (NOS) specific pharmacological
inhibitors and siRNAs. Indeed, NO donor
sodium nitroprusside (SNP) also enhanced imatinib-mediated
apoptosis of Bcr-Abl? cells.
Conclusion NAC enhances imatinib-induced apoptosis
of Bcr-Abl? cells by endothelial nitric oxide synthasemediated
production of nitric oxide
Involvement of ROS in Chlorogenic Acid-Induced Apoptosis of Bcr-Abl+ CML Cells
Chlorogenic acid (Chl) has been reported to possess a wide range of biological and pharmacological
properties including induction of apoptosis of Bcr-Abl+ chronic myeloid leukemia (CML) cell lines and
clinical leukemia samples via inhibition of Bcr-Abl phosphorylation. Here we studied the mechanisms of
action of Chl in greater detail. Chl treatment induced an early accumulation of intracellular reactive
oxygen species (ROS) in Bcr-Abl+ cells leading to downregulation of Bcr-Abl phosphorylation and
apoptosis. Chl treatment upregulated death receptor DR5 and induced loss of mitochondrial membrane
potential accompanied by release of cytochrome c from the mitochondria to the cytosol. Pharmacological
inhibition of caspase-8 partially inhibited apoptosis, whereas caspase-9 and pan-caspase inhibitor
almost completely blocked the killing. Knocking down DR5 using siRNA completely attenuated Chlinduced
caspase-8 cleavage but partially inhibited apoptosis. Antioxidant NAC attenuated Chl-induced
oxidative stress-mediated inhibition of Bcr-Abl phosphorylation, DR5 upregulation, caspase activation
and CML cell death. Our data suggested the involvement of parallel death pathways that converged in
mitochondria. The role of ROS in Chl-induced death was confirmed with primary leukemia cells fromCML
patients in vitro as well as in vivo in nude mice bearing K562 xenografts. Collectively, our results establish
the role of ROS for Chl-mediated preferential killing of Bcr-Abl+ cells
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Hydroxychavicol, a Piper betle leaf component, induces apoptosis of CML cells through mitochondrial reactive oxygen species-dependent JNK and endothelial nitric oxide synthase activation and overrides imatinib resistance
Alcoholic extract of Piper betle (Piper betle L.) leaves was recently found to induce apoptosis of CML cells expressing wild type and mutated Bcr-Abl with imatinib resistance phenotype. Hydroxy-chavicol (HCH), a constituent of the alcoholic extract of Piper betle leaves, was evaluated for anti-CML activity. Here, we report that HCH and its analogues induce killing of primary cells in CML patients and leukemic cell lines expressing wild type and mutated Bcr-Abl, including the T315I mutation, with minimal toxicity to normal human peripheral blood mononuclear cells. HCH causes early but transient increase of mitochondria-derived reactive oxygen species. Reactive oxygen species-dependent persistent activation of JNK leads to an increase in endothelial nitric oxide synthase-mediated nitric oxide generation. This causes loss of mitochondrial membrane potential, release of cytochrome c from mitochondria, cleavage of caspase 9, 3 and poly-adenosine diphosphate-ribose polymerase leading to apoptosis. One HCH analogue was also effective in vivo in SCID mice against grafts expressing the T315I mutation, although to a lesser extent than grafts expressing wild type Bcr-Abl, without showing significant bodyweight loss. Our data describe the role of JNK-dependent endothelial nitric oxide synthase-mediated nitric oxide for anti-CML activity of HCH and this molecule merits further testing in pre-clinical and clinical settings
Granulocyte–macrophage colony-stimulating factor drives monocytes to CD14low CD83+ DCSIGN– interleukin-10-producing myeloid cells with differential effects on T-cell subsets
Granulocyte–macrophage colony-stimulating factor (GM-CSF) has long been found to have growth-promoting effects on multipotent haematopoietic lineages, specifically granulocytes and macrophages. GM-CSF combined with interleukin-4 (IL-4) drives monocytes to become myeloid dendritic cells (mDCs) in vitro. We report that culturing human monocytes with GM-CSF alone generates myeloid cells (GM-Mono) that have lower expression of CD14 than monocytes and that fail to express DC-SIGN. GM-Monos, however, express CD83 and the transcription factor PU.1, although at a lower level than the conventional mDCs generated in the presence of GM-CSF and IL-4. On stimulation with tumour necrosis factor-α, interferon-γ and anti-CD40 monoclonal antibody, the GM-Monos predominantly produced IL-10 but were less efficient in IL-12 production. In a primary allogeneic mixed lymphocyte reaction, GM-Monos induced hyporesponsiveness and IL-10-biased cytokine production in CD4+ T cells. In fresh mixed lymphocyte reaction, GM-Monos inhibited conventional mDC-induced allogeneic CD4+ T-cell proliferation. GM-Mono-induced inhibition of allogeneic CD4+ T-cell proliferation was partially attributed to IL-10. Interestingly, GM-Monos neither induced hyporesponsiveness in allogeneic CD8+ T cells nor inhibited conventional mDC-induced allogeneic CD8+ T-cell proliferation. Taken together, we characterize monocyte-derived CD14low CD83+ cells generated by GM-CSF that can induce tolerance or stimulation of T cells depending on T-cell subsets