Identification of novel inhibitors against UDP-galactopyranose mutase to combat leishmaniasis.

Leishmania, a protozoan parasite that causes leishmaniasis, affects 1-2 million people every year worldwide. Leishmaniasis is a vector born disease and characterized by a diverse group of clinical syndromes. Current treatment is limited because of drug resistance, high cost, poor safety, and low efficacy. The urgent need for potent agents against Leishmania has led to significant advances in the development of novel antileishmanial drugs. ß-galactofuranose (ß-Galf) is an important component of Leishmanial cell surface matrix and plays a critical role in the pathogenesis of parasite. UDP-galactopyranose mutase (UGM) converts UDP-galactopyranose (UDP-Galp) to UDP-galactofuranose (UDP-Galf) which acts as the precursor for ß-Galf synthesis. Due to its absence in human, this enzyme is selected as the potential target in search of new antileishmanial drugs. Three dimensional protein structure model of Leishmania major UGM (LmUGM) has been homology modeled using Trypanosoma cruzi UGM (TcUGM) as a template. The stereochemistry was validated further. We selected already reported active compounds from PubChem database to target the LmUGM. Three compounds (6064500, 44570814, and 6158954) among the top hit occupied the UDP binding site of UGM suggested to work as a possible inhibitor for it. In vitro antileishmanial activity assay was performed with the top ranked inhibitor, 6064500. The 6064500 molecule has inhibited the growth of Leishmania donovani promastigotes significantly. Further, at similar concentrations it has exhibited significantly lesser toxicity than standard drug miltefosine hydrate in mammalian cells.

Screening of Novel Inhibitors Against Leishmania donovani Calcium ion Channel to Fight Leishmaniasis.

Leishmania is an intracellular protozoan parasite which causes Leishmaniasis, a global health problem affecting millions of people throughout 89 different countries in the world. The current treatment which includes use of amphotericin B, antimonials, and others has major drawbacks due to toxicity, resistance, and extraordinary high cost. So there is an urgent need of development of new drug targets to fight against leishmaniasis. In this regard we have selected Leishmania donovani Ca2+ ion channel (Ld-CC) as potential drug target. Ld-CC regulates concentration of Ca2+ ions which is involved in several functions like flagellar motion, mitochondrial oxidative metabolism and entry inside the macrophages. Since Ld-CC has not been characterised yet, we performed homology modelling of Leishmania donovani Ca2+ ion channel (Ld-CC) and docking studies of ligand library against this channel. 542 compound library of National Cancer Institute (NCI) diversity 3 dataset selected for screening studies. The ligands ZINC17287336 and ZINC29590262 were selected as best energy conformers because they show highest binding affinity towards its target (Ld-CC). They interact with the active site residues in the pocket of Ld-CC which suggests that the docked conformations are good and acceptable. Moreover, these two selected compounds also have relatively high binding affinity than nifedipine and verapamil, known human calcium channel blockers which had been reported to have mild anti-leishmanial activity. Among these two top screened inhibitors the ligand ZINC29590262 shows poor binding affinity towards the Human voltagedependent L-type calcium channel subunit alpha-1C in comparison to the Ld-CC. Therefore, we proposed this ligand as the best inhibitor which shows 40% more binding affinity with Ld-CC than the human-VDCC. These results suggest that our screened ligand ZINC29590262 could act as novel drug and may show much better antileishmanial activity.

Combined immunotherapy with whole tumor lysate-pulsed interleukin-15-activated dendritic cells and cucurbitacin I promotes strong CD8(+) T-cell responses and cures highly aggressive lymphoma.

BACKGROUND AIMS: Dendritic cell (DC)-based therapies could be important strategies for lymphoma treatment. METHODS: AKR/J mice with Dalton's lymphoma were treated with recombinant interleukin-15 (rIL-15)-activated autologous DCs and pulsed with whole tumor cell lysates in the presence or absence of suboptimal doses of the STAT3 inhibitor cucurbitacin I. One group of treated mice received an additional dose of rIL-15 to boost the DC-based adoptive cell therapy (ACT). Kaplan-Meier survival analysis and multiple immunologic and enzymatic parameters were assessed to demonstrate the efficacy of the vaccination protocol. RESULTS: Therapy with tumor lysate-pulsed, rIL-15-activated DCs plus cucurbitacin I significantly prolongs the survival of tumor-bearing mice but fails to provide a complete cure. Additional treatment of vaccinated mice with rIL-15 dramatically improves the therapeutic efficacy and provides a lifelong cure with no relapse. DCs derived from the surviving vaccinated mice regained their anti-tumor potential against the lymphoma cells with respect to growth inhibition and cytotoxicity. Both cluster of differentiation (CD)4(+) and CD8(+) T cells were mobilized in metastatic organs of successfully vaccinated mice in large numbers and demonstrated antigen-specific proliferation and tumor cell cytotoxicity. ACT also augments DC function by upregulating tumor necrosis factor-related apoptosis-inducing ligand and tumor necrosis factor-α expression. In addition, combinatorial immunotherapy restores the levels of antioxidant enzymes and liver function enzyme activities that are severely repressed in untreated tumor-bearing mice. CONCLUSIONS: Effective vaccination for a complete cure against aggressive lymphoma requires DC-based ACT in combination with chemotherapy and cytokine therapy.

Tumor antigen-/cytokine-pulsed dendritic cells in therapy against lymphoma.

Adoptive cell therapy using dendritic cells (DCs) is a strategy to deliver tumor antigens in cancer immunotherapy. Co-delivery of antigens to DC with essential components like genes encoding cytokines, chemokines, and other molecules or stimulation with recombinant cytokines is a potential method for designing an effective tumor vaccine protocol. Here, we describe the stimulation of purified splenic- or bone marrow-derived DC with recombinant interleukin-15 (IL-15) in the presence of intact soluble antigen from metastatic lymphoma tumor cells in an experimental animal model.

CD47 augments Fas/CD95-mediated apoptosis.

Fas (CD95) mediates apoptosis of many cell types, but the susceptibility of cells to killing by Fas ligand and anti-Fas antibodies is highly variable. Jurkat T cells lacking CD47 (integrin-associated protein) are relatively resistant to Fas-mediated death but are efficiently killed by Fas ligand or anti-Fas IgM (CH11) upon expression of CD47. Lack of CD47 impairs events downstream of Fas activation including caspase activation, poly-(ADP-ribose) polymerase cleavage, cytochrome c release from mitochondria, loss of mitochondrial membrane potential, and DNA cleavage. Neither CD47 signaling nor raft association of CD47 is required to enable Fas apoptosis. CH11 induces association of Fas and CD47. Primary T cells from CD47-null mice are also protected from Fas-mediated killing relative to wild type T cells. Thus CD47 associates with Fas upon its activation and augments Fas-mediated apoptosis.

Generation of CD8+ cytotoxic T lymphocytes against breast cancer cells by stimulation with mammaglobin-A-pulsed dendritic cells.

Mammaglobin-A is exclusively expressed by breast cancer cells. Thus, mammaglobin-A-specific T cell immune responses may be useful for the design of new breast cancer-specific immunotherapies. We show herein that CD8+ T cells generated against recombinant mammaglobin-A-pulsed dendritic cells display a marked cytotoxic activity against mammaglobin-A-positive breast cancer cell lines. This study indicates the immunotherapeutic potential of this novel antigen for the treatment of breast cancer.

Identification of HLA-A3-restricted CD8+ T cell epitopes derived from mammaglobin-A, a tumor-associated antigen of human breast cancer.

Mammaglobin-A is highly overexpressed in breast cancer cell lines and primary breast tumors. This pattern of expression is restricted to mammary epithelium and metastatic breast tumors. Thus, mammaglobin-A-specific T cell immune responses may provide an important approach for the design of breast cancer-specific immunotherapy. The purpose of our study was to define the T cell-mediated immune response to mammaglobin-A. We determined that the frequency of mammaglobin-A-reactive CD8+ and CD4+ T cells in breast cancer patients is significantly higher than that observed in healthy female controls using limiting dilution analyses (p = 0.026 and p = 0.02, respectively). We identified 8 mammaglobin-A-derived 9-mer peptides with the highest binding affinity for the HLA-A3 molecule (Mam-A3.1-8) using a computer-assisted analysis of the mammaglobin-A protein sequence. Subsequently, we determined that CD8+ T cells from breast cancer patients reacted to peptides Mam-A3.1 (23-31, PLLENVISK), Mam-A3.3 (2-10, KLLMVLMLA), Mam-A3.4 (55-63, TTNAIDELK) and Mam-A3.8 (58-66, AIDELKECF) using an IFN-gamma enzyme-linked immunospot assay. A CD8+ T cell line generated in vitro against HLA-A*0301-transfected TAP-deficient T2 cells loaded with these peptides showed significant cytotoxic activity against the Mam-A3.1 peptide. This CD8+ T cell line showed a significant HLA-A3-restricted cytotoxic activity against mammaglobin-A-positive but not mammaglobin-A-negative breast cancer cells. In summary, our study identified four HLA-A3-restricted mammaglobin-A-derived epitopes naturally expressed by breast cancer cells, indicating the immunotherapeutic potential of this novel antigen for the treatment and prevention of breast cancer.

Human dendritic cell mediated cytotoxicity against breast carcinoma cells in vitro.

Dendritic cells (DC) are an important subset of antigen-presenting cells characterized by their potent capacity to activate immunologically naïve T cells. However, their role in effector function in tumor resistance is less well characterized. We report here that activated human peripheral blood DC acquire a potent antitumor effect against breast cancer cell lines in vitro, leading to growth inhibition and apoptosis of the tumor cell. The antitumor effect of DC was augmented by proinflammatory stimuli induced by lipopolysaccharide (LPS) treatment. Tumor necrosis factor alpha (TNF-alpha) produced after DC activation was responsible for the antitumor activity of DC. Interferon-gamma, interleukin-15, or LPS treatment of DC markedly augmented the effector function of DC against most of the breast cells, indicating heterogeneity of the tumor and its susceptibility to cytokine-mediated damage. Treatment of LPS-activated DC or cell-free supernatant with anti-human TNF-alpha significantly reduces the antitumor effect against the tumor cells tested. These results suggest that in addition to their predominant role as immune regulatory cells, DC could serve as innate effector cells in tumor immunity.