The STING agonist, DMXAA, reduces tumor vessels and enhances mesothelioma tumor antigen presentation yet blunts cytotoxic T cell function in a murine model.

We assessed the murine Stimulator of Interferon Genes (STING) agonist, DMXAA, for anti-mesothelioma potential using the AE17-sOVA model that expresses ovalbumin (OVA) as a neo tumor antigen. Dose response experiments alongside testing different routes of administration identified a safe effective treatment regimen that induced 100% cures in mice with small or large tumors. Three doses of 25mg/kg DMXAA given intra-tumorally every 9 days induced tumor regression and long-term survival (>5 months). Re-challenge experiments showed that tumor-free mice developed protective memory. MTT and propidium-iodide assays showed that DMXAA exerted direct cytotoxic effects at doses >1mg/ml on the murine AE17 and AB1 mesothelioma cell lines. In-vivo studies using a CFSE-based in-vivo proliferation assay showed that DMXAA improved tumor-antigen presentation in tumor-draining lymph nodes, evidenced by OVA-specific OT-1 T cells undergoing more divisions. An in-vivo cytotoxic T lymphocyte (CTL) assay showed that DMXAA blunted the lytic quality of CTLs recognizing the dominant (SIINFEKL) and a subdominant (KVVRFDKL) OVA epitopes. DMXAA reduced tumor vessel size in-vivo and although the proportion of T cells infiltrating tumors reduced, the proportion of tumor-specific T cells increased. These data show careful dosing and treatment protocols reduce mesothelioma cell viability and modulate tumor vessels such that tumor-antigen specific CTLs access the tumor site. However, attempts to enhance DMXAA-induced anti-tumor responses by combination with an agonist anti-CD40 antibody or IL-2 reduced efficacy. These proof-of-concept data suggest that mesothelioma patients could benefit from treatment with a STING agonist, but combination with immunotherapy should be cautiously undertaken.

Re-evaluating pretomanid analogues for Chagas disease: Hit-to-lead studies reveal both in vitro and in vivo trypanocidal efficacy.

Phenotypic screening of a 900 compound library of antitubercular nitroimidazole derivatives related to pretomanid against the protozoan parasite Trypanosoma cruzi (the causative agent for Chagas disease) identified several structurally diverse hits with an unknown mode of action. Following initial profiling, a first proof-of-concept in vivo study was undertaken, in which once daily oral dosing of a 7-substituted 2-nitroimidazooxazine analogue suppressed blood parasitemia to low or undetectable levels, although sterile cure was not achieved. Limited hit expansion studies alongside counter-screening of new compounds targeted at visceral leishmaniasis laid the foundation for a more in-depth assessment of the best leads, focusing on both drug-like attributes (solubility, metabolic stability and safety) and maximal killing of the parasite in a shorter timeframe. Comparative appraisal of one preferred lead (58) in a chronic infection mouse model, monitored by highly sensitive bioluminescence imaging, provided the first definitive evidence of (partial) curative efficacy with this promising nitroimidazooxazine class.

Elderly dendritic cells respond to LPS/IFN-γ and CD40L stimulation despite incomplete maturation.

There is evidence that dendritic cells (DCs) undergo age-related changes that modulate their function with their key role being priming antigen-specific effector T cells. This occurs once DCs develop into antigen-presenting cells in response to stimuli/danger signals. However, the effects of aging on DC responses to bacterial lipopolysaccharide (LPS), the pro-inflammatory cytokine interferon (IFN)-γ and CD40 ligand (CD40L) have not yet been systematically evaluated. We examined responses of blood myeloid (m)DC1s, mDC2s, plasmacytoid (p)DCs, and monocyte-derived DCs (MoDCs) from young (21-40 years) and elderly (60-84 years) healthy human volunteers to LPS/IFN-γ or CD40L stimulation. All elderly DC subsets demonstrated comparable up-regulation of co-stimulatory molecules (CD40, CD80 and/or CD86), intracellular pro-inflammatory cytokine levels (IFN-γ, tumour necrosis factor (TNF)-α, IL-6 and/or IL-12), and/or secreted cytokine levels (IFN-α, IFN-γ, TNF-α, and IL-12) to their younger counterparts. Furthermore, elderly-derived LPS/IFN-γ or CD40L-activated MoDCs induced similar or increased levels of CD8+ and CD4+ T cell proliferation, and similar T cell functional phenotypes, to their younger counterparts. However, elderly LPS/IFN-γ-activated MoDCs were unreliable in their ability to up-regulate chemokine (IL-8 and monocyte chemoattractant protein (MCP)-1) and IL-6 secretion, implying an inability to dependably induce an inflammatory response. A key age-related difference was that, unlike young-derived MoDCs that completely lost their ability to process antigen, elderly-derived MoDCs maintained their antigen processing ability after LPS/IFN-γ maturation, measured using the DQ-ovalbumin assay; this response implies incomplete maturation that may enable elderly DCs to continuously present antigen. These differences may impact on the efficacy of anti-pathogen and anti-tumour immune responses in the elderly.

Green tea polyphenol "epigallocatechin-3-gallate", differentially induces apoptosis in CLL B-and T-Cells but not in healthy B-and T-Cells in a dose dependant manner.

B-cell chronic lymphocytic leukaemia (CLL) is characterized by an accumulation of CD5-positive monoclonal B-cells due in large part to a failure of apoptosis. The ability to study CLL B-cells in vitro has always been a challenge and hampered by the low viability of the CLL B-cells in cell culture systems. In this study, we present a multicellular cell culture system to maintain CLL B-cells viable in culture for 60h in the presence of a stromal cell feeder layer in combination with a whole white blood cell preparation. Using this optimized system, we tested and showed that the addition of epigallocatechin-3-gallate (EGCG) at concentrations ranging from 25 to 100µg/ml induced apoptosis in CLL B-cells whilst not affecting healthy control B-cells. Moreover, the results showed that in contrast to healthy controls, T-cells from CLL patients underwent apoptosis in the presence of EGCG. This study demonstrated that the combination of a cell feeder layer with a whole white blood cell preparation maintained B-cell viability in vitro over an extended period of time. In addition, the study showed that EGCG differentially induces apoptosis in CLL B-and T-Cells but not in healthy B-and T-Cells in a dose dependent manner.

Human mesothelioma induces defects in dendritic cell numbers and antigen-processing function which predict survival outcomes.

Mesothelioma is an almost invariably fatal tumor with chemotherapy extending survival by a few months. One immunotherapeutic strategy is to target dendritic cells (DCs), key antigen-presenting cells involved in antigen presentation, to induce antigen-specific T cell responses. However, DC-targeting will only be effective if DCs are fit-for-purpose, and the functional status of DCs in mesothelioma patients was not clear. We found that mesothelioma patients have significantly decreased numbers of circulating myeloid (m)DC1 cells, mDC2 cells and plasmacytoid (p)DCs relative to healthy age and gender-matched controls. Blood monocytes from patients could not differentiate into immature monocyte-derived DCs (MoDCs), indicated by a significantly reduced ability to process antigen and reduced expression of costimulatory (CD40, CD80 and CD86) and MHC (HLA-DR) molecules, relative to controls. Activation of mesothelioma-derived MoDCs with LPS+/-IFNγ generated partially mature MoDCs, evident by limited upregulation of the maturation marker, CD83, and the costimulatory markers. Attempts to rescue mesothelioma-derived DC function using CD40Ligand(L) also failed, indicated by maintenance of antigen-processing capacity and limited upregulation of CD40, CD83, CD86 and HLA-DR. These data suggest that mesothelioma patients have significant numerical and functional DC defects and that their reduced capacity to process antigen and reduced expression of costimulatory molecules could induce anergized/tolerized T cells. Nonetheless, survival analyses revealed that individuals with mesothelioma and higher than median levels of mDC1s and/or whose MoDCs matured in response to LPS, IFNγ or CD40L lived longer, implying their selection for DC-targeting therapy could be promising especially if combined with another treatment modality.

Two analogues of fenarimol show curative activity in an experimental model of Chagas disease.

Chagas disease, caused by the protozoan parasite Trypanosoma cruzi (T. cruzi), is an increasing threat to global health. Available medicines were introduced over 40 years ago, have undesirable side effects, and give equivocal results of cure in the chronic stage of the disease. We report the development of two compounds, 6 and (S)-7, with PCR-confirmed curative activity in a mouse model of established T. cruzi infection after once daily oral dosing for 20 days at 20 mg/kg 6 and 10 mg/kg (S)-7. Compounds 6 and (S)-7 have potent in vitro activity, are noncytotoxic, show no adverse effects in vivo following repeat dosing, are prepared by a short synthetic route, and have druglike properties suitable for preclinical development.

Selection and optimization of hits from a high-throughput phenotypic screen against Trypanosoma cruzi.

BACKGROUND: Inhibitors of Trypanosoma cruzi with novel mechanisms of action are urgently required to diversify the current clinical and preclinical pipelines. Increasing the number and diversity of hits available for assessment at the beginning of the discovery process will help to achieve this aim. RESULTS: We report the evaluation of multiple hits generated from a high-throughput screen to identify inhibitors of T. cruzi and from these studies the discovery of two novel series currently in lead optimization. Lead compounds from these series potently and selectively inhibit growth of T. cruzi in vitro and the most advanced compound is orally active in a subchronic mouse model of T. cruzi infection. CONCLUSION: High-throughput screening of novel compound collections has an important role to play in diversifying the trypanosomatid drug discovery portfolio. A new T. cruzi inhibitor series with good drug-like properties and promising in vivo efficacy has been identified through this process.

CD40-activated B cells contribute to mesothelioma tumor regression.

Targeting CD40, a member of the tumor necrosis factor superfamily, using agonist antibodies (Abs) produces dramatic antitumor effects. Indeed, high-dose intravenous anti-CD40 Ab 'licenses' dendritic cells (DCs) that instruct activated CD8(+) cytotoxic T cells to leave lymph nodes (LNs) and penetrate the mesothelioma tumor microenvironment. However, toxic side effects and the potential of an 'overwhelmed' immune response warrant an alternative approach. In this study, we show that injecting lower doses of anti-CD40 Ab directly into the tumor bed avoided toxic side effects and prolonged survival in 60% of mice, with most cured. Unexpectedly, DCs in tumors and LNs 'disappeared', CD8(+) tumor-specific T-cell numbers and function were not enhanced, and T cells did not infiltrate regressing tumors. CD4(+) or CD8(+) depletion only marginally hindered anti-CD40 Ab efficacy implying another effector mechanism. B-cell numbers significantly increased in tumors, draining LNs and spleens during intratumoral anti-CD40 Ab treatment. CD40 targeting had no effect on splenic B-1 cells, obliterated marginal zone B cells and promoted follicular (FO) B-cell activity. Adoptive transfer of tumor antigen-experienced, CD40-activated B cells, or their immunoglobulin products, which recognized autoantigens on mesothelioma cells, protected against tumor challenge. Finally, studies using B-cell knockout mice showed that successful treatment of established tumors required the presence of B cells. Thus, these data suggest that CD40-activated FO B cells can become an important component of an effective antitumor immune response.

Myeloid leukemia factor 1 associates with a novel heterogeneous nuclear ribonucleoprotein U-like molecule.

Myeloid leukemia factor 1 (MLF1) is an oncoprotein associated with hemopoietic lineage commitment and acute myeloid leukemia. Here we show that Mlf1 associated with a novel binding partner, Mlf1-associated nuclear protein (Manp), a new heterogeneous nuclear ribonucleoprotein (hnRNP) family member, related to hnRNP-U. Manp localized exclusively in the nucleus and could redirect Mlf1 from the cytoplasm into the nucleus. The nuclear content of Mlf1 was also regulated by 14-3-3 binding to a canonical 14-3-3 binding motif within the N terminus of Mlf1. Significantly Mlf1 contains a functional nuclear export signal and localized primarily to the nuclei of hemopoietic cells. Mlf1 was capable of binding DNA, and microarray analysis revealed that it affected the expression of several genes, including transcription factors. In summary, this study reveals that Mlf1 translocates between nucleus and cytoplasm, associates with a novel hnRNP, and influences gene expression.