Single-cell analysis tools for drug discovery and development

The genetic, functional or compositional heterogeneity of healthy and diseased tissues presents major challenges in drug discovery and development. Such heterogeneity hinders the design of accurate disease models and can confound the interpretation of biomarker levels and of patient responses to specific therapies. The complex nature of virtually all tissues has motivated the development of tools for single-cell genomic, transcriptomic and multiplex proteomic analyses. Here, we review these tools and assess their advantages and limitations. Emerging applications of single cell analysis tools in drug discovery and development, particularly in the field of oncology, are discussed

Interleukin-15 augments oxidative metabolism and fungicidal activity of human monocytes against Paracoccidioides brasiliensis

Interleukin (IL)-15 is a pleiotropic cytokine that regulates the proliferation and survival of many cell types. IL-15 is produced by monocytes and macrophages against infectious agents and plays a pivotal role in innate and adaptive immune responses. This study analyzed the effect of IL-15 on fungicidal activity, oxidative metabolism and cytokine production by human monocytes challenged in vitro with Paracoccidioides brasiliensis (Pb18), the agent of paracoccidioidomycosis. Peripheral blood monocytes were pre-incubated with IL-15 and then challenged with Pb18. Fungicidal activity was assessed by viable fungi recovery from cultures after plating on brain-heart infusion-agar. Superoxide anion (O2-), hydrogen peroxide (H2O2), tumour necrosis factor-alpha (TNF-α), IL-6, IL-15 and IL-10 production by monocytes were also determined. IL-15 enhanced fungicidal activity against Pb18 in a dose-dependent pattern. This effect was abrogated by addition of anti-IL-15 monoclonal antibody. A significant stimulatory effect of IL-15 on O2- and H2O2 release suggests that fungicidal activity was dependent on the activation of oxidative metabolism. Pre-treatment of monocytes with IL-15 induced significantly higher levels of TNF-α, IL-10 and IL-15 production by cells challenged with the fungus. These results suggest a modulatory effect of IL-15 on pro and anti-inflammatory cytokine production, oxidative metabolism and fungicidal activity of monocytes during Pb18 infection

Proteins of Leishmania (Viannia) shawi confer protection associated with Th1 immune response and memory generation

<p>Abstract</p> <p>Background</p> <p><it>Leishmania (Viannia) shawi </it>parasite was first characterized in 1989. Recently the protective effects of soluble leishmanial antigen (SLA) from <it>L. (V.) shawi </it>promastigotes were demonstrated using BALB/c mice, the susceptibility model for this parasite. In order to identify protective fractions, SLA was fractionated by reverse phase HPLC and five antigenic fractions were obtained.</p> <p>Methods</p> <p>F1 fraction was purified from L. (V.) shawi parasite extract by reverse phase HPLC. BALB/c mice were immunized once a week for two consecutive weeks by subcutaneous routes in the rump, using 25 μg of F1. After 1 and 16 weeks of last immunization, groups were challenged in the footpad with L. (V.) shawi promastigotes. After 2 months, those same mice were sacrificed and parasite burden, cellular and humoral immune responses were evaluated.</p> <p>Results</p> <p>The F1 fraction induced a high degree of protection associated with an increase in IFN-γ, a decrease in IL-4, increased cell proliferation and activation of CD8⁺T lymphocytes. Long-term protection was acquired in F1-immunized mice, associated with increased CD4⁺central memory T lymphocytes and activation of both CD4⁺and CD8⁺T cells. In addition, F1-immunized groups showed an increase in IgG2a levels.</p> <p>Conclusions</p> <p>The inductor capability of antigens to generate memory lymphocytes that can proliferate and secrete beneficial cytokines upon infection could be an important factor in the development of vaccine candidates against American Tegumentary Leishmaniasis.</p

Production of TNF-α, nitric oxide and hydrogen peroxide by macrophages from mice with paracoccidioidomycosis that were fed a linseed oil-enriched diet

Omega-3 polyunsaturated fatty acids (n-3 PUFA) can modulate the immune system and their primary effect is on macrophage function. Paracoccidioidomycosis (PCM) is an endemic systemic mycosis in Latin America that is caused by the dimorphic fungus Paracoccidioides brasiliensis (Pb). Macrophages are the main defence against this pathogen and have microbicidal activity that is dependent on interferon-Γ and tumour necrosis factor (TNF)-α. These cytokines stimulate the synthesis of nitric oxide (NO) and hydrogen peroxide (H2O2), leading to the death of the fungus. To study the effect of n-3 PUFA on the host immune response during experimental PCM, macrophages that were obtained from animals infected with Pb18 and fed a diet enriched by linseed (LIN) oil were cultured and challenged with the fungus in vitro. The macrophage function was analysed based on the concentrations of TNF-α, NO and H2O2. LIN oil seems to influence the production of TNF-α during the development of disease. A diet enriched with LIN oil influences the microbicidal activity of the macrophages by inducing the production of cytokines and metabolites such as NO and H2O2, predominantly in the chronic phase of infection

Stimulation of bovine monocyte-derived macrophages with lipopolysaccharide, interferon-ɣ, Interleukin-4 or Interleukin-13 does not induce detectable changes in nitric oxide or arginase activity

Background: Bacterial lipopolysaccharide and interferon-γ stimulation of rodent macrophages in vitro induces up-regulation of inducible nitric oxide synthase, whereas interleukin-4 stimulation results in increased activity of arginase-1. Thus different stimulants result in differing macrophage phenotypes, appropriate for responses to a range of pathogens. The current study was conducted in order to determine whether bovine macrophages derived from monocytes and spleen respond similarly. Results: Lipopolysaccharide and interferon-γ did not induce detectable increases in nitric oxide production by bovine monocyte-derived or splenic macrophages in vitro. Similarly, interleukin-4 and interleukin-13 did not affect arginase activity. However, changes in transcription of genes coding for these products were detected. Conclusion: Differences between macrophage activation patterns exist between cattle and other species and these differences may occur during the post-transcription phase

Recent insights into targeting the IL-6 cytokine family in inflammatory diseases and cancer

The IL-6 family of cytokines consists of IL-6, IL-11, IL-27, IL-31, oncostatin M (OSM), leukaemia inhibitory factor (LIF), ciliary neurotrophic factor (CNTF), cardiotrophin 1 (CT-1) and cardiotrophin-like cytokine factor 1 (CLCF1). Membership of this cytokine family is defined by usage of common β-receptor signalling subunits, which activate various intracellular signalling pathways. Each IL-6 family member elicits responses essential to the physiological control of immune homeostasis, haematopoiesis, inflammation, development and metabolism. Accordingly, distortion of these cytokine activities often promotes chronic disease and cancer; the pathological importance of this is exemplified by the successful treatment of certain autoimmune conditions with drugs that target the IL-6 pathway. Here, we discuss the emerging roles for IL-6 family members in infection, chronic inflammation, autoimmunity and cancer and review therapeutic strategies designed to manipulate these cytokines in disease

Paracoccidioides brasiliensis Uses Endogenous and Exogenous Arachidonic Acid for PGE(x) Production

Paracoccidioides brasiliensis is the agent of paracoccidioidomycosis, the most prevalent deep mycosis in Latin America. Production of eicosanoids during fungal infections plays a critical role on fungal biology as well as on host immune response modulation. The purpose of our study was to assess whether P. brasiliensis strains with different degree of virulence (Pb18, Pb265, Bt79, Pb192) produce prostaglandin E-x (PGE(x)). Moreover, we asked if P. brasiliensis could use exogenous sources of arachidonic acid (AA), as well as metabolic pathways dependent on cyclooxygenase (COX) enzyme, as reported for mammalian cells. A possible association between this prostanoid and fungus viability was also assessed. Our results showed that all strains, independently of their virulence, produce high PGE(x) levels on 4 h culture that were reduced after 8 h. However, in both culture times, higher prostanoid levels were detected after supplementation of medium with exogenous AA. Treatment with indomethacin, a COX inhibitor, induced a reduction on PGEx, as well as in fungus viability. The data provide evidence that P. brasiliensis produces prostaglandin-like molecules by metabolizing either endogenous or exogenous AA. Moreover, the results suggest the involvement of these mediators on fungal viability

Identification of new pillared-layered carbon nitride materials at high pressure

The compression of the layered carbon nitride C6N9H3·HCl was studied experimentally and with density functional theory (DFT) methods. This material has a polytriazine imide structure with Cl(-) ions contained within C12N12 voids in the layers. The data indicate the onset of layer buckling accompanied by movement of the Cl(-) ions out of the planes beginning above 10-20 GPa followed by an abrupt change in the diffraction pattern and c axis spacing associated with formation of a new interlayer bonded phase. The transition pressure is calculated to be 47 GPa for the ideal structures. The new material has mixed sp(2)-sp(3) hybridization among the C and N atoms and it provides the first example of a pillared-layered carbon nitride material that combines the functional properties of the graphitic-like form with improved mechanical strength. Similar behavior is predicted to occur for Cl-free structures at lower pressures