Carbon Monoxide Modulation of Microglia-Neuron Communication: Anti-Neuroinflammatory and Neurotrophic Role

Background Microglia, the ‘resident immunocompetent cells’ of the central nervous system (CNS), are key players in innate immunity, synaptic refinement and homeostasis. Dysfunctional microglia contribute heavily to the creation of a toxic inflammatory milieu, a driving factor in the pathophysiology of several CNS disorders. Strategies for modulation of microglial function are required to tackle exacerbated tissue inflammation. Carbon monoxide (CO) is an endogenous gaseous molecule, produced by the degradation of haem, which presents several biological functions, namely anti-inflammatory, anti-apoptotic, pro-homeostatic and cytoprotective.Methods A novel molybdenum based CO-releasing molecule ALF826 was used for the assessment of neuron-microglia remote communication. Primary cultures of rat microglia and neurons, or BV-2 microglial mouse cell line and CAD neuronal mouse cell line were used to study microglia to neuron interaction. An approach based on microglial derived conditioned media in neuronal culture was applied.Results Medium derived from CO-treated microglia provided indirect neuroprotection against inflammation by limiting lipopolysaccharide (LPS)-induced expression of reactivity markers (CD11b), production of reactive oxygen species (ROS) and secretion of inflammatory factors (TNF-α, nitrites). This consequently prevented neuronal cell death and maintained neuronal morphology. In contrast, in the absence of inflammatory stimulus, conditioned media from CO-treated microglia improved neuronal morphological complexity, which is an indirect manner of assessing neuronal function. Likewise, microglial medium also prevented neuronal cell death induced by pro-oxidant tert-Butyl hydroperoxide (t-BHP). ALF826 treatment reinforced microglia secretion of Interleukin-10 (IL-10) and adenosine, mediators which may be involved in providing protection against t-BHP stress in this remote communication model. Chemical inhibition of adenosine receptors A2A and A1 reverted CO-derived neuroprotective effect, further highlighting a role for CO in regulating neuron-microglia communication via purinergic signalling.Conclusions Our findings indicate that CO has a modulatory role on microglia-to-neuron communication, promoting neuroprotection in a non-cell autonomous manner. In conclusion, CO-induced neuroprotection is afforded (i) by blocking exacerbated microglial inflammation and (ii) by the microglial release of neurotrophic factors. For the first time it is described CO improvement of microglial neurotrophism under non-inflammatory conditions.

The Central THαβ Immunity Associated Cytokine: IL-10 Has a Strong Anti-Tumor Ability Toward Established Cancer Models In Vivo and Toward Cancer Cells In Vitro

Immunotherapy is a promising new approach for cancer treatment. In this study, I propose to use the THαβ-mediated immune response for cancer treatment. The THαβ-mediated immune response is activated by IL-10 and IL-15. Thus, I used IL-10 and-15 as therapeutic agents in the 4T1 cell line, which is a mouse cell line of breast cancer, and the NXS2 cell line, which is a mouse cell line of neuroblastoma. Cells from 4T1 and NXS2 were subcutaneously inoculated in wild type BALB/c female mice and AJ mice, respectively, and administered cytokines or an antibody treatment at various dosages. My results showed that IL-10 and IL-15 administration led to reduction in tumor volume and increase in survival. However, traditional TH1 cytokine IFN-γ administration led to increase in tumor volume and decline in survival. Antibody treatment in conjunction with IL-10 was not significantly better than IL-10, due to the expression of GD2 on immune cells. Moreover, an anti-GD2 antibody inhibited the immune cells themselves. Additionally, I found that IL-10 was directly toxic to tumor cells in vitro. Thus, I conclude that the THαβ immunological pathway is a good treatment strategy for cancer.

The tetraspanin Tspan15 is an essential subunit of an ADAM10 scissor complex

A disintegrin and metalloprotease 10 (ADAM10) is a transmembrane protein essential for embryonic development, and its dysregulation underlies disorders such as cancer, Alzheimer's disease, and inflammation. ADAM10 is a “molecular scissor” that proteolytically cleaves the extracellular region from >100 substrates, including Notch, amyloid precursor protein, cadherins, growth factors, and chemokines. ADAM10 has been recently proposed to function as six distinct scissors with different substrates, depending on its association with one of six regulatory tetraspanins, termed TspanC8s. However, it remains unclear to what degree ADAM10 function critically depends on a TspanC8 partner, and a lack of monoclonal antibodies specific for most TspanC8s has hindered investigation of this question. To address this knowledge gap, here we designed an immunogen to generate the first monoclonal antibodies targeting Tspan15, a model TspanC8. The immunogen was created in an ADAM10-knockout mouse cell line stably overexpressing human Tspan15, because we hypothesized that expression in this cell line would expose epitopes that are normally blocked by ADAM10. Following immunization of mice, this immunogen strategy generated four Tspan15 antibodies. Using these antibodies, we show that endogenous Tspan15 and ADAM10 co-localize on the cell surface, that ADAM10 is the principal Tspan15-interacting protein, that endogenous Tspan15 expression requires ADAM10 in cell lines and primary cells, and that a synthetic ADAM10/Tspan15 fusion protein is a functional scissor. Furthermore, two of the four antibodies impaired ADAM10/Tspan15 activity. These findings suggest that Tspan15 directly interacts with ADAM10 in a functional scissor complex.

Cytotoxic effects of Ammi visnaga volatile oil on some cancer cell lines

Ammi visnaga a herbal plant is rich with important active constituents that make the plant to be described in traditional medicine and among them is the volatile oil. The oil extracted by distillation and then study it's cytotoxic effect on two cell line: The human pelvic rhanbdomyosarcoma (RD) and The mouse cell line( L20B) which expresses the genes for human cellular receptor for Polio viruses. Results showed that there were potent toxic effects on both cell lines RD&L20B specially at the concentration (100,50 and 25)µl/ml of the essential oil ,then decreased as the oil concentrations decreased.