Viral and bacterial pathogens cause inflammation via Toll-like receptor (TLR) signaling. We have shown that effective responses to LPS may depend on cooperative interactions between TLR-expressing leukocytes and TLR-negative tissue cells. The aim of this work was to determine the roles of such networks in response to agonists of TLRs associated with antiviral and autoimmune responses. The TLR3 agonist poly(I:C) activated epithelial cells, primary endothelial cells, and two types of primary human smooth muscle cells (airway [ASMC] and vascular) directly, while the TLR7/8 agonist R848 required the presence of leukocytes to activate ASMC. In keeping with these data, ASMC expressed TLR3 but not TLR7 or TLR8. Activation of ASMC by poly(I:C) induced a specific cytokine repertoire characterized by induction of CXCL10 generation and the potential to recruit mast cells. We subsequently explored the ability of TLR agonists to cooperate in the induction of inflammation. Dual stimulation with LPS and poly(I:C) caused enhanced cytokine generation from epithelial and smooth muscle cells when in the presence of leukocytes. Thus, inflammatory responses to pathogens are regulated by networks in which patterns of TLR expression and colocalization of tissue cells and leukocytes are critical.

Inappropriate plateletactivation is afeatureofacute and chronic diseasess uch as disseminated intravascular coagulation (DIC) and atherosclerosis. Since proinflammatorym icrobial-derived agonists can be involved in the pathogenesiso ft hese diseases, we examined the potential role ofTLR4 (mediating responsesto LPS) andTLR2(which responds to bacteriallipopeptides)inplateleta ctivation. Our datas uggestedl ow-level expression of TLR2 andTLR4 on platelets, determinedbyflowcytometry,and we also observed expression of TLR4 on amegakaryocytic cell line by bothflowcytometry and immunohistochemistry. Stimulation of the platelets with the TLR4 agonist LPS, and thes ynthetic TLR2 agonist Pam 3 CSK 4 ,r esulted in no plateleta ggregation,noincrease in CD62P surfaceexpression andnoincrease Keywords Platelets, TLR, LPS, atherosclerosis in thec ytosolic concentration of Ca 2+ .The TLRa gonists were also unablet od irectlya ctivate platelets primedw ith epinephrine,orpretreatedwith alow concentration ofADP or PAF. Pretreatment of platelets with LPSo rP am 3 CSK 4 also failedt o modulate thep lateletr esponset os ubmaximal concentrations of the classical plateletagonists ADP and PAF. We conclude that theTLRagonists LPS andPam 3 CSK 4 have no direct effect on plateleta ctivation and that plateletTLRsm ay be ar emnant from megakaryocytes.TLR2 and TLR4 agonists arethought to have a significant role in diseasessuch as atherosclerosis and DIC, but our research suggests that this is through am echanism other than directplateletactivation or by modification of plateletresponsestootheragonists.

Lipopolysaccharide (LPS) has long been known to enhance innate and adaptive immune responses; however, its extreme toxicity precludes its use in clinical settings. The combined toxicity and adjuvanticity of LPS have contributed to the view that immunological adjuvants need to be highly inflammatory to be maximally effective. Here, we compared the effects of LPS with its less-toxic derivatives, monophosphoryl lipid A (MPL) and a chemical mimetic, RC529, on CD4+ T cell clonal expansion, long-term survival, and T helper cell type 1 (Th1) differentiation. We found that LPS, MPL, and RC529 had similar effects on CD4+ T cell clonal expansion, cell division, and ex vivo survival. Analysis of the ability of activated CD4+ T cells to produce interferon-gamma following a 21-day immunization and challenge protocol with LPS and MPL resulted in similar Th1 differentiation. In contrast, we found that LPS was more effective in promoting long-term CD4+ T cell responses, as we recovered nearly sixfold more cells following immunization/challenge as compared with treatment with MPL. Our results indicate that low-inflammation adjuvants, such as MPL and RC529, are capable of enhancing short-term CD4+ T cell clonal expansion and Th1 differentiation, but inflammatory signaling aids in the long-term retention of antigen-specific T cells.

The component failures and self-reported accidents (some potentially dangerous) have implications for the NHS, manufacturers, prescribers and users. EPIOCs enhance the independence of severely physically disabled individuals in the community and assist carers by lessening dependence and enhancing social interaction. This study demonstrated the need for follow-up of EPIOC users and chairs.