The cellular mechanism of corticosteroid resistance in asthma

Chronic asthmatics norrally respond to oral corticosteroid therapy with an increase in their ventilatory function. However, Grant and his co- workers identified a small proportion of severe chronic asthmatics who did not improve their ventilatory function when given oral corticosteroid. These patients were termed "corticosteroid resistant ". This thesis describes the in vitro and in vivo effects of the corticosteroid, Ilethylprednisolone (INS), on monocytes and Tcells from Corticosteroid Sensitive (CS) and Corticosteroid Resistant (CR) asthmatics.In vitro colony formation by mononuclear cells (MNC) from CS asthmatics was found to be significantly more inhibited by 10⁻⁸ N MPS than was colony formation by MNC from CR asthmatics. This in vitro assay of corticosteroid sensitivity was subsequently used diagnostically to discriuina_te between CS and CR asthmatics. It was demonstrated that the unresponsiveness of MNC from CR asthmatics to MPS in vitro was attributable to the monocytes from these patients. Defects in the corticosteroid sensitivity of T-cells, eosinophils and polymorphs were also observed in vivo and in vitro; their relationship to the monocyte defect was not clarified.Monocytes are thought to affect the Type 1 hypersensitivity reaction seen in asthma by the production of many factors. Two of these factors are of particular relevance to this thesis: lipomodulin, which inhibits prostanoid synthesis and is induced by corticosteroid and Interleukin 1, which supports T- lymphocyte proliferation and is inhibited by corticosteroid. There is therefore reason to believe that the defect in corticosteroid responsiveness demonstrated in monocytes from CR asthmatics in vitro could reduce the therapeutic efficacy of these drugs in patients with this condition.If this is so, these findings emphasise the importance of the actions of corticosteroid on monocyte function in the commoner CS asthma. Further investigation of this phenomenon may have wider implications for other developmental and pathological processes

La doctrina eckhartiana del desasimiento en San Lucas 10 (38-42)

The Gospel of Saint Luke reveals Jesus' visit Martha and Mary ‘s home. In his sermon number 86 Eckhart vindicates Marta over her sister Mary, rebelling himself against the traditional claim of contemplation over action. Eckhart says that Marta’s dignified life is the life where our souls are one with God in via. In the first part I will explain the Eckhartian detachment doctrine. In a second part I will expose Marta's vindication in the Eckhartian reading of the Gospel and show that it is possible because she lives according to the detachment doctrine.El evangelio de San Lucas revela la visita de Jesús al hogar de Marta y María. Eckhart en su sermón 86 reivindica a Marta sobre su hermana María rebelándose así a la tradicional reivindicación de la contemplación por sobre la acción. Responderá Eckhart al reivindicar a Marta que esa vida digna es la vida donde nuestras almas son unas con Dios in vía. En una primera parte expondré la doctrina eckhartiana del desasimiento. En una segunda parte expondré la reivindicación de Marta en la lectura eckhartiana del evangelio y finalmente mostraré que se debe a que ella logra el ideal de unión mística que propone la doctrina eckhartiana del desasimiento

Chemical genetic analyses of quantitative changes in Cdk1 activity during the human cell cycle

Cyclin-dependent kinase 1 (Cdk1) controls cell proliferation and is inhibited by promising anticancer agents, but its mode of action and the consequences of its inhibition are incompletely understood. Cdk1 promotes S- and M-phases during the cell-cycle but also suppresses endoreduplication, which is associated with polyploidy and genome instability. The complexity of Cdk1 regulation has made it difficult to determine whether these different roles require different thresholds of kinase activity and whether the surge of activity as inhibitory phosphates are removed at mitotic onset is essential for cell proliferation. Here, we have used chemical genetics in a human cell line to address these issues. We rescued cells lethally depleted of endogenous Cdk1 with an exogenous Cdk1 conferring sensitivity to one ATP analogue inhibitor (1NMPP1) and resistance to another (RO3306). At no 1NMPP1 concentration was mitosis in rescued clones prevented without also inducing endoreduplication, suggesting that these two key roles for Cdk1 are not simply controlled by different Cdk1 activity thresholds. We also rescued RO3306-resistant clones using exogenous Cdk1 without inhibitory phosphorylation sites, indicating that the mitotic surge of Cdk1 activity is dispensable for cell proliferation. These results suggest that the basic mammalian cycle requires at least some qualitative changes in Cdk1 activity and that quantitative increases in activity need not be rapid. Furthermore, the viability of cells that are unable to undergo rapid Cdk1 activation, and the strong association between endoreduplication and impaired proliferation, may place restrictions on the therapeutic use of a Cdk1 inhibitors

Comparative studies of the preparation of immunoliposomes with the use of two bifunctional coupling agents and investigation of in vitro immunoliposome-target cell binding by cytofluorometry and electron microscopy

The two coupling agents SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate) and SATA (N-succinimidyl-S-acetylthioacetate) were compared in their efficiency and feasibility to couple monoclonal antibodies (Abs) via thioether linkage to liposomes functionalized by various lipophilic maleimide compounds like Full-size image methyl ester (MP-PL), N-(3-maleimidopropionyl)phosphatidylethanolamide (MP-PE), Full-size image methyl ester (EMC-PL), and N-(6-maleimidocaproyl)phosphatidylethanolamine (EMC-PE). The composition of the liposomes was soy phosphatidylcholine (SPC), cholesterol, maleimide compounds and -tocopherol (1:0.2:0.02:0.01, mol parts), plus N4-oleylcytosine arabinoside (NOAC) as cytostatic prodrug (0.2 mol parts) and a new, lipophilic and highly fluorescent dye N,N′-bis(1-hexylheptyl)-3,4:9,10-perylenebis(dicarboximide) (BHPD, 0.006 mol parts). From the maleimide derivatives MP-PL was the most effective in terms of preservation of the coupling activity in dependence of liposome storage. The coupling of the monoclonal A B8-24.3 (mouse IgG2b, MHC class I, anti H-2kb) and IB16-6 (rat IgG2a, anti B16 mouse melanoma) to the drug carrying liposomes was more effective and easier to accomplish with SATA as compared to SPDP. Coupling rates of 60–65% were obtained with SATA at molar ratios of 12 SATA:1 Ab:40 maleimide spacer groups on the surface of one liposome. The highest coupling rates with SPDP were obtained at the ratio of 24 SPDP:1 Ab:40 liposomal maleimide groups, with an Ab binding efficiency of only 20–25%. The optimal in vitro binding conditions to specific target cells (EL4 for B8-24.3-liposomes and B16-F10 for IB16-6-liposomes) were determined by cytofluorometric measurement of the liposomal BHPD fluorescence with SATA linked Abs. Optimal immunoliposome binding to specific epitopes on the target cells was achieved with 1–2 Ab molecules coupled to one liposome, with immunoliposome concentrations of 20–130 nM and with a small incubation volume of 0.3–0.4 ml. The specificity of the binding of B8-24.3-liposomes to EL4 target cells was visualized by scanning electron microscopy. Antibody mediated endocytic uptake of immunoliposomes could be demonstrated by transmission electron microscopy

Leveraging the tolerogenic potential of TNF-α and regulatory B cells in organ transplantation

A subset of B-cells with tolerogenic functions, termed B-regulatory cells or Bregs, is characterized by the expression of anti-inflammatory/tolerogenic cytokines, namely IL-10, TGF-β, and IL-35, that contribute to their regulatory functions. Breg regulation favors graft acceptance within a tolerogenic milieu. As organ transplantation invariably triggers inflammation, new insights into the crosstalk between cytokines with dual properties and the inflamed milieu are needed to tailor their function toward tolerance. Using TNF-α as a proxy of dual-function cytokines involved in immune-related diseases and transplantation settings, the current review highlights the multifaceted role of TNF-α. It focuses on therapeutic approaches that have revealed the complexity of TNF-α properties tested in clinical settings where total TNF-α inhibition has proven ineffective and often detrimental to clinical outcomes. To improve the efficacy of current TNF-α inhibiting therapeutics, we propose a three-prong strategy to upregulate the tolerogenic pathway engaging the TNFR2 receptor while simultaneously inhibiting the inflammatory mechanisms associated with TNFR1 engagement. When combined with additional administrations of Bregs-TLR that activate Tregs, this approach may become a potential therapeutic in overcoming transplant rejection and promoting graft tolerance

Immunomodulatory effect of canine periodontal ligament stem cells on allogenic and xenogenic peripheral blood mononuclear cells

Purpose: The aim of this study was to investigate the immunomodulatory effects of canine periodontal ligament stem cells on allogenic and xenogenic immune cells in vitro. Methods: Mixed cell cultures consisting of canine stem cells (periodontal ligament stem cells and bone marrow stem cells) and allogenic canine/xenogenic human peripheral blood mononuclear cells (PBMCs) were established following the addition of phytohemagglutinin. The proliferation of PBMCs was evaluated using the MTS assay. The cell division of PBMCs was analyzed using the CFSE assay. The apoptosis of PBMCs was assessed using the trypan blue uptake method. Results: Periodontal ligament stem cells and bone marrow stem cells inhibited the proliferation of allogenic and xenogenic PBMCs. Both periodontal ligament stem cells and bone marrow stem cells suppressed the cell division of PBMCs despite the existence of a mitogen. No significant differences in the percentages of apoptotic PBMCs were found among the groups. Conclusions: Canine periodontal ligament stem cells have an immunomodulatory effect on allogenic and xenogenic PBMCs. This effect is not a product of apoptosis of PBMCs but is caused by the inhibition of cell division of PBMCs.