Increased cell proliferation in spleen and lymph nodes peripheral to contact allergen application site
ABSTRACT The local lymph node assay (LLNA) is widely used to identify chemicals that are contact sensitizers. The assay involves dosing mice with the chemical on both ears and pooling the superficial parotid lymph nodes for assessment of lymphocyte proliferation as a marker of sensitization. The present study explored potential reduction in animal usage by dosing one ear with the allergen and the other with vehicle-only. The respective draining lymph nodes were processed separately for tritiated thymidine ((3)H-TdR) incorporation. Cell proliferation in proper axillary and renal nodes, as well as in the spleen was also assessed. Cross-contamination of the chemicals from the dosed ears to other parts of the body via preening was prevented by dosing restrained animals and washing off the residual chemical with saline after 4h. Dosing the left ear with 0.02% oxazolone (OX) on unrestrained animals resulted in marked cell proliferation in its draining lymph node (stimulation index, SI=12.8) and in the lymph node draining the contra-lateral vehicle-dosed ear (SI=6), as well as the proper axillary lymph nodes (SI=3.3). Increased (3)H-TdR incorporation was not observed in the renal lymph nodes (SI=1.1). Similar stimulation of cells was observed in the lymph node draining the ear contra-lateral to the 30% hexylcinnamaldehyde (HCA)-dosed ear. Increased proliferative activity was observed in contra-lateral draining lymph nodes of restrained mice demonstrating that these results cannot be attributed to cross-contamination of adjacent skin. A significant increase in proliferation of splenocytes was also observed. It is concluded that dermal application of a contact allergen, as exemplified by OX and HCA, may induce cell proliferation in the neighboring lymph nodes and spleen indicative of hapten and/or haptenated proteins diffusing through the skin to peripheral nodes and the blood to produce systemic sensitization. It is also possible that lymphatic capillaries may communicate between the left and right side of the mouse head. Thus the contra-lateral draining superficial parotid node cannot be used as a control for application of contact allergen to a single ear in a modified LLNA.
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ABSTRACT: Thymosin-alpha1 (Talpha1) is indicated for the treatment of certain viral infections, including hepatitis B and C, and cancers, such as melanoma. In this paper, the fusion genes encoding human serum albumin (HSA) and Talpha1 with (rHSA-L-Talpha1) and without a linker peptide (rHSA-Talpha1) were constructed and overexpressed in P. pastoris. Through the process of ion interaction chromatography (Q-Sepharose F.F), hydrophobic interaction chromatography (Phenyl Sepharose HP) and affinity chromatography (Blue Sepharose F.F), the purity of fusion proteins was greater than 97%. In contrast to the reactivity of normal spleen cells to Con A, the data of in vitro murine spleen lymphocytes proliferation experiment suggested that spleen cells achieved a higher degree of T cell maturation after rHSA-L-Talpha1, rHSA-Talpha1 and Talpha1 treatments, respectively. Moreover, rHSA-L-Talpha1, rHSA-Talpha1 and Talpha1 can also antagonize dexamethasone-induced apoptosis of thymocyte sub-populations. In hydrocortisone-induced immunosuppression mice (in vivo experiments), after subcutaneous injections with two fusion proteins and Talpha1 for seven consecutive days, the net increment of body weight, the spleen index and the thymus index were significantly improved. Simultaneously, the increase in SOD level and the decrease in MDA level in plasma were observed. The pharmacokinetic data of rHSA-L-Talpha1 and rHSA-Talpha1 administered in rats showed an improved pharmacokinetic profile with a conspicuous prolonged half life. The analysis of bioactivity and pharmacokinetics suggested that fusion proteins rHSA-L-Talpha1 and rHSA-Talpha1 were new drug candidates.Cancer Immunology and Immunotherapy 09/2010; 59(9):1335-45. DOI:10.1007/s00262-010-0862-9 · 3.94 Impact Factor
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ABSTRACT: BACKGROUND AND PURPOSE: The intermediate-conductance Ca(2+) -activated K(+) channel KCa 3.1 modulates the Ca(2+) response through the control of the membrane potential in the immune system. We investigated the role of KCa 3.1 on the pathogenesis of delayed-type hypersensitivity (DTH) in auricular lymph node (ALN) CD4(+) T-lymphocytes of oxazolone (Ox)-induced DTH model mice. EXPERIMENTAL APPROACH: The expression patterns of KCa 3.1 and its possible transcriptional regulators were compared among ALN T-lymphocytes of three groups [non-sensitized (Ox-/-), Ox-sensitized but non-challenged (Ox+/-), and Ox-sensitized and -challenged (Ox+/+)] using real-time PCR, Western blotting and flow cytometry. KCa 3.1 activity was measured by whole-cell patch clamp and the voltage-sensitive dye imaging. The effects of KCa 3.1 blockade were examined by the administration of selective KCa 3.1 blockers. KEY RESULTS: Significant up-regulation of KCa 3.1a was observed in CD4(+) T-lymphocytes of Ox+/- and Ox+/+, without any evident changes in the expression of the dominant-negative form, KCa 3.1b. Negatively correlated with this, the repressor element-1 silencing transcription factor, REST was significantly down-regulated. Pharmacological blockade of KCa 3.1 resulted in an accumulation of the CD4(+) T-lymphocytes of Ox+/+ at the G0 /G1 phase of the cell cycle, and also significantly recovered not only the pathogenesis of DTH, but also the changes in the KCa 3.1 expression and activity in the CD4(+) T-lymphocytes of Ox+/- and Ox+/+. CONCLUSIONS AND IMPLICATIONS: The up-regulation of KCa 3.1a in conjunction with the down-regulation of REST may be involved in CD4(+) T-lymphocyte proliferation in the ALNs of DTH model mice, and KCa 3.1 may be an important target for therapeutic intervention in allergy diseases such as DTH.British Journal of Pharmacology 04/2013; 169(5). DOI:10.1111/bph.12215 · 4.99 Impact Factor