Systemic administration of interferon-γ-expressing plasmid reduces late allergic bronchitis in a mouse model of asthma

Asthma might be caused by a helper T(Th)2 immune response. We hypothesized that the systemic administration of the Th1 cytokines may reduce the Th2 type late asthmatic response (LAR). We examined the effect of the intraperitoneal injection of interferon(IFN)-γ-expressing plasmid, a Th1 cytokine, or interleukin(IL)-4-expressing plasmid, a Th2 cytokine, at the time of sensitization on a mouse model of asthma induced by ovalbumin in BALB/c mice. We demonstrated that the IFN-γ-expressing plasmid reduced the LAR, whereas the IL-4-expressing plasmid enhanced the LAR as compared with the saline or plasmid-only treated group. The present study suggests that the systemic administration of IFN-γ-expressing plasmid may have a modulating ability of Th1/Th2 balance to down-regulate Th2 response by a mutual inhibitory mechanism between Th1 and Th2 cells, leading to the reduction of the LAR

Origin of Enriched Regulatory T Cells in Patients Receiving Combined Kidney-Bone Marrow Transplantation to Induce Transplantation Tolerance

We examined tolerance mechanisms in patients receiving HLA-mismatched combined kidney-bone marrow transplantation (CKBMT) that led to transient chimerism under a previously published nonmyeloablative conditioning regimen (Immune Tolerance Network study 036). Polychromatic flow cytometry and high-throughput sequencing of T cell receptor-β hypervariable regions of DNA from peripheral blood regulatory T cells (Tregs) and CD4 non-Tregs revealed marked early enrichment of Tregs (CD3(+) CD4(+) CD25(high) CD127(low) Foxp3(+) ) in blood that resulted from peripheral proliferation (Ki67(+) ), possibly new thymic emigration (CD31(+) ), and, in one tolerant subject, conversion from non-Tregs. Among recovering conventional T cells, central memory CD4(+) and CD8(+) cells predominated. A large proportion of the T cell clones detected in posttransplantation biopsy specimens by T cell receptor sequencing were detected in the peripheral blood and were not donor-reactive. Our results suggest that enrichment of Tregs by new thymic emigration and lymphopenia-driven peripheral proliferation in the early posttransplantation period may contribute to tolerance after CKBMT. Further, most conventional T cell clones detected in immunologically quiescent posttransplantation biopsy specimens appear to be circulating cells in the microvasculature rather than infiltrating T cells.status: publishe

C57BL/6 mice are more susceptible to antigen‐induced pulmonary eosinophilia than BALB/c mice, irrespective of systemic T helper 1/T helper 2 responses

Inflammatory response differences between C57BL/6 and BALB/c mice following ovalbumin (OVA) sensitization and a single challenge were investigated. Serum immunoglobulin (Ig)E and IgG1 levels were higher in C57BL/6 mice than in BALB/c mice. In contrast, IgG2a levels in C57BL/6 mice were lower than in BALB/c mice. Furthermore, the number of eosinophils infiltrating into lungs in C57BL/6 mice was significantly higher than in BALB/c mice after OVA challenge. The levels of the T helper 2 (Th2)‐type cytokines interleukin (IL)‐4 and IL‐5, generated in challenged C57BL/6 lung tissue, were also higher than in BALB/c lung tissue. The participation of IL‐4 and IL‐5 in the induction of eosinophil infiltration into the lungs was confirmed in both strains of mice by injection of anti‐IL‐4 and anti‐IL‐5 monoclonal antibodies (mAbs). However, following OVA stimulation, in vitro IL‐4 and IL‐5 production in splenocyte cultures from C57BL/6 mice was lower than in splenocyte cultures from BALB/c mice. These results indicate that C57BL/6 mice induce Th2‐type responses in the lungs, while BALB/c mice induce T helper 1 (Th1)‐type responses in the lungs, despite considerable production of IL‐4 and IL‐5 from splenocytes. Therefore, local immune responses are more important in the induction of allergic inflammation in the lungs and are different from systemic immune responses, which are thought to depend on genetic background

High-Level Production, Solubilization and Purification of Synthetic Human GPCR Chemokine Receptors CCR5, CCR3, CXCR4 and CX3CR1

Chemokine receptors belong to a class of integral membrane G-protein coupled receptors (GPCRs) and are responsible for transmitting signals from the extracellular environment. However, the structural changes in the receptor, connecting ligand binding to G-protein activation, remain elusive for most GPCRs due to the difficulty to produce them for structural and functional studies. We here report high-level production in E.coli of 4 human GPCRs, namely chemokine receptors (hCRs) CCR5, CCR3, CXCR4 and CX3CR1 that are directly involved in HIV-1 infection, asthma and cancer metastasis. The synthetic genes of CCR5, CCR3, CXCR4 and CX3CR1 were synthesized using a two-step assembly/amplification PCR method and inserted into two different kinds of expression systems. After systematic screening of growth conditions and host strains, TB medium was selected for expression of pEXP-hCRs. The low copy number pBAD-DEST49 plasmid, with a moderately strong promoter tightly regulated by L-arabinose, proved helpful for reducing toxicity of expressed membrane proteins. The synthetic Trx-hCR fusion genes in the pBAD-DEST49 vector were expressed at high levels in the Top10 strain. After a systematic screen of 96 detergents, the zwitterionic detergents of the Fos-choline series (FC9-FC16) emerged as the most effective for isolation of the hCRs. The FC14 was selected both for solubilization from bacterial lysates and for stabilization of the Trx-hCRs during purification. Thus, the FC-14 solubilized Trx-hCRs could be purified using size exclusion chromatography as monomers and dimers with the correct apparent MW and their alpha-helical content determined by circular dichroism. The identity of two of the expressed hCRs (CCR3 and CCR5) was confirmed using immunoblots using specific monoclonal antibodies. After optimization of expression systems and detergent-mediated purification procedures, we achieved large-scale, high-level production of 4 human GPCR chemokine receptor in a two-step purification, yielding milligram quantities of CCR5, CCR3, CXCR4 and CX3CR1 for biochemical, biophysical and structural analysis