The anti-inflammatory effects of IL-1 inhibition are well established but most studies have focused on the endogenous IL-1 receptor antagonist (IL-1Ra) protein. However, IL1ra has a very short plasma half-life and hundred-fold excess over IL-1 is required to obtain a 50% inhibition of IL-1 responses. Another naturally occurring inhibitor, soluble IL-1 receptor type II (sIL-1R2) may offer a higher IL-1 neutralising activity and several studies have emphasised its potential function as a regulatory "decoy" for IL1b.
Objectives
To construct soluble IL-1 receptor type II chimeric protein with an IgG heavy chain and to evaluate its therapeutic potential in a murine arthritis model using a gene therapy approach.
Methods
The murine IL-1R2 extracellular domain was cloned using a RT-PCR protocol from total cytoplasmic RNA, sequenced and a chimeric expressing gene constructed by the addition of a XbaI fragment encoding the constant and hinge regions of a murine IgG1 heavy chain. The IL-1R2IgG coding insert was subcloned into an E1-deleted adenoviral shuttle expression plasmid under the control of a CMV promoter and the resulting plasmid used to construct a standard first generation E1-deleted recombinant adenovirus (Ad-sIL1R2 Ig) using standard homologous recombination techniques in 293 cells. Stock viral preparations were produced in 293 cells and purified by ultracentrifugation in two CsCl2 gradients. Viral titer (pfu/ml) was determined by plaque-assay. An EGFP (enhanced green fluorescent protein) expressing recombinant adenovirus (Ad-EGFP) was prepared using a similar protocol. Expression was confirmed by Western blotting using transient expression on HeLa cells under serum free-conditions. The therapeutic effects of this approach was tested in the acute murine antigen-induced arthritis model (AIA).
Results
Western blotting under reducing and nonreducing conditions confirmed expression and dimerization of the chimeric protein. More importantly, adenovirus-mediated systemic expression of sIL1R2 Ig suppressed acute inflammation in vivo in murine AIA. Mice were injected into the tail vein with 109 pfu Ad-sIL1R2Ig or Ad-EGFP three days before induction of arthritis by intraarticular injection of 100μg mBSA into the right knee. The left knee was injected with PBS as a control. Joint inflammation determined by 99technetium pertechnetate uptake69 was statistically significantly decreased at day 3 and 7 post-injection in animal treated with Ad-sIL1R2 Ig as compared to Ad-EGFP (10 animals per group, 2 experiments) but proteoglycan depletion of cartilage did not appear to be diminished.
Conclusion
Our study demonstrated the value of a soluble IL1R2Ig chimeric protein has a IL1 inhibitor in vivo. It also demonstrated adenovirus-mediated gene expression allow for sufficient level of expression. But our preliminary data also raise new questions of the respective values and roles of IL1 or TNFa inhibition regarding acute inflammation and cartilage degradation.
We developed a new phage-display based approach, the Large Fragment Phage Display (LFPD), that can be used for mapping conformational epitopes on target molecules of immunological interest. LFPD uses a simplified and more effective phage-display approach in which only a limited set of larger fragments (about 100 aa in length) are expressed on the phage surface. Using the human HER2 oncoprotein as a target, we identified novel B-cell conformational epitopes. The same homologous epitopes were also detected in rat HER2 and all corresponded to the epitopes predicted by computational analysis (PEPITO software), showing that LFPD gives reproducible and accurate results. Interestingly, these newly identified HER2 epitopes seem to be crucial for an effective immune response against HER2-overexpressing breast cancers and might help discriminating between metastatic breast cancer and early breast cancer patients. Overall, the results obtained in this study demonstrated the utility of LFPD and its potential application to the detection of conformational epitopes on many other molecules of interest, as well as, the development of new and potentially more effective B-cell conformational epitopes based vaccines.
Abstract Because IL-1β plays an important role in inflammation in human and murine arthritis, we investigated the contribution of the inflammasome components ASC, NALP-3, IPAF, and caspase-1 to inflammatory arthritis. We first studied the phenotype of ASC-deficient and wild-type mice during Ag-induced arthritis (AIA). ASC−/− mice showed reduced severity of AIA, decreased levels of synovial IL-1β, and diminished serum amyloid A levels. In contrast, mice deficient in NALP-3, IPAF, or caspase-1 did not show any alteration of joint inflammation, thus indicating that ASC associated effects on AIA are independent of the classical NALP-3 or IPAF inflammasomes. Because ASC is a ubiquitous cytoplasmic protein that has been implicated in multiple cellular processes, we explored other pathways through which ASC may modulate inflammation. Ag-specific proliferation of lymph node and spleen cells from ASC-deficient mice was significantly decreased in vitro, as was the production of IFN-γ, whereas IL-10 production was enhanced. TCR ligation by anti-CD3 Abs in the presence or absence of anti-CD28 Abs induced a reduction in T cell proliferation in ASC−/− T cells compared with wild-type ones. In vivo lymph node cell proliferation was also significantly decreased in ASC−/− mice, but no effects on apoptosis were observed either in vitro or in vivo in these mice. In conclusion, these results strongly suggest that ASC modulates joint inflammation in AIA through its effects on cell-mediated immune responses but not via its implication in inflammasome formation.
Gonadotropin hormones undergo important dynamic changes during life. Their rise during puberty stimulates gonadal steroid secretion, triggering the development of secondary sexual characteristics and the acquisition of fertility. The full spectrum of possible mutations and polymorphisms in the human gonadotropins and in their receptor genes has been described in recent years. Patients harboring these mutations display a very wide range of phenotypes affecting all aspects of the reproductive axis. An important insight provided by the careful study of these patients lies in the striking gender differences in the phenotypes associated with a given mutation. As a result, the careful study of these rare patients has allowed us to better define the respective roles of luteinizing hormone and follicle-stimulating hormone in normal human pubertal development and in the achievement of full fertility potential in either males or females. In this work, we describe briefly the known mutations in the genes for both gonadotropins and their receptors, and discuss their genotype/phenotype correlations in light of these important gender differences.
C1q deficiency strongly predisposes to the development of systemic lupus erythematosus in humans and mice. We used the model of accelerated nephrotoxic nephritis in C1q-deficient mice to explore the mechanisms behind these associations. C1q-deficient mice developed severe glomerular thrombosis within 4 days of induction of disease, whereas wild-type mice developed mild injury. These findings suggest that C1q protects from immune-mediated glomerular injury. This exacerbated thrombosis was also seen in mice triply deficient in C1q, factor B, and C2, excluding a major pathogenic role for the alternative pathway of complement in this phenomenon. However, these mice did not develop elevated creatinine levels. No exacerbation of accelerated nephrotoxic nephritis was observed in mice doubly deficient in factor B and C2, suggesting a protective role for C1q against renal inflammation that is proximal to C2 activation. There were increased murine IgG deposits, neutrophil numbers, and apoptotic cells in the glomeruli of C1q-deficient mice compared with wild-type mice. Renal expression of genes encoding procoagulant proteins was also enhanced in C1q-deficient mice. The increased IgG deposits and apoptotic cells in the glomeruli of C1q-deficient mice suggest that the exacerbation of disease may be due to a defect in the clearance of immune complexes and/or apoptotic cells from their kidneys.
