Month: February 2020

Next, we sought to elucidate the mechanism by which TBPTALE in combination with VP64-TALEs robustly activated silent IL-2 gene expression in non-immune cells. We wanted to know if these artificially constructed TALE activators were able to activate the IL-2 gene expression by directly binding to the chromatin as reported by others or if chromatin remodeling was necessary. Within naı ¨ve T-cells, the IL-2 chromatin architecture is maintained in an inaccessible state, formed by nucleosome accumulation within the proximal promoter region, which masks TCR specific response elements. Consequently, nucleosome masking of these elements prevents TFs from binding to their cognate target sites and inhibits IL-2 gene transcription. However, upon T-cell stimulation, the IL-2 promoter is remodeled to an accessible state, which accommodates multiple TFs binding such as NFAT, AP-1, Oct-1 and NF-kB family members. Thus, chromatin remodeling accompanied by specific TFs is required to initiate IL-2 gene expression. Previous studies have shown that resting T-cells can position a distinct nucleosome between 60 to 200 bp upstream of the TSS and renders the IL-2 promoter inactive. During T-cell activation, this distinct nucleosome is subsequently displaced and accompanies increased DNase I hypersensitivity followed by ensuing IL-2 gene activation. To investigate the IL-2 promoter chromatin architecture in the presence of TBP-TALE and VP64-TALE activators in non-immune cells, we co-transfected 293FT cells with the most active combination of TALE activators or with empty vector control followed by CHART-PCR as previously described. CHART-PCR quantitatively measures the accessibility of a particular DNA region to DNase I cleavage as measured by real time qPCR. In concept, regions of open or relaxed chromatin DNA are more sensitive to DNase I cleavage, whereas regions of closed chromatin DNA are resistant to cleavage. Hence, we hypothesized that in the presence of TALE activators, the IL-2 promoter would be more sensitive to DNase I cleavage than that of empty vector control. As shown in Figure 7A, a series of primers were designed to probe the accessibility status of different loci within the IL-2 promoter region. The results from the CHART-PCR revealed that the combination of AD’CF TALEs but not empty vector control increased DNase I hypersensitivity across the IL-2 promoter with a significant increase observed in regions probed by primer sets 2 and 3. Interestingly, both primer sets amplified genomic regions either within the vicinity of a distinctly positioned nucleosome found in resting T-cells or within the TSS. Together, these data demonstrate that TBPTALE together with VP64-TALEs have either displaced or repositioned the nucleosome outside the detection range to allow for exclusive access of TALE activators to the IL-2 promoter regulatory regions, which controls gene activation.

We observed in our patients when they underwent HFR as compared to HD. Interestingly, similar values of percent decrease in plasma p-cresol concentration after HD were recently reported by Meert et al.. Therefore, our data suggest that HFR could perform better than HD in p-cresol removal though this conclusion remains to be confirmed in larger studies specifically designed to address this point. Finally, regarding classical hemodiafiltration, a previous work showed that total p-cresol concentration decreases by 40% after post-dilution and 42% after pre-dilution HDF but no comparative study with HFR has ever been performed. Although the HFR cartridge retained IL-6, we did not observe any significant change in serum concentrations of this cytokine when we compared blood samples collected before and following a single HFR session. This finding could be explained considering that the actual amount of IL-6 removed during a single HFR is presumably small. Based on concentration in UF and on the value of Quf in the HFR system, we estimated that less than 10% of circulating IL-6 could be removed by the cartridge. Remarkably, no additional IL-6 removal can occur in the diffusive stage of the HFR apparatus because free IL-6 cannot be dialyzed by the low flux membrane of its filter. It is likely that the small amount of IL-6 removed by HFR could be entirely replaced either by the new synthesis of this cytokine or by its release from tissues. It has been suggested, indeed, that in inflamed patients, circulating IL-6 remains high despite its very short plasma half-life because it is produced at a very high rate. Under this respect we should consider that all our patients showed a remarkable systemic inflammation as demonstrated by the high serum concentrations of hsCRP. It should be emphasized at this point, however, that such a marked systemic inflammation is not the most typical finding in ESRD patients that, instead, usually present only a moderate inflammatory state known as ‘‘microinflammation’’. We can speculate, thus, that HFR that was ineffective against the high IL-6 concentration of our markedly inflamed patients could perform better in the average ESRD patient with microinflammation. Our findings suggest that HFR does not differ from other dialysis systems that have all been shown ineffective in lowering the circulating levels of IL-6 and/or of other cytokines. Specifically, IL-6 levels did not change after a single HD session, as shown by Tarakc¸iog˘lu et al. and as also observed by us in the present paper. In addition, continuous hemofiltration was also ineffective in lowering circulating IL-6 levels in patients with systemic inflammatory response syndrome. After measuring the very small amounts of several cytokines such as TNFa and IL1 that are removed by low-volume HF, Van Bommel et al. estimated that a UF volumes of at least 50 L/day should be needed to clear the plasma from these molecules using hemofiltration.

In adults, cells within the organ of Corti normally secrete soluble factors to suppress remodeling of cochlear bone by osteoclasts and osteoblasts. The importance of this crosstalk is evident in bone syndromes where these pathways are disrupted. For example, otosclerosis and osteogenesis imperfecta tarda are characterized by sensorineural, conductive, and mixed forms of hearing loss. Patients with fibrous dysplasia of bone have lesions containing dense fibro-cellular infiltrate and increased trabecular bone formation. FD is caused by activating mutations in the Gs G-protein coupled receptor signaling pathway, which increases cyclic adenosine monophosphate levels. As many as 20% of FD patients have hearing loss. Disrupted bone remodeling leading to the overgrowth of temporal bones is thought to contribute to this progressive hearing loss in FD patients, which also can be either conductive, sensorineural, or both. However, the traditional methods of discriminating conductive vs. sensorineural hearing loss require intact bone physical properties to make that distinction. Thus our goal was to elucidate the mechanisms responsible for hearing loss in fibrous dysplasia. In this study, we used a transgenic mouse model of increased Gs-GPCR signaling to better define the mechanisms by which FD causes hearing loss. The complexity of the GNAS locus and embryonic lethality of constitutively-active Gsa signaling preclude direct genetic modifications to introduce the classical GNAS mutations that cause fibrous dysplasia. Since GPCRs signal through a limited number of canonical pathways including Gs and Gi that ultimately regulate intracellular cAMP levels, we developed a method of inducing regulated Gs signaling in cells by engineered receptors such as RASSLs. RASSLs are powerful tools for studying GPCR signaling as they no longer respond to endogenous hormones but can be activated by synthetic small-molecule ligands. In addition, RASSLs are small genes easily expressed in constructs and transgenes allowing precise spatial and temporal regulation. RASSLs have proven to be useful for dissecting GPCR signaling in complex tissues including bone, brain, and heart. In addition, we consistently observed MMP-13 staining in the pericellular matrix and intracellular space, in addition to the bone matrix staining that is typically observed. This altered MMP-13 localization in the severe FD mice may indicate that Rs1 expression is associated with a protein processing defect or change in the localization of bone matrix proteins, and may account for a decrease in bone matrix maturation we observed previously in long bones. Surprisingly, while the FD-like lesions were easily identified in the bony structures surrounding the cochlea, the most severe lesions largely spared the cochlea itself. We speculate that this protection may result from the unique regulation of bone remodeling in the cochlea which is very limited relative to bone remodeling.