Examining cellular responses to reconstituted antibody protein liquids.

Protein ionic liquids (PIL) are a new class of biologic stabilizers designed to protect the functionality and extend the shelf-life of biotechnological and therapeutic agents making them more readily available, and resistant to austere environments. Protein biorecognition elements such as monoclonal antibodies are commonly utilized therapeutics that require the robust stabilization offered by PILs, but biocompatibility remains an important issue. This study has focused on characterizing the biocompatibility of an antibody based PIL by exposing multiple cells types to a cationized immunoglobulin suspended in an anionic liquid (IgG-IL). The IgG-IL caused no significant alterations in cellular health for all three cell types with treatments < 12.5 µg/mL. Concentrations ≥ 12.5 µg/mL resulted in significant necrotic cell death in A549 and HaCaT cells, and caspase associated cell death in HepG2 cells. In addition, all cells displayed evidence of oxidative stress and IL-8 induction in response to IgG-IL exposures. Therapeutic Ig can be utilized with a wide dose range that extends into concentrations we have found to exhibit cytotoxicity raising a toxicity concern and a need for more extensive understanding of the biocompatibility of IgG-ILs.

Replication stress at microsatellites causes DNA double-strand breaks and break-induced replication.

Short tandemly repeated DNA sequences, termed microsatellites, are abundant in the human genome. These microsatellites exhibit length instability and susceptibility to DNA double-strand breaks (DSBs) due to their tendency to form stable non-B DNA structures. Replication-dependent microsatellite DSBs are linked to genome instability signatures in human developmental diseases and cancers. To probe the causes and consequences of microsatellite DSBs, we designed a dual-fluorescence reporter system to detect DSBs at expanded (CTG/CAG) n and polypurine/polypyrimidine (Pu/Py) mirror repeat structures alongside the c-myc replication origin integrated at a single ectopic chromosomal site. Restriction cleavage near the (CTG/CAG)100 microsatellite leads to homology-directed single-strand annealing between flanking AluY elements and reporter gene deletion that can be detected by flow cytometry. However, in the absence of restriction cleavage, endogenous and exogenous replication stressors induce DSBs at the (CTG/CAG)100 and Pu/Py microsatellites. DSBs map to a narrow region at the downstream edge of the (CTG)100 lagging-strand template. (CTG/CAG) n chromosome fragility is repeat length-dependent, whereas instability at the (Pu/Py) microsatellites depends on replication polarity. Strikingly, restriction-generated DSBs and replication-dependent DSBs are not repaired by the same mechanism. Knockdown of DNA damage response proteins increases (Rad18, polymerase (Pol) η, Pol κ) or decreases (Mus81) the sensitivity of the (CTG/CAG)100 microsatellites to replication stress. Replication stress and DSBs at the ectopic (CTG/CAG)100 microsatellite lead to break-induced replication and high-frequency mutagenesis at a flanking thymidine kinase gene. Our results show that non-B structure-prone microsatellites are susceptible to replication-dependent DSBs that cause genome instability.

Hydrogen peroxide modulates immunoglobulin expression by targeting the 3'Igh regulatory region through an NFκB-dependent mechanism.

Reactive oxygen species such as hydrogen peroxide (H(2)O(2)) appear to play a role in signal transduction in immune cells and have been shown to be synthesized upon antigen-mediated activation and to facilitate cellular activation in B- and T-cells. However, an effect of H(2)O(2) on B-cell function (i.e. immunoglobulin (Ig) expression) has been less well-characterized. The effects of H(2)O(2) exposure on lymphocytes may be partly mediated by oxidative modulation of the NFκB signal transduction pathway, which also plays a role in Ig heavy chain (Igh) gene expression. Igh transcription in B lymphocytes is an essential step in antibody production and is governed through a complex interaction of several regulatory elements, including the 3'Igh regulatory region (3'IghRR). Utilizing an in vitro mouse B-cell line model, this study demonstrates that exposure to low µM concentrations of H(2)O(2) can enhance 3'IghRR-regulated transcriptional activity and Igh gene expression, while either higher concentrations of H(2)O(2) or the expression of a degradation resistant inhibitory κB (IκBα super-repressor) can abrogate this effect. Furthermore, suppressive H(2)O(2) concentrations increased protein levels of the p50 NFκB sub-unit, IκBα, and an IκBα immunoreactive band which was previously characterized as an IκBα cleavage product exhibiting stronger inhibitory function than native IκBα. Taken together, these observations suggest that exposure of B lymphocytes to H(2)O(2) can alter Igh transcriptional activity and Ig expression in a complex biphasic manner which appears to be mediated by NFκB and altered 3'IghRR activity. These results may have significant implications to disease states previously associated with the 3'IghRR.

Diverse chemicals including aryl hydrocarbon receptor ligands modulate transcriptional activity of the 3'immunoglobulin heavy chain regulatory region.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), a known disruptor of B-cell differentiation and a ligand for the aryl hydrocarbon receptor (AhR), induces binding of the AhR to dioxin responsive elements (DRE) in sensitive genes. The Ig heavy chain (IgH) gene is a sensitive target of TCDD and may be transcriptionally inhibited by TCDD through inhibition of the 3'IgH transcriptional regulatory region (3'IgHRR). While the 3'IgHRR contains binding sites for several transcription factors, two DRE motifs were also identified which may be responsible for TCDD-induced inhibition of 3'IgHRR activation and may implicate the AhR as an important regulator of IgH expression. The objectives of the present study were to determine if 3'IgHRR modulation is limited to TCDD or if structurally diverse chemicals (AhR ligands and non-AhR ligands) from environmental, industrial, dietary or pharmaceutical origin are also capable of modulating the 3'IgHRR and to verify a correlation between effects on a stable 3'IgHRR reporter and the endogenous IgH protein. Utilizing a CH12.LX mouse B-cell line that stably expresses a 3'IgHRR-regulated transgene, we identified an inhibition of both 3'IgHRR activation and IgH protein expression by the non-dioxin AhR activators indolo(3,2-b)carbazole, primaquine, carbaryl, and omeprazole which followed a rank order potency for AhR activation supporting a role of the AhR in the transcriptional regulation of the 3'IgHRR and IgH expression. However, modulation of the 3'IgHRR and IgH expression was not limited to AhR activators or to suppressive effects. Hydrogen peroxide and terbutaline had an activating effect and benzyl isothiocyanate was inhibitory. These chemicals are not known to influence the AhR signaling pathway but have been previously shown to modulate humoral immunity and/or transcription factors that regulate the 3'IgHRR. Taken together these results implicate the 3'IgHRR as a sensitive immunological target and are the first to identify altered 3'IgHRR activation by a diverse range of chemicals.

Transgenic expression of Spi-C impairs B-cell development and function by affecting genes associated with BCR signaling.

Spi-C is an Ets family transcription factor closely related to PU.1 and Spi-B. Expression of Spi-C is developmentally regulated in the B-cell lineage, but its function remains unknown. To determine the function of Spi-C in B-cell development, we generated mice expressing a B-cell-specific Spi-C transgene under the control of the IgH intronic enhancer. Spi-C transgenic mice had 50% fewer B cells than wild-type littermates. Flow cytometric analyses showed that splenic transitional B cells and bone marrow pre-B or immature B cells from transgenic mice were dramatically reduced compared with those of wild type. Both nonspecific and Ag-specific serum IgM levels were significantly increased in transgenic mice, while serum IgG levels were significantly decreased compared with wild type. Spi-C transgenic B cells proliferated poorly after stimulation by anti-IgM or anti-CD40 in vitro, although they responded normally to LPS stimulation. Using real-time RT-PCR, we found that several BCR signaling-related mediators were downregulated at pre-B-cell and mature B-cell stages in transgenic mice, while an inhibitor of BCR signaling was upregulated. Taken together, these data indicate that ectopic expression of Spi-C can impair B-cell development and function by affecting genes associated with BCR signaling.