Jennifer F. Harris

Nuclear Accumulation of cRel following C-Terminal phosphorylation by TBK1/IKKε

The NF-κB transcription factors are key regulators of immunomodulatory, cell cycle, and developmental gene regulation. NF-κB activity is mainly regulated through the phosphorylation of IκB by the IκB kinase (IKK) complex IKKαβγ, leading to proteasome-mediated degradation of IκB, nuclear translocation of NF-κB dimers, DNA binding, and gene induction. Additionally, direct posttranslational modifications of NF-κB p65 and cRel subunits involving C-terminal phosphorylation has been demonstrated. The noncanonical IKK-related homologs, TNFR-associated factor family member-associated NF-κB activator (TANK)-binding kinase (TBK)1 and IKKε, are also thought to play a role in NF-κB regulation, but their functions remain unclear. TBK1 and IKKε were recently described as essential regulators of IFN gene activation through direct phosphorylation of the IFN regulatory factor-3 and -7 transcription factors. In the present study, we sought to determine whether IKKε and TBK1 could modulate cRel activity via phosphorylation. TBK1 and IKKε directly phosphorylate the C-terminal domain of cRel in vitro and in vivo and regulate nuclear accumulation of cRel, independently of the classical IκB/IKK pathway. IκBα degradation is not affected, but rather IKKε-mediated phosphorylation of cRel leads to dissociation of the IκBα-cRel complex. These results illustrate a previously unrecognized aspect of cRel regulation, controlled by direct IKKε/TBK1 phosphorylation.

Hollow fiber integrated microfluidic platforms for in vitro Co-culture of multiple cell types.

This study demonstrates a rapid prototyping approach for fabricating and integrating porous hollow fibers (HFs) into microfluidic device. Integration of HF can enhance mass transfer and recapitulate tubular shapes for tissue-engineered environments. We demonstrate the integration of single or multiple HFs, which can give the users the flexibility to control the total surface area for tissue development. We also present three microfluidic designs to enable different co-culture conditions such as the ability to co-culture multiple cell types simultaneously on a flat and tubular surface, or inside the lumen of multiple HFs. Additionally, we introduce a pressurized cell seeding process that can allow the cells to uniformly adhere on the inner surface of HFs without losing their viabilities. Co-cultures of lung epithelial cells and microvascular endothelial cells were demonstrated on the different platforms for at least five days. Overall, these platforms provide new opportunities for co-culturing of multiple cell types in a single device to reconstruct native tissue micro-environment for biomedical and tissue engineering research.

Microscale 3D Liver Bioreactor for In Vitro Hepatotoxicity Testing under Perfusion Conditions

The accurate prediction of hepatotoxicity demands validated human in vitro models that can close the gap between preclinical animal studies and clinical trials. In this study we investigated the response of primary human liver cells to toxic drug exposure in a perfused microscale 3D liver bioreactor. The cellularized bioreactors were treated with 5, 10, or 30 mM acetaminophen (APAP) used as a reference substance. Lactate production significantly decreased upon treatment with 30 mM APAP (p < 0.05) and ammonia release significantly increased in bioreactors treated with 10 or 30 mM APAP (p < 0.0001), indicating APAP-induced dose-dependent toxicity. The release of prostaglandin E2 showed a significant increase at 30 mM APAP (p < 0.05), suggesting an inflammatory reaction towards enhanced cellular stress. The expression of genes involved in drug metabolism, antioxidant reactions, urea synthesis, and apoptosis was differentially influenced by APAP exposure. Histological examinations revealed that primary human liver cells in untreated control bioreactors were reorganized in tissue-like cell aggregates. These aggregates were partly disintegrated upon APAP treatment, lacking expression of hepatocyte-specific proteins and transporters. In conclusion, our results validate the suitability of the microscale 3D liver bioreactor to detect hepatotoxic effects of drugs in vitro under perfusion conditions.

IL-13 in LPS-Induced Inflammation Causes Bcl-2 Expression to Sustain Hyperplastic Mucous cells

Exposure to lipopolysaccharides (LPS) causes extensive neutrophilic inflammation in the airways followed by mucous cell hyperplasia (MCH) that is sustained by the anti-apoptotic protein, Bcl-2. To identify inflammatory factor(s) that are responsible for Bcl-2 expression, we established an organ culture system consisting of airway epithelial tissue from the rat nasal midseptum. The highest Muc5AC and Bcl-2 expression was observed when organ cultures were treated with brochoalveolar lavage (BAL) fluid harvested from rats 10 h post LPS instillation. Further, because BAL harvested from rats depleted of polymorphonuclear cells compared to controls showed increased Bcl-2 expression, analyses of cytokine levels in lavages identified IL-13 as an inducer of Bcl-2 expression. Ectopic IL-13 treatment of differentiated airway epithelial cells increased Bcl-2 and MUC5AC expression in the basal and apical regions of the cells, respectively. When Bcl-2 was blocked using shRNA or a small molecule inhibitor, ABT-263, mucous cell numbers were reduced due to increased apoptosis that disrupted the interaction of Bcl-2 with the pro-apoptotic protein, Bik. Furthermore, intranasal instillation of ABT-263 reduced the LPS-induced MCH in bik+/+ but not bik−/− mice, suggesting that Bik mediated apoptosis in hyperplastic mucous cells. Therefore, blocking Bcl-2 function could be useful in reducing IL-13 induced mucous hypersecretion.

A microfluidic method to measure bulging heights for bulge testing of polydimethylsiloxane (PDMS) and polyurethane (PU) elastomeric membranes

Thin and flexible elastomeric membranes are frequently used in many microfluidic applications including microfluidic valves and organs-on-a-chip. The elastic properties of these membranes play an important role in the design of such microfluidic devices. Bulge testing, which is a common method to characterize the elastic behavior of these membranes, involves direct observation of the changes in the bulge height in response to a range of applied pressures. Here, we report a microfluidic approach to measure the bulging height of elastic membranes to replace direct observation of the bulge height under a microscope. Bulging height is measured by tracking the displacement of a fluid inside a microfluidic channel, where the fluid in the channel was designed to be directly in contact with the elastomeric membrane. Polydimethylsiloxane (PDMS) and polyurethane (PU) membranes with thickness 12–35 μm were fabricated by spin coating for bulge testing using both direct optical observation and the microfluidic method. Bulging height determined from the optical method was subject to interpretation by the user, whereas the microfluidic approach provided a simple but sensitive method for determining the bulging height of membranes down to a few micrometers. This work validates the proof of principle that uses microfluidics to accurately measure bulging height in conventional bulge testing for polydimethylsiloxane (PDMS) and polyurethane (PU)eElastomeric membranes.