Travis L. Shiba

Activation of noncanonical NF-κB requires coordinated assembly of a regulatory complex of the adaptors cIAP1, cIAP2, TRAF2, TRAF3 and the kinase NIK

Recent studies suggest that nuclear factor κB-inducing kinase (NIK) is suppressed through constitutive proteasome-mediated degradation regulated by TRAF2, TRAF3 and cIAP1 or cIAP2. Here we demonstrated that the degradation of NIK occurs upon assembly of a regulatory complex through TRAF3 recruitment of NIK and TRAF2 recruitment of cIAP1 and cIAP2. In contrast to TRAF2 and TRAF3, cIAP1 and cIAP2 seem to play redundant roles in the degradation of NIK, as inhibition of both cIAPs was required for noncanonical NF-κB activation and increased survival and proliferation of primary B lymphocytes. Furthermore, the lethality of TRAF3 deficiency in mice could be rescued by a single NIK gene, highlighting the importance of tightly regulated NIK.

Regulation of antiviral responses by a direct and specific interaction between TRAF3 and Cardif

Upon recognition of viral infection, RIG‐I and Helicard recruit a newly identified adapter termed Cardif, which induces type I interferon (IFN)‐mediated antiviral responses through an unknown mechanism. Here, we demonstrate that TRAF3, like Cardif, is required for type I interferon production in response to intracellular double‐stranded RNA. Cardif‐mediated IFNα induction occurs through a direct interaction between the TRAF domain of TRAF3 and a TRAF‐interaction motif (TIM) within Cardif. Interestingly, while the entire N‐terminus of TRAF3 was functionally interchangeable with that of TRAF5, the TRAF domain of TRAF3 was not. Our data suggest that this distinction is due to an inability of the TRAF domain of TRAF5 to bind the TIM of Cardif. Finally, we show that preventing association of TRAF3 with this TIM by mutating two critical amino acids in the TRAF domain also abolishes TRAF3‐dependent IFN production following viral infection. Thus, our findings suggest that the direct and specific interaction between the TRAF domain of TRAF3 and the TIM of Cardif is required for optimal Cardif‐mediated antiviral responses.

Negative feedback in noncanonical NF-kappaB signaling modulates NIK stability through IKKalpha-mediated phosphorylation.

Feedback inhibition of the central regulator of noncanonical NF-κB signaling is mediated by its target kinase. Activation and Inhibition through IKKα Details about the activation and termination of the noncanonical NF-κB pathway, which is involved in processes such as B cell maturation and bone development, still continue to emerge (see the Perspective by Sun). Central to this pathway is NF-κB–inducing kinase (NIK), which activates inhibitor of κB kinase α (IKKα). IKKα phosphorylates an NF-κB precursor protein called p100, promoting its processing to the mature p52 subunit, which then interacts with the RelB subunit to form the noncanonical NF-κB complex. Under resting conditions, NIK is constitutively degraded; however, after stimulation of receptors such as B cell–activating factor receptor (BAFF-R) and lymphotoxin β receptor (LTβR), NIK accumulates and initiates noncanonical signaling. Razani et al. observed an increase in the steady-state abundance of NIK in cells from IKKα-deficient mice and from mice that have a variant NIK protein that is incapable of binding to IKKα. They identified NIK as a target of IKKα and showed that phosphorylation of NIK by IKKα resulted in its destabilization and the inhibition of noncanonical signaling. Further understanding of the regulation of NIK may help in the development of therapies that specifically target noncanonical NF-κB signaling. Canonical and noncanonical nuclear factor κB (NF-κB) signaling are the two basic pathways responsible for the release of NF-κB dimers from their inhibitors. Enhanced NF-κB signaling leads to inflammatory and proliferative diseases; thus, inhibitory pathways that limit its activity are critical. Whereas multiple negative feedback mechanisms control canonical NF-κB signaling, none has been identified for the noncanonical pathway. Here, we describe a mechanism of negative feedback control of noncanonical NF-κB signaling that attenuated the stabilization of NF-κB–inducing kinase (NIK), the central regulatory kinase of the noncanonical pathway, induced by B cell–activating factor receptor (BAFF-R) and lymphotoxin β receptor (LTβR). Inhibitor of κB (IκB) kinase α (IKKα) was previously thought to lie downstream of NIK in the noncanonical NF-κB pathway; we showed that phosphorylation of NIK by IKKα destabilized NIK. In the absence of IKKα-mediated negative feedback, the abundance of NIK increased after receptor ligation. A form of NIK with mutations in the IKKα-targeted serine residues was more stable than wild-type NIK and resulted in increased noncanonical NF-κB signaling. Thus, in addition to the regulation of the basal abundance of NIK in unstimulated cells by a complex containing tumor necrosis factor receptor–associated factor (TRAF) and cellular inhibitor of apoptosis (cIAP) proteins, IKKα-dependent destabilization of NIK prevents the uncontrolled activity of the noncanonical NF-κB pathway after receptor ligation.

Tissue-Engineered Vocal Fold Mucosa Implantation in Rabbits

Objective To assess phonatory function and wound healing of a tissue-engineered vocal fold mucosa (TE-VFM) in rabbits. An “artificial” vocal fold would be valuable for reconstructing refractory scars and resection defects, particularly one that uses readily available autologous cells and scaffold. This work implants a candidate TE-VFM after resecting native epithelium and lamina propria in rabbits. Study Design Prospective animal study. Setting Research laboratory. Subjects and Methods Rabbit adipose-derived stem cells were isolated and cultured in three-dimensional fibrin scaffolds to form TE-VFM. Eight rabbits underwent laryngofissure, unilateral European Laryngologic Society type 2 cordectomy, and immediate reconstruction with TE-VFM. After 4 weeks, larynges were excised, phonated, and examined by histology. Results Uniform TE-VFM implants were created, with rabbit mesenchymal cells populated throughout fibrin hydrogels. Rabbits recovered uneventfully after implantation. Phonation was achieved in all, with mucosal waves evident at the implant site. Histology after 4 weeks showed resorbed fibrin matrix, continuous epithelium, and mildly increased collagen relative to contralateral unoperated vocal folds. Elastic fiber appearance was highly variable. Inflammatory cell infiltrate was limited to animals receiving sex-mismatched implants. Conclusion TE-VFMs were successfully implanted into 8 rabbits, with minor evidence of scar formation and immune reaction. Vibration was preserved 4 weeks after resecting and reconstructing the complete vocal fold cover layer. Further studies will investigate the mechanism and durability of improvement. TE-VFM with autologous cells is a promising new approach for vocal fold reconstruction.

Dynamics of phonatory posturing at phonation onset

In speech and singing, the intrinsic laryngeal muscles set the prephonatory posture prior to the onset of phonation. The timing and shape of the prephonatory glottal posture can directly affect the resulting phonation type. We investigated the dynamics of human laryngeal phonatory posturing.

Photodocumentation of the Development of Type I Posterior Glottic Stenosis after Intubation Injury

Bilateral vocal fold immobility may result from bilateral recurrent laryngeal nerve paralysis or physiologic insults to the airway such as glottic scars. The progression of mucosal injury to granulation tissue, and then posterior glottis stenosis, is an accepted theory but has not been photodocumented. This paper presents serial images from common postintubation injury to less common posterior glottic stenosis with interarytenoid synechia.

Adipose-Derived Mesenchymal Stromal Cells Persist in Tissue-Engineered Vocal Fold Replacement in Rabbits

Objectives: Cell therapies using mesenchymal stromal cells (MSCs) have been proposed as a promising new tool for the treatment of vocal fold scarring. However, the mechanisms by which MSCs promote healing as well as their duration of survival within the host vocal fold have yet to be defined. The aim of this work was to assess the persistence of embedded MSCs within a tissue-engineered vocal fold mucosal replacement in a rabbit model of vocal fold injury. Methods: Male rabbit adipose-derived MSCs were embedded within a 3-dimensional fibrin gel, forming the cell-based outer vocal fold replacement. Four female rabbits underwent unilateral resection of vocal fold epithelium and lamina propria and reconstruction with cell-based outer vocal fold replacement implantation. Polymerase chain reaction and fluorescent in situ hybridization for the sex-determining region of the Y chromosome (SRY-II) in the sex-mismatched donor-recipient pairs sought persistent cells after 4 weeks. Results: A subset of implanted male cells was detected in the implant site at 4 weeks. Many SRY-II-negative cells were also detected at the implant site, presumably representing native female cells that migrated to the area. No SRY-II signal was detected in contralateral control vocal folds. Conclusions: The emergent tissue after implantation of a tissue-engineered outer vocal fold replacement is derived both from initially embedded adipose-derived stromal cells and infiltrating native cells. Our results suggest this tissue-engineering approach can provide a well-integrated tissue graft with prolonged cell activity for repair of severe vocal fold scars.

Development of a Tissue-Engineered Vocal Fold Cover Replacement from Rabbit Adipose Stem Cells

Objectives: Tissue engineering of a vocal fold replacement is a promising potential treatment for severe vocal fold scarring. Development and testing of such a tissue construct is an ongoing project. We have previously demonstrated that human adipose-derived stem cells (ASC) can produce a bilayered construct with suitable properties for implantation and phonation. This phase of the project developed a similar construct for pre-clinical animal studies, using rabbit cells. Methods: Rabbit ASCs were isolated, cultured, and embedded in fibrin gels under air-liquid interface conditions and with epidermal growth factor. After culture periods of 1 to 4 weeks, constructs were harvested, sectioned, and examined with immunohistochemistry. Results: Rabbit cells attached and survived within fibrin gels. Differentiation of the cells to epithelial and mesenchymal lineages was determined by microscopic markers. Deposition of extracellular matrix and basement membrane proteins was also examined. Conclusions: Rabbit ASCs are suitable for use in a tissue-engineered vocal fold replacement. This model will be used in future implantation trials in rabbits.