Differentiated fibrocytes assume a functional mesenchymal phenotype with regenerative potential.

Fibrocytes (FCs) are hematopoietic lineage cells that migrate to sites of injury, transition to a mesenchymal phenotype, and help to mediate wound repair. Despite their relevance to human fibrotic disorders, there are few data characterizing basic FC biology. Herein, using proteomic, bioenergetic, and bioengineering techniques, we conducted deep phenotypic characterization of differentiating and mature FCs. Differentiation was associated with metabolic reprogramming that favored oxidative phosphorylation. Mature FCs had distinct proteomes compared to classic mesenchymal cells, formed functional stromae that supported epithelial maturation during in vitro organotypic culture, and exhibited in vivo survival and self-tolerance as connective tissue isografts. In an in vitro scratch assay, FCs promoted fibroblast migration and wound closure by paracrine signaling via the chemokine CXCL8 (interleukin-8). These findings characterize important aspects of FC differentiation and show that, in addition to their role in wound healing, FCs hold potential as an easily isolated autologous cell source for regenerative medicine.

Bioengineered vocal fold mucosa for voice restoration.

Patients with voice impairment caused by advanced vocal fold (VF) fibrosis or tissue loss have few treatment options. A transplantable, bioengineered VF mucosa would address the individual and societal costs of voice-related communication loss. Such a tissue must be biomechanically capable of aerodynamic-to-acoustic energy transfer and high-frequency vibration and physiologically capable of maintaining a barrier against the airway lumen. We isolated primary human VF fibroblasts and epithelial cells and cocultured them under organotypic conditions. The resulting engineered mucosae showed morphologic features of native tissue, proteome-level evidence of mucosal morphogenesis and emerging extracellular matrix complexity, and rudimentary barrier function in vitro. When grafted into canine larynges ex vivo, the mucosae generated vibratory behavior and acoustic output that were indistinguishable from those of native VF tissue. When grafted into humanized mice in vivo, the mucosae survived and were well tolerated by the human adaptive immune system. This tissue engineering approach has the potential to restore voice function in patients with otherwise untreatable VF mucosal disease.

Immune privilege of the CNS is not the consequence of limited antigen sampling.

Central nervous system (CNS) immune privilege is complex, and it is still not understood how CNS antigens are sampled by the peripheral immune system under steady state conditions. To compare antigen sampling from immune-privileged or nonprivileged tissues, we created transgenic mice with oligodendrocyte or gut epithelial cell expression of an EGFP-tagged fusion protein containing ovalbumin (OVA) antigenic peptides and tested peripheral anti-OVA peptide-specific sentinel OT-I and OT-II T cell activation. We report that oligodendrocyte or gut antigens are sampled similarly, as determined by comparable levels of OT-I T cell activation. However, activated T cells do not access the CNS under steady state conditions. These data show that afferent immunity is normally intact as there is no barrier at the antigen sampling level, but that efferent immunity is restricted. To understand how this one-sided surveillance contributes to CNS immune privilege will help us define mechanisms of CNS autoimmune disease initiation.

Cross-sample validation provides enhanced proteome coverage in rat vocal fold mucosa.

The vocal fold mucosa is a biomechanically unique tissue comprised of a densely cellular epithelium, superficial to an extracellular matrix (ECM)-rich lamina propria. Such ECM-rich tissues are challenging to analyze using proteomic assays, primarily due to extensive crosslinking and glycosylation of the majority of high M(r) ECM proteins. In this study, we implemented an LC-MS/MS-based strategy to characterize the rat vocal fold mucosa proteome. Our sample preparation protocol successfully solubilized both proteins and certain high M(r) glycoconjugates and resulted in the identification of hundreds of mucosal proteins. A straightforward approach to the treatment of protein identifications attributed to single peptide hits allowed the retention of potentially important low abundance identifications (validated by a cross-sample match and de novo interpretation of relevant spectra) while still eliminating potentially spurious identifications (global single peptide hits with no cross-sample match). The resulting vocal fold mucosa proteome was characterized by a wide range of cellular and extracellular proteins spanning 12 functional categories.

E-cadherin and transglutaminase-1 epithelial barrier restoration precedes type IV collagen basement membrane reconstruction following vocal fold mucosal injury.

The vocal fold epithelium is critical to upper airway immunologic defense and water/ion transport; therefore, any form of physical trauma or insult increases the vulnerability of this structure to functional impairment and pathogen invasion/infection. In this study, we examined the reestablishment of epithelial and basement membrane barrier structures in a well-established rat model of vocal fold mucosal injury. We observed active cell recruitment culminating in peak hyperplasia at 3 days postinjury, the establishment of robust E-cadherin+ and transglutaminase-1+ biochemical barrier signals along the epithelial surface by 3 days postinjury, and the persistent absence of a type IV collagen+ basement membrane at 7 days postinjury. The distinct spatial and temporal immunoactivity of these molecules is consistent with a programmed repair process driving the restoration of vocal fold mucosal integrity and permeability. These data may inform future efforts to optimize functional mucosal recovery postinjury and avoid undesirable events such as barrier compromise or epithelial metaplasia.

Pulsed dye laser-induced inflammatory response and extracellular matrix turnover in rat vocal folds and vocal fold fibroblasts.

BACKGROUND AND OBJECTIVES: Disruption of the vocal fold extracellular matrix (ECM) can induce a profound and refractory dysphonia. Pulsed dye laser (PDL) irradiation has shown early promise as a treatment modality for disordered ECM in patients with chronic vocal fold scar; however, there are limited data addressing the mechanism by which this laser energy might induce cellular and extracellular changes in vocal fold tissues. In this study, we examined the inflammatory and ECM modulating effects of PDL irradiation on normal vocal fold tissues and cultured vocal fold fibroblasts (VFFs). STUDY DESIGN/MATERIALS AND METHODS: We evaluated the effects of 585 nm PDL irradiation on inflammatory cytokine and collagen/collagenase gene transcription in normal rat vocal folds in vivo (3-168 hours following delivery of approximately 39.46 J/cm(2) fluence) and VFFs in vitro (3-72 hours following delivery of 4.82 or 9.64 J/cm(2) fluence). We also examined morphological vocal fold tissue changes 3 hours, 1 week, and 1 month post-irradiation. RESULTS: PDL irradiation altered inflammatory cytokine and procollagen/collagenase expression at the transcript level, both in vitro and in vivo. Additionally, PDL irradiation induced an inflammatory repair process in vivo that was completed by 1 month with preservation of normal tissue morphology. CONCLUSIONS: PDL irradiation can modulate ECM turnover in phenotypically normal vocal folds. Additional work is required to determine if these findings extend to disordered ECM, such as is seen in vocal fold scar. Lasers Surg. Med. 41:585-594, 2009. (c) 2009 Wiley-Liss, Inc.

Intracerebral Mycobacterium bovis bacilli Calmette-Guerin infection-induced immune responses in the CNS.

To study whether cerebral mycobacterial infection induces granuloma and protective immunity similar to systemic infection, we intracerebrally infected mice with Mycobacterium bovis bacilli Calmette-Guerin. Granuloma and IFN-gamma(+)CD4(+) T cell responses are induced in the central nervous system (CNS) similar to periphery, but the presence of IFN-gammaIL-17 double-positive CD4(+) T cells is unique to the CNS. The major CNS source of TNF-alpha is microglia, with modest production by CD4(+) T cells and macrophage. Protective immunity is accompanied by accumulation of Foxp3(+)CD4(+) T cells and PD-L2(+) dendritic cells, suggesting that both inflammatory and anti-inflammatory responses develop in the CNS following mycobacterial infection.