Altered protein conformation and lower stability of the dystrophic transforming growth factor beta-induced protein mutants.

PURPOSE: Transforming growth factor beta-induced protein (TGFBIp) is a widely expressed extracellular matrix protein that plays roles in cell adhesion and migration, differentiation, apoptosis, bone morphogenesis, and carcinogenesis. Mutations of TGFBIp have been linked to stromal corneal dystrophies, a group of protein conformational diseases characterized by abnormal protein aggregations in the cornea. However, the underlying pathogenic mechanism remains elusive due to a lack of insight into the molecular properties of the disease-causing mutants. In the current study, we applied spectroscopic tools to compare the conformation and protein stability of recombinant wild-type (WT) TGFBIp to two dystrophic mutants, R124C and R555W. METHODS: A serum-free expression system was used to produce the recombinant TGFBIp proteins. Fluorescence and far-ultraviolet circular dichroism spectroscopies were used to compare WT and dystrophic mutants under various conditions. RESULTS: Our results showed that dystrophic mutants were processed differentially by the expressing cells and produced different proteolytic fragment patterns by proteolysis. Intrinsic tryptophan fluorescence studies revealed moderate shifts in the emission maxima and increased quenching by iodide ion of mutant TGFBIp, suggesting a different conformation than WT protein. Denaturation experiments indicated a difference in protein stability between WT and mutant proteins. Under oxidizing conditions, the mutants produced higher 1-anilinonaphthalene-8-sulfonic acid and thioflavin T fluorescence signals than the WT, indicating increased protein unfolding and fibril formation, respectively. Finally, far-ultraviolet circular dichroism spectroscopy revealed that WT TGFBIp undergoes concentration-dependent conformational changes; similar experiments were not possible on mutant TGFBIp, which remained soluble only at low concentrations. CONCLUSIONS: Our study provides new evidence for the pathogenic mechanism of dystrophic mutants. Although mutant TGFBIp has moderate but consistent structural perturbations, other factors such as oxidation or degradation may be required to cause the phenotypic abnormal aggregations.

Halofuginone down-regulates Smad3 expression and inhibits the TGFbeta-induced expression of fibrotic markers in human corneal fibroblasts.

PURPOSE: Due to its ability to disrupt transforming growth factor beta (TGF-ß) signaling, halofuginone has been successfully used to treat various fibrotic disorders. Here we investigated the antifibrotic potential of halofuginone in human corneal fibroblasts. METHODS: Human corneal fibroblasts were isolated from human donor corneas for in vitro experiments. TGF-ß was used to stimulate pro-fibrotic responses from corneal fibroblasts under halofuginone treatment. The expression of alpha smooth muscle actin (α-SMA) and fibronectin was analyzed by western blots. Phalloidin toxin was used to stain cultures for stress fiber assemblies. Quantitative reverse transcription PCR (qRT-PCR) and immunostaining were used to analyze the expression of type I collagen mRNA and protein, respectively. The expression of Smad2, Smad3, phospho-Smad2, and phospho-Smad3 was determined by western blots. RESULTS: Halofuginone was well tolerated by human corneal fibroblasts up to 10 ng/ml as demonstrated by a cell viability assay. At this concentration, TGF-ß-induced expression of the fibrotic markers α-SMA, fibronectin, and type I collagen was significantly reduced. Interestingly, under our experimental conditions, halofuginone treatment led to reduced protein expression of Smad3, which was both dose- and time-dependent. CONCLUSIONS: Our results suggest that halofuginone may exert its antifibrotic effects in the cornea via a novel molecular mechanism and may be used as an antifibrotic agent for corneal fibrosis treatment.

Characterizing Participant Perceptions about Smoking-Cessation Pharmacotherapy and E-Cigarettes from Facebook Smoking-Cessation Support Groups.

The prevalence of smoking among young adults aged 19-28 years old in the United States persists at rates of 14.3%. Young adults underutilize pharmacotherapy for smoking cessation, and the use of e-cigarettes has increased. We analyzed comments from online smoking-cessation support groups to understand young-adult smokers' views of pharmacotherapy and e-cigarettes, to provide a more in-depth insight into the underutilization of pharmacotherapy. A qualitative analysis was performed on comments about pharmacotherapy and e-cigarettes from participants enrolled in online smoking-cessation support groups in 2016-2020. A codebook was developed with a deductive approach to code the comments, followed by thematic analysis. Eighteen themes were identified, with four dominant themes: interest, benefit, knowledge, and flavor. Participants expressed less interest in both nicotine-replacement therapy and e-cigarettes; moreover, they expressed unfamiliarity with and misconceptions about pharmacotherapy, and recognized the enticing flavors of e-cigarettes. Participants often felt e-cigarettes were not useful for smoking cessation, but the flavors of e-cigarettes were appealing for use. Participants had mixed opinions about the use of e-cigarettes for smoking cessation, but predominantly felt e-cigarettes were not useful for smoking cessation. The use of social media may be an effective way to address misconceptions about pharmacotherapy for smoking cessation and increase willingness to accept assistance.

Searching the literature using medical subject headings versus text word with PubMed.

OBJECTIVE/HYPOTHESIS: This study was conducted to investigate the performance of two search strategies in the retrieval of information from the National Library of Medicine (NLM) on otolaryngology-head and neck surgery related conditions and diagnoses using PubMed. METHODS: Two search strategies-one based on the use of Medical Subject Headings (MeSH) and the second based on text word searching-were compared. RESULTS: The MeSH search provided a more efficient search than the text word search. CONCLUSIONS: Head and neck surgeons can most efficiently search the NLM using PubMed as a search engine by initiating the search with MeSH terms. Once a key article is identified, the searcher should use the "Related Articles" feature.

Inhibition of nuclear factor-kappaB and target genes during combined therapy with proteasome inhibitor bortezomib and reirradiation in patients with recurrent head-and-neck squamous cell carcinoma.

PURPOSE: To examine the effects the proteasome inhibitor bortezomib (VELCADE) on transcription factor nuclear factor-kappaB (NF-kappaB) and target genes and the feasibility of combination therapy with reirradiation in patients with recurrent head-and-neck squamous cell carcinoma (HNSCC). METHODS AND MATERIALS: The tolerability and response to bortezomib 0.6 mg/m2 and 0.9 mg/m2 given twice weekly concurrent with daily reirradiation to 50-70 Gy was explored. Blood proteasome inhibition and NF-kappaB-modulated cytokines and factors were measured. Proteasome inhibition, nuclear localization of NF-kappaB phospho-p65, apoptosis, and expression of NF-kappaB-modulated mRNAs were compared in serial biopsies from accessible tumors. RESULTS: The maximally tolerated dose was exceeded, and study was limited to 7 and 2 patients, respectively, given bortezomib 0.6 mg/m2 and 0.9 mg/m2/dose with reirradiation. Grade 3 hypotension and hyponatremia were dose limiting. Mucositis was Grade 3 or less and was delayed. The mean blood proteasome inhibition at 1, 24, and 48 h after 0.6 mg/m2 was 32%, 16%, and 7% and after 0.9 mg/m2 was 56%, 26%, and 14%, respectively. Differences in proteasome and NF-kappaB activity, apoptosis, and expression of NF-kappaB-modulated cell cycle, apoptosis, and angiogenesis factor mRNAs were detected in 2 patients with minor tumor reductions and in serum NF-kappaB-modulated cytokines in 1 patient with a major tumor reduction. CONCLUSIONS: In combination with reirradiation, the maximally tolerated dose of bortezomib was exceeded at a dose of 0.6 mg/m2 and the threshold of proteasome inhibition. Although this regimen with reirradiation is not feasible, bortezomib induced detectable differences in NF-kappaB localization, apoptosis, and NF-kappaB-modulated genes and cytokines in tumor and serum in association with tumor reduction, indicating that other schedules of bortezomib combined with primary radiotherapy or reirradiation may merit future investigation.

Inferior turbinate augmentation with auricular cartilage for the treatment of empty nose syndrome.

Empty nose syndrome (ENS) is a potential complication of excessive resection of turbinate tissue. Patients with ENS complain of nasal obstruction despite a widely patent nasal cavity. Various implants, including autologous bone and biomaterials, have been used to reduce the width of the nasal cavity. Implantation of these grafts, however, has been limited by extrusion, infection, and resorption. We introduce a novel surgical technique that uses autologous auricular cartilage to augment the turbinate and to restore the natural airflow patterns of the nasal cavity. We present a representative case of ENS caused by excessive inferior turbinate reduction that was improved by turbinate augmentation with autologous auricular cartilage.

Corneal wound healing is compromised by immunoproteasome deficiency.

Recent studies have revealed roles for immunoproteasome in regulating cell processes essential for maintaining homeostasis and in responding to stress and injury. The current study investigates how the absence of immunoproteasome affects the corneal epithelium under normal and stressed conditions by comparing corneas from wildtype (WT) mice and those deficient in two immunoproteasome catalytic subunits (lmp7(-/-)/mecl-1(-/-), L7M1). Immunoproteasome expression was confirmed in WT epithelial cells and in cells of the immune system that were present in the cornea. More apoptotic cells were found in both corneal explant cultures and uninjured corneas of L7M1 compared to WT mice. Following mechanical debridement, L7M1 corneas displayed delayed wound healing, including delayed re-epithelialization and re-establishment of the epithelial barrier, as well as altered inflammatory cytokine production compared to WT mice. These results suggest that immunoproteasome plays an important role in corneal homeostasis and wound healing.

In vivo implantation of tissue-engineered human nasal septal neocartilage constructs: a pilot study.

OBJECTIVE: To determine the in vivo biocompatibility of septal neocartilage constructs developed in vitro by an alginate intermediate step. STUDY DESIGN: Prospective, animal model. SETTING: Research laboratory. SUBJECTS AND METHODS: A murine model was used to examine the maturation of neocartilage constructs in vivo. Chondrocytes collected from patients undergoing septoplasty were expanded in monolayer and suspended in alginate beads for 3-dimensional culture in media containing human serum and growth factors. After in vitro incubation for 5 weeks, the constructs were implanted in the dorsum of athymic mice for 30 and 60 days (n = 9). After the mice were sacrificed, the constructs were recovered for assessment of their morphological, histochemical, biochemical, and biomechanical properties. RESULTS: The mice survived and tolerated the implants well. Infection and extrusion were not observed. Neocartilage constructs maintained their general shape and size and demonstrated cell viability after implantation. The implanted constructs were firm and opaque, sharing closer semblance to native septal tissue relative to the gelatinous, translucent preimplant constructs. Histochemical staining with hematoxylin and eosin (H&E) revealed that the constructs exhibited distinct morphologies characteristic of native tissue, which were not observed in preimplant constructs. DNA and type II collagen increased with duration of implantation, whereas type I collagen and glycoaminoglycans (GAG) decreased. Mechanical testing of a 60-day implanted construct demonstrated characteristics similar to native human septal cartilage. CONCLUSIONS: Neocartilage constructs are viable in an in vivo murine model. The histologic, biochemical, and biomechanical features of implanted constructs closely resemble native septal tissue when compared with preimplant constructs.

Culture of human septal chondrocytes in a rotary bioreactor.

OBJECTIVES: (1) To show that extracellular matrix deposition in 3-dimensional culture of human septal chondrocytes cultured in a rotary bioreactor is comparable to the deposition achieved under static culture conditions. (2) To demonstrate that the biomechanical properties of human septal chondrocytes cultured in a bioreactor are enhanced with time and are analogous to beads cultured under static culture. STUDY DESIGN: Prospective, basic science. SETTING: Research laboratory. METHODS: Human septal chondrocytes from 9 donors were expanded in monolayer and seeded in alginate beads. The beads were cultured in a rotary bioreactor for 21 days in media supplemented with growth factors and human serum, using static culture as the control. Biochemical and biomechanical properties of the beads were measured. RESULTS: Glycosaminoglycan (GAG) accumulation significantly increased during 2 measured time intervals, 0 to 21 days and 10 to 21 days (P < .01). No significant difference was seen between the static and bioreactor conditions. Substantial type II collagen production was demonstrated in the beads terminated at day 21 of culture in both conditions. In addition, the biomechanical properties of the beads were significantly improved at 21 days in comparison to beads from day 0. CONCLUSION: Human septal chondrocytes cultured in alginate beads exhibit significant matrix deposition and improved biomechanical properties after 21 days. Alginate bead diameter and stiffness positively correlated with GAG and type II collagen accretion. Matrix production in beads is supported by the use of a rotary bioreactor.

Growth of human septal chondrocytes in fibrin scaffolds.

BACKGROUND: Tissue engineering of nasal septal cartilage has been the focus of research owing to its superior structural rigidity and ease of harvest. In vitro constructs formed from septal chondrocytes using fibrin glue within a polyglycolic acid (PGA) scaffold have been shown to be viable, but their cellular growth and expression of differentiated features still have not been quantified. In this study, we evaluated cellular proliferation and production of cartilaginous extracellular matrix (ECM) components in fibrin glue preparations within a PGA scaffold. METHODS: Human chondrocytes were expanded for one passage in monolayer in culture medium. The cells were then grown in (1) fibrinogen, (1/2)x-thrombin, (1/2)x (F/2:T/2); (2) fibrinogen, 1/10x-thrombin, 1/10x (F/10:T/10); (3) fibrinogen, 1x-thrombin, 1/100x (F/1:T/100). RESULTS: Cellular proliferation and glycosaminoglycan (GAG) production per cell were highest in the F/2:T/2 preparations. Greater proliferation was seen in chondrocyte-fibrin composites seeded onto the PGA scaffold when compared with chondrocytes seeded onto the PGA scaffold alone. No significant difference in GAG production was seen. CONCLUSION: The addition of fibrin glue to chondrocytes seeded onto a PGA scaffold results in increased cellular proliferation while maintaining production of ECM components. Long-term stable fibrin gels in combination with PGA scaffolds may facilitate generation of cartilaginous tissue for use in reconstructive surgery.

Cartilage outgrowth in fibrin scaffolds.

BACKGROUND: Fibrin glue has been a favorable hydrogel in cartilage tissue engineering, but implantation of chondrocyte-fibrin suspensions have resulted in volume loss. In this study, human septal cartilage chips were seeded onto a fibrin scaffold, and cellular proliferation and production of cartilaginous extracellular matrix (ECM) were evaluated. METHODS: Human septal cartilage was diced into cartilage chips and encased with and without fibrin glue. Four conditions were initially tested for DNA content and glycosaminoglycan (GAG) production: (1) control medium in tissue culture, (2) control medium with fibrin glue, (3) collagenase-supplemented medium in tissue culture, and (4) collagenase-supplemented medium seeded in fibrin glue. Cartilage chips cultured in collagenase-treated medium were then seeded onto either cell culture plates, suspended in alginate, or mixed with fibrin. Cellular proliferation, GAG production, and histochemistry were evaluated. RESULTS: Fibrin preparations increased cellular proliferation and DNA content. GAG levels were highest in collagenase-treated samples encased in fibrin. Cartilage chips treated with collagenase showed increased cellular proliferation in the fibrin preparations compared with preparations without fibrin. GAG increased with the addition of fibrin when compared with explant. Histochemistry revealed increased GAG accumulation in the regions between the cartilage chips with the addition of fibrin. CONCLUSION: Adding fibrin glue to collagenase-treated cartilage chips results in increased proliferation and maintains ECM production and, therefore, may facilitate generation of cartilaginous tissue for use in reconstructive surgery.

Nuclear factor-KappaB as a common target and activator of oncogenes in head and neck squamous cell carcinoma.

Head and neck squamous cell carcinomas exhibit alterations in cell proliferation, survival (apoptosis), migration, angiogenesis and inflammation. The transcription factor nuclear factor-KappaB integrates multiple signals and regulates expression of multiple genes involved in these phenotypic responses, suggesting the hypothesis that nuclear factor-KappaB is an important molecular switch for development of head and neck squamous cell carcinoma. Nuclear factor-KappaB has been found to be constitutively activated, and a common target and activator of oncogenes in cancer. Because of its important role, activation of nuclear factor-KappaB by the proteasome and other signal molecules may provide targets for molecular therapy of squamous cell carcinoma and other cancers.