Tissue-Engineered Implant for Hemilaryngectomy Reconstruction with Recurrent Laryngeal Nerve Injury.

OBJECTIVE: Laryngeal cancer resections often require excision of portions of the larynx along with sacrifice of the ipsilateral recurrent laryngeal nerve (RLN). In such cases, there are no reconstructive options that reliably restore laryngeal function, rendering patients with severe functional impairment. To address this unmet clinical need, we extend our evaluation of a 3-implant mucosal, muscle, cartilage reconstruction approach aimed at promoting functional laryngeal restoration in a porcine hemilaryngectomy model with ipsilateral RLN transection. METHODS: Six Yucatan mini-pigs underwent full-thickness hemilaryngectomies with RLN transection followed by transmural reconstruction using fabricated collagen polymeric mucosal, muscle, and cartilage replacements. To determine the effect of adding therapeutic cell populations, subsets of animals received collagen muscle implants containing motor-endplate-expressing muscle progenitor cells (MEEs) and/or collagen cartilage implants containing adipose stem cell (ASC)-derived chondrocyte-like cells. Acoustic vocalization and laryngeal electromyography (L-EMG) provided functional assessments and histopathological analysis with immunostaining was used to characterize the tissue response. RESULTS: Five of six animals survived the 4-week postoperative period with weight gain, airway maintenance, and audible phonation. No tracheostomy or feeding tube was required. Gross and histological assessments of all animals revealed implant integration and regenerative remodeling of airway mucosa epithelium, muscle, and cartilage in the absence of a material-mediated foreign body reaction or biodegradation. Early voice and L-EMG data were suggestive of positive functional outcomes. CONCLUSION: Laryngeal reconstruction with collagen polymeric mucosa, muscle, and cartilage replacements may provide effective restoration of function after hemilaryngectomy with RLN transection. Future preclinical studies should focus on long-term functional outcomes. LEVEL OF EVIDENCE: NA Laryngoscope, 2024.

Scaffold-Forming Collagen and Motor-Endplate Expressing Muscle Cells for Porcine Laryngoplasty.

OBJECTIVE: Vocal fold paralysis impairs quality of life, and no curative injectable therapy exists. We evaluated injection of a novel in situ polymerizing (scaffold-forming) collagen in the presence and absence of muscle-derived motor-endplate expressing cells (MEEs) to promote medialization and recurrent laryngeal nerve (RLN) regeneration in a porcine model of unilateral vocal fold paralysis. METHODS: Twelve Yucatan minipigs underwent right RLN transection. Autologous muscle progenitor cells were isolated from muscle biopsies, differentiated, and induced to MEEs. Three weeks after RLN injury, animals received injections of collagen, collagen containing MEEs, or saline into the paralyzed right vocal fold. Stimulated laryngeal electromyography and acoustic vocalization were used for function assessments. Larynges were harvested and underwent histologic, gene expression, and further quantitative analyses. RESULTS: Injections were well-tolerated, with the collagen scaffold showing immunotolerance and collagen-encapsulated MEEs remaining viable. Collagen-treated paralyzed vocal folds showed increased laryngeal adductor muscle volumes relative to that of the uninjured side, with those receiving MEEs and collagen showing the highest volumes. Muscles injected with MEEs and collagen demonstrated increased expression of select neurotrophic (BDNF and NTN1), motor-endplate (DOK7, CHRNA1, and MUSK), and myogenic (MYOG and MYOD) related genes relative to saline controls. CONCLUSION: In a porcine model of unilateral vocal fold paralysis, injection of in situ polymerizing collagen in the absence and presence of MEEs enhanced laryngeal adductor muscle volume, modulated expression of neurotrophic and myogenic factors, and avoided adverse material-mediated immune responses. Further study is needed to determine long-term functional outcomes with this novel therapeutic approach. LEVEL OF EVIDENCE: NA Laryngoscope, 2024.

Nerve Conduction Studies of the Larynx: Porcine Model and Human Application.

OBJECTIVE: There is a paucity of information in the literature about how electromyography (EMG) with nerve conduction studies (NCS) can be utilized in the field of neurolaryngology. The goal of this study was two-part: (1) to identify the NCS test parameters that best reflect underlying neurolaryngeal pathophysiology in a porcine model, and (2) to determine if comparable NCS parameters can be used to assess clinical laryngeal denervation injuries in patients. METHODS: Yukatan minipigs underwent general anesthesia with EMG and recurrent laryngeal NCS assessment. Recurrent laryngeal nerve (RLN) transection injury and uninjured RLN cases were included to determine NCS neuropathologic and normative findings, respectively. Our porcine analytic model was then applied to a clinical scenario of seven patients with RLN injury. RESULTS: The porcine model was able to differentiate and characterize normative and neuropathologic NCS changes. Clinical outcomes suggest that RLN NCS can be performed clinically and is overall well tolerated by in patients. Findings may serve as a useful tool to differentiate normal nerve function, neuropraxia, axonotmesis, and irreversible RLN injuries. CONCLUSION: This novel study is the first to demonstrate the feasibility and applicability of EMG with NCS in the evaluation of vocal fold function in a porcine model in addition to the clinical setting. Further studies are needed to better understand the clinical utility and predictive value in recovery of vocal fold paralysis.

Early Changes in Porcine Larynges Following Injection of Motor-Endplate Expressing Muscle Cells for the Treatment of Unilateral Vocal Fold Paralysis.

OBJECTIVES: No curative injectable therapy exists for unilateral vocal fold paralysis. Herein, we explore the early implications of muscle-derived motor-endplate expressing cells (MEEs) for injectable vocal fold medialization after recurrent laryngeal nerve (RLN) injury. METHODS: Yucatan minipigs underwent right RLN transection (without repair) and muscle biopsies. Autologous muscle progenitor cells were isolated, cultured, differentiated, and induced to form MEEs. Three weeks after the injury, MEEs or saline were injected into the paralyzed right vocal fold. Outcomes including evoked laryngeal electromyography (LEMG), laryngeal adductor pressure, and acoustic vocalization data were analyzed up to 7 weeks post-injury. Harvested porcine larynges were examined for volume, gene expression, and histology. RESULTS: MEE injections were tolerated well, with all pigs demonstrating continued weight gain. Blinded analysis of videolaryngoscopy post-injection revealed infraglottic fullness, and no inflammatory changes. Four weeks after injection, LEMG revealed on average higher right distal RLN activity retention in MEE pigs. MEE-injected pigs on average had vocalization durations, frequencies, and intensities higher than saline pigs. Post-mortem, the MEE-injected larynges revealed statistically greater volume on quantitative 3D ultrasound, and statistically increased expression of neurotrophic factors (BDNF, NGF, NTF3, NTF4, NTN1) on quantitative PCR. CONCLUSIONS: Minimally invasive MEE injection appears to establish an early molecular and microenvironmental framework to encourage innate RLN regeneration. Longer follow-up is needed to determine if early findings will translate into functional contraction. LEVEL OF EVIDENCE: NA Laryngoscope, 134:272-282, 2024.

Low host immune pressure may be associated with the development of hepatocellular carcinoma: a longitudinal analysis of complete genomes of the HBV 1762T, 1764A mutant.

Background: It has been reported that hepatitis B virus (HBV) double mutations (A1762T, G1764A) are an aetiological factor of hepatocellular carcinoma (HCC). However, it is unclear who is prone to develop HCC, among those infected with the mutant. Exploring HBV quasispecies, which are strongly influenced by host immune pressure, may provide more information about the association of viral factors and HCC. Materials and methods: Nine HCC cases and 10 controls were selected from the Long An cohort. Serum samples were collected in 2004 and 2019 from subjects with HBV double mutations and the complete genome of HBV was amplified and sequenced using next-generation sequencing (NGS). Results: The Shannon entropy values increased from 2004 to 2019 in most cases and controls. There was no significant difference in mean intrahost quasispecies genetic distances between cases and controls. The change in the values of mean intrahost quasispecies genetic distances of the controls between 2004 and 2019 was significantly higher than that of the cases (P<0.05). The viral loads did not differ significantly between cases and controls in 2004(p=0.086) but differed at diagnosed in 2019 (p=0.009). Three mutations occurring with increasing frequency from 2004 to 2019 were identified in the HCC cases, including nt446 C→G, nt514 A→C and nt2857T→C. Their frequency differed significantly between the cases and controls (P<0.05). Conclusions: The change in the values of mean intrahost quasispecies genetic distances in HCC was smaller, suggesting that HBV in HCC cases may be subject to low host immune pressure. Increasing viral loads during long-term infection are associated with the development of HCC. The novel mutations may increase the risk for HCC.

Impact of Needle Selection on Survival of Muscle-Derived Cells When Used for Laryngeal Injections.

Objective: To describe how differing injector needles and delivery vehicles impact Autologous Muscle-Derived Cell (AMDC) viability when used for laryngeal injection. Methods: In this study, adult porcine muscle tissue was harvested and used to create AMDC populations. While controlling cell concentration (1 × 107 cells/ml), AMDCs including Muscle Progenitor Cells (MPCs) or Motor Endplate Expressing Cells (MEEs) were suspended in either phosphate-buffered saline or polymerizable (in-situ scaffold forming) type I oligomeric collagen solution. Cell suspensions were then injected through 23- and 27-gauge needles of different lengths at the same rate (2 ml/min) using a syringe pump. Cell viability was measured immediately after injection and 24- and 48-hours post-injection, and then compared to baseline cell viability prior to injection. Results: The viability of cells post-injection was not impacted by needle length or needle gauge but was significantly impacted by the delivery vehicle. Overall, injection of cells using collagen as a delivery vehicle maintained the highest cell viability. Conclusion: Needle gauge, needle length, and delivery vehicle are important factors that can affect the viability of injected cell populations. These factors should be considered and adapted to improve injectable MDC therapy outcomes when used for laryngeal applications.

PTP4A2 promotes lysophagy by dephosphorylation of VCP/p97 at Tyr805.

Overexpression of PTP4A phosphatases are associated with advanced cancers, but their biological functions are far from fully understood due to limited knowledge about their physiological substrates. VCP is implicated in lysophagy via collaboration with specific cofactors in the ELDR complex. However, how the ELDR complex assembly is regulated has not been determined. Moreover, the functional significance of the penultimate and conserved Tyr805 phosphorylation in VCP has not been established. Here, we use an unbiased substrate trapping and mass spectrometry approach and identify VCP/p97 as a bona fide substrate of PTP4A2. Biochemical studies show that PTP4A2 dephosphorylates VCP at Tyr805, enabling the association of VCP with its C-terminal cofactors UBXN6/UBXD1 and PLAA, which are components of the ELDR complex responsible for lysophagy, the autophagic clearance of damaged lysosomes. Functionally, PTP4A2 is required for cellular homeostasis by promoting lysophagy through facilitating ELDR-mediated K48-linked ubiquitin conjugate removal and autophagosome formation on the damaged lysosomes. Deletion of Ptp4a2 in vivo compromises the recovery of glycerol-injection induced acute kidney injury due to impaired lysophagy and sustained lysosomal damage. Taken together, our data establish PTP4A2 as a critical regulator of VCP and uncover an important role for PTP4A2 in maintaining lysosomal homeostasis through dephosphorylation of VCP at Tyr805. Our study suggests that PTP4A2 targeting could be a potential therapeutic approach to treat cancers and other degenerative diseases by modulating lysosomal homeostasis and macroautophagy/autophagy.Abbreviations: AAA+: ATPases associated with diverse cellular activities; AKI: acute kidney injury; CBB: Coomassie Brilliant Blue; CRISPR: clustered regularly interspaced short palindromic repeats; ELDR: endo-lysosomal damage response; GFP: green fluorescent protein; GST: glutathione S-transferase; IHC: immunohistochemistry; IP: immunoprecipitation; LAMP1: lysosomal-associated membrane protein 1; LC-MS: liquid chromatography-mass spectrometry; LGALS3/Gal3: galectin 3; LLOMe: L-leucyl-L-leucine methyl ester; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MEF: mouse embryonic fibroblast; PLAA: phospholipase A2, activating protein; PTP4A2: protein tyrosine phosphatase 4a2; PUB: NGLY1/PNGase/UBA- or UBX-containing protein; PUL: PLAP, Ufd3, and Lub1; TFEB: transcription factor EB; UBXN6/UBXD1: UBX domain protein 6; UPS: ubiquitin-proteasome system; VCP/p97: valosin containing protein; VCPIP1: valosin containing protein interacting protein 1; YOD1: YOD1 deubiquitinase.

Eliciting and Characterizing Porcine Vocalizations: When Pigs Fly.

BACKGROUND/OBJECTIVES: While voice-related therapeutic interventions are often researched preclinically in the porcine model, there are no well-established methods to induce porcine glottic phonation. Described approaches, such as training animals to phonate for positive reinforcement are time-consuming and plagued by inherent variability in the type of phonation produced and contamination of background noise. Thus, a reliable method of assessing glottic phonation in the porcine model is needed. METHODS: In this study, we have created a novel pulley-based apparatus with harness for "pig-lifting" with surrounding acoustic insulation and high-directional microphone with digital recorder for recording phonation. Praat and Matlab were used to analyze all porcine vocalizations for fundamental frequency (F0), intensity, duration of phonation and cepstral peak prominence (CPP). Glottic phonation was detected using F0 (≥2000 hz), duration (≥3 seconds) and researcher perceptual judgment. Partial-glottic phonations were also analyzed. Reliability between researcher judgment and acoustic measures for glottic phonation detection was high. RESULTS: Acoustic analysis demonstrated that glottic and partial-glottic phonation was consistently elicited, with no formal training of the minipigs required. Glottic vocalizations increased with multiple lifts. Glottic phonation continued to be elicited after multiple days but became less frequent. Glottic and partial-glottic phonations had similar CPP values over the 6 experimental days. CONCLUSION: Our cost-effective, reliable method of inducing and recording glottic phonation in the porcine model may provide a cost effective, preclinical tool in voice research.

Analysis of entire hepatitis B virus genomes reveals reversion of mutations to wild type in natural infection, a 15 year follow-up study.

It has been reported that some mutations in the genome of hepatitis B virus (HBV) may predict the outcome of the virus infection. However, evolutionary data derived from long-term longitudinal analysis of entire HBV genomes using next generation sequencing (NGS) remain rare. In this study, serum samples were collected from asymptomatic hepatitis B surface antigen (HBsAg) carriers from a long-term prospective cohort. The entire HBV genome was amplified by polymerase chain reaction (PCR) and sequenced using NGS. Twenty-eight time series serum samples from nine subjects were successfully analysed. The Shannon entropy (Sn) ranged from 0 to 0.89, with a median value of 0.76, and the genetic diversity (D) ranged from 0 to 0.013, with a median value of 0.004. Intrahost HBV viral evolutionary rates ranged from 2.39E-04 to 3.11E-03. Double mutations at nt1762(A â†’ T) and 1764(G â†’ A) and a stop mutation at nt1896(G â†’ A) were seen in all sequences from subject BO129 in 2007. However, in 2019, most sequences were wild type at these positions. Deletions between nt 2920-3040 were seen in all sequences from subject TS115 in 2007 and 2013 but these were not present in 2004 or 2019. Some sequences from subject CC246 had predicted escape substitutions (T123N, G145R) in the surface protein in 2004, 2013 and 2019 but none of the sequences from 2007 had these changes. In conclusion, HBV mutations may revert to wild type in natural infection. Clinicians should be wary of predicting long-term prognoses on the basis of the presence of mutations.

Infection with Hepatitis B Virus May Increase the Serum Concentrations of Osteopontin.

BACKGROUND: Serum osteopontin (OPN) concentrations were found to be significantly increased in patients infected with hepatitis B virus (HBV) and patients with hepatocellular carcinoma (HCC). OBJECTIVE: The aim of this study was to determine the association among HCC, OPN, and HBV. METHODS: Two hundred and forty-one subjects were recruited and divided into 6 groups: healthy controls, asymptomatic HBsAg carriers, HBsAg (-) patients with other tumors, HBsAg (+) chronic liver disease patients, HBsAg (+) patients with HCC, and HBsAg (-) patients with HCC or liver cirrhosis (LC). Serum concentrations of OPN and HBsAg were measured and analyzed. RESULTS: OPN concentrations in the HBsAg (+) HCC group were significantly higher than the healthy control group and the HBsAg (-) patients with other cancers (both p = 0.0001). The OPN concentrations of the HBsAg (-) patients with HCC or LC also did not differ significantly from those of the healthy control group (p = 0.075). There is a correlation between the titer of HBsAg and concentrations of OPN in all 3 HBsAg (+) groups (all p values <0.05). CONCLUSIONS: Infection with HBV may increase the serum concentrations of OPN. The association of OPN and HCC may be not attributable to tumor development per se but, rather, to HBV infection.

Laryngeal Reconstruction Using Tissue-Engineered Implants in Pigs: A Pilot Study.

OBJECTIVE/HYPOTHESIS: There are currently no treatments available that restore dynamic laryngeal function after hemilaryngectomy. We have shown that dynamic function can be restored post hemilaryngectomy in a rat model. Here, we report in a first of its kind, proof of concept study that this previously published technique is scalable to a porcine model. STUDY DESIGN: Animal study. METHODS: Muscle and fat biopsies were taken from three Yucatan minipigs. Muscle progenitor cells (MPCs) and adipose stem cells (ASCs) were isolated and cultured for 3 weeks. The minipigs underwent a left laterovertical partial laryngectomy sparing the left arytenoid cartilage and transecting the recurrent laryngeal nerve. Each layer was replaced with a tissue-engineered implant: 1) an acellular mucosal layer composed of densified Type I oligomeric collagen, 2) a skeletal muscle layer composed of autologous MPCs and aligned oligomeric collagen differentiated and induced to express motor endplates (MEE), and 3) a cartilage layer composed of autologous ASCs and densified oligomeric collagen differentiated to cartilage. Healing was monitored at 2 and 4 weeks post-op, and at the 8 week study endpoint. RESULTS: Animals demonstrated appropriate weight gain, no aspiration events, and audible phonation. Video laryngoscopy showed progressive healing with vascularization and re-epithelialization present at 4 weeks. On histology, there was no immune reaction to the implants and there was complete integration into host tissue with nerve and vascular ingrowth. CONCLUSIONS: This pilot study represents a first in which a transmural vertical partial laryngectomy was performed and successfully repaired with a customized, autologous stem cell-derived multi-layered tissue-engineered implant. LEVEL OF EVIDENCE: NA Laryngoscope, 131:2277-2284, 2021.

Seroconversion of hepatitis B surface antigen among those with previously successful immune response in Southern China.

Recommendations promoted worldwide have suggested a period of protection lasting more than 20 years against hepatitis B (HB) following primary immunization. Starting in 1987, universal HB vaccinations were carried out in Long An County, Guangxi Province, one of the earliest counties in which plasma-derived HB vaccine was delivered to newborns across China. Data collection targeted toward understanding the long term (26-33 years since primary immunization) immune effects of the plasma-derived HB vaccine was conducted in 2015; a second data collection was carried out in 2019 to assess seroconversion in the same cohort. This study qualitatively compared positive vs negative results and quantitatively assessed HB biomarkers - HB surface antigen (HBsAg), antibody to HBsAg (anti-HBs), HB e-antigen (HBeAg), antibody to HBeAg (anti-HBe), and antibody to HB core antigen (anti-HBc) - in serum 26-33 years after the full initial course of HB vaccination, then analyzed anti-HBs seroconversion using the two-phase sampling method in the same cohort and calculated the anti-HBs seroconversion rate from 2015 to 2019. The protective sero-conversion rate (anti-HBs ≥10mIU/mL) was 37.6% (192/511); the HBsAg-positive rate was 5.3% (27/511); the anti-HBs mean geometric titer (GMT) was 11.1 mIU/mL. Among the 143 participants involved in both 2015 and 2019 data collections, the seroconversion rate was 3.5% (5/143); two individuals had protective anti-HBs levels in 2015. These findings indicate that anti-HBs status can be seroconverted to a protective concentration level 4 years earlier in a high HBV epidemic region. The role of genomic mutations and the disappearance of immune memory and seroconversion should be investigated.

Role of intraganglionic transmission in the trigeminovascular pathway.

Migraine is triggered by poor air quality and odors through unknown mechanisms. Activation of the trigeminovascular pathway by environmental irritants may occur via activation of transient receptor potential ankyrin 1 (TRPA1) receptors on nasal trigeminal neurons, but how that results in peripheral and central sensitization is unclear. The anatomy of the trigeminal ganglion suggests that noxious nasal stimuli are not being transduced to the meninges by axon reflex but likely through intraganglionic transmission. Consistent with this concept, we injected calcitonin gene-related peptide, adenosine triphosphate, or glutamate receptor antagonists or a gap junction channel blocker directly and exclusively into the trigeminal ganglion and blocked meningeal blood flow changes in response to acute nasal TRP agonists. Previously, we observed chronic sensitization of the trigeminovascular pathway after acrolein exposure, a known TRPA1 receptor agonist. To explore the mechanism of this sensitization, we utilized laser dissection microscopy to separately harvest nasal and meningeal trigeminal neuron populations in the absence or presence of acrolein exposure. mRNA levels of neurotransmitters important in migraine were then determined by reverse transcription polymerase chain reaction. TRPA1 message levels were significantly increased in meningeal cell populations following acrolein exposure compared to room air exposure. This was specific to TRPA1 message in meningeal cell populations as changes were not observed in either nasal trigeminal cell populations or dorsal root ganglion populations. Taken together, these data suggest an important role for intraganglionic transmission in acute activation of the trigeminovascular pathway. It also supports a role for upregulation of TRPA1 receptors in peripheral sensitization and a possible mechanism for chronification of migraine after environmental irritant exposure.

Motor endplate-expressing cartilage-muscle implants for reconstruction of a denervated hemilarynx.

OBJECTIVE: Tissue engineering of the larynx requires a complex, multiple tissue layer design. Additionally, spontaneous reinnervation of the larynx after recurrent laryngeal nerve (RLN) injury is often disorganized, resulting in subpar function. This study investigates use of tissue-engineered cartilage and motor endplate-expressing (MEE) tissue-engineered skeletal muscle implants for laryngeal reconstruction and the promotion of organized reinnervation after RLN injury. METHODS: F344 rat primary muscle progenitor cells (MPCs) were isolated. Three-dimensional muscle constructs were created by encapsulating MPCs in type I oligomeric collagen under passive tension. Constructs were then cultured in differentiation medium (MPC control constructs) or induced to form motor endplates (MEE constructs) with neurotrophic agents. Three-dimensional cartilage constructs were created with adipose stem cells differentiated in chondrocyte medium. The muscle and cartilage constructs were implanted into surgically created myochondral defects in the F344 rat larynx with injured or intact (control) RLN. At 1-, 3-, and 6-month timepoints, videolaryngoscopy, electromyography (EMG), histology, and immunohistochemistry were used to assess outcomes. RESULTS: At all timepoints, cartilage-muscle implants were well integrated into host tissue. Functionally, there was increased vocal fold adduction and EMG activity in nerve-injured rats treated with the MEE constructs when compared to those treated with the MPC control constructs. Motor endplate-expressing constructs had increased myofiber cross-sectional area compared to MPC control constructs. CONCLUSION: Although our laboratory previously demonstrated that muscle and cartilage constructs could be used separately for hemilaryngeal reconstruction, this study suggests combining them with the modification of MEEs rather than MPCs, resulting in improved muscle recovery after recurrent laryngeal nerve injury. LEVEL OF EVIDENCE: NA Laryngoscope, 129:1293-1300, 2019.

Induction of chronic migraine phenotypes in a rat model after environmental irritant exposure.

Air pollution is linked to increased emergency department visits for headache and migraine patients frequently cite chemicals or odors as headache triggers, but the association between air pollutants and headache is not well understood. We previously reported that chronic environmental irritant exposure sensitizes the trigeminovascular system response to nasal administration of environmental irritants. Here, we examine whether chronic environmental irritant exposure induces migraine behavioral phenotypes. Male rats were exposed to acrolein, a transient receptor potential channel ankyrin-1 (TRPA1) agonist, or room air by inhalation for 4 days before meningeal blood flow measurements, periorbital cutaneous sensory testing, or other behavioral testing. Touch-induced c-Fos expression in trigeminal nucleus caudalis was compared in animals exposed to room air or acrolein. Spontaneous behavior and olfactory discrimination was examined in open-field testing. Acrolein inhalation exposure produced long-lasting potentiation of blood flow responses to a subsequent TRPA1 agonist and sensitized cutaneous responses to mechanical stimulation. C-Fos expression in response to touch was increased in trigeminal nucleus caudalis in animals exposed to acrolein compared with room air. Spontaneous activity in an open-field and scent preference behavior was different in acrolein-exposed compared with room air-exposed animals. Sumatriptan, an acute migraine treatment blocked acute blood flow changes in response to TRPA1 or transient receptor potential vanilloid receptor-1 agonists. Pretreatment with valproic acid, a prophylactic migraine treatment, attenuated the enhanced blood flow responses observed after acrolein inhalation exposures. Environmental irritant exposure yields an animal model of chronic migraine in which to study mechanisms for enhanced headache susceptibility after chemical exposure.

Use of autologous adipose-derived mesenchymal stem cells for creation of laryngeal cartilage.

OBJECTIVES/HYPOTHESIS: Adipose-derived mesenchymal stem cells (ASCs) are an exciting potential cell source for tissue engineering because cells can be derived from the simple excision of autologous fat. This study introduces a novel approach for tissue-engineering cartilage from ASCs and a customized collagen oligomer solution, and demonstrates that the resultant cartilage can be used for laryngeal cartilage reconstruction in an animal model. STUDY DESIGN: Basic science experimental design. METHODS: ASCs were isolated from F344 rats, seeded in a customized collagen matrix, and cultured in chondrogenic differentiation medium for 1, 2, and 4 weeks until demonstrating cartilage-like characteristics in vitro. Large laryngeal cartilage defects were created in the F344 rat model, with the engineered cartilage used to replace the cartilage defects, and the rats followed for 1 to 3 months. Staining examined cellular morphology and cartilage-specific features. RESULTS: In vitro histological staining revealed rounded chondrocyte-appearing cells evenly residing throughout the customized collagen scaffold, with positive staining for cartilage-specific markers. The cartilage was used to successfully repair large cartilaginous defects in the rat model, with excellent functional results. CONCLUSIONS: This study is the first study to demonstrate, in an animal model, that ASCs cultured in a unique form of collagen oligomer can create functional cartilage-like grafts that can be successfully used for partial laryngeal cartilage replacement. LEVEL OF EVIDENCE: NA. Laryngoscope, 128:E123-E129, 2018.

Safety assessment of xylan by a 90-day feeding study in rats.

Xylans are present naturally in various plants and have important uses in nutrition, food, novel material and biotechnology; however, to date, data regarding their systemic toxicity and safety evaluation is still limited. This study investigated the potential toxicity of xylan from sugarcane bagasse by a subchronic toxicity study in rats. A total of 80 male and female rats were fed with diets containing 10%, 5%, 2.5% and 0% (control) xylan for 90 days. A toxicological assessment was performed including mortality, body and organ weights, food consumption, blood biochemistry, hematology, urinalysis, gross necropsy and histopathological examinations. There were no signs of toxicity and treatment-related changes in rats treated with xylan. The no-observed-adverse-effect levels (NOAEL) of xylan were 9.0 g kg-1 bw for males and 10.6 g kg-1 bw for females of rats under this experimental condition, respectively.

Protein Tyrosine Phosphatase PRL2 Mediates Notch and Kit Signals in Early T Cell Progenitors.

The molecular pathways regulating lymphoid priming, fate, and development of multipotent bone marrow hematopoietic stem and progenitor cells (HSPCs) that continuously feed thymic progenitors remain largely unknown. While Notch signal is indispensable for T cell specification and differentiation, the downstream effectors are not well understood. PRL2, a protein tyrosine phosphatase that regulates hematopoietic stem cell proliferation and self-renewal, is highly expressed in murine thymocyte progenitors. Here we demonstrate that protein tyrosine phosphatase PRL2 and receptor tyrosine kinase c-Kit are critical downstream targets and effectors of the canonical Notch/RBPJ pathway in early T cell progenitors. While PRL2 deficiency resulted in moderate defects of thymopoiesis in the steady state, de novo generation of T cells from Prl2 null hematopoietic stem cells was significantly reduced following transplantation. Prl2 null HSPCs also showed impaired T cell differentiation in vitro. We found that Notch/RBPJ signaling upregulated PRL2 as well as c-Kit expression in T cell progenitors. Further, PRL2 sustains Notch-mediated c-Kit expression and enhances stem cell factor/c-Kit signaling in T cell progenitors, promoting effective DN1-DN2 transition. Thus, we have identified a critical role for PRL2 phosphatase in mediating Notch and c-Kit signals in early T cell progenitors. Stem Cells 2017;35:1053-1064.

Role of phosphatase of regenerating liver 1 (PRL1) in spermatogenesis.

The PRL phosphatases are oncogenic when overexpressed but their in vivo biological function is less well understood. Previous gene deletion study revealed a role for PRL2 in spermatogenesis. We report here the first knockout mice lacking PRL1, the most related homolog of PRL2. We found that loss of PRL1 does not affect spermatogenesis and reproductive ability of male mice, likely due to functional compensation by the relatively higher expression of PRL2 in the testes. However, PRL1-/-/PRL2+/- male mice show testicular atrophy phenotype similar to PRL2-/- mice. More strikingly, deletion of one PRL1 allele in PRL2-/- male mice causes complete infertility. Mechanistically, the total level of PRL1 and PRL2 is negatively correlated with the PTEN protein level in the testis and PRL1+/-/PRL2-/- mice have the highest level of PTEN, leading to attenuated Akt activation and increased germ cell apoptosis, effectively halting spermatozoa production. These results provide the first evidence that in addition to PRL2, PRL1 is also required for spermatogenesis by downregulating PTEN and promoting Akt signaling. The ability of the PRLs to suppress PTEN expression underscores the biochemical basis for their oncogenic potential.

Novel Anticancer Agents Based on Targeting the Trimer Interface of the PRL Phosphatase.

Phosphatase of regenerating liver (PRL) oncoproteins are phosphatases overexpressed in numerous types of human cancer. Elevated levels of PRL associate with metastasis and poor clinical outcomes. In principle, PRL phosphatases offer appealing therapeutic targets, but they remain underexplored due to the lack of specific chemical probes. In this study, we address this issue by exploiting a unique property of PRL phosphatases, namely, that they may function as homotrimers. Starting from a sequential structure-based virtual screening and medicinal chemistry strategy, we identified Cmpd-43 and several analogs that disrupt PRL1 trimerization. Biochemical and structural analyses demonstrate that Cmpd-43 and its close analogs directly bind the PRL1 trimer interface and obstruct PRL1 trimerization. Cmpd-43 also specifically blocks the PRL1-induced cell proliferation and migration through attenuation of both ERK1/2 and Akt activity. Importantly, Cmpd-43 exerted potent anticancer activity both in vitro and in vivo in a murine xenograft model of melanoma. Our results validate a trimerization-dependent signaling mechanism for PRL and offer proof of concept for trimerization inhibitors as candidate therapeutics to treat PRL-driven cancers. Cancer Res; 76(16); 4805-15. ©2016 AACR.