Prognostic and predictive significance of long interspersed nucleotide element-1 methylation in advanced-stage colorectal cancer.

BACKGROUND: Hypomethylation of Long Interspersed Nucleotide Element-1 (LINE-1) is associated with worse prognosis in colorectal cancer (CRC). However, little is known about the relevance of this marker for the prognosis and response to chemotherapy of metastatic and recurrent (advanced-stage) CRC. Our aim was therefore to investigate whether tumor LINE-1 hypomethylation correlates with patient survival and with response to 5-fluorouracil (5-FU)/ oxaliplatin (FOLFOX) chemotherapy in advanced-stage CRC. METHODS: The study included 40 CRC patients who developed metastasis or local recurrence after surgery and subsequently underwent FOLFOX therapy. Progression-free and overall survival were estimated using the Kaplan-Meier method. LINE-1 methylation levels in formalin-fixed and paraffin-embedded primary tumor tissues were measured by MethyLight assay and correlated with patient survival. In vitro analyses were also conducted with human colon cancer cell lines having different LINE-1 methylation levels to examine the effects of 5-FU and oxaliplatin on LINE-1 activity and DNA double-strand-breaks. RESULTS: Patients with LINE-1 hypomethylation showed significantly worse progression-free (median: 6.6 vs 9.4 months; P = 0.02) and overall (median: 16.6 vs 23.2 months; P = 0.01) survival following chemotherapy compared to patients with high methylation. LINE-1 hypomethylation was an independent factor for poor prognosis (P = 0.018) and was associated with a trend for non-response to FOLFOX chemotherapy. In vitro analysis showed that oxaliplatin increased the LINE-1 score in LINE-1-expressing (hypomethylated) cancer cells, thereby enhancing and prolonging the effect of 5-FU against these cells. This finding supports the observed correlation between tumor LINE-1 methylation and response to chemotherapy in CRC patients. CONCLUSIONS: Tumor LINE-1 hypomethylation is an independent marker of poor prognosis in advanced-stage CRC and may also predict non-response to combination FOLFOX chemotherapy. Prospective studies are needed to optimize the measurement of tumor LINE-1 methylation and to confirm its clinical impact, particularly as a predictive marker.

Sustained Effects of Capsaicin Infusion into the Oropharynx on Swallowing in Perfused Rats.

OBJECTIVES: To examine the sustained effects of oropharyngeal capsaicin stimulation on the regulation of swallowing, we recorded the swallowing-related nerve activities during continuous infusion of capsaicin solution into the oropharynx. METHODS: In 33 in situ perfused brainstem preparation of rats, we recorded the activities of the vagus, hypoglossal, and phrenic nerves during fictive swallowing. The interburst intervals (IBIs) of the swallowing-related nerves during sequential pharyngeal swallowing (sPSW) elicited by electrical stimulation of the superior laryngeal nerve (SLN) during concurrent capsaicin stimulation of 10, 1, and 0.1 µM (n = 28) were compared with those during oropharyngeal infusion of saline (control) (n = 5). RESULTS: The IBIs during SLN-induced sPSW were reduced at 5 min after initiation of continuous infusion of 10 and 1 µM capsaicin solution. The IBIs showed significant decreases to -25.8 ± 6.9%, -25.9 ± 5.3, -18.3 ± 3.7, and -12.0 ± 1.6 at 30 min following 1 µM capsaicin stimulation at SLN stimulus conditions at 5 Hz of 1.2 times threshold, 10 Hz of 40 µA, 5 Hz of 60 µA, and 10 Hz of 60 µA, respectively. Continuous capsaicin stimulation of 0.1 µM solution did not show significant sustained effects. CONCLUSION: Pharmacological stimulation of capsaicin could provide time-dependent effects on the likelihood of swallowing, particularly subserving sustained facilitation of swallowing reflex with appropriate concentration of capsaicin. LEVEL OF EVIDENCE: NA Laryngoscope, 134:305-314, 2024.

Transient Receptor Potential Ankyrin 1 Channel Alters Transforming Growth Factor Beta 1/Smad Signaling in Rat Vocal Fold Fibroblasts.

OBJECTIVES: Vocal fold scar remains a therapeutic challenge. Vocal fold fibroblasts (VFFs) secrete extracellular matrix (ECM), and transforming growth factor-beta 1 (TGF-ß1)-mediated fibroblast to myofibroblast differentiation is central to the development of fibrosis. The transient receptor potential (TRP) channel superfamily is a group of nonselective cation channels, and activation of TRP ankyrin 1 (TRPA1) channel has been shown to have antifibrotic effects through TGF-ß1/Smad signaling in various organs. This study aimed to elucidate expression of TRPA1 and the impact of TRPA1 activation on TGF-ß1/Smad signaling in VFFs. METHODS: Vocal folds were dissected from 10-week-old, male Sprague-Dawley rats and primary VFFs were established. TRPA1 was examined in VFFs and lamina propria via immunostaining. VFFs were treated with allyl isothiocyanate (AITC, TRP channel agonist, 10-5 M) ± TGF-ß1 (10 ng/ml) ± A-967079 (selective TRPA1 channel antagonist, 5.0 × 10-7 M) for 4 or 24 h. Trpa1, Smad3, Smad7, Col1a1, Acta2, and Has1 mRNA expression were quantified via qPCR. RESULTS: TRPA1 was expressed in cultured VFFs and the lamina propria. TGF-ß1 administration significantly increased Trpa1 compared to control. AITC alone did not alter Smad3, Smad7, Acta2, or ECM related genes. However, the combination of AITC and TGF-ß1 significantly increased Smad3 and decreased Smad7 and Acta2 compared to TGF-ß1 alone; A-967079 significantly reduced this response. CONCLUSIONS: VFFs expressed TRPA1, and the activation of TRPA1 regulated TGF-ß1/Smad signaling in VFFs. These findings provide preliminary insights into potential anti-fibrotic mechanisms of TRPA1 activation through TGF-ß1/Smad signaling in VFFs. LEVEL OF EVIDENCE: NA Laryngoscope, 134:4593-4598, 2024.

A dysphagia model with denervation of the pharyngeal constrictor muscles in guinea pigs: functional evaluation of swallowing.

Introduction: Swallowing impairment is a crucial issue that can lead to aspiration, pneumonia, and malnutrition. Animal models are useful to reveal pathophysiology and to facilitate development of new treatments for dysphagia caused by many diseases. The present study aimed to develop a new dysphagia model with reduced pharyngeal constriction during pharyngeal swallowing. Methods: We analyzed the dynamics of pharyngeal swallowing over time with the pharyngeal branches of the vagus nerve (Ph-X) bilaterally or unilaterally transected, using videofluoroscopic assessment of swallowing in guinea pigs. We also evaluated the detailed anatomy of the pharyngeal constrictor muscles after the denervation. Results: Videofluoroscopic examination of swallowing showed a significant increase in the pharyngeal area during swallowing after bilateral and unilateral sectioning of the Ph-X. The videofluoroscopy also showed significantly higher pharyngeal transit duration for bilateral and unilateral section groups. The thyropharyngeal muscle on the sectioned side was significantly thinner than that on the intact side. In contrast, the thickness of the cricopharyngeal muscles on the sectioned and intact sides were not significantly different. The mean thickness of the bilateral thyropharyngeal muscles showed a linear correlation to the pharyngeal area and pharyngeal transit duration. Discussion: Data obtained in this study suggest that denervation of the Ph-X could influence the strength of pharyngeal contraction during pharyngeal swallowing in relation to thickness of the pharyngeal constrictor muscles, resulting in a decrease in bolus speed. This experimental model may provide essential information (1) for the development of treatments for pharyngeal dysphagia and (2) on the mechanisms related to the recovery process, reinnervation, and nerve regeneration following injury and swallowing impairment possibly caused by medullary stroke, neuromuscular disease, or surgical damage from head and neck cancer.

[A case of recurrent breast cancer with multiple bone metastasis successfully treated with S-1 and zoledronic acid therapy].

For a case of recurrent breast cancer with multiple bone metastasis, an oral pyrimidine fluoride-based anti-cancer drug S -1, and zoledronic acid(a third-generation bisphosphonate formulation), were prescribed in experiments to test their efficacy. S-1 was prescribed orally at doses of 100 mg/day(twice)over a 4-week period with a cessation period of 2 weeks. Zoledronic acid was commenced with a strict administration of 4 mg every 4 weeks, taken intravenously. At the conclusion of the 1st S-1 course, the tumor markers began to improve to a certain extent with an improvement in the symptoms. Following the 16 courses, there was no abnormal accumulation detected by PET. Now, at the end of the 21st course, the treatment is being continued, and there has been no recurrence of inflammation or reoccurring growth detected. No adverse effects of more than Grade 1 occurred during the elapsed process. As a form of combined therapy, we can expect zoledronic acid to be a good anti-cancer drug because of its effectiveness and tolerability in treating recurring breast cancer.

Complementary Effect of Transcutaneous Cervical Stimulation by Interferential Current on Functional Dysphonia.

OBJECTIVES: Functional dysphonia (FD) varies in terms of vocal behavior and treatment efficacy. So-called hypofunctional dysphonia is characterized by insufficient subglottal pressure which causes a lack of driving power needed to vibrate the vocal folds leading to weak voice or aphonia in severe cases. While voice therapy is the initial treatment, some patients fail to respond to it. Interferential current (IFC) stimulation has been used as part of rehabilitation by physical therapists to reduce the progressive pain. IFC stimulation has also been developed as a laryngeal sensory stimulation device to modify the swallowing function by triggering swallowing reflex. Many researchers have shown recently in animal studies that laryngeal afferent inputs, such as vocal fold vibrations, subglottic pressure, flow rate, and vocal fold location affect vocal motor pattern and voice quality. However, IFC stimulation as a laryngeal afferent has not been verified. Herein, we assessed the effects of IFC stimulation to the neck on difficult functional dysphonia. METHODS: Six patients with refractory FD with insufficient subglottic pressure were assessed in this study. All six cases were females and two of them presented with aphonia. All cases were initially treated by voice therapy (VTx) such as flow phonation, water resistance therapy, or tube phonation for 2 months to increase subglottic pressure; however, this resulted in poor improvement in voice. We additionally performed VTx with concurrent application of IFC stimulation to the neck for 3 months, and the effects on voice were evaluated. RESULTS: VTx with IFC stimulation resulted in improved voice in all cases, demonstrating the improvement in maximum phonation time, subglottic pressure, and voice handicap index-10. CONCLUSIONS: Results from this clinical study suggest that VTx with IFC stimulation may be useful for adjusting vocal function in patients with FD caused by insufficient subglottic pressure.

Effects of Repeated Intracordal Glucocorticoid Injection on the Histology and Gene Expression of Rat Vocal Folds.

OBJECTIVES: Local injection of glucocorticoids (GCs) into the vocal folds has been used for treating the vocal fold lesions. While the positive effects on vocal fold nodules, polyps, or scarring have been clinically reported, some concern remains around the potential adverse effects such as vocal fold atrophy, and the mechanisms remain unclear. The present study examined the histology and gene expression of locally injected GC into the vocal folds in rats. METHODS: Thirteen-week-old male Sprague-Dawley rats were used in the experiments. Triamcinolone acetonide (TAA) or saline were administered repeatedly to the right vocal folds at a weekly interval, and rats were euthanized one week after the last administration for histological examination. Genetic examination was assessed hyaluronic acid (HA) metabolism at 1 or 3 days after a single TAA injection by quantitative real-time polymerase chain reaction (qRT-PCR). RESULTS: The group which underwent four TAA injections showed a significant decrease in HA in the lamina propria (LP), thickness of the LP and total cell numbers of the LP compared with the saline group. In contrast, there was no significant difference in the area of collagen accumulation and the thyroarytenoid muscle, although there was a tendency of atrophy of the muscle. After single injection of TAA, qRT-PCR showed a significant decrease in the expression of HA synthases, Has2 and Has3. CONCLUSIONS: The current animal study first demonstrates that repeated intracordal injection of GCs may lead to atrophy of vocal folds caused by decrease of deposition of HA in the LP and decrease of gene expression of Has.

Tamoxifen Alters TGF-ß1/Smad Signaling in Vocal Fold Injury.

OBJECTIVES: Effective treatments for vocal fold fibrosis remain elusive. Tamoxifen (TAM) is a selective estrogen receptor modulator and was recently reported to have antifibrotic actions. We hypothesized that TAM inhibits vocal fold fibrosis via altered transforming growth factor beta 1 (TGF-ß1) signaling. Both in vitro and in vivo approaches were employed to address this hypothesis. METHODS: In vitro, vocal fold fibroblasts were treated with TAM (10-8 or 10-9 M) ± TGF-ß1 (10 ng/ml) to quantify cell proliferation. The effects of TAM on genes related to fibrosis were quantified via quantitative real-time polymerase chain reaction. In vivo, rat vocal folds were unilaterally injured, and TAM was administered by oral gavage from pre-injury day 5 to post-injury day 7. The rats were randomized into two groups: 0 mg/kg/day (sham) and 50 mg/kg/day (TAM). Histological changes were examined on day 56 to assess tissue architecture. RESULTS: TAM (10-8 M) did not affect Smad3, Smad7, Acta2, or genes related to extracellular matrix metabolism. TAM (10-8 or 10-9 M) + TGF-ß1, however, significantly increased Smad7 and Has3 expression and decreased Col1a1 and Acta2 expression compared to TGF-ß1 alone. In vivo, TAM significantly increased lamina propria area, hyaluronic acid concentration, and reduced collagen deposition compared to sham treatment. CONCLUSIONS: TAM has antifibrotic potential via the regulation of TGF-ß1/Smad signaling in vocal fold injury. These findings provide foundational data to develop innovative therapeutic options for vocal fold fibrosis. LEVEL OF EVIDENCE: NA Laryngoscope, 133:2248-2254, 2023.

Induction of motor neuron differentiation by transduction of Olig2 protein.

Olig2 protein, a member of the basic helix-loop-helix transcription factor family, was introduced into the mouse embryonic carcinoma cell line P19 for induction of motor neuron differentiation. We show that Olig2 protein has the ability to permeate the cell membrane without the addition of a protein transduction domain (PTD), similar to other basic helix-loop-helix transcription factors such as MyoD and NeuroD2. Motor neuron differentiation was evaluated for the elongation of neurites and the expression of choline acetyltransferase (ChAT) mRNA, a differentiation marker of motor neurons. By addition of Olig2 protein, motor neuron differentiation was induced in P19 cells.

LINE-1 methylation shows little intra-patient heterogeneity in primary and synchronous metastatic colorectal cancer.

BACKGROUND: Long interspersed nucleotide element 1 (LINE-1) hypomethylation is suggested to play a role in the progression of colorectal cancer (CRC). To assess intra-patient heterogeneity of LINE-1 methylation in CRC and to understand its biological relevance in invasion and metastasis, we evaluated the LINE-1 methylation at multiple tumor sites. In addition, the influence of stromal cell content on the measurement of LINE-1 methylation in tumor tissue was analyzed. METHODS: Formalin-fixed paraffin-embedded primary tumor tissue was obtained from 48 CRC patients. Matched adjacent normal colon tissue, lymph node metastases and distant metastases were obtained from 12, 18 and 7 of these patients, respectively. Three different areas were microdissected from each primary tumor and included the tumor center and invasive front. Normal mucosal and stromal cells were also microdissected for comparison with the tumor cells. The microdissected samples were compared in LINE-1 methylation level measured by multicolor MethyLight assay. The assay results were also compared between microdissected and macrodissected tissue samples. RESULTS: LINE-1 methylation within primary tumors showed no significant intra-tumoral heterogeneity, with the tumor center and invasive front showing identical methylation levels. Moreover, no difference in LINE-1 methylation was observed between the primary tumor and lymph node and distant metastases from the same patient. Tumor cells showed significantly less LINE-1 methylation compared to adjacent stromal and normal mucosal epithelial cells. Consequently, LINE-1 methylation was significantly lower in microdissected samples compared to macrodissected samples. A trend for less LINE-1 methylation was also observed in more advanced stages of CRC. CONCLUSIONS: LINE-1 methylation shows little intra-patient tumor heterogeneity, indicating the suitability of its use for molecular diagnosis in CRC. The methylation is relatively stable during CRC progression, leading us to propose a new concept for the association between LINE-1 methylation and disease stage.

[A case of liver metastases of sigmoid colon cancer treated effectively by oral UFT/LV].

The patient was a 77-year-old woman with multiple liver metastases of sigmoid colon cancer. She underwent low anterior resection for sigmoid colon cancer. After surgery, she selected oral administration of UFT and LV for liver metastases and multiple lymph node metastases. After two courses, the liver metastases had markedly diminished. Thirty-two months later, liver metastases had disappeared on computer tomography. This therapy was continued for five years, and recurrences are no longer shown. Severe adverse effects were not observed. Oral anti-cancer drugs can serve as effective therapy for advanced colorectal cancer of old patients.

Effects of Voice Therapy for Dysphonia due to Tension Imbalance in Unilateral Vocal Fold Paralysis and Paresis.

OBJECTIVES: Medialization procedures, such as type I thyroplasty, arytenoid adduction, and vocal fold injection, are popular treatments for dysphonia due to unilateral vocal fold paralysis (UVFP). However, dysphonia occasionally persists after medialization procedures owing to tension imbalance. This tension imbalance causes diplophonia, asymmetry and aperiodic vibrational flutter in travelling wave motion. Currently, there is no established treatment for tension imbalance. We herein report two cases with residual dysphonia due to tension imbalance following medialization for chronic UVFP, and another case presenting with dysphonia due to tension imbalance following chronic unilateral vocal fold paresis. METHODS: Three patients underwent voice therapy using flow phonation to facilitate increased airflow management in speech as well as forward oral resonance by focusing on balanced airflow. Phonatory outcomes were evaluated using stroboscopic findings, aerodynamic and acoustic measures, as well as self-rating. RESULTS: Aerodynamic assessments, acoustic findings and self-ratings improved in all three cases after voice therapy. Stroboscopic findings prior to voice therapy showed asymmetric vibration with glottic gap, which was improved after voice therapy. Fundamental frequency (F0) also increased post-therapy. CONCLUSIONS: In a previous canine study, it was shown that enhanced breath support with expiratory airflow resulted in increased F0, suggesting that enhanced breath support could increase vocal fold tension. The increased F0 achieved in the present cases following voice therapy may increase vocal fold tension with breath support. Thus, voice therapy using flow phonation may be effective for supporting vocal fold tension and improving dysphonia due to tension imbalance following UVFP and paresis.

Effect of pharmacological inhibition of the pontine respiratory group on swallowing interneurons in the dorsal medulla oblongata.

OBJECTIVES: To examine the role of neurons of the pontine respiratory group (PRG) overlapping with the Kölliker-Fuse nucleus in the regulation of swallowing, we compared the activity of swallowing motor activities and interneuron discharge in the dorsal swallowing group in the medulla before and after pharmacological inhibition of the PRG. METHODS: In 23 in situ perfused brainstem preparation of rats, we recorded the activities of the vagus (VNA), hypoglossal (HNA), and phrenic nerves (PNA), and swallowing interneurons of the dorsal medulla during fictive swallowing elicited by electrical stimulation of the superior laryngeal nerve or oral water injection. Subsequently, respiratory- and swallow-related motor activities and single unit cell discharge were assessed before and after local microinjection of the GABA-receptor agonist muscimol into the area of PRG ipsilateral to the recording sites of swallowing interneurons. RESULTS: After muscimol injection, the amplitude and duration of swallow-related VNA bursts decreased to 71.3 ± 2.84 and 68.1 ± 2.76 % during electrically induced swallowing and VNA interburst intervals during repetitive swallowing decreased. Similar effects were observed for swallowing-related HNA. The swallowing motor activity was similarly qualitatively altered during physiologically induced swallowing. All 23 neurons were changed in either discharge duration or frequency after PRG inhibition, however, the general discharge patterns in relation to the motor output remained unchanged. CONCLUSION: Descending synaptic inputs from PRG provide control of the primary laryngeal sensory gate and synaptic activity of the PRG partially determine medullary cell and cranial motor nerve activities that govern the pharyngeal stage of swallowing.

The Effects of Amniotic Membrane Transplantation on Vocal Fold Regeneration.

OBJECTIVES/HYPOTHESIS: Vocal fold (VF) scar and sulcus cause severe vocal problems, but optimal methods have not been established. Total replacement of the mucosa is required particularly for cases in which the whole lamina propria is occupied by severe fibrosis and vibratory function is totally lost. The amniotic membrane (AM) has been proven to have regenerative potential, as it contains stem cells and growth factors. The current study investigated the biocompatibility and effects of AM for regeneration of the VF mucosa. STUDY DESIGN: In vitro and in vivo studies. METHODS: Vocal fold fibroblasts (VFFs) from 13 Sprague-Dawley rats were seeded on AM and subjected to histology and immunohistochemistry, and gene expressions in the VFFs on AM were examined in in vitro study. Twelve New Zealand White rabbits were used in in vivo study. VFs were stripped down and were reconstructed with AM. The regenerative effects were examined 3 months later by histological examination. RESULTS: In vitro study indicated VFFs survived on AM and stained positively for Ki67, vimentin, and fibronectin. Gene expressions of Has1, Has2, and Hgf were significantly increased in the VFFs on AM compared with the other groups. The in vivo study indicated AM-transplanted VFs showed a significantly higher density of hyaluronic acid and lower density of collagen compared with sham VFs. CONCLUSIONS: The current preliminary study suggests biocompatibility and possible regenerative effects of AM for VFs. LEVEL OF EVIDENCE: NA Laryngoscope, 132:2017-2025, 2022.

Firing characteristics of swallowing interneurons in the dorsal medulla during physiologically induced swallowing in perfused brainstem preparation in rats.

Oropharyngeal swallowing is centrally mediated by a swallowing central pattern generator (Sw-CPG) in the medulla oblongata. The activity of the Sw-CPG depends on the sensory inputs determined by physical and chemical bolus properties. Here we investigate the sensory-motor integration during swallowing arising from different sensory sources. To do so we electrically stimulated the superior laryngeal nerve and we triggered swallowing with oral injections of distilled water or capsaicin solution and extracellularly recorded from swallowing interneurons in arterially perfused brainstem preparations of rats. We recorded the activities of 40 neurons, while monitoring the motor activities of the phrenic, vagal and hypoglossal nerves. Eighteen neurons responded to electrical stimulation of the ipsilateral superior laryngeal nerve, and 6 neurons were excited by oral fluid injection, while 16 non-respiratory neurons did not receive afferent inputs to either electrical or physiological stimuli. The cellular activities displayed by swallowing interneurons during electrical and physiological stimulation of pharyngeal and laryngeal afferent input reveal complex adaptations of the timing of firing patterns and frequencies. The modulation of neuronal activity is likely to contribute to the coordination of efficient bolus transfer during the pharyngeal stage of swallowing.

Long interspersed nuclear element-1 hypomethylation is a potential biomarker for the prediction of response to oral fluoropyrimidines in microsatellite stable and CpG island methylator phenotype-negative colorectal cancer.

We investigated the clinical value of methylation of long interspersed nuclear element-1 (LINE-1) for the prognosis of colorectal cancer (CRC) and for the survival benefit from adjuvant chemotherapy with oral fluoropyrimidines. LINE-1 methylation in tumor DNA was measured by quantitative methylation-specific PCR in 155 samples of stage II and stage III CRC. The presence of microsatellite instability and CpG island methylator phenotype (CIMP) were assessed and 131 microsatellite stable/CIMP- cases were selected for survival analysis, of which 77 patients had received postoperative adjuvant chemotherapy with oral fluoropyrimidines. The CRC cell lines were used to investigate possible mechanistic links between LINE-1 methylation and effects of 5-fluorouracil (5-FU). High LINE-1 methylation was a marker for better prognosis in patients treated by surgery alone. Patients with low LINE-1 methylation who were treated with adjuvant chemotherapy survived longer than those treated by surgery alone, suggestive of a survival benefit from the use of oral fluoropyrimidines. In contrast, a survival benefit from chemotherapy was not observed for patients with high LINE-1 methylation. The CRC cell lines treated with 5-FU showed increased expression of LINE-1 mRNA. This was associated with upregulation of the phospho-histone H2A.X in cells with low LINE-1 methylation, but not in cells with high LINE-1 methylation. The 5-FU-mediated induction of phospho-histone H2A.X, a marker of DNA damage, was inhibited by knockdown of LINE-1. These results suggest that LINE-1 methylation is a novel predictive marker for survival benefit from adjuvant chemotherapy with oral fluoropyrimidines in CRC patients. This finding could be important for achieving personalized chemotherapy.

Physiological Effects of Voice Therapy for Aged Vocal Fold Atrophy Revealed by EMG Study.

OBJECTIVES: Age-related voice changes are characterized as breathy, weak and strained, and a deterioration in vocal function in the elderly has been putatively linked to a reduced intensity of speech. They contribute to undesirable voice changes known as presbyphonia. These changes are caused by histological alterations in the lamina propria of the vocal fold mucosa and atrophy of the thyroarytenoid muscle, as well as by decreased respiratory support. There are several clinical studies on presbylarynx dysphonia showing the effectiveness of voice therapy. However, physiological changes of the presbylarynx following voice therapy have not been verified. The purpose of this prospective study was to demonstrate the clinical effectiveness of voice therapy for rehabilitating presbylarynx dysphonia, using vocal function assessments and thyroarytenoid muscular activity detection on laryngeal electromyography (LEMG). METHODS: 10 patients who were diagnosed with aged vocal fold atrophy from ages 60 to 87 years (mean age: 72 years) underwent approximately 12 weeks of voice therapy, mainly using forward-focused voice and vocal resistance training. Stroboscopic examination, aerodynamic assessment, acoustic analysis, Voice Handicap Index (VHI)-10, and LEMG were performed pre- and post-voice therapy. Vocal fold vibratory amplitude (VFVA) was measured by image analysis from the stroboscopic examinations. Turns analysis during steady phonation on LEMG was also assessed. RESULTS: Maximum phonation time, subglottic pressure, jitter, shimmer, VFVA, and VHI-10 significantly improved after voice therapy. The number of turns per second on LEMG also significantly increased. CONCLUSION: Our data suggest that voice therapy may improve vocal function and thyroarytenoid muscle activity in patients with aged vocal fold atrophy.

Thymidylate synthase locus LOH in combination with genotype has prognostic and predictive significance in colorectal cancer.

The aim of the current study was to investigate the prognostic and predictive significance of polymorphisms in the thymidylate synthase (TS) gene, alongside the loss of heterozygocity (LOH) at this gene locus in patients with colorectal cancer. Genotyping was carried out for a variable number tandem repeat (VNTR) polymorphism in the TS 5'-untranslated region, a G/C single nucleotide polymorphism (SNP) located within this VNTR, and for TS LOH status in 246 colorectal cancer and paired normal DNA samples. The results were analyzed in relation to clinicopathological features, including the prognostic and predictive significance of TS genotype in patients who underwent curative surgery. Complete VNTR, SNP and LOH information for TS was obtained in 226 cases. No significant associations were observed between normal tissue TS genotype status and clinicopathological features. LOH of TS was observed in 58% of tumor samples and was associated with poor prognosis independently of clinical stage. Cases exhibiting TS LOH were classified into the three groups of 2R/loss, 3G/loss and 3C/loss. Patients with 3C/loss genotype status had poor outcomes when treated by surgery alone, but their survival was similar to patients with other genotypes following Fluorouracil (5-FU)-based adjuvant chemotherapy. The results suggested that LOH of the TS locus may be a significant prognostic factor in colorectal cancer, with the genotype of the residual allele also demonstrating an influence on prognosis. In conclusion, LOH status should be considered when TS genotype is explored as a potential prognostic and predictive marker for 5-FU-based adjuvant chemotherapy in colorectal cancer.

Intracordal Injection of Basic Fibroblast Growth Factor in 100 Cases of Vocal Fold Atrophy and Scar.

OBJECTIVES/HYPOTHESIS: Vocal fold atrophy, scar, and sulcus reduce the vibratory function of the vocal fold mucosa, which causes severe refractory dysphonia. We have reported encouraging preliminary results using an intracordal injection of basic fibroblast growth factor (bFGF) and showed improvement in phonatory parameters and voice. The present study summarizes our experience with 100 cases of stiffened vocal folds that were treated with bFGF injections. STUDY DESIGN: Retrospective chart review with Interstitial Review Board (IRB) approval. METHODS: Local injection of bFGF was performed in 100 cases of vocal fold pathology, which included 43 cases of vocal fold atrophy, 41 cases with scar, and 16 cases with sulcus. Ten micrograms of bFGF were injected into the vocal folds under topical anesthesia 4 times in each patient. Therapeutic outcomes were examined with maximum phonation time (MPT), voice handicap index-10 (VHI-10), and GRBAS scale. RESULTS: MPT, VHI-10, and GRBAS scores significantly improved in all pathology groups. An improvement on the VHI-10 greater than five points was observed in 82% of atrophy cases, 78% of scar cases, and 67% of sulcus cases. Improvement on the VHI-10 was significantly better in the atrophy group than the scar or sulcus groups. The mild/moderate cases of scar and sulcus showed better improvement than severe cases. CONCLUSIONS: The current large case series indicates positive effects of intracordal injection of bFGF for improvement of voice with no severe adverse events. The effects appeared best for cases of atrophy, while the treatment of severe scar and sulcus requires further improvement. LEVEL OF EVIDENCE: 4 Laryngoscope, 131:2059-2064, 2021.

Anti-inflammatory and Antioxidant Effects of Japanese Herbal Medicine Kyoseihatekigan on Vocal Fold Wound Healing.

OBJECTIVES: The Japanese herbal medicine kyoseihatekigan (KHG) has been used to alleviate the symptoms of croaky voice and globus hystericus, and each of its components has anti-inflammatory and antioxidant effects. However, the mechanisms underlying these beneficial actions of KHG on the vocal folds remain largely unknown. We examined the effects of KHG on rat vocal fold wound healing and assessed its anti-inflammatory and antioxidant properties. STUDY DESIGN: Animal model. METHODS: The vocal folds of Sprague-Dawley rats were unilaterally injured under endoscopy. Rats were divided into three groups based on KHG dosing from pre injury day 4 to post injury day 3: 0 mg/kg/day (sham group), 500 mg/kg/day (1% KHG group) and 1000 mg/kg/day (2% KHG group). Histologic changes were examined to assess the degree of inflammation and oxidative stress at day 3, and fibrosis at day 56. In addition, gene expression related to pro-inflammatory cytokines and transforming growth factor-beta1 (TGF-ß1) signaling was examined by quantitative real-time polymerase chain reaction (qPCR). RESULTS: Histologic analysis showed that the 1% and 2% KHG treatments significantly decreased cell infiltration and the 4-hydroxy-2-nonenalx-immunopositive area, and increased hyaluronic acid at day 3. Both KHG treatments significantly decreased fibrosis at day 56. qPCR revealed that mRNA of interleukin-1ß and cyclooxygenase-2 were significantly suppressed at day 1 and TGF-ß1 mRNA was significantly downregulated at day 5 in both KHG groups. CONCLUSIONS: The current findings suggest that KHG has anti-inflammatory and antioxidant effects in the early phase of vocal fold wound healing, which can lead to better wound healing with less scar formation.