Laryngopharyngeal reflux (LPR) induces a differential damage effect on several anatomic sites within the larynx and hypopharynx; therefore, an in vitro model is needed for each anatomic site. This study aimed to establish a primary culture method for human laryngeal and hypopharyngeal epithelial cells derived from multiple anatomic sites. Surgical mucosa specimens were treated with a two-step enzymatic strategy to establish a primary culture. Of the 46 samples, primary cultivation was achieved successfully with 36 samples, and the positive ratio was 78.3%. In addition, flow cytometry revealed that these primary cells were epithelial cells with a purity of 94.9%. The proliferative ability was confirmed by positive staining for Ki-67. Laryngeal and hypopharyngeal epithelial cells from multiple sites exhibited similar epithelial morphology and positive cytokeratin expression. These cells can be cultured to passage 4. In summary, we successfully established the in vitro epithelial model of larynx and hypopharynx subsites, which may potentially be used as a platform for reflux research, especially for site-specific damage effect.
This study aimed to evaluate if laryngopharyngeal reflux (LPR) plays a role as a risk factor for vocal fold polyps (VFPs), and if pepsin is associated with higher oxidative DNA damage of VFPs in the presence of LPR. Thirty patients with VFPs were recruited between 2017 and 2018. Prior to surgery, a laryngoscopy was performed on all subjects to evaluate VFPs. Polyp tissue and saliva samples were obtained scrupulously. Hematoxylin-eosin staining was performed for pathologic analysis. Immunohistochemistry and ELISA were used to detect pepsin in tissue and saliva of VFP patients. 8-OHdG and p-H2AX expression was detected to measure oxidative DNA damage in tissue. DNA damage was investigated in human immortalized laryngeal epithelial cells exposed to pepsin. The pepsin concentration in saliva was significantly higher (t = 2.38, P = .024) in the pepsin positive group. There was no significant difference in pepsin expression at different sites and pathological subtypes of VFPs. The levels of 8-OHdG and p-H2AX were significantly higher in the pepsin positive group and positively correlated with the tissue expression of pepsin. The concentration of pepsin in saliva also showed a significant correlation with 8-OHdG levels. Expression of 8-OHdG and p-H2AX, and tail moment of the comet assay were elevated in human immortalized laryngeal epithelial cells following treatment with pepsin. Patients with VFPs have higher levels of oxidative DNA damage in the presence of pepsin reflux. Pepsin may induce DNA damage in laryngeal epithelial cells and participate in the pathogenesis of VFPs.
Abstract Background and aims In 2008, the International Association for the Study of Pain Special Interest Group on Neuropathic Pain (NeuPSIG) proposed a clinical grading system to help identify patients with neuropathic pain (NeP). We previously applied this classification system, along with two NeP screening tools, the painDETECT (PD-Q) and Leeds Assessment of Neuropathic Symptoms and Signs pain scale (LANSS), to identify NeP in patients with neck/upper limb pain. Both screening tools failed to identify a large proportion of patients with clinically classified NeP, however a limitation of our study was the use of a single clinician performing the NeP classification. In 2016, the NeuPSIG grading system was updated with the aim of improving its clinical utility. We were interested in field testing of the revised grading system, in particular in the application of the grading system and the agreement of interpretation of clinical findings. The primary aim of the current study was to explore the application of the NeuPSIG revised grading system based on patient records and to establish the inter-rater agreement of detecting NeP. A secondary aim was to investigate the level of agreement in detecting NeP between the revised NeuPSIG grading system and the LANSS and PD-Q. Methods In this retrospective study, two expert clinicians (Specialist Pain Medicine Physician and Advanced Scope Physiotherapist) independently reviewed 152 patient case notes and classified them according to the revised grading system. The consensus of the expert clinicians’ clinical classification was used as “gold standard” to determine the diagnostic accuracy of the two NeP screening tools. Results The two clinicians agreed in classifying 117 out of 152 patients (ICC 0.794, 95% CI 0.716–850; κ 0.62, 95% CI 0.50–0.73), yielding a 77% agreement. Compared to the clinicians’ consensus, both LANSS and PD-Q demonstrated limited diagnostic accuracy in detecting NeP (LANSS sensitivity 24%, specificity 97%; PD-Q sensitivity 53%, specificity 67%). Conclusions The application of the revised NeP grading system was feasible in our retrospective analysis of patients with neck/upper limb pain. High inter-rater percentage agreement was demonstrated. The hierarchical order of classification may lead to false negative classification. We propose that in the absence of sensory changes or diagnostic tests in patients with neck/upper limb pain, classification of NeP may be further improved using a cluster of clinical findings that confirm a relevant nerve lesion/disease, such as reflex and motor changes. The diagnostic accuracy of LANSS and PD-Q in identifying NeP in patients with neck/upper limb pain remains limited. Clinical judgment remains crucial to diagnosing NeP in the clinical practice. Implications Our observations suggest that in view of the heterogeneity in patients with neck/upper limb pain, a considerable amount of expertise is required to interpret the revised grading system. While the application was feasible in our clinical setting, it is unclear if this will be feasible to apply in primary health care settings where early recognition and timely intervention is often most needed. The use of LANSS and PD-Q in the identification of NeP in patients with neck/upper limb pain remains questionable.
The benefits of adding anti-EGFR therapy to conventional chemoradiotherapy (CRT) for nasopharyngeal carcinoma (NPC) remain uncertain, possibly because only a subgroup of patients show better outcome. To address this issue, we compared the efficacy of CRT plus cetuximab (CTX) or nimotuzumab (NTZ) to CRT alone for stage II-IVb NPC and examined possible prognostic indicators, including tumour EGFR and VEGR expression levels.This retrospective study enrolled 1812 patients at initial NPC diagnosis at Nanfang Hospital Affiliated to Southern Medical University between January 2005 and December 2015. The cetuximab or nimotuzumab plus CRT group (CRT+NTZ/CTX) and the conventional chemoradiotherapy group (CRT) were matched by propensity scoring at 1:5, yielding 282 patients at clinical stage II-IVb with 47 in the CRT+NTZ/CTX group and 235 in the CRT group.The endpoints of the present study were locoregional relapse-free survival (LRFS), distant metastasis-free survival (DMFS), overall survival (OS). Immunohistochemistry (IHC) was used to investigate EGFR and VEGF expression levels in 31 patients of the CRT+NTZ/CTX group.There were no significant differences in LRFS, DMFS and OS, haematologic toxicity reactions, and gastrointestinal reactions between CRT+NTZ/CTX and CRT groups. There was a positive correlation between EGFR and VEGF expression levels. Among CRT+NTZ/CTX patients, those with high EGFR and VEGF expression levels exhibited better DMFS.Addition of anti-EGFR to cisplatin-based CRT appears to benefit only a subset of stage II-IVb NPC patients, those with elevated EGFR and VEGF expression levels.
To evaluate the relationship between pepsin induced by laryngopharyngeal reflux and laryngeal carcinoma.Patients with vocal cord leukoplakia(n=18) and laryngeal carcinoma(n=21) encountered in Nanfang Hospital between December 2012 and April 2014 were included and sixteen healthy volunteers were recruited as control. Laryngeal biopsy specimens were taken from the patients with laryngeal carcinoma, or vocal cord leukoplakia and control subjects for the immunohistochemical study of pepsin. The correlation between pepsin expression and reflux events of 24 hour multichannel intraluminal impedance-pH monitoring (MII-pH) was analyzed.The patients with laryngeal carcinoma showed the highest expression of pepsin, followed by the patients with vocal cord leukoplakia and control subjects, with significant difference among the three groups (in strong positive expression, the constituent ratio of each group are 0/16、1/18 and 4/21, P<0.01). The presence of pepsin was associated with upright and total laryngopharyngeal acid reflux (P<0.05), including acid reflux episodes, the percentage of times that the pH was below four, the percentages of acid reflux time and average acid removal time. There was a significant correlation between the pepsin level and the esophageal acid reflux parameters (P<0.05) except supine the percentage of time that the pH was below four.Pepsin expression in laryngeal tissue increases in patients with vocal cord leukoplakia and laryngeal carcinoma, contributing to the development of laryngopharyngeal carcinogenesis.
Nearly 700 haematopoietic stem cell transplants have been performed at the Singapore General Hospital since 1985. Over the years, conditioning regimens, indications, choices of hematopoietic stem cells and transplant outcomes have evolved in tandem with advances and trends in the medical and scientific community. Such practices vary between countries and even between institutions within the same city. Up to September 2005, there were 76 nonmyeloablative transplants and 573 myeloablative transplants. In our own center, bone marrow was the chosen source of hematopoietic stem cells (HSC) in between 100% of patients between the years 1985 to 1987, but fell to 70.5 in 1995 and 11.1% in the last one year. The percentage of patients transplanted for the various illnesses was: acute leukemia 66.7% in 1985, 42.9% in 1995, and 41.9% in 2005; chronic myeloid leukemia 33.3% in 1985, 16.7% and 3.2% in 2005; multiple myeloma 0% in 1985, 7.1% in 1995 and 22.6% in 2005; lymphoma 0% in 1985, 2.4% in 1995, and 25.8% in 2005. One hundred day transplant related mortality all the patients, which was 100% in 1985 when the program started, fell to 35.7% in 1995, 22.5% in 2001, then stabilising at about 28% in the last few years, as increasingly more complex cases were taken on. For autologous transplants, however, the transplant related mortality has remained between 3 to 5%. In this presentation of the evolving trends in HSCT observed within our own institution, we also postulate on the future directions that our center and, possibly, other Asian transplant centers will take in the coming decade. Nearly 700 haematopoietic stem cell transplants have been performed at the Singapore General Hospital since 1985. Over the years, conditioning regimens, indications, choices of hematopoietic stem cells and transplant outcomes have evolved in tandem with advances and trends in the medical and scientific community. Such practices vary between countries and even between institutions within the same city. Up to September 2005, there were 76 nonmyeloablative transplants and 573 myeloablative transplants. In our own center, bone marrow was the chosen source of hematopoietic stem cells (HSC) in between 100% of patients between the years 1985 to 1987, but fell to 70.5 in 1995 and 11.1% in the last one year. The percentage of patients transplanted for the various illnesses was: acute leukemia 66.7% in 1985, 42.9% in 1995, and 41.9% in 2005; chronic myeloid leukemia 33.3% in 1985, 16.7% and 3.2% in 2005; multiple myeloma 0% in 1985, 7.1% in 1995 and 22.6% in 2005; lymphoma 0% in 1985, 2.4% in 1995, and 25.8% in 2005. One hundred day transplant related mortality all the patients, which was 100% in 1985 when the program started, fell to 35.7% in 1995, 22.5% in 2001, then stabilising at about 28% in the last few years, as increasingly more complex cases were taken on. For autologous transplants, however, the transplant related mortality has remained between 3 to 5%. In this presentation of the evolving trends in HSCT observed within our own institution, we also postulate on the future directions that our center and, possibly, other Asian transplant centers will take in the coming decade.
ObjectiveLaryngopharyngeal reflux (LPR) causes laryngopharyngeal hypersensitivity and laryngeal muscle hyperfunction, which may result in hard voice onset in patients with LPR. The purpose of this study is to examine the incidence of hard voice onset in patients with LPR and the effects of hard voice onset on objective voice function in patients with LPR.MethodsForty patients with confirmed LPR were enrolled in the LPR group, and 40 healthy subjects were enrolled in the non-LPR group. Subjects underwent laryngeal high-speed videoendoscopy, and the presence or absence of hard voice onset in each subject was determined by two experienced laryngologists based on whether glottal closure was complete or incomplete before vocal fold vibration. Based on the results, the subjects with LPR were divided into a hard voice onset group and a non-hard voice onset group. The voice onset time (VOT) was measured and compared between the hard and non-hard voice onset groups within the LPR group. Laryngeal aerodynamic assessment was also carried out on the LPR group, and subglottal pressure, phonation threshold pressure (PTP), glottal resistance, and mean flow rate were measured. The voice acoustic signals of subjects were additionally analyzed in the LPR group, and the fundamental frequency, jitter, shimmer, and noise-harmony ratio were determined. The kappa statistic, chi-square test and independent-samples t test in SPSS were used for statistical analysis.ResultsThe two laryngologists had substantial inter-rater consistency on the evaluation of hard voice onset and non-hard voice onset, with a kappa statistic of 0.71. In the LPR group, 42.5% of patients had hard voice onset (17/40), significantly more than in the non-LPR group (8/40, 20%) (P < 0.05). The VOT in the LPR group was significantly longer than in the non-LPR group (P < 0.05). Within the LPR group, the VOT, PTP and shimmer were significantly greater in the hard voice onset group than in the non-hard voice onset group (all P < 0.05). The other laryngeal aerodynamic parameters and acoustic parameters were not significantly different between the hard voice onset group and the non-hard voice onset group (P > 0.05).ConclusionChanges in vocal production may occur in LPR patients, causing them to be more susceptible to hard voice onset. The patients with hard voice onset require longer VOT and greater PTP to initiate phonation.
Primary laryngeal epithelial cells are essential to exploring the mechanisms of laryngeal and voice disorders; however, they are difficult to study and apply because of their limited life span. The purpose of this study was to develop a stable and reliable in vitro model for the comprehensive study of the pathogenesis of laryngeal and voice diseases. The pLVTHM-Bmi1 plasmid was constructed and used to immortalize primary laryngeal epithelial cells by lentiviral infection. The expressions of Bmi1, human telomerase reverse transcriptase (hTERT), p53, and pRB pathway proteins were detected by western blotting. Functional characteristics of the immortalized cell lines were verified by cell senescence β-galactosidase staining, 5-ethynyl-2'-deoxyuridine cell proliferation test, and flow cytometry. We successfully introduced Bmi into human subglottic (hSG) cells and human ventricle (hV) cells. Both the human immortalized subglottic Bmi1 (hSG-Bmi1) cell line and the human immortalized ventricle Bmi1 (hV-Bmi1) cell line maintained normal epithelial morphology and divided successfully after more than 20 culture passages. As Bmi1 was overexpressed in these cells, the expression of human telomerase reverse transcriptase (hTERT) and phosphorylated Rb increased while p16 and p21 decreased. Following Bmi1-mediated immortalization, cell senescence decreased significantly, and cell proliferation was accelerated. Tumor formation was not observed for hSG, hV, or hSG-Bmi1, and hV-Bmi1 cells in nude mice. hSG-Bmi1 cells dominated by stratified squamous epithelium and hV-Bmi1 cells dominated by columnar cells were established. The new cell lines lay a foundation for the study of the pathogenic mechanisms of laryngeal and voice diseases.
Abstract Background Since Immune response, nutritional status and Epstein–Barr Virus (EBV) DNA status have been confirmed to be relevant to the prognosis of patients with nasopharyngeal carcinoma (NPC), we believe that the combination of these factors is of great value for improving the predictive ability. LA (lymphocytes × albumin), a novel indicator, had not been studied yet in NPC. We combined it with EBV DNA and used nomograms to increase the accuracy of prognosis. Methods A total of 688 NPC patients were retrospectively reviewed and further divided into training and validation cohort randomly. Kaplan–Meier analyses were used to to distinguish the different survival outcomes. Multivariate Cox analyses were used to identify the independent prognostic factors for progression-free survival (PFS) and overall survival (OS). Calibration curves, concordance indexes (C-indexes) and decision curve analyses (DCA) were used to evaluate the nomograms’ predictive value. Results Patients with low LA and positive EBV DNA correlated with poorer 5-year PFS and OS (all P < 0.005). In multivariate Cox analyses, LA and EBV DNA were both confirmed to be independent prognostic factors for PFS and OS (all P < 0.05). Prognostic nomograms incorporating LA and EBV DNA achieved ideal C-indexes of 0.69 (95% CI: 0.65–0.73) and 0.77 (95% CI: 0.71–0.82) in the prediction of PFS and OS. Otherwise, the calibration curves and DCA curves also revealed that our nomograms had pleasant predictive power. Conclusions LA is a novel and powerful biomarker for predicting clinical outcomes in NPC. Our nomograms based on LA and EBV DNA can predict individual prognosis more accurately and effectively.
'/%3e%3cuse xlink:href='%23a' transform='rotate(23.036 .093 25.536)'/%3e%3cuse xlink:href='%23a' transform='rotate(45.87 1.273 16.18)'/%3e%3cuse xlink:href='%23a' transform='rotate(69.945 .996 12.078)'/%3e%3cuse xlink:href='%23a' transform='rotate(20.66 -19.689 31.932)'/%3e%3c/svg%3e)
'/%3e%3cuse xlink:href='%23a' transform='translate(288.889 -214.211)'/%3e%3cuse xlink:href='%23a' transform='translate(108 342.749)'/%3e%3cuse xlink:href='%23a' transform='translate(469.189 342.749)'/%3e%3c/svg%3e)
'/%3e%3c/defs%3e%3cpath fill='%23012169' d='M0 0h10080v5040H0z'/%3e%3cpath stroke='%23fff' stroke-width='.6' d='m0 0 6 3m0-3L0 3' clip-path='url(%23b)' transform='scale(840)'/%3e%3cpath stroke='%23e4002b' stroke-width='.4' d='m0 0 6 3m0-3L0 3' clip-path='url(%23c)' transform='scale(840)'/%3e%3cpath stroke='%23fff' stroke-width='840' d='M2520 0v2520M0 1260h5040'/%3e%3cpath stroke='%23e4002b' stroke-width='504' d='M2520 0v2520M0 1260h5040'/%3e%3cg fill='%23fff'%3e%3cuse xlink:href='%23d' x='2520' y='3780'/%3e%3cuse xlink:href='%23a' x='7560' y='4200'/%3e%3cuse xlink:href='%23a' x='6300' y='2205'/%3e%3cuse xlink:href='%23a' x='7560' y='840'/%3e%3cuse xlink:href='%23a' x='8680' y='1869'/%3e%3cuse xlink:href='%23e' x='8064' y='2730'/%3e%3c/g%3e%3c/svg%3e)