Regulation of Glycosylation in Bone Metabolism.

Glycosylation plays a crucial role in the maintenance of homeostasis in the body and at the onset of diseases such as inflammation, neurodegeneration, infection, diabetes, and cancer. It is also involved in bone metabolism. N- and O-glycans have been shown to regulate osteoblast and osteoclast differentiation. We recently demonstrated that ganglio-series and globo-series glycosphingolipids were essential for regulating the proliferation and differentiation of osteoblasts and osteoclasts in glycosyltransferase-knockout mice. Herein, we reviewed the importance of the regulation of bone metabolism by glycoconjugates, such as glycolipids and glycoproteins, including our recent results.

Utility of bronchoscopically obtained frozen cytology pellets for next-generation sequencing.

BACKGROUND: Next-generation sequencing (NGS) is essential for lung cancer treatment. It is important to collect sufficient tissue specimens, but sometimes we cannot obtain large enough samples for NGS analysis. We investigated the yield of NGS analysis by frozen cytology pellets using an Oncomine Comprehensive Assay or Oncomine Precision Assay. METHODS: We retrospectively enrolled patients with lung cancer who underwent bronchoscopy at Kobe University Hospital and were enrolled in the Lung Cancer Genomic Screening Project for Individualized Medicine. We investigated the amount of extracted DNA and RNA and determined the NGS success rates. We also compared the amount of DNA and RNA by bronchoscopy methods. To create the frozen cytology pellets, we first effectively collected the cells and then quickly centrifuged and cryopreserved them. RESULTS: A total of 132 patients were enrolled in this study between May 2016 and December 2022; of them, 75 were subjected to frozen cytology pellet examinations and 57 were subjected to frozen tissue examinations. The amount of DNA and RNA obtained by frozen cytology pellets was nearly equivalent to frozen tissues. Frozen cytology pellets collected by endobronchial ultrasound-guided transbronchial needle aspiration yielded significantly more DNA than those collected by transbronchial biopsy methods. (P < 0.01) In RNA content, cytology pellets were not inferior to frozen tissue. The success rate of NGS analysis with frozen cytology pellet specimens was comparable to the success rate of NGS analysis with frozen tissue specimens. CONCLUSIONS: Our study showed that frozen cytology pellets may have equivalent diagnostic value to frozen tissue for NGS analyses. Bronchial cytology specimens are usually used only for cytology, but NGS analysis is possible if enough cells are collected to create pellet specimens. In particular, the frozen cytology pellets obtained by endobronchial ultrasound-guided transbronchial needle aspiration yielded sufficient amounts of DNA. TRIAL REGISTRATION: This was registered with the University Medical Hospital Information Network in Japan (UMINCTR registration no. UMIN000052050).

A stress sensor, IRE1α, is required for bacterial-exotoxin-induced interleukin-1ß production in tissue-resident macrophages.

Cholera toxin (CT), a bacterial exotoxin composed of one A subunit (CTA) and five B subunits (CTB), functions as an immune adjuvant. CTB can induce production of interleukin-1ß (IL-1ß), a proinflammatory cytokine, in synergy with a lipopolysaccharide (LPS), from resident peritoneal macrophages (RPMs) through the pyrin and NLRP3 inflammasomes. However, how CTB or CT activates these inflammasomes in the macrophages has been unclear. Here, we clarify the roles of inositol-requiring enzyme 1 alpha (IRE1α), an endoplasmic reticulum (ER) stress sensor, in CT-induced IL-1ß production in RPMs. In RPMs, CTB is incorporated into the ER and induces ER stress responses, depending on GM1, a cell membrane ganglioside. IRE1α-deficient RPMs show a significant impairment of CT- or CTB-induced IL-1ß production, indicating that IRE1α is required for CT- or CTB-induced IL-1ß production in RPMs. This study demonstrates the critical roles of IRE1α in activation of both NLRP3 and pyrin inflammasomes in tissue-resident macrophages.

The lipid globotriaosylceramide promotes germinal center B cell responses and antiviral immunity.

Influenza viruses escape immunity owing to rapid antigenic evolution, which requires vaccination strategies that allow for broadly protective antibody responses. We found that the lipid globotriaosylceramide (Gb3) expressed on germinal center (GC) B cells is essential for the production of high-affinity antibodies. Mechanistically, Gb3 bound and disengaged CD19 from its chaperone CD81, permitting CD19 to translocate to the B cell receptor complex to trigger signaling. Moreover, Gb3 regulated major histocompatibility complex class II expression to increase diversity of T follicular helper and GC B cells reactive with subdominant epitopes. In influenza infection, elevating Gb3, either endogenously or exogenously, promoted broadly reactive antibody responses and cross-protection. These data demonstrate that Gb3 determines the affinity and breadth of B cell immunity and has potential as a vaccine adjuvant.

Globo-series Gb4 activates ERK and promotes the proliferation of osteoblasts.

OBJECTIVES: Globo-series Gb4 (globoside) is involved in the immune system and disease pathogenesis. We recently reported that systemic Gb4 deficiency in mice led to decreased bone formation due to a reduction in osteoblast number. However, it remains unclear whether Gb4 expressed in osteoblasts promotes their proliferation. Therefore, we investigated the role of Gb4 in osteoblast proliferation in vitro. METHODS: We examined osteoblast proliferation in Gb3 synthase knockout mice lacking Gb4. We investigated the effects of Gb4 synthase knockdown in the mouse osteoblast cell line MC3T3-E1 on its proliferation. Furthermore, we administered Gb4 to MC3T3-E1 cells in which Gb4 was suppressed by a glucosylceramide synthase (GCS) inhibitor and evaluated its effects on their proliferation. To elucidate the mechanisms by which Gb4 promotes osteoblast proliferation, the phosphorylated extracellular signal-regulated kinases 1 and 2 (ERK1/2) levels were measured in MC3T3-E1 cells. RESULTS: Osteoblast proliferation was lower in Gb3 synthase knockout mice lacking Gb4 than in wild-type mice. Proliferation was inhibited by Gb4 synthase knockdown in MC3T3-E1 cells. Furthermore, the administration of Gb4 to MC3T3-E1 cells, in which a GCS inhibitor suppressed Gb4, promoted their proliferation. Moreover, it increased the phosphorylated ERK1/2 levels in MC3T3-E1 cells. CONCLUSIONS: Our results suggest that Gb4 expressed in osteoblasts promotes their proliferation through ERK1/2 activation.

The lipid Gb3 promotes germinal center B cell responses and anti-viral immunity.

Influenza viruses escape immunity due to rapid antigenic evolution, which requires vaccination strategies that allow for broadly protective antibody responses. Here, we demonstrate that the lipid globotriaosylceramide (Gb3) expressed on germinal center (GC) B cells is essential for the production of high-affinity antibodies. Mechanistically, Gb3 binds and disengages CD19 from its chaperone CD81 for subsequent translocation to the B cell receptor (BCR) complex to trigger signaling. Abundance of Gb3 amplifies the PI3-kinase/Akt/Foxo1 pathway to drive affinity maturation. Moreover, this lipid regulates MHC-II expression to increase diversity of T follicular helper (Tfh) and GC B cells reactive with subdominant epitopes. In influenza infection, Gb3 promotes broadly reactive antibody responses and cross-protection. Thus, we show that Gb3 determines affinity as well as breadth in B cell immunity and propose this lipid as novel vaccine adjuvant against viral infection. One Sentence Summary: Gb3 abundance on GC B cells selects antibodies with high affinity and broad epitope reactivities, which are cross-protective against heterologous influenza infection.

Identification and Analysis of Monoclonal Antibodies with Neutralizing Activity against Diverse SARS-CoV-2 Variants.

We identified neutralizing monoclonal antibodies against severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) variants (including Omicron variants BA.5 and BA.2.75) from individuals who received two doses of mRNA vaccination after they had been infected with the D614G virus. We named them MO1, MO2, and MO3. Among them, MO1 showed particularly high neutralizing activity against authentic variants: D614G, Delta, BA.1, BA.1.1, BA.2, BA.2.75, and BA.5. Furthermore, MO1 suppressed BA.5 infection in hamsters. A structural analysis revealed that MO1 binds to the conserved epitope of seven variants, including Omicron variants BA.5 and BA.2.75, in the receptor-binding domain of the spike protein. MO1 targets an epitope conserved among Omicron variants BA.1, BA.2, and BA.5 in a unique binding mode. Our findings confirm that D614G-derived vaccination can induce neutralizing antibodies that recognize the epitopes conserved among the SARS-CoV-2 variants. IMPORTANCE Omicron variants of SARS-CoV-2 acquired escape ability from host immunity and authorized antibody therapeutics and thereby have been spreading worldwide. We reported that patients infected with an early SARS-CoV-2 variant, D614G, and who received subsequent two-dose mRNA vaccination have high neutralizing antibody titer against Omicron lineages. It was speculated that the patients have neutralizing antibodies broadly effective against SARS-CoV-2 variants by targeting common epitopes. Here, we explored human monoclonal antibodies from B cells of the patients. One of the monoclonal antibodies, named MO1, showed high potency against broad SARS-CoV-2 variants including BA.2.75 and BA.5 variants. The results prove that monoclonal antibodies that have common neutralizing epitopes among several Omicrons were produced in patients infected with D614G and who received mRNA vaccination.

Fourth mRNA vaccination increases cross-neutralizing antibody titers against SARS-CoV-2 variants, including BQ.1.1 and XBB, in a very elderly population.

BACKGROUND: Omicron variants with immune evasion have emerged, and they continue to mutate rapidly, raising concerns about the weakening of vaccine efficacy, and the very elderly populations are vulnerable to Coronavirus Disease 2019 (COVID-19). Therefore, to investigate the effect of multiple doses of mRNA vaccine for the newly emerged variants on these populations, cross-neutralizing antibody titers were examined against Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) variants, including BQ.1.1 and XBB. METHODS: Blood samples were taken from residents at four long-term care facilities in Hyogo prefecture, Japan (median age, 91 years), after 3rd (n = 67) and 4th (n = 48) mRNA vaccinations, from April to October 2022. A live virus microneutralization assay was performed to determine the neutralizing antibody titers in participants' sera. RESULTS: After 3rd vaccination, cross-neutralizing antibody prevalence against conventional (D614G) virus, Delta, Omicron BA.2, BA.5, BA.2.75, BQ.1.1, and XBB were 100%, 97%, 81%, 51%, 67%, 4%, and 21%, respectively. After 4th vaccination, the antibody positivity rates increased to 100%, 100%, 98%, 79%, 92%, 31%, and 52%, respectively. The 4th vaccination significantly increased cross-neutralizing antibody titers against all tested variants. CONCLUSION: The positivity rates for BQ.1.1 and XBB increased after 4th vaccination, although the titer value was lower than those of BA.5 and BA.2.75. Considering the rapid mutation of viruses and the efficacy of vaccines, it may be necessary to create a system that can develop vaccines suitable for each epidemic in consideration of the epidemic of the virus.

Extracellular vesicles released from ganglioside GD2-expressing melanoma cells enhance the malignant properties of GD2-negative melanomas.

Exosomes (small extracellular vesicles: EVs) have attracted increasing attention from basic scientists and clinicians since they play important roles in cell-to-cell communication in various biological processes. Various features of EVs have been elucidated regarding their contents, generation and secretion mechanisms, and functions in inflammation, regeneration, and cancers. These vesicles are reported to contain proteins, RNAs, microRNAs, DNAs, and lipids. Although the roles of individual components have been rigorously studied, the presence and roles of glycans in EVs have rarely been reported. In particular, glycosphingolipids in EVs have not been investigated to date. In this study, the expression and function of a representative cancer-associated ganglioside, GD2, in malignant melanomas was investigated. Generally, cancer-associated gangliosides have been shown to enhance malignant properties and signals in cancers. Notably, EVs derived from GD2-expressing melanomas enhanced the malignant phenotypes of GD2-negative melanomas, such as cell growth, invasion, and cell adhesion, in a dose-dependent manner. The EVs also induced increased phosphorylation of signaling molecules such as EGF receptor and focal adhesion kinase. These results suggest that EVs released from cancer-associated ganglioside-expressing cells exert many functions that have been reported as a function of these gangliosides and regulate microenvironments, including total aggravation of heterogeneous cancer tissues, leading to more malignant and advanced cancer types.

Disordered testosterone transport in mice lacking the ganglioside GM2/GD2 synthase gene.

Genetic disruption of glycosyltransferases has provided clear information on the roles of their reaction products in the body. Our group has studied the function of glycosphingolipids by genetic engineering of glycosyltransferases in cell culture and in mice, which has demonstrated both expected and unexpected results. Among these findings, aspermatogenesis in ganglioside GM2/GD2 synthase knockout mice was one of the most surprising and intriguing results. There were no sperms in testis, and multinuclear giant cells were detected instead of spermatids. Although serum levels of testosterone in the male mice were extremely low, testosterone accumulated in the interstitial tissues, including Leydig cells, and seemed not to be transferred into the seminiferous tubules or vascular cavity from Leydig cells. This was considered to be the cause of aspermatogenesis and low serum levels of testosterone. Patients with a mutant GM2/GD2 synthase gene (SPG26) showed similar clinical signs, not only in terms of the neurological aspects, but also in the male reproductive system. The mechanisms for testosterone transport by gangliosides are discussed here based on our own results and reports from other laboratories.

Possible regulation of ganglioside GD3 synthase gene expression with DNA methylation in human glioma cells.

Gangliosides are expressed in nervous systems and some neuroectoderm-derived tumors at high levels and play pivotal roles. However, mechanisms for the regulation of glycosyltransferase genes responsible for the ganglioside synthesis are not well understood. In this study, we analyzed DNA methylation patterns of promoter regions of GD3 synthase (ST8SIA1) as well as mRNA levels and ganglioside expression using human glioma cell lines. Among 5 cell lines examined, 4 lines showed changes in the expression levels of related genes after treatment with 5-aza-dC. LN319 showed up-regulation of St8sia1 and increased b-series gangliosides after 5-aza-dC treatment, and an astrocytoma cell line, AS showed high expression of ST8SIA1 and b-series gangliosides persistently before and after 5-Aza-2'-deoxycytidine treatment. Using these 2 cell lines, DNA methylation patterns of the promoter regions of the gene were analyzed by bisulfite-sequencing. Consequently, 2 regions that were methylated before 5-Aza-2'-deoxycytidine treatment were demethylated in LN319 after the treatment, while those regions were persistently demethylated in AS. These 2 regions corresponded with sites defined as promoter regions by Luciferase assay. Taken together, it was suggested that ST8SIA1 gene is regulated by DNA methylation at the promoter regions, leading to the regulation of tumor phenotypes.

Ganglioside GM3 prevents high fat diet-induced hepatosteatosis via attenuated insulin signaling pathway.

Gangliosides, sialic acid-containing glycosphingolipids, are widely involved in regulations of signal transductions to control cellular functions. It has been suggested that GM3, the simplest structure among gangliosides, is involved in insulin resistance, whereas it remains unclear whether insulin signaling diminished by GM3 actually aggravates the pathological conditions in metabolic disorders. Moreover, the functional roles of gangliosides in the regulation of insulin signaling have not yet been fully elucidated in liver or hepatocytes despite that it is one of the major insulin-sensitive organs. To understand physiological roles of GM3 in metabolic homeostasis in liver, we conducted a high fat diet (HFD) loading experiment using double knockout (DKO) mice of GM2/GD2 synthase and GD3 synthase, which lack all gangliosides except GM3, as well as wild-type (WT) mice. DKO mice were strikingly resistant to HFD-induced hepatosteatosis, and hepatic lipogenesis-related molecules including insulin signaling components were down-regulated in HFD-fed DKO. Furthermore, we established primary hepatocyte cultures from DKO and WT mice, and examined their responses to insulin in vitro. Following insulin stimulation, DKO hepatocytes expressing GM3 showed attenuated expression and/or activations in the downstream components compared with WT hepatocytes expressing GM2. While insulin stimulation induced lipogenic proteins in hepatocytes from both genotypes, their expression levels were lower in DKO than in WT hepatocytes after insulin treatment. All our findings suggest that the modified gangliosides, i.e., a shift to GM3 from GM2, might exert a suppressive effect on lipogenesis by attenuating insulin signaling at least in mouse hepatocytes, which might result in protection of HFD-induced hepatosteatosis.

Attenuation of Bone Formation through a Decrease in Osteoblasts in Mutant Mice Lacking the GM2/GD2 Synthase Gene.

The ganglioside GD1a has been reported to promote the differentiation of mesenchymal stem cells to osteoblasts in cell culture systems. However, the involvement of gangliosides, including GD1a, in bone formation in vivo remains unknown; therefore, we herein investigated their roles in GM2/GD2 synthase-knockout (GM2/GD2S KO) mice without GD1a. The femoral cancellous bone mass was analyzed using three-dimensional micro-computed tomography. A histomorphometric analysis of bone using hematoxylin and eosin (HE) and tartrate-resistant acid phosphatase was performed to examine bone formation and resorption, respectively. Calcein double labeling was also conducted to evaluate bone formation. Although no significant differences were observed in bone mass or resorption between GM2/GD2S KO mice and wild-type (WT) mice, analyses of the parameters of bone formation using HE staining and calcein double labeling revealed less bone formation in GM2/GD2S KO mice than in WT mice. These results suggest that gangliosides play roles in bone formation.

Assessment of Neutralizing Antibody Response Against SARS-CoV-2 Variants After 2 to 3 Doses of the BNT162b2 mRNA COVID-19 Vaccine.

Importance: Although 2 and 3 doses of vaccine have been implemented against the SARS-CoV-2 pandemic, the level of immunity achieved by these additional vaccinations remains unclear. Objective: To investigate the induction of neutralizing antibodies against the SARS-CoV-2 Omicron variant after 2 and 3 doses of the BNT162b2 messenger RNA (mRNA) vaccine among recipients of different ages. Design, Setting, and Participants: A cohort study was conducted from June 1, 2021, to January 12, 2022, among 82 physicians at Kobe University Hospital who had received 2 doses of the BNT162b2 mRNA vaccine. Main Outcomes and Measures: The rates of positive test results and the titers of neutralizing antibodies against the Omicron variant after 2 and 3 doses of the vaccine were compared with those against other variants and compared among 3 age groups (≤38 years [younger age group], 39-58 years [intermediate age group], and ≥59 years [older age group]). Results: A total of 82 physicians (71 men [87%]; median age, 44 years [IQR, 33-58 years]) participated; 31 (38%) were in the younger age group, 32 (39%) were in the intermediate age group, and 19 (23%) were in the older age group. At 2 months after 2 doses of the vaccine, 23 participants (28%) had neutralizing antibodies against the Omicron variant, with a titer of 1.3 (95% CI, 1.2-1.4), which was 11.8-fold (95% CI, 9.9-13.9) lower than the titer against the D614G variant and the lowest among the variants tested. Although the titer of the neutralizing antibody against the Delta variant tended to be low among the older age group (2.9 [95% CI, 2.0-4.1]), the titers of the neutralizing antibody against the Omicron variant were low among all age groups (younger age group, 1.3 [95% CI, 1.1-1.6]; intermediate age group, 1.3 (95% CI, [95% CI, 1.1-1.5]; and older age group, 1.2 [95% CI, 1.0-1.4]). At 7 months after 2 doses of the vaccine, 5 participants (6%) had the neutralizing antibody against the Omicron variant, but after the booster (third dose) vaccination, all 72 participants who received the booster had the neutralizing antibody, and the titer was 41 (95% CI, 34-49), much higher than that at 7 months after 2 doses of the vaccine (1.0 [95% CI, 1.0-1.1]). This increase in titers was observed regardless of age groups; the titers were 44 (95% CI, 32-59) among the younger age group, 44 (95% CI, 32-59) among the intermediate age group, and 30 (95% CI, 22-41) among the older age group. Conclusions and Relevance: In this cohort study of 82 Japanese participants, 2 doses of the BNT162b2 mRNA vaccine did not induce sufficient neutralizing antibody against the Omicron variant. However, booster vaccination was associated with induction of a high level of neutralizing antibodies against the Omicron variant, irrespective of the recipient's age.

Randomized single-blind comparative study of the midazolam/pethidine combination and midazolam alone during bronchoscopy.

BACKGROUND: Bronchoscopy can be a distress for the patient. There have been few studies on the combination of sedatives and opioids. The aim of this study was to demonstrate the usefulness and safety of administration of the combination of midazolam and pethidine during bronchoscopy. METHODS: In this prospective randomized single (patient)-blind study, we randomly assigned 100 patients who were scheduled to undergo bronchoscopy biopsy to receive treatment with either the midazolam/pethidine combination (combination group) or midazolam alone (midazolam group) during examinations. After the end of bronchoscopy, patients completed a questionnaire and the visual analogue scale was measured. The primary outcome was the patients' acceptance of re-examination assessed by visual analogue scale. We also assessed pain levels, vital signs, midazolam use, xylocaine use, and adverse events. Univariate analyses were performed using Fisher's exact test for categorical data, and the t-test or Mann-Whitney test was carried out for analysis of numeric data. All P-values were two-sided, and values < 0.05 were considered statistically significant. RESULTS: We analyzed 47 patients in the combination group and 49 patients in the midazolam group. The primary outcome was a good trend in the combination group, but not significantly different (3.82 ± 2.3 in combination group versus 4.17 ± 2.75 in midazolam alone, P = 0.400). In the combination group, the visual analog scale score for pain during bronchoscopy was significantly lower (1.10 ± 1.88 versus 2.13 ± 2.42, P = 0.022), and the sedation level score per the modified observer's assessment of alertness/sedation scale was significantly deeper (3.49 ± 0.98 versus 3.94 ± 1.03, P = 0.031). Maximal systolic blood pressure during testing was significantly lower (162.39 ± 23.45 mmHg versus 178.24 ± 30.24 mmHg, P = 0.005), and the number of additional administrations of midazolam was significantly lower (2.06 ± 1.45 versus 2.63 ± 1.35, P = 0.049). There were also significantly fewer adverse events (30 versus 41, P = 0.036). CONCLUSIONS: The combination uses of midazolam and pethidine for sedation resulted in significant improvements in the pain, blood pressure, additional use of midazolam, and safety during bronchoscopy among patients. TRIAL REGISTRATION: This study was registered in the University Medical Hospital Information Network in Japan (UMINCTR Registration number: UMIN000032230 , Registered: 13/April/2018).

Cross-Neutralizing Activity Against Omicron Could Be Obtained in SARS-CoV-2 Convalescent Patients Who Received Two Doses of mRNA Vaccination.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant omicron is now under investigation. We evaluated cross-neutralizing activity against omicron in coronavirus disease 2019 (COVID-19) convalescent patients (n = 23) who had received 2 doses of an mRNA vaccination (BNT162b2 or mRNA-1273). Intriguingly, after the second vaccination, the neutralizing antibody titers of subjects against SARS-CoV-2 variants, including omicron, all became seropositive, and significant fold-increases (21.1-52.0) were seen regardless of the disease severity. Our findings thus demonstrate that 2 doses of mRNA vaccination to SARS-CoV-2 convalescent patients can induce cross-neutralizing activity against omicron.

Large-scale serosurveillance of COVID-19 in Japan: Acquisition of neutralizing antibodies for Delta but not for Omicron and requirement of booster vaccination to overcome the Omicron's outbreak.

Continuous appearance of SARS-CoV-2 variants and mass vaccination have been intricately influencing on the COVID-19 situation. To elucidate the current status in Japan, we analyzed totally 2,000 sera in August (n = 1,000) and December (n = 1,000) 2021 collected from individuals who underwent a health check-up. The anti-N seropositive rate were 2.1% and 3.9% in August and December 2021, respectively, demonstrating a Delta variant endemic during that time; it was approximately twofold higher than the rate based on the PCR-based diagnosis. The anti-S seropositive rate was 38.7% in August and it reached 90.8% in December, in concordance with the vaccination rate in Japan. In the December cohort, 78.7% of the sera showed neutralizing activity against the Delta variant, whereas that against the Omicron was much lower at 36.6%. These analyses revealed that effective immunity against the Delta variant was established in December 2021, however, prompt three-dose vaccination is needed to overcome Omicron's outbreak.

Esophageal Diverticulum - Indications and Efficacy of Therapeutic Endoscopy.

Objective Esophageal diverticulum is rare, and the concomitance of esophageal motility disorders (EMDs) and the efficacy of novel endoscopic treatment have not been investigated in Japan. Methods An examination including high-resolution manometry (HRM) was performed for patients with both EMDs and epiphrenic diverticulum. EMD-related epiphrenic diverticulum and Zenker's diverticulum were treated using salvage peroral endoscopic myotomy (s-POEM) and endoscopic diverticulotomy, respectively. Results Six cases of epiphrenic diverticulum were diagnosed in this study. Among 125 patients with achalasia and spastic disorders, concomitant epiphrenic diverticulum was observed in 4 (3.2%). Of these, three showed a normal lower esophageal sphincter pressure on HRM, although gastroscopy and esophagography revealed typical findings of an impaired lower esophageal sphincter relaxation. These four patients were successfully treated with s-POEM, and the Eckardt score improved from 6.3 to 0.25 at 32.5 (range: 13-56) months of follow-up, with equivalent treatment efficacy to that observed for achalasia and spastic disorders without epiphrenic diverticulum. In contrast, the two remaining cases of epiphrenic diverticulum had normal esophageal motility. Six cases of Zenker's diverticulum were diagnosed, and endoscopic diverticulotomy was successfully performed in all. The dysphagia score decreased from 2.8 to 0.17 at 14.8 (range: 2-36) months of follow-up. Overall, 12 endoscopic treatments were performed for esophageal diverticulum; no adverse events were observed. Conclusion In epiphrenic diverticulum patients, concomitant EMDs are not rare and should be carefully diagnosed. A normal lower esophageal sphincter pressure on HRM does not always mean a normal lower esophageal sphincter relaxation. S-POEM and endoscopic diverticulotomy are effective minimally invasive treatment options for EMD-related epiphrenic diverticulum and Zenker's diverticulum.