Prognostic impact of shift to low visceral fat mass after neoadjuvant chemotherapy in patients with esophageal cancer.

BACKGROUND: Based on the JCOG1109 trial, it is suggested that the combination of docetaxel, cisplatin, and 5-fluorouracil (DCF) could potentially become a standard neoadjuvant chemotherapy regimen, alongside the conventional 5-fluorouracil and cisplatin (CF) therapy, for esophageal cancer. However, there are few reports on the impact of body composition changes associated with neoadjuvant chemotherapy on prognosis. AIM: Our study aimed to explore the effect of different neoadjuvant chemotherapy regimens on body composition during treatment and the impacts of body composition changes on their prognosis. METHODS AND RESULTS: This is a retrospective study of 215 patients with advanced thoracic esophageal cancer who had surgery after neoadjuvant chemotherapy from 2013 to 2019. Computed tomography scans were performed before and after neoadjuvant chemotherapy to assess body composition. Skeletal muscle mass index (SMI) was calculated by dividing total skeletal muscle mass at the 3rd lumbar level by the square of height, while visceral and subcutaneous fat masses were measured at the level of umbilicus. Patients in the lowest 25% of both sexes were classified into the low visceral fat and low subcutaneous fat groups, respectively. Of the patients enrolled, 178 were male and 37 were female. Among them, 91 had clinical Stage II disease, and 124 had clinical Stage III disease. Additionally, 146 patients received neoadjuvant chemotherapy CF, and 69 received neoadjuvant chemotherapy DCF. Comparing the DCF and CF groups, the DCF group consisted of significantly younger patients (p < .01), a higher proportion of males (p = .03), and a greater number of clinical Stage III cases (p < .01). However, although percent change in SMI and visceral fat mass was not significantly different between two regimens, percent change in subcutaneous fat mass was significant in the DCF group. The major prognostic factors for patients undergoing surgery after neoadjuvant chemotherapy for thoracic esophageal cancer were clinical Stage III, transition to low visceral fat, and response rating (SD/PD), while the specific neoadjuvant chemotherapy regimen did not significantly influence the outcomes. CONCLUSION: This study suggests that prevention of the shift to low visceral fat throughout the neoadjuvant chemotherapy process should improve patient outcomes.

Characterization of anisotropic pore structure and dense selective layer of capillary membranes for long-term ECMO by cross-sectional ion-milling method.

Since the COVID-19 pandemic of 2020-2023, extracorporeal membrane oxygenator (ECMO) has attracted considerable attention worldwide. It is expected that ECMO with long-term durability is put into practical use in order to prepare for next emerging infectious diseases and to facilitate manufacturing for novel medical devices. Polypropylene (PP) and polymethylpentene (PMP) capillary membranes are currently the mainstream for gas exchange membrane for ECMO. ECMO support days for COVID-19-related acute hypoxemic respiratory failure have been reported to be on average for 14 or 24 days. It is necessary to improve opposing functions such that promoting the permeation of oxygen and carbon dioxide and inhibiting the permeation of water vapor or plasma to develop sufficient durability for long-term use. For this purpose, accurately controlling the anisotropy of the pore structure of the entire cross section and functions of capillary membrane is significant. In this study, we focused on the cross-sectional ion-milling (CSIM) method, to precisely clarify the pore structure of the entire cross section of capillary membrane for ECMO, because there is less physical stress on the porous structure applied during the preparation of cross-sectional samples of porous capillary membranes. We attempted to observe the cross sections of commercially available PMP membranes using the CSIM method. As a result, we succeeded in fabricating fine-scale flat cross-sectional samples of PMP capillary membranes. The pore structures and the degree of anisotropy of the cross sections are quantitatively clarified. The achievements and the approaches of this study are being applied to the development of next-generation gas exchange membranes.

Parallel metabolic pathway engineering for aerobic 1,2-propanediol production in Escherichia coli.

The demand for the essential commodity chemical 1,2-propanediol (1,2-PDO) is on the rise, as its microbial production has emerged as a promising method for a sustainable chemical supply. However, the reliance of 1,2-PDO production in Escherichia coli on anaerobic conditions, as enhancing cell growth to augment precursor availability remains a substantial challenge. This study presents glucose-based aerobic production of 1,2-PDO, with xylose utilization facilitating cell growth. An engineered strain was constructed capable of exclusively producing 1,2-PDO from glucose while utilizing xylose to support cell growth. This was accomplished by deleting the gloA, eno, eda, sdaA, sdaB, and tdcG genes for 1,2-PDO production from glucose and introducing the Weimberg pathway for cell growth using xylose. Enhanced 1,2-PDO production was achieved via yagF overexpression and disruption of the ghrA gene involved in the 1,2-PDO-competing pathway. The resultant strain, PD72, produced 2.48 ± 0.15 g L-1 1,2-PDO with a 0.27 ± 0.02 g g-1-glucose yield after 72 h cultivation. Overall, this study demonstrates aerobic 1,2-PDO synthesis through the isolation of the 1,2-PDO synthetic pathway from the tricarboxylic acid cycle.

Efficacy of a novel traction method: outside-lesion clip-thread method for gastric endoscopic submucosal dissection of lesions of the greater curvature of the upper/middle stomach (with video).

BACKGROUND: Gastric endoscopic submucosal dissection (ESD) for lesions located on the greater curvature of the upper and middle (U/M) third of the stomach remains challenging, even for experienced endoscopists. Accordingly, we have developed a novel traction technique, termed the outside-lesion clip-thread method (O-CTM). In this method, a clip thread is attached to the healthy mucosa outside the circumferential incision line, and traction is applied to bring the scope and lesion into proximity for ESD. Here, we assessed the efficacy of ESD using the O-CTM compared to ESD without the O-CTM. METHODS: We retrospectively reviewed data from 63 consecutive patients who underwent gastric ESD for 63 lesions located on the greater curvature of the U/M third of the stomach between September 2015 and April 2024. The primary outcome was the operation time, and secondary outcomes were resection speed, en bloc resection, R0 resection and complications in the O-CTM and without O-CTM ESD groups. RESULTS: Of the 63 included lesions, 37 were resected without the O-CTM between September 2015 and June 2022 (without O-CTM group), and 26 lesions were resected using the O-CTM between July 2022 and April 2024 (O-CTM group). The O-CTM group had significantly shorter operation times (40 min vs. 77 min, p = 0.01) than the without O-CTM group. The resection speed was also significantly faster (20.1 mm2/min vs. 11.3 mm2/min, p = 0.02). No significant differences in en bloc resection rate, R0 resection rate, and complications were observed. CONCLUSIONS: Gastric ESD using O-CTM is beneficial when compared with the ESD without O-CTM in reducing operation time and improving resection speeds for treating lesions located on the greater curvature of the U/M region.

Enhanced production of isobutyl and isoamyl acetate using Yarrowia lipolytica.

Short-chain esters, particularly isobutyl acetate and isoamyl acetate, hold significant industrial value due to their wide-ranging applications in flavors, fragrances, solvents, and biofuels. In this study, we demonstrated the biosynthesis of acetate esters using Yarrowia lipolytica as a host by feeding alcohols to the yeast culture. Initially, we screened for optimal alcohol acyltransferases for ester biosynthesis in Y. lipolytica. Strains of Y. lipolytica expressing atf1 from Saccharomyces cerevisiae, produced 251 or 613 mg/L of isobutyl acetate or of isoamyl acetate, respectively. We found that introducing additional copies of ATF1 enhanced ester production. Furthermore, by increasing the supply of acetyl-CoA and refining the culture conditions, we achieved high production of isoamyl acetate, reaching titers of 3404 mg/L. We expanded our study to include the synthesis of a range of acetate esters, facilitated by enriching the culture medium with various alcohols. This study underscores the versatility and potential of Y. lipolytica in the industrial production of acetate esters.

Multicenter retrospective analysis of complications and risk factors in endoscopic resection for esophageal cancer across Japan.

BACKGROUND: Endoscopic resection (ER) is a minimally invasive treatment for esophageal cancer that sometimes causes complications. To understand the real-world incidence and risk factors for these complications, a nationwide survey was conducted across Japan. METHODS: This retrospective multicenter study included patients who underwent ER for esophageal cancer from April 2017 to March 2018 (2017 complication analysis) and April 2021 to March 2022 (2021 complication analysis). The study assessed the complication rates and conducted risk factor analyses for endoscopic submucosal dissection (ESD) using data for these patients, with exclusions based on specific criteria to ensure data accuracy. RESULTS: In the 2021 complication analysis, there were two mortalities highly likely attributable (0.03%) to ER and one mortality possibly attributable (0.01%) to ER. Intraoperative perforation, delayed bleeding, and pneumonia occurred in 137 cases (1.8%), 44 cases (0.6%), and 130 cases (1.7%), respectively. In the multivariate analysis for complications after ESD, low ER volume of the facility was an independent risk factor for perforation, while lesion location in the cervical or upper thoracic esophagus was an independent factor for reduced risk of perforation. Age ≥ 80 years was a risk factor for pneumonia, while use of traction techniques was a factor for reduced risk of pneumonia. Lesions located in the middle thoracic esophagus had a lower risk of stricture, and the risk of stricture increased as the circumferential extent of the lesion increased. CONCLUSIONS: This large-scale study provided detailed insights into the complications associated with esophageal ER and identified significant risk factors.

Amplified Type I Interferon Response in Sjögren's Disease via Ectopic Toll-Like Receptor 7 Expression in Salivary Gland Epithelial Cells Induced by Lysosome-Associated Membrane Protein 3.

OBJECTIVE: Lysosome-associated membrane protein 3 (LAMP3) misexpression in salivary gland epithelial cells plays a causal role in the development of salivary gland dysfunction and autoimmunity associated with Sjögren's disease (SjD). This study aimed to clarify how epithelial LAMP3 misexpression is induced in SjD. METHODS: To explore upstream signaling pathways associated with LAMP3 expression, we conducted multiple RNA sequencing analyses of minor salivary glands from patients with SjD, submandibular glands from a mouse model of SjD, and salivary gland epithelial cell lines. A hypothesis generated by the RNA sequencing analyses was further tested by in vitro and in vivo assays with gene manipulation. RESULTS: Transcriptome analysis suggested LAMP3 expression was associated with enhanced type I interferon (IFN) and IFNγ signaling pathways in patients with SjD. In vitro studies showed that type I IFN but not IFNγ stimulation could induce LAMP3 expression in salivary gland epithelial cells. Moreover, we discovered that LAMP3 overexpression could induce ectopic Toll-like receptor 7 (TLR-7) expression and type I IFN production in salivary gland epithelial cells both in vitro and in vivo. TLR-7 knockout mice did not develop any SjD-related symptoms following LAMP3 induction. CONCLUSION: Epithelial LAMP3 misexpression can be induced through enhanced type I IFN response in salivary glands. In addition, LAMP3 can promote type I IFN production via ectopic TLR-7 expression in salivary gland epithelial cells. This positive feedback loop can contribute to maintaining LAMP3 misexpression and amplifying type I IFN production in salivary glands, which plays an essential role in the pathophysiology of SjD.

The balance between nuclear import and export of NLRC5 regulates MHC class I transactivation.

Major histocompatibility complex (MHC) class I molecules play an essential role in regulating the adaptive immune system by presenting antigens to CD8 T cells. CITA (MHC class I transactivator), also known as NLRC5 (NLR family, CARD domain-containing 5), regulates the expression of MHC class I and essential components involved in the MHC class I antigen presentation pathway. While the critical role of the nuclear distribution of NLRC5 in its transactivation activity has been known, the regulatory mechanism to determine the nuclear localization of NLRC5 remains poorly understood. In this study, a comprehensive analysis of all domains in NLRC5 revealed that the regulatory mechanisms for nuclear import and export of NLRC5 coexist and counterbalance each other. Moreover, GCN5 (general control non-repressed 5 protein), a member of HATs (histone acetyltransferases), was found to be a key player to retain NLRC5 in the nucleus, thereby contributing to the expression of MHC class I. Therefore, the balance between import and export of NLRC5 has emerged as an additional regulatory mechanism for MHC class I transactivation, which would be a potential therapeutic target for the treatment of cancer and virus-infected diseases.

Novel Ureteral Stent Catheterization Technique for Treating Hyperchloremic Metabolic Acidosis After Total Pelvic Exenteration.

BACKGROUND/AIM: Hyperchloremic metabolic acidosis after total pelvic exenteration (TPE) is relatively rare. Urinary diversion of the ileal conduit during TPE can result in increased urine reabsorption leading to hyperchloremic metabolic acidosis. We developed a new technique for the retrograde catheterization of a ureteral stent into an ileal conduit to treat hyperchloremic metabolic acidosis. CASE REPORT: A 70-year-old man underwent TPE for locally recurrent rectal cancer. Multiple episodes of complications, such as hyperchloremia and metabolic acidosis, occurred. Effective drainage of urine from the ileal conduit is crucial. With collaboration between an endoscopist and a radiologist, we developed a novel method for retrograde catheterization of the ureteral stent into an ileal conduit for hyperchloremic metabolic acidosis after TPE. The patient's condition quickly improved after the procedure. CONCLUSION: Our novel technique of retrograde catheterization of a ureteral stent into an ileal conduit for hyperchloremic metabolic acidosis could be adopted worldwide, as it is effective and safe.

Skeletal Muscle Quality and Quantity Affect Prognosis after Neoadjuvant Chemotherapy with a Triple Regimen of Docetaxel/Cisplatin/5-FU in Patients with Esophageal Cancer.

The purpose of this study was to identify factors associated with the prognosis after docetaxel, cisplatin, and 5-fluorouracil (DCF) neoadjuvant chemotherapy (NAC) in patients with advanced esophageal squamous cell carcinoma (ESCC) undergoing surgical resection. We retrospectively examined a total of 100 patients who received neoadjuvant DCF therapy for ESCC at our institution between 2011 and 2020. The psoas muscle index (PMI) was calculated from the psoas muscle area at the L3 vertebral level, and the intramuscular adipose tissue content (IMAC) was calculated from the mean CT value of the multifidus muscle and from four points of subcutaneous fat. The median PMI value was 6.11 cm2/m2 (range, 3.12-11.07 cm2/m2) in men and 3.65 cm2/m2 (range, 2.70-6.82 cm2/m2) in women. The median IMAC was -0.426 (range, -0.079--0.968) in men and -0.359 (range, -0.079--0.671) in women. Based on the PMI, IMAC, and other patient factors, factors associated with NAC-DCF postoperative survival were identified using multivariate Cox regression analysis. A high IMAC was significantly related to overall survival after surgery (p = 0.005, hazard ratio 2.699). A comparison of Kaplan-Meier curves showed that the 5-year survival rate was 76.5% in the low IMAC group and 42.7% in the high IMAC group (log-rank test; p = 0.001). A low IMAC was associated with good survival outcomes and was an independent prognostic factor in patients with cStage II/III ESCC who were treated with the NAC-DCF regimen and underwent surgical resection.

p-Nitrobenzoate production from glucose by utilizing p-aminobenzoate N-oxygenase: AurF.

Nitroaromatic compounds are widely used in industry, but their production is associated with issues such as the hazardousness of the process and low regioselectivity. Here, we successfully demonstrated the production of p-nitrobenzoate (PNBA) from glucose by constructing p-aminobenzoate N-oxygenase AurF-expressing E. coli. We generated this strain, which we named PN-1 by disrupting four genes involved in PNBA degradation: nfsA, nfsB, nemA, and azoR. We then expressed AurF from Streptomyces thioluteus in this strain, which resulted in the production of 945 mg/L PNBA in the presence of 1 g/L p-aminobenzoate. Direct production of PNBA from glucose was achieved by co-expressing the pabA, pabB, and pabC, as well as aurF, resulting in the production of 393 mg/L PNBA from 20 g/L glucose. To improve the PNBA titer, we disrupted genes involved in competing pathways: pheA, tyrA, trpE, pykA, and pykF. The resultant strain PN-4Ap produced 975 mg/L PNBA after 72 h of cultivation. These results highlight the potential of using microorganisms to produce other nitroaromatic compounds.

Triacetic acid lactone production using 2-pyrone synthase expressing Yarrowia lipolytica via targeted gene deletion.

An environmentally sustainable world can be realized by using microorganisms to produce value-added materials from renewable biomass. Triacetic acid lactone (TAL) is a high-value-added compound that is used as a precursor of various organic compounds such as food additives and pharmaceuticals. In this study, we used metabolic engineering to produce TAL from glucose using an oleaginous yeast Yarrowia lipolytica. We first introduced TAL-producing gene 2-pyrone synthase into Y. lipolytica, which enabled TAL production. Next, we increased TAL production by engineering acetyl-CoA and malonyl-CoA biosynthesis pathways by redirecting carbon flux to glycolysis. Finally, we optimized the carbon and nitrogen ratios in the medium, culminating in the production of 4078 mg/L TAL. The strategy presented in this study had the potential to improve the titer and yield of polyketide biosynthesis.

Mediation analysis unveils a carcinogenic effect of ADH1B rs1229984 through mechanisms other than change in drinking intensity: oesophageal cancer case-control study.

BACKGROUND: Ingested alcohol is predominantly oxidized to acetaldehyde by alcohol dehydrogenase 1B (ADH1B), and acetaldehyde is further oxidized to acetate mainly by aldehyde dehydrogenase 2 (ALDH2). Although alcohol consumption is a convincing risk factor for oesophageal cancer, the role of ADH1B rs1229984 (His48Arg), the single-nucleotide polymorphism associated with slow alcohol metabolism, in oesophageal cancer development is unclear. Because this single-nucleotide polymorphism is associated with both increased risk of oesophageal cancer and drinking intensity, its association with oesophageal cancer might operate either through a direct pathway independently of drinking intensity, via an indirect pathway mediated by drinking intensity, or both. METHODS: To disentangle these different pathways, we applied a mediation analysis to an oesophageal cancer case-control study (600 cases and 865 controls) by defining the ADH1B Arg allele and alcohol consumption as exposure and mediator, respectively, and decomposed the total-effect odds ratio of the ADH1B Arg allele into direct- and indirect-effect odds ratio. RESULTS: The ADH1B Arg allele was associated with oesophageal cancer risk through pathways other than change in drinking intensity (direct-effect odds ratio, 2.03; 95% confidence interval, 1.41-2.92), in addition to the indirect pathway mediated by drinking intensity (indirect-effect odds ratio, 1.27; 95% confidence interval, 1.05-1.53). Further analyses by stratifying genotypes of ALDH2 rs671 (Glu504Lys), the functional single-nucleotide polymorphism that strongly attenuates the enzymatic activity, showed significant direct-effect odds ratio within each stratum. CONCLUSIONS: These results indicate that ADH1B Arg allele contributes to oesophageal cancer risk by slowing alcohol breakdown, in addition to its effect on the amount of alcohol consumed.

A mixed infection involving Bacteroides denticanum, Lactobacillus salivarius, and Streptococcus anginosus as causative agents of abscess around a pharyngo-esophageal anastomosis and acute vertebral osteomyelitis: Identification by ribosomal RNA sequencing of bacterial isolates.

"Bacteroides denticanum" is an anaerobic, non-spore-forming, gram-negative bacterium with a rod morphology typical of canine, ovine, and macropod oral flora. There is only one report of bloodstream infection caused by "B. denticanum" from a dog bite in human. Here, we report a case with no history of animal contact who developed an abscess caused by "B. denticanum" around a pharyngo-esophageal anastomosis after undergoing balloon dilatation procedure for stenosis following laryngectomy. The patient was a 73-year-old man with laryngeal cancer, esophageal cancer, hyperuricemia, dyslipidemia, and hypertension with a 4-week history of cervical pain, sore throat, and fever. Computed tomography showed fluid collection on the posterior pharyngeal wall. Matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) identified Bacteroides pyogenes, Lactobacillus salivarius, and Streptococcus anginosus from abscess aspiration. 16S ribosomal RNA sequencing re-identified the Bacteroides species as "B. denticanum". T2-weighted magnetic resonance images showed a high signal intensity adjacent to the anterior vertebral body of C3-C7. The diagnosis was peripharyngeal esophageal anastomotic abscess and acute vertebral osteomyelitis caused by "B. denticanum", L. salivarius, and S. anginosus. The patient was treated with sulbactam ampicillin intravenously for 14 days and then switched to oral amoxicillin with clavulanic acid for 6 weeks. To our knowledge, this is the first report of a human infection caused by "B. denticanum" without a history of animal contact. Despite remarkable advancements facilitated by MALDI-TOF MS in microbiological diagnosis, the accurate identification of novel, emerging, or uncommon microorganisms and comprehending their pathogenicity, suitable therapy, and follow up necessitate sophisticated molecular approaches.

Lysosome-Associated Membrane Protein 3 Induces Lysosome-Dependent Cell Death by Impairing Autophagic Caspase 8 Degradation in the Salivary Glands of Individuals With Sjögren's Disease.

OBJECTIVE: Lysosome-associated membrane protein 3 (LAMP3) overexpression is implicated in the development and progression of Sjögren's disease (SjD) by inducing lysosomal membrane permeabilization (LMP) and apoptotic cell death in salivary gland epithelium. The aim of this study was to clarify the molecular details of LAMP3-induced lysosome-dependent cell death and to test lysosomal biogenesis as a therapeutic intervention. METHODS: Human labial minor salivary gland biopsies were analyzed using immunofluorescence staining for LAMP3 expression levels and galectin-3 puncta formation, a marker of LMP. Expression level of caspase 8, an initiator of LMP, was determined by Western blotting in cell culture. Galectin-3 puncta formation and apoptosis were evaluated in cell cultures and a mouse model treated with glucagon-like peptide 1 receptor (GLP-1R) agonists, a known promoter of lysosomal biogenesis. RESULTS: Galectin-3 puncta formation was more frequent in the salivary glands of SjD patients compared to control glands. The proportion of galectin-3 puncta-positive cells was positively correlated with LAMP3 expression levels in the glands. LAMP3 overexpression increased caspase 8 expression, and knockdown of caspase 8 decreased galectin-3 puncta formation and apoptosis in LAMP3-overexpressing cells. Inhibition of autophagy increased caspase 8 expression, while restoration of lysosomal function using GLP-1R agonists decreased caspase 8 expression, which reduced galectin-3 puncta formation and apoptosis in both LAMP3-overexpressing cells and mice. CONCLUSION: LAMP3 overexpression induced lysosomal dysfunction, resulting in lysosome-dependent cell death via impaired autophagic caspase 8 degradation, and restoring lysosomal function using GLP-1R agonists could prevent this. These findings suggested that LAMP3-induced lysosomal dysfunction is central to disease development and is a target for therapeutic intervention in SjD.

Helicobacter pylori, Homologous-Recombination Genes, and Gastric Cancer.

BACKGROUND: Helicobacter pylori infection is a well-known risk factor for gastric cancer. However, the contribution of germline pathogenic variants in cancer-predisposing genes and their effect, when combined with H. pylori infection, on the risk of gastric cancer has not been widely evaluated. METHODS: We evaluated the association between germline pathogenic variants in 27 cancer-predisposing genes and the risk of gastric cancer in a sample of 10,426 patients with gastric cancer and 38,153 controls from BioBank Japan. We also assessed the combined effect of pathogenic variants and H. pylori infection status on the risk of gastric cancer and calculated the cumulative risk in 1433 patients with gastric cancer and 5997 controls from the Hospital-based Epidemiologic Research Program at Aichi Cancer Center (HERPACC). RESULTS: Germline pathogenic variants in nine genes (APC, ATM, BRCA1, BRCA2, CDH1, MLH1, MSH2, MSH6, and PALB2) were associated with the risk of gastric cancer. We found an interaction between H. pylori infection and pathogenic variants in homologous-recombination genes with respect to the risk of gastric cancer in the sample from HERPACC (relative excess risk due to the interaction, 16.01; 95% confidence interval [CI], 2.22 to 29.81; P = 0.02). At 85 years of age, persons with H. pylori infection and a pathogenic variant had a higher cumulative risk of gastric cancer than noncarriers infected with H. pylori (45.5% [95% CI, 20.7 to 62.6] vs. 14.4% [95% CI, 12.2 to 16.6]). CONCLUSIONS: H. pylori infection modified the risk of gastric cancer associated with germline pathogenic variants in homologous-recombination genes. (Funded by the Japan Agency for Medical Research and Development and others.).

LAMP3 transfer via extracellular particles induces apoptosis in Sjögren's disease.

Sjögren's disease (SjD) is an autoimmune disease that affects exocrine tissues and is characterized by increased apoptosis in salivary and lacrimal glands. Although the pathogenic mechanism triggering SjD is not well understood, overexpression of lysosome-associated membrane protein 3 (LAMP3) is associated with the disease in a subset of SjD patients and the development of SjD-like phenotype in mice. In this study, histological analysis of minor salivary glands of SjD patients suggested that LAMP3-containing material is being ejected from cells. Follow-on in vitro experiments with cells exposed to extracellular particles (EPs) derived from LAMP3-overexpressing cells showed increased apoptosis. Proteomics identified LAMP3 as a major component of EPs derived from LAMP3-overexpressing cells. Live-cell imaging visualized release and uptake of LAMP3-containing EPs from LAMP3-overexpressing cells to naïve cells. Furthermore, experiments with recombinant LAMP3 protein alone or complexed with Xfect protein transfection reagent demonstrated that internalization of LAMP3 was required for apoptosis in a caspase-dependent pathway. Taken together, we identified a new role for extracellular LAMP3 in cell-to-cell communication via EPs, which provides further support for targeting LAMP3 as a therapeutic approach in SjD.

Salivary gland LAMP3 mRNA expression is a possible predictive marker in the response to hydroxychloroquine in Sjögren's disease.

Hydroxychloroquine (HCQ) is a lysosomotropic agent that is commonly used for treating Sjögren's disease (SjD). However, its efficacy is controversial because of the divergent response to the drug among patients. In a subgroup of SjD patients, lysosome-associated membrane protein 3 (LAMP3) is elevated in expression in the salivary glands and promotes lysosomal dysregulation and lysosome-dependent apoptotic cell death. In this study, chloroquine (CQ) and its derivative HCQ were tested for their ability to prevent LAMP3-induced apoptosis, in vitro and on a mouse model of SjD. In addition, efficacy of HCQ treatment was retrospectively compared between high LAMP3 mRNA expression in minor salivary glands and those with LAMP3 mRNA levels comparable with healthy controls. Study results show that CQ treatment stabilized the lysosomal membrane in LAMP3-overexpressing cells via deactivation of cathepsin B, resulting in decreased apoptotic cell death. In mice with established SjD-like phenotype, HCQ treatment also significantly decreased apoptotic cell death and ameliorated salivary gland hypofunction. Retrospective analysis of SjD patients found that HCQ tended to be more effective in improving disease activity index, symptom severity and hypergammaglobulinemia in patients with high LAMP3 expression compared those with normal LAMP3 expression. Taken together, these findings suggested that by determining salivary gland LAMP3 mRNA level, a patient's response to HCQ treatment could be predicted. This finding may provide a novel strategy for guiding the development of more personalized medicine for SjD.