The balance of inflammation is critical to the repair of spinal cord injury (SCI), which is one of the most devastating traumas in human beings. Inflammatory cytokines, the direct mediators of local inflammation, have differential influences on the repair of the injured spinal cord. Some inflammatory cytokines are demonstrated beneficial to spinal cord repair in SCI models, while some detrimental. Various animal researches have revealed that local delivery of therapeutic agents efficiently regulates inflammatory cytokines and promotes repair from SCI. Quite a few clinical studies have also shown the promotion of repair from SCI through regulation of inflammatory cytokines. However, local delivery of a single agent affects only a part of the inflammatory cytokines that need to be regulated. Meanwhile, different individuals have differential profiles of inflammatory cytokines. Therefore, future studies may aim to develop personalized strategies of locally delivered therapeutic agent cocktails for effective and precise regulation of inflammation, and substantial functional recovery from SCI. It plays a crucial role in the transmission of motor and sensory information, as well as in the coordination of certain reflexes. The importance of its functions makes SCI one of the most disastrous traumas. SCI is the damage to the spinal cord that results in alterations of its functions, including loss of sensory, motor and visceral functions.The incidence of SCI varies from 3.6 to 195.4 patients per million in different countries worldwide.After SCI, a series of inflammatory responses are activated. The disruption of the blood‐spinal cord barrier (BSCB) leads to the accumulation of various inflammatory cytokines.Therapeutic agents can be used for the regulation of inflammatory cytokines to promote repair from SCI. However, the fact that the therapeutic agents have difficulty crossing the BSCB restricts the treatments of spinal cord diseases. The BSCB consists of tight junctions and capillary endothelial cells without intracellular fenestrations2TablesThe inflammatory cytokines beneficial and detrimental to SCI repair will be discussed. The criterion by which a certain inflammatory cytokine is determined to be beneficial or detrimental is the direct evidence of functional recovery in vivo. The actions of the beneficial and detrimental inflammatory cytokines are summarized in 2.12.1.1EPO, also known as hematopoietin or hemopoietin, was initially discovered as a glycoprotein hormone that controls erythropoiesis.+ cells and enhanced expression of brain‐derived neurotrophic factor (BDNF).One of the most important mechanisms of action might be apoptosis blockade and tissue preservation.EPO also limited inflammation,Neuroregeneration is another crucial effect of EPO. EPO decreased phosphacan,EPO has other beneficial effects as well, such as promoting angiogenesis and restoration of vascular integrity,2.1.2G‐CSF is a 19.6 kDa cytokineUrdzíková et al. first found that G‐CSF improved functional recovery after SCI in rats2.1.3GM‐CSF is a 14.2 kDa hematopoietic factorHa et al. first demonstrated that GM‐CSF improves functional outcome after rat contusive SCI.2.1.4IFN‐β is one of the type I interferons, which were identified and named because they “interfere” with viral infections. IFN‐β also has antiangiogenic, antiproliferative, immunomodulatory, and cell differentiation activities.IFN‐β was first demonstrated to promote functional recovery in a rat contusive SCI model.2.1.5IFN‐γ, one of the type II interferons, has the major activity of immunoregulation. It can be produced by mitogenically or antigenically stimulated lymphocytes.IFN‐γ was first found to enhance hindlimb function in a mouse contusive SCI model.2.1.6IL‐4 is an antiinflammatory cytokine produced by activated T‐lymphocytes.IL‐4 was found to regulate acute macrophage activation and confine secondary cavity formation after SCI.2.1.7IL‐10 is also an antiinflammatory cytokine. IL‐10 can be synthesized by Th2, monocytes/macrophages, astrocytes, and microgliaIL‐10 has been demonstrated to promote functional recovery in SCI models in rats2.1.8IL‐12 is a 70‐kDa heterodimeric cytokine produced by dendritic cells (DCs), macrophages, monocytes, and B cells.Yaguchi et al. demonstrated in a mouse SCI model that IL‐12, secreted from DCs, promoted functional recovery.2.1.9IL‐33 belongs to the IL‐1 cytokine family.+/CD4+ cells, and M1 polarization, increased M2 polarization and T‐regulatory cells, and induced a shift toward Th2. Thus, IL‐33 decreased demyelination and tissue loss, as well as astrogliosis.IL‐33 was shown to reduce secondary injury and improve functional recovery in a mouse contusive SCI model.2.1.10SDF‐1, also known as CXCL12, is a member of the CXC chemokine subfamily. SDF‐1 has activities in cell migration, proliferation, differentiation and survival,Zendedel et al. first corroborated that SDF‐1 improved functional recovery in a rat contusion model.2.22.2.1CXCL10, also known as interferon‐inducible protein 10 kDa (IP‐10), is a CXC chemokine. CXCL10 inhibits angiogenesisGonzalez et al. first showed that antibody neutralization of CXCL10 enhanced functional recovery in a mouse dorsal hemisection SCI model.2.2.2IL‐1 is a soluble cytokine produced by various types of cells, including monocytes and macrophages. IL‐1 has the ability to activate T cells and facilitate the host response to infection.IL‐1 receptor antagonist (IL‐1ra), a selective endogenous receptor antagonist, blocks the actions of IL‐1α and IL‐1β.There are also investigations distinguishing IL‐1α from IL‐1β. Tonai et al. found that injection of exogenous IL‐1α into the spinal cord increased production of cyclooxygenase‐2 and eicosanoid, and enhanced migration of polymorphonuclear leukocytes in a rat compression SCI model.IL‐1β, a 17‐kDa protein, is one of the most extensively studied proinflammatory cytokines. Liu et al. first demonstrated that IL‐1β impaired locomotion recovery in a rat glutamate‐induced SCI model.2.2.3IL‐17, also known as IL‐25, is mainly produced by T cells and their precursors. IL‐17 is closely related to the origin of a type 2 immune response, which is characterized by the differentiation of Th2 and the production of the type 2 cytokines.Hill et al. first showed that IL‐17 hindered functional recovery in a mouse contusive SCI model.2.2.4TNF‐α is a pleiotropic pro‐inflammatory cytokine that can be produced by activated macrophages as well as other mammalian mononuclear leukocytes.TNF‐α has been demonstrated to dampen functional recovery in rat and mouse SCI models.3FigureLocal delivery means that the therapeutic agents are administered directly into the lesion site or that an agent administration system is constructed for local application. In comparison with systemic administration, local delivery overcomes the BSCB and thus improves the concentration of the local agent with less damage to other sensitive organs.3.1Administrating agents around or in the lesion site can achieve sufficient therapeutic effects of regulating inflammatory cytokines and promoting injury repair. The directly administered agents include inhibitors, cytokines and other therapeutic agents.3.1.1Several investigations have used antibodies to directly inhibit the levels of certain inflammatory cytokines. Local applications of anti‐TNF‐α antibodies reduced apoptosis,Antagonists are also important inhibitors. Intrathecal injections of IL‐1ra decreased apoptosis after rat SCI.3.1.2In the SCI epicenter, directly administered cytokines include EPO, G‐CSF, and VEGF. The intrathecal injection of recombinant human EPO (rhEPO) was demonstrated to enhance the SDF‐1/CXCR4 axis in transplanted bone marrow stromal cells (BMSCs), thus increasing their migration toward the lesion site.3.1.3Moreover, there are other therapeutic agents, including chondroitinase ABC, Annexin A1 (ANXA1) and 11‐dehydrosinulariolide. Multiple lumbar puncture injections of chondroitinase ABC promoted IL‐10 expression and inhibited IL‐12b expression after SCI.3.2Sustained release forms are designed to release agents at a controllable rate in order to maintain a constant drug concentration for a period of time. To regulate inflammatory cytokines after SCI, sustained agent release can be achieved mainly through two methods: biomaterial systems and pumps.3.2.1In SCI models, the topically applied biomaterial systems involve gelatin sponges, hyaluronan methylcellulose (HAMC), and biomaterial particles.Gelatin Sponge: Gelatin sponges are sterile sponges with porous structure that work primarily as hemostatic agents. They are pH‐neutral and biodegradable and, therefore, also used for local delivery.Gelatin Sponge: Particles: Both microspheres and nanoparticles have been applied in animal models. Transplanting chitosan‐based atorvastatin calcium microspheres into the SCI site attenuated the expression of TNF‐α, IL‐1β and IL‐6, and improved functional outcomes.FigureGelatin Sponge: HAMC: The University of Toronto developed a new biomaterial called hydrogel of HAMC as a drug carrier for SCI treatment.3.2.2Osmotic pumps are small, infusion pumps for administrating agents in animals. They are convenient, cost‐effective and capable of maintaining drug levels.Cytokines: Intrathecal administration of SDF‐1α by osmotic pumps reduced the levels of inflammatory cytokines,Inhibitors: In rat SCI models, EGFR blockers from osmotic pumps depressed IL‐1β and TNF‐α production, inhibited microglia/astrocyte activation and glial scar/cavity formation, and promoted axonal outgrowth with functional recovery.Other Therapeutic Agents: Metabotropic glutamate receptor 5 (mGluR5) has neuroprotective properties. mGluR5 agonist treatment through pumps reduced TNF‐α, microglial activation and lesion volume and improved functional recovery.3.3Recently, cell transplantation has been one of the hotspots in SCI repair. Transplanted cells can release cytokines/chemokines and influence the inflammatory response.3.3.1Mesenchymal Stem Cells (MSCs): MSCs are mainly derived from bone marrow and may have the potential ability to reduce the acute inflammatory response. Cells transplanted directly into the lesion site in SCI animal models reduce the expression of TNF‐α,Compared to direct transplantation, alternative methods could enhance the therapeutic effects of MSCs. MSCs encapsulated with alginate microencapsulation could shift the phenotype of macrophages from M1 to M2 shown by evaluation of the expression of CD206 and IL‐10, attenuate the expression of IL‐1β, IP‐10, and macrophage inflammatory protein 1‐α (MIP1‐α), and increase IL‐6 expression, thus promoting tissue repair.Neural Stem Cells (NSCs): NSCs are self‐renewing cells that can generate neurons, oligodendrocytes, and astrocytes.Dental Stem Cells: Dental stem cells include dental pulp stem cells (DPSCs) and dental follicle stem cells (DFSCs), representing another possible way to treat SCI. Both DPSCs and DFSCs significantly inhibited expression of IL‐1β to reduce inflammation damage,Blood Stem Cells: Takahashi et al. used G‐CSF to mobilize peripheral blood stem cells (PBSCs) from peripheral blood, and PBSC intraspinal transplantation promoted angiogenesis and serotonergic fiber sparing, preserved myelin, and improved function recovery.3.3.2Olfactory Ensheathing Cells (OECs): OECs replace necrotic or apoptotic neural cells and help remyelination and neurotrophin secretion.Other Somatic Cells: Skin‐coincubated macrophages injected into the caudal border of the lesion elevated the secretion of IL‐1β and BDNF, reduced the secretion of TNF‐α and showed meaningful recovery in rats.3.4Gene therapy refers to the techniques and strategies to genetically transform or modify cells for healing or attenuating disease conditions. Various animal experiments have shown that regulation of inflammatory cytokines and repair from SCI is facilitated by local gene therapy, including liposome‐mediated and virus‐mediated gene therapy.3.4.1Ito et al. built a liposome‐mediated IFN‐β gene delivery system and applied it locally after SCI. This gene therapy inhibited glial scar formation and promoted functional recovery by deactivating the MEK‐ERK pathway.3.4.2Viral vector injections are also used for expressing specific proteins in targeted areas.HSV is neurotropic, lives without replication, has a large genome and is easily operated in tissue culture.Lentivirus is the most widely used virus in gene therapy studies aimed at inflammatory cytokines after SCI. Local applications of lentivirus‐mediated regulation of both TNF‐αAdenovirus is also widely applied for local inflammatory cytokines in SCI. Local adenovirus‐mediated RNAi of IL‐1β decreased TNF‐α and neuronal loss, and improved locomotor function.Poliovirus vectors have the capacity for self‐amplification and for expressing foreign proteins without infecting other cells. Jackson et al. intrathecally injected poliovirus vectors to express IL‐10 in SCI animals.4FigureQuite a few promising clinical studies have tested different treatments for SCI.TableHere, we summarized and discussed all of the relevant clinical studies of SCI repair through regulation of inflammatory cytokines Table3. Acco4.1Subcutaneous injection is a shot given into the subcutis. This form of administration is used to give small amounts of certain kinds of medicine. At present, clinical trials with subcutaneous injections of inflammatory cytokines, including G‐CSF and EPO, have shown promise.4.1.1−1 day−1) subcutaneously for 5 days.Derakhshanrad et al. recruited 19 patients, all of whom received G‐CSF (5 µg kg4.1.2−1) for 3 days as an addition to her medications. The next day, her pain was relieved, and lower limb strength recovered gradually. After one year, she walked without help and returned to her previous job.In another case, a 42‐year‐old woman showed low response to routine drug treatment after cervical discectomy that directly led to SCI.−1) subcutaneously three times a week for 6 weeks.−1 was not enough to significantly improve patient prognosis.Additionally, rhEPO administration has had a positive impact on SCI patients with pressure ulcers (PUs) associated with Anemia of Chronic Disease (ACD).4.2Intravenous injection is a method that infuses liquid substances directly into the vein, through which they are then carried by blood circulation and brought into effect systemically. Currently, intravenously administered agents being examined in clinical trials include G‐CSF and EPO.4.2.1−1 day−1) intravenously for 5 days, while another 10 patients received 10 µg kg−1 day−1. None of the 15 patients showed side effects, and treatment resulted in neurological improvements in both motor and sensory functions. Along with the above research, this team also obtained similar results in patients with acute SCI caused by falls and road trauma, as well as athletic injury.In 2012, Sakuma et al. reported that G‐CSF administration was safe and had neuroprotective effects.In consideration of the influence of different injury sites, this group continued a further trial only on patients with cervical injury.In addition, compared with high‐dose methylprednisolone sodium succinate (MPSS) treatment, intravenous administration of G‐CSF produced significantly higher ASIA motor scores.4.2.2−1 EPO by chemotherapy infusion pump over 30 min and showed improved motor function and shorter recovery time, including functional class and ambulation.In clinical studies, EPO has also been administered intravenously. Ten paraparetic patients with malignant extradural spinal cord compression (MESCC) received 1500 IU kg−1) of rhEPO, expression of the EPO receptor or the route of administration.However, in another trial, administration of rhEPO together with MP did not improve the functional outcome of patients with traumatic cervical SCI, which might be due to insufficient dosage  injury and found that combined administration of 1000 IU kg4.4Two methods of local delivery have been applied in clinical trials of SCI: intrathecal delivery and intraspinal injection. These trials have been registered , but currently they have no posted results.Intrathecal delivery is a very important means of local administration and is relatively convenient as it can be done by lumbar puncture. BMSCs, adipose‐derived MSCs and umbilical cord‐derived MSCs have been utilized respectively in the intrathecal delivery of 6 trials . The outcome measures include TNF‐α, TGF‐β, IL‐1β, IL‐6, iNOS, IL‐10, subsets of T‐lymphocytes, and C‐reactive protein.There are two clinical trials using intraspinal injection (NCT01046786 and NCT02917291). The investigators at the Chinese University of Hong Kong and The University of Hong Kong slowly injected cord blood mononuclear cells into the posterior grey matter after laminectomy and opening of the dura. In the other trial, the intramedullary injection of allogeneic adipose‐derived MSCs was used.5Current studies demonstrate that regulation of inflammatory cytokines plays a significant role in SCI repair both in animal models and clinical trials. Nevertheless, two problems exist: enabling the efficacy of agents at small dosages and reducing side effects. Local delivery is showing outstanding advantages in solving these problems. Local delivery of therapeutic agents and personalized therapy have great potential in the future.5.1−1−1.We have learned that subcutaneous or intravenous injections of inflammatory cytokines are widely used for the clinical treatment of SCI. As mentioned above, EPO administration of 1500Moreover, a large number of animal experiments have revealed that local delivery of therapeutic agents can regulate inflammatory cytokines and promote functional recovery, which further illustrates that local agent delivery can promote SCI repair in the clinical setting. Local, but not systemic administration of TNF inhibitor, was therapeutic for traumatic SCI in mice.At the same time, SCI could occur in any spinal cord site, so local delivery is more accurate and economic than systemic administration.5.2In systemic agent delivery, high doses of agents produce side effects. In clinical trials of SCI, the restriction of the blood–brain barrier and short half‐life of IL‐10 required a high dose of IL‐10 in systemic delivery,5.3low/CD16+) and M2‐dominant subpopulations (CD14high/CD16+). They found that the M1‐dominant subpopulation showed higher levels of IL‐12 and CXCL10 in the plasma, while the M2‐dominant subpopulation presented with more IL‐10, IL‐15, and IL‐7. Therefore, future studies may aim to develop personalized strategies for locally delivered therapeutic agent cocktails for effective and precise regulation of inflammation and substantial functional recovery from SCI.Local delivery of a single agent only affects a small portion of the inflammatory cytokines, and different individuals have differential profiles of inflammatory cytokines. Huang et al. found that inflammatory cytokine profiles were related to monocyte (MO) phenotypes in SCI patients.The authors declare no conflict of interest.


SS) is salivary gland (SG) hypofunction. The inability of resident SG stem cells (SGSCs) to maintain homeostasis and saliva production has never been explained and limits our comprehension of mechanisms underlying primary SS. The present study was undertaken to investigate the role of salivary gland stem cells in hyposalivation in primary SS.A major characteristic of the autoimmune disease primary Sjögren's syndrome (SGSCs were isolated from parotid biopsy samples from controls and patients classified as having primary SS or incomplete primary SS, according to the American College of Rheumatology/European League Against Rheumatism criteria. Self‐renewal and differentiation assays were used to determine SGSC regenerative potential, RNA was extracted for sequencing analysis, single telomere length analysis was conducted to determine telomere length, and frozen tissue samples were used for immunohistochemical analysis.SGSCs isolated from primary SS parotid gland biopsy samples were regeneratively inferior to healthy control specimens. We demonstrated that SGSCs from samples from patients with primary SS are not only lower in number and less able to differentiate, but are likely to be senescent, as revealed by telomere length analysis, RNA sequencing, and immunostaining. We further found that SGSCs exposed to primary SS–associated proinflammatory cytokines we induced to proliferate, express senescence‐associated genes, and subsequently differentiate into intercalated duct cells. We also localized p16+ senescent cells to the intercalated ducts in primary SS SG tissue, suggesting a block in SGSC differentiation into acinar cells.SGSCs in primary SS, and also the first demonstration of a linkage between an autoimmune disease and a parenchymal premature‐aging phenotype. The knowledge garnered in this study indicates that disease‐modifying antirheumatic drugs used to treat primary SS are not likely to restore saliva production, and should be supplemented with fresh SGSCs to recover saliva production.This study represents the first characterization of  Between 0.4 and 3.1 million people in the US have been diagnosed as having the autoimmune disease primary Sjögren's syndrome (SS) In healthy SGs, homeostasis is maintained by proliferation and differentiation of tissue‐resident SGSCs. According to one of the prevailing views in the field, these cells reside in striated ducts, from where they differentiate first toward intercalated ducts and subsequently to acinar cells For healthy control specimens, biopsy samples of parotid SG tissue were obtained from donors  who were treated for a squamous cell carcinoma of the oral cavity. In these patients, an elective head and neck dissection procedure was performed. During this procedure, a parotid SG is exposed and removed. This tissue does not contain malignant cells, as oral squamous cell carcinoma does not disseminate to the parotid SG. To obtain study samples from patients with primary and incomplete primary SS, specimens were obtained during the routine diagnostic evaluation for primary SS. Patients were classified as having primary SS if they fulfilled the 2016 American College of Rheumatology (ACR)/European League Against Rheumatism (EULAR) criteria M, for 30 minutes at 37°C. Forty milligrams of tissue was processed per 1 ml buffer volume; total volume was adjusted according to biopsy sample weight. Digested cells were collected by centrifugation, washed twice in HBSS/1% BSA solution, and passed through 100‐μm cell strainers (BD Biosciences).Parotid SG biopsy samples harvested from oral squamous cell cancer patients with healthy parotid gland tissue and samples obtained from patients with primary SS and incomplete primary SS after surgery were processed in Hanks’ balanced salt solution (HBSS) containing 1% bovine serum albumin . Biopsy samples were digested mechanically using a gentleMACS dissociator (Miltenyi Biotec) or manually with scissors and simultaneously digested in HBSS/1% BSA buffer containing 0.63 mg/ml type II collagenase (Invitrogen) and 0.5 mg/ml hyaluronidase (Sigma‐Aldrich), as well as calcium chloride at a final concentration of 6.25 mM dexamethasone (Sigma‐Aldrich), 10 μM Rho kinase inhibitor (Abcam), 5 μM transforming growth factor β inhibitor , and 12.5 ng/ml Noggin (PeproTech). A total of 800,000 primary isolate cells were resuspended in 25 μl of SGSC medium combined with 50 μl of basement membrane Matrigel (BD Biosciences) and deposited in the center of 12‐well tissue culture plates. After the gels were allowed to solidify (20 minutes at 37°C), 1 ml of stem cell medium was added per well. After 3–5 days of culture, the formed primary spheres were released from Matrigel by incubation in 1 mg/ml Dispase (Sigma) (1 hour at 37°C). Primary spheres of a minimum size of 50 μM were counted and used to establish primary sphere yield per milligram of biopsy material. To correct primary sphere yield for the site of biopsy, the primary sphere yields for healthy control samples and primary SS samples were multiplied by factors of 4.1 and 11.95, respectively. Multiplication factors were derived from the yield of primary spheres isolated from the SGSC‐rich area, as described by van Luijk et al The resultant cell suspensions were collected again by centrifugation and resuspended in SGSC medium consisting of 40% Dulbecco's modified Eagle's medium/F‐12 medium, penicillin/streptomycin antibiotics (Invitrogen), Glutamax (Invitrogen), 50% Wnt‐3a–conditioned medium, 10% R‐spondin–conditioned medium , 20 ng/ml epidermal growth factor (Sigma‐Aldrich), 20 ng/ml fibroblast growth factor 2 (Sigma‐Aldrich), N2 (Invitrogen), 10 mg/ml insulin (Sigma‐Aldrich), 1 mA total of 100 μl of cell suspension obtained after SGSC isolation was added into a cytospin funnel, after prewetting of coated microscope slides with 1% BSA/phosphate buffered saline (PBS) solution. After centrifugation at 300 revolutions per minute for 2 minutes, slides were air dried and fixed with 4% paraformaldehyde (PFA) at room temperature for 20 minutes. Hematoxylin and eosin staining was then performed according to standard protocols. The number of acinar and ductal cells was determined by capturing images of 3 areas of the cytospot per sample. The total cell number in each area was determined by counting hematoxylin‐stained nuclei. Acinar cells were identified by characteristic triangular morphology and predominant hematoxylin staining. Ductal cells were identified by heavily eosin‐stained cytoplasm. The proportion of each cell type was expressed as the percentage of total cells. For CD45+ cell quantification, cytospots were fixed as described above, then permeabilized in 100% ethanol (20 minutes at −20°C), washed in PBS, and then incubated in mouse anti‐CD45 antibody (Dako) (1 hour at room temperature), diluted 1:100 in 1% BSA containing 0.05% Tween. Following PBS washing, Alexa Fluor 488–conjugated goat anti‐mouse secondary antibody was added onto cytospots at 1:300 dilution in 1% BSA/0.05 PBS containing Tween and incubated at room temperature for 1 hour. Following final PBS washes, nuclei were counterstained with DAPI, and cytospots were visualized using a Leica 6000 Series microscope.M EDTA (staining buffer), containing a maximum of 1 million cells. Staining was performed for 20 minutes on ice. Cells were collected by centrifugation and resuspended in staining buffer for analysis with an LSRII flow cytometer (BD Bioscience).After isolation, cell suspensions were dispersed to single cells. Cells were immunolabeled with antibodies against the following human proteins, conjugated to fluorophores as indicated: eFluor 660–conjugated epithelial cell adhesion molecule (EpCAM) , phycoerythrin (PE)–Cy5–conjugated CD45 , BUV737‐conjugated CD19 , allophycocyanin (APC)–eF700–conjugated CD3 , PE‐Cy7–conjugated CD56 , APC‐eF780–conjugated CD4 , PE‐Cy7–CD24 , and fluorescein isothiocyanate –conjugated Ki‐67 . For intranuclear staining for Ki‐67, a Foxp3 Transcription Factor Buffer Set was used, according to the instructions of the manufacturer (eBioscience). Staining for K14 and smooth muscle actin (SMA) was performed in 2 steps using rabbit anti‐human K14  and mouse anti‐human SMA  and Alexa Fluor 647–conjugated secondary antibody (1:300). Antibodies were added in a total volume of 100 μl 0.5% BSA/PBS with 2 mM magnesium sulfate and 50 μg/ml DNase (both from Sigma) into cell suspension to prevent cell clumping. Collected CD45+ cells were harvested into stem cell medium collected by centrifugation and plated into Matrigel as described above. The gating strategy for flow cytometric analysis and FACS is shown in Arthritis & Rheumatology web site at http://onlinelibrary.wiley.com/doi/10.1002/art.40659/abstract.Living–dead discrimination was performed using 80 ng/ml propidium iodide (ThermoFisher). For FACS sorting of EpCAM+ cells from SG isolate, staining was performed as described above, with the addition of 0.16 cells per ml in SGSC medium. Twenty‐five microliters of this cell solution was combined with 50‐μl volumes of basement membrane Matrigel and deposited in the center of 12‐well tissue culture plates. After the Matrigel was solidified for 20 minutes at 37°C, gels were covered in stem cell medium as described above. Organoids appeared 2–3 days after seeding of single cells in Matrigel. Ten days after seeding, Matrigel was dissolved by incubation with Dispase enzyme as described above. Organoids >50 μM in diameter were enumerated, cells were processed to a single‐cell suspension using 0.05% trypsin–EDTA, and cell numbers were determined. These data were used to generate the organoid formation efficiency and population doublings. Population doublings (pds) were calculated according to the following formula:Following the release of primary spheres from Matrigel as described above, cells were dispersed to form single‐cell suspensions using 0.05% trypsin–EDTA (Invitrogen), enumerated, and concentrations adjusted to 0.4 × 10Encapsulation in Matrigel was repeated to generate the next passage. This cycle was repeated 4 times (4 passages). At the end of each passage, an image of the cells was captured using an Olympus CKX53 microscope and DP2‐SAL software.M isoproterenol. Mature organoid formation was monitored over a 2‐week period.For mature organoid formation assays, organoid cultures were supplemented with 1 μTotal RNA was extracted from stem cells using an Absolutely RNA Miniprep Kit , according to the instructions of the manufacturer (Agilent). The integrity of RNA was examined with an Agilent 2100 Bioanalyzer. Subsequent sequencing was performed using a SMART‐Seq v4 Ultra Low Input RNA Kit  and a Nextera XT DNA Library Prep Kit  according to the instructions of the manufacturers, and prepared DNA libraries were sequenced on a HiSeq 2500 system. Data quality assessment was performed to understand the main source of variability, and differential expression analysis and visualization were performed in R . The MetaCore pathway database was used for pathway enrichment analysis; data can be accessed via the NCBI Sequence Reads Archive (accession no. PRJNA506620).Cytokines were purchased as follows: human interleukin‐6 (IL‐6) , human interferon‐α (IFNα) , and human tumor necrosis factor (TNF)  and reconstituted according to the instructions of the manufacturers. Dilutions for coculture with cytokines were performed in such a manner that the volume of cytokine added to the medium was always 1% of the total medium volume. The medium was refreshed 2 times within the passage (days 3 and 6), in parallel with control cultures.M and fixed in 2% PFA for 5 minutes. Staining for all samples was performed from this point, using a Tyramide Signal Amplification Kit according to the instructions of the manufacturer (ThermoFisher). After hydrogen peroxide blocking and general blocking, primary antibodies were incubated with organoids, mature organoids, or tissue sections overnight in PBS at 4°C. Dilutions of primary antibodies used for immunostaining were as follows: rabbit anti‐human amylase (1:100) , rabbit anti‐human aquaporin 5, rabbit anti‐human EpCAM, mouse anti‐human IL‐6 receptor (1:100) , mouse anti‐human TNF receptor type I , rabbit anti‐human IFNα receptor (1:100) , mouse anti‐human p16 (1:100) , and mouse anti‐human SMA (1:100) . Nuclear counterstaining was performed with Hoechst 33342, at a 1:300 dilution from 10 mg/ml stock solution, for 10 minutes at room temperature. Immunostainings were visualized using a Leica TCS SP8 confocal laser scanning microscope and Leica Application Suite software.Mature organoids were released from Matrigel using Dispase, collected in round‐bottomed 96‐well plates, and fixed in 2% PFA for 10 minutes. Frozen tissue sections were cut at a thickness of 8 μM Telorette 2 linker was added to 10 ng of purified genomic DNA in a final volume of 40 μl per sample. Multiple polymerase chain reactions (PCRs) were performed for each test DNA in 10‐μl volumes, incorporating 250 pg of DNA, 0.5 μM telomere‐adjacent and Teltail primers, 75 mM Tris HCl (pH 8.8), 20 mM (NH4)2SO4, 0.01% Tween 20, 1.5 mM MgCl2, and 0.5 units of a 10:1 mixture of Taq (ABgene) and Pwo polymerase . The reactions were processed in a Tetrad2 Thermal Cycler (Bio‐Rad). DNA fragments were resolved by 0.5% Tris–acetate–EDTA agarose gel electrophoresis and identified by Southern hybridization with a random‐primed a‐33P‐labeled (PerkinElmer) TTAGGG repeat probe, together with probes specific for the 1 kb (Stratagene) and 2.5 kb (Bio‐Rad) molecular weight markers. Hybridized fragments were detected using a Typhoon FLA 9500 Phosphorimager . The molecular weights of the DNA fragments were calculated using a Phoretix 1D Quantifier (Nonlinear Dynamics).DNA was extracted from human SGSCs using a QIAamp DNA Micro Kit (Qiagen). Single telomere length analysis (STELA) was carried out at the XpYp telomere as described previously by Capper et al 15‐18 primers, 1.0 mM dNTPs, 1× Reaction Buffer, 20 units of RiboLock, and 200 units of RevertAid reverse transcriptase  in a total volume of 20 μl per reaction. The cDNA product was diluted 10‐fold in water and used at this concentration for qPCR. Qualitative PCR was performed using SsoAdvanced Universal SYBR Green qPCR Master Mix (Bio‐Rad), with primers at a final concentration of 500 nM from a 10 μM stock. Diluted cDNA (2.5 μl) was used per reaction, and all reactions were performed in triplicate in a total volume of 10 μl. Primer sequences are shown in Arthritis & Rheumatology web site at http://onlinelibrary.wiley.com/doi/10.1002/art.40659/abstract. A 2‐step qPCR cycle with a Bio‐Rad iCycler qPCR machine was used for target amplification, according to the instructions of the manufacturer for SSoAdvanced Universal SYBR Green Master Mix, and CFX Manager was used for analysis.Total RNA was extracted from cultured cells using an RNeasy Microkit, including DNase incubation, according to the instructions of the manufacturer (Qiagen). One microgram of total RNA was reverse transcribed to complementary DNA (cDNA) using 0.5 μg of oligo(dT)Arthritis & Rheumatology web site at http://onlinelibrary.wiley.com/doi/10.1002/art.40659/abstract). As previously reported for minor SGs (23), we detected a high proportion of B cells and a predominance of CD4+ T cells within the flow cytometry–defined CD45+ fraction of the biopsy isolate (http://onlinelibrary.wiley.com/doi/10.1002/art.40659/abstract). SGSCs are EpCAMhigh in nature. The number of both spheres generated per EpCAMhigh cell and yield of spheres per milligram of biopsy sample was significantly lower (a 10‐fold difference) in samples from patients with primary SS compared to the healthy samples .We began by isolating SGSCs from parotid SG biopsy samples from control patients with healthy SGs and patients with primary SS fulfilling the ACR/EULAR classification criteria http://onlinelibrary.wiley.com/doi/10.1002/art.40659/abstract), supporting the notion that infiltration does not determine SG function. We have previously demonstrated that SGSC yield decreases with age and that more SGSCs are present closest to the facial nerve in the parotid gland http://onlinelibrary.wiley.com/doi/10.1002/art.40659/abstract). Stem cells are classically defined by their ability to proliferate and differentiate. When SGSCs from primary SS biopsies were cultured as organoids to assess their proliferation capacity, we observed a significantly (up to 5 times) lower self‐renewal capability compared to healthy samples , indicating that the sole presence of CD45+ cells in SGs of patients with primary SS is not responsible for the regenerative deficits observed. Primary sphere yield was not correlated with focus score (lymphocytic infiltration)  but do not have a positive lymphocyte focus score. Also, the patients with incomplete primary SS were not taking medication known to cause dry mouth symptoms, they all had recorded symptoms of dry eyes and mouth associated with primary SS development, and they did not fulfill the ACR/EULAR criteria. We consider these features indicative of early SG pathology development in primary SS. When SGSCs were isolated from the patients with incomplete primary SS we observed that the primary sphere yield from a small proportion (2 of 10) of these biopsy samples was markedly greater than the median of the healthy control samples  . As shown in Figures Arthritis & Rheumatology web site at http://onlinelibrary.wiley.com/doi/10.1002/art.40659/abstract). The mean length of the lowest 10% of telomeres in the SGSCs of healthy control samples was significantly greater (5.58 kb) compared to primary SS SGSCs (3.10 kb)  and DNA replication . When healthy control SGSCs from passages 1–4 were incubated with a cocktail of proinflammatory cytokines at concentrations matching those found in the serum of patients with primary SS  http://onlinelibrary.wiley.com/doi/10.1002/art.40659/abstract).Primary SS is an autoimmune disease associated with the glandular presence of classic proinflammatory cytokines, as exemplified by IFNα, TNF, and IL‐6 http://onlinelibrary.wiley.com/doi/10.1002/art.40659/abstract), we also showed that SGSCs resident in the basal layer of striated ducts (BSD cells) were responsible for the proliferation observed (http://onlinelibrary.wiley.com/doi/10.1002/art.40659/abstract). We also suggest that proinflammatory cytokines induce differentiation of BSD cells into intercalated ducts (ID cells) , inhibiting cell cycle (E2F1 and CDKN2D), and promoting senescence (p16 and p21) was up‐regulated Figure C. ThrougThe origins of hyposalivation development in primary SS have never been fully elucidated, although many studies have now firmly established that its development cannot be fully explained by the extent of lymphocytic infiltration First, the parenchymal epithelium, e.g., the non‐stem ductal cells and saliva‐producing acinar cells in primary SS, have been demonstrated to undergo enhanced levels of apoptosis, from sources intrinsic and extrinsic to the cells themselves. Extrinsically, the action of cytokines, cytotoxic T cells, and natural killer cells all promote apoptosis Second, as we have demonstrated here, proinflammatory cytokines exert a direct effect on proliferation of SGSCs. In other model systems, and most extensively in the well‐characterized intestinal stem cell niche, proinflammatory cytokines have also been shown to exert a proliferative effect, mediated by modulation of the stem cell–associated Wnt, Notch, and Yes‐associated protein/transcriptional co‐activator with PDZ‐binding motif (YAP/TAZ) pathways, suggesting that cross‐talk between stem cells and the elements of the immune system may underlie many disease manifestations One prevailing theory regarding stem cells dictates that under healthy conditions, SGSCs reside in the striated ducts, proliferate and differentiate into intercalated ducts, and then finally into saliva‐producing acinar cells. We have demonstrated the presence of senescent cells in intercalated ducts of primary SS SGs. This presence suggests a blockade in the ability of SGSCs to further differentiate into acinar cells, presumably due to having reached their regenerative limit, similar to the poor mature organoid differentiation potential we demonstrated in vitro. Clinically, our data suggest that screening patient SGs or saliva for senescence biomarker expression may indicate the extent of SGSC exhaustion. We further suggest that clinical interventions aimed at preventing hyposalivation development need to occur before the appearance of high levels of senescent markers in SGs or saliva. The present study findings also suggest, critically, that effective interventions to cure established hyposalivation by targeting the inflammatory process are not likely to involve only immune signal blockade, but rather the replenishment of SGSC stocks in conjunction with resolving the inflammation. Plausible strategies include the use of induced pluripotent stem cell technologies in the manufacture of SGSCs.In conclusion, we have demonstrated for the first time an aging phenotype as a potential causative agent for the lack of SG repair in the autoimmune disease primary SS, and link this finding to possible future clinical strategies.All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be submitted for publication. Dr. Pringle had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.Pringle, Wang, Verstappen, Terpstra, Zhang, He, Patel, Jones, Baird, Spijkervet, Vissink, Bootsma, Coppes, Kroese.Pringle, Wang, Verstappen, Terpstra, Zhang, He, Patel, Jones.Pringle.Bristol‐Myers Squibb had no role in the study design, the data interpretation, the writing of the manuscript, or the decision to submit the manuscript for publication. Bristol‐Myers Squibb did perform RNASeq data analysis. Publication of this article was not contingent upon approval by Bristol‐Myers Squibb. Click here for additional data file.


Aim of this review is to propose an acceptable surgical indication for conversion surgery in patients with initially unresectable (UR) pancreatic ductal adenocarcinoma (PDAC) by considering desirable outcomes, including resectability, overall survival (OS), and disease‐free survival (DFS). A comprehensive literature search of PubMed was conducted through July 15, 2019. Eligible studies were those reporting on patients with UR‐PDAC who underwent surgery. We excluded case reports with fewer than 10 patients, insufficient descriptions of survival data, and palliative surgery. When patients with UR‐PDAC with no progression after chemo(radiation) therapy were offered surgical exploration, resectability and median survival time (MST) of those who underwent conversion surgery ranged from 20% to 69%  and from 19.5 to 33 months , respectively. When conversion surgery was carried out in patients with expected margin‐negative resection or with clinical response by Response Evaluation Criteria In Solid Tumors (RECIST), resectability and MST ranged from 18% to 27%  and from 21 to 35.3 months , respectively. Among patients who underwent conversion surgery based on clinical response and decreased CA19‐9 level after multimodal treatment, resectability and MST ranged from 2% to 24%  and from 24.1 to 64 months , respectively. Decreased CA19‐9 level was a predictor of resectability, OS and DFS by multivariate analysis. In conclusion, decision‐making for conversion surgery based on clinical response and decreased CA19‐9 level after multimodal treatment may be appropriate. With regard to desirable outcomes of OS and DFS, conversion surgery may provide improved survival for patients with initial UR‐PDAC. Decision‐making for conversion surgery based on clinical response and decreased CA19‐9 level after multimodal treatment may be appropriate. With regard to desirable outcomes of OS and DFS, conversion surgery may provide improved survival for patients with initial UR‐PDAC. The following search terms were used: “ AND ”. Finally, the reference lists of eligible studies were assessed manually to detect any potentially relevant articles .2.2Eligible studies were those reporting on patients with histologically confirmed unresectable PDAC who underwent surgery after multimodal therapy, including chemotherapy/radiation therapy. Exclusion criteria were as follows: (i) irrelevant studies, (ii) editorials and letters to the editor, (iii) non‐English articles, (iv) case reports including fewer than 10 patients undergoing surgical resection, (v) insufficient description of survival data, and (vi) studies involving treatment mainly by ablative or non‐surgical technologies.2.3Two authors (S.S. and T.Y.) conducted data extraction. Variables of interest included general study characteristics , regimens of multimodal therapy and percentages of patients who received them, surgical indication, OS and DFS, and predictive factors for surgical outcome. Data were tabulated when possible. Discordant judgment was resolved by discussion and consensus.33.1Following the initial algorithm and the successive steps of the selection process, including screening of the titles and abstracts, six review articles3.2Several articles reported that conversion surgery could be carried out safely despite a high incidence of portal vein or arterial resection, ranging from 8% to 81%.3.3Seventeen articles reported a surgical indication for conversion surgery in patients with initial UR‐PDAC.3.43.4.1P = .02) which was better than RECIST‐regression criteria. Lee et alFive articles identified prognostic factors for resectability in patients with initial UR‐PDAC.3.4.2Eleven articles reported prognostic factors for OS in patients with initial UR‐PDAC.10 (CA19‐9), log10 (tumor size), CA19‐9‐lowering rate, and tumor size‐lowering rate were predictive factors for OS. Klaiber et alReni et al10 CA1‐9, log10Two of five articles4On imaging studies, the majority of PDAC is classified as UR disease. The 5‐year OS rate remains less than 10% as a result of the high proportion of UR‐PDAC. In past decades, there have been several developments in the treatment of UR‐PDAC. Implementation of modern chemotherapy regimens, such as FOLFIRINOX and gemcitabine + nab‐paclitaxel, has provided better MST of 8‐12 months, even in metastatic PDAC,Actual resectability of conversion surgery is difficult to quantify due to varying populations in each institution, because candidates for conversion surgery are generally centralized in high‐volume centers. There is a possibility of publication bias or patients’ selection bias in the selected articles. In a review article, Suker et alIn the present review, the surgical indication for conversion surgery remains unclear, and it differed in each institution. One institution had strict criteria, as follows: tumor shrinkage to R/BR status, decreased levels of tumor marker, maintenance of performance status, and a long interval between initial treatment and surgical resection.Optimal selection criteria for surgical exploration or resection remains controversial in patients with initial UR‐PDAC. Four of five articles that identified predictors of resectability showed that decreased CA19‐9 level was a predictor.When patients with initial UR‐PDAC experience significant tumor shrinkage  to R/BR status, decision‐making for conversion surgery is easy. However, decisions are still controversial in patients showing stable disease after multimodal therapy, because it is difficult to differentiate whether viable tumor tissue is present on contrast‐enhanced CT imaging.4.1Recent chemotherapy regimens, such as FOLFIRINOX and gem + nab‐PTX, provide better MST of 24.2 months in UR‐LAMoreover, the optimal timing between initial treatment and surgical resection, an accurate method to evaluate tumor remission, and the type/duration of multimodal therapy are still under investigation. Several prospective studies are now in progress.5Number of candidates for conversion surgery is now increasing with the introduction of modern chemotherapy regimens; however, the actual clinical benefits of resection have not yet been fully investigated. Although conversion surgery can improve long‐term survival in patients with UR‐PDAC, the early recurrence rate should be recognized. There are still several problems to be resolved in this area, and prospective studies will be needed to explore the clinical benefit of conversion surgery. An appropriate surgical indication for achieving desirable outcomes can definitely provide further improved survival and early recurrence rates. Therefore, novel biomarkers predicting resectability, OS and DFS should be investigated in the near future.Conflicts of Interest: Authors declare no conflicts of interest for this article.


The objective of the study is to investigate the relation between pretreatment depressive symptoms (DS) and the course of DS during the first year after cancer diagnosis, and overall survival among people with head and neck cancer (HNC).Data from the Head and Neck 5000 prospective clinical cohort study were used. Depressive symptoms were measured using the Hospital Anxiety and Depression Scale (HADS) pretreatment, at 4 and 12‐month follow‐up. Also, socio‐demographic, clinical, lifestyle, and mortality data were collected. The association between before start of treatment DS (HADS‐depression > 7) and course  and survival was investigated using Cox regression. Unadjusted and adjusted analyses were performed.In total, 384 of the 2144 persons (18%) reported pretreatment DS. Regarding DS course, 63% never had DS, 16% recovered, and 20% had persistent/recurrent/late DS. People with pretreatment DS had a higher risk of earlier death than people without DS (hazard ratio (HR) = 1.65; 95% confidence interval (CI) 1.33‐2.05), but this decreased after correcting for socio‐demographic, clinical, and lifestyle‐related factors . Regarding the course of DS, people with persistent/recurrent/late DS had a higher risk of earlier death , while people who recovered had a comparable risk  as the reference group who never experienced DS. After correcting for socio‐demographic and clinical factors, people with persistent/recurrent/late DS still had a higher risk of earlier death .Pretreatment DS and persistent/recurrent/late DS were associated with worse survival among people with HNC. The study was approved by the National Research Ethics Committee  was used to assess psychological distress , level of DS (HADS‐D), and level of anxiety symptoms (HADS‐A) before the start of treatment, and at 4 and 12‐month follow‐up.Study‐specific questions were used to measure pretreatment tobacco use and alcohol consumption. Tobacco use was categorized as current smoker, former smoker, or never smoked.2.3International Classification of Diseases (ICD) 2010 diagnosis, intended and actual received treatment, Adult Comorbidity Evaluation (ACE‐27), TNM‐stage, and human papilloma virus (HPV) status. Human papilloma virus status was based on serology data, and defined as positive where HPV16E6 was positive (>1000 median fluorescence intensity).Clinical information was abstracted from the hospital information system and patients' notes by research nurses. Clinical information included the primary 2.4t‐test analyses were used to analyze differences between groups.All analyses were performed using the IBM Statistical Package for the Social Science (SPSS) version 23 . Chi‐square tests and independent samples To assess the association between pretreatment DS and overall survival, a series of Cox regression analyses were performed. At first, minimally adjusted analyses adjusted for age and gender were performed. Analyses were performed in the total population as well as in people with oral cavity, HPV‐positive oropharyngeal, and HPV‐negative oropharyngeal and laryngeal cancer separately. Survival time was defined as days from date of consent to censoring or date of death. Besides these minimally adjusted analyses, we investigated whether potential associations remained after adjusting for socio‐demographic and clinical factors. Also, Cox regression analyses adjusted for lifestyle‐related factors were performed. Previous literature hypothesized that lifestyle may mediate the association between depression or DS and survival.Besides analyses on the association between pretreatment DS and survival, unadjusted and adjusted Cox regression analyses were performed using the course of DS in the first year after diagnosis as potential associated factor. For these analyses, people needed to have, besides the previously discussed eligibility criteria, complete HADS‐D at 4 and 12‐month follow‐up, and complete information on actual received treatment. All people were classified according to their course of DS3The HADS‐D score of the total study population n = 2144) was on average 4.0 . Eighteen percent (n = 384) had pretreatment DS   and HPV‐negative oropharyngeal  and laryngeal cancer  with DS, compared to people without DS, while no such association was found among people with HPV‐positive oropharyngeal cancer  . In total, 123 (10%) people died during this follow‐up period, of whom 66 never had DS (9%), 18 had recovered from DS (9%), and 39 had persistent/recurrent/late DS (16%). Using people who never experienced DS as a reference group, it was found that those with persistent/recurrent/late DS had a HR of 2.04 (95% CI 1.36‐3.05), while people who recovered from DS had a comparable hazard as the reference group   this association. Also, tumor‐related or patient‐related biomarkers are hypothesized to mediate this association.5Results of this study indicate that people with pretreatment DS as well as persistent/recurrent/late DS are at increased risk of earlier death. Further research is needed on potential pathways via which depression or DS may influence survival.None.


Both periodontal disease and benign prostatic hyperplasia are age-related diseases that affect millions of people worldwide. Hence, this study aimed to investigate the association between periodontal disease and the risk of benign prostatic hyperplasia.A total of 4930 participants were selected from an available health examination that was carried out in 2017, only males were considered for further analysis. All eligible males were divided into benign prostatic hyperplasia and normal groups, the benign prostatic hyperplasia group was then divided into prostate volume ≤ 60 g and > 60 g subgroups; all their periodontal status was extracted and then into normal (CPI score of 0), periodontal disease (CPI score between 1 and 4), and periodontitis (CPI score between 3 and 4) groups. The correlation between periodontal disease and benign prostatic hyperplasia was investigated using logistic regression analyses and greedy matching case-control analysis. Subgroup analysis based on prostate volume was also performed. All analyses were conducted with SAS 9.4 software.OR = 1.68, 95% CI: 1.26–2.24), and individuals with periodontitis showed a higher risk . In addition, among matched cases and controls, this association remained robust . Subgroup analysis revealed that periodontal disease significantly increased benign prostate hyperplasia risk as well , and there was a higher risk in the group with a prostate volume greater than 60 g.A total of 2171 males were selected for this analysis. The presence of periodontal disease significantly increased the risk of benign prostatic hyperplasia by 1.68 times (Periodontal disease is significantly and positively associated with an increased risk of benign prostatic hyperplasia. Further validation studies should be performed to explore the relationship between periodontal treatment and benign prostate hyperplasia. Periodontal disease is a complex polymicrobial inflammation and a global burden disease (GBD), and periodontal disease mainly includes gingivitis and periodontitis. In 2015, the incidence of severe chronic periodontitis reached 616 million cases around the world , 4, arteBenign prostatic hyperplasia (BPH) is defined as unregulated proliferation of connective tissue, smooth muscle and glandular epithelium within the prostate transition zone and is one of the most common diseases in humans. It is estimated that the doubling time of BPH growth is 4.5 years among individuals between the ages of 31 and 50 and 10 years among those between 51 and 70 years old . A meta-OR) = 1.50, 95% CI, 1.05–2.10]  . In 201705–2.10] . TherefoWe used the Strengthening the Reporting of Observation Studies in Epidemiology (STROBE)  statemenWith a retrospective design, this study adopted all available data from a health examination at Henan University in 2017 . This stThe largest anteroposterior , transverse , and cephalocaudal  prostate diameters were detected for BPH patients using transabdominal B-ultrasonography. A normal prostate was directly recorded without information on those measurements. Prostate volume (PV) was obtained using the prostate ellipsoid formula: PV = π/6 × [H (cm) × W (cm) × L (cm)]. Then, the BPH group was divided into PV ≤60 g and > 60 g subgroups. Clinical oral examinations of the participants in the dental chair were performed by dentists using a headlamp, a mouth mirror, and a periodontal probe. Periodontal status was assessed using the Community Periodontal Index (CPI), and maximal CPI score of sextants ranged from 0 to 4: 0 referred to healthy periodontal tissue, 1 to the appearance of bleeding, 2 to the existence of calculus, 3 to pocket depth of 4–5 mm, and 4 to pocket depth ≥ 4 mm , 28. A C2). Systolic blood pressure  and diastolic blood pressure  were measured, and hypertension referred to SBP ≥140 mmHg, DBP ≥90 mmHg or use of medicine for hypertension. Covariates for the analyses included age, BMI, hypertension status, fasting blood glucose , serum lipid composition , erythrocyte sedimentation rate , C-reactive protein , urea nitrogen , uric acid , creatinine (μmol/L), and proteinuria.Covariates were selected based on originally recorded data and the current knowledge of potential influencers of BPH and periodontal disease. The weight and height of each participant were measured, and body mass index (BMI) was calculated as weight in kilograms divided by height in meters squared (kg/mt tests (or Wilcoxon rank sum tests) for continuous variables and chi-squared tests (or Fisher exact tests) for categorical variables.Basic characteristics were summarized for the overall sample as well as for the normal and BPH groups. Categorical variables are shown as counts (percentages), while continuous variables are shown as the means ± standard deviations or medians (interquartile ranges) based on results from the normal distribution test. Comparisons between the normal and BPH groups were conducted using two independent sample OR, 95% CI and corresponding P value for simplification.The association between periodontal diseases and BPH was examined with univariable and multivariable logistic regressions. Further and detailed evaluations were performed according to the CPI scores individually. Three models were established stepwise. Specifically, model 1 was constructed without adjustment; model 2 was adjusted for age, BMI, and blood pressure status; and model 3 adopted additional adjustments for FBG, TC, TGs, LDL-C, HDL-C, ESR, UN, UA, creatinine, and proteinuria in addition to the variables adjusted in model 2. Statistical presentation includes P < 0.05 was considered significant.Sensitivity analysis was performed to detect whether differences in age, BMI, or blood pressure status between the normal and BPH groups influenced the final results. We matched cases and controls using greedy matching age ± 5 years, BMI ±2, and the same blood pressure status (hypertension and normotension). If the results of the sensitivity analysis were similar to the original results, associations were robust; otherwise, we adopted matched results. Subgroup analysis was conducted on the basis of PV to explore specific effects in groups with different PV sizes. All statistical analyses were calculated with SAS 9.4 software. Tests on statistical significance were two-sided, and 2 in the BPH group and 25.20 ± 3.00 kg/m2 in the normal group; the mean PV was 41.03 ± 18.11 g in the BPH group. Age, BMI and blood pressure status showed statistically significant differences between the two groups (P < 0.05). The baseline characteristics of all of these participants are presented in Table Finally, 2171 participants were included in this analysis after excluding females and those not undergoing periodontal or prostate status examinations. The selection process is shown in Fig. OR = 0.92, 95% CI: 0.74–1.13), but it was shown that periodontitis might increase the risk of BPH . Adjusted analysis using model 2 revealed that periodontal disease  and periodontitis  were both significantly associated with an increased risk of BPH (P < 0.05). Adjusted analysis adopting model 3 also achieved similar results .Table P > 0.05). However, periodontitis was significantly associated with an increased risk of BPH in both groups (P < 0.05). After adjustment, both model 2 and model 3 analyses revealed a significant positive relationship of periodontal disease with BPH onset (P < 0.05). However, the disease risk in the PV > 60 g group was higher than that in the PV ≤60 g group.Table OR = 4.83, 95% CI: 2.57–9.07) and periodontal disease  significantly increased BPH susceptibility. Compared to the overall analysis findings, these results demonstrated that the association of periodontal disease with BPH was robust.We matched cases and controls using greedy matching age ± 5 years, BMI ±2, and the same blood pressure status (hypertension and normotension). Finally, 662 participants were enrolled in the analysis, involving 331 cases and 331 controls. Table In our study, periodontitis was associated with an increased risk of BPH in both crude and adjusted analyses; periodontal disease had no independent relationship with BPH in crude analysis, but statistical significance appeared after adjustments and subgroup analysis. Moreover, when we performed sensitivity analyses using the greedy matching method , the association of periodontal disease with BPH remained the same.OR = 1.50, 95% CI: 1.05–2.10) in males younger than 70 years old after adjustments. Then, they reported subgroup results for Asians , individuals of European descent , a Hispanic population , an unknown ethnicity group , and others . Our results, based on Asians, confirmed their findings, revealing a significant positive association between the two diseases .Investigating the association between periodontal disease and BPH risk is an interesting topic. In 2013, Boland et al.  performeP < 0.001) than those without MetS. Boland and colleagues [OR = 1.4, 95% CI: 1.20–1.55), hypertension , obesity , and lipid conditions . These comorbidities might increase the risk of BPH among people in the United States [Our study revealed that periodontal disease was probably an independent risk factor for BPH. BPH is strongly associated with age , as are lleagues  also repd States . In our d States . Hyperlid States , and ESRd States  and BMI d States  might inPorphyromonasgingivalis (Pg), Prevotella intermedia (Pi), Treponema denticola (Td), and Escherichia coli (E. coli) in expressed prostatic secretions from patients with both periodontal disease and chronic prostatitis or BPH and found at least one oral pathogen in 9 out of 10 BPH patients in their prostatic secretions [The primary mechanism of periodontal disease promoting BPH origination and progression may be associated with inflammation and accompanied by possible influences from oral bacteria. The oral cavity presents a potential reservoir for pathogens, including bacteria and viruses , 37, andcretions . This ficretions , while pcretions , pretermcretions , cogniticretions  and respcretions  but not Neither smoking nor alcohol consumption status was included in our data, so we could not analyze their potential influences in this study. However, a recent systematic review and meta-analysis based on 44,100 subjects showed no significant association between cigarette smoking and BPH risk for either ex-smokers or current smokers . AnotherTo our knowledge, this was the first study on the association between periodontal disease and BPH risk in Chinese people. Our study revealed a significant robust relationship between increased BPH risk and periodontal disease. This relationship may be a consequence of periodontal and prostatic inflammation. According to these results, we recommend that old males pay more attention to their oral health, especially BPH patients developing relevant symptoms. Periodontal treatment, which is easy and inexpensive, should be included in algorithms to predict BPH surgery risk in future studies.


The relationship between obesity and adverse health is well established, but little is known about the contribution of DNA methylation to obesity-related health outcomes. This study tests associations between an epigenetic score for body mass index (BMI) and health-related, cognitive, psychosocial and lifestyle outcomes in the Lothian Birth Cohort 1936. This study also tests whether these associations are independent of phenotypic BMI.n = 892). Weights for the epigenetic BMI score were derived using penalised regression on methylation data from unrelated Generation Scotland participants (n = 2562). Associations were tested for replication in an independent sample: the Lothian Birth Cohort 1921 (n = 433).Analyses were conducted using data from the Lothian Birth Cohort 1936 (R2 = 0.1), greater body weight (R2 = 0.06), greater time taken to walk 6 m, poorer lung function and poorer general physical health , greater levels of triglycerides (R2 = 0.09), greater %total HbA1c (R2 = 0.06), lower levels of high-density lipoprotein cholesterol , higher HDL ratio , lower health-related quality of life, physical inactivity, and greater social deprivation . The epigenetic BMI score (per SD) was also associated with type 2 diabetes , cardiovascular disease  and high blood pressure . Associations were replicated for BMI (R2 = 0.06), body weight (R2 = 0.04), health-related quality of life (R2 = 0.02), HbA1c (R2 = 0.07) and triglycerides .A higher epigenetic BMI score was associated with higher BMI (We observed and replicated associations between an epigenetic score for BMI and variables related to poor physical health and metabolic syndrome. Regression models with both epigenetic and phenotypic BMI scores as predictors accounted for a greater proportion of variance in all outcome variables than either predictor alone, demonstrating independent and additive effects of epigenetic and phenotypic BMI scores. Through recent advances in genomic analysis, we are now able to test whether obesity-related adverse health and disease may, in part, influence or be influenced by epigenetic changes, with the ultimate aim of more accurately predicting health outcomes.Body mass is a complex trait that demonstrates a high degree of variance in the general population . It is dSeveral recent studies have suggested links between BMI and DNA methylation: epigenome-wide association studies (EWAS) have identified CpG sites associated with BMI at loci involved in lipid and lipoprotein metabolism, blood lipid levels and inflammatory pathways –4. Shah A growing body of research examining the associations between DNA methylation and health assumes that exposures to adverse environmental stimuli induce epigenetic changes, which increase a genetically mediated risk of disease . If it iThe Lothian Birth Cohorts 1921 (LBC1921) and 1936 (LBC1936) are follow-up studies of the Scottish Mental Surveys of 1932 and 1947, respectively . These sEthical permissions for the LBC1921 were obtained from the Lothian Research Ethics Committee (LREC/1998/4/183). Permissions for the LBC1936 were obtained from the Multi-Centre Research Ethics Committee for Scotland (MREC/01/0/56) and the Lothian Research Ethics Committee (LREC/2003/2/29). Written informed consent was obtained from all participants.p > 0.01 for >5% of samples), low quality , low call rate (p < 0.01 for <95% of probes), and samples with a poor match between genotypes and single nucleotide polymorphism (SNP) control probes, and with incorrectly predicted sex.DNA methylation data were assessed in whole blood samples from the LBC1921 and LBC1936 using the Illumina HumanMethylation450 BeadChip . Full details of sample preparation and methylation typing have been reported previously . In brieThe regression weights for the LBC1921 and LBC1936 BMI epigenetic signatures were derived from an independent cohort—Generation Scotland: the Scottish Family Health Study GS; , 13). Fu. Fu13]).Physical health2), time taken to walk 6 m (s), grip strength  and lung function . A measure of general physical health was also calculated using the first unrotated component of a principal component analysis of 6m walk time, grip strength and lung function.Physical health variables included height (cm), weight (kg), BMI . The scCognitive abilityVariables measuring cognitive ability included the following subtests of the Wechsler Adult Intelligence Scale III : SymbolPsychosocial factorsPersonality traits were measured using the 50-item version of the International Personality Item Pool questionnaire . This qBloods and biomarkersBlood samples were taken to assess glycated haemoglobin , fibrinogen (g/l), C-reactive protein (mg/l), triglycerides (mmol/l), total cholesterol (mmol/l) and high-density lipoprotein cholesterol . A HDL ratio was calculated by dividing HDL by total cholesterol. Total cholesterol levels were adjusted for individuals on lipid-lowering medications. Systolic and diastolic blood pressure, both sitting and standing, was measured at our research facility with an Omron 705IT monitor . Blood pressure scores were adjusted for individuals taking antihypertensive medications.Lifestyle factorsContinuous outcome measures formed six categories:Social deprivation was measured using the Scottish Index of Multiple Deprivation . The SIParticipants reported their disease history during a structured interview with a member of the LBC research team. Self-reported disease history variables were binary and included: high blood pressure, type 1 diabetes mellitus, type 2 diabetes mellitus, high cholesterol, cardiovascular disease, leg pain, poor blood circulation, stroke, neoplasm, health issues related to the thyroid, Parkinson’s disease, arthritis and allergies.Descriptive statistics for all continuous outcome variables from LBC1936 and LBC1921 are presented in Table R2 statistic represents the proportion of variance in the outcome variable that can be accounted for by the epigenetic BMI score (see model 1), phenotypic BMI score (model 2), and both epigenetic BMI and phenotypic BMI scores together (model 3). To estimate the proportion of variance accounted for by these predictors without the effect of covariates, the R2 statistic represents the difference between the R2 of the null model (see below) and the R2 of models 1, 2 and 3, respectively. Age at the time of testing and sex were included as covariates in all models. Height was included as an additional covariate in models for time taken to walk 6 m, forced expiratory volume (lung function) and general physical health. The models are as follows:Linear regression models were used to investigate the relationship between the epigenetic BMI score and outcome variables. For each model, the Model 1: variable of interest ~ epigenetic BMI score + age at time of testing + sex.Model 2: variable of interest ~ phenotypic BMI + age at time of testing + sex.Model 3: variable of interest ~ epigenetic BMI score + phenotypic BMI + age at time of testing + sex.Null model: variable of interest ~ age at time of testing + sex.p < 0.05/n_tests = 0.00026). Analyses were carried out in R version 3.4.0 [Logistic regressions were conducted for self-reported disease history variables as these had binary outcomes (disease/no disease). Similar to the models above, logistic regressions were carried out for each variable to test whether epigenetic BMI, phenotypic BMI, and an additive model with both epigenetic BMI score and phenotypic BMI as predictor variables, were associated with disease history. For all analyses carried out using LBC1936 data, a Bonferroni corrected level of significance was used  as a predictor in the additive model together with epigenetic BMI score and phenotypic BMI (model 4).Model 4: variable of interest ~ epigenetic BMI score + phenotypic BMI + polygenic BMI score + age at time of testing + sex.α = 0.05). For all analyses carried out using LBC1921 data, a Bonferroni corrected level of significance was used (p < 0.05/n_tests = 0.0045).Significant associations between epigenetic BMI score and outcome variables were tested for replication in an independent sample, the LBC1921. Prior to testing, we carried out power analyses based on the effect sizes of associations in the LBC1936, to assess whether the sample size for each variable in the LBC1921 was large enough to detect a significant effect , with a mean age of 69.5 years.Analyses of the LBC1936 included 892 individuals  are presented here—full results are presented in Table Only associations with a p < 2 × 10–16) and accounted for 10% of its variation. Increases in epigenetic BMI score also correlated with poorer performance on other physical health variables: as epigenetic BMI score increased by 1 SD, time taken to walk 6 m increased by 0.14 s , body weight increased by 0.24 kg , forced expiratory volume decreased by 0.13 l , and general physical health decreased by 0.13 SDs .The results of the linear regression models showed that epigenetic BMI was strongly associated with phenotypic BMI , triglycerides , and HDL ratio , and lower levels of HDL cholesterol .Regarding blood and biomarker variables, epigenetic BMI score was associated with higher levels of %total HbA1c , lower scores on the Scottish Index of Multiple Deprivation , and lower levels of self-reported physical activity .Finally, epigenetic BMI score was associated with lower physical health-related quality of life , greater body weight , poorer general physical health , higher levels of %total HbA1c , higher levels of triglycerides , higher HDL ratio , lower levels of HDL cholesterol , poorer physical health-related quality of life , and lower levels of self-reported physical activity . With the exception of triglycerides, phenotypic BMI accounted for a greater or equal amount of variance of all outcome variables, relative to epigenetic BMI.Phenotypic BMI was associated with greater time taken to walk 6 m  was positively associated with type 2 diabetes , cardiovascular disease , and high blood pressure . Phenotypic BMI was associated with type 2 diabetes  and high blood pressure . In an additive model with both epigenetic and phenotypic BMI scores as predictors, epigenetic BMI score was associated with type 2 diabetes  and cardiovascular disease  - all associations significant at n = 263, 60.7%), with a mean age of 79.1 years. Where possible, variables that associated with epigenetic BMI score in the LBC1936 (n = 15) were tested for replication in the LBC1921. Some variables were not available in the LBC1921 (n = 5) and we did not have sufficient statistical power to detect the effects for several others (n = 2). Only associations with a p value < 0.0045 (i.e. after Bonferroni correction) are presented in Table Analyses of the LBC1921 included 433 individuals  and together, predictors accounted for 18% of the variation in phenotypic BMI.Tables Using DNA methylation data, we constructed an epigenetic score for BMI to test its association with health, cognitive and lifestyle outcomes in the LBC1936. We found that a higher  epigenetic score for BMI is associated with poorer physical health , higher levels of HbA1c and triglycerides, higher HDL ratio, lower HDL cholesterol, lower physical health-related quality of life, lower self-reported physical activity and greater social deprivation. We replicated several of these associations  in an independent, older sample, the LBC1921. Our analysis of the LBC1936 also showed that epigenetic BMI score is associated with self-reported type 2 diabetes, cardiovascular disease and high blood pressure: health outcomes for which obesity is a major risk factor. An association between an epigenetic score for BMI and type 2 diabetes, identified via clinical diagnosis or HbA1c ≥6.5%, has previously been reposted by Wahl et al. [In almost all analyses, phenotypic BMI accounted for a greater proportion of variance in outcome variables than epigenetic BMI, but phenotypic and epigenetic BMI scores together accounted for a greater proportion of variance still. This additive effect suggests that epigenetic and phenotypic BMI scores account for at least partially independent proportions of variance. It is possible that some of these partially independent associations are driven by correlations between the CpG probes used to create the epigenetic BMI score, and the outcome variables of interest. This could mean that our epigenetic score for BMI is capturing the downstream effects of obesity, such as biological processes associated with poor metabolic health, which phenotypic BMI  does not account for directly.We found that an epigenetic score for BMI accounted for 10% of the variance in phenotypic BMI, a higher proportion than that reported by Shah et al. , whose e0.5 (WHT.5R) are better predictors of whole body fat percentage and visceral adipose tissue mass than BMI [Our results demonstrate that BMI (and epigenetic BMI) is associated with obesity-related adverse health and thus, is a clinically relevant metric for measuring body mass. However, the BMI has been criticised due to its assumption that a low body mass equates to metabolic health. Dvorak et al.  found ththan BMI . DespiteThat our replication sample (the LBC1921) was smaller than our discovery sample (the LBC1936) is a limitation of this study. While we were able to replicate five out of eight associations with epigenetic BMI score in the LBC1936, the smaller sample size of the LBC1921 prevented us from testing a greater number of associations due to a lack of power. A further limitation of this study is the inability to make strong directional inferences; it is impossible to tell whether differential DNA methylation is a mediator of the causal disease pathway, or occurs in response to disease processes. Recent progress has been made in this area using a Mendelian Randomisation approach , which tHIF3A . The results across each method indicated that a higher BMI causally influenced higher HIF3A methylation, suggesting that alterations in DNA methylation are a downstream effect of obesity. Further longitudinal studies and studies of naturalistic interventions are needed to elucidate the direction of causal association between obesity and DNA methylation, and to provide mechanistic insight into the pathophysiology of obesity-related disease.A recent study by Richmond et al.  combinedple EWAS , 32), inWe have demonstrated that an epigenetic score for BMI is associated with poorer physical health, poorer health-related quality of life, biomarkers for metabolic syndrome, physical inactivity, social deprivation and major diseases for which obesity is a risk factor. We replicated the associations between BMI, body weight, HbA1c, triglycerides and physical health-related quality of life in an independent, older cohort. An epigenetic score for BMI based on DNA methylation increased the amount of variance in health outcomes accounted for by phenotypic BMI score alone, demonstrating the value in using epigenetic information for constructing more accurate risk prediction for obesity-related biomarkers, health conditions and disease.Supplementary TablesAppendix 1


Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive and lethal cancers lacking valid prognostic biomarkers. As an essential component of a large ribonucleoprotein complex, U Three Protein 14a (UTP14a) might play important roles in human tumorigenesis. However, the clinical significance and functions of UTP14a in ESCC still remain unclear.From September 2009 to August 2015, 210 patients with ESCC of the thoracic esophagus underwent thoracoscopic esophagectomy in our institute. The corresponding 210 tissue samples and 30 cancer‐distant mucosa (CDM) samples were tested for UTP14a expression by immunohistochemical staining. The long‐term survival was analyzed by the Kaplan–Meier method and Cox proportional hazards regression analyses. CCK8, cell colony formation, cell cycle, apoptosis, cell invasion, and wound healing assays were carried out with ECA109 cells to evaluate the effects of UTP14a on ESCC in vitro.UTP14a was positively expressed in 88.1% (185/210) of the ESCC samples. UTP14a expression in ESCC was significantly higher than in CDM, as further confirmed by Western blot analysis. High expression of UTP14a in ESCC correlated significantly with tumor invasive depth (pT stage), which predicts poor disease‐free survival and disease‐specific survival, as indicated by the log‐rank test and Cox proportional hazards regression analysis. Additionally, our in vitro experiments further demonstrated that knockdown of UTP14a inhibits cell proliferation and invasion in ECA109 cells.Our results suggest that UTP14a is aberrantly expressed in ESCC, plays a critical role in cancer progression and could be a potential prognosis predictor of ESCC. High expression of UTP14a in ESCC correlated significantly with tumor invasive depth and predicts poor survival; in vitro experiments demonstrated that UTP14a inhibits cell proliferation and invasion.The results suggest that UTP14a could be a potential prognosis predictor of ESCC.Esophageal cancer (EC) is one of the most aggressive and lethal cancers in patients, and it ranks seventh in terms of incidence and sixth in mortality globally.et al. unpublished data).In our previous research, we found that wild‐type p53‐induced gene 1 (WIG‐1) could effectively reverse the malignant phenotype, suppress cell proliferation, and induce G0/G1 cell cycle arrest and apoptosis in EC109 cells under zinc deficiency conditions.et al.UTP14a is an essential component of a large ribonucleoprotein complex bound to the U3 small nucleolar RNA. It plays a key role in ribosome biogenesis and 18S rRNA synthesis.In this study, we investigated the expression of UTP14a in ESCC and cancer‐distant mucosa (CDM), evaluated the relationship between UTP14a expression and clinicopathological characteristics and survival time of patients with ESCC, and examined whether UTP14a is a potential prognostic biomarker for ESCC.From September 2009 to August 2015, 210 patients with ESCC of the thoracic esophagus underwent thoracoscopic esophagectomy (McKeown) in the Department of Thoracic Surgery, Daping Hospital, Army Medical University . All surgeries were performed by the same surgeon (W.G.). The postoperative TNM stage was classified in accordance with the eighth edition of the American Joint Committee on Cancer (AJCC) staging protocol.Exclusion criteria was as follows: (i) neoadjuvant therapy, (ii) postoperative death , (iii) loss to follow‐up, (iv) noncurative (R1 or R2) resection (tumor‐free margin <1 mm), and (v) death due to causes unrelated to ESCC.The esophageal cancer cell line ECA‐109 was obtained from GeneChem  in 2017. The cell line was tested and authenticated by short tandem repeat (STR) analysis as described in the ANSI Standard (ASN‐0002) by the American Type Culture Collection (ATCC) Standards Development Organization (SDO) in 2017.A tissue microarray containing a total of 210 formalin‐fixed and paraffin‐embedded tissue samples was constructed, and immunohistochemistry (IHC) was performed as previously described.15 Each slide was evaluated by the intensity of the cytoplasmic staining  and the proportion of stained cells , and the sum ranging from 0 (the minimum score) to six (the maximum score) was the final immunoreactive score, classified as negative expression (0), weak expression (1–2), moderate expression (3–4), and strong expression (5–6). We considered the final immunoreactive score was 1 or higher to be positive.All slides were assessed by two pathologists  without any knowledge of the clinicopathological features. Disagreements were resolved by discussion. A semiquantitative scoring system in considering the staining intensity and proportion was used to estimate the expression of UTP14a as described previously.Proteins from freshly frozen tissue samples were extracted using the T‐PER Tissue Protein Extraction kit , separated on SDS‐PAGE , and transferred onto a polyvinylidene difluoride (PVDF) membrane  for Western blot analysis. The membrane was then incubated with rabbit anti‐UTP14a polyclonal antibody . GAPDH  was used as an internal control. Protein bands were analyzed with Quantity One software.−ΔΔCt method.Total RNA was extracted from cells using Trizol reagent  and reverse‐transcribed into cDNA with the M‐MLV reagent kit . qRT‐PCR analysis was conducted with SYBR Master Mixture  to measure the expression of UTP14a. GAPDH was used as an internal control, and the relative mRNA expression of UTP14a mRNA was calculated using the 2UTP14a primers: forward, 5'‐AATAAAACCGCACAAGTCC‐3′, and reverse, 5'‐ACAGGGGTCAGTAAAGGGT‐3′; product size: 218 bp.GAPDH primers: forward, 5'‐TGACTTCAACAGCGACACCCA‐3′, and reverse, 5'‐CACCCTGTTGCTGTAGCCAAA‐3′; product size: 121 bp.GV248 lentivirus was used to express small interfering RNAs (siRNAs) targeting the UTP14a sequence (Genbank no. NM_006649) (UTP14a‐siRNA lentivirus). A nontargeting sequence was purchased from GeneChem  and used as a negative control (NC). The template of the siRNA was 5'‐GAGCAGCTGCGGAAGGTTAAT‐3′. The sequences were cloned into the GV248 lentivirus  to generate the lentiviral vectors. ECA109 cells were infected with UTP14a‐siRNA lentivirus and NC lentivirus. The interference efficiency of the template was measured by qRT‐PCR.The proliferation of ECA109 cells infected with LV‐UTP14A‐RNAi lentivirus (KD) and NC lentivirus was assessed by cell colony formation and cell counting kit 8 (CCK8) assays.In the cell formation assay, ECA109 cells suspended in DMEM  containing 10% fetal bovine serum were plated in six‐well plates at 1000 cells/well. The plates were incubated for 14 days and colonies with more than 50 cells were counted. The cells were then stained by crystal violet dye  and counted with a microscope. Each sample was done in triplicate, and the experiments were repeated three times.The CCK8 assay was carried out following the manufacturer's instructions . Absorbance of each well was quantified at 450 nm by an enzyme‐linked immunosorbent assay (ELISA) microplate reader, and cell growth was assayed every day for five days.Cells were infected with LV‐UTP14A‐RNAi lentivirus (KD) or NC lentivirus. The various cell cycle phases were determined using propidium iodide . Cell populations in the G0/G1 phase, S phase, and G2/M phase were measured using flow cytometry and plotted against cell volume. Data are reported as mean ± standard deviation of three independent experiments. Cell apoptosis was assayed by staining with Annexin V‐APC , following the manufacturer's instructions, and counting the cells by flow cytometry.Cell invasion was studied by using BioCoat Matrigel Invasion Chambers . After 72 hours, cells migrating through the membrane were stained by Giemsa  and counted with a microscope. Nine random visual fields (×200 magnification) were chosen for each membrane, and the results expressed as number of migratory cells per field. Data are reported as the mean ± standard deviation of three independent experiments.The Celigo cytometer  provided a tool to directly measure cell migration for label‐free or fluorescently labeled cells. We chose a 96‐well format, and the wound was created by a square mask. After 24 hours, the entire well was imaged, and the number of cells or percentage of confluence within the previously cleared region was measured. The migration rate is reported as migrated cell surface area (24 hours)/wound area (0 hours) × 100%. Data are reported as the mean ± standard deviation of five independent experiments.All 210 patients were followed‐up in the outpatient clinic after surgery every three months during the first year and every six months until the fifth year; subsequent follow‐ups were performed annually.t‐test and the χ2 test, quantitative variables were compared by one‐way analysis of variance (ANOVA). Patient survival curves were constructed by the Kaplan–Meier method. The log‐rank test was used to compare survival differences between groups for each variable. Univariate and multivariate Cox proportional hazards regression analyses were performed to identify potential prognostic factors. All statistical calculations were performed using SPSS statistical software, version 22.0 . A P‐value of <0.05 was considered statistically significant.Numerical data are expressed as the mean ± standard deviation. Continuous and categorical variables were compared by using the Student P < 0.001, Table P = 0.002; Fig As shown in Fig P = 0.005) and advanced TNM stage (P = 0.001). However, no significant correlation was found between UTP14a expression and clinical characteristics such as tumor location, differentiation grades, and postoperative lymphatic dissemination stage (pN stage).Among the 210 patients, 164 were male, and the male:female ratio was 3.6:1 . In Fig P = 0.012 and P = 0.003, respectively), and high UTP14a expression may indicate poor prognosis.The DFS rate was 82.1% at one year, 58.4% at three years, 48.1% at five years, and 39.6% at eight years, whereas the DSS rate was 90.8% at one year, 63.0% at three years, 51.9% at five years, and 41.5% at eight years . The results of the cell apoptosis assay, after five days of cell culture, are shown in Fig P = 0.098).As shown in Fig et al.et al., unpublished data). UTP is encoded by U3 snoRNA, which is found predominantly in the nucleolus.WIG‐1, which encodes an RNA zinc finger protein, is known to be a p53 target gene. Our previous research found that WIG‐1 could effectively reverse the malignant phenotype, suppress cell proliferation, and induce G0/G1 cell cycle arrest and apoptosis of EC109 cells under zinc deficiency conditions.In this study, UTP14a was positively expressed in 88.1% of the ESCC samples, and 30% of CDM samples exhibited positive expression; Western blot analysis further confirmed that the protein levels of UTP14a were significantly higher in ESCC. Nucleolar proteins participate in ribosome biogenesis; some studies further demonstrated that nucleolar proteins may interact with many tumor suppressors and proto‐oncogenes, and may be associated with many types of cancer.et al., who found that UTP14a interacts with p53; overexpression of UTP14a could lead to p53 protein degradation, and knockdown of UTP14a could cause cell cycle arrest and apoptosis in HeLa cells.et al.et al. demonstrated that UTP14a acts as a ubiquitin E3 ligase for tumor suppressor retinoblastoma (RB); overexpression of UTP14a leads to protein degradation of both p53 and RB, and causes cancer cell proliferation.In our previous research, we found that UTP14a is a downstream molecular target of WIG‐1 and may play an important role in the p53 pathway. This was confirmed by Hu All 210 patients in this study were operated on by the same surgeon using a uniform surgical approach so that the results were more reliable. The five‐year DFS and DSS of these patients were 48.1% and 51.9%, respectively, and the eight‐year DFS and DSS were 39.6% and 41.5%, respectively. High expression of UTP14a in ESCC was significantly correlated with the pT stage and pTNM stage; overexpression of UTP14a indicated an increased risk of tumor proliferation and invasion. Meanwhile, from the Kaplan–Meier survival curves and the log‐rank test, we found that high UTP14a expression predicts poor DFS and DSS. Data from the Worldwide Esophageal Cancer Collaboration database show that the pTNM stage is strongly associated with survival,Although our samples were retrospectively collected from a single institution, the database in our department and the patient follow‐up system were well established, and the surgical procedures, pathologic examinations, and patient follow‐up were highly uniform throughout the entire study period. Additionally, all patients enrolled in this study had ESCC and the operations were performed by a single surgeon using a uniform surgical approach, with standard exclusion and inclusion criteria. Therefore, we believe that our results are robust and valid.In conclusion, our results suggest that UTP14a expression is correlated with tumor proliferation and invasion; higher expression of UTP14a is a predictor of poor prognosis in patients with ESCC. Further studies with larger cohorts are needed to validate our results, and it will be interesting and useful to investigate the underlying mechanisms.There were no potential conflicts of interest, including specific financial interests and relationships in our manuscript, and the authors have no conflicts of interest.