Healthcare 5.0: A secure and distributed network for system informatics in medical surgery.

INTRODUCTION: Health records serve not only as a database of a patient's health history and treatment process but also as a crucial tool for doctors to diagnose and treat patients. However, the storage and sharing of these records are sensitive issues as they involve maintaining patient privacy and ensuring data transparency, security, and interoperability between different parties. Challenges to achieving these goals in the current surgical process can impact the allocation of medical resources and surgical outcomes. METHODS: This article proposes a healthcare 5.0 framework for medical surgery that deploys a secure and distributed network using Blockchain to demonstrate transactions between different parties in the orthopedic surgery process. The proposed network uses the Hyperledger Composer platform for deployment, and a patient-doctor-supplier orthopedic surgery network is designed and implemented to enable the safe sharing of medical records. RESULTS: A benchmarking tool was implemented for analyzing different scenarios of applying blockchain technology to orthopedic surgery. The application of blockchain technology to orthopedic surgery presents a promising solution for data sharing and supply chain management in the field. The integration of blockchain with cloud storage and hybrid encryption ensures secure and efficient storage of Electronic Health Record (EHR) and Personal Health Record (PHR) data. By leveraging the tamper-proof nature of blockchain and addressing concerns regarding centralized data storage, this scenario demonstrates enhanced security, improved access efficiency, and privacy protection in medical data sharing. CONCLUSIONS: The article demonstrates the feasibility of using an IoT-based blockchain network in orthopedic surgery, which can reduce medical errors and improve data interoperability among different parties. This unique application of blockchain enables secure sharing of medical records, ensuring transparency, security, and interoperability. The network design may also be applicable to other surgeries and medical applications in the future.

Oral Lactobacillus zeae exacerbates the pathological manifestation of periodontitis in a mouse model.

INTRODUCTION: The worldwide prevalence of periodontitis is considerably high, and its pathogenic mechanisms must be investigated and understood in order to improve clinical treatment outcomes and reduce the disease prevalence and burden. The exacerbation of the host immune system induced by oral microbial dysbiosis and the subsequent tissue destruction are the hallmarks of the periodontitis. However, the oral bacteria involved in periodontitis are not fully understood. We used the Oxford Nanopore Technologies (ONT) sequencing system to analyze metagenomic information in subgingival dental plaque from periodontitis and non-periodontitis patients. The number of Lactobacillus zeae (L. zeae) in the periodontitis patients was 17.55-fold higher than in the non-periodontitis patients, suggesting that L. zeae is a novel periodontitis-associated pathogen. Although several Lactobacillus species are used in vivo as probiotics to treat periodontitis and compete with Porphyromonas gingivalis (P. gingivalis), the roles of L. zeae in periodontitis progression, and the relationship between L. zeae and P. gingivalis needs to be investigated. METHODS: Both L. zeae and P. gingivalis were inoculated in the ligature-implant site of periodontitis mice. We collected mouse gingival crevicular fluid to analyze inflammatory cytokine secretion using a multiplex assay. Intact or sliced mouse maxilla tissue was used for micro-computed tomography analysis or hematoxylin and eosin staining, immunohistochemistry, and tartrate-resistant acid phosphatase staining to evaluate alveolar bone loss, neutrophil infiltration, and osteoclast activation, respectively. RESULTS: We observed that L. zeae competed with P. gingivalis, and it increased inflammatory cytokine secretion at the ligature-implant site. Similar to P. gingivalis, L. zeae promoted ligature-induced neutrophile infiltration, osteoclast activation, and alveolar bone loss. DISCUSSION: We, therefore, concluded that L. zeae accelerated the progression of periodontitis in the ligature-induced periodontitis mouse model.

A combination of all-trans retinoic acid derivative and COX-2 inhibitor has anticancer effects in human pharyngeal carcinoma cells.

Backgrounds and aims: Carcinogenesis is characterized by an unlimited growth of cells exacerbated by Cox-2 overexpression. Cox-2 inhibitors have been proven effective in preventing and treating tumors. In our previous studies, we found that 4-Amino-2-Trifluoromethylphenyl Retinate (ATPR) induces cell apoptosis and inhibits cell proliferation to exhibit anti-cancer properties. The use of ATRA as well as Cox-2 inhibitors in clinical settings can cause adverse reactions. It is unknown what the effects and mechanisms of co-administration of ATPR and Cox-2 inhibitors are. Results: A combination of ATPR and Cox-2 inhibitors, Celecoxib, inhibited pharyngeal cancer cell proliferation in vitro and induced apoptosis. The cell cycle was arrested at G0/G1 by activating P53 and CDNA1. By activating MAPK/JNK pathways, ATPR and Celecoxib led to intrinsic and extrinsic apoptosis in pharyngeal cancer cells. ATPR/Celecoxib combined treatment suppressed tumor growth in the pharyngeal cancer cell-derived xenograft mouse model by increasing the number of apoptotic cells. The expression of the RARA and PTGS2 genes was significantly increased in tumor tissue compared to non-tumor tissue in the clinical analysis of the head and neck squamous cell carcinoma dataset. An association was found between this and the level of intrinsic apoptotic signals. Furthermore, a survival analysis conducted over a period of five years indicated that higher levels of RARA expression were associated with a better clinical outcome. Conclusion: ATPR and celecoxib inhibit the proliferation of cancer cells as well as induce apoptosis. Co-administration of ATPR and Cox-2 inhibitors has the potential to be a novel treatment plan for cancer.

Controlled release of Clenbuterol from a hydroxyapatite carrier for the treatment of Alzheimer's Disease.

BACKGROUND: Alzheimer's disease is a neurodegenerative disorder, and Aß aggregation is considered to be the central process implicated in its pathogenesis. Current treatments are faced by challenges such as serious side effects and reduced drug bioavailability. In this study, we developed a drug delivery system for intramuscular injection that uses cellular activity to achieve constant and long-term drug release. METHODS: Synthesized mesoporous hydroxyapatite (SHAP) was prepared via co-precipitation, and hydrophobic surface modification using stearic acid was then used to load clenbuterol by physical absorption, thus creating the drug delivery system. Clenbuterol release was achieved through cellular activity, with macrophage uptake triggering lysosome/endosome disruption, cytoplasmic release, extracellular exocytosis, and subsequent systemic circulation. RESULTS: We found that clenbuterol-loaded SHAP enabled sustained release for more than 2 weeks and effectively modulated inflammation, reduced Aß oligomer-induced toxicity, and prevented Aß aggregation. CONCLUSIONS: Our findings suggest that treatment with clenbuterol loaded in this SHAP delivery system could be a promising strategy for treating Alzheimer's disease.

The synthesis and evaluation of thiolated alginate as the barrier to block nutrient absorption on small intestine for body-weight control.

Obesity is the most common health concern all over the world. However, till now, there is no promising way to manage obesity or body-weight control. The aim of the study is to develop an edible gel as a health supplement that temporarily attaches to the mucus of the intestines, forming an absorption barrier to block the nutrients. We modify the alginate with the thiol group as thiolated alginate (TA) that may stay on the mucosa layer for a much longer time to reduce nutrient absorption. In this study, the TA is synthesized successfully and proved a good mucosal adhesion to serve as a barrier for nutrient absorption both in vitro and in vivo. The results of in vivo imaging system (IVIS) show that the synthesized TA can be exiled from the gastrointestinal tract within 24 h. The animal study shows that the TA by daily oral administration can effectively reduce body weight and fat deposition. The biosafety is evaluated in vitro at the cellular level, based on ISO-10993, and further checked by animal study. We do believe that the TA could have a greater potential to be developed into a safe health supplement to manage obesity and for body-weight control.

Metastasis and immunosuppression promoted by mtDNA and PD-L1 in extracellular vesicles are reversed by WGP ß-glucan in oral squamous cell carcinoma.

The suppressive regulatory T cells (Treg) are frequently upregulated in cancer patients. This study aims to demonstrate the hypothesis that arecoline could induce the secretion of mitochondrial (mt) DNA D-loop and programmed cell death-ligand 1 (PD-L1) in extracellular vesicles (EVs), and attenuate T-cell immunity by upregulated Treg cell numbers. However, the immunosuppression could be reversed by whole glucan particle (WGP) ß-glucan in oral squamous cell (OSCC) patients. Arecoline-induced reactive oxygen specimen (ROS) production and cytosolic mtDNA D-loop were analyzed in OSCC cell lines. mtDNA D-loop, PD-L1, IFN-γ, and Treg cells were also identified for the surgical specimens and sera of 60 OSCC patients. We demonstrated that higher mtDNA D-loop, PD-L1, and Treg cell numbers were significantly correlated with larger tumor size, nodal metastasis, advanced clinical stage, and areca quid chewing. Furthermore, multivariate analysis confirmed that higher mtDNA D-loop levels and Treg cell numbers were unfavorable independent factors for survival. Arecoline significantly induced cytosolic mtDNA D-loop leakage and PD-L1 expression, which were packaged by EVs to promote immunosuppressive Treg cell numbers. However, WGP ß-glucan could elevate CD4+ and CD8+ T-cell numbers, mitigate Treg cell numbers, and promote oral cancer cell apoptosis. To sum up, arecoline induces EV production carrying mtDNA D-loop and PD-L1, and in turn elicits immune suppression. However, WGP ß-glucan potentially enhances dual effects on T-cell immunity and cell apoptosis and we highly recommend its integration with targeted and immune therapies against OSCC.

Vapor-Induced Pore-Forming Atmospheric-Plasma-Sprayed Zinc-, Strontium-, and Magnesium-Doped Hydroxyapatite Coatings on Titanium Implants Enhance New Bone Formation-An In Vivo and In Vitro Investigation.

OBJECTIVES: Titanium implants are regarded as a promising treatment modality for replacing missing teeth. Osteointegration and antibacterial properties are both desirable characteristics for titanium dental implants. The aim of this study was to create zinc (Zn)-, strontium (Sr)-, and magnesium (Mg)-multidoped hydroxyapatite (HAp) porous coatings, including HAp, Zn-doped HAp, and Zn-Sr-Mg-doped HAp, on titanium discs and implants using the vapor-induced pore-forming atmospheric plasma spraying (VIPF-APS) technique. METHODS: The mRNA and protein levels of osteogenesis-associated genes such as collagen type I alpha 1 chain (COL1A1), decorin (DCN), osteoprotegerin (TNFRSF11B), and osteopontin (SPP1) were examined in human embryonic palatal mesenchymal cells. The antibacterial effects against periodontal bacteria, including Porphyromonas gingivalis and Prevotella nigrescens, were investigated. In addition, a rat animal model was used to evaluate new bone formation via histologic examination and micro-computed tomography (CT). RESULTS: The ZnSrMg-HAp group was the most effective at inducing mRNA and protein expression of TNFRSF11B and SPP1 after 7 days of incubation, and TNFRSF11B and DCN after 11 days of incubation. In addition, both the ZnSrMg-HAp and Zn-HAp groups were effective against P. gingivalis and P. nigrescens. Furthermore, according to both in vitro studies and histologic findings, the ZnSrMg-HAp group exhibited the most prominent osteogenesis and concentrated bone growth along implant threads. SIGNIFICANCE: A porous ZnSrMg-HAp coating using VIPF-APS could serve as a novel technique for coating titanium implant surfaces and preventing further bacterial infection.

Third-generation sequencing-selected Scardovia wiggsiae promotes periodontitis progression in mice.

BACKGROUNDS: Periodontitis is an oral-bacteria-directed disease that occurs worldwide. Currently, periodontal pathogens are mostly determined using traditional culture techniques, next-generation sequencing, and microbiological screening system. In addition to the well-known and cultivatable periodontal bacteria, we aimed to discover a novel periodontal pathogen by using DNA sequencing and investigate its role in the progression of periodontitis. OBJECTIVE: This study identified pathogens from subgingival dental plaque in patients with periodontitis by using the Oxford Nanopore Technology (ONT) third-generation sequencing system and validated the impact of selected pathogen in periodontitis progression by ligature-implanted mice. METHODS: Twenty-five patients with periodontitis and 25 healthy controls were recruited in this study. Subgingival plaque samples were collected for metagenomic analysis. The ONT third-generation sequencing system was used to confirm the dominant bacteria. A mouse model with ligature implantation and bacterial injection verified the pathogenesis of periodontitis. Neutrophil infiltration and osteoclast activity were evaluated using immunohistochemistry and tartrate-resistant acid phosphatase assays in periodontal tissue. Gingival inflammation was evaluated using pro-inflammatory cytokines in gingival crevicular fluids. Alveolar bone destruction in the mice was evaluated using micro-computed tomography and hematoxylin and eosin staining. RESULTS: Scardovia wiggsiae (S. wiggsiae) was dominant in the subgingival plaque of the patients with periodontitis. S. wiggsiae significantly deteriorated ligature-induced neutrophil infiltration, osteoclast activation, alveolar bone destruction, and the secretion of interleukin-6, monocyte chemoattractant protein-1, and tumor necrosis factor-α in the mouse model. CONCLUSION: Our metagenome results suggested that S. wiggsiae is a dominant flora in patients with periodontitis. In mice, the induction of neutrophil infiltration, proinflammatory cytokine secretion, osteoclast activation, and alveolar bone destruction further verified the pathogenic role of S. wiggsiae in the progress of periodontitis. Future studies investigating the metabolic interactions between S. wiggsiae and other periodontopathic bacteria are warranted.

Tacrolimus-binding protein FKBP8 directs myosin light chain kinase-dependent barrier regulation and is a potential therapeutic target in Crohn's disease.

OBJECTIVE: Intestinal barrier loss is a Crohn's disease (CD) risk factor. This may be related to increased expression and enzymatic activation of myosin light chain kinase 1 (MLCK1), which increases intestinal paracellular permeability and correlates with CD severity. Moreover, preclinical studies have shown that MLCK1 recruitment to cell junctions is required for tumour necrosis factor (TNF)-induced barrier loss as well as experimental inflammatory bowel disease progression. We sought to define mechanisms of MLCK1 recruitment and to target this process pharmacologically. DESIGN: Protein interactions between FK506 binding protein 8 (FKBP8) and MLCK1 were assessed in vitro. Transgenic and knockout intestinal epithelial cell lines, human intestinal organoids, and mice were used as preclinical models. Discoveries were validated in biopsies from patients with CD and control subjects. RESULTS: MLCK1 interacted specifically with the tacrolimus-binding FKBP8 PPI domain. Knockout or dominant negative FKBP8 expression prevented TNF-induced MLCK1 recruitment and barrier loss in vitro. MLCK1-FKBP8 binding was blocked by tacrolimus, which reversed TNF-induced MLCK1-FKBP8 interactions, MLCK1 recruitment and barrier loss in vitro and in vivo. Biopsies of patient with CD demonstrated increased numbers of MLCK1-FKBP8 interactions at intercellular junctions relative to control subjects. CONCLUSION: Binding to FKBP8, which can be blocked by tacrolimus, is required for MLCK1 recruitment to intercellular junctions and downstream events leading to immune-mediated barrier loss. The observed increases in MLCK1 activity, MLCK1 localisation at cell junctions and perijunctional MLCK1-FKBP8 interactions in CD suggest that targeting this process may be therapeutic in human disease. These new insights into mechanisms of disease-associated barrier loss provide a critical foundation for therapeutic exploitation of FKBP8-MLCK1 interactions.

Glutamine induces remodeling of tight junctions in Caco-2 colorectal cancer cell.

Malignant cells often exhibit significant metabolic alterations, including the utilization of different nutrients to meet energetic and biosynthetic demands. Recent studies have shown that glutamine can support primary colorectal tumor growth and also serve as an alternate energy source during distant metastasis under glucose-limited conditions. However, the overall effects of glutamine on cancer cell physiology are not completely understood. In this study, we investigated how glutamine impacts epithelial integrity in colorectal cancer cells under glucose deprivation. Human colorectal cancer (Caco-2) cells were grown to confluency in transwells and cultured in glucose/pyruvate-free DMEM with various glutamine concentrations (0-50 mM). Cell viability was assessed, and monolayer integrity was examined in terms of transepithelial resistance (TER) and paracellular permeability. Tight junction (TJ) component proteins were examined by immunofluorescence staining and Western blotting. A dose-dependent decrease in TER was observed in Caco-2 cells, but paracellular permeability was not affected after 24 h incubation with glutamine. At the same time, the TJ proteins, zonula occludens (ZO)-1 and Claudin-1, showed lateral undulations and punctate staining patterns accompanied by enlargement of cellular and nuclear sizes. Furthermore, decreased protein levels of ZO-1, but not claudin-1, were found in detergent-insoluble cellular fractions. Notably, the decreased TER and alterations in TJ structure were not associated with cell viability changes. Moreover, the addition of glutamate, which is produced by the first step of glutamine catabolism, had no impact on TER. Our results suggested that the enteral glutamine may play an important role in the regulation of TJ dynamics in colorectal cancer cells.

Experimental Realization of a Fermionic Spin-Momentum Lattice.

We experimentally realize a spin-momentum lattice with a homogeneously trapped Fermi gas. The lattice is created via cyclically rotated atom-laser couplings between three bare atomic spin states, and are such that they form a triangular lattice in a synthetic spin-momentum space. We demonstrate the lattice and explore its dynamics with spin- and momentum-resolved absorption imaging. This platform will provide new opportunities for synthetic spin systems and the engineering of topological bands. In particular, the use of three spin states in two spatial dimensions would allow the simulation of synthetic magnetic fields of high spatial uniformity, which would lead to ultranarrow Chern bands that support robust fractional quantum Hall states.

Tight junction proteins occludin and ZO-1 as regulators of epithelial proliferation and survival.

Epithelial cells are the first line of mucosal defense. In the intestine, a single layer of epithelial cells must establish a selectively permeable barrier that supports nutrient absorption and waste secretion while preventing the leakage of potentially harmful luminal materials. Key to this is the tight junction, which seals the paracellular space and prevents unrestricted leakage. The tight junction is a protein complex established by interactions between members of the claudin, zonula occludens, and tight junction-associated MARVEL protein (TAMP) families. Claudins form the characteristic tight junction strands seen by freeze-fracture microscopy and create paracellular channels, but the functions of ZO-1 and occludin, founding members of the zonula occludens and TAMP families, respectively, are less well defined. Recent studies have revealed that these proteins have essential noncanonical (nonbarrier) functions that allow them to regulate epithelial apoptosis and proliferation, facilitate viral entry, and organize specialized epithelial structures. Surprisingly, neither is required for intestinal barrier function or overall health in the absence of exogenous stressors. Here, we provide a brief overview of ZO-1 and occludin canonical (barrier-related) functions, and a more detailed examination of their noncanonical functions.

Engineered cell-laden thermosensitive poly(N-isopropylacrylamide)-immobilized gelatin microspheres as 3D cell carriers for regenerative medicine.

Several studies have focused on using cell carriers to solve the problem of mesenchymal stem cell expansion on regenerative medicine. However, the disadvantages of using prolonged enzymatic treatment and low cell harvest efficiency still trouble researchers. In this study, PNIPAAm-immobilized gelatin microspheres (abbreviated as GNMS) were synthesized using a simple power-driven flow-focusing microinjection system. The developed thermosensitive GNMS can allow easier harvesting of cells from the microspheres, requiring only 10 â€‹min of low-temperature treatment and 5 â€‹min of trypsin treatment. The developed GNMS was characterized by Fourier-transform infrared spectroscopy, optical microscopy, and scanning electron microscopy. Further, live/dead staining, F-actin staining, and PrestoBlue cell viability assays were used to evaluate cytotoxicity, cell morphology, cell proliferation, and harvest efficiency. The gene expression of stem cell markers was determined by real-time quantitative PCR (Q-PCR) analysis to investigate the stemness and phenotypic changes in Wharton's jelly-derived mesenchymal stem cells. The results showed that the engineered cell-laden thermosensitive GNMS could significantly increase the cell harvest rate with over 99% cell survival rate and no change in the cell phenotype. Thus, the described strategy GNMS could be the suitable 3D cell carriers in the therapeutic application and opens new avenues for regenerative medicine.

High expression of tight junction protein 1 as a predictive biomarker for bladder cancer grade and staging.

Tight junction proteins 1-3 (TJP1-3) are components of tight junctions that can link transmembrane proteins to the actin cytoskeleton, and their incidence directly correlates to metastasis. However, the role of the TJP family in bladder cancer has not been adequately evaluated. In this study, we evaluated the genetic changes, mRNA and protein expressions of the target genes of the TJP family in bladder cancer patients using online database and immunohistochemistry, respectively. We found that TJP1 was amplified in bladder cancer tissue and that the protein expression levels were significantly associated with age (p = 0.03), grade (p = 0.007), and stage (p = 0.011). We also examined the correlation between TJP1 and other high-frequency mutation genes using TIMER. TJP1 mRNA levels were positively correlated with TTN and RYR3 mRNA levels in bladder cancer tissue. Taken together, TJP1 expression is associated with poor clinical outcomes in patients with bladder cancer and can be a useful predictive biomarker for bladder cancer staging.

Redox/pH-Responsive 2-in-1 Chimeric Nanoparticles for the Co-Delivery of Doxorubicin and siRNA.

The co-delivery of chemotherapy drugs and gene-suppressing small interfering RNA (siRNA) show promise for cancer therapy. The key to the clinical realization of this treatment model will be the development of a carrier system enabling the simultaneous delivery ("co-delivery" instead of combinatorial delivery) of chemotherapy and siRNA agents to cancer. In this study, a co-delivery system was developed from two individual components to form one integrated nanovehicle through a redox-sensitive thiol-disulfide bond for the synergistic delivery of chemotherapy and RNA silencing: doxorubicin (Dox)-loaded N,O-carboxymethyl chitosan (NOCC) complex with a thiolated hyaluronic acid (HA-SH) nanocarrier and dopamine (Dopa)-conjugated thiolated hyaluronic acid (SH-HA-Dopa)-coated calcium phosphate (CaP)-siRNA nanocarrier. The 2-in-1 chimeric nanoparticles (NPs) were structurally stable together in the storage environment and in the circulation. This smart system selectively releases Dox and siRNA into the cytosol. Furthermore, equipped with the tumor-targeting component HA, the co-delivery system shows specific targeting and high cellular uptake efficiency by receptor-mediated endocytosis. In summary, these dual-responsive (redox and pH), tumor-targeting smart 2-in-1 chimeric NPs show promise to be employed in functional co-delivery and tumor therapy.

SERPINE2 Overexpression Is Associated with Poor Prognosis of Urothelial Carcinoma.

Recent studies have reported that SERPINE2 contributes to the development of various cancers. However, its association with urothelial carcinoma (UC) remains unclear. In this study, data on urinary bladder UC (UBUC) cases from The Cancer Genome Atlas (TCGA) database were used to investigate the prognostic value of SERPINE2 mRNA expression. Then, SERPINE2 expression was analyzed with tissue microarrays constructed from 117 upper tract UC (UTUC) and 84 UBUC tissue specimens using immunohistochemical staining. Results were compared to clinicopathologic data by multivariate analysis. In the TCGA database, high SERPINE2 mRNA expression indicated a poor prognosis in patients with UBUC. Furthermore, Mann-Whitney U test showed that high SERPINE2 immunoexpression was significantly associated with adverse pathologic parameters including invasion, high grade, coexistence of UC in situ, and advanced pT stage (all p < 0.05, except for a marginal association with high-grade UBUC, p = 0.066). Kaplan-Meier analysis revealed that high SERPINE2 expression was associated with worse overall survival (OS; UTUC, p = 0.003; UBUC, p = 0.014) and disease-free survival (UTUC, p = 0.031; UBUC, p = 0.033). Moreover, multivariate analysis identified high SERPINE2 expression as an independent prognostic factor for OS (UTUC, p = 0.002; UBUC, p = 0.024). Taken together, our findings demonstrated that increased SERPINE2 expression is associated with adverse pathologic features and may serve as a prognostic biomarker for UC.

Current Development and Application of Anaerobic Glycolytic Enzymes in Urothelial Cancer.

Urothelial cancer is a malignant tumor with metastatic ability and high mortality. Malignant tumors of the urinary system include upper tract urothelial cancer and bladder cancer. In addition to typical genetic alterations and epigenetic modifications, metabolism-related events also occur in urothelial cancer. This metabolic reprogramming includes aberrant expression levels of genes, metabolites, and associated networks and pathways. In this review, we summarize the dysfunctions of glycolytic enzymes in urothelial cancer and discuss the relevant phenotype and signal transduction. Moreover, we describe potential prognostic factors and risks to the survival of clinical cancer patients. More importantly, based on several available databases, we explore relationships between glycolytic enzymes and genetic changes or drug responses in urothelial cancer cells. Current advances in glycolysis-based inhibitors and their combinations are also discussed. Combining all of the evidence, we indicate their potential value for further research in basic science and clinical applications.

The Tight Junction Protein ZO-1 Is Dispensable for Barrier Function but Critical for Effective Mucosal Repair.

BACKGROUNDS & AIMS: Increased permeability is implicated in the pathogenesis of intestinal disease. In vitro and in vivo studies have linked down-regulation of the scaffolding protein ZO-1, encoded by the TJP1 gene, to increased tight junction permeability. This has not, however, been tested in vivo. Here, we assessed the contributions of ZO-1 to in vivo epithelial barrier function and mucosal homeostasis. METHODS: Public Gene Expression Omnibus data sets and biopsy specimens from patients with inflammatory bowel disease (IBD) and healthy control individuals were analyzed. Tjp1f/f;vil-CreTg mice with intestinal epithelial-specific ZO-1 knockout (ZO-1KO.IEC) mice and Tjp1f/f mice littermates without Cre expression were studied using chemical and immune-mediated models of disease as well as colonic stem cell cultures. RESULTS: ZO-1 transcript and protein expression were reduced in biopsy specimens from patients with IBD. Despite mildly increased intestinal permeability, ZO-1KO.IEC mice were healthy and did not develop spontaneous disease. ZO-1KO.IEC mice were, however, hypersensitive to mucosal insults and displayed defective repair. Furthermore, ZO-1-deficient colonic epithelia failed to up-regulate proliferation in response to damage in vivo or Wnt signaling in vitro. ZO-1 was associated with centrioles in interphase cells and mitotic spindle poles during division. In the absence of ZO-1, mitotic spindles failed to correctly orient, resulting in mitotic catastrophe and abortive proliferation. ZO-1 is, therefore, critical for up-regulation of epithelial proliferation and successful completion of mitosis. CONCLUSIONS: ZO-1 makes critical, tight junction-independent contributions to Wnt signaling and mitotic spindle orientation. As a result, ZO-1 is essential for mucosal repair. We speculate that ZO-1 down-regulation may be one cause of ineffective mucosal healing in patients with IBD.

Differentiating Between Tight Junction-Dependent and Tight Junction-Independent Intestinal Barrier Loss In Vivo.

The intestinal barrier is an essential component of innate host defense. The single layer of epithelial cells that line the intestine must balance barrier function with both active, transcellular and diffusive, paracellular transport. Tight junctions, which link adjacent cells, form a selectively permeable seal that defines both paracellular transport and barrier properties. Molecules can cross tight junctions by either of two distinct routes, termed pore and the leak pathways, that differ in capacity, charge-selectivity, size-selectivity, and responses to physiological and pathophysiological stimuli. A third intestinal permeability route, the unrestricted pathway, reflects loss of the epithelial barrier, as occurs with mucosal damage, is independent of paracellular and transcellular pathways, and is neither charge- nor size-selective.The most commonly used approach for measuring intestinal permeability in vivo involves gavage of FITC-4 kDa dextran and analysis of the quantity recovered in serum. Unfortunately, this method cannot distinguish between leak and unrestricted pathways, as 4 kDa dextran can cross both. Moreover, 4 kDa dextran is too large to cross the pore pathway and, therefore, provides no information regarding this paracellular flux route. Here we describe a multiplex method that allows simultaneous, independent analysis of each pathway.