Interleukin-22 enhanced the mucosal barrier and inhibited the invasion of Salmonella enterica in human-induced pluripotent stem cell-derived small intestinal epithelial cells.

Human-induced pluripotent stem cell-derived small intestinal epithelial cell (hiPSC-SIEC) monolayers are useful in vitro models for evaluating the gut mucosal barrier; however, their reactivity to cytokines, which are closely related to the regulation of mucosal barrier function, remains unclear. Interleukin (IL)-22 is a cytokine that contributes to regulate the mucosal barrier in the intestinal epithelia. Using microarray and gene set enrichment analysis, we found that hiPSC-SIEC monolayers activate the immune response and enhance the mucosal barrier in response to IL-22. Moreover, hiPSC-SIEC monolayers induced the gene expression of antimicrobials, including the regenerating islet-derived protein 3 family. Furthermore, IL-22 stimulation upregulated Mucin 2 secretion and gene expression of an enzyme that modifies sugar chains, suggesting alteration of the state of the mucus layer of hiPSC-SIEC monolayers. To evaluate its physiological significance, we measured the protective activity against Salmonella enterica subsp. enterica infection in hiPSC-SIEC monolayers and found that prestimulation with IL-22 reduced the number of viable intracellular bacteria. Collectively, these results suggest that hiPSC-SIEC monolayers enhance the mucosal barrier and inhibit infection by pathogenic bacteria in response to IL-22, as previously reported. These results can contribute to the further application of hiPSC-SIECs in evaluating mucosal barriers.

Yogurt starter strains ameliorate intestinal barrier dysfunction via activating AMPK in Caco-2 cells.

Lactic acid bacteria (LAB) are commonly used probiotics that improve human health in various aspects. We previously reported that yogurt starter strains, Lactobacillus delbrueckii subsp. bulgaricus 2038 and Streptococcus thermophilus 1131, potentially enhance the intestinal epithelial barrier function by inducing the expression of antimicrobial peptides in the small intestine. However, their effects on physical barrier functions remain unknown. In this study, we found that both strains ameliorated the decreased trans-epithelial resistance and the increased permeability of fluorescein isothiocyanate-dextran induced by tumor necrosis factor (TNF)-α and interferon (IFN)-γ in Caco-2 cells. We also demonstrated that LAB prevented a decrease in the expression and disassembly of tight junctions (TJs) induced by TNF-α and IFN-γ. To assess the repair activity of TJs, a calcium switch assay was performed. Both strains were found to promote the reassembly of TJs, and their activity was canceled by the inhibitor of AMP-activated protein kinase (AMPK). Moreover, these strains showed increased AMPK phosphorylation. These observations suggest that the strains ameliorated physical barrier dysfunction via the activation of AMPK. The activities preventing barrier destruction induced by TNF-α and IFN-γ were strain-dependent. Several strains containing L. bulgaricus 2038 and S. thermophilus 1131 significantly suppressed the barrier impairment, and L. bulgaricus 2038 showed the strongest activity among them. Our findings suggest that the intake of L. bulgaricus 2038 and S. thermophilus 1131 is a potential strategy for the prevention and repair of leaky gut.

The Anterior Position of the Hip Center of Rotation Is Related to Anterior Cup Protrusion Length and Symptomatic Iliopsoas Impingement in Primary Total Hip Arthroplasty.

BACKGROUND: Iliopsoas impingement (IPI) is an important complication after total hip arthroplasty (THA), and anterior cup protrusion is believed to be its main cause; however, the relationship between the hip center of rotation (COR) and symptomatic IPI or cup protrusion remains poorly understood. Therefore, the present study investigated these relationships. METHODS: The medical records of 138 patients who underwent unilateral primary THA were retrospectively reviewed. There were 8 patients (5.8%) who had symptomatic IPI. The COR and cup protrusion length measured with 2 methods were assessed on computed tomography. Risk factors for symptomatic IPI and the relationship between the COR and protrusion length were evaluated. RESULTS: Logistic regression analyses showed that anteroposterior position of the COR, sagittal cup protrusion length (SCPL) at the COR, and both axial and SCPLs at the most anterior margin of the cup were related to symptomatic IPI. Multivariable regression analyses showed that acetabular offset was related to axial protrusion length at the COR, and anteroposterior position of the COR was related to both axial and sagittal protrusion lengths at the most anterior margin of the cup. CONCLUSION: Anterior position of the cup was related to symptomatic IPI and both axial and sagittal protrusion lengths at the most anterior margin of the cup. Anterior reaming and cup protrusion should be avoided as much as possible to prevent symptomatic IPI.

High risk of elevated metal concentrations with 9/10-mm stem trunnions and highly cross-linked polyethylene grafted with poly(2-methacryloyloxyethyl phosphorylcholine) in total hip arthroplasty.

BACKGROUND: The risks of metal release due to fretting and corrosion at the head-neck junction and consequent adverse local tissue reaction (ALTR) have concerns in metal-on-polyethylene (MoP) total hip arthroplasty (THA). Although trunnions have become thinner in diameter to increase the range of motion, it has remained unclear whether this change affects metal release and ALTR in vivo. This study aimed to investigate serum metal concentrations and the prevalence of ALTR in MoP THA with a 9/10-mm stem trunnion. PATIENTS AND METHODS: A consecutive series of 37 hips that underwent THA using MoP grafted with poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC) with a 9/10-mm trunnion stem were retrospectively reviewed. Serum metal levels were assessed and compared with those in MoP THA with a 10/12-mm trunnion stem. ALTR was diagnosed with serum metal levels and cross-sectional images. The factors associated with serum metal levels were also assessed. RESULTS: The median serum cobalt and chromium levels were 1.5 µg/L and 1.0 µg/L in the 9/10-mm group and 0.2 µg/L and 0.4 µg/L in the 10/12-mm group, respectively. ALTR was found in 5 hips of 3 patients. Revision surgery was performed in 4 hips, and all stem trunnions and femoral heads showed severe corrosion. Postoperative walking ability was associated with serum metal levels. CONCLUSION: It was found that a 9/10-mm stem trunnion with MoP grafted with PMPC had high risks of metal release in primary THA. Careful follow-up and cross-sectional imaging are needed to detect ALTR for early revision.

Stem-bone contact patterns of a long straight tapered uncemented stem for primary THA.

INTRODUCTION: Despite excellent long-term survival, total hip arthroplasty (THA) can fail due to aseptic loosening, dislocations, sepsis and periprosthetic fractures, all of which remain considerably burdensome. Aseptic loosening is one of the main causes of THA failure, often due to osteolysis, stress shielding and/or lack of primary stability. This study aimed to investigate stem-bone contact patterns of a long straight-tapered uncemented stem following primary THA, and to determine whether these contact patterns are related to preoperative femoral morphology and whether they influence postoperative outcomes. MATERIALS AND METHODS: The authors reviewed a continuous series of 60 hips (55 patients) that underwent primary THA using the Corail® stem (DePuy, Leeds, UK). Patients were evaluated pre- and post-operatively using the Japanese Orthopaedic Association (JOA) score. Computed-tomography (CT) scans were performed preoperatively to assess femoral bone morphology, and immediate postoperatively to assess stem-bone contact patterns. Postoperative radiographs were performed to calculate the Engh score. Regression analyses were performed to determine associations of postoperative JOA and Engh score with 27 independent variables. RESULTS: Forty-nine patients (54 hips) were assessed at 31 ± 8 months, with a JOA score of 92.9 ± 8.1 and an Engh score of 21.2 ± 1.9. Six patients (6 hips) were lost-to-follow-up. There were no revisions and only one complication (recurrent dislocation). Stem-bone contact patterns were associated with preoperative femoral morphology (sagittal CFI [p = 0.006], femoral offset [p = 0.028], and NSA [p = 0.022]), but were not associated with either postoperative JOA or postoperative Engh score. CONCLUSIONS: The stem-bone contact patterns of a long straight-tapered uncemented stem are related to preoperative femoral morphology, but do not influence short-term postoperative outcomes. Contact patterns were related to preoperative femoral offset, NSA, and sagittal CFI, but not coronal CFI. Surgeons should, therefore, consider sagittal morphology for surgical planning and templating, in addition to the conventional parameters of coronal morphology.

Higher incidence of aseptic loosening caused by a lower canal filling ratio with a modified modular stem in total hip arthroplasty.

PURPOSE: Although a cementless modular prosthesis has shown reliable results, cases of unstable fixation and revision due to aseptic loosening were observed in our institute. The purpose of this study was to clarify the causes of unstable fixation of the prosthesis. METHODS: A total of 144 patients (154 hips) who underwent total hip arthroplasty using the modular prosthesis were retrospectively investigated. For the cohort study, 97 patients (104 hips) were included. The femoral component survival rate and sleeve fixation were assessed at a minimum follow-up of 5 years. Patients were divided into 2 groups, including stable and unstable fixation groups, by sleeve fixation. Clinical and radiographic outcomes were compared. RESULTS: The Kaplan-Meier survival rate at 9 years was 93% with revision for any reason as the endpoint in study cohort. The reasons for revision were recurrent dislocation (1 hip) and aseptic loosening of the stem (5 hips). A total of 88 hips (84.6%) showed stable fixation, and 16 hips (15.4%) showed unstable fixation at final follow-up. There was no significant difference in clinical outcomes between the 2 groups at final follow-up. The canal flare index was significantly higher, and the canal filling ratio was significantly lower in the unstable fixation group. CONCLUSION: Although the modified modular prosthesis was useful for treating anatomically difficult patients, we need to pay attention to both proximal/distal mismatch of the intramedullary canal and the canal filling ratio to achieve stable fixation and good long-term results.

Patellar facet ratio affects knee pain, stair climbing and stair descent after TKA without patellar resurfacing.

PURPOSE: To determine whether knee pain or functional impairment after total knee arthroplasty (TKA) without patellar resurfacing are correlated with preoperative patellar morphology or postoperative patellar orientation. The hypotheses were that patellar shape, increased tilt and lateral displacement would be associated with pain and functional impairment. METHODS: From a consecutive series of 152 knees that received a cemented postero-stabilized TKA, the Oxford Knee Score (OKS) and the Knee injury and Osteoarthritis Outcome Score (KOOS) were collected at a minimum follow-up of 12 months. Uni- and multi-variable linear regression analyses were performed to determine associations between the collected clinical scores and patient demographics and patellar morphology, measured from pre- and post-operative frontal, lateral and skyline view radiographs. RESULTS: The OKS was 75 ± 23, whereas the KOOS pain, stair climbing, and descent were respectively 77 ± 24, 3.9 ± 1.1 and 3.8 ± 1.2. OKS was not associated with any radiographic outcomes, whereas KOOS pain was better for knees with larger medial patellar facets. The KOOS stair climbing and descent were also better for knees with larger medial patellar facets. CONCLUSION: The findings of this study partly confirm the hypotheses that pain and functional impairments after TKA without patellar resurfacing are associated with patellar shape. No association was revealed between postoperative patellar orientation and function nor pain. Quantitative consideration of patellar congruency could therefore prevent pain and improve function after TKA without patellar resurfacing. LEVEL OF EVIDENCE: Retrospective study, Level III.

Lactobacillus delbrueckii subsp. bulgaricus 2038 and Streptococcus thermophilus 1131 Induce the Expression of the REG3 Family in the Small Intestine of Mice via the Stimulation of Dendritic Cells and Type 3 Innate Lymphoid Cells.

Accumulating evidence clarifies that intestinal barrier function, for example, by the mucus layer, antimicrobial peptides, immune systems, and epithelial tight junctions, plays crucial roles in maintaining our health. We reported previously that yogurt fermented with Lactobacillus delbrueckii subsp. bulgaricus 2038 and Streptococcus thermophilus 1131 induced the gene expression of the regenerating family member 3 (REG3) family, which encodes antimicrobial peptides in the small intestine, although it was unclear how the yogurt activated the intestinal cells related to it. Here, we evaluated the cytokine production from the intestinal immune cells stimulated by these strains in vitro and in vivo to elucidate the mechanism for the induction of the REG3 family by the yogurt. The results showed that stimulation by both strains induced interleukin (IL)-23 production from bone marrow-derived dendritic cells (DCs) and IL-22 production from small intestinal lamina propria (LP) cells. In addition, oral administration of these strains to mice increased IL-23p19+ LPDCs and IL-22+ type 3 innate lymphoid cells and induced the expression of Reg3g in small intestinal tissue. Moreover, we showed that the activities for the induction of IL-23 by DCs were strain dependent on L. bulgaricus and S. thermophilus and that S. thermophilus 1131, which is the predominant species in the yogurt, exhibited relatively higher activity compared to the other strains of S. thermophilus. Our findings suggested that these yogurt starter strains, L. bulgaricus 2038 and S. thermophilus 1131, have the potential to maintain and improve intestinal barrier function by stimulating immune cells in the LP.

Antibiotics suppress colon tumorigenesis through inhibition of aberrant DNA methylation in an azoxymethane and dextran sulfate sodium colitis model.

Chronic inflammation is involved in the development of colon cancer by inducing mutations and aberrant DNA methylation in colon epithelial cells. Furthermore, there is growing evidence that colonic microbiota modulates the inflammation response in the host and influences colon tumorigenesis. However, the influence of colonic microbiota on aberrant DNA methylation remains unknown. Here, we show the effect of colonic microbes on DNA methylation and tumorigenicity using a mouse model of human ulcerative colitis. Mice treated with azoxymethane (AOM) and dextran sulfate sodium (DSS) showed an increase in degree of colitis, as estimated by body weight, occult blood, and stool consistency/diarrhea at 2 weeks after treatment, but treatment with antibiotics markedly reduced the severity of the colitis. Although mucosal hyperplasia and increased inflammation-related genes were observed in the colonic epithelial cells of the AOM/DSS-treated mice, treatment with antibiotics abrogated these changes. In addition, treatment with antibiotics significantly decreased the number of mucosal nodules from 5.9 ± 5.3 to 0.2 ± 0.6 (P < .01) and area of occupancy from 50.1 ± 57.4 to 0.5 ± 1.4 mm2 (P < .01). Aberrant DNA methylation of three marker CpG islands (Cbln4, Fosb, and Msx1) was induced by AOM/DSS treatment in colonic mucosae, but this increase was suppressed by 50%-92% (P < .05) with antibiotic treatment. Microbiome analysis showed that this change was associated with a decrease of the Clostridium leptum subgroup. These data indicate that antibiotics suppressed tumorigenesis through inhibition of aberrant DNA methylation induced by chronic inflammation.

FEAT enhances INSL3 expression in testicular Leydig cells.

FEAT, the protein encoded by methyltransferase-like 13 (METTL13), is aberrantly upregulated in most human cancers and potently drives tumorigenesis in vivo; however, its role in normal tissues remains elusive. Immunoblotting has displayed weak FEAT expression in normal human tissues, including the testis. Here, we found that FEAT is expressed in fetal and adult Leydig cells in the testis. FEAT knockdown using siRNA increased primary cilia formation in MA-10 Leydig tumor cells, accompanied by enhanced 5' adenosine monophosphate-activated protein kinase (AMPK) activation. Immunofluorescence analyses of FEAT-silenced MA-10 cells showed diminished insulin-like factor 3 (INSL3) expression. A male Mettl13+/- mouse developed bilateral intraabdominal cryptorchidism, suggesting defective INSL3 production by fetal Leydig cells. Leydig cells from the mouse showed markedly decreased INSL3 protein by immunohistochemistry. Together, these results suggest that FEAT facilitates the INSL3 production in testicular Leydig cells that is essential for transabdominal testis migration.

Effects of long-term intake of a yogurt fermented with Lactobacillus delbrueckii subsp. bulgaricus 2038 and Streptococcus thermophilus 1131 on mice.

The gut is an extremely complicated ecosystem where micro-organisms, nutrients and host cells interact vigorously. Although the function of the intestine and its barrier system weakens with age, some probiotics can potentially prevent age-related intestinal dysfunction. Lactobacillus delbrueckii subsp. bulgaricus 2038 and Streptococcus thermophilus 1131, which are the constituents of LB81 yogurt, are representative probiotics. However, it is unclear whether their long-term intake has a beneficial influence on systemic function. Here, we examined the gut microbiome, fecal metabolites and gene expression profiles of various organs in mice. Although age-related alterations were apparent in them, long-term LB81 yogurt intake led to an increased Bacteroidetes to Firmicutes ratio and elevated abundance of the bacterial family S24-7 (Bacteroidetes), which is known to be associated with butyrate and propanoate production. According to our fecal metabolite analysis to detect enrichment, long-term LB81 yogurt intake altered the intestinal metabolic pathways associated with propanoate and butanoate in the mice. Gene ontology analysis also revealed that long-term LB81 yogurt intake influenced many physiological functions related to the defense response. The profiles of various genes associated with antimicrobial peptides-, tight junctions-, adherens junctions- and mucus-associated intestinal barrier functions were also drastically altered in the LB81 yogurt-fed mice. Thus, long-term intake of LB81 yogurt has the potential to maintain systemic homeostasis, such as the gut barrier function, by controlling the intestinal microbiome and its metabolites.

Immunogenic FEAT protein circulates in the bloodstream of cancer patients.

BACKGROUND: FEAT is an intracellular protein that potently drives tumorigenesis in vivo. It is only weakly expressed in normal human tissues, including the testis. In contrast, FEAT is aberrantly upregulated in most human cancers. The present study was designed to investigate whether FEAT is applicable to tumor immunotherapy and whether FEAT is discernible in the bloodstream as a molecular biomarker of human cancers. METHODS: Two mouse FEAT peptides with predicted affinities for major histocompatibility complex H-2Kb and H-2Db were injected subcutaneously into C57BL/6 mice before subcutaneous transplantation of isogenic B16-F10 melanoma cells. Intracellular localization of FEAT was determined by immunogold electron microscopy. Immunoprecipitation was performed to determine whether FEAT was present in blood from cancer patients. A sandwich enzyme-linked immunosorbent assay was used to measure FEAT concentrations in plasma from 30 cancer patients and eight healthy volunteers. RESULTS: The vaccination experiments demonstrated that FEAT was immunogenic, and that immune responses against FEAT were induced without deleterious side effects in mice. Electron microscopy revealed localization of FEAT in the cytoplasm, mitochondria, and nucleus. Immunoprecipitation identified FEAT in the blood plasma from cancer patients, while FEAT was not detected in plasma exosomes. Plasma FEAT levels were significantly higher in the presence of cancers. CONCLUSIONS: These findings suggest that FEAT is a candidate for applications in early diagnosis and prevention of some cancers.

The miR-199a/Brm/EGR1 axis is a determinant of anchorage-independent growth in epithelial tumor cell lines.

In epithelial cells, miRNA-199a-5p/-3p and Brm, a catalytic subunit of the SWI/SNF complex were previously shown to form a double-negative feedback loop through EGR1, by which human cancer cell lines tend to fall into either of the steady states, types 1 [miR-199a(-)/Brm(+)/EGR1(-)] and 2 [miR-199a(+)/Brm (-)/EGR1(+)]. We show here, that type 2 cells, unlike type 1, failed to form colonies in soft agar, and that CD44, MET, CAV1 and CAV2 (miR-199a targets), all of which function as plasma membrane sensors and can co-localize in caveolae, are expressed specifically in type 1 cells. Single knockdown of any of them suppressed anchorage-independent growth of type 1 cells, indicating that the miR-199a/Brm/EGR1 axis is a determinant of anchorage-independent growth. Importantly, two coherent feedforward loops are integrated into this axis, supporting the robustness of type 1-specific gene expression and exemplifying how the miRNA-target gene relationship can be stably sustained in a variety of epithelial tumors.

Association of CD209 polymorphisms with tuberculosis in an Indonesian population.

Tuberculosis (TB) caused by Mycobacterium tuberculosis is a major cause of morbidity and mortality worldwide. Thus far, many candidate genes have been investigated for their possible association with TB. Dendritic cell-specific ICAM-3 grabbing nonintegrin (DC-SIGN) encoded by CD209 is the major receptor of M tuberculosis on human dendritic cells. Previous studies reported inconsistent results on the association between CD209 polymorphisms and TB. We examined whether 9 single nucleotide polymorphisms (SNPs) of CD209 are associated with TB in 2 southeast Asian populations (Indonesian and Vietnamese) by Fisher's exact test. The SNP at -939 in the promoter region exhibited a significant association with TB in Indonesian (GG vs GA + AA, p = 0.0051, odds ratio [OR] = 0.68, 95% confidence interval [CI] = 0.52-0.89) but not in Vietnamese populations. Further extensive studies are required to confirm the contribution of CD209 polymorphisms to TB susceptibility.

Use of the kinase inhibitor analog 1NM-PP1 reveals a role for Toxoplasma gondii CDPK1 in the invasion step.

Toxoplasma gondii CDPK1 (TgCDPK1) was found to be the target of the toxoplasmocidal compound 1NM-PP1. When TgCDPK1 was mutated at position 128 from glycine to methionine, resistance was gained. Inhibition of gliding motility without inhibition of micronemal secretion by 1NM-PP1 suggests a function for TgCDPK1 in gliding motility.