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Background and Aim E6011 is a humanized monoclonal antibody targeting fractalkine (FKN), a CX3C chemokine, which regulates leukocyte trafficking during inflammation. We evaluated the safety and pharmacokinetic profile of E6011 in patients with Crohn's disease (CD) and also performed preliminary pharmacodynamic (PD) and efficacy assessments. Methods This study included a 12‐week multiple ascending dose (MAD) phase (2, 5, 10, and 15 mg/kg intravenously every 2 weeks, n = 6, 8, 7, and 7, respectively) and a 40‐week Extension phase (n = 12) at the same dose as the MAD phase. Serum E6011, serum total FKN (free soluble FKN and E6011‐FKN complex) as a PD marker and CD activity index were evaluated. The primary outcome was safety assessment in the MAD phase. Results Twenty‐seven (96%) of 28 patients had previously been treated with anti‐tumor necrosis factor α agents. During the MAD phase, adverse events (AEs) occurred in 18 (64%). The most common AE was nasopharyngitis (five patients, 18%). No severe AEs occurred. Serious AEs occurred in three patients, progression of CD in two, and anemia in one. Serum E6011 concentrations increased dose‐dependently after infusion and reached a plateau around 4–6 weeks. Serum total FKN rose simultaneously. Five (18%) patients developed anti‐E6011 antibodies during the study. Overall, clinical response and clinical remission were observed at Week 12 in 40% (10/25) and 16% (4/25) of active CD patients, respectively. Conclusion E6011 was well‐tolerated and might be effective in CD patients. These findings need to be clarified in a randomized controlled study.

To evaluate the safety and efficacy of E6011, a novel humanized anti-fractalkine monoclonal antibody, in patients with active rheumatoid arthritis (RA) with an inadequate response to biological disease-modifying antirheumatic drugs (DMARDs).Active RA patients inadequately responding to biological DMARDs were randomly assigned to placebo or E6011 400-mg group at a 1:1 ratio, and administered E6011 at weeks 0, 1, 2, and subsequently every 2 weeks. Primary endpoint was American College of Rheumatology (ACR)20 response at week 12.Of 64, 33 received placebo, 31 received E6011 400-mg. The ACR20 response rate at week 12 (non-responder imputation) was 27.3% and 22.6% in the placebo and E6011 groups, respectively. ACR50, ACR70 response rates at week 12 were 3.0%, 0% in the placebo and 9.7%, 3.2% in the E6011 group. Exploratory PK exposure analysis revealed that the effect of E6011 tended to be clearer in patients with higher serum trough E6011 concentration. E6011 was well tolerated with no notable safety concerns.E6011 400-mg was well tolerated but had no clear efficacy at week 12 in RA patients with inadequate response to biologics. Further investigations are warranted to determine the optimal clinical dose and evaluation period for E6011.

Organoids derived from epithelial tumors have recently been utilized as a preclinical model in basic and translational studies. This model is considered to represent the original tumor in terms of 3D structure, genetic and cellular heterogeneity, but not tumor microenvironment. In this study, we established organoids and paired cancer-associated fibroblasts (CAFs) from surgical specimens of colorectal carcinomas (CRCs), and evaluated gene expression profiles in organoids with and without co-culture with CAFs to assess interactions between tumor cells and CAFs in tumor tissues. We found that the expression levels of several genes, which are highly expressed in original CRC tissues, were downregulated in organoids but re-expressed in organoids by co-culturing with CAFs. They comprised immune response- and external stimulus-related genes, e.g., REG family and dual oxidases (DUOXs), which are known to have malignant functions, leading tumor cells to proliferative and/or anti-apoptotic states and drug resistant phenotypes. In addition, the degree of differential induction of REG1 and DUOX2 in the co-culture system varied depending on CAFs from each CRC case. In conclusion, the co-culture system of CRC organoids with paired CAFs was able to partially reproduce the tumor microenvironment.

Human induced pluripotent stem cells (hiPSCs) are useful starting materials for the generation of cell therapy products, due to their pluripotency and ability to self-renew. Quality control of hiPSCs is extremely important in creating a stable supply of hPSC-derived products. Previously we identified an hiPSC-specific lectin probe, rBC2LCN, which binds specifically to α1,2-fucosylated glycan and recognizes podocalyxin (PODXL) as a glycoprotein ligand. In this study, we produced monoclonal antibodies (mAbs) specific for α1,2-fucosylated PODXL expressed on hiPSCs. PODXL was recombinantly expressed in fucosyltransferase 1 (FUT1)-transfected HEK293, followed by immunization into mice. Monoclonal antibodies, which bind to PODXL/FUT1-transfected cells, but not to cells transfected with only one of PODXL or FUT1, were screened by flow cytometry. The two mAbs generated (179-6B8C9 and 179-7E12E10), termed α1,2-fucosylated PODXL-specific mAbs (FpMabs), showed binding specificity to PODXL/FUT1-transfected cells. The FpMabs bound to hiPSCs but never to human adipose-derived mesenchymal stem cells, human dermal fibroblasts, or hiPSC-derived mesoderm. Altogether, FpMabs are highly specific probes for hiPSCs, which might be a powerful tool for the characterization of hiPSCs used in regenerative medicine.

Introduction: Rheumatic diseases are inflammatory diseases that damage target organs via multiple subsets of immune cells. Fractalkine (FKN) acts as chemoattractant as well as adhesion molecule. It...

Background Yolk sac tumors (YSTs) of the pancreas are extremely rare, and no drug responsiveness data are available regarding YSTs. Methods We report a pancreatic YST in a 70-year-old woman, and its chemotherapeutic responsiveness based on clinical records and evaluation of a patient-derived xenograft (PDX) line of the YST. Results The YST was an 11-cm, solid mass located in the pancreatic tail. Histologically, the tumor showed medullary proliferation of tumor cells, with a variety of growth patterns including microcystic/reticular, endodermal sinus, and hepatoid patterns. Immunohistochemically, the tumor cells were positive for Sall4, glypican-3, and alpha-fetoprotein. We administered VIP (etoposide, ifosfamide, cisplatin) chemotherapy for a recurrent liver tumor, and obtained complete pathological remission. A drug-response assay using the PDX line from this YST revealed that both VIP and gemcitabine effectively inhibit tumor growth. Conclusions These results suggest that differential diagnosis of YST from adenocarcinoma is important for selecting appropriate chemotherapy.

Diffuse-type gastric carcinoma (DGC) has a poor prognosis due to its rapid diffusive infiltration and frequent peritoneal dissemination. DGC is associated with massive fibrosis caused by aberrant proliferation of cancer-associated fibroblasts (CAFs). Previously, we reported that direct heterocellular interactions between cancer cells and CAFs is important for the peritoneal dissemination of DGC. In this study, we aimed to identify and target the molecules that mediate such heterocellular interactions. Monoclonal antibodies (mAbs) against intact DGC cells were generated and subjected to high-throughput screening to obtain several mAbs that inhibit the adhesion of DGC cells to CAFs. Immunoprecipitation and mass spectrometry revealed that all mAbs recognized integrin α5 complexed with integrin β1. Blocking integrin α5 in DGC cells or fibronectin, a ligand of integrin α5β1, deposited on CAFs abrogated the heterocellular interaction. Administration of mAbs or knockout of integrin α5 in DGC cells suppressed their invasion led by CAFs in vitro and peritoneal dissemination in a mouse xenograft model. Altogether, these findings demonstrate that integrin α5 mediates the heterotypic cancer cell-fibroblast interaction during peritoneal dissemination of DGC and may thus be a therapeutic target.

Gastric cancer is heavily influenced by aberrant DNA methylation that alters multiple cancer-related pathways, and may respond to DNA demethylating agents, such as 5-aza-2′-deoxycytidine (5-aza-dC). Here, we aimed to analyze whether 5-aza-dC can sensitize gastric cancer cells to clinically used cytotoxic drugs. Ten gastric cancer cell lines were treated with 5-aza-dC for 72 h and their growth was analyzed by conducting WST assay. In vivo effect of the drugs was analyzed using xenografts of OCUM-2 M/SN38 cells. Genome-wide expression and DNA methylation analyses were conducted using microarrays, and biological functions were identified through ingenuity pathway analysis. The cell lines most resistant to SN38 (an active metabolite of irinotecan), CDDP, PTX, and 5-FU, were identified. 5-Aza-dC pre-treatment of the resistant cell lines decreased the IC50 values for SN38 (TMK1, 226.4 nM to 32.91 nM; 44As3, 128.2 nM to 19.32 nM; OCUM2 M/SN38, 74.43 nM to 16.47 nM) and CDDP (TMK1, 5.05 µM to 2.26 µM; OCUM2 M, 10.79 µM to 2.77 µM), but not PTX and 5-FU. The reactivation of apoptosis-related genes, such as RUNX3, PYCARD, TNF, FAS, and FASLG, was induced by pre-treatment with 5-aza-dC, and the DNA demethylation of promoter CpG islands of RUNX3 and PYCARD was confirmed. In a xenograft model with OCUM2 M/SN38, treatment with 5-aza-dC before irinotecan showed markedly enhanced tumor suppression. Epigenetic priming with 5-aza-dC can improve the sensitivity of gastric cancer cells to SN38 and CDDP.

The organoid culture technique has been recently applied to modeling carcinogenesis in several organs. To further explore its potential and gain novel insights into tumorigenesis, we here investigated whether pancreatic ductal adenocarcinoma (PDA) could be generated as subcutaneous tumors in immunocompromised nude mice, by genetic engineering of normal organoids. As expected, acute induction of KrasG12Din vitro occasionally led to development of tiny nodules compatible with early lesions known as pancreatic intraepithelial neoplasia (PanIN). KrasG12D-expressing cells were enriched after inoculation in the subcutis, yet proved rather declined during culture, suggesting that its advantage might depend on surrounding environments. Depletion of growth factors or concurrent Trp53 deletion resulted in its robust enrichment, invariably leading to development of PanIN or large high-grade adenocarcinoma, respectively, consistent with in vivo mouse studies for the same genotype. Progression from PanIN was also recapitulated by subsequent knockdown of common tumor suppressors, whereas the impact of Tgfbr2 deletion was only partially recapitulated, illustrating genotype-dependent requirement of the pancreatic niche for tumorigenesis. Intriguingly, analysis of tumor-derived organoids revealed that KrasG12D-expressing cells with spontaneous deletion of wild-type Kras were positively selected and exhibited an aging-related mutation signature in nude mice, mirroring the pathogenesis of human PDA, and that the sphere-forming potential and orthotopic tumorigenicity in syngenic mice were significantly augmented. These observations highlighted the relevance of the subcutis of nude mice in promoting PDA development despite its ectopic nature. Taken together, pancreatic carcinogenesis could be considerably recapitulated with organoids, which would probably serve as a novel disease model.

Chemokine systems modulate inflammatory and immune responses in inflammatory bowel disease (IBD). The colons of IBD patients show increased levels of fractalkine (FKN) and high numbers of FKN receptor-positive (CX3CR1+) cells; however, the FKN–CX3CR1 axis’s role in intestinal inflammation, especially in intravascular leukocyte behaviors, still remains unclear. Here, we show that interruption of the FKN–CX3CR1 axis by anti-FKN monoclonal antibody (mAb) ameliorates murine colitis through regulation of intravascular monocyte behaviors in murine colitis models. FKN expression was detectable in vascular endothelium and CX3CR1+ macrophages accumulated in the mucosal lamina propria and submucosa of the inflamed colons. CD115+ monocytes tethered to the venous endothelium and expressed pro-inflammatory mediators. The anti-FKN mAb improved colitis symptoms, markedly reduced pro-inflammatory factors in the colon, maintained blood vessel integrity and reduced tethered monocytes in the inflamed veins. Intravital imaging revealed that CD115+Gr-1low/− monocytes crawled on the apical surfaces of venous endothelium, and anti-FKN mAb rapidly dislodged the crawling monocytes and inhibited their patrolling behavior. These findings suggest that the FKN–CX3CR1 axis triggers the patrolling behavior of crawling monocytes on the venous endothelium of inflamed colons, and accelerates the subsequent leukocyte activation and infiltration by locally producing inflammatory cytokines and chemokines. The mAb also ameliorated symptoms in another IBD model, T-cell-transferred colitis. Blocking the FKN–CX3CR1 axis with an anti-FKN mAb considerably inhibits the colitis-triggered inflammatory cascades, which may be an alternative strategy to treat IBD.

The use of patient‐derived xenografts (PDXs) has recently attracted attention as a drug discovery platform with a high predictive clinical efficacy and a preserved tumor heterogeneity. Given the racial differences in genetic variations, it would be desirable to establish a PDX library from Japanese cancer patients on a large scale. We thus tried to construct the Japanese PDX (J‐PDX) library with a detailed clinical information for further clinical utilization. Between August 2018 and May 2020, a total of 1126 cancer specimens from 1079 patients were obtained at the National Cancer Center Hospital and National Cancer Center Hospital East, Japan, and were immediately transplanted to immunodeficient mice at the National Cancer Center Research Institute. A total of 298 cross‐cancer PDXs were successfully established. The time to engraftment varied greatly by cancer subtypes, especially in the first passage. The engraftment rate was strongly affected by the clinical stage and survival time of the original patients. Approximately 1 year was needed from tumor collection to the time when coclinical trials were conducted to test the clinical utility. The 1‐year survival rates of the patients who were involved in establishing the PDX differed significantly, from 95.6% for colorectal cancer to 56.3% for lung cancer. The J‐PDX library consisting of a wide range of cancer subtypes has been successfully established as a platform for drug discovery and development in Japan. When conducting coclinical trials, it is necessary to consider the target cancer type, stage, and engraftment rate in light of this report., We have successfully established a PDX library derived from Japanese cancer patients. In less than 2 years, we have registered more than 1000 specimens and established nearly 300 PDXs. We will further enrich the library and use it as a platform to accelerate drug discovery in Japan.

Objective To evaluate the efficacy and safety of E6011, a humanized IgG2 monoclonal antibody against human fractalkine (FKN), in a phase II, double-blind, placebo-controlled study in rheumatoid arthritis (RA) patients. Methods Patients with moderate-to-severe RA who had an inadequate response to methotrexate were randomly assigned to a placebo group or to E6011 100-mg, 200-mg, or 400/200-mg groups at a 2:1:2:2 ratio. During the 24-week period, patients received the study drug subcutaneously at weeks 0, 1, and 2 and then once every 2 weeks. The primary end point was the American College of Rheumatology 20% improvement criteria (ACR20) response rate at week 12. Results Study drugs were administered to 190 patients (placebo, n = 54; E6011 100 mg, n = 28; E6011 200 mg, n = 54; E6011 400/200 mg, n = 54), and 169 patients completed treatment. A significant difference from placebo was not found in ACR20 response rates at week 12 (37.0% [placebo], 39.3% [100 mg], 48.1% [200 mg], and 46.3% [400/200 mg], using nonresponder imputation). As a secondary end point, ACR20 response rate in the 200-mg and 400/200-mg groups attained statistical significance at week 24 (35.2% [placebo], 39.3% [100 mg], 53.7% [200 mg], and 57.4% [400/200 mg]). Subsequent exploratory subgroup analysis revealed greater efficacy of E6011, particularly in patients with a higher baseline proportion of CD16+ monocytes; ACR20 response rates in this patient subgroup at week 24 were 30.0% (placebo), 46.7% (100 mg), 57.7% (200 mg), and 69.6% (400/200 mg). E6011 administered for 24 weeks was well tolerated. Conclusion This is the first evidence that E6011, a novel cell trafficking inhibitor targeting the FKN-CX3 CR1 interaction, is modestly effective with 24 weeks of treatment in RA patients, although the primary end point was not met.

Obesity, type 2 diabetes mellitus (T2DM) and aging are associated with pancreatic cancer risk, but the mechanisms of pancreatic cancer development caused by these factors are not clearly understood. Syrian golden hamsters are susceptible to N‐nitrosobis(2‐oxopropyl)amine (BOP)‐induced pancreatic carcinogenesis. Aging, BOP treatment and/or a high‐fat diet cause severe and scattered fatty infiltration (FI) of the pancreas with abnormal adipokine production and promote pancreatic ductal adenocarcinoma (PDAC) development. The KK‐A y mouse, a T2DM model, also develops severe and scattered FI of the pancreas. Treatment with BOP induced significantly higher cell proliferation in the pancreatic ducts of KK‐A y mice, but not in those of ICR and C57BL/6J mice, both of which are characterized by an absence of scattered FI. Thus, we hypothesized that severely scattered FI may be involved in the susceptibility to PDAC development. Indeed, severe pancreatic FI, or fatty pancreas, is observed in humans and is associated with age, body mass index (BMI) and DM, which are risk factors for pancreatic cancer. We analyzed the degree of FI in the non‐cancerous parts of PDAC and non‐PDAC patients who had undergone pancreatoduodenectomy by histopathology and demonstrated that the degree of pancreatic FI in PDAC cases is significantly higher than that in non‐PDAC controls. Moreover, the association with PDAC is positive, even after adjusting for BMI and the prevalence of DM. Accumulating evidence suggests that pancreatic FI is involved in PDAC development in animals and humans, and further investigations to clarify the genetic and environmental factors that cause pancreatic FI are warranted.

Fractalkine (CX3CL1/FKN) is a chemokine that regulates chemotaxis and adhesion of CX3C chemokine receptor 1 (CX3CR1)-expressing inflammatory cells. We conducted the first phase 1/2, open-label, multiple ascending dose study of E6011, a humanized anti-FKN monoclonal antibody, in Japanese rheumatoid arthritis (RA) patients (clinicaltrial.gov identifier: NCT02196558).Active RA patients with an inadequate response or intolerance to methotrexate or tumor necrosis factor (TNF) inhibitor received E6011 at week 0, 1, 2, and thereafter every 2 weeks for 12 weeks.Twelve, 15, and 10 subjects were enrolled in the 100, 200, and 400 mg cohorts, respectively. No severe adverse events (AEs) or deaths occurred, and no major differences were observed in the incidence or severity of AEs across the cohorts. Serum E6011 concentrations increased dose dependently. American College of Rheumatology (ACR) 20, 50, and 70 responses at week 12 were 75.0%, 33.3%, and 8.3% in the 100 mg cohort; 66.7%, 20.0%, and 13.3% in the 200 mg cohort; and 60.0%, 30.0%, and 20.0% in the 400 mg cohort, respectively.E6011 appeared to be safe and well tolerated in RA patients during this 12-week treatment period, suggesting that E6011 has an effective clinical response in active RA patients.

Hepatocyte growth factor (HGF) is an endogenously expressed bioactive substance that has a strong anti-apoptotic effect. In this study, we biochemically and histologically characterized the effects of rh-HGF on in vitro human hepatocyte injury and mouse acute liver failure (ALF) models, both of which were induced by antibody-mediated Fas signaling. rh-HGF inhibited intracellular caspase-3/7 activation and cytokeratin 18 (CK-18) fragment release in both models. Histologically, rh-HGF dramatically suppressed parenchymal damage and intrahepatic hemorrhage. Among the laboratory parameters, prothrombin time (PT) was strongly preserved by rh-HGF, and PT was well correlated with the degree of intrahepatic hemorrhage. These results showed that the anti-apoptotic effect of rh-HGF on hepatocytes coincided strikingly with the suppression of intrahepatic hemorrhage. PT was considered to be the best parameter that correlated with the intrahepatic hemorrhages associated with hepatocellular damage. The action of rh-HGF might derive not only from its anti-apoptosis effects on liver parenchymal cells but also from its stabilization of structural and vasculature integrity.

To assess the preclinical efficacy and mechanism of action of an anti-CXCultured human dermal fibroblasts were used to evaluate the direct effect of anti-CXAnti-CXAnti-CX

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

Inactivation of the Adenomatous polyposis coli (APC) gene is an initiating and the most relevant event in most sporadic cases of colorectal cancer, providing a rationale for using Apc-mutant mice as the disease model. Whereas carcinogenesis has been observed only at the organism level, the recent development of the organoid culture technique has enabled long-term propagation of intestinal stem cells in a physiological setting, raising the possibility that organoids could serve as an alternative platform for modeling colon carcinogenesis. Indeed, it is demonstrated in the present study that lentivirus-based RNAi-mediated knockdown of Apc in intestinal organoids gave rise to subcutaneous tumors upon inoculation in immunodeficient mice. Reconstitution of common genetic aberrations in organoids resulted in development of various lesions, ranging from aberrant crypt foci to full-blown cancer, recapitulating multi-step colorectal tumorigenesis. Due to its simplicity and utility, similar organoid-based approaches have been applied to both murine and human cells in many investigations, to gain mechanistic insight into tumorigenesis, to validate putative tumor suppressor genes or oncogenes, and to establish preclinical models for drug discovery. In this review article, we provide a multifaceted overview of these types of approaches that will likely accelerate and advance research on colon cancer.

E6011 is a novel humanized antifractalkine (FKN) monoclonal antibody being developed as a therapeutic target for Crohn's disease, rheumatoid arthritis, and primary biliary cholangitis. This study was a randomized, double-blind, placebo-controlled single-ascending-dose study of intravenous administration of E6011 (0.0006-10 mg/kg) in healthy Japanese adult men (n = 64). The starting dose was the minimum anticipated biological effect level (MABEL). MABEL was estimated by extrapolating results of a pharmacokinetic/pharmacodynamic (PK/PD) model relating E6011 exposure and suppression of free soluble FKN using data obtained from cynomolgus monkeys. Safety assessments consisted of monitoring and recording adverse events, laboratory tests, vital signs, intensive electrocardiograms, and chest x-rays. Blood samples to determine PK, PD (serum total FKN concentration), and serum anti-E6011 antibody were collected. Noncompartmental analysis was used to derive PK parameters. Single intravenous infusions of E6011 were safe and well tolerated in healthy subjects. Serum E6011 concentrations showed triphasic elimination. An increase in serum total FKN concentration was observed, confirming target engagement. The dose strategy for patient studies is to select regimens that will attain a minimum serum E6011 exposure of 10 μg/mL, identified as the minimum concentration needed to saturate the target-mediated elimination pathway. Model-based drug development from preclinical stage was successful in identifying dose regimens for clinical testing.

Background/Aim: To evaluate bone mineral density (BMD) in Japanese patients with prostate cancer (PCa) after administering androgen deprivation therapy (ADT) for 2 years. Patients and Methods: A total of 84 Japanese patients with PCa were enrolled in this study during the period 2008-2011. BMD was measured by dual energy X-ray absorptiometry, every 6 months. The fracture risk assessment tool (FRAX) score was calculated before starting ADT. We evaluated the change in BMD over a 2-year period and the relationship between this change, the FRAX score, and the estimated glomerular filtration rate (eGFR). Results: Compared to baseline, BMD decreased by 2.50% at 6 months after ADT, by 4.28% after 12 months, by 5.34% after 18 months, and by 6.16% after 2 years (all p

Small intestinal mononuclear cells that express CX3CR1 (CX3CR1

Background Adipokines are bioactive hormones secreted by adipose tissues. Resistin, an adipokine, plays important roles in the regulation of insulin resistance and inflammation. Resistin levels are known to be increased in the serum and synovial fluid of rheumatoid arthritis (RA) patients. However, the pathogenic role of resistin in RA has not yet been elucidated. Methods The expression of resistin and adenylate cyclase-associated protein 1 (CAP1), a receptor for resistin, was examined immunohistochemically in synovial tissue. CAP1 expression in in vitro cultured fibroblast-like synoviocytes (FLSs) was assessed with a reverse transcription-polymerase chain reaction (PCR) and western blotting. The gene expression of resistin-stimulated FLSs was evaluated by RNA sequencing (RNA-Seq) and quantitative real-time PCR. Concentrations of chemokine (C-X-C motif) ligand (CXCL) 8, chemokine (C-C motif) ligand (CCL) 2, interleukin (IL)-1β, IL-6 and IL-32 in culture supernatants were measured by enzyme-linked immunosorbent assay. Small interfering RNA (siRNA) for CAP1 was transfected into FLSs in order to examine inhibitory effects. Results The expression of resistin and CAP1 in synovial tissue was stronger in RA than in osteoarthritis (OA). Resistin was expressed by macrophages in the RA synovium, while CAP1 was expressed by macrophages, FLSs and endothelial cells. In vitro cultured RA FLSs also expressed CAP1. RNA-Seq revealed that the expression levels of 18 molecules were more than twofold higher in resistin-stimulated FLSs than in unstimulated FLSs. Seven chemokines, CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL8, and CCL2, were included among the 18 molecules. Increases induced in the expression of CXCL1, CXCL8, and CCL2 by the resistin stimulation were confirmed by real-time PCR. The stimulation with resistin increased the protein levels of CXCL8 and CCL2 produced by RA FLSs, and the upregulated expression of CXCL8 was inhibited by the abrogation of CAP1 by siRNA for CAP1. Production of IL-6 by FLSs was also increased by resistin. Expression of IL-1β and IL-32 was not detected by ELISA. Conclusions Resistin contributes to the pathogenesis of RA by increasing chemokine production by FLSs via CAP1 in synovial tissue.

The current definitive treatment for acute or chronic liver condition, that is, cirrhosis, is liver transplantation from a limited number of donors, which might cause complications after donation. Hence, bone marrow stem cell transplantation has been developed, but the risk of carcinogenesis remains. We have recently developed a protocol for hepatic differentiation of CD117(+) stem cells from human exfoliated deciduous teeth (SHED). In the present study, we examine whether SHED hepatically differentiated (hd) in vitro could be used to treat acute liver injury (ALI) and secondary biliary cirrhosis. The CD117(+) cell fraction was magnetically separated from SHED and then differentiated into hepatocyte-like cells in vitro. The cells were transplanted into rats with either ALI or induced secondary biliary cirrhosis. Engraftment of human liver cells was determined immunohistochemically and by in situ hybridization. Recovery of liver function was examined by means of histochemical and serological tests. Livers of transplanted animals were strongly positive for human immunohistochemical factors, and in situ hybridization confirmed engraftment of human hepatocytes. The tests for recovery of liver function confirmed the presence of human hepatic markers in the animals' blood serum and lack of fibrosis and functional integration of transplanted human cells into livers. No evidence of malignancy was found. We show that in vitro hdSHED engraft morphologically and functionally into the livers of rats having acute injury or secondary biliary cirrhosis. SHED are readily accessible adult stem cells, capable of proliferating in large numbers before differentiating in vitro. This makes SHED an appropriate and safe stem cell source for regenerative medicine.

Osteopontin (OPN) is a secreted phosphoglycoprotein, and is a transcriptional target of aberrant Wnt signaling. OPN is upregulated in human colon cancers, and is suggested to enhance cancer progression. In this study, the effect of deficiency of OPN on intestinal tumor development in Apc-deficient Min mice was investigated. At 16 weeks of age, the number of small intestinal polyps in Min/OPN(+/−) and Min/OPN(−/−) mice was lower than that of Min/OPN(+/+) mice. Colorectal tumor incidences and multiplicities in Min/OPN(+/−) and Min/OPN(−/−) mice were significantly lower than those in Min/OPN(+/+) mice, being 48% and 0.6 ± 0.8, 50% and 0.8 ± 0.9 vs. 80% and 1.6 ± 1.7, respectively. OPN expression in colorectal tumors was strongly upregulated in Min/OPN(+/+) compared to adjacent non-tumor parts, but was decreased in Min/OPN(+/−) and not detected in Min/OPN(−/−). Targets of OPN, matrix metalloproteinases (MMPs)-3, -9, and -13 were lowered by OPN deficiency. Macrophage marker F4/80 in colorectal tumors was also lowered by OPN deficiency. MMP-9 expression was observed in tumor cells and tumor-infiltrating neutrophils. These results indicate that induction of OPN by aberrant Wnt signaling could enhance colorectal tumor development in part by upregulation of MMP-3, -9, and -13 and infiltration of macrophage and neutrophils. Suppression of OPN expression could contribute to tumor prevention, but complete deficiency of OPN may cause some adverse effects.

The chemokine fractalkine (CX3C chemokine ligand 1; CX3CL1) and its receptor CX3CR1 are involved in the pathogenesis of several diseases, including inflammatory bowel diseases such as Crohn's disease and ulcerative colitis, rheumatoid arthritis, hepatitis, myositis, multiple sclerosis, renal ischemia, and atherosclerosis. There are no orally available agents that modulate the fractalkine/CX3CR1 axis. [(3

Aim To determine the differences in stem cell properties, in hepatic differentiation and in the effects of hydrogen sulphide (H2S) on hepatic differentiation between human bone marrow stem cells (hBMC) and stem cells from human exfoliated primary tooth pulp (SHED). Methodology CD117+ cells were magnetically separated and subjected to hepatic differentiation. CD117+ cell lineages were characterized for transcription factors indicative of stem cells by qRT-PCR. For the last 9 days of the differentiation, the test cells were exposed to 0.1 ng mL−1 H2S. Immunocytochemistry and flow cytometry of albumin, alpha-fetoprotein and carbamoyl phosphate synthetase were carried out after differentiation. Urea concentration and glycogen synthesis were also determined. Results Genes expressed in SHED were also expressed in BMC. No difference in expression level of hepatic markers was shown by immunofluorescence. SHED showed more positive cells than hBMC (P

Aim: Adult stem cells cannot proliferate to produce enough cells for human transplantation with keeping stem cell characteristics shown in the primary culture. We established a novel culture protocol using human dental pulp stem cells (DPSCs) that can produce quantities sufficient for human transplantation. The present study assessed differentiation of DPSCs toward a pancreatic lineage in serum-free conditions, which is essential for safe transplantation. Materials & methods: CD117+ stem cells were separated from human exfoliated deciduous teeth (stem cells from human exfoliated deciduous teeth; SHED) and adult DPSCs. The cells were characterized with real-time reverse-transcription PCR for a panel of embryonal lineage markers. Results: 82 out of 84 markers were expressed in different levels in SHED or DPSCs. After pancreatic differentiation in vitro, we found expression of pancreatic-specific endocrine markers insulin, glucagon, somatostatin and pancreatic polypeptide, and exocrine marker amylase-2a in both cultures. We also found reprogramming in both cell cultures mimicking the embryonal stages of development of the pancreas. Transcription factors PDX1, HHEX, MNX1, NEUROG3, PAX4, PAX6 and NKX6-1, crucial markers for the pancreatic development, were all activated. Expression of these factors strongly implies that the cells differentiated toward a distinguished pancreatic lineage. Conclusion: Our results show that CD117+ SHED and DPSCs are capable of differentiation toward all functional endocrine and exocrine subsets of pancreatic cells in serum-free conditions. SHED and DPSCs may therefore have great potential for future cell therapy of pancreatic disorders.

Animal models for human colorectal cancer recapitulate multistep carcinogenesis that is typically initiated by activation of the Wnt pathway. Although potential roles of both genetic and environmental modifiers have been extensively investigated in vivo, it remains elusive whether epithelial cells definitely require interaction with stromal cells or microflora for tumor development. Here we show that tumor development could be simply induced independently of intestinal microenvironment, even with WT murine primary intestinal cells alone. We developed an efficient method for lentiviral transduction of intestinal organoids in 3D culture. Despite seemingly antiproliferative effects by knockdown of adenomatous polyposis coli ( APC ), we managed to reproducibly induce APC -inactivated intestinal organoids. As predicted, these organoids were constitutively active in the Wnt signaling pathway and proved tumorigenic when injected into nude mice, yielding highly proliferative tubular epithelial glands accompanied by prominent stromal tissue. Consistent with cellular transformation, tumor-derived epithelial cells acquired sphere formation potential, gave rise to secondary tumors on retransplantation, and highly expressed cancer stem cell markers. Inactivation of p53 or phosphatase and tensin homolog deleted from chromosome 10, or activation of Kras, promoted tumor development only in the context of APC suppression, consistent with earlier genetic studies. These findings clearly indicated that genetic cooperation for intestinal tumorigenesis could be essentially recapitulated in intestinal organoids without generating gene-modified mice. Taken together, this in vitro model for colon cancer described herein could potentially provide unique opportunities for carcinogenesis studies by serving as a substitute or complement to the currently standard approaches.

Lung cancer is one of the leading causes of cancer-related deaths worldwide, including Japan. Although computed tomography (CT) can detect small lung lesions such as those appearing as ground glass opacity, it cannot differentiate between malignant and non-malignant lesions. Previously, we have shown that single photon emission computed tomography (SPECT) imaging using (111)In-1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid-cyclo-(Arg-Gly-Asp-D-Phe-Lys) (DOTA-c(RGDfK)), an imaging probe of αvβ3 integrin, is useful for the early detection of pancreatic cancer in a hamster pancreatic carcinogenesis model. In this study, we aimed to assess the usefulness of SPECT/CT with (111)In-DOTA-c(RGDfK) for the evaluation of the malignancy of lung cancer.Lung tumors were induced by a single intraperitoneal injection (250 mg/kg) of urethane in male A/J mice. Twenty-six weeks after the urethane treatment, SPECT was performed an hour after injection of (111)In-DOTA-c(RGDfK). Following this, the radioactivity ratios of tumor to normal lung tissue were measured by autoradiography (ARG) in the excised lung samples. We also examined the expression of αvβ3 integrin in mouse and human lung samples.Urethane treatment induced 5 hyperplasias, 41 adenomas and 12 adenocarcinomas in the lungs of 8 A/J mice. SPECT with (111)In-DOTA-c(RGDfK) could clearly visualize lung nodules, though we failed to detect small lung nodules like adenoma and hyperplasias (adenocarcinoma: 66.7%, adenoma: 33.6%, hyperplasia: 0.0%). ARG analysis revealed significant uptake of (111)In-DOTA-c(RGDfK) in all the lesions. Moreover, tumor to normal lung tissue ratios increased along with the progression of carcinogenesis. Histopathological examination using human lung tissue samples revealed clear up-regulation of αvβ3 integrin in well-differentiated adenocarcinoma (Noguchi type B and C) rather than atypical adenomatous hyperplasia.Although there are some limitations in evaluating the malignancy of small lung tumors using (111)In-DOTA-c(RGDfK), SPECT with (111)In-DOTA-c(RGDfK) might be a useful non-invasive imaging approach for evaluating the characteristics of lung tumors in mice, thus showing potential for use in humans.

Fractalkine is a CX3C chemokine that exists in both membrane-bound and soluble forms. Interaction between fractalkine and its unique receptor (CX3CR1) induces cell adhesion, chemotaxis, crawling, "accessory cell" activity, and survival. The serum level of fractalkine is elevated in patients with rheumatoid arthritis (RA) and is correlated with disease activity. Peripheral blood CD16+ monocytes and a subset of T cells express CX3CR1, while fractalkine is expressed on fibroblast-like synoviocytes and endothelial cells in the synovial tissue of patients with RA. Fractalkine expression is enhanced by tumor necrosis factor-α and interferon-γ, and it promotes the migration of monocytes, T cells, and osteoclast precursors into RA synovial tissue. Fractalkine also induces the production of inflammatory mediators by macrophages, T cells, and fibroblast-like synoviocytes. Moreover, fractalkine promotes angiogenesis and osteoclastogenesis. In an animal model of RA, arthritis was improved by the abrogation of fractalkine. Recently, a clinical trial of an anti-fractalkine monoclonal antibody for the treatment of RA commenced in Japan. We review the multiple roles of fractalkine in the pathogenesis of RA and its potential as a therapeutic target for this disease.

Background and Objective Hydrogen sulfide ( H2S ) is a volatile sulfur compound responsible for physiological halitosis. H2S was also reported as having periodontal pathologic activities. Gingival crevicular epithelium is the first barrier against periodontal pathogens and their products; oral keratinocyte stem cells OKSCs play key roles in maintaining this barrier. The p53 pathway is responsible for regulating key biological events. Increased apoptosis and cell-cycle arrest of DNA repair can affect keratinocyte stem cells, having a direct impact on the architecture of the oral epithelial tissue. However, the link between H2S , p53 activity and OKSCs has not yet been fully explored. The main objective of the present study was to explore the implications of the p53 pathway in OKSCs following exposure to H2S . Material and Methods OKSCs were isolated from human gingival epithelium and incubated with physiological levels of H2S for 24 and 48 h. Apoptosis and the mitochondrial membrane potential were detected using flow cytometry. Cytochrome c, total p53, phosphorylated p53 and caspase activity were assessed using specific ELISAs. p53 Pathway gene activity was assayed using quantitative RT-PCR. Results The levels of apoptosis were significantly increased following incubation in the presence of H2S, especially after 48 h (36.95 ± 1.91% vs. 4.77 ± 0.74%). Caspases 9 and 3 were activated, whereas caspase-8 activity remained low. Total p53 activity and particularly phosphorylated p53 at serine 46, were significantly enhanced compared with controls (47.11 ± 9.84 units/mL vs. 1.5 ± 0 units/mL and 32.22 ± 10.23 units/mL vs. 0.15 ± 0 units/mL, respectively, at 48 h). Among p53 pathway genes, apoptosis-related genes [i.e. phosphatase and tensin homolog ( PTEN ), B-cell CLL/lymphoma 2 ( BCL2), sirtuin 3 ( SIRT3) and caspases]) were dramatically increased when compared with controls. Moreover, cell-cycle progression genes [i.e. E2F transcription factor (E2F) family and histone deacetylase ( HDAC )] and DNA-repair genes [i.e. growth arrest and DNA-damage-inducible, gamma ( GADD45G ) family and serine/threonine-protein kinase Chk2 ( CHEK2)] were also increased. Conclusion Following incubation with H2S , OKSCs express multiple p53-associated genes, including programmed cell death, cell-cycle control and DNA-repair genes.

Untreated dental caries will eventually lead to pulpal inflammation. Although much is known regarding the interaction of microbial antigens and the immunologic defense systems of pulp, many aspects of the pathogenesis of pulpitis are not fully understood. The relationship between human pulp stem cells (HPSCs) and the pathogenesis of pulpitis remains among the poorly understood areas. Many of the invading bacteria are known to produce considerable amounts of hydrogen sulfide (H(2)S), which causes apoptosis in some tissues. The aims of this study were to determine whether H(2)S causes apoptosis in HPSCs and to examine its signaling pathway.Stem cells were isolated from human dental pulp and incubated with 50 ng/mL H(2)S for 48 hours. To detect apoptosis, the cells were analyzed by using flow cytometry. The mitochondrial signaling pathway was examined by determining mitochondrial membrane depolarization. Activation of the key apoptotic enzymes caspase-9, caspase-8, and caspase-3 was assessed by using enzyme-linked immunosorbent assay. Release of cytochrome C from mitochondria was also determined.The number of apoptotic cells increased significantly with H(2)S treatment from 1.6% to 16.3% (P.01). Significant increases were also measured in the amounts of caspase-9 and caspase-3 and in cytochrome C release (all P.01) and in mitochondrial membrane depolarization (P.05), whereas caspase-8 activity was not found.H(2)S causes apoptosis in HPSCs by activating the mitochondrial pathway. It is suggested that H(2)S might be one of the factors modifying the pathogenesis of pulpitis by causing loss of viability of HPSCs through apoptosis.

It is well known that hydrogen sulfide (H(2)S), the main substance causing physiologic halitosis, is also involved in the etiology of periodontitis. Gingival crevicular epithelium is the first barrier against periodontal pathogens and their products; keratinocyte stem cells play key roles in maintaining this barrier. An increased apoptotic process can affect keratinocyte stem cells, having a direct impact on oral epithelial tissue architecture. Our objective is to determine whether H(2)S induces apoptosis in human keratinocyte stem cells.Apoptosis levels; p53 activity; reactive oxygen species; mitochondrial membrane depolarization; cytochrome C release; and caspase-9, -8, and -3 were assessed using enzyme-linked immunosorbent assay and flow cytometry. Genomic DNA damage was examined using single-cell gel electrophoresis. Real-time polymerase chain reaction was used for Bax detection.The percentage of apoptotic cells was significantly increased (20.5% +/- 1.6% versus 4.5% +/- 1.1% at 24 hours and 37.8% +/- 5.4% versus 4.8% +/- 0.9% at 48 hours; P0.05, respectively; n = 5). Mitochondrial membrane potential was collapsed and reactive oxygen species levels were significantly increased compared to their control groups. At each time point the amount of released cytochrome C into the cytosol was significantly increased. Caspase-9 and -3 activities were significantly increased (P0.05), whereas caspase-8 remained inactive. After both 24 and 48 hours, total and phosphorylated p53 levels were significantly increased.We conclude that H(2)S can induce apoptosis in human keratinocyte stem cells, a key component of the epithelial barrier, following DNA damage and p53 activation.

Oral malodorous compounds including hydrogen sulfide (H2S) are causative agents of periodontitis because the toxicities are similar to that of cyanate. Previous studies demonstrated that volatile sulfur compounds (VSCs) were highly toxic to periodontal tissues, causing a large reduction in the amount of collagen in human gingival fibroblasts and extracellular matrix as well as, for example, apoptosis, immunologic responses, and matrix metalloproteinase production. The objective of this study was to determine the effect of H2S on the proliferation of osteoblasts and a signaling transduction pathway through the mitogen-activated protein kinase (MAPK).Normal human osteoblasts (NHOst) and murine osteoblasts (cell line MC3T3-E1) were incubated with H2S. Cell proliferation was assessed by measuring [3H]thymidine incorporation. The effects of H2S on the signal transduction pathways, the MAPK cascade, that control cell proliferation were evaluated in NHOst by determining extracellular signal-regulated kinase (ERK)1/2 and p38 phosphorylation with a Western blot analysis.After incubating NHOst with H2S for 24 hours, [3H]thymidine incorporation into the DNA significantly decreased dose-dependently with H(2)S. At a concentration of 100 ng/ml H2S, [3H]thymidine incorporation decreased 79% compared to the control. Similar results were obtained from MC3T3-E1. The phosphorylation of ERK1/2 and p38 was increased by H2S at 10 minutes after starting the treatment and then decreased time dependently. The activation of ERK1/2 and p38 induced by H2S was inhibited by the specific inhibitor of MAPK/ERK kinase ([MEK]; U0126) or p38 (SB203580).H2S inhibited the proliferation of human osteoblastic cells through the MAPK pathway.

The effect of sodium fluoride on the growth of two continuous human cell lines, i.e. HeLa cells and human conjunctiva clone 1-5C-4 cells, was studied. The growth of HeLa cells and clone 1-5C-4 cells was arrested nearly completely by the addition of 0.95 and 1,90 mM of sodium fluoride, respectively. DNA synthesis in HeLa cells, determined by incorporation of 3H-thymidine, was not affected appreciably for the first 24-hr period after the addition of sodium fluoride. Markedly reduced incorporation, however, occurred during the next 24-hr period. Thus, there was a discrepancy between the immediate cessation of cell division and the delayed suppression of DNA synthesis. On the other hand, a suppressive effect of sodium fluoride on protein synthesis determined by 14C-leucine incorporation was evident already during the first 24-hr period. The results indicate that the inhibition of protein synthesis is the main cause of growth inhibition.

Background and Objective: Volatile sulfur compounds are the main cause of halitosis. Hydrogen sulfide is one of these volatile sulfur compounds and the principal malodorous compound in physiological halitosis. Periodontally pathogenic activities of hydrogen sulfide have been previously reported. Hydrogen sulfide induces apoptotic cell death in aorta smooth muscle cells and in other tissues. Apoptosis plays an important role in the onset and progress of periodontitis. The objective of this study was to determine whether hydrogen sulfide causes apoptosis in human gingival fibroblasts. Material and methods: Necrotic cells were detected using a lactate dehydrogenase assay. Apoptosis was ascertained using a histone-complexed DNA fragment assay and flow cytometry. The level of caspase 3, a key enzyme in apoptotic signaling, was also measured, and the effects of hydrogen sulfide on reactive oxygen species and superoxide dismutase were assessed. DNA damage caused by hydrogen sulfide was examined by means of single-cell gel electrophoresis. Results: After 72 h of incubation with 100 ng/mL of hydrogen sulfide, necrosis was found in less than 10% of human gingival fibroblasts, whereas apoptosis was significantly increased (p

Purpose: Dentistry is a major resource for the treatment of halitosis, therefore dental professionals must also pay attention to their own oral malodour for professional courtesy. However, oral malodour among dental professionals has not yet been investigated. In this study, the diurnal changes in oral malodour in dental-office workers were determined, and preventative measures were assessed. Methods: Diurnal changes in the levels of volatile sulphur compounds (VSCs), which are the main cause of oral malodour, in mouth air were determined with a gas chromatograph specially designed for such analysis and the effects of several preventive measures were evaluated. Results: High concentrations of VSCs in mouth air persisted during the morning and decreased after lunch. Tongue-cleaning followed by tooth brushing decreased VSCs dramatically. Further measures such as eating breakfast, drinking tea or using zinc mouthwash significantly decreased VSCs, but the effects were limited in dental hygienists who suffered from persistent oral malodour, especially in the afternoon. Conclusion: Eating breakfast, cleaning the tongue followed by brushing the teeth and zinc chloride mouthwash were very effective in preventing oral malodour in dental-office workers; however, the effectiveness of these preventive measures was limited in dental hygienists.

Background CX3CL1/Fractalkine (FKN) has been reported to play important roles in various inflammatory diseases. We examined the role of FKN and its receptor CX3CR1 in T-cell migration in the inflammatory bowel diseases (IBDs), ulcerative colitis (UC) and Crohn's disease (CD). Methods CX3CR1 expression on peripheral CD4(+) cells from normal controls (NL n = 24) and IBD patients (UC n = 28, CD n = 26) was examined using flow cytometry. CX3CR1(+)CD4(+) T cells were further characterized for surface antigens, cytokine production, and cytotoxic granule release by flow cytometry and ELISA. FKN expression in 53 colonic biopsy specimens (UC n = 20, CD n = 23, NL n = 10) was analyzed by quantitative PCR and immunohistochemistry. Isolated lamina propria and intraepithelial lymphocytes were also analyzed by flow cytometry (UC n = 10, CD n = 10, NL n = 6). Results CX3CR1(+)CD4(+) cells were increased in IBD while they were virtually absent in controls. Upregulation of CX3CR1 on CD4(+) T cells was positively correlated with disease activity. These unique T cells expressed markers for both effector memory and cytotoxic cells. Interestingly, CX3CR1 was expressed on CD4(+) T cells lacking CD28. CX3CR1(+)CD28(-)CD4(+) cells produced more IFN-gamma and TNF-alpha than CX3CR1(-) counterparts and released cytotoxic granules. FKN mRNA was upregulated in inflamed colonic tissues and robust expression of FKN was immunohistochemically observed on epithelial cells. Although CX3CR1(+) CD4(+) cells could not be detected in the gut, CD28(-)CD4(+) cells were found in IBD mainly as intraepithelial lymphocytes. Conclusions FKN/CX3CR1 may contribute to the pathogenesis of IBD through the emergence of unique CX3CR1(+)CD28(-)CD4(+) T cells that can act both as proinflammatory and cytotoxic cells.

We previously identified the carbohydrate determinant sialyl 6-sulfo Lewis X (Le(x)) as the major L-selectin ligand on high endothelial venules of peripheral lymph nodes. In this study, we examined the distribution of the sialyl 6-sulfo Le(x) determinant among peripheral lymphocytes. The determinant was expressed on a subset of helper memory T and NK cells. The helper memory T cells expressing sialyl 6-sulfo Le(x) were CD45RO(bright+) PSGL-1(high+) CCR4+ L-selectin+ CCR7+ but did not express alpha4beta7 integrin or CCR9, indicating that they were the skin-homing population of central memory T cells. The T-cell subset significantly expressed mRNA for 6-sulfotransferase HEC-GlcNAc6ST and fucosyltransferase Fuc-T VII, responsible for the synthesis of sialyl 6-sulfo Le(x). Characteristics of the T-cell population were similar to those previously described for cutaneous lymphocyte-associated antigen (CLA)-positive T cells defined by the HECA-452 or 2F3 antibody. The binding of the T-cell subset with the specific anti-sialyl 6-sulfo Le(x) antibody G152 was almost completely abrogated by HECA-452 or 2F3. Binding of recombinant E-, P-, and L-selectins to the T-cell subset was significantly inhibited by G152 and by HECA-452 antibodies. We propose that CLA, which is expressed without any activation stimuli on peripheral skin-homing helper memory T cells in healthy persons, is at least partly the sialyl 6-sulfo Le(x) determinant.

Necl-5/Tage4/poliovirus receptor/CD155 has been shown to be the poliovirus receptor and to be up-regulated in rodent and human carcinoma. We have found previously that mouse Necl-5 regulates cell motility. We show here that mouse Necl-5 is furthermore involved in the regulation of cell proliferation. Studies using a specific antibody against Necl-5 and a dominant negative mutant of Necl-5 revealed that Necl-5 enhanced the serum-induced proliferation of NIH3T3, Swiss3T3, and mouse embryonic fibroblast cells. Necl-5 enhanced the serum-induced activation of the Ras-Raf-MEK-ERK signaling, up-regulated cyclins D2 and E, and down-regulated p27(Kip1), eventually shortening the period of the G(0)/G(1) phase of the cell cycle in NIH3T3 cells. Necl-5 similarly enhanced the platelet-derived growth factor-induced activation of the Ras-Raf-MEK-ERK signaling and shortened the period of the G(0)/G(1) phase of the cell cycle in NIH3T3 cells. Necl-5 acted downstream of the platelet-derived growth factor receptor and upstream of Ras. Moreover, up-regulated Necl-5 was involved at least partly in the enhanced proliferation of transformed cells including NIH3T3 cells transformed by an oncogenic Ras or v-Src. These results indicate that Necl-5 plays roles not only in cell motility but also in cell proliferation.

Chronic gastritis is frequently associated with infection of Helicobacter pylori and characterized by tissue infiltration of neutrophils, lymphocytes, and plasma cells. To address the mechanism of lymphocyte infiltration in chronic gastritis, we examined the expression of chemokines and their receptors using frozen sections of chronic gastritis, obtained from 23 patients who underwent gastrectomy for gastric cancer. By immunohistochemistry, lymphocytes in inflamed gastric mucosa expressed CCR5 abundantly, CXCR3 less frequently, and CCR4 sparsely. The numbers of CCR5(+) cells, which were composed of mainly CD8(+) and partly CD4(+) T cells, were positively correlated with the degree of neutrophil infiltration, and decreased in areas with intestinal metaplasia or mucosal atrophy. RANTES/CCL5, one of the ligands of CCR5, was localized mainly in CD8(+) and partly CD4(+) T cells with a characteristic dotted pattern, and such lymphocytes were most densely distributed around the neck region of gastric glands. In situ hybridization confirmed the expression of CCL5 mRNA in these cells, and immunoelectron microscopy revealed localization of CCL5 in the membrane-bound granules, which most probably corresponded to the cytolytic granules of cytotoxic T cells. The numbers of CCL5(+) lymphocytes showed a close correlation with the degree of neutrophil infiltration and markedly decreased in intestinal metaplasia. In conclusion, our data suggest that, together with neutrophils, CCL5(+) T cells, presumably activated cytotoxic T cells, would play important roles in the active inflammatory process of chronic gastritis. Our data also suggest a self-recruiting mechanism involving CCR5 and CCL5 for tissue accumulation of such T cells.

Neuroblastoma (NBL), one of the most common childhood solid tumors, has a distinct nature in different prognostic subgroups. However, the precise mechanism underlying this phenomenon remains largely unknown. To understand the molecular and genetic bases of neuroblastoma, we have generated its cDNA libraries and identified a human ortholog of tubulin tyrosine ligase gene (hTTL/Nbla0660) as a differentially expressed gene at high levels in a favorable subset of the tumor. Tubulin is subjected to several types of evolutionarily conserved posttranslational modification, including tyrosination and detyrosination. Tubulin tyrosine ligase catalyzes ligation of the tyrosine residue to the COOH terminus of the detyrosinated form of alpha-tubulin. The measurement of hTTL mRNA expression in 74 primary neuroblastomas by quantitative real-time reverse transcription-PCR revealed that its high expression was significantly associated with favorable stages (1, 2 and 4s; p = 0.0069), high TrkA expression (p = 0.002), a single copy of MYCN (p < 0.00005), tumors found by mass screening (p = 0.0042), nonadrenal origin (p = 0.0042) and good prognosis (p = 0.023). The log-rank test showed that high expression of hTTL was an indicator of favorable prognosis (p = 0.026). Immunohistochemical analysis using specific antibodies generated by us demonstrated that tyrosinated tubulin (Tyr-tubulin), detyrosinated tubulin (Glu-tubulin) and hTTL as well as Delta2-tubulin were positive in favorable tumors, whereas only Delta2-tubulin was positive in the tumors with MYCN amplification. In an RTBM1 neuroblastoma cell line, hTTL was increased after treating the cells with bone morphogenetic protein 2 (BMP2) or all-trans retinoic acid (RA), which induced neuronal differentiation. These results suggest that the deregulated tubulin tyrosination/detyrosination cycle caused by decreased expression of hTTL is associated with inhibition of neuronal differentiation and enhancement of cell growth in the primary neuroblastomas with poor outcome.

Fractalkine (now also called CX3CL1) is a unique chemokine that functions not only as a chemoattractant but also as an adhesion molecule and is expressed on endothelial cells activated by proinflammatory cytokines, such as interferon-γ and tumor necrosis factor-α. The fractalkine receptor, CX3CR1, is expressed on cytotoxic effector lymphocytes, including natural killer (NK) cells and cytotoxic T lymphocytes, which contain high levels of intracellular perforin and granzyme B, and on macrophages. Soluble fractalkine causes migration of NK cells, cytotoxic T lymphocytes, and macrophages, whereas the membrane-bound form captures and enhances the subsequent migration of these cells in response to secondary stimulation with other chemokines. Furthermore, stimulation through membrane-bound fractalkine activates NK cells, leading to increased cytotoxicity and interferon-γ production. Recently, accumulating evidence has shown that fractalkine is involved in the pathogenesis of various clinical disease states or processes, such as atherosclerosis, glomerulonephritis, cardiac allograft rejection, and rheumatoid arthritis. In addition, polymorphisms in CX3CR1, which reduce its binding activity to fractalkine, have been reported to increase the risk of HIV disease and to reduce the risk of coronary artery disease. This review will examine new concepts underlying fractalkine-mediated leukocyte migration and tissue damage, focusing primarily on the pathophysiological roles of fractalkine in various clinical conditions, especially in atherosclerosis and vascular injury.

Malignant transformation of cells causes disruption of cell-cell adhesion, enhancement of cell motility, and invasion into surrounding tissues. Nectins have both homophilic and heterophilic cell-cell adhesion activities and organize adherens junctions in cooperation with cadherins. We examined here whether Tage4, which was originally identified to be a gene overexpressed in colon carcinoma and has a domain structure similar to those of nectins, is involved in cell adhesion and/or migration. Tage4 heterophilically trans-interacted with nectin-3, but not homophilically with Tage4. Expression of Tage4 was markedly elevated in NIH3T3 cells transformed by an oncogenic Ki-Ras (V12Ras-NIH3T3 cells) as compared with that of wild-type NIH3T3 cells. trans-Interaction of Tage4 with nectin-3 enhanced motility of V12Ras-NIH3T3 cells. Tage4 did not bind afadin, a nectin- and actin filament-binding protein that connects nectins to the actin cytoskeleton and cadherins through catenins. Thus, Tage4 heterophilically trans-interacts with nectin-3 and regulates cell migration. Tage4 is tentatively re-named here nectin-like molecule-5 (necl-5) on the basis of its function and domain structure similar to those of nectins.

Objective Rheumatoid arthritis (RA) is characterized by chronic inflammation of multiple joints. Large numbers of T cells, which produce type 1 cytokines, infiltrate into RA synovium. Chemokines and chemokine receptors are considered to contribute to the T cell infiltration. In this study, we examined the role of CX3CL1/fractalkine and its receptor CX3C chemokine receptor 1 (CX3CR1) in the T cell migration into RA synovium. Methods Using flow cytometry, immunohistochemistry, and reverse transcription–polymerase chain reaction, we analyzed CX3CR1 expression by peripheral blood and synovial T cells, and CX3CL1 expression in synovium from patients with RA. Cytokine and cytotoxic molecule expression by CX3CR1-positive T cells was analyzed by flow cytometry. Results CX3CR1 expression by peripheral CD4+ and CD8+ T cells was up-regulated in RA patients. The peripheral CD4+ and CD8+ T cells expressing CX3CR1 predominantly produced interferon-γ and tumor necrosis factor α, and expressed cytotoxic molecules such as granzyme A and perforin. Furthermore, CX3CR1+,CD3+ T cells infiltrated into RA synovium. CX3CL1, the unique ligand of CX3CR1, was expressed by endothelial cells and synoviocytes in RA synovium, but not in osteoarthritis synovium. Conclusion Our findings suggest that the interactions of CX3CL1 and CX3CR1 might contribute to the accumulation of CX3CR1+ T cells expressing type 1 cytokines and possessing cytotoxic granules in RA synovium.

Fractalkine is a unique CX(3)C chemokine/mucin hybrid molecule that functions like selectins in inducing the capture of receptor-expressing cells. Because of the importance of tyrosine sulfation for ligand binding of the selectin ligand PSGL1, we tested the role of tyrosine sulfation for CX(3)CR1 function in cell adhesion. Tyrosine residues 14 and 22 in the N terminus of CX(3)CR1 were mutated to phenylalanine and stably expressed on K562 cells. Cells expressing CX(3)CR1-Y14F were competent in signal transduction but defective in capture by and firm adhesion to immobilized fractalkine under physiologic flow conditions. In static binding assays, CX(3)CR1-Y14F mutants had a 2-4-fold decreased affinity to fractalkine compared with wild type CX(3)CR1. By surface plasmon resonance measurements of fractalkine binding to biosensor chip-immobilized cell membranes, CX(3)CR1-Y14F mutants had a 100-fold decreased affinity to fractalkine. CX(3)CR1-expressing cell membranes treated with arylsulfatase to desulfate tyrosine residues also showed a 100-fold decreased affinity for fractalkine. Finally, synthesized, sulfated N-terminal CX(3)CR1 peptides immobilized on biosensor chips showed a higher affinity for fractalkine than non-sulfated peptides. Thus, we conclude that sulfation of tyrosine 14 enhances the function of CX(3)CR1 in cell capture and firm adhesion. Further, tyrosine sulfation may represent a general mechanism utilized by molecules that function in the rapid capture of circulating leukocytes.

Worldwide, the most important concern in the treatment of sexually transmitted infections is the increase in antimicrobial resistant Neisseria gonorrhoeae strains including resistance to cephalosporins, penicillins, fluoroquinolones or macrolides. To investigate the trends of antimicrobial susceptibility among N. gonorrhoeae strains isolated from male patients with urethritis, a Japanese surveillance committee conducted the second nationwide surveillance study. Urethral discharge was collected from male patients with urethritis at 26 medical facilities from March 2012 to January 2013. Of the 151 specimens, 103 N. gonorrhoeae strains were tested for susceptibility to 20 antimicrobial agents. None of the strains was resistant to ceftriaxone, but the minimum inhibitory concentration (MIC) 90% of ceftriaxone increased to 0.125 μg/ml, and 11 (10.7%) strains were considered less susceptible with an MIC of 0.125 μg/ml. There were 11 strains resistant to cefixime, and the MICs of these strains were 0.5 μg/ml. The distributions of the MICs of fluoroquinolones, such as ciprofloxacin, levofloxacin and tosufloxacin, were bimodal. Sitafloxacin, a fluoroquinolone, showed strong activity against all strains, including strains resistant to other three fluoroquinolones, such as ciprofloxacin, levofloxacin and tosufloxacin. The azithromycin MICs in 2 strains were 1 μg/ml.

Oncogenic fusion of the RET (rearranged during transfection) gene was recently identified as a novel driver gene aberration not only for the development of thyroid carcinoma but also of lung adenocarcinoma, the most frequent histological type of lung cancer. This study constructed and analyzed transgenic mice expressing KIF5B-RET, the predominant form of RET fusion gene specific for lung adenocarcinoma, under the control of the SPC (surfactant protein C) gene promoter. The mice expressed the KIF5B-RET fusion gene specifically in lung alveolar epithelial cells, and developed multiple tumors in the lungs. Treatment of the transgenic mice with vandetanib, which is a RET tyrosine kinase inhibitor approved by the U.S. Food and Drug Administration for the treatment of thyroid carcinoma, for 8 or 20 weeks led to a marked reduction in the number of lung tumors (3.3 versus 0 and 6.5 versus 0.2 per tissue section, respectively; P < 0.01, t-test). The results suggest that the RET fusion functions as a driver for the development of lung tumors, whose growth is inhibited by RET tyrosine kinase inhibitors.

Objectives The aim of the present study was to characterize molecular targets for the prevention/diagnosis of pancreatic cancer using a chemically induced hamster pancreatic carcinogenesis model, in which background injuries to the parenchyma, for example, chronic pancreatitis or acinar atrophy, are limited. Methods Gene expression profiles in atypical hyperplasias were first investigated using a microarray technique. Immunohistochemical analyses of early lesions and invasive ductal carcinoma (IDC) were then conducted for MUC1, of which mRNA levels were prominent among the up-regulated genes, in contrast with the coexpression of epithelial-mesenchymal transition (EMT)-related proteins. Results Immunohistochemistry for MUC1 cytoplasmic domain (MUC1-CD), which was not detected in normal-like pancreatic ducts, was positive in the apical surfaces of the epithelia of hyperplasias with and without atypia and IDC areas with distinct tubular patterns. In contrast, cytoplasmic/nuclear positivity for MUC1-CD was observed in the invasive front of IDCs. The coexpression of EMT-related proteins, such as slug and vimentin, with cytoplasmic/nuclear MUC1-CD was also detected. Conclusions Alterations in the expression and subcellular localization of MUC1 represent a biphasic phenomenon, and the latter may be associated with EMT in pancreatic carcinogenesis in hamsters, which indicates that changes in MUC1 are important targets for pancreatic cancer prevention and chemotherapy.

Recent studies have suggested an association of Helicobacter pylori and iron-deficiency anemia (IDA). This is a report of six cases of IDA associated with H. pylori gastritis.Six patients with IDA were studied (5 boys and 1 girl; mean age 13.6 years; range 13-15 years). Five had a medical history of long-standing IDA and of oral iron supplementation at outpatient clinics. The anemia recurred after the iron therapy had been discontinued. The sixth patient was admitted to the hospital with severe IDA. An extensive work-up was ordered that included technetium-99m (99mTc) scans for Meckel's diverticulum, total colonoscopy, and gastrointestinal endoscopy. After biopsy-based H. pylori test results were confirmed to be positive, anti-H. pylori therapy consisting of lansoprazole, clarithromycin, and metronidazole was administered for 2 weeks with no iron supplementation. The hematologic profile and iron status were assessed periodically after the end of the eradication regimen.Upper gastrointestinal endoscopy revealed a marked antral nodularity but no evidence of bleeding lesions in all the patients. Given the histology and the fact that rapid urease test results were positive, chronic active gastritis with H. pylori was diagnosed in all these cases. H. pylori was successfully eradicated in all the patients. There was no evidence of IDA in any of the follow-up examinations between 27 and 50 months after anti-H. pylori therapy.H. pylori infection may be involved in cases of IDA of unknown origin, and the eradication of H. pylori can be associated with the resolution of anemia.