Identification of small peptides and glycinamide that inhibit melanin synthesis using a positional scanning synthetic peptide combinatorial library.

BACKGROUND: Antimelanogenic peptides are potentially useful to treat hyperpigmentation, but many peptides have limited application because of high cost and/or low activity. OBJECTIVES: To identify small and potent peptide inhibitors of cellular melanin synthesis that are useful for cosmetic and medical applications. METHODS: A positional scanning synthetic tetrapeptide combinatorial library was used for screening of potentially active peptides. Antimelanogenic activities of the peptide pools and individual peptides were evaluated in B16-F10 melanoma cells and human epidermal melanocytes treated with alpha-melanocyte-stimulating hormone (α-MSH). RESULTS: Predicted active tetrapeptide sequences were R-(F/L)-(C/W)-(G/R)-NH2 . Of the individual tetrapeptides tested, D3 (RFWG-NH2 ) and D5 (RLWG-NH2 ) exhibited high antimelanogenic activities. Tetrapeptide D9 (FRWG-NH2 ) with a sequence identical to that of a portion of α-MSH also showed antimelanogenic activity. Of the tripeptides tested, E5 (FWG-NH2 ), E6 (LWG-NH2 ) and E7 (RWG-NH2 ) were relatively more active. Dipeptide F1 (WG-NH2 ) and monopeptide G1 (G-NH2 , glycinamide) retained activity, but G2 (Ac-G-NH2 ) and G3 (glycine) did not. The antimelanogenic activities of peptides D3, E5, F1 and G1 were verified in α-MSH-stimulated human epidermal melanocytes. Commercially available G-NH2 ·HCl suppressed the phosphorylation levels of cAMP-responsive element binding protein, protein levels of microphthalmia-associated transcription factor and tyrosinase, l-tyrosine hydroxylase activity of tyrosinase, and the melanin levels in stimulated cells. CONCLUSIONS: Small peptides, including glycinamide and tryptophanyl glycinamide, are potent antimelanogenic agents with potential value for the treatment of skin hyperpigmentation.

Crepidiastrum denticulatum Extract Ameliorates Kidney Ischemia-Reperfusion Injury in Mice.

BACKGROUND: Crepidiastrum denticulatum (CD) is a well-known, traditionally consumed vegetable in Korea, which was recently reported to contain bioactive compounds with detoxification and antioxidant properties. Ischemia-reperfusion injury (IRI) is a major problem after renal transplantation. Furthermore, inflammatory responses to IRI exacerbate the resultant renal injury. In the present study, we investigated whether CD extract exhibits renoprotective effects against IR-induced acute kidney injury in mice. MATERIALS AND METHODS: Renal IRI was induced in male C57BL/6 mice by bilateral renal pedicle occlusion for 30 minutes followed by reperfusion for 48 hours. CD extract (75 mg/kg) was administered orally 5 days before IRI. RESULTS: Treatment with CD extract significantly decreased blood urea nitrogen and serum creatinine levels as well as kidney tubular injury. CD also prevented IRI-induced renal glutathione depletion and increased malondialdehyde levels. Western blotting and reverse transcriptase polymerase chain reaction indicated that CD extract significantly attenuates inducible nitric oxide synthase and toll-like receptor 2/4 protein levels 48 h after IRI. The expression of tumor necrosis factor-α and interleukin-1ß was significantly decreased in the CD extract treatment group. CONCLUSION: CD extract improved acute renal IRI through its antioxidant and anti-inflammatory effects. These findings suggest that CD extract is a potential therapeutic agent for acute ischemia-induced renal damage.

Reliability and validity of the Atopic Dermatitis Symptom Score (ADSS).

BACKGROUND: We have developed the Atopic Dermatitis Symptom Score (ADSS) by which patients or parents can easily assess and record AD symptoms on a daily basis in a smartphone application. The aim of this study was to evaluate the reliability and validity of the ADSS. METHODS: We enrolled 307 children and adolescents with AD. Parents or caregivers were asked to record daily symptoms of the patients (itching, sleep disturbance, erythema, dryness, oozing, and edema) using a scale of 0-4. Statistical analyses consisted of the test-retest reliability, concurrent validity, minimal clinically important difference (MCID), responsiveness, floor or ceiling effects, and screening accuracy. Receiver-operating characteristic analyses were conducted to evaluate the ADSS cutoff point for predicting severe AD (SCORing AD [SCORAD] ≥40). RESULTS: Test-retest reliability between daytime and night-time ADSS was good (intraclass correlation coefficient, 0.82 [95% CI: 0.70-0.90]). An increase in ADSS was significantly associated with an increase in SCORAD (r = 0.64, P < .0001) (concurrent validity). The MCID was 4.1 points for the ADSS. There was a significant association between changes in ADSS and SCORAD (r = 0.56, P < .0001), indicating good responsiveness. At the optimal ADSS cutoff value of 7.0, sensitivity, specificity, and positive and negative predictive values were 88.4%, 78.6%, 21.1%, and 99.1%, respectively (screening accuracy). CONCLUSIONS: The ADSS can be a useful tool for self-assessment of skin symptoms in children with AD.

Revision anatomical reconstruction of the lateral ligaments of the ankle augmented with suture tape for patients with a failed Broström procedure.

AIMS: The aim of this prospective study was to evaluate the intermediate-term outcomes after revision anatomical ankle ligament reconstruction augmented with suture tape for a failed modified Broström procedure. PATIENTS AND METHODS: A total of 30 patients with persistent instability of the ankle after a Broström procedure underwent revision augmented with suture tape. Of these, 24 patients who were followed up for more than two years were included in the study. There were 13 men and 11 women. Their mean age was 31.8 years (23 to 44). The mean follow-up was 38.5 months (24 to 56) The clinical outcome was assessed using the Foot and Ankle Outcome Score (FAOS) and the Foot and Ankle Ability Measure (FAAM) score. The stability of the ankle was assessed using stress radiographs. RESULTS: The mean FAOS and FAAM scores improved significantly to 87.5 (73 to 94) and 85.1 (70 to 95) points at final follow-up, respectively (p < 0.001). The mean angle of talar tilt and anterior talar translation improved significantly to 2.8° (0° to 6°) and 4.1 mm (2 to 7) at final follow-up, respectively (p < 0.001). Side to side comparison in stress radiographs at final follow-up showed no significant difference. The revision failed in one patient who underwent a further revision using allograft tendon. CONCLUSION: The revision modified Broström procedure augmented with suture tape is an effective form of treatment for recurrent instability of the ankle following a failed Broström procedure. This technique provides reliable stability and satisfactory clinical outcomes at intermediate-term follow-up. Cite this article: Bone Joint J 2017;99-B:1183-9.

Technical note: Induction of pluripotent stem cell-like cells from chicken feather follicle cells.

Pluripotent stem cells including embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC) are regarded as representative tools for conservation of animal genetic resources. Although ESC have been established from chicken, it is very difficult to obtain enough embryos for isolation of stem cells for avian conservation in most wild birds. Therefore, the high feasibility of obtaining the pluripotent cell is most important in avian conservation studies. In this study, we generated induced pluripotent stem cell-like cells (iPSLC) from avian Feather Follicular cells (FFC). Avian FFC are one of the most easily accessible cell sources in most avian species, and their reprogramming into pluripotent stem cells can be an alternative system for preservation of avian species. Intriguingly, FFC had mesenchymal stromal cells (MSC)-like characteristics with regard to gene expression, protein expression, and adipocyte differentiation. Subsequently, we attempted to generate iPSLC from FFC using retroviral vectors. The FFC-iPSLC can proliferate with the stem pluripotent property and differentiate into several types of cells in vitro. Our results suggest that chicken FFC are an alternative cell source for avian cell reprogramming into pluripotent stem cells. This experimental strategy should be useful for conservation and restoration of endangered or high-value avian species without sacrificing embryos.

Major Histocompatibilty Complex-Restricted Adaptive Immune Responses to CT26 Colon Cancer Cell Line in Mixed Allogeneic Chimera.

BACKGROUND: Although the induction of mixed allogeneic chimera shows promising clinical tolerance results in organ transplantation, its clinical relevance as an anti-cancer therapy is yet unknown. We introduced a mixed allogenic chimera setting with the use of a murine colon cancer cell line, CT26, by performing double bone marrow transplantation. METHODS: We analyzed donor- and recipient-restricted anti-cancer T-cell responses, and phenotypes of subpopulations of T cells. The protocol involves challenging 1 × 105 cells of CT26 cells intra-hepatically on day 50 after bone marrow transplantation, and, by use of CT26 lysates and an H-2Ld-restricted AH1 pentamer, flow cytometric analysis was performed to detect the generation of cancer-specific CD4+ and CD8+ T cells at various time points. RESULTS: We found that immunocompetence against tumors depends heavily on cancer-specific CD8+ T-cell responses in a major histocompatibility complex-restricted manner; the evidence was further supported by the increase of interferon-γ-secreting CD4+ T cells. Moreover, we demonstrated that during the effector immune response to CT26 cancer challenge, there was a presence of central memory cells (CD62LhiCCR7+) as well as effector memory cells (CD62LloCCR7-). Moreover, mixed allogeneic chimeras (BALB/c to C56BL/6 or vice versa) showed similar or heightened immune responses to CT26 cells compared with that of wild-type mice. CONCLUSIONS: Our results suggest that the responses of primary immunocompetency and of pre-existing memory T cells against allogeneic cancer are sustained and preserved long-term in a mixed allogeneic chimeric environment.

Effects of dietary methyl sulfonyl methane (MSM) supplementation on growth performance, nutrient digestibility, meat quality, excreta microbiota, excreta gas emission, and blood profiles in broilers.

A 29-d trial was conducted to evaluate the effects of dietary methyl sulfonyl methane (MSM) supplementation on growth performance, meat quality, nutrient digestibility, excreta microbiota, excreta gas emission, and blood profiles in broilers. A total of 816 1-day-old male Ross 308 broilers (44 ± 0.44 g) were assigned to 4 dietary treatments, composed of 12 replicates with 17 birds per replicate. The 4 treatments were: 1) CON, basal diet; 2) S1, CON + 0.05% MSM; 3) S2, CON + 0.10% MSM; 4) S3, CON + 0.20% MSM. In the current study, body weight (BW) on d 14 and 29 showed significant improvement as dietary MSM increased from 0.05% to 0.20% (P < 0.05). During d 1 to 14 and overall, higher (P < 0.05) body weight gain (BWG) and lower feed conversion ratio (FCR) were observed in broilers fed MSM diets. Between d 15 and 29, higher (P < 0.05) BWG was observed in broilers fed MSM diets. Redness (a*) was increased linearly (P < 0.05) in broilers fed MSM diets. On d 3, 5, and 7, drip loss was decreased linearly (P < 0.05) in broilers fed MSM diets. Lactobacillus and E. coli were effected linearly (P < 0.05) in broilers fed MSM diets. Alanine aminotransferase (ALT), white blood cells (WBC) and lymphocytes were improved linearly (P < 0.05) in broilers fed MSM diets. In conclusion, dietary supplementation MSM has positive effects on growth performance, meat quality, excreta microbiota, and blood profiles in broilers.

The T-ALL related gene BCL11B regulates the initial stages of human T-cell differentiation.

The initial stages of T-cell differentiation are characterized by a progressive commitment to the T-cell lineage, a process that involves the loss of alternative (myelo-erythroid, NK, B) lineage potentials. Aberrant differentiation during these stages can result in T-cell acute lymphoblastic leukemia (T-ALL). However, the mechanisms regulating the initial stages of human T-cell differentiation are obscure. Through loss of function studies, we showed BCL11B, a transcription factor recurrently mutated T-ALL, is essential for T-lineage commitment, particularly the repression of NK and myeloid potentials, and the induction of T-lineage genes, during the initial stages of human T-cell differentiation. In gain of function studies, BCL11B inhibited growth of and induced a T-lineage transcriptional program in T-ALL cells. We found previously unknown differentiation stage-specific DNA binding of BCL11B at multiple T-lineage genes; target genes showed BCL11B-dependent expression, suggesting a transcriptional activator role for BCL11B at these genes. Transcriptional analyses revealed differences in the regulatory actions of BCL11B between human and murine thymopoiesis. Our studies show BCL11B is a key regulator of the initial stages of human T-cell differentiation and delineate the BCL11B transcriptional program, enabling the dissection of the underpinnings of normal T-cell differentiation and providing a resource for understanding dysregulations in T-ALL.

Reduced expression of the epidermal growth factor signaling system in preeclampsia.

INTRODUCTION: The epidermal growth factor (EGF) signaling system regulates trophoblast differentiation, and its disruption could contribute to perinatal disease. We hypothesized that this pathway is altered in preeclampsia, a disorder associated with trophoblast apoptosis and failure to invade and remodel the uterine spiral arteries. METHODS: Six EGF family peptides and a truncated EGF receptor splice variant (p110/EGFR) were examined using immunohistochemistry in the trophoblast of placentas (N = 76) from women with preeclampsia, and compared to placentas from women of similar gestational age (GA) with preterm labor (PTL) or small for gestational age (SGA) fetuses, as well as normal term placentas. EGF, transforming growth factor-α (TGFA), and heparin-binding EGF-like growth factor (HBEGF) were evaluated using ELISA in maternal plasma from another 20 pregnancies with or without preeclampsia. Cell death was evaluated in the HTR-8/SVneo human cytotrophoblast cell line using TUNEL to evaluate the protective effects of EGF peptides. RESULTS: Trophoblast HBEGF, TGFA, and EGF were significantly reduced in preeclampsia compared to PTL and SGA, while p110/EGFR accumulated significantly on the surface of the chorionic villi (p < 0.05). Plasma EGF levels were significantly decreased in preeclamptic patients, compared to non-preeclamptic patients (p < 0.05). HBEGF, EGF, TGFA, epiregulin, and betacellulin each blocked cytotrophoblast cell death in vitro (p < 0.05). DISCUSSION: Three members of the EGF family are dysregulated in placentas with preeclampsia, whereas p110/EGFR, a potential EGF receptor antagonist, is overexpressed. These findings are consistent with the concept that disruption of the EGF signaling system contributes to aberrant trophoblast development associated with preeclampsia.

Reliability of tumour grade 1 and endometrioid cell type on preoperative endometrial biopsy.

The aim of this study was to evaluate the reliability of using tumour grade and cell type on preoperative endometrial biopsy for the selection of patients for conservative hormone treatment. We retrospectively reviewed results of 643 patients with endometrial carcinoma for tumour grade and 817 for tumour cell type who underwent endometrial biopsy followed by surgery. Of the 357 patients with a grade 1 tumour on preoperative endometrial biopsy, 58 (16.2%) were upgraded based on a final pathology report from hysterectomy specimens. For grade 1, the preoperative endometrial biopsy showed a sensitivity of 80.4%, a specificity of 78.6%, a positive predictive value (PPV) of 83.8% and a negative predictive value (NPV) of 74.5%. Of the 672 patients with the endometrioid cell type on preoperative biopsy, 46 (5.6%) showed a different cell type on final pathology. For the endometrioid cell type, preoperative endometrial biopsy had a sensitivity of 91.3%, a specificity of 64.9%, a PPV of 93.2% and an NPV of 58.6%. This weak predictive value should be considered when selecting patients for conservative hormone treatment.

Metabolic tumour volume and total lesion glycolysis, measured using preoperative 18F-FDG PET/CT, predict the recurrence of endometrial cancer.

OBJECTIVE: To investigate the prognostic value of metabolic tumour volume (MTV) and total lesion glycolysis (TLG), measured by preoperative positron emission tomography and computerised tomography (PET/CT), in women with endometrial cancer. DESIGN: Retrospective cohort study. SETTING: A tertiary referral centre. POPULATION: Women with endometrial cancer who underwent preoperative (18)F-FDG PET/CT in the period 2004-2009. METHODS: Clinicopathological data for 84 women with endometrial cancer were reviewed from medical records. Cox proportional hazards modelling identified recurrence predictors. The receiver operating characteristic (ROC) curve was used to determine the cut-off value for predicting recurrence. MAIN OUTCOME MEASURE: Disease-free survival (DFS). RESULTS: The number of patients with International Federation of Gynecology and Obstetrics (FIGO) stages were: I (58); II (11); III (13); and IV (2). The median DFS was 48 (1-85) months. By univariate analysis, DFS was significantly associated with FIGO stage, histology, peritoneal cytology, myometrial invasion, nodal metastasis, serum CA-125, MTV, and TLG. Using multivariate analysis, the MTV (P = 0.010; hazard ratio, HR = 1.010; 95% confidence interval, 95% CI = 1.002-1.018) and TLG (P = 0.024; HR = 1.001; 95% CI = 1.000-1.002) were associated with DFS. The area under the ROC curve was 0.679 (95% CI = 0.505-0.836) after discriminating for recurrence using an MTV cut-off value of 17.15 ml. Regarding TLG, the cut-off value was 56.43 g and the area under the ROC plot was 0.661 (95% CI = 0.501-0.827). Kaplan-Meier survival graphs demonstrated a significant difference in DFS between groups categorised using the cut-off values for MTV and TLG (P < 0.022 for MTV and P < 0.047 for TLG, by log-rank test). CONCLUSIONS: Preoperative MTV and TLG could be independent prognostic factors predicting the recurrence of endometrial cancer.

An important role of tumor necrosis factor receptor-2 on natural killer T cells on the development of dsRNA-enhanced Th2 cell response to inhaled allergens.

BACKGROUND: Recent evidence indicates that TNF-α is a key mediator of the development of dsRNA-enhanced Th2 cell response to inhaled allergens. Natural killer T (NKT) cells may be a candidate source of Th2-polarizing cytokines. OBJECTIVE: The objective of this study was to evaluate the role of lung NKT cells on the development of TNF-α-mediated Th2 cell response. METHODS: A virus-associated asthma mouse model was generated by the administration of ovalbumin (OVA, 75 µg) and poly[I:C] (0.1 µg). Role of NKT and type I NKT cells was evaluated using CD1d- and Jα18-deficient mice. TNF-α receptors (TNFRs) were antagonized by using TNFR blocking peptides. RESULTS: The number of infiltrated NKT cells was increased in a virus-associated asthma mouse model. Increase in Th2 and Th17 cytokine levels in wild-type mice were abolished in both CD1d- and Jα18-deficient mice. In vitro co-culture experiments with alveolar macrophages and NKT cells showed that TNF-α produced by macrophages in the presence of poly[I:C] acts on NKT cells, inducing production of Th2-polarizing cytokines. Moreover, the induction of Th2-polarizing cytokines by poly[I:C] or recombinant TNF-α was impaired in both CD1d- and Jα18-deficient mice and that the above effect was reversed by a TNF-α receptor-2 (TNFR2) blocking peptide, but not by a TNFR1 blocker. CONCLUSIONS: These findings suggest that NKT cells play a key role in the development of Th2 cell response to inhaled allergens and that TNF-α produced by alveolar macrophages induces Th2 cell response, via TNFR2 on NKT cells.

Preoperative nomogram for the identification of lymph node metastasis in early cervical cancer.

BACKGROUND: The objective of this study is to construct a preoperative nomogram predicting lymph node metastasis (LNM) in early-cervical cancer patients. METHODS: Between 2009 and 2012, 493 early-cervical cancer patients received hysterectomy and pelvic/para-aortic lymphadenectomy. Patients who were diagnosed during 2009-2010 were assigned to a model-development cohort (n=304) and the others were assigned to a validation cohort (n=189). A multivariate logistic model was created from preoperative clinicopathologic data, from which a nomogram was developed and validated. A predicted probability of LNM<5% was defined as low risk. RESULTS: Age, tumour size assessed by magnetic resonance imaging, and LNM assessed by positron emission tomography/computed tomography were independent predictors of nodal metastasis. The nomogram incorporating these three predictors demonstrated good discrimination and calibration (concordance index=0.878; 95% confidence interval (CI), 0.833-0.917). In the validation cohort, the discrimination accuracy was 0.825 (95% CI, 0.736-0.895). In the model-development cohort, 34% of them were classified as low risk and negative predictive value (NPV) was 99.0%. In the validation cohort, 38% were identified as low risk and NPV was 95.8%. Integrating the model-development and validation cohorts, negative likelihood ratio was 0.094 (95% CI, 0.036-0.248). CONCLUSION: A robust nomogram predicting LNM in early cervical cancer was developed. This model may improve clinical trial design and help physicians to decide whether lymphadenectomy should be performed.

Small-molecule inhibition of CBP/catenin interactions eliminates drug-resistant clones in acute lymphoblastic leukemia.

Drug resistance in acute lymphoblastic leukemia (ALL) remains a major problem warranting new treatment strategies. Wnt/catenin signaling is critical for the self-renewal of normal hematopoietic progenitor cells. Deregulated Wnt signaling is evident in chronic and acute myeloid leukemia; however, little is known about ALL. Differential interaction of catenin with either the Kat3 coactivator CREBBP (CREB-binding protein (CBP)) or the highly homologous EP300 (p300) is critical to determine divergent cellular responses and provides a rationale for the regulation of both proliferation and differentiation by the Wnt signaling pathway. Usage of the coactivator CBP by catenin leads to transcriptional activation of cassettes of genes that are involved in maintenance of progenitor cell self-renewal. However, the use of the coactivator p300 leads to activation of genes involved in the initiation of differentiation. ICG-001 is a novel small-molecule modulator of Wnt/catenin signaling, which specifically binds to the N-terminus of CBP and not p300, within amino acids 1-110, thereby disrupting the interaction between CBP and catenin. Here, we report that selective disruption of the CBP/ß- and γ-catenin interactions using ICG-001 leads to differentiation of pre-B ALL cells and loss of self-renewal capacity. Survivin, an inhibitor-of-apoptosis protein, was also downregulated in primary ALL after treatment with ICG-001. Using chromatin immunoprecipitation assay, we demonstrate occupancy of the survivin promoter by CBP that is decreased by ICG-001 in primary ALL. CBP mutations have been recently identified in a significant percentage of ALL patients, however, almost all of the identified mutations reported occur C-terminal to the binding site for ICG-001. Importantly, ICG-001, regardless of CBP mutational status and chromosomal aberration, leads to eradication of drug-resistant primary leukemia in combination with conventional therapy in vitro and significantly prolongs the survival of NOD/SCID mice engrafted with primary ALL. Therefore, specifically inhibiting CBP/catenin transcription represents a novel approach to overcome relapse in ALL.

Minimally invasive surgery for serosa-positive gastric cancer (pT4a) in patients with preoperative diagnosis of cancer without serosal invasion.

BACKGROUND: Although surgeons normally use minimally invasive surgery (MIS) for patients with early gastric cancer, in Korea and Japan the procedure is also indicated for serosa-negative tumors. Serosal invasion is regarded to be a potential risk factor for peritoneal dissemination as a result of the effect of pneumoperitoneum and tumor manipulation during the operation. We compared operative outcomes between MIS and conventional open surgery for serosa-involved advanced gastric cancer patients who had a preoperative diagnosis of cancer without serosal invasion. METHODS: A total of 61 patients (39 patients treated by MIS and 22 by open surgery) treated between 2003 and 2009 who were first diagnosed preoperatively as serosa negative on the basis of computed tomography, endoscopy, and endoscopic ultrasound but then diagnosed as serosa positive upon final pathology were studied. We retrospectively compared recurrence and survival between the two treatment groups. RESULTS: Clinicopathologic characteristics, clinical stage, extent of surgery, and short-term operative outcome did not differ between the groups. 5-year overall survival (73.5 vs. 67.5 %, p = 0.518, respectively) and disease-free survival (67.8 vs. 54.2 %, p = 0.296, respectively) were comparable between the MIS and open surgery groups. There were recurrences in 12 patients in the MIS group and 11 patients in the open surgery group, with a median follow-up period of 64 months. Recurrence patterns did not differ between the groups; moreover, MIS did not increase peritoneal recurrences compared to open surgery (42.0 vs. 54.5 %, p = 0.537, respectively). In multivariate analyses, the type of surgery was not an independent prognostic factor. CONCLUSIONS: Similar survival and recurrence patterns were observed in advanced gastric cancer patients preoperatively diagnosed as serosa negative who were treated either by MIS or open surgery. MIS may be safely applied in patients with serosa-positive tumors.

Upregulation of CXCR4 is functionally crucial for maintenance of stemness in drug-resistant non-small cell lung cancer cells.

The hypothesis of cancer stem cells has been proposed to explain the therapeutic failure in a variety of cancers including lung cancers. Previously, we demonstrated acquisition of epithelial-mesenchymal transition, a feature highly reminiscent of cancer stem-like cells, in gefitinib-resistant A549 cells (A549/GR). Here, we show that A549/GR cells contain a high proportion of CXCR4+ cells that are responsible for having high potential of self-renewal activity in vitro and tumorigenicity in vivo. A549/GR cells exhibited strong sphere-forming activity and high CXCR4 expression and SDF-1α secretion compared with parent cells. Pharmacological inhibition (AMD3100) and/or siRNA transfection targeting CXCR4 significantly suppressed sphere-forming activity in A549 and A549/GR cells, and in various non-small cell lung cancer (NSCLC) cell lines. A549/GR cells showed enhanced Akt, mTOR and STAT3 (Y705) phosphorylation. Pharmacological inhibition of phosphatidyl inositol 3-kinase or transfection with wild-type PTEN suppressed phosphorylation of Akt, mTOR and STAT3 (Y705), sphere formation, and CXCR4 expression in A549/GR cells, whereas mutant PTEN enhanced these events. Inhibition of STAT3 by WP1066 or siSTAT3 significantly suppressed the sphere formation, but not CXCR4 expression, indicating that STAT3 is a downstream effector of CXCR4-mediated signaling. FACS-sorted CXCR4+ A549/GR cells formed many large spheres, had self-renewal capacity, demonstrated radiation resistance in vitro and exhibited stronger tumorigenic potential in vivo than CXCR4- cells. Lentiviral-transduction of CXCR4 enhanced sphere formation and tumorigenicity in H460 and A549 cells, whereas introduction of siCXCR4 suppressed these activities in A549/GR cells. Our data indicate that CXCR4+ NSCLC cells are strong candidates for tumorigenic stem-like cancer cells that maintain stemness through a CXCR4-medated STAT3 pathway and provide a potential therapeutic target for eliminating these malignant cells in NSCLC.

Proposed mechanism in the change of cellular composition in the outer medullary collecting duct during potassium homeostasis.

Potassium depletion (K⁺-D) induces hypertrophy and hyperplasia of collecting duct cells, and potassium repletion (K⁺-R) induces regression of these changes. The purpose of this study was to examine the time courses of the changes in cellular composition, the origin of intercalated cells (ICs) and the mechanism responsible for these changes. SD rats received K⁺-depleted diets for 1, 7, or 14 days. After K⁺-D for 14 days some of the rats received normal diets for 1, 3, 5, or 7 days. In the inner stripe of the outer medulla, K⁺-D increased significantly the number and proportion of H⁺-ATPase-positive ICs, but decreased the proportion of H⁺-ATPase-negative principal cells (PCs). However, proliferation was limited to H⁺-ATPase-negative PCs. During K⁺-R, the cellular composition was recovered to control level. Apoptosis increased during K⁺-R and exclusively limited in H⁺-ATPase-negative PCs. Double immunolabeling with antibodies to PC and IC markers identified both cells negative or positive for all markers during both K⁺-D and K⁺-R. Electron microscopic observation showed that ultrastructure of AE1-positive some cells were similar to AE1-negative some cells during K⁺-R. LC3 protein expression increased significantly and autophagic vacuoles appeared particularly in PCs on days 14 of K⁺-D and in ICs on days 3 of K⁺-R. These results suggest that PCs and ICs may interconvert in response to changes in dietary K+ availability and that autophagic pathways may be involved in the interconversion.

TNF-alpha is a key mediator in the development of Th2 cell response to inhaled allergens induced by a viral PAMP double-stranded RNA.

BACKGROUND: Viral pathogen-associated molecular patterns, such as dsRNA, disrupt airway tolerance to inhaled allergens. Specifically, the Th2 and Th17 cell responses are induced by low-dose dsRNA and the Th1-dominant response by high-dose dsRNA. OBJECTIVE: In this model, we evaluate the role of TNF-α in the development of adaptive immune dysfunction to inhaled allergens induced by airway sensitization with dsRNA-containing allergens. METHODS: A virus-associated asthma mouse model was generated via simultaneous airway administration of ovalbumin (OVA) and low (0.1 µg) or high (10 µg) doses of polyinosine-polycytidylic acid (poly[I:C]). The effect of TNF-α on Th2 airway inflammation was evaluated using TNF-α-deficient mice and recombinant TNF-α. RESULTS: TNF-α production was enhanced by airway exposure to low and high doses of poly[I:C]. After airway sensitization with OVA plus low-dose poly[I:C], TNF-α-deficient mice exhibited less OVA-induced airway inflammation than did wild-type (WT) mice. However, this did not occur upon sensitization with high-dose poly[I:C]. In terms of T-cell response, the production of IL-4 from lung T cells after OVA challenge was enhanced by airway sensitization with OVA plus low-dose poly[I:C] in WT mice, and this phenotype was inhibited by the absence of TNF-α. Moreover, the Th2 cell response induced by sensitization with OVA plus low-dose poly[I:C], which was abolished in TNF-α-deficient mice, was restored in these mice upon addition of recombinant TNF-α. CONCLUSION: The results of this study suggest that TNF-α produced by airway exposure to low-dose dsRNA is a key mediator in the development of Th2 cell response to inhaled allergens.

Fyn positively regulates the activation of DAP12 and FcRγ-mediated costimulatory signals by RANKL during osteoclastogenesis.

Osteoclasts (OCs) are the only bone-resorbing cells and are critically involved in various bone-associated diseases, including osteoporosis and rheumatoid arthritis. Differentiation of OCs from bone marrow macrophage cells (BMMs) is regulated by RANK and the adaptor protein (DAP12/FcRγ)-mediated costimulatory signals. However, it is unknown how RANKL/RANK signal stimulates phosphorylation of DAP12/FcRγ to initiate the costimulatory signals. As reported here, we found that OC differentiation and acquisition of bone resorption capacity were suppressed in RANKL-stimulated Fyn(-/-) or Fyn-siRNA-transfected BMMs, but could be restored by overexpression of Fyn kinase in Fyn(-/-) BMMs. However, the RANKL-stimulated proliferation of BMMs was unaffected by the absence of Fyn. In addition, RANKL-stimulated Fyn(-/-) BMMs no longer exhibited the optimal induction of typical OC markers such as NFATc1, c-Fos, c-Src, TRAF6, and cathepsin K or costimulatory signals such as the activating phosphorylations of Syk, PLCγ2, and Gab2. These were restored by overexpression of Fyn in Fyn(-/-) BMMs. Immunoprecipitation studies also indicated that the adaptor proteins DAP12/FcRγ and Syk interacted with RANK during RANKL stimulation in BMMs in a Fyn-dependent manner. Phosphorylation of the DAP12/FcRγ and the recruitment of Syk by DAP12/FcRγ were suppressed in Fyn(-/-) BMMs. This is the first demonstration that Fyn relays the initial RANK/RANKL signal to the ITAM-containing adaptors DAP12/FcRγ for OC differentiation.