Sequential control of myeloid cell proliferation and differentiation by cytokine receptor-based chimeric antigen receptors.

As chimeric antigen receptor (CAR)-T cell therapy has been recently applied in clinics, controlling the fate of blood cells is increasingly important for curing blood disorders. In this study, we aim to construct proliferation-inducing and differentiation-inducing CARs (piCAR and diCAR) with two different antigen specificities and express them simultaneously on the cell surface. Since the two antigens are non-cross-reactive and exclusively activate piCAR or diCAR, sequential induction from cell proliferation to differentiation could be controlled by switching the antigens added in the culture medium. To demonstrate this notion, a murine myeloid progenitor cell line 32Dcl3, which proliferates in an IL-3-dependent manner and differentiates into granulocytes when cultured in the presence of G-CSF, is chosen as a model. To mimic the cell fate control of 32Dcl3 cells, IL-3R-based piCAR and G-CSFR-based diCAR are rationally designed and co-expressed in 32Dcl3 cells to evaluate the proliferation- and differentiation-inducing functions. Consequently, the sequential induction from proliferation to differentiation with switching the cytokine from IL-3 to G-CSF is successfully replaced by switching the antigen from one to another in the CARs-co-expressing cells. Thus, piCAR and diCAR may become a platform technology for sequentially controlling proliferation and differentiation of various cell types that need to be produced in cell and gene therapies.

Proposal of index to evaluate caregivers' fear of care recipient falls.

This study aimed to identify evaluation items that can be used to create an index to evaluate caregivers' fear of care recipient falls. A three-round Delphi method was conducted with medical professionals engaged in discharge support for patients with fall-related fractures. In the first round, a working group brainstormed evaluation items. In the second and third rounds, opinions of medical professionals were quantified and evaluation items were refined. The Delphi method showed convergence of opinion with Kendall's W of 0.561 in the third round. Of the 109 evaluation items pooled in the first round, the consensus was reached on the importance of 19 items and one more item was additionally included. The 20 items may be useful for creating an index that sensitively measures caregivers' fear of care recipient falls.

Cell fate-inducing CARs orthogonally control multiple signaling pathways.

While chimeric antigen receptor (CAR) has been clinically applied in T cell-mediated cancer therapy, CARs that combinatorially control general cell fates have yet to be practical. In this study, we aim to control multiple cell fates simultaneously by combinatorial activation of multiple cell fate-inducing CARs (cfiCARs). We construct CARs that transduce two different signals using two antigen-specific CARs. The CARs are co-expressed pairwise on the cell surface to let the two antigens act alone or in combination, thus arbitrarily control multiple cell fates. We utilize signaling domains derived from natural receptors (gp130, receptor activator of nuclear factor κB [RANK], c-Fms, and Fas) and tyrosine motif-engineered artificial signaling domains that preferentially recruit target signaling molecules (Shc and STAT3) for inducing specific cell fates by the CARs. Consequently, the signaling molecules downstream of these receptors are activated orthogonally by the corresponding CAR-specific antigens. Furthermore, two completely different cell fates (proliferation and death) are successfully controlled simultaneously or sequentially over time by adding the two antigens, demonstrating proliferation- and apoptosis-inducing CARs (piCAR and aiCAR). Thus, cfiCARs will be applied for delineating the basic principle of cell fate control and improving the efficacy of cell therapy, which would significantly contribute to cell biology and future medicine.

Tailoring minimal synthetic receptors to reconstitute signaling properties through multiple tyrosine motifs.

As intracellular signal transduction is important for determining cell fate, artificial control of signaling properties through engineered receptors is attractive in the fields of synthetic biology and cell therapy. In this study, we tailored minimal synthetic receptors to reconstitute signaling properties by incorporating multiple tyrosine motifs. The size of molecular parts including the linker between the tyrosine motifs was minimized as much as possible to create the minimal synthetic receptors. By combining the membrane localization signal sequence, a mutant of FK506-binding protein, a JAK-binding domain, tyrosine motifs, and linkers, we successfully reconstituted simple receptor chains that were activated by dimerization via a synthetic small-molecule ligand capable of membrane permeation. Furthermore, up to four signaling molecules of interest were able to be recruited and activated by the minimal synthetic receptors. Thus, the tailored minimal synthetic receptors could be utilized to analyze the role of specific signaling molecules/pathways in controlling cell fate and to efficiently induce specific cell fate for therapeutic applications in the future.

Cytokine Profiles of Tear Fluid From Patients With Pediatric Lacrimal Duct Obstruction.

Purpose: This study evaluated the cytokine levels in unilateral tear samples from both sides in patients with pediatric lacrimal duct obstruction. Methods: Fifteen cases of unilateral lacrimal duct obstruction (mean, 26.9 ± 28.7 months old) were enrolled in this study. Tear samples were collected separately from the obstructed side and the intact side in each case before surgery, which was performed under general anesthesia or sedation. The levels of IL-2, IL-4, IL-6, IL-10, TNF, IFN-γ, and IL-17A then were measured in each tear sample. A receiver operating characteristic (ROC) curve was constructed for the IL-6 levels in the tears. We also measured the postoperative tear fluid levels of IL-6 in those cases from which tear fluid samples could be collected after the surgery. Results: Only the IL-6 concentration was significantly higher on the lacrimal duct-obstructed sides, compared to the control sides (P < 0.001). An ROC curve analysis for the IL-6 levels in tears showed a high value for discriminating the lacrimal duct-obstructed side from the control side (area under the ROC curve [AUC], 0.99; 95% confidence interval [CI], 0.968-1). Significant decrease of the tear fluid IL-6 levels was observed in the seven cases from which tear fluid samples also could be collected after the surgery (P = 0.016). Conclusions: The IL-6 level in tear fluid was significantly higher on the sides with lacrimal duct obstruction, compared to the control sides, and could be a biomarker for pediatric lacrimal duct obstruction.

[Sequential Chemoradiotherapy for Advanced Head and Neck Cancer: A Clinical Study with 33 Cases].

A total of 33 patients with advanced head and neck cancer (AHNC) treated with sequential chemoradiotherapy (SCRT) were retrospectively evaluated at Gunma University Hospital between 2009 and 2011. The regimen of SCRT was docetaxel, cisplatin, and fluorouracil (TPF)-based induction chemotherapy (ICT), accompanied by docetaxel and cisplatin-based concurrent chemoradiotherapy (CCRT), and oral administration of TS-1 after that. The response rate was 61%, the 3-year overall survival rate was 42%, the non-tumor-bearing survival rate was 27%, and the tumor-bearing survival rate was 15%. Fourteen of 33 patients were tumor-free, and their 3-year overall survival rate was surprisingly 86%. On the other hand, 3-year overall survival rate in the remaining 19 patients was significantly low. To select good response cases for ICT was important. In such cases, TPF should be applied repeatedly, which achieved a 61% response rate even in AHNC. A long-term TS-1 oral medication suppressed cancer regrowth and contributed to long-term survival.

The ApoA-I mimetic peptide FAMP promotes recovery from hindlimb ischemia through a nitric oxide (NO)-related pathway.

BACKGROUND/OBJECTIVE: HDL has various atheroprotective functions and improves endothelial function. Apolipoprotein A-I (apoA-I) is a major protein of HDL and plays a crucial role in HDL functions. We developed a novel apoA-I mimetic peptide, FAMP (Fukuoka University ApoA-I Mimetic Peptide). It is unclear whether an apoA-I mimetic peptide can promote neovascularization in vivo. Here, we investigated the effect of FAMP on endothelial nitric oxide synthase (eNOS) activation and angiogenesis in a murine hindlimb ischemia model. METHODS AND RESULTS: Intramuscular administration of FAMP significantly enhanced blood flow recovery and increased capillary density in the ischemic limb of mice fed a high-cholesterol diet (HCD). In a gait analysis, FAMP ameliorated functional recovery compared with that in the control group. FAMP significantly activated Akt, ERK, and eNOS phosphorylation in endothelial cells, and improved the migratory functions of human aortic endothelial cells (HAECs). LY294002, an inhibitor of phosphatidylinositol 3-kinase (PI3K), significantly inhibited the activation of eNOS by FAMP. FAMP had no beneficial effects on blood flow recovery in eNOS(-/-) mice. CONCLUSIONS: FAMP promoted recovery from hindlimb ischemia through a nitric oxide (NO)-related pathway by activation of a PI3K/Akt pathway. FAMP may become a new therapeutic agent for the future clinical treatment of critical limb ischemia (CLI).

Extranodal soft tissue Rosai-Dorfman disease of the head and neck and its diagnostic difficulty.

Rosai-Dorfman disease (RDD) is a rare proliferative histiocytic disorder that is characterized by persistent massive lymphadenopathy mimicking malignant tumors. Extranodal RDD is uncommon, and more severe fibrosis and fewer histiocytes in lesions make the diagnosis of RDD more difficult than that of nodal RDD. We herein described a 31-year-old male patient with isolated soft tissue RDD of the head and neck. The patient was referred to our hospital with a right neck mass. Computed tomography (CT) scans showed a diffuse enhanced tumor with an unclear border in the right side of the neck. 18F-fluorodeoxyglucose positron emission tomography (PET)/CT revealed high uptake in the corresponding lesion. Second wide local excisional biopsy led to a diagnosis of RDD, and immunohistochemistry was useful for diagnosing RDD. A systemic treatment with steroids improved his symptoms, including the neck mass. Physicians need to consider the diagnostic difficulty associated with extranodal soft tissue RDD as well as its rarity.

Separation efficiency of a microfluidic sperm sorter to minimize sperm DNA damage.

OBJECTIVE: To evaluate whether microfluidic sperm sorters (MFSSs) allow effective recovery of sorted motile sperm without DNA damage compared with the centrifugation and swim-up procedure. DESIGN: Experimental laboratory study. All participants completed questionnaires regarding previous and/or current diseases, surgery, reproductive experiences, lifestyle factors, and date of the preceding ejaculation. SETTING: University research laboratory. PATIENT(S): Male volunteers were recruited without setting conditions. Semen samples from healthy volunteers (n = 37) were collected in sterile containers by masturbation. INTERVENTION(S): Flow cytometric measurement and sperm chromatin structure assay analysis of DNA damage after sperm preparation using MFSS and the centrifugation and swim-up procedure. MAIN OUTCOME MEASURE(S): Efficacy and efficiency of sperm preparation, correlation between sperm DNA fragmentation index (DFI) and semen parameters, and relationship between basic characteristics and DFI after the centrifugation and swim-up procedure. RESULT(S): Final sperm concentration and motility were significantly different between the centrifugation and swim-up procedure and MFSS sperm preparations. A significantly lower sperm DNA fragmentation rate was detected with MFSS compared with the centrifugation and swim-up procedure use. No correlation was observed between DFI and smoking or drinking, but significant correlations were observed between DFI and medication use and sexual abstinence duration. CONCLUSION(S): MFSSs can be used to efficiently and reliably prepare sperm compared with the centrifugation and swim-up procedure. Further research on the clinical use of MFSSs is required to evaluate the safety and usefulness of this device.

[Prediction of Post-operative Lymph Node Metastasis with a Molecular Biological Test in Head and Neck Cancer].

We assessed herein the post-operative lymph node metastasis in head and neck cancer, using the One-step nucleotide amplification (OSNA) method targeting matrix metalloproteinase 7 (MMP-7). Compared with the pathological test, the molecular biological test revealed more lymph node metastasis, resulting in poor prognosis. Six cases, of which the number of lymph node metastasis was the same between pathological and molecular biological test, survived. On the other hand, three of four cases, in which number of lymph node metastasis in the molecular biological test were larger than the pathological test, died from metastasis. We concluded that the pathological test underestimated metastasis, and OSNA with MMP-7 was useful for the prediction of post-operative lymph node metastasis.

Intestinal myofibroblast TRPC6 channel may contribute to stenotic fibrosis in Crohn's disease.

BACKGROUND: Intestinal fibrosis is a frequent complication of Crohn's disease (CD) and often leads to detrimental stricture formation. Myofibroblasts play active roles in mediating fibrotic changes in various tissues. We investigated whether transient receptor potential channels in intestinal myofibroblasts are involved in CD-associated intestinal fibrosis. METHODS: An intestinal myofibroblast cell line (InMyoFibs) was stimulated with transforming growth factor-ß1 (TGF-ß1) to model excessive fibrosis. Biopsy samples from nonstenotic or stenotic intestinal regions from patients with CD were used for quantitative comparisons of transient receptor potential channel and fibrosis-associated factor expression levels. RESULTS: TGF-ß1 treatment transformed spindle-shaped InMyoFibs into filament-shaped cells with enhanced α-actin stress fiber formation, transient receptor potential canonical (TRPC) 4 and TRPC6 messenger RNA and protein expression, and basal- and agonist-induced Ca influxes. TGF-ß1 also enhanced the formation of TRPC6/smooth muscle α-actin, TRPC6/N-cadherin, and TRPC4/N-cadherin coimmunoprecipitates. Inhibition of TRPC6 in InMyoFibs by RNA interference or dominant-negative mutations suppressed TGF-ß1-induced Ca influxes, stress fiber formation, and smooth muscle α-actin expression, but increased COL1A1, interleukin (IL)-10, and IL-11 expression, as well as Smad-2, ERK, and p38-MAPK phosphorylation. Similar increases in phosphorylation levels were observed with TRPC and calcineurin inhibitors. In stenotic areas in patients with CD, TRPC6, ACTA2 (smooth muscle α-actin), CDH2 (N-cadherin), COL1A1, IL-10, and IL-11 were significantly increased. CONCLUSIONS: These results suggest that augmented Ca influxes due to TRPC6 upregulation facilitate stress fiber formation and strengthen cell-cell interactions by negatively regulating the synthesis of antifibrotic factors in TGF-ß1-treated myofibroblasts. Similar changes observed in stenotic areas of patients with CD suggest the therapeutic significance of targeting TRPC6.

A case of pyriform sinus fistula infection with double tracts.

Pyriform sinus fistula is a rare clinical entity and the precise origin remains controversial. The fistula is discovered among patients with acute suppurative thyroiditis or deep neck infection of the left side of the neck and is usually located in the left pyriform sinus. To the best of our knowledge, only a single tract has been reported to be responsible for pyriform sinus fistula infection. We present a case of a 13-year-old female patient with a pyriform sinus fistula that caused a deep infection of the left side of the neck and showed double-tract involvement discovered during surgical resection of the entire fistula. Both tracts arose around the pyriform sinus and terminated at the upper portion of the left lobe of the thyroid.

Molecular hierarchy of heparin-binding EGF-like growth factor-regulated angiogenesis in triple-negative breast cancer.

Heparin-binding EGF-like growth factor (HB-EGF) is one of several proangiogenic factors and represents a possible therapeutic target for patients with triple-negative breast cancer (TNBC). However, the role of HB-EGF in promoting tumor aggressiveness in TNBC remains unclear. To investigate specific genes and pathways involved in TNBC tumorigenesis, we profiled gene expression changes in two TNBC cell lines under two-dimensional culture (2DC) and three-dimensional culture (3DC) and in a tumor xenograft model. We identified simultaneous upregulation of HB-EGF, VEGFA, and angiopoietin-like 4 (ANGPTL4) in 3DC and tumor xenografts, compared with 2DC. We show that HB-EGF regulates the expression of VEGFA or ANGPTL4 via transcriptional regulation of hypoxia-inducible factor-1α and NF-κB. Furthermore, suppression of VEGFA or ANGPTL4 expression enhanced HB-EGF expression, highlighting a unique regulatory loop underlying this angiogenesis network. Targeted knockdown of HB-EGF significantly suppressed tumor formation in a TNBC xenograft model, compared with individual knockdown of either VEGFA or ANGPTL4, by reducing the expression of both VEGFA and ANGPTL4. In patients with TNBC, VEGFA or ANGPTL4 expression was also significantly correlated with HB-EGF expression. Low concentrations of exogenously added HB-EGF strongly activated the proliferation of endothelial cells, tube formation, and vascular permeability in blood vessels, in a similar fashion to high doses of VEGFA and ANGPTL4. Taken together, these results suggest that HB-EGF plays a pivotal role in the acquisition of tumor aggressiveness in TNBC by orchestrating a molecular hierarchy regulating tumor angiogenesis.

Metronomic doxifluridine chemotherapy combined with the anti-angiogenic agent TNP-470 inhibits the growth of human uterine carcinosarcoma xenografts.

Uterine carcinosarcoma is a highly aggressive gynecological neoplasm that responds poorly to conventional chemotherapy and radiotherapy. Metronomic chemotherapy is accepted as a new approach for cancer treatment, and its underlying mechanism seems to involve the suppression of angiogenesis. However, the efficacy of metronomic and anti-angiogenic therapies against uterine carcinosarcoma is unknown. The anti-angiogenic effect of doxifluridine was assessed in vitro using human umbilical vein endothelial cells (HUVEC) co-cultured with FU-MMT-1 human uterine carcinosarcoma cells. The antitumor and anti-angiogenic effects of metronomic doxifluridine (delivered via oral gavage) in combination with TNP-470 were evaluated in vivo. Tumor vascularity was assessed by contrast-enhanced color Doppler ultrasound, laser Doppler and microvessel density staining. Doxifluridine suppressed tube formation of HUVEC and vascular endothelial growth factor production by FU-MMT-1 cells. Metronomic doxifluridine alone significantly suppressed tumor growth compared with the untreated (control) group, while metronomic doxifluridine in combination with TNP-470 significantly inhibited tumor growth compared with each treatment alone. A significant reduction of intratumoral vascularity was observed in FU-MMT-1 xenografts following treatment with metronomic doxifluridine in combination with TNP-470, as compared with each treatment alone. Intestinal bleeding was only observed when the maximum tolerated dose of doxifluridine was administered in combination with TNP-470. Metronomic doxifluridine chemotherapy in combination with TNP-470 might be effective for uterine carcinosarcoma without marked toxicity, possibly acting via its potent anti-angiogenic effects. Clinical studies are needed to evaluate the safety and efficacy of this treatment in humans.

Metronomic irinotecan chemotherapy combined with ultrasound irradiation for a human uterine sarcoma xenograft.

Metronomic chemotherapy is the frequent administration of low doses of chemotherapeutic agents targeting tumor-associated endothelial cells. We examined the efficacy of metronomic irinotecan combined with low-intensity ultrasound (US) in human uterine sarcoma and evaluated its antiangiogenesis mechanism by measuring the circulating endothelial progenitor cells (CEP), a surrogate marker of angiogenesis. A human uterine sarcoma cell line, FU-MMT-3, was used in the present study because this tumor is one of the most malignant neoplasms of human solid tumors and it also has a high angiogenesis property. The combination of low-dose irinotecan and US irradiation significantly inhibited the tube formation of HUVEC and vascular endothelial growth factor expression of tumor cells in vitro. The FU-MMT-3 xenografts in nude mice were treated using US at a low intensity (2.0 w/cm(2), 1 MHz) for 4 min three times per week each after the intraperitoneal administration of irinotecan; this treatment was continued for 5 weeks. The tumor vascularity was assessed by contrast-enhanced color Doppler US in real time. The combination treatment significantly inhibited the mobilization of CEP and intratumoral vascularity compared with the control. This combination therapy showed a significant reduction in tumor volume, resulting in a significant prolongation of survival, in comparison with each treatment alone. These results suggest that the effect of metronomic chemotherapy for human uterine sarcoma was accelerated by US irradiation in vivo and this combination might therefore be potentially effective for new cancer therapy.

Contribution of Ca(2+) -dependent protein kinase C in the spinal cord to the development of mechanical allodynia in diabetic mice.

In this paper, we directly demonstrate, for the first time, the activation of Ca(2+)-dependent protein kinase C (PKC) in the spinal cord of diabetic mice. In streptozotocin (STZ)-treated (200 mg/kg, i.v.) diabetic mice, hypersensitivity (allodynia) to mechanical stimulation appeared 7 d after STZ injection. This mechanical allodynia was inhibited by intrathecal injection of the PKC inhibitors 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7) and calphostin C, but not the protein kinase A inhibitor N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89). The activity of membrane-associated Ca(2+)-dependent PKC in the spinal cords of STZ-induced diabetic mice was significantly higher than that observed in non-diabetic mice. These results suggest that activation of Ca(2+)-dependent PKC in the spinal cord, contributes to the mechanical allodynia in the pain associated with diabetic neuropathy.

Expression of thymidylate synthase and dihydropyrimidine dehydrogenase in adenoid cystic carcinoma of the head and neck: correlation with clinical outcome.

The purpose of this study was to estimate the possibility of using thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD), and p53 as predictive values of clinical outcome in adenoid cystic carcinoma (ACC). The expressions of TS, DPD, and p53 were examined with immunohistochemistry in 27 ACC patients, and the association with clinicopathological factors was determined. Cases with high DPD expression had significantly higher distant metastasis rates compared to those with low DPD expression (p=0.001), whereas neither TS nor p53 expression showed any significant correlation to clinicopathological factors. Interestingly, six of 14 early-stage patients had distant metastases and all of their tumors showed high DPD expression. Kaplan-Meier analysis revealed that a solid histological pattern and distant metastasis correlated with a poor prognosis. In early-stage patients, whose tumor was completely resected, those with high TS or DPD expression had a worse prognosis compared to those with low expression, but the difference did not reach statistical significance (TS, p=0.178; DPD, p=0.251). Our results suggest that assessment of DPD expression in ACC may be a useful tool in determining the mode of treatment as well as evaluating clinical outcome.