Non-canonical NFKB signaling endows suppressive function through FOXP3-dependent regulatory T cell program.

Regulatory T cells (Tregs) play a central role in modulating adaptive immune responses in humans and mice. The precise biological role of non-canonical nuclear factor 'κ-light-chain-enhancer' of activated B cells (NFKB) signaling in human Tregs has yet to be fully elucidated. To gain insight into this process, a Treg-like cell line (MT-2) was genetically modified using CRISPR/Cas9. Interestingly, NFKB2 knockout MT-2 cells exhibited downregulation of FOXP3, while NFKB1 knockout did not. Additionally, mRNA expression of FOXP3-dependent molecules was significantly reduced in NFKB2 knockout MT-2 cells. To better understand the functional role of the NFKB signaling, the NFKB1/NFKB2 loci of human primary Tregs were genetically edited using CRISPR/Cas9. Similar to MT-2 cells, NFKB2 knockout human Tregs displayed significantly reduced FOXP3 expression. Furthermore, NFKB2 knockout human Tregs showed downregulation of FOXP3-dependent molecules and a diminished suppressive function compared to wild-type and NFKB1 knockout Tregs. These findings indicate that non-canonical NFKB signaling maintains a Treg-like phenotype and suppressive function in human Tregs through the FOXP3-dependent regulatory T cell program.

Effect of Thymidine Phosphorylase Gene Demethylation on Sensitivity to 5-Fluorouracil in Colorectal Cancer Cells.

BACKGROUND/AIM: Chemotherapy is used for recurrent and metastatic colorectal cancer, but the response rate of 5-fluorouracil (5-FU), the standard treatment for colorectal cancer, is low. We hypothesized that thymidine phosphorylase (TYMP) expression, a rate-limiting activating enzyme of 5-FU, is regulated by methylation of the gene promoter region, and demethylation of TYMP would increase sensitivity to 5-FU. MATERIALS AND METHODS: HCT116 colon cancer cells were treated with 5-aza-2'-deoxycytidine, a demethylating agent, and changes in TYMP transcription and sensitivity to 5-FU were evaluated. RESULTS: TYMP expression increased over 54-fold in HCT116 transfected with TYMP. The cytotoxicity of 5-FU increased up to 5.5-fold. In comparison, in HCT116 treated with 5-aza-2'-deoxycytidine, TYMP expression increased 5.8-fold. However, the cytotoxicity of 5-FU remained unchanged. CONCLUSION: Demethylating agent alone did not promote the cytotoxicity of 5-FU against colorectal cancer. To further increase the sensitivity to 5-FU, combination with adjuvant therapy focusing on metabolic pathways other than the TYMP pathway appear necessary.

Gelatin Sponge as an Anchorage for Three-dimensional Culture of Colorectal Cancer Cells.

BACKGROUND: Compared to two-dimensional cultures, three-dimensional (3D) cultures have many advantages in cancer studies. Nevertheless, their implementation is unsatisfactory. This study aimed to develop an anchorage-dependent 3D culture model for colorectal cancer research. MATERIALS AND METHODS: Human HCT116, DLD-1 and SW620 colorectal cell lines were cultured in a gelatin sponge, and its applicability for morphological examination was studied. RESULTS: The resulting specimens were suitable for scanning electron microscopy, transmission electron microscopy, and immunohistochemical examination. HCT116 formed smaller structures and migrated through the pores of the sponge. DLD-1 formed larger structures with tight cell-to-cell adhesion. SW620 also formed large structures but small clustered cells tended to attach to the anchorage more favorably. Immunohistochemical staining demonstrated phosphorylated yes-associated protein (YAP) localized near the attachment site in HCT116 cells. CONCLUSION: Because the gelatin sponge provided suitable anchorage and the cultured cells formed distinguishable 3D structures, this method may be useful for further colorectal cancer research.

Measurement and differentiation of banana juice scent using an electronic nose FF-2A.

BACKGROUND: Banana juice is becoming a popular beverage in Japan and the number of soft-drink stands or shops that take great care and pride in the quality of their products has been increasing. This study aims to measure the scent of banana juice from different brands using the electronic (e-) nose FF-2A in order to identify the characteristics, time-related changes, and the differences among them. METHODS: We standardized the scent value of banana juice measured using FF-2A and determined the absolute value in three different shops. We compared the similarities in samples from each shop with axis data created using standardized measurement. With FF-2A we identified the scent common to all banana juice samples from the composite scent and numerically showed the similarity to the reference gas. RESULTS: The juices from each shop had their own characteristics and we were able to identify the difference between some of these. The response of FF-2A varied according to the increase/decrease in the number of characteristic molecules measured by GC-MS such as overtime fluctuations in the gas. These data were shown along with the differences between the various banana juices. CONCLUSIONS: FF-2A was able to identify the scent of banana juice at each banana shop as well as time-related changes. By combining GC-MS, we were able to evaluate scent components that changed over time. The results using the electronic nose may prove useful for objective evaluation and comparison of scent with other types of juices.

Thyroid storm associated with Graves' disease covered by diabetic ketoacidosis: A case report.

BACKGROUND: Thyroid storm is a condition in which multiple organ dysfunction results from failure of the compensatory mechanisms of the body owing to excessive thyroid hormone activity induced by some factors in patients with thyrotoxicosis. While diabetic ketoacidosis (DKA) is an important trigger for thyroid storm, simultaneous development of DKA and thyroid storm is rare. CASE PRESENTATION: A 59-year-old woman with no history of either diabetes mellitus or thyroid disease presented to our hospital because of developing nausea, vomiting and diarrhea for 2 days. Physical examination showed mild disturbance of consciousness, fever, and tachycardia. There were no other signs of thyrotoxicosis. Laboratory studies revealed elevation of random blood glucose and glycosylated hemoglobin, strongly positive of urine acetone, and metabolic acidosis. Since DKA was diagnosed, we initiated the patient on treatment with administration of insulin and adequate fluid replacement. Although the hyperglycemia and acidosis were immediately relieved, the disturbance of consciousness and tachycardia remained persistent. Levels of FT3 and FT4 were extremely high and TSH was below the detectable limit. TRAb was positive. The thyroid storm score of Burch & Wartofsky was 75/140, and the thyroid storm diagnostic criteria of the Japan Thyroid Association were satisfied. Oral administration of thiamazole, potassium iodide and propranolol resulted in immediate relief of the tachycardia. DISCUSSION: We encountered a case of thyroid storm associated with Graves' disease covered by DKA. Thyroid storm and DKA are both potentially fatal, and the prognosis varies depending on whether or not these conditions are detected and treated sufficiently early. The thyroid storm diagnostic criteria prepared in 2008 by the Japan Thyroid Association are very simple as compared to the Burch & Wartofsky scoring system for thyroid storm. The Japanese criteria may be useful in the diagnosis of this condition since they enable clinicians to identify a broad range of cases with thyroid storm. When dealing with cases of DKA or thyroid storm, it seems essential to bear in mind the possibility of the coexistence of these two diseases.