Possible Role of miR-375-3p in Cervical Lymph Node Metastasis of Oral Squamous Cell Carcinoma.

No clinically useful predictors of latent cervical lymph node metastasis (LNM) in early oral squamous cell carcinoma (OSCC) are available. In this study, we focused on the microRNAs (miRNAs) involved in the expression of numerous genes and explored those associated with latent cervical LNM in early OSCC (eOSCC). First, microarray and RT-PCR analyses revealed a significant downregulation of miR-375-3p expression in primary eOSCC tissues with latent cervical LNM. Next, we examined the effects of miR-375-3p mimics on the growth and migration of four human OSCC cell lines that do not express miR-375-3p. The overexpression of miR-375-3p significantly suppressed the cell proliferation and migration of human OSCC cells in vitro. Furthermore, miR-375-3p mimics markedly inhibited the subcutaneously xenografted human OSCC tumors. Finally, we found the genes involved in the PI3K-AKT pathway and cell migration as target gene candidates of miR-375-3p in human OSCC cells. These findings suggest that miR-375-3p functions as a tumor suppressive-miRNA in OSCC and may serve as a potential biomarker for the prediction of latent cervical LNM in eOSCC and a useful therapeutic target to suppress OSCC progression.

MicroRNA-1289 Functions as a Novel Tumor Suppressor in Oral Squamous Cell Carcinoma.

Recently, numerous tumor-suppressive microRNAs (TS-miRs) have been identified in human malignancies. Here, we attempted to identify novel TS-miRs in oral squamous cell carcinoma (OSCC). First, we transfected human OSCC cells individually with 968 synthetic miRs mimicking human mature miRs individually, and the growth of these cells was evaluated using the WST-8 assay. Five miR mimics significantly reduced the cell growth rate by less than 30%, and the miR-1289 mimic had the most potent growth inhibitory effect among these miRs. Subsequently, we assessed the in vivo growth-inhibitory effects of miR-1289 using a mouse model. The administration of the miR-1289 mimic-atelocollagen complex significantly reduced the size of subcutaneously xenografted human OSCC tumors. Next, we investigated the expression of miR-1289 in OSCC tissues using reverse transcription-quantitative PCR. The expression level of miR-1289 was significantly lower in OSCC tissues than in the adjacent normal oral mucosa. Furthermore, 15 genes were identified as target genes of miR-1289 via microarray and Ingenuity Pathway Analysis (IPA) microRNA target filtering. Among these genes, the knockdown of magnesium transporter 1 (MAGT1) resulted in the most remarkable cell growth inhibition in human OSCC cells. These results suggested that miR-1289 functions as a novel TS-miR in OSCC and may be a useful therapeutic tool for patients with OSCC.

Use of Leflunomide as an Antiviral Agent with Everolimus for BK Virus Nephropathy Patients After Kidney Transplantation: A Case Series.

BACKGROUND BK virus nephropathy (BKVN) is the major cause of transplant renal dysfunction. However, a specific antiviral agent to treat it does not exist. One therapeutic option is to reduce use of immunosuppression drugs, which can cause allograft rejection. Leflunomide has both antiviral and immunosuppressive effects, and clinical research has demonstrated its clinical efficacy against BKVN. However, a phase II randomized trial did not support this effect. Therefore, the efficacy of leflunomide remains controversial. CASE REPORT We examined 4 BKVN patients whose Cr levels stabilized with leflunomide therapy. BKVN was confirmed by a kidney biopsy 7-16 months after transplantation. The Cr levels in 3 cases continued to increase after the reduction of immunosuppression drugs, then leflunomide was administered. In 1 case, leflunomide was administered when the immunosuppression drugs were reduced. In all of the cases, mycophenolate mofetil was replaced with everolimus, and tacrolimus was replaced with cyclosporine A. The maintenance doses of leflunomide were 20 mg/day, and leflunomide was used as an antiviral agent for 3 months. In all of the cases, Cr levels and plasma BKV-PCR loads improved after the administration of leflunomide. Renal function was stable without BKVN recurrence or allograft rejection over 3 years after transplantation. CONCLUSIONS Our 4 cases show that short-term use of leflunomide during the active phase of BKVN and a combination of leflunomide and everolimus may be effective against BKVN.

Early Steroid Withdrawal Protocol With Basiliximab and Rituximab in ABO-Incompatible Kidney Transplant Recipients.

OBJECTIVES: Corticosteroids remain an important component of immunosuppressive regimens in high-risk kidney transplants. In this study, we investigated the efficacy of early steroid withdrawal with basiliximab and rituximab in ABO-blood type incompatible (ABO-i) recipients of kidney transplants. METHODS: Between 2008 and 2019, 15 patients underwent ABO-i kidney transplantation. Seven of the 15 patients were treated with a steroid maintenance protocol and the remaining 8 with an early steroid withdrawal protocol. The immunosuppressive protocol consisted of tacrolimus, mycophenolate mofetil, and methylprednisolone (MP), with basiliximab administered as induction therapy. Rituximab was administered as a single 200-mg dose 1 to 4 weeks before kidney transplantation. Two to 4 sessions of either double-filtration plasmapheresis or regular plasmapheresis or both were performed to remove anti-AB antibodies before transplantation. During surgery, MP was administered at a dose of 500 mg; thereafter, the dosage was tapered rapidly, and the drug was discontinued on day 14 post transplant. RESULTS: In the steroid maintenance group, 2 patients experienced acute antibody-mediated rejection (AMR). One patient with severe AMR had graft loss on postoperative day 4. Patient and graft survival rates in the steroid maintenance group were 100% and 86%, respectively. MP was successfully withdrawn in the steroid withdrawal group. In this group, there was no biopsy-proven rejection. Patient and graft survival rates were 100%, and when last measured, serum creatinine level ± SD was 1.6 ± 0.8 mg/dL. CONCLUSIONS: Our protocol successfully enabled the early withdrawal of steroids in recipients of ABO-i grafts; however, further follow-up is necessary to confirm our results.

Successful cadaveric kidney transplantation in an extended-hours hemodialysis patient with long-term hemodialysis vintage for 297 months.

The patient was a 41-year-old male who had been maintained on extended-hours hemodialysis for 297 months. Despite of long-term hemodialysis vintage, he had no vascular calcification and ectopic calcification. His kidney graft did not experience rejection or other complications 18 months after the cadaveric kidney transplant. Previous reports indicated that graft survival of extended-hours hemodialysis patients did not differ from conventional hemodialysis. However, the dialysis periods in these reports were much shorter than our case. Therefore, extended-hours hemodialysis in long-term dialysis patients may improve renal transplant outcomes in the countries where the waiting time for kidney transplant is long.

High low-density lipoprotein cholesterol level is the independent risk factor of primary patency rate of arteriovenous fistula.

OBJECTIVES: An arteriovenous fistula is the first choice of vascular access in dialysis patients. However, the correlations between patient factors and the arteriovenous fistula patency rate remain unclear. Therefore, we examined the effect of dialysis patient factors on arteriovenous fistula patency rate. METHODS: This study included 101 patients who received maintenance dialysis and used arteriovenous fistula for vascular access at Atami Hospital, International University of Health and Welfare in July 2018. A retrospective review was performed from the time of arteriovenous fistula creation to July 2018, and the primary and secondary arteriovenous fistula patency rates were investigated. The patency rate was calculated using the Kaplan-Meier method, and risk factor analysis was performed using Cox proportional hazards regression analysis. RESULTS: The primary patency rate of arteriovenous fistula was 71.2% at one year and 43.0% at five years, and the secondary patency rate was 92.7% at one year and 79.8% at five years. In the multivariate analysis, high low-density lipoprotein cholesterol (LDL-C) level and a history of diabetes were considered significant risk factors (HR 1.023, p value <0.01 and HR 2.550, p value <0.01, respectively). A log rank test was conducted on the groups of patients with LDL <90 mg/dl and LDL ≥90 mg/dl, and the <90 mg/dl group resulted in a good primary patency rate (p value 0.0327). CONCLUSIONS: High LDL-C level was considered the independent risk factors of arteriovenous fistula primary patency rate.

Low-dose In Situ Perfusion With Euro-Collins Solution Is Effective for the Procurement of Marginal Kidney Grafts From Donation After Circulatory Death Donors.

We have adopted a modified method to resuscitate kidneys from donation after circulatory death (DCD) donors with the use of Euro-Collins (EC) solution instead of University of Wisconsin solution. This study aimed to evaluate kidney transplantation (KTx) outcomes of DCD procured with low-dose in situ perfusion using EC solution. PATIENTS AND METHODS: KTx was performed in 8 adults. Kidney grafts were procured following in situ perfusion with approximately 1 L of EC solution and preserved in the solution. The kidney donor profile index value was 88% ± 21%. The terminal creatinine level of the donors was 5.5 ± 3.4 mg/dL. Of the 8 donors, 6 experienced oligoanuria prior to graft procurement. RESULTS: The mean age of the recipients and the hemodialysis vintage were 50 ± 10 years and 161 ± 25 months, respectively. The warm and cold ischemic times were 8.3 ± 7.9 minutes and 8.7 ± 4.3 hours, respectively. All grafts functioned after a delayed graft function of 10.6 ± 6.9 days (2-25 days). There was neither immediate graft function nor primary nonfunction. The patient and graft survivals were both 100% with a terminal creatinine level of 1.3 ± .5 mg/dL. CONCLUSIONS: Kidney grafts procured from DCD donors with a high kidney donor profile index value demonstrated good renal function with an excellent midterm outcome. Low-dose in situ perfusion with EC solution is effective for the procurement of marginal kidney grafts from DCD donors under optimal conditions such as a relatively shorter preservation time.

Transdermal delivery of 40-nm silk fibroin nanoparticles.

Transdermal administration of drugs improves their bioavailability and is capable of systemic and local treatment. To improve the skin permeability of drugs, nano-sized systems have attracted attention as drug carriers for transdermal drug delivery system. We considered that silk fibroin composed of a crystalline region with many hydrophobic amino acids and an amorphous region with many hydrophilic amino acids was useful as a carrier for transdermal administration of a drug because of the balance between hydrophilicity and hydrophobicity. In this study, silk fibroin nanoparticles with mean volume diameters of 42.3 nm were successfully prepared, and storage stability was confirmed by storing the nanoparticle suspension at 4, 32, and 37 °C for a week. At any storage temperature, the mean volume diameter and standard deviation were stable. The polydispersity indexes were 0.19-0.23, and no specific trends were observed. Then, to investigate the transdermal delivery route of the silk fibroin nanoparticles, skin permeability in vivo was evaluated using mice. Six hours after administration, fluorescent substances were observed in the dermis in addition to the stratum corneum, hair follicles and the epidermis around them. This result indicated that fibroin nanoparticles with the mean volume diameter of 40-nm penetrated the stratum corneum and was delivered deep into the skin. Therefore, it was suggested that small nanoparticles prepared using silk fibroin are useful for drug delivery to the dermis.

Thrombotic microangiopathy caused by oral contraceptives in a kidney transplant recipient.

Thrombotic microangiopathy (TMA) after kidney transplantation has various aetiologies, including acute antibody-mediated rejection, bacterial or viral infection and immunosuppressive drugs, particularly calcineurin inhibitors. We present the case of a 28-year-old woman who developed TMA 30 months after the transplantation of an ABO-incompatible kidney from a living unrelated donor. The patient developed a sudden onset of allograft renal dysfunction and became uremic. She was transferred to our institution from a community hospital with strongly suspected acute allograft rejection. Intensive treatments for both T- and B-cell mediated acute rejection, including steroid pulse therapy, double-filtration plasmapheresis, antithymocyte globulin (1.5 mg/kg × 14 days) and rituximab (100 mg), were initiated during haemodialysis. However, her renal allograft function did not improve. Histopathological analysis 8 days after the treatment indicated TMA, despite the absence of apparent acute T-cell- or acute antibody-mediated rejection. There were no symptoms of infectious diseases, such as intestinal haemorrhagic colitis or viral infection. We concluded that the use of oral contraceptives, which had been initiated 3 weeks before TMA onset for the treatment of irregular vaginal bleeding, was the aetiologic agent.

Indium-catalyzed annulation of 3-aryl- and 3-heteroarylindoles with propargyl ethers: synthesis and photoluminescent properties of aryl- and heteroaryl[c]carbazoles.

Treatment of 3-aryl- and 3-heteroarylindoles with propargyl ethers under indium catalysis successfully provided aryl- and heteroaryl[c]carbazoles, which were found to be more efficient emitters compared with the corresponding [a]-analogs.

Two adjacent cis-regulatory elements are required for ecdysone response of ecdysone receptor (EcR) B1 transcription.

Three distinct classes of nuclear receptors, EcR, E75, and HR3, are key regulators in the ecdysone-inducible gene activation cascade in insects. The transcription of these genes is induced by ecdysone (20E) differently, although the detailed mechanisms underlying their responses to 20E are largely unknown. We identified ecdysone response elements (EcREs) present in the promoters of genes coding BmEcR-B1, BmE75-A, and BHR3-B isoforms from Bombyx mori employing luciferase reporter assays in an ecdysteroid-responsive cultured cell line, NIAS-Bm-aff3 (aff3). The EcRE of BmEcR-B1 at -2800 comprises of two adjacent elements separated by 5 bp, E1 (15 bp) and E2 (21 bp), both of which are required for the 20E response. Further analysis using electrophoretic mobility shift assays showed that E1 binds to the EcR/USP heterodimer and that E2 may bind to the E-box (CACGTG) binding factor such as bHLH protein. The unique E1+E2-type EcRE is also detected in the promoter upstream regions of EcR-B1 from seven lepidopteran species studied. In contrast, both a 20 bp EcRE identified in the promoter of BmE75-A and a 18 bp EcRE identified in the BHR3-B promoter, contained only E1-type EcR/USP binding element but the E2 type element was not in the promoter regions of these genes. The combination of presence of the E2 element or other cis-regulatory elements in promoter regions explains the different 20E response of each class of nuclear receptor genes. Furthermore, the E1+E2 structure for EcR-B1 can be involved in a possible cross-talk between ecdysteroid and other regulatory pathways.

Primary omental pleomorphic liposarcoma identified at autopsy in a living donor kidney transplant recipient.

Metastatic and recurrent tumors of the omentum are common, but primary omental pleomorphic liposarcoma (POPL) is an extremely rare type of solid omental tumor. We describe the case of a patient with POPL who received a renal transplant from a living donor. Despite good allograft function, the volume of peritoneal fluid gradually increased. An exploratory laparotomy could not be performed because the patient was obese and developed hemodynamic instability. Therefore, a shunt was placed between the peritoneal cavity and the internal jugular vein using the Denver® shunt system; however, the patient died of respiratory insufficiency. On the basis of the autopsy results, we diagnosed the patient's condition as POPL. We speculated that the malignancy did not originate directly from the donor cells. We report POPL in a living donor renal transplant recipient.

Influence of particle design on oral absorption of poorly water-soluble drug in a silica particle-supercritical fluid system.

The physicochemical characteristics and oral absorption of a poorly water-soluble drug, K-832, adsorbed onto porous silica (Sylysia 350), were compared with those of K-832 adsorbed onto non-porous silica (Aerosil 200). K-832 and silica were treated with supercritical CO(2) (scCO(2)) to produce K-832-Sylysia 350 and K-832-Aerosil 200 formulations. Scanning electron microscopy, polarizing microscopy, powder X-ray diffraction, and differential scanning calorimetry results suggested that K-832 mainly existed in an amorphous state in both formulations. The specific surface area of both formulations was much larger than that of pure K-832 crystals. The dissolution rate of K-832 from both formulations was considerably greater than that from corresponding physical mixtures due to rapid wetting of the hydrophilic carrier surfaces and amorphous state, the dissolution from the K-832-Sylysia 350 formulation being the fastest. In vivo absorption tests on the two formulations indicated no significant differences in their peak concentration (C(max)) and the area under their plasma concentration-time curve (AUC), while the concentrations of K-832 in the K-832-Sylysia 350 formulation were significantly higher than those in the K-832-Aerosil 200 formulation 1 h and 1.5 h after administration of these formulations (p<0.05). This could be attributed to the different dispersion states of K-832 in the formulations due to their different three-dimensional structures (porous and non-porous). In physical stability tests, the amorphous drugs in both formulations were stable at room temperature for at least 14 months. Thus, the absorption of poorly water-soluble drugs could be greatly improved by adsorption onto porous silica using scCO(2).

Stability of amorphous drug, 2-benzyl-5-(4-chlorophenyl)-6-[4-(methylthio)phenyl]-2H-pyridazin-3-one, in silica mesopores and measurement of its molecular mobility by solid-state (13)C NMR spectroscopy.

This study evaluated the physical stability and molecular mobility of a poorly water-soluble amorphous drug, 2-benzyl-5-(4-chlorophenyl)-6-[4-(methylthio)phenyl]-2H-pyridazin-3-one (K-832), adsorbed onto silica mesopores. K-832-Sylysia 740 and K-832-Sylysia 350 formulations, prepared by adsorbing K-832 onto porous silica Sylysia 740 (2.5-nm-diameter pores) and Sylysia 350 (21-nm-diameter pores) and stored at 60°C/80%RH (open and closed conditions), were investigated. Differential scanning calorimetry revealed that crystallization of K-832 in the K-832-Sylysia 350 formulation stored at 60°C/80%RH (open and closed conditions) was faster than that of the other formulation stored under identical conditions. Raman spectroscopy revealed shifts to higher wavenumbers in the K-832-Sylysia 350 and K-832-Sylysia 740 formulations (1497 and 1493 cm(-1), respectively) in comparison to amorphous K-832 (1481 cm(-1)); however, no distinct differences were observed in the spectra of the two formulations. Solid-state (13)C NMR spectroscopy revealed a difference in spin-lattice relaxation time in the rotating frame (T(1ρ)) between the two formulations, suggesting the lower molecular mobility of K-832 in the 2.5-nm-diameter pores than in the 21-nm-diameter pores. Thus, the crystallization rate of amorphous K-832 in the K-832-Sylysia 740 formulation was much slower. These results will be useful in estimating the physical stability of amorphous drugs in mesopores.

Enhancement of dissolution rate and oral absorption of a poorly water-soluble drug, K-832, by adsorption onto porous silica using supercritical carbon dioxide.

The aim of this study was to enhance the dissolution rate and oral absorption of a poorly water-soluble drug, 2-benzyl-5-(4-chlorophenyl)-6-[4-(methylthio)phenyl]-2H-pyridazin-3-one (K-832) by adsorbing it onto the porous silica Sylysia 350 using supercritical CO(2) (scCO(2)) as a solvent. K-832-silica formulations were prepared using scCO(2) or dichloromethane (DCM) as the solvent (K-832-silica scCO(2) and K-832-silica DCM). Scanning electron microscopy, polarizing microscopy, differential scanning calorimetry, and powder X-ray diffraction observations revealed that in both formulations, K-832 existed mainly in an amorphous state. In a dissolution test, 70.2% and 13.3% of K-832 were released from K-832-silica scCO(2) and K-832-silica DCM, respectively, within 5min, whereas only 2.3% of K-832 was released from a physical mixture within 120min. Results of an in vivo absorption test showed that the area under the plasma concentration-time curve and peak concentration of K-832-silica scCO(2) were 8.3- and 13.3-fold greater than those of K-832 crystal, whereas the corresponding values of K-832-silica DCM were 5.0- and 8.3-fold greater than those of K-832 crystal. These results suggest that the method of using scCO(2) as the solvent is effective in enhancing the dissolution rate and oral absorption of poorly water-soluble drugs because it does not require a toxic solvent and surfactant.

Protein and gene expression of tumor-associated antigen RCAS1 in esophageal squamous cell carcinoma.

RCAS1 (receptor-binding cancer antigen expressed on SiSo cells) is expressed on the tumor cell membrane and induces apoptosis on infiltrated immune lymphocytes. RCAS1 has been reported to correlate with the escape of tumor cells from host immune surveillance, and with poor prognosis. However, the clinical importance of RCAS1 protein and gene expression in esophageal squamous cell carcinoma (ESCC) has not been well investigated. In the present study, RCAS1 gene and protein expression levels were evaluated and compared with clinical findings in 67 patients with ESCC. Expression levels of RCAS1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) messenger RNAs (mRNAs) from tumors and non-cancerous epithelia were analyzed quantitatively by real-time reverse transcriptase polymerase chain reaction (RT-PCR). RCAS1 protein expression was analyzed by immunohistochemistry. The mean RCAS1/GAPDH ratio of tumors (0.7) was not different from that of non-cancerous epithelia (0.7, p=0.715). RCAS1 immunoreactivity was detected in 19 tumors (28.4%). The mean RCAS1/GAPDH ratio of tumors with RCAS1 protein positive (0.6) did not differ from tumors without RCAS1 expression (0.8, p=0.131). RCAS1 gene and protein expressions did not correlate with tumor size, depth of invasion, lymph node metastasis, or patient prognosis. Thus, RCAS1 gene or protein expression may not correlate with tumor progression in ESCC.

Detection of cancer cells in the peripheral blood of gastric cancer patients.

The existence of occult metastasis in peripheral blood has been reported in various tumors. However, in gastric cancer (GC), this metastasis has not been well analyzed. In the present study, to identify circulating cancer cells in patients with GC, peripheral blood samples from GC patients were investigated. Total RNA was extracted from 1.5 ml peripheral blood from 55 patients with GC, from 34 non-cancer patients, and from 10 healthy volunteers. Carcinoembryonic antigen (CEA), cytokeratin 19 (CK19), and 20 (CK20) messenger RNA (mRNA) were used as probes to detect GC cells in the blood samples using real-time reverse transcriptase polymerase chain reaction (RT-PCR). CEA and CK19 mRNA expression were not detected in the 40 healthy volunteers and non-cancer patients, while 2 of the 40 showed CK20 mRNA expression. In 55 patients with GC, CK19 mRNA was not detected and CEA mRNA was detected in only one case (1.8%) with stage IV. While CK20 mRNA expression was observed in 15 cases (27.3%) and even in stage I, 8 of 24 (33.3%) showed CK20 mRNA expression. Thus, the specificity of CK20 marker may be low. Even though the sensitivity of CEA marker is low, CEA may be a more reliable marker than cytokeratins for detection of cancer cells in GC patient's peripheral blood.