Development of a cellular model to study CCR8 signaling in tumor-infiltrating regulatory T cells.

The human CC chemokine receptor 8 (CCR8) is specifically expressed on tumor-infiltrating regulatory T cells (TITRs) and is a promising drug target for cancer immunotherapy. However, the role of CCR8 signaling in TITR biology and the effectiveness of CCR8 small molecule antagonists as TITR-targeting immunotherapy remain subjects of ongoing debate. In this work, we generated a novel cellular model of TITRs by culturing peripheral blood mononuclear cell-derived regulatory T cells in medium containing tumor cell-conditioned medium, CD3/CD28 activator, interleukin-2 and 1α,25-dihydroxyvitamin D3. This cellular model (named TITR mimics) highly and stably expressed a series of TITR signature molecules, including CCR8, FOXP3, CD30, CD39, CD134, CD137, TIGIT and Tim-3. Moreover, TITR mimics displayed robust in vitro immunosuppressive activity. To unravel the functional role of CCR8 in TITR mimics, a chemotaxis assay was performed showing strong and CCR8-specific migration toward CCL1, the natural chemokine agonist of CCR8. However, either stimulation (with CCL1) or blocking (with the small molecule antagonist NS-15) of CCR8 signaling did not affect the immunosuppressive activity, proliferation and survival of TITR mimics. Collectively, our work provides a method for the generation of TITR mimics in vitro, which can be used to study TITR biology and to evaluate drug candidates targeting TITRs. Furthermore, our findings suggest that CCR8 signaling primarily regulates migration of these cells.

Ultrasensitive detection of tumor-derived small extracellular vesicles based on nonlinear hybridization chain reaction fluorescence signal amplification and immunomagnetic separation.

Small extracellular vesicles (sEVs) have attracted wide attention as a promising tumor biomarker. However, sensitive and selective detection of sEVs is challenging due to the low levels of sEVs in the early stage of cancers. Herein, a novel fluorescent sensor was developed for the detection of sEVs with high sensitivity and selectivity based on nonlinear hybridization chain reaction (nHCR) signal amplification and immunomagnetic separation. Firstly, sEVs were captured and enriched by CD63 antibody conjugated magnetic beads via antibody-antigen reactions. Then, cholesterol-modified DNA probes were anchored spontaneously on lipid membranes of sEVs through efficient hydrophobic interactions between the cholesterol moiety and the phospholipid bilayer of sEVs. The simultaneous recognition of the transmembrane protein and the phospholipid bilayer structure of the sEVs could effectively eliminate interferences from free proteins. The sticky ends of the cholesterol-modified DNA probes acted as the initiator to trigger nHCR to form a hyperbranched network of DNA structure that could recruit more fluorescent signal molecules for signal amplification. Under the optimal conditions, the nHCR-based strategy showed high sensitivity for the detection of sEVs with a limit of detection of 80 particles per µL. In addition, the as-constructed method was successfully applied for the analysis of clinical samples. It provides a sensitive and selective platform for the isolation and detection of sEVs in the early diagnosis of cancers.

Correlation between tumor markers and type 2 diabetes mellitus complications and their related influencing factors.

BACKGROUND: This study aims to investigate the correlation between type 2 diabetes mellitus (T2DM) and the tumor markers/biochemical parameters of patients, as well as the related factors leading to complications. METHODS: A total of 150 T2DM patients in our hospital were included as the research group, and 80 healthy persons were matched in the normal control group. The levels of tumor markers (CA199, CEA, CA153, CA125, AFP) and body mass index (BMI), waist-to-hip ratio (WHR), blood pressure (BP), fasting plasma glucose (FPG), glycosylated hemoglobin (HbA1c), urine microalbumin (M-ALB), and triglyceride (TG) in the two groups were determined. Based on the complications status of T2DM patients, the patients were further divided into a complication-free group (patients with simple diabetes) and complication group. Univariate analysis was performed between patients with and without complications. RESULTS: The levels of serum CA199, CEA, and CA125 in the study group were significantly higher than those in the normal control group (all P<0.0001), and the levels of BMI, WHR, systolic BP (SBP), diastolic BP (DBP), FPG, HbA1c, M-ALB, and TG in the study group were significantly higher than those in the control group (all P<0.05). Tumor markers CA199, CEA, and CA125 were positively correlated with BMI, WHR, BP, FPG, HbA1c, M-ALB, and TG. Smoking, family history of diabetes, combined hypertension, hyperlipemia, course of disease, CA199, CEA, CA153, CA125, AFP, SBP, DBP, FPG, HbA1c, M-ALB, and TG were the influencing factors of complications in T2DM patients. CONCLUSIONS: Relevant indicators of T2DM patients with complications should be fully evaluated clinically, and long-term follow-up observation should be conducted, so as to reduce the occurrence of complications.

An Ultrasensitive Strand Displacement Signal Amplification-Assisted Synchronous Fluorescence Assay for Surface Proteins of Small Extracellular Vesicle Analysis and Cancer Identification.

Small extracellular vesicles (sEVs), often known as exosomes, are expected to be a promising biomarker for the early diagnosis of cancer because they carry enriched proteins that originated from parent cells. Profiling surface proteins of sEVs offers non-invasive access for the early diagnosis of cancer. However, it remains challenging to simultaneously detect surface proteins of sEVs with desired sensitivity. Herein, a dual color DNA nanodevice based on toehold-mediated DNA strand displacement signal amplification and the synchronous fluorescence technique has been developed for simultaneous analysis of surface proteins of sEVs with high sensitivity. As for the DNA nanodevice-based system, the nanoconjugates of aptamer-magnetic beads can recognize surface proteins of sEVs and lead to the release of single-stranded DNA. Then, the released DNA can trigger toehold-mediated DNA strand displacement for signal amplification. In this system, a CD63 aptamer and MUC1 aptamer were used as recognition elements for the detection of surface proteins of sEVs isolated from cancer cells. Under the optimal conditions, the corresponding proteins of sEVs were simultaneously determined with ultrasensitivity by the synchronous fluorescence method. Also, the detection limits of sEVs by two surface proteins were 67 particles/µL by CD63 and 37 particles/µL by MUC1. Of note, the as-constructed method can be applied to recognize sEVs from different tumor cell lines (SGC7901, HepG2, and MCF-7 cells). Furthermore, the system has been successfully applied to precisely identify cancer patients from healthy people by serum analysis. The strategy demonstrates great potential applications in the early diagnosis of cancer.

A novel label-free electrochemiluminescence aptasensor using a tetrahedral DNA nanostructure as a scaffold for ultrasensitive detection of organophosphorus pesticides in a luminol-H2O2 system.

In this work, a new type of Au-tetrahedral DNA nanostructure (Au-TDN) was originally proposed and successfully applied in an electrochemiluminescence aptasensor to detect organophosphorus pesticides (Ops). The aptamers modified with -SH could be covalently bonded with gold nanoparticles (AuNPs) to form a tetrahedron structure, and there were independent probes at each vertex of the tetrahedron, which could increase the probability of specific binding with Ops. The originally designed structure could not only maintain a stable tetrahedral configuration, but also combined with the target to improve the sensitivity of the sensor. Meanwhile, silver nanoparticles (AgNPs) could catalyze the chemical reaction between luminol and H2O2 to generate a variety of intermediates called reactive oxygen species (ROS) for signal enhancement. Factors that had important influences on the aptasensor, such as the concentration of Au-TDN, the incubation time, and the pH value of the buffer, were optimized in this trial. According to the final results, the limit of detection (LOD) of 3 pg mL-1 (S/N = 3) for methyl parathion, the LOD of 0.3 pg mL-1 (S/N = 3) for parathion and the LOD of 0.03 pg mL-1 (S/N = 3) for phoxim were obtained, respectively. Moreover, the novel tetrahedral structure could be replaced by different types of aptamers to expand its application range and lay a foundation for the development of portable rapid detection devices for pesticide residues.

Long-term outcomes of "open iridectomy" for secondary anterior chamber epithelial iris cysts.

Epithelial cysts run a high risk of recurrence and conversion to sheet-like ingrowth after surgical intervention. In this retrospective study, we introduced a modified iridectomy for treatment of secondary epithelial iris cysts (EICs) in the anterior chamber. Twenty-nine patients (29 eyes) aged 2-61 years received "open iridectomy" for EICs between April 1995 and July 2019. After viscodissection, most of the cyst wall was cut using a 20-gauge aspiration cutter via a 2.5-mm clear corneal incision. The residue closely adhering to the iris stroma was remained to avoid photophobia and diplopia. At 3 months, best corrected visual acuity was ≥ 20/100 in 55.5% (15/27, except two pediatric patients with poor cooperation) of patients. Among the eight patients suffering partial corneal edema preoperatively, six patients received surgery treatment at 3-6.5 months, and the cornea in the other two patients became transparent after medication. In a mean follow-up of 47.4 months, recurrence occurred in 3 patients at 7, 37, and 118 months, respectively. The percentage of treatment success was 96%, 87%, and 65% at 1, 5, and 10 years, respectively. "Open iridectomy" was effective for EICs, with a minimal invasion, less damage to the corneal endothelium, and a low recurrence rate.

pH-Sensitive Dye-Based Nanobioplatform for Colorimetric Detection of Heterogeneous Circulating Tumor Cells.

The efficient capture and sensitive detection of circulating tumor cells (CTCs) play a vital role in cancer diagnosis and prognosis. However, CTCs in the peripheral blood are very rare and heterogeneous, which make them difficult to isolate and detect. Herein, a novel colorimetric nanobioplatform was successfully developed for the highly efficient capture and highly sensitive detection of heterogeneous CTCs, which consisted of two parts: the multivalent aptamer-modified gold nanoparticles as the capture unit and two kinds of aptamer-functionalized pH-sensitive allochroic dyes (thymolphthalein and curcumin) @ molybdenum disulfide nanoflakes (MoS2 NFs) acting as the visual simultaneous detection of heterogeneous CTCs. Using MCF-7 and HeLa cells as the CTC models, the capture unit can effectively isolate the CTCs due to the multivalent probe with improved affinity. The two allochroic dyes can display obvious color changes under alkaline conditions (pH 12.5) in the presence of MCF-7 and HeLa cells, which provided a rapid and sensitive strategy for visualizing simultaneous detection of heterogeneous CTCs as low as 5 cells mL-1. This nanoplatform possessed a high sensitivity toward CTC detection owing to high dye loading capacity of MoS2 NFs and allochroic dyes with excellent pH sensitivity. It can successfully distinguish and quantitatively detect the targeted heterogeneous CTCs from numerous interfering cells in diluted whole blood. It can also be used to detect CTCs from lysed blood samples from cancer patients, indicating promising application for cancer diagnosis.

Indications for penetrating keratoplasty and anterior lamellar keratoplasty during 2010-2017.

AIM: To review the indications of penetrating keratoplasty (PK) and anterior lamellar keratoplasty (ALK) at Qingdao Eye Hospital, Shandong Eye Institute, Qingdao, China, from 2010 to 2017. METHODS: The data of all patients undergoing PK or ALK from January 2010 to December 2017 was retrospectively reviewed, with the indications during 2010-2013 and 2014-2017 compared. RESULTS: A total of 1869 eyes were included, among which 1405 eyes (75.2%) had PK and 464 eyes (24.8%) had ALK. The leading indications were suppurative keratitis (36.8%), keratoconus (15.5%), herpes keratitis (13.1%), and regraft (10.5%). In eyes undergoing PK, the top four indications were suppurative keratitis (38.7%), herpes keratitis (15.3%), keratoconus (12.6%), and regraft (12.5%) during 2014-2017, with the proportion of suppurative keratitis and herpes keratitis decreased while regraft and keratoconus increased compared with 2010-2013. In eyes with ALK, suppurative keratitis (30.8%), keratoconus (24.1%), corneal dystrophies and degenerations (10.6%), and corneal dermoid tumor (9.7%) were the top four indications, and there was no significant difference for the proportion of each indication between 2010-2013 and 2014-2017. CONCLUSION: Suppurative keratitis is the most common indication for PK and ALK at Qingdao Eye Hospital during 2010-2017, followed by keratoconus, herpes keratitis, and regraft. In eyes treated with PK, the proportion of suppurative keratitis and herpes keratitis decrease while regraft and keratoconus increase during 2014-2017 compared with 2010-2013.

Codon optimization significantly enhanced the expression of human 37-kDa iLRP in Escherichia coli.

37-kDa immature laminin receptor protein (iLRP), the precursor of 67-kDa laminin receptor protein (LRP), is overexpressed on the surface of most cancer cells and recognized as a universal tumor antigen. The role makes it a potential target for cancer immunotherapy, which has been well-studied. Our study aimed to produce high quality of human iLRP in bacteria so that the needs in research of its clinical application could be met. The powerful system for heterologous protein expression, pET system was used. Two types of DNA sequences encoding the same amino acid sequences were separately cloned into the vector pET30a(+). One of the resulting vectors includes the wild-type iLRP, and other one includes the codon-optimized iLRP. The expression by both genes was then compared in Escherichia coli BL21(DE3). Our results revealed that the performance of codon optimization was crucial for the expression of human iLRP in Escherichia coli. The yield was significantly enhanced up to 300 mg/L of bacterial culture by this approach.

N-myc downstream-regulated gene 1 inhibits the proliferation and invasion of hepatocellular carcinoma cells via the regulation of integrin ß3.

N-myc downstream-regulated gene 1 (NDRG1) is a multifunctional protein associated with carcinogenesis and tumor progression. The function of NDRG1 in hepatocellular carcinoma (HCC) cells remains controversial. The present study investigated the role of NDRG1 in HCC as well as its molecular mechanism using a range of techniques, including western blot analysis, cellular proliferation test, wound healing assay and Transwell assay. In HCC, the levels of NDRG1 expression were highest in the cytoplasm, followed by the membrane, and were lowest in the nucleus. NDRG1 was revealed to inhibit the proliferation and invasion of BEL7402 cells, which facilitated the hypothesis that NDRG1 expression levels may be lower in cell line with a high metastatic potential compared with those in cell lines with a low metastatic potential. However, the present study identified that NDRG1 expression was higher in detached BEL7402 cells and MHCC-97H cells compared with that in attached BEL7402 cells and MHCC-97L cells. Thus, this finding was contrary to what was expected, suggesting that NDRG1 overexpression in the HCC with a high metastatic potential may be the compensatory mechanism. The human HCC BEL7402 cell line demonstrated a significant increase in the capability of motility, invasion and cellular proliferation following NDRG1-short hairpin RNA transfection. Integrin ß3 (ITGB3) protein expression was increased in NDRG1-downregulated BEL7402 cells and SMMC7721 cells compared with that in the control cells. The present study suggested that NDRG1 may be a potential anti-tumor target for the treatment of patients with HCC. A potential mechanism for these roles of NDRG1 is by regulating ITGB3 expression; however, this requires additional investigation.

Diagnostic Value of Serum hsa-mir-92a in Patients with Cervical Cancer.

BACKGROUND: Hsa-mir-92a acts as an onco-miRNA and may contribute to the progression and invasion of cervical cancer, which is a common malignant tumor in women worldwide. The objective of the present study was to evaluate whether serum hsa-mir-92a could serve as a diagnostic biomarker for cervical cancer patients. METHODS: The expression levels of hsa-mir-92a were analyzed in the serum of patients with CIN I, CIN II, CIN III, cervical cancer Ia - IIa and compared with those of the control group samples. The receiver operating characteristic (ROC) curve was used to evaluate the diagnostic significance of cervical cancer. RESULTS: It was found that the expression of hsa-miR-92a in the serum of individuals with CIN or cervical cancer was significantly higher than that in healthy volunteers (p < 0.01). The expression of hsa-miR-92a was higher in the serum of patients with advanced stage and cervical cancer than those with early stage. ROC analysis revealed that the cutoff value of hsa-mir-92a was 1.52 for the diagnosis of CIN and cervical cancer. The sensitivity and specificity were 69.6% and 80.4%, respectively, and an area under the curve (AUC) was 0.83. CONCLUSIONS: hsa-mir-92a up-regulation was associated with cervical cancer and the serum level of hsa-mir-92a could be used as an independent marker for the diagnosis of cervical cancer.

Increased expression of MyD88 and association with paclitaxel resistance in breast cancer.

MyD88 was reported to be associated with paclitaxel sensitivity in lung cancer; however, its roles in breast cancer remain unclear. The objective of this study is to investigate the expression and function of MyD88 in breast cancer. Immunohistochemistry (IHC) was used to analyze the expression of MyD88 in both breast cancer tissues and adjacent normal tissues. Real-time PCR and Western blots were further used to measure the messenger RNA (mRNA) and protein expression. The proliferation was assessed by WST-1. Flow cytometry was used to measure the cell cycle and apoptosis. The transwell assay was used to observe the change of migration and invasion of transfected cells. In breast cancer tissues, the expression of MyD88 was significantly higher than that in tumor-adjacent normal tissues (P < 0.001). MyD88 expression was found to be associated with the differentiation stages (P = 0.019). Kaplan-Meier survival curves showed statistically significant difference on survival in patients with high expression of MyD88 compared with those with normal expression of MyD88 (P = 0.018). Knockdown of MyD88 reduced the proliferation, migration, and invasion of MCF-7 cells and increased the sensitivity of MCF-7 cells to paclitaxel treatment through the inhibition of activation of NF-κB via PI3K/Akt. Our data indicate that MyD88 may be a potential target molecule to be used in diagnosis and treatment of breast cancer.

Ursolic Acid Inhibits the Proliferation of Gastric Cancer Cells by Targeting miR-133a.

Ursolic acid (UA), a potential chemotherapeutic agent, has the properties of inhibition of the growth of many human cancer cell lines. Whether UA can inhibit the growth and metastasis of human gastric cancer cells remains unknown. In this study, it was found that UA inhibited the growth and metastasis of human gastric cancer cells in vitro. Our results showed the increase of the percent of apoptotic cells and G1 phase, the inhibition of cell migrations well as the decrease of the expression of Bax, caspase 3 and Bcl-2 in BGC-823 cells after the treatment with UA. Real-time quantitative PCR analysis showed that UA treatment upregulated the level of miR-133a in BGC-823 cells. Overexpression of miR-133a increased the G1 phase of cell cycle and decreased Akt1 expression in BGC-823 cells. These outcomes might be secondary to the increased expression of miR-133a after the treatment with UA.

Anti-Sp17 monoclonal antibody-doxorubicin conjugates as molecularly targeted chemotherapy for ovarian carcinoma.

Sperm protein 17 (Sp17) is selectively overexpressed in several human malignancies including ovarian carcinoma, but is absent or expressed at low levels in most normal tissues. Previous work from our group characterized an anti-Sp17 monoclonal antibody (clone 3C12) and showed that it specifically targeted tumor cells. In this report, we investigated whether a novel immunoconjugate containing 3C12 linked to the chemotherapeutic agent doxorubicin [(DOX) Adriamycin] had antitumor activity against ovarian cancer cell lines and tumor models. DOX was conjugated to 3C12 using a linker, and the specificity of 3C12-DOX was examined in Sp17-positive SKOV3 and Sp17-negative COC2 ovarian cancer cells using cell-based ELISA and internalization assays. The cytotoxicity of 3C12-DOX was assessed with the MTT assay, and its therapeutic effectiveness was evaluated in immunodeficient mice bearing SKOV3 cells. In vitro, the 3C12-DOX immunoconjugate specifically bound to and was internalized by Sp17-positive SKOV3 cells but did not bind to Sp17-negative cells. Treatment with 3C12-DOX (0.001 to 10 µg/mL) decreased the viability of SKOV3 cells in a Sp17-specific manner. In vivo, 3C12-DOX (3 mg/kg) induced the regression of established SKOV3 xenograft tumors in BALB/c mice compared with control treatment. The antitumor effects of 3C12-DOX were significantly associated with the induction of apoptosis in tumor cells. In addition, 3C12-DOX showed no observable adverse effects or toxicity when compared with DOX alone in mice bearing ovarian tumor xenografts. Our findings suggest that 3C12-DOX may be a potential antibody-drug conjugate for clinical development.

Surveillance study of species distribution, antifungal susceptibility and mortality of nosocomial candidemia in a tertiary care hospital in China.

BACKGROUND: Bloodstream infections due to Candida species cause significant morbidity and mortality, and the epidemiology of Candida infection is changing. Surveillance for candidemia is necessary to detect trends in species distribution and antifungal resistance. METHODS: The medical and electronic records of all patients who had candidemia at the authors' hospital from 2009 to 2011 were reviewed for demographic data and clinical information, including the infecting Candida species, resistance to antifungals and survival, and the presence of risk factors associated with candidemia. RESULTS: A total of 133 distinct episodes of candidemia were identified over the study period. The annual incidence of candidemia ranged between 0.71 and 0.85 cases/1000 hospital discharges. The most frequent Candida species were C. tropicalis (28.6%), followed by C. albicans (23.3%) and C. parapsilosis (19.5%). The rates of susceptibility to antifungal agents were as followed: voriconazole (97.8%), itraconazole (69.5%), fluconazole (46.1%), ketoconazole (38.9%). Out of 131 evaluable patients, 34 (26.0%) died within 30 days from the onset of candidemia. C. tropicalis candidemia was associated with the highest mortality rate (44.7%). Regarding the crude mortality in the different units, patients in Hemato-Oncology ward had the highest mortality rate (66.7%), followed by patients in cardiovascular wards and ICU (57.1% and 25.6%, respectively). Predictors of 30-day mortality were identified by uni- and multivariate analyses. Complicated abdominal surgery, presence of central venous catheter (CVC), neutropenia, candidemia due to C. tropicalis and poor treatment with fluconazole were significantly associated with the 30-day mortality. Presence of CVC (odds ratio[OR] = 4.177; 95% confidence interval [CI] = 1.698 to 10.278; P = 0.002) was the only independent predictor for mortality in the multivariate analysis. CONCLUSION: This report provides baseline data for future epidemiological and susceptibility studies and for the mortality rates associated with candidemia in our hospital. The knowledge of the local epidemiological trends in Candida species isolated in blood cultures is important to guide therapeutic choices.

Synthesis of water soluble quantum dots for monitoring carrier-DNA nanoparticles in plant cells.

Fluorescent quantum dots (QDs) have shown great promise for use as biolabels in cell and animal biology and more recently in plant sciences. An important use of QDs is for monitoring the dynamics, intracellular trafficking, and fate of carrier-DNA nanocomplexes in cell transfection and potentially in plant transformation. In this study, a low cost aqueous procedure has been developed to efficiently prepare biocompatible QDs for monitoring nanoparticle-mediated gene transfer in conjunction with molecular breeding of Jatropha curcas. Water-soluble CdSe nanoparticles were synthesized by self-assembly using L-Cysteine as stabilizer and optimal synthesis scheme established by fluorescence spectroscopy. The QDs were used to label chitosan-DNA nanoparticles via electrostatic interaction and the resultant QD-labeled chitosan-DNA complexes were shown to have superior fluorescence properties with red shift of emission and absorption spectra relative to the CdSe QDs alone. This system is being explored as a superior alternative to Agrobacterium-mediated genetic transformation of Jatropha curcas cells. PCR amplification of the full length of the carried reporter gene (GFP) suggests that the DNA was not digested in Jatropha curcas cells transfected with CdSe/CS-DNA complexes. Furthermore, GFP gene expression in the transfected callus cells, as evidenced by fluorescence detection, suggests that the target DNA was integrated into the plant genome.