A simultaneous strategy with multiple-signal amplification and self-calibration for ultrasensitive assay of miRNA-21 based on 3D MNPs-IL-rGO-AuNPs.

MicroRNA-21 (miRNA-21) is a significant biomarker for the development and progression of diverse cancers but is present in relatively low concentrations. Detecting such low-abundance molecules accurately can be challenging, especially in early-stage cancers where the concentration may be even lower. Herein, a self-calibration biosensing platform based on 3D novel MNPs-IL-rGO-AuNPs nanocomposites was successfully established for the ultrasensitive detection of miRNA-21. Duplex-specific nuclease (DSN) was introduced to recognize perfectly matched duplexes and trigger target recycling, enhancing the specificity and sensitivity of the biosensor. DSN-assisted target recycling, in conjunction with magnetic separation enrichment and high-performance MNPs-IL-rGO-AuNPs, collectively formed a multiple-signal amplification strategy. The obtained biosensor could output dual signals in both electrochemical and fluorescent modes, enabling self-correcting detection to enhance the accuracy. The obtained dual-mode biosensor prepared exhibited a wide detection range from 5 fM to 100 nM with a remarkably low LOD of 1.601 fM. It accomplished the sensitive evaluation of miRNA-21 in total RNA extracted from various human cancer cell lines and normal cell lines. Additionally, the greatly satisfactory outcomes in the analysis of human serum samples suggested that the proposed biosensor was a powerful screening candidate in early clinical diagnosis of cancer.

Analysis of the clinical factors affecting excellent response of Iodine-131 treatment for pulmonary metastases from differentiated thyroid cancer.

Background: Iodiene-131 (131I) treatment is the primary therapeutic approach for imaging 131I-avid pulmonary metastases. The response to radioiodine (RAI) treatment is an important prognostic factor in patients with pulmonary metastases from differentiated thyroid cancer (DTC). Patients who achieve an excellent response (ER) to 131I treatment show significantly reduced disease-related mortality. This study aimed to retrospectively analyse the clinical data and therapeutic effects of 131I treatment in patients with DTC and pulmonary metastases and to screen out the clinical factors affecting ER. Materials and methods: The study included a total of 75 patients with exclusively Iodine-131 avid (131I-avid) pulmonary metastases who underwent 131I treatment. Relevant clinical data for these patients were collected. Following treatment, the status of DTC metastatic lesions was categorized as follows: excellent response (ER), biochemical incomplete response (BIR), structural incomplete response (SIR), or indeterminate response (IDR). Gender, age at diagnosis, pathological type, stages (TNM), stimulated thyroglobulin (sTg) value before initial 131I treatment, metastatic nodule size, and type of post-treatment whole body scan (Rx-WBS) were recorded. Mono-factor analysis and binary logistic regression analyses were used to identify the factors that might affect the ER in DTC pulmonary metastases. The receiver operating characteristic (ROC) curve of the sTg value was used to predict the ER of 131I treatment. Results: All 75 patients with exclusively 131I-avid pulmonary metastases received 131I treatment and underwent follow-up. Out of the 75 patients, 26 achieved ER, resulting in an excellent response rate of 34.7 % (26/75). Among them, 25 (25/26, 96.2 %) achieved an ER after undergoing two rounds of 131I treatment. Binary logistic regression analysis showed that the factors influencing DTC pulmonary metastases excellent response were lower sTg levels [odds ratio (OR) = 0.998, P < 0.001], micronodular metastases (OR = 0.349, P = 0.001) and focal distribution on Rx-WBS imaging (OR = 0.113, P = 0.001). The area under the ROC curve for sTg value predicting ER was 0.876, and the cut-off value was 26.84 ng/mL, with a sensitivity and specificity of 87.9 % and 80.3 %, respectively. Conclusions: 131I treatment is effective for 131I-avid pulmonary metastases of DTC. Some patients who underwent 131I treatment achieved ER. Most patients with ER were obtained after two rounds of 131I treatments. Patients with sTg values before initial 131I treatment lower than 26.84 ng/mL, micronodular metastases, and focal distribution on Rx-WBS imaging were more likely to achieve ER.

Tubastatin A potently inhibits GPX4 activity to potentiate cancer radiotherapy through boosting ferroptosis.

Ferroptosis, an iron-dependent lipid peroxidation-driven programmed cell death, is closely related to cancer therapy. The development of druggable ferroptosis inducers and their rational application in cancer therapy are critical. Here, we identified Tubastatin A, an HDAC6 inhibitor as a novel druggable ferroptosis inducer through large-scale drug screening. Tubastatin A directly bonded to GPX4 and inhibited GPX4 enzymatic activity through biotin-linked Tubastatin A putdown and LC/MS analysis, which is independent of its inhibition of HDAC6. In addition, our results showed that radiotherapy not only activated Nrf2-mediated GPX4 transcription but also inhibited lysosome-mediated GPX4 degradation, subsequently inducing ferroptosis tolerance and radioresistance in cancer cells. Tubastatin A overcame ferroptosis resistance and radioresistance of cancer cells by inhibiting GPX4 enzymatic activity. More importantly, Tubastatin A has excellent bioavailability, as demonstrated by its ability to significantly promote radiotherapy-induced lipid peroxidation and tumour suppression in a mouse xenograft model. Our findings identify a novel druggable ferroptosis inducer, Tubastatin A, which enhances radiotherapy-mediated antitumor effects. This work provides a compelling rationale for the clinical evaluation of Tubastatin A, especially in combination with radiotherapy.

The predictive value of total-body PET/CT in non-small cell lung cancer for the PD-L1 high expression.

Purpose: Total-body positron emission tomography/computed tomography (PET/CT) provides faster scanning speed, higher image quality, and lower injected dose. To compensate for the shortcomings of the maximum standard uptake value (SUVmax), we aimed to normalize the values of PET parameters using liver and blood pool SUV (SUR-L and SUR-BP) to predict programmed cell death-ligand 1 (PD-L1) expression in non-small cell lung cancer (NSCLC) patients. Materials and methods: A total of 138 (104 adenocarcinoma and 34 squamous cell carcinoma) primary diagnosed NSCLC patients who underwent 18F-FDG-PET/CT imaging were analyzed retrospectively. Immunohistochemistry (IHC) analysis was performed for PD-L1 expression on tumor cells and tumor-infiltrating immune cells with 22C3 antibody. Positive PD-L1 expression was defined as tumor cells no less than 50% or tumor-infiltrating immune cells no less than 10%. The relationships between PD-L1 expression and PET parameters (SUVmax, SUR-L, and SUR-BP) and clinical variables were analyzed. Statistical analysis included χ2 test, receiver operating characteristic (ROC), and binary logistic regression. Results: There were 36 patients (26%) expressing PD-L1 positively. Gender, smoking history, Ki-67, and histologic subtype were related factors. SUVmax, SUR-L, and SUR-BP were significantly higher in the positive subset than those in the negative subset. Among them, the area under the curve (AUC) of SUR-L on the ROC curve was the biggest one. In NSCLC patients, the best cutoff value of SUR-L for PD-L1-positive expression was 4.84 (AUC = 0.702, P = 0.000, sensitivity = 83.3%, specificity = 54.9%). Multivariate analysis confirmed that age and SUR-L were correlated factors in adenocarcinoma (ADC) patients. Conclusion: SUVmax, SUR-L, and SUR-BP had utility in predicting PD-L1 high expression, and SUR-L was the most reliable parameter. PET/CT can offer reference to screen patients for first-line atezolizumab therapy.

Elevated DLL3 in stomach cancer by tumor-associated macrophages enhances cancer-cell proliferation and cytokine secretion of macrophages.

Background: The notch signal pathway is important in the development of both tumor-associated macrophages (TAMs) and stomach cancer, but how Notch signaling affects TAMs in stomach cancer is barely understood. Methods: The expressions of Notch1, Notch2, Notch3, Notch4, hes family bHLH transcription factor 1 (Hes1), and delta-like canonical Notch ligand 3 (DLL3) were detected by Western blot and the expressions of interleukin (IL)-10, IL-12, and IL1-ß were detected using enzyme-linked immunosorbent assay after the co-culture of macrophages and stomach-cancer cells. The proliferation and migration of cancer cells were detected using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and scratch assay, respectively, and the cell cycle was detected using Annexin V/propidium iodide assay. The protein interactions with DLL3 were detected using co-immunoprecipitation and mass spectrometry. Results: The co-culture of macrophages and stomach-cancer cells MKN45 and BGC823 could enhance cell proliferation accompanied by the activation of Notch1/Notch2 signaling and upregulation of DLL3. Notch signaling gamma-secretase inhibitor (DAPT) blocked this process. The overexpression of DLL3 in stomach-cancer cells could promote the proliferation of cancer cells, enhance the activation of Notch1/Notch2 signaling, induce the expression of IL-33, lead to the degradation of galectin-3-binding protein (LG3BP) and heat shock cognate 71 kDa protein (HSPA8), and result in elevated IL-1ß, IL-12, and IL-10 secretion by macrophages. Higher expression of DLL3 or IL-33 could lead to a lower survival rate based on University of California, Santa Cruz Xena Functional Genomics Explorer and The Cancer Genome Atlas data set. Conclusions: This is evidence that DLL3 regulates macrophages in stomach cancer, suggesting that DLL3 may be a novel and potential target for stomach-cancer therapy.

PKCßII phosphorylates ACSL4 to amplify lipid peroxidation to induce ferroptosis.

The accumulation of lipid peroxides is recognized as a determinant of the occurrence of ferroptosis. However, the sensors and amplifying process of lipid peroxidation linked to ferroptosis remain obscure. Here we identify PKCßII as a critical contributor of ferroptosis through independent genome-wide CRISPR-Cas9 and kinase inhibitor library screening. Our results show that PKCßII senses the initial lipid peroxides and amplifies lipid peroxidation linked to ferroptosis through phosphorylation and activation of ACSL4. Lipidomics analysis shows that activated ACSL4 catalyses polyunsaturated fatty acid-containing lipid biosynthesis and promotes the accumulation of lipid peroxidation products, leading to ferroptosis. Attenuation of the PKCßII-ACSL4 pathway effectively blocks ferroptosis in vitro and impairs ferroptosis-associated cancer immunotherapy in vivo. Our results identify PKCßII as a sensor of lipid peroxidation, and the lipid peroxidation-PKCßII-ACSL4 positive-feedback axis may provide potential targets for ferroptosis-associated disease treatment.

Assembly of fungal mycelium-carbon nanotube composites and their application in pyrene removal.

Polycyclic aromatic hydrocarbons (PAHs) have been known for decades to threaten human health. Various physical, chemical and biological methods have been developed to remove PAHs from different matrices. Microbial biodegradation processes are thought to be effective and environmentally friendly, but the low bioavailability of PAHs and their slow removal rate often limit the application of biodegradation. In this study, novel self-assembled PAH-degrading fungal mycelium (Penicillium oxalicum SYJ-1)-carbon nanotube (CNT) composites were applied for pyrene removal. The addition of CNTs did not affect the growth of strain SYJ-1 and promoted the total PAH removal efficiency. The composite could completely remove pyrene at 20 mg L-1 within 48 h, while the sole fungus and CNTs alone could only remove 72% and 80% of pyrene at 72 h, respectively. A cytochrome P450 inhibition experiment, together with degradation product identification and transcriptomic analysis, suggested that an intracellular PAH transformation pathway was employed by strain SYJ-1. The versatility of this assembly approach was also confirmed by adding different nanomaterials and using them to remove different pollutants. This study provides a strategy of coupling the chemical adsorption and biodegradation capacity of inorganic nanomaterials and microorganisms as composites to treat hydrophobic substrates in restricted bioreactor.

FUT8-mediated aberrant N-glycosylation of B7H3 suppresses the immune response in triple-negative breast cancer.

Most patients with triple negative breast cancer (TNBC) do not respond to anti-PD1/PDL1 immunotherapy, indicating the necessity to explore immune checkpoint targets. B7H3 is a highly glycosylated protein. However, the mechanisms of B7H3 glycosylation regulation and whether the sugar moiety contributes to immunosuppression are unclear. Here, we identify aberrant B7H3 glycosylation and show that N-glycosylation of B7H3 at NXT motif sites is responsible for its protein stability and immunosuppression in TNBC tumors. The fucosyltransferase FUT8 catalyzes B7H3 core fucosylation at N-glycans to maintain its high expression. Knockdown of FUT8 rescues glycosylated B7H3-mediated immunosuppressive function in TNBC cells. Abnormal B7H3 glycosylation mediated by FUT8 overexpression can be physiologically important and clinically relevant in patients with TNBC. Notably, the combination of core fucosylation inhibitor 2F-Fuc and anti-PDL1 results in enhanced therapeutic efficacy in B7H3-positive TNBC tumors. These findings suggest that targeting the FUT8-B7H3 axis might be a promising strategy for improving anti-tumor immune responses in patients with TNBC.

Dynamic monitoring of active calcification in atherosclerosis by 18F-NaF PET imaging.

The objective was to dynamically monitor the progression of atherosclerotic plaques in ApoE-/- mice with 18F-NaF PET imaging. The ApoE-/- mice were used to develop atherosclerosis models, and the C57BL/6 J mice were used as control. 18F-NaF PET was performed when the mice were 12, 20, and 30 weeks of age. Serum lipids and lipoproteins profiles, inflammatory cytokines, and calcification factors were tested by ELISA. The lipid distribution, morphology, and calcification of plaque were evaluated by Oil Red O, HE, and alizarin red staining. The correlation between imaging and the extent of calcification was analyzed by Pearson correlation analysis. The uptake of 18F-NaF in the aorta was gradually increased with each weekly extension. Compared with the ApoE-/- mice at the age of 12 weeks and 20 weeks, the levels of lipoprotein, inflammatory cytokines, and calcification factors were higher at 30 weeks. In Oil Red O, HE, and alizarin red staining, the extent of the lipid area and calcification increased with time. The correlation analysis showed that the uptake of 18F-NaF in the aorta was related to the extent of calcification. 18F-NaF may dynamically monitor the progression of atherosclerotic plaques and ongoing microcalcification formation.

MAPK1/3 kinase-dependent ULK1 degradation attenuates mitophagy and promotes breast cancer bone metastasis.

The function of mitophagy in cancer is controversial. ULK1 is critical for induction of macroautophagy/autophagy and has a more specific role in mitophagy in response to hypoxia. Here, we show that ULK1 deficiency induces an invasive phenotype of breast cancer cells under hypoxia and increases osteolytic bone metastasis. Mechanistically, ULK1 depletion attenuates mitophagy ability during hypoxia. As a result, the accumulation of damaged, ROS-generating mitochondria leads to activation of the NLRP3 inflammasome, which induces abnormal soluble cytokines secretion, then promotes the differentiation and maturation of osteoclasts, and ultimately results in bone metastasis. Notably, phosphorylation of ULK1 by MAPK1/ERK2-MAPK3/ERK1 kinase triggers its interaction with BTRC and subsequent K48-linked ubiquitination and proteasome degradation. Also, a clearly negative correlation between the expression levels of ULK1 and p-MAPK1/3 was observed in human breast cancer tissues. The MAP2K/MEK inhibitor trametinib is sufficient to restore mitophagy function via upregulation of ULK1, leading to inhibition of NLRP3 inflammasome activation, thereby reduces bone metastasis. These results indicate that ULK1 knockout-mediated mitophagy defect promotes breast cancer bone metastasis and provide evidence to explore MAP2K/MEK- MAPK1/3 pathway inhibitors for therapy, especially in cancers displaying low levels of ULK1.Abbreviations: ATG: autophagy-related; Baf A1: bafilomycin A1; BTRC/ß-TrCP: beta-transducin repeat containing E3 ubiquitin protein ligase; CHX: cycloheximide; CM: conditioned media; FBXW7/FBW7: F-box and WD repeat domain containing 7; MAPK1: mitogen-activated protein kinase 1; MTDR: MitoTracker Deep Red; mtROS: mitochondrial reactive oxygen species; microCT: micro-computed tomography; mtROS: mitochondrial reactive oxygen species; OCR: oxygen consumption rate; SQSTM1: sequestosome 1; ACP5/TRAP: acid phosphatase, tartrate resistant; ULK1: unc-51 like autophagy activating kinase 1.

Notch 1 Is Involved in CD4+ T Cell Differentiation Into Th1 Subtype During Helicobacter pylori Infection.

Helicobacter pylori infection induces CD4+ T differentiation cells into IFN-γ-producing Th1 cells. However, the details of mechanism underlying this process remain unclear. Notch signal pathway has been reported to regulate the differentiation of CD4+ T cells into Th1 subtype in many Th1-mediated inflammatory disorders but not yet in H. pylori infection. In the present study, the mRNA expression pattern of CD4+ T cells in H. pylori-infected patients differed from that of healthy control using Human Signal Transduction Pathway Finder RT2 Profiler PCR Array, and this alteration was associated with Notch signal pathway, as analyzed by Bioinformation. Quantitative real-time PCR showed that the mRNA expression of Notch1 and its target gene Hes-1 in CD4+ T cells of H. pylori-infected individuals increased compared with the healthy controls. In addition, the mRNA expression of Th1 master transcription factor T-bet and Th1 signature cytokine IFN-γ was both upregulated in H. pylori-infected individuals and positively correlated with Notch1 expression. The increased protein level of Notch1 and IFN-γ were also observed in H. pylori-infected individuals confirmed by flow cytometry and ELISA. In vitro, inhibition of Notch signaling decreased the mRNA expression of Notch1, Hes-1, T-bet, and IFN-γ, and reduced the protein levels of Notch1 and IFN-γ and the secretion of IFN-γ in CD4+ T cells stimulated by H. pylori. Collectively, this is the first evidence that Notch1 is upregulated and involved in the differentiation of Th1 cells during H. pylori infection, which will facilitate exploiting Notch1 as a therapeutic target for the control of H. pylori infection.

Features of IgG4-related lung disease on 18F-FDG PET/computed tomography imaging.

OBJECTIVES: The aim of the study was to summarize the features of immunoglobulin G4-related lung disease (IgG4-RLD) on fluorine 18-fluorodeoxyglucose (F-FDG) PET/computed tomography (CT). METHODS: In this retrospective case series, 12 consecutive patients (9 men and 3 women, mean age 55.4 ± 13.7 years) with IgG4-RLD were included. The clinicopathological information and features of F-FDG PET/CT imaging were analyzed. RESULTS: Six (50%) patients had pulmonary involvement alone and six (50%) patients had extrapulmonary involvement with intense F-FDG uptake. Pulmonary manifestations included mass (25%, 3/12), solid nodule (solitary 25%, 3/12; multiple 50%, 6/12), multiple ground-glass opacities (GGOs) (50%, 6/12), thickening of alveolar interstitium (50%, 6/12), and thickening of bronchovascular bundle (33.3%, 4/12). The maximum standardized uptake value (SUVmax) of the solid nodules and masses, multiple GGOs, bronchovascular bundle and the thickening of septa was 4.0 ± 2.5, 2.3 ± 1.8, 1.4 ± 0.6, and 0.9 ± 0.5, respectively. The SUVmax statistically significant linear association with the diameter of masses or solid nodules (P value = 0.03), but no significant inverse linear association (P value = 0.06) with the concentration of serum IgG4 concentration. CONCLUSIONS: The image patterns of IgG4-RLD on F-FDG PET/CT are varying. Multiple pulmonary manifestations or multiple organ involvement, especially in combination with elevated levels of serum IgG and IgG4, may help to make the diagnosis. A potential major application of PET-CT would be evaluation of response to treatment, and the impact of PET/CT on IgG4-RLD management is worth investigating further in the future.

Disruption of super-enhancer-driven tumor suppressor gene RCAN1.4 expression promotes the malignancy of breast carcinoma.

BACKGROUND: Super-enhancers (SEs) play a crucial role in cancer, which is often associate with activated oncogenes. However, little is known about how SEs facilitate tumour suppression. Individuals with Down syndrome exhibit a remarkably reduced incidence of breast cancer (BC), moving the search for tumor suppressor genes on human chromosome 21 (HSA21). In this study, we aim to identify and explore potential mechanisms by which SEs are established for tumor suppressor RCAN1.4 on HSA21 in BC. METHODS: In silico analysis and immunohistochemical staining were used to assess the expression and clinical relevance of RCAN1.4 and RUNX3 in BC. Function experiments were performed to evaluate the effects of RCAN1.4 on the malignancy of breast carcinoma in vitro and in vivo. ChIP-seq data analysis, ChIP-qPCR, double-CRISPR genome editing, and luciferase reporter assay were utilized to confirm RUNX3 was involved in regulating RCAN1.4-associated SE in BC. The clinical value of co-expression of RCAN1.4 and RUNX3 was evaluated in BC patients. RESULTS: Here, we characterized RCAN1.4 as a potential tumour suppressor in BC. RCAN1.4 loss promoted tumour metastasis to bone and brain, and its overexpression inhibited tumour growth by blocking the calcineurin-NFATc1 pathway. Unexpectedly, we found RCAN1.4 expression was driven by a ~ 23 kb-long SE. RCAN1.4-SEdistal was sensitive to BRD4 inhibition, and its deletion decreased RCAN1.4 expression by over 90% and induced the malignant phenotype of BC cells. We also discovered that the binding sites in the SE region of RCAN1.4 were enriched for consensus sequences of transcription factor RUNX3. Knockdown of RUNX3 repressed the luciferase activity and also decreased H3K27ac enrichment binding at the SE region of RCAN1.4. Furthermore, abnormal SE-driven RCAN1.4 expression mediated by RUNX3 loss could be physiologically significant and clinically relevant in BC patients. Notably, we established a prognostic model based on RCAN1.4 and RUNX3 co-expression that effectively predicted the overall survival in BC patients. CONCLUSIONS: These findings reveal an important role of SEs in facilitating tumour suppression in BC. Considering that the combination of low RCAN1.4 and low RUNX3 expression has worse prognosis, RUNX3-RCAN1.4 axis maybe a novel prognostic biomarker and therapeutic target for BC patients.

Synthesis and evaluation of 18F labeled crizotinib derivative [18F]FPC as a novel PET probe for imaging c-MET-positive NSCLC tumor.

c-MET-positive NSCLC is an important subtype accounting for about 5%~22% of lung cancer. NSCLC patients with activating c-MET are intensively sensitive to c-MET selective receptor tyrosine kinase (RTK) inhibitors, so we aimed to develop a specific PET probe targeting to c-MET-positive NSCLC for potential patients screened by PET/CT. Herein, PET tracer 18F-radiolabeled crizotinib derivative ([18F]FPC) was successfully achieved through a simple one-step 18F-labeling method. [18F]FPC PET imaging on c-MET-positive (as well as blocking group) and negative NSCLC models were further evaluated, and results showed that [18F]FPC was effective as a PET imaging probe that targeted c-MET-positive tumor. Therefore, [18F]FPC could be a potential PET imaging probe for NSCLC tumor which was sensitive to c-MET-TKIs. By virtue of this property, it will benefit NSCLC patients for c-MET-TKI treatment.

18F-FDG maximum standard uptake value predicts PD-L1 expression on tumor cells or tumor-infiltrating immune cells in non-small cell lung cancer.

OBJECTIVES: Programmed cell death-ligand 1 (PD-L1) is expressed on tumor cells (TC) and tumor-infiltrating immune cells (IC). We conducted a retrospective study to investigate the relationship between PD-L1 expression on TC/IC and 18F-FDG uptake in patients with surgically resected non-small cell lung cancer (NSCLC). METHODS: Total 362 NSCLC patients (297 adenocarcinoma and 65 squamous cell carcinoma) who underwent preoperative 18F-FDG-PET/CT imaging were analyzed retrospectively. Immunohistochemistry analysis was performed for PD-L1 expression on TC and IC in NSCLC specimens with 28-8 antibody. The cut-off value of 5% for defining PD-L1 positivity was determined according to previous trials. The association between PD-L1 expression and clinicopathological variables were analyzed, including age, gender, smoking status, tumor diameter, lymph node metastasis, stage and the maximum standardized uptake value (SUVmax). RESULTS: PD-L1 positive expression was 50.8% (184/362) in NSCLC patients. Its positive expression on TC and IC were 24.3% (88/362) and 42.5% (154/362), respectively. SUVmax was significantly higher in patients with PD-L1 positive expression on TC or IC than that with negative. Multivariate analysis demonstrated that PD-L1 expression were correlated with SUVmax. The best cut-off value of SUVmax for PD-L1 expression on TC/IC was 8.5 [area under the curve (AUC) = 0.607, 95% CI 0.549-0.665, P = 0.001, sensitivity 50.5% and specificity 71.4%] determined by ROC curve. CONCLUSION: High SUVmax is linked to PD-L1 expression on TC and IC in our patients with surgically resected non-small cell lung cancer. 18F-FDG-PET/CT imaging may be used to predict the PD-L1 expression on TC and IC in NSCLC patients.

Correlation of PD-L1 expression on tumor cell and tumor infiltrating immune cell with 18F-fluorodeoxyglucose uptake on PET/computed tomography in surgically resected pulmonary adenocarcinoma.

OBJECTIVE: The uptake of F-fluorodeoxyglucose (F-FDG) PET/computed tomography (CT) is known to be linked to programmed death ligand 1 (PD-L1) expression on tumor cells (TC). However, the association between PD-L1 expression on immune cells (IC) and F-FDG accumulation is still unclear. Here, we conducted a clinicopathological study to investigate the relationship between PD-L1 expression on TC/IC and F-FDG uptake in patients with surgically resected pulmonary adenocarcinoma (ADC). METHODS: A total of 450 ADC patients who underwent preoperative F-FDG-PET/CT imaging were analyzed retrospectively. Immunohistochemistry analysis was performed for PD-L1 expression on TC and IC in ADC specimens with SP142. PD-L1 expression was performed on whole-tissue sections and given scores (0/1/2/3) according to percent of PD-L1 cells in TC and IC. RESULTS: Compared to TC0 and IC0, PD-L1 positive expression was 90.4% (407/450) in ADC specimens. Both PD-L1 expression score on TC and IC were associated with maximum standardized uptake (SUVmax). SUVmax augmented with increasing PD-L1 expression (TC0 and IC0, 4.3 ± 3.4; TC or IC1/2/3, 7.7 ± 5.6; TC or IC2/3, 8.1 ± 5.6; TC or IC3, 8.4 ± 5.4). The best cut-off value of PD-L1 expression, determined by receiver operating characteristic curve, was 5.1 for TC or IC1/2/3 [area under the curve (AUC) = 0.713, sensitivity 62.2%, specificity 72.1%]. Multivariate analysis demonstrated that TC or IC1/2/3 subset was correlated with histological subtype, PD-1 expression on IC and SUVmax. CONCLUSION: High SUVmax is associated with PD-L1 expression on TC and IC in surgically resected pulmonary ADC. F-FDG-PET/CT imaging can be a potential tool to evaluate PD-L1 expression in pulmonary ADC.

Group B Streptococcus DNA Copy Numbers Measured by Digital PCR Correlates with Perinatal Outcomes.

Group B Streptococcus (GBS) is a one of the main causes of perinatal disease, yet the method for GBS detection, broth-enriched culture, is time-consuming and has low sensitivity and accuracy. We aimed to develop a GBS digital PCR (GBS-dPCR) assay for detecting GBS colonization. More rapid and accurate detection of GBS colonization could increase GBS diagnosis and treatment closer to delivery. A single-center, retrospective, case-controlled study was performed. A total of 182 rectovaginal swabs from pregnant women, who were undergoing prenatal screening by broth-enriched culture, were evaluated using GBS-dPCR targeting the cfb gene of GBS. Pregnant women with GBS colonization were followed up for correlation analysis between GBS DNA copy numbers and perinatal outcomes. The results of the GBS-dPCR assay were compared to those from the broth-enriched culture, which is the gold standard for GBS detection. The sensitivity and specificity of GBS-dPCR were 98% and 92.5%, respectively. By discrepant result analysis, the specificity of GBS-dPCR was raised to 97.4%. The incidence of premature rupture of membrane (PROM) and neonatal infection were statistically significantly positively correlated with GBS DNA copy numbers. GBS-dPCR has the advantage of directly detecting GBS colonization from swabs with high specificity and sensitivity, while reducing turnaround time (<4 h). Analysis of clinical samples with GBS-dPCR shows that GBS DNA copy numbers are positively correlated with the incidence of PROM and neonatal infection, suggesting that dPCR is a promising method for detection of GBS colonization during pregnancy.

P2X7 PET Radioligand 18F-PTTP for Differentiation of Lung Tumor from Inflammation.

Site-specific imaging agents play a key role in tumor targeting, but only a few agents are currently available for inflammation targeting. Since the P2X7 receptor (P2X7R) is a promising molecular target for inflammation, we evaluated the potential value of the 18F-labeled tracer 18F-PTTP (5-{[2-Chloro-3-(trifluoromethyl)phenyl]carbonyl}-1-pyrimidin-2-yl-4,5,6,7-tetrahydro-1H-[1,2,3]triazolo[4,5-c]pyridin) for targeting P2X7Rs and thus differentiating inflammation from tumors. Methods: The radioligand 18F-PTTP was achieved by a 1-step 18F-trifluoromethylation reaction. The binding affinity of the ligand for P2X7R and its stability were evaluated in vitro. Blood pharmacokinetics tests and biodistribution studies were performed in vivo. Dynamic 18F-PTTP small-animal PET/CT imaging was performed for 60 min on A549 tumor-bearing mice and inflammation-model mice for targeting differentiation. Results:18F-PTTP was afforded with decay-corrected radiochemical yields of 2.5%-7.0%, specific activity of 296-370 MBq/µmol, and radiochemical purity over 95%. 18F-PTTP showed excellent stability in 0.9% NaCl and 0.1% bovine serum albumin, good affinity to RAW264.7 cells, and rapid blood clearance in mice. In inflammation-model mice, uptake of 18F-PTTP peaked at 5 min after injection and kept at an imageable level till 30 min, whereas no significant radioactivity uptake was found in tumor grafts till 1 h after injection. The specificity of 18F-PTTP was verified by blocking studies and histologic analysis. Conclusion: The current study provides compelling data that 18F-PTTP is a novel radioligand targeting P2X7R and has potential to screen new drugs, quantify peripheral inflammation, and distinguish inflammation from certain solid tumors.

Identification of Two Lpp20 CD4+ T Cell Epitopes in Helicobacter pylori-Infected Subjects.

Antigen-specific CD4+ T cells play an essential role in effective immunity against Helicobacter pylori (H. pylori) infection. Lpp20, a conserved lipoprotein of H. pylori, has been investigated as one of major protective antigens for vaccination strategies. Our previous study identified two H-2d-restricted CD4+ T cell epitopes within Lpp20 and an epitope vaccine based on these epitopes was constructed, which protected mice in prophylactic and therapeutic vaccination against H. pylori infection. Immunodominant CD4+ T cell response is an important feature of antiviral, antibacterial, and antitumor cellular immunity. However, while many immunodominant HLA-restricted CD4+ T cell epitopes of H. pylori protective antigens have been identified, immunodominant HLA-restricted Lpp20 CD4+ T cell epitope has not been elucidated. In this study, a systematic method was used to comprehensively evaluate the immunodominant Lpp20-specific CD4+ T cell response in H. pylori-infected patients. Using in vitro recombinant Lpp20 (rLpp20)-specific expanded T cell lines from H. pylori-infected subjects and 27 18mer overlapping synthetic peptides spanned the whole Lpp20 protein, we have shown that L55-72 and L79-96 harbored dominant epitopes for CD4+ T cell responses. Then the core sequence within these two 18mer dominant epitopes was screened by various extended or truncated 13mer peptides. The immunodominant epitope was mapped to L57-69 and L83-95. Various Epstein-Barr virus (EBV) transformed B lymphoblastoid cell lines (B-LCLs) with different HLA alleles were used as antigen presenting cell (APC) to present peptides to CD4+ T cells. The restriction molecules were determined by HLA class-antibody blocking. L57-69 was restricted by DRB1-1501 and L83-95 by DRB1-1602. The epitopes were recognized on autologous dendritic cells (DCs) loaded with rLpp20 but also those pulsed with whole cell lysates of H. pylori (HP-WCL), suggesting that these epitopes are naturally processed and presented by APC. CD4+ T cells were isolated from H. pylori-infected patients and stimulated with L57-69 and L83-95. These two epitopes were able to stimulate CD4+ T cell proliferation. This study may be of value for the future development of potential H. pylori vaccine.

[Over-expression of human Notch ligand Delta-like 3 promotes proliferation of human gastric cancer cells in vitro].

OBJECTIVE: To construct a eukaryotic expression plasmid carrying human full-length Notch ligand Delta-like 3 (DLL3) gene and study the effect of DLL3 knockdown and overexpression on the proliferation of gastric cancer cells in vitro. METHODS: Human full-length DLL3 gene was amplified by PCR and cloned into the eukaryotic expression vector pCMV-Tag4. After verification by restriction enzymes and sequencing, the recombinant DLL3/pCMV-Tag4 vector was transiently transfected into HEK293T cells, in which the expressions of human DLL3 mRNA and protein were detected using real-time quantitative PCR and Western blotting, respectively. The expression of DLL3 in normal gastric epithelial cells and gastric cancer cell lines was detected by qRT-PCR and Western blotting. DLL3/pCMV-Tag4 was transfected into 3 gastric cancer cell lines, and their proliferation was assessed with MTT assay. Human gastric cancer cells MGC803 and MKN45 were also transfected with a specific human DLL3-siRNA to assess the effect of DLL3 down-expression on the cell proliferation. RESULTS: The recombinant eukaryotic expression vector DLL3/pCMV-Tag4 was successfully constructed and human full-length DLL3 was expressed in HEK293T cells. MTT assay showed that DLL3 over-expression obviously promoted the proliferation and down-regulation of DLL3 inhibited the proliferation of the gastric cancer cells. CONCLUSIONS: DLL3 overexpression can promote the proliferation of gastric cancer cells in vitro, and down-regulation of DLL3 inhibits the proliferation of gastrc cancer cells, which provides a novel strategy for targeted thrapy of gastric cancer.