Triggering receptor expressed on myeloid cells (TREM) like transcript-1 (TLT-1) reveals platelet activation in preeclampsia.

Triggering receptor expressed on myeloid cells (TREM) like transcript-1 (TLT-1) is a membrane protein receptor found in α-granules of megakaryocytes and platelets. Upon platelet activation TLT-1 is rapidly relocated to the surface of platelets. In plasma, a soluble form of TLT-1 (sTLT-1) is present. Plasma levels of sTLT-1 are significantly elevated in thrombotic diseases. In the present study, we investigated to whether TLT-1 reflects platelet activation in pregnant women with preeclampsia. We studied 30 preeclamptic patients who were matched with 30 normotensive pregnant women and 30 non-pregnant controls. Basal TLT-1, P-selectin, and CD63 expressions on platelets were analyzed with the use of flow-cytometry (FCM). Platelet reactivity was induced by thrombin receptor activation peptide and determined by FCM. Plasma concentrations of sTLT-1 and soluble P-selectin (sP-selectin) were measured by an enzyme-linked immunosorbent assay. Results show that basal platelet expression of TLT-1, P-selectin and CD63 were increased in women with preeclampsia (PE) compared with normotensive pregnant women (NP). Platelets from PE women and NP women were more responsive compared to from nonpregnant women controls (NC), and which was demonstrated by increased expression of TLT-1, P-selectin, and CD63 upon stimulation in vitro. Plasma concentration of sTLT-1 was greater in PE women compared to NP women and NC women. Plasma sP-selectin level was higher in pregnant women than in nonpregnant women, but there were no significant differences between PE and NP women. In summary, our results revealed that platelet activation is prominent in preeclampsia, TLT-1 reflects platelet activation and may be a useful indicator for preeclampsia.

Impact of multiple negative charges on blood clearance and biodistribution characteristics of 99mTc-labeled dimeric cyclic RGD peptides.

This study sought to evaluate the impact of multiple negative charges on blood clearance kinetics and biodistribution properties of (99m)Tc-labeled RGD peptide dimers. Bioconjugates HYNIC-P6G-RGD2 and HYNIC-P6D-RGD2 were prepared by reacting P6G-RGD2 and P6D-RGD2, respectively, with excess HYNIC-OSu in the presence of diisopropylethylamine. Their IC50 values were determined to be 31 ± 5 and 41 ± 6 nM, respectively, against (125)I-echistatin bound to U87MG glioma cells in a whole-cell displacement assay. Complexes [(99m)Tc(HYNIC-P6G-RGD2)(tricine)(TPPTS)] ((99m)Tc-P6G-RGD2) and [(99m)Tc(HYNIC-P6D-RGD2)(tricine)(TPPTS)] ((99m)Tc-P6D-RGD2) were prepared in high radiochemical purity (RCP > 95%) and specific activity (37-110 GBq/µmol). They were evaluated in athymic nude mice bearing U87MG glioma xenografts for their biodistribution. The most significant difference between (99m)Tc-P6D-RGD2 and (99m)Tc-P6G-RGD2 was their blood radioactivity levels and tumor uptake. The initial blood radioactivity level for (99m)Tc-P6D-RGD2 (4.71 ± 1.00%ID/g) was ∼5× higher than that of (99m)Tc-P6G-RGD2 (0.88 ± 0.05%ID/g), but this difference disappeared at 60 min p.i. (99m)Tc-P6D-RGD2 had much lower tumor uptake (2.20-3.11%ID/g) than (99m)Tc-P6G-RGD2 (7.82-9.27%ID/g) over a 2 h period. Since HYNIC-P6D-RGD2 and HYNIC-P6G-RGD2 shared a similar integrin αvß3 binding affinity (41 ± 6 nM versus 31 ± 5 nM), the difference in their blood activity and tumor uptake is most likely related to the nine negative charges and high protein binding of (99m)Tc-P6D-RGD2. Despite its low uptake in U87MG tumors, the tumor uptake of (99m)Tc-P6D-RGD2 was integrin αvß3-specific. SPECT/CT studies were performed using (99m)Tc-P6G-RGD2 in athymic nude mice bearing U87MG glioma and MDA-MB-231 breast cancer xenografts. The SPECT/CT data demonstrated the tumor-targeting capability of (99m)Tc-P6G-RGD2, and its tumor uptake depends on the integrin αvß3 expression levels on tumor cells and neovasculature. It was concluded that the multiple negative charges have a significant impact on the blood clearance kinetics and tumor uptake of (99m)Tc-labeled dimeric cyclic RGD peptides.

FITC-conjugated cyclic RGD peptides as fluorescent probes for staining integrin αvß3/αvß5 in tumor tissues.

This study sought to evaluate FITC-conjugated cyclic RGD peptides (FITC-RGD2, FITC-3P-RGD2, and FITC-Galacto-RGD2) as fluorescent probes for in vitro assays of integrin αvß3/αvß5 expression in tumor tissues. FITC-RGD2, FITC-3P-RGD2, and FITC-Galacto-RGD2 were prepared, and their integrin αvß3/αvß5 binding affinity was determined using the displacement assay against (125)I-echistatin bound to U87MG glioma cells. IC50 values of FITC-Galacto-RGD2, FITC-3P-RGD2, and FITC-RGD2 were calculated to be 28 ± 8, 32 ± 7, and 89 ± 17 nM, respectively. The integrin αvß3/αvß5 binding affinity followed a general trend: FITC-Galacto-RGD2 ∼ FITC-3P-RGD2 > FITC-RGD2. The xenografted tumor-bearing models were established by subcutaneous injection of 5 × 10(6) tumor cells into shoulder flank (U87MG, A549, HT29, and PC-3) or mammary fat pad (MDA-MB-435) of each athymic nude mouse. Three to six weeks after inoculation, the tumor size was 0.1-0.3 g. Tumors were harvested for integrin αvß3/αvß5 staining, as well as hematoxylin and eosin (H&E) staining. Six human carcinoma tissues (colon cancer, pancreatic cancer, lung adenocarcinoma, squamous cell lung cancer, gastric cancer, and esophageal cancer) were obtained from recently diagnosed cancer patients. Human carcinoma slides were deparaffinized in xylene, rehydrated with ethanol, and then used for integrin αvß3/αvß5 staining, as well as H&E staining. It was found that the tumor staining procedures with FITC-conjugated cyclic RGD peptides were much simpler than those with the fluorescence-labeled integrin αvß3 antibodies. Since FITC-RGD2, FITC-3P-RGD2, and FITC-Galacto-RGD2 were able to co-localize with the fluorescence-labeled integrin ß3 antibody, their tumor localization and tumor cell binding are integrin αvß3-specific. Quantification of the fluorescent intensity in five xenografted tumors (U87MG, MDA-MB-435, A549, HT29, and PC-3) and six human carcinoma tissues revealed an excellent linear relationship between the relative integrin αvß3/αvß5 expression levels determined with FITC-Galacto-RGD2 and those obtained with the fluorescence-labeled anti-human integrin ß3 antibody. There was also an excellent linear relationship between the tumor uptake (%ID/g) of (99m)Tc-3P-RGD2 (an integrin αvß3/αvß5-targeted radiotracer) and the relative integrin αvß3/αvß5 expression levels from the quantification of fluorescent intensity in the tumor tissues stained with FITC-Galacto-RGD2. These results suggest that FITC-conjugated cyclic RGD peptides might be useful to correlate the in vitro findings with the in vivo imaging data from an integrin αvß3/αvß5-targeted radiotracer. The results from this study clearly showed that the FITC-conjugated cyclic RGD peptides (particularly FITC-3P-RGD2 and FITC-Galacto-RGD2) are useful fluorescent probes for assaying relative integrin αvß3/αvß5 expression levels in tumor tissues.

Development of kit formulations for (99m) TcN-MPO: a cationic radiotracer for myocardial perfusion imaging.

The objective of this study was to develop a kit formulation for [(99m) TcN(mpo)(PNP5)](+) (MPO = 2-mercaptopyridine oxide), ((99m) TcN-MPO) to support its clinical evaluations as a SPECT radiotracer. Radiolabeling studies were performed using three different formulations (two-vial formulation and single-vial formulations with/without SnCl2 ) to explore the factors influencing radiochemical purity (RCP) of (99m) TcN-MPO. We found that the most important factor affecting the RCP of (99m) TcN-MPO was the purity of PNP5. (99m) TcN-MPO was prepared >98% RCP (n = 20) using the two-vial formulation. For single-vial formulations with/without SnCl2 , ß-cyclodextrin (ß-CD) is particularly useful as a stabilizer for PNP5. The RCP of (99m) TcN-MPO was 95-98% using ß-CD, but its RCP was only 90-93% with γ-cyclodextrin (γ-CD). It seems that PNP5 fits better into the inner cavity of ß-CD, which forms more stable inclusion complex than γ-CD in the single-vial formulations. The results from biodistribution and imaging studies in Sprague-Dawley rats clearly demonstrated biological equivalence of three different formulations. Single photon-emission computed tomography data suggested that high quality images could be obtained at 0-30-min post-injection without significant interference from the liver radioactivity. Considering the ease for (99m) Tc-labeling and high RCP of (99m) TcN-MPO, the non-SnCl2 single-vial formulation is an attractive choice for future clinical studies.

Monitoring tumor response to linifanib therapy with SPECT/CT using the integrin αvß3-targeted radiotracer 99mTc-3P-RGD2.

The objective of this study was to determine the utility of (99m)Tc-3P-Arg-Gly-Asp (RGD2) single photon emission computed tomography (SPECT)/computed tomography (CT) for noninvasive monitoring of integrin αvß3-expression response to antiangiogenic treatment with linifanib. Linifanib or vehicle therapy was carried out in female athymic nu/nu mice bearing U87MG glioma (high αvß3 expression) or PC-3 prostate (low αvß3 expression) tumors at 12.5 mg/kg twice daily. The average tumor volume was 180 ± 90 mm(3) the day prior to baseline SPECT/CT. Longitudinal (99m)Tc-3P-RGD2 SPECT/CT imaging was performed at baseline (-1 day) and days 1, 4, 11, and 18. Tumors were harvested at all imaging time points for histopathological analysis with H&E and immunohistochemistry. A significant difference in tumor volumes between vehicle- and linifanib-treated groups was observed after 4 days of linifanib therapy in the U87MG model. The percent injected dose (%ID) tumor uptake of (99m)Tc-3P-RGD2 peaked in the vehicle-treated group at day 11, while the %ID/cm(3) tumor uptake decreased slowly over the whole study period. During the first 2 days of linifanib treatment, a rapid decrease in both %ID/cm(3) tumor uptake and tumor/muscle ratios of (99m)Tc-3P-RGD2 was observed, followed by a slow decrease until day 18. No decrease in tumor uptake of (99m)Tc-3P-RGD2 or tumor volume was observed for either treatment group in the PC-3 model. Changes in tumor vasculature were confirmed by histopathological H&E analysis and immunohistochemistry. Longitudinal imaging using (99m)Tc-3P-RGD2 SPECT/CT may be a useful tool for monitoring the downstream biologic effects of linifanib therapy.

Evaluation of K(HYNIC)(2) as a bifunctional chelator for (99m)Tc-labeling of small biomolecules.

This study sought to evaluate K(HYNIC)(2) (K = lysine and HYNIC = 6-hydrazinonicotinyl) as a bifunctional chelator for (99m)Tc-labeling of biomolecule. In this study, four K(HYNIC)(2)-conjugated cyclic RGD peptides, K(HYNIC)(2)-RGD(2) (RGD(2) = E[c(RGDfK)](2)), K(HYNIC)(2)-3G-RGD(2) (3G-RGD(2) = Gly-Gly-Gly-E[Gly-Gly-Gly-c(RGDfK)](2)), K(HYNIC)(2)-2P-RGD(2) (2P-RGD(2) = E[PEG4-c(RGDfK)](2), and PEG(4) = 15-amino-4,7,10,13-tetraoxapentadecanoic acid), and K(HYNIC)(2)-3P-RGD(2) (3P-RGD(2) = PEG4-E[PEG4-c(RGDfK)]2) were prepared, and evaluated for their integrin αvß3 binding affinity. IC(50) values were determined to be 47 ± 2, 35 ± 2, 37 ± 2, 85 ± 2, and 422 ± 15 nM for K(HYNIC)(2)-2P-RGD(2), K(HYNIC)(2)-3P-RGD(2), K(HYNIC)(2)-3G-RGD(2), K(HYNIC)(2)-RGD(2), and c(RGDyK), respectively, against (125)I-echistatin bound to U87MG cells. Macrocyclic complexes [(99m)Tc(K(HYNIC)(2)-RGD(2))(tricine)] (1), [(99m)Tc(K(HYNIC)(2)-3G-RGD(2))(tricine)] (2), [(99m)Tc(K(HYNIC)(2)-2P-RGD(2))(tricine)] (3), and [(99m)Tc(K(HYNIC)(2)-3P-RGD(2))(tricine)] (4) were prepared, and evaluated in athymic nude mice bearing U87MG glioma xenografts for their tumor targeting capability and biodistribution. It was found that 1-4 all had high solution stability and more than two isomers, as evidenced by the presence of multiple radiometric peaks in their radio-HPLC chromatograms. The tumor uptake of 1-4 was 3.78 ± 0.81, 7.46 ± 1.68, 9.74 ± 1.65, and 8.59 ± 1.52%ID/g, respectively, which was completely consistent with trend of integrin α(v)ß(3) binding affinity for cyclic RGD peptides. Replacing [(99m)Tc(HYNIC)(tricine)(TPPTS)] (TPPTS = trisodium triphenylphosphine-3,3',3"-trisulfonate) with [(99m)Tc(K(HYNIC)(2))(tricine)] had little impact on radiotracer tumor uptake; but it had significant effect on the uptake of radiotracer in kidneys, lungs, and spleen. The tumor was clearly visualized by SPECT/CT with excellent contrast in a glioma-bearing mouse administered with 4. K(HYNIC)(2) would be particularly useful for (99m)Tc-labeling of small biomolecules with one or more disulfide linkages.

(99m)Tc-Galacto-RGD2: a novel 99mTc-labeled cyclic RGD peptide dimer useful for tumor imaging.

This study sought to evaluate [(99m)Tc(HYNIC-Galacto-RGD2)(tricine)(TPPTS)] ((99m)Tc-Galacto-RGD2: HYNIC = 6-hydrazinonicotinyl; Galacto-RGD2 = Glu[cyclo[Arg-Gly-Asp-D-Phe-Lys(SAA-PEG2-(1,2,3-triazole)-1-yl-4-methylamide)]]2 (SAA = 7-amino-L-glycero-L-galacto-2,6-anhydro-7-deoxyheptanamide, and PEG2 = 3,6-dioxaoctanoic acid); and TPPTS = trisodium triphenylphosphine-3,3',3″-trisulfonate) as a new radiotracer for tumor imaging. Galacto-RGD2 was prepared via the copper(I)-catalyzed 1,3-dipolar azide-alkyne Huisgen cycloaddition. HYNIC-Galacto-RGD2 was prepared by reacting Galacto-RGD2 with sodium succinimidyl 6-(2-(2-sulfonatobenzaldehyde)hydrazono)nicotinate (HYNIC-OSu) in the presence of diisopropylethylamine, and was evaluated for its integrin αvß3 binding affinity against (125)I-echistatin bound to U87MG glioma cells. The IC50 value for HYNIC-Galacto-RGD2 was determined to be 20 ± 2 nM. (99m)Tc-Galacto-RGD2 was prepared in high specific activity (∼ 185 GBq/µmol) and high radiochemical purity (>95%), and was evaluated in athymic nude mice bearing U87MG glioma xenografts for its tumor-targeting capability and biodistribution. The tumor uptake of (99m)Tc-Galacto-RGD2 was 10.30 ± 1.67, 8.37 ± 2.13, 6.86 ± 1.33, and 5.61 ± 1.52%ID/g at 5, 30, 60, and 120 min p.i., respectively, which was in agreement with high integrin αvß3 expression on glioma cells and neovasculature. Its lower uptake in intestines, lungs, and spleen suggests that (99m)Tc-Galacto-RGD2 has advantages over (99m)Tc-3P-RGD2 ([(99m)Tc(HYNIC-3P-RGD2)(tricine)(TPPTS)]: 3P-RGD2 = PEG4-E[PEG4-c(RGDfK)]2; PEG4 = 15-amino-4,7,10,13-tetraoxapentadecanoic acid) for imaging tumors in the chest and abdominal regions. U87MG tumors were readily detected by SPECT and the tumor uptake of (99m)Tc-Galacto-RGD2 was integrin αvß3-specific. (99m)Tc-Galacto-RGD2 also had very high metabolic stability. On the basis of results from this study, it was concluded that (99m)Tc-Galacto-RGD2 is an excellent radiotracer for imaging integrin αvß3-positive tumors and related metastases.

Forkhead box L2 is a target of miR-133b and plays an important role in the pathogenesis of non-small cell lung cancer.

BACKGROUND: Forkhead box L2 (FOXL2) has been recognized as a transcription factor in the progression of many malignancies, but its role in non-small cell lung cancer (NSCLC) remains unclear. This research clarified on the role of FOXL2 and the specific molecular mechanism in NSCLC. METHODS: RNA and protein levels were detected by quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting assays. Cell proliferation was examined by cell counting kit-8 (CCK-8) and clonogenic assays. Transwell and wound healing assays were used to detect cell invasion and migration. Cell cycle alterations were assessed by flow cytometry. The relationship between FOXL2 and miR-133b was verified by dual-luciferase reporter assays. In vivo metastasis was monitored in the tail vein-injected mice. RESULTS: FOXL2 was upregulated in NSCLC cells and tissues. Downregulation of FOXL2 restrained cell proliferation, migration, and invasion and arrested the cell cycle of NSCLC cells. Moreover, FOXL2 promoted the epithelial-mesenchymal transition (EMT) process of NSCLC cells by inducing the transforming growth factor-ß (TGF-ß)/Smad signaling pathway. miR-133b directly targeted the 3'-UTR of FOXL2 and negatively regulated FOXL2 expression. Knockdown of FOXL2 blocked metastasis in vivo. CONCLUSIONS: miR-133b downregulates FOXL2 by targeting the 3'-UTR of FOXL2, thereby inhibiting cell proliferation, EMT and metastasis induced by the TGF-ß/Smad signaling pathway in NSCLC. FOXL2 may be a potential molecular target for treating NSCLC.

Effects of concurrent TP53 mutations on the efficacy and prognosis of targeted therapy for advanced EGFR mutant lung adenocarcinoma.

BACKGROUND: How concurrent TP53 mutations affect targeted therapy of advanced Epidermal Growth Factor Receptor (EGFR) mutant lung adenocarcinoma remains controversial, particularly the deep classification of TP53 mutations. METHODS: This study retrospectively analyzed the clinical data of advanced EGFR mutant lung adenocarcinoma patients treated with EGFR-tyrosine kinase inhibitors (TKIs) in the First Affiliated Hospital of Soochow University. The survival rates were compared using Log-rank tests. Potential prognostic factors were identified using multivariate Cox hazard regression models. RESULTS: Total 156 advanced lung adenocarcinoma patients treated with EGFR-TKIs were included in this study. Multivariate analysis showed that male [hazard rate (HR): 1.537, 95% confidence interval (CI): 1.055-2.240, P = 0.025], brain metastasis (HR: 1.707, 95%CI: 1.086-2.682, P = 0.020) and concurrent TP53 mutations (HR: 1.569, 95%CI: 1.051-2.341, P = 0.028) were independent negative predictors of progression-free survival (PFS). EGFR L858R mutations (HR: 2.475, 95%CI: 1.443-4.248, p = 0.001), smoking history (HR: 2.530, 95%CI: 1.352-4.733, P = 0.004) and concurrent TP53 mutations (HR: 2.326, 95%CI: 1.283-4.218, P = 0.005) were associated with worse survival. Further analysis revealed that mutations in TP53 exons 4, 5 and 8 (P<0.05), missense mutations (P = 0.006) and nondisruptive mutations (P<0.001) were associated with shorter PFS, whereas mutations in TP53 exons 5 and 7 (P<0.05), missense mutations and non-missense mutations (P = 0.006; P = 0.007), disruptive mutations and nondisruptive mutations (P = 0.013; P = 0.013) were all associated with poorer survival times. In addition, the PFS and overall survival (OS) of nondisruptive mutations in exon 7 were worse than those in other exons (P = 0.041; P<0.001). CONCLUSIONS: Concurrent TP53 mutations conferred worse EGFR-TKIs efficacy and prognosis in advanced EGFR mutant lung adenocarcinoma and the effects of different TP53 mutation types were heterogeneous.

Development of an ELISA method for testing VWF ristocetin cofactor activity with improved sensitivity and reliability in the diagnosis of von Willebrand disease.

OBJECTIVES: Current methods in assessing von Willebrand factor (VWF) ristocetin cofactor activity for Von Willebrand's disease (VWD) diagnosis include platelet agglutination by aggregometer or macroscopic slide examination, which are both time-consuming with suboptimal interassay and intra-assay variation. The purpose of this study is to establish a sensitive assay to detect VWF:RCo activity and evaluate its performance in VWD diagnosis. METHODS: We have established a sensitive VWF:RCo-ELISA method using a monoclonal antibody, SZ-151, to immobilize the recombinant fragment of platelet glycoprotein Ib (rfGPIbα). VWF was captured by rfGPIbα in the presence of ristocetin, and then detected by HRP-conjugated rabbit anti-human VWF IgG. We tested the VWF:RCo level by this VWF:RCo-ELISA in 25 patients with different types of VWD and 36 healthy donors, and compared this method to a previously reported ELISA using 2D4 coating antibody. RESULTS: The sensitivity of VWF:RCo-ELISA was greatly improved with this assay (0.008 IU/dL compared to 0.031 IU/dL by 2D4 antibody). The interassay and intra-assay coefficient variation were 8% and 12%, respectively. The mean values (ranges) of VWF:RCo in patients with type 1, type 2A, type 2B, type 2M, and type 3 of VWD and control group are 31.8 (22.3-56.9), 4.8 (0.6-11.8), 8.6 (1.6-19.7), 3.9 (1.0-6.8), 1.0 (0.5-1.6), and 91.5 (47.3-169.2) IU/dL, respectively. The corresponding ratios (ranges) of VWF:RCo/VWF:Ag are 0.83 (0.70-1.16), 0.27 (0.08-0.58), 0.31 (0.15-0.40), 0.18 (0.14-0.21), 0.52 (0.13-1.19), and 0.92 (0.62-1.26). CONCLUSION: The VWF:RCo-ELISA using monoclonal anti-rfGPIbα antibody SZ-151 showed improved sensitivity and reliability in detecting VWF:RCo activity, and its clinical application would facilitate the diagnosis and classification of VWD.

Detection of pulmonary embolism with 99mTc-labeled F(ab)2 fragment of anti-P-selectin monoclonal antibody in dogs.

Pulmonary embolism is a common and potentially life-threatening condition, and its correct diagnosis is highly desirable before anticoagulant therapy is initiated. However, the safe and accurate diagnosis of acute pulmonary embolism remains a challenge. Single photon emission computed tomography (SPECT) is a highly sensitive scintigraphic imaging technique. Pulmonary embolism can be detected by SPECT with (99m)Tc-labeled imaging agents that bind to components present predominantly on thromboemboli. P-selectin is an adhesion glycoprotein that is expressed in platelets and endothelial cells. P-selectin on activated platelets is a suitable biomarker of the active thrombus process. The objective of this study was to evaluate (99m)Tc-labeled F(ab)(2) fragment of anti-P-selectin monoclonal antibody SZ51, (99m)Tc-SZ51-F(ab)(2), for imaging pulmonary embolism in beagle canines. SZ51 was digested to F(ab)(2) fragment, named SZ51-F(ab)(2), and its specific binding to P-selectin on either human or canine platelets was verified by flow cytometry assay. In each dog, an 18-gauge catheter was inserted into left or right pulmonary artery, and a two-stranded spiral stainless-steel coil (20 mm) was inserted through catheter. At 30 min after coil placement, X-ray angiography was performed to document the pulmonary embolism and the locations of the coil. After intravenous injection of (99m)Tc-SZ51-F(ab)(2), experimental thrombi in dogs could be consistently visualized for 2-3 hours by SPECT. Pulmonary embolism showed higher uptake of (99m)Tc-SZ51-F(ab)(2). The present study suggests that (99m)Tc-SZ51-F(ab)(2) may be a promising agent for detecting pulmonary embolism.

Flagellar Hook Protein FlgE Induces Microvascular Hyperpermeability via Ectopic ATP Synthase ß on Endothelial Surface.

We previously demonstrated the immunostimulatory efficacy of Pseudomonas aeruginosa flagellar hook protein FlgE on epithelial cells, presumably via ectopic ATP synthases or subunits ATP5B on cell membranes. Here, by using recombinant wild-type FlgE, mutant FlgE (FlgEM; bearing mutations on two postulated critical epitopes B and F), and a FlgE analog in pull-down assay, Western blotting, flow cytometry, and ELISA, actual bindings of FlgE proteins or epitope B/F peptides with ATP5B were all confirmed. Upon treatment with FlgE proteins, human umbilical vein endothelial cells (HUVECs) and SV40-immortalized murine vascular endothelial cells manifested decreased proliferation, migration, tube formation, and surface ATP production and increased apoptosis. FlgE proteins increased the permeability of HUVEC monolayers to soluble large molecules like dextran as well as to neutrophils. Immunofluorescence showed that FlgE induced clustering and conjugation of F-actin in HUVECs. In Balb/c-nude mice bearing transplanted solid tumors, FlgE proteins induced a microvascular hyperpermeability in pinna, lungs, tumor mass, and abdominal cavity. All effects observed in FlgE proteins were partially or completely impaired in FlgEM proteins or blocked by pretreatment with anti-ATP5B antibodies. Upon coculture of bacteria with HUVECs, FlgE was detectable in the membrane and cytosol of HUVECs. It was concluded that FlgE posed a pathogenic ligand of ectopic ATP5B that, upon FlgE-ATP5B coupling on endothelial cells, modulated properties and increased permeability of endothelial layers both in vitro and in vivo. The FlgE-ectopic ATP5B duo might contribute to the pathogenesis of disorders associated with bacterial infection or ectopic ATP5B-positive cells.

Survival and prognostic factors for patients with malignant central airway obstruction following airway metallic stent placement.

BACKGROUND: There have been many studies on the effectiveness and complications of airway stent, but few had focused on factors that affect survival after stent placement. This study intended to assess the factors associated with the survival in patients with malignant central airway obstruction (MCAO) after airway metallic stent placement. METHODS: The clinical data of adult MCAO patients who underwent stent placement form February 2003 to June 2017 in the First Affiliated Hospital of Soochow University in China were retrospectively analyzed. The survival rates were compared using Log-rank tests. Potential prognostic factors were identified using multivariate Cox hazard regression models. RESULTS: Total 102 MCAO patients were included in this study. The median survival time of these patients after airway metallic stent placement was 4.1 months. Multivariate analysis showed that MCAO patients receiving radiotherapy [hazard ratio (HR) 0.554; 95% confidence interval (CI): 0.308-0.999] or chemoradiotherapy (HR 0.251; 95% CI: 0.126-0.499) after stenting had better prognosis. However, ECOG PS ≥3 score prior to the stenting (HR 2.193; 95% CI: 1.364-3.526) and stents placed in both trachea and main bronchus (HR 2.458; 95% CI: 1.384-4.366) were associated with worse survival. CONCLUSIONS: In our results, survival of MCAO patients after airway metallic stenting was related to ECOG PS score prior to the stenting, the site of stent placement and we have hereby proposed for the first time that having opportunity to receive radiotherapy or chemoradiotherapy after stenting contribute to better prognosis.

68Ga-labeled dimeric and trimeric cyclic RGD peptides as potential PET radiotracers for imaging gliomas.

68Ga-labeled cyclic RGD dimers and trimer were evaluated as PET radiotracers. It was found that the linker group had little impact on αvß3 binding affinity of RGD dimers, which share similar αvß3 binding affinity with the RGD trimer despite of their different multiplicity. Biodistribution properties of 68Ga radiotracers depend on RGD peptides and radiometal chelates. Among the 68Ga radiotracers evaluated, 68Ga-I2P-RGD2 has the best tumor uptake with good tumor-to-background ratios, and is a good PET radiotracer for imaging gliomas.

Mutations of genes including DNMT3A detected by next-generation sequencing in thyroid cancer.

More than 90% of thyroid cancer belongs to the papillary and follicular thyroid carcinomas based on pathological subtypes. Papillary and follicular thyroid carcinoma are generally associated with a good prognosis. In contrast, other pathological subtypes such as poorly-differentiated and anaplastic thyroid carcinoma (PDTC and ATC) have a poor clinical outcome with a short life expectancy. To identify the genetic variations and biomarkers that may potentially distinguish the aggressive form of thyroid cancer, we performed a retrospective analysis of the formalin-fixed paraffin-embedded tumor samples from 50 patients who mainly displayed aggressive thyroid cancer using next-generation sequencing of 416 solid tumor-related genes. We adopted extensive bioinformatic analysis to vigorously remove germline single-nucleotide polymorphism and systematic sequencing errors, and report here that mutation in DNMT3A gene was significantly enriched in patients with PDTC or ATC.

Imaging paclitaxel (chemotherapy)-induced tumor apoptosis with 99mTc C2A, a domain of synaptotagmin I: a preliminary study.

PURPOSE: To evaluate the dynamics and feasibility of imaging non-small cell lung cancer (NSCLC) apoptosis induced by paclitaxel treatment using 99mTc-labeled C2A domain of synaptotagmin I in a mouse model. MATERIALS AND METHODS: H460 tumor-bearing mice were treated with intravenous paclitaxel, and 12, 24, 48 and 72 h later, 99mTc-C2A-GST was injected intravenously, and planar images were acquired at 2, 4 and 6 h postinjection on a dual-head gamma camera fitted with a pinhole collimator. Tumor-to-normal tissue ratios (T/NT) were calculated by ROI analysis and reflected specific binding of 99mTc-C2A-GST. Mice were sacrificed after 6-h imaging; caspase-3 as apoptosis executer was determined by flow cytometry; DNA fragmentation was analyzed by terminal deoxynucleotidytransferase mediated dUTP nick-end labeling (TUNEL) assay. Whereas nonspecific accumulation was estimated using inactivated C2A-GST. The imaging data were correlated with TUNEL-positive nuclei and caspase-3 activity. RESULTS: T/NT significantly increased after paclitaxel inducement, whereas it was low in untreated tumors (T/NT=1.24+/-0.23). In terms of % ID/g, activity in Group 2 (12 h), Group 3 (24 h), Group 4 (48 h) and Group 5 (72 h) after the treatment was 2.05+/-0.20, 3.02+/-1.01, 3.17+/-1.16 and 3.96+/-1.72, respectively. Whereas in the nontreated group, Group 1 % ID/g was 1.21+/-0.51. The radiotracer uptake was positively correlated to the apoptotic index (r=0.70, P<.01), as well as caspase-3 activity (r=0.75, P<.01). CONCLUSION: This study addresses the dynamics and feasibility of imaging non-small cell lung tumor apoptosis using 99mTc-labeled C2A.

Engineering a novel protease-based Exendin-4 derivative for type 2 antidiabetic therapeutics.

To develop an effective long-acting antidiabetic agent, we designed a novel Exendin-4 derivative (termed LEx4) containing an albumin-binding domain (ABD), a protease-cleavable linker and a native Exendin-4. Here, we present the LEx4 with balanced glucoregulatory activity and prolonged in vivo activity. As a first step, the LEx4 with purity more than 99% was prepared. Microscale thermophoresis (MST) results demonstrated that LEx4 associates with rat and monkey serum albumin with high-affinity (Ka = 1.26 × 106 M-1 and 1.52 × 106 M-1, respectively). Then the stability test in vitro showed the enhanced antiproteolytic ability of LEx4 in rat and human plasma compared to native Exendin-4. Oral glucose tolerance test (OGTT) in type 2 diabetic mice showed the glucose-lowering efficacy of LEx4 was clearly dosage-dependent within 25-250 nmol/kg. In addition, the protracted antidiabetic effects of LEx4 were further confirmed by both multiple OGTTs and hypoglycemic efficacies test in type 2 diabetic mice. In Sprague Dawley (SD) rats, LEx4 also showed 3.3-fold longer elimination half-life (t1/2) than native Exendin-4. Furthermore, once daily injection of LEx4 to db/db mice achieved long-term beneficial effects on body weight, blood biochemical values, glucose tolerance and pancreatic tissue. We believe LEx4 has superior pharmaceutical potential as a therapeutic drug to against type-2 diabetes mellitus (T2DM) based on these results. This strategy of albumin binding is also applicable to other bioactive peptides for development of long-acting therapeutic drugs.

SPECT imaging of myocardial infarction using 99mTc-labeled C2A domain of synaptotagmin I in a porcine ischemia-reperfusion model.

INTRODUCTION: The C2A domain of synaptotagmin I recognizes necrotic and apoptotic cells by binding to exposed anionic phospholipids. The goal is to explore the potential imaging utility of 99mTc-labeled C2A in the detection of acute cardiac cell death in a porcine model that resembles human cardiovascular physiology. METHODS: Ischemia (20-25 min) was induced in pigs (M/F, 20-25 kg) using balloon angioplasty. 99mTc-C2A-GST (n=7) or 99mTc-BSA (n=2) was injected intravenously 1-2 h after reperfusion. Noninfarct animals were injected with 99mTc-C2A-GST (n=4). SPECT images were acquired at 3 and 6 h postinjection. Cardiac tissues were analyzed to confirm the presence of cell death. RESULTS: Focal uptake was detected in five out of seven subjects at 3 h and in all infarct subjects at 6 h postinjection but not in infarct animals injected with 99mTc-BSA or in noninfarct animals with 99mTc-C2A-GST. Gamma counting of infarct versus normal myocardium yielded a 10.2+/-5.7-fold elevation in absolute radioactivity, with histologically confirmed infarction. CONCLUSIONS: We present data on imaging myocardial cell death in the acute phase of infarction in pigs. C2A holds promise and warrants further development as an infarct-avid molecular probe.

[Technetium-99m labeled synaptotagmin I C2A detection of paclitaxel-induced apoptosis in non-small cell lung cancer].

UNLABELLED: Objective To evaluate the efficacy of 99mTc-labeled C2A probe in detection of apoptosis of non-small cell lung cancer (NSCLC) cells after chemotherapy. METHODS: Imaging studies were performed in NSCLC H460-bearing mice. The mice were divided into 2 groups: the paclitaxel-treated group and control group. 99mTc-C2A was injected intravenously at 12, 24, 48 and 72 h after chemotherapy. Images were acquired at 3 h and 6 h after injection using a pinhole collimator. The regions of interest (ROI) were drawn in tumor area and contralateral nomal tissue, and the ratio of T/NT were caculated. The tumor sections were stained by HE and TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-nick-end labeling) staining to confirm the presence of apoptosis. Activated caspase-3 was also analyzed with flow cytometry. RESULTS: Little uptake of 99mTc-C2A was found in baseline images, but tumor uptake increased very much after chemotherapy, the T/NT ratio was 1.79 +/- 0.34, 2.23 +/- 0.33 and 2.78 +/- 0.34, respectively. The T/NT ratio of control was 1.48 +/- 0.23. Tumor uptake (% ID/g) of 99mTc-C2A in chemotherapy groups were 2.82 +/- 0.90, 3.13 +/- 0.48 and 3.52 +/- 1.18, respectively. Tumor uptake (% ID/g) in the control group was 1.21 +/- 0.51. It in paclitaxel-treatment groups were 2.82 +/- 0.90, 3.13 +/- 0.48 and 3.51 +/- 1.18, respectively, significantly higher than that in untreated mice. Furthermore, the uptake of 99mTc-C2A correlated well with apoptotic index (r = 0.56, P < 0.01), and activated caspase-3 (r = 0.59, P < 0.01). CONCLUSION: Our preliminary results demonstrated that 99mTc-C2A imaging in vivo for detection of cell death in solid tumors is feasible and well correlated with TUNEL staining and activated caspase-3. The C2A holds promise and warrants further development as a molecular probe to early predict cancer treatment efficacy.

[Noninvasive detection of experimental vulnerable atherosclerotic plaques with 99Tc m labeled C2A domain of synaptotagmin I].

OBJECTIVE: (99)Tc(m) labeled C2A domain of synaptotagmin I ((99)Tc(m)-Syt I-C2A) is used for noninvasive detection of vulnerable atherosclerotic plaque. METHODS: Recombinant C2A domain of synaptotagmin I, overexpressed in E. Coli, was thiolated with 2-iminothiolane (2-IT) and labeled with (99)Tc(m). Atherosclerotic plaques were produced in 5 rabbits by deendothelialization of the abdominal aorta and the rabbits were fed with cholesterol diet for 3 months. Three rabbits not manipulated served as normal controls. All animals were injected with (99)Tc(m)-Syt I-C2A and underwent in vivo imaging thereafter. Aortas were then explanted for ex vivo imaging and histological characterization. RESULTS: In deendothelialized animals, intense radio-uptake in abdominal aorta, showed by gamma camera at 2 h after injection, was visualized and T/B was 3.25 +/- 0.51 by ROI measurement, quantitative uptake ratio of abdominal aortas with atherosclerotic lesions to thoracic aortas was 8.39 +/- 1.74 in ex vivo imaging. The mean uptake in specimens of abdominal aortas with lesions was 12.6-fold higher than in control abdominal aortas, and 10.2-fold higher than in thoracic aortas of deendothelialized animals by gamma-counter. CONCLUSION: (99)Tc(m)-Syt I-C2A has a high affinity for vulnerable atherosclerotic plaque and is a suitable a gent for the noninvasive detection of vulnerable atherosclerotic plaque.