Circulating Tumor-Macrophage Fusion Cells and Circulating Tumor Cells Complement Non-Small-Cell Lung Cancer Screening in Patients With Suspicious Lung-RADS 4 Nodules.

PURPOSE: Low-dose computed tomography (LDCT) screening of high-risk patients decreases lung cancer-related mortality. However, high false-positive rates associated with LDCT result in unnecessary interventions. To distinguish non-small-cell lung cancer (NSCLC) from benign nodules, in the present study, we integrated cellular liquid biomarkers in patients with suspicious lung nodules (lung cancer screening reporting and data system [Lung-RADS] 4). METHODS: Prospectively, 7.5 mL of blood was collected from 221 individuals (training set: 90 nonscreened NSCLC patients, 74 high-risk screening patients with no/benign nodules [Lung-RADS 1-3], and 20 never smokers; validation set: 37 patients with suspicious nodules [Lung-RADS 4]). Circulating tumor cells (CTCs), CTC clusters, and tumor-macrophage fusion (TMF) cells were identified by blinded analyses. Screening patients underwent a median of two LDCTs (range, 1-4) with a median surveillance time of 30 (range, 11-50) months. RESULTS: In the validation set of 37 Lung-RADS 4 patients, all circulating cellular biomarker counts (P < .005; Wilcoxon test) and positivity rates were significantly higher in 23 biopsy-proven NSCLC patients (CTCs: 23 of 23 [100%], CTC clusters: 6 of 23 [26.1%], and TMF cells: 15 of 23 [65.2%]) than in 14 patients with biopsy-proven benign nodules (6 of 14 [42.9%], 0 of 14 [0%], and 2 of 14 [14.3%]). On the basis of cutoff values from the training set, logistic regression with receiver operating characteristic and area under the curve analyses demonstrated that CTCs (sensitivity: 0.870, specificity: 1.0, and area under the curve: 0.989) and TMF cells (0.652; 0.880; 0.790) complement LDCT in diagnosing NSCLC in Lung-RADS 4 patients. CONCLUSION: Cellular liquid biomarkers have a potential to complement LDCT interpretation of suspicious Lung-RADS 4 nodules to distinguish NSCLC from benign lung nodules. A future prospective, large-scale, multicenter clinical trial should validate the role of cellular liquid biomarkers in improving diagnostic accuracy in high-risk patients with Lung-RADS 4 nodules.

Combinatorial inhibition of Angiotensin converting enzyme, Neutral endopeptidase and Aminopeptidase N by N-methylated peptides alleviates blood pressure and fibrosis in rat model of dexamethasone-induced hypertension.

Angiotensin converting enzyme (ACE), neutral endopeptidase (NEP) and aminopeptidase N (APN) are responsible for generation of vasoactive peptides that regulates vasoconstriction, vasodilation and natriuresis, which altogether regulate blood pressure. Cumulative inhibition of ACE, NEP and APN effectively blocks the progression of respective pathways. In this study, N-methylated peptide inhibitors F-N(Me)H-L, V-N(Me)F-R and R-N(Me)V-Y were synthesized against ACE, NEP and APN respectively, using their respective physiological substrates. F-N(Me)H-L inhibited ACE activity with an IC50 of 83 nmol/L, V-N(Me)F-R inhibited NEP activity with an IC50 of 1.173 µmol/L and R-N(Me)V-Y inhibited APN activity with an IC50 of 3.94 nmol/L respectively. Further, the anti-hypertensive effect of N-methylated peptides was evaluated using rat model of dexamethasone-induced hypertension. Individual peptides and their cocktail treatment were started from day 6 of the study period and blood pressure was measured on every alternate day during 15 day study. Administration of F-N(Me)H-L (138 ± 3 mmHg) and cocktail of all the three peptides at a dose of 100 mg/kg significantly reduced systolic blood pressure (SBP) compared to dexamethasone group (SBP of Groups-dexamethasone; (167 ± 5 mmHg), F-N(Me)H-L (138 ± 3 mmHg), and Cocktail (122 ± 3 mmHg). Anti-hypertensive, anti-hypertrophic and anti-fibrotic effects of N-methylated peptides and cocktail was further reflected by the decreased levels of circulating Ang II and increased ANP levels in sera of hypertensive rats along with decrease in collagen deposition in heart and kidney. Though, ACE inhibition is adequate to reduce SBP, targeting NEP and APN along with ACE is beneficial in tackling hypertension and associated fibrosis of heart.

Active-site directed peptide l-Phe-d-His-l-Leu inhibits angiotensin converting enzyme activity and dexamethasone-induced hypertension in rats.

Hypertension is the fundamental cause of cardiovascular and cerebrovascular disorders. Several natural and synthetic peptides are being used as antihypertensive agents, which target angiotensin converting enzyme (ACE), the master regulator of angiotensin (Ang) II production. In this study, we have evaluated ACE-inhibitory potential of the tripeptide l-Phenylalanyl-d-Histidyl-l-Leucine (l-Phe-d-His-l-Leu) in vitro and its antihypertensive effect in rat model of dexamethasone-induced hypertension. l-Phe-d-His-l-Leu was custom-designed by changing the configuration of penultimate amino acid residue (histidine) from C-terminal of Ang I, the site at which ACE acts upon and generates Ang II. l-Phe-d-His-l-Leu effectively inhibited ACE activity in a dose-dependent and competitive manner with an IC50 of 53.32 ± 0.13 nmol/L. Both fluorescence spectra and circular dichroism data revealed the direct interaction between l-Phe-d-His-l-Leu and ACE. In addition, molecular docking studies revealed the strong interaction of l-Phe-d-His-l-Leu with the critical active site amino acid residues of ACE. Further, the administration of l-Phe-d-His-l-Leu resulted in decrease in blood pressure (142 ± 3 mmHg) compared to dexamethasone alone group (167 ± 2 mmHg). Besides, l-Phe-d-His-l-Leu decreased the levels of circulating Ang II, and reduced fibrosis in heart and kidney, as evidenced by decreases in collagen deposition. Thus, the strategy of incorporation of d-amino acids in ACE-inhibitory peptides could be valuable in the development of antihypertensive drugs.

EC-PIII, a novel non-hemorrhagic procoagulant metalloproteinase: Purification and characterization from Indian Echis carinatus venom.

Procoagulant snake venom toxins find extensive use as reagents in laboratory tests and diagnostic kits. In the present study we report a novel P-III class procoagulant SVMP, EC-PIII from Echis carinatus venom. EC-PIII was purified using a combination of gel-filtration and anion-exchange chromatography. It has a molecular mass of 110kDa and is a dimeric protein as determined by SDS-PAGE. DLS results show that the protein is homogenous and stable in solution. Peptide mass fingerprinting revealed that the peptides obtained show high homology to the other members of SVMP family. The enzymatic studies revealed that EC-PIII shows protease activity and is inhibited by metalloproteinase inhibitors such as EDTA. EC-PIII exhibits procoagulant effect under in-vitro conditions. Local toxicity studies revealed that EC-PIII is devoid of hemorrhagic as well as myotoxic activities. This is the first report of a non-hemorrhagic SVMP to be identified from Indian Echis carinatus venom. EC-PIII can find potential use in diagnostic and other therapeutic uses owing to its biochemical and pharmacological properties.

Dimethyl ester of bilirubin exhibits anti-inflammatory activity through inhibition of secretory phospholipase A2, lipoxygenase and cyclooxygenase.

Overproduction of arachidonic acid (AA) mediated by secretory phospholipase A2 group IIA (sPLA2IIA) is a hallmark of many inflammatory disorders. AA is subsequently converted into pro-inflammatory eicosanoids through 5-lipoxygenase (5-LOX) and cyclooxygenase-1/2 (COX-1/2) activities. Hence, inhibition of sPLA2IIA, 5-LOX and COX-1/2 activities is critical in regulating inflammation. We have previously reported unconjugated bilirubin (UCB), an endogenous antioxidant, as sPLA2IIA inhibitor. However, lipophilic UCB gets conjugated in liver with glucuronic acid into hydrophilic conjugated bilirubin (CB). Since hydrophobicity is pre-requisite for sPLA2IIA inhibition, conjugation reduces the efficacy of UCB. In this regard, UCB was chemically modified and derivatives were evaluated for sPLA2IIA, 5-LOX and COX-1/2 inhibition. Among the derivatives, BD1 (dimethyl ester of bilirubin) exhibited âˆ¼ 3 fold greater inhibitory potency towards sPLA2IIA compared to UCB. Both UCB and BD1 inhibited human 5-LOX and COX-2 activities; however only BD1 inhibited AA induced platelet aggregation. Molecular docking studies demonstrated BD1 as better inhibitor of aforesaid enzymes than UCB and other endogenous antioxidants. These data suggest that BD1 exhibits strong anti-inflammatory activity through inhibition of AA cascade enzymes which is of great therapeutic importance.

Progressive Hemorrhage and Myotoxicity Induced by Echis carinatus Venom in Murine Model: Neutralization by Inhibitor Cocktail of N,N,N',N'-Tetrakis (2-Pyridylmethyl) Ethane-1,2-Diamine and Silymarin.

Viperbite is often associated with severe local toxicity, including progressive hemorrhage and myotoxicity, persistent even after the administration of anti-snake venom (ASV). In the recent past, investigations have revealed the orchestrated actions of Zn2+ metalloproteases (Zn2+MPs), phospholipase A2s (PLA2s) and hyaluronidases (HYs) in the onset and progression of local toxicity from the bitten site. As a consequence, venom researchers and medical practitioners are in deliberate quest of potent molecules alongside ASV to tackle the brutal local manifestations induced by aforesaid venom toxins. Based on these facts, we have demonstrated the protective efficacy of inhibitor cocktail containing equal ratios of N,N,N',N'-tetrakis (2-pyridylmethyl) ethane-1,2-diamine (TPEN) and silymarin (SLN) against progressive local toxicity induced by Echis carinatus venom (ECV). In our previous study we have shown the inhibitory potentials of TPEN towards Zn2+MPs of ECV (IC50: 6.7 µM). In this study we have evaluated in vitro inhibitory potentials of SLN towards PLA2s (IC50: 12.5 µM) and HYs (IC50: 8 µM) of ECV in addition to docking studies. Further, we have demonstrated the protection of ECV induced local toxicity with 10 mM inhibitor cocktail following 15, 30 min (for hemorrhage and myotoxicity); 60 min (for hemorrhage alone) of ECV injection in murine model. The histological examination of skin and thigh muscle sections taken out from the site of ECV injection substantiated the overall protection offered by inhibitor cocktail. In conclusion, the protective efficacy of inhibitor cocktail is of high interest and can be administered locally alongside ASV to treat severe local toxicity.

Inhibitory potential of three zinc chelating agents against the proteolytic, hemorrhagic, and myotoxic activities of Echis carinatus venom.

Viperbites undeniably cause local manifestations such as hemorrhage and myotoxicity involving substantial degradation of extracellular matrix (ECM) at the site of envenomation and lead to progressive tissue damage and necrosis. The principle toxin responsible is attributed to snake venom metalloproteases (SVMPs). Treatment of such progressive tissue damage induced by SVMPs has become a challenging task for researchers and medical practitioners who are in quest of SVMPs inhibitors. In this study, we have evaluated the inhibitory potential of three specific zinc (Zn(2+)) chelating agents; N,N,N',N'-tetrakis (2-pyridylmethyl) ethane-1,2-diamine (TPEN), diethylene triamine pentaacetic acid (DTPA), tetraethyl thiuram disulfide (TTD) on Echis carinatus venom (ECV) induced hemorrhage and myotoxicity. Amongst them, TPEN has high affinity for Zn(2+) and revealed potent inhibition of ECV metalloproteases (ECVMPs) in vitro (IC50: 6.7 µM) compared to DTPA and TTD. The specificity of TPEN towards Zn(2+) was confirmed by spectral and docking studies. Further, TPEN, DTPA, and TTD completely blocked the hemorrhagic and myotoxic activities of ECV in a dose dependent manner upon co-injection; whereas, only TPEN successfully neutralized hemorrhage and myotoxicity following independent injection. Histological examinations revealed that TPEN effectively prevents degradation of dermis and basement membrane surrounding the blood vessels in mouse skin sections. TPEN also prevents muscle necrosis and accumulation of inflammatory cells at the site of ECV injections. In conclusion, a high degree of structural and functional homology between mammalian MMPs and SVMPs suggests that specific Zn(2+) chelators currently in clinical practice could be potent first aid therapeutic agents in snakebite management, particularly for local tissue damage.