Prognostic significance of annexin A2 and tumor associated macrophages (TAMs) in metastatic renal cell carcinoma and their relation to Sunitinib resistance.

Sunitinib, an antiangiogenic tyrosine kinase inhibitor, is the main treatment for metastatic renal cell carcinoma (mRCC). Development of resistance is a major obstacle against therapy success. The aim of this study was to assess annexin A2 and CD163+ tumor associated macrophages (TAMs) immunohistochemical expression in 50 mRCC cases as regard to patients' prognosis and Sunitinib response. Also, to assess the correlation between annexin A2 and TAMs expression. High annexin A2 expression and TAMs density were associated with serum calcium level (P = 0.024 and 0.037, respectively), larger tumor size (P < 0.001), high tumor grade (P = 0.014 and <0.001, respectively), and the presence of tumor necrosis (P < 0.001). High annexin A2 and TAMs expressions were related to shorter patients' overall survival (P = 0.009 and 0.001, respectively) and progression-free survival (P = 0.003 and 0.001, respectively). Annexin A2 was correlated with TAMs density (r = 0.890). Annexin A2 and TAMs are associated with poor prognostic parameters in mRCC patients, including high nuclear grade, increased tumor size, and the presence of tumor necrosis, together with shorter patients' survivals and poor response to Sunitinib. Annexin A2 expression is correlated with TAMs density suggesting immunomodulatory role of annexin A2.

COVID-19 Plasma Extracellular Vesicles Increase the Density of Lipid Rafts in Human Small Airway Epithelial Cells.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus is the causative agent of the COVID-19 disease. COVID-19 viral infection can affect many cell types, including epithelial cells of the lungs and airways. Extracellular vesicles (EVs) are released by virtually all cell types, and their packaged cargo allows for intercellular communication, cell differentiation, and signal transduction. Cargo from virus-infected cells may include virally derived metabolites, miRNAs, nucleic acids, and proteins. We hypothesized that COVID-19 plasma EVs can induce the formation of signaling platforms known as lipid rafts after uptake by normal human small airway epithelial cells (SAECs). Circulating EVs from patients with or without COVID-19 were characterized by nanoparticle tracking analysis, Western blotting using specific antibodies, and transmission electron microscopy. Primary cultures of normal human small airway epithelial cells were challenged with EVs from the two patient groups, and lipid raft formation was measured by fluorescence microscopy and assessed by sucrose density gradient analysis. Collectively, our data suggest that circulating EVs from COVID-19-infected patients can induce the formation of lipid rafts in normal human small airway epithelial cells. These results suggest the need for future studies aimed at investigating whether the increased density of lipid rafts in these cells promotes viral entry and alteration of specific signaling pathways in the recipient cells.

New tilomisole-based benzimidazothiazole derivatives as anti-inflammatory agents: Synthesis, in vivo, in vitro evaluation, and in silico studies.

New tilomisole-based benzimidazothiazole derivatives were designed and synthesized in this work. Their anti-inflammatory activity was assessed through the in vivo carrageenan rat paw edema model, and the in vitro COX inhibition assay. Compounds 13, 20, 30, 40, 43, and 46 demonstrated values of inhibition of induced edema in the in vivo assay comparable to celecoxib. All the synthesized compounds expressed their activity on COX-2 enzyme more than COX-1, proving their advantageous selectivity. In addition, compounds 13, 16, 20, 25, and 46 displayed lower IC50 values than celecoxib as a reference drug against COX-2 enzyme; having values of 0.09, 13.87, 32.28, 33.01, and 5.18 nM respectively vs 40.00 nM for celecoxib. Particularly, the most active compound (13) with its extreme potency (400 folds more potent than celecoxib) exhibited a notable high selectivity index (SI = 159.5). In silico studies, including ADMET prediction, compliance to Lipinski's rule of five, and molecular docking into the active site of both COX isozymes were conducted for the synthesized compounds. The results suggested that these compounds are good candidates for orally active drugs, and docking revealed higher number of interactions with COX-2 for 13 as the most active compound compared with COX-1 reflecting its advantageous selectivity and explaining its extreme potency.

New benzimidazothiazole derivatives as anti-inflammatory, antitumor active agents: Synthesis, in-vitro and in-vivo screening and molecular modeling studies.

A new series of benzimidazothiazole derivatives has been synthesized. The structure of the products was confirmed by spectroscopic techniques such as IR, NMR and mass spectroscopy. The tested compounds were evaluated for their anti-inflammatory activity either in vitro through the COX enzyme inhibition assay, or in vivo through carrageenan paw edema technique. Results revealed that compound 25 and 29 represented the most active ones among the entire series with % inhibition 72.19, 72.07 for COX-1, and 87.46, 87.38 for COX-2, respectively. Interestingly, all synthesized compounds exhibited IC50 values less than both reference drugs celecoxib and naproxen, indicating their superior potency. For compound 25, it showed about 340 and 198 times more potent than celecoxib and naproxen respectively as COX-1 inhibitor (IC50 value 0.044 vs. 15.000 and 8.700 µM), and 10 and 115 times more potent than the same drugs as COX-2 inhibitor (IC50 value 4.52 vs. 40.00 and 520.00 nM). The antitumor activity of the products was also evaluated and the results obtained are consistent with those obtained by the anti-inflammatory screening where compounds 25 and 29 proved to be the most active ones among the other compounds with %GI ranging from 31.5 to 62.5% and they exhibited the lowest IC50 values as well. The ADMET analysis of the tested compounds was also performed in addition to the molecular modeling studies that included flexible alignment, surface and electrostatic maps in addition to the Lipinisk's rule of five.

Readability and Comprehensibility of Patient Information Leaflets for Antidiabetic Medications in Qatar.

Background:The readability and comprehensibility of the patient information leaflets (PILs) provided with antidiabetic medications are of questionable standards; this issue negatively affects adherence to drug therapy, especially in patients with limited literacy skills. Objective: To evaluate the readability and comprehensibility of PILs supplied with medications used for the treatment of type 2 diabetes mellitus in Qatar. Methods: All PILs of the antidiabetic medications in Qatar were evaluated using the Flesch Reading Ease (FRE) score for readability. The Flesch-Kincaid Grade Level, Gunning-Fog Index, and SMOG Grading were used to estimate the comprehensibility of PILs in terms of school grade levels. Results: A total of 45 PILs were evaluated: 32 (71.1%) PILs of brand-name products and 13 (28.9%) for generics. Nine (20%) of the PILs were in English only; 8 (17.8%) were in English, Arabic, and French; and 28 (62.2%) were in English and Arabic. The mean FRE score was 37.71 (±15.85), and the most readable PIL had FRE score of 62. The mean scores for the comprehensibility evaluations were 10.96 (±2.67), 15.02 (±2.52), and 11.41 (±1.6) for the Flesch-Kincaid Grade Level, Gunning-Fog Index, and SMOG Grading, respectively. The most commonly used antidiabetic medication was metformin with 1372.9 (±552.9) as PILs' mean number of words. Conclusion: Only 2.2% of PILs had acceptable readability scores. All PILs could be comprehended by at least an 11th grade student, which exceeds the recommended grade level for health-related materials. Approximately 20% of these PILs were in English only and were not readable by most patients.

Functional and metabolomic analysis of urinary extracellular vesicles from juvenile mice with renal compensatory hypertrophy.

Unilateral nephrectomy, a procedure reducing kidney mass, triggers a compensatory response in the remaining kidney, increasing its size and function to maintain a normal glomerular filtration rate (GFR). Recent research has highlighted the role of extracellular vesicles (EVs) in renal physiology and disease, although their involvement in unilateral nephrectomy has been underexplored. In this study, unilateral nephrectomy was performed on young mice, and urinary extracellular vesicles (uEVs) characterization and cargo were analyzed. Kidney volume increased significantly post-nephrectomy, demonstrating compensatory hypertrophy. Serum creatinine, cystatin C, and urinary electrolytes concentrations were similar in both nephrectomized and control groups. Western blot analysis revealed upregulation of sodium-glucose cotransporter 2 (SGLT2) and sodium chloride cotransporter (NCC), and downregulation of sodium­potassium-chloride co-transporter (NKCC2) and epithelial sodium channel (ENaC) in the nephrectomized group. Metabolomic analysis of uEVs showed an enrichment of certain metabolites, including citrate and stachydrine. Interestingly, uEVs from the nephrectomized group demonstrated a protective effect, downregulating signal transducer and activator of transcription 3 (STAT3) and reducing reactive oxygen species (ROS) in renal proximal cells, compared to uEVs from the control group. This study suggests that uEVs contain bioactive components capable of inducing protective, anti-inflammatory, anti-fibrinolytic, and antioxidative effects in renal cells. These findings contribute to our understanding of uEVs' role in renal compensatory mechanisms after unilateral nephrectomy and may hold promise for future therapeutic interventions in renal diseases.

Liquid vs dried blood matrices: Application to longitudinal monitoring of androstenedione, testosterone, and IGF-1 by LC-MS-based techniques.

BACKGROUND: Dried blood spots have recently been approved by the World Anti-Doping Agency as an alternative biological matrix for testing of doping substances. However, their use is limited to the detection of non-threshold compounds without a Minimum Reporting Level due to the numerous issues related to quantitative analyses and the limitation on testing capabilities of a haemolysed matrix. AIM: In this study androstenedione, testosterone and IGF-1 were longitudinally monitored in four different blood matrices to evaluate the potential of liquid capillary blood as an alternative matrix for quantitative determination in doping control analysis. METHODOLOGY: The analytical protocols developed to pretreat 20 µL of the blood matrices selected were based: i) for testosterone and androstenedione, on supported liquid extraction for liquid blood matrices, and on ultrasonication in the presence of methanol for dried blood matrices; ii) for IGF-1, proteins precipitation followed by evaporation of the supernatant was used to pretreat both liquid and dried blood matrices. The detection for all the target analytes was performed using liquid chromatography coupled to mass spectrometry. The analytical workflows, once optimized, were fully validated according to the requirements of World Anti-Doping Agency and ISO 17025 standard and used for the analysis of venous (serum) and capillary (liquid plasma and dried whole blood collected using either volumetric or non-volumetric devices) blood samples collected from 7 healthy subjects. RESULTS: The validation results showed satisfactory performance as related to specificity, sensitivity, matrix effects, linearity, accuracy, and precision in all the blood matrices evaluated despite the limited volume of sample used. The analysis of the different blood matrices collected from the subjects showed non-significant differences between the levels of testosterone and androstenedione measured in dried (fixed volume collected) and liquid matrices. An acceptable underestimation (lower than 15 %) was observed in capillary plasma compared to venous serum. The testosterone/androstenedione ratio was similar in all the blood matrices considered (bias lower than 5 %), indicating this parameter was not affected by either the blood matrix or collection device selected. For IGF-1, the levels measured in liquid blood matrices differed significantly (bias higher than 20 %) from those measured in dried whole blood matrices, suggesting haemolyzed blood might represent a challenge for the determination of macromolecules, mainly due to the complexity of the whole blood matrix in comparison to plasma/serum. NOVELTY: The outcomes of our study suggest that liquid capillary blood might open new avenues to blood microsampling in doping control field. It represents an efficient alternative to overcome the issues related to venous blood and dried blood spot sampling. Furthermore, it also allows greater frequency of blood sampling, with minor discomfort and without needing a phlebotomist, for analyses that can only be performed in blood samples, with an increased probability to detect and report Adverse Analytical Finding.

Evaluation of the prognostic significance of receptor tyrosine kinase-like orphan receptor 1 (ROR1) in lung carcinoma and its relation to lymphangiogenesis and epithelial mesenchymal transition.

Exploring the carcinogenic mechanisms of lung carcinoma helps to discover novel prognostic biomarkers and develop new therapeutic options to improve patient's survival. Receptor Tyrosine Kinase-Like Orphan Receptor 1 (ROR1), a transmembrane protein, contributes to cancer progression and metastasis; via stimulation of epithelial mesenchymal transition (EMT) and promotion of angiogenesis. This makes ROR1 an important target for tumor therapy. This study aimed to evaluate expression of ROR1, E-cadherin (a marker of EMT), and D2-40 (a marker of lymphangiogenesis) in lung carcinoma and associate their expressions with the available clinicopathological parameters and patients' survival. Immunohistochemical staining using ROR1, E-cadherin, and D2-40 was performed for 78 cases of lung carcinoma. Kaplan-Meier survival curves and Cox-regression analysis were done. High ROR1 expression was detected in 46.2% of cases. Significant relations were found between high ROR1 expression and larger tumor size (P < 0.001), poorly differentiated tumors (P = 0.001), advanced tumor stages (P < 0.001), positive lymph nodal status (P < 0.001), decreased E-cadherin expression (P < 0.001), and high lymphovascular density (LVD) (P < 0.001). Patients' progression free survival (PFS) and overall survival (OS) were shorter with high ROR1 expression. High ROR1 expression, high LVD, large tumor size, and adenocarcinoma histopathological type were independent risk factors for OS in lung carcinoma patients. High ROR1 expression is associated with poor prognostic parameters in lung carcinoma patients including higher grade, advanced stages, high LVD, epithelial mesenchymal transition, as well as decreased PFS and OS.

Immunohistochemical based molecular subtypes of muscle-invasive bladder cancer: association with HER2 and EGFR alterations, neoadjuvant chemotherapy response and survival.

Muscle-invasive bladder cancers (MIBCs) is a group of molecularly heterogonous diseases that could be stratified into subtypes with distinct clinical courses and sensitivities to chemotherapy. Clinical application of molecular subtypes could help in prediction of neoadjuvant chemotherapy (NAC) responders. Immunohistochemical (IHC) markers such as GATA3, cytokeratin (CK) 5/6, and p53 are associated with these subtypes and are widely available. Human epidermal growth factor receptor 2 (HER2) and epidermal growth factor receptor (EGFR) are mutated in multiple cancers including MIBC and are potential therapeutic targets. HER2/EGFR status of MIBC subtypes has not been investigated. Tissue microarrays (TMAs) were constructed from transurethral resection of the bladder tumor (TURB) specimens and stained with GATA3,CK5/6,p53 and HER2 in addition to Quantitative Reverse Transcription PCR for detection of EGFR gene. Of the total cases, 45% were luminal, 36.7% basal and 18.3% p53 wild subtype (p53-WT). Univariate analysis showed that overall survival (OS) and disease-free progression survival (DFS) were significantly longer for luminal subtype. In multivariate analysis, molecular subtype, HER2 status and LV invasion were independent prognostic factors for DFS and OS. Basal subtype showed a significantly better response to NAC. HER2 expression was significantly higher in luminal while EGFR expression was significantly higher in basal subtype. Kaplan-Meier survival curves revealed a significant longer OS and DFS for HER2 negative than positive cases. MIBC can be stratified using a simple IHC panel [GATA3,CK5/6,P53] into clinically relevant prognostic molecular subtypes. Basal tumors are aggressive and respond well to NAC while luminal have better OS. P53-WT tumors are chemoresistant and require further treatments. HER2 and EGFR are potential therapeutic targets for molecular subtypes of MIBC where luminal tumors are more likely to benefit from HER2 and basal from EGFR directed therapies.

Utility of serum neuropilin-1 and angiopoietin-2 as markers of hepatocellular carcinoma.

This study aimed to assess the diagnostic value of two serum angiogenetic markers neuropilin-1 (NRP-1) and angiopoietin-2 (ANG-2) in patients with hepatocellular carcinoma (HCC) and their relation to tumor characteristics. 149 subjects were recruited and divided into 50 patients with recently diagnosed HCC, 49 patients with cirrhosis on top of hepatitis C virus infection, and 50 healthy subjects. Serum NRP-1 and ANG-2 were estimated by ELISA. Alpha-fetoprotein (AFP) levels were measured using fluorescence immunoassay. Serum NRP-1 and ANG-2 levels were significantly higher in patients with HCC (2221.8±1056.6 pg/mL and 3018.5±841.4 pg/mL) than healthy subjects (219.3±61.8 pg/mL and 2007.7±904.8 pg/mL) and patients with cirrhosis (1108.9±526.6 pg/mL and 2179.1±599.2 pg/mL), respectively. In multivariate logistic regression analysis, NRP-1 and AFP were the only independent factors of HCC development and correlated positively with each other (r=0.781, p<0.001). Receiver operating characteristic curve analysis showed that the area under the curve (AUC) of NRP-1 was higher than that of ANG-2 in discriminating HCC from patients with cirrhosis (0.801 vs 0.748, p=0.250) and healthy subjects (0.992 vs 0.809, p<0.001). The AUC of NRP-1 was detected to be increased (0.994) when combined estimation with AFP was performed. Elevated serum NRP-1 and ANG-2 levels were detected in patients with HCC with tumor numbers >3, tumor size ≥5 cm, tumor stages B/C according to the Barcelona Clinic Liver Cancer staging system, vascular invasion, and distant metastasis. In conclusion, NRP-1 is a potential serological marker for HCC diagnosis and is better than ANG-2. It is feasible to be estimated in combination with AFP to enhance its diagnostic power. High serum NRP-1 and ANG-2 levels are associated with advanced HCC tumor characteristics.

A Prospective Study Comparing Two-Time Points of Thyroid Hormone Replacement during the Holy Month of Ramadan.

BACKGROUND: Muslims all over the world fast during the month of Ramadan from dawn until dusk. There is little data regarding the best timing of levothyroxine intake during the month of Ramadan where taking it on an empty stomach represents a challenge to most patients. Our study aims to compare two-time points of levothyroxine intake during Ramadan in terms of change in thyroid stimulating hormone (TSH), compliance, and convenience. STUDY DESIGN AND METHODS: This was an open-label, randomized, prospective trial. Adult patients known to have primary hypothyroidism with stable TSH for the last 6 months who intended to fast during the month of Ramadan were invited to participate in this prospective study. The study took place during Ramadan of H1438 (May-June 2017). All patients were randomly assigned to two groups. In group A (n= 50) patients took levothyroxine 30 minutes before breaking the fast at sunset (iftar), and in group B (n= 46) patients took it 30 minutes before an early morning meal before sunrise (suhour). RESULTS: TSH levels increased in both group A (from 1.99 to 3.28 mIU/L) and group B (from 1.54 to 3.28 mIU/L) after Ramadan fasting. There was no difference between the two groups. Compliance with intake instructions, all of the time, was reported in 41.6% of group A and 35.7% of group B patients. In both the groups, 95% of patients said it was convenient for them to take the medication at the assigned time. CONCLUSION: Choosing an optimal time for levothyroxine intake during the month of Ramadan remains a challenge. The current study did not provide any evidence on ideal time and dose of levothyroxine administration during fasting to manage hypothyroidism. Studies with a larger number of patients need to be done to further explore this issue.