Effect of lipid composition on incorporation of trastuzumab-PEG-lipid into nanoliposomes by post-insertion method: physicochemical and cellular characterization

Context: Anti-HER2 immunoliposomes are promising nanotechnology based systems for active targeting of breast tumors, which depends on the amount of incorporated antibody.

Objective/Aim: In this work, we investigated the possible effect of lipid composition on the incorporation of trastuzumab-PEG-PE micelles into nanoliposomes and on their subsequent specific cellular targeting.

Materials and methods: Trastuzumab (anti-HER2 monoclonal antibody) was monothiolated and conjugated to maleimide-PEG-PE micelles. Liposomes of different lipid compositions were prepared by the thin layer hydration. Trastuzumab-PEG-PE micelles were incorporated into the liposomes by the post-insertion method. The percentage of lipid mixing was determined based on fluorescence resonance energy transfer. Cellular binding and uptake of rhodamine-labeled immunoliposomes were studied in SKBR-3 (HER2⁺⁺⁺) and MCF-7 (HER2⁺) cells. Also, antitumor cell activity of the immunoliposomes was compared to free trastuzumab and the liposomes.

Results: The lipid mixing of trastuzumab-PEG-PE micelles depended on the liposome composition. The immunoliposomes containing DPPC, cholesterol and PEG-PE showed prominent lipid mixing. The lipid mixing was consistent with the cell binding results which showed an efficient and specific binding of the immunoliposomes to SKBR-3 cells. Antitumor cell activity of the immunoliposomes in SKBR-3, unlike MCF-7 cells, depended on the content of trastuzumab.

Discussion: Cholesterol and PEG-PE in the liposome composition are prerequisites for a successful lipid mixing due to their ability to facilitate fusion. The higher lipid mixing results in higher antibody incorporation and consequently higher targeted cell binding.

Conclusions: The lipid mixing depends on the liposome composition, which reflects targeted cell binding of the immunoliposomes.     

Mutation in the beta3 subunit of Guanine nucleotide-binding protein (GNB3) gene is not associated with Type II diabetes mellitus risk: a case–control study of a North Indian population

Context: Genetics play a major role in development and pathophysiology of Type 2 diabetes mellitus (T2DM).

Objective: To asses the association of Guanine nucleotide-binding protein (GNB3) (C825T) gene's polymorphism with T2DM.

Materials and methods: A case–control study including 400 North Indians was performed using Polymerase Chain Reaction–Restriction Fragment Length Polymorphism (PCR-RFLP) approach to analyze genetic polymorphism.

Results: No significant difference was observed in genotype and allele frequencies of GNB3 gene on comparing cases with controls.

Discussion: Our study is in agreement with studies on Polish, Japanese, Hispanic-American and Danish populations who observed no significant association between GNB3 (C825T) polymorphism and T2DM.

Conclusion: GNB3 (C825T) polymorphism is not associated with T2DM.     

Tumor delivery of liposomal doxorubicin prepared with poly-L-glutamic acid as a drug-trapping agent

Context: Poly-l-glutamic acid (PGA) is an anionic polymer with a large number of carboxyl groups that can interact electrostatically with cationic drugs such as doxorubicin (DOX).

Objective: For stable encapsulation of DOX into liposomes, we prepared triethylamine (TEA)-PGA-liposomes using PGA as an internal trapping agent.

Methods: We prepared TEA-PGA-liposomes by remote loading of DOX with a TEA gradient into preformed liposomes prepared with 1, 2, or 4?mg/mL PGA (molecular weights 4800, 9800, and 20 500), and evaluated their biodistribution and antitumor effects on Lewis lung carcinoma (LLC) tumor-bearing mice.

Results: TEA-PGA-liposomes using the higher the molecular weight or concentration of PGA showed a slower release of DOX from the liposomes. TEA-PGA-liposomes prepared with a high concentration of PGA could enhance DOX accumulation in tumors and prolonged DOX circulation in the serum, indicating that DOX may be retained stably in the liposomal interior by interaction with PGA. Furthermore, injection of TEA-PGA-liposomes prepared with 4?mg/mL of PGA₉₈₀₀ or 2?mg/mL PGA₂₀₅₀₀ strongly inhibited tumor growth in LLC tumor-bearing mice.

Conclusions: PGA may be a potential trapping agent for liposomal DOX for tumor drug delivery.     

Novel ortho ester-based,pH-sensitive cationic lipid for gene delivery in vitro and in vivo

Context: Cationic lipoplexes are less toxic than viral gene vectors and more convenient to prepare but their efficiencies of gene delivery are generally lower.

Objective: To develop ortho ester-based, pH-sensitive lipoplexes for efficient gene delivery both in cultured cells and in vivo.

Materials and methods: A novel cationic and acid-labile lipid (DOC) containing a cationic headgroup and a cholesterol-derived lipid tail joined together by an acid-labile ortho ester linker was designed and synthesized. DOC was formulated into liposomes with the conical helper lipid DOPE, and then into lipoplexes with plasmid DNA encoding a luciferase reporter gene. The physicochemical properties of the lipoplexes (size, surface charge and pH-sensitivity) were characterized. Gene delivery by DOC/DOPE/DNA lipoplexes was also evaluated in CV-1 cells and in CD-1 mice following intratracheal injection. Lipoplexes consisting of the acid-stable cationic lipid DC-Chol were characterized as a control.

Results: DOC formed cationic lipoplexes with DOPE and DNA. After incubation at acidic pH 4.6, DOC/DOPE/DNA lipoplexes lost their positive charges and aggregated with one another as a result of DOC hydrolysis. Both in CV-1 cell culture and in CD-1 mice, DOC/DOPE/DNA lipoplexes increased the luciferase gene expression by 5- to 10-fold compared with the analogous but acid-stable DC-Chol/DOPE/DNA lipoplexes.

Discussion and conclusion: Incorporation of an acid-labile ortho ester linker into a cationic lipid is a viable approach to enhance gene delivery by the corresponding lipoplexes both in cultured cells and in vivo.     

Sleep-disordered breathing is associated with higher carboxymethyllysine level in elderly women but not elderly men in the cardiovascular health study

Context: Carboxymethyl-lysine (CML) results from oxidative stress and has been linked to cardiovascular disease.

Objective: The objective of this study is to investigate the association between sleep-disordered breathing (SDB) – a source of oxidative stress – and CML.

Materials and methods: About 1002 participants in the Cardiovascular Health Study (CHS) were studied.

Results: Women with SDB had significantly higher CML concentration compared with those without SDB (OR?=?1.63, 95%CI?=?1.03–2.58, p?=?0.04). The association was not significant among men.

Discussion: SDB was associated with CML concentration among elderly women but not men in the Cardiovascular Health Study.

Conclusion: Accumulation of CML may be an adverse health consequence of SDB     

H19 non-coding RNA in urine cells detects urothelial carcinoma: a pilot study

Context: Urothelial carcinoma (UC) is common and highly recurrent. Diagnosis and follow-up involve invasive cystoscopies.

Objective: To evaluate H19 RNA in urine cells as diagnostic tool for UC.

Materials and methods: RT-PCR analysis of urine samples from healthy volunteers and UC patients.

Results: H19 RNA was unequivocally detected in the urine of 90.5% of patients and 25.9% of controls. H19 copies were three orders of magnitude higher in patients. Receiver operating characteristic analysis showed an area under the curve of 0.933.

Conclusions: This pilot study shows that urinary cell H19 is a highly sensitive test for UC and pending verification could transform patient management.     

Differential expression of circulating vascular cell adhesion molecule-1 in subjects with coronary artery disease and cardiac syndrome X without known diabetes mellitus

Context: Inflammation is one of the mechanisms underlying cardiac syndrome X (CSX).

Objectives: Few studies have compared the expression of inflammatory or adhesion molecules between coronary artery disease (CAD) versus CSX.

Materials and methods: Ninety-two CSX and 145 CAD subjects without known diabetes mellitus underwent coronary angiogram for angina.

Results: Vascular cell adhesion molecule (VCAM)-1 (median, 507 versus 431?ng/ml, p?=?0.001) was significantly higher in the CAD group. In the binary regression, VCAM-1 was a significant differential factor for CAD versus CSX.

Discussion and conclusion: Adhesion molecules might be implicated in the differential expression of macro versus microvascular coronary disease.

Trial registration number: NCT01198730 at https://clinicaltrials.gov     

Pre-fledging survival in a Yellow-legged Gull Larus michahellis population in northern Iberia is mostly determined by hatching date

Capsule: Pre-fledging survival in a Yellow-legged Gull Larus michahellis population in northern Iberia is negatively correlated to hatching date.

Aims: To explore which factors have more importance in determining the pre-fledging daily survival rates in a Yellow-legged Gull population from northern Iberia. Specifically, we tested for the effect of hatching date and order, body size and condition and meteorological conditions on pre-fledging survival.

Methods: Cormack–Jolly–Seber models with mixtures were used to model daily survival rates.

Results: Daily survival rates were mostly negatively affected by hatching date.

Conclusions: Hatching date was the most important factor affecting survival of chicks during the pre-fledging period in a Yellow-legged Gull colony from northern Iberia.     

Release kinetics of high-sensitivity cardiac troponins I and T and troponin T upstream open reading frame peptide (TnTuORF) in clinically induced acute myocardial infarction

Context: Troponin T upstream open reading frame peptide (TnTuORF) may be useful as a novel biomarker in acute cardiac syndromes.

Objective: The study examined the early release kinetics of TnTuORF.

Materials and methods: We analyzed the time course of the release of cardiac troponins I and T and TnTuORF in patients (n?=?31) with hypertrophic obstructive cardiomyopathy undergoing transcoronary ablation of septal hypertrophy (TASH).

Results: Fifteen minutes after TASH, the levels of both troponins increased significantly (cTnT median: 18?ng/L versus 27?ng/L; cTnI median: 15?ng/L versus 25?ng/L). TnTuORF showed no variation.

Discussion: We observed a significantly greater increase in cTnI compared with cTnT.

Conclusion: Our results demonstrate that troponin assays allow early detection of myocardial injury, whereas TnTuORF levels remain unchanged in this setting.     

Predictors of leptin concentration and association with cardiovascular risk in patients with coronary artery disease: results from the AtheroGene study

Context: Leptin is produced in white adipose tissue, but also in human coronary atherosclerotic lesions.

Objective: The aim of this study is to assess the prognostic value of leptin in patients with proven coronary artery disease (CAD) (N?=?1907).

Methods: AtheroGene is a contemporary CAD cohort study (N?=?3229). Median follow-up time was 3.8 (Quartile 1/3 with 2.8/4.9) years.

Results: Leptin concentration was associated with a hazard ratio (HR) for the fully adjusted model of HR?=?1.32 in women but was not significant in men. The endpoint cardiovascular death and non-fatal myocardial infarction was observed in 167 patients.

Conclusion: In women with known CAD, increased leptin concentration is useful for predicting cardiovascular death and non-fatal myocardial infarction.     

Riboflavin as an independent and accurate biomarker for adherence in a randomized double-blind and placebo-controlled clinical trial

Background: Medication adherence is critical for success of clinical trials.

Objective: To assess oral riboflavin is an adherence marker.

Methods: Riboflavin was incorporated into active treatment and placebo pills for a clinical trial lasting for 2 years.

Results: The accuracy (area under the receiver operating curve) of urinary riboflavin was 0.91 as a binary classifier of adherence, and was similar or better than for two active study ingredients daidzein (0.92) and genistein (0.87) (all p?<?0.0001). Decreased adherence over time was similar in the two study groups.

Conclusion: Riboflavin is an accurate and useful biomarker for study pill ingestion.     

Bacterial pili,with emphasis on Mycobacterium tuberculosis curli pili: potential biomarkers for point-of care tests and therapeutics

Context: Novel biomarkers are essential for developing rapid diagnostics and therapeutic interventions

Objective: This review aimed to highlight biomarker characterisation and assessment of unique bacterial pili.

Methods: A PubMed search for bacterial pili, diagnostics, vaccine and therapeutics was performed, with emphasis on the well characterised pili.

Results: In total, 46 papers were identified and reviewed.

Conclusion: Extensive analyses of pili enabled by advanced nanotechnology and whole genome sequencing provide evidence that they are strong biomarker candidates. Mycobacterium tuberculosis curli pili are emphasised as important epitopes for the development of much needed point-of-care diagnostics and therapeutics.     

Circulating levels of apoptotic markers and oxidative stress parameters in women with polycystic ovary syndrome: a case-controlled descriptive study

Context: Apoptotic dysregulation plays a role in the pathogenesis of polycystic ovary syndrome (PCOS).

Objective: To evaluate circulatory apoptotic markers and oxidative stress in patients with PCOS.

Materials and methods: Forty-four women with PCOS, and 44 healthy women as controls were enrolled in the study. Oxidative stress parameters and caspases levels were measured in serum.

Results: The caspase 9 level was significantly lower and related with oxidant status in patients with PCOS, while the circulating levels of caspases 3 and 7 were statistically similar in both groups.

Discussion: This study is the first report demonstrating the circulating levels of apoptotic markers and their relationship with oxidant status in PCOS.

Conclusion: The circulating caspase 9 and oxidant status might contribute to apoptotic dysregulation in PCOS.     

Association between polymorphisms in IL21 gene and risk for sepsis

Context: Sepsis is now the leading cause of death in the noncardiovascular intensive care unit (ICU).

Objective: To investigate whether polymorphisms in IL21 gene contribute to sepsis susceptibility.

Materials and methods: Three single-nucleotide polymorphisms of IL21 (rs907715, rs2055979, rs12508721) were genotyped by TaqMan assay in patients with sepsis and control subjects.

Results: Polymorphisms rs2055979 and rs12508721 in IL21 were more frequent in sepsis patients compared to general population. But allele frequency of rs907715 was not significantly different between sepsis patients and control subjects.

Conclusion: Polymorphisms in IL21 may be associated with sepsis risk.     

Micronucleus frequency and content in healthy relatives of cancer patients

Objective: To compare chromosomal damage in lymphocytes of individuals who did or did not report first-degree relatives with cancer.

Materials and methods: Cases and controls (68 each) were matched for sex, age and radon exposure. Chromosomal damage was quantified as frequency of micronucleus-containing cells and proportion of centromere-free micronuclei.

Results: Individuals not reporting cancer in their families showed lower values of both the frequency of micronucleus-containing cells (n.s.) and the proportion of centromere-free micronuclei (p?<?0.05) in some subgroups, but not in all.

Conclusion: The chromosomal damage observed may be due to inheritable genomic instability, but environmental influences cannot be excluded.     

Blood markers of inflammation and endothelial dysfunction in cardioembolic stroke: systematic review and meta-analysis

Context: Various processes including inflammation and endothelial dysfunction have been implicated in the pathogenesis of cardioembolic (CE) strokes.

Objective: To review the evidence and investigate the association between immune-inflammatory biomarkers and CE strokes versus other stroke subtypes.

Methods: We systematically reviewed the literature (sources: MEDLINE, web-based register http://stroke-biomarkers.com, reference lists) with quality assessment and meta-analysis of selected articles.

Results: The most consistent association was found between C-reactive protein (CRP) and CE strokes when compared to other stroke subtypes (standardized mean difference 0.223 (0.116, 0.343); p?<?0.001)

Conclusions: Our findings confirm a possible association between selected inflammatory biomarkers and CE stroke.     

The effects of nifedipine on respiratory mechanics investigated by theend-inflation occlusion method in the rat

Context: Calcium channel blockers may theoretically exhibit relaxing effects not only on vascular smooth muscle but also on airway smooth muscle.

Objective: To investigate possible effects of nifedipine on respiratory mechanics in the rat.

Methods: Respiratory system mechanical parameters were measured by the end-inflation occlusion method in the rat in vivo before and after the intraperitoneal administration of nifedipine.

Results: We found that nifedipine affects respiratory mechanics, inducing a reduction of airway resistance and of respiratory system elastance, probably because of a relaxing action on airway and parenchimal smooth muscle cells.

Conclusion: Should these results be further confirmed by human investigations, a possible role of nifedipine in pharmacological respiratory system’s diseases treatment may be suggested.     

Prognostic significance of insulin-like growth factor-I serum levels in acute decompensation of cirrhosis

Context: IGF-I serum levels are suppressed in cirrhosis, but its prognostic significance is unknown.

Objectives: To investigate the prognostic value of IGF-I in patients admitted for acute decompensation of cirrhosis.

Materials and methods: Cohort study that included 103 patients. IGF-I was measured by enzyme-linked immunosorbent assay (ELISA).

Results: Ninety-day mortality was 26.2% and it was independently associated with MELD, age and IGF-I. The Kaplan–Meier survival probability at 90 days was 94.3% in patients with IGF-I?≥13?ng/mL and 63.2% for patients with IGF-I?<13?ng/mL (p?=?.001).

Discussion and conclusion: IGF-I levels are independently associated with mortality in acute decompensation of cirrhosis.     

Impact of CT-apelin and NT-proBNP on identifying non-responders to cardiac resynchronization therapy

Context: Assessment of response to cardiac resynchronization therapy (CRT) is essential.

Objective: To assess the predictive value of CT-apelin together with NT-proBNP in patients undergoing CRT.

Methods: Serum CT-apelin and NT-proBNP were measured by ELISA before, and six months after CRT. Primary endpoint was non-response (<4% increase in LVEF) after six months.

Results: From 81 patients, 15 proved to be non-responders. Six-month CT-apelin was superior compared to NT-proBNP in identifying non-responders by multivariate ROC (CT-apelin: p?=?0.01, NT-proBNP: p?=?0.13) and by logistic regression (CT-apelin: p?=?0.01, NT-proBNP: p?=?0.41) analyses.

Conclusion: Six-month CT-apelin might be a valuable novel biomarker in identifying non-responders to CRT that was superior to NT-proBNP.     

Biomarker discovery for drug-induced phospholipidosis: phenylacetylglycine to hippuric acid ratio in urine and plasma as potential markers

Context: Drug-induced phospholipidosis is one of the significant concerns in drug development, especially in safety assessment and noninvasive diagnostic tool is highly desirable.

Objective: The objective of this study is to explored novel biomarkers for phospholipidosis using a metabolomic approach.

Method: NMR spectrometry and LC/MS/MS analyses were applied to urine and plasma of rats administrated cationic amphiphilic drugs.

Results: The phenylacetylglycine to hippuric acid ratio in plasma was increased in time and dose-dependent manners; and it was well correlated with histopathological observation.

Conclusion: The plasma phenylacetylglycine to hippuric acid ratio is a potential marker in monitoring drug-induced phospholipidosis.