Trypanotoxic activity of thiosemicarbazone iron chelators.

Only a few drugs are available for treating sleeping sickness and nagana disease; parasitic infections caused by protozoans of the genus Trypanosoma in sub-Saharan Africa. There is an urgent need for the development of new medicines for chemotherapy of these devastating diseases. In this study, three newly designed thiosemicarbazone iron chelators, TSC24, Dp44mT and 3-AP, were tested for in vitro activity against bloodstream forms of Trypanosoma brucei and human leukaemia HL-60 cells. In addition to their iron chelating properties, TSC24 and Dp44mT inhibit topoisomerase IIα while 3-AP inactivates ribonucleotide reductase. All three compounds exhibited anti-trypanosomal activity, with minimum inhibitory concentration (MIC) values ranging between 1 and 100 µM and 50% growth inhibition (GI50) values of around 250 nM. Although the compounds did not kill HL-60 cells (MIC values >100 µM), TSC24 and Dp44mT displayed considerable cytotoxicity based on their GI50 values. Iron supplementation partly reversed the trypanotoxic and cytotoxic activity of TSC24 and Dp44mT but not of 3-AP. This finding suggests possible synergy between the iron chelating and topoisomerase IIα inhibiting activity of the compounds. However, further investigation using separate agents, the iron chelator deferoxamine and the topoisomerase II inhibitor epirubicin, did not support any synergy for the interaction of iron chelation and topoisomerase II inhibition. Furthermore, TSC24 was shown to induce DNA degradation in bloodstream forms of T. brucei indicating that the mechanism of trypanotoxic activity of the compound is topoisomerase II independent. In conclusion, the data support further investigation of thiosemicarbazone iron chelators with dual activity as lead compounds for anti-trypanosomal drug development

Serum opsonin ficolin-A enhances host-fungal interactions and modulates cytokine expression from human monocyte-derived macrophages and neutrophils following Aspergillus fumigatus challenge.

Invasive aspergillosis is a devastating invasive fungal disease associated with a high mortality rate in the immunocompromised, such as leukaemia patients, transplant patients and those with HIV/AIDS. The rodent serum orthologue of human L-ficolin, ficolin-A, can bind to and opsonize Aspergillus fumigatus, the pathogen that causes invasive aspergillosis, and may participate in fungal defence. Using human monocyte-derived macrophages and neutrophils isolated from healthy donors, we investigated conidial association and fungal viability by flow cytometry and microscopy. Additionally, cytokine production was measured via cytometric bead arrays. Ficolin-A opsonization was observed to significantly enhance association of conidia, while also inhibiting hyphal growth and contributing to increased fungal killing following incubation with monocyte-derived macrophages and neutrophils. Additionally, ficolin-A opsonization was capable of manifesting a decrease in IL-8, IL-1β, IL-6, IL-10 and TNF-α production from MDM and IL-1β, IL-6 and TNF-α from neutrophils 24 h post-infection. In conclusion, rodent ficolin-A is functionally comparable to human L-ficolin and is capable of modulating the innate immune response to A. fumigatus, down-regulating cytokine production and could play an important role in airway immunity

Plasma Vascular Endothelial Growth Factor Concentration and Alveolar Nitric Oxide as Potential Predictors of Disease Progression and Mortality in Idiopathic Pulmonary Fibrosis.

BACKGROUND: Declining lung function signifies disease progression in idiopathic pulmonary fibrosis (IPF). Vascular endothelial growth factor (VEGF) concentration is associated with declining lung function in 6 and 12-month studies. Alveolar nitric oxide concentration (CANO) is increased in patients with IPF, however its significance is unclear. This study investigated whether baseline plasma VEGF concentration and CANO are associated with disease progression or mortality in IPF. METHODS: 27 IPF patients were studied (maximum follow-up 65 months). Baseline plasma VEGF concentration, CANO and pulmonary function tests (PFTs) were measured. PFTs were performed the preceding year and subsequent PFTs and data regarding mortality were collected. Disease progression was defined as one of: death, relative decrease of ≥10% in baseline forced vital capacity (FVC) % predicted, or relative decrease of ≥15% in baseline single breath diffusion capacity of carbon monoxide (TLCO-SB) % predicted. RESULTS: Plasma VEGF concentration was not associated with progression-free survival or mortality. There was a trend towards shorter time to disease progression and death with higher CANO. CANO was significantly higher in patients with previous declining versus stable lung function. CONCLUSION: The role of VEGF in IPF remains uncertain. It may be of value to further investigate CANO in IPF. KEYWORDS: alveolar nitric oxide; idiopathic pulmonary fibrosis; vascular endothelial growth facto

The Effect of Cellular Coenzyme Q10 Deficiency on Lysosomal Acidification

Coenzyme Q10 (CoQ10) deficiency currently represents the only treatable mitochondrial disorder, however, little is known about how it may affect other organelles. The lysosome has been found to have a large concentration of CoQ10 localised at its membrane; additionally, it has been suggested that it plays a role in the normal acidification of the lysosomal lumen. As a result, in this study we assessed the effect of CoQ10 deficiency on lysosomal acidification. In order to investigate this, a neuronal cell model of CoQ10 deficiency was established via the treatment of SH-SY5Y cells with para-aminobenzoic acid (PABA). This method works through the competitive inhibition of the CoQ10 biosynthetic pathway enzyme, CoQ2. A single 1 mM (5 days) treatment with PABA resulted in a decrease of up to 58% in cellular CoQ10 (p < 0.05). It was found that this resulted in a significant decrease in fluorescence of both the LysoSensor (23%) and LysoTracker (35%) probes used to measure lysosomal pH (p < 0.05). It was found that subsequent treatment with CoQ10 (5 µM, 3 days) was able to restore cellular CoQ10 concentration (p < 0.005), which was associated with an increase in fluorescence from both probes to around 90% of controls (p < 0.05), suggesting a restoration of lysosomal pH. This study provides insights into the association between lysosomal pH and cellular CoQ10 status and the possibility that a deficit in the status of this isoprenoid may result in an impairment of lysosomal acidification

Ficolins and the Recognition of Pathogenic Microorganisms: An Overview of the Innate Immune Response and Contribution of Single Nucleotide Polymorphisms

Ficolins are innate pattern recognition receptors (PRR) and play integral roles within the innate immune response to numerous pathogens throughout the circulation, as well as within organs. Pathogens are primarily removed by direct opsonisation following the recognition of cell surface carbohydrates and other immunostimulatory molecules or via the activation of the lectin complement pathway, which results in the deposition of C3b and the recruitment of phagocytes. In recent years, there have been a number of studies implicating ficolins in the recognition and removal of numerous bacterial, viral, fungal, and parasitic pathogens. Moreover, there has been expanding evidence highlighting that mutations within these key immune proteins, or the possession of particular haplotypes, enhance susceptibility to colonization by pathogens and dysfunctional immune responses. This review will therefore encompass previous knowledge on the role of ficolins in the recognition of bacterial and viral pathogens, while acknowledging the recent advances in the immune response to fungal and parasitic infections. Additionally, we will explore the various genetic susceptibility factors that predispose individuals to infection

Modulation of mitochondrial activity in HaCaT keratinocytes by the cell penetrating peptide Z-Gly-RGD(DPhe)-mitoparan.

OBJECTIVE: Biologically active cell penetrating peptides (CPPs) are an emerging class of therapeutic agent. The wasp venom peptide mastoparan is an established CPP that modulates mitochondrial activity and triggers caspase-dependent apoptosis in cancer cells, as does the mastoparan analogue mitoparan (mitP). Mitochondrial depolarisation and activation of the caspase cascade also underpins the action of dithranol, a topical agent for treatment of psoriasis. The effects of a potent mitP analogue on mitochondrial activity were therefore examined to assess its potential as a novel approach for targeting mitochondria for the treatment of psoriasis. RESULTS: In HaCaT keratinocytes treated with the mitP analogue Z-Gly-RGD(DPhe)-mitP for 24 h, a dose-dependent loss of mitochondrial activity was observed using the methyl-thiazolyl-tetrazolium (MTT) assay. At 10 μmol L-1, MTT activity was less than 30% that observed in untreated cells. Staining with the cationic dye JC-1 suggested that Z-Gly-RGD(DPhe)-mitP also dissipated the mitochondrial membrane potential, with a threefold increase in mitochondrial depolarisation levels. However, caspase activity appeared to be reduced by 24 h exposure to Z-Gly-RGD(DPhe)-mitP treatment. Furthermore, Z-Gly-RGD(DPhe)-mitP treatment had little effect on overall cell viability. Our findings suggest Z-Gly-RGD(DPhe)-mitP promotes the loss of mitochondrial activity but does not appear to evoke apoptosis in HaCaT keratinocytes

The serum opsonin L-ficolin is detected in lungs of human transplant recipients following fungal infections and modulates inflammation and killing of Aspergillus fumigatus.

BACKGROUND: Invasive aspergillosis (IA) is a life-threatening systemic fungal infection in immunocompromised individuals that is caused by Aspergillus fumigatus. The human serum opsonin, L-ficolin, has been observed to recognize A. fumigatus and could participate in fungal defense. METHODS: Using lung epithelial cells, primary human monocyte-derived macrophages (MDMs), and neutrophils from healthy donors, we assessed phagocytosis and killing of L-ficolin-opsonized live A. fumigatus conidia by flow cytometry and microscopy. Additionally, cytokines were measured by cytometric bead array, and L-ficolin was measured in bronchoalveolar lavage (BAL) fluid from lung transplant recipients by enzyme-linked immunosorbent assay. RESULTS: L-ficolin opsonization increased conidial uptake and enhanced killing of A. fumigatus by MDMs and neutrophils. Opsonization was also shown to manifest an increase in interleukin 8 release from A549 lung epithelial cells but decreased interleukin 1β, interleukin 6, interleukin 8, interleukin 10, and tumor necrosis factor α release from MDMs and neutrophils 24 hours after infection. The concentration of L-ficolin in BAL fluid from patients with fungal infection was significantly higher than that for control subjects (P = .00087), and receiving operating characteristic curve analysis highlighted the diagnostic potential of L-ficolin for lung infection (area under the curve, 0.842; P < .0001). CONCLUSIONS: L-ficolin modulates the immune response to A. fumigatus. Additionally, for the first time, L-ficolin has been demonstrated to be present in human lungs

Toxicological assessment of nanoparticle interactions with the pulmonary system.

Nanoparticle(NP)-based materials have breakthrough applications in many fields of life, such as in engineering, communications and textiles industries; food and bioenvironmental applications; medicines and cosmetics, etc. Biomedical applications of NPs are very active areas of research with successful translation to pharmaceutical and clinical uses overcoming both pharmaceutical and clinical challenges. Although the attractiveness and enhanced applications of these NPs stem from their exceptional properties at the nanoscale size, i.e. 1-1000 nm, they exhibit completely different physicochemical profiles and, subsequently, toxicological profiles from their parent bulk materials. Hence, the clinical evaluation and toxicological assessment of NPs interactions within biological systems are continuously evolving to ensure their safety at the nanoscale. The pulmonary system is one of the primary routes of exposure to airborne NPs either intentionally, via aerosolized nanomedicines targeting pulmonary pathologies such as cancer or asthma, or unintentionally, via natural NPs and anthropogenic (man-made) NPs. This review presents the state-of-the-art, contemporary challenges, and knowledge gaps in the toxicological assessment of NPs interactions with the pulmonary system. It highlights the main mechanisms of NP toxicity, factors influencing their toxicity, the different toxicological assessment methods and their drawbacks, and the recent NP regulatory guidelines based on literature collected from the research pool of NPs interactions with lung cell lines, in vivo inhalation studies, and clinical trials

Adjunctive treatment with oral AKL1, a botanical nutraceutical, in chronic obstructive pulmonary disease

Purpose: The objective of this pilot trial was to evaluate the safety and efficacy of AKL1, a patented botanical formulation containing extracts of Picrorhiza kurroa, Ginkgo biloba, and Zingiber officinale, as add-on therapy for patients with chronic obstructive pulmonary disease (COPD) and chronic cough. Patients and methods: This randomized, double-blind, placebo-controlled trial enrolled male and female patients .18 years old with COPD and Leicester Cough Questionnaire (LCQ) score of ,18. The 10-week study period comprised a 2-week single-blind placebo run-in period followed by add-on treatment with AKL1 or placebo twice daily for 8 weeks. The primary study endpoint was the change from week 0 to week 8 in cough-related health status, as assessed by the LCQ. Results: Of 33 patients enrolled, 20 were randomized to AKL1 and 13 to placebo. Patients included 19 (58%) men and 14 (42%) women of mean (standard deviation [SD]) age of 67 (9.4) years; 15 (45%) patients were smokers and 16 (49%) were ex-smokers. The mean (SD) change from baseline in LCQ score at 8 weeks was 2.3 (4.9) in the AKL1 group and 0.6 (3.7) in the placebo group, with mean difference in change of 1.8 (95% confidence interval: -1.5 to 5.1; P=0.28). The St George’s Respiratory Questionnaire score improved substantially in the AKL1 treatment group by a mean (SD) of -7.7 (11.7) versus worsening in the placebo group (+1.5 [9.3]), with mean difference in change of -9.2 (95% confidence interval: -19.0 to 0.6; P=0.064). There were no significant differences between treatment groups in change from baseline to week 8 in other patient-reported measures, lung function, or the 6-minute walk distance. Conclusion: Further study is needed with a larger patient population and over a longer duration to better assess the effects of add-on therapy with AKL1 in COPD

Screening disability insurance applications

This paper investigates the effects of stricter screening of disability insurance applications. A large-scale experiment was setup where in two of the 26 Dutch regions case workers of the disability insurance administration were instructed to screen applications more stringently. The empirical results show that stricter screening reduces long-term sickness absenteeism and disability insurance applications. We find evidence for direct effects of stricter screening on work resumption during the period of sickness absence and for self-screening by potential disability insurance applicants. Stricter screening seems to improve targeting efficiency, without inducing negative spillover effects to the inflow into unemployment insurance. The costs of stricter screening are only a small fraction of the monetary benefits.Disability insurance; experiment; policy evaluation; sickness absenteeism; self-screening