Fungal Integrated Histomolecular Diagnosis Using Targeted Next-Generation Sequencing on Formalin-Fixed Paraffin-Embedded Tissues.

Histopathology is the gold standard for fungal infection (FI) diagnosis, but it does not provide a genus and/or species identification. The objective of the present study was to develop targeted next-generation sequencing (NGS) on formalin-fixed tissue samples (FTs) to achieve a fungal integrated histomolecular diagnosis. Nucleic acid extraction was optimized on a first group of 30 FTs with Aspergillus fumigatus or Mucorales infection by macrodissecting the microscopically identified fungal-rich area and comparing Qiagen and Promega extraction methods through DNA amplification by A. fumigatus and Mucorales primers. Targeted NGS was developed on a second group of 74 FTs using three primer pairs (ITS-3/ITS-4, MITS-2A/MITS-2B, and 28S-12-F/28S-13-R) and two databases (UNITE and RefSeq). A prior fungal identification of this group was established on fresh tissues. Targeted NGS and Sanger sequencing results on FTs were compared. To be valid, the molecular identifications had to be compatible with the histopathological analysis. In the first group, the Qiagen method yielded a better extraction efficiency than the Promega method (100% and 86.7% of positive PCRs, respectively). In the second group, targeted NGS allowed fungal identification in 82.4% (61/74) of FTs using all primer pairs, in 73% (54/74) using ITS-3/ITS-4, in 68.9% (51/74) using MITS-2A/MITS-2B, and in 23% (17/74) using 28S-12-F/28S-13-R. The sensitivity varied according to the database used (81% [60/74] using UNITE compared to 50% [37/74] using RefSeq [P = 0.000002]). The sensitivity of targeted NGS (82.4%) was higher than that of Sanger sequencing (45.9%; P < 0.00001). To conclude, fungal integrated histomolecular diagnosis using targeted NGS is suitable on FTs and improves fungal detection and identification.

Sarcoidosis and the mTOR, Rac1, and Autophagy Triad.

Sarcoidosis is an enigmatic multisystem disease characterized by the development and accumulation of granulomas: a compact collection of macrophages that have differentiated into epithelioid cells and which are associated with T helper (Th)1 and Th17 cells. Although no single causative factor has been shown to underlie sarcoidosis in humans, its etiology has been related to microbial, environmental, and genetic factors. We examine how these factors play a role in sarcoidosis pathogenesis. Specifically, we propose that dysfunction of mTOR, Rac1, and autophagy-related pathways not only hampers pathogen or nonorganic particle clearance but also participates in T cell and macrophage dysfunction, driving granuloma formation. This concept opens new avenues for potentially treating sarcoidosis and may serve as a blueprint for other granulomatous disorders.

Chronic Exposure to Secondary Organic Aerosols Causes Lung Tissue Damage.

Recently, secondary organic aerosols (SOAs) emerged as a predominant component of fine particulate matter. However, the pathogenic mechanism(s) of SOAs are still poorly understood. Herein, we show that chronic exposure of mice to SOAs resulted in lung inflammation and tissue destruction. Histological analyses found lung airspace enlargement associated with massive inflammatory cell recruitment predominated by macrophages. Concomitant with such cell influx, our results found changes in the levels of a series of inflammatory mediators in response to SOA. Interestingly, we observed that the expression of the genes encoding for TNF-α and IL-6 increased significantly after one month of exposure to SOAs; mediators that have been largely documented to play a role in chronic pulmonary inflammatory pathologies. Cell culture studies confirmed these in vivo findings. Of importance as well, our study indicates increased matrix metalloproteinase proteolytic activity suggesting its contribution to lung tissue inflammation and degradation. Our work represents the first in vivo study, which reports that chronic exposure to SOAs leads to lung inflammation and tissue injury. Thus, we hope that these data will foster new studies to enhance our understanding of the underlying pathogenic mechanisms of SOAs and perhaps help in the design of therapeutic strategies against SOA-mediated lung injury.

Pathogenic Mechanisms of Secondary Organic Aerosols.

Air pollution represents a major health problem and an economic burden. In recent years, advances in air pollution research has allowed particle fractionation and identification of secondary organic aerosol (SOA). SOA is formed from either biogenic or anthropogenic emissions, through a mass transfer from the gaseous mass to the particulate phase in the atmosphere. They can have deleterious impact on health and the mortality of individuals with chronic inflammatory diseases. The pleiotropic effects of SOA could involve different and interconnected pathogenic mechanisms ranging from oxidative stress, inflammation, and immune system dysfunction. The purpose of this review is to present recent findings about SOA pathogenic roles and potential underlying mechanisms focusing on the lungs; the latter being the primary exposed organ to atmospheric pollutants.

Weight loss and abnormal lung inflammation in mice chronically exposed to secondary organic aerosols.

Secondary organic aerosols (SOAs) have emerged recently as a major component of fine particulate matter. Cell culture studies revealed a role for SOAs in cell oxidative stress, toxicity and inflammation and only a few studies investigated short-term SOA exposure in animal models. Here, mice were chronically exposed to naphthalene-derived SOAs for one and two months. Weight monitoring indicated a marked mass loss, especially in females, following chronic exposure to SOAs. Significantly, a cytokine antibody microarray approach revealed SOA-induced abnormal lung inflammation similar to that seen in cigarette smoke-induced chronic obstructive pulmonary disease (COPD). This in vivo study testifies to the pathogenic role of sub-chronic SOA exposure on human health.

Characterization of Lung Inflammatory Response to Aspergillus fumigatus Spores.

The airway exposure to Aspergillus fumigatus spores (AFsp) is associated with an inflammatory response, potentially leading to allergic and/or chronic pulmonary aspergillosis. The aim of our study is to better understand the host response, first in vitro, then in vivo, following the chronic exposure of mice to AFsp. We investigated the inflammatory response to AFsp in cell mono- and co-culture systems with murine macrophages and alveolar epithelial cells. The mice were subjected to two intranasal instillations using 105 AFsp. Their lungs were processed for inflammatory and histopathological analyses. In cell culture, the gene expressions significantly increased for TNF-α, CXCL-1, CXCL-2, IL-1ß, IL-1α and GM-CSF in macrophages, with these increases being limited for TNF-α, CXCL-1 and IL-1α in epithelial cells. In co-culture, increases in the TNF-α, CXCL-2 and CXCL-1 gene expressions were observed to be associated with increased protein levels. The in vivo lung histological analyses of mice challenged by AFsp showed cellular infiltrates in the peribronchial and/or alveolar spaces. A Bio-Plex approach on the bronchoalveolar lavage revealed significant increases in the protein secretion of selected mediators of the challenged mice compared to the unchallenged mice. In conclusion, the exposure to AFsp resulted in a marked inflammatory response of macrophages and epithelial cells. These inflammatory findings were confirmed in mouse models associated with lung histologic changes.

Nanocapsules containing Saussurea lappa essential oil: Formulation, characterization, antidiabetic, anti-cholinesterase and anti-inflammatory potentials.

Plant-based remedies have been widely used for the management of variable diseases due to their safety and less side effects. In the present study, we investigated Saussurea lappa CB. Clarke. (SL) given its largely reported medicinal effects. Specifically, our objective was to provide an insight into a new polymethyl methacrylate based nanocapsules as carriers of SL essential oil and characterize their biologic functions. The nanoparticles were prepared by nanoprecipitation technique, characterized and analyzed for their cytotoxicity, anti-inflammatory, anti-Alzheimer and antidiabetic effects. The results revealed that the developed nanoparticles had a diameter around 145 nm, a polydispersity index of 0.18 and a zeta potential equal to +45 mV and they did not show any cytotoxicity at 25 µg·mL-1. The results also showed an anti-inflammatory activity (reduction in metalloprotease MMP-9 enzyme activity and RNA expression of inflammatory cytokines: TNF-α, GM-CSF and IL1ß), a high anti-Alzheimer's effect (IC50 around 25.0 and 14.9 µg·mL-1 against acetylcholinesterase and butyrylcholinesterase, respectively), and a strong antidiabetic effect (IC50 were equal to 22.9 and 75.8 µg·mL-1 against α-amylase and α-glucosidase, respectively). Further studies are required including the in vivo studies (e.g., preclinical), the pharmacokinetic properties, the bioavailability and the underlying associated metabolic pathways.

Magnetic Resonance Imaging of Protease-Mediated Lung Tissue Inflammation and Injury.

Pulmonary inflammation usually involves strong neutrophil recruitment with a marked release of proteases such as neutrophil elastase (NE). Noninvasive in vivo assessment of unregulated elastase activity in the lungs would provide a valuable diagnostic tool. Here, it is proposed to use Overhauser-enhanced magnetic resonance imaging (OMRI) in mice where inflammation was induced by the instillation of lipopolysaccharide (LPS). OMRI contrast in the lungs was generated by a dedicated NE free radical substrate. The free radical decayed more rapidly in LPS-treated mouse lungs than in control mice, indicating the occurrence of increased proteolysis under inflammation. Preclinical detection of abnormal proteolysis opens the way for new diagnosis modality and antiprotease testing in vivo.

Human serum albumin nanoparticles as nanovector carriers for proteins: Application to the antibacterial proteins "neutrophil elastase" and "secretory leukocyte protease inhibitor".

The use of proteins and defined amino acid sequences as therapeutic drugs have gained a certain interest in the past decade. However, protein encapsulation within protein nanoparticles was never endeavored. For this reason, human serum albumin (HSA) nanoparticles were prepared by nanoprecipitation method. The process was optimized, and particles were obtained with a size of 120 nm and zeta potential of -25 mV. Neutrophil elastase (NE) and secretory leukocyte protease inhibitor (SLPI) were encapsulated separately within HSA nanoparticles. Gel electrophoresis and western blot studies demonstrate the successful encapsulation and the stability of the particles. On the other hand, enzymatic assays show that encapsulated NE lost its proteolytic activity, whereas encapsulated SLPI maintained its inhibitory property. In addition, the antibacterial studies showed that both formulations were able to drastically reduce bacterial growth of Pseudomonas aeruginosa. This work showed the possibility of using both NE and SLPI as anti-bacterial agents through encapsulation within HSA nanoparticles.

Neutrophil Elastase Activity Imaging: Recent Approaches in the Design and Applications of Activity-Based Probes and Substrate-Based Probes.

The last few decades of protease research has confirmed that a number of important biological processes are strictly dependent on proteolysis. Neutrophil elastase (NE) is a critical protease in immune response and host defense mechanisms in both physiological and disease-associated conditions. Particularly, NE has been identified as a promising biomarker for early diagnosis of lung inflammation. Recent studies have shown an increasing interest in developing methods for NE activity imaging both in vitro and in vivo. Unlike anatomical imaging modalities, functional molecular imaging, including enzymatic activities, enables disease detection at a very early stage and thus constitutes a much more accurate approach. When combined with advanced imaging technologies, opportunities arise for measuring imbalanced proteolytic activities with unprecedented details. Such technologies consist in building the highest resolved and sensitive instruments as well as the most specific probes based either on peptide substrates or on covalent inhibitors. This review outlines strengths and weaknesses of these technologies and discuss their applications to investigate NE activity as biomarker of pulmonary inflammatory diseases by imaging.

Essential Oils-Loaded Polymer Particles: Preparation, Characterization and Antimicrobial Property.

In the last few years, essential oils (EOs) derived from plants have aroused great interest due to their well-known antimicrobial activity. Unfortunately, they present several limitations in their use, such as photosensitivity, temperature sensitivity, high volatility, and poor water solubility. The encapsulation technique represents a good solution to these problems and ensures protection of the functional properties of essential oils. In this work, bergamot essential oil (BEO) and sweet orange essential oil (OEO) loaded-Eudragit® RS 100 (EuRS100) nanoparticles (NPs) were prepared by using the nanoprecipitation technique. We obtained nanoparticles characterized by a mean diameter of 57 to 208 nm and a positive surface charge (39 to 74 mV). The antibacterial activity of the obtained systems against Escherichia coli was in vitro investigated. We demonstrated that both orange and bergamot essential oils were successfully encapsulated and our nanoparticles have good antibacterial activity. Finally, in order to evaluate the potential applicability of OEONps to prolong fresh orange juice shelf-life, survival of E. coli during a storage period of one week at 25 °C was investigated: Orange essential oil-loaded nanoparticles (OEONPs) have been able to prolong the orange juice shelf life.

HoxA9 transforms murine myeloid cells by a feedback loop driving expression of key oncogenes and cell cycle control genes.

Ectopic expression of the oncogenic transcription factor HoxA9 is a major cause of acute myeloid leukemia (AML). Here, we demonstrate that HoxA9 is a specific substrate of granule proteases. Protease knockout allowed the comprehensive determination of genome-wide HoxA9 binding sites by chromatin immunoprecipitation sequencing in primary murine cells and a human AML cell line. The kinetics of enhancer activity and transcription rates in response to alterations of an inducible HoxA9 were determined. This permitted identification of HoxA9-controlled enhancers and promoters, allocation to their respective transcription units, and discrimination against HoxA9-bound, but unresponsive, elements. HoxA9 triggered an elaborate positive-feedback loop that drove expression of the complete Hox-A locus. In addition, it controlled key oncogenic transcription factors Myc and Myb and directly induced the cell cycle regulators Cdk6 and CyclinD1, as well as telomerase, drawing the essential blueprint for perturbation of proliferation by leukemogenic HoxA9 expression.

Protein-Based Nanoparticle Preparation via Nanoprecipitation Method.

Nanoparticles are nowadays largely investigated in the field of drug delivery. Among nanoparticles, protein-based particles are of paramount importance since they are natural, biodegradable, biocompatible, and nontoxic. There are several methods to prepare proteins containing nanoparticles, but only a few studies have been dedicated to the preparation of protein- based nanoparticles. Then, the aim of this work was to report on the preparation of bovine serum albumin (BSA)-based nanoparticles using a well-defined nanoprecipitation process. Special attention has been dedicated to a systematic study in order to understand separately the effect of each operating parameter of the method (such as protein concentration, solvent/non-solvent volume ratio, non-solvent injection rate, ionic strength of the buffer solution, pH, and cross-linking) on the colloidal properties of the obtained nanoparticles. In addition, the mixing processes (batch or drop-wise) were also investigated. Using a well-defined formulation, submicron protein-based nanoparticles have been obtained. All prepared particles have been characterized in terms of size, size distribution, morphology, and electrokinetic properties. In addition, the stability of nanoparticles was investigated using Ultraviolet (UV) scan and electrophoresis, and the optimal conditions for preparing BSA nanoparticles by the nanoprecipitation method were concluded.

An elastase activity reporter for Electronic Paramagnetic Resonance (EPR) and Overhauser-enhanced Magnetic Resonance Imaging (OMRI) as a line-shifting nitroxide.

Pulmonary inflammatory diseases are a major burden worldwide. They have in common an influx of neutrophils. Neutrophils secrete unchecked proteases at inflammation sites consequently leading to a protease/inhibitor imbalance. Among these proteases, neutrophil elastase is responsible for the degradation of the lung structure via elastin fragmentation. Therefore, monitoring the protease/inhibitor status in lungs non-invasively would be an important diagnostic tool. Herein we present the synthesis of a MeO-Suc-(Ala)2-Pro-Val-nitroxide, a line-shifting elastase activity probe suitable for Electron Paramagnetic Resonance spectroscopy (EPR) and Overhauser-enhanced Magnetic Resonance Imaging (OMRI). It is a fast and sensitive neutrophil elastase substrate with Km = 15 ±â€¯2.9 µM, kcat/Km = 930,000 s-1 M-1 and Km = 25 ±â€¯5.4 µM, kcat/Km = 640,000 s-1 M-1 for the R and S isomers, respectively. These properties are suitable to detect accurately concentrations of neutrophil elastase as low as 1 nM. The substrate was assessed with broncho-alveolar lavages samples derived from a mouse model of Pseudomonas pneumonia. Using EPR spectroscopy we observed a clear-cut difference between wild type animals and animals deficient in neutrophil elastase or deprived of neutrophil Elastase, Cathepsin G and Proteinase 3 or non-infected animals. These results provide new preclinical ex vivo and in vivo diagnostic methods. They can lead to clinical methods to promote in time lung protection.

Combination of PCR and dual nanoparticles for detection of Plasmodium falciparum.

Highly reactive particle-based DNA amplification was developed for the detection of the Pfg377 gene from P. falciparum gametocytes using functional magnetic latex particles (MLPs) and quantum dots encapsulated polymer particles (QDs-PPs). Firstly, MLPs were prepared from the precipitation of iron oxide, polymerization using initiator, and adsorption of aminodextran (AMD) so as to provide amino-functionalized MLPs. Furthermore, amino-containing polymer particles (PPs) were prepared by emulsifier-free polymerization and encapsulated with fluorescent quantum dots (QDs) for use as a signaling support. Subsequently, poly(maleic anhydride-alt-methyl vinyl ether) (PMAMVE) copolymer was effectively used for rapid and simple grafting of amino-modified DNA primers onto the surface of amino-functionalized particles thereby providing a promising method for particle immobilization. Herein, primer-grafted particles were applied in the amplification of the Pfg377 gene using the PCR approach. After amplification, PCR products containing PMAMVE-grafted MLPs and QDs-PPs were separated using a magnet and examined via a fluorescence microscope. PMAMVE-grafted particles were not found to inhibit the PCR reaction while facilitating efficient fluorescent detection of the PCR product. Results showed high sensitivity and specificity for the detection of amplified Pfg377 gene within only a few steps. This procedure represents a novel improvement to the post-amplification analysis.