Advances in 3D Bioprinting for Cancer Biology and Precision Medicine: From Matrix Design to Application.

The tumor microenvironment is highly complex owing to its heterogeneous composition and dynamic nature. This makes tumors difficult to replicate using traditional 2D cell culture models that are frequently used for studying tumor biology and drug screening. This often leads to poor translation of results between in vitro and in vivo and is reflected in the extremely low success rates of new candidate drugs delivered to the clinic. Therefore, there has been intense interest in developing 3D tumor models in the laboratory that are representative of the in vivo tumor microenvironment and patient samples. 3D bioprinting is an emerging technology that enables the biofabrication of structures with the virtue of providing accurate control over distribution of cells, biological molecules, and matrix scaffolding. This technology has the potential to bridge the gap between in vitro and in vivo by closely recapitulating the tumor microenvironment. Here, a brief overview of the tumor microenvironment is provided and key considerations in biofabrication of tumor models are discussed. Bioprinting techniques and choice of bioinks for both natural and synthetic polymers are also outlined. Lastly, current bioprinted tumor models are reviewed and the perspectives of how clinical applications can greatly benefit from 3D bioprinting technologies are offered.

Slower degradation rate of cytarabine in blood samples from acute myeloid leukemia by comparison with control samples.

PURPOSE: Cytarabine, a key chemotherapy agent for acute myeloid leukemia (AML) treatment, is deaminated into inactive uracil-arabinoside by cytidine deaminase. This deamination leads to samples stability issues with respect to clinical pharmacokinetic trials. The aim of our study was to study in vitro cytarabine stability in blood samples obtained from AML patients. METHODS: Cytarabine quantification was performed using a fully validated LC/MS/MS method. In vitro cytarabine stability was assessed at room temperature over 24 h in samples coming from 14 AML patients and 7 control patients (CTRL) with no hematological malignancy. In vitro concentrations versus time data were analyzed using a noncompartmental approach. RESULTS: Cytarabine in vitro area under the curve (AUCIVlast) was 22-fold higher in AML samples as compared to CTRL samples (AML mean (standard deviation (SD)), 51,829 (27,004) h ng/mL; CTRL mean (SD), 2356 (1250) h ng/mL, p = 0.00019). This increase was associated with a prolonged in vitro degradation half-life (t1/2IVdeg AML mean (SD), 15 (11.8) h; CTRL mean (SD), 0.36 (0.37) h, p = 0.0033). Multiple linear regression analysis showed that AML diagnosis significantly influenced t1/2IVdeg and AUCIVlas relationship. CONCLUSION: Cytarabine stability is higher in AML than in CTRL samples. The absence of correlation between t1/2IVdeg and AUCIVlast in AML samples suggests that in vitro cytarabine degradation in AML is complex. These results open perspectives including the evaluation of the clinical relevance and the involved molecular mechanisms.

Drug-induced vitiligo: a case/non-case study in Vigibase® , the WHO pharmacovigilance database.

Vitiligo is a common depigmenting disorder ensuing the loss of epidermal melanocytes. It is a multifactorial disease with immunological, genetic and environmental factors including drug exposure. The purpose of the study was to investigate the drugs and therapeutic subclasses associated with vitiligo occurrence reported in VigiBase® , the WHO pharmacovigilance database. A case/non-case study was carried out by defining cases as vitiligo reports and non-cases as all other reports. The reporting odds ratio (ROR) was calculated for the 'suspected' drugs and drug classes according to ATC level 4. During the study period, 741 cases of vitiligo were registered. Mean age was 49 ± 20 years. The disproportionality analysis showed an association between vitiligo and pembrolizumab (ROR 116.9, 95% Confidence Interval (CI) 94.8, 144.3), nivolumab (ROR 22.6, 95% CI 15.8, 32.4), ipilimumab (ROR 41.7, 95% CI 25.0, 69.7), imiquimod (ROR 152.8, 95% CI 103.0, 226.7), adalimumab (ROR 3.8, 95% CI 2.5,5.8), infliximab (ROR 2.6, 95% CI 1.65, 4.01), alemtuzumab (ROR 27.8, 95% CI 17.6, 43.9), and ustekinumab (ROR 9.3, 95% CI 5.6, 15.6). Concerning the pharmacological classes ATC level 4, a significant association was found with monoclonal antibodies, interferons, selective immunosuppressants, TNF-alpha inhibitors, interleukin inhibitors, and topical antivirals. This study confirmed the expected associations between vitiligo and immune checkpoint inhibitors and strengthened the emerging signal about the association between vitiligo and imiquimod, TNF-alpha inhibitors and interferons. New signals were shown with selective immunosuppressants including alemtuzumab and interleukin inhibitors.

Drug interactions related to self-medication: a French pharmacovigilance database study.

Self-medication (SM) is a common practice perceived by patients as harmless which can, however, entail health risks. The aim of the study was to identify drug-drug interactions (DDIs) involving SM drugs leading to adverse drug reactions (ADRs) in the National French Pharmacovigilance Database. All ADR reports from 1 January 1985 to 31 July 312018, coded as 'interaction' and 'self-medication', were selected and studied. Patient characteristics, the level and type of interaction, and the therapeutic classes of the drugs were examined. Adverse drug reactions were analysed and classified according to the system organ classes of the Medical Dictionary for Regulatory Activities. One hundred and three reports totalling 158 ADRs (71% severe cases) were included; 153 DDIs (59.5% pharmacodynamic) involving 234 drugs were identified. The latter included 119 SM drugs (51% available on prescription), mainly analgaesics, anti-inflammatory drugs, dietary supplements and antibiotics. Haemostasis disorders and renal failure were the most frequently reported ADRs. The analysis of reference documents raised concerns on the lack of information provided by package leaflets. In conclusion, the present study highlights the risks of medically unapproved re-use of prescription drugs or the consumption of dietary supplements without monitoring possible interactions and ADRs. Patient awareness could be improved by more regular updates of medication package inserts.

Promising silicones modified with cationic biocides for the development of antimicrobial medical devices.

We have tested silicones containing 2% or 5% of the cationic biocides polyhexamethylene guanidine dodecylbenzenesulfonate (PHMG-DBS), 1-octyl-3-methylimidazolium tetrafluoroborate (OMIM-BF4) or 1-dodecyl-3-methylimidazolium tetrafluoroborate (DMIM-BF4) against the major relevant bacterial and yeast species in health care-associated infections (HCAI). Study conducted according to the international standard ISO 22196 revealed that silicones containing 2% or 5% DMIM-BF4 or 5% PHMG-DBS presented the highest antimicrobial activity, leading to a logarithmic growth reduction of 3.03 to 6.46 and 3.65 to 4.85 depending on the bacterial or fungal species. Heat-pretreated silicones containing 2% DMIM-BF4 kept a high activity, with at least a 3-log reduction in bacterial growth, except against P. aeruginosa where there was only a 1.1-log reduction. After 33days, the release ratio of cationic biocide from silicone films containing 5% of DMIM-BF4 was found to be 5.6% in pure water and 1.9% in physiological saline solution, respectively. No leaching of PHMG-DBS polymeric biocide was detected under the same conditions. These results demonstrate unambiguously that silicones containing 2% DMIM-BF4 or 5% PHMG-DBS present high antimicrobial activity, as well as high leaching resistance and therefore may be good candidates for the development of safer medical devices.

A Multifaceted Study of Scedosporium boydii Cell Wall Changes during Germination and Identification of GPI-Anchored Proteins.

Scedosporium boydii is a pathogenic filamentous fungus that causes a wide range of human infections, notably respiratory infections in patients with cystic fibrosis. The development of new therapeutic strategies targeting S. boydii necessitates a better understanding of the physiology of this fungus and the identification of new molecular targets. In this work, we studied the conidium-to-germ tube transition using a variety of techniques including scanning and transmission electron microscopy, atomic force microscopy, two-phase partitioning, microelectrophoresis and cationized ferritin labeling, chemical force spectroscopy, lectin labeling, and nanoLC-MS/MS for cell wall GPI-anchored protein analysis. We demonstrated that the cell wall undergoes structural changes with germination accompanied with a lower hydrophobicity, electrostatic charge and binding capacity to cationized ferritin. Changes during germination also included a higher accessibility of some cell wall polysaccharides to lectins and less CH3/CH3 interactions (hydrophobic adhesion forces mainly due to glycoproteins). We also extracted and identified 20 GPI-anchored proteins from the cell wall of S. boydii, among which one was detected only in the conidial wall extract and 12 only in the mycelial wall extract. The identified sequences belonged to protein families involved in virulence in other fungi like Gelp/Gasp, Crhp, Bglp/Bgtp families and a superoxide dismutase. These results highlighted the cell wall remodeling during germination in S. boydii with the identification of a substantial number of cell wall GPI-anchored conidial or hyphal specific proteins, which provides a basis to investigate the role of these molecules in the host-pathogen interaction and fungal virulence.

Draft Genome Sequence of the Pathogenic Fungus Scedosporium apiospermum.

The first genome of one species of the Scedosporium apiospermum complex, responsible for localized to severe disseminated infections according to the immune status of the host, will contribute to a better understanding of the pathogenicity of these fungi and also to the discovery of the mechanisms underlying their low susceptibility to current antifungals.

Cell wall modifications during conidial maturation of the human pathogenic fungus Pseudallescheria boydii.

Progress in extending the life expectancy of cystic fibrosis (CF) patients remains jeopardized by the increasing incidence of fungal respiratory infections. Pseudallescheria boydii (P. boydii), an emerging pathogen of humans, is a filamentous fungus frequently isolated from the respiratory secretions of CF patients. It is commonly believed that infection by this fungus occurs through inhalation of airborne conidia, but the mechanisms allowing the adherence of Pseudallescheria to the host epithelial cells and its escape from the host immune defenses remain largely unknown. Given that the cell wall orchestrates all these processes, we were interested in studying its dynamic changes in conidia as function of the age of cultures. We found that the surface hydrophobicity and electronegative charge of conidia increased with the age of culture. Melanin that can influence the cell surface properties, was extracted from conidia and estimated using UV-visible spectrophotometry. Cells were also directly examined and compared using electron paramagnetic resonance (EPR) that determines the production of free radicals. Consistent with the increased amount of melanin, the EPR signal intensity decreased suggesting polymerization of melanin. These results were confirmed by flow cytometry after studying the effect of melanin polymerization on the surface accessibility of mannose-containing glycoconjugates to fluorescent concanavalin A. In the absence of melanin, conidia showed a marked increase in fluorescence intensity as the age of culture increased. Using atomic force microscopy, we were unable to find rodlet-forming hydrophobins, molecules that can also affect conidial surface properties. In conclusion, the changes in surface properties and biochemical composition of the conidial wall with the age of culture highlight the process of conidial maturation. Mannose-containing glycoconjugates that are involved in immune recognition, are progressively masked by polymerization of melanin, an antioxidant that is commonly thought to allow fungal escape from the host immune defenses.

Proteolytic cleavage of the long pentraxin PTX3 in the airways of cystic fibrosis patients.

The prototypic long pentraxin PTX3, a soluble pattern recognition receptor, plays an important role in innate defense against selected pathogens by favoring their elimination and the initiation of protective responses. PTX3 has notably beneficial effects in mice infected with Aspergillus fumigatus and Pseudomonas aeruginosa. Cystic fibrosis (CF), a severe inherited autosomal recessive disease, is characterized by recurrent lung infections, especially by these two pathogens. We thus hypothesized that the status of PTX3 may be altered in CF patients. Level and integrity of PTX3 were analyzed in the sputum samples from 51 CF patients and 7 patients with chronic obstructive pulmonary disease (COPD). The levels of PTX3 were increased in serums from CF patients, but low in their respiratory secretions. PTX3 concentrations in sputum samples were dramatically lower in CF patients than in COPD patients. The low concentration of PTX3 resulted from a proteolysis cleavage by elastase and A. fumigatus proteases. Interestingly, the N-ter domain of PTX3, involved in protection against A. fumigatus, is preferentially degraded by these proteases. These results indicate that the selective proteolysis of PTX3 in the CF lung may explain, in part, the recurrent lung infections by PTX3-sensitive pathogens in CF patients.

Photodegradation of the Mycobacterium ulcerans toxin, mycolactones: considerations for handling and storage.

BACKGROUND: Mycolactones are toxins secreted by M. ulcerans, the etiological agent of Buruli ulcer. These toxins, which are the main virulence factors of the bacilli, are responsible for skin lesions. Considering their specificity for M. ulcerans and their presence in skin lesions even at early stages, mycolactones are promising candidates for the development of a diagnostic tool for M. ulcerans infection. Stability of purified mycolactones towards light and heat has not yet been investigated, despite the importance of such parameters in the selection of strategies for a diagnosis tool development. In this context, the effects of UV, light and temperature on mycolactone stability and biological activity were studied. METHODOLOGY/PRINCIPAL FINDINGS: To investigate the effect of these physical parameters, mycolactones were exposed to different wavelengths in several solvents and temperatures. Structural changes and biological activity were monitored. Whilst high temperature had no effect on mycolactones, UV irradiation (UV-A, UV-B and UV-C) and sunlight exposure caused a considerable degradation, as revealed by LC-MS and NMR analysis, correlated with a loss of biological activity. Moreover, effect of UVs on mycolactone caused a photodegradation rather than a phototransformation due to the identification of degradation product. CONCLUSION/SIGNIFICANCE: This study demonstrates the high sensitivity of mycolactones to UVs as such it defines instructions for storage and handling.

Cell surface properties of two differently virulent strains of Acinetobacter baumannii isolated from a patient.

The aim of this study was to unravel, by focusing on cell surface properties, the underlying virulence factors contributing to the difference in the pathogenicity observed in two Acinetobacter baumannii strains isolated from the same patient. The two strains were phenotypically different: (i) a mucoid strain (AB-M), highly virulent in a mouse model of pneumonia, and (ii) a nonmucoid strain (AB-NM), moderately virulent in the same model. The study of the cell surface properties included the microbial adhesion to solvents method, the measurement of the electrophoretic mobility of bacteria, the analysis of biofilm formation by calcofluor white staining, the adherence to silicone catheters, and scanning electron microscopy. The AB-NM strain was more hydrophobic, more adherent to silicone catheters, and produced more biofilm than the AB-M strain. Scanning electron microscopy showed bacterial cells with a rough surface and the formation of large cell clusters for AB-NM whereas the AB-M strain had a smooth surface and formed only a few cell clusters. Contrary to the results of most previous studies, cell surface properties were not correlated to the virulence described in our experimental model, indicating that mechanisms other than adherence may be involved in the expression of A. baumannii virulence.