Design and preclinical testing of an anti-CD41 CAR T cell for the treatment of acute megakaryoblastic leukaemia

Acute megakaryoblastic leukaemia (AMkL) is a rare subtype of acute myeloid leukaemia (AML) representing 5% of all reported cases, and frequently diagnosed in children with Down syndrome. Patients diagnosed with AMkL have low overall survival and have poor outcome to treatment, thus novel therapies such as CAR T cell therapy could represent an alternative in treating AMkL. We investigated the effect of a new CAR T cell which targets CD41, a specific surface antigen for M7-AMkL, against an in vitro model for AMkL, DAMI Luc2 cell line. The performed flow cytometry evaluation highlighted a percentage of 93.8% CAR T cells eGFP-positive and a limited acute effect on lowering the target cell population. However, the interaction between effector and target (E:T) cells, at a low ratio, lowered the cell membrane integrity, and reduced the M7-AMkL cell population after 24 h of co-culture, while the cytotoxic effect was not significant in groups with higher E:T ratio. Our findings suggest that the anti-CD41 CAR T cells are efficient for a limited time spawn and the cytotoxic effect is visible in all experimental groups with low E:T ratio.     

The Interplay Between NSAIDs and Candida albicans on the Gastrointestinal Tract of Guinea Pigs

Recent studies suggest that Candida albicans colonization is associated with several gastrointestinal inflammatory disorders and is also responsible for the delay in ulcer healing. No data are reported about the effects of C. albicans on the nonsteroidal anti-inflammatory drugs (NSAIDs)-induced necroinflammatory lesions. On the other hand, beneficial effects of NSAIDs regarding the colonization potential with C. albicans have been reported. Our aim was to investigate whether the association between NSAIDs and C. albicans could potentially induce necroinflammatory lesions in the guinea pigs gastric and enteral mucosa. Three interventional groups of 11 guinea pigs each were investigated after 5 days of receiving indomethacin, C. albicans or the association of both. C. albicans and necroinflammatory lesions were graded based on histological examinations. Statistical analysis used Mann–Whitney nonparametric test. NSAIDs did not significantly decrease C. albicans colonization grades on gastrointestinal mucosa. Administration of indomethacin subsequent to C. albicans determined significantly more severe necroinflammatory lesions compared to group that only received C. albicans. The association of NSAIDs and C. albicans did not cause significantly more severe degenerative or inflammatory lesions compared to the administration of only NSAIDs in this experimental model. Associations between NSAIDs and C. albicans caused significantly more severe necroinflammatory injuries than the lesions produced by C. albicans, without enhancing the mucosal injury or inflammation caused by NSAIDs.     

Chiral Separation of the Clinically Important Compounds Fucose and Pipecolic Acid Using CE: Determination of the Most Effective Chiral Selector

In this study, simple electrophoretic methods were developed for the chiral separation of the clinically important compounds fucose and pipecolic acid. In recent years, these analytes, and particularly their individual enantiomers, have attracted considerable attention due to their role in biological functions and disorders. The detectability and sensitivity of pipecolic acid and fucose were improved by reacting them with fluorenylmethyloxycarbonyl chloride (FMOC‐Cl) and 5‐amino‐2‐naphthalene‐sulfonic acid (ANSA), respectively. The enantioseparation conditions were optimized by initially investigating the type of the chiral selector. Different chiral selectors, such as polymeric surfactants and cyclodextrins, were used and the most effective ones were determined with regard to resolution and analysis time. A 10‐mM β‐cyclodextrin was able to separate the enantiomers of ANSA‐DL‐fucose and the polymeric surfactant poly(sodium N‐undecanoyl‐LL‐leucine‐valinate) was able to separate the enantiomers of FMOC‐DL‐pipecolic acid, with resolution values of 3.45 and 2.78, respectively. Additional parameters, such as the concentration and the pH of the background electrolyte (BGE), the concentration of the chiral selector, and the addition of modifiers were examined in order to optimize the separations. The addition of the chiral ionic liquid D‐alanine tert‐butyl ester lactate into the BGE was also investigated, for the first time, in order to improve resolution of the enantiomers. Chirality 25:556–560, 2013. © 2013 Wiley Periodicals, Inc.     

Novel materials based on DNA‐CTMA and lanthanide (Ce3+, Pr3+)

New, deoxyribonucleic acid (DNA) based compounds, functionalized with hexadecyltrimethylammonium chloride (CTMA) and lanthanide hydroxide nanoparticles were synthesized. The spectral measurements suggest that between the DNA‐CTMA complex and the lanthanide (III) ions a chemical interaction takes place. The obtained materials exhibit an improved fluorescence efficiency, showing a potential interest for application in photonics, and more particularly, in light emitting devices. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 613–617, 2016.     

In vitro differentiation of human mesenchymal stem cells to epithelial lineage

Our study examined whether human bone marrow-derived MSCs are able to differentiate, in vitro, into functional epithelial-like cells. MSCs were isolated from the sternum of 8 patients with different hematological disorders. The surface phenotype of these cells was characterized.To induce epithelial differentiation, MSCs were cultured using Epidermal Growth Factor, Keratinocyte Growth Factor, Hepatocyte Growth Factor and Insulin-like growth Factor-II. Differentiated cells were further characterized both morphologically and functionally by their capacity to express markers with specificity for epithelial lineage. The expression of cytokeratin 19 was assessed by immunocytochemistry, and cytokeratin 18 was evaluated by quantitative RT-PCR (Taq-man). The data demonstrate that human MSCs isolated from human bone marrow can differentiate into epithelial-like cells and may thus serve as a cell source for tissue engineering and cell therapy of epithelial tissue.     

Gene and protein expression in the epididymis of infertile c-ros receptor tyrosine kinase-deficient mice

Transgenic male mice bearing inactive mutations of the receptor tyrosine kinase c-ros lack the initial segment of the epididymis and are infertile. Several techniques were applied to determine differences in gene expression in the epididymal caput of heterozygous fertile (HET) and infertile homozygous knockout (KO) males that may explain the infertility. Complementary DNA arrays, gene chips, Northern and Western blots, and immunohistochemistry indicated that some proteins were downregulated, including the initial segment/proximal caput-specific genes c-ros, cystatin-related epididymal-spermatogenic (CRES), and lipocalin mouse epididymal protein 17 (MEP17), whereas other caput-enriched genes (glutathione peroxidase 5, a disintegrin and metalloproteinase [ADAM7], bone morphogenetic proteins 7 and 8a, A-raf, CCAAT/enhancer binding protein beta, PEA3) were unchanged. Genes normally absent from the initial segment (gamma-glutamyltranspeptidase, prostaglandin D2 synthetase, alkaline phosphatase) were expressed in the undifferentiated proximal caput of the KO. More distally, lipocalin 2 (24p3), CRISP1 (formerly MEP7), PEBP (MEP9), and mE-RABP (MEP10) were unchanged in expression. Immunohistochemistry and Western blots confirmed the absence of CRES in epididymal tissue and fluid and the continued presence of CRES in spermatozoa of the KO mouse. The glutamate transporters EAAC1 (EAAT3) and EAAT5 were downregulated and upregulated, respectively. The genes of over 70 transporters, channels, and pores were detected in the caput epididymidis, but in the KO, only three were downregulated and six upregulated. The changes in these genes could affect sperm function by modifying the composition of epididymal fluid and explain the infertility of the KO males. These genes may be targets for a posttesticular contraceptive.     

Selectivity and environmental variations in herbivory by Orthoptera

The aim of this study is 1 degree) to quantify environmental changes in herbivory due to Orthoptera on two perennial grasses and 2 degrees) to assess the processes involved in the control of herbivory. Herbivory varies strongly according to shade, drought and mowing, and is positively related to vapour pressure deficit and temperature. Besides the hypothesis of a trophic control of herbivory, our results are consistent with a microclimatic control of herbivory by Orthoptera. The coexistence of different hypothesis of herbivory control may depend on the studied system and specifically on the type of herbivore involved.     

Chemical Composition and Antipathogenic Activity of Artemisia annua Essential Oil from Romania

The essential oil extracted by hydrodistillation from Romanian Artemisia annua aerial parts was characterized by GC/MS analysis, which allowed the identification of 94.64% of the total oil composition. The main components were camphor (17.74%), α‐pinene (9.66%), germacrene D (7.55%), 1,8‐cineole (7.24%), trans‐β‐caryophyllene (7.02%), and artemisia ketone (6.26%). The antimicrobial activity of this essential oil was evaluated by determining the following parameters: minimal inhibitory concentration (MIC), minimal bactericidal concentration (MBC), minimal fungicidal concentration (MFC), and minimal biofilm eradication concentration (MBEC). Moreover, the soluble virulence factors were quantified with different biochemical substrates incorporated in the culture media. The reference and resistant, clinical strains proved to be susceptible to the A. annua oil, with MICs ranging from 0.51 to 16.33 mg/ml. The tested essential oil also showed good antibiofilm activity, inhibiting both the initial stage of the microbial cell adhesion to the inert substratum and the preformed mature biofilm. When used at subinhibitory concentrations, the essential oil proved to inhibit the phenotypic expression of five soluble virulence factors (hemolysins, gelatinase, DNase, lipases, and lecithinases). Briefly, the present results showed that the A. annua essential oil contained antimicrobial compounds with selective activity on Gram‐positive and Gram‐negative bacterial strains as well as on yeast strains and which also interfere with the expression of cell‐associated and soluble virulence factors.     

Development and Evaluation of New Microemulsion-Based Hydrogel Formulations for Topical Delivery of Fluconazole

The aim of the present investigation was to develop and evaluate microemulsion-loaded hydrogels (MEHs) for the topical delivery of fluconazole (FZ). The solubility of FZ in oils, surfactants and cosurfactants was evaluated to identify the components of the microemulsion. The pseudo-ternary phase diagrams were constructed using the novel phase diagram by micro-plate dilution method. Carbopol EDT 2020 was used to convert FZ-loaded microemulsions into gel form without affecting their structure. The selected microemulsions were assessed for globule size, zeta potential and polidispersity index. Besides this, the microemulsion-loaded hydrogel (MEH) formulations were evaluated for drug content, pH, rheological properties and in vitro drug release through synthetic membrane and excised pig ear skin in comparison with a conventional hydrogel. The optimised MEH FZ formulations consisting of FZ 2%, Transcutol P 11.5% and 11%, respectively, as oil phase, Lansurf SML 20-propyleneglycol 52% and 50%, respectively, as surfactant–cosurfactant (2:1), Carbopol EDT 2020 1.5% as gelling agent and water 34.5% and 37%, respectively, showed highest flux values and high release rate values, and furthermore, they had low surfactant content. The in vitro FZ permeation through synthetic membrane and excised pig ear skin from the studied MEHs was best described by the zero-order and first-order models. Finally, the optimised MEH FZ formulations showed similar or slightly higher antifungal activity as compared to that of conventional hydrogel and Nizoral® cream, respectively. The results suggest the potential use of developed MEHs as vehicles for topical delivery of FZ, encouraging further in vitro and in vivo evaluation.KEY WORDS: fluconazole, in vitro skin permeation, microemulsion, microemulsion-loaded hydrogel, topical     

Absolute quantification of DcR3 and GDF15 from human serum by LC‐ESI MS

Biomarkers are widely used in clinical diagnosis, prognosis and therapy monitoring. Here, we developed a protocol for the efficient and selective enrichment of small and low concentrated biomarkers from human serum, involving a 95% effective depletion of high‐abundant serum proteins by partial denaturation and enrichment of low‐abundant biomarkers by size exclusion chromatography. The recovery of low‐abundance biomarkers was above 97%. Using this protocol, we quantified the tumour markers DcR3 and growth/differentiation factor (GDF)15 from 100 μl human serum by isotope dilution mass spectrometry, using ¹⁵N metabolically labelled and concatamerized fingerprint peptides for the both proteins. Analysis of three different fingerprint peptides for each protein by liquid chromatography electrospray ionization mass spectrometry resulted in comparable concentrations in three healthy human serum samples (DcR3: 27.23 ± 2.49 fmol/ml; GDF15: 98.11 ± 0.49 fmol/ml). In contrast, serum levels were significantly elevated in tumour patients for DcR3 (116.94 ± 57.37 fmol/ml) and GDF15 (164.44 ± 79.31 fmol/ml). Obtained data were in good agreement with ELISA and qPCR measurements, as well as with literature data. In summary, our protocol allows the reliable quantification of biomarkers, shows a higher resolution at low biomarker concentrations than antibody‐based strategies, and offers the possibility of multiplexing. Our proof‐of‐principle studies in patient sera encourage the future analysis of the prognostic value of DcR3 and GDF15 for colon cancer patients in larger patient cohorts.