Bio-Sprayed/Threaded Microalgae Remain Viable and Indistinguishable from Controls.

Microalgae are increasingly playing a significant role in many areas of research and development. Recent studies have demonstrated their ability to aid wound healing by their ability to generate oxygen, aiding the healing process. Bearing this in mind, the capability to spray/spin deposit microalgae in suspension (solution) or compartmentalize living microalgae within architectures such as fibers/scaffolds and beads, would have significance as healing mechanisms for addressing a wide range of wounds. Reconstructing microalgae-bearing architectures as either scaffolds or beads could be generated via electric field (bio-electrospraying and cell electrospinning) and non-electric field (aerodynamically assisted bio-jetting/threading) driven technologies. However, before studying the biomechanical properties of the generated living architectures, the microalgae exposed to these techniques must be interrogated from a molecular level upward first, to establish these techniques, have no negative effects brought on the processed microalgae. Therefore these studies, demonstrate the ability of both these jetting and threading technologies to directly handle living microalgae, in suspension or within a polymeric suspension, safely, and form algae-bearing architectures such as beads and fibers/scaffolds.

Phenotyping clonal populations of glioma stem cell reveals a high degree of plasticity in response to changes of microenvironment.

The phenotype of glioma-initiating cells (GIC) is modulated by cell-intrinsic and cell-extrinsic factors. Phenotypic heterogeneity and plasticity of GIC is an important limitation to therapeutic approaches targeting cancer stem cells. Plasticity also presents a challenge to the identification, isolation, and propagation of purified cancer stem cells. Here we use a barcode labelling approach of GIC to generate clonal populations over a number of passages, in combination with phenotyping using the established stem cell markers CD133, CD15, CD44, and A2B5. Using two cell lines derived from isocitrate dehydrogenase (IDH)-wildtype glioblastoma, we identify a remarkable heterogeneity of the phenotypes between the cell lines. During passaging, clonal expansion manifests as the emergence of a limited number of barcoded clones and a decrease in the overall number of clones. Dual-labelled GIC are capable of forming traceable clonal populations which emerge after as few as two passages from mixed cultures and through analyses of similarity of relative proportions of 16 surface markers we were able to pinpoint the fate of such populations. By generating tumour organoids we observed a remarkable persistence of dominant clones but also a significant plasticity of stemness marker expression. Our study presents an experimental approach to simultaneously barcode and phenotype glioma-initiating cells to assess their functional properties, for example to screen newly established GIC for tumour-specific therapeutic vulnerabilities.

Activation priming and cytokine polyfunctionality modulate the enhanced functionality of low-affinity CD19 CAR T cells.

We recently described a low-affinity second-generation CD19 chimeric antigen receptor (CAR) CAT that showed enhanced expansion, cytotoxicity, and antitumor efficacy compared with the high-affinity (FMC63-based) CAR used in tisagenlecleucel, in preclinical models. Furthermore, CAT demonstrated an excellent toxicity profile, enhanced in vivo expansion, and long-term persistence in a phase 1 clinical study. To understand the molecular mechanisms behind these properties of CAT CAR T cells, we performed a systematic in vitro characterization of the transcriptomic (RNA sequencing) and protein (cytometry by time of flight) changes occurring in T cells expressing low-affinity vs high-affinity CD19 CARs following stimulation with CD19-expressing cells. Our results show that CAT CAR T cells exhibit enhanced activation to CD19 stimulation and a distinct transcriptomic and protein profile, with increased activation and cytokine polyfunctionality compared with FMC63 CAR T cells. We demonstrate that the enhanced functionality of low-affinity CAT CAR T cells is a consequence of an antigen-dependent priming induced by residual CD19-expressing B cells present in the manufacture.

High expression of the ectonucleotidase CD39 on T cells from the inflamed site identifies two distinct populations, one regulatory and one memory T cell population.

The ectonucleotidase CD39 has recently been described as being highly expressed on regulatory Foxp3(+) CD4 T cells. Through hydrolysis of proinflammatory extracellular ATP, CD39 activity represents a newly described mechanism of regulatory T cell action. We report a novel population of human CD4 T cells that express CD39 yet are Foxp3 negative. These cells produce the proinflammatory cytokines IFN-gamma and IL-17 and fail to suppress proliferation; however, they still have high ATP hydrolysis activity. In the inflammatory site in human juvenile idiopathic arthritis, the CD39(+)Foxp3(-) population is greatly increased compared with peripheral blood of patients or healthy controls. We also show that cells expressing the AMPase CD73 are less frequent in the joint than in blood. To our knowledge, this is the first study to describe and characterize CD39 function on CD4 T cells from the target site in a human autoinflammatory condition. Our data suggest that in human CD4(+) T cells from the inflamed site, CD39 can be highly expressed on two populations, one regulatory and the other of a memory phenotype.

Cellular uptake and cytotoxic impact of chemically functionalized and polymer-coated carbon nanotubes.

The impact of nanomaterials such as carbon nanotubes on biological matter is a topic of increasing interest and concern and requires a multifaceted approach to be resolved. A modified cytotoxic (lactate dehydrogenase (LDH)) assay is developed in an attempt to offer a valid and reliable methodology for screening carbon nanotube toxicity in vitro. Two of the most widely used types of surface-modified multiwalled carbon nanotubes (MWNTs) are tested: ammonium-functionalized MWNTs (MWNT-NH3+ ) and Pluronic F127 coated MWNTs (MWNT:F127). Chemically functionalized MWNTs show significantly greater cellular uptake into lung epithelial A549 cells compared to the non-covalently Pluronic F127-coated MWNTs. In spite of this, MWNT:F127 exhibit enhanced cytotoxicity according to the modified LDH assay. The validity of the modified LDH assay is further validated by direct comparison with other less reliable or accurate cytotoxicity assays. These findings indicate the reliability of the modified LDH assay as a screening tool to assess carbon nanotube cytotoxicity and illustrate that high levels of carbon nanotube cellular internalization do not necessarily lead to adverse responses.

Cells with dendritic cell morphology and immunophenotype, binuclear morphology, and immunosuppressive function in dendritic cell cultures.

Culturing of human peripheral blood CD14 positive monocytes is a method for generation of dendritic cells (DCs) for experimental purposes or for use in clinical grade vaccines. When culturing human DCs in this manner for clinical vaccine production, we noticed that 5-10% of cells within the bulk culture were binuclear or multiple nuclear, but had typical dendritic cell morphology and immunophenotype. We refer to the cells as binuclear cells in dendritic cell cultures (BNiDCs). By using single cell PCR analysis of mitochondrial DNA polymorphisms we demonstrated that approximately 20-25% of cells in DC culture undergo a fusion event. Flow sorted BNiDC express low HLA-DR and IL-12p70, but high levels of IL-10. In mixed lymphocyte reactions, purified BNiDC suppressed lymphocyte proliferation. Blockade of dendritic cell-specific transmembrane protein (DC-STAMP) decreased the number of binuclear cells in DC cultures. BNiDC represent a potentially tolerogenic population within DC preparations for clinical use.

Enhanced cellular internalization and gene silencing with a series of cationic dendron-multiwalled carbon nanotube:siRNA complexes.

One of the major obstacles to the clinical development of gene silencing by small interfering RNA (siRNA) is its effective cytoplasmic delivery. Carbon nanotubes have been proposed as novel nanomaterials that can offer significant advantages for the intracellular delivery of nucleic acids, such as siRNA. We recently demonstrated in a proof-of-principle study that amino-functionalized multiwalled carbon nanotubes (f-MWNT) can effectively deliver in vivo an siRNA sequence, triggering cell apoptosis that results in human lung xenograft eradication and prolonged survival. In the present study, we demonstrate how a newly synthesized series of polycationic dendron-MWNT constructs with a precisely tailored number of amino functions (dendron generations) can complex and effectively deliver double-stranded siRNA to achieve gene silencing in vitro. A systematic comparison between the f-MWNT series in terms of cellular uptake, cytotoxicity, and siRNA complexation is offered. Significant improvement in siRNA delivery with the dendron-MWNT conjugates is shown, and gene silencing was obtained in 2 human cell lines using 2 different siRNA sequences. The study reveals that through f-MWNT structure-biological function analysis novel nanotube-based siRNA transfer vectors can be designed with minimal cytotoxicity and effective delivery and gene-silencing capabilities.

Biologic predictors of extension of oligoarticular juvenile idiopathic arthritis as determined from synovial fluid cellular composition and gene expression.

OBJECTIVE: To identify biomarkers in the first synovial fluid (SF) aspirate obtained from children with oligoarticular juvenile idiopathic arthritis (JIA), which could be used to identify children whose disease is likely to extend to a more severe phenotype. METHODS: Patients with recent-onset oligoarticular JIA were identified and grouped according to those whose mild disease persisted (persistent disease) or those whose disease would extend from a mild to more severe phenotype (extended-to-be disease) at 1 year after diagnosis. Flow cytometry was used to delineate differences in the mononuclear cell populations between the first blood sample and first SF aspirate from the same patient and between outcome (persistent versus extended-to-be) groups. Proportions of lymphocytes in the joint were modeled on chemotaxis of lymphocytes to CCL5, using Transwell migration assays. Levels of CCL5 in the SF were quantified by enzyme-linked immunosorbent assay. RNA profiles of SF mononuclear cells were compared between groups using the Affymetrix GeneChip hybridization protocol and hierarchical clustering analyses. RESULTS: Compared with peripheral blood mononuclear cells, SF mononuclear cells displayed an expansion of CD8+ T cells, reduced proportion of B cells, and expansion of CD16- natural killer cells. The lower CD4:CD8 ratio in the SF was recapitulated in vitro by the observed migration of blood T cells in response to CCL5. Synovial CCL5 levels were higher in children whose disease extended to a more severe phenotype. The CD4:CD8 ratio in the SF was significantly lower in patients with extended-to-be oligoarticular JIA (0.57 compared with 0.90 in the persistent disease group, difference 0.33, 95% confidence interval 0.04-0.62; P = 0.009). Gene expression profiling revealed that 344 genes were >1.5-fold differentially expressed between outcome groups (P < 0.05), and these included genes associated with inflammation and macrophage differentiation, which showed increased levels in patients with extended disease at 1 year, and genes associated with immune regulation, which showed increased levels in patients with persistent disease at 1 year. CONCLUSION: Analyses of the proportions of synovial lymphocytes, levels of CCL5, and differential gene expression yielded potential biomarkers with which to predict the likelihood of extension of oligoarticular JIA to a more severe disease phenotype.

Selective targeting of B cells with agonistic anti-CD40 is an efficacious strategy for the generation of induced regulatory T2-like B cells and for the suppression of lupus in MRL/lpr mice.

We have previously reported that IL-10(+) regulatory B cells, known to play an important role in controlling autoimmunity and inflammatory disorders, are contained within the transitional 2 immature (T2) B cell pool (T2 Bregs). Therapeutic strategies facilitating their enrichment or enhancing their suppressive activity are highly attractive. In this study, we report that agonistic anti-CD40 specifically targets T2 B cells and enriches Bregs upon short-term in vitro culture. Although transfer of unmanipulated T2 B cells, isolated from mice with established lupus, failed to confer protection to diseased mice, transfer of in vitro anti-CD40-generated T2 B cells (T2-like-Bregs) significantly improved renal disease and survival by an IL-10-dependent mechanism. T2-like-Bregs readily accumulated in the spleen after transfer, suppressed Th1 responses, induced the differentiation of IL-10(+)CD4(+)T cells, and conveyed a regulatory effect to CD4(+)T cells. In addition, in vivo administration of agonistic anti-CD40, currently on trial for the treatment of cancer, halted and reversed established lupus. Taken together, our results suggest a novel cellular approach for the amelioration of experimental lupus.

Molecular characterisation of post-bio-electrosprayed human brain astrocytoma cells.

Bio-electrospraying (BES) is a method for directly jetting living cells under conditions that allow their distribution in the x, y, and z axes. Previous work has been focused on achieving jetting in stable cone-jet mode, which is required for precision placement, and these studies have demonstrated that there are no significant effects of bio-electrospraying on cell morphology or viability. In this work, we examine the biological properties of bio-electrosprayed cells using assays of cellular function that range from the molecular level through to integrated cellular systems, and include proteomics, signal transduction, cell growth and proliferation, and the characterisation of apoptotic blebs. From these molecular methods, we have determined that bio-electrospraying, under the electric field conditions used to achieve stable cone-jet mode, causes no alterations to the biological properties and function of the cells being jetted. Bio-electrosprayed and control cells had similar viability, proliferation properties and virtually indistinguishable cell cycle profiles. The biophysical properties of large conducting (BK) potassium channels were unchanged, as were the pharmacological responses of the endogenous muscarinic and exogenous P2Y(11) receptors, both of which are cell surface receptors of the 7TM superfamily. Proteomic analyses revealed that although three proteins had subtle differences in expression level between bio-electrosprayed and control cells, none of these fold differences was above the 1.5-fold cut-off threshold required for further analyses. These findings support the further development of bio-electrosprays as a viable technology for a wide diversity of tissue engineering, regenerative biology, advanced cellular therapeutics and medicinal applications, having significance in the clinic.

Bone marrow-derived IFN-producing killer dendritic cells account for the tumoricidal activity of unpulsed dendritic cells.

The CD11c(int)B220(+)NK1.1(+)CD49(+) subset of cells has recently been described as IFN-producing killer dendritic cells (IKDC), which share phenotypic and functional properties of dendritic cells and NK cells. Herein we show that bone marrow-derived murine dendritic cell preparations contain abundant CD11c(int)B220(+)NK1.1(+)CD49(+) cells, the removal of which results in loss of tumoricidal activity of unpulsed dendritic cells in vivo. Moreover, following s.c. injection, as few as 5 x 10(3) highly pure bone marrow-derived IKDC cells are capable of shrinking small contralateral syngeneic tumors in C57BL/6 mice, but not in immunodeficient mice, implying the obligate involvement of host effector cells in tumor rejection. Our data suggest that bone marrow-derived IKDC represent a population that has powerful tumoricidal activity in vivo.

In Utero Gene Therapy (IUGT) Using GLOBE Lentiviral Vector Phenotypically Corrects the Heterozygous Humanised Mouse Model and Its Progress Can Be Monitored Using MRI Techniques.

In utero gene therapy (IUGT) to the fetal hematopoietic compartment could be used to treat congenital blood disorders such as ß-thalassemia. A humanised mouse model of ß-thalassemia was used, in which heterozygous animals are anaemic with splenomegaly and extramedullary hematopoiesis. Intrahepatic in utero injections of a ß globin-expressing lentiviral vector (GLOBE), were performed in fetuses at E13.5 of gestation. We analysed animals at 12 and 32 weeks of age, for vector copy number in bone marrow, peripheral blood liver and spleen and we performed integration site analysis. Compared to noninjected heterozygous animals IUGT normalised blood haemoglobin levels and spleen weight. Integration site analysis showed polyclonality. The left ventricular ejection fraction measured using magnetic resonance imaging (MRI) in treated heterozygous animals was similar to that of normal non-ß-thalassemic mice but significantly higher than untreated heterozygous thalassemia mice suggesting that IUGT ameliorated poor cardiac function. GLOBE LV-mediated IUGT normalised the haematological and anatomical phenotype in a heterozygous humanised model of ß-thalassemia.

Publisher Correction: In Utero Gene Therapy (IUGT) Using GLOBE Lentiviral Vector Phenotypically Corrects the Heterozygous Humanised Mouse Model and Its Progress Can Be Monitored Using MRI Techniques.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Flow Cytometric Detection of G0 in Live Cells by Hoechst 33342 and Pyronin Y Staining.

Hoechst 33342 and Pyronin Y double staining can be used to measure DNA and RNA content in live cells by flow cytometry. Quiescent cells at G0 phase have the same amount of DNA as cells at G1 phase but lower RNA levels compared to proliferating cells. Therefore, resting cells in G0 phase can be distinguished from proliferating cells in G1, S, and G2 M phases. This chapter describes a protocol for double staining of live cells with Hoechst 33342 and Pyronin Y. Combined with immunophenotyping of intact and live cells Hoechst 33342 and Pyronin Y staining is a powerful noninvasive method for the analysis and isolation of quiescent cells from any defined cell population.

A novel method for ABO-incompatible heart transplantation.

BACKGROUND: Since 1996, ABO-incompatible heart transplantation has been undertaken by performing whole-body plasma exchange to remove isohemagglutinins using the cardiopulmonary bypass (CPB) circuit at the time of transplantation. This requires large volumes of donated blood and blood products, causes hemodynamic instability during the exchange transfusion, and limits practical use to small children. We sought to determine the efficacy of anti-A/B immunoadsorption within the CPB circuit on removal of isohemagglutinins in an ex vivo setting before its use clinically. METHODS: An anti-A/B immunoadsorption column was placed into a CPB circuit mimicking a typical ABO-incompatible transplant patient, which had been primed with type O whole human blood. Samples were taken for determination of isohemagglutinin titers following each plasma volume pass through the anti-A/B immunoadsorption column. RESULTS: There was a linear decrease of at least 1 dilution seen in both anti-A and anti-B IgG and IgM antibodies with each plasma volume pass through the column. This predictable removal allowed the formulation of selection criteria for ABO-incompatible heart transplantation given the reciprocal of titer and patient weight. This degree of predictability allowed us to use it successfully in the clinical setting, reducing antibodies to an undetectable level during ABO-incompatible heart transplantation. CONCLUSIONS: The incorporation of an anti-A/B immunoadsorption column into the extracorporeal circuit reduces allogeneic blood product requirement for ABO-incompatible heart transplantation, while providing efficacious removal of anti-A and anti-B isohemagglutinins. This can be undertaken within the time period of CPB before graft reperfusion and expands the potential recipient pool to larger patients with higher isohemagglutinin titers.

High monocyte to lymphocyte ratio is associated with impaired protection after subcutaneous administration of BCG in a mouse model of tuberculosis.

Background: The only available tuberculosis (TB) vaccine, Bacillus Calmette-Guérin (BCG), has variable efficacy. New vaccines are therefore urgently needed. Why BCG fails is incompletely understood, and the tools used for early assessment of new vaccine candidates do not account for BCG variability. Taking correlates of risk of TB disease observed in human studies and back-translating them into mice to create models of BCG variability should allow novel vaccine candidates to be tested early in animal models that are more representative of the human populations most at risk. Furthermore, this could help to elucidate the immunological mechanisms leading to BCG failure. We have chosen the monocyte to lymphocyte (ML) ratio as a correlate of risk of TB disease and have back-translated this into a mouse model. Methods: Four commercially available, inbred mouse strains were chosen. We investigated their baseline ML ratio by flow cytometry; extent of BCG-mediated protection from Mtb infection by experimental challenge; vaccine-induced interferon gamma (IFNγ) response by ELISPOT assay; and tissue distribution of BCG by plating tissue homogenates. Results: The ML ratio varied significantly between A/J, DBA/2, C57Bl/6 and 129S2 mice. A/J mice showed the highest BCG-mediated protection and lowest ML ratio, while 129S2 mice showed the lowest protection and higher ML ratio. We also found that A/J mice had a lower antigen specific IFNγ response than 129S2 mice. BCG tissue distribution appeared higher in A/J mice, although this was not statistically significant. Conclusions: These results suggest that the ML ratio has an impact on BCG-mediated protection in mice, in alignment with observations from clinical studies. A/J and 129S2 mice may therefore be useful models of BCG vaccine variability for early TB vaccine testing. We speculate that failure of BCG to protect from TB disease is linked to poor tissue distribution in a ML high immune environment.

The effect of calorie restriction on mouse skeletal muscle is sex, strain and time-dependent.

Loss of skeletal muscle mass and function occurs with increasing age. Calorie restriction (CR) increases the lifespan of C57Bl/6 mice, but not in the shorter-lived DBA/2 strain. There is some evidence that calorie restriction reduces or delays many of the age-related defects that occur in rodent skeletal muscle. We therefore investigated the effect of short (2.5 month) and longer term (8.5 and 18.5 months) CR on skeletal muscle in male and female C57Bl/6 and DBA/2 mice. We found that short-term CR increased the satellite cell number and collagen VI content of muscle, but resulted in a delayed regenerative response to injury.Consistent with this, the in vitro proliferation of satellite cells derived from these muscles was reduced by CR. The percentage of stromal cells, macrophages, hematopoietic stem cells and fibroadipogenic cells in the mononucleated cell population derived from skeletal muscle was reduced by CR at various stages. But overall, these changes are neither consistent over time, nor between strain and sex. The fact that changes induced by CR do not persist with time and the dissimilarities between the two mouse strains, combined with sex differences, urge caution in applying CR to improve skeletal muscle function across the lifespan in humans.

Mycobacterium leprae Activates Toll-Like Receptor-4 Signaling and Expression on Macrophages Depending on Previous Bacillus Calmette-Guerin Vaccination.

Toll-like receptor (TLR)-1 and TLR2 have been shown to be receptors for Mycobacterium leprae (M. leprae), yet it is unclear whether M. leprae can signal through alternative TLRs. Other mycobacterial species possess ligands for TLR4 and genetic association studies in human populations suggest that people with TLR4 polymorphisms may be protected against leprosy. Using human embryonic kidney (HEK)-293 cells co-transfected with TLR4, we demonstrate that M. leprae activates TLR4. We used human macrophages to show that M. leprae stimulation of cytokine production is diminished if pre-treated with TLR4 neutralizing antibody. TLR4 protein expression was up-regulated on macrophages derived from non-bacillus Calmette-Guerin (BCG) vaccinated healthy volunteers after incubation with M. leprae, whereas it was down-regulated in macrophages derived from BCG-vaccinated donors. Finally, pre-treatment of macrophages derived from BCG-naive donors with BCG reversed the effect of M. leprae on TLR4 expression. This may be a newly described phenomenon by which BCG vaccination stimulates "non-specific" protection to the human immune system.

Improved intracellular delivery of peptide- and lipid-nanoplexes by natural glycosides.

Targeted nanocarriers undergo endocytosis upon binding to their membrane receptors and are transported into cellular compartments such as late endosomes and lysosomes. In gene delivery the genetic material has to escape from the cellular compartments into the cytosol. The process of endosomal escape is one of the most critical steps for successful gene delivery. For this reason synthetic lipids with fusogenic properties such as 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) are integrated into the nanocarriers. In this study we show that a natural, plant derived glycoside (SO1861) from Saponaria officinalis L. greatly improves the efficacy of lipid based as well as non-lipid based targeted nanoplexes consisting of a targeted K16 peptide with a nucleic acid binding domain and plasmid-DNA, minicircle-DNA or small interfering RNA (siRNA). By confocal live cell imaging and single cell analyses, we demonstrate that SO1861 augments the escape of the genetic cargo out of the intracellular compartments into the cytosol. Co-localisation experiments with fluorescence labelled dextran and transferrin indicate that SO1861 induces the release of the genetic cargo out of endosomes and lysosomes. However, the transduction efficacy of a lentivirus based gene delivery system was not augmented. In order to design receptor-targeted nanoplexes (LPD) with improved functional properties, SO1861 was integrated into the lipid matrix of the LPD. The SO1861 sensitized LPD (LPDS) were characterized by dynamic light scattering and transmission electron microscopy. Compared to their LPD counterparts the LPDS-nanoplexes showed a greatly improved gene delivery. As shown by differential scanning calorimetry SO1861 can be easily integrated into the lipid bilayer of glycerophospholipid model membranes. This underlines the great potential of SO1861 as a new transfection multiplier for non-viral gene delivery systems.

Molecular signature of human amniotic fluid stem cells during fetal development.

Mid-gestation c-KIT(+) amniotic fluid stem cells (AFSC) have an intermediate phenotype between embryonic and adult stem cells and are easy to reprogram to pluripotency. We previously showed that 1st trimester AFSC can be reprogrammed to functional pluripotency in a transgene-free approach. Despite both parental populations sharing a common phenotype, expressing CD29, CD44, CD73, CD90, CD105, SSEA4 and OCT4, 2nd trimester AFSC, contrary to 1st trimester cells, do not express NANOG, SSEA3, TRA-1-60 and TRA-1-81, and have slower growth kinetics. Here, we used the Illumina Beadstudio microarray platform to analyse the transcriptome of 1st and 2nd trimester AFSC and show a unique 1st trimester AFSC-specific gene expression signature consisting of 366 genes and a larger set of 603 genes common with hESC compared to 496 genes overlapping between 2nd trimester AFSC and hESC. We conclude that both populations are related but distinct to each other as well as to hESC.