技术资料

Chlamydia infection and disease are endemic in free-ranging koalas. Antibiotics remain the front line treatment for Chlamydia in koalas,despite their rates of treatment failure and adverse gut dysbiosis outcomes. A Chlamydia vaccine for koalas has shown promise for replacing antibiotic treatment in mild ocular Chlamydia disease. In more severe disease presentations that require antibiotic intervention,the effect of vaccinating during antibiotic use is not currently known. This study investigated whether a productive immune response could be induced by vaccinating koalas during antibiotic treatment for Chlamydia-induced cystitis. Plasma IgG antibody levels against the C. pecorum major outer membrane protein (MOMP) dropped during antibiotic treatment in both vaccinated and unvaccinated koalas. Post-treatment,IgG levels recovered. The IgG antibodies from naturally-infected,vaccinated koalas recognised a greater proportion of the MOMP protein compared to their naturally-infected,unvaccinated counterparts. Furthermore,peripheral blood mononuclear cell gene expression revealed an up-regulation in genes related to neutrophil degranulation in vaccinated koalas during the first month post-vaccination. These findings show that vaccination of koalas while they are being treated with antibiotics for cystitis can result in the generation of a productive immune response,in the form of increased and expanded IgG production and host response through neutrophil degranulation. View Publication

Aim of work was to locate a simple,reproducible protocol for uniform seeding and optimal cellularization of biodegradable patch minimizing the risk of structural damages of patch and its contamination in long-term culture. Two seeding procedures are exploited,namely static seeding procedures on biodegradable and biocompatible patches incubated as free floating (floating conditions) or supported by CellCrownTM insert (fixed conditions) and engineered by porcine bone marrow MSCs (p-MSCs). Scaffold prototypes having specific structural features with regard to pore size,pore orientation,porosity,and pore distribution were produced using two different techniques,such as temperature-induced precipitation method and electrospinning technology. The investigation on different prototypes allowed achieving several implementations in terms of cell distribution uniformity,seeding efficiency,and cellularization timing. The cell seeding protocol in stating conditions demonstrated to be the most suitable method,as these conditions successfully improved the cellularization of polymeric patches. Furthermore,the investigation provided interesting information on patches' stability in physiological simulating experimental conditions. Considering the in vitro results,it can be stated that the in vitro protocol proposed for patches cellularization is suitable to achieve homogeneous and complete cellularizations of patch. Moreover,the protocol turned out to be simple,repeatable,and reproducible. View Publication

Hepatocyte death during acetaminophen (APAP) intoxication elicits a reactive inflammatory response,with hepatic recruitment of neutrophils and monocytes,which further aggravates liver injury. Neutrophil elastase (NE),secreted by activated neutrophils,carries degradative and cytotoxic functions and maintains a proinflammatory state. We investigated NE as a therapeutic target in acetaminophen-induced liver injury (AILI). C57BL/6 mice were administered a toxic dose of APAP,2 h prior to receiving the NE inhibitor sivelestat,N-acetylcysteine (NAC),or a combination therapy,and were euthanized after 24 and 48 h. Upon APAP overdose,neutrophils and monocytes infiltrate the injured liver,accompanied by increased levels of NE. Combination therapy of NAC and sivelestat significantly limits liver damage,as evidenced by lower serum transaminase levels and less hepatic necrosis compared to mice that received APAP only,and this to a greater extent than NAC monotherapy. Lower hepatic expression of proinflammatory markers was observed in the combination treatment group,and flow cytometry revealed significantly less monocyte influx in livers from mice treated with the combination therapy,compared to untreated mice and mice treated with NAC only. The potential of NE to induce leukocyte migration was confirmed in vitro. Importantly,sivelestat did not impair hepatic repair. In conclusion,combination of NE inhibition with sivelestat and NAC dampens the inflammatory response and reduces liver damage following APAP overdose. This strategy exceeds the standard of care and might represent a novel therapeutic option for AILI. View Publication

We have designed and validated a set of robust and non-toxic protocols for directly evaluating the properties of engineered neural tissue. These protocols characterize the mechanical properties of engineered neural tissues and measure their electrophysical activity. The protocols obtain elastic moduli of very soft fibrin hydrogel scaffolds and voltage readings from motor neuron cultures. Neurons require soft substrates to differentiate and mature,however measuring the elastic moduli of soft substrates remains difficult to accurately measure using standard protocols such as atomic force microscopy or shear rheology. Here we validate a direct method for acquiring elastic modulus of fibrin using a modified Hertz model for thin films. In this method,spherical indenters are positioned on top of the fibrin samples,generating an indentation depth that is then correlated with elastic modulus. Neurons function by transmitting electrical signals to one another and being able to assess the development of electrical signaling serves is an important verification step when engineering neural tissues. We then validated a protocol wherein the electrical activity of motor neural cultures is measured directly by a voltage sensitive dye and a microplate reader without causing damage to the cells. These protocols provide a non-destructive method for characterizing the mechanical and electrical properties of living spinal cord tissues using novel biosensing methods. View Publication

Gut organoids are stem cell derived 3D models of the intestinal epithelium that are useful for studying interactions between enteric pathogens and their host. While the organoid model has been used for both bacterial and viral infections,this is a closed system with the luminal side being inaccessible without microinjection or disruption of the organoid polarization. In order to overcome this and simplify their applicability for transepithelial studies,permeable membrane based monolayer approaches are needed. In this paper,we demonstrate a method for generating a monolayer model of the human fetal intestinal polarized epithelium that is fully characterized and validated. Proximal and distal small intestinal organoids were used to generate 2D monolayer cultures,which were characterized with respect to epithelial cell types,polarization,barrier function,and gene expression. In addition,viral replication and bacterial translocation after apical infection with enteric pathogens Enterovirus A71 and Listeria monocytogenes were evaluated,with subsequent monitoring of the pro-inflammatory host response. This human 2D fetal intestinal monolayer model will be a valuable tool to study host-pathogen interactions and potentially reduce the use of animals in research. View Publication

Microelectrode material and cell culture medium have significant roles in the signal-to-noise ratio and cell well-being in in vitro electrophysiological studies. Here,we report an ion beam assisted e-beam deposition (IBAD) based process as an alternative titanium nitride (TiN) deposition method for sputtering in the fabrication of state-of-the-art TiN microelectrode arrays (MEAs). The effects of evaporation and nitrogen flow rates were evaluated while developing the IBAD TiN deposition process. Moreover,the produced IBAD TiN microelectrodes were characterized by impedance,charge transfer capacity (CTC) and noise measurements for electrical properties,AFM and SEM for topological imaging,and EDS for material composition. The impedance (at 1 kHz) of brand new 30 $\mu$m IBAD TiN microelectrodes was found to be double but still below 100 k$\Omega$ compared with commercial reference MEAs with sputtered TiN microelectrodes of the same size. On the contrary,the noise level of IBAD TiN MEAs was lower compared with that of commercial sputtered TiN MEAs in equal conditions. In CTC IBAD TiN electrodes (3.3 mC/cm2) also outperformed the sputtered counterparts (2.0 mC/cm2). To verify the suitability of IBAD TiN microelectrodes for cell measurements,human pluripotent stem cell (hPSC)-derived neuronal networks were cultured on IBAD TiN MEAs and commercial sputtered TiN MEAs in two different media: neural differentiation medium (NDM) and BrainPhys (BPH). The effect of cell culture media to hPSC derived neuronal networks was evaluated to gain more stable and more active networks. Higher spontaneous activity levels were measured from the neuronal networks cultured in BPH compared with those in NDM in both MEA types. However,BPH caused more problems in cell survival in long-term cultures by inducing neuronal network retraction and clump formation after 1-2 weeks. In addition,BPH was found to corrode the Si3N4 insulator layer more than NDM medium. The developed IBAD TiN process gives MEA manufacturers more choices to choose which method to use to deposit TiN electrodes and the medium evaluation results remind that not only electrode material but also insulator layer and cell culturing medium have crucial role in successful long term MEA measurements. View Publication

Efficient generation of antibodies by B cells is one of the prerequisites of protective immunity. B cell activation by cognate antigens via B cell receptors (BCRs),or pathogen-associated molecules through pattern-recognition receptors,such as Toll-like receptors (TLRs),leads to transcriptional and metabolic changes that ultimately transform B cells into antibody-producing plasma cells or memory cells. BCR signaling and a number of steps downstream of it rely on coordinated action of cellular membranes and the actin cytoskeleton,tightly controlled by concerted action of multiple regulatory proteins,some of them exclusive to B cells. Here,we dissect the role of Missing-In-Metastasis (MIM),or Metastasis suppressor 1 (MTSS1),a cancer-associated membrane and actin cytoskeleton regulating protein,in B cell-mediated immunity by taking advantage of MIM knockout mouse strain. We show undisturbed B cell development and largely normal composition of B cell compartments in the periphery. Interestingly,we found that MIM-/- B cells are defected in BCR signaling in response to surface-bound antigens but,on the other hand,show increased metabolic activity after stimulation with LPS or CpG. In vivo,MIM knockout animals exhibit impaired IgM antibody responses to immunization with T cell-independent antigen. This study provides the first comprehensive characterization of MIM in B cells,demonstrates its regulatory role for B cell-mediated immunity,as well as proposes new functions for MIM in tuning receptor signaling and cellular metabolism,processes,which may also contribute to the poorly understood functions of MIM in cancer. View Publication

Human pluripotent stem cells including induced pluripotent stem cells (iPSCs) and embryonic stem cells hold great promise for cell-based therapies,but safety concerns that complicate consideration for routine clinical use remain. Installing a safety switch" based on the inducible caspase-9 (iCASP9) suicide gene system should offer added control over undesirable cell replication or activity. Previous studies utilized lentiviral vectors to integrate the iCASP9 system into T cells and iPSCs. This method results in random genomic insertion of the suicide switch and inefficient killing of the cells after the switch is "turned on" with a small molecule (eg AP1903). To improve the safety and efficiency of the iCASP9 system for use in iPSC-based therapy we precisely installed the system into a genomic safe harbor the AAVS1 locus in the PPP1R12C gene. We then evaluated the efficiencies of different promoters to drive iCASP9 expression in human iPSCs. We report that the commonly used EF1$\alpha$ promoter is silenced in iPSCs and that the endogenous promoter of the PPP1R12C gene is not strong enough to drive high levels of iCASP9 expression. However the CAG promoter induces strong and stable iCASP9 expression in iPSCs and activation of this system with AP1903 leads to rapid killing and complete elimination of iPSCs and their derivatives including MSCs and chondrocytes in vitro. Furthermore iPSC-derived teratomas shrank dramatically or were completely eliminated after administration of AP1903 in mice. Our data suggest significant improvements on existing iCASP9 suicide switch technologies and may serve as a guide to other groups seeking to improve the safety of stem cell-based therapies." View Publication

Leukocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase plays a key role in host defense and immune regulation. Genetic defects in NADPH oxidase result in chronic granulomatous disease (CGD),characterized by recurrent bacterial and fungal infections and aberrant inflammation. Key drivers of hyper-inflammation induced by fungal cell walls in CGD are still incompletely defined. Here,we found that CGD (CYBB-null) neutrophils produced higher amounts of leukotriene B4 (LTB4) in vitro following activation with zymosan or Immune complexes,as compared to wild type (WT) neutrophils. This correlated with increased calcium influx in CGD neutrophils,which is restrained in WT neutrophils by the electrogenic activity of the NADPH oxidase. Increased LTB4 generation by CGD neutrophils was also augmented by paracrine cross-talk with the LTB4 receptor BLT1. CGD neutrophils formed more numerous and larger clusters in the presence of zymosan in vitro compared to WT,which was also LTB4- and BLT1-dependent. In zymosan-induced lung inflammation,focal neutrophil infiltrates were increased in CGD compared to WT mice and associated with higher LTB4 levels. Inhibiting LTB4 synthesis or antagonizing the BLT1 receptor following zymosan challenge reduced lung neutrophil recruitment in CGD to WT levels. Thus,LTB4 was the major driver of excessive neutrophilic lung inflammation in CGD mice in the early response to fungal cell walls,likely by a dysregulated feed-forward loop involving amplified neutrophil production of LTB4. This study identifies neutrophil LTB4 generation as a target of NADPH oxidase regulation,which could potentially be exploited therapeutically to reduce excessive inflammation in CGD. View Publication

Activation of RIPK1 controls TNF-mediated apoptosis,necroptosis and inflammatory pathways1. Cleavage of human and mouse RIPK1 after residues D324 and D325,respectively,by caspase-8 separates the RIPK1 kinase domain from the intermediate and death domains. The D325A mutation in mouse RIPK1 leads to embryonic lethality during mouse development2,3. However,the functional importance of blocking caspase-8-mediated cleavage of RIPK1 on RIPK1 activation in humans is unknown. Here we identify two families with variants in RIPK1 (D324V and D324H) that lead to distinct symptoms of recurrent fevers and lymphadenopathy in an autosomal-dominant manner. Impaired cleavage of RIPK1 D324 variants by caspase-8 sensitized patients' peripheral blood mononuclear cells to RIPK1 activation,apoptosis and necroptosis induced by TNF. The patients showed strong RIPK1-dependent activation of inflammatory signalling pathways and overproduction of inflammatory cytokines and chemokines compared with unaffected controls. Furthermore,we show that expression of the RIPK1 mutants D325V or D325H in mouse embryonic fibroblasts confers not only increased sensitivity to RIPK1 activation-mediated apoptosis and necroptosis,but also induction of pro-inflammatory cytokines such as IL-6 and TNF. By contrast,patient-derived fibroblasts showed reduced expression of RIPK1 and downregulated production of reactive oxygen species,resulting in resistance to necroptosis and ferroptosis. Together,these data suggest that human non-cleavable RIPK1 variants promote activation of RIPK1,and lead to an autoinflammatory disease characterized by hypersensitivity to apoptosis and necroptosis and increased inflammatory response in peripheral blood mononuclear cells,as well as a compensatory mechanism to protect against several pro-death stimuli in fibroblasts. View Publication

INTRODUCTION The stem cell portion of the dental pulp derived cultures (DPSCs) showed a higher resistance to cytotoxic effect of restorative dental materials compared to pulpal fibroblasts (DPFs). Here,we aimed to compare the expression of some drug resistant genes between these cells. METHODS AND MATERIALS To separate DPSCs from DPFs,we used magnetic cell sorting technique based on CD146 expression. To assess the stem cell properties,the positive and negative portions underwent colony forming assays and were induced to be differentiated into the adipocytes,osteoblasts,hepatocytes,and neural cells. Cell surface antigen panels were checked using immune fluorescence and flow-cytometry techniques. The mRNA expression of 14 ABC transporters including ABCA2,ABCB1,ABCB11,ABCC1,ABCC2,ABCC3,ABCC4,ABCC5-2,ABCC5-4,ABCC5-13,ABCC6,ABCC10,ABCC11,and ABCG2 genes was assessed,using quantitative RT-PCR technique. RESULTS Only the CD146 positive portion could be differentiated into the desired fates,and they formed higher colonies (16.7 ± 3.32 vs. 1.7 ± 1.67,p {\textless} .001). The cell surface antigen panels were the same,except for CD146 and STRO-1 markers which were expressed only in the positive portion. Among the ABC transporter genes studied,the positive portion showed a higher expression (approximately two-fold) of ABCA2,ABCC5-13,and ABCC5-2 genes. CONCLUSION Dental pulp stem cells which can be separated from dental pulp fibroblasts based on CD146 expression,express higher levels of some drug resistance genes which probably accounts for their features of more resistance to cytotoxic effects of some dental materials. This needs to be more validated in future. View Publication

Increasing occurrence of moderate to severe intrauterine adhesion (IUA) is seriously affecting the quality of human life. The aim of the study was to establish IUA models in nonhuman primates and to explore the dual repair effects of human umbilical cord-derived mesenchymal stem cells (huMSCs) loaded on autocrosslinked hyaluronic acid gel (HA-GEL) on endometrial damage and adhesion. Here,we recorded the menstrual cycle data in detail with uterine cavities observed and endometrial tissues detected after intervention,and the thicker endometria,decreased amount of fibrotic formation,increased number of endometrium glands,etc.,suggested that both HA-GEL and huMSC/HA-GEL complexes could partially repair IUA caused by mechanical injury,but huMSC/HA-GEL complex transplantation had notable dual repair effects: a reliable antiadhesion property and the promotion of endometrial regeneration. View Publication