Drosophila as an In Vivo Model for Human Neurodegenerative Disease

With the increase in the ageing population, neurodegenerative disease is devastating to families and poses a huge burden on society. The brain and spinal cord are extraordinarily complex: they consist of a highly organized network of neuronal and support cells that communicate in a highly specialized manner. One approach to tackling problems of such complexity is to address the scientific questions in simpler, yet analogous, systems. The fruit fly, Drosophila melanogaster, has been proven tremendously valuable as a model organism, enabling many major discoveries in neuroscientific disease research. The plethora of genetic tools available in Drosophila allows for exquisite targeted manipulation of the genome. Due to its relatively short lifespan, complex questions of brain function can be addressed more rapidly than in other model organisms, such as the mouse. Here we discuss features of the fly as a model for human neurodegenerative disease. There are many distinct fly models for a range of neurodegenerative diseases; we focus on select studies from models of polyglutamine disease and amyotrophic lateral sclerosis that illustrate the type and range of insights that can be gleaned. In discussion of these models, we underscore strengths of the fly in providing understanding into mechanisms and pathways, as a foundation for translational and therapeutic research.     

Attenuated T Cell Responses to a High-Potency Ligand In Vivo

αβ T cell receptor (TCR) recognition of foreign peptides bound to major histocompatibility complex (pMHC) molecules on the surface of antigen presenting cells is a key event in the initiation of adaptive cellular immunity. In vitro, high-affinity binding and/or long-lived interactions between TCRs and pMHC correlate with high-potency T cell activation. However, less is known about the influence of TCR/pMHC interaction parameters on T cell responses in vivo. We studied the influence of TCR/pMHC binding characteristics on in vivo T cell immunity by tracking CD4⁺ T cell activation, effector, and memory responses to immunization with peptides exhibiting a range of TCR/pMHC half-lives and in vitro T cell activation potencies. Contrary to predictions from in vitro studies, we found that optimal in vivo T cell responses occur to ligands with intermediate TCR/pMHC half-lives. The diminished in vivo responses we observed to the ligand exhibiting the longest TCR/pMHC half-life were associated with attenuation of intracellular signaling, expansion, and function over a broad range of time points. Our results reveal a level of control over T cell activation in vivo not recapitulated in in vitro assays and highlight the importance of considering in vivo efficacy of TCR ligands as part of vaccine design.     

The Resistance of Retroviral Vectors Produced from Human Cells to Serum Inactivation In Vivo and In Vitro Is Primate Species Dependent

The ability to deliver genes as therapeutics requires an understanding of the vector pharmacokinetics similar to that required for conventional drugs. A first question is the half-life of the vector in the bloodstream. Retroviral vectors produced in certain human cell lines differ from vectors produced in nonhuman cell lines in being substantially resistant to inactivation in vitro by human serum complement (F. L. Cosset, Y. Takeuchi, J. L. Battini, R. A. Weiss, and M. K. Collins, J. Virol. 69:7430-7436, 1995). Thus, use of human packaging cell lines (PCL) may produce vectors with longer half-lives, resulting in more-efficacious in vivo gene therapy. However, survival of human PCL-produced vectors in vivo following systemic administration has not been explored. In this investigation, the half-lives of retroviral vectors packaged by either canine D17 or human HT1080 PCL were measured in the bloodstreams of macaques and chimpanzees. Human PCL-produced vectors exhibited significantly higher concentrations of circulating biologically active vector at the earliest time points measured (>1, 000-fold in chimpanzees), as well as substantially extended half-lives, compared to canine PCL-produced vectors. In addition, the circulation half-life of human PCL-produced vector was longer in chimpanzees than in macaques. This was consistent with in vitro findings which demonstrated that primate serum inactivation of vector produced from human PCL increased with increasing phylogenetic distance from humans. These results establish that in vivo retroviral vector half-life correlates with in vitro resistance to complement. Furthermore, these findings should influence the choice of animal models used to evaluate retroviral-vector-based therapies.     

Homing of Regulatory T Cells to Human Skin Is Important for the Prevention of Alloimmune-Mediated Pathology in an In Vivo Cellular Therapy Model

Regulatory T cell (Treg) therapy for immune modulation is a promising therapeutic strategy for the treatment and prevention of autoimmune disease and graft-versus-host disease (GvHD) after bone marrow transplantation. However, Treg are heterogeneous and express a variety of chemokine receptor molecules. The optimal subpopulation of Treg for therapeutic use may vary according to the pathological target. Indeed, clinical trials of Treg for the prevention of GvHD where the skin is a major target of the anti-host response have employed Treg derived from a variety of different sources. We postulated that for the effective treatment of GvHD-related skin pathology, Treg must be able to migrate to skin in order to regulate local alloimmune responses efficiently. To test the hypothesis that different populations of Treg display distinct efficacy in vivo based on their expression of tissue-specific homing molecules, we evaluated the activity of human Treg derived from two disparate sources in a model of human skin transplantation. Treg were derived from adult blood or cord blood and expanded in vitro. While Treg from both sources displayed similar in vitro suppressive efficacy, they exhibited marked differences in the expression of skin homing molecules. Importantly, only adult-derived Treg were able to prevent alloimmune-mediated human skin destruction in vivo, by virtue of their improved migration to skin. The presence of Treg within the skin was sufficient to prevent its alloimmune-mediated destruction. Additionally, Treg expressing the skin homing cutaneous lymphocyte antigen (CLA) were more efficient at preventing skin destruction than their CLA-deficient counterparts. Our findings highlight the importance of the careful selection of an effective subpopulation of Treg for clinical use according to the pathology of interest.     

Resistance of Human Liver Mesenchymal Stem Cells to FAS-Induced Cell Death

Mesenchymal stem cells (MSCs) have a pronounced therapeutic potential in various pathological conditions. Though therapeutic effects of MSC transplantation have been studied for a long time, the underlying mechanisms are still not clear. It has been shown that transplanted MSCs are rapidly eliminated, presumably by apoptosis. As the mechanisms of MSC apoptosis are not fully understood, in the present work we analyzed MSC sensitivity to Fas-induced apoptosis using MSCs isolated from the biopsies of liver fibrosis patients (L-MSCs). The level of cell death was analyzed by flow cytometry in the propidium iodide test. The luminescent ATP assay was used to measure cellular ATP levels; and the mitochondrial membrane potential was assessed using the potential-dependent dye JC-1. We found that human L-MSCs were resistant to Fas-induced cell death over a wide range of FasL and anti-Fas mAb concentrations. At the same time, intrinsic death signal inducers CoCl₂ and staurosporine caused apoptosis of L-MSCs in a dose-dependent manner. Despite the absence of Fas-induced cell death treatment of L-MSCs with low concentrations of FasL or anti-Fas mAb resulted in a cellular ATP level decrease, while high concentrations of the inducers caused a decline of the mitochondrial membrane potential. Pre-incubation of L-MSCs with the pro-inflammatory cytokine TNF-α did not promote L-MSC cell death. Our data indicate that human L-MSCs have increased resistance to receptor-mediated cell death even under inflammatory conditions.     

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological deficiencies. GVHD is caused by mature alloreactive T cells present in the bone marrow graft that are infused into the recipient and cause damage to host organs. However, in mice, T cells must be added to the bone marrow inoculum to cause GVHD. Although extensive work has been done to characterize T cell responses post transplant, bioluminescent imaging technology is a non-invasive method to monitor T cell trafficking patterns in vivo. Following lethal irradiation, recipient mice are transplanted with bone marrow cells and splenocytes from donor mice. T cell subsets from L2G85.B6 (transgenic mice that constitutively express luciferase) are included in the transplant. By only transplanting certain T cell subsets, one is able to track specific T cell subsets in vivo, and based on their location, develop hypotheses regarding the role of specific T cell subsets in promoting GVHD at various time points. At predetermined intervals post transplant, recipient mice are imaged using a Xenogen IVIS CCD camera. Light intensity can be quantified using Living Image software to generate a pseudo-color image based on photon intensity (red = high intensity, violet = low intensity). Between 4-7 days post transplant, recipient mice begin to show clinical signs of GVHD. Cooke et al.¹ developed a scoring system to quantitate disease progression based on the recipient mice fur texture, skin integrity, activity, weight loss, and posture. Mice are scored daily, and euthanized when they become moribund. Recipient mice generally become moribund 20-30 days post transplant. Murine models are valuable tools for studying the immunology of GVHD. Selectively transplanting particular T cell subsets allows for careful identification of the roles each subset plays. Non-invasively tracking T cell responses in vivo adds another layer of value to murine GVHD models.     

Directed In Vitro Myogenesis of Human Embryonic Stem Cells and Their In Vivo Engraftment

Development of human embryonic stem cell (hESC)-based therapy requires derivation of in vitro expandable cell populations that can readily differentiate to specified cell types and engraft upon transplantation. Here, we report that hESCs can differentiate into skeletal muscle cells without genetic manipulation. This is achieved through the isolation of cells expressing a mesodermal marker, platelet-derived growth factor receptor-α (PDGFRA), following embryoid body (EB) formation. The ESC-derived cells differentiated into myoblasts in vitro as evident by upregulation of various myogenic genes, irrespective of the presence of serum in the medium. This result is further corroborated by the presence of sarcomeric myosin and desmin, markers for terminally differentiated cells. When transplanted in vivo, these pre-myogenically committed cells were viable in tibialis anterior muscles 14 days post-implantation. These hESC-derived cells, which readily undergo myogenic differentiation in culture medium containing serum, could be a viable cell source for skeletal muscle repair and tissue engineering to ameliorate various muscle wasting diseases.     

Regulation of T Cell Motility In Vitro and In Vivo by LPA and LPA2

Lysophosphatidic acid (LPA) and the LPA-generating enzyme autotaxin (ATX) have been implicated in lymphocyte trafficking and the regulation of lymphocyte entry into lymph nodes. High local concentrations of LPA are thought to be present in lymph node high endothelial venules, suggesting a direct influence of LPA on cell migration. However, little is known about the mechanism of action of LPA, and more work is needed to define the expression and function of the six known G protein-coupled receptors (LPA 1–6) in T cells. We studied the effects of 18∶1 and 16∶0 LPA on naïve CD4+ T cell migration and show that LPA induces CD4+ T cell chemorepulsion in a Transwell system, and also improves the quality of non-directed migration on ICAM-1 and CCL21 coated plates. Using intravital two-photon microscopy, lpa2−/− CD4+ T cells display a striking defect in early migratory behavior at HEVs and in lymph nodes. However, later homeostatic recirculation and LPA-directed migration in vitro were unaffected by loss of lpa2. Taken together, these data highlight a previously unsuspected and non-redundant role for LPA2 in intranodal T cell motility, and suggest that specific functions of LPA may be manipulated by targeting T cell LPA receptors.     

The Reversal Effects of 3-Bromopyruvate on Multidrug Resistance In Vitro and In Vivo Derived from Human Breast MCF-7/ADR Cells

Purpose

P-glycoprotein mediated efflux is one of the main mechanisms for multidrug resistance in cancers, and 3-Bromopyruvate acts as a promising multidrug resistance reversal compound in our study. To test the ability of 3-Bromopyruvate to overcome P-glycoprotein-mediated multidrug resistance and to explore its mechanisms of multidrug resistance reversal in MCF-7/ADR cells, we evaluate the in vitro and in vivo modulatory activity of this compound.

Methods

The in vitro and in vivo activity was determined using the MTT assay and human breast cancer xenograft models. The gene and protein expression of P-glycoprotein were determined using real-time polymerase chain reaction and the Western blotting technique, respectively. ABCB-1 bioactivity was tested by fluorescence microscopy, multi-mode microplate reader, and flow cytometry. The intracellular levels of ATP, HK-II, and ATPase activity were based on an assay kit according to the manufacturer’s instructions.

Results

3-Bromopyruvate treatment led to marked decreases in the IC₅₀ values of selected chemotherapeutic drugs [e.g., doxorubicin (283 folds), paclitaxel (85 folds), daunorubicin (201 folds), and epirubicin (171 folds)] in MCF-7/ADR cells. 3-Bromopyruvate was found also to potentiate significantly the antitumor activity of epirubicin against MCF-7/ADR xenografts. The intracellular level of ATP decreased 44%, 46% in the presence of 12.5.25 µM 3-Bromopyruvate, whereas the accumulation of rhodamine 123 and epirubicin (two typical P-glycoprotein substrates) in cells was significantly increased. Furthermore, we found that the mRNA and the total protein level of P-glycoprotein were slightly altered by 3-Bromopyruvate. Moreover, the ATPase activity was significantly inhibited when 3-Bromopyruvate was applied.

Conclusion

We demonstrated that 3-Bromopyruvate can reverse P-glycoprotein-mediated efflux in MCF-7/ADR cells. Multidrug resistance reversal by 3-Bromopyruvate occurred through at least three approaches, namely, a decrease in the intracellular level of ATP and HK-II bioactivity, the inhibition of ATPase activity, and the slight decrease in P-glycoprotein expression in MCF-7/ADR cells.     

Rotating Cell Culture Systems for Human Cell Culture: Human Trophoblast Cells as a Model

The field of human trophoblast research aids in understanding the complex environment established during placentation. Due to the nature of these studies, human in vivo experimentation is impossible. A combination of primary cultures, explant cultures and trophoblast cell lines¹ support our understanding of invasion of the uterine wall² and remodeling of uterine spiral arteries^3,4 by extravillous trophoblast cells (EVTs), which is required for successful establishment of pregnancy. Despite the wealth of knowledge gleaned from such models, it is accepted that in vitro cell culture models using EVT-like cell lines display altered cellular properties when compared to their in vivo counterparts^5,6. Cells cultured in the rotating cell culture system (RCCS) display morphological, phenotypic, and functional properties of EVT-like cell lines that more closely mimic differentiating in utero EVTs, with increased expression of genes mediating invasion (e.g. matrix metalloproteinases (MMPs)) and trophoblast differentiation^7,8,9. The Saint Georges Hospital Placental cell Line-4 (SGHPL-4) (kindly donated by Dr. Guy Whitley and Dr. Judith Cartwright) is an EVT-like cell line that was used for testing in the RCCS.The design of the RCCS culture vessel is based on the principle that organs and tissues function in a three-dimensional (3-D) environment. Due to the dynamic culture conditions in the vessel, including conditions of physiologically relevant shear, cells grown in three dimensions form aggregates based on natural cellular affinities and differentiate into organotypic tissue-like assemblies^10,11,12 . The maintenance of a fluid orbit provides a low-shear, low-turbulence environment similar to conditions found in vivo. Sedimentation of the cultured cells is countered by adjusting the rotation speed of the RCCS to ensure a constant free-fall of cells. Gas exchange occurs through a permeable hydrophobic membrane located on the back of the bioreactor. Like their parental tissue in vivo, RCCS-grown cells are able to respond to chemical and molecular gradients in three dimensions (i.e. at their apical, basal, and lateral surfaces) because they are cultured on the surface of porous microcarrier beads. When grown as two-dimensional monolayers on impermeable surfaces like plastic, cells are deprived of this important communication at their basal surface. Consequently, the spatial constraints imposed by the environment profoundly affect how cells sense and decode signals from the surrounding microenvironment, thus implying an important role for the 3-D milieu¹³.We have used the RCCS to engineer biologically meaningful 3-D models of various human epithelial tissues^7,14,15,16. Indeed, many previous reports have demonstrated that cells cultured in the RCCS can assume physiologically relevant phenotypes that have not been possible with other models^10,17-21. In summary, culture in the RCCS represents an easy, reproducible, high-throughput platform that provides large numbers of differentiated cells that are amenable to a variety of experimental manipulations. In the following protocol, using EVTs as an example, we clearly describe the steps required to three-dimensionally culture adherent cells in the RCCS.     

Clearance of Human IgG1-Sensitised Red Blood Cells In Vivo in Humans Relates to the In Vitro Properties of Antibodies from Alternative Cell Lines

We previously produced a recombinant version of the human anti-RhD antibody Fog-1 in the rat myeloma cell line, YB2/0. When human, autologous RhD-positive red blood cells (RBC) were sensitised with this IgG1 antibody and re-injected, they were cleared much more rapidly from the circulation than had been seen earlier with the original human-mouse heterohybridoma-produced Fog-1. Since the IgG have the same amino acid sequence, this disparity is likely to be due to alternative glycosylation that results from the rat and mouse cell lines. By comparing the in vitro properties of YB2/0-produced Fog-1 IgG1 and the same antibody produced in the mouse myeloma cell line NS0, we now have a unique opportunity to pinpoint the cause of the difference in ability to clear RBC in vivo. Using transfected cell lines that express single human FcγR, we showed that IgG1 made in YB2/0 and NS0 cell lines bound equally well to receptors of the FcγRI and FcγRII classes but that the YB2/0 antibody was superior in FcγRIII binding. When measuring complexed IgG binding, the difference was 45-fold for FcγRIIIa 158F, 20-fold for FcγRIIIa 158V and approximately 40-fold for FcγRIIIb. The dissimilarity was greater at 100-fold in monomeric IgG binding assays with FcγRIIIa. When used to sensitise RBC, the YB2/0 IgG1 generated 100-fold greater human NK cell antibody-dependent cell-mediated cytotoxicity and had a 10³-fold advantage over the NS0 antibody in activating NK cells, as detected by CD54 levels. In assays of monocyte activation and macrophage adherence/phagocytosis, where FcγRI plays major roles, RBC sensitised with the two antibodies produced much more similar results. Thus, the alternative glycosylation profiles of the Fog-1 antibodies affect only FcγRIII binding and FcγRIII-mediated functions. Relating this to the in vivo studies confirms the importance of FcγRIII in RBC clearance.     

Salinomycin Inhibits Proliferation and Induces Apoptosis of Human Hepatocellular Carcinoma Cells In Vitro and In Vivo

The anti-tumor antibiotic salinomycin (Sal) was recently identified as a selective inhibitor of breast cancer stem cells; however, the effect of Sal on hepatocellular carcinoma (HCC) is not clear. This study aimed to determine the anti-tumor efficacy and mechanism of Sal on HCC. HCC cell lines (HepG2, SMMC-7721, and BEL-7402) were treated with Sal. Cell doubling time was determinated by drawing growth curve, cell viability was evaluated using the Cell Counting Kit 8. The fraction of CD133⁺ cell subpopulations was assessed by flow cytometry. We found that Sal inhibits proliferation and decreases PCNA levels as well as the proportion of HCC CD133⁺cell subpopulations in HCC cells. Cell cycle was analyzed using flow cytometry and showed that Sal caused cell cycle arrest of the various HCC cell lines in different phases. Cell apoptosis was evaluated using flow cytometry and Hoechst 33342 staining. Sal induced apoptosis as characterized by an increase in the Bax/Bcl-2 ratio. Several signaling pathways were selected for further mechanistic analyses using real time-PCR and Western blot assays. Compared to control, β-catenin expression is significantly down-regulated upon Sal addition. The Ca²⁺ concentration in HCC cells was examined by flow cytometry and higher Ca²⁺ concentrations were observed in Sal treatment groups. The anti-tumor effect of Sal was further verified in vivo using the hepatoma orthotopic tumor model and the data obtained showed that the size of liver tumors in Sal-treated groups decreased compared to controls. Immunohistochemistry and TUNEL staining also demonstrated that Sal inhibits proliferation and induces apoptosis in vivo. Finally, the role of Sal on in vivo Wnt/β-catenin signaling was evaluated by Western blot and immunohistochemistry. This study demonstrates Sal inhibits proliferation and induces apoptosis of HCC cells in vitro and in vivo and one potential mechanism is inhibition of Wnt/β-catenin signaling via increased intracellular Ca²⁺ levels.     

Induction of G1 Cell Cycle Arrest in Human Glioma Cells by Salinomycin Through Triggering ROS-Mediated DNA Damage In Vitro and In Vivo

Chemotherapy has always been one of the most effective ways in combating human glioma. However, the high metastatic potential and resistance toward standard chemotherapy severely hindered the chemotherapy outcomes. Hence, searching effective chemotherapy drugs and clarifying its mechanism are of great significance. Salinomycin an antibiotic shows novel anticancer potential against several human tumors, including human glioma, but its mechanism against human glioma cells has not been fully elucidated. In the present study, we demonstrated that salinomycin treatment time- and dose-dependently inhibited U251 and U87 cells growth. Mechanically, salinomycin-induced cell growth inhibition against human glioma was mainly achieved by induction of G1-phase arrest via triggering reactive oxide species (ROS)-mediated DNA damage, as convinced by the activation of histone, p53, p21 and p27. Furthermore, inhibition of ROS accumulation effectively attenuated salinomycin-induced DNA damage and G1 cell cycle arrest, and eventually reversed salinomycin-induced cytotoxicity. Importantly, salinomycin treatment also significantly inhibited the U251 tumor xenograft growth in vivo through triggering DNA damage-mediated cell cycle arrest with involvement of inhibiting cell proliferation and angiogenesis. The results above validated the potential of salinomycin-based chemotherapy against human glioma.     

Characterization of In Vivo Dlg1 Deletion on T Cell Development and Function

Background

The polarized reorganization of the T cell membrane and intracellular signaling molecules in response to T cell receptor (TCR) engagement has been implicated in the modulation of T cell development and effector responses. In siRNA-based studies Dlg1, a MAGUK scaffold protein and member of the Scribble polarity complex, has been shown to play a role in T cell polarity and TCR signal specificity, however the role of Dlg1 in T cell development and function in vivo remains unclear.

Methodology/Principal Findings

Here we present the combined data from three independently-derived dlg1-knockout mouse models; two germline deficient knockouts and one conditional knockout. While defects were not observed in T cell development, TCR-induced early phospho-signaling, actin-mediated events, or proliferation in any of the models, the acute knockdown of Dlg1 in Jurkat T cells diminished accumulation of actin at the IS. Further, while Th1-type cytokine production appeared unaffected in T cells derived from mice with a dlg1germline-deficiency, altered production of TCR-dependent Th1 and Th2-type cytokines was observed in T cells derived from mice with a conditional loss of dlg1 expression and T cells with acute Dlg1 suppression, suggesting a differential requirement for Dlg1 activity in signaling events leading to Th1 versus Th2 cytokine induction. The observed inconsistencies between these and other knockout models and siRNA strategies suggest that 1) compensatory upregulation of alternate gene(s) may be masking a role for dlg1 in controlling TCR-mediated events in dlg1 deficient mice and 2) the developmental stage during which dlg1 ablation begins may control the degree to which compensatory events occur.

Conclusions/Significance

These findings provide a potential explanation for the discrepancies observed in various studies using different dlg1-deficient T cell models and underscore the importance of acute dlg1 ablation to avoid the upregulation of compensatory mechanisms for future functional studies of the Dlg1 protein.