Theiler’s murine encephalomyelitis virus infection induces a redistribution of heat shock proteins 70 and 90 in BHK-21 cells,and is inhibited by novobiocin and geldanamycin

Theiler’s murine encephalomyelitis virus (TMEV) is a positive-sense RNA virus belonging to the Cardiovirus genus in the family Picornaviridae. In addition to other host cellular factors and pathways, picornaviruses utilise heat shock proteins (Hsps) to facilitate their propagation in cells. This study investigated the localisation of Hsps 70 and 90 in TMEV-infected BHK-21 cells by indirect immunofluorescence and confocal microscopy. The effect of Hsp90 inhibitors novobiocin (Nov) and geldanamycin (GA) on the development of cytopathic effect (CPE) induced by infection was also examined. Hsp90 staining was uniformly distributed in the cytoplasm of uninfected cells but was found concentrated in the perinuclear region during late infection where it overlapped with the signal for non-structural protein 2C within the viral replication complex. Hsp70 redistributed into the vicinity of the viral replication complex during late infection, but its distribution did not overlap with that of 2C. Inhibition of Hsp90 by GA and Nov had a negative effect on virus growth over a 48-h period as indicated by no observable CPE in treated compared to untreated cells. 2C was detected by Western analysis of GA-treated infected cell lysates at doses between 0.01 and 0.125 μM, suggesting that processing of viral precursors was not affected in the presence of this drug. In contrast, 2C was absent in cell lysates of Nov-treated cells at doses above 10 μM, although CPE was evident 48 hpi. This is the first study describing the dynamic behaviour of Hsps 70 and 90 in TMEV-infected cells and to identify Hsp90 as an important host factor in the life cycle of this virus.     

Dynamic changes and molecular analysis of cell death in the spinal cord of SJL mice infected with the BeAn strain of Theiler’s murine encephalomyelitis virus

Theiler’s murine encephalomyelitis (TME) is caused by the TME virus (TMEV) and represents an important animal model for multiple sclerosis (MS). Oligodendroglial apoptosis and reduced apoptotic elimination of encephalitogenic leukocytes seem to participate in autoimmune demyelination in MS. The present study quantified apoptotic cells in BeAn–TMEV-induced spinal cord white matter lesions at 14, 42, 98, and 196 days post infection (dpi) using immunostaining. Apoptotic cells were identified by transmission electron microscopy and double-immunofluorescence. The mRNA expression of apoptosis-related genes was investigated using microarray analysis. Oligodendroglial apoptosis was already detected in the predemyelinating phase at 14 dpi. Apoptotic cell numbers peaked at 42 dpi and decreased until 196 dpi partly due to reduced T cell apoptosis. In addition to genes involved in the classical pathways of apoptosis induction, microarray analysis detected the expression of genes related to alternative mechanisms of cell death such as pyroptosis, necroptosis, and endoplasmic reticulum stress. Consequently, oligodendroglial apoptosis is involved in the initiation of the TME demyelination process, whereas the development of apoptosis resistance of T cells potentially favors the maintenance of inflammation and myelin loss.     

Proteome analysis of virus–host cell interaction: rabies virus replication in Vero cells in two different media

The use of Vero cells for rabies vaccine production was recommended from the WHO in 2005. A controlled production process is necessary to reduce the risk of contaminants in the product. One step towards this is to turn away from animal-derived components (e.g. serum, trypsin, bovine serum albumin) and face a production process in animal component-free medium. In this study, a proteomic approach was applied, using 2-D differential gel electrophoresis and mass spectrometry to compare rabies virus propagation in Vero cells under different cultivation conditions in microcarrier culture. Protein alterations were investigated for uninfected and infected Vero cells over a time span from 1 to 8 days post-infection in two different types of media (serum-free versus serum-containing media). For mock-infected cells, proteins involved in stress response, redox status, protease activity or glycolysis, and protein components in the endoplasmic reticulum were found to be differentially expressed comparing both cultivation media at all sampling points. For virus-infected cells, additionally changes in protein expression involved in general cell regulation and in calcium homeostasis were identified under both cultivation conditions. The fact that neither of these additional proteins was identified for cells during mock infection, but similar protein expression changes were found for both systems during virus propagation, indicates for a specific response of the Vero cell proteome on rabies virus infection.     

Sulindac induces apoptotic cell death in susceptible human breast cancer cells through, at least in part, inhibition of IKKβ

Sulindac is a non-steroidal anti-inflammatory agent with anti-tumor activities that include the induction of apoptosis in various cancer cells and the inhibition malignant transformation. However, the molecular mechanisms underlying these effects are unclear. Recently, it has been shown that sulindac can inhibit NF-κB activation. Here, we demonstrate that sulindac induces apoptotic cell death in susceptible human breast cancer cells through, at least in part, inhibition of IKKβ activity. More specifically, when we compared two different human breast cancer cell lines, Hs578T, which has relatively low basal IKKβ activity, and MDA-MB231, which has relatively high basal IKKβ activity, we found that MDA-MB231 was markedly more sensitive to sulindac-induced apoptosis than Hs578T. This was associated with greater caspase-3 and -9 activity in sulindac-treated MDA-MB231 cells. Using a combination of chemical kinase inhibitors and siRNA-mediated knockdown of specific kinases, we found that sulindac inhibits IKKβ, which, in turn, leads to the p38 MAPK-dependent activation of JNK1. Together, these findings suggest that sulindac induces apoptosis in susceptible human breast cancer cells through, at least in part, the inhibition of IKKβ and the subsequent p38 MAPK-dependent activation of JNK1. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. A-Mi Seo and Seoug-Woo Hong have contributed equally.     

Migration and differentiation of transplanted enteric neural crest-derived cells in murine model of Hirschsprung’s disease

Stem cell therapy offers the potential of rebuilding the enteric nervous system (ENS) in the aganglionic bowel of patients with Hirschsprung’s disease. P0-Cre/Floxed-EGFP mice in which neural crest-derived cells express EGFP were used to obtain ENS stem/progenitor cells. ENS stem/progenitor cells were transplanted into the bowel of Ret^−/− mouse, an animal model of Hirschsprung’s disease. Immunohistochemical analysis was performed to determine whether grafted cells gave rise to neurons in the recipient bowel. EGFP expressing neural crest-derived cells accounted for 7.01 ± 2.52 % of total cells of gastrointestinal tract. ENS stem/progenitor cells were isolated using flow cytometry and expanded as neurosphere-like bodies (NLBs) in a serum-free culture condition. Some cells in NLBs expressed neural crest markers, p75 and Sox10 and neural stem/progenitor cells markers, Nestin and Musashi1. Multipotency of isolated ENS stem/progenitor cells was determined as they differentiated into neurons, glial cells, and myofibloblasts in culture. When co-cultured with explants of hindgut of Ret^−/− mice, ENS stem/progenitor cells migrated into the aganglionic bowel and gave rise to neurons. ENS stem/progenitor cells used in this study appear to be clinically relevant donor cells in cell therapy to treat Hirschsprung’s disease capable of colonizing the affected bowel and giving rise to neurons.     

Composite mathematical modeling of calcium signaling behind neuronal cell death in Alzheimer’s disease

Background

Alzheimer’s disease (AD) is a progressive neurological disorder, recognized as the most common cause of dementia affecting people aged 65 and above. AD is characterized by an increase in amyloid metabolism, and by the misfolding and deposition of β-amyloid oligomers in and around neurons in the brain. These processes remodel the calcium signaling mechanism in neurons, leading to cell death via apoptosis. Despite accumulating knowledge about the biological processes underlying AD, mathematical models to date are restricted to depicting only a small portion of the pathology.

Results

Here, we integrated multiple mathematical models to analyze and understand the relationship among amyloid depositions, calcium signaling and mitochondrial permeability transition pore (PTP) related cell apoptosis in AD. The model was used to simulate calcium dynamics in the absence and presence of AD. In the absence of AD, i.e. without β-amyloid deposition, mitochondrial and cytosolic calcium level remains in the low resting concentration. However, our in silico simulation of the presence of AD with the β-amyloid deposition, shows an increase in the entry of calcium ions into the cell and dysregulation of Ca ²⁺ channel receptors on the Endoplasmic Reticulum. This composite model enabled us to make simulation that is not possible to measure experimentally.

Conclusions

Our mathematical model depicting the mechanisms affecting calcium signaling in neurons can help understand AD at the systems level and has potential for diagnostic and therapeutic applications.

     

WIN induces apoptotic cell death in human colon cancer cells through a block of autophagic flux dependent on PPARγ down-regulation

Cannabinoids have been reported to possess anti-tumorigenic activity in cancer models although their mechanism of action is not well understood. Here, we show that the synthetic cannabinoid WIN55,212-2 (WIN)-induced apoptosis in colon cancer cell lines is accompanied by endoplasmic reticulum stress induction. The formation of acidic vacuoles and the increase in LC3-II protein indicated the involvement of autophagic process which seemed to play a pro-survival role against the cytotoxic effects of the drug. However, the enhanced lysosomal membrane permeabilization (LMP) blocked the autophagic flux after the formation of autophagosomes as demonstrated by the accumulation of p62 and LC3, two markers of autophagic degradation. Data also provided evidence for a role for nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ) in cannabinoid signalling. PPARγ expression, at both protein and mRNA levels, was significantly down-regulated after WIN treatment and its inhibition, either by specific antagonists or by down-regulation via gene silencing, induced effects on cell viability as well as on ER stress and autophagic markers similar to those obtained in the presence of WIN. Moreover, the observation that the increase in p62 level and the induction of LMP were also modified by PPARγ antagonists seemed to indicate that PPARγ down-regulation was crucial to determinate the block of autophagic flux, thus confirming the critical role of PPARγ in WIN action. In conclusion, at our knowledge, our results are the first to show that the reduction of PPARγ levels contributes to WIN-induced colon carcinoma cell death by blocking the pro-survival autophagic response of cells.     

Effects of microwave exposure and Gemcitabine treatment on apoptotic activity in Burkitt’s lymphoma (Raji) cells

Abstract

We investigated the effects of 1.8?MHz Global System for Mobile Communications (GSM)-modulated microwave (MW) radiation on apoptotic level and cell viability of Burkitt’s lymphoma (Raji) cells with or without Gemcitabine, which exhibits cell phase specificity, primarily killing cells undergoing DNA synthesis (S-phase). Raji cells were exposed to 1.8?GHz GSM-modulated MW radiation at a specific absorption rate (SAR) of 0.350?W/kg in a CO₂ incubator. The duration of the exposure was 24?h. The amount of apoptotic cells was analyzed using Annexin V-FITC and propidium iodide (PI) staining with flow cytometer. The apoptotic activity of MW exposed Raji cells was increased significantly. In addition, cell viability of exposed samples was significantly decreased. Combined exposure of MW and Gemcitabine increased the amount of apoptotic cells than MW radiation alone. Moreover, viability of MW?+?Gemcitabine exposed cells was lower than that of cells exposed only to MW. These results demonstrated that MW radiation exposure and Gemcitabine treatment have a synergistic effect on apoptotic activity of Raji cells.     

Expression profile of the stem cell markers in human Hertwig’s epithelial root sheath/Epithelial rests of Malassez cells

Hertwig’s epithelial root sheath/Epithelial rests of Malassez (HERS/ERM) cells are unique epithelial cells in the periodontal ligament. They remain in periodontal tissues through-out the adult life, and it is expected that their functional role is to maintain the homeostasis of the periodontium through reciprocal interactions with other periodontal cells. In this study, we investigated whether HERS/ERM cells have primitive stem cell characteristics: those of embryonic stem cells as well as of epithelial stem cells. Primary HERS/ERM cells had typical epithelial cell morphology and characteristics and they maintained for more than five passages. They expressed epithelial stem cell-related genes: ABCG2, ANp63, p75, EpCAM, and Bmi-1. Moreover, the expression of embryonic stem cell markers such as Oct-4, Nanog, and SSEA-4 were detected. Next, we investigated whether the expression of these stem cell markers was maintained during the sub-culture process. HERS/ERM cells showed different expression levels of these stemness genes at each passage, but their expression was maintained throughout the passages. Taken together, our data suggest that a primary culture of HERS/ERM cells contains a population of primitive stem cells that express epithelial stem cell markers and embryonic stem cell markers. Furthermore, these cell populations were maintained during the sub-culturing process in our culture conditions. Therefore, our findings suggest that there is a strong possibility of accomplishing cementum tissue engineering with HERS/ERM cells.     

Anti-inflammatory and anti-apoptotic effects of (RS)-glucoraphanin bioactivated with myrosinase in murine sub-acute and acute MPTP-induced Parkinson’s disease

This study was focused on the possible neuroprotective role of (R_S)-glucoraphanin, bioactivated with myrosinase enzyme (bioactive R_S-GRA), in an experimental mouse model of Parkinson’s disease (PD). R_S-GRA is one of the most important glucosinolates, a thiosaccharidic compound found in Brassicaceae, notably in Tuscan black kale seeds.R_S-GRA was extracted by one-step anion exchange chromatography, further purified by gel-filtration and analyzed by HPLC. Following, pure R_S-GRA was characterized by ¹H and ¹³C NMR spectrometry and the purity was assayed by HPLC analysis of the desulfo-derivative according to the ISO 9167-1 method. The obtained purity has been of 99%.To evaluate the possible pharmacological efficacy of bioactive R_S-GRA (administrated at the dose of 10 mg/kg, ip +5 μl/mouse myrosinase enzyme), C57BL/6 mice were used in two different sets of experiment (in order to evaluate the neuroprotective effects in different phases of the disease), according to an acute (2 injections · 40 mg/kg MPTP) and a sub-acute (5 injections · 20 mg/kg MPTP) model of PD.Behavioural test, body weight changes measures and immunohistochemical localization of the main PD markers were performed and post-hoc analysis has shown as bioactive R_S-GRA is able to reduce dopamine transporter degradation, tyrosine hydroxylase expression, IL-1β release, as well as the triggering of neuronal apoptotic death pathway (data about Bax/Bcl-2 balance and dendrite spines loss) and the generation of radicalic species by oxidative stress (results focused on nitrotyrosine, Nrf2 and GFAP immunolocalization). These effects have been correlated with the release of neurotrophic factors, such as GAP-43, NGF and BDNF, that, probably, play a supporting role in the neuroprotective action of bioactive R_S-GRA. Moreover, after PD-induction mice treated with bioactive R_S-GRA are appeared more in health than animals that did not received the treatment both for phenotypic behaviour and for general condition (movement coordination, presence of tremors, nutrition).Overall, our results suggest that bioactive R_S-GRA can protect neurons against the neurotoxicity involved in PD via an anti-apoptotic/anti-inflammatory action.     

LncRNA17A regulates autophagy and apoptosis of SH-SY5Y cell line as an in vitro model for Alzheimer’s disease

Considerable evidence suggest that a variety of Long-non-coding RNAs (LncRNAs) are widely implicated in several neurodegenerative disorders. The present study aims to investigate the regulatory effect of LncRNA 17A in an in vitro model of Alzheimer’s disease (AD). AD cell model was established by treating the SH-SY5Y cells with amyloid β peptide 1-42, and then the cells were transfected with 17A shRNA and pcDNA-17A. Apoptosis, migration, invasion and ELISA assays were performed to investigate the effect of differentiated 17A expression level on AD cell line. It was determined that 17A-overexpressing promotes autophagy, induces neurodegenration and deactivates GABAB signaling. In conclusion, our results demonstrated that the dysregulation of LncRNA 17A was involved in cellular functions and biological processes of neuroblastoma cells in an AD cell model, shedding light on the diagnostic value and therapeutic potential of LncRNA 17A for AD intervention.     

Cytokine-mediated induction of human immunodeficiency virus (HIV) expression and cell death in chronically infected U1 cells: do tumor necrosis factor alpha and gamma interferon selectively kill HIV-infected cells?

Infection with several DNA or RNA viruses induces a state of increased sensitivity to cell lysis mediated by tumor necrosis factor (TNF), particularly in the presence of gamma interferon (IFN-gamma). Infection of human cells with the human immunodeficiency virus (HIV) may induce a similar phenomenon. However, TNF and IFN-gamma are known upregulators of HIV replication, raising the question of the potential role of these cytokines in the selective elimination of cells infected with this virus. The present study demonstrates that chronically infected U1 cells were killed with much greater efficiency by costimulation with TNF-alpha and IFN-gamma than their uninfected parental cell line U937. However, synergistic induction of viral expression also occurred in U1 cells as a consequence of treatment with the two cytokines. Cell death in U1 cells was not caused by the massive production of virions, in that costimulation with glucocorticoid hormones and TNF-alpha or IFN-gamma resulted in high levels of virion production without cytopathicity. To investigate the nature of the selective cytotoxic effect observed in U1 cells costimulated with TNF-alpha plus IFN-gamma, a panel of uninfected cell clones was generated by limiting dilution of U937 cells and tested for response to TNF-alpha and/or IFN-gamma. In contrast to the uncloned bulk parental U937 cell line, most uninfected cell clones showed a very high susceptibility to being killed by TNF-alpha and IFN-gamma. Similar findings were obtained when both infected U1 cells and several uninfected U937 cell clones were costimulated with an anti-Fas monoclonal antibody in the presence of IFN-gamma, although, unlike cells stimulated with TNF-alpha, cells treated with anti-Fas antibody did not express virus. Therefore, the increased susceptibility to cytokine-mediated lysis observed in cell lines infected with HIV is likely due to the selection of preexisting cell clones rather than viral infection.     

Phagocytic functions of microglial cells in the central nervous system and their importance in two neurodegenerative diseases: multiple sclerosis and Alzheimer’s disease

Microglial cells are the resident phagocytic cells of the central nervous system (CNS). They possess a wide range of receptors allowing them to identify and internalize numerous pathogens. We will discuss here the role of the most important receptors of microglia involved in non-opsonin-dependent phagocytosis (mannose receptor, β-glucan receptor, scavenger receptor) and that of receptors involved in the opsonin-dependent phagocytosis, namely the complement 3 (CR3) and the Fcγ receptors (FcγR). First, the molecular and cellular mechanisms induced when these receptors are conducting a phagocytic event are presented. In the second part, we will discuss the role these receptors may play in multiple sclerosis and Alzheimer’s disease, in the elimination by phagocytosis of myelin and beta amyloid peptide respectively. The first two authors contributed equally to this work.     

IL-10 secreting CD21+ B cells are present in jejunal Peyer’s patches of sheep during fetal development

We recently reported a novel interleukin-10 (IL-10)-secreting CD21⁺ B cell population in jejunal Peyer’s patches (JPP) of sheep with a regulatory function (B_regs) suppressing Toll-like receptor 9 (TLR9)-induced cytokine responses. However, little is known about the development of these cells. Therefore, we investigate their existence in JPP cells from fetal and newborn lambs. CD21⁺ B cells were purified from JPP cells by magnetic cell sorting and subsequently stimulated with the TLR9 agonist, CpG ODN (CpG oligodeoxynucleotide). Lymphocyte proliferative responses, cytokine production (IL-10, IL-12 and interferon-γ [INF-γ]) and antibody secretion were assayed. We found that fetal and neonatal CD21⁺ B cells spontaneously secreted high levels of IL-10 regardless of CpG stimulation but that these cells did not produce any IL-12 or INF-γ upon stimulation with CpG. The observed responses are consistent with those previously reported for B_regs characterized in JPP of older lambs. Surprisingly, unlike in older lambs, fetal and neonatal JPP CD21⁺ B cells proliferated in response to CpG stimulation. Our investigations of fetal and neonatal lambs provide evidence for the development of IL-10-secreting CD21⁺ B cells in PPs prior to antigen exposure.     

Neuroprotective effects of human mesenchymal stem cells on neural cultures exposed to 6-hydroxydopamine: implications for reparative therapy in Parkinson’s disease

Stem cell (SC) transplantation represents a promising tool to treat neurodegenerative disorders, such as Parkinson's disease (PD), but positive therapeutic outcomes require elucidation of the biological mechanisms involved. Therefore, we investigated human Mesenchymal SCs (hMSCs) ability to protect murine differentiated Neural SCs (mdNSCs) against the cytotoxic effects of 6-hydroxydopamine (6-OHDA) in a co-culture model mimicking the in vivo neurovascular niche. The internalization of 6-OHDA mainly relies on its uptake by the dopamine active transporter (DAT), but its toxicity could also involve other pathways. We demonstrated that mdNSCs consistently expressed DAT along the differentiative process. Exposure to 6-OHDA did not affect hMSCs, but induced DAT-independent apoptosis in mdNSCs with generation of reactive oxygen species and caspases 3/7 activation. The potential neuroprotective action of hMSCs on mdNSCs exposed to 6-OHDA was tested in different co-culture conditions, in which hMSCs were added to mdNSCs prior to, simultaneously, or after 6-OHDA treatment. In the presence of the neurotoxin, the majority of mdNSCs acquired an apoptotic phenotype, while co-cultures with hMSCs significantly increased their survival (up to 70%) in all conditions. Multiplex human angiogenic array analysis on the conditioned media demonstrated that cytokine release by hMSCs was finely modulated. Moreover, sole growth factor addition yielded a similar neuroprotective effect on mdNSCs. In conclusion, our findings demonstrate that hMSCs protect mdNSCs against 6-OHDA neurotoxicity, and rescue cells from ongoing neurodegeneration likely through the release of multiple cytokines. Our findings provide novel insights for the development of therapeutic strategies designed to counteract the neurodegenerative processes of PD.     

B cell subsets and dysfunction of regulatory B cells in IgG4-related diseases and primary Sj?gren’s syndrome: the similarities and differences

Introduction

IgG4-related disease (IgG4-RD) is a multisystem-involved autoimmune disease. Abnormally activated and differentiated B cells may play important roles. Regulatory B cells (Breg) are newly defined B cell subgroups with immunosuppressive functions. In this study, we investigated the differences of B cell subsets, the expressions of co-stimulatory molecules on B cells, and the function of Breg cells in patients with IgG4-RD, primary Sjögren’s syndrome (pSS) as well as in healthy controls (HC).

Methods

Newly diagnosed IgG4-RD patients (n = 48) were enrolled, 38 untreated pSS patients and 30 healthy volunteers were recruited as disease and healthy controls. To analyze B cell subsets and B cell activity, PBMCs were surface stained and detected by flow cytometry. The function of Breg cells was tested by coculturing isolated CD19 + CD24^hiCD38^hi Breg cells with purified CD4 + CD25- T cells. Serum cytokines were measured by ELISA and cytometric bead array. Relationship between clinical data and laboratory findings were analyzed as well.

Results

Compared with pSS patients and HC, IgG4-RD patients had a lower frequency of peripheral Breg cells. Interestingly, CD19 + CD24-CD38^hi B cell subsets were significantly higher in peripheral B cells from IgG4-RD patients than in pSS patients and HC, which correlated with serum IgG4 levels. The expression of BAFF-R and CD40 on B cells was significantly lower in IgG4-RD patients compared with those in pSS patients and HC. Unlike HC, Breg cells from pSS patients lacked suppressive functions.

Conclusions

B cells in patients with IgG4-RD and pSS display a variety of abnormalities, including disturbed B cell subpopulations, abnormal expression of key signaling molecules, co-stimulatory molecules, and inflammatory cytokines. In addition, a significantly increased B cell subset, CD19 + CD24-CD38^hi B cells, may play an important role in the pathogenesis of IgG4-RD.