Lidia Szulc-Dąbrowska

Graphene and carbon nanocompounds: biofunctionalization and applications in tissue engineering

In tissue engineering, the possibility of a comprehensive restoration of the tissue, structure or a portion of the organ is largely determined by the type of material used. A wide range of materials such as graphene and other carbon nanocompounds which have different physical and chemical properties can be expected to react differently upon contact with biomolecules, cells and tissues. This mini-review describes the current knowledge on biocompatibility of graphene and its derivatives with a variety of mammalian cells, such as osteoblasts, neuroendocrine cells, fibroblasts NIH/3T3 line, PMEFs (primary mouse embryonic fibroblasts), stem cells and neurons. The results from different studies give hope for the possibility of graphene to be used in the regeneration of almost all tissues, including neural tissue implants or in the form of neural chips, which may allow in the future treatment of degenerative diseases and injuries of the central nervous system.

Modulation of NF-кB transcription factor activation by Molluscum contagiosum virus proteins.

Molluscum contagiosum virus is a human and animal dermatotropic pathogen, which causes a severe disease in immunocompromised individuals. MCV belongs to the Poxviridae family whose members exert immunomodulatory effects on the host antiviral response. Poxviruses interfere with cell signaling pathways that lead to the activation of nuclear factor кB, a pleiotropic transcription factor which is crucial for regulation of the immune response, the cell cycle and apoptosis. In resting cells, NF-κB is present in the cytoplasm, where it is associated with inhibitor κB. Upon stimulation by activators, such as proinflammatory cytokines and bacterial or viral products, the inhibitory protein undergoes phosphorylation and proteasomal degradation. NF-κB, in turn, translocates to the nucleus, where it regulates the transcription of various genes that are essential for processes mentioned above. Since poxviruses replicate exclusively in the cell cytoplasm, NF-кB became a good target for poxviral immunomodulation. MCV encodes various proteins which interfere with the signaling pathways that lead to the activation of NF-κB. Ligand inhibitor encoded by MCV, MC54, binds interleukin-18 and inhibits interferon-γ production. Other MCV proteins, MC159 and MC160, belong to intracellular inhibitors of NF-κB and are members of viral FLICE-inhibitory proteins (vFLIPs). MC159 protein encoded by MCV was shown to inhibit apoptosis of virus-infected cells. Such interactions serve immune evasion and are responsible for the persistence of MCV.

Crosstalk between autophagy and apoptosis in RAW 264.7 macrophages infected with ectromelia orthopoxvirus.

Several studies have provided evidence that complex relationships between autophagic and apoptotic cell death pathways occur in cancer and virus-infected cells. Previously, we demonstrated that infection of macrophages with Moscow strain of ectromelia virus (ECTV-MOS) induces apoptosis under in vitro and in vivo conditions. Here, we found that autophagy was induced in RAW 264.7 cells during infection with ECTV-MOS. Silencing of beclin 1, an autophagy-related gene, reduced the percentage of late apoptotic cells in virus-infected RAW 264.7 macrophages. Pharmacological modulation of autophagy by wortmannin (inhibitor) or rapamycin (inductor) did not affect or cause increased apoptosis in ECTV-MOS-infected RAW 264.7 cells, respectively. Meantime, blocking apoptosis by a pan-caspase inhibitor, Z-VAD-FMK, increased the formation of autophagosomes in infected macrophages. Taken together, three important points arise from our study. First, autophagy may co-occur with apoptosis in RAW 264.7 cells exposed to ECTV-MOS. Second, at later stages of infection, autophagy may partially participate in the execution of macrophage cell death by enhancing apoptosis. Third, when apoptosis is blocked infected macrophages undergo increased autophagy. Our results provide new information about the relationship between autophagy and apoptosis in ECTV-MOS-infected macrophages.

Limfocyty Th17 w zakażeniach bakteryjnych

Th17 cells are a relatively newly discovered subpopulation of helper CD4+ T lymphocytes. It has been shown that these cells may contribute to tissue damage during certain inflammatory and autoimmune diseases and also play an important role in antitumor and antimicrobial, particularly antibacterial, immunity. Bacteria stimulate the Th17 response through several Toll-like (TLR), NOD-like (NLR) and C-type lectin (CLR) receptors. When activated, Th17 lymphocytes produce several cytokines, mainly interleukin (IL)-17 and chemokines, that further attract and activate phagocytes to mediate bacterial clearance. Thus Th17 cells contribute to induction of host protective immunity, particularly against extracellular bacterial pathogens: Staphylococcus aureus, Streptococcus pneumoniae and Klebsiella pneumoniae. Furthermore, numerous studies indicate the importance of Th17 lymphocytes in immunity against intracellular bacteria such as Francisella tularensis and Chlamydia muridarum. In this case, the protective immune response is mediated mainly through stimulation of local dendritic cell (DC) function for establishing a Th1 immune response, indispensable for controlling intracellular infectious agents. However, deregulation of the Th17/IL17 response during bacterial infections may lead to profound pathologies. As a result, Th17 cells participate in chronic inflammatory diseases, leading to tissue destruction and favoring tumor development. This article summarizes current understanding of the bacteriainduced Th17 response in the context of the protective immune response and immunopathology.

Modulation of proinflammatory NF-κB signaling by ectromelia virus in RAW 264.7 murine macrophages

Macrophages are antigen-presenting cells (APCs) that play a crucial role in the innate immune response and may be involved in both clearance and spread of viruses. Stimulation of macrophages via Toll-like receptors (TLRs) results in activation of nuclear factor κB (NF-κB) and synthesis of proinflammatory cytokines. In this work, we show modulation of proinflammatory NF-κB signaling by a member of the family Poxviridae, genus Orthopoxvirus – ectromelia virus (ECTV) – in RAW 264.7 murine macrophages. ECTV interfered with p65 NF-κB nuclear translocation induced by TLR ligands such as lipopolysaccharide (LPS) (TLR4), polyinosinic-polycytidylic acid (poly(I:C)) (TLR3) and diacylated lipopeptide Pam2CSK4 (TLR2/6). We observed that ECTV modulates phosphorylation of Ser32 of inhibitor of κB (IκBα) and Ser536 of p65. Interference of ECTV with TLR signaling pathways implied that proinflammatory cytokine synthesis was inhibited. Our studies provide new insights into the strategies of proinflammatory signaling modulation by orthopoxviruses during their replication cycle in immune cells. Understanding important immune interactions between viral pathogens and APCs might contribute to the identification of drug targets and the development of vaccines.

Quantitative immunophenotypic analysis of antigen-presenting cells involved in ectromelia virus antigen presentation in BALB/c and C57BL/6 mice.

During mousepox in resistant (C57BL/6) or susceptible (BALB/c) strains of mice, stimulation of Th1 or Th2 cytokine immune response, respectively, is observed. Because mechanisms of different polarization of T cells remain elusive, in this study, we quantitatively assessed the phenotype of antigen-presenting cells (APCs) involved in ectromelia virus (ECTV) antigen presentation and cluster formation with effector cells in secondary lymphoid organs of BALB/c and C57BL/6 mice. We showed that both strains of mice display similar dynamics and kinetics of viral antigen presentation by CD11c(+) , CD11b(+) , and CD19(+) cells. CD11c(+) and CD11b(+) cells highly participated in viral antigen presentation during all stages of mousepox, whereas CD19(+) cells presented viral peptides later in infection. The main population of dendritic cells (DCs) engaged in ECTV antigen presentation and cell junction formation with effector cells was a population of myeloid CD11b(+) DCs (mDCs). We suggest that, on the one hand, ECTV may differentially affect the functions of APCs depending on the strain of mice. On the other hand, we suggest that some types of APCs, such as mDCs or other DCs subsets, have different abilities to direct the shape of immune response depending on the host resistance to mousepox.

Biocompatibility of pristine graphene monolayer: Scaffold for fibroblasts

The aim of the present study was to evaluate the cytotoxicity of pristine graphene monolayer and its utility as a scaffold for murine fibroblast L929 cell line. Cell viability, morphology, cytoskeleton architecture (microfilaments and microtubules), cell adhesion and migration into the scratch-wound area were determined using pristine graphene-coated microscopic slides. We found that fibroblasts cultured on pristine graphene monolayer exhibited changes in cell attachment, motility and cytoskeleton organization. Graphene was found to have no cytotoxicity on L929 fibroblasts and increased cell adhesion and proliferation within 24 h of culture. The area of cells growing on graphene was comparable to the area of fibroblasts cultured on glass. Migration of cells on the surface of graphene substrate appeared to be more regular in comparison to uncoated glass surface, however in both control (glass) and experimental (graphene) groups the scratch wound was closed after 48 h of culture. Taken together, our results indicate that pristine graphene monolayer is non-toxic for murine subcutaneous connective tissue fibroblasts and could be beneficial for recovery of damaged tissues after injury. These studies could be helpful in evaluating biocompatibility of graphene, which still remains ambiguous.

Functional paralysis of GM-CSF–derived bone marrow cells productively infected with ectromelia virus

Ectromelia virus (ECTV) is an orthopoxvirus responsible for mousepox, a lethal disease of certain strains of mice that is similar to smallpox in humans, caused by variola virus (VARV). ECTV, similar to VARV, exhibits a narrow host range and has co-evolved with its natural host. Consequently, ECTV employs sophisticated and host-specific strategies to control the immune cells that are important for induction of antiviral immune response. In the present study we investigated the influence of ECTV infection on immune functions of murine GM-CSF–derived bone marrow cells (GM-BM), comprised of conventional dendritic cells (cDCs) and macrophages. Our results showed for the first time that ECTV is able to replicate productively in GM-BM and severely impaired their innate and adaptive immune functions. Infected GM-BM exhibited dramatic changes in morphology and increased apoptosis during the late stages of infection. Moreover, GM-BM cells were unable to uptake and process antigen, reach full maturity and mount a proinflammatory response. Inhibition of cytokine/chemokine response may result from the alteration of nuclear translocation of NF-κB, IRF3 and IRF7 transcription factors and down-regulation of many genes involved in TLR, RLR, NLR and type I IFN signaling pathways. Consequently, GM-BM show inability to stimulate proliferation of purified allogeneic CD4+ T cells in a primary mixed leukocyte reaction (MLR). Taken together, our data clearly indicate that ECTV induces immunosuppressive mechanisms in GM-BM leading to their functional paralysis, thus compromising their ability to initiate downstream T-cell activation events.

[MAVS protein and its interactions with hepatitis A, B and C viruses].

Mitochondrial antiviral signaling protein (MAVS) transmits activation signal of type I interferon (IFN) gene transcription in the molecular intracellular pathway, which depends on the protein encoded by retinoic acid inducible gene I (RIG-I) or melanoma differentiation-associated protein-5 (MDA-5). MAVS, as a signal molecule, performs an essential function in the development of an antiviral immune response. The molecule of MAVS consists of two domains: the N-terminal domain and the C-terminal domain. The N-terminal end of MAVS contains the caspase activation and recruitment domain (CARD). CARD is responsible for MAVS interaction with RIG-I and MDA-5, which act as cytosolic sensors detecting foreign viral genetic material in the host cell. After binding to viral RNA, RIG-I or MDA-5 activates MAVS and transmits the signal of IFN type I gene expression. The C-terminal transmembrane domain (TM) of MAVS anchors the protein to the outer mitochondrial membrane. In this paper interactions between MAVS and hepatitis virus type A (HAV), type B (HBV) and type C (HCV) are presented. Mechanisms of indirect activation of MAVS by viral DNA and RNA, as well as the strategies of HAV, HBV and HCV for blocking of the intracellular signaling pathway at the level of MAVS, are described.

Strategies of NF-κB signaling modulation by ectromelia virus in BALB/3T3 murine fibroblasts.

Nuclear factor κB (NF-κB) is a pleiotropic transcription factor that regulates the expression of immune response genes. NF-κB signaling can be disrupted by pathogens that prevent host immune response. In this work, we examined the influence of ectromelia (mousepox) virus (ECTV) on NF-κB signaling in murine BALB/3T3 fibroblasts. Activation of NF-κB via tumor necrosis factor (TNF) receptor 1 (TNFR1) in these cells induces proinflammatory cytokine secretion. We show that ECTV does not recruit NF-κB to viral factories or induce NF-κB nuclear translocation in BALB/3T3 cells. Additionally, ECTV counteracts TNF-α-induced p65 NF-κB nuclear translocation during the course of infection. Inhibition of TNF-α-induced p65 nuclear translocation was also observed in neighboring cells that underwent fusion with ECTV-infected cells. ECTV inhibits the key step of NF-κB activation, i.e. Ser32 phosphorylation and degradation of inhibitor κBα (IκBα) induced by TNF-α. We also observed that ECTV prevents TNF-α-induced Ser536 of p65 phosphorylation in BALB/3T3 cells. Studying TNFR1 signaling provides information about regulation of inflammatory response and cell survival. Unraveling poxviral immunomodulatory strategies may be helpful in drug target identification as well as in vaccine development.