55 research outputs found
Inhibition of C5aR1 as a promising approach to treat taxane-induced neuropathy
: Chemotherapy-induced peripheral neuropathy (CIPN) is a common side effect of several antitumor agents resulting in progressive and often irreversible damage of peripheral nerves. In addition to their known anticancer effects, taxanes, including paclitaxel, can also induce peripheral neuropathy by activating microglia and astrocytes, which release pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α), interleukin 1-beta (IL-1ÎČ), and chemokine (C-C motif) ligand 2 (CCL-2). All these events contribute to the maintenance of neuropathic or inflammatory response. Complement component 5a (C5a)/C5a receptor 1 (C5aR1) signaling was very recently shown to play a crucial role in paclitaxel-induced peripheral neuropathy. Our recent findings highlighted that taxanes have the previously unreported property of binding and activating C5aR1, and that C5aR1 inhibition by DF3966A is effective in preventing paclitaxel-induced peripheral neuropathy (PIPN) in animal models. Here, we investigated if C5aR1 inhibition maintains efficacy in reducing PIPN in a therapeutic setting. Furthermore, we characterized the role of C5aR1 activation by paclitaxel and the CIPN-associated activation of nod-like receptor (NLR) family pyrin domain containing 3 (NLRP3) inflammasome. Our results clearly show that administration of the C5aR1 inhibitor strongly reduced cold and mechanical allodynia in mice when given both during the onset of PIPN and when neuropathy is well established. C5aR1 activation by paclitaxel was found to be a key event in the induction of inflammatory factors in spinal cord, such as TNF-α, ionized calcium-binding adapter molecule 1 (Iba-1), and glial fibrillary acidic protein (GFAP). In addition, C5aR1 inhibition significantly mitigated paclitaxel-induced inflammation and inflammasome activation by reducing IL-1ÎČ and NLRP3 expression at both sciatic and dorsal root ganglia level, confirming the involvement of inflammasome in PIPN. Moreover, paclitaxel-induced upregulation of C5aR1 was significantly reduced by DF3966A treatment in central nervous system. Lastly, the antinociceptive effect of C5aR1 inhibition was confirmed in an in vitro model of sensory neurons in which we focused on receptor channels usually activated upon neuropathy. In conclusion, C5aR1 inhibition is proposed as a therapeutic option with the potential to exert long-term protective effect on PIPN-associated neuropathic pain and inflammation
Phenotype Sequencing: Identifying the Genes That Cause a Phenotype Directly from Pooled Sequencing of Independent Mutants
Random mutagenesis and phenotype screening provide a powerful method for dissecting microbial functions, but their results can be laborious to analyze experimentally. Each mutant strain may contain 50â100 random mutations, necessitating extensive functional experiments to determine which one causes the selected phenotype. To solve this problem, we propose a âPhenotype Sequencingâ approach in which genes causing the phenotype can be identified directly from sequencing of multiple independent mutants. We developed a new computational analysis method showing that 1. causal genes can be identified with high probability from even a modest number of mutant genomes; 2. costs can be cut many-fold compared with a conventional genome sequencing approach via an optimized strategy of library-pooling (multiple strains per library) and tag-pooling (multiple tagged libraries per sequencing lane). We have performed extensive validation experiments on a set of E. coli mutants with increased isobutanol biofuel tolerance. We generated a range of sequencing experiments varying from 3 to 32 mutant strains, with pooling on 1 to 3 sequencing lanes. Our statistical analysis of these data (4099 mutations from 32 mutant genomes) successfully identified 3 genes (acrB, marC, acrA) that have been independently validated as causing this experimental phenotype. It must be emphasized that our approach reduces mutant sequencing costs enormously. Whereas a conventional genome sequencing experiment would have cost 1200. In fact, our smallest experiments reliably identified acrB and marC at a cost of only 340
The Lipid Transfer Protein CERT Interacts with the Chlamydia Inclusion Protein IncD and Participates to ER-Chlamydia Inclusion Membrane Contact Sites
Bacterial pathogens that reside in membrane bound compartment manipulate the host cell machinery to establish and maintain their intracellular niche. The hijacking of inter-organelle vesicular trafficking through the targeting of small GTPases or SNARE proteins has been well established. Here, we show that intracellular pathogens also establish direct membrane contact sites with organelles and exploit non-vesicular transport machinery. We identified the ER-to-Golgi ceramide transfer protein CERT as a host cell factor specifically recruited to the inclusion, a membrane-bound compartment harboring the obligate intracellular pathogen Chlamydia trachomatis. We further showed that CERT recruitment to the inclusion correlated with the recruitment of VAPA/B-positive tubules in close proximity of the inclusion membrane, suggesting that ER-Inclusion membrane contact sites are formed upon C. trachomatis infection. Moreover, we identified the C. trachomatis effector protein IncD as a specific binding partner for CERT. Finally we showed that depletion of either CERT or the VAP proteins impaired bacterial development. We propose that the presence of IncD, CERT, VAPA/B, and potentially additional host and/or bacterial factors, at points of contact between the ER and the inclusion membrane provides a specialized metabolic and/or signaling microenvironment favorable to bacterial development
Structure of a bacterial type III secretion system in contact with a host membrane in situ
Many bacterial pathogens of animals and plants use a conserved type III secretion system
(T3SS) to inject virulence effector proteins directly into eukaryotic cells to subvert host
functions. Contact with host membranes is critical for T3SS activation, yet little is known
about T3SS architecture in this state or the conformational changes that drive effector
translocation. Here we use cryo-electron tomography and sub-tomogram averaging to derive
the intact structure of the primordial Chlamydia trachomatis T3SS in the presence and absence
of host membrane contact. Comparison of the averaged structures demonstrates a marked
compaction of the basal body (4 nm) occurs when the needle tip contacts the host cell
membrane. This compaction is coupled to a stabilization of the cytosolic sorting platformâ
ATPase. Our findings reveal the first structure of a bacterial T3SS from a major human
pathogen engaged with a eukaryotic host, and reveal striking âpump-actionâ conformational
changes that underpin effector injection
Chlamydia trachomatis Co-opts the FGF2 Signaling Pathway to Enhance Infection
The molecular details of Chlamydia trachomatis binding, entry, and spread are incompletely understood, but heparan sulfate proteoglycans (HSPGs) play a role in the initial binding steps. As cell surface HSPGs facilitate the interactions of many growth factors with their receptors, we investigated the role of HSPG-dependent growth factors in C. trachomatis infection. Here, we report a novel finding that Fibroblast Growth Factor 2 (FGF2) is necessary and sufficient to enhance C. trachomatis binding to host cells in an HSPG-dependent manner. FGF2 binds directly to elementary bodies (EBs) where it may function as a bridging molecule to facilitate interactions of EBs with the FGF receptor (FGFR) on the cell surface. Upon EB binding, FGFR is activated locally and contributes to bacterial uptake into non-phagocytic cells. We further show that C. trachomatis infection stimulates fgf2 transcription and enhances production and release of FGF2 through a pathway that requires bacterial protein synthesis and activation of the Erk1/2 signaling pathway but that is independent of FGFR activation. Intracellular replication of the bacteria results in host proteosome-mediated degradation of the high molecular weight (HMW) isoforms of FGF2 and increased amounts of the low molecular weight (LMW) isoforms, which are released upon host cell death. Finally, we demonstrate the in vivo relevance of these findings by showing that conditioned medium from C. trachomatis infected cells is enriched for LMW FGF2, accounting for its ability to enhance C. trachomatis infectivity in additional rounds of infection. Together, these results demonstrate that C. trachomatis utilizes multiple mechanisms to co-opt the host cell FGF2 pathway to enhance bacterial infection and spread
Cholesterol-Dependent Anaplasma phagocytophilum Exploits the Low-Density Lipoprotein Uptake Pathway
In eukaryotes, intracellular cholesterol homeostasis and trafficking are tightly regulated. Certain bacteria, such as Anaplasma phagocytophilum, also require cholesterol; it is unknown, however, how this cholesterol-dependent obligatory intracellular bacterium of granulocytes interacts with the host cell cholesterol regulatory pathway to acquire cholesterol. Here, we report that total host cell cholesterol increased >2-fold during A. phagocytophilum infection in a human promyelocytic leukemia cell line. Cellular free cholesterol was enriched in A. phagocytophilum inclusions as detected by filipin staining. We determined that A. phagocytophilum requires cholesterol derived from low-density lipoprotein (LDL), because its replication was significantly inhibited by depleting the growth medium of cholesterol-containing lipoproteins, by blocking LDL uptake with a monoclonal antibody against LDL receptor (LDLR), or by treating the host cells with inhibitors that block LDL-derived cholesterol egress from late endosomes or lysosomes. However, de novo cholesterol biosynthesis is not required, since inhibition of the biosynthesis pathway did not inhibit A. phagocytophilum infection. The uptake of fluorescence-labeled LDL was enhanced in infected cells, and LDLR expression was up-regulated at both the mRNA and protein levels. A. phagocytophilum infection stabilized LDLR mRNA through the 3âČ UTR region, but not through activation of the sterol regulatory element binding proteins. Extracellular signalâregulated kinase (ERK) was up-regulated by A. phagocytophilum infection, and inhibition of its upstream kinase, MEK, by a specific inhibitor or siRNA knockdown, reduced A. phagocytophilum infection. Up-regulation of LDLR mRNA by A. phagocytophilum was also inhibited by the MEK inhibitor; however, it was unclear whether ERK activation is required for LDLR mRNA up-regulation by A. phagocytophilum. These data reveal that A. phagocytophilum exploits the host LDL uptake pathway and LDLR mRNA regulatory system to accumulate cholesterol in inclusions to facilitate its replication
Structural and Topographic Dynamics of Pulmonary Histopathology and Local Cytokine Profiles in Paracoccidioides brasiliensis Conidia-Infected Mice
Paracoccidioidomycosis (PCM), an endemic fungal infection of pulmonary origin resulting in severe disseminated disease, occurs in rural areas of most South American countries and presents several clinical forms. The infection is acquired by inhalation of specific fungal propagules, called conidia. Considering the difficulties encountered when studying the infection in humans, this work was done in mice infected by inhalation of infective fungal conidia thus mimicking the human natural infection. The lungs of mice were sequentially studied by histopathological and multiplex cytokine methods from 2 h to 16 weeks after infection to verify the course of the disease. The mycosis presented different morphologic aspects during the course of time, affecting several pulmonary compartments. Otherwise and based on the analysis of 30 cytokines, the immune response also showed heterogeneous responses, which were up or down regulated depending on the time of infection. By recognizing the different stages that correspond to the evolution of pulmonary lesions, the severity (benign, chronic or fibrotic) of the disease could be predicted and the probable prognosis of the illness be inferred
Acapsular Staphylococcus aureus with a non-functional agr regains capsule expression after passage through the bloodstream in a bacteremia mouse model
Selection pressures exerted on Staphylococcus aureus by host factors during infection may lead to the emergence of regulatory phenotypes better adapted to the infection site. Traits convenient for persistence may be fixed by mutation thus turning these mutants into microevolution endpoints. The feasibility that stable, non-encapsulated S. aureus mutants can regain expression of key virulence factors for survival in the bloodstream was investigated. S. aureus agr mutant HU-14 (IS256 insertion in agrC) from a patient with chronic osteomyelitis was passed through the bloodstream using a bacteriemia mouse model and derivative P3.1 was obtained. Although IS256 remained inserted in agrC, P3.1 regained production of capsular polysaccharide type 5 (CP5) and staphyloxanthin. Furthermore, P3.1 expressed higher levels of asp23/SigB when compared with parental strain HU-14. Strain P3.1 displayed decreased osteoclastogenesis capacity, thus indicating decreased adaptability to bone compared with strain HU-14 and exhibited a trend to be more virulent than parental strain HU-14. Strain P3.1 exhibited the loss of one IS256 copy, which was originally located in the HU-14 noncoding region between dnaG (DNA primase) and rpoD (sigA). This loss may be associated with the observed phenotype change but the mechanism remains unknown. In conclusion, S. aureus organisms that escape the infected bone may recover the expression of key virulence factors through a rapid microevolution pathway involving SigB regulation of key virulence factors.Fil: Suligoy Lozano, Carlos Mauricio. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Oficina de CoordinaciĂłn Administrativa Houssay. Instituto de Investigaciones en MicrobiologĂa y ParasitologĂa MĂ©dica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones en MicrobiologĂa y ParasitologĂa MĂ©dica; ArgentinaFil: DĂaz, RocĂo E.. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Oficina de CoordinaciĂłn Administrativa Houssay. Instituto de Investigaciones en MicrobiologĂa y ParasitologĂa MĂ©dica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones en MicrobiologĂa y ParasitologĂa MĂ©dica; ArgentinaFil: Gehrke, Ana-katharina Elsa. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas; Argentina. Universidad MaimĂłnides. Ărea de Investigaciones BiomĂ©dicas y BiotecnolĂłgicas. Centro de Estudios BiomĂ©dicos, BiotecnolĂłgicos, Ambientales y de DiagnĂłstico; ArgentinaFil: Ring, Natalie. University of Edinburgh; Reino UnidoFil: Yebra, Gonzalo. University of Edinburgh; Reino UnidoFil: Alves, Joana. University of Edinburgh; Reino UnidoFil: GĂłmez, Marisa Ileana. Universidad MaimĂłnides. Ărea de Investigaciones BiomĂ©dicas y BiotecnolĂłgicas. Centro de Estudios BiomĂ©dicos, BiotecnolĂłgicos, Ambientales y de DiagnĂłstico; ArgentinaFil: Wendler, Sindy. UniversitĂ€tsklinikum Jena Und Medizinische FakultĂ€t; AlemaniaFil: Fitzgerald, J. Ross. University of Edinburgh; Reino UnidoFil: Tuchscherr, Lorena. Jena University Hospital; AlemaniaFil: Löffler, Bettina. Jena University Hospital; AlemaniaFil: Sordelli, Daniel Oscar. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Oficina de CoordinaciĂłn Administrativa Houssay. Instituto de Investigaciones en MicrobiologĂa y ParasitologĂa MĂ©dica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones en MicrobiologĂa y ParasitologĂa MĂ©dica; ArgentinaFil: Noto Llana, Mariangeles. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Oficina de CoordinaciĂłn Administrativa Houssay. Instituto de Investigaciones en MicrobiologĂa y ParasitologĂa MĂ©dica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones en MicrobiologĂa y ParasitologĂa MĂ©dica; ArgentinaFil: Buzzola, Fernanda Roxana. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Oficina de CoordinaciĂłn Administrativa Houssay. Instituto de Investigaciones en MicrobiologĂa y ParasitologĂa MĂ©dica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones en MicrobiologĂa y ParasitologĂa MĂ©dica; Argentin
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Valutazione dellâefficacia della lamotrigina come farmaco aggiunto nelle epilessie farmacoresistenti dellâetĂ evolutiva: risultati di uno studio aperto
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