Mitochondrial alterations triggered by repeated exposure to fine (PM2.5-0.18) and quasi-ultrafine (PM0.18) fractions of ambient particulate matter.

Nowadays ambient particulate matter (PM) levels still regularly exceed the guideline values established by World Health Organization in most urban areas. Numerous experimental studies have already demonstrated the airway toxicity of the fine fraction of PM (FP), mainly triggered by oxidative stress-induced airway inflammation. However, only few studies have actually paid close attention to the ultrafine fraction of PM (UFP), which is likely to be more easily internalized in cells and more biologically reactive. Mitochondria are major endogenous sources of reactive oxygen species (ROS) through oxidative metabolism, and coordinate many critical cellular signaling processes. Mitochondria have been often studied in the context of PM toxicity and generally associated with apoptosis activation. However, little is known about the underlying adaptation mechanisms that could occur following exposure at sub-apoptotic doses of ambient PM. Here, normal human bronchial epithelial BEAS-2B cells were acutely or repeatedly exposed to relatively low doses (5 µg.cm-2) of FP (PM2.5-0.18) or quasi-UFP (Q-UFP; PM0.18) to better access the critical changes in mitochondrial morphology, functions, and dynamics. No significant cytotoxicity nor increase of apoptotic events were reported for any exposure. Mitochondrial membrane potential (ΔΨm) and intracellular ATP content were also not significantly impaired. After cell exposure to sub-apoptotic doses of FP and notably Q-UFP, oxidative phosphorylation was increased as well as mitochondrial mass, resulting in increased production of mitochondrial superoxide anion. Given this oxidative boost, the NRF2-ARE signaling pathway was significantly activated. However, mitochondrial dynamic alterations in favor of accentuated fission process were observed, in particular after Q-UFP vs FP, and repeated vs acute exposure. Taken together, these results supported mitochondrial quality control and metabolism dysfunction as an early lung underlying mechanism of toxicity, thereby leading to accumulation of defective mitochondria and enhanced endogenous ROS generation. Therefore, these features might play a key role in maintaining PM-induced oxidative stress and inflammation within lung cells, which could dramatically contribute to the exacerbation of inflammatory chronic lung diseases. The prospective findings of this work could also offer new insights into the physiopathology of lung toxicity, arguably initiate and/or exacerbate by acutely and rather repeated exposure to ambient FP and mostly Q-UFP.

Air pollution-derived PM2.5 impairs mitochondrial function in healthy and chronic obstructive pulmonary diseased human bronchial epithelial cells.

In order to clarify whether the mitochondrial dysfunction is closely related to the cell homeostasis maintenance after particulate matter (PM2.5) exposure, oxidative, inflammatory, apoptotic and mitochondrial endpoints were carefully studied in human bronchial epithelial BEAS-2B, normal human bronchial epithelial (NHBE) and chronic obstructive pulmonary disease (COPD)-diseased human bronchial epithelial (DHBE) cells acutely or repeatedly exposed to air pollution-derived PM2.5. Some modifications of the mitochondrial morphology were observed within all these cell models repeatedly exposed to the highest dose of PM2.5. Dose- and exposure-dependent oxidative damages were reported in BEAS-2B, NHBE and particularly COPD-DHBE cells acutely or repeatedly exposed to PM2.5. Nuclear factor erythroid 2-p45 related factor 2 (NRF2) gene expression and binding activity, together with the mRNA levels of some NRF2 target genes, were directly related to the number of exposures for the lowest PM2.5 dose (i.e., 2 µg/cm2), but, surprisingly, inversely related to the number of exposures for the highest dose (i.e., 10 µg/cm2). There were dose- and exposure-dependent increases of both nuclear factor kappa-B (NF-κB) binding activity and NF-κB target cytokine secretion in BEAS-2B, NHBE and particularly COPD-DHBE cells exposed to PM2.5. Mitochondrial ROS production, membrane potential depolarization, oxidative phosphorylation, and ATP production were significantly altered in all the cell models repeatedly exposed to the highest dose of PM2.5. Collectively, our results indicate a cytosolic ROS overproduction, inducing oxidative damage and activating oxygen sensitive NRF2 and NF-kB signaling pathways for all the cell models acutely or repeatedly exposed to PM2.5. However, one of the important highlight of our findings is that the prolonged and repeated exposure in BEAS-2B, NHBE and in particular sensible COPD-DHBE cells further caused an oxidative boost able to partially inactivate the NRF2 signaling pathway and to critically impair mitochondrial redox homeostasis, thereby producing a persistent mitochondrial dysfunction and a lowering cell energy supply.

RIP3 is downregulated in human myeloid leukemia cells and modulates apoptosis and caspase-mediated p65/RelA cleavage.

The receptor-interacting protein kinase 3 (RIP3) associates with RIP1 in a necrosome complex that can induce necroptosis, apoptosis, or cell proliferation. We analyzed the expression of RIP1 and RIP3 in CD34+ leukemia cells from a cohort of patients with acute myeloid leukemia (AML) and CD34+ cells from healthy donors. RIP3 expression was significantly reduced in most AML samples, whereas the expression of RIP1 did not differ significantly. When re-expressed in the mouse DA1-3b leukemia cell line, RIP3 induced apoptosis and necroptosis in the presence of caspase inhibitors. Transfection of RIP3 in the WEHI-3b leukemia cell line or in the mouse embryonic fibroblasts also resulted in increased cell death. Surprisingly, re-expression of a RIP3 mutant with an inactive kinase domain (RIP3-kinase dead (RIP3-KD)) induced significantly more and earlier apoptosis than wild-type RIP3 (RIP3-WT), indicating that the RIP3 kinase domain is an essential regulator of apoptosis/necroptosis in leukemia cells. The induced in vivo expression of RIP3-KD but not RIP3-WT prolonged the survival of mice injected with leukemia cells. The expression of RIP3-KD induced p65/RelA nuclear factor-κB (NF-κB) subunit caspase-dependent cleavage, and a non-cleavable p65/RelA D361E mutant rescued these cells from apoptosis. p65/RelA cleavage appears to be at least partially mediated by caspase-6. These data indicate that RIP3 silencing in leukemia cells results in suppression of the complex regulation of the apoptosis/necroptosis switch and NF-κB activity.

Sstr2A: a relevant target for the delivery of genes into human glioblastoma cells using fiber-modified adenoviral vectors.

Glioblastomas are the most aggressive of the brain tumors occurring in adults and children. Currently available chemotherapy prolongs the median survival time of patients by only 4 months. The low efficiency of current treatments is partly owing to the blood-brain barrier, which restricts the penetration of most drugs into the central nervous system. Locoregional treatment strategies thus become mandatory. In this context, viral tools are of great interest for the selective delivery of genes into tumoral cells. Gliomas express high levels of type 2 somatostatin receptors (sstr2A), pinpointing them as suitable targets for the improvement of transduction efficiency in these tumors. We designed a new adenoviral vector based on the introduction of the full-length somatostatin (SRIF (somatotropin release-inhibiting factor)) sequence into the HI loop of the HAdV fiber protein. We demonstrate that (i) HAdV-5-SRIF uptake into cells is mediated by sstr2A, (ii) our vector drives high levels of gene expression in cells expressing endogenous sstr2A, with up to 65-fold enhancement and (iii) low doses of HAdV-5-SRIF are sufficient to infect high-grade human primary glioblastoma cells. Adenoviral vectors targeting SRIF receptors might thus represent a promising therapeutic approach to brain tumors.

Influence of chimeric human-bovine fibers on adenoviral uptake by liver cells and the antiviral immune response.

Human adenoviruses (HAdV) are widely used for in vitro and in vivo gene transfer. Viral hepatotropism, inflammatory responses and neutralization by pre-existing antibodies (NAbs) are obstacles for clinical applications of HAdV vectors. Although the multifactorial events leading to innate HAdV toxicity are far from being elucidated, there is a consensus that the majority of intravenously injected-HAdV vectors is sequestered by Kuppfer cells, probably independently of coagulation factors. In this study, we show that the adenoviral-associated humoral and innate cytokine immune responses are significantly reduced when HAdV-5 vector carrying human bovine chimeric fibers (HAdV-5-F2/BAdV-4) is intravenously injected into mice. Fiber pseudotyping modified its interaction with blood coagulation factors, as FIX and FX no longer mediate the infection of liver cells by HAdV-5-F2/BAdV-4. As a consequence, at early time points post-infection, several cytokines and chemokines (IFN-gamma, IL-6, IP-10, MCP-1, RANTES and MP1beta) were found to be present at lower levels in the plasma of mice that had been intravenously injected with HAdV-5-F2/BAdV-4 compared with mice injected with the parental vector HAdV-5. Moreover, genetic modification of the fiber allowed HAdV-5-F2/BAdV-4 to partially escape neutralization by NAbs.

Galanin modulates the activity of proopiomelanocortin neurons in the isolated mediobasal hypothalamus of the male rat.

It has become apparent that galanin as well as proopiomelanocortin-derived peptides, such as beta-endorphin, play an important role in the hypothalamic circuitry that regulates neuroendocrine functions and appetite behavior. We have recently shown that GalR1 and GalR2 galanin receptor mRNAs are expressed in proopiomelanocortin neurons of the arcuate nucleus, suggesting a direct modulatory action of galanin on the proopiomelanocortin neuronal system. In the present study, we investigated the effect of galanin on beta-endorphin release and proopiomelanocortin mRNA expression from male rat mediobasal hypothalamic fragments incubated ex vivo. Galanin induced a decrease of spontaneous beta-endorphin release within the first 30-60 min of incubation and this effect was blocked by the galanin receptor antagonist galantide. Co-incubation of galanin with FK-506 (tacrolimus), a calcineurin inhibitor, suppressed the inhibitory effect of galanin on beta-endorphin release, suggesting that calcineurin is involved in the galanin-evoked decrease in beta-endorphin release. Measurement of beta-endorphin levels in the tissues at the end of the incubation period (120 min) revealed that galanin caused a two-fold increase of beta-endorphin peptide concentration in the mediobasal hypothalamic tissues. Concurrently, galanin induced an increase in the mean density of silver grains overlying proopiomelanocortin neurons after 60 min of incubation, an effect antagonized by galantide. Finally, reverse transcription-polymerase chain reaction analysis revealed that the mRNAs for the three galanin receptor subtypes (i.e. GalR1, GalR2, and GalR3) were expressed in the incubated mediobasal hypothalamic fragments. Taken as a whole, our results indicate that galanin plays a modulatory role on proopiomelanocortin neurons and this interrelation contributes to the elucidation of the neural circuitry that controls, among others, gonadotropin-releasing hormone function.

Molecular characterization of TgMIC5, a proteolytically processed antigen secreted from the micronemes of Toxoplasma gondii.

During invasion of host cells, Toxoplasma gondii discharges the contents of small, apically located secretory organelles called micronemes. Micronemal proteins are known to be necessary for both parasite motility and invasion of host cells. To further define the contents of Toxoplasma micronemes, we used cell fractionation and secretion-modulating drugs to identify six novel, putative micronemal proteins. In this paper we describe preliminary characterization of one of these novel proteins, TgMIC5. Molecular cloning and DNA sequence analysis of the TgMIC5 cDNA and gene revealed that it encodes a previously identified immunodominant antigen called H4. TgMIC5 also possesses a consensus sequence unique to members of the parvulin family of peptidyl-prolyl cis-trans isomerases (PPIases). TgMIC5 is expressed as a preproprotein, which is proteolytically processed to a proprotein by signal peptidase before being further processed to a mature protein of 22 kDa. Using a combination of protein secretion experiments, immunofluorescence and immunoelectron microscopy, we demonstrated that TgMIC2 is stored in the micronemes of T. gondii tachyzoites before it is secreted into the surrounding medium. Based on its homology with parvulin-like PPIases, TgMIC5 may assist in the folding of other micronemal proteins that function in invasion of host cells by T. gondii tachyzoites.

Neospora caninum: identification of 19-, 38-, and 40-kDa surface antigens and a 33-kDa dense granule antigen using monoclonal antibodies.

Neospora caninum, a coccidian parasite closely related to Toxoplasma gondii, can infect a broad host range and is regarded as an important cause of bovine abortion worldwide. In the present study, four antigens of N. caninum were partially characterized using monoclonal antibodies. Immunofluorescence of viable tachyzoites as well as the immunoprecipitation of antigens extracted from tachyzoites previously labeled by surface biotinylation revealed that three of these antigens with apparent molecular weights of 40, 38, and 19 kDa are located in the outer surface membrane of this parasite stage. Further evidence for the surface localization of the 38-kDa antigen was obtained by immunoelectron microscopy. In addition to the surface molecules, an antigen located in dense granules and in the tubular network of the parasitophorous vacuole was detected by another monoclonal antibody. When tachyzoite antigens separated under nonreducing conditions were probed on Western blots, this antibody reacted mainly with a 33-kDa antigen. Immunohistochemical analysis of infected tissue sections indicated that the 33-kDa dense granule antigen is present in both tachyzoites and bradyzoites, while the 38-kDa surface antigen from tachyzoites seems to be absent in bradyzoites.

Identification and heterologous expression of a new dense granule protein (GRA7) from Toxoplasma gondii.

Immunoscreening of an expression library constructed with Toxoplasma gondii tachyzoite mRNA with sera from toxoplasmosis-positive humans has led to the identification of a new parasite antigen. Sequence analysis of the gene encoding this antigen allowed the calculation of the theoretical molecular mass (25,857 Da) and showed that the protein contains a putative signal sequence. The C-terminal region contains two hydrophobic regions, the last of which has the characteristics of a membrane-spanning domain. When the protein was heterologously expressed in E. coli and tested by Western blot, it reacted with the human sera originally used for screening. The new antigen also reacted with a monoclonal antibody raised against the entire parasite. Ultrastructural analysis showed that the protein is localized in the dense granules. After host cell invasion, the protein is secreted into the vacuolar network, the parasitophorous vacuole membrane, and into extensions protruding in the cytoplasm. Therefore, it is suggested to designate this new dense granule protein GRA7, following the established nomenclature for this protein family.

The MIC1 microneme protein of Toxoplasma gondii contains a duplicated receptor-like domain and binds to host cell surface.

The cDNA encoding the Toxoplasma gondii microneme protein MIC1 and the corresponding gene have been cloned and sequenced. The MIC1 gene contains three introns. The cDNA encodes a 456 amino acid (aa) sequence, with a typical signal sequence and no other trans-membrane domain. The protein contains a tandemly duplicated domain with conservation of cysteines and presents distant homology with the Plasmodium sp. microneme protein TRAP-SSP2. The MIC1 protein from tachyzoite lysates and a PMAL recombinant expressing the N-terminal duplicated domain of the protein bound to the surface of putative host cells, suggesting a possible involvement of MIC1 in host cell binding/recognition.

Molecular cloning of the Toxoplasma gondii sag4 gene encoding an 18 kDa bradyzoite specific surface protein.

An 18 kDa bradyzoite specific surface protein of Toxoplasma gondii (T. gondii) has been purified by affinity chromatography with a specific monoclonal antibody using parasites grown in vitro under conditions inducing the biosynthesis of bradyzoite specific proteins. N-terminal and internal amino acid sequences obtained by microsequencing enabled us to design degenerate oligonucleotides. A fragment of 187 bp was amplified by polymerase chain reaction (PCR). It was used as a probe to clone a 4 kb-Bam HI fragment encompassing the gene encoding the 18 kDa protein. Nucleotide sequence analysis revealed a single open reading frame of 516 nucleotides encoding a 172 amino acid protein. The deduced amino acid sequence matched perfectly the peptides microsequenced from the native protein. The N-terminal hydrophobic region was found to possess the characteristics of a signal peptide of 27 amino acids. The hydrophobic C-terminal part could represent a signal for a glycan-phosphoinositide anchor. The full-length cDNA was also isolated and both the 5' and 3' untranslated regions were determined. Reverse transcriptase-PCR (RT-PCR) using p18-specific primers showed a stage-specific expression of this gene. Comparison of the nucleic acid sequence and the predicted amino acid sequence with databases did not reveal significant homology with known genes or proteins. This gene is proposed to be named sag4, according to the existing T. gondii nomenclature.

Non-specific potentiation of T- and B-lymphocyte proliferation at the early stage of infection by Schistosoma mansoni: role of factors secreted by the larvae.

The response of rat lymphocytes to schistosomula released products (SRP) was examined. SRP non-specifically activated lymphocytes by potentiating their proliferative response to PHA, Con A or LPS. The parasite factor involved was dialysable and heat stable. The addition of SRP to cultures containing nylon-wool non-adherent lymph node cells resulted in a significant enhancement of cell proliferation. The effect of SRP on athymic nude (Nu/Nu) and litter mate (Nu/+) control rat cells indicated an effect on the proliferation of both B and T lymphocytes. SRP acted in a dose-dependent manner and its action was observed as early as the beginning of cell division. This corresponds to the in vivo situation, since at the early stage of infection increased proliferative responses of the lymph node cells to mitogens were observed. The adjuvant effect of SRP could partly explain the regulation of the cellular immune response observed during S. mansoni infection by the parasite itself and could represent one of the mechanisms involved in immunity to reinfection that is under the control of the parasite.