Uninfected Macrophages Research Articles

Impulsive application to design effective therapies against Cutaneous Leishmaniasis under mathematical perceptive

Leishmaniasis is one type of infectious diseases that is generated by different parasites of the Leishmania. The disease spreads in mammals (both human and animal hosts) through the bites of various infected sand fly. species. Leishmania parasites have two important developmental stages in their life cycle such as Promastigote (the proliferative form found in the lumen of the female sand fly) and another one is Amastigote (the proliferative form found inside several types of mammalian host cells). Here we have considered a mathematical model consisting susceptible (uninfected) macrophage cell, early stage infected macrophage cell (Promastigote), late stage infected macrophage cell (Amastigote) and parasite population. We have applied drugs like Sodium Stibogluconate and Meglumine Antimoniate for reducing parasite population in impulsive way under mathematical framework. Our analytical and numerical results justify that controlling parasite population by applying drugs in impulsive way gives realistic outcome. Also we have established the efficiency of the drug dose that contributes a greater effect on the system moving towards the healthy situation. https://doi.org/10.28919/cmbn/3037

Pharmacological inhibition of p110δ subunit of PI3K confers protection against experimental leishmaniasis.

This study aimed to evaluate the immuno-prophylactic and -therapeutic effect of p110δ-specific pharmacological inhibitors (CAL-101 and IC87114), either alone or in combination with amphotericin B, against experimental cutaneous leishmaniasis (CL) and visceral leishmaniasis (VL). Female BALB/c mice were infected intravenously with Leishmania donovani or subcutaneously with Leishmania major Prophylactic treatment was initiated 24 h prior to infection, whereas therapeutic treatments with or without amphotericin B were initiated either 1 week or 2 weeks post-infection. At different times post-infection, mice were sacrificed and parasite burden, regulatory T cell (Treg) numbers and cytokine production were assessed in the liver, spleen, draining lymph nodes and footpads. In addition, direct cytolytic effects of the inhibitors on parasite growth in axenic cultures and inside infected and uninfected macrophages were also assessed. Prophylactic and therapeutic administration of p110δ pharmacological inhibitors significantly reduced cutaneous lesion (in CL) and parasite burdens (in VL and CL) in the spleens, livers and footpads of infected mice. The reduction in parasite burden was associated with a concomitant reduction in Treg numbers and cytokine production by liver, spleen and lymph node cells. Combined low-dose CAL-101 and amphotericin B therapy caused complete clearance of parasites in mice infected with L. donovani CONCLUSIONS: Our studies clearly show a novel therapeutic option for leishmaniasis based on CAL-101 monotherapy or CAL-101 and amphotericin B combination therapy. These observations have important and direct implications for antimicrobial immunotherapy and drug/vaccine development against leishmaniasis.

Macrophage Epithelial Reprogramming Underlies Mycobacterial Granuloma Formation and Promotes Infection

Mycobacterium tuberculosis infection in humans triggers formation of granulomas, which are tightly organized immune cell aggregates that are the central structure of tuberculosis. Infected and uninfected macrophages interdigitate, assuming an altered, flattened appearance. Although pathologists have described these changes for over a century, the molecular and cellular programs underlying this transition are unclear. Here, using the zebrafish-Mycobacterium marinum model, we found that mycobacterial granuloma formation is accompanied by macrophage induction of canonical epithelial molecules and structures. We identified fundamental macrophage reprogramming events that parallel E-cadherin-dependent mesenchymal-epithelial transitions. Macrophage-specific disruption of E-cadherin function resulted in disordered granuloma formation, enhanced immune cell access, decreased bacterial burden, and increased host survival, suggesting that the granuloma can also serve a bacteria-protective role. Granuloma macrophages in humans with tuberculosis were similarly transformed. Thus, during mycobacterial infection, granuloma macrophages are broadly reprogrammed by epithelial modules, and this reprogramming alters the trajectory of infection and the associated immune response.

Caveolin-1-mediated endocytic pathway is involved in classical swine fever virus Shimen infection of porcine alveolar macrophages

Macrophages are at the frontline of defense against pathogenic microorganisms. However, very little is known about the cell invasion mechanism of classical swine fever virus (CSFV) Shimen strain. To elucidate the infective mechanism of this important pathogen, we screened deep-sequencing data derived from macrophages infected with CSFV Shimen and uninfected macrophages, and identified a role of caveolin-1 (CAV1). Digital gene expression profiling showed that CAV1 was differentially expressed in CSFV Shimen-infected macrophages. Quantitative polymerase chain reaction and western blot analyses showed that the transcription and translation of CAV1 were significantly up-regulated in CSFV Shimen-infected macrophages. In addition, immunofluorescent confocal microscopy analysis suggested that CAV1 was temporally colocalized with CSFV E2 throughout the course of the infection. Through the overexpression of recombinant CAV1 or the silencing of CAV1 expression using small interfering RNA in macrophages, we demonstrated that CAV1 expression is beneficial for the replication of CSFV Shimen. However, RNA silencing of CAV1 did not prevent viral replication, which may indicate that CSFV can also enter macrophages by other mechanisms. Our findings suggest that CAV1-mediated endocytosis is advantageous for productive CSFV Shimen infection in macrophages, providing a new insight into the mechanisms of evasion of host immunity for successful viral infection.

Activity of a novel sulfonamide compound 2-nitro-N-(pyridin-2-ylmethyl)benzenesulfonamide against Leishmania donovani.

New treatments for visceral leishmaniasis, caused by Leishmania donovani, are needed to overcome sustained toxicity, cost, and drug resistance. The aim of this study was to evaluate the therapeutic effects of 2-nitro-N-(pyridin-2-ylmethyl)benzenesulfonamide (2NB) against promastigote and amastigote forms of L. donovani and examine its effect in combination with amphotericin B (AmB) against AmB-resistant clinical isolates. Effects were assessed against extracellular promastigotes in vitro and intracellular amastigotes in L. donovani-infected macrophages. Levels of inducible nitric oxide and Th1 and Th2 cytokines were measured in infected 2NB-treated macrophages, and levels of reactive oxygen species and NO were measured in 2NB-treated macrophages. 2NB was active against promastigotes and intracellular amastigotes with 50% inhibitory concentration values of 38.5±1.5 µg/mL and 86.4±2.4 µg/mL, respectively. 2NB was not toxic to macrophages. Parasite titer was reduced by >85% in infected versus uninfected macrophages at a 2NB concentration of 120 µg/mL. The parasiticidal activity was associated with increased levels of Th1 cytokines, NO, and reactive oxygen species. Finally, 2NB increased the efficacy of AmB against AmB-resistant L. donovani. These results demonstrate 2NB to be an antileishmanial agent, opening up a new avenue for the development of alternative chemotherapies against visceral leishmaniasis.

Latex protein extracts from Calotropis procera with immunomodulatory properties protect against experimental infections with Listeria monocytogenes

BackgroundThe latex from the medicinal plant Calotropis procera is often used in folk medicine against infectious and inflammatory diseases. PurposeIn this study, we investigate a protein fraction with immunomodulatory properties, named LPPI, against experimental infections, in vitro and in vivo, with a virulent strain of Listeria monocytogenes. Study designLPPI was exposed to cultured macrophages or Swiss mice and then challenged with L. monocytogenes. MethodsPeritoneal macrophages were obtained from Swiss mice, and cultured in 96-well microplates. Soluble latex proteins (LP) were subjected to fractionation by ion-exchange chromatography. The major peak (LPPI) was added into wells at 10 or 100µg/ml. Albumin (100µg/ml) was used for comparison between protein treatments. After incubation for 1h at 5% CO2/ 37°C, the supernatant was discarded and 0.2ml of L. monocytogenes overnight culture was added in the wells. Following 4h and 24h infection, the cytokine mRNA expression was evaluated as well as the number of intracellular colony forming units. Swiss mice (n=16) were injected intraperitoneally (i.p.) with LPPI (5 and 10mg/kg) while the control mice received albumin (10mg/kg) or LP (10mg/kg). After 24h, all animal groups were challenged with L. monocytogenes (106 CFU/ ml), also by i.p. route. ResultsLPPI was not toxic to uninfected macrophages (pMØ) and significantly increased mRNA expression of TNF-α, IL-6, IL-1β and iNOS. Following infection, cell viability was reduced by 50% in albumin-treated pMØ (control); but only 17% in pMØ treated with LPPI at 100µg/ml. In this case, LPPI increased expression of TNF-α and IL-6 whereas the number of bacterial colony-forming units was reduced 100-fold in comparison to control groups. Swiss mice pretreated with LPPI showed dose-dependent survival rates that reached 80%, while mice that received albumin died 1–3 days after infection. After 24h infection, leukocyte migration to the infectious foci was high in LPPI-treated mice whereas the number of viable bacteria in the peritoneal fluid, liver and bloodstream were significantly reduced. ConclusionWe conclude that LPPI present immunomodulatory properties that are beneficial for prevention of systemic bacterial infections caused by the intracellular bacteria L. monocytogenes.

Identification and Analysis of the Porcine MicroRNA in Porcine Cytomegalovirus-Infected Macrophages Using Deep Sequencing.

Porcine cytomegalovirus (PCMV; genus Cytomegalovirus, subfamily Betaherpesvirinae, family Herpesviridae) is an immunosuppressive virus that mainly inhibits the immune function of T lymphocytes and macrophages, which has caused substantial damage in the farming industry. In this study, we obtained the miRNA expression profiles of PCMV-infected porcine macrophages via high-throughput sequencing. The comprehensive analysis of miRNA profiles showed that 239 miRNA database-annotated and 355 novel pig-encoded miRNAs were detected. Of these, 130 miRNAs showed significant differential expression between the PCMV-infected and uninfected porcine macrophages. The 10 differentially expressed pig-encoded miRNAs were further determined by stem-loop reverse-transcription polymerase chain reaction, and the results were consistent with the high-throughput sequencing. Gene Ontology analysis of the target genes of miRNAs in PCMV-infected porcine macrophages showed that the differentially expressed miRNAs are mainly involved in immune and metabolic processes. This is the first report of the miRNA transcriptome in porcine macrophages and an analysis of the miRNA regulatory mechanisms during PCMV infection. Further research into the regulatory mechanisms of miRNAs during immunosuppressive viral infections should contribute to the treatment and prevention of immunosuppressive viruses.

Trogocytosis-associated cell to cell spread of intracellular bacterial pathogens.

Macrophages are myeloid-derived phagocytic cells and one of the first immune cell types to respond to microbial infections. However, a number of bacterial pathogens are resistant to the antimicrobial activities of macrophages and can grow within these cells. Macrophages have other immune surveillance roles including the acquisition of cytosolic components from multiple types of cells. We hypothesized that intracellular pathogens that can replicate within macrophages could also exploit cytosolic transfer to facilitate bacterial spread. We found that viable Francisella tularensis, as well as Salmonella enterica bacteria transferred from infected cells to uninfected macrophages along with other cytosolic material through a transient, contact dependent mechanism. Bacterial transfer occurred when the host cells exchanged plasma membrane proteins and cytosol via a trogocytosis related process leaving both donor and recipient cells intact and viable. Trogocytosis was strongly associated with infection in mice, suggesting that direct bacterial transfer occurs by this process in vivo.

Melittin induces in vitro death of Leishmania (Leishmania) infantum by triggering the cellular innate immune response.

BackgroundApis mellifera venom, which has already been recommended as an alternative anti-inflammatory treatment, may be also considered an important source of candidate molecules for biotechnological and biomedical uses, such as the treatment of parasitic diseases.MethodsAfricanized honeybee venom from Apis mellifera was fractionated by RP-C18-HPLC and the obtained melittin was incubated with promastigotes and intracellular amastigotes of Leishmania (L.) infantum. Cytotoxicity to mice peritoneal macrophages was evaluated through mitochondrial oxidative activity. The production of anti- and pro-inflammatory cytokines, NO and H2O2 by macrophages was determined.ResultsPromastigotes and intracellular amastigotes were susceptible to melittin (IC50 28.3 μg.mL−1 and 1.4 μg.mL−1, respectively), but also showed mammalian cell cytotoxicity with an IC50 value of 5.7 μg.mL−1. Uninfected macrophages treated with melittin increased the production of IL-10, TNF-α, NO and H2O2. Infected melittin-treated macrophages increased IL-12 production, but decreased the levels of IL-10, TNF-α, NO and H2O2.ConclusionsThe results showed that melittin acts in vitro against promastigotes and intracellular amastigotes of Leishmania (L.) infantum. Furthermore, they can act indirectly on intracellular amastigotes through a macrophage immunomodulatory effect.

Physalis angulata induces death of promastigotes and amastigotes of Leishmania (Leishmania) amazonensis via the generation of reactive oxygen species

Leishmaniasis are a neglected group of emerging diseases that have been found in 98 countries and are caused by protozoa of the genus Leishmania. The therapy for leishmaniasis causes several side effects and leads to drug-resistant strains. Natural products from plants have exhibited activities against Leishmania in various experimental models. Physalis angulata is a widely used plant in popular medicine, and in the literature it has well-documented leishmanicidal activity. However, its mechanism of action is still unknown. Thus, this study aims to evaluate the mechanism driving the leishmanicidal activity of an aqueous extract of P. angulata root (AEPa). AEPa was effective against both promastigotes and intracellular amastigote forms of Leishmania amazonensis. This effect was mediated by an increase of reactive oxygen species (ROS), but not of nitric oxide (NO). The increased production of ROS induces cell death by phenotypes seems by apoptosis cell death in Leishmania, but not autophagy or necrosis. In addition, morphological analysis of macrophages showed that AEPa induced a high number of cytoplasmic projections, increased the volume of cytoplasm and number of vacuoles, caused cytoskeleton alterations and resulted in high spreading ability. AEPa also promoted superoxide anion (O2−) production in both uninfected macrophages and those infected with Leishmania. Therefore, these results revealed that AEPa causes cell death by phenotypes seems by apoptosis cell death in L. amazonensis and modulates macrophage activation through morphofunctional alterations and O2− generation to induce Leishmania death.

Chlamydia pneumoniae Clinical Isolate from Gingival Crevicular Fluid: A Potential Atherogenic Strain.

Chlamydia pneumoniae has been associated to atherosclerotic cardiovascular diseases. The aim of our study was to characterize, for the first time, a C. pneumoniae strain isolated from the gingival crevicular fluid of a patient with chronic periodontitis, described as a risk factor for cardiovascular diseases. C. pneumoniae isolate was characterized and compared to the respiratory AR-39 strain by VD4-ompA genotyping and by investigating the intracellular growth in epithelial and macrophage cell lines and its ability to induce macrophage-derived foam cells. Inflammatory cytokine levels were determined in the gingival crevicular fluid sample. C. pneumoniae isolate showed a 99% similarity with the AR-39 strain in the VD4-ompA gene sequence and shared a comparable growth kinetic in epithelial cells and macrophages, as evidenced by the infectious progeny and by the number of chlamydial genomic copies. C. pneumoniae isolate significantly increased the number of foam cells as compared to uninfected and LDL-treated macrophages (45 vs. 6%, P = 0.0065) and to the AR-39 strain (45 vs. 30%, P = 0.0065). Significantly increased levels of interleukin 1-β (2.1 ± 0.3 pg/μL) and interleukin 6 (0.6 ± 0.08 pg/μL) were found. Our results suggest that C. pneumoniae may harbor inside oral cavity and potentially be atherogenic, even though further studies will be needed to clarify the involvement of C. pneumoniae in chronic periodontitis as a risk factor for cardiovascular diseases.

Cellular miR-130b inhibits replication of porcine reproductive and respiratory syndrome virus in vitro and in vivo.

MicroRNAs (miRNAs) can impact viral infections by binding to sequences with partial complementarity on viral RNA transcripts, usually resulting in the repression of virus replication. In the present study, we identified a potential binding site for miR-130 in the 5′ untranslated region (bps 155-162) of the porcine reproductive and respiratory syndrome virus (PRRSV) genome. We found that the delivery of multiple miR-130 family mimics, especially miR-130b, resulted in inhibition of PRRSV replication in vitro. miR-130 was effective in inhibiting the replication of multiple type 2 PRRSV strains, but not against vSHE, a classical type 1 strain. miR-130 over-expression did not induce IFN-α or TNF-α expression in either uninfected or PRRSV-infected porcine alveolar macrophages. Results from luciferase reporter assays indicated that miR-130 directly targeted the PRRSV 5′ UTR. Intranasal inoculation of piglets with miR-130b exhibited antiviral activity in vivo and partially protected piglets from an otherwise lethal challenge with HP-PRRSV strain vJX143. Overall, these results demonstrate the importance of the miR-130 family in modulating PRRSV replication and also provide a scientific basis for using cellular miRNAs in anti-PRRSV therapies.

Interacting partners of macrophage-secreted cathepsin B contribute to HIV-induced neuronal apoptosis.

HIV-1 infection of macrophages increases cathepsin B secretion and induces neuronal apoptosis, but the molecular mechanism remains unclear. We identified macrophage-secreted cathepsin B protein interactions extracellularly and their contribution to neuronal death in vitro. Cathepsin B was immunoprecipitated from monocyte-derived macrophage supernatants after 12 days postinfection. The cathepsin B interactome was identified by label-free tandem mass spectrometry and compared with uninfected supernatants. Proteins identified were validated by western blot. Neurons were exposed to macrophage-conditioned media in presence or absence of antibodies against cathepsin B and interacting proteins. Apoptosis was measured using TUNEL labeling. Immunohistochemistry of postmortem brain tissue samples from healthy, HIV-infected and Alzheimer's disease patients was performed to observe the ex-vivo expression of the proteins identified. Nine proteins co-immunoprecipitated differentially with cathepsin B between uninfected and HIV-infected macrophages. Serum amyloid P component (SAPC)-cathepsin B interaction increased in HIV-infected macrophage supernatants, while matrix metalloprotease 9 (MMP-9)-cathepsin B interaction decreased. Pre-treatment of HIV-infected macrophage-conditioned media with antibodies against cathepsin B and SAPC decreased neuronal apoptosis. The addition of MMP-9 antibodies was not neuro-protective SAPC was overexpressed in postmortem brain tissue from HIV-positive neurocognitive impaired patients compared with HIV positive with normal cognition and healthy controls, although MMP-9 expression was similar in all tissues. Inhibiting SAPC-cathepsin B interaction protects against HIV-induced neuronal death and may help to find alternative treatments for HIV-associated neurocognitive disorders.

Immunometabolism within the tuberculosis granuloma: amino acids, hypoxia, and cellular respiration.

Tuberculosis (TB) granulomas are compact, organized agglomerations of infected and uninfected macrophages, T cells, neutrophils, and other immune cells. Within the granuloma, several unique metabolic adaptations occur to modify the behavior of immune cells, potentially favoring bacterial persistence balanced with protection against immunopathology. These include the induction of arginase-1 in macrophages to temper nitric oxide (NO) production and block T cell proliferation, inhibition of oxygen-requiring NO production in hypoxic regions, and induction of tryptophan-degrading enzymes that modify T cell proliferation and function. The spatial and time-dependent organization of granulomas further influences immunometabolism, for example through lactate production by activated macrophages, which can induce arginase-1. Although complex, the metabolic changes in and around TB granulomas can be potentially modified by host-directed therapies. While elimination of the TB bacilli is often the goal of any anti-TB therapy, host-directed approaches must also account for the possibility of immunopathologic damage to the lung.

Nrf2: new insight in cell apoptosis.

Nrf2: new insight in cell apoptosis.

Trophoblast-macrophage crosstalk on human extravillous under Toxoplasma gondii infection

IntroductionThe interaction between human extravillous trophoblasts and macrophages has an important role in implantation and placentation. However, any dysfunction in this communication system is associated with pregnancy pitfalls, and a Toxoplasma gondii infection can be a potential problem in this crosstalk. Therefore, the aim of this study was to assess the influence of infected macrophages on cytokine production and the incidence of apoptosis in T. gondii-infected extravillous trophoblast cells. MethodsHTR-8/SVneo cells were treated with supernatant from macrophages infected or not by T. gondii (conditioned medium) in order to analyze apoptosis and cytokine production in comparison to uninfected control conditions. ResultsThe IL-6 secretion by HTR-8/SVneo cells increased synergistically by treatment with conditioned medium and T. gondii infection. The apoptosis index of HTR-8/SVneo cells was also upregulated by treatment with conditioned medium and infection. In addition, a low expression of Fas/CD95 and a high soluble FasL release were observed during infection, although no significant change was observed in the proliferation of T. gondii. DiscussionThe parasite modulates the high apoptosis index in HTR-8/SVneo cells in order to favor its establishment inside its host cells. On the other hand, the conditioned medium from uninfected macrophages restores the apoptosis rates, although the effect of the infection seems to be stronger. In conclusion, our results showed that T. gondii infection in human extravillous trophoblasts is able to modulate the trophoblast-macrophage crosstalk.

High-content assay for measuring intracellular growth of Leishmania in human macrophages.

Leishmania species are sandfly-transmitted protozoan parasites that cause a spectrum of diseases, ranging from localized skin lesions to fatal visceral disease, in more than 12 million people worldwide. These parasites primarily target macrophages in their mammalian hosts and proliferate as non-motile amastigotes in the phagolysosomal compartment of these cells. High-throughput screens for measuring Leishmania growth within this intracellular niche are needed to identify host and parasite factors that are required for virulence and to identify new drug candidates. Here we describe the development of a new high-content imaging method for quantifying the intracellular growth of Leishmania mexicana parasites in THP-1 macrophages. Wild-type parasites were pre-stained with the fluorescent dye CellTracker(™) Orange CMRA and used to infect THP-1 macrophages in 384-well plates. Infected and uninfected macrophages were subsequently stained with CellTracker Green CMFDA, allowing accurate quantitation of the number of parasites per macrophage using separate detector channels. We validated this method for use in high-content drug screening by examining the dose dependence of known anti-leishmanial drugs on intracellular growth. Unlike previous protocols, this method does not require the generation of transgenic fluorescent or bioluminescent parasite lines and can be readily adapted for screening different Leishmania species, strains, or mutant lines in a wide range of phagocytic host cell types.

Bacterial Membrane Vesicles Mediate the Release of Mycobacterium tuberculosis Lipoglycans and Lipoproteins from Infected Macrophages.

Mycobacterium tuberculosis is an intracellular pathogen that infects lung macrophages and releases microbial factors that regulate host defense. M. tuberculosis lipoproteins and lipoglycans block phagosome maturation, inhibit class II MHC Ag presentation, and modulate TLR2-dependent cytokine production, but the mechanisms for their release during infection are poorly defined. Furthermore, these molecules are thought to be incorporated into host membranes and released from infected macrophages within exosomes, 40-150-nm extracellular vesicles that derive from multivesicular endosomes. However, our studies revealed that extracellular vesicles released from infected macrophages include two distinct, largely nonoverlapping populations: one containing host cell markers of exosomes (CD9, CD63) and the other containing M. tuberculosis molecules (lipoglycans, lipoproteins). These vesicle populations are similar in size but have distinct densities, as determined by separation on sucrose gradients. Release of lipoglycans and lipoproteins from infected macrophages was dependent on bacterial viability, implicating active bacterial mechanisms in their secretion. Consistent with recent reports of extracellular vesicle production by bacteria (including M. tuberculosis), we propose that bacterial membrane vesicles are secreted by M. tuberculosis within infected macrophages and subsequently are released into the extracellular environment. Furthermore, extracellular vesicles released from M. tuberculosis-infected cells activate TLR2 and induce cytokine responses by uninfected macrophages. We demonstrate that these activities derive from the bacterial membrane vesicles rather than exosomes. Our findings suggest that bacterial membrane vesicles are the primary means by which M. tuberculosis exports lipoglycans and lipoproteins to impair effector functions of infected macrophages and circulate bacterial components beyond the site of infection to regulate immune responses by uninfected cells.

Cellular pharmacokinetics and intracellular activity of the novel peptide deformylase inhibitor GSK1322322 against Staphylococcus aureus laboratory and clinical strains with various resistance phenotypes: studies with human THP-1 monocytes and J774 murine macrophages.

GSK1322322 is a peptide deformylase inhibitor active against Staphylococcus aureus strains resistant to currently marketed antibiotics. Our aim was to assess the activity of GSK1322322 against intracellular S. aureus using an in vitro pharmacodynamic model and, in parallel, to examine its cellular pharmacokinetics and intracellular disposition. For intracellular activity analysis, we used an established model of human THP-1 monocytes and tested one fully susceptible S. aureus strain (ATCC 25923) and 8 clinical strains with resistance to oxacillin, vancomycin, daptomycin, macrolides, clindamycin, linezolid, or moxifloxacin. Uptake, accumulation, release, and subcellular distribution (cell fractionation) of [(14)C]GSK1322322 were examined in uninfected murine J774 macrophages and uninfected and infected THP-1 monocytes. GSK1322322 demonstrated a uniform activity against the intracellular forms of all S. aureus strains tested, disregarding their resistance phenotypes, with a maximal relative efficacy (E max) of a 0.5 to 1 log10 CFU decrease compared to the original inoculum within 24 h and a static concentration (C s) close to its MIC in broth. Influx and efflux were very fast (<5 min to equilibrium), and accumulation was about 4-fold, with no or a minimal effect of the broad-spectrum eukaryotic efflux transporter inhibitors gemfibrozil and verapamil. GSK1322322 was recovered in the cell-soluble fraction and was dissociated from the main subcellular organelles and from bacteria (in infected cells). The results of this study show that GSK1322322, as a typical novel deformylase inhibitor, may act against intracellular forms of S. aureus. They also suggest that GSK1322322 has the ability to freely diffuse into and out of eukaryotic cells as well as within subcellular compartments.

Microparticles released from Mycobacterium tuberculosis-infected human macrophages contain increased levels of the type I interferon inducible proteins including ISG15.

Microparticles (MPs) are small membranous particles (100-1000 nm) released under normal steady-state conditions and are thought to provide a communication network between host cells. Previous studies demonstrated that Mycobacterium tuberculosis (M. tb) infection of macrophages increased the release of MPs, and these MPs induced a proinflammatory response from uninfected macrophages in vitro and in vivo following their transfer into uninfected mice. To determine how M. tb infection modulates the protein composition of the MPs, and if this contributes to their proinflammatory properties, we compared the proteomes of MPs derived from M. tb-infected (TBinf-MP) and uninfected human THP-1 monocytic cells. MP proteins were analyzed by GeLC-MS/MS with spectral counting revealing 68 proteins with statistically significant differential abundances. The 42 proteins increased in abundance in TBinf-MPs included proteins associated with immune function (7), lysosomal/endosomal maturation (4), vesicular formation (12), nucleosome proteins (4), and antigen processing (9). Prominent among these were the type I interferon inducible proteins, ISG15, IFIT1, IFIT2, and IFIT3. Exposure of uninfected THP-1 cells to TBinf-MPs induced increased gene expression of isg15, ifit1, ifit2, and ifit3 and the release of proinflammatory cytokines. These proteins may regulate the proinflammatory potential of the MPs and provide candidate biomarkers for M. tb infection.