Leptin impairs myogenesis in C2C12 cells through JAK/STAT and MEK signaling pathways.

Reduced lean body mass in genetically obese (ob/ob) or anorectic/cachectic subjects prompted us to verify the hypothesis whether leptin, white adipose tissue cytokine, might be a negative organizer of myogenesis. Recombinant leptin (100 ng/mL) stimulated mitogenesis together with the raise in T(202/)Y(204)P-ERK1/2 protein expression. Concomitantly, it impaired cell viability and muscle fiber formation from C2C12 mouse myoblasts. Detailed acute and chronic studies with the use of metabolic inhibitors revealed that both JAK/STAT3 and MEK/MAPK but not PI3-K/AKT/GSK-3ß signaling pathways were activated by leptin, and that STAT3 (Y(705)P-STAT3) and MEK (T(202/)Y(204)P-ERK1/2) mediate these effects. In contrary, insulin evoked PI3-K-dependent phosphorylation of AKT (S(473)) and GSK-3ß (S(9)) and insulin surpassed leptin-dependent inhibition of myogenic differentiation in PI3-K-dependent manner. GSK-3ß seems to play dual role in muscle development. Insulin-dependent effect on GSK-3ß (S(9)P-GSK-3ß) led to accelerated myotube construction. In contrary, leptin through MEK-dependent manner caused GSK-3ß phosphorylation (Y(216)P-GSK-3ß) with resultant drop in myoblast fusion. Summing up, partially opposite effects of insulin and leptin on skeletal muscle growth emphasize the importance of interplay between these cytokines. They determine how muscle mass is gained or lost.

[Lipid rafts in Alzheimer's disease]. / Tratwy lipidowe w chorobie Alzheimera.

Forty years after Singer and Nicolson (1972) announced the fluid mosaic membrane model a number of new facts caused updating of their historic view. Plasma membrane is not uniform in state of matter, i.e. fluid portion is represented by glycerophospholipids spontaneously mounted into lipid bilayer in disordered manner (Ld - liquid disordered). In such membrane numerous nanodomains (millions in single cell) known as lipid rafts (TL) and caveolae contain sphingolipids and cholesterol as well as lipid modified integral membrane proteins. Nanodomains are more rigid, denser portion of plasma membrane (Lo - liquid ordered). Nanodomains are buoyant in fluid portion of membrane and have tendency to coalesce into larger platforms to form signalosomes essential for signal transduction. TL constitutively express certain proteins (alpha subunits of heterotrimeric G proteins, secretases, caveolins, flotillin). There are other proteins found in TL after additional lipid modifications (palmitoylation, myristoylation). With regard to dementia, retrospective studies carried out in human beings point to cholesterol and TL as crucial factors in etiopathogenesis of Alzheimer's disease (AD). Chronic administration of statins to patients significantly reduced the incidence of AD. This article is intended to make closer view into the molecular basis of AD and sheds more light on possible causal links between TL and AD.

Verapamil-induced autophagy-like process in colon adenocarcinoma COLO 205 cells; the ultrastructural studies.

BACKGROUND: Verapamil (Ver) is a well known, worldwide used drug to correct cardiac arrhythmias. The main Ver target is the L-type calcium channel. Modulation of calcium homeostasis vaulted Ver into use in medical applications. METHODS: To examine COLO 205 cells morphology after Ver treatment, an electron microscopy technique was used. RESULTS: This study shows ultrastructural evidence that Ver initiates autophagy-like process in human colon adenocarcinoma COLO 205 cells. TEM photographs revealed the presence of differently developed autophagic vacuoles in response to Ver administration. Furthermore, extensive ultrastructural cell alterations confirmed that cancer cells died via necrosis or apoptosis, as demonstrated by ruptured plasma membrane or condensed chromatin, respectively. CONCLUSIONS: It is the evidence that apoptosis resistant COLO 205 cells are overruled by autophagy-like process. Autophagy-like cell death could be a promising venue to delete cancer cells. Ver appears to be a new potentially effective anticancer compound.

A novel mechanism of non-Aß component of Alzheimer's disease amyloid (NAC) neurotoxicity. Interplay between p53 protein and cyclin-dependent kinase 5 (Cdk5).

The non-Aß component of Alzheimer's disease (AD) amyloid (NAC) is produced from the precursor protein NACP/α-synuclein (ASN) by till now unknown mechanism. Previous study showed that like ASN, NAC peptide induced oxidative/nitrosative stress and apoptosis. Our present study focused on the mechanisms of PC12 cells death evoked by NAC peptide, with particular consideration on the role of p53 protein. On the basis of molecular and transmission electron microscopic (TEM) analysis it was found that exogenous NAC peptide (10 µM) caused mitochondria dysfunction, enhanced free radical generation, and induced both apoptotic and autophagic cell death. Morphological and immunocytochemical evidence from TEM showed marked changes in expression and in translocation of proapoptotic protein Bax. We also observed time-dependent enhancement of Tp53 gene expression after NAC treatment. Free radicals scavenger N-tert-butyl-alpha-phenylnitrone (PBN, 1 mM) and p53 inhibitor (α-Pifithrin, 20 µM) significantly protected PC12 cells against NAC peptide-evoked cell death. In addition, exposure to NAC peptide resulted in higher expression of cyclin-dependent kinase 5 (Cdk5), one of the enzymes responsible for p53 phosphorylation and activation. Concomitantly, we observed the increase of expression of Cdk5r1 and Cdk5r2 genes, coding p35 and p39 peptides that are essential regulators of Cdk5 activity. Moreover, the specific Cdk5 inhibitor (BML-259, 10 µM) protected large population of cells against NAC-evoked cell death. Our findings indicate that NAC peptide exerts its toxic effect by activation of p53/Cdk5 and Bax-dependent apoptotic signaling pathway.

Blood-brain barrier permeability differentiates Sadowski mouse lines of high and low stress-induced analgesia: electron microscopy analysis.

The blood-brain barrier (BBB) forms a filtering system between peripheral circulating blood and the central nervous system. Pathological leakage of the BBB is probably responsible for various dysfunctions and diseases. Over twenty years ago, Sadowski et al. separated two lines of mouse, one with high sensitivity (HA) and the other with low sensitivity (LA) to stress-induced analgesia (SIA). We propose that leakage of the BBB is responsible for the difference in SIA of the "Sadowski mouse" model. The presented BBB electron microscopy structural analysis of both lines provided evidence for this hypothesis. Up to now, a good natural animal model of differences of BBB permeability is not known. The "Sadowski mouse" may fulfil this deficiency.

Systemic administration of lipopolysaccharide induces molecular and morphological alterations in the hippocampus.

A systemic inflammatory reaction may have detrimental effects on the organism, including the central nervous system. Previous studies have indicated that lipopolysaccharide (LPS)-evoked systemic inflammation induces pathological alterations in the mouse midbrain, especially in the substantia nigra. The aim of the present study was to investigate whether the hippocampus is also affected after an intraperitoneal (i.p.) injection of LPS. We focussed on the dynamics of proinflammatory gene expression and the processes leading to neuronal cell death. A systemic inflammatory response in C57BL/6 mice was induced by an i.p. injection of LPS (1mg/kg b.w.). The genetic, biochemical and morphological alterations were analysed up to 96h after LPS administration using quantitative PCR, immunochemical, immunocytochemical and electron microscopic methods. Real-time PCR analysis indicated an altered expression of several genes, mainly responsible for arachidonic acid release and metabolism, in the hippocampus 96h after the systemic administration of LPS. Three hours after LPS treatment, the level of mRNA for iNOS, COX-2 and TNFα was increased; then, after 6-24h, it rose for TLR4 and cPLA2. The expression of 5-LOX and 12-LOX was increased at 12-24 and 24-48h after LPS injection, respectively. Our data demonstrate for the first time the sequential activation of the expression of several pro-inflammatory genes responsible for the maintenance of the inflammatory response. Moreover, the electron microscopy studies presented the stimulation of apoptosis-inducing factor (AIF)-mediated death signalling and cathepsin B-related autophagy or necrosis. These biochemical and morphological alterations in the hippocampus, which were induced by systemic inflammation, may be responsible for the impairment of cognition function observed previously.

Ultrastructural evidence of amyloid beta-induced autophagy in PC12 cells.

Herein we demonstrate that PC12 cells overexpress human amyloid beta precursor protein bearing double Swedish mutation(AbetaPPsw), showing the phenotype characteristic for Alzheimer's disease (AD). Examination of cells at ultrastructural level revealed the intracellular presence of peptide aggregates. Furthermore, autophagy induction was found to be a hallmark of amyloid beta-induced cytotoxicity. Importantly, autophagic vacuoles were co-localized within amyloid beta (Abeta) deposits. This suggests the involvement of autophagy in amyloid beta-elicited cell degeneration.

Generation of functional neural artificial tissue from human umbilical cord blood stem cells.

Stem cell-based regenerative neurology is an emerging concept for treatment of diseases of central nervous system. Among variety of proposed procedures, one of the most promising is refilling of cystic cavities of injured brain parenchyma with artificial neural tissue. Recent studies revealed that after allogenic transplantation in rodents these tissue-engineered entities were shown efficient in repair of hypoxic/ischemic brain injury. Human umbilical cord blood (HUCB) was recognized to be an efficient and noncontroversial source of neural stem cells (NSC). The main purpose of this study was to generate HUCB-derived neural artificial tissue and investigate their functional properties. Neural organoids formed on human-originated biodegradable scaffolds within 3 weeks and resembled niche structure where immature stem cells (Oct4+ and Sox2+) and proliferating neuroblasts (Nestin+, GFAP+, and Ki67+) were present. Such aggregates were placed on multi-electrode chips and differentiated toward mature neurons (TUJ1+ and MAP2+). These three-dimensional aggregates in contrast to two-dimensional cultures formed functional circuits and generated spontaneous field/action potentials. Our results indicate that three-dimensional environment facilitates maturation of HUCB-derived NSC what should be considered regarding regenerative medicine application.

Organic cation/carnitine transporter OCTN3 is present in astrocytes and is up-regulated by peroxisome proliferators-activator receptor agonist.

In the brain beta-oxidation, which takes place in astrocytes, is not a major process of energy supply. Astrocytes synthesize important lipid metabolites, mainly due to the processes taking place in peroxisomes. One of the compounds necessary in the process of mitochondrial beta-oxidation and export of acyl moieties from peroxisomes is l-carnitine. Two Na-dependent plasma membrane carnitine transporters were shown previously to be present in astrocytes: a low affinity amino acid transporter B(0,+) and a high affinity cation/carnitine transporter OCTN2. The expression of OCTN2 is known to increase in peripheral tissues upon the stimulation of peroxisome proliferators-activator receptor alpha (PPARalpha), a nuclear receptor known to up-regulate several enzymes involved in fatty acid metabolism. The present study was focused on another high affinity carnitine transporter-OCTN3, its presence, regulation and activity in astrocytes. Experiments using the techniques of real-time PCR, Western blot and immunocytochemistry analysis demonstrated the expression of octn3 in rat astrocytes and, out of two rat sequences ascribed as similar to mouse OCTN3, XM_001073573 was found in these cells. PPARalpha activator-2-[4-chloro-6-[(2,3-dimethylphenyl)amino]-2-pyrimidinyl]thio]acetic acid (WY-14,643) stimulated by 50% expression of octn3, while, on the contrary to peripheral tissues, it did not change the expression of octn2. This observation was correlated with an increased Na-independent activity of carnitine transport. Analysis by transmission electron microscopy showed an augmented intracellular localization of OCTN3 upon PPARalpha stimulation, mainly in peroxisomes, indicating a physiological role of OCTN3 as peroxisomal membrane transporter. These observations point to an important role of OCTN3 in peroxisomal fatty acid metabolism in astrocytes.

Alzheimer's disease genetic mutation evokes ultrastructural alterations: correlation to an intracellular Abeta deposition and the level of GSK-3beta-P(Y216) phosphorylated form.

Herein we demonstrate that PC12 cells, which overexpress human wild-type amyloid-beta precursor protein (AbetaPPwt) or AbetaPP bearing double Swedish mutation (AbetaPPsw), reveal phenotype characteristic for Alzheimer's disease (AD). The examination of cell ultrastructure showed the presence of peptide aggregates within the cells, activation of endosomal-lysosomal system and extensive exocytosis. Furthermore, the autophagy induction was also characteristic hallmark of amyloid-beta-induced cytotoxicity. Morphological changes were positively correlated with the extent of phosphorylated glycogen synthase kinase-3beta (phospho-Tyr(216)-GSK-3beta, GSK-3beta-P(Y216)). The activity of GSK-3beta is believed to cause tau protein hyper-phosphorylation, increased amyloid-beta production and local plaque-associated microglial-mediated inflammatory responses. All of them are symptomatic for AD. In our studies, the highly significant Y216 phosphorylation and over-expression of total GSK-3beta were observed in AbetaPPsw-transfected PC12 cells. In addition, the immuocytochemical analysis showed co-localization of GSK-3beta-P(Y216) and amyloid-beta deposits. Thus, our data support a functional role of GSK-3beta in AbetaPP processing, further implicating this kinase in the amyloid-beta-dependent pathogenesis.

Association of protein kinase C delta and phospholipid scramblase 3 in hippocampal mitochondria correlates with neuronal vulnerability to brain ischemia.

Recent findings support the idea that mitochondrial integrity plays an important role in the propagation of excitotoxic ischemic signal and PKC is implicated in the regulation of mitochondrial membranes properties. One of the targets of PKC delta is phospholipid scramblase 3 (PLSCR3), an enzyme responsible for cardiolipin translocation from the inner to outer mitochondrial membrane. To get an insight into in vivo mechanism by which PKC delta mediates ischemia/reperfusion injury of hippocampal neurons, we examined the effects of transient brain ischemia in gerbil on association of PKC delta with mitochondria isolated from ischemia-vulnerable (CA1) and ischemia-resistant regions, and interactions between PKC delta and PLSCR3. Postischemic, biphasic and brain region-specific translocation of PKC delta to mitochondria was observed. First peak was at 30-60 min of reperfusion and the second was observed after 72-96 h following ischemia. PKC delta was translocated to mitochondria only in CA1 region. The PLSCR3 mRNA and protein was detected in brain by RT-PCR and sequence analysis, Western blotting and immunocytochemistry in electron microscopy (EM). Co-immunoprecipitation and double-labeled immuno-EM showed association of PKC delta and PLSCR3 in postischemic CA1 mitochondria. Additionally, the amount of tBid associated with mitochondria was elevated 96 h following ischemia. Our data suggest that in the postischemic brain PKC delta co-localizes with PLSCR3 in mitochondria and this event might influence the mitochondrial membranes architecture and delayed neurons degeneration.

Impaired growth hormone-releasing hormone neurons ultrastructure and peptide accumulation in the arcuate nucleus of mosaic mice with altered copper metabolism.

A progressive decrease in body weight and retarded linear growth observed in mosaic male mice with the mutation linked to X-chromosome (Atp7a(mo-ms)) raised the question whether hypophysiotropic growth axis activity may be affected in these animals. A pathologically developed median eminence ultrastructure with very low somatostatin accumulation as well as an intensive phagocytosis of growth hormone cells observed in the anterior pituitary gland raised the question whether hypothalamic growth hormone-releasing hormone (GHRH) neuronal network is also affected in mosaic mice. In this study an arcuate nucleus GHRH neurons ultrastructure as well as GHRH peptide accumulation in normal and mutant mice were compared. An electron microscopic immunocytochemical method with colloidal-gold labeling was applied to compare the ultrastructural morphology of GHRH neuron and intracellular GHRH peptide distribution. Mosaic mice exhibited a pathologically developed ultrastructure of arcuate nucleus GHRH neurons, defective intracellular peptide localization as well as reduced peptide storage. Obtained results support the crucial role of unaltered copper metabolism in physiological development of hypophysiotropic growth axis activity. Consequently, a pathologically developed GHRH hypothalamic network may impact progressive decrease in body weight and retarded length growth observed in mosaic male mice.

Staphylococcal cysteine protease staphopain B (SspB) induces rapid engulfment of human neutrophils and monocytes by macrophages.

Abstract Circulating neutrophils and monocytes constitute the first line of antibacterial defence, which is responsible for the phagocytosis and killing of microorganisms. Previously, we have described that the staphylococcal cysteine proteinase staphopain B (SspB) cleaves CD11b on peripheral blood phagocytes, inducing the rapid development of features of atypical cell death in protease-treated cells. Here, we report that exposure of phagocytes to SspB critically impairs their antibacterial functions. Specifically, SspB blocks phagocytosis of Staphylococcus aureus by both neutrophils and monocytes, represses their chemotactic activity and induces extensive, nonphlogistic clearance of SspB-treated cells by macrophages. The proteinase also cleaves CD31, a major repulsion ('do not-eat-me') signal, on the surface of neutrophils. We suggest that both proteolytic degradation of repulsion signals and induction of 'eat-me' signals on the surface of leukocytes are responsible for the observed intensive phagocytosis of SspB-treated neutrophils by human monocyte-derived macrophages. Collectively, this may lead to the depletion of functional neutrophils at the site of infection, thus facilitating staphylococcal colonisation and spreading.

Sodium butyrate sensitizes human colon adenocarcinoma COLO 205 cells to both intrinsic and TNF-alpha-dependent extrinsic apoptosis.

Overexpression of cFLIP protein seems to be critical in the antiapoptotic mechanism of immune escape of human COLO 205 colon adenocarcinoma cells. Actually, cFLIP appears to inhibit the death receptor ligand-mediated cell death. Application of the metabolic inhibitor sodium butyrate (NaBt), short-chain volatile fatty acid, sensitized COLO 205 cells to TNF-alpha-mediated apoptosis. Western-blot analysis revealed that the susceptibility of human COLO 205 cells to apoptogenic stimuli resulted from time-dependent reduction in cFLIP and simultaneous up-regulation of TNF-R1 protein levels. Additionally, the combined TNF-alpha and NaBt treatment caused cleavage of Bid and caspase-9 activation, as well as cytochrome c release from mitochondria. Thus, the evidence of this study indicates that NaBt facilitates the death receptor signal evoked by TNF-alpha. Moreover, NaBt alone initiated intrinsic apoptosis, that in turn was abolished by intracellular BCL-2 delivery. It confirms the involvement of mitochondria in the proapoptotic activity of NaBt. The activation of mitochondrial pathway was substantiated by up-regulated expression of BAK with concomitant reduction of antiapoptotic BCL-x(L), XIAP and survivin proteins. These findings suggest that NaBt could represent a good candidate for the new therapeutic strategy aimed to improve chemo- and immunotherapy of colon cancer.

IGF-I, EGF, and sex steroids regulate autophagy in bovine mammary epithelial cells via the mTOR pathway.

Mammary gland growth and involution are based on a dynamic equilibrium between proliferation and apoptosis of mammary epithelial cells (MEC). The main type of cell death responsible for bovine mammary gland involution is apoptosis, but MEC also exhibit morphological features of autophagy. The present study has been undertaken in order to examine factors, which are responsible for the regulation of autophagy in bovine MEC. We used a model of in vitro mammary gland involution known to be dependent on fetal bovine serum (FBS) deficiency in the culture of bovine BME-UV1 cells. We investigated the effects of insulin-like growth factor-1 (IGF-I) and epidermal growth factor (EGF) signaling, as well as sex steroids and rapamycin (a specific inhibitor of mammalian target of rapamycin, mTOR, kinase) on autophagy in the MEC line BME-UV1. Our main focus was on the role of mTOR in the regulation of autophagy by growth factors and hormones. Laser scanning cytometry, electron microscopy, Western-blot analysis, GFP-LC3 reporter-based expression analysis, and LysoTracker Green-related fluorescence were used to determine the activity of autophagy in BME-UV1 cells. We found that FBS deficiency induced both autophagy and apoptosis with the highest intensity of both processes after 48h of MEC exposure to the deficient medium (0.5% FBS). Addition of IGF-I or/and EGF to the FBS-deficient medium clearly diminished autophagy. We also show that IGF-I and EGF are involved in the activation of mTOR in bovine MEC, whereas inhibition of mTOR by rapamycin abrogated the suppressive effects of IGF-I and EGF on autophagy. This suggests that mTOR links IGF-I and EGF signaling in inhibiting the autophagy pathways. Contrary to IGF-I and EGF, 17beta-estradiol and progesterone exerted stimulatory effects on autophagy in bovine MEC. At the same time we observed a suppressive effect of both steroids on mTOR activation/phosphorylation. In conclusion, autophagy in bovine MEC undergoes complex regulation, where its activity is controlled by survival pathways dependent on IGF-I and EGF, which are involved in suppression of autophagy, and by pregnancy steroids, which act as inducers of the process.

Impact of oxygenation of bioartificial liver using perfluorocarbon emulsion perftoran on metabolism of human hepatoma C3A cells.

One of the most important challenges in bioartificial liver designed for patients suffering from acute liver failure is oxygenation of cells within the bioreactor. The aim of this study was to evaluate the impact of oxygenation of bioartificial liver using perfluorocarbon (PFC) emulsion on the metabolic activity of hepatic cells in vitro. Mineral fibers coated with collagen type I were used as scaffolds for hepatic cells. Significantly higher total protein synthesis by hepatic C3A cells cultured in the bioreactor for 24 hours, in the group treated with medium supplemented with PFC emulsion, was observed in comparison with medium without PFC. Albumin production increased in the group treated with PFC after 1 hour of perfusion and was continuously, statistically, significantly higher during perfusion then the control group. In conclusion, the use of oxygen carriers, such as the PFC emulsion, can significantly improve synthetic performance of the bioreactor. Mineral fibers coated with extracellular proteins may serve as support for hepatic cells in the bioreactor.

Spongy polyethersulfone membrane for hepatocyte cultivation: studies on human hepatoma C3A cells.

There are different types of membranes used for hepatocyte cultivation. In our studies, spongy polyethersulfone (PES) membranes were examined as a support for hepatic cell cultivation in vitro. The extended surface of the membranes allows to introduce a high cell number especially in three-dimensional gel structure. Scanning electron microscopy analysis indicated that C3A cells used in our experiments grew well on PES membranes forming microvilli characteristic for normal hepatocytes. Analysis of cell viability proved that spongy PES membrane is well tolerated by J774 macrophages and did not stimulate nitric oxide synthesis. Bile canalicular structures were observed in fluorescence microscopy after F-actin staining with tetramethyl rhodamine iso-thiocyanate (TRITC)-phalloidin. The C3A cells showed high affinity to the PES membranes and adhered to almost 90% during the initial 24 h of incubation. Albumin production increased during static culture from the value of 805.2 +/- 284.4 (ng/24 h/initial 10(6) cells) during the first days, to 2017.6 +/- 505.9 (ng/24 h/initial 10(6) cells) after 10 days of culture. In conclusion, the spongy PES membranes can be used as scaffold for hepatocyte cultivation, especially for the creation of three-dimensional environments.

Molecular basis of parthenolide-dependent proapoptotic activity in cancer cells.

This review outlines the molecular events that accompany the anti-tumor action of parthenolide (PN). Parthenolide (PN) is naturally derived compound, isolated from plant Tanacetum parthenium. PN has been previously shown to withdraw cells from cell cycle or to promote cell differentiation, and finally to induce programmed cell death. Recent advances in molecular biology indicate that this sesquiterpene lactone might evoke the above-mentioned effects by indirect action on genes. PN was shown to inhibit NF-kappaB- and STATs-mediated antiapoptotic gene transcription. On one hand, the proapoptotic activity of PN includes stimulation of intrinsic apoptotic pathway with the higher level of intracellular ROS and modifications of Bcl-2 family proteins (conformational changes of Bak and Bax, Bid cleavage). On the other hand, PN amplifies the apoptotic signal through the sensitization of cancer cells to extrinsic apoptosis, induced by TNF-alpha. These effects are specific to tumor cells. Unique properties of PN make this agent a promising metabolic inhibitor to retard tumorigenesis and to suppress tumor growth.

Bisindolylmaleimide IX facilitates extrinsic and initiates intrinsic apoptosis in TNF-alpha-resistant human colon adenocarcinoma COLO 205 cells.

Human COLO 205 colon adenocarcinoma cells are immune to extrinsic apoptosis induced by immunomodulatory cytokines. Among the antiapoptotic mechanisms responsible for the immune escape, the overexpression of the cFLIP protein seems to be critical. cFLIP appears to inhibit the TNF-alpha-induced death receptor signal. The application of the metabolic inhibitor bisindolylmaleimide IX (Bis-IX), known as a potent PKC repressor, sensitized COLO 205 cells to TNF-alpha-mediated apoptosis. The Western-blot analysis revealed that the susceptibility of human COLO 205 cells to apoptogenic stimuli resulted from time-dependent reduction in cFLIP(L) and TRADD protein levels. At the same time, the level of FADD protein was up-regulated. Additionally, the combined TNF-alpha and Bis-IX treatment caused cleavages of Bid and procaspase-9, as well as cytochrome c release. Thus, the evidence of this study indicates that Bis-IX facilitates the death receptor signal mediated by TNF-R1. Moreover, Bis-IX alone initiated intrinsic apoptosis, which could be abolished by Bcl-2 delivery. It heralds the involvement of mitochondria in caspase-8-independent intrinsic apoptosis. In turn, the treatment with bisindolylmaleimide III (Bis-III) did not assist TNF-alpha-dependent apoptosis.

A potential new pathway for Staphylococcus aureus dissemination: the silent survival of S. aureus phagocytosed by human monocyte-derived macrophages.

Although considered to be an extracellular pathogen, Staphylococcus aureus is able to invade a variety of mammalian, non-professional phagocytes and can also survive engulfment by professional phagocytes such as neutrophils and monocytes. In both of these cell types S. aureus promptly escapes from the endosomes/phagosomes and proliferates within the cytoplasm, which quickly leads to host cell death. In this report we show that S. aureus interacted with human monocyte-derived macrophages in a very different way to those of other mammalian cells. Upon phagocytosis by macrophages, S. aureus persisted intracellularly in vacuoles for 3-4 days before escaping into the cytoplasm and causing host cell lysis. Until the point of host cell lysis the infected macrophages showed no signs of apoptosis or necrosis and were functional. They were able to eliminate intracellular staphylococci if prestimulated with interferon-gamma at concentrations equivalent to human therapeutic doses. S. aureus survival was dependent on the alternative sigma factor B as well as the global regulator agr, but not SarA. Furthermore, isogenic mutants deficient in alpha-toxin, the metalloprotease aureolysin, protein A, and sortase A were efficiently killed by macrophages upon phagocytosis, although with different kinetics. In particular alpha-toxin was a key effector molecule that was essential for S. aureus intracellular survival in macrophages. Together, our data indicate that the ability of S. aureus to survive phagocytosis by macrophages is determined by multiple virulence factors in a way that differs considerably from its interactions with other cell types. S. aureus persists inside macrophages for several days without affecting the viability of these mobile cells which may serve as vehicles for the dissemination of infection.