Polyene macrolide antifungal drugs trigger interleukin-1β secretion by activating the NLRP3 inflammasome.

The use of antimycotic drugs in fungal infections is based on the concept that they suppress fungal growth by a direct killing effect. However, amphotericin and nystatin have been reported to also trigger interleukin-1β (IL-1β) secretion in monocytes but the molecular mechanism is unknown. Here we report that only the polyene macrolides amphotericin B, nystatin, and natamycin but none of the tested azole antimycotic drugs induce significant IL-1β secretion in-vitro in dendritic cells isolated from C57BL/6 mouse bone marrow. IL-1β release depended on Toll-like receptor-mediated induction of pro-IL-1β as well as the NLRP3 inflammasome, its adaptor ASC, and caspase-1 for enzymatic cleavage of pro-IL-1β into its mature form. All three drugs induced potassium efflux from the cells as a known mechanism for NLRP3 activation but the P2X7 receptor was not required for this process. Natamycin-induced IL-1β secretion also involved phagocytosis, as cathepsin activation as described for crystal-induced IL-1β release. Together, the polyene macrolides amphotericin B, nystatin, and natamycin trigger IL-1β secretion by causing potassium efflux from which activates the NLRP3-ASC-caspase-1. We conclude that beyond their effects on fungal growth, these antifungal drugs directly activate the host's innate immunity.

Measurement of the mechanical power of walking by satellite positioning system (GPS).

PURPOSE: This descriptive article illustrates the application of Global Positioning System (GPS) professional receivers in the field of locomotion studies. The technological challenge was to assess the external mechanical work in outdoor walking. METHODS: Five subjects walked five times during 5 min on an athletic track at different imposed stride frequency (from 70-130 steps x min(-1)). A differential GPS system (carrier phase analysis) measured the variation of the position of the trunk at 5 Hz. A portable indirect calorimeter recorded breath-by-breath energy expenditure. RESULTS: For a walking speed of 1.05 +/- 0.11 m x s(-1), the vertical lift of the trunk (43 +/- 14 mm) induced a power of 46.0 +/- 20.4 W. The average speed variation per step (0.15 +/- 0.03 m x s(-1)) produced a kinetic power of 16.9 +/- 7.2 W. As compared with commonly admitted values, the energy exchange (recovery) between the two energy components was low (39.1 +/- 10.0%), which induced an overestimated mechanical power (38.9 +/- 18.3 W or 0.60 W x kg(-1) body mass) and a high net mechanical efficiency (26.9 +/- 5.8%). CONCLUSION: We assumed that the cause of the overestimation was an unwanted oscillation of the GPS antenna. It is concluded that GPS (in phase mode) is now able to record small body movements during human locomotion, and constitutes a promising tool for gait analysis of outdoor unrestrained walking. However, the design of the receiver and the antenna must be adapted to human experiments and a thorough validation study remains to be conducted.

Myelin basic protein content of aggregating rat brain cell cultures treated with cytokines and/or demyelinating antibody: effects of macrophage enrichment.

The demyelinative potential of the cytokines interleukin-1 alpha (IL-1 alpha), interferon-gamma (IFN-gamma), and tumor necrosis factor-alpha (TNF-alpha) has been investigated in myelinating aggregate brain cell cultures. Treatment of myelinated cultures with these cytokines resulted in a reduction in myelin basic protein (MBP) content. This effect was additively increased by anti-myelin/oligodendrocyte glycoprotein (alpha-MOG) in the presence of complement. Qualitative immunocytochemistry demonstrated that peritoneal macrophages, added to the fetal telencephalon cell suspensions at the start of the culture period, successfully integrated into aggregate cultures. Supplementing the macrophage component of the cultures in this fashion resulted in increased accumulation of MBP. The effect of IFN-gamma on MBP content of cultures was not affected by the presence of macrophages in increased numbers.

Diagnostic immunologique du cancer

Two different types of immunological reaction are of assistance in the diagnosis of cancer: The first is detection of a weak immunological response of the patient toward his own tumor cells. Unfortunately the currently available techniques for the demonstration of humoral or cellular immunological reaction against autologous tumor cells are not reproducible enough to be recommended as routine clinical tests. Secondly, it is possible to use antisera, obtained by immunization of animals with human tumor extracts, for the detection of substances released into the blood by the tumor cells. The two major antigens associated with human cancer that can be measured in the blood by very sensitive immunological methods are the alphafetoprotein (AFP) and the carcinoembryonic antigen (CEA). It is very important for the physician to be fully alive to the usefulness and limitations of such tests in order to interpret them correctly. Clinical situations in which the measurement of AFP and CEA can provide useful information are reviewed.

Wound-healing gene family expression differences between fetal and foreskin cells used for bioengineered skin substitutes.

For tissue engineering, several cell types and tissues have been proposed as starting material. Allogenic skin products available for therapeutic usage are mostly developed with cell culture and with foreskin tissue of young individuals. Fetal skin cells offer a valuable solution for effective and safe tissue engineering for wounds due to their rapid growth and simple cell culture. By selecting families of genes that have been reported to be implicated in wound repair and particularly for scarless fetal wound healing including transforming growth factor-beta (TGF-beta) superfamily, extracellular matrix, and nerve/angiogenesis growth factors, we have analyzed differences in their expression between fetal skin and foreskin cells, and the same passages. Of the five TGF-beta superfamily genes analyzed by real-time reverse transcription-polymerase chain reaction, three were found to be significantly different with sixfold up-regulated for TGF-beta2, and 3.8-fold for BMP-6 in fetal cells, whereas GDF-10 was 11.8-fold down-regulated. For nerve growth factors, midkine was 36-fold down-regulated in fetal cells, and pleiotrophin was 4.76-fold up-regulated. We propose that fetal cells present technical and therapeutic advantages compared to foreskin cells for effective cell-based therapy for wound management, and overall differences in gene expression could contribute to the degree of efficiency seen in clinical use with these cells.

M-protein and other intrinsic virulence factors of Streptococcus pyogenes are encoded on an ancient pathogenicity island.

BACKGROUND: The increasing number of completely sequenced bacterial genomes allows comparing their architecture and genetic makeup. Such new information highlights the crucial role of lateral genetic exchanges in bacterial evolution and speciation. RESULTS: Here we analyzed the twelve sequenced genomes of Streptococcus pyogenes by a naïve approach that examines the preferential nucleotide usage along the chromosome, namely the usage of G versus C (GC-skew) and T versus A (TA-skew). The cumulative GC-skew plot presented an inverted V-shape composed of two symmetrical linear segments, where the minimum and maximum corresponded to the origin and terminus of DNA replication. In contrast, the cumulative TA-skew presented a V-shape, which segments were interrupted by several steep slopes regions (SSRs), indicative of a different nucleotide composition bias. Each S. pyogenes genome contained up to nine individual SSRs, encompassing all described strain-specific prophages. In addition, each genome contained a similar unique non-phage SSR, the core of which consisted of 31 highly homologous genes. This core includes the M-protein, other mga-related factors and other virulence genes, totaling ten intrinsic virulence genes. In addition to a high content in virulence-related genes and to a peculiar nucleotide bias, this SSR, which is 47 kb-long in a M1GAS strain, harbors direct repeats and a tRNA gene, suggesting a mobile element. Moreover, its complete absence in a M-protein negative group A Streptococcus natural isolate demonstrates that it could be spontaneously lost, but in vitro deletion experiments indicates that its excision occurred at very low rate. The stability of this SSR, combined to its presence in all sequenced S. pyogenes sequenced genome, suggests that it results from an ancient acquisition. CONCLUSION: Thus, this non-phagic SSR is compatible with a pathogenicity island, acquired before S. pyogenes speciation. Its potential excision might bear relevance for vaccine development, because vaccines targeting M-protein might select for M-protein-negative variants that still carry other virulence determinants.

Remaining rod activity mediates visual behavior in adult Rpe65-/- mice.

Purpose: C57/Bl6, Cpfl1-/- (Cone photoreceptors function loss 1; pure rod function), Gnat1alpha-/- (rod alpha-transducin; pure cone function) and Rpe65-/-;Rho-/- double knock-out mice were studied in order to distinguish the respective contributions of the different photoreceptor (PR) systems that enable light perception and mediate a visual reflex in adult Rpe65-/- mice using a simple behavioural procedure. Methods: Visual function was estimated using a rotating automatized optomotor drum covered with vertical black and white stripes at spatial frequencies of 0.025 to 0.5 cycles per degree (cpd) in both photopic and scotopic conditions. To evaluate the contribution as well as the light intensity threshold of each PR system, we tested the mouse strains with different luminances. Results: Stripe rotation elicits head movements in wild-type (WT) animals in photopic and scotopic conditions depending on the spatial frequency, whereas Cpfl1-/- mice show a reduced activity in the photopic condition and Gnat1alpha-/- mice an almost absent response in the scotopic condition. Interestingly, a robust visual response is obtained with Rpe65-/- knockout mice at 0.075 cpd and 0.1 cpd in the photopic condition. The residual rod function in the Rpe65-/- animals was demonstrated by testing Rpe65-/-;Rho-/- mice that present no response in photopic conditions. Conclusions: The optomotor test is a simple method to estimate the visual function, and to evaluate the respective contributions of rod and cone systems. Using this test, we demonstrate that in Rpe65-/- mice, devoid of functional cones and of detectable 11-cis-retinal protein, rods mimic in part the cone function by mediating vision in photopic conditions.

Iontophoresis of dexamethasone in the treatment of endotoxin-induced-uveitis in rats.

The purpose of this study was to evaluate the efficacy of a Coulomb Controlled Iontophoresis system (CCI) in the local delivery of corticosteroids for the treatment of uveitis. The therapeutic efficacy of Dexamethasone (Dex) administered by CCI was compared to systemic injection and to topical application with the iontophoresis apparatus in the absence of electrical current. The evaluation was done in the treatment of the endotoxin-induced uveitis (EIU) model, and in the effect on TNF gene expression in the iris/ciliary body as well as in the retina and on TNF levels in aqueous humor and vitreous. Dex was administered either at the time of LPS injection or 5 hours later. For iontophoresis, we used a 1 ml reservoir-electrode covering the cornea, the limbus, and the first millimeter of the sclera. The applied electrical current was of 400 microA during four minutes with a total surface charge of 0.4 C cm-2. EIU was evaluated by clinical examination, by counts of intraocular inflammatory cells on histological sections, and by measuring the protein levels in the aqueous humor and in the vitreous. The TNF-alpha gene expression in the iris and ciliary body, and in the retina was evaluated by RT-PCR. The systemic effect of Dex delivered by CCI was evaluated on the level of serum TNF-alpha in EIU. Our results demonstrated that local administration of Dex by CCI inhibited anterior and posterior signs of intraocular inflammation as effectively as systemic administration, with no effect on systemic level of TNF. In the anterior and posterior segments of the eye, the protein exudation. TNF levels and the cellular infiltration were inhibited. The TNF-alpha gene expression was inhibited in the anterior as well as the posterior segment of the eye. No clinical nor histological damage were caused by the CCI apparatus. In conclusion, CCI administration of Dex allows for a therapeutic effect on the posterior as well as the anterior segment of the eye, and may present a viable alternative to systemic administration of glucocorticoids in severe ocular inflammations.

Increased vulnerability to kainic acid-induced epileptic seizures in mice underexpressing the scaffold protein Islet-Brain 1/JIP-1.

Islet-Brain 1, also known as JNK-interacting protein-1 (IB1/JIP-1) is a scaffold protein mainly involved in the regulation of the pro-apoptotic signalling cascade mediated by c-Jun-N-terminal kinase (JNK). IB1/JIP-1 organizes JNK and upstream kinases in a complex that facilitates JNK activation. However, overexpression of IB1/JIP-1 in neurons in vitro has been reported to result in inhibition of JNK activation and protection against cellular stress and apoptosis. The occurrence and the functional significance of stress-induced modulations of IB1/JIP-1 levels in vivo are not known. We investigated the regulation of IB1/JIP-1 in mouse hippocampus after systemic administration of kainic acid (KA), in wild-type mice as well as in mice hemizygous for the gene MAPK8IP1, encoding for IB1/JIP-1. We show here that IB1/JIP-1 is upregulated transiently in the hippocampus of normal mice, reaching a peak 8 h after seizure induction. Heterozygous mutant mice underexpressing IB1/JIP-1 showed a higher vulnerability to the epileptogenic properties of KA, whereas hippocampal IB1/JIP-1 levels remained unchanged after seizure induction. Subsequently, an increasing activation of JNK in the 8 h following seizure induction was observed in IB1/JIP-1 haploinsufficient mice, which also underwent more severe excitotoxic lesions in hippocampal CA3, as assessed histologically 3 days after KA administration. Taken together, these data indicate that IB1/JIP-1 in hippocampus participates in the regulation of the neuronal response to excitotoxic stress in a level-dependent fashion.

Thioredoxin-interacting protein links oxidative stress to inflammasome activation.

The NLRP3 inflammasome has a major role in regulating innate immunity. Deregulated inflammasome activity is associated with several inflammatory diseases, yet little is known about the signaling pathways that lead to its activation. Here we show that NLRP3 interacted with thioredoxin (TRX)-interacting protein (TXNIP), a protein linked to insulin resistance. Inflammasome activators such as uric acid crystals induced the dissociation of TXNIP from thioredoxin in a reactive oxygen species (ROS)-sensitive manner and allowed it to bind NLRP3. TXNIP deficiency impaired activation of the NLRP3 inflammasome and subsequent secretion of interleukin 1beta (IL-1beta). Akin to Txnip(-/-) mice, Nlrp3(-/-) mice showed improved glucose tolerance and insulin sensitivity. The participation of TXNIP in the NLRP3 inflammasome activation may provide a mechanistic link to the observed involvement of IL-1beta in the pathogenesis of type 2 diabetes.

Epithelium-mesenchyme interactions control the activity of peroxisome proliferator-activated receptor beta/delta during hair follicle development.

Hair follicle morphogenesis depends on a delicate balance between cell proliferation and apoptosis, which involves epithelium-mesenchyme interactions. We show that peroxisome proliferator-activated receptor beta/delta (PPARbeta/delta) and Akt1 are highly expressed in follicular keratinocytes throughout hair follicle development. Interestingly, PPARbeta/delta- and Akt1-deficient mice exhibit similar retardation of postnatal hair follicle morphogenesis, particularly at the hair peg stage, revealing a new important function for both factors in the growth of early hair follicles. We demonstrate that a time-regulated activation of the PPARbeta/delta protein in follicular keratinocytes involves the up-regulation of the cyclooxygenase 2 enzyme by a mesenchymal paracrine factor, the hepatocyte growth factor. Subsequent PPARbeta/delta-mediated temporal activation of the antiapoptotic Akt1 pathway in vivo protects keratinocytes from hair pegs against apoptosis, which is required for normal hair follicle development. Together, these results demonstrate that epithelium-mesenchyme interactions in the skin regulate the activity of PPARbeta/delta during hair follicle development via the control of ligand production and provide important new insights into the molecular biology of hair growth.

Thyroid function in severely traumatized patients with or without head injury.

The pattern of thyroid function changes following severe trauma was assessed prospectively in 35 patients during the first 5 days after injury. Patients were divided into 2 groups to evaluate the effect of head injury: group I, patients with severe head injury; group II, patients with multiple injuries without head injury. The results demonstrate a low T3 and low T4 syndrome throughout the study, with decreases in both total and free levels of T3 and T4, normal or increased rT3 levels, and normal TSH levels. The presence of severe head injury was associated with lower levels of TSH and free T3. Mortality was 37%. Survival was associated with higher TSH and T3 levels, but not with higher T4 levels. TSH levels exceeding 1 mU/l on the first day were only observed in survivors. These findings show that a typical low T3 and low T4 syndrome is present after severe trauma in patients with multiple injury as well as with head injury. Primary hypothyroidism can be excluded, pituitary or hypothalamic hypothyroidism is likely in these patients.

Inhibition of fas death signals by FLIPs.

The death receptor Fas is a member of the tumor necrosis factor receptor family; upon interaction with its ligand it efficiently activates caspases and induces apoptosis. Despite abundant Fas surface expression, however, Fas death-signals are frequently interrupted. Many viruses express antiapoptotic proteins, including caspase inhibitors, Bcl-2 homologues and death-effector-domain-containing proteins that are termed FLIPs (FLICE [Fas-associated death-domain-like IL-1beta-converting enzyme]-inhibitory proteins). Cellular homologues of these inhibitors have been identified. Cellular FLIPs structurally resemble caspase-8 except that they lack proteolytic activity. FLIPs are highly expressed in tumor cells, T lymphocytes and healthy, but not injured, myocytes; this suggests a critical role of FLIPs as endogenous modulators of apoptosis.

Triggering of Bcl-2-related pathway is associated with apoptosis of photoreceptors in Rpe65-/- mouse model of Leber's congenital amaurosis.

Mutations in RPE65 protein is characterized by the loss of photoreceptors, although the molecular pathways triggering retinal cell death remain largely unresolved. The role of the Bcl-2 family of proteins in retinal degeneration is still controversial. However, alteration in Bcl-2-related proteins has been observed in several models of retinal injury. In particular, Bax has been suggested to play a crucial role in apoptotic pathways in murine glaucoma model as well as in retinal detachment-associated cell death. We demonstrated that Bcl-2-related signaling pathway is involved in Rpe65-dependent apoptosis of photoreceptors during development of the disease. Pro-apoptotic Bax alpha and beta isoforms were upregulated in diseased retina. This was associated with a progressive reduction of anti-apoptotic Bcl-2, reflecting imbalanced Bcl-2/Bax ratio as the disease progresses. Moreover, specific translocation of Bax beta from cytosol to mitochondria was observed in Rpe65-deficient retina. This correlated with the initiation of photoreceptor cell loss at 4 months of age, and further increased during disease development. Altogether, these data suggest that Bcl-2-apoptotic pathway plays a crucial role in Leber's congenital amaurosis disease. They further highlight a new regulatory mechanism of Bax-dependent apoptosis based on regulated expression and activation of specific isoforms of this protein.

The fusion protein of wild-type canine distemper virus is a major determinant of persistent infection.

The wild-type A75/17 canine distemper virus (CDV) strain induces a persistent infection in the central nervous system but infects cell lines very inefficiently. In contrast, the genetically more distant Onderstepoort CDV vaccine strain (OP-CDV) induces extensive syncytia formation. Here, we investigated the roles of wild-type fusion (F(WT)) and attachment (H(WT)) proteins in Vero cells expressing, or not, the canine SLAM receptor by transfection experiments and by studying recombinants viruses expressing different combinations of wild-type and OP-CDV glycoproteins. We show that low fusogenicity is not due to a defect of the envelope proteins to reach the cell surface and that H(WT) determines persistent infection in a receptor-dependent manner, emphasizing the role of SLAM as a potent enhancer of fusogenicity. However, importantly, F(WT) reduced cell-to-cell fusion independently of the cell surface receptor, thus demonstrating that the fusion protein of the neurovirulent A75/17-CDV strain plays a key role in determining persistent infection.