Phenylbutyrate Is Bacteriostatic against Mycobacterium tuberculosis and Regulates the Macrophage Response to Infection,Synergistically with 25-Hydroxy-Vitamin D?

Adjunctive vitamin D treatment for pulmonary tuberculosis enhances resolution of inflammation but has modest effects on bacterial clearance. Sodium 4-phenylbutyrate (PBA) is in clinical use for a range of conditions and has been shown to synergise with vitamin D metabolites to upregulate cathelicidin antimicrobial peptide (CAMP) expression. We investigated whether clinically attainable plasma concentrations of PBA (0.4-4mM) directly affect Mycobacterium tuberculosis (Mtb) growth and human macrophage and PBMC response to infection. We also tested the ability of PBA to enhance the immunomodulatory actions of the vitamin D metabolite 25(OH)D₃ during infection and synergistically inhibit intracellular Mtb growth. PBA inhibited Mtb growth in broth with an MIC₉₉ of 1mM, which was reduced to 0.25mM by lowering pH. During human macrophage infection, PBA treatment restricted Mtb uptake, phagocytic receptor expression and intracellular growth in a dose-dependent manner. PBA independently regulated CCL chemokine secretion and induced expression of the antimicrobial LTF (lactoferrin), the anti-inflammatory PROC (protein C) and multiple genes within the NLRP3 inflammasome pathway. PBA co-treatment with 25(OH)D₃ synergistically modulated expression of numerous vitamin D-response genes, including CAMP, CYP24A1, CXCL10 and IL-37. This synergistic effect was dependent on MAPK signalling, while the effect of PBA on LTF, PROC and NLRP3 was MAPK-independent. During PBA and 25(OH)D₃ co-treatment of human macrophages, in the absence of exogenous proteinase 3 (PR3) to activate cathelicidin, Mtb growth restriction was dominated by the effect of PBA, while the addition of PR3 enhanced growth restriction by 25(OH)D₃ and PBA co-treatment. This suggests that PBA augments vitamin D–mediated cathelicidin-dependent Mtb growth restriction by human macrophages and independently induces antimicrobial and anti-inflammatory action. Therefore through both host-directed and bacterial-directed mechanisms PBA and vitamin D may prove an effective combinatorial adjunct therapy for tuberculosis to both resolve immunopathology and enhance bacterial clearance.     

Control of Cell Survival and Proliferation by Mammalian Eukaryotic Initiation Factor 4B

Translation initiation plays an important role in cell growth, proliferation, and survival. The translation initiation factor eIF4B (eukaryotic initiation factor 4B) stimulates the RNA helicase activity of eIF4A in unwinding secondary structures in the 5′ untranslated region (5′UTR) of the mRNA in vitro. Here, we studied the effects of eIF4B depletion in cells using RNA interference (RNAi). In agreement with the role of eIF4B in translation initiation, its depletion resulted in inhibition of this step. Selective reduction of translation was observed for mRNAs harboring strong to moderate secondary structures in their 5′UTRs. These mRNAs encode proteins, which function in cell proliferation (Cdc25C, c-myc, and ODC [ornithine decarboxylase]) and survival (Bcl-2 and XIAP [X-linked inhibitor of apoptosis]). Furthermore, eIF4B silencing led to decreased proliferation rates, promoted caspase-dependent apoptosis, and further sensitized cells to camptothecin-induced cell death. These results demonstrate that eIF4B is required for cell proliferation and survival by regulating the translation of proliferative and prosurvival mRNAs.Targeting the translation initiation pathway is emerging as a potential therapy for inhibiting cancer cell growth (35, 38). Ribosome recruitment to the 5′ ends of eukaryotic mRNAs proceeds via translation initiation mechanisms that are dependent either on the 5′ cap structure (m⁷GpppN, where N is any nucleotide) or an internal ribosome entry site (IRES). The majority of translation initiation events in eukaryotes are mediated through cap-dependent translation whereby the 40S ribosomal subunit is recruited to the vicinity of the mRNA 5′ cap structure by the eukaryotic initiation factor 4F (eIF4F) complex. eIF4F is comprised of eIF4E (the cap-binding subunit), eIF4A (an RNA helicase), and eIF4G (a large scaffolding protein for eIF4E, eIF4A, and other initiation factors). Once assembled at the 5′ cap, the 40S ribosomal subunit in association with several initiation factors scans the 5′ untranslated region (5′UTR) of the mRNA until it encounters a start codon in a favorable context, followed by polypeptide synthesis (37).Early in vitro studies have shown that the initiation factor eIF4B acts to potentiate ribosome recruitment to the mRNA (3, 45). eIF4B stimulates translation of both capped and uncapped mRNAs in vitro (1, 36). This function is exerted through stimulation of the helicase activity of eIF4A (43), possibly through direct interactions with eIF4A (44) or with mRNA, the ribosome-associated eIF3, and 18S rRNA (28, 29, 44). Thus, eIF4B is thought to form auxiliary bridges between the mRNA and the 40S ribosomal subunit. Toeprinting studies using mammalian eIF4B underscored its importance in the assembly of the 48S initiation complex, especially on mRNAs harboring secondary structures in the 5′UTRs (11).In vivo studies of eIF4B are limited. Ectopic expression of eIF4B in cultured Drosophila melanogaster cells and in developing eye imaginal discs stimulated cell proliferation (16). Enhanced cell proliferation is most likely mediated by increased translation of a subset of mRNAs, since knockdown of Drosophila eIF4B by RNA interference (RNAi) caused a modest reduction in global translation but compromised the survival of insect cells grown under low serum conditions (16). Studies of eIF4B in mammalian cells yielded contradictory results. Transient overexpression of eIF4B stimulated translation initiation in a phosphorylation-dependent manner in some cells (18, 49) while inhibiting translation in others (30, 31, 41). These differences might be attributed to disparate levels of eIF4B overexpression.To address the physiological role of eIF4B in mRNA translation in the cell, RNAi knockdown of eIF4B was used here. We demonstrate that eIF4B is required for optimal translation. Importantly, the translation of mRNAs bearing structured 5′UTRs, such as the cell cycle regulators Cdc25C, c-myc, and ODC (ornithine decarboxylase), and the antiapoptotic factors Bcl-2 and XIAP (X-linked inhibitor of apoptosis), was reduced as a result of eIF4B silencing by RNAi. Furthermore, eIF4B silencing promoted caspase-dependent apoptosis. Thus, we show that mammalian eIF4B is required for cell proliferation and survival, whereby it acts by regulating the translation of a functionally related subset of mRNAs.     

Urinary levels of 2,3-dinor-6-oxo-PGF1 alpha: a reliable index of the production of PGI2 in the spontaneously hypertensive rat

The urinary levels of 2,3-dinor-6-oxo-PGF1 alpha (PGI2-M), a major metabolite of PGI2, are determined by the balance between the amount of PGI2 synthesized and the extent of its further metabolic oxidation. The purpose of the present study was to determine if the urinary excretion of PGI2-M can be used as a reliable index of the in vivo production of PGI2 in both normal Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR). This involved the exclusion of differences in metabolism between these two strains of rats. In order to do so, we monitored the urinary excretion of PGI2-M during paired intravenous infusions of 6-oxo-PGF1 alpha (the stable product of the spontaneous hydrolysis of PGI2) in conscious, unrestrained SHR and WKY rats aged 12-15 weeks, in doses ranging from 250 to 700 ng. In one experiment, PGI2 was infused instead of 6-oxo-PGF1 alpha. The results of these experiments indicate that SHR and WKY rats are equal with regard to the transformation of 6-oxo-PGF1 alpha and PGI2 into PGI2-M. For both groups, there is a good correlation between the amount of 6-oxo-PGF1 alpha infused and the amount of PGI2-M excreted in urine. These observations confirm the validity of using the urinary levels of 2,3-dinor-6-oxo-PGF1 alpha as an index of PGI2 production in both WKY and SHR. In addition, they support the conclusions drawn from our previous studies, namely that SHR do not produce more PGI2 than WKY rats in vivo, contrary to the situation prevailing in vitro.     

Typology of environmental conditions at the onset of winter phytoplankton blooms in a shallow macrotidal coastal ecosystem, Arcachon Bay (France)

Phytoplankton dynamics were assessed in the macrotidal ecosystemof Arcachon Bay through high-frequency surveys over a 5-yearperiod in order to characterize typology of environmental conditionsat the onset of the productive period. Temporal variations ofhydrological and biological parameters were examined in externaland internal waters of the lagoon, during the winter–springperiods from 1999 to 2003. An additional survey was performedduring winter–spring 2005 in order to study the verticalstructure of the water column. The occurrence of winter phytoplanktonblooms between January and March emerged as a recurrent event.The early onset of the productive period is influenced by thebiological functioning of adjacent Bay of Biscay oceanic waters.It is hypothesized that under a propitious hydrodynamic regime,phytoplankton inocula from the Bay of Biscay enter in the ArcachonBay where cells presumably find favourable conditions for theirfast development. The timing of the onset of those winter bloomsin Arcachon Bay seems to be mainly influenced by the presenceof anticyclonic weather conditions (associated with an increasein incident irradiance) during late winter (i.e. by February),while the water column does not show any particular stabilizationnor stratification liable to facilitate the onset of these blooms.Moreover, these winter blooms dominated by diatoms led to anearly nutrient depletion which could have inevitable consequenceson the structuration of the food web during spring and summer.     

A focused antibody library for selecting scFvs expressed at high levels in the cytoplasm

Background  

Intrabodies are defined as antibody molecules which are ectopically expressed inside the cell. Such intrabodies can be used to visualize or inhibit the targeted antigen in living cells. However, most antibody fragments cannot be used as intrabodies because they do not fold under the reducing conditions of the cell cytosol and nucleus.     

The Wagon Wheel Illusions and models of orientation selection

We present an analysis of the Wagon Wheel Illusions—classic psychophysical phenomena—in the context of a neural network model of orientation selectivity in the visual system. We find that both the continuous Wagon Wheel Illusion (c-WWI) and the standard, stroboscopic Wagon Wheel Illusion (WWI) can be explained by a recurrent model in which the cortex provides both excitatory and inhibitory feedback to a weakly tuned input from the lateral geniculate nucleus. Comparison of data from recent psychophysics experiments with theoretical predictions derived from the network dynamics leads to excellent agreement. Conversely, this agreement confirms the validity of the model and highlights the fact that the Wagon Wheel Illusion can serve as a useful probe of the human striate cortex. We find that the WWI results from phase-locking in the visual system and that a circle map determines the dynamics of the illusion. Furthermore, our results suggest that the c-WWI is a consequence of continuous processing and cannot be used to support claims of discrete processing by the visual system.     

A Functional Whole Blood Assay to Measure Viability of Mycobacteria,using Reporter-Gene Tagged BCG or M.Tb (BCG lux/M.Tb lux)

Functional assays have long played a key role in measuring of immunogenicity of a given vaccine. This is conventionally expressed as serum bactericidal titers. Studies of serum bactericidal titers in response to childhood vaccines have enabled us to develop and validate cut-off levels for protective immune responses and such cut-offs are in routine use. No such assays have been taken forward into the routine assessment of vaccines that induce primarily cell-mediated immunity in the form of effector T cell responses, such as TB vaccines. In the animal model, the performance of a given vaccine candidate is routinely evaluated in standardized bactericidal assays, and all current novel TB-vaccine candidates have been subjected to this step in their evaluation prior to phase 1 human trials. The assessment of immunogenicity and therefore likelihood of protective efficacy of novel anti-TB vaccines should ideally undergo a similar step-wise evaluation in the human models now, including measurements in bactericidal assays.Bactericidal assays in the context of tuberculosis vaccine research are already well established in the animal models, where they are applied to screen potentially promising vaccine candidates. Reduction of bacterial load in various organs functions as the main read-out of immunogenicity. However, no such assays have been incorporated into clinical trials for novel anti-TB vaccines to date.Although there is still uncertainty about the exact mechanisms that lead to killing of mycobacteria inside human macrophages, the interaction of macrophages and T cells with mycobacteria is clearly required. The assay described in this paper represents a novel generation of bactericidal assays that enables studies of such key cellular components with all other cellular and humoral factors present in whole blood without making assumptions about their relative individual contribution. The assay described by our group uses small volumes of whole blood and has already been employed in studies of adults and children in TB-endemic settings. We have shown immunogenicity of the BCG vaccine, increased growth of mycobacteria in HIV-positive patients, as well as the effect of anti-retroviral therapy and Vitamin D on mycobacterial survival in vitro. Here we summarise the methodology, and present our reproducibility data using this relatively simple, low-cost and field-friendly model.

Note: Definitions/Abbreviations

BCG lux = M. bovis BCG, Montreal strain, transformed with shuttle plasmid pSMT1 carrying the luxAB genes from Vibrio harveyi, under the control of the mycobacterial GroEL (hsp60) promoter. CFU = Colony Forming Unit (a measure of mycobacterial viability).Download video file.^{(51M, mov)}     

Major histocompatibility genes in the Lake Tana African large barb species flock: evidence for complete partitioning of class II B, but not class I, genes among different species

The 16 African large barb fish species of Lake Tana inhabit different ecological niches, exploit different food webs and have different temporal and spatial spawning patterns within the lake. This unique fish species flock is thought to be the result of adaptive radiation within the past 5 million years. Previous analyses of major histocompatibility class II B exon 2 sequences in four Lake Tana African large barb species revealed that these sequences are indeed under selection. No sharing of class II B alleles was observed among the four Lake Tana African large barb species. In this study we analysed the class II B exon 2 sequences of seven additional Lake Tana African large barb species and African large barbs from the Blue Nile and its tributaries. In addition, the presence and variability of major histocompatibility complex class I UA exon 3 sequences in six Lake Tana and Blue Nile African large barb species was analysed. Phylogenetic lineages are maintained by purifying or neutral selection on non-peptide binding regions. Class II B intron 1 and exon 2 sequences were not shared among the different Lake Tana African large barb species or with the riverine barb species. In contrast, identical class I UA exon 3 sequences were found both in the lacustrine and riverine barb species. Our analyses demonstrate complete partitioning of class II B alleles among Lake Tana African large barb species. In contrast, class I alleles remain for the large part shared among species. These different modes of evolution probably reflect the unlinked nature of major histocompatibility genes in teleost fishes.Electronic Supplementary Material Supplementary material is available for this article at .An erratum to this article can be found at     

Blockade of PI3Kgamma suppresses joint inflammation and damage in mouse models of rheumatoid arthritis

Phosphoinositide 3-kinases (PI3K) have long been considered promising drug targets for the treatment of inflammatory and autoimmune disorders as well as cancer and cardiovascular diseases. But the lack of specificity, isoform selectivity and poor biopharmaceutical profile of PI3K inhibitors have so far hampered rigorous disease-relevant target validation. Here we describe the identification and development of specific, selective and orally active small-molecule inhibitors of PI3Kgamma (encoded by Pik3cg). We show that Pik3cg(-/-) mice are largely protected in mouse models of rheumatoid arthritis; this protection correlates with defective neutrophil migration, further validating PI3Kgamma as a therapeutic target. We also describe that oral treatment with a PI3Kgamma inhibitor suppresses the progression of joint inflammation and damage in two distinct mouse models of rheumatoid arthritis, reproducing the protective effects shown by Pik3cg(-/-) mice. Our results identify selective PI3Kgamma inhibitors as potential therapeutic molecules for the treatment of chronic inflammatory disorders such as rheumatoid arthritis.