Apolipoprotein E controls Dectin-1-dependent development of monocyte-derived alveolar macrophages upon pulmonary ß-glucan-induced inflammatory adaptation.

The lung is constantly exposed to the outside world and optimal adaptation of immune responses is crucial for efficient pathogen clearance. However, mechanisms that lead to lung-associated macrophages' functional and developmental adaptation remain elusive. To reveal such mechanisms, we developed a reductionist model of environmental intranasal ß-glucan exposure, allowing for the detailed interrogation of molecular mechanisms of pulmonary macrophage adaptation. Employing single-cell transcriptomics, high-dimensional imaging and flow cytometric characterization paired with in vivo and ex vivo challenge models, we reveal that pulmonary low-grade inflammation results in the development of apolipoprotein E (ApoE)-dependent monocyte-derived alveolar macrophages (ApoE+CD11b+ AMs). ApoE+CD11b+ AMs expressed high levels of CD11b, ApoE, Gpnmb and Ccl6, were glycolytic, highly phagocytic and produced large amounts of interleukin-6 upon restimulation. Functional differences were cell intrinsic, and myeloid cell-specific ApoE ablation inhibited Ly6c+ monocyte to ApoE+CD11b+ AM differentiation dependent on macrophage colony-stimulating factor secretion, promoting ApoE+CD11b+ AM cell death and thus impeding ApoE+CD11b+ AM maintenance. In vivo, ß-glucan-elicited ApoE+CD11b+ AMs limited the bacterial burden of Legionella pneumophilia after infection and improved the disease outcome in vivo and ex vivo in a murine lung fibrosis model. Collectively these data identify ApoE+CD11b+ AMs generated upon environmental cues, under the control of ApoE signaling, as an essential determinant for lung adaptation enhancing tissue resilience.

The estimated burden of fungal disease in South Africa

Background. With a population of 56.5 million, over 7 million persons living with HIV, one of the world's highest rates of tuberculosis (TB) and a large proportion of the population living in poverty, South Africa (SA)'s fungal disease burden is probably substantial and broad in scope.Objectives. To estimate the burden of fungal disease in SA.Methods. Using total and at-risk populations and national, regional and occasionally global data, we estimated the incidence and prevalence of the majority of fungal diseases in SA.Results. Estimates for the annual incidence of HIV-related life-threatening fungal disease include cryptococcal meningitis (8 357 cases), Pneumocystis pneumonia (4 452 cases) and endemic mycoses (emergomycosis, histoplasmosis and blastomycosis, with 100, 60 and 10 cases per year, respectively). We estimate 3 885 cases of invasive aspergillosis annually. The annual burden of candidaemia and Candida peritonitis is estimated at 5 421 and 1 901 cases, respectively. The epidemic of pulmonary TB has probably driven up the prevalence of chronic pulmonary aspergillosis to 99 351 (175.8/100 000), perhaps the highest in the world. Fungal asthma probably affects >100 000 adults. Mucosal candidiasis is common, with an annual prevalence estimated at 828 666 and 135 289 oral and oesophageal cases, respectively, complicating HIV infection alone (estimates in other conditions not made), and over a million women are estimated to be affected by recurrent vulvovaginal candidiasis each year. Tinea capitis in children is common and conservatively estimated at >1 000 000 cases. The inoculation mycoses sporotrichosis, chromoblastomycosis and eumycetoma occur occasionally (with 40, 40 and 10 cases estimated, respectively). Overall, we estimate that over 3.2 million South Africans are afflicted by a fungal disease each year (7.1% of the population).Conclusions. Significant numbers of South Africans are estimated to be affected each year by fungal infections, driven primarily by the syndemics of HIV, TB and poverty. These estimates emphasise the need for better epidemiological data, and for improving the diagnosis and management of these diseases

Fungal-derived cues promote ocular autoimmunity through a Dectin-2/Card9-mediated mechanism.

Uveitis (intraocular inflammation) is a leading cause of loss of vision. Although its aetiology is largely speculative, it is thought to arise from complex genetic-environmental interactions that break immune tolerance to generate eye-specific autoreactive T cells. Experimental autoimmune uveitis (EAU), induced by immunization with the ocular antigen, interphotoreceptor retinoid binding protein (IRBP), in combination with mycobacteria-containing complete Freund's adjuvant (CFA), has many clinical and histopathological features of human posterior uveitis. Studies in EAU have focused on defining pathogenic CD4+ T cell effector responses, such as those of T helper type 17 (Th17) cells, but the innate receptor pathways precipitating development of autoreactive, eye-specific T cells remain poorly defined. In this study, we found that fungal-derived antigens possess autoimmune uveitis-promoting function akin to CFA in conventional EAU. The capacity of commensal fungi such as Candida albicans or Saccharomyces cerevisae to promote IRBP-triggered EAU was mediated by Card9. Because Card9 is an essential signalling molecule of a subgroup of C-type lectin receptors (CLRs) important in host defence, we evaluated further the proximal Card9-activating CLRs. Using single receptor-deficient mice we identified Dectin-2, but not Mincle or Dectin-1, as a predominant mediator of fungal-promoted uveitis. Conversely, Dectin-2 activation by α-mannan reproduced the uveitic phenotype of EAU sufficiently, in a process mediated by the Card9-coupled signalling axis and interleukin (IL)-17 production. Taken together, this report relates the potential of the Dectin-2/Card9-coupled pathway in ocular autoimmunity. Not only does it contribute to understanding of how innate immune receptors orchestrate T cell-mediated autoimmunity, it also reveals a previously unappreciated ability of fungal-derived signals to promote autoimmunity.

Fungal Recognition and Host Defense Mechanisms.

Fungi have emerged as premier opportunistic microbes of the 21st century, having a considerable impact on human morbidity and mortality. The huge increase in incidence of these diseases is largely due to the HIV pandemic and use of immunosuppressive therapies, underscoring the importance of the immune system in defense against fungi. This article will address how the mammalian immune system recognizes and mounts a defense against medically relevant fungal species.

CD4(+) T-cell survival in the GI tract requires dectin-1 during fungal infection.

Dectin-1 is an innate antifungal C-type lectin receptor necessary for protective antifungal immunity. We recently discovered that Dectin-1 is involved in controlling fungal infections of the gastrointestinal (GI) tract, but how this C-type lectin receptor mediates these activities is unknown. Here, we show that Dectin-1 is essential for driving fungal-specific CD4(+) T-cell responses in the GI tract. Loss of Dectin-1 resulted in abrogated dendritic cell responses in the mesenteric lymph nodes (mLNs) and defective T-cell co-stimulation, causing substantial increases in CD4(+) T-cell apoptosis and reductions in the cellularity of GI-associated lymphoid tissues. CD8(+) T-cell responses were unaffected by Dectin-1 deficiency. These functions of Dectin-1 have significant implications for our understanding of intestinal immunity and susceptibility to fungal infections.

Genomic interaction between ER and HMGB2 identifies DDX18 as a novel driver of endocrine resistance in breast cancer cells.

Breast cancer resistance to endocrine therapies such as tamoxifen and aromatase inhibitors is a significant clinical problem. Steroid receptor coactivator-1 (SRC-1), a coregulatory protein of the oestrogen receptor (ER), has previously been shown to have a significant role in the progression of breast cancer. The chromatin protein high mobility group box 2 (HMGB2) was identified as an SRC-1 interacting protein in the endocrine-resistant setting. We investigated the expression of HMGB2 in a cohort of 1068 breast cancer patients and found an association with increased disease-free survival time in patients treated with endocrine therapy. However, it was also verified that HMGB2 expression could be switched on in endocrine-resistant tumours from breast cancer patients. To explore the function of this poorly characterized protein, we performed HMGB2 ChIPseq and found distinct binding patterns between the two contexts. In the resistant setting, the HMGB2, SRC-1 and ER complex are enriched at promoter regions of target genes, with bioinformatic analysis indicating a switch in binding partners between the sensitive and resistant phenotypes. Integration of binding and gene expression data reveals a concise set of target genes of this complex including the RNA helicase DDX18. Modulation of DDX18 directly affects growth of tamoxifen-resistant cells, suggesting that it may be a critical downstream effector of the HMGB2:ER complex. This study defines HMGB2 interactions with the ER complex at specific target genes in the tamoxifen-resistant setting.

Intravenous administration of human embryonic stem cell-derived neural precursor cells attenuates cuprizone-induced central nervous system (CNS) demyelination.

AIMS: Previous studies have demonstrated the therapeutic potential for human embryonic stem cell-derived neural precursor cells (hES-NPCs) in autoimmune and genetic animal models of demyelinating diseases. Herein, we tested whether intravenous (i.v.) administration of hES-NPCs would impact central nervous system (CNS) demyelination in a cuprizone model of demyelination. METHODS: C57Bl/6 mice were fed cuprizone (0.2%) for 2 weeks and then separated into two groups that either received an i.v. injection of hES-NPCs or i.v. administration of media without these cells. After an additional 2 weeks of dietary cuprizone treatment, CNS tissues were analysed for detection of transplanted cells and differences in myelination in the region of the corpus callosum (CC). RESULTS: Cuprizone-induced demyelination in the CC was significantly reduced in mice treated with hES-NPCs compared with cuprizone-treated controls that did not receive stem cells. hES-NPCs were identified within the brain tissues of treated mice and revealed migration of transplanted cells into the CNS. A limited number of human cells were found to express the mature oligodendrocyte marker, O1, or the astrocyte marker, glial fibrillary acidic protein. Reduced apoptosis and attenuated microglial and astrocytic responses were also observed in the CC of hES-NPC-treated mice. CONCLUSIONS: These findings indicated that systemically administered hES-NPCs migrated from circulation into a demyelinated lesion within the CNS and effectively reduced demyelination. Observed reductions in astrocyte and microglial responses, and the benefit of hES-NPC treatment in this model of myelin injury was not obviously accountable to tissue replacement by exogenously administered cells.

Self-assembly of a designed amyloid peptide containing the functional thienylalanine unit.

The self-assembly of a peptide based on a sequence from the amyloid beta peptide but incorporating the non-natural amino acid beta-2-thienylalanine (2-Thi) has been investigated in aqueous and methanol solutions. The peptide AAKLVFF was used as a design motif, replacing the phenylalanine residues (F) with 2-Thi units to yield (2-Thi)(2-Thi)VLKAA. The 2-Thi residues are expected to confer interesting electronic properties due to charge delocalization and pi-stacking. The peptide is shown to form beta-sheet-rich amyloid fibrils with a twisted morphology, in both water and methanol solutions at sufficiently high concentration. The formation of a self-assembling hydrogel is observed at high concentration. Detailed molecular modeling using molecular dynamics methods was performed using NOE constraints provided by 2D-NMR experiments. The conformational and charge properties of 2-Thi were modeled using quantum mechanical methods, and found to be similar to those previously reported for the beta-3-thienylalanine analogue. The molecular dynamics simulations reveal well-defined folded structures (turn-like) in dilute aqueous solution, driven by self-assembly of the hydrophobic aromatic units, with charged lysine groups exposed to water.

Influence of the solvent on the self-assembly of a modified amyloid beta peptide fragment. II. NMR and computer simulation investigation.

The conformation of a model peptide AAKLVFF based on a fragment of the amyloid beta peptide Abeta16-20, KLVFF, is investigated in methanol and water via solution NMR experiments and molecular dynamics computer simulations. In previous work, we have shown that AAKLVFF forms peptide nanotubes in methanol and twisted fibrils in water. Chemical shift measurements were used to investigate the solubility of the peptide as a function of concentration in methanol and water. This enabled the determination of critical aggregation concentrations. The solubility was lower in water. In dilute solution, diffusion coefficients revealed the presence of intermediate aggregates in concentrated solution, coexisting with NMR-silent larger aggregates, presumed to be beta-sheets. In water, diffusion coefficients did not change appreciably with concentration, indicating the presence mainly of monomers, coexisting with larger aggregates in more concentrated solution. Concentration-dependent chemical shift measurements indicated a folded conformation for the monomers/intermediate aggregates in dilute methanol, with unfolding at higher concentration. In water, an antiparallel arrangement of strands was indicated by certain ROESY peak correlations. The temperature-dependent solubility of AAKLVFF in methanol was well described by a van't Hoff analysis, providing a solubilization enthalpy and entropy. This pointed to the importance of solvophobic interactions in the self-assembly process. Molecular dynamics simulations constrained by NOE values from NMR suggested disordered reverse turn structures for the monomer, with an antiparallel twisted conformation for dimers. To model the beta-sheet structures formed at higher concentration, possible model arrangements of strands into beta-sheets with parallel and antiparallel configurations and different stacking sequences were used as the basis for MD simulations; two particular arrangements of antiparallel beta-sheets were found to be stable, one being linear and twisted and the other twisted in two directions. These structures were used to simulate circular dichroism spectra. The roles of aromatic stacking interactions and charge transfer effects were also examined. Simulated spectra were found to be similar to those observed experimentally (in water or methanol) which show a maximum at 215 or 218 nm due to pi-pi* interactions, when allowance is made for a 15-18 nm red-shift that may be due to light scattering effects.

Pathogens in Crassostrea ariakensis and other Asian oyster species: implications for non-native oyster introduction to Chesapeake Bay.

With the drastic decline of eastern oyster Crassostrea virginica populations in the Chesapeake Bay due to over-fishing, diseases and habitat destruction, there is interest in Maryland and Virginia in utilizing the non-native oyster species Crassostrea ariakensis for aquaculture, fishery resource enhancement, and ecological restoration. The International Council for the Exploration of the Sea (ICES) recommends that non-native species be examined for ecological, genetic and disease relationships in the native range prior to a deliberate introduction to a new region. Therefore, a pathogen survey of C. ariakensis and other sympatric oyster species was conducted on samples collected in the PR China, Japan and Korea using molecular diagnostics and histopathology. Molecular assays focused on 2 types of pathogens: protistan parasites in the genus Perkinsus and herpesviruses, both with known impacts on commercially important molluscan species around the world, including Asia. PCR amplification and DNA sequence data from the internal transcribed spacer region of the rRNA gene complex revealed the presence of 2 Perkinsus species not currently found in USA waters: P. olseni and an undescribed species. In addition, 3 genetic strains of molluscan herpesviruses were detected in oysters from several potential C. ariakensis broodstock acquisition sites in Asia. Viral gametocytic hypertrophy, Chlamydia-like organisms, a Steinhausia-like microsporidian, Perkinsus sp., Nematopsis sp., ciliates, and cestodes were also detected by histopathology.

Early-onset sepsis with Staphylococcus auricularis in an extremely low-birth weight infant - an uncommon pathogen.

Coagulase-negative staphylococci (CoNS) are often dismissed as a contaminant of blood cultures and are rarely considered as an etiology of perinatally acquired infections. We describe a case of early-onset sepsis with Staphylococcus auricularis in an extremely low-birth weight infant.

Episodic temporal generalization: a developmental study.

Groups of 5-year-olds, 10-year-olds, and adults completed either an episodic temporal generalization task, in which no stimuli were repeated, or a repeated standard temporal generalization task, in which there was a fixed standard that was repeated on every trial. Significant developmental improvements were found on both tasks. In both tasks, gradients of performance over two different stimulus ranges superimposed well when plotted on the same relative scale. Performance was similar for the adults and 10-year-olds across tasks, but the 5-year-olds performed better on the repeated standard task. These findings suggest that perceptual processes are a source of scalar variability in timing, and that there are developmental changes in levels of such variability.

Macrophage receptors and immune recognition.

Macrophages express a broad range of plasma membrane receptors that mediate their interactions with natural and altered-self components of the host as well as a range of microorganisms. Recognition is followed by surface changes, uptake, signaling, and altered gene expression, contributing to homeostasis, host defense, innate effector mechanisms, and the induction of acquired immunity. This review covers recent studies of selected families of structurally defined molecules, studies that have improved understanding of ligand discrimination in the absence of opsonins and differential responses by macrophages and related myeloid cells.

PTX3 function as an opsonin for the dectin-1-dependent internalization of zymosan by macrophages.

Pentraxin 3 (PTX3) is a tumor necrosis factor and interleukin-1beta-stimulated gene that encodes a long PTX with proinflammatory activity. Here, we show that peritoneal macrophages derived from PTX3 transgenic (Tg) mice express higher levels of PTX3 mRNA than macrophages from wild-type (WT) mice, at basal level as well as upon stimulation with zymosan (Zy). Macrophages from Tg mice also showed improved opsonin-independent phagocytosis of Zy particles and the yeast form of the fungus Paracoccidioides brasiliensis. In the case of P. brasiliensis, an enhanced microbicidal activity accompanied by higher production of nitric oxide was also observed in macrophages from Tg mice. Using fluorescein-activated cell sorter analysis and reverse transcriptase-polymerase chain reaction, we demonstrated that basal level of Toll-like receptor-6 and Zy-induced dectin-1 expression was slightly but consistently higher in macrophages from Tg mice than in macrophages from WT mice. Recombinant (r)PTX3 protein binds to Zy particles as well as to yeast cells of P. brasiliensis and addition of rPTX3, to a culture of WT-derived macrophages containing Zy leads to an increase in the phagocytic index, which parallels that of Tg-derived macrophages, demonstrating the opsonin-like activity of PTX3. It is important that blockade of dectin-1 receptor inhibited the phagocytosis of Zy particles by WT and PTX3 Tg macrophages, pointing out the relevant role of dectin-1 as the main receptor involved in Zy uptake. Our results provide evidence for a role of PTX3 as an important component of the innate-immune response and as part of the host mechanisms that control fungal recognition and phagocytosis.

Duration judgements in patients with schizophrenia.

BACKGROUND: The ability to encode time cues underlies many cognitive processes. In the light of schizophrenic patients' compromised cognitive abilities in a variety of domains, it is noteworthy that there are numerous reports of these patients displaying impaired timing abilities. However, the timing intervals that patients have been evaluated on in prior studies vary considerably in magnitude (e.g. 1 s, 1 min, 1 h etc.). METHOD: In order to obviate differences in abilities in chronometric counting and place minimal demands on cognitive processing, we chose tasks that involve making judgements about brief durations of time (< 1 s). RESULTS: On a temporal generalization task, patients were less accurate than controls at recognizing a standard duration. The performance of patients was also significantly different from controls on a temporal bisection task, in which participants categorized durations as short or long. Although time estimation may be closely intertwined with working memory, patients' working memory as measured by the digit span task did not correlate significantly with their performance on the duration judgement tasks. Moreover, lowered intelligence scores could not completely account for the findings. CONCLUSIONS: We take these results to suggest that patients with schizophrenia are less accurate at estimating brief time periods. These deficits may reflect dysfunction of biopsychological timing processes.

Deoxyarteannuin B, dihydro-deoxyarteannuin B and trans-5-hydroxy-2-isopropenyl-5-methylhex-3-en-1-ol from Artemisia anuua.

The amorphane sesquiterpenes, deoxyarteannuin B and dihydro-deoxyarteannuin B, were isolated from Artemisia annua and their structures mainly determined by two-dimensional NMR spectoscopic analyses. The irregular monoterpene, trans-5-hydroxy-2-isopropenyl-5-methylhex-3-en-1-ol, was also characterized in the same way, and its structure was confirmed by synthesis from lavandulol. All of these natural products are suggested to be formed by autoxidation reactions. Full assignments of the 1H and 13C resonances for the known natural products epi-deoxyarteannuin B and isoannulide, determined by the same methodology, are also reported.

Two routes to [11C-carbonyl]organo-isocyanates utilizing [11C]phosgene ([11C]organo-isocyanates from [11C]phosgene).

Two generic radiosynthetic routes for the preparation of [11C-carbonyl]isocyanates have been developed. Reaction of N-organo-sulfinylamines; RNSO, (R = Me, Et, allyl, cyclohexyl and phenyl) with [11C]phosgene gave the corresponding [11C-carbonyl]isocyanates in good radiochemical yield (53-68%) from [11C]phosgene (decay corrected) in ca 16 min from EOB. Alternatively, the reaction of [11C]phosgene with N,N'-organo-ureas; (RNH)(2)CO, (R = Me, Et, Pr and phenyl) also gave the corresponding [11C-carbonyl]isocyanates in moderate radiochemical yield (9-37%) from [11C]phosgene (decay corrected) in ca 16 min from EOB. For identification, the [11C-carbonyl]organo-isocyanates were derivatized with 1-(2-methoxyphenyl)piperazine in situ to [11C-carbonyl]carboxamides and the position of radiolabelling in the carbonyl group confirmed by [11/13C]co-labeling and subsequent carbon-13 NMR spectroscopy.

The ESAT-6 gene cluster of Mycobacterium tuberculosis and other high G+C Gram-positive bacteria.

BACKGROUND: The genome of Mycobacterium tuberculosis H37Rv has five copies of a cluster of genes known as the ESAT-6 loci. These clusters contain members of the CFP-10 (lhp) and ESAT-6 (esat-6) gene families (encoding secreted T-cell antigens that lack detectable secretion signals) as well as genes encoding secreted, cell-wall-associated subtilisin-like serine proteases, putative ABC transporters, ATP-binding proteins and other membrane-associated proteins. These membrane-associated and energy-providing proteins may function to secrete members of the ESAT-6 and CFP-10 protein families, and the proteases may be involved in processing the secreted peptide. RESULTS: Finished and unfinished genome sequencing data of 98 publicly available microbial genomes has been analyzed for the presence of orthologs of the ESAT-6 loci. The multiple duplicates of the ESAT-6 gene cluster found in the genome of M. tuberculosis H37Rv are also conserved in the genomes of other mycobacteria, for example M. tuberculosis CDC1551, M. tuberculosis 210, M. bovis, M. leprae, M. avium, and the avirulent strain M. smegmatis. Phylogenetic analyses of the resulting sequences have established the duplication order of the gene clusters and demonstrated that the gene cluster known as region 4 (Rv3444c-3450c) is ancestral. Region 4 is also the only region for which an ortholog could be found in the genomes of Corynebacterium diphtheriae and Streptomyces coelicolor. CONCLUSIONS: Comparative genomic analysis revealed that the presence of the ESAT-6 gene cluster is a feature of some high-G+C Gram-positive bacteria. Multiple duplications of this cluster have occurred and are maintained only within the genomes of members of the genus Mycobacterium.

Characterization of the human beta -glucan receptor and its alternatively spliced isoforms.

beta-1,3-d-Glucans are biological response modifiers with potent effects on the immune system. A number of receptors are thought to play a role in mediating these responses, including murine Dectin-1, which we recently identified as a beta-glucan receptor. In this study we describe the characterization of the human homologue of this receptor and show that it is structurally and functionally similar to the mouse receptor. The human beta-glucan receptor is a type II transmembrane receptor with a single extracellular carbohydrate recognition domain and an immunoreceptor tyrosine activation motif in its cytoplasmic tail. The human beta-glucan receptor is widely expressed and functions as a pattern recognition receptor, recognizing a variety of beta-1,3- and/or beta-1,6-linked glucans as well as intact yeast. In contrast to the murine receptor, the human receptor mRNA is alternatively spliced, resulting in two major (A and B) and six minor isoforms. The two major isoforms differ by the presence of a stalk region separating the carbohydrate recognition domain from the transmembrane region and are the only isoforms that are functional for beta-glucan binding. The human receptor also binds T-lymphocytes at a site distinct from the beta-glucan binding site, indicating that this receptor can recognize both endogenous and exogenous ligands.