The lncRNA LUCAT1 is elevated in inflammatory disease and restrains inflammation by regulating the splicing and stability of NR4A2.

The nuclear long non-coding RNA LUCAT1 has previously been identified as a negative feedback regulator of type I interferon and inflammatory cytokine expression in human myeloid cells. Here, we define the mechanistic basis for the suppression of inflammatory gene expression by LUCAT1. Using comprehensive identification of RNA-binding proteins by mass spectrometry as well as RNA immunoprecipitation, we identified proteins important in processing and alternative splicing of mRNAs as LUCAT1-binding proteins. These included heterogeneous nuclear ribonucleoprotein C, M, and A2B1. Consistent with this finding, cells lacking LUCAT1 have altered splicing of selected immune genes. In particular, upon lipopolysaccharide stimulation, the splicing of the nuclear receptor 4A2 (NR4A2) gene was particularly affected. As a consequence, expression of NR4A2 was reduced and delayed in cells lacking LUCAT1. NR4A2-deficient cells had elevated expression of immune genes. These observations suggest that LUCAT1 is induced to control the splicing and stability of NR4A2, which is in part responsible for the anti-inflammatory effect of LUCAT1. Furthermore, we analyzed a large cohort of patients with inflammatory bowel disease as well as asthma and chronic obstructive pulmonary disease. In these patients, LUCAT1 levels were elevated and in both diseases, positively correlated with disease severity. Collectively, these studies define a key molecular mechanism of LUCAT1-dependent immune regulation through post-transcriptional regulation of mRNAs highlighting its role in the regulation of inflammatory disease.

Investigating pyroptosis as a mechanism of L. major cell-to-cell spread in the human BLaER1 infection model.

Leishmania is the causative agent of the tropical neglected disease leishmaniasis and infects macrophages as its definitive host cell. In order to sustain and propagate infections, Leishmania parasites have to complete cycles of exit and re-infection. Yet, the mechanism driving the parasite spread to other cells remains unclear. Recent studies reported pro-inflammatory monocytes as replicative niche of Leishmania major and showed prolonged expression of IL-1ß at the site of infection, indicating an activation of the NLRP3 inflammasome and pointing toward pyroptosis as a possible mechanism of parasite spread. To address the species-specific inflammasome activation of human cells, we characterized the BLaER1 monocytes as a model for L. major infection. We found that BLaER1 monocytes support infection and activation by Leishmania parasites to the same extent as primary human macrophages. Harnessing the possibilities of this infection model, we first showed that BLaER1 GSDMD-/- cells, which carry a deletion of the pore-forming protein gasdermin D, are more resistant to pyroptotic cell death and, concomitantly, display a strongly delayed release of intracellular parasite. Using that knockout in a co-incubation assay in comparison with wild-type BLaER1 cells, we demonstrate that impairment of the pyroptosis pathway leads to lower rates of parasite spread to new host cells, thus, implicating pyroptotic cell death as a possible exit mechanism of L. major in pro-inflammatory microenvironments.

Nonreducing Sugar Scaffold Enables the Development of Immunomodulatory TLR4-specific LPS Mimetics with Picomolar Potency.

Innate immune defense mechanisms against infection and cancer encompass the modulation of pattern recognition receptor (PRR)-mediated inflammation, including upregulation of various transcription factors and the activation of pro-inflammatory pathways important for immune surveillance. Dysfunction of PRRs-mediated signaling has been implicated in cancer and autoimmune diseases, while the overactivation of PRRs-driven responses during infection can lead to devastating consequences such as acute lung injury or sepsis. We used crystal structure-based design to develop immunomodulatory lipopolysaccharide (LPS) mimetics targeting one of the ubiquitous PRRs, toll-like receptor 4 (TLR4). Taking advantage of an exo-anomeric conformation and specific molecular shape of synthetic nonreducing ß,ß-diglucosamine, which was investigated by NMR, we developed two sets of Lipid A mimicking glycolipids capable of either potently activating innate immune responses or inhibiting pro-inflammatory signaling. Stereoselective 1,1'-glycosylation towards fully orthogonally protected nonreducing GlcNß(1↔1')ßGlcN followed by stepwise assembly of differently functionalised phosphorylated glycolipids provided biologically active molecules that were evaluated for their ability to trigger or to inhibit cellular innate immune responses. Two LPS mimetics, identified as potent TLR4-specific inducers of the intracellular signaling pathways, serve as vaccine adjuvant- and immunotherapy candidates, while anionic glycolipids with TLR4-inhibitory potential hold therapeutic promise for the management of acute or chronic inflammation.

Exploring Species-Specificity in TLR4/MD-2 Inhibition with Amphiphilic Lipid A Mimicking Glycolipids.

The Toll-like receptor 4 (TLR4)/myeloid differentiation factor 2 (MD-2) complex is a key receptor of the innate immune system and a major driver of inflammation that is responsible for the multifaceted defense response to Gram-negative infections. However, dysfunction in the tightly regulated mechanisms of TLR4-mediated signaling leads to the uncontrolled upregulation of local and systemic inflammation, often resulting in acute or chronic disease. Therefore, the TLR4/MD-2 receptor complex is an attractive target for the design and development of anti-inflammatory therapies which aim to control the unrestrained activation of TLR4-mediated signaling. Complex structure-activity relationships and species-specificity behind ligand recognition by the TLR4/MD-2 complex complicate the development of MD-2-specific TLR4 antagonists. The restriction of the conformational flexibility of the disaccharide polar head group is one of the key structural features of the newly developed lipid A-mimicking glycophospholipids, which are potential inhibitors of TLR4-mediated inflammation. Since phosphorylation has a crucial influence on MD-2-ligand interaction, glycolipids with variable numbers and positioning of phosphate groups were synthesized and evaluated for their ability to inhibit TLR4-mediated pro-inflammatory signaling in human and murine immune cells. A bis-phosphorylated glycolipid was found to have nanomolar antagonist activity on human TLR4 while acting as a partial agonist on murine TLR4. The glycolipid inhibited mTLR4/MD-2-mediated cytokine release, acting as an antagonist in the presence of lipopolysaccharide (LPS), but at the same time induced low-level cytokine production.

Lipid A Mimetics Based on Unnatural Disaccharide Scaffold as Potent TLR4 Agonists for Prospective Immunotherapeutics and Adjuvants.

TLR4 is a key pattern recognition receptor that can sense pathogen- and danger- associated molecular patterns to activate the downstream signaling pathways which results in the upregulation of transcription factors and expression of interferons and cytokines to mediate protective pro-inflammatory responses involved in immune defense. Bacterial lipid A is the primary TLR4 ligand with very complex, species-specific, and barely predictable structure-activity relationships. Given that therapeutic targeting of TLR4 is an emerging tool for management of a variety of human diseases, the development of novel TLR4 activating biomolecules other than lipid A is of vast importance. We report on design, chemical synthesis and immunobiology of novel glycan-based lipid A-mimicking molecules that can activate human and murine TLR4-mediated signaling with picomolar affinity. Exploiting crystal structure - based design we have created novel disaccharide lipid A mimetics (DLAMs) where the inherently flexible ß(1→6)-linked diglucosamine backbone of lipid A is exchanged with a conformationally restrained non-reducing ßGlcN(1↔1')ßGlcN scaffold. Excellent stereoselectivity in a challenging ß,ß-1,1' glycosylation was achieved by tuning the reactivities of donor and acceptor molecules using protective group manipulation strategy. Divergent streamlined synthesis of ß,ß-1,1'-linked diglucosamine-derived glycolipids entailing multiple long-chain (R)-3- acyloxyacyl residues and up two three phosphate groups was developed. Specific 3D-molecular shape and conformational rigidity of unnatural ß,ß-1,1'-linked diglucosamine combined with carefully optimized phosphorylation and acylation pattern ensured efficient induction of the TLR4-mediated signaling in a species-independent manner.

2'-O-methylation within prokaryotic and eukaryotic tRNA inhibits innate immune activation by endosomal Toll-like receptors but does not affect recognition of whole organisms.

Bacterial RNA has emerged as an important activator of innate immune responses by stimulating Toll-like receptors TLR7 and TLR8 in humans. Guanosine 2'-O-methylation at position 18 (Gm18) in bacterial tRNA was shown to antagonize tRNA-induced TLR7/8 activation, suggesting a potential role of Gm18 as an immune escape mechanism. This modification also occurs in eukaryotic tRNA, yet a physiological immune function remained to be tested. We therefore set out to investigate the immune modulatory role of Gm18 in both prokaryotic and eukaryotic microorganisms, Escherichia coli and Saccharomyces cerevisiae, and in human cells. Using RiboMethSeq analysis we show that mutation of trmH in E. coli, trm3 in S. cereviase, and CRISPR/Cas9-induced knockout of TARBP1 in H. sapiens results in loss of Gm18 within tRNA. Lack of Gm18 across the kingdoms resulted in increased immunostimulation of peripheral blood mononuclear cells when activated by tRNA preparations. In E. coli, lack of 2'-O-methyltransferase trmH also enhanced immune stimulatory properties by whole cellular RNA. In contrast, lack of Gm18 in yeasts and human cells did not affect immunostimulation by whole RNA preparations. When using live E. coli bacteria, lack of trmH did not affect overall immune stimulation although we detected a defined TLR8/RNA-dependent gene expression signature upon E. coli infection. Together, these results demonstrate that Gm18 is a global immune inhibitory tRNA modification across the kingdoms and contributes to tRNA recognition by innate immune cells, but as an individual modification has insufficient potency to modulate recognition of the investigated microorganisms.

The Lipid A from the Lipopolysaccharide of the Phototrophic Bacterium Rhodomicrobium vannielii ATCC 17100 Revisited.

The structure of lipid A from lipopolysaccharide (LPS) of Rhodomicrobium vannielii ATCC 17100 (Rv) a phototrophic, budding bacterium was re-investigated using high-resolution mass spectrometry, NMR, and chemical degradation protocols. It was found that the (GlcpN)-disaccharide lipid A backbone was substituted by a GalpA residue that was connected to C-1 of proximal GlcpN. Some of this GalpA residue was ß-eliminated by alkaline de-acylation, which indicated the possibility of the presence of another so far unidentified substituent at C-4 in non-stoichiometric amounts. One Manp residue substituted C-4' of distal GlcpN. The lipid A backbone was acylated by 16:0(3-OH) at C-2 of proximal GlcpN, and by 16:0(3-OH), i17:0(3-OH), or 18:0(3-OH) at C-2' of distal GlcpN. Two acyloxy-acyl moieties that were mainly formed by 14:0(3-O-14:0) and 16:0(3-O-22:1) occupied the distal GlcpN of lipid A. Genes that were possibly involved in the modification of Rv lipid A were compared with bacterial genes of known function. The biological activity was tested at the model of human mononuclear cells (MNC), showing that Rv lipid A alone does not significantly stimulate MNC. At low concentrations of toxic Escherichia coli O111:B4 LPS, pre-incubation with Rv lipid A resulted in a substantial reduction of activity, but, when higher concentrations of E. coli LPS were used, the stimulatory effect was increased.

RNA is taking its Toll: Impact of RNA-specific Toll-like receptors on health and disease.

RNA-sensing Toll-like receptors (TLRs) are often described as antiviral receptors of the innate immune system. However, the past decade has shown that the function and relevance of these receptors are far more complex. They were found to be essential for the detection of various bacterial, archaeal, and eukaryotic microorganisms and facilitate the discrimination between dead and living microbes. The cytokine and interferon response profile that is triggered has the potential to improve the efficacy of next-generation vaccines and may prevent the development of asthma and allergy. Nevertheless, the ability to recognize foreign RNA comes with a cost as also damaged host cells can release nucleic acids that might induce an inappropriate immune response. Thus, it is not surprising that RNA-sensing TLRs play a key role in various autoimmune diseases. However, promising new inhibitors and antagonists are on the horizon to improve their treatment.

Endosomal recognition of Lactococcus lactis G121 and its RNA by dendritic cells is key to its allergy-protective effects.

BACKGROUND: Bacterial cowshed isolates are allergy protective in mice; however, the underlying mechanisms are largely unknown. We examined the ability of Lactococcus lactis G121 to prevent allergic inflammatory reactions. OBJECTIVE: We sought to identify the ligands and pattern recognition receptors through which L lactis G121 confers allergy protection. METHODS: L lactis G121-induced cytokine release and surface expression of costimulatory molecules by untreated or inhibitor-treated (bafilomycin and cytochalasin D) human monocyte-derived dendritic cells (moDCs), bone marrow-derived mouse dendritic cells (BMDCs), and moDC/naive CD4+ T-cell cocultures were analyzed by using ELISA and flow cytometry. The pathology of ovalbumin-induced acute allergic airway inflammation after adoptive transfer of BMDCs was examined by means of microscopy. RESULTS: L lactis G121-treated murine BMDCs and human moDCs released TH1-polarizing cytokines and induced TH1 T cells. Inhibiting phagocytosis and endosomal acidification in BMDCs or moDCs impaired the release of TH1-polarizing cytokines, costimulatory molecule expression, and T-cell activation on L lactis G121 challenge. In vivo allergy protection mediated by L lactis G121 was dependent on endosomal acidification in dendritic cells (DCs). Toll-like receptor (Tlr) 13-/- BMDCs showed a weak response to L lactis G121 and were unresponsive to its RNA. The TH1-polarizing activity of L lactis G121-treated human DCs was blocked by TLR8-specific inhibitors, mediated by L lactis G121 RNA, and synergistically enhanced by activation of nucleotide-binding oligomerization domain-containing protein (NOD) 2. CONCLUSION: Bacterial RNA is the main driver of L lactis G121-mediated protection against experimentally induced allergy and requires both bacterial uptake by DCs and endosomal acidification. In mice L lactis G121 RNA signals through TLR13; however, the most likely intracellular receptor in human subjects is TLR8.

Biological effects of carbon black nanoparticles are changed by surface coating with polycyclic aromatic hydrocarbons.

BACKGROUND: Carbon black nanoparticles (CBNP) are mainly composed of carbon, with a small amount of other elements (including hydrogen and oxygen). The toxicity of CBNP has been attributed to their large surface area, and through adsorbing intrinsically toxic substances, such as polycyclic aromatic hydrocarbons (PAH). It is not clear whether a PAH surface coating changes the toxicological properties of CBNP by influencing their physicochemical properties, through the specific toxicity of the surface-bound PAH, or by a combination of both. METHODS: Printex®90 (P90) was used as CBNP; the comparators were P90 coated with either benzo[a]pyrene (BaP) or 9-nitroanthracene (9NA), and soot from acetylene combustion that bears various PAHs on the surface (AS-PAH). Oxidative stress and IL-8/KC mRNA expression were determined in A549 and bronchial epithelial cells (16HBE14o-, Calu-3), mouse intrapulmonary airways and tracheal epithelial cells. Overall toxicity was tested in a rat inhalation study according to Organization for Economic Co-operation and Development (OECD) criteria. Effects on cytochrome monooxygenase (Cyp) mRNA expression, cell viability and mucociliary clearance were determined in acute exposure models using explanted murine trachea. RESULTS: All particles had similar primary particle size, shape, hydrodynamic diameter and ζ-potential. All PAH-containing particles had a comparable specific surface area that was approximately one third that of P90. AS-PAH contained a mixture of PAH with expected higher toxicity than BaP or 9NA. PAH-coating reduced some effects of P90 such as IL-8 mRNA expression and oxidative stress in A549 cells, granulocyte influx in the in vivo OECD experiment, and agglomeration of P90 and mucus release in the murine trachea ex vivo. Furthermore, P90-BaP decreased particle transport speed compared to P90 at 10 µg/ml. In contrast, PAH-coating induced IL-8 mRNA expression in bronchial epithelial cell lines, and Cyp mRNA expression and apoptosis in tracheal epithelial cells. In line with the higher toxicity compared to P90-BaP and P90-9NA, AS-PAH had the strongest biological effects both ex vivo and in vivo. CONCLUSIONS: Our results demonstrate that the biological effect of CBNP is determined by a combination of specific surface area and surface-bound PAH, and varies in different target cells.

Toll-Like Receptor 2 Recognizes Orientia tsutsugamushi and Increases Susceptibility to Murine Experimental Scrub Typhus.

Scrub typhus is a potentially lethal infection that is caused by the obligate intracellular bacterium Orientia tsutsugamushi The roles of Toll-like receptor 2 (TLR2) and TLR4 in innate recognition of O. tsutsugamushi have not been elucidated. By overexpression of TLR2 or TLR4 in HEK293 cells, we demonstrated that TLR2, but not TLR4, recognizes heat-stable compounds of O. tsutsugamushi that were sensitive to treatment with sodium hydroxide, hydrogen peroxide, and proteinase K. TLR2 was required for the secretion of tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) by dendritic cells. In an intradermal mouse infection model, TLR2-deficient mice did not show impaired control of bacterial growth or reduced survival. Moreover, after intraperitoneal infection, TLR2-deficient mice were even more resistant to lethal infection than C57BL/6 wild-type mice, which showed stronger symptoms and lower survival rates during the convalescent phase. Compared to the time of reduction of bacterial loads in TLR2-deficient mice, the reduction of bacterial loads in infected organs was accelerated in wild-type mice. The higher mortality of wild-type mice was associated with increased concentrations of serum alkaline phosphatase but not aspartate aminotransferase. The transcription of mRNA for TNF-α and IL-6 decreased more rapidly in peritoneum samples from wild-type mice than in those from TLR2-deficient mice and was therefore not a correlate of increased susceptibility. Thus, although TLR2 is an important mediator of the early inflammatory response, it is dispensable for protective immunity against O. tsutsugamushi Increased susceptibility to O. tsutsugamushi infection in TLR2-competent mice rather suggests a TLR2-related immunopathologic effect.

Allergy protection by cowshed bacteria - recent findings and future prospects.

Since the first publication in 1999, numerous epidemiologic studies provided strong evidence that frequent contact to a traditional farm environment in early life protects children from the development of allergic airway diseases. These consistent findings prompted enormous efforts to identify and characterize the potential causative factors and the underlying immunologic mechanisms in experimental studies. The cumulating evidence for the role of the cowshed-associated bacterial flora led to enhanced efforts not only to identify the relevant species but also to examine their specific immunomodulatory capacity, the bacterial components involved, and particularly the cellular and molecular mechanisms of their interaction with the immune system. We review here the methods applied to identify relevant bacterial species, the species which emerged thereof, and the similarities and differences in their mode of action as revealed so far. We further consider the impact of the current knowledge on worthwhile clinical application and reflect on the required next steps to foster the translation of the encouraging scientific progress which has been made in recent years.

Dermatophagoides pteronyssinus major allergen 1 activates the innate immune response of the fruit fly Drosophila melanogaster.

Some allergens with relevant protease activity have the potential to directly interact with host structures. It remains to be elucidated whether this activity is relevant for developing their allergenic properties. The major goal of this study was to elucidate whether allergens with a strong protease activity directly interact with modules of the innate immune system, thereby inducing an immune response. We chose Drosophila melanogaster for our experiments to prevent the results from being influenced by the adaptive immune system and used the armamentarium of methods available for the fly to study the underlying mechanisms. We show that Dermatophagoides pteronyssinus major allergen 1 (Der p 1), the major allergen of the house dust mite, efficiently activates various facets of the Drosophila innate-immune system, including both epithelial and systemic responses. These responses depend on the immune deficiency (IMD) pathway via activation of the NF-κB transcription factor Relish. In addition, the major pathogen associated molecular pattern recognizing receptor of the IMD pathway, peptidoglycan recognition protein-LC, was necessary for this response. We showed that Der p 1, which has cysteine protease activity, cleaves the ectodomain of peptidoglycan recognition protein-LC and, thus, activates the IMD pathway to induce a profound immune response. We conclude that the innate immune response to this allergen-mediated proteolytic cleavage represents an ancient type of danger signaling that may be highly relevant for the primary allergenicity of compounds such as Der p 1.

Total Synthesis of Five Lipoteichoic acids of Clostridium difficile.

The emergence of hypervirulent resistant strains have made Clostridium difficile a notorious nosocomial pathogen and has resulted in a renewed interest in preventive strategies, such as vaccines based on (synthetic) cell wall antigens. Recently, the structure of the lipoteichoic acid (LTA) of this species has been elucidated. Additionally, this LTA was found to induce the formation of protective antibodies against C. difficile in rabbits and mice. The LTA from C. difficile is isolated as a microheterogenous mixture, differing in size and composition, impeding any structure-activity relationship studies. To ensure reliable biological results, pure and well-defined synthetic samples are required. In this work the total synthesis of LTAs from C. difficile with defined chain length is described and the initial biological results are presented.

Isolation of Enterococcus faecium NM113, Enterococcus faecium NM213 and Lactobacillus casei NM512 as novel probiotics with immunomodulatory properties.

Probiotics, defined as living bacteria that are beneficial for human health, mainly function through their immunomodulatory abilities. Hence, these microorganisms have proven successful for treating diseases resulting from immune deregulation. The aim of this study was to find novel candidates to improve on and complement current probiotic treatment strategies. Of 60 lactic acid bacterial strains that were isolated from fecal samples of healthy, full-term, breast-fed infants, three were chosen because of their ability to activate human immune cells. These candidates were then tested with regard to immunomodulatory properties, antimicrobial effects on pathogens, required pharmacological properties and their safety profiles. To identify the immunomodulatory structures of the selected isolates, activation of specific innate immune receptors was studied. The three candidates for probiotic treatment were assigned Enterococcus faecium NM113, Enterococcus faecium NM213 and Lactobacillus casei NM512. Compared with the established allergy-protective strain Lactococcus lactis G121, these isolates induced release of similar amounts of IL-12, a potent inducer of T helper 1 cells. In addition, all three neonatal isolates had antimicrobial activity against pathogens. Analysis of pharmacological suitability showed high tolerance of low pH, bile salts and pancreatic enzymes. In terms of safe application in humans, the isolates were sensitive to three antibiotics (chloramphenicol, tetracycline and erythromycin). In addition, the Enterococcus isolates were free from the four major virulence genes (cylA, agg, efaAfs and ccf). Moreover, the isolates strongly activated Toll-like receptor 2, which suggests lipopeptides as their active immunomodulatory structure. Thus, three novel bacterial strains with great potential as probiotic candidates and promising immunomodulatory properties have here been identified and characterized.

Exploring the human archaeome: its relevance for health and disease, and its complex interplay with the human immune system.

This Review aims to coalesce existing knowledge on the human archaeome, a less-studied yet critical non-bacterial component of the human microbiome, with a focus on its interaction with the immune system. Despite a largely bacteria-centric focus in microbiome research, archaea present unique challenges and opportunities for understanding human health. We examine the archaeal distribution across different human body sites, such as the lower gastrointestinal tract (LGT), upper aerodigestive tract (UAT), urogenital tract (UGT), and skin. Variability in archaeal composition exists between sites; methanogens dominate the LGT, while Nitrososphaeria are prevalent on the skin and UAT. Archaea have yet to be classified as pathogens but show associations with conditions such as refractory sinusitis and vaginosis. In the LGT, methanogenic archaea play critical metabolic roles by converting bacterial end-products into methane, correlating with various health conditions, including obesity and certain cancers. Finally, this work looks at the complex interactions between archaea and the human immune system at the molecular level. Recent research has illuminated the roles of specific archaeal molecules, such as RNA and glycerolipids, in stimulating immune responses via innate immune receptors like Toll-like receptor 8 (TLR8) and 'C-type lectin domain family 4 member E' (CLEC4E; also known as MINCLE). Additionally, metabolic by-products of archaea, specifically methane, have demonstrated immunomodulatory effects through anti-inflammatory and anti-oxidative pathways. Despite these advancements, the mechanistic underpinnings of how archaea influence immune activity remain a fertile area for further investigation.

Recurrent Phases of Strict Protein Limitation Inhibit Tumor Growth and Restore Lifespan in A Drosophila Intestinal Cancer Model.

Diets that restrict caloric or protein intake offer a variety of benefits, including decreasing the incidence of cancer. However, whether such diets pose a substantial therapeutic benefit as auxiliary cancer treatments remains unclear. We determined the effects of severe protein depletion on tumorigenesis in a Drosophila melanogaster intestinal tumor model, using a human RAF gain-of-function allele. Severe and continuous protein restriction significantly reduced tumor growth but resulted in premature death. Therefore, we developed a diet in which short periods of severe protein restriction alternated cyclically with periods of complete feeding. This nutritional regime reduced tumor mass, restored gut functionality, and rescued the lifespan of oncogene-expressing flies to the levels observed in healthy flies on a continuous, fully nutritious diet. Furthermore, this diet reduced the chemotherapy-induced stem cell activity associated with tumor recurrence. Transcriptome analysis revealed long-lasting changes in the expression of key genes involved in multiple major developmental signaling pathways. Overall, the data suggest that recurrent severe protein depletion effectively mimics the health benefits of continuous protein restriction, without undesired nutritional shortcomings. This provides seminal insights into the mechanisms of the memory effect required to maintain the positive effects of protein restriction throughout the phases of a full diet. Finally, the repetitive form of strict protein restriction is an ideal strategy for adjuvant cancer therapy that is useful in many tumor contexts.

Inhibition of tumorigenesis driven by different Wnt proteins requires blockade of distinct ligand-binding regions by LRP6 antibodies.

Disregulated Wnt/beta-catenin signaling has been linked to various human diseases, including cancers. Inhibitors of oncogenic Wnt signaling are likely to have a therapeutic effect in cancers. LRP5 and LRP6 are closely related membrane coreceptors for Wnt proteins. Using a phage-display library, we identified anti-LRP6 antibodies that either inhibit or enhance Wnt signaling. Two classes of LRP6 antagonistic antibodies were discovered: one class specifically inhibits Wnt proteins represented by Wnt1, whereas the second class specifically inhibits Wnt proteins represented by Wnt3a. Epitope-mapping experiments indicated that Wnt1 class-specific antibodies bind to the first propeller and Wnt3a class-specific antibodies bind to the third propeller of LRP6, suggesting that Wnt1- and Wnt3a-class proteins interact with distinct LRP6 propeller domains. This conclusion is further supported by the structural functional analysis of LRP5/6 and the finding that the Wnt antagonist Sclerostin interacts with the first propeller of LRP5/6 and preferentially inhibits the Wnt1-class proteins. We also show that Wnt1 or Wnt3a class-specific anti-LRP6 antibodies specifically block growth of MMTV-Wnt1 or MMTV-Wnt3 xenografts in vivo. Therapeutic application of these antibodies could be limited without knowing the type of Wnt proteins expressed in cancers. This is further complicated by our finding that bivalent LRP6 antibodies sensitize cells to the nonblocked class of Wnt proteins. The generation of a biparatopic LRP6 antibody blocks both Wnt1- and Wnt3a-mediated signaling without showing agonistic activity. Our studies provide insights into Wnt-induced LRP5/6 activation and show the potential utility of LRP6 antibodies in Wnt-driven cancer.

The structure of the extracellular teichoic acids from the allergy-protective bacterium Lactococcus lactis G121.

The Gram-positive bacterium Lactococcus lactis G121 is a farm isolate that protects mice from ovalbumin-induced asthma. To understand the molecular mechanisms of such allergy-protective properties, the isolation and characterization of cell envelope constituents is crucial. Here, structural analyses of the extracellular teichoic acid (EC TA) from L. lactis G121 are presented. Extraction with 0.9% saline afforded a crude TA fraction. Consecutive size exclusion chromatography on Biogel P60 and P10 matrix was performed to purify the sample. Chemical component analyses, high-resolution electrospray ionization Fourier-transformed ion cyclotron mass spectrometry, and nuclear magnetic resonance spectroscopy were conducted for structural elucidation. The EC TA was a poly(glycosylglycerol phosphate) molecule with a repeating unit of -6)-[ß-D-Glcp-(1→3)-][α-D-GlcpNAc-(1→4)-]α-D-GalpNAc-(1→3)-ß-D-GlcpNAc-(1→2)-glycerol-(1-P-).