The CATERPILLER protein Monarch-1 is an antagonist of toll-like receptor-, tumor necrosis factor α-, and Mycobacterium tuberculosis-induced pro-inflammatory signals

The CATERPILLER (CLR, also NOD and NLR) proteins share structural similarities with the nucleotide binding domain (NBD)-leucine-rich repeat (LRR) superfamily of plant disease-resistance (R) proteins and are emerging as important immune regulators in animals. CLR proteins contain NBD-LRR motifs and are linked to a limited number of distinct N-terminal domains including transactivation, CARD (caspase activation and recruitment), and pyrin domains (PyD). The CLR gene, Monarch-1/Pypaf7, is expressed by resting primary myeloid/monocytic cells, and its expression in these cells is reduced by Toll-like receptor (TLR) agonists tumor necrosis factor (TNF) α and Mycobacterium tuberculosis. Monarch-1 reduces NFκB activation by TLR-signaling molecules MyD88, IRAK-1 (type I interleukin-1 receptor-associated protein kinase), and TRAF6 (TNF receptor (TNFR)-associated factor) as well as TNFR signaling molecules TRAF2 and RIP1 but not the downstream NFκB subunit p65. This indicates that Monarch-1 is a negative regulator of both TLR and TNFR pathways. Reducing Monarch-1 expression with small interference RNA in myeloid/monocytic cells caused a dramatic increase in NFκB activation and cytokine expression in response to TLR2/TLR4 agonists, TNFα, or M. tuberculosis infection, suggesting that Monarch-1 is a negative regulator of inflammation. Because Monarch-1 is the first CLR protein that interferes with both TLR2 and TLR4 activation, the mechanism of this interference is significant. We find that Monarch-1 associates with IRAK-1 but not MyD88, resulting in the blockage of IRAK-1 hyperphosphorylation. Mutants containing the NBD-LRR or PyD-NBD also blocked IRAK-1 activation. This is the first example of a CLR protein that antagonizes inflammatory responses initiated by TLR agonists via interference with IRAK-1 activation

Sialyl Residues Modulate LPS-Mediated Signaling through the Toll-Like Receptor 4 Complex

We previously reported that neuraminidase (NA) pretreatment of human PBMCs markedly increased their cytokine response to lipopolysaccharide (LPS). To study the mechanisms by which this occurs, we transfected HEK293T cells with plasmids encoding TLR4, CD14, and MD2 (three components of the LPS receptor complex), as well as a NFκB luciferase reporting system. Both TLR4 and MD2 encoded by the plasmids are α-2,6 sialylated. HEK293T cells transfected with TLR4/MD2/CD14 responded robustly to the addition of LPS; however, omission of the MD2 plasmid abrogated this response. Addition of culture supernatants from MD2 (sMD2)-transfected HEK293T cells, but not recombinant, non-glycosylated MD2 reconstituted this response. NA treatment of sMD2 enhanced the LPS response as did NA treatment of the TLR4/CD14-transfected cell supplemented with untreated sMD2, but optimal LPS-initiated responses were observed with NA-treated TLR4/CD14-transfected cells supplemented with NA-treated sMD2. We hypothesized that removal of negatively charged sialyl residues from glycans on the TLR4 complex would hasten the dimerization of TLR4 monomers required for signaling. Co-transfection of HEK293T cells with separate plasmids encoding either YFP- or FLAG-tagged TLR4, followed by treatment with NA and stimulation with LPS, led to an earlier and more robust time-dependent dimerization of TLR4 monomers on co-immunoprecipitation, compared to untreated cells. These findings were confirmed by fluorescence resonance energy transfer (FRET) analysis. Overexpression of human Neu1 increased LPS-initiated TLR4-mediated NFκB activation and a NA inhibitor suppressed its activation. We conclude that (1) sialyl residues on TLR4 modulate LPS responsiveness, perhaps by facilitating clustering of the homodimers, and that (2) sialic acid, and perhaps other glycosyl species, regulate MD2 activity required for LPS-mediated signaling. We speculate that endogenous sialidase activity mobilized during cell activation may play a role in this regulation

Canagliflozin and renal outcomes in type 2 diabetes and nephropathy

BACKGROUND Type 2 diabetes mellitus is the leading cause of kidney failure worldwide, but few effective long-term treatments are available. In cardiovascular trials of inhibitors of sodium–glucose cotransporter 2 (SGLT2), exploratory results have suggested that such drugs may improve renal outcomes in patients with type 2 diabetes. METHODS In this double-blind, randomized trial, we assigned patients with type 2 diabetes and albuminuric chronic kidney disease to receive canagliflozin, an oral SGLT2 inhibitor, at a dose of 100 mg daily or placebo. All the patients had an estimated glomerular filtration rate (GFR) of 30 to <90 ml per minute per 1.73 m2 of body-surface area and albuminuria (ratio of albumin [mg] to creatinine [g], >300 to 5000) and were treated with renin–angiotensin system blockade. The primary outcome was a composite of end-stage kidney disease (dialysis, transplantation, or a sustained estimated GFR of <15 ml per minute per 1.73 m2), a doubling of the serum creatinine level, or death from renal or cardiovascular causes. Prespecified secondary outcomes were tested hierarchically. RESULTS The trial was stopped early after a planned interim analysis on the recommendation of the data and safety monitoring committee. At that time, 4401 patients had undergone randomization, with a median follow-up of 2.62 years. The relative risk of the primary outcome was 30% lower in the canagliflozin group than in the placebo group, with event rates of 43.2 and 61.2 per 1000 patient-years, respectively (hazard ratio, 0.70; 95% confidence interval [CI], 0.59 to 0.82; P=0.00001). The relative risk of the renal-specific composite of end-stage kidney disease, a doubling of the creatinine level, or death from renal causes was lower by 34% (hazard ratio, 0.66; 95% CI, 0.53 to 0.81; P<0.001), and the relative risk of end-stage kidney disease was lower by 32% (hazard ratio, 0.68; 95% CI, 0.54 to 0.86; P=0.002). The canagliflozin group also had a lower risk of cardiovascular death, myocardial infarction, or stroke (hazard ratio, 0.80; 95% CI, 0.67 to 0.95; P=0.01) and hospitalization for heart failure (hazard ratio, 0.61; 95% CI, 0.47 to 0.80; P<0.001). There were no significant differences in rates of amputation or fracture. CONCLUSIONS In patients with type 2 diabetes and kidney disease, the risk of kidney failure and cardiovascular events was lower in the canagliflozin group than in the placebo group at a median follow-up of 2.62 years

Expression, purification and characterisation of antimicrobial peptides of human and bovine lactoferrins : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry at Massey University, Palmerston North, New Zealand

Lactoferrin (Lf). a basic. ~80 kDa iron-binding glycoprotein, is a member of the transferrin family. It is present in the milk and other secretory fluids of many, but not all, mammalian vertebrates. Several biological functions have been ascribed to this protein. These include bactcriostastis, modulation of the inflammatory response, iron nutrition, a role as an anti-oxidant and regulation of myelopoiesis. Full-length human lactoferrin has been expressed in BHK cells, many strains of Aspergillus and with limited success in Saccharomyces cerevisiae. The main aim at the start of this project was to express full-length human lactoferrin (hLF) cDNA in the yeast Kluyveromyces lactis on whey-based media. Yeasts of the genus Kluyveromyces have been used for many years in the food industry and are classified as "Generally Regarded As Safe" (GRAS) organisms. K. lactis has impressive secretion capabilities and can grow on whey-based media (which is abundantly available in New Zealand). Attempts were made to sub-clone full-length hLF cDNA into the K. lactis vector, pEPS1 and the S. cerevisiae vector, pYEXS1 and to express the protein. The establishment of stable insert-carrying constructs of these yeast vectors in E. coli turned out to be an unattainable goal. Direct transformation of the ligation mix into K. lactis produced transformants, which secreted human lactoferrin protein products into the media as assessed by the lactoferrin ELISA assay. The secretion of hLF protein products by recombinant K. lactis continued for few generations, but gradually stopped. There are no known reports on the use of these vectors for the expression of any mammalian proteins in yeast. Lactoferrin has antimicrobial activity against a broad range of Gram-negative and Gram-positive bacteria and against fungi. Originally, the antimicrobial effect of lactoferrin was attributed to its ability to tightly sequester two atoms of iron and hence inhibit microbial growth through nutritional deprivation of iron. Recently, an N-terminal peptide called lactoferricin, isolated from the acid-pepsin hydrolysate of lactoferrin was shown to have greater antimicrobial activity than the intact protein. Currently, the only way to obtain native lactoferricins is to isolate the peptides from the acid pepsin-hydrolysate of lactoferrin, which gives very low yields, or to synthesise them by protein chemical methods, which is very expensive on a large scale. So, heterologous expression of both human and bovine lactoferricins in E. coli was attempted in this study. Synthetic DNA fragments encoding both human and bovine lactoferricins and 3'-end variants of these fragments were sub-cloned into E. coli expression vectors. pPROEXHTa, pET-15b and pGEX-4T1. The constructs were designed to express lactoferricins either as poly-His- or as GST-fusion proteins. In all cases the fusion proteins were expressed as inclusion bodies. The inclusion bodies were urea solubilised and purified on appropriate affinity resins. However, none of the recombinant proteins remained soluble after the urea was removed and therefore could not be further characterised. A similar situation was encountered by other investigators who attempted to express cationic peptides in E. coli. Both lactoferrin and lactoferricin have been shown to bind to the lipid A portion of the bacterial cell wall lipopolysaccharide (LPS), induce the release of LPS and kill the bacteria. In this work, five different E. coli strains were shown to have different susceptibility to native lactoferricin B. Transmission electron microscopy studies of the E. coli strains treated with lactoferricin B revealed considerable differences in their membrane ultrastructure. The most susceptible E. coli strain showed a direct outer membrane dislocation and effect on the cytoplasmic contents. A relatively resistant E. coli strain showed an initial formation of 'membrane blisters'. However, after a long exposure to lactoferricin B, a few cells of this strain showed an outer membrane dislocation and effect on the cytoplasmic contents. The formation of 'membrane blisters' might allow the relatively resistant strain of E. coli to reduce the lethal action of lactoferricin B

Differential Activation of Human TLR4 by Escherichia coli and Shigella flexneri 2a Lipopolysaccharide: Combined Effects of Lipid A Acylation State and TLR4 Polymorphisms on Signaling

International audienceThe lipid A of LPS activates TLR4 through an interaction with myeloid differentiation protein-2 (MD-2) and the degree of lipid A acylation affects TLR4 responsiveness. Two TLR4 single nucleotide polymorphisms (Asp299Gly and Thr399Ile) have been associated with LPS hyporesponsiveness. We hypothesized that the combination of hypoacylation and these single nucleotide polymorphisms would exhibit a compounded effect on TLR4 signaling. HEK293T transfectants expressing wild-type or polymorphic TLR4 were stimulated with Escherichia coli (predominantly hexaacylated lipid A) or Shigella flexneri 2a (a mixture of hexaacylated, pentaacylated, and predominantly tetraacylated lipid A) LPS, or hexaacylated vs pentaacylated synthetic lipid As. NF-kappaB-reporter activity was significantly lower in response to S. flexneri 2a than E. coli LPS and further decreased in polymorphic transfectants. Neither hexaacylated nor pentaacylated synthetic lipid A induced NF-kappaB activity in wild-type transfectants under the identical transfection conditions used for LPS; however, increasing human MD-2 expression rescued responsiveness to hexaacylated lipid A only, while murine MD-2 was required to elicit a response to pentaacylated lipid A. Adherent PBMC of healthy volunteers were also compared for LPS-induced TNF-alpha, IL-6, IL-1beta, and IL-10 production. Cytokine levels were significantly lower (approximately 20-90%) in response to S. flexneri than to E. coli LPS/lipid A and PBMC from polymorphic individuals secreted decreased cytokine levels in response to both LPS types and failed to respond to pentaacylated lipid A. Thus, the combination of acylation state and host genetics may significantly impact vaccine immunogenicity and/or efficacy, whether LPS is an integral component of a whole organism vaccine or included as an adjuvant

Multi-laboratory validation of the xMAP-Food Allergen Detection Assay: A multiplex, antibody-based assay for the simultaneous detection of food allergens.

The increasing prevalence of individuals with multiple food allergies and the need to distinguish between foods containing homologous, cross-reactive proteins have made the use of single-analyte antibody-based methods (e.g., ELISAs) sometimes insufficient. These issues have resulted in the need to conduct multiple analyses and sometimes employ orthogonal methods like mass spectrometry or DNA-based methods for confirmatory purposes. The xMAP Food Allergen Detection Assay (xMAP FADA) was developed to solve this problem while also providing increased throughput and a modular design suitable for adapting to changes in analytical needs. The use of built-in redundancy provides the xMAP FADA with built-in confirmatory analytical capability by including complementary antibody bead sets and secondary analytical end points (e.g., ratio analysis and multi-antibody profiling). A measure of a method's utility is its performance when employed by analysts of varying expertise in multiple laboratory environments. To gauge this aspect, a multi-laboratory validation (MLV) was conducted with 11 participants of different levels of proficiency. The MLV entailed the analysis of incurred food samples in four problematic food matrices, meat sausage, orange juice, baked muffins, and dark chocolate. Except for a couple of instances, involving two confirmatory components in the analysis of baked muffins, the allergenic foods were detected by all participants at concentrations in the analytical samples comparable to ≤ 10 μg/g in the original food sample. In addition, despite high levels of inter-lab variance in the absolute intensities of the responses, the intra-laboratory reproducibility was sufficient to support analyses based on the calibration standards and direct comparison controls (DCCs) analyzed alongside the samples. In contrast, ratio analyses displayed inter-laboratory %CV (RSDR) values < 20%; presumably because the ratios are based on inherent properties of the antigenic elements. The excellent performance of the xMAP FADA when performed by analysts of varying proficiency indicates a reliability sufficient to meet analytical needs

Vibrio cholerae Flagellins Induce Toll-Like Receptor 5-Mediated Interleukin-8 Production through Mitogen-Activated Protein Kinase and NF-κB Activation▿

Vaccine reactogenicity has complicated the development of safe and effective live, oral cholera vaccines. Δctx Vibrio cholerae mutants have been shown to induce inflammatory diarrhea in volunteers and interleukin-8 (IL-8) production in cultured intestinal epithelial cells. Bacterial flagellins are known to induce IL-8 production through Toll-like receptor 5 (TLR5). Since the V. cholerae genome encodes five distinct flagellin proteins, FlaA to FlaE, with homology to conserved TLR5 recognition regions of Salmonella FliC, we hypothesized that V. cholerae flagellins may contribute to IL-8 induction through TLR5 and mitogen-activated protein kinase (MAPK) signaling. Each purified recombinant V. cholerae flagellin induced IL-8 production in T84 intestinal epithelial cells and also induced nuclear factor kappa B (NF-κB) activation in HEK293T/TLR5 transfectants, which was blocked by cotransfection with a TLR5 dominant-negative construct, demonstrating TLR5 specificity. Supernatants derived from ΔflaAC and ΔflaEDB mutants induced IL-8 production in HT-29 intestinal epithelial cells and in HEK293T cells overexpressing TLR5, whereas ΔflaABCDE supernatants induced significantly less IL-8 production, demonstrating the contribution of multiple flagellins in IL-8 induction. NF-κB activation by ΔflaABCDE supernatants was partially restored by flaA or flaAC complementation. Western analysis confirmed the presence of V. cholerae flagellins in culture supernatants. Purified recombinant V. cholerae FlaA activated the MAPKs p38, c-jun N-terminal kinase (JNK), and extracellular regulated kinase (ERK) in T84 cells. FlaA-induced IL-8 production in T84 cells was inhibited by the p38 inhibitor in combination with either the JNK or ERK inhibitors. Collectively, these data suggest that V. cholerae flagellins are present in culture supernatants and can induce TLR5- and MAPK-dependent IL-8 secretion in host cells