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Carriers are often an essential element of drug delivery, bestowing attributes to their cargo such as biocompatibility, enhanced delivery, extended half-life and efficacy as well as mediating specific targeting at a tissue, cell or intracellular level. Liposomes and lipid-based carriers have been investigated for decades for this purpose, many achieving clinical approval including products such as Doxil® and Myocet™. Large-scale compound screens are routinely carried out in the field of drug discovery; however, less work has been done on harnessing high-throughput methods for carrier material screening. Screening the interaction of drug carriers and materials with cells is particularly critical for the development of emerging therapies, including biomedicines, in order to facilitate the development of safe and efficient drug products. Herein, a range of liposomes of neutral, anionic and cationic charge and others that are surface-modified with mannose residues were screened for cell interaction, toxicity and immune reactivity in THP-1-derived macrophages using a high-throughput format. Liposomes were seen to be efficacious in a concentration-dependent and, for mannosylated liposomes, mannosylated cholesterol linker length-dependent manner. [ABSTRACT FROM AUTHOR]

Neuronal survival and plasticity critically depend on constitutive activity of the transcription factor nuclear factor-kB (NF-kB). We here describe a role for a small intracellular fibroblast growth factor homologue, the fibroblast growth factor homologous factor 1 (FHF1/ FGF12), in the regulation of NF-kB activity in mature neurons. FHFs have previously been described to control neuronal excitability, and mutations in FHF isoforms give rise to a form of progressive spinocerebellar ataxia. Using a protein-array approach, we identified FHF1b as a novel interactor of the canonical NF-kB modulator IKKγ/NEMO. Co-immunoprecipitation, pull-down and GAL4-reporter experiments, as well as proximity ligation assays, confirmed the interaction of FHF1 and NEMO and demonstrated that a major site of interaction occurred within the axon initial segment. Fhf1 gene silencing strongly activated neuronal NF-kB activity and increased neurite lengths, branching patterns and spine counts in mature cortical neurons. The effects of FHF1 on neuronal NF-kB activity and morphology required the presence of NEMO. Our results imply that FHF1 negatively regulates the constitutive NF-kB activity in neurons. [ABSTRACT FROM AUTHOR]

The article offers information on the role of MHC molecules and Toll like receptors (TLRs) in the immune system of human body. It states that TLRs are inherited immune receptors that causes the production of inflammatory cytokines. It also mentions about the importance of a nonreceptor tyrosine kinase, Bruton's tyrosine kinase (Btk) for immune response.

Signal transduction processes activated by Toll-like receptors (TLRs) include the important transcription factor NF-κB and 2 MAP kinases, p38 and Jun N-terminal kinase. These signals ultimately give rise to increased expression of a multitude of pro-inflammatory proteins. Receptor-proximal proteins involved in signalling by all TLRs include the adapter MyD88, 3 IRAKs (IRAK-4, IRAK and IRAK-2), Tollip, Traf-6 and TAK-1. Differences between signals generated by TLRs are emerging, with both TLR4 and TLR2 signalling requiring an additional adapter termed MyD88-adapter-like (Mal; also known as TIRAP). MyD88 and Mal both have a homologous Toll/IL-1 receptor (TIR) domain although they differ in their N-termini, with MyD88 possessing a death domain. In addition, structural models reveal marked differences in surface charges which, when taken with surface charge differences between TLR2 and TLR4 TIR domains, may indicate that TLR4 but not TLR2 recruits Mal directly. Another difference is that Mal can become phosphorylated. Future studies on Mal will reveal specificities in signal transduction by different TLRs, which may ultimately provide molecular explanations for specificities in the innate immune response to infection. [ABSTRACT FROM PUBLISHER]

Systemic lupus erythematosus (SLE) patients are 90% women and over three times more likely to die of cardiovascular disease than women in the general population. Chest pain with no obstructive cardiac disease is associated with coronary microvascular disease (CMD), where narrowing of the small blood vessels can lead to ischemia, and frequently reported by SLE patients. Using whole blood RNA samples, we asked whether gene signatures discriminate SLE patients with coronary microvascular dysfunction (CMD) on cardiac MRI (n=4) from those without (n=7) and whether any signaling pathway is linked to the underlying pathobiology of SLE CMD. RNA-seq analysis revealed 143 differentially expressed (DE) genes between the SLE and healthy control (HC) groups, with virus defense and interferon (IFN) signaling being the key pathways identified as enriched in SLE as expected. We next conducted a comparative analysis of genes differentially expressed in SLE-CMD and SLE-non-CMD relative to HC samples. Our analysis highlighted differences in IFN signaling, RNA sensing and ADP-ribosylation pathways between SLE-CMD and SLE-non-CMD. This is the first study to investigate possible gene signatures associating with CMD in SLE, and our data strongly suggests that distinct molecular mechanisms underly vascular changes in CMD and non-CMD involvement in SLE., Competing Interests: Competing Interest Statement: Authors declare that there are no competing interests.

Background: Women with SLE have an elevated risk of cardiovascular disease. Many women with SLE frequently report chest pain in the absence of obstructive coronary artery disease (CAD) due to coronary microvascular dysfunction (CMD), a form of ischemia with no obstructive CAD. Echocardiographic studies have shown that SLE patients have reduced left ventricular (LV) function, which may also correlate with higher SLE disease activity scores. As such, we used cardiac magnetic resonance imaging (cMRI) to investigate the relationship between SLE, related inflammatory biomarkers, and cardiac function in female SLE patients., Methods: We performed stress cMRI in women with SLE and chest pain with no obstructive CAD (n=13, all met ACR 1997 criteria,) and reference controls (n=22) using our published protocol. We evaluated LV function, tissue characterization (T1 mapping, ECV), and delayed enhancement, using CV142 software (Circle Cardiovascular Imaging Inc, Calgary, AB, Canada). Myocardial perfusion reserve index (MPRI) was calculated using our published protocol. SLEDAI and SLICC Damage Index (DI) were calculated per validated criteria. Serum samples were analyzed for inflammatory markers and autoantibodies. Wilcoxon rank-sum test was performed on clinical values with CMD and no CMD SLE subjects, and on cMRI values with all SLE subjects and controls. Correlation analysis was done on clinical values, and cMRI values on all SLE subjects., Results: Overall, 40% of SLE subjects had MPRI values < 1.84, consistent with CMD. Compared to controls, SLE subjects had significantly lower LVEF, and higher LVESVi and LVMi. Corresponding to this, radial, longitudinal, and circumferential strain were significantly lower in the SLE subjects. In correlation analysis of serum inflammatory biomarkers to cMRI values in the SLE subjects, SLICC DI was related to worse cardiac function (lower radial, circumferential and longitudinal strain) and higher T1 time. Additionally, fasting insulin and ESR were negatively correlated with LVMi. Fasting insulin also negatively correlated with ECV. CRP had a positive association with LVESV index and CI and a negative association with longitudinal strain., Conclusions: Among women with SLE with chest pain and no obstructive CAD, 40% have CMD. While evaluations of known inflammatory markers (such as CRP and ESR) predictably correlated with decreased cardiac function, our study found that decreased fasting insulin levels as a novel marker of diminished LV function. In addition, low insulin levels were observed to correlate with increased LVMi and ECV, suggesting a cardioprotective effect of insulin in SLE patients. We also noted that SLICC DI, an assessment of SLE damage, correlates with cardiac dysfunction in SLE. Our findings underline the potential of non-invasive cMRI as a tool for monitoring cardiovascular function in SLE, particularly in patients with high SLICC DI, ESR and CRP and low fasting insulin levels., Competing Interests: The authors have no disclosures to report.

Background: The placenta, composed of chorionic villi, changes dramatically across gestation. Understanding differences in ongoing pregnancies are essential to identify the role of chorionic villi at specific times in gestation and develop biomarkers and prognostic indicators of maternal- fetal health., Methods: The normative mRNA profile is established using next-generation sequencing of 124 first trimester and 43 third trimester human placentas from ongoing healthy pregnancies. Stably expressed genes not different between trimesters and with low variability are identified. Differential expression analysis of first versus third trimester adjusted for fetal sex is performed, followed by a subanalysis with 23 matched pregnancies to control for subject variability using the same genetic and environmental background., Results: Placenta expresses 14,979 mRNAs above sequencing noise (TPM>0.66), with 1,545 stably expressed genes across gestation. Differentially expressed genes account for 86.7% of genes in the full cohort (FDR<0.05). Fold changes highly correlate between the full cohort and subanalysis (Pearson = 0.98). At stricter thresholds (FDR<0.001, fold change>1.5), there are 6,941 differentially expressed protein coding genes (3,206 upregulated in first and 3,735 upregulated in third trimester)., Conclusion: This is the largest mRNA atlas of healthy human placenta across gestation, controlling for genetic and environmental factors, demonstrating substantial changes from first to third trimester in chorionic villi. Specific differences and stably expressed genes may be used to understand the specific role of the chorionic villi throughout gestation and develop first trimester biomarkers of placental health that transpire across gestation, which can be used for future development of biomarkers in maternal-fetal disease.

Background: Women with SLE have an elevated risk of CVD morbidity and mortality and frequently report chest pain in the absence of obstructive CAD. Echocardiographic studies often demonstrate reduced LV function, correlating with higher disease activity. We used cardiac MRI (cMRI) to investigate the relationship between SLE, related inflammatory biomarkers and cardiac function in female SLE patients., Methods: Women with SLE reporting chest pain with no obstructive CAD (n=13) and reference controls (n=22) were evaluated using stress-rest cMRI to measure LV structure, function, tissue characteristics, and myocardial perfusion reserve index (MPRI). Coronary microvascular dysfunction (CMD) was defined as MPRI <1.84. Serum samples were analyzed for inflammatory markers. Relationships between clinical and cMRI values of SLE subjects were assessed, and groups were compared., Results: 40% of SLE subjects had MPRI < 1.84 on cMRI. Compared to controls, SLE subjects had higher LV volumes and mass and lower LV systolic function. SLICC DI was related to worse cardiac function and higher T1. CRP was related to higher cardiac output and a trend to better systolic function, while ESR and fasting insulin were related to lower LV mass. Lower fasting insulin levels correlated with increased ECV., Conclusions: Among our female SLE cohort, 40% had CMD, and SLICC DI correlated with worse cardiac function and diffuse fibrosis. Higher inflammatory markers and low insulin levels may associate with LV dysfunction. Our findings underline the potential of non-invasive cMRI as a tool for monitoring cardiovascular function in SLE patients.

Objectives: A cross-sectional study was conducted in 270 Chinese patients with ankylosing spondylitis (AS) in order to identify potential risk factors for severity of spinal structural damage., Methods: Two hundred seventy AS patients fulfilled the Modified New York Criteria. Computed tomography (CT) was used to scan sacroiliac and hip joints, and radiography was used to scan anteroposterior and lateral lumbar spine, as well as lateral cervical spine. Bath Ankylosing Spondylitis Radiology Index and modified Stoke Ankylosing Spondylitis Spine Score (mSASSS) were scored in duplicate., Results: One hundred eighty-three patients had low mSASSS (mSASSS, <10), and 87 patients had high mSASSS (mSASSS, ≥10). Univariate analysis revealed that AS age of onset, body mass index (BMI), smoking duration, duration of symptoms, diagnostic delay, hip involvement, and sacroiliitis grade were significantly associated with the risk of having high mSASSS after adjustment (all p's < 0.05). Hip involvement interacted significantly with BMI and smoking duration in a graded manner. Particularly, relative to patients with low BMI-negative hip involvement, those with high BMI-negative hip involvement, low BMI-positive hip involvement, and high BMI-positive hip involvement had a 1.94-fold, 3.29-fold, and 5.07-fold increased risk of high mSASSS (95% confidence interval, 0.84-4.47, 1.37-7.89, and 1.97-13.06, p = 0.118, 0.008, and 0.001, respectively). Finally, a nomogram graph based on 7 significant risk factors was generated with substantial prediction accuracy (concordance index, 0.906)., Conclusions: We have identified 7 potential risk factors for the severity of spinal structural damage in Chinese AS patients. Importantly, positive hip involvement, combined with high BMI or long smoking duration, was associated with a remarkably increased risk of having severe spinal structural damage., Competing Interests: The authors declare no conflict of interest., (Copyright © 2020 Wolters Kluwer Health, Inc. All rights reserved.)

This study sought to identify potential therapeutic targets in herpes simplex keratitis (HSK) patients with active and inactive infection by investigating peripheral cytokine production. Peripheral blood mononuclear cells (PBMCs) and serum were prepared from healthy controls and HSK patients during active infection or following treatment (inactive infection). Serum antibody titres were determined by ELISA. Protein expression levels were analysed by Western blot. Cytokine levels were determined by multiplex ELISA. Active corneal herpes simplex virus type 1 (HSV-1) infection resulted in significantly elevated peripheral levels of IL-1β in HSK patients compared to healthy controls, and remained significantly increased following treatment. Elevated production of IL-1β in inactive patients was associated with significantly increased levels of IRF3 and STAT1, key proteins involved in promoting anti-viral immune responses. Our data suggest that inflammation persists beyond the period that it is clinically evident and that enhanced peripheral production of IL-1β may have implications for HSV-1 viral clearance in active and inactive HSK patients.

This study defines a critical role for Btk in regulating TLR4-induced crosstalk between antigen presenting cells (APCs) and natural killer (NK) cells. Reduced levels of IL-12, IL-18 and IFN-γ were observed in Btk-deficient mice and ex vivo generated macrophages and dendritic cells (DCs) following acute LPS administration, whilst enhanced IL-10 production was observed. In addition, upregulation of activation markers and antigen presentation molecules on APCs was also impaired in the absence of Btk. APCs, by virtue of their ability to produce IL-12 and IL-18, are strong inducers of NK-derived IFN-γ. Co-culture experiments demonstrate that Btk-deficient DCs were unable to drive wild-type or Btk-deficient NK cells to induce IFN-γ production, whereas these responses could be restored by exogenous administration of IL-12 and IL-18. Thus Btk is a critical regulator of APC-induced NK cell activation by virtue of its ability to regulate IL-12 and IL-18 production in response to acute LPS administration., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Autoimmune diseases, such as systemic lupus erythematosus and rheumatoid arthritis, result from a loss of tolerance to self-antigens and immune-mediated injury precipitated by the overproduction of type I IFN and inflammatory cytokines. We have identified the inositol 5' phosphatase SHIP-1 as a negative regulator of TLR3-induced type I IFN production. SHIP-1-deficient macrophages display enhanced TLR-induced IFN-beta production, and overexpression of SHIP-1 negatively regulates the ability of TLR3 and its adaptor, Toll/IL-1 receptor domain-containing adaptor-inducing IFN-beta, to induce IFN-beta promoter activity, indicating that SHIP-1 negatively regulates TLR-induced IFN-beta production. Further dissection of the IFN-beta pathway implicates TANK-binding kinase 1 (TBK1) as the target for SHIP-1. Critically, in the absence of SHIP-1, TBK1 appears to be hyperphosphorylated both in unstimulated cells and following TLR3 stimulation. In addition, TBK1 appears to be constitutively associated with Toll/IL-1 receptor domain-containing adaptor-inducing IFN-beta and TNFR-associated factor 3 in SHIP-1 deficient cells, whereas in wild-type cells this association is inducible following TLR3 stimulation. In support of a role for SHIP-1 in regulating complex formation, confocal microscopy demonstrates that TBK1 distribution in the cell is significantly altered in SHIP-1-deficient cells, with more prominent endosomal staining observed, compared with wild-type controls. Taken together, our results point to SHIP-1 as a critical negative regulator of IFN-beta production downstream of TLR3 through the regulation of TBK1 localization and activity.

Two-dimensional (2-D) gel electrophoresis concerted with protein identification by mass spectrometry (MS) is an extremely powerful method for comparative expression profiling of complex protein samples such as cell lysates. The highly resolutive 2-D electrophoresis allows the separation of heterogeneous protein samples on the basis of isoelectric point (pI), molecular mass (Mr), solubility, and relative abundance ((1) J Biol Chem 250: 4007-4021, 1975; (2) Electrophoresis 14: 1067-1073, 1993). Consequently, it provides a comprehensive view of a proteome state ((3) Electrophoresis 21: 1037-1053, 2000), where variations in protein expression levels, isoforms, or post-translational modifications (e.g. phosphorylation) can be highlighted and investigated ((4) Electrophoresis 21: 2196-2208, 2000). Furthermore, this allows the identification of biological markers that characterize a specific physiological or pathological background of a cell or a tissue ((5) Proteomics 1: 397-408, 2001; (6) J Bacteriol 179: 7595-7599, 1997). In this way one can compare the effects of a stimulus or drug on cells or tissue, or more importantly, analyse the effects of disease on the expression level of proteins. Relatively recently, conventional 2-D gel electrophoresis has been combined with protein labelling strategies using up to three different fluorescent dyes to allow comparative analysis of different protein samples within a single 2-D gel platform. In this technique, termed differential in-gel electrophoresis (DIGE), samples are labelled separately then combined and run on the same 2D gel minimizing experimental variation and greatly facilitating spot matching. When three CyDyes (Cy2, Cy3, and Cy5) have been used, three images of the gel are captured then superposed to localize the differentially regulated spots on the 2-D gel using image analysis software. This is an extremely powerful tool in comparative proteomics as these dyes provide a linear response to protein concentration up to five orders of magnitude and great sensitivity with detection down to 125 pg of a single protein, which is less than needed for MS identification. In this chapter, we describe the basic methods for protein labelling, optimization of the isoelectrofocusing parameters for the first dimension (where proteins are separated according to their isoelectric point (pI)), sodium-dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) separation for the second dimension (based on molecular weight (MW)), and different post-staining protocols of the 2-D gel and protein preparation for mass spectrometry identification.

Purification of protein complexes and identification of the constituent components therein have been made relatively simple by the recent advances in proteomics. Uniting good biochemical and protein chemistry techniques with protein identification by mass spectrometry (MS) has resulted in advances in this field that are unprecedented. Our knowledge of Toll-like receptor (TLR) biology has been considerably advanced through the use of such techniques, with key intermediates such as TRAF3, TANK, RIP1 all being identified using proteomic strategies. Applying these techniques to key questions in TLR -biology will undoubtedly serve to further advance the field.

Induction of type I IFNs is a fundamental cellular response to both viral and bacterial infection. The role of the transcription factor IRF3 is well established in driving this process. However, equally as important are cellular mechanisms for turning off type I IFN production to limit this response. In this respect, IRF3 has previously been shown to be targeted for ubiquitin-mediated degradation postviral detection to turn off the IFN-beta response. In this study, we provide evidence that the E3 ligase Ro52 (TRIM21) targets IRF3 for degradation post-pathogen recognition receptor activation. We demonstrate that Ro52 interacts with IRF3 via its C-terminal SPRY domain, resulting in the polyubiquitination and proteasomal degradation of the transcription factor. Ro52-mediated IRF3 degradation significantly inhibits IFN-beta promoter activity, an effect that is reversed in the presence of the proteasomal inhibitor MG132. Specific targeting of Ro52 using short hairpin RNA rescues IRF3 degradation following polyI:C-stimulation of HEK293T cells, with a subsequent increase in IFN-beta production. Additionally, shRNA targeting of murine Ro52 enhances the production of the IRF3-dependent chemokine RANTES following Sendai virus infection of murine fibroblasts. Collectively, this demonstrates a novel role for Ro52 in turning off and thus limiting IRF3-dependent type I IFN production by targeting the transcription factor for polyubiquitination and subsequent proteasomal degradation.