Reduction in PA28αβ activation in HD mouse brain correlates to increased mHTT aggregation in cell models

Huntington’s disease is an autosomal dominant heritable disorder caused by an expanded CAG trinucleotide repeat at the N-terminus of the Huntingtin (HTT) gene. Lowering the levels of soluble mutant HTT protein prior to aggregation through increased degradation by the proteasome would be a therapeutic strategy to prevent or delay the onset of disease. Native PAGE experiments in HdhQ150 mice and R6/2 mice showed that PA28αβ disassembles from the 20S proteasome during disease progression in the affected cortex, striatum and hippocampus but not in cerebellum and brainstem. Modulating PA28αβ activated proteasomes in various in vitro models showed that PA28αβ improved polyQ degradation, but decreased the turnover of mutant HTT. Silencing of PA28αβ in cells lead to an increase in mutant HTT aggregates, suggesting that PA28αβ is critical for overall proteostasis, but only indirectly affects mutant HTT aggregation

Estimation of interdomain flexibility of N-terminus of factor H using residual dipolar couplings

Characterization of segmental flexibility is needed to understand the biological mechanisms of the very large category of functionally diverse proteins, exemplified by the regulators of complement activation, that consist of numerous compact modules or domains linked by short, potentially flexible, sequences of amino acid residues. The use of NMR-derived residual dipolar couplings (RDCs), in magnetically aligned media, to evaluate interdomain motion is established but only for two-domain proteins. We focused on the three N-terminal domains (called CCPs or SCRs) of the important complement regulator, human factor H (i.e. FH1-3). These domains cooperate to facilitate cleavage of the key complement activation-specific protein fragment, C3b, forming iC3b that no longer participates in the complement cascade. We refined a three-dimensional solution structure of recombinant FH1-3 based on nuclear Overhauser effects and RDCs. We then employed a rudimentary series of RDC datasets, collected in media containing magnetically aligned bicelles (disk-like particles formed from phospholipids) under three different conditions, to estimate interdomain motions. This circumvents a requirement of previous approaches for technically difficult collection of five independent RDC datasets. More than 80% of conformers of this predominantly extended three-domain molecule exhibit flexions of < 40 °. Such segmental flexibility (together with the local dynamics of the hypervariable loop within domain 3), could facilitate recognition of C3b via initial anchoring and eventual reorganization of modules to the conformation captured in the previously solved crystal structure of a C3b:FH1-4 complex

Toll-like receptor signaling adapter proteins govern spread of neuropathic pain and recovery following nerve injury in male mice.

BackgroundSpinal Toll-like receptors (TLRs) and signaling intermediaries have been implicated in persistent pain states. We examined the roles of two major TLR signaling pathways and selected TLRs in a mononeuropathic allodynia.MethodsL5 spinal nerve ligation (SNL) was performed in wild type (WT, C57BL/6) male and female mice and in male Tlr2-/-Tlr3-/-, Tlr4-/-, Tlr5-/-, Myd88-/-, Triflps2, Myd88/Triflps2, Tnf-/-, and Ifnar1-/- mice. We also examined L5 ligation in Tlr4-/- female mice. We examined tactile allodynia using von Frey hairs. Iba-1 (microglia) and GFAP (astrocytes) were assessed in spinal cords by immunostaining. Tactile thresholds were analyzed by 1- and 2-way ANOVA and the Bonferroni post hoc test was used.ResultsIn WT male and female mice, SNL lesions resulted in a persistent and robust ipsilateral, tactile allodynia. In males with TLR2, 3, 4, or 5 deficiencies, tactile allodynia was significantly, but incompletely, reversed (approximately 50%) as compared to WT. This effect was not seen in female Tlr4-/- mice. Increases in ipsilateral lumbar Iba-1 and GFAP were seen in mutant and WT mice. Mice deficient in MyD88, or MyD88 and TRIF, showed an approximately 50% reduction in withdrawal thresholds and reduced ipsilateral Iba-1. In contrast, TRIF and interferon receptor null mice developed a profound ipsilateral and contralateral tactile allodynia. In lumbar sections of the spinal cords, we observed a greater increase in Iba-1 immunoreactivity in the TRIF-signaling deficient mice as compared to WT, but no significant increase in GFAP. Removing MyD88 abrogated the contralateral allodynia in the TRIF signaling-deficient mice. Conversely, IFNβ, released downstream to TRIF signaling, administered intrathecally, temporarily reversed the tactile allodynia.ConclusionsThese observations suggest a critical role for the MyD88 pathway in initiating neuropathic pain, but a distinct role for the TRIF pathway and interferon in regulating neuropathic pain phenotypes in male mice

Uptake of oxLDL and IL-10 production by macrophages requires PAFR and CD36 recruitment into the same lipid rafts

Macrophage interaction with oxidized low-density lipoprotein (oxLDL) leads to its differentiation into foam cells and cytokine production, contributing to atherosclerosis development. In a previous study, we showed that CD36 and the receptor for platelet-activating factor (PAFR) are required for oxLDL to activate gene transcription for cytokines and CD36. Here, we investigated the localization and physical interaction of CD36 and PAFR in macrophages stimulated with oxLDL. We found that blocking CD36 or PAFR decreases oxLDL uptake and IL-10 production. OxLDL induces IL-10 mRNA expression only in HEK293T expressing both receptors (PAFR and CD36). OxLDL does not induce IL-12 production. The lipid rafts disruption by treatment with βCD reduces the oxLDL uptake and IL-10 production. OxLDL induces co-immunoprecipitation of PAFR and CD36 with the constitutive raft protein flotillin-1, and colocalization with the lipid raft-marker GM1-ganglioside. Finally, we found colocalization of PAFR and CD36 in macrophages from human atherosclerotic plaques. Our results show that oxLDL induces the recruitment of PAFR and CD36 into the same lipid rafts, which is important for oxLDL uptake and IL-10 production. This study provided new insights into how oxLDL interact with macrophages and contributing to atherosclerosis development

Epstein-Barr Virus-Encoded BARF1 Protein is a Decoy Receptor for Macrophage Colony Stimulating Factor and Interferes with Macrophage Differentiation and Activation

Epstein-Barr virus (EBV), like many other persistent herpes viruses, has acquired numerous mechanisms for subverting or evading immune surveillance. This study investigates the role of secreted EBV-encoded BARF1 protein (sBARF1) in creating an immune evasive microenvironment. Wild-type consensus BARF1 was expressed in the human 293 cell line and purified. This native hexameric sBARF1 had inhibitory capacity on macrophage colony stimulating factor (M-CSF)-stimulated, and not on granulocyte macrophage-colony stimulating factor (GM-CSF)-stimulated growth and differentiation of myeloid cells. Antibodies specific to hexameric sBARF1 were able to block this effect. M-CSF was shown to interact with sBARF1 via the protruding N-terminal loops involving Val38 and Ala84. Each BARF1 hexamer was capable of binding three M-CSF dimers. Mutations in the BARF1 loops greatly affected M-CSF interaction, and showed loss of growth inhibition. Analysis of the activation state of the M-CSF receptor c-fms and its downstream kinase pathways showed that sBARF1 prevented M-CSF-induced downstream phosphorylation. Since M-CSF is an important factor in macrophage differentiation, the effect of sBARF1 on the function of monocyte-derived macrophages was evaluated. sBARF1 affected overall survival and morphology and significantly reduced expression of macrophage differentiation surface markers such as CD14, CD11b, CD16, and CD169. Macrophages differentiating in the presence of sBARF1 showed impaired responses to lipopolysaccharide and decreased oxygen radical formation as well as reduced phagocytosis of apoptotic cells. In conclusion, EBV sBARF1 protein is a potent decoy receptor for M-CSF, hampering the function and differentiation of macrophages. These results suggest that sBARF1 contributes to the modulation of immune responses in the microenvironment of EBV-positive carcinoma

Chlamydia trachomatis Test-of-Cure Cannot Be Based on a Single Highly Sensitive Laboratory Test Taken at Least 3 Weeks after Treatment

Current test-of-cure practice in patients with Chlamydia trachomatis (Ct) infection is to confirm cure with a single test taken at least 3 weeks after treatment. Effectiveness of single-time-point testing however lacks a scientific evidence basis and the high sensitivity of laboratory assays nowadays in use for this purpose may compromise the clinical significance of their results. Prospectively following 59 treated Ct infections, administering care as usual, the presence of Ct plasmid DNA and rRNA was systematically assessed by multiple time-sequential measurements, i.e. on 18 samples taken per patient during 8 weeks following treatment with a single dose of 1000 mg Azythromycin. A high proportion (42%) of Ct infections tested positive on at least one of the samples taken after 3 weeks. Patients' test results showed substantial inter-individual and intra-individual variation over time and by type of NAAT used. We demonstrated frequent intermittent positive patterns in Ct test results over time, and strongly argue against current test-of-cure practice

Lampe1: An ENU-Germline Mutation Causing Spontaneous Hepatosteatosis Identified through Targeted Exon-Enrichment and Next-Generation Sequencing

Using a small scale ENU mutagenesis approach we identified a recessive germline mutant, designated Lampe1 that exhibited growth retardation and spontaneous hepatosteatosis. Low resolution mapping based on 20 intercrossed Lampe1 mice revealed linkage to a ∼14 Mb interval on the distal site of chromosome 11 containing a total of 285 genes. Exons and 50 bp flanking sequences within the critical region were enriched with sequence capture microarrays and subsequently analyzed by next-generation sequencing. Using this approach 98.1 percent of the targeted DNA was covered with a depth of 10 or more reads per nucleotide and 3 homozygote mutations were identified. Two mutations represented intronic nucleotide changes whereas one mutation affected a splice donor site in intron 11–12 of Palmitoyl Acetyl-coenzyme A oxygenase-1 (Acox1), causing skipping of exon 12. Phenotyping of Acox1Lampe1 mutants revealed a progression from hepatosteatosis to steatohepatitis, and ultimately hepatocellular carcinoma. The current approach provides a highly efficient and affordable method to identify causative mutations induced by ENU mutagenesis and animal models relevant to human pathology

Integrin CD11b positively regulates TLR4-induced signalling pathways in dendritic cells but not in macrophages.

Tuned and distinct responses of macrophages and dendritic cells to Toll-like receptor 4 (TLR4) activation induced by lipopolysaccharide (LPS) underpin the balance between innate and adaptive immunity. However, the molecule(s) that confer these cell-type-specific LPS-induced effects remain poorly understood. Here we report that the integrin α(M) (CD11b) positively regulates LPS-induced signalling pathways selectively in myeloid dendritic cells but not in macrophages. In dendritic cells, which express lower levels of CD14 and TLR4 than macrophages, CD11b promotes MyD88-dependent and MyD88-independent signalling pathways. In particular, in dendritic cells CD11b facilitates LPS-induced TLR4 endocytosis and is required for the subsequent signalling in the endosomes. Consistent with this, CD11b deficiency dampens dendritic cell-mediated TLR4-triggered responses in vivo leading to impaired T-cell activation. Thus, by modulating the trafficking and signalling functions of TLR4 in a cell-type-specific manner CD11b fine tunes the balance between adaptive and innate immune responses initiated by LPS