Micro‐RNA‐155‐mediated control of heme oxygenase 1 (HO‐1) is required for restoring adaptively tolerant CD4+ T‐cell function in rodents

T cells chronically stimulated by a persistent antigen often become dysfunctional and lose effector functions and proliferative capacity. To identify the importance of micro‐RNA‐155 (miR‐155) in this phenomenon, we analyzed mouse miR‐155‐deficient CD4⁺ T cells in a model where the chronic exposure to a systemic antigen led to T‐cell functional unresponsiveness. We found that miR‐155 was required for restoring function of T cells after programmed death receptor 1 blockade. Heme oxygenase 1 (HO‐1) was identified as a specific target of miR‐155 and inhibition of HO‐1 activity restored the expansion and tissue migration capacity of miR‐155^?/? CD4⁺ T cells. Moreover, miR‐155‐mediated control of HO‐1 expression in CD4⁺ T cells was shown to sustain in vivo antigen‐specific expansion and IL‐2 production. Thus, our data identify HO‐1 regulation as a mechanism by which miR‐155 promotes T‐cell‐driven inflammation.     

Requirement for interleukin‐1 to drive brain inflammation reveals tissue‐specific mechanisms of innate immunity

The immune system is implicated in a wide range of disorders affecting the brain and is, therefore, an attractive target for therapy. Interleukin‐1 (IL‐1) is a potent regulator of the innate immune system important for host defense but is also associated with injury and disease in the brain. Here, we show that IL‐1 is a key mediator driving an innate immune response to inflammatory challenge in the mouse brain but is dispensable in extracerebral tissues including the lung and peritoneum. We also demonstrate that IL‐1α is an important ligand contributing to the CNS dependence on IL‐1 and that IL‐1 derived from the CNS compartment (most likely microglia) is the major source driving this effect. These data reveal previously unknown tissue‐specific requirements for IL‐1 in driving innate immunity and suggest that IL‐1‐mediated inflammation in the brain could be selectively targeted without compromising systemic innate immune responses that are important for resistance to infection. This property could be exploited to mitigate injury‐ and disease‐associated inflammation in the brain without increasing susceptibility to systemic infection, an important complication in several neurological disorders.     

New assay to detect low‐affinity interactions and characterization of leukocyte receptors for collagen including leukocyte‐associated Ig‐like receptor‐1 (LAIR‐1)

Leukocyte activity is controlled by numerous interactions between membrane receptors and ligands on the cell surface. These interactions are of low affinity making detection difficult. We developed a sensitive assay that could readily detect extremely weak interactions such as that between CD200 and the activating receptor CD200RLa (K_d>500 μM) at the protein level. We used the new technology to screen for interactions of inhibitory receptors for collagens. We confirmed that both human and mouse leukocyte‐associated Ig‐like receptor‐1, and in addition the related inhibitory leukocyte Ig‐like receptor subfamily B member 4 (CD85K, Gp49B), bound collagen specifically, whereas other cell surface proteins gave no binding. The monomeric affinities of the interactions were then determined to allow comparison with other leukocyte interactions and indicate conditions when these interactions might lead to inhibitory signals.     

Effect of Temperature on the Mobility of Core‐Shell‐Type Nanoparticles Composed of Poly(γ‐benzyl‐L‐glutamate) and Poly(N‐isopropylacrylamide)

Summary: Core‐shell‐type nanoparticles composed of PBLG and PNIPAAm were prepared in an attempt to study the effects of temperature on the dynamic behavior of temperature‐sensitive polymeric shell, PNIPAAm, in the nanoparticles by ¹H NMR spectroscopy. Spin‐lattice relaxation time (T₁) and line halfwidth in D₂O and CDCl₃ were measured to monitor the change of the chain mobility of PNIPAAm in the GN nanoparticles within the temperature range encompassing its LCST. The molecular motion of PBLG segment in GN nanoparticles in CDCl₃ was also examined and compared with that of the PNIPAAm. The morphology, size distribution, and effect of temperature on the sizes of the GN nanoparticles were also investigated. The temperature dependence of T₁ and line halfwidth suggests that the N‐isopropyl group turns gradually into the solid‐like aggregates at temperatures higher than the LCST of PNIPAAm due to the collapsed coil‐globule transition. T₁ values of N‐isopropyl groups decreased with increasing PBLG content in GN diblock copolymer, suggesting the mobility of PNIPAAm chain, as the shell, becomes more restricted at higher PBLG core content.

Changes in the mobility of PNIPAAm shell in the core‐shell‐type nanoparticles composed of PBLG and PNIPAAm.     

Comparing P300 modulations: Target‐to‐target interval versus infrequent nontarget‐to‐nontarget interval in a three‐stimulus task

This study examined temporal determinants of the P300 component of the ERP in a three‐stimulus visual oddball task. Frequent standards, with equiprobable targets and infrequent nontargets, were utilized. We tested whether the infrequent nontarget‐to‐nontarget interval (infrequent NNI) influences P300 amplitudes and latencies analogously to the target‐to‐target interval (TTI). EEG was recorded from 27 participants, and response time and P300 effects of TTIs and infrequent NNIs were assessed. Increases in TTI augmented target P300 amplitudes and decreased latencies and response times. However, this modulation of P300 amplitude was weak for manipulations of infrequent NNI. P300 latencies increased initially before decreasing across infrequent NNI levels. Together, these findings support the notion that the P300 has an underlying temporal mechanism that is modulated by motivationally significant events. Theoretical implications are discussed.     

High‐mobility group box 1 released by autophagic cancer‐associated fibroblasts maintains the stemness of luminal breast cancer cells

Cancer stem cells/cancer‐initiating cells (CICs) and their microenvironmental niche play a vital role in malignant tumour recurrence and metastasis. Cancer‐associated fibroblasts (CAFs) are major components of the niche of breast cancer‐initiating cells (BCICs), and their interactions may profoundly affect breast cancer progression. Autophagy has been considered to be a critical process for CIC maintenance, but whether it is involved in the cross‐talk between CAFs and CICs to affect tumourigenesis and pathological significance has not been determined. In this study, we found that the presence of CAFs containing high levels of microtubule‐associated protein 1 light chain 3 (LC3II), a marker of autophagosomes, was associated with more aggressive luminal human breast cancer. CAFs in human luminal breast cancer tissues with high autophagy activity enriched BCICs with increased tumourigenicity. Mechanistically, autophagic CAFs released high‐mobility group box 1 (HMGB1), which activated its receptor, Toll‐like receptor (TLR) 4, expressed by luminal breast cancer cells, to enhance their stemness and tumourigenicity. Furthermore, immunohistochemistry of 180 luminal breast cancers revealed that high LC3II/TLR4 levels predicted an increased relapse rate and a poorer prognosis. Our findings demonstrate that autophagic CAFs play a critical role in promoting the progression of luminal breast cancer through an HMGB1–TLR4 axis, and that both autophagy in CAFs and TLR4 on breast cancer cells constitute potential therapeutic targets. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

MLH1‐mediated recruitment of FAN1 to chromatin for the induction of apoptosis triggered by O6‐methylguanine

O⁶‐Methylguanines (O⁶‐meG), which are produced in DNA by the action of alkylating agents, are mutagenic and cytotoxic, and induce apoptosis in a mismatch repair (MMR) protein‐dependent manner. To understand the molecular mechanism of O⁶‐meG‐induced apoptosis, we performed functional analyses of FANCD2 and FANCI‐associated nuclease 1 (FAN1), which was identified as an interacting partner of MLH1. Immunoprecipitation analyses showed that FAN1 interacted with both MLH1 and MSH2 after treatment with N‐methyl‐N‐nitrosourea (MNU), indicating the formation of a FAN1‐MMR complex. In comparison with control cells, FAN1‐knockdown cells were more resistant to MNU, and the appearances of a sub‐G₁ population and caspase‐9 activation were suppressed. FAN1 formed nuclear foci in an MLH1‐dependent manner after MNU treatment, and some were colocalized with both MLH1 foci and single‐stranded DNA (ssDNA) created at damaged sites. Under the same condition, FANCD2 also formed nuclear foci, although it was dispensable for the formation of FAN1 foci and ssDNA. MNU‐induced formation of ssDNA was dramatically suppressed in FAN1‐knockdown cells. We therefore propose that FAN1 is loaded on chromatin through the interaction with MLH1 and produces ssDNA by its exonuclease activity, which contributes to the activation of the DNA damage response followed by the induction of apoptosis triggered by O⁶‐meG.     

Expression of cyclooxygenase‐2 in intestinal goblet cells of pre‐diabetic NOD mice

Cyclooxygenase, the rate‐limiting enzyme in prostaglandin synthesis, is expressed in constitutive (COX‐1) and inducible (COX‐2) isoforms. The COX‐2 has been proposed to be involved in development of autoimmune type 1 diabetes (T1D). We examined COX‐2 expression in the gut‐associated lymphoid tissue (GALT), and found COX‐2 was strongly expressed in goblet cells of non‐obese diabetic (NOD) mice at the apical villi at the age of 2.5 weeks, clearly before the onset of insulitis, while the expression in the control BALB/c mice was weak or absent at all ages (P < 0.001). Lipopolysaccharide (LPS) given intraperitoneally slightly increased COX‐2 expression in the goblet cells and epithelium of both NOD and BALB/c mice. High‐resolution confocal microscopy showed that the surroundings of the goblet cells contained no COX‐2, implying that the enzyme is synthesized by the goblet cells. The COX‐2 is secreted from goblet cells into the intestinal lumen along with mucins. The COX‐2 concentration in the goblet cell of BALB/c and especially of NOD mice was markedly higher than that in the intraepithelial lymphocytes or lamina propria macrophages. High mucin COX‐2 from goblet cells may increase luminal prostaglandin synthesis, alter epithelial permeability, modulate intestinal immune responses and modify functional properties of the lymphocytes in the GALT, which all may be important for the initiation of the autoimmune phenomenon in the NOD mice.     

TXNDC5 contributes to rheumatoid arthritis by down‐regulating IGFBP1 expression

The thioredoxin domain‐containing 5 (TXNDC5) gene is associated with susceptibility to rheumatoid arthritis (RA) and exhibits increased expression in the synovial tissues. TXNDC5 is also associated strongly with diabetes, a metabolic disease characterized by interrupted insulin signalling. This study investigated whether TXNDC5 contributes to RA via the insulin signalling pathway. In this study, RA synovial fibroblast‐like cells (RASFs) transfected with an anti‐TXNDC5 small interfering RNA (siRNA) were analysed with an insulin signaling pathway RT² profiler polymerase chain reaction (PCR) array and an insulin resistance RT² profiler PCR array. The PCR arrays detected significantly increased expression of insulin‐like growth factor binding protein 1 (IGFBP1) in RASFs with suppressed TXNDC5 expression. The result was verified using real‐time PCR and Western blot analyses. Significantly elevated IGFBP1 expression and decreased interleukin (IL)‐6 secretion were also detected in culture medium of transfected RASFs. Furthermore, decreased IGFBP1 mRNA and protein expression levels were detected in RA synovial tissues. Additionally, significantly increased apoptosis and decreased cell proliferation and cell migration were observed in RASFs transfected with the anti‐TXNDC5 siRNA, whereas transfection with the anti‐IGFBP1 siRNA or a mixture of the anti‐IGFBP1 and anti‐TXNDC5 siRNAs restored normal cell proliferation, migration and IL‐6 level in RASFs. Insulin‐like growth factor (IGF) has potent prosurvival and anti‐apoptotic functions, and IGFBP1 can suppress IGF activity. Based on the results of the present study, we suggest that TXNDC5 contributes to abnormal RASF proliferation, migration and IL‐6 production by inhibiting IGFBP1 expression.     

Counter‐Anion Effect on the Properties of Anion‐Conducting Polymer Electrolyte Membranes Prepared by Radiation‐Induced Graft Polymerization

Graft‐type anion‐conducting polymer electrolyte membranes (AEMs) are prepared by the radiation‐induced graft polymerization of chloromethylstyrene into poly(ethylene‐co‐tetrafluoroethylene) (ETFE) films and subsequent quaternization with trimethylamine. AEMs in the hydroxide form (AEM‐OH) are prepared by immersing the chloride form (AEM‐Cl) in 1 M potassium hydroxide (KOH) solution, followed by KOH and washing with nitrogen‐saturated water to prevent bicarbonate formation (AEM‐HCO₃). The AEM‐OH shows conductivity and water uptake four and two times higher than AEM‐Cl and ‐HCO₃ and is thermally and chemically less stable, resulting in the tendency to absorb water and to convert to the bicarbonate form.

     

Synthesis and Characterization of Comb‐Like Copolymers Based on Poly(ε‐caprolactone) and Poly(α‐olefin)

Comb‐like copolymers based on a polyolefin backbone of poly(10‐undecene‐1‐ol) (PUol) with poly(ε‐caprolactone) (PCL) side chains are synthesized in two steps. After synthesis of PUol by metallocene‐catalyzed polymerization, the side‐chain hydroxyl functionalities of this polar polyolefin are used as an initiator for the ring‐opening polymerization (ROP) of ε‐caprolactone (CL). In this context, copolymers with different lengths of PCL grafts are prepared. The chemical structure and the composition of the synthesized copolymers are characterized by ¹H and ¹³C NMR spectroscopy. It is shown that the hydroxyl end groups of PUol act effectively as initiating sites for the CL ROP. Size‐exclusion chromatography (SEC) measurements confirm the absence of non‐attached PCL and the expected increase in molar mass after grafting. The thermal and decomposition behaviors are investigated by DSC and thermogravimetric analysis (TGA). The effect of the length of the PCL grafts on the crystallization behavior of the comb‐like copolymers is investigated by DSC and wide‐angle X‐ray scattering (WAXS).

     

Peritoneal cavity B‐1a cells promote peripheral CD4+ T‐cell activation

Innate‐like murine B‐1a cells are well known for their ability to secrete natural IgM. Their non‐Ab mediated functions, including Ag presentation to CD4⁺ T cells, are less well explored. Using combined adoptive transfer experiments with peptide‐pulsed peritoneal cavity (PerC)‐derived B‐1a cells and CFSE‐labeled T cells, we show that B‐1a cells present Ag to CD4⁺ T cells from the periphery in vivo. In vitro characterization, using co‐cultures in which B‐1a or splenic B cells presented whole OVA protein to OVA‐specific Tg T cells, shows that B‐1a cells differentially promote intracellular cytokine‐expressing T cells. PerC‐derived B‐1a cells increase the percentage of IL‐10‐producing T cells along with IL‐4‐ and IFN‐γ‐producing CD4⁺ T cells. These data suggest that B cells in the PerC have the potential to influence peripheral immune responses without the necessity to migrate out of this location. This, to our knowledge previously undescribed, immuno‐logical pathway potentially plays a role in the presentation of gut microbiota‐derived Ags to peripheral T cells.     

Nano‐particle vaccination combined with TLR‐7 and ‐9 ligands triggers memory and effector CD8+ T‐cell responses in melanoma patients

Optimal vaccine strategies must be identified for improving T‐cell vaccination against infectious and malignant diseases. MelQbG10 is a virus‐like nano‐particle loaded with A‐type CpG‐oligonucleotides (CpG‐ODN) and coupled to peptide_16–35 derived from Melan‐A/MART‐1. In this phase IIa clinical study, four groups of stage III‐IV melanoma patients were vaccinated with MelQbG10, given (i) with IFA (Montanide) s.c.; (ii) with IFA s.c. and topical Imiquimod; (iii) i.d. with topical Imiquimod; or (iv) as intralymph node injection. In total, 16/21 (76%) patients generated ex vivo detectable Melan‐A/MART‐1‐specific T‐cell responses. T‐cell frequencies were significantly higher when IFA was used as adjuvant, resulting in detectable T‐cell responses in all (11/11) patients, with predominant generation of effector‐memory‐phenotype cells. In turn, Imiquimod induced higher proportions of central‐memory‐phenotype cells and increased percentages of CD127⁺ (IL‐7R) T cells. Direct injection of MelQbG10 into lymph nodes resulted in lower T‐cell frequencies, associated with lower proportions of memory and effector‐phenotype T cells. Swelling of vaccine site draining lymph nodes, and increased glucose uptake at PET/CT was observed in 13/15 (87%) of evaluable patients, reflecting vaccine triggered immune reactions in lymph nodes. We conclude that the simultaneous use of both Imiquimod and CpG‐ODN induced combined memory and effector CD8⁺ T‐cell responses.     

IGFBP‐6 plays a role as an oncosuppressor gene in NPC pathogenesis through regulating EGR‐1 expression

Nasopharyngeal carcinoma (NPC) is prevalent in south‐eastern Asia, particularly southern China, Singapore and Taiwan. The aim of this study was to identify the pivotal genes that may be altered during NPC progression. Using cDNA microarray analysis, we compared the expression of 18 genes between NPC and normal nasomucosal cells. qRT–PCR analysis found the expression of IBFBP‐6 in NPC cell lines and immunolocalization of IGFBP‐6 in NPC to be very weak. To explore the effects of IGFBP‐6 on NPC tumour growth, we constructed inducible plasmids containing full‐length IGFBP‐6 cDNA (pBIG2i‐IGFBP‐6) and established pBIG2i‐IGFBP‐6‐transfected NPC stable cell lines (NPC‐TW01‐pBIG2i‐IGFBP‐6). We then performed functional analysis of the IGFBP‐6 in cell lines in vitro and in vivo. Over‐expression of IGFBP‐6 significantly suppressed the proliferation, invasion and metastatic activity of NPC cells and increased their apoptosis. We found the EGR‐1, caspase‐1 and TSP‐1 genes to be markedly up‐regulated when NPC‐pBIG2i‐IGFBP‐6 was treated with doxycycline. Knocking down EGR‐1 with EGR‐1 siRNA resulted in a decrease in expression of caspase‐1, TSP‐1 and EGR‐1 but not the expression of IGFBP‐6. However, in knockdown cells the unchanged expression of IGFBP‐6 did not inhibit the migration of NPC cells. Chromatin immunoprecipitation and luciferase reporter assay experiments showed that IGFBP‐6 bound the EGR‐1 promoter regions and activated EGR‐1 promoter. We conclude that IGFBP‐6 can regulate the progression of NPC by regulating the expression of EGR‐1. These results suggest that IGFBP‐6 could be used as a new target in NPC therapy. Copyright © 2010 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Highly Air‐Stable Thieno[3,2‐b]thiophene‐Thiophene‐Thiazolo[5,4‐d]thiazole‐Based Polymers for Light‐Emitting Diodes

A series of highly air‐stable, low‐bandgap poly(3‐alkylthiophene)s containing electron‐rich thieno[3,2‐b]thiophene and electron‐deficient thiazolo[5,4‐d]thiazole rings were synthesized by the Stille coupling reaction. The polymers exhibited good thermal stability and solubility with excellent film forming properties when drop‐ or spin‐cast from solution. A strong absorption at 564–568 nm and a shoulder at 614–616 nm were observed. The optical bandgap of the polymers was found to be 1.82–1.85 eV. The IP of the polymers was found to be 5.62–5.65 eV. All polymers showed strong fluorescent emission both in solution and in the solid state. EL devices were fabricated using the polymers as an emissive layer and red emission was observed with the emission range of 649–679 nm.