The linker histone H1.2 is a novel component of the nucleolar organizer regions.

The nucleoli accumulate rRNA genes and are the sites of rRNA synthesis and rRNA assembly into ribosomes. During mitosis, nucleoli dissociate, but nucleolar remnants remain on the rRNA gene loci, forming distinct nucleolar organizer regions (NORs). Little is known about the composition and structure of NORs, but upstream binding factor (UBF) has been established as its master organizer. In this study, we sought to establish new proteins in NORs. Using UBF-Sepharose to isolate UBF-binding proteins, we identified histone H1.2 as a candidate partner but were puzzled by this observation, given that UBF is known to be located predominantly in nucleoli, whereas H1.2 distributed broadly among the chromatins in interphase nuclei. We then examined cells undergoing mitosis and saw that both H1.2 and UBF were recruited into NORs in this state, reconciling the results of our UBF pulldowns. Inhibiting rRNA synthesis in interphase nuclei also induced NOR-like structures containing both UBF and H1.2. When chromosomes were isolated and spread on coverslips, NORs appeared separated from the chromosomes containing both UBF and H1.2. After chromosomes were fragmented by homogenization, intact NORs remained visible. Results collectively suggest that NORs are independent structures and that the linker histone H1.2 is a novel component of this structure.

Broad Susceptibility of Nucleolar Proteins and Autoantigens to Complement C1 Protease Degradation.

Anti-nuclear autoantibodies, which frequently target the nucleoli, are pathogenic hallmarks of systemic lupus erythematosus (SLE). Although the causes of these Abs remain broad and ill-defined, a genetic deficiency in C1 complex (C1qC1r2C1s2) or C4 is able to induce these Abs. Considering a recent finding that, in dead cells, nucleoli were targeted by C1q and two nucleolar autoantigens were degraded by C1r/C1s proteases, we considered that C1 could help protect against antinuclear autoimmunity by broadly degrading nucleolar proteins or autoantigens. Nucleoli were isolated to homogeneity and structurally defined. After C1 treatment, cleaved nucleolar proteins were identified by proteomic two-dimensional fluorescence difference gel electrophoresis and mass spectrometry, and further verified by Western blotting using specific Abs. The extent of nucleolar autoantigen degradation upon C1 treatment was estimated using SLE patient autoantibodies. The isolated nucleoli were broadly reactive with SLE patient autoantibodies. These nucleoli lacked significant autoproteolysis, but many nucleolar proteins and autoantigens were degraded by C1 proteases; >20 nucleolar proteins were identified as C1 cleavable. These were further validated by Western blotting using specific Abs. The broad autoantigenicity of the nucleoli may attribute to their poor autoproteolysis, causing autologous immune stimulation upon necrotic exposure. However, C1q targets at these nucleoli to cause C1 protease activation and the cleavage of many nucleolar proteins or autoantigens. This may represent one important surveillance mechanism against antinuclear autoimmunity because C1 genetic deficiency causes anti-nuclear autoantibodies and SLE disease.

The axis of complement C1 and nucleolus in antinuclear autoimmunity.

Antinuclear autoantibodies (ANA) are heterogeneous self-reactive antibodies that target the chromatin network, the speckled, the nucleoli, and other nuclear regions. The immunological aberration for ANA production remains partially understood, but ANA are known to be pathogenic, especially, in systemic lupus erythematosus (SLE). Most SLE patients exhibit a highly polygenic disease involving multiple organs, but in rare complement C1q, C1r, or C1s deficiencies, the disease can become largely monogenic. Increasing evidence point to intrinsic autoimmunogenicity of the nuclei. Necrotic cells release fragmented chromatins as nucleosomes and the alarmin HMGB1 is associated with the nucleosomes to activate TLRs and confer anti-chromatin autoimmunogenecity. In speckled regions, the major ANA targets Sm/RNP and SSA/Ro contain snRNAs that confer autoimmunogenecity to Sm/RNP and SSA/Ro antigens. Recently, three GAR/RGG-containing alarmins have been identified in the nucleolus that helps explain its high autoimmunogenicity. Interestingly, C1q binds to the nucleoli exposed by necrotic cells to cause protease C1r and C1s activation. C1s cleaves HMGB1 to inactive its alarmin activity. C1 proteases also degrade many nucleolar autoantigens including nucleolin, a major GAR/RGG-containing autoantigen and alarmin. It appears that the different nuclear regions are intrinsically autoimmunogenic by containing autoantigens and alarmins. However, the extracellular complement C1 complex function to dampen nuclear autoimmunogenecity by degrading these nuclear proteins.

The GAR/RGG motif defines a family of nuclear alarmins.

The nucleus is the target of autoantibodies in many diseases, which suggests intrinsic nuclear adjuvants that confer its high autoimmunogenicity. Nucleolin (NCL) is one abundant nucleolar autoantigen in systemic lupus erythematosus (SLE) patients and, in lupus-prone mice, it elicits autoantibodies early. With purified NCL, we observed that it was a potent alarmin that activated monocytes, macrophages and dendritic cells and it was a ligand for TLR2 and TLR4. NCL released by necrotic cells also exhibited alarmin activity. The NCL alarmin activity resides in its glycine/arginine-rich (GAR/RGG) motif and can be displayed by synthetic GAR/RGG peptides. Two more GAR/RGG-containing nucleolar proteins, fibrillarin (FBRL) and GAR1, were also confirmed to be novel alarmins. Therefore, the GAR/RGG alarmin motif predicts a family of nucleolar alarmins. The apparent prevalence of nucleolar alarmins suggests their positive contribution to tissue homeostasis by inducing self-limiting tissue inflammation with autoimmunity only occurring when surveillance is broken down.

Intense versus standard regimens of intermittent occlusion therapy for unilateral moderate amblyopia in children: study protocol for a randomized controlled trial.

BACKGROUND: We reported that in our previous study that wearing intermittent occlusion therapy glasses (IO-therapy) for 4 hours (h) was non-inferior to patching for 2 h in 3 to 8-year-old children with amblyopia. We hypothesize that an intense regimen of 12-h IO-therapy per day for 4 weeks could be as effective as the standard regimen of 4-h IO-therapy per day for 12 weeks in treating moderate amblyopia in 3 to 8-year-old children. METHODS/DESIGN: A total of 56 children between 3 and 8 years of age with amblyopia in association with anisometropia and/or strabismus will be enrolled. All participants will be prescribed IO-therapy glasses (Amblyz™), set at 30-s opaque/transparent intervals (i.e., occluded 50% of wear time). They will be randomized to receive the standard regimen for 12 weeks or the intense regimen for 4 weeks. Adherence to using the IO-therapy glasses will be objectively monitored in each participant by means of a microsensor dose monitor. The primary study objective is to compare the effectiveness of an intense regimen to a standard regimen of IO-therapy in 3 to 8-year-old children with moderate amblyopia. The secondary study objectives are to determine whether adherence differs between an intense regimen and a standard regimen of IO-therapy, and to determine the dose-response relationship of IO-therapy. DISCUSSION: In addition to testing the effectiveness, this study will test for the first time the association between treatment adherence and the visual outcome of IO-therapy, which will enhance our understanding of the dose-response relationship of IO-therapy. If an intense regimen is shown to be effective, it would alter amblyopia treatment strategies and improve visual outcomes. TRIAL REGISTRATION: ClinicalTrials.gov: NCT02767856. Registered on 10 May 2016.

Feasibility of monitoring compliance with intermittent occlusion therapy glasses for amblyopia treatment.

BACKGROUND: Liquid crystal glasses use an intermittent occlusion technique and may improve compliance compared to adhesive patches. Previous studies support the effectiveness of intermittent occlusion therapy (IO therapy) glasses for amblyopia treatment. However, objective compliance for these glasses has not been measured. The purpose of this study was to investigate the feasibility of using a microsensor to monitor objective compliance with IO therapy glasses. METHODS: Children 3 to ≤8 years of age with unilateral amblyopia were enrolled. All subjects had optimal refractive correction (if needed) for at least 5 weeks without improvement. Subjects were prescribed IO therapy glasses, set at 30-second opaque/transparent intervals (ie, occluded 50% of wear time). Wear time was prescribed according to amblyopia severity. For each patient, objective compliance with the IO therapy glasses was monitored by means of a microsensor. RESULTS: A total of 13 subjects returned with microsensor data. Compliance varied among and within individuals. General compliance averaged 51.6% (range, 10%-97%). Mean daily compliance decreased slightly over time. On average, patients' visual acuity improved 0.14 ± 0.15 logMAR (range, -0.1 to 0.5 logMAR). No parents reported that their child had social concerns related to the attached microsensor. CONCLUSIONS: Objective compliance with IO therapy glasses can be monitored by a simple microsensor reliably. In our study cohort, objective compliance with IO therapy glasses varied among individuals, but on average it declined slightly over time.

Characterisation of a human antibody that potentially links cytomegalovirus infection with systemic lupus erythematosus.

Human cytomegalovirus (HCMV) is a ubiquitous herpesvirus that has been linked with the development of systemic lupus erythematosus (SLE). Thus far, molecular mimicry has been implicated as the principal mechanism that explains this association. In this study, we characterise a potential alternative process whereby HCMV contributes to SLE. In a cohort of SLE patients, we show a significant association between HCMV infection and SLE through a human antibody response that targets UL44. UL44 is an obligate nuclear-resident, non-structural viral protein vital for HCMV DNA replication. The intracellular nature of this viral protein complicates its targeting by the humoral response - the mechanism remains unresolved. To characterise this response, we present a thorough molecular analysis of the first human monoclonal antibody specific for UL44 derived from a HCMV seropositive donor. This human antibody immunoprecipitates UL44 from HCMV-infected cells together with known nuclear-resident SLE autoantigens - namely, nucleolin, dsDNA and ku70. We also show that UL44 is redistributed to the cell surface during virus-induced apoptosis as part of a complex with these autoantigens. This phenomenon represents a potential mechanism for the bystander presentation of SLE autoantigens to the humoral arm of our immune system under circumstances that favour a break in peripheral tolerance.