Search

Ryan M Walk,1,2 Steven T Elliott,2 Felix C Blanco,2 Jason A Snyder,2 Ashley M Jacobi,3 Scott D Rose,3 Mark A Behlke,3 Aliasger K Salem,4 Stanislav Vukmanovic,2 Anthony D Sandler21Department of Surgery, Walter Reed Army Medical Center, Washington, DC, USA; 2Sheikh Zayed Institute for Pediatric Surgical Innovation, Children's National Medical Center, Washington, DC, USA; 3Integrated DNA Technologies, Coralville, IA, USA; 4Division of Pharmaceutics, University of Iowa, Iowa City, IA, USAAbstract: Toll-like receptor (TLR) agonists represent potentially useful cancer vaccine adjuvants in their ability to stimulate antigen-presenting cells (APCs) and subsequently amplify the cytotoxic T-cell response. The purpose of this study was to characterize APC responses to TLR activation and to determine the subsequent effect on lymphocyte activation. We exposed murine primary bone marrow-derived macrophages to increasing concentrations of agonists to TLRs 2, 3, 4, and 9. This resulted in a dose-dependent increase in production of not only tumor necrosis factor–alpha (TNF-α), a surrogate marker of the proinflammatory response, but also interleukin 10 (IL-10), a well-described inhibitory cytokine. Importantly, IL-10 secretion was not induced by low concentrations of TLR agonists that readily produced TNF-α. We subsequently stimulated lymphocytes with anti-CD3 antibody in the presence of media from macrophages activated with higher doses of TLR agonists and observed suppression of interferon gamma release. Use of both IL-10 knockout macrophages and IL-10 small-interfering RNA (siRNA) ablated this suppressive effect. Finally, IL-10 siRNA was successfully used to suppress CpG-induced IL-10 production in vivo. We conclude that TLR-mediated APC stimulation can induce a paradoxical inhibitory effect on T-cell activation mediated by IL-10.Keywords: toll-like receptors, innate immunity, IL-10

Systemic lupus erythematosus (SLE) can be a severe and potentially life-threatening disease that often represents a therapeutic challenge because of its heterogeneous organ manifestations. Only glucocorticoids, chloroquine and hydroxychloroquine, azathioprine, cyclophosphamide and very recently belimumab have been approved for SLE therapy in Germany, Austria and Switzerland. Dependence on glucocorticoids and resistance to the approved therapeutic agents, as well as substantial toxicity, are frequent. Therefore, treatment considerations will include ‘off-label’ use of medication approved for other indications. In this consensus approach, an effort has been undertaken to delineate the limits of the current evidence on therapeutic options for SLE organ disease, and to agree on common practice. This has been based on the best available evidence obtained by a rigorous literature review and the authors’ own experience with available drugs derived under very similar health care conditions.Preparation of this consensus document included an initial meeting to agree upon the core agenda, a systematic literature review with subsequent formulation of a consensus and determination of the evidence level followed by collecting the level of agreement from the panel members. In addition to overarching principles, the panel have focused on the treatment of major SLE organ manifestations (lupus nephritis, arthritis, lung disease, neuropsychiatric and haematological manifestations, antiphospholipid syndrome and serositis).This consensus report is intended to support clinicians involved in the care of patients with difficult courses of SLE not responding to standard therapies by providing up-to-date information on the best available evidence. [ABSTRACT FROM AUTHOR]

OBJECTIVE: To understand the effects of long-term BLyS inhibition in human systemic lupus erythematosus (SLE). METHODS: Seventeen patients with SLE who were enrolled in a clinical trial of belimumab, a BLyS-specific inhibitor, plus standard of care therapy were studied. Phenotypic analysis of lymphocytes was performed using flow cytometry. Circulating antibody-secreting cells were enumerated using enzyme-linked immunospot assay. Serum was analyzed by enzyme-linked immunosorbent assay using an antibody that recognizes products of the V(H)4-34 gene. Lymphocyte counts, Ig levels, and anti-double-stranded DNA antibody levels were available as part of the clinical trial analyses. RESULTS: Samples were collected on days 0, 84, 168, 365, and 532 and after day 730. The total number of B cells started to decrease from baseline between days 84 and 168. This was due to a decrease in naive and transitional B cells. CD27+IgD+ memory B cells and plasmablasts decreased only after 532 days, whereas CD27+IgD- memory B cells were not affected, and there were no changes in T cells. Serum IgM levels began to decline between days 84 and 168, but there were no changes in serum levels of IgG, IgG anti-DNA antibodies, or V(H)4-34 antibodies during the study. SLE patients had more IgM-, IgG-, and autoantibody-producing B cells than did normal controls on day 0. There was only a modest decrease in the frequency of total IgM-producing, but not IgG-producing, cells on days 365 and 532, consistent with the phenotypic and serologic data. CONCLUSION: Our data confirm the dependence of newly formed B cells on BLyS for survival in humans. In contrast, memory B cells and plasma cells are less susceptible to selective BLyS inhibition. [ABSTRACT FROM AUTHOR]

Objectives: Monitoring of peripheral B-cell subsets in patients with systemic lupus erythematosus (SLE) revealed an activity-related expansion of CD27(++)CD20(-)CD19(dim) Ig-secreting cells. A similar subset has also been identified 6-8 days after tetanus/diphtheria vaccination in normal individuals and in patients with infectious disease.Methods: This subset was analysed further focussing on the HLA-DR surface expression in a cohort of 25 patients with SLE.Results: This study revealed that 86% (range 59-97%) of CD27(++)CD20(-)CD19(dim) cells express high levels of HLA-DR, are also expanded in the bone marrow, and represent plasmablasts enriched with anti-dsDNA secreting cells. The remaining CD27(++)CD20(-)CD19(dim) cells were HLA-DR(low) and represent mature plasma cells. Importantly, HLA-DR(high) plasmablasts showed a closer correlation with lupus activity and anti-dsDNA levels than the previously identified CD27(++)CD20(-)CD19(dim) cells.Conclusion: HLA-DR(high)CD27(++)CD20(-)CD19(dim) plasmablasts represent a more precise indicator of lupus activity and suggest that there is an overproduction or lack of negative selection of these cells in SLE. [ABSTRACT FROM AUTHOR]

OBJECTIVE: Analysis of peripheral B cell subsets in patients with systemic lupus erythematosus (SLE) has provided evidence of specific alterations, such as an expansion of CD27++ plasma cells/blasts and transitional B cells. However, memory B cells in lupus have not been thoroughly investigated, and only recently a CD27- memory B cell subset was identified in the peripheral blood of lupus patients. Focusing on CD27- B cells, this study aimed to identify abnormalities in peripheral B cell subsets in patients with SLE. METHODS: Three independent cohorts of lupus patients were used to characterize CD27- memory B cells, using multiparameter flow cytometry and single-cell reverse transcription-polymerase chain reaction of heavy-chain transcripts. RESULTS: We identified a homogeneous subset of CD27-,IgD-,CD95+ memory B cells with an activated phenotype that was increased in patients with disease flares and that correlated with disease activity and serologic abnormalities. In contrast, the entire subset of CD27-,IgD- B cells was found to be heterogeneous, did not correlate significantly with lupus activity, and was also increased in patients with bacterial infections. CONCLUSION: We conclude that CD95 is a useful marker to identify CD27- memory B cells with an activated phenotype, which might serve as a biomarker for lupus activity and as a target of further investigations aiming to elucidate the pathogenic potential of these cells and the mechanisms involved in the generation as well as regulation of this CD27-,IgD-,CD95+ memory B cell subset. [ABSTRACT FROM AUTHOR]

OBJECTIVE: Both the genesis and outgrowth of extranodal marginal-zone B cell lymphomas (MZLs) of the mucosa-associated lymphoid tissue (MALT) type are generally thought to represent antigen-driven processes. We undertook this study to analyze lymphoma progression and dissemination outside of the MALT-type lesions. METHODS: Histopathologic and Ig heavy- and light-chain variable-region gene (V(H/L)) analyses were performed in sequential tissue samples from a patient with primary Sjögren's syndrome (SS) with glandular (parotid) manifestations and subsequent nodal dissemination of a low-grade MZL. RESULTS: This MZL expressed a CD20+,CD27+,sIgM/kappa+,IgD-,CD5-,CD10-,Bcl-6-,CD23-,p53-,p21-, MDM2- phenotype and mutated V(H)1-69/D2-21/J(H)4alpha-V(kappa)A27/J(kappa)2 Ig rearrangements. Notably, circulating lymphoma cells from the parotid glands occurred transiently in the patient's blood, as detected by single-cell polymerase chain reaction. In addition, 2 minor B cell clones (clones 2 and 3, with V(H)3-07/D3-22/J(H)3b-V(lambda)3L/J(lambda)2/3 and V(H)3-64/D3-03/J(H)2-V(kappa)A19/J(kappa)2 rearrangements, respectively) were also detected in the parotid glands and blood, and 1 of these (clone 2) was also detected in the lymph nodes. Ig V(H/L) analyses revealed ongoing (antigen-driven) mutations of the glandular lymphoma rearrangements, but an invariant mutation pattern of their nodal counterparts. CONCLUSION: These data indicate coexpansion and transient (re)circulation of the lymphoma clone and 2 additional glandular B cell clones in a primary SS-associated extranodal MZL. Combined histologic and molecular features of the nodal lymphoma subclone reflect a process of 'follicular colonization' that eventually froze the mutation machinery after accumulation of additional (antigen-driven) Ig V(H/L) mutations. [ABSTRACT FROM AUTHOR]

Recent accumulated evidence suggests that prolactin (PRL) is an important immunomodulator and plays a part in the pathogenesis of systemic lupus erythematosus (SLE). The current study assessed the frequency of hyperprolactinaemia in patients with SLE and its association with defined clinical manifestations or serological abnormalities. PRL levels were analysed in 60 patients with SLE including a follow-up of 20 patients, 18 patients with rheumatic autoimmune diseases other than SLE (AID) and in 47 normal healthy subjects (NHS) using ELISA. Clinical manifestations and disease activity (ECLAM) were recorded. Autoantibodies (anti-dsDNA, anti-CL) were determined by standard techniques. In all, 28.3% of the patients with SLE had raised serum PRL. Their PRL levels (17.4±15.1 ng/ml, P<0.0001) and those of patients with AID (13.1±10.3 ng/ml, P<0.001) were significantly higher compared to NHS (6.3±3.2 ng/ml). Anti-dsDNA (rs=0.3, P=0.04) and anti-CL antibody titres (IgG; rs=0.3, P=0.03) correlated with PRL level. Furthermore, elevated erythrocyte sedimentation rate (ESR), anaemia, decrease in C3, fatigue, fever and renal involvement were associated with hyperprolactinaemia. These results were confirmed by follow-up examinations. Moderate hyperprolactinaemia is present in a subset of patients with SLE and serum PRL correlates with clinical and serological disease activity. [ABSTRACT FROM AUTHOR]

CRISPR-mediated transcriptional activation (CRISPRa) is a powerful technology for inducing gene expression from endogenous loci with exciting applications in high throughput gain-of-function genomic screens and the engineering of cell-based models. However, current strategies for generating potent, stable, CRISPRa-competent cell lines present limitations for the broad utility of this approach. Here, we provide a high-efficiency, self-selecting CRISPRa enrichment strategy, which combined with piggyBac transposon technology enables rapid production of CRISPRa-ready cell populations compatible with a variety of downstream assays. We complement this with an optimized guide RNA scaffold that significantly enhances CRISPRa functionality. Finally, we describe a synthetic guide RNA tool set that enables transient, population-wide gene activation when used with the self-selecting CRISPRa system. Taken together, this versatile platform greatly enhances the potential for CRISPRa across a wide variety of cellular contexts., (© 2023. The Author(s).)

Chimeric antigen receptor (CAR) redirected T cells are potent therapeutic options against hematological malignancies. The current dominant manufacturing approach for CAR T cells depends on retroviral transduction. With the advent of gene editing, insertion of a CD19-CAR into the T cell receptor (TCR) alpha constant ( TRAC ) locus using adeno-associated viruses for gene transfer was demonstrated, and these CD19-CAR T cells showed improved functionality over their retrovirally transduced counterparts. However, clinical-grade production of viruses is complex and associated with extensive costs. Here, we optimized a virus-free genome-editing method for efficient CAR insertion into the TRAC locus of primary human T cells via nuclease-assisted homology-directed repair (HDR) using CRISPR-Cas and double-stranded template DNA (dsDNA). We evaluated DNA-sensor inhibition and HDR enhancement as two pharmacological interventions to improve cell viability and relative CAR knockin rates, respectively. While the toxicity of transfected dsDNA was not fully prevented, the combination of both interventions significantly increased CAR knockin rates and CAR T cell yield. Resulting TRAC -replaced CD19-CAR T cells showed antigen-specific cytotoxicity and cytokine production in vitro and slowed leukemia progression in a xenograft mouse model. Amplicon sequencing did not reveal significant indel formation at potential off-target sites with or without exposure to DNA-repair-modulating small molecules. With TRAC -integrated CAR + T cell frequencies exceeding 50%, this study opens new perspectives to exploit pharmacological interventions to improve non-viral gene editing in T cells., Competing Interests: As part of a collaboration agreement between Charité Universitätsmedizin Berlin and Integrated DNA Technologies (IDT), IDT provided certain reagents (HDR enhancer v.2 and TRAC sgRNA used in some experiments) and performed GUIDE-seq analysis, HDR-enhancing small-molecule screen in Jurkat cells, and targeted sequencing of potential off-target sites. R.T., B.T., M.L.S., G.L.K., and A.M.J. are employees of IDT, which offers reagents for sale similar to some of the compounds described in the manuscript. Products and tools supplied by IDT are for research use only and not intended for diagnostic or therapeutic purposes. Purchaser and/or user are solely responsible for all decisions regarding the use of these products and any associated regulatory or legal obligations. Lonza GmbH provided 96-well 4D-Nucleofector unit and some nucleofection reagents. A.W. and L. Akyüz are part-time employees of CheckImmune GmbH. A.R. and U.E.H. filed a patent application WO 2017211900A1 “Chimeric antigen receptor and CAR T cells that bind BCMA” related to the work with the BCMA-CAR disclosed in this paper. A.R. and U.E.H. have received research funding from Fate Therapeutics for work unrelated to the data generated in the manuscript., (© 2022 The Author(s).)

CRISPR systems enable targeted genome editing in a wide variety of organisms by introducing single- or double-strand DNA breaks, which are repaired using endogenous molecular pathways. Characterization of on- and off-target editing events from CRISPR proteins can be evaluated using targeted genome resequencing. We characterized DNA repair fingerprints that result from non-homologous end joining (NHEJ) after double-stranded breaks (DSBs) were introduced by Cas9 or Cas12a for >500 paired treatment/control experiments. We found that building biological understanding of the repair into a novel analysis tool (CRISPAltRations) improved the quality of the results. We validated our software using simulated, targeted amplicon sequencing data (11 guide RNAs [gRNAs] and 603 on- and off-target locations) and demonstrated that CRISPAltRations outperforms other publicly available software tools in accurately annotating CRISPR-associated indels and homology-directed repair (HDR) events. We enable non-bioinformaticians to use CRISPAltRations by developing a web-accessible, cloud-hosted deployment, which allows rapid batch processing of samples in a graphical user interface (GUI) and complies with HIPAA security standards. By ensuring that our software is thoroughly tested, version controlled, and supported with a user interface (UI), we enable resequencing analysis of CRISPR genome editing experiments to researchers no matter their skill in bioinformatics., Competing Interests: G.K., A.M.J., M.S.M., R.T., G.R.R., N.R., H.L., L.T., M.S., Y.W., and M.A.B. are employees or paid contractors/consultants of Integrated DNA Technologies (IDT), which sells reagents used or similar to those used in this manuscript. M.M., K.F., and R.N. are employees of Illumina Inc., which provides a productized cloud-computing platform for doing NGS analysis., (© 2021 Integrated DNA Technologies, Inc.)

Aging drives pathological accumulation of proteins such as tau, causing neurodegenerative dementia disorders like Alzheimer's disease. Previously we showed loss of function mutations in the gene encoding the poly(A) RNA binding protein SUT-2/MSUT2 suppress tau-mediated neurotoxicity in C. elegans neurons, cultured human cells, and mouse brain, while loss of PABPN1 had the opposite effect (Wheeler et al., 2019). Here we found that blocking poly(A) tail extension with cordycepin exacerbates tauopathy in cultured human cells, which is rescued by MSUT2 knockdown. To further investigate the molecular mechanisms of poly(A) RNA-mediated tauopathy suppression, we examined whether genes encoding poly(A) nucleases also modulated tauopathy in a C. elegans tauopathy model. We found that loss of function mutations in C. elegans ccr-4 and panl-2 genes enhanced tauopathy phenotypes in tau transgenic C. elegans while loss of parn-2 partially suppressed tauopathy. In addition, loss of parn-1 blocked tauopathy suppression by loss of parn-2. Epistasis analysis showed that sut-2 loss of function suppressed the tauopathy enhancement caused by loss of ccr-4 and SUT-2 overexpression exacerbated tauopathy even in the presence of parn-2 loss of function in tau transgenic C. elegans. Thus sut-2 modulation of tauopathy is epistatic to ccr-4 and parn-2. We found that human deadenylases do not colocalize with human MSUT2 in nuclear speckles; however, expression levels of TOE1, the homolog of parn-2, correlated with that of MSUT2 in post-mortem Alzheimer's disease patient brains. Alzheimer's disease patients with low TOE1 levels exhibited significantly increased pathological tau deposition and loss of NeuN staining. Taken together, this work suggests suppressing tauopathy cannot be accomplished by simply extending poly(A) tails, but rather a more complex relationship exists between tau, sut-2/MSUT2 function, and control of poly(A) RNA metabolism, and that parn-2/TOE1 may be altered in tauopathy in a similar way., (Copyright © 2020. Published by Elsevier Inc.)

Harnessing CRISPR-Cas9 technology for cancer therapeutics has been hampered by low editing efficiency in tumors and potential toxicity of existing delivery systems. Here, we describe a safe and efficient lipid nanoparticle (LNP) for the delivery of Cas9 mRNA and sgRNAs that use a novel amino-ionizable lipid. A single intracerebral injection of CRISPR-LNPs against PLK1 (sgPLK1-cLNPs) into aggressive orthotopic glioblastoma enabled up to ~70% gene editing in vivo, which caused tumor cell apoptosis, inhibited tumor growth by 50%, and improved survival by 30%. To reach disseminated tumors, cLNPs were also engineered for antibody-targeted delivery. Intraperitoneal injections of EGFR-targeted sgPLK1-cLNPs caused their selective uptake into disseminated ovarian tumors, enabled up to ~80% gene editing in vivo, inhibited tumor growth, and increased survival by 80%. The ability to disrupt gene expression in vivo in tumors opens new avenues for cancer treatment and research and potential applications for targeted gene editing of noncancerous tissues., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

Genome editing of human cluster of differentiation 34 + (CD34 + ) hematopoietic stem and progenitor cells (HSPCs) holds great therapeutic potential. This study aimed to optimize on-target, ex vivo genome editing using the CRISPR-Cas9 system in CD34 + HSPCs and to create a clear workflow for precise identification of off-target effects. Modified synthetic guide RNAs (gRNAs), either 2-part gRNA or single-guide RNA (sgRNA), were delivered to CD34 + HSPCs as part of ribonucleoprotein (RNP) complexes, targeting therapeutically relevant genes. The addition of an Alt-R electroporation enhancer (EE), a short, single-stranded oligodeoxynucleotide (ssODN), significantly increased editing efficiency in CD34 + HSPCs. Notably, similar editing improvement was observed when excess gRNA over Cas9 protein was used, providing a DNA-free alternative suitable for therapeutic applications. Furthermore, we demonstrated that sgRNA may be preferable over 2-part gRNA in a locus-specific manner. Finally, we present a clear experimental framework suitable for the unbiased identification of bona fide off-target sites by Genome-Wide, Unbiased Identification of Double-Strand Breaks (DSBs) Enabled by Sequencing (GUIDE-seq), as well as subsequent editing quantification in CD34 + HSPCs using rhAmpSeq. These findings may facilitate the implementation of genome editing in CD34 + HSPCs for research and therapy and can be adapted for other hematopoietic cells., (© 2020 The Author(s).)

Inconsistent activity limits the use of CRISPR-Cas9 in zebrafish. We show supernumerary guanine nucleotides at the 5' ends of single guide RNAs (sgRNAs) account for diminished CRISPR-Cas9 activity in zebrafish embryos. Genomic sequences can be targeted consistently with extremely high efficiency using Cas9 ribonucleoproteins (RNPs) containing either a sgRNA molecule or a synthetic crRNA:tracrRNA duplex that perfectly matches the protospacer target site. Following injection of zebrafish eggs with such RNPs, virtually every copy of a targeted locus harbors an induced indel mutation. Loss of gene function is often complete, as F0 embryos closely resemble true null mutants without detectable non-specific effects. Mosaicism is sufficiently low in F0 embryos that cell non-autonomous gene functions can be probed effectively and redundant activities of genes can be uncovered when two genes are targeted simultaneously. Finally, heritable deletion mutations of at least 50 kbp can be readily induced using pairs of duplex guide RNPs targeted to a single chromosome., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Translation of the CRISPR-Cas9 system to human therapeutics holds high promise. However, specificity remains a concern especially when modifying stem cell populations. We show that existing rationally engineered Cas9 high-fidelity variants have reduced on-target activity when using the therapeutically relevant ribonucleoprotein (RNP) delivery method. Therefore, we devised an unbiased bacterial screen to isolate variants that retain activity in the RNP format. Introduction of a single point mutation, p.R691A, in Cas9 (high-fidelity (HiFi) Cas9) retained the high on-target activity of Cas9 while reducing off-target editing. HiFi Cas9 induces robust AAV6-mediated gene targeting at five therapeutically relevant loci (HBB, IL2RG, CCR5, HEXB, and TRAC) in human CD34 + hematopoietic stem and progenitor cells (HSPCs) as well as primary T cells. We also show that HiFi Cas9 mediates high-level correction of the sickle cell disease (SCD)-causing p.E6V mutation in HSPCs derived from patients with SCD. We anticipate that HiFi Cas9 will have wide utility for both basic science and therapeutic genome-editing applications.

CRISPR/Cas9 can be used for precise genetic knock-in of epitope tags into endogenous genes, simplifying experimental analysis of protein function. However, Cas9-assisted epitope tagging in primary mammalian cell cultures is often inefficient and reliant on plasmid-based selection strategies. Here, we demonstrate improved knock-in efficiencies of diverse tags (V5, 3XFLAG, Myc, HA) using co-delivery of Cas9 protein pre-complexed with two-part synthetic modified RNAs (annealed crRNA:tracrRNA) and single-stranded oligodeoxynucleotide (ssODN) repair templates. Knock-in efficiencies of ~5-30%, were achieved without selection in embryonic stem (ES) cells, neural stem (NS) cells, and brain-tumor-derived stem cells. Biallelic-tagged clonal lines were readily derived and used to define Olig2 chromatin-bound interacting partners. Using our novel web-based design tool, we established a 96-well format pipeline that enabled V5-tagging of 60 different transcription factors. This efficient, selection-free and scalable epitope tagging pipeline enables systematic surveys of protein expression levels, subcellular localization, and interactors across diverse mammalian stem cells., Competing Interests: PD, BS, KM, GM, EO, NQ, AT, ML, CP, MK, CB, RF, RB, GM, Av, ST, JK, SP No competing interests declared, AJ employed by Integrated DNA Technologies (IDT), who sells reagents similar to some described herein. IDT is, however, not a publicly traded company and the authors do not own any shares or equity in IDT. No other authors have any financial interests or relationships with IDT; nor do they own any shares or equity, MB Mark A Behlke: employed by Integrated DNA Technologies (IDT), who sells reagents similar to some described herein. IDT is, however, not a publicly traded company and the authors do not own any shares or equity in IDT. No other authors have any financial interests or relationships with IDT; nor do they own any shares or equity, (© 2018, Dewari et al.)

Objective: To evaluate the clinical efficacy and safety of off-label biological therapies in patients with ANCA-associated vasculitis (AAV) and non-ANCA-associated small-vessel vasculitis (nAAV) in clinical practice., Methods: The German Registry in Autoimmune Diseases 2 (GRAID2) is a national, retrospective, non-interventional, multicentre observational study (August 2006 until December 2013) on patients with autoimmune diseases refractory to standard immunosuppressive therapy treated with off-label biologicals., Results: Data from 64 patients (20.6% of all GRAID2 patients) were collected: 54 patients (84.4%) had ANCA-associated vasculitis (AAV) and 10 patients (15.6%) had non-ANCA-associated small-vessel vasculitis (nAAV). Of the AAV patients, 96.3% were treated off-label with rituximab (RTX) and 3.7% with tumor necrosis factor alpha (TNFα)-inhibitors. Of patients with nAAV, 30% were treated with RTX, 60% with TNFα-inhibitors, and 10% with tocilizumab. The main reasons for off-label biological treatment in AAV patients were pulmonary, renal, or ear, nose, and throat involvement. These manifestations clearly improved in most patients after off-label biological therapy was initiated. Daily glucocorticoid dosage could be reduced. The off-label biological therapy was generally well tolerated. In AAV patients, 4.18 severe infections per 100 patient years were observed. There was one death in the nAAV group caused by fungal infection and ileus. A correlation between this fatality and RTX treatment was regarded as possible., Conclusion: Safety and efficacy of off-label RTX-treatment in AAV-patients could be assessed in the GRAID2 data. Results point to good efficacy and safety of RTX in this special patient cohort and support the approval of RTX for AAV induction therapy.

Background: Conditional knockout mice and transgenic mice expressing recombinases, reporters, and inducible transcriptional activators are key for many genetic studies and comprise over 90% of mouse models created. Conditional knockout mice are generated using labor-intensive methods of homologous recombination in embryonic stem cells and are available for only ~25% of all mouse genes. Transgenic mice generated by random genomic insertion approaches pose problems of unreliable expression, and thus there is a need for targeted-insertion models. Although CRISPR-based strategies were reported to create conditional and targeted-insertion alleles via one-step delivery of targeting components directly to zygotes, these strategies are quite inefficient., Results: Here we describe Easi-CRISPR (Efficient additions with ssDNA inserts-CRISPR), a targeting strategy in which long single-stranded DNA donors are injected with pre-assembled crRNA + tracrRNA + Cas9 ribonucleoprotein (ctRNP) complexes into mouse zygotes. We show for over a dozen loci that Easi-CRISPR generates correctly targeted conditional and insertion alleles in 8.5-100% of the resulting live offspring., Conclusions: Easi-CRISPR solves the major problem of animal genome engineering, namely the inefficiency of targeted DNA cassette insertion. The approach is robust, succeeding for all tested loci. It is versatile, generating both conditional and targeted insertion alleles. Finally, it is highly efficient, as treating an average of only 50 zygotes is sufficient to produce a correctly targeted allele in up to 100% of live offspring. Thus, Easi-CRISPR offers a comprehensive means of building large-scale Cre-LoxP animal resources.

Genome editing using the CRISPR/Cas9 system requires the presence of guide RNAs bound to the Cas9 endonuclease as a ribonucleoprotein (RNP) complex in cells, which cleaves the host cell genome at sites specified by the guide RNAs. New genetic material may be introduced during repair of the double-stranded break via homology dependent repair (HDR) if suitable DNA templates are delivered with the CRISPR components. Early methods used plasmid or viral vectors to make these components in the host cell, however newer approaches using recombinant Cas9 protein with synthetic guide RNAs introduced directly as an RNP complex into cells shows faster onset of action with fewer off-target effects. This approach also enables use of chemically modified synthetic guide RNAs that have improved nuclease stability and reduces the risk of triggering an innate immune response in the host cell. This article provides detailed methods for genome editing using the RNP approach with synthetic guide RNAs using lipofection or electroporation in mammalian cells or using microinjection in murine zygotes, with or without addition of a single-stranded HDR template DNA., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

We previously reported that delivery of a microRNA-138 mimic or siRNA against SIN3A to cultured cystic fibrosis (ΔF508/ΔF508) airway epithelia partially restored ΔF508-cystic fibrosis transmembrane conductance regulator (CFTR)-mediated cAMP-stimulated Cl - conductance. We hypothesized that dissecting this microRNA-138/SIN3A-regulated gene network would identify individual proteins contributing to the rescue of ΔF508-CFTR function. Among the genes in the network, we rigorously validated candidates using functional CFTR maturation and electrolyte transport assays in polarized airway epithelia. We found that depletion of the ubiquitin ligase SYVN1, the ubiquitin/proteasome system regulator NEDD8, or the F-box protein FBXO2 partially restored ΔF508-CFTR-mediated Cl - transport in primary cultures of human cystic fibrosis airway epithelia. Moreover, knockdown of SYVN1, NEDD8, or FBXO2 in combination with corrector compound 18 further potentiated rescue of ΔF508-CFTR-mediated Cl - conductance. This study provides new knowledge of the CFTR biosynthetic pathway. It suggests that SYVN1 and FBXO2 represent two distinct multiprotein complexes that may degrade ΔF508-CFTR in airway epithelia and identifies a new role for NEDD8 in regulating ΔF508-CFTR ubiquitination., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Background: Accumulating evidence suggests that the C-C chemokine ligand 2 (CCL2, or monocyte chemoattractant protein 1) acts as a neuromodulator in the central nervous system through its binding to the C-C chemokine receptor 2 (CCR2). Notably, it is well established that the CCL2/CCR2 axis plays a key role in neuron-glia communication as well as in spinal nociceptive transmission. Gene silencing through RNA interference has recently emerged as a promising avenue in research and drug development, including therapeutic management of chronic pain. In the present study, we used 27-mer Dicer-substrate small interfering RNA (DsiRNA) targeting CCR2 and assessed their ability to reverse the nociceptive behaviors induced by spinal CCL2 injection or following intraplantar injection of complete Freund's adjuvant., Results: To this end, we first developed high-potency DsiRNAs designed to target different sequences distributed across the rat CCR2 (rCCR2) messenger RNA. For optimization, methyl groups were added to the two most potent DsiRNA candidates (Evader and M7 2'-O-methyl modified duplexes) in order to improve in vivo duplex stability and to reduce potential immunostimulatory activity. Our results demonstrated that all modified candidates formulated with the cell-penetrating peptide reagent Transductin showed strong RNAi activity following intrathecal delivery, exhibiting >50% rCCR2 knockdown in lumbar dorsal root ganglia. Accordingly, we found that these DsiRNA duplexes were able to reduce spinal microglia activation and were effective at blocking CCL2-induced mechanical hypersensitivity. Along with similar reductions of rCCR2 messenger RNA, both sequences and methylation patterns were similarly effective in inhibiting the CCL2 nociceptive action for the whole seven days testing period, compared to mismatch DsiRNA. DsiRNAs against CCR2 also reversed the hypernociceptive responses observed in the complete Freund's adjuvant-induced inflammatory chronic pain model., Conclusion: Altogether, these results validate CCR2 as a an appropriate molecular target for pain control and demonstrate that RNAi-based gene therapy represent an highly specific alternative to classical pharmacological approaches to treat central pathologies such as chronic pain., (© The Author(s) 2016.)

Background: In an earlier study we developed a unique strategy allowing us to specifically eliminate antigen-specific murine B cells via their distinct B cell receptors using a new class of fusion proteins. In the present work we elaborated our idea to demonstrate the feasibility of specifically addressing and eliminating human memory B cells., Results: The present study reveals efficient adaptation of the general approach to selectively target and eradicate human memory B cells. In order to demonstrate the feasibility we engineered a fusion protein following the principle of recombinant immunotoxins by combining a model antigen (tetanus toxoid fragment C, TTC) for B cell receptor targeting and a truncated version of Pseudomonas aeruginosa exotoxin A (ETA') to induce apoptosis after cellular uptake. The TTC-ETA' fusion protein not only selectively bound to a TTC-reactive murine B cell hybridoma cell line in vitro but also to freshly isolated human memory B cells from immunized donors ex vivo. Specific toxicity was confirmed on an antigen-specific population of human CD27(+) memory B cells., Conclusions: This protein engineering strategy can be used as a generalized platform approach for the construction of therapeutic fusion proteins with disease-relevant antigens as B cell receptor-binding domains, offering a promising approach for the specific depletion of autoreactive B-lymphocytes in B cell-driven autoimmune diseases.

Hypothesis: Droplet wettability and mobility play an important role in dropwise condensation heat transfer. Heat exchangers and heat pipes operate at liquid-vapor saturation. We hypothesize that the wetting behavior of liquid water on microstructures surrounded by pure water vapor differs from that for water droplets in air., Experiments: The static and dynamic contact angles and contact angle hysteresis of water droplets were measured in air and pure water vapor environments inside a pressure vessel. Pressures ranged from 60 to 1000 mbar, with corresponding saturation temperatures between 36 and 100°C. The wetting behavior was studied on four hydrophobic surfaces: flat Teflon-coated, micropillars, micro-scale meshes, and nanoparticle-coated with hierarchical micro- and nanoscale roughness., Findings: Static advancing contact angles are 9° lower in the water vapor environment than in air on a flat surface. One explanation for this reduction in contact angles is water vapor adsorption to the Teflon. On microstructured surfaces, the vapor environment has little effect on the static contact angles. In all cases, variations in pressure and temperature do not influence the wettability and mobility of the water droplets. In most cases, advancing contact angles increase and contact angle hysteresis decreases when the droplets are sliding or rolling down an inclined surface., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Much is known about the how Gβγ subunits regulate effectors in response to G protein-coupled receptor stimulation. However, there is still a lot we don't know about how specific combinations of Gβ and Gγ are wired into different signalling pathways. Here, using an siRNA screen for different Gβ and Gγ subunits, we examined an endogenous M3 muscarinic receptor signalling pathway in HEK 293 cells. We observed that Gβ(4) subunits were critical for calcium signalling and a downstream surrogate measured as ERK1/2 MAP kinase activity. A number of Gγ subunits could partner with Gβ(4) but the best coupling was seen via Gβ(4)γ(1). Intriguingly, knocking down Gβ(1) actually increased signalling through the M3-mAChR most likely via an increase in Gβ(4) levels. We noted that Gβ(1) occupies the promoter of Gβ(4) and may participate in maturation of its mRNA. This highlights a new role for Gβγ signalling beyond their canonical roles in cellular signalling., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Introduction: Disease activity and therapy show an impact on cellular and serological parameters in patients with systemic lupus erythematosus (SLE). This study was performed to compare the influence of mycophenolate mofetil (MMF) and cyclophosphamide (CYC) therapy on these parameters in patients with flaring, organ-threatening disease., Methods: SLE patients currently receiving CYC (n = 20), MMF (n = 25) or no immunosuppressive drugs (n = 22) were compared using a cross-sectional design. Median disease activity and daily corticosteroid dose were similar in these treatment groups. Concurrent medication, organ manifestations, and disease activity were recorded, and cellular and serological parameters were determined by routine diagnostic tests or flow cytometric analysis. In addition follow-up data were obtained from different sets of patients (CYC n = 24; MMF n = 23)., Results: Although both drugs showed a significant effect on disease activity and circulating B cell subsets, only MMF reduced circulating plasmablasts and plasma cells as well as circulating free light chains within three months of induction therapy. Neither MMF nor CYC were able to reduce circulating memory B cells. MMF lowered IgA levels more markedly than CYC. We did not observe a significant difference in the reduction of IgG levels or anti-dsDNA antibodies comparing patients receiving MMF or CYC. In contrast to MMF, induction therapy with CYC was associated with a significant increase of circulating CD8+ effector T cells and plasmacytoid dendritic cells (PDCs) after three months., Conclusions: The results indicate differences between MMF and CYC with regard to the mechanism of action. MMF, but not CYC, treatment leads to a fast and enduring reduction of surrogate markers of B cell activation, such as circulating plasmablasts, plasma cells and free light chains but a comparable rate of hypogammaglobulinemia.

Splice switching oligonucleotides (SSOs) induce alternative splicing of pre-mRNA and typically employ chemical modifications to increase nuclease resistance and binding affinity to target pre-mRNA. Here we describe a new SSO non-base modifier (a naphthyl-azo group, "ZEN™") to direct exon exclusion in mutant dystrophin pre-mRNA to generate functional dystrophin protein. The ZEN modifier is placed near the ends of a 2'-O-methyl (2'OMe) oligonucleotide, increasing melting temperature and potency over unmodified 2'OMe oligonucleotides. In cultured H2K cells, a ZEN-modified 2'OMe phosphorothioate (PS) oligonucleotide delivered by lipid transfection greatly enhanced dystrophin exon skipping over the same 2'OMePS SSO lacking ZEN. However, when tested using free gymnotic uptake in vitro and following systemic delivery in vivo in dystrophin deficient mdx mice, the same ZEN-modified SSO failed to enhance potency. Importantly, we show for the first time that in vivo activity of anionic SSOs is modelled in vitro only when using gymnotic delivery. ZEN is thus a novel modifier that enhances activity of SSOs in vitro but will require improved delivery methods before its in vivo clinical potential can be realized.

Objective: To determine whether certain rheumatic diseases will affect the fetal thymus diameter when compared to uncomplicated singleton pregnancies. Additionally, we created a reference chart for fetal thymus size in healthy singleton pregnancies from 19 to 37 weeks of gestation., Methods: Sonographic fetal thymus size was retrospectively evaluated in 190 healthy pregnant women, and 84 pregnancies of mothers suffering from systemic lupus erythematosus, antiphospholipid syndrome (APS), or Sjögren's syndrome between 19 and 37 weeks of gestation. These fetuses were matched one-to-one for gestational age with control fetuses. The thymic-thoracic ratio (TT-ratio) was defined as the quotient of the anteroposterior thymic and the intrathoracic mediastinal diameter., Results: Rheumatic diseases often affect pregnancy outcome, especially in case of primary APS. The TT ratio of fetuses of mothers suffering from rheumatic disease was equal to controls (P=0.807)., Conclusions: Ours is the first study to assess the correlation of fetal thymus size in high-risk pregnancies with rheumatic diseases in comparison to controls. Women with rheumatic diseases deal with pregnancy complications more frequently than controls. Our data suggest that maternal rheumatic diseases do not affect the fetal thymus size.

Felty’s syndrome is a rare variant of severe seropositive rheumatoid arthritis with neutropenia and splenomegaly. It is difficult to treat and associated with a poor prognosis due to the substantial risk of infections. This article presents the case of a patient with refractory disease who responded to rituximab with permanent normalization of neutrophil counts. Repeated infusions were necessary to induce and maintain remission.

The role of Gβγ subunits in Kir3 channel gating is well characterized. Here, we have studied the role of Gβγ dimers during their initial contact with Kir3 channels, prior to their insertion into the plasma membrane. We show that distinct Gβγ subunits play an important role in orchestrating and fine-tuning parts of the Kir3 channel life cycle. Gβ1γ2, apart from its role in channel opening that it shares with other Gβγ subunit combinations, may play a unique role in protecting maturing channels from degradation as they transit to the cell surface. Taken together, our data suggest that Gβ1γ2 prolongs the lifetime of the Kir3.1/Kir3.2 heterotetramer, although further studies would be required to shed more light on these early Gβγ effects on Kir3 maturation and trafficking.

RNA-specific adenosine deaminase (ADAR)-mediated adenosine-to-inosine (A-to-I) editing is a critical arm of the antiviral response. However, mechanistic insights into how A-to-I RNA editing affects viral infection are lacking. We posited that inosine incorporation into RNA facilitates sensing of nonself RNA by innate immune sensors and accordingly investigated the impact of inosine-modified RNA on Toll-like receptor 7 and 8 (TLR7/8) sensing. Inosine incorporation into synthetic single-stranded RNA (ssRNA) potentiated tumor necrosis factor alpha (TNF-α) or alpha interferon (IFN-α) production in human peripheral blood mononuclear cells (PBMCs) in a sequence-dependent manner, indicative of TLR7/8 recruitment. The effect of inosine incorporation on TLR7/8 sensing was restricted to immunostimulatory ssRNAs and was not seen with inosine-containing short double-stranded RNAs or with a deoxy-inosine-modified ssRNA. Inosine-mediated increase of self-secondary structure of an ssRNA resulted in potentiated IFN-α production in human PBMCs through TLR7 recruitment, as established through the use of a TLR7 antagonist and Tlr7-deficient cells. There was a correlation between hyperediting of influenza A viral ssRNA and its ability to stimulate TNF-α, independent of 5'-triphosphate residues, and involving Adar-1. Furthermore, A-to-I editing of viral ssRNA directly enhanced mouse Tlr7 sensing, when present in proportions reproducing biologically relevant levels of RNA editing. Thus, we demonstrate for the first time that inosine incorporation into immunostimulatory ssRNA can potentiate TLR7/8 activation. Our results suggest a novel function of A-to-I RNA editing, which is to facilitate TLR7/8 sensing of phagocytosed viral RNA.

MicroRNAs (miRNAs) are increasingly recognized as important posttranscriptional regulators of gene expression, and changes in their actions can contribute to disease states. Little is understood regarding miRNA functions in the airway epithelium under normal or diseased conditions. We profiled miRNA expression in well-differentiated primary cultures of human cystic fibrosis (CF) and non-CF airway epithelia, and discovered that miR-509-3p and miR-494 concentrations were increased in CF epithelia. Human non-CF airway epithelia, transfected with the mimics of miR-509-3p or miR-494, showed decreased cystic fibrosis transmembrane conductance regulator (CFTR) expression, whereas their respective anti-miRs exerted the opposite effect. Interestingly, the two miRNAs acted cooperatively in regulating CFTR expression. Upon infecting non-CF airway epithelial cells with Staphylococcus aureus, or upon stimulating them with the proinflammatory cytokines TNF-α or IL-1β, we observed an increased expression of both miRNAs and a concurrent decrease in CFTR expression and function, suggesting that inflammatory mediators may regulate these miRNAs. Transfecting epithelia with anti-miRs for miR-509-3p and miR-494, or inhibiting NF-κB signaling before stimulating cells with TNFα or IL-1β, suppressed these responses, suggesting that the expression of both miRNAs was responsive to NF-κB signaling. Thus, miR-509-3p and miR-494 are dynamic regulators of CFTR abundance and function in normal, non-CF airway epithelia.

Management of painful peripheral neuropathies remains challenging, since patients with chronic pain respond poorly to the available pharmacopeia. In recent years, the G-protein-coupled receptor neurotensin (NT) type 2 (NTS2) emerged as an attractive target for treating transitory pain states. To date, however, there is no evidence for its role in the regulation of chronic peripheral neuropathies. Here, we found that NTS2 receptors were largely localized to primary afferent fibers and superficial dorsal horns. Changes in the time course of the gene expression profile of NT, NTS1, and NTS2 were observed over a 28-d period following the sciatic nerve constriction [chronic constriction injury (CCI) model]. We next determined the effects of central delivery of selective-NTS2 agonists to CCI-treated rats on both mechanical allodynia (evoked withdrawal responses) and weight-bearing deficits (discomfort and quality-of-life proxies). The NTS2 analogs JMV431, levocabastine, and β-lactotensin were all effective in reducing ongoing tactile allodynia in CCI-treated rats. Likewise, amitriptyline, pregabalin, and morphine significantly attenuated CCI-induced mechanical hypersensitivity. NTS2 agonists were also efficient in reversing weight-bearing and postural deficits caused by nerve damage, unlike reference analgesics currently used in the clinic. Thus, NTS2 agonists may offer new treatment avenues for limiting pain associated with peripheral neuropathies and improve functional rehabilitation and well-being.

Polarized and pseudostratified primary airway epithelia present barriers that significantly reduce their transfection efficiency and the efficacy of RNA interference oligonucleotides. This creates an impediment in studies of the airway epithelium, diminishing the utility of loss-of-function as a research tool. Here we outline methods to introduce RNAi oligonucleotides into primary human and porcine airway epithelia grown at an air-liquid interface and difficult-to-transfect transformed epithelial cell lines grown on plastic. At the time of plating, we reverse transfect small-interfering RNA (siRNA), Dicer-substrate siRNA, or microRNA oligonucleotides into cells by use of lipid or peptide transfection reagents. Using this approach we achieve significant knockdown in vitro of hypoxanthine-guanine phosphoribosyltransferase, IL-8, and CFTR expression at the mRNA and protein levels in 1-3 days. We also attain significant reduction of secreted IL-8 in polarized primary pig airway epithelia 3 days posttransfection and inhibition of CFTR-mediated Cl⁻ conductance in polarized air-liquid interface cultures of human airway epithelia 2 wk posttransfection. These results highlight an efficient means to deliver RNA interference reagents to airway epithelial cells and achieve significant knockdown of target gene expression and function. The ability to reliably conduct loss-of-function assays in polarized primary airway epithelia offers benefits to research in studies of epithelial cell homeostasis, candidate gene function, gene-based therapeutics, microRNA biology, and targeting the replication of respiratory viruses.

Latently infected cell reservoirs represent the main barrier to HIV eradication. Combination antiretroviral therapy (cART) effectively blocks viral replication but cannot purge latent provirus. One approach to HIV eradication could include cART to block new infections plus an agent to activate latent provirus. NF-κB activation induces HIV expression, ending latency. Before activation, IκB proteins sequester NF-κB dimers in the cytoplasm. Three canonical IκBs, IκBα, IκBβ, and IκBε, exist, but the IκB proteins' role in HIV activation regulation is not fully understood. We studied the effects on HIV activation of targeting IκBs by single and pairwise small interfering RNA (siRNA) knockdown. After determining the relative abundance of the IκBs, the relative abundance of NF-κB subunits held by the IκBs, and the kinetics of IκB degradation and resynthesis following knockdown, we studied HIV activation by IκB knockdown, in comparison with those of known HIV activators, tumor necrosis factor alpha (TNF-α), tetradecanoyl phorbol acetate (TPA), and trichostatin A (TSA), in U1 monocytic and J-Lat 10.6 lymphocytic latently infected cells. We found that IκBα knockdown activated HIV in both U1 and J-Lat 10.6 cells, IκBβ knockdown did not activate HIV, and, surprisingly, IκBε knockdown produced the most HIV activation, comparable to TSA activation. Our data show that HIV reactivation can be triggered by targeting two different IκB proteins and that IκBε may be an effective target for HIV latency reactivation in T-cell and macrophage lineages. IκBε knockdown may offer attractive therapeutic advantages for HIV activation because it is not essential for mammalian growth and development and because new siRNA delivery strategies may target siRNAs to HIV latently infected cells.

Interleukin-1 (IL-1)-mediated diseases are caused by an inappropriately high production and release of IL-1 beta which results in a multitude of symptoms, e.g. arthritis, exanthema, conjunctivitis, serositis, fever and loss of hearing. If IL-1-mediated diseases remain unrecognized or are recognized and treated too late, long-term complications, such as amyloidosis may occur. In recent years the diagnostic and therapeutic options with respect to IL-1-mediated diseases have drastically improved. These diseases often manifesting in childhood can now be treated with monoclonal antibodies against IL-1 or with IL-1 receptor antagonists. Increased IL-1 secretion does not only play a role in relatively rare hereditary diseases, such as cryopyrin-associated periodic fever syndromes or familial Mediterranean fever but also in widespread diseases, such as gout or type 2 diabetes. This article will focus on pathogenic, diagnostic and therapeutic aspects of IL-1-mediated inflammatory diseases.

In the present study, contact angle hysteresis and sliding behavior of water droplets on parallel, periodic microgrooved brass surfaces are investigated experimentally for enhancement of water drainage and compared to that on flat baseline surfaces. The surfaces (a total of 17 microgrooved samples, with a range of groove depth of 22 to 109 μm, pillar width of 26 to 190 μm, and groove width of 103 and 127 μm) are fabricated using a mechanical micromachining process. The wetting state and shape/elongation of deposited water droplets, anisotropy of the contact angle hysteresis, and the drainage behavior of water droplets on the microgrooved surfaces are found to be strongly dependent on the topography of the groove geometry, which is analyzed in detail. The wetting state is found to be Wenzel for microgrooved surfaces with very low aspect ratio (<0.2) and narrow pillars (pillar width to groove width ratio of ≈0.2), and also for the two deepest grooved surfaces of two different sample series, all of which exhibit high contact angle hysteresis. Mechanisms of the advancing and receding motions are identified. The critical sliding angle (the angle from horizontal at incipient motion of the advancing confluence) for the microgrooved surfaces is found to be significantly smaller than for flat surfaces. The sliding angle exhibits significant groove geometry dependence and is found to increase with pillar width and decrease with groove depth. The findings of this study may be useful in a broad range of applications where water retention plays an important role.

B cells are able to present antigen, secrete cytokines and differentiate into (auto)antibody secreting cells and are therefore considered as an important therapeutic target in patients with autoantibody-mediated autoimmune disease. Benefits and limitations of B-cell-directed therapies and unmet medical needs are discussed in this minireview. B cell targeting broadens our armamentarium available to treat SLE and other connective tissue diseases. But further research addressing unmet medical needs is required and refractory patients receiving B cell-directed off-label therapeutics should be enrolled in registries to collect information on the value and safety of these drugs in rare autoimmune diseases., (© Georg Thieme Verlag KG Stuttgart · New York.)

Production of functional proteins requires multiple steps, including gene transcription and posttranslational processing. MicroRNAs (miRNAs) can regulate individual stages of these processes. Despite the importance of the cystic fibrosis transmembrane conductance regulator (CFTR) channel for epithelial anion transport, how its expression is regulated remains uncertain. We discovered that miRNA-138 regulates CFTR expression through its interactions with the transcriptional regulatory protein SIN3A. Treating airway epithelia with an miR-138 mimic increased CFTR mRNA and also enhanced CFTR abundance and transepithelial Cl(-) permeability independent of elevated mRNA levels. An miR-138 anti-miR had the opposite effects. Importantly, miR-138 altered the expression of many genes encoding proteins that associate with CFTR and may influence its biosynthesis. The most common CFTR mutation, ΔF508, causes protein misfolding, protein degradation, and cystic fibrosis. Remarkably, manipulating the miR-138 regulatory network also improved biosynthesis of CFTR-ΔF508 and restored Cl(-) transport to cystic fibrosis airway epithelia. This miRNA-regulated network directs gene expression from the chromosome to the cell membrane, indicating that an individual miRNA can control a cellular process more broadly than recognized previously. This discovery also provides therapeutic avenues for restoring CFTR function to cells affected by the most common cystic fibrosis mutation.

Regulation of gene expression using small interfering RNA (siRNA) is a promising strategy for research and treatment of numerous diseases. In this study, we develop and characterize a delivery system for siRNA composed of polyethylenimine (PEI), polyethylene glycol (PEG), and mannose (Man). Cationic PEI complexes and compacts siRNA, PEG forms a hydrophilic layer outside of the polyplex for steric stabilization, and mannose serves as a cell binding ligand for macrophages. The PEI-PEG-mannose delivery system was constructed in two different ways. In the first approach, mannose and PEG chains are directly conjugated to the PEI backbone. In the second approach, mannose is conjugated to one end of the PEG chain and the other end of the PEG chain is conjugated to the PEI backbone. The PEI-PEG-mannose delivery systems were synthesized with 3.45-13.3 PEG chains and 4.7-3.0 mannose molecules per PEI. The PEI-PEG-Man-siRNA polyplexes displayed a coarse surface in Scanning Electron Microscopy (SEM) images. Polyplex sizes were found to range from 169 to 357 nm. Gel retardation assays showed that the PEI-PEG-mannose polymers are able to efficiently complex with siRNA at low N/P ratios. Confocal microscope images showed that the PEI-PEG-Man-siRNA polyplexes could enter cells and localized in the lysosomes at 2h post-incubation. Pegylation of the PEI reduced toxicity without any adverse reduction in knockdown efficiency relative to PEI alone. Mannosylation of the PEI-PEG could be carried out without any significant reduction in knockdown efficiency relative to PEI alone. Conjugating mannose to PEI via the PEG spacer generated superior toxicity and gene knockdown activity relative to conjugating mannose and PEG directly onto the PEI backbone., (Copyright © 2011 Elsevier B.V. All rights reserved.)

25/27 Base duplex RNAs that are substrates for Dicer have been demonstrated to enhance RNA interference (RNAi) potency and efficacy. Since the target sites are not always equally susceptible to suppression by small interfering RNA (siRNA), not all 27-mer duplexes that are processed into the corresponding conventional siRNAs show increased potency. Thus random designing of Dicer-substrate siRNAs (DsiRNAs) may generate siRNAs with poor RNAi due to unpredictable Dicer processing. Previous studies have demonstrated that the 3'-overhang affects dicing cleavage site and the orientation of Dicer entry. Moreover, an asymmetric 27-mer duplex having a 3' two-nucleotide overhang and 3'-DNA residues on the blunt end has been rationally designed to obtain greater efficacy. This asymmetric structure directs dicing to predictably yield a single primary cleavage product. In the present study, we analyzed the in vitro and intracellular dicing patterns of chemically synthesized duplex RNAs with different 3'-overhangs. Consistent with previous studies, we observed that Dicer preferentially processes these RNAs at a site 21-22 nucleotide (nt) from the two-base 3'-overhangs. We also observed that the direction and ability of human Dicer to generate siRNAs can be partially or completely blocked by DNA residues at the 3'-termimi. To examine the effects of various 3'-end modifications on Dicer processing in cells, we employed Illumina Deep sequencing analyses to unravel the fates of the asymmetric 27-mer duplexes. To validate the strand selection process and knockdown capabilities we also conducted dual-luciferase psiCHECK reporter assays to monitor the RNAi potencies of both the "sense" (S) and "antisense" (AS) strands derived from these DsiRNAs. Consistent with our in vitro Dicer assays, the asymmetric duplexes were predictably processed into desired primary cleavage products of 21-22-mers in cells. We also observed the trimming of the 3' end, especially when DNA residues were incorporated into the overhangs and this trimming ultimately influenced the Dicer-cleavage site and RNAi potency. Moreover, the observation that the most efficacious strand was the most abundant revealed that the relative frequencies of each "S" or "AS" strand are highly correlated with the silencing activity and strand selectivity. Collectively, our data demonstrate that even though the only differences between a family of DsiRNAs was the 3' two-nuclotide overhang, dicing polarity and strand selectivity are distinct depending upon the sequence and chemical nature of this overhang. Thus, it is possible to predictably control dicing polarity and strand selectivity via simply changing the 3'-end overhangs without altering the original duplex sequence. These optimal design features of 3'-overhangs might provide a facile approach for rationally designing highly potent 25/27-mer DsiRNAs.

The preparation of nanofluids is very important to their thermophysical properties. Nanofluids with the same nanoparticles and base fluids can behave differently due to different nanofluid preparation methods. The agglomerate sizes in nanofluids can significantly impact the thermal conductivity and viscosity of nanofluids and lead to a different heat transfer performance. Ultrasonication is a common way to break up agglomerates and promote dispersion of nanoparticles into base fluids. However, research reports of sonication effects on nanofluid properties are limited in the open literature. In this work, sonication effects on thermal conductivity and viscosity of carbon nanotubes (0.5 wt%) in an ethylene glycol-based nanofluid are investigated. The corresponding effects on the agglomerate sizes and the carbon nanotube lengths are observed. It is found that with an increased sonication time/energy, the thermal conductivity of the nanofluids increases nonlinearly, with the maximum enhancement of 23% at sonication time of 1,355 min. However, the viscosity of nanofluids increases to the maximum at sonication time of 40 min, then decreases, finally approaching the viscosity of the pure base fluid at a sonication time of 1,355 min. It is also observed that the sonication process not only reduces the agglomerate sizes but also decreases the length of carbon nanotubes. Over the current experimental range, the reduction in agglomerate size is more significant than the reduction of the carbon nanotube length. Hence, the maximum thermal conductivity enhancement and minimum viscosity increase are obtained using a lengthy sonication, which may have implications on application.

Introduction: Clinical trials revealed a high efficacy of mycophenolate mofetil (MMF)in inducing and maintaining remission in patients with class III-V-lupus nephritis. Also extrarenal manifestations respond to MMF treatment. However, few attempts have been undertaken to delineate its mechanism of action in systemic lupus erythematosus (SLE) a disease characterized by enhanced B cell activation., Methods: Clinical and paraclinical parameters of 107 patients with SLE were recorded consecutively and analyzed retrospectively. Patients were divided into treatment groups (MMF: n=39, azathioprine (AZA) n=30 and controls without immunosuppressive therapy n=38). To further delineate the effect of mycophenolic acid (MPA) on naive and memory B cells in vitro assays were performed., Results: Although patients taking AZA flared more frequently than patients on MMF or controls, the analysis of clinical parameters did not reveal significant differences.However, profound differences in paraclinical parameters were found. B cell frequencies and numbers were significantly higher in patients taking MMF compared to those on AZA but lower numbers and frequencies of plasmablasts were detected compared to AZA-treated patients or controls. Notably, MMF treatment was associated with a significantly higher frequency and number of transitional B cells as well as naive B cells compared to AZA treatment. Differences in T cell subsets were not significant. MPA abrogated in vitro proliferation of purified B cells completely but had only moderate impact on B cell survival., Conclusions: The thorough inhibition of B cell activation and plasma cell formation by MMF might explain the favorable outcomes of previous clinical trials in patients with SLE, since enhanced B cell proliferation is a hallmark of this disease.