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There is intense clinical interest in the potential effects of platelet-rich plasma (PRP) for the treatment of osteoarthritis (OA). This study tested the hypotheses that (i) "lower" levels of the inflammatory mediators (IMs), interleukin-1β, and tumor necrosis factor α (TNF-α) and (ii) "higher" levels of the growth factors (GFs), insulin-like growth factor 1, and transforming growth factor β1 within leukocyte-poor PRP correlate with more favorable chondrocyte and macrophage responses in vitro. Samples were collected from 10 "healthy" young male (23-33 years old) human subjects (H-PRP) and nine older (62-85 years old) male patients with severe knee OA (OA-PRP). The samples were separated into groups of "high" or "low" levels of IM and GF based on multiplex cytokine and enzyme-linked immunosorbent assay data. Three-dimensional (3D) alginate bead chondrocyte cultures and monocyte-derived macrophage cultures were treated with 10% PRP from donors in different groups. Gene expression was analyzed by quantitative polymerase chain reaction. Contrary to our hypotheses, the effect of PRP on chondrocytes and macrophages was mainly influenced by the age and disease status of the PRP donor as opposed to the IM or GF groupings. While H-PRP showed similar effects on expression of chondrogenic markers (Col2a1 and Sox9) as the negative control group (p > 0.05), OA-PRP decreased chondrocyte expression of Col2a1 and Sox-9 messenger RNA by 40% and 30%, respectively (Col2a1, p = 0.015; Sox9, p = 0.037). OA-PRP also upregulated TNF-α and matrix metallopeptidase 9 (p < 0.001) gene expression in macrophages while H-PRP did not. This data suggests that PRP from older individuals with OA contain factors that may suppress chondrocyte matrix synthesis and promote macrophage inflammation in vitro. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1760-1770, 2019.

Purpose: The economic implications of brachial plexus injuries (BPI) in the United States are not well understood. The purpose of our study was to quantify the direct costs associated with surgical treatment of BPI after traumatic injury in adults, which would enable future study of the societal value of surgical reconstruction. Methods: Using an administrative database of patients with commercial insurance, a cohort of patients aged 18 to 64 years with BPI treated surgically from 2007 to 2015 was assembled and assessed for index admission associated with BPI surgery and all payments toward claims (including medical, surgical, therapy, and pharmacy claims) for 1 year after surgery. Results: Among 189 patients undergoing surgery for BPI, median direct payments were $38,816 (interquartile range: $18,209 to $72,411; minimum: $3,512; maximum: $732,641). Conclusions: Relative to recently published data for the indirect cost of traumatic BPI (median: $801,723), direct payments for 1 year after surgical treatment represent 4.6% of the total long-term cost of BPI. In the context of existing literature demonstrating cost-effectiveness in models of BPI surgical care, our data suggest that surgery and other interventions to maximize return to work after traumatic BPI in adults may be beneficial to society. Type of study/level of evidence: Economic and Decision Analyses IV. Key words: Brachial plexus injury, Cost of surgery, Surgical reconstruction, Trauma

Multiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system (CNS), with characteristic inflammatory lesions and demyelination. The clinical benefit of cell-depleting therapies targeting CD20 has emphasized the role of B cells and autoantibodies in MS pathogenesis. We previously introduced an enzyme-linked immunospot spot (ELISpot)-based assay to measure CNS antigen-specific B cells in the blood of MS patients and demonstrated its usefulness as a predictive biomarker for disease activity in measuring the successful outcome of disease-modifying therapies (DMTs). Here we used a planar protein array to investigate CNS-reactive antibodies in the serum of MS patients as well as in B cell culture supernatants after polyclonal stimulation. Anti-CNS antibody reactivity was evident in the sera of the MS cohort, and the antibodies bound a heterogeneous set of molecules, including myelin, axonal cytoskeleton, and ion channel antigens, in individual patients. Immunoglobulin reactivity in supernatants of stimulated B cells was directed against a broad range of CNS antigens. A group of MS patients with a highly active B cell component was identified by the ELISpot assay. Those antibody reactivities remained stable over time. These assays with protein arrays identify MS patients with a highly active B cell population with antibodies directed against a swathe of CNS proteins.

OBJECTIVE Rheumatoid arthritis (RA) is characterized by the presence of anti-citrullinated protein antibodies (ACPAs); nevertheless, the origin, specificity, and functional properties of ACPAs remain poorly understood. The aim of this study was to characterize the evolution of ACPAs by sequencing the plasmablast antibody repertoire at serial time points in patients with established RA. METHODS Blood samples were obtained at up to 4 serial time points from 8 individuals with established RA who were positive for ACPAs by the anti-cyclic citrullinated peptide test. CD19+CD3-IgD-CD14-CD20-CD27+CD38++ plasmablasts were isolated by single-cell sorting and costained with citrullinated peptide tetramers to identify ACPA-expressing plasmablasts. Cell-specific oligonucleotide barcodes were utilized, followed by large-scale sequencing and bioinformatics analysis, to obtain error-corrected, paired heavy- and light-chain antibody gene sequences for each B cell. RESULTS Bioinformatics analysis revealed 170 persistent plasmablast lineages in the RA blood, of which 19% included multiple isotypes. Among IgG- and IgA-expressing plasmablasts, significantly more IgA-expressing than IgG-expressing persistent lineages were observed (P < 0.01). Shared complementarity-determining region 3 sequence motifs were identified across subjects. A subset of the plasmablast lineages included members derived from later time points with divergent somatic hypermutations that encoded antibodies that bind an expanded set of citrullinated antigens. Furthermore, these recombinant, differentially mutated plasmablast antibodies formed immune complexes that stimulated higher macrophage production of tumor necrosis factor (TNF) compared to antibodies representing earlier time point-derived lineage members that were less mutated. CONCLUSION These findings demonstrate that established RA is characterized by a persistent IgA ACPA response that exhibits ongoing affinity maturation. This observation suggests the presence of a persistent mucosal antigen that continually promotes the production of IgA plasmablasts and their affinity maturation and epitope spreading, thus leading to the generation of ACPAs that bind additional citrullinated antigens and more potently stimulate macrophage production of TNF.

The development of rheumatoid factor (RF) and/or anti-citrullinated protein antibodies (ACPAs) can be observed years prior to clinical diagnosis of rheumatoid arthritis (RA). Nevertheless, the interaction between these two autoantibodies and their combined effect on development of RA is unclear. We measured RF, cytokines, and ACPA subtypes in serial pre-clinical serum samples collected from 83 US veterans who all developed RA. Levels of cytokines and ACPAs were compared between the following groups: anti-cyclic citrullinated peptide (anti-CCP)-/RF- (double negative), anti-CCP+/RF-, anti-CCP-/RF+, or anti-CCP+/RF+ (double-positive). The double-positive subgroup had significantly higher levels of 20 inflammatory cytokines and 29 ACPA reactivities, and the shortest interval, 1.3 years, between the preclinical sample timepoint and diagnosis of RA. Thus, the combined presence of ACPAs and RF is associated with a more rapid progression to RA, suggesting that anti-CCP+/RF+ individuals have a more advanced preclinical disease state and that the onset of RA may be imminent.

Objective Rheumatoid arthritis (RA) is characterized by the activation of B cells that produce anti-citrullinated protein antibodies (ACPAs) and rheumatoid factors (RFs), but the mechanisms by which tolerance is broken in these B cells remain incompletely understood. We undertook this study to investigate whether ACPA+ and RF+ B cells break tolerance through distinct molecular mechanisms. Methods We developed antigen-tetramers to isolate ACPA+ and RF+ B cells and performed single-cell RNA sequencing on 2,349 B cells from 6 RA patients and 1 healthy donor to analyze their immunoglobulin repertoires and transcriptional programs. Prominent immunoglobulins were expressed as monoclonal antibodies and tested for autoantigen reactivity. Results ACPA+ and RF+ B cells were enriched in the peripheral blood of RA patients relative to healthy controls. Characterization of patient-derived monoclonal antibodies confirmed ACPA and RF targeting of tetramer-specific B cells at both antigen-inexperienced and affinity-matured B cell stages. ACPA+ B cells used more class-switched isotypes and exhibited more somatic hypermutations relative to RF+ B cells, and these differences were accompanied by down-regulation of CD72 and up-regulation of genes that promote class-switching and T cell-dependent responses. In contrast, RF+ B cells expressed transcriptional programs that stimulate rapid memory reactivation through multiple innate immune pathways. Coexpression analysis revealed that ACPA+ and RF+ B cell-enriched genes belong to distinct transcriptional regulatory networks. Conclusion Our findings suggest that ACPA+ and RF+ B cells are imprinted with distinct transcriptional programs, which suggests that these autoantibodies associated with increased inflammation in RA arise from 2 different molecular mechanisms.

Seasonal influenza vaccines elicit antibody responses that can prevent infection, but their efficacy is reduced in the elderly. While a subset of elderly individuals can still mount sufficient vaccine-induced antibody responses, little is known about the properties of the vaccine-induced antibody repertoires in elderly as compared to young responders. To gain insights into the effects of aging on influenza vaccine-induced antibody responses, we used flow cytometry and a cell-barcoding method to sequence antibody heavy and light chain gene pairs expressed by individual blood plasmablasts generated in response to influenza vaccination in elderly (aged 70 – 89) and young (aged 20 – 29) responders. We found similar blood plasmablast levels in the elderly and young responders seven days post vaccination. Informatics analysis revealed increased clonality, but similar heavy chain V(D)J gene usage in the elderly as compared to young vaccine responders. Although the elderly responders exhibited decreased antibody sequence diversity and fewer consequential mutations relative to young responders, recombinant antibodies from elderly responders bound a broader range of influenza strain HAs. Thus elderly influenza vaccine responders mount plasmablast responses with restricted diversity but with an increased breadth of binding across influenza strains. Our results suggest that the ability to generate plasmablast responses encoding cross-strain binding antibodies likely represents a mechanism important to vaccine responses in the elderly.

Background: Autologous platelet-rich plasma (PRP) is widely used for a variety of clinical applications. However, clinical outcome studies have not consistently shown positive effects. The composition of PRP differs based on many factors. An improved understanding of factors influencing the composition of PRP is important for the optimization of PRP use. Hypothesis: Age and sex influence the PRP composition in healthy patients. Study Design: Controlled laboratory study. Methods: Blood from 39 healthy patients was collected at a standardized time and processed into leukocyte-poor PRP within 1 hour of collection using the same laboratory centrifuge protocol and frozen for later analysis. Eleven female and 10 male patients were “young” (aged 18-30 years), while 8 male and 10 female patients were “older” (aged 45-60 years). Thawed PRP samples were assessed for cytokine and growth factor levels using a multiplex assay and enzyme-linked immunosorbent assay. The platelet count and high-sensitivity C-reactive protein levels were measured. Two-way analysis of variance determined age- and sex-based differences. Results: Platelet and high-sensitivity C-reactive protein concentrations were similar in PRP between the groups ( P = .234). Male patients had higher cytokine and growth factor levels in PRP compared with female patients for inflammatory cytokines such as interleukin–1 beta (IL-1β) (9.83 vs 7.71 pg/mL, respectively; P = .008) and tumor necrosis factor–alpha (TNF-α) (131.6 vs 110.5 pg/mL, respectively; P = .048); the anti-inflammatory IL-1 receptor antagonist protein (IRAP) (298.0 vs 218.0 pg/mL, respectively; P < .001); and growth factors such as fibroblast growth factor–basic (FGF-basic) (237.9 vs 194.0 pg/mL, respectively; P = .01), platelet-derived growth factor (PDGF-BB) (3296.2 vs 2579.3 pg/mL, respectively; P = .087), and transforming growth factor–beta 1 (TGF-β1) (118.8 vs 92.8 ng/mL, respectively; P = .002). Age- but not sex-related differences were observed for insulin-like growth factor–1 (IGF-1) ( P < .001). Age and sex interaction terms were not significant. While mean differences were significant, there was also substantial intragroup variability. Conclusion: This study in healthy patients shows differences in the composition of PRP between men and women, with sex being a greater factor than age. There was also proteomic variability within the groups. These data support a personalized approach to PRP treatment and highlight the need for a greater understanding of the relationships between proteomic factors in PRP and clinical outcomes. Clinical Relevance: Variability in the proteomic profile of PRP may affect tissue and clinical responses to treatment. These data suggest that clinical studies should account for the composition of PRP used.

Objective Overweight/obesity and anti-citrullinated protein antibodies (ACPA) increase rheumatoid arthritis (RA) risk. We investigated the relationship between body mass index (BMI) and ACPA, tested for an interaction between BMI and ACPA for RA risk, and examined effects of BMI and ACPA on time to RA diagnosis. Design Within the Nursesʼ Health Studies, blood samples were collected before diagnosis from medical record-confirmed incident RA cases and matched controls. Multiplex assays measured 7 ACPA subtypes (biglycan, clusterin, enolase, fibrinogen, histone 2A, histone 2B, and vimentin). Logistic regression analyses tested the association of BMI and ACPA and for a multiplicative interaction between BMI groups (≥25 vs. 2 ) and ACPA positivity (≥2 vs. HLA -shared epitope. In case-only analyses, log-rank tests compared time from blood draw to RA onset by cross-classified BMI/ACPA status. Results Among 255 pre-RA cases and 778 matched controls, 15.7% vs. 2.1% ( p p 2 unit BMI: 1.03 (95% CI: 0.97–1.09)]. However, there was a multiplicative interaction between elevated BMI and the presence of ≥2 ACPA for RA risk ( p = 0.027). Women with BMI≥25kg/m 2 and ≥2 ACPA had OR 22.7 (95% CI: 6.64–77.72) for RA. Median time to RA differed by BMI/ACPA group (overall log-rank p 2 [45.0 months, IQR: 17.5–72.5] and longest in women with 2 [125.0 months, IQR: 72.0–161.0] (pairwise log-rank p = 0.002). Conclusion Elevated BMI and presence of ACPA interacted to increase RA risk. Time to RA onset was shortest among overweight/obese women with ≥2 ACPA.

Osteoarthritis (OA) is the leading cause of joint failure, yet the underlying mechanisms remain elusive, and no approved therapies that slow progression exist. Dysregulated integrin function was previously implicated in OA pathogenesis. However, the roles of integrin α(V)β(3) and the integrin-associated receptor CD47 in OA remain largely unknown. Here, transcriptomic and proteomic analyses of human and murine osteoarthritic tissues revealed dysregulated expression of α(V)β(3), CD47, and their ligands. Using genetically deficient mice and pharmacologic inhibitors, we showed that α(V)β(3), CD47, and the downstream signaling molecules Fyn and FAK are crucial to OA pathogenesis. MicroPET/CT imaging of a mouse model showed elevated ligand-binding capacities of integrin α(V)β(3) and CD47 in osteoarthritic joints. Further, our in vitro studies demonstrated that chondrocyte breakdown products, derived from articular cartilage of individuals with OA, induced α(V)β(3)/CD47-dependent expression of inflammatory and degradative mediators, and revealed the downstream signaling network. Our findings identify a central role for dysregulated α(V)β(3) and CD47 signaling in OA pathogenesis and suggest that activation of α(V)β(3) and CD47 signaling in many articular cell types contributes to inflammation and joint destruction in OA. Thus, the data presented here provide a rationale for targeting α(V)β(3), CD47, and their signaling pathways as a disease-modifying therapy.

Osteoarthritis is characterized by articular cartilage breakdown, and emerging evidence suggests that dysregulated innate immunity is likely involved. Here, we performed proteomic, transcriptomic, and electron microscopic analyses to demonstrate that mast cells are aberrantly activated in human and murine osteoarthritic joint tissues. Using genetic models of mast cell deficiency, we demonstrate that lack of mast cells attenuates osteoarthritis in mice. Using genetic and pharmacologic approaches, we show that the IgE/FcεRI/Syk signaling axis is critical for the development of osteoarthritis. We find that mast cell-derived tryptase induces inflammation, chondrocyte apoptosis, and cartilage breakdown. Our findings demonstrate a central role for IgE-dependent mast cell activation in the pathogenesis of osteoarthritis, suggesting that targeting mast cells could provide therapeutic benefit in human osteoarthritis.Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter).

Rheumatoid arthritis (RA) is characterized by the production of anti-citrullinated protein antibodies (ACPAs). To gain insights into the relationship between ACPA-expressing B cells in peripheral blood (PB) and synovial tissue (ST), we sequenced the B cell repertoire in paired PB and ST samples from five individuals with established, ACPA+ RA. Bioinformatics analysis of paired heavy- and light-chain sequences revealed clonally-related family members shared between PB and ST. ST-derived antibody repertoires exhibited reduced diversity and increased normalized clonal family size compared to PB-derived repertoires. Functional characterization showed that seven recombinant antibodies (rAbs) expressed from subject-derived sequences from both compartments bound citrullinated antigens and immune complexes (ICs) formed using one ST-derived rAb stimulated macrophage TNF-α production. Our findings demonstrate B cell trafficking between PB and ST in subjects with RA and ST repertoires include B cells that encode ACPA capable of forming ICs that stimulate cellular responses implicated in RA pathogenesis.

The correlation between the Food and Drug Administration–cleared C6 enzyme immunoassay (EIA) C6 index values and a diagnosis of Lyme disease has not been examined. We used pooled patient-level data from 5 studies of adults and children with Lyme disease and control subjects who were tested with the C6 EIA. We constructed a receiver operating characteristic curve using regression clustered by study and measured the area under the curve (AUC) to examine the accuracy of the C6 index values in differentiating between patients with noncutaneous Lyme disease and control subjects. In the 4821 included patients, the C6 index value had excellent ability to distinguish between patients with noncutaneous Lyme disease and control subjects [AUC 0.99; 95% confidence interval (CI) 0.99–1.00]. An index value cut point of ≥3.0 had a sensitivity of 90.9% (95% CI, 87.8–93.3) and specificity of 99.0% (95% CI, 98.6–99.2%) for Lyme disease.

Objective Anti-citrullinated protein antibodies (ACPAs) are a hallmark of rheumatoid arthritis (RA). While epitope spreading of the serum ACPA response is believed to contribute to RA pathogenesis, little is understood regarding how this phenomenon occurs. This study was undertaken to analyze the antibody repertoires of individuals with RA to gain insight into the mechanisms leading to epitope spreading of the serum ACPA response in RA. Methods Plasmablasts from the blood of 6 RA patients were stained with citrullinated peptide tetramers to identify ACPA-producing B cells by flow cytometry. Plasmablasts were single-cell sorted and sequenced to obtain antibody repertoires. Sixty-nine antibodies were recombinantly expressed, and their anticitrulline reactivities were characterized using a cyclic citrullinated peptide enzyme-linked immuosorbent assay and synovial antigen arrays. Thirty-six mutated antibodies designed either to represent ancestral antibodies or to test paratope residues critical for binding, as determined from molecular modeling studies, were also tested for anticitrulline reactivities. Results Clonally related monoclonal ACPAs and their shared ancestral antibodies each exhibited differential reactivity against citrullinated antigens. Molecular modeling identified residues within the complementarity-determining region loops and framework regions predicted to be important for citrullinated antigen binding. Affinity maturation resulted in mutations of these key residues, which conferred binding to different citrullinated epitopes and/or increased polyreactivity to citrullinated epitopes. Conclusion These results demonstrate that the different somatic hypermutations accumulated by clonally related B cells during affinity maturation alter the antibody paratope to mediate epitope spreading and polyreactivity of the ACPA response in RA, suggesting that these may be key properties that likely contribute to the pathogenicity of ACPAs.

Therapeutics based on transcription factors have the potential to revolutionize medicine but have had limited clinical success as a consequence of delivery problems. The delivery of transcription factors is challenging because it requires the development of a delivery vehicle that can complex transcription factors, target cells and stimulate endosomal disruption, with minimal toxicity. Here, we present a multifunctional oligonucleotide, termed DARTs (DNA assembled recombinant transcription factors), which can deliver transcription factors with high efficiency in vivo. DARTs are composed of an oligonucleotide that contains a transcription-factor-binding sequence and hydrophobic membrane-disruptive chains that are masked by acid-cleavable galactose residues. DARTs have a unique molecular architecture, which allows them to bind transcription factors, trigger endocytosis in hepatocytes, and stimulate endosomal disruption. The DARTs have enhanced uptake in hepatocytes as a result of their galactose residues and can disrupt endosomes efficiently with minimal toxicity, because unmasking of their hydrophobic domains selectively occurs in the acidic environment of the endosome. We show that DARTs can deliver the transcription factor nuclear erythroid 2-related factor 2 (Nrf2) to the liver, catalyse the transcription of Nrf2 downstream genes, and rescue mice from acetaminophen-induced liver injury.

Background Granulomatosis with polyangiitis (GPA) is a disease characterized by inflammation in small blood vessels, leading to significant organ damage and ultimately great morbidity and mortality. The presence of anti-neutrophil cytoplasmic antibodies (ANCAs) is a hallmark of the disease, and thought to directly play a role in pathogenesis by activating primed neutrophils. The two major antigens that ANCAs are believed to recognize are myeloperoxidase (MPO) and proteinase 3 (PR3). Although anti-MPO antibodies are directly pathogenic when transferred or induced in animal models, the evidence is less clear for PR3. Clinically, it is debated whether levels of anti-PR3 correlate with disease activity. Therefore, we believe that anti-PR3 antibodies are not the primary drivers of disease in patients with PR3+ ANCA-vasculitis (i.e. GPA), and that novel autoantibodies may play a role in disease pathogenesis. Objectives The objective of this work is to identify the human autoantigen targets of novel autoantibodies identified in GPA, and to test their association with disease. The goal is to discover novel autoantigens that will ultimately lead to new diagnostics and therapeutics. Methods Cell-barcode-enabled antibody repertoire sequencing was performed on blood plasmablasts (antibody-producing cells formed during an immune response) from five PR3+ ANCA-vasculitis patients with GPA treated with rituximab in the RAVE trial. Phylogenetic trees were bioinformatically created in order to identify clonal families of plasmablasts. Antibodies representing clonal families of plasmablasts were recombinantly expressed and are being tested using human protein arrays and ELISAs to determine their antigen specificity. Results All five PR3+ ANCA-vasculitis patients sequenced from the RAVE trial achieved complete remission but subsequently flared. Plasmablasts were isolated at the baseline flare, at remission and at the post-rituximab flare. Phylograms of the antibody repertoires revealed clonal families of affinity-matured antibodies that share heavy and light chain VJ usage. A total of 24 representative antibodies were selected for recombinant expression, including representative antibodies derived from clonal families: (1) shared across patients at baseline flare and/or post-rituximab flare (n=5), (2) present at baseline flare and post-rituximab flare (n=7) [persistent clone or same clone came back], (3) present at baseline flare or post-rituximab flare (n=6), (4) present in remission and post-rituximab flare (n=5) [achieves remission despite clone presence], (5) present in remission (but not flare) (n=1) [patient in remission despite clone presence]. None of the 24 antibodies bind PR3 in an ELISA and thus these antibodies do not represent ANCAs. Antigen discovery to determine the targets of these recombinant plasmablast antibodies is ongoing. Conclusions Plasmablasts from patients with GPA in the RAVE trial were sequenced and antibody repertoires were generated. Clonal families of plasmablasts, including those expressing antibodies possessing shared CDR sequences across multiple patients, were identified and recombinantly expressed. None of the 24 antibodies bound to PR3, suggesting these are not ANCAs and are potentially novel autoantibodies. Identification of the antigen targets of these antibodies is ongoing. Acknowledgements The Immune Tolerance Network Training Program in Adult and Pediatric Rheumatology 2T32AR050942–11 Disclosure of Interest None declared

Rheumatoid arthritis (RA) is characterized by synovial joint inflammation and by development of pathogenic humoral and cellular autoimmunity to citrullinated proteins. Neutrophil extracellular traps (NETs) are a source of citrullinated autoantigens and activate RA synovial fibroblasts (FLS), cells crucial in joint damage. We investigated the molecular mechanisms by which NETs promote proinflammatory phenotypes in FLS, and whether these interactions generate pathogenic anti-citrulline adaptive immune responses. NETs containing citrullinated peptides are internalized by FLS through a RAGE-TLR9 pathway promoting FLS inflammatory phenotype and their upregulation of MHC class II. Once internalized, arthritogenic NET-peptides are loaded into FLS MHC class II and presented to Ag-specific T cells. HLADRB1*0401 transgenic mice immunized with mouse FLS loaded with NETs develop antibodies specific to citrullinated forms of relevant RA autoantigens implicated in RA pathogenesis as well as cartilage damage. These results implicate FLS as mediators in RA pathogenesis, through the internalization and presentation of NET citrullinated peptides to the adaptive immune system leading to pathogenic autoimmunity and cartilage damage.

ObjectivesWhile various monocyte chemokine systems are increased in expression in osteoarthritis (OA), the hierarchy of chemokines and chemokine receptors in mediating monocyte/macrophage recruitment to the OA joint remains poorly defined. Here, we investigated the relative contributions of the CCL2/CCR2 versus CCL5/CCR5 chemokine axes in OA pathogenesis.MethodsCcl2-, Ccr2-, Ccl5- and Ccr5-deficient and control mice were subjected to destabilisation of medial meniscus surgery to induce OA. The pharmacological utility of blocking CCL2/CCR2 signalling in mouse OA was investigated using bindarit, a CCL2 synthesis inhibitor, and RS-504393, a CCR2 antagonist. Levels of monocyte chemoattractants in synovial tissues and fluids from patients with joint injuries without OA and those with established OA were investigated using a combination of microarray analyses, multiplexed cytokine assays and immunostains.ResultsMice lacking CCL2 or CCR2, but not CCL5 or CCR5, were protected against OA with a concomitant reduction in local monocyte/macrophage numbers in their joints. In synovial fluids from patients with OA, levels of CCR2 ligands (CCL2, CCL7 and CCL8) but not CCR5 ligands (CCL3, CCL4 and CCL5) were elevated. We found that CCR2+ cells are abundant in human OA synovium and that CCR2+ macrophages line, invade and are associated with the erosion of OA cartilage. Further, blockade of CCL2/CCR2 signalling markedly attenuated macrophage accumulation, synovitis and cartilage damage in mouse OA.ConclusionsOur findings demonstrate that monocytes recruited via CCL2/CCR2, rather than by CCL5/CCR5, propagate inflammation and tissue damage in OA. Selective targeting of the CCL2/CCR2 system represents a promising therapeutic approach for OA.

Objective To characterize the expression of malondialdehdye-acetaldehyde (MAA) adducts and anti-MAA antibody in articular tissues and serum of patients with RA. Methods Paired sera and SF were examined from 29 RA and 13 OA patients. Anti-MAA antibody, RF, ACPA and total immunoglobulin were quantified. SF-serum measures were compared within and between disease groups. The presence and co-localization of MAA, citrulline and select leukocyte antigens in RA and OA synovial tissues were examined using immunohistochemistry. Results Circulating and SF anti-MAA antibody concentrations were higher in RA vs OA by 1.5- to 5-fold. IgG (P < 0.001), IgM (P = 0.006) and IgA (P = 0.036) anti-MAA antibodies were higher in paired RA SF than serum, differences not observed for total immunoglobulin, RF or ACPA. In RA synovial tissues, co-localization of MAA with citrulline and CD19+ or CD27+ B cells was demonstrated and was much higher in magnitude than MAA or citrulline co-localization with T cells, monocytes, macrophages or dendritic cells (P < 0.01). Conclusion Anti-MAA antibodies are present in higher concentrations in the RA joint compared with sera, a finding not observed for other disease-related autoantibodies. Co-localization of MAA and citrulline with mature B cells, coupled with the local enrichment of anti-MAA immune responses, implicates MAA-adduct formation in local autoantibody production.

There is great need for the development of an efficient delivery method of macromolecules, including nucleic acids, proteins, and peptides, to cell cytoplasm without eliciting toxicity or changing cell behavior. High-aspect ratio nanomaterials have addressed many challenges present in conventional methods, such as cell membrane passage and endosomal degradation, and have shown the feasibility of efficient high-throughput macromolecule delivery with minimal perturbation of cells. This review describes the recent advances of in vitro and in vivo physical macromolecule delivery with high-aspect ratio nanostructured materials and summarizes the synthesis methods, material properties, relevant applications, and various potential directions.

Kunwoo Lee,1,2 Pengzhi Yu,3 Nithya Lingampalli,1 Hyun Jin Kim,1 Richard Tang,1 Niren Murthy1,2 1Department of Bioengineering, University of California, Berkeley, CA,USA; 2UC Berkeley and UCSF Joint Graduate Program in Bioengineering, Berkeley/San Francisco, CA, USA; 3Gladstone Institute of Cardiovascular Disease, San Francisco, CA, USA Abstract: The treatment of myocardial infarction is a major challenge in medicine due to the inability of heart tissue to regenerate. Direct reprogramming of endogenous cardiac fibroblasts into functional cardiomyocytes via the delivery of transcription factor mRNAs has the potential to regenerate cardiac tissue and to treat heart failure. Even though mRNA delivery to cardiac fibroblasts has the therapeutic potential, mRNA transfection in cardiac fibroblasts has been challenging. Herein, we develop an efficient mRNA transfection in cultured mouse cardiac fibroblasts via a polyarginine-fused heart-targeting peptide and lipofectamine complex, termed C-Lipo and demonstrate the partial direct reprogramming of cardiac fibroblasts towards cardiomyocyte cells. C-Lipo enabled the mRNA-induced direct cardiac reprogramming due to its efficient transfection with low toxicity, which allowed for multiple transfections of Gata4, Mef2c, and Tbx5 (GMT) mRNAs for a period of 2 weeks. The induced cardiomyocyte-like cells had α-MHC promoter-driven GFP expression and striated cardiac muscle structure from a-actinin immunohistochemistry. GMT mRNA transfection of cultured mouse cardiac fibroblasts via C-Lipo significantly increased expression of the cardiomyocyte marker genes, Actc1, Actn2, Gja1, Hand2, and Tnnt2, after 2 weeks of transfection. Moreover, this study provides the first direct evidence that the stoichiometry of the GMT reprogramming factors influence the expression of cardiomyocyte marker genes. Our results demonstrate that mRNA delivery is a potential approach for cardiomyocyte generation. Keywords: direct cardiac reprogramming, cardiac fibroblast transfection, mRNA transfection, cardiac regeneration

T otal H eart T ransplant : A M odern O verview B S J Nithya Lingampalli More than 5 million people are diagnosed with congestive heart failure (CHF) every year, and this number rises by about 500,000 more every year. CHF is a multifaceted problem that is becoming increasingly prevalent in our society and is associated with end- stage heart disease (Norman, 2006). This final stage of heart failure is usually characterized by a myocardial infarction, a heart attack. Cardiac transplants are the only option for the long-term treatment of severe cases that lead to irreparable biventricular failure, which is a failure of both ventricles of the heart. This condition effectively handicaps the heart’s ability to pump blood through the body (Copeland, 2004). Since there are many complications associated with existing procedures, there is a constant push to improve the success of treatment for congestive heart failure. the most effective treatment for heart failure as they involve only natural human tissue, and the process of getting onto a waitlist is tightly restricted. Placement on the waitlist still does not guarantee treatment because 25% of patients on a waitlist die before they are able to receive a donor heart (Dunning, 1997). This lag in receiving a donor heart is due to the process by which the donors are selected. The donor heart is procured from an individual that has been declared brain dead. Their organ can be used only if they have previously given consent, or their family has provided the consent on their behalf, for their heart to be donated. Before the organ can be donated, two surgeons that are not affiliated with the patient must declare it suitable for transplant, and only then does the heart qualify for transplantation. The heart is then kept “M ore than 5 million people are diagnosed with congestive heart failure every year , and this number rises by about 500,000 more every year .” Since a cardiac transplant is currently the only option available for patients in the final stages of heart failure, multiple devices have been developed to replace all the components of the heart (valves, chambers, and the pacemaker) before total failure. The three types of procedures currently available are a donor transplant, left ventricle assist devices (LVAD), and total artificial heart implantation (TAH) (Dunning, In the case of donor transplant, the heart of a donor is isolated and placed into the patient’s body after most of their original heart is removed. The process of obtaining a donor heart and having transplant surgery is very long and complicated due to the many criteria that must be met before the surgery can proceed. To be considered for a donor transplant, the patient must go through extensive tests proctored by cardiologists, nurses, and health workers to ensure that they will be able to survive the procedure and abide by the restrictive post-operative requirements. Once a patient is determined to be a suitable candidate, they are placed on a waiting list with thousands of other similar patients. Then begins the waiting game, as the patient must now wait until it is finally their turn to receive a donor heart. Although donor heart transplants are beating through dopamine support and mechanical ventilation. Information regarding the blood type and health of the heart is entered into the waiting list database. The database then uses an algorithm that takes into account blood type, body size, and length of waiting period to select the most suitable candidate for the organ. Interestingly, race and gender are kept out of the consideration, although gender may affect the size requirement of the donor heart required. Once a suitable candidate has been identified, surgery itself proceeds very quickly because the donor heart is only capable of a successful transplant a few hours after harvesting (Cleveland Clinic). Given that the donor heart has a certain blood type, there is a chance of immunosuppression or rejection by the patient’s body. Although every effort is made to ensure that the donor and patient blood types are as similar as possible, there are a multitude of genes that affect blood type and composition and it is impossible to control for all blood factors. Therefore, even though a donor and patient are classified as having the same blood type, the composition of their blood may vary enough to cause immune rejection. Immunosuppression is the process by which the 19 • B erkeley S cientific J ournal • S ynthetics • S pring 2014 • V olume 18 • I ssue 2

T he D angers of S leep D eprivation Nithya Lingampalli B S J During President Ronald Reagan’s term as president, he went on a state visit to Paraguay. As cameras were documenting the occasion and broadcasting it for the world to see, a surprising shot was included. When the cameras focused on Reagan during the Paraguayan president’s speech, he was asleep! His wife quickly woke him up, and then he ascended the stage to deliver his address. However, when he started speaking, he said that he was very happy to be in Uruguay while everyone else smiled politely (Schneider, 2014). This instance illustrates a national health problem, often unrecognized as a severe issue, that many suffer from on a daily basis. Sleep deprivation, either a chronic or transient state, is when the lack of the requisite amount of sleep for upkeep causes the body to experience many physical and mental problems. According to data from the National Health Interview Survey, “nearly 30% of adults reported an average of ≤6 hours of sleep per day in 2005-2007. In 2009, only 31% of high school students reported getting at least 8 hours of sleep on an average school night” (Insufficient Sleep Is a Public Health Epidemic, 2014). Children appear to be most affected by sleep deprivation as a recent study from Boston College suggests that, “70 to 80 percent of school-aged children fall short of experts’ recommendations,” a higher percentage than any other country in the world (Liese, 2014). “According to data from the National Health Interview Survey, “nearly 30% of adults reported an average of ≤6 hours of sleep per day in 2005-2007. In 2009, only 31% of high school students reported getting at least 8 hours of sleep on an average school night” To put the severity of the problem into perspective, the recommended amount of sleep, as suggested by The National Institute of Health, is at least 10 hours for school-age children, 9-10 hours for teens, and 7-8 hours for adults (Insufficient Sleep Is a Public Health Epidemic, 2014). Although there is a variation from these standard times between individuals due to different body requirements, these represent the average amount of time necessary based upon a large sample of the population, and thus serve as a baseline to evaluate individual situations. We see that regardless of the age group, people are falling short of their recommended sleep quota by two hours or more, often on a daily basis. The biological mechanism of sleep deprivation deals with the reduced amount of REM and deep sleep stages, hallmarks of healthy sleep. Normal sleep is divided into “Some of the most interesting [causes] are broad scale societal factors such as the pervasive presence of technology and extremely busy schedules.” two stages, non-rapid eye movement (NREM) and rapid eye movement (REM). NREM sleep is further divided into three sub stages (N1, N2, and N3) that progress sequentially and represent an increasing degree of deep sleep. Stage N3, also known as delta-wave sleep, is the deepest stage of sleep and requires a very strong stimulus to be broken Although many areas of the brain are involved in sleep regulation, the hypothalamus seems to be the key component (Stevens, 2013). There are many causes, both direct and indirect, that contribute to sleep deprivation. Some of the most interesting are broad scale societal factors such as the pervasive presence of technology and extremely busy schedules. Access to technology has never been as ubiquitous as it is today, and its influence is growing even more in our daily lives. Phones, tablets, laptops, television, MP3 players, and many more devices are always within reach, at any time of the day. They are alluring due to the vast amount of information they offer a portal to. As a result, many people find it increasingly difficult to separate themselves from their devices, and often use them right until the moment they sleep, or sometimes fall asleep using them. However, using technological devices right before bed, or even some time before, has been shown to negatively impact the ease and quality of sleep. The light from the devices suppresses the levels of melatonin, a hormone that signals to the brain to 21 • B erkeley S cientific J ournal • E xtremes • F all 2014 • V olume 19 • I ssue 1

Cancer Due to Prolonged Inflammation Nithya Lingampalli B S J Inflammation has long been accepted as the most standard, and perhaps the most effective, response that the body has in place to fight both external and internal pathogens. What mechanisms are activated when your body is invaded by pathogens? How do these mechanisms interact with other pathways in the body? Scientists have considered these questions for years in order to determine exactly how we fight diseases and how we can amplify our body’s defense system. Inflammation has long been accepted as the most standard, and perhaps the most effective, response that the body has in place to fight both external and internal pathogens. This mechanism is considered useful because it activates whenever a threat is present, “Trouble can arise when these immune cells have been genetically damaged or attacked by mutagenic cells (initiated cells). When this DNA mutation occurs, the immune cells continue to proliferate at the site of an injury even though they are no longer needed (Coussens and Werb). “ http:// web.mit. edu/ Figure 1: Endoscopic biopsy showing granulomatous inflammation of the colon in a case of Crohn’s disease. and becomes inactive upon restoring health. However, in some instances this inflammation continues to propagate, now harming cells and threatening to expand throughout the body. This prolonged inflammation gives rise to multiples forms of cancer. In this article we will consider the mechanism of inflammation and how it is activated in the body. To understand how this mechanism can become 1 • B erkeley S cientific J ournal • S tress • F all 2013 • V olume 18 • I ssue 1

L ight as a H ealing M echanism O Nithya Lingampalli absorb different wavelengths of light at various intensities, yielding diverse physiological responses. By studying these varying physiological effects, doctors can then learn how to manipulate a patient’s body with the strategic exposure to light (Cocilovo and Rosen, 1999). Over time, the literature on phototherapy continued to grow as various researches sought to explain how and why it worked. While Ghadiali’s initial Spectro-Chrome Theory was widely accepted as an explanation for phototherapy’s effectiveness, another intriguing postulate was presented through research. The human eye contains a large concentration of light-sensitive molecules called flavins that have long been known to play a part in light absorption, and thus color perception. Research soon unveiled that these flavins also existed in various parts of our body including our skin, organs, blood and bones. This distribution of flavins throughout our entire body may be the mechanism through which we are able to absorb light and have varying physiological responses to it (Starwynn, 2009). A similar theory stems from the fact that certain nerve clusters, often targeted by acupuncturists, also seem to be involved in light absorption. Since these nerve clusters can be manipulated to influence human “Light-sensitive molecules called flavins are concentrated in our eyes ... it was discovered that these flavins also existed in various parts of our body including our skin, organs, blood and bones.” B erkeley S cientific J ournal • C olors • F all 2012 • V olume 17 • I ssue 1 • 1 B S J ver the centuries, various human populations have made use of sunlight’s reputed healing qualities. The ancient Greeks built healing temples in which the incoming sunlight would hit various colored jewels such as emeralds and rubies on the temple’s roof, sending colored beams throughout the interior of the temple. The Romans color-diagnosed their patients and placed them into rooms that radiated a specific color. Even the Chinese used color as a manifestation of cosmic energy that could be used to heal internal organs (Day, 2008). But why is it that different colors of light have diverse physiological effects, increasing heart rate and speeding up skin cell regeneration, on the body? It was not until an accident in 1876 that extensive phototherapy research began. A doctor named Dinshah Ghadiali was astonished when he encountered a woman that was dying from intractable dysentery that no medication would cure. Out of other options, Ghadiali shined a violet light on the patient’s body as a final effort to save her, and the curative effect of the light was so drastic that she was up and walking after only three days. This incident prompted him to research the applications of phototherapy and subsequently develop the Spectro-Chrome Theory, which postulates that the body contained different concentrations of various minerals which all absorbed light differently. As a result, different areas of the body

Objectives. The aim was to investigate the effects of nicotine on neutrophil extracellular traps (NETs) formation in current and non-smokers and on a murine model of RA. Methods. We compared spontaneous and phorbol 12-myristate 13-acetate-induced NETosis between current and non-smokers by DNA release binding. Nicotine-induced NETosis from non-smokers was assessed by DNA release binding, NET-specific (myeloperoxidase (MPO)–DNA complex) ELISA and real-time fluorescence microscopy. We also used immunofluorescent staining to detect nicotinic acetylcholine receptors (nAChRs) on neutrophils and performed a functional analysis to assess the role of nAChRs in nicotine-induced NETosis. Finally, we investigated the effects of systemic nicotine exposure on arthritis severity and NETosis in the CIA mouse model. Results. Neutrophils derived from current smokers displayed elevated levels of spontaneous and phorbol 12-myristate 13-acetate-induced NETosis. Nicotine induced dose-dependent NETosis in ex vivo neutrophils from healthy non-smokers, and co-incubation with ACPA-immune complexes or TNF-α facilitated a synergistic effect on NETosis. Real-time fluorescence microscopy revealed robust formation of NET-like structures in nicotine-exposed neutrophils. Immunofluorescent staining demonstrated the presence of the α7 subunit of the nAChR on neutrophils. Stimulation of neutrophils with an α7-specific nAChR agonist induced NETosis, whereas pretreatment with an nAChR antagonist attenuated nicotine-induced NETosis. Nicotine administration to mice with CIA exacerbated inflammatory arthritis, with higher plasma levels of NET-associated MPO–DNA complex. Conclusion. We demonstrate that nicotine is a potent inducer of NETosis, which may play an important role in accelerating arthritis in the CIA model. This study generates awareness of and the mechanisms by which nicotine-containing products, including e-cigarettes, may have deleterious effects on patients with RA.

Osteoarthritis (OA), the most common form of arthritis in the world, is characterized by articular cartilage breakdown in synovial joints. There is evidence of low-grade inflammatory responses in OA, but their contribution to OA pathogenesis is unclear. Studies have demonstrated that dysregulated complement activity is involved in OA pathogenesis. The complement component, C1q, is known to regulate inflammation via mechanisms involving apoptotic cell clearance and macrophage activation. Thus, we hypothesized that C1q is a key negative regulator of inflammatory mechanisms in OA. Using a combination of NanoString-based mRNA quantification and Luminex-based analyses for cytokine profiling, we found that differentiation of human monocyte-derived macrophages in C1q-depleted serum followed by stimulation with cartilage debris resulted in enhanced pro-inflammatory (e.g., IL1β) and reduced anti-inflammatory (e.g,. IL10) cytokine expression and secretion compared to cells grown in normal human serum. To definitively establish a regulatory role for C1q in OA, we used a well-established destabilization of the medial meniscus (DMM) model of knee OA in genetically-deficient mice. We found that knees of C1q-deficient mice subjected to DMM developed worse OA pathologies including exacerbated cartilage damage, osteophyte formation and synovitis relative to C1q-sufficient mice. Our data suggest that C1q deficiency exacerbates inflammation and cartilage damage through mechanisms involving dysregulated macrophage activation. Understanding the precise mechanisms underlying C1q-mediated regulation of inflammation may lead to the identification of novel targets for the treatment of inflammatory diseases including OA.