Improving Clinical Trials for Anticomplement Therapies in Complement-Mediated Glomerulopathies: Report of a Scientific Workshop Sponsored by the National Kidney Foundation.

Blocking the complement system as a therapeutic strategy has been proposed for numerous glomerular diseases but presents myriad questions and challenges, not the least of which is demonstrating efficacy and safety. In light of these potential issues and because there are an increasing number of anticomplement therapy trials either planned or under way, the National Kidney Foundation facilitated an all-virtual scientific workshop entitled "Improving Clinical Trials for Anti-Complement Therapies in Complement-Mediated Glomerulopathies." Attended by patient representatives and experts in glomerular diseases, complement physiology, and clinical trial design, the aim of this workshop was to develop standards applicable for designing and conducting clinical trials for anticomplement therapies across a wide spectrum of complement-mediated glomerulopathies. Discussions focused on study design, participant risk assessment and mitigation, laboratory measurements and biomarkers to support these studies, and identification of optimal outcome measures to detect benefit, specifically for trials in complement-mediated diseases. This report summarizes the discussions from this workshop and outlines consensus recommendations.

Occupational trichloroethylene exposure and antinuclear antibodies: a cross-sectional study in China.

OBJECTIVES: There has been concern over the possible risk of autoimmune diseases from exposure to trichloroethylene (TCE), an industrial solvent and common pollutant near hazardous waste sites. Studies of TCE-exposed lupus-prone mouse strains have reported increases in serum antinuclear antibodies (ANAs), a marker of autoimmunity, and autoimmune pathologic changes, while epidemiologic studies have provided limited support for an association between TCE exposure and scleroderma. To investigate exposure-related biologic evidence of autoimmunity in humans, we measured ANA levels in sera from a cross-sectional study of TCE-exposed (n=80) and TCE-unexposed (n=96) workers in Guangdong, China. METHODS: Full-shift personal air exposure measurements for TCE were taken prior to blood collection. Serum ANAs were detected by immunofluorescence on HEp-2 cells. We calculated ORs and 95% CI relating levels of TCE exposure (categorised using tertiles as cut-points) and ANA positivity (1+ intensity at 1:320 dilution) using multivariable logistic regression. RESULTS: Samples from 16 of 176 participants were ANA-positive. We found higher levels of TCE exposure (concentrations>17.27 ppm) to be associated with an elevated odds of ANA positivity (OR 4.7, 95% CI 1.3 to 16.8) compared with unexposed controls. This association remained after excluding two subjects with diagnosed autoimmune disease (OR 4.5, 95% CI 1.2 to 16.2). We did not observe an association with ANAs at lower exposure levels. CONCLUSIONS: Our findings, to our knowledge the first direct human evidence of an association between TCE exposure and systemic autoimmunity, provide biologic plausibility to epidemiologic evidence relating TCE and autoimmune disease.

Antinuclear Antibodies Are Associated with an Increased Risk of Diffuse Large B-Cell Lymphoma.

Immune dysregulation is thought to increase the risk of non-Hodgkin lymphoma (NHL), but the evidence varies by subtype. We evaluated whether antinuclear antibodies (ANA), double-stranded DNA antibodies (anti-dsDNA), and extractable nuclear antigen antibodies (anti-ENA) were associated with the risk of common NHL subtypes in a nested case-control study. The autoantibodies were tested in serum collected years prior to NHL diagnosis in 832 cases and 809 controls from the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial. Logistic regression was used to determine odds ratios (ORs) and 95% confidence intervals (95% CI) for the association with NHL risk. No association was observed between ANA positivity and NHL risk overall (OR: 1.18, 95% CI: 0.88-1.58); however, ANA positivity was associated with an increased risk of diffuse large B-cell lymphoma (DLBCL) (OR: 1.83, 95% CI: 1.15-2.91), with 19.7% of cases and 12.2% of controls testing positive. The presence of either anti-ENA or anti-dsDNA was associated with an increased risk of NHL (OR: 2.93, 95% CI: 1.18-7.28), particularly DLBCL (OR: 3.51, 95% CI: 1.02-12.0) and marginal zone lymphoma (OR: 8.86, 95% CI: 1.26-62.0). Our study demonstrates that autoantibodies are associated with an elevated risk of DLBCL, providing support for autoimmunity as a risk factor.

Sex and Age-Related Differences in Complement Factors Among Patients With Intermediate Age-Related Macular Degeneration.

Purpose: Age-related macular degeneration (AMD) is an acquired degenerative disease of the retina classified into early, intermediate, and advanced AMD. A key factor in the pathogenesis of AMD is the complement system. The interaction of age and sex with the complement system may affect the risk of developing AMD. The purpose of this study was to determine if there were sex-specific differences in levels of complement factors among patients with the intermediate phenotype of AMD (iAMD) and explore the correlation between age and complement proteins. Methods: We studied complement factors in patients with iAMD and controls without AMD. Nonparametric, rank-based linear regressions including a sex by AMD interaction were used to compare levels for each analyte. Correlations between age and complement proteins were evaluated using the Spearman rank correlation coefficient. Results: We found significantly higher levels of factor B and factor I in females compared with males with iAMD, whereas no differences were seen in complement levels in male and female controls. The ratios of Ba/factor B, C3a/C3, C4b/C4, and C5a/C5 were not different in males and females with iAMD. Conclusions: We demonstrate disparities in a subset of systemic complement factors between females and males with iAMD, but apparent complement turnover as measured by ratios of activation fragments to intact molecules was not different between these groups. The results suggest that complement system levels, including complement regulator factor I, exhibits sex-related differences in patients with iAMD and highlights that stratification by sex might be helpful in the interpretation of clinical trials of anticomplement therapy.

Association of Systemic Inflammatory Factors with Progression to Advanced Age-related Macular Degeneration.

PURPOSE: Age-related macular degeneration (AMD) is a leading cause of vision loss in the elderly. The role of systemic inflammation in AMD remains unclear specifically in patients with intermediate AMD (iAMD). We sought to determine whether systemic inflammation was associated with future iAMD progression. METHODS: Combinations of 27 circulating inflammatory markers including complement factors, cytokines, chemokines, and high-sensitivity C-reactive protein (hsCRP) were evaluated in iAMD patients recruited into a Colorado AMD registry. Systemic inflammatory markers were combined using principal component analysis. Risk factors for AMD progression were evaluated using Cox regression models. RESULTS: This study included 99 subjects with iAMD, 21 of which progressed to advanced AMD. Two principal components (PCs) were identified that contributed to the risk of progression to advanced AMD, after adjusting for age and bilateral reticular pseudodrusen. The strongest associated PC was explained largely by the pro-inflammatory cytokine TNFα and the anti-inflammatory IL1ra antagonist of IL1. The additional PC was largely explained by IL6, IL8, C3 and factor D in the positive direction and CRP, MCP1, factor B and factor I in the negative direction. CONCLUSION: When evaluated through multivariate analyses, combinations of biomarkers distinguished patients who did and did not progress to future advanced AMD. Increased risk could result from different combinations of analyte levels indicating a complex relationship rather than a simple increase in a few markers. This suggests that studying systemic inflammation in iAMD can provide insights into early pathologic events and potentially identify patients at highest risk for the development of severe AMD.

Association of Sputum Neutrophil Extracellular Trap Subsets With IgA Anti-Citrullinated Protein Antibodies in Subjects at Risk for Rheumatoid Arthritis.

OBJECTIVE: Mechanisms leading to anti-citrullinated protein antibody (ACPA) generation in rheumatoid arthritis (RA) are hypothesized to originate in the lung. We undertook this study to understand associations between neutrophil extracellular trap (NET) formation in the lung and local ACPA generation in subjects at risk of developing RA. METHODS: Induced sputum was collected from 49 subjects at risk of developing RA, 12 patients with RA, and 18 controls. Sputum neutrophils were tested for ex vivo NET formation, and sputum-induced NET formation of control neutrophils was measured using immunofluorescence imaging. Sputum macrophages were tested for ex vivo endocytosis of apoptotic and opsonized cells. Levels of ACPA, NET remnants, and inflammatory proteins were quantified in sputum supernatant. RESULTS: Spontaneous citrullinated histone H3 (Cit-H3)-expressing NET formation was higher in sputum neutrophils from at-risk subjects and RA patients compared to controls (median 12%, 22%, and 0%, respectively; P < 0.01). In at-risk subjects, sputum IgA ACPA correlated with the percentage of neutrophils that underwent Cit-H3+ NET formation (r = 0.49, P = 0.002) and levels of Cit-H3+ NET remnants (r = 0.70, P < 0.001). Reduced endocytic capacity of sputum macrophages was found in at-risk subjects and RA patients compared to controls. Using a mediation model, we found that sputum inflammatory proteins were associated with sputum IgA ACPA through a pathway mediated by Cit-H3+ NET remnants. Sputum-induced Cit-H3+ NET formation also correlated with sputum levels of interleukin-1ß (IL-1ß), IL-6, and tumor necrosis factor in at-risk subjects, suggesting a causal relationship. CONCLUSION: These data support a potential mechanism for mucosal ACPA generation in subjects at risk of developing RA, whereby inflammation leads to increased citrullinated protein-expressing NETs that promote local ACPA generation.

Combinations of Anticyclic Citrullinated Protein Antibody, Rheumatoid Factor, and Serum Calprotectin Positivity Are Associated With the Diagnosis of Rheumatoid Arthritis Within 3 Years.

OBJECTIVE: To evaluate the prevalence of elevations of anti-cyclic citrullinated peptide-3 (anti-CCP3) antibody, rheumatoid factor IgM (RF-IgM) and serum calprotectin (sCP) in pre-rheumatoid arthritis (RA) as well as the diagnostic accuracies of these biomarkers for the timing of diagnosis of future RA. METHODS: A total of 215 RA cases, each with approximately three pre-RA diagnoses and one post-RA diagnosis serum sample, and controls were identified from the Department of Defense Serum Repository. All case samples and a single sample from each control subject were tested for anti-CCP3 (IgG), RF-IgM, and sCP. The diagnostic accuracies of biomarkers for future RA were evaluated. RESULTS: Anti-CCP3, RF-IgM, and sCP were elevated in pre-RA, with anti-CCP3 and sCP significantly elevated compared with RF-IgM at the earliest time points. Within the cases, the combination of anti-CCP3 and RF-IgM positivity had a positive predictive value (PPV) of 35.6% for a diagnosis of RA in 3 years or less, which is significantly higher than the PPV of 18.7% for anti-CCP3 positivity alone (P < 0.001). A combination of anti-CCP3, RF-IgM, and sCP had the highest PPV (53.0%) for a diagnosis of RA in 3 years or less; however, this was not significantly higher than the PPV for anti-CCP3 and RF-IgM positivity (P = 0.248). CONCLUSION: Anti-CCP3, RF-IgM, and sCP are elevated in pre-RA; furthermore, combinations of elevations of these biomarkers are more commonly seen in the period of less than or equal to 3 years to diagnosis. This may be considered in creating inclusion criteria in prevention trials in RA. In addition, the biologic relationships of these biomarkers in pre-RA need exploration.

A Toolkit and Framework for Optimal Laboratory Evaluation of Individuals with Suspected Primary Immunodeficiency.

Knowledge related to the biology of inborn errors of immunity and associated laboratory testing methods continues to expand at a tremendous rate. Despite this, many patients with inborn errors of immunity suffer for prolonged periods of time before identification of their underlying condition, thereby delaying appropriate care. Understanding that test selection and optimal evaluation for patients with recurrent infections or unusual patterns of inflammation can be unclear, we present a document that distills relevant clinical features of immunologic disease due to inborn errors of immunity and related appropriate and available test options. This document is intended to serve the practicing clinical immunologist and, in turn, patients by describing best available test options for initial and expanded immunologic evaluations across the disease spectrum. Our goal is to demystify the process of evaluating patients with suspected immune dysfunction and to enable more rapid and accurate diagnosis of such individuals.

Nicotine-mediated signals modulate cell death and survival of T lymphocytes.

The capacity of nicotine to affect the behavior of non-neuronal cells through neuronal nicotinic acetylcholine receptors (nAChRs) has been the subject of considerable recent attention. Previously, we showed that exposure to nicotine activates the nuclear factor of activated T cells (NFAT) transcription factor in lymphocytes and endothelial cells, leading to alterations in cellular growth and vascular endothelial growth factor production. Here, we extend these studies to document effects of nicotine on lymphocyte survival. The data show that nicotine induces paradoxical effects that might alternatively enforce survival or trigger apoptosis, suggesting that depending on timing and context, nicotine might act both as a survival factor or as an inducer of apoptosis in normal or transformed lymphocytes, and possibly other non-neuronal cells. In addition, our results show that, while having overlapping functions, low and high affinity nAChRs also transmit signals that promote distinct outcomes in lymphocytes. The sum of our data suggests that selective modulation of nAChRs might be useful to regulate lymphocyte activation and survival in health and disease.

Core flexibility of a truncated metazoan mitochondrial tRNA.

Secondary and tertiary structures of tRNAs are remarkably preserved from bacteria to humans, the notable exception being the mitochondrial (m) tRNAs of metazoans, which often deviate substantially from the canonical cloverleaf (secondary) or 'L'-shaped (tertiary) structure. Many metazoan mtRNAs lack either the TpsiC (T) or dihydrouridine (D) loops of the canonical cloverleaf, which are known to confer structural rigidity to the folded structure. Thus, the absence of canonical TpsiC-D interactions likely results in greater dispersion of anticodon-acceptor interstem angle than for canonical tRNAs. To test this hypothesis, we have assessed the dispersion of the anticodon-acceptor angle for bovine mtRNA(Ser)(AGY), which lacks the canonical D arm and is thus incapable of forming stabilizing interarm interactions. Using the method of transient electric birefringence (TEB), and by changing the helical torsion angle between a core mtRNA bend and a second bend of known angle/rigidity, we have demonstrated that the core of mtRNA(Ser)(AGY) has substantially greater flexibility than its well-characterized canonical counterpart, yeast cytoplasmic tRNA(Phe). These results suggest that increased flexibility, in addition to a more open interstem angle, would allow both noncanonical and canonical mtRNAs to utilize the same protein synthetic apparatus.

Subvisible Particles in IVIg Formulations Activate Complement in Human Serum.

When administered intravenously, various particles and nanomedicines activate complement, potentially leading to infusion reactions and other adverse drug reactions. Particles form within formulations of therapeutic proteins due to stresses incurred during shipping, handling, and administration to patients. In this study, IVIg solutions were stored in multiple types of vials and prefilled syringes and exposed to agitation and freeze-thaw stresses to generate particles. The stressed samples were added to human serum to determine whether these particles activated complement. Subvisible IVIg particles ranging in size between 2 and 10 microns activated complement in a fashion that was linearly dependent on the number of particles dosed, whereas little correlation was found between doses of larger particles (>10 microns) and complement activation. Activation of complement by subvisible particles of IVIg followed the alternative pathway, as shown by the release of complement cascade factor Bb and the production of the anaphylatoxins C3a and C5a without generation of C4a. The number and the morphology of subvisible particles formed depended on the applied stress, formulation, and on the container material. But the capacity of the 2- to 10-micron-sized particles to activate complement in human serum appeared to depend only on particle concentration.

DEHP Nanodroplets Leached From Polyvinyl Chloride IV Bags Promote Aggregation of IVIG and Activate Complement in Human Serum.

Concerns regarding the impact of subvisible particulate impurities on the safety and efficacy of therapeutic protein products have led manufacturers to implement strategies to minimize protein aggregation and particle formation during manufacturing, storage, and shipping. However, once these products are released, manufacturers have limited control over product handling. In this work, we investigated the effect of di(2-ethylhexyl) phthalate (DEHP) nanodroplets generated in polyvinyl chloride (PVC) bags of intravenous (IV) saline on the stability and immunogenicity of IV immunoglobulin (IVIG) formulations. We showed that PVC IV bags containing saline can release DEHP droplets into the solution when agitated or transported using a pneumatic tube transportation system in a clinical setting. We next investigated the effects of emulsified DEHP nanodroplets on IVIG stability and immunogenicity. IVIG adsorbed strongly to DEHP nanodroplets, forming a monolayer. In addition, DEHP nanodroplets accelerated IVIG aggregation in agitated samples. The immunogenicity of DEHP nanodroplets and IVIG aggregates generated in these formulations were evaluated using an in vitro assay of complement activation in human serum. The results suggested DEHP nanodroplets shed from PVC IV bags could reduce protein stability and induce activation of the complement system, potentially contributing to adverse immune responses during the administration of therapeutic proteins.

Systemic activation of the complement system in patients with advanced age-related macular degeneration.

PURPOSE: To examine the role of systemic activation of the complement system (assessed by levels of circulating C3a, Ba, and sC5b-9) in patients (n = 122) with advanced age-related macular degeneration, geographic atrophy, and neovascular age-related macular degeneration, compared with cataract controls (n = 27). METHODS: Plasma complement factors were measured using enzyme-linked immunosorbent assays. Statistical analysis included univariate and multivariate logistic regression (p < 0.05). RESULTS: Adjusted for age, the odds ratios of C3a and sC5b-9 for any advanced age-related macular degeneration were 1.78 (95% confidence interval = 1.16-2.73, p < 0.01) and 1.20 (95% confidence interval = 1.04-1.39, p = 0.01), respectively. We found a significantly elevated adjusted odds ratio of C3a (adjusted odds ratio = 1.71, 95% confidence interval = 1.12-2.60, p = 0.01) and sC5b-9 (adjusted odds ratio = 1.22, 95% confidence interval = 1.04-1.43, p = 0.01) for neovascular age-related macular degeneration. Adjusted for age, neither C3a, sC5b-9, nor Ba were associated with geographic atrophy. CONCLUSION: We suggest a role for elevated plasma levels of C3a and sC5b-9 in patients with neovascular age-related macular degeneration. The study's results reinforce the need for more investigation to assess the impact of therapeutic interventions targeted at the complement signaling pathways in age-related macular degeneration.

Cigarette tar phenols impede T cell cycle progression by inhibiting cyclin-dependent kinases.

Cigarette smoking causes profound suppression of pulmonary T cell responses, which is associated with increased susceptibility to respiratory tract infections and decreased tumor surveillance. We previously demonstrated that the phenolic compounds in cigarette tar inhibit blastogenesis and interfere with human T cell cycle progression. To identify the mechanism by which cell cycle arrest occurs, we examined the effects of these compounds on cyclin-dependent kinases (Cdk) that control the G0/G1 transition. We found that hydroquinone inhibited induction of Cdk4 and Cdk6 kinase activities by >80%, while catechol and phenol were markedly less potent. HQ did not affect mitogenic induction of the Cdk6 protein, but inhibited expression of cyclin D3 by >90% resulting in a dramatic reduction in proper Cdk6/Cyclin D3 complex formation.

Variation of the acceptor-anticodon interstem angles among mitochondrial and non-mitochondrial tRNAs.

A cloverleaf secondary structure and the concomitant "L"-shaped tertiary conformation are considered the paradigm for tRNA structure, since there appears to be very little deviation from either secondary or tertiary structural forms among the more than one dozen canonical (cloverleaf) tRNAs that have yielded to crystallographic structure determination. However, many metazoan mitochondrial tRNAs deviate markedly from the canonical secondary structure, and are often highly truncated (i.e. missing either a dihydrouridine or a TPsiC arm). These departures from the secondary cloverleaf form call into question the universality of the tertiary (L-shaped) conformation, suggesting that other structural constraints may be at play for the truncated tRNAs. To examine this issue, a set of 11 tRNAs, comprising mitochondrial and non-mitochondrial, and canonical and non-canonical species, has been examined in solution using the method of transient electric birefringence. Apparent interstem angles have been determined for each member of the set, represented as transcripts in which the anticodon and acceptor stems have each been extended by approximately 70 bp of duplex RNA helix. The measurements demonstrate much more variation in global structure than had been supposed on the basis of crystallographic analysis of canonical tRNAs. In particular, the apparent acceptor-anticodon interstem angles are more obtuse for the metazoan mitochondrial tRNAs that are truncated (missing either a dihydrouridine or a TPsiC arm) than for the canonical (cloverleaf) tRNAs. Furthermore, the magnesium dependence of this interstem angle differs for the set of truncated tRNAs compared to the canonical species.

NFATc2-mediated repression of cyclin-dependent kinase 4 expression.

The calcineurin-regulated transcription factor, nuclear factor of activated T cells (NFAT), controls many aspects of T cell function. Here, we demonstrate that the calcineurin/NFAT pathway negatively regulates the expression of cyclin-dependent kinase 4 (CDK4). A canonical NFAT binding site was identified and found to be sensitive to calcium signals, FK506/CsA, and histone deacetylase activity and to not require AP-1. Ectopic expression of NFATc2 inhibited the basal activity of the human CDK4 promoter. Additionally, both calcineurin Aalpha(-/-) and NFATc2(-/-) mice had elevated protein levels of CDK4, confirming a negative regulatory role for the calcineurin/NFAT pathway. This pathway may thus regulate the expression of CDK4 at the transcriptional level and control how cells re-enter a resting, nonproliferative state.

Nicotine activates nuclear factor of activated T cells c2 (NFATc2) and prevents cell cycle entry in T cells.

We used primary peripheral blood T cells, a population that exists in G(0) and can be stimulated to enter the cell cycle synchronously, to define more precisely the effects of nicotine on pathways that control cell cycle entry and progression. Our data show that nicotine decreased the ability of T cells to transit through the G(0)/G(1) boundary (acquire competence) and respond to progression signals. These effects were due to nuclear factor of activated T cells c2 (NFATc2)-dependent repression of cyclin-dependent kinase 4 (CDK4) expression. Growth arrest at the G(0)/G(1) boundary was further enforced by inhibition of cyclin D2 expression and by increased expression and stabilization of p27Kip1. Intriguingly, T cells from habitual users of tobacco products and from NFATc2-deficient mice constitutively expressed CDK4 and were resistant to the antiproliferative effects of nicotine. These results indicate that nicotine impairs T cell cycle entry through NFATc2-dependent mechanisms and suggest that, in the face of chronic nicotine exposure, selection may favor cells that can evade these effects. We postulate that cross talk between nicotinic acetylcholine receptors and growth factor receptor-activated pathways offers a novel mechanism by which nicotine may directly impinge on cell cycle progression. This offers insight into possible reasons that underlie the unique effects of nicotine on distinct cell types and identifies new targets that may be useful control tobacco-related diseases.

Canine malignant hemangiosarcoma as a model of primitive angiogenic endothelium.

Hemangiosarcoma (HSA) is a common untreatable cancer of dogs that resembles human angiosarcoma. Detailed studies of these diseases have been historically hindered by the paucity of suitable reagents. Here, we show that expression of CD117 (c-Kit) can distinguish primitive (malignant) from mature (benign) proliferative endothelial lesions, and we describe eight independent cell lines derived from canine HSA explants. Endothelial origin was confirmed by sustained expression of surface CD105 (endoglin), CD146 (MUC18), and CD51/CD61 (alpha(v)beta(3) integrin). The cell lines showed anchorage-independent growth and were motile and invasive when cultured on a basement membrane matrix. They required endothelial growth factors for growth and survival, and they could be induced to form tubular structures resembling blood vessels when cultured under low calcium conditions. The formation of vessel-like structures was blocked by nicotine, and restored by FK506, suggesting that 'nuclear factor of activated T cells' activity prevents differentiation of these cells. In summary, these cell lines represent a unique and novel resource to improve our understanding of endothelial cell biology in general and canine HSA in particular.