Plasma exchange and glucocorticoid dosing for patients with ANCA-associated vasculitis: a clinical practice guideline.

CLINICAL QUESTIONS: What is the role of plasma exchange and what is the optimal dose of glucocorticoids in the first 6 months of therapy of patients with antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV)? This guideline was triggered by the publication of a new randomised controlled trial. CURRENT PRACTICE: Existing guideline recommendations vary regarding the use of plasma exchange in AAV and lack explicit recommendations regarding the tapering regimen of glucocorticoids during induction therapy. RECOMMENDATIONS: The guideline panel makes a weak recommendation against plasma exchange in patients with low or low-moderate risk of developing end stage kidney disease (ESKD), and a weak recommendation in favour of plasma exchange in patients with moderate-high or high risk of developing ESKD. For patients with pulmonary haemorrhage without renal involvement, the panel suggests not using plasma exchange (weak recommendation). The panel made a strong recommendation in favour of a reduced dose rather than standard dose regimen of glucocorticoids, which involves a more rapid taper rate and lower cumulative dose during the first six months of therapy. HOW THIS GUIDELINE WAS CREATED: A guideline panel including patients, a care giver, clinicians, content experts, and methodologists produced these recommendations using GRADE and in adherence with standards for trustworthy guidelines. The recommendations are based on two linked systematic reviews. The panel took an individual patient perspective in the development of recommendations. THE EVIDENCE: The systematic review of plasma exchange identified nine randomised controlled trials (RCTs) that enrolled 1060 patients with AAV. Plasma exchange probably has little or no effect on mortality or disease relapse (moderate and low certainty). Plasma exchange probably reduces the one year risk of ESKD (approximately 0.1% reduction in those with low risk, 2.1% reduction in those with low-moderate risk, 4.6% reduction in those with moderate-high risk, and 16.0% reduction in those with high risk or requiring dialysis) but increases the risk of serious infections (approximately 2.7% increase in those with low risk, 4.9% increase in those with low-moderate risk, 8.5% increase in those with moderate-high risk, to 13.5% in high risk group) at 1 year (moderate to high certainty). The guideline panel agreed that most patients with low or low-moderate risk of developing ESKD would consider the harms to outweigh the benefits, while most of those with moderate-high or high risk would consider the benefits to outweigh the harms. For patients with pulmonary haemorrhage without kidney involvement, based on indirect evidence, plasma exchange may have little or no effect on death (very low certainty) but may have an important increase in serious infections at 1 year (approximately 6.8% increase, low certainty). The systematic review of different dose regimens of glucocorticoids identified two RCTs at low risk of bias with 704 and 140 patients respectively. A reduced dose regimen of glucocorticoid probably reduces the risk of serious infections by approximately 5.9% to 12.8% and probably does not increase the risk of ESKD at the follow-up of 6 months to longer than 1 year (moderate certainty for both outcomes). UNDERSTANDING THE RECOMMENDATION: The recommendations were made with the understanding that patients would place a high value on reduction in ESKD and less value on avoiding serious infections. The panel concluded that most (50-90%) of fully informed patients with AAV and with low or low-moderate risk of developing ESKD with or without pulmonary haemorrhage would decline plasma exchange, whereas most patients with moderate-high or high risk or requiring dialysis with or without pulmonary haemorrhage would choose to receive plasma exchange. The panel also inferred that the majority of fully informed patients with pulmonary haemorrhage without kidney involvement would decline plasma exchange and that all or almost all (≥90%) fully informed patients with AAV would choose a reduced dose regimen of glucocorticoids during the first 6 months of therapy.

Impact of baseline clinical and radiological features on outcome of chronic rhinosinusitis in granulomatosis with polyangiitis.

BACKGROUND: Granulomatosis with polyangiitis (GPA) causes a recurring inflammation in nose and paranasal sinuses that clinically resembles chronic rhinosinusitis (CRS) of other aetiologies. While sinonasal inflammation is not among the life-threatening features of GPA, patients report it to have major negative impact on quality of life. A relatively large proportion of GPA patients have severe CRS with extensive damage to nose and sinus structures evident by CT, but risk factors for severe CRS development remain largely unknown. In this study, we aimed to identify clinical and radiological predictors of CRS-related damage in GPA. METHODS: We included GPA patients who had clinical data sets from time of diagnosis, and two or more paranasal sinus CT scans obtained ≥12 months apart available for analysis. We defined time from first to last CT as the study observation period, and evaluated CRS development across this period using CT scores for inflammatory sinus bone thickening (osteitis), bone destructions, and sinus opacifications (here defined as mucosal disease). In logistic regression, we applied osteitis as main outcome measure for CRS-related damage. RESULTS: We evaluated 697 CT scans obtained over median 5 years observation from 116 GPA patients. We found that 39% (45/116) of the GPA patients remained free from CRS damage across the study observation period, while 33% (38/116) had progressive damage. By end of observation, 32% (37/116) of the GPA patients had developed severe osteitis. We identified mucosal disease at baseline as a predictor for osteitis (odds ratio 1.33), and we found that renal involvement at baseline was less common in patients with severe osteitis at last CT (41%, 15/37) than in patients with no osteitis (60%, 27/45). CONCLUSIONS: In this largely unselected GPA patient cohort, baseline sinus mucosal disease associated with CRS-related damage, as measured by osteitis at the end of follow-up. We found no significant association with clinical factors, but the data set indicated an inverse relationship between renal involvement and severe sinonasal affliction.

Impact of Paranasal Sinus Surgery in Granulomatosis With Polyangiitis: A Longitudinal Computed Tomography Study.

OBJECTIVES/HYPOTHESIS: Severe chronic rhinosinusitis (CRS) in patients with granulomatosis with polyangiitis (GPA) failing medical therapies can be treated with paranasal sinus surgery. Whether this surgery protects from progressive sinonasal damage remains unknown. Here, we aimed to analyze time-dependent relations between sinus surgeries and computed tomography (CT) imaging features in the CRS of GPA. STUDY DESIGN: Longitudinal observational study. METHODS: We assessed CRS features including bone thickening by global osteitis scoring scale, bone erosions, and mucosal thickening by Lund-Mackay scores in serial paranasal sinus CT scans (742 CT scans in total) from a cohort of 127 well-characterized GPA patients. Data on sinonasal surgical procedures were from a mandatory national registry and from chart review. We defined the time from baseline CT to last CT as the study observation period in each patient. Datasets were analyzed by linear mixed models. RESULTS: We found that 23/127 cohort patients had one or more paranasal sinus surgical procedures, and 96% of these (22/23) had osteitis by CT after surgery. In patients with nasal surgery alone or no surgery, we identified osteitis in 7/11 (64%) and 45/93 (48%), respectively. During the observation period of a median of 5 years, 38 patients had progression of their sinus osteitis, with the highest annual osteitis progression rates observed around the time of surgery. CONCLUSIONS: In this cohort, paranasal sinus surgery was associated with prevalence, severity, and progression rate of sinus osteitis, indicating that sinus surgery does not reduce the bone damage development in the CRS of GPA. LEVEL OF EVIDENCE: 4 Laryngoscope, 130: E460-E468, 2020.

Development of CT-based methods for longitudinal analyses of paranasal sinus osteitis in granulomatosis with polyangiitis.

BACKGROUND: Even though progressive rhinosinusitis with osteitis is a major clinical problem in granulomatosis with polyangiitis (GPA), there are no studies on how GPA-related osteitis develops over time, and no quantitative methods for longitudinal assessment. Here, we aimed to identify simple and robust CT-based methods for capture and quantification of time-dependent changes in GPA-related paranasal sinus osteitis and compare performance of the methods under study in a largely unselected GPA cohort. METHODS: GPA patients (n = 121) with ≥3 paranasal CT scans obtained ≥12 months apart and control patients not having GPA or rhinosinusitis (n = 15) were analysed by: (i) Global osteitis scoring scale (GOSS), originally developed for chronic rhinosinusitis; (ii) Paranasal sinus volume by manual segmentation; (iii) Mean maxillary and sphenoid diameter normalised to landmark distances (i.e. diameter ratio measurement, DRM). RESULTS: Time-dependent changes in GPA-related osteitis were equally well measured by the simple DRM and the labour-intensive volume method while GOSS missed ongoing changes in cases with extensive osteitis. GOSS at last CT combined with DRM identified three distinct patient groups: (i) The no osteitis group, who had no osteitis and no change in DRM from baseline CT to last CT (45/121 GPA patients and 15/15 disease controls); (ii) Stable osteitis group, with presence of osteitis, but no change in DRM across time (31 GPA); (iii) Progressive osteitis, defined by declining DRM (45 GPA). CONCLUSIONS: We suggest DRM and GOSS as complementary methods for capturing, classifying and quantifying time-dependent changes in GPA-related osteitis.

Mortality and causes of death across the systemic connective tissue diseases and the primary systemic vasculitides.

Objectives: Studies assessing relative mortality risks across the spectrum of systemic inflammatory rheumatic diseases are largely missing. In this study, we wanted to estimate standard mortality ratios (SMRs) and causes of death in an ethnically homogeneous cohort covering all major CTDs and primary systemic vasculitides (PSVs). Methods: We prospectively followed all incident CTD and PSV cases included in the Norwegian CTD and vasculitis registry (NOSVAR) between 1999 and 2015. Fifteen controls for each patient matched for sex and age were randomly drawn from the Norwegian National Population Registry. Causes of death were obtained from the National Cause of Death Register, death certificates and hospital charts. Results: The cohort included 2140 patients (1534 with CTD, 606 with PSV). During a mean follow-up time of 9 years, 279 of the patients (13%) died, compared with 2864 of 32 086 (9%) controls (P < 0.001). Ten years after diagnosis, the lowest survival was 60% in dcSSc, 73% in anti-synthetase syndrome (ASS) and 75% in lcSSc. In the CTD group, the highest SMRs were observed in dcSSc (SMR 5.8) and ASS (SMR 4.1). In the PSV group, Takayasu arteritis (SMR 2.5) and ANCA-associated vasculitis (SMR 1.5) had the highest SMRs. Major causes of death were cardiovascular disease (CTD 27%, PSV 28%), neoplasms (CTD 25%, PSV 27%), chronic respiratory disease (CTD 20%, PSV10%) and infections (CTD 9%, PSV 16%). Conclusion: We observed premature deaths across the spectrum of CTDs and PSVs, with highest SMRs in dcSSc and ASS. The overall mortality was highest in the CTD group.

One science-driven approach for the regulatory implementation of alternative methods: A multi-sector perspective.

EU regulations call for the use of alternative methods to animal testing. During the last decade, an increasing number of alternative approaches have been formally adopted. In parallel, new 3Rs-relevant technologies and mechanistic approaches have increasingly contributed to hazard identification and risk assessment evolution. In this changing landscape, an EPAA meeting reviewed the challenges that different industry sectors face in the implementation of alternative methods following a science-driven approach. Although clear progress was acknowledged in animal testing reduction and refinement thanks to an integration of scientifically robust approaches, the following challenges were identified: i) further characterization of toxicity pathways; ii) development of assays covering current scientific gaps, iii) better characterization of links between in vitro readouts and outcome in the target species; iv) better definition of alternative method applicability domains, and v) appropriate implementation of the available approaches. For areas having regulatory adopted alternative methods (e.g., vaccine batch testing), harmonised acceptance across geographical regions was considered critical for broader application. Overall, the main constraints to the application of non-animal alternatives are the still existing gaps in scientific knowledge and technological limitations. The science-driven identification of most appropriate methods is key for furthering a multi-sectorial decrease in animal testing.

Frequencies of borderline pulmonary hypertension before and after the DETECT algorithm: results from a prospective systemic sclerosis cohort.

Objective: The DETECT algorithm was developed for screening patients with SSc at high risk of pulmonary arterial hypertension (PAH). We evaluated the impact of this algorithm in a SSc population. Methods: Patients from the unselected, prospective Oslo University Hospital SSc study were divided into the Early and DETECT cohorts, respectively, depending on whether an incident right heart catheterization (RHC) was performed before (2009-13) or after (2014-17) the DETECT algorithm was instituted. A PAH diagnosis and patient risk stratification (low, intermediate and high risk) were performed according to 2015 European Society of Cardiology guidelines. Results: At the time of the incident RHC, PAH frequency was similar between the DETECT (15/84 with PAH; 18%) and Early (16/77; 21%) cohorts, but more patients had borderline pulmonary hypertension (PH) in the DETECT (31%) than in the Early (17%) cohort. PAH risk levels were distributed differently. In the DETECT cohort, 27% and 27% were at low and high risk, respectively, at the time of PAH diagnosis. In the Early cohort, 19 and 44% were at low and high risk, respectively. A follow-up RHC, performed after [mean (SD)] 2.4 (1.8) years, showed that 39% of patients with borderline PH in the Early cohort had developed PAH. Conclusion: The DETECT algorithm did not alter PAH incidence in this unselected SSc population. However, it appeared to affect the risk distribution at the time of PAH diagnosis and increased the frequency of borderline PH cases. These findings may translate into novel opportunities for earlier PAH treatment and, possibly, prevention.

L-selectin--a dynamic regulator of leukocyte migration.

The leukocytic cell adhesion receptor L-selectin mediates the initial step of the adhesion cascade, the capture and rolling of leukocytes on endothelial cells. This event enables leukocytes to migrate out of the vasculature into surrounding tissues during inflammation and immune surveillance. Distinct domains of L-selectin contribute to proper leukocyte migration. In this review, we discuss the contributions of these domains with respect to L-selectin function: the regulation by serine phosphorylation of the cytoplasmic tail, the role of the transmembrane domain in receptor positioning on the cell surface as well as the N-glycosylation of the extracellular part and the identification of novel binding partners.

bZIP-Type transcription factors CREB and OASIS bind and stimulate the promoter of the mammalian transcription factor GCMa/Gcm1 in trophoblast cells.

One of the master regulators of placental cell fusion in mammals leading to multi-nucleated syncytiotrophoblasts is the transcription factor GCMa. Recently, we proved that the cAMP-driven protein kinase A signaling pathway is fundamental for up-regulation of GCMa transcript levels and protein stability. Here, we show that Transducer of Regulated CREB activity (TORC1), the human co-activator of cAMP response element-binding protein (CREB), but not a dominant-negative CREB mutant, significantly up-regulates the GCMa promoter. We identified potential cAMP response element (CRE)-binding sites within the GCMa promoter upstream of the transcriptional start site. Only the CRE site at -1337 interacted strongly with CREB in promoter mapping experiments. The characterization of GCMa promoter mutants and additional bZIP-type family members demonstrated that also old astrocyte specifically-induced substance (OASIS) is able to stimulate GCMa transcription. Knockdown of endogenous CREB or OASIS in BeWo cells decreased endogenous GCMa mRNA level and activity. Overexpression of TORC1 or OASIS in choriocarcinoma cells led to placental cell fusion, accompanied by placental expression of gap junction forming protein connexin-43. Further, we show that CREB expression is replaced by OASIS expression around E12.5 suggesting that a sequential order of bZIP-type family members ensures a high rate of GCMa transcription throughout placentation.

Identification of integrin-alpha4, Rb1, and syncytin a as murine placental target genes of the transcription factor GCMa/Gcm1.

Members of the GCM (glial cells missing) transcription factor family have been shown to act as master regulators in different cells during mammalian and fly development being responsible for processes including gliogenesis, hematopoiesis, placental formation, and development of the parathyroidea. In the central nervous system of flies, several target genes for GCM have been reported, namely repo, pointed, and tramtrack. In mammals, two GCM genes are known (GCMa and GCMb), but the knowledge of their target genes is very limited. Here, we present for the first time a global approach aimed to identify GCMa target genes. We found 66 genes up-regulated and 11 genes down-regulated in GCMa-deficient chorionic tissue of mice at embryonic day 9.5. Moreover, we verified by quantitative reverse transcription-PCR all 11 down-regulated genes. The two most strongly down-regulated genes, integrin-alpha4 and retinoblastoma (Rb1), were further analyzed by promoter studies. Additionally, we identified down-regulation of the murine syncytin A gene, which is fundamental for syncytiotrophoblast formation. Finally, we proved strong down-regulation of integrin-alpha4 and Rb1 transcript levels by in situ hybridization in murine GCMa-deficient placentae at embryonic day 9.5. Our data demonstrate for the first time that genes encoding key regulators of placental tissue formation and architecture are regulated by GCMa.

Interaction, cooperative promoter modulation, and renal colocalization of GCMa and Pitx2.

The transcription factor GCMa is a member of a new small family of transcription factors with a conserved zinc-containing DNA-binding domain. All members of this transcription factor family play crucial roles as master regulators during development. GCMa is restricted to placenta during development and to kidney and thymus at postnatal stages. It is essential for the formation of the placental labyrinth and as a consequence for survival of the embryo from mid-embryogenesis onwards. Here, we identify Pitx transcription factors as GCMa-interacting proteins. We show that Pitx proteins interact via their conserved homeodomain with the DNA-binding domain of GCMa. As a consequence, Pitx proteins and GCMa exhibit cooperative DNA binding. Furthermore, Pitx proteins influence GCMa-dependent promoter activation in a cell-specific manner. One of the three Pitx paralogues in mice, Pitx2, is the predominant Pitx member present in the placenta and colocalizes on the cellular level with GCMa in the kidney. This is the first description of a regulatory cross-talk between a transcription factor of the GCM family and a homeodomain protein.

Mitogen-activated 3p kinase is active in the nucleus.

The MAPK-activated kinase 3pK (chromosome 3p kinase), also known as MAPKAPK-3, is a member of a family of kinases that are activated by more than one mitogen-activated protein kinase (MAPK). 3pK is unique since it was shown to be activated by three members of the MAPK family, namely extracellular-signal-regulated kinase (ERK), p38, and Jun-N-terminal kinase (JNK). Accordingly, 3pK is highly activated both by mitogens and by stress-inducing agents or proinflammatory cytokines. Studies utilizing dominant interfering mutants and pharmacological agents revealed that upon mitogenic stimulation, 3pK is exclusively activated via the classical MAPK cascade, while stress-induced activation of 3pK is mainly mediated by p38. The mechanism defining the specificity of kinase action in response to mitogenic versus stress activation remains unknown. Here we show that 3pK is transported to the cytoplasm upon both stress and mitogenic stimulation. While kinetics of nuclear export are similar in both situations, the activation pattern differs substantially. In the mitogenic situation, active 3pK remains in the nucleus for a significant time and there may fulfill mitogen-specific functions. These data not only show that nuclear export of the kinase is mechanistically uncoupled from its activation, but also provide a novel mechanism by which cells may modulate enzyme activity toward a stimulus-specific response.

The interaction of protein kinase C isozymes alpha, iota, and theta with the cytoplasmic domain of L-selectin is modulated by phosphorylation of the receptor.

The leukocyte adhesion molecule L-selectin has an important role in the initial steps of leukocyte extravasation during inflammation and lymphocyte homing. Its cytoplasmic domain is involved in signal transduction after L-selectin cross-linking and in the regulation of receptor binding activity in response to intracellular signals. However, the signaling events occurring at the level of the receptor are largely unknown. This study therefore addressed the question of whether protein kinases associate with the cytoplasmic domain of the receptor and mediate its phosphorylation. Using a glutathione S-transferase fusion protein of the L-selectin cytoplasmic domain, we isolated a kinase activity from cellular extracts of the human leukemic Jurkat T-cell line that phosphorylated L-selectin on serine residues. This kinase showed characteristics of the protein kinase C (PKC) family. Moreover, the Ca(2+)-independent PKC isozymes theta and iota were found associated with the cytoplasmic domain of L-selectin. Pseudosubstrate inhibitors of these isozymes abolished phosphorylation of the cytoplasmic domain, demonstrating that these kinases are responsible for the phosphorylation. Analysis of proteins specifically bound to the phosphorylated cytoplasmic tail of L-selectin revealed that PKCalpha and -theta are strongly associated with the phosphorylated cytoplasmic domain of L-selectin. Binding of these isozymes to L-selectin was also found in intact cells after phorbol ester treatment inducing serine phosphorylation of the receptor. Furthermore, stimulation of Jurkat T-cells by CD3 cross-linking induced association of PKCalpha and -theta with L-selectin, indicating a role of these kinases in the regulation of L-selectin through the T-cell receptor complex. The phosphorylation-regulated association of PKC isozymes with the cytoplasmic domain of L-selectin indicates an important role of this kinase family in L-selectin signal transduction.

Conservation and variation of structure and function in a newly identified GCM homolog from chicken.

Glial cell missing (GCM) proteins constitute a small family of transcription factors with two members each described in Drosophila and several mammalian species. Here, we report the identification of a GCM homolog from chicken. Although the exon-intron structure is well conserved between chicken GCM and other family members, sequence similarity is largely restricted to the DNA-binding GCM-domain (residues 24-176). In accord with the high degree of sequence conservation within the GCM-domain, the chicken GCM protein has a DNA-binding specificity similar to that of other GCM proteins. Like other GCM proteins, it is located to the nucleus and can act as a transcriptional activator despite the strong divergence in sequences outside the GCM-domain. The chicken GCM protein contains two transactivation domains with cell-specific function, one immediately following the DNA-binding domain, the other at its extreme carboxy terminus. Intriguingly, chicken GCM is expressed only transiently during embryogenesis and is restricted exclusively to extraembryonic tissues where it was detected in close vicinity to embryonic blood vessels. Taking the extraembryonic expression of chicken GCM and mammalian GCMa into account, it is tempting to speculate that a conserved extraembryonic function exists for GCM proteins in birds and mammals.

Structural requirements for nuclear localization of GCMa/Gcm-1.

GCM proteins constitute a small transcription factor family. Nuclear localization of Drosophila GCM is mediated by a typical bipartite nuclear localization sequence (NLS) close to the DNA-binding GCM domain. Here, we have analyzed nuclear localization of the mammalian GCM proteins. Whereas GCMb/Gcm-2 contained a classical bipartite NLS, nuclear localization of GCMa/Gcm-1 was mediated by two regions without resemblance to known NLS, one corresponding to the amino-terminal part of the GCM domain, the second defined as a tyrosine-and-proline-rich carboxy-terminal region. Nuclear import was counteracted by an amino-terminal nuclear export activity. This complex regulation of subcellular localization has important implications for GCMa/Gcm-1 function.

Structure of the GCM domain-DNA complex: a DNA-binding domain with a novel fold and mode of target site recognition.

Glia cell missing (GCM) transcription factors form a small family of transcriptional regulators in metazoans. The prototypical Drosophila GCM protein directs the differentiation of neuron precursor cells into glia cells, whereas mammalian GCM proteins are involved in placenta and parathyroid development. GCM proteins share a highly conserved 150 amino acid residue region responsible for DNA binding, known as the GCM domain. Here we present the crystal structure of the GCM domain from murine GCMa bound to its octameric DNA target site at 2.85 A resolution. The GCM domain exhibits a novel fold consisting of two domains tethered together by one of two structural Zn ions. We observe the novel use of a beta-sheet in DNA recognition, whereby a five- stranded beta-sheet protrudes into the major groove perpendicular to the DNA axis. The structure combined with mutational analysis of the target site and of DNA-contacting residues provides insight into DNA recognition by this new type of Zn-containing DNA-binding domain.