Additional file 2: of Valaciclovir to prevent Cytomegalovirus mediated adverse modulation of the immune system in ANCA-associated vasculitis (CANVAS): study protocol for a randomised controlled trial

CANVAS Version 1.4 of the participant information sheet. (PDF 293 kb

Additional file 3: of Valaciclovir to prevent Cytomegalovirus mediated adverse modulation of the immune system in ANCA-associated vasculitis (CANVAS): study protocol for a randomised controlled trial

CANVAS – Consent Version 1.4 of the consent form. (PDF 139 kb

Additional file 1: of Valaciclovir to prevent Cytomegalovirus mediated adverse modulation of the immune system in ANCA-associated vasculitis (CANVAS): study protocol for a randomised controlled trial

Complete SPIRIT checklist. (DOC 122 kb

Additional file 4: of Valaciclovir to prevent Cytomegalovirus mediated adverse modulation of the immune system in ANCA-associated vasculitis (CANVAS): study protocol for a randomised controlled trial

CANVAS – Lab Manual Version 3.0 of the trial laboratory manual. (DOCX 181 kb

Patient demographics, hematological and biochemical variables according to CMV seropositivity.

*<p>Log transformed prior to analysis. Data are frequency (percentage), mean±standard deviation or median (interquartile range). Data analysed using unpaired two-tailed Student's t-test or Pearson's χ². CMV, cytomegalovirus; eGFR, estimated glomerular filtration rate; hsCRP, high sensitive C-reactive protein; SBP, systolic blood pressure; DBP, diastolic blood pressure; AIx, augmentation index; AIx₇₅, augmentation index adjusted to heart rate of 75 bpm; AoD, aortic distensibility.</p

Patient demographics for 60 patient pairs matched for gender and age.

*<p>log transformed before analysis. CMV, cytomegalovirus; eGFR, estimated glomerular filtration rate; hsCRP, high sensitive C-reactive protein; SBP, systolic blood pressure; DBP, diastolic blood pressure; AIx, augmentation index; AIx₇₅, augmentation index adjusted to heart rate of 75 bpm; PWV, pulse wave velocity; AoD, aortic distensibility.</p

Review of early immune response to SARS-CoV-2 vaccination among patients with CKD

Background: The effects of the coronavirus disease-2019 (COVID-19) pandemic, particularly among those with chronic kidney disease (CKD), who commonly have defects in humoral and cellular immunity, and the efficacy of vaccinations against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) are uncertain. Methods: To inform public health and clinical practice, we synthesized published studies and preprints evaluating surrogate measures of immunity after SARS-CoV-2 vaccination in patients with CKD, including those receiving dialysis or with a kidney transplant. Results: We found 35 studies (28 published, 7 preprints), with sample sizes ranging from 23 to 1140 participants and follow-up ranging from 1 week to 1 month after vaccination. Seventeen of these studies enrolled a control group. In the 22 studies of patients receiving dialysis, the development of antibodies was observed in 18% to 53% after 1 dose and in 70% to 96% after 2 doses of mRNA vaccine. In the 14 studies of transplant recipients, 3% to 59% mounted detectable humoral or cellular responses after 2 doses of mRNA vaccine. After vaccination, there were a few reported cases of relapse or de novo glomerulonephritis, and acute transplant rejection, suggesting a need for ongoing surveillance. Conclusion: Studies are needed to better evaluate the effectiveness of SARS-CoV-2 vaccination in these populations. Rigorous surveillance is necessary for detection of long-term adverse effects in patients with autoimmune disease and transplant recipients. For transplant recipients and those with suboptimal immune responses, alternate vaccination platforms and strategies should be considered. As additional data arise, the NephJC COVID-19 page will continue to be updated (http://www.nephjc.com/news/covid-vaccine)

PR3 and Elastase Alter PAR1 Signaling and Trigger vWF Release via a Calcium-Independent Mechanism from Glomerular Endothelial Cells

<div><p>Neutrophil proteases, proteinase-3 (PR3) and elastase play key roles in glomerular endothelial cell (GEC) injury during glomerulonephritis. Endothelial protease-activated receptors (PARs) are potential serine protease targets in glomerulonephritis. We investigated whether PAR1/2 are required for alterations in GEC phenotype that are mediated by PR3 or elastase during active glomerulonephritis. Endothelial PARs were assessed by flow cytometry. Thrombin, trypsin and agonist peptides for PAR1 and PAR2, TFLLR-NH₂ and SLIGKV-NH_2, respectively, were used to assess alterations in PAR activation induced by PR3 or elastase. Endothelial von Willebrand Factor (vWF)release and calcium signaling were used as PAR activation markers. Both PR3 and elastase induced endothelial vWF release, with elastase inducing the highest response. PAR1 peptide induced GEC vWF release to the same extent as PR3. However, knockdown of PARs by small interfering RNA showed that neither PAR1 nor PAR2 activation caused PR3 or elastase-mediated vWF release. Both proteases interacted with and disarmed surface GEC PAR1, but there was no detectable interaction with cellular PAR2. Neither protease induced a calcium response in GEC. Therefore, PAR signaling and serine protease-induced alterations in endothelial function modulate glomerular inflammation via parallel but independent pathways.</p> </div

PR3 and elastase induce vWF release via a PAR-independent mechanism.

<p>vWF release from untreated GEC in medium alone and endothelial cells after exposure for 2 hr to 100 µM PAR1ap (TFLLR-NH₂) or 100 µM PAR2ap (SLIGKV-NH₂) or 1 µg/ml (34.5 nM) PR3 was measured (Fig. 1A). Data were expressed as mean ± SEM, n = 4. Statistic tests used for Fig. 1A were paired T-tests. For the siRNA knockdown experiments of Fig. 1B, confluent GEC were incubated for 4 hr. Three controls were used (1) Optimem medium (Medium alone; open bars) or (2) Optimem medium containing 0.2% Lipofectamine RNAiMax without siRNA (Control LF; diagonal line bars) or with (3) 20 nM non-silencing scrambled Stealth™ RNAi (Control siRNA; horizontal line bars). PAR expression was silenced by using 20 nM of Stealth™ RNAi against PAR1 (PAR1 siRNA; black bars), or PAR2 (PAR2 siRNA; cross-hatched bars), or both PAR1 and PAR2 (PAR1+2 siRNA; vertical line bars). The vWF release induced by 1 µg/ml PR3 (34.5 nM) or elastase (33.9 nM) were assessed 48 hr after siRNA treatment (Fig. 1B). Statistic test used for Fig. 1B was a Two-way ANOVA p = 0.0001 comparing vWF release from untreated cells vs. cells treated with PR3 and elastase under all four siRNA conditions.</p

The immediate effect of PR3 or elastase on PAR signaling induced by other stimuli.

<p>Fig. 4A–C show the effect of a 10 min pre-treatment of GEC with PR3 (34.5 nM = 1 µg/ml PR3(1) or 172 nM = 5 µg/ml PR3(5)) or elastase (33.9 nM = 1 µg/ml HNE(1), or 169.5 nM = 5 µg/ml HNE(5)), on subsequent PAR1 calcium signaling induced by either thrombin (Fig. 4A) or PAR1ap (Fig. 4B) and on subsequent trypsin activation of PAR2 (Fig. 4C). Baseline calcium levels in untreated cells were recorded (control). The bars marked ‘HBH’ show responses after addition of buffer alone (Hanks balanced salt solution +20 mM HEPES) in the absence of any protease during a 10 min pre-treatment period, followed by stimulation of the cells with either thrombin (Fig. 4A) or PAR1ap (Fig. 4B) or PAR2 (Fig. 4C) were used as positive controls for each experiment. Fig. 4A–C show data from 3–4 independent experiments (mean ± SEM).</p