Familial vasculitides: granulomatosis with polyangitis and microscopic polyangitis in two brothers with differing anti-neutrophil cytoplasm antibody specificity

Anti-neutrophil cytoplasm antibody (ANCA)-associated vasculitis (AAV) is a group of rare autoimmune diseases. Although the aetiology of AAV is uncertain, it is likely that genetic and environmental factors contribute. We report the unusual case of two brothers presenting with AAV with differing clinical pictures and differing ANCA specificity. There is a recently identified difference in genetic risk factors associated with ANCA specificity, making it surprising that first-degree relatives develop AAV with differing clinical and serological features. Our report illustrates the complex aetiology of AAV and suggests that further research on the interaction of genetic and environmental factors is needed

P2X7 receptor antagonism ameliorates renal dysfunction in a rat model of sepsis

Sepsis is a major clinical problem associated with significant organ dysfunction and high mortality. The ATP‐sensitive P2X7 receptor activates the NLRP3 inflammasome and is a key component of the innate immune system. We used a fluid‐resuscitated rat model of fecal peritonitis and acute kidney injury (AKI) to investigate the contribution of this purinergic receptor to renal dysfunction in sepsis. Six and 24 h time‐points were chosen to represent early and established sepsis, respectively. A selective P2X7 receptor antagonist (A‐438079) dissolved in dimethyl sulfoxide (DMSO) was infused 2 h following induction of sepsis. Compared with sham‐operated animals, septic animals had significant increases in heart rate (−1(−4 to 8)% vs. 21(12–26)%; P = 0.003), fever (37.4(37.2–37.6)°C vs. 38.6(38.2–39.0)°C; P = 0.0009), and falls in serum albumin (29(27–30)g/L vs. 26(24–28); P = 0.0242). Serum IL‐1β (0(0–10)(pg/mL) vs. 1671(1445–33778)(pg/mL); P < 0.001) and renal IL‐1β (86(50–102)pg/mg protein vs. 200 (147–248)pg/mg protein; P = 0.0031) were significantly elevated in septic compared with sham‐operated animals at 6 h. Serum creatinine was elevated in septic animals compared with sham‐operated animals at 24 h (23(22–25) μmol/L vs. 28 (25–30)μmol/L; P = 0.0321). Renal IL‐1β levels were significantly lower in A‐438079‐treated animals compared with untreated animals at 6 h (70(55–128)pg/mg protein vs. 200(147–248)pg/mg protein; P = 0.021). At 24 h, compared with untreated animals, A‐438079‐treated animals had more rapid resolution of tachycardia (22(13–36)% vs. −1(−6 to 7)%; P = 0.019) and fever (39.0(38.6–39.1)°C vs. 38.2(37.6–38.7)°C; P < 0.024), higher serum albumin (23(21–25)g/L vs. (27(25–28)g/L); P = 0.006), lower arterial lactate (3.2(2.5–4.3)mmol/L vs. 1.4(0.9–1.8)mmol/L; P = 0.037), and lower serum creatinine concentrations (28(25–30)μmol/L vs. 22(17–27)μmol/L; P = 0.019). P2X7A treatment ameliorates the systemic inflammatory response and renal dysfunction in this clinically relevant model of sepsis‐related AKI

Severe COVID-19 is associated with endothelial activation and abnormal glycosylation of von Willebrand factor in patients undergoing hemodialysis

Background: A major clinical feature of severe coronavirus diease 2019 (COVID-19) is microvascular thrombosis linked to endothelial cell activation. Consistent with this, a number of studies have shown that patients with severe COVID-19 have highly elevated plasma levels of von Willebrand Factor (VWF) that may contribute to the prothrombotic phenotype. In the current study, we investigated the extent of endothelial activation in patients receiving hemodialysis who had either mild or severe COVID-19. Methods: Plasma VWF, ADAMTS-13, angiopoietin-2 (Ang2), and syndecan-1 levels were determined by ELISA. The sialic acid content of VWF was investigated using a modified ELISA to measure elderberry bark lectin, specific for sialic acid residues, binding to VWF. Results: Patients receiving hemodialysis with severe COVID-19 had significantly higher plasma levels of VWF and lower ADAMTS-13. VWF levels peaked and were sustained during the first 10 days after positive confirmation of infection. While Ang2 trended toward being higher in severely ill patients, this did not reach significance; however, severely ill patients had significantly higher soluble syndecan-1 levels, with high levels related to risk of death. Finally, higher VWF levels in severely ill patients were correlated with lower VWF sialic acid content. Conclusions: Severe COVID-19 in patients undergoing hemodialysis is associated with both acute and sustained activation of the endothelium, leading to alteration of the VWF/ADAMTS-13 axis. Lower VWF sialic acid content represents altered VWF processing and further confirms the disturbance caused to the endothelium in COVID-19

Lack of seroresponse to SARS-CoV-2 booster vaccines given early post-transplant in patients primed pre-transplantation

SARS-CoV-2 vaccines are recommended pre-transplantation, however, waning immunity and evolving variants mandate booster doses. Currently there no data to inform the optimal timing of booster doses post-transplant, in patients primed pre-transplant. We investigated serial serological samples in 204 transplant recipients who received 2 or 3 SARS-CoV-2 vaccines pre-transplant. Spike protein antibody concentrations, [anti-S], were measured on the day of transplantation and following booster doses post-transplant. In infection-naïve patients, post-booster [anti-S] did not change when V3 (1st booster) was given at 116(78-150) days post-transplant, falling from 122(32-574) to 111(34-682) BAU/ml, p=0.78. Similarly, in infection-experienced patients, [anti-S] on Day-0 and post-V3 were 1090(133-3667) and 2207(650-5618) BAU/ml respectively, p=0.26. In patients remaining infection-naïve, [anti-S] increased post-V4 (as 2nd booster) when given at 226(208-295) days post-transplant, rising from 97(34-1074) to 5134(229-5680) BAU/ml, p=0.0016. Whilst in patients who had 3 vaccines pre-transplant, who received V4 (as 1st booster) at 82(49-101) days post-transplant, [anti-S] did not change, falling from 981(396-2666) to 871(242-2092) BAU/ml, p=0.62. Overall, infection pre-transplant and [anti-S] at the time of transplantation predicted post-transplant infection risk. As [Anti-S] fail to respond to SARS-CoV-2 booster vaccines given early post-transplant, passive immunity may be beneficial to protect patients during this period

Syk activation in circulating and tissue innate immune cells in antineutrophil cytoplasmic antibody-associated vasculitis

OBJECTIVE: Syk is a cytoplasmic protein tyrosine kinase that plays a role in signaling via B cell and Fc receptors (FcR). FcR engagement and signaling via Syk is thought to be important in antineutrophil cytoplasm antibody (ANCA) IgG-mediated neutrophil activation. This study was undertaken to investigate the role of Syk in ANCA-induced myeloid cell activation and vasculitis pathogenesis. METHODS: Phosphorylation of Syk in myeloid cells from healthy controls and ANCA-associated vasculitis (AAV) patients was analyzed using flow cytometry. The effect of Syk inhibition on myeloperoxidase (MPO)-ANCA IgG activation of cells was investigated using functional assays (interleukin-8 and reactive oxygen species production) and targeted gene analysis with NanoString. Total and phosphorylated Syk at sites of tissue inflammation in patients with AAV was assessed using immunohistochemistry and RNAscope in situ hybridization. RESULTS: We identified increased phosphorylated Syk at critical activatory tyrosine residues in blood neutrophils and monocytes from patients with active AAV compared to patients with disease in remission or healthy controls. Syk was phosphorylated in vitro following MPO-ANCA IgG stimulation, and Syk inhibition was able to prevent ANCA-mediated cellular responses. Using targeted gene expression analysis, we identified up-regulation of FcR- and Syk-dependent signaling pathways following MPO-ANCA IgG stimulation. Finally, we showed that Syk is expressed and phosphorylated in tissue leukocytes at sites of organ inflammation in AAV. CONCLUSION: These findings indicate that Syk plays a critical role in MPO-ANCA IgG-induced myeloid cell responses and that Syk is activated in circulating immune cells and tissue immune cells in AAV; therefore, Syk inhibition may be a potential therapeutic option

Characterisation of an enhanced preclinical model of experimental MPO-ANCA autoimmune vasculitis

Experimental autoimmune vasculitis (EAV) is a model of antineutrophil cytoplasm antibody (ANCA)-associated vasculitis (AAV) induced by immunisation of susceptible rat strains with myeloperoxidase (MPO). Animals develop circulating MPO-ANCA, pulmonary haemorrhage and glomerulonephritis, although renal injury is mild and recovers spontaneously without treatment. In this study we aimed to augment the severity of glomerulonephritis. Following induction of EAV on day 0, a sub-nephritogenic dose of nephrotoxic serum (NTS) containing heterologous antibodies to glomerular basement membrane was administered on day 14. This resulted in a significant increase in disease severity at day 28 compared to MPO immunisation alone - with more urinary abnormalities, infiltrating glomerular leucocytes, and crescent formation that progressed to glomerular and tubulointerstitial scarring by day 56, recapitulating important features of human disease. Importantly, the glomerulonephritis remained pauci-immune, and was strictly dependent on the presence of autoimmunity to MPO, as there was no evidence of renal disease following administration of sub-nephritogenic NTS alone or after immunisation with a control protein in place of MPO. Detailed phenotyping of glomerular leucocytes identified an early infiltrate of non-classical monocytes following NTS administration that, in the presence of autoimmunity to MPO, may initiate the subsequent influx of classical monocytes which augment glomerular injury. We also showed that this model can be used to test novel therapeutics by using a small molecule kinase inhibitor (fostamatinib) that rapidly attenuated both glomerular and pulmonary injury over a four-day treatment period. We believe that this enhanced model of MPO-AAV will prove useful for the study of glomerular leucocyte behaviour and novel therapeutics in AAV in the future. This article is protected by copyright. All rights reserved

Multi-omics identify falling LRRC15 as a COVID-19 severity marker and persistent pro-thrombotic signals in convalescence

Patients with end-stage kidney disease (ESKD) are at high risk of severe COVID-19. Here, we perform longitudinal blood sampling of ESKD haemodialysis patients with COVID-19, collecting samples pre-infection, serially during infection, and after clinical recovery. Using plasma proteomics, and RNA-sequencing and flow cytometry of immune cells, we identify transcriptomic and proteomic signatures of COVID-19 severity, and find distinct temporal molecular profiles in patients with severe disease. Supervised learning reveals that the plasma proteome is a superior indicator of clinical severity than the PBMC transcriptome. We show that a decreasing trajectory of plasma LRRC15, a proposed co-receptor for SARS-CoV-2, is associated with a more severe clinical course. We observe that two months after the acute infection, patients still display dysregulated gene expression related to vascular, platelet and coagulation pathways, including PF4 (platelet factor 4), which may explain the prolonged thrombotic risk following COVID-19

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibody lateral flow assay for antibody prevalence studies following vaccination: a diagnostic accuracy study

Background: Lateral flow immunoassays (LFIAs) are able to achieve affordable, large scale antibody testing and provide rapid results without the support of central laboratories. As part of the development of the REACT programme extensive evaluation of LFIA performance was undertaken with individuals following natural infection. Here we assess the performance of the selected LFIA to detect antibody responses in individuals who have received at least one dose of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine. Methods: This was a prospective diagnostic accuracy study. Sampling was carried out at renal outpatient clinic and healthcare worker testing sites at Imperial College London NHS Trust. Two cohorts of patients were recruited; the first was a cohort of 108 renal transplant patients attending clinic following two doses of SARS-CoV-2 vaccine, the second cohort comprised 40 healthcare workers attending for first SARS-CoV-2 vaccination and subsequent follow up. During the participants visit, finger-prick blood samples were analysed on LFIA device, while paired venous sampling was sent for serological assessment of antibodies to the spike protein (anti-S) antibodies. Anti-S IgG was detected using the Abbott Architect SARS-CoV-2 IgG Quant II CMIA. A total of 186 paired samples were collected. The accuracy of Fortress LFIA in detecting IgG antibodies to SARS-CoV-2 compared to anti-spike protein detection on Abbott Assay Results: The LFIA had an estimated sensitivity of 92.0% (114/124; 95% confidence interval [CI] 85.7% to 96.1%) and specificity of 93.6% (58/62; 95% CI 84.3% to 98.2%) using the Abbott assay as reference standard (using the threshold for positivity of 7.10 BAU/ml) Conclusions: Fortress LFIA performs well in the detection of antibody responses for intended purpose of population level surveillance but does not meet criteria for individual testing