Remote ischaemic conditioning: defining critical criteria for success-report from the 11th Hatter Cardiovascular Workshop.

The Hatter Cardiovascular Institute biennial workshop, originally scheduled for April 2020 but postponed for 2 years due to the Covid pandemic, was organised to debate and discuss the future of Remote Ischaemic Conditioning (RIC). This evolved from the large multicentre CONDI-2-ERIC-PPCI outcome study which demonstrated no additional benefit when using RIC in the setting of ST-elevation myocardial infarction (STEMI). The workshop discussed how conditioning has led to a significant and fundamental understanding of the mechanisms preventing cell death following ischaemia and reperfusion, and the key target cyto-protective pathways recruited by protective interventions, such as RIC. However, the obvious need to translate this protection to the clinical setting has not materialised largely due to the disconnect between preclinical and clinical studies. Discussion points included how to adapt preclinical animal studies to mirror the patient presenting with an acute myocardial infarction, as well as how to refine patient selection in clinical studies to account for co-morbidities and ongoing therapy. These latter scenarios can modify cytoprotective signalling and need to be taken into account to allow for a more robust outcome when powered appropriately. The workshop also discussed the potential for RIC in other disease settings including ischaemic stroke, cardio-oncology and COVID-19. The workshop, therefore, put forward specific classifications which could help identify so-called responders vs. non-responders in both the preclinical and clinical settings.

Comparison of outcomes using the rituximab originator MabThera with the biosimilar Truxima in patients with ANCA-associated vasculitis.

OBJECTIVES: The use of rituximab (MabThera®), an anti-CD20 monoclonal antibody, is the most significant development in the management of anti-neutrophil cytoplasm antibody (ANCA)-associated vasculitis (AAV) since the introduction of cytotoxic therapy in 1950. Truxima® is the first anti-CD20 biosimilar approved for the same indications, and has been available in the UK since 2017. Significant cost savings have been reported when switching to biosimilars, which could lead to greater patient access to such treatment. Therefore, it is important to know whether patients' clinical and laboratory parameters respond equally well to biosimilars as to reference medicines, tested in clinical trials. METHOD: We retrospectively reviewed the clinical outcomes and laboratory parameters in 257 consecutive patients treated with anti-CD20 depletion therapy using MabThera or Truxima, for induction and maintenance of remission, in two tertiary renal centres between 2010 and 2019. RESULTS: We demonstrated no difference between patients treated with MabThera or Truxima in rates of remission, relapse, and hospitalization with infection when used for either induction or maintenance of remission of AAV. In one hospital subgroup analysis, we showed comparable levels of hypogammaglobulinaemia, B-cell depletion, and frequency of infusion reactions, with no significant differences. CONCLUSION: The efficacy and safety of the rituximab biosimilar Truxima are not inferior to the originator MabThera in patients with AAV. Truxima represents a cheaper and safe therapeutic alternative that could increase patient access to rituximab.

Sirolimus use in patients with subglottic stenosis in the context of granulomatosis with polyangiitis (GPA), suspected GPA, and immunoglobulin G4-related disease.

Objective: Subglottic stenosis (SGS) is a severe, life-threatening disease found in immune-mediated diseases such as granulomatosis with polyangiitis (GPA) and in rare cases of immunoglobulin G4 (IgG4)-related disease. It can result in persistent airway compromise due to the fibrotic response following inflammation. Standard management involves repeated endoscopic interventions to dilate the airway, and tracheostomy is occasionally required. In addition, immunosuppression remains a cornerstone of therapy aimed at controlling the underlying inflammatory disease; however, cumulative dosing leads to significant adverse effects. We present five cases of predominantly anti-neutrophil cytoplasmic antibody-negative GPA and a case of IgG4-related disease with SGS, in whom we evaluated the long-term utility of sirolimus, which has beneficial anti-proliferative and fibrotic effects, in the management of their disease. Method: We conducted a retrospective review of a cohort of patients with SGS at a tertiary vasculitis unit. These patients were treated with sirolimus, in addition to conventional medical and endoscopic treatment. Clinical symptoms, frequency and time to endoscopic intervention pre- and post-treatment, additional rescue therapy, and any adverse effects were recorded and analysed. Results: Six patients were treated with sirolimus and followed for up to 9 years; two discontinued the drug owing to adverse effects, early on. In the remaining four patients, glucocorticoids were withdrawn, and the frequency of endoscopic intervention was reduced. One patient on sirolimus required rituximab therapy for disease flare. Conclusion: Sirolimus may be a therapeutic option for some patients with severe SGS, allowing steroid withdrawal and resulting in a positive adverse effect profile.

Renal transplantation for lupus nephritis: non-adherence and graft survival.

OBJECTIVES: Poor adherence to immunosuppressive treatment is common in patients with systemic lupus erythematosus and may identify those with lupus nephritis (LN) who have a poorer prognosis. Non-adherence has also been reported to be a potential adverse outcome predictor in renal transplantation (rTp). We investigated whether non-adherence is associated with increased rTp graft rejection and/or failure in patients with LN. METHODS: Patients with LN undergoing rTp in two major London hospitals were retrospectively included. Medical and electronic records were reviewed for documented concerns of non-adherence as well as laboratory biochemical drug levels. The role of non-adherence and other potential predictors of graft rejection/failure including demographics, comorbidities, age at systemic lupus erythematosus and LN diagnosis, type of LN, time on dialysis prior to rTp and medication use were investigated using logistic regression. RESULTS: Out of 361 patients with LN, 40 had rTp. During a median follow-up of 8.7 years, 17/40 (42.5%) of these patients had evidence of non-adherence. A total of 12 (30.0%) patients experienced graft rejection or failure or both. In the adherent group 2/23 (8.7%) had graft rejection, whilst in the non-adherent this rose to 5/17 (29.4%, p = 0.11). Graft failure was seen in 5/23 (21.7%) patients from the adherent group and 4/17 (23.5%) in the non-adherent group ( p = 0.89). Non-adherent patients had a trend towards increased graft rejection, hazard ratio 4.38, 95% confidence interval = 0.73-26.12, p = 0.11. Patients who spent more time on dialysis prior to rTp were more likely to be adherent to medication, p = 0.01. CONCLUSION: Poor adherence to immunosuppressive therapy is common and has been shown to associate with a trend towards increased graft failure in patients with LN requiring rTp. This is the first paper to report that shorter periods on dialysis prior to transplantation might lead to increased non-adherence in lupus patients.

An analysis of lymphocyte phenotype after steroid avoidance with either alemtuzumab or basiliximab induction in renal transplantation.

Several studies have analyzed the phenotype of repopulated T-lymphocytes following alemtuzumab induction; however there has been less scrutiny of the reconstituted B-cell compartment. In the context of a randomized controlled trial (RCT) comparing alemtuzumab induction with tacrolimus monotherapy against basiliximab induction with tacrolimus and mycophenolate mofetil (MMF) therapy in renal transplantation, we analyzed the peripheral B- and T-lymphocyte phenotypes of patients at a mean of 25 +/- 2 months after transplantation. We examined the relationship between peripheral lymphocyte phenotype and graft function. Patients who received alemtuzumab had significantly higher numbers of B cells including naïve, transitional and regulatory subsets. In contrast, the CD4(+) T-cell compartment was dominated by a memory cell phenotype. Following either basiliximab or alemtuzumab induction patients with lower numbers of B cells or B subsets had significantly worse graft function. For alemtuzumab there was also a correlation between these subsets the stability of graft function and the presence of HLA-specific antibodies. These results demonstrate that a significant expansion of regulatory type B cells is associated with superior graft function and that this pattern is more common after alemtuzumab induction. This phenomenon requires further prospective study to see whether this phenotype could be used to customize immunotherapy.

ANCA-stimulated neutrophils release BLyS and promote B cell survival: a clinically relevant cellular process.

OBJECTIVES: To determine a role for antineutrophil cytoplasmic antibody (ANCA)-activated neutrophils in promoting B cell survival through the release of B lymphocyte stimulator (BLyS). METHODS: Neutrophil BLyS expression was measured by flow cytometry. Concentrations of BLyS in cell supernatants and donor serum samples were measured by ELISA. Cell survival assays were carried out using an L3055 cell line and viability measured by flow cytometry. RESULTS: Tumour necrosis factor α and formyl-Met-Leu-Phe (fMLP) treatment of non-primed neutrophils and treatment of primed neutrophils with anti-PR3 ANCA IgG resulted in a significant increase in surface expression of BLyS within 30 min which returned to basal levels by 2 h. Supernatants from ANCA-stimulated neutrophils were shown to contain increased levels of BLyS and to promote the survival of the centroblast cell line L3055. Serum BLyS concentrations are increased in patients with active ANCA-associated systemic vasculitis and these levels are increased further following 1-3 months of treatment with rituximab. CONCLUSIONS: ANCA specifically causes the release of BLyS from activated neutrophils which can support B cell survival in vitro. The presence of serum BLyS in active disease and its increase following B cell depletion suggest it is an important factor in disease pathogenesis and may facilitate disease relapse.

ANCA-associated vasculitis is linked to carriage of the Z allele of α1 antitrypsin and its polymers.

BACKGROUND: Small studies have linked α1 antitrypsin (α1AT) deficiency to patients with antineutrophil cytoplasmic antibodies (ANCA)-associated vasculitis (AAV). OBJECTIVE: To test the validity and the mechanism of this association between α1AT and AAV. METHODS: The distribution of α1AT deficiency alleles Z and S was compared between 856 White Europeans with AAV and 1505 geographic and ethnically matched healthy controls. Genotyping was performed by allelic discrimination assay. RESULTS: were compared between cases and controls using χ(2) tests. The serum and renal biopsies for α1AT polymers were compared using the polymer-specific 2C1 antibody. The role of α1AT polymers in promoting inflammation was investigated by examining their ability to prime neutrophils for ANCA activation as assessed by CD62L shedding, superoxide production and myeloperoxidase degranulation. Results The Z but not the S allele was over-represented in the patients compared with controls (HR=2.25, 95% CI 1.60 to 3.19). Higher concentrations of polymers of α1AT were detected in serum from patients carrying the Z allele than in those not carrying the Z allele (median (IQR) 1.40 (0.91-3.32) mg/dl vs 0.17 (0.06-0.28) mg/dl, p<0.001); polymers of α1AT were also seen in the renal biopsy of a patient with vasculitic glomerulonephritis. Polymers of α1AT primed neutrophils with CD62L shedding and increased superoxide production following ANCA activation. Carriage of the Z allele was not associated with disease severity, survival or relapse. CONCLUSIONS: The Z but not the S deficiency allele is associated with AAV. Polymers of α1AT are present in the serum and glomeruli of at least some patients with the Z allele, which may promote inflammation through priming of neutrophils.

Wegener's granulomatosis presenting as acute systemic vasculitis following 20 years of limited tracheobronchial disease.

OBJECTIVE: We report a patient with a 20-year history of apparently idiopathic airways stenoses, who presented with an antineutrophil cytoplasmic antibody (ANCA) associated, acute, systemic vasculitis with necrotising glomerulonephritis, subsequently diagnosed as Wegener's granulomatosis. METHODS: We present a case report and a review of the world literature on airway stenosis in Wegener's granulomatosis. RESULTS: To our knowledge, this is the first report of Wegener's granulomatosis manifesting as local airway disease for such a prolonged period, before transforming into a systemic vasculitis. CONCLUSIONS: This case highlights the need for physicians to be alert to the possibility of Wegener's granulomatosis as a cause of apparently idiopathic airway stenosis, and to be aware that systemic disease may occur in very long-standing, limited Wegener's granulomatosis.

Results of endoscopic surgery and intralesional steroid therapy for airway compromise due to tracheobronchial Wegener's granulomatosis.

BACKGROUND: Upper airway compromise due to tracheobronchial stenosis commonly occurs in patients with Wegener's granulomatosis (WG). There is at present no consensus on the optimal management of this life threatening condition. OBJECTIVE: To assess the results of laryngo-tracheo-bronchoscopy, intralesional steroid therapy, laser surgery and dilatation in managing obstructive tracheobronchial WG. METHODS: Records of 18 previously untreated stridulous patients with obstructive tracheobronchial WG, treated between 2004 and 2006, were prospectively recorded on an airway database and retrospectively reviewed. Information about patient and lesion characteristics and treatment details were recorded. Treatment progress was illustrated using a timeline plot, and intervention-free intervals were calculated with actuarial analysis. RESULTS: There were nine males and the average age at presentation was 40 (16) years (range 13-74). There were 13 patients with tracheal and five with tracheal and bronchial lesions. The average tracheal lesion height was 8 (3) mm, located 23 (9) mm below the glottis. There were 1, 10 and 7 Myer-Cotton grade I, II and III lesions, respectively. Mean intervention-free interval following minimally invasive treatment was 26 (2.8) months. Following endobronchial therapy, the median intervention-free interval was 22 months (p>0.8 vs tracheal lesions). No patient required a tracheostomy or endoluminal stenting. CONCLUSIONS: Intralesional steroid therapy and conservative endoluminal surgery is an effective strategy for treating airway compromise due to active tracheal and bronchial WG, obviating the need for airway bypass or stenting. We recommend the combination of endotracheal dilatation, conservative laser surgery and steroid therapy as the standard of care for treating airway compromise due to obstructive tracheobronchial WG.

Effect of targeted disruption of STAT4 and STAT6 on the induction of experimental autoimmune encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) is mediated by myelin-specific CD4(+) T cells secreting Th1 cytokines, while recovery from disease is associated with expression of Th2 cytokines. Investigations into the role of individual cytokines in disease induction have yielded contradictory results. Here we used animals with targeted deletion of the STAT4 or STAT6 genes to determine the role of these signaling molecules in EAE. The STAT4 pathway controls the differentiation of cells into a Th1 phenotype, while the STAT6 pathway controls the differentiation of cells into a Th2 phenotype. We found that mice deficient in STAT4 are resistant to the induction of EAE, with minimal inflammatory infiltrates in the central nervous system. In contrast, STAT6-deficient mice, which have a predominantly Th1 phenotype, experience a more severe clinical course of EAE as compared with wild-type or STAT4 knockout mice. In addition, adoptive transfer studies confirm the regulatory functions of a Th2 environment in vivo. These novel data indicate that STAT4 and STAT6 genes play a critical role in regulating the autoimmune response in EAE.

CD28-independent costimulation of T cells in alloimmune responses.

T cell costimulation by B7 molecules plays an important role in the regulation of alloimmune responses. Although both B7-1 and B7-2 bind CD28 and CTLA-4 on T cells, the role of B7-1 and B7-2 signaling through CTLA-4 in regulating alloimmune responses is incompletely understood. To address this question, we transplanted CD28-deficient mice with fully allogeneic vascularized cardiac allografts and studied the effect of selective blockade of B7-1 or B7-2. These mice reject their grafts by a mechanism that involves both CD4(+) and CD8(+) T cells. Blockade of CTLA-4 or B7-1 significantly accelerated graft rejection. In contrast, B7-2 blockade significantly prolonged allograft survival and, unexpectedly, reversed the acceleration of graft rejection caused by CTLA-4 blockade. Furthermore, B7-2 blockade prolonged graft survival in recipients that were both CD28 and CTLA-4 deficient. Our data indicate that B7-1 is the dominant ligand for CTLA-4-mediated down-regulation of alloimmune responses in vivo and suggest that B7-2 has an additional receptor other than CD28 and CTLA-4 to provide a positive costimulatory signal for T cells.

Transplant accommodation in highly sensitized patients: a potential role for Bcl-xL and alloantibody.

Transplantation of renal allografts into recipients with circulating anti-HLA antibodies results in hyperacute rejection. In some cases, however, antibodies return without causing harm; this phenomenon has been termed 'accommodation'. We have investigated this process in human allotransplantation. We removed anti-HLA antibodies by immunoadsorption in seven highly sensitized dialysis patients who subsequently underwent renal transplantation. Immunohistochemistry of renal biopsies for IgG and antiapoptotic proteins was performed. We also developed a model of 'accommodation' using anti-HLA antibodies eluted from sensitized patients and incubated with human umbilical vein endothelial cells (HUVECs) at different concentrations. Their effect on HUVEC phenotype was then analysed. Anti-donor antibody returned in 4/7 patients, without evidence of hyperacute rejection. Three out of four of these 'accommodated' grafts showed specific endothelial up-regulation of Bcl-xL and 2/2 tested positive for endothelial IgG deposition. HUVECs incubated with subsaturating concentrations of anti-HLA antibody showed increased expression of Bcl-xL, were rendered refractory to endothelial cell activation and became resistant to complement-mediated lysis. In contrast, HUVECs incubated with saturating concentrations underwent activation and expressed low levels of Bcl-xL. In conclusion, endothelial Bcl-xL expression defines the accommodation process in human allografts and this phenotype may be initiated by exposure of endothelium to low concentrations of anti-donor HLA antibodies.

Improving the therapeutic monitoring of cyclosporin A.

The narrow therapeutic window of cyclosporin A (CsA) and the variable pharmacokinetics of the traditional preparation, CsA-SIM (Sandimmune), have made it difficult to establish its optimal use. The introduction of a microemulsion preparation, CsA-ME (Neoral), with less variable pharmacokinetics, has made it possible to attempt to define its use more closely. One hundred and one renal allograft recipients were converted from CsA-SIM to CsA-ME. Absorption was monitored using a standard pharmacokinetic 'profile' measuring 'whole blood' CsA levels at several time points following drug administration. Areas under the resulting time-versus-CsA concentration curve (AUC) were calculated. Surprisingly, many patients showed very little fluctuation in 'whole blood' levels after administration of the conventional preparation: 31% had a difference between trough (to) and maximal levels of < 100 micrograms/L. On microemulsion cyclosporin, there was much better correlation between AUC and several parameters incorporating elements of trough and peak values. The best correlation was with t0 + (t1/4), where t1 represents the concentration at 1 h following administration, (r2 = 0.779 for microemulsion cyclosporin). The use of this parameter is a practical possibility in an out-patient setting. The very common, but under-recognised, pattern of almost flat absorption profiles in patients on conventional CsA suggests that the use of CsA in numerous clinical contexts should be reviewed, since CsA immunosuppression may previously have been inadequately monitored.