ANCA-associated vasculitis: pathogenesis, novel markers of the disease and emerging therapies.

Much has been learned in recent years on the pathogenesis of ANCA-associated small-vessel vasculitis. The interaction of primed neutrophils with ANCA and endothelial cells is crucial to the disease. Next we gained a better understanding, from animal models, of the pathogenetic importance of the ANCA antibody. Very recent evidence provides intriguing data regarding the link between infection and vasculitis, LAMP-2 antibodies as novel markers, and NETs as a novel pathogenetic mechanism. It remains to be seen whether others are able to corroborate these findings and whether testing for LAMP-2 antibodies will become part of the clinical routine in vasculitis. Recent years also saw the emergence of various new markers of endothelial damage and the disease itself, such as circulating endothelial cells and endothelial microparticles. These novel markers correlate well with disease activity; they may well complement traditional diagnostic tools, such as ANCA testing. Preliminary evidence has provided some insight into the balance between endothelial damage and repair. Exciting preliminary data also indicate that circulating endothelial cells may not only be markers of disease activity but that these cells may have pathogenetic importance in their own right. These findings may have profound implications for the pathogenesis of vasculitis and vascular disease in general. Recent years also saw the publication of a number of seminal studies for the treatment of ANCA-associated vasculitis. We now have a much better understanding of the role of pulse intravenous cyclophosphamide and plasma exchange than ten or even five years ago. Further studies must now show whether plasma exchange is also beneficial for less severely ill patients with AASV. Finally, as ever, it is hoped that further progress in understanding the disease pathogenesis will also provide more tailored and less toxic therapies.

Diagnostic role of endothelial microparticles in vasculitis.

OBJECTIVE: Endothelial cells play a central pathogenetic role in ANCA-associated small-vessel vasculitis (AAV). Circulating endothelial cells (CECs), as a marker of endothelial damage, have been shown to be elevated in vasculitis. More recently, endothelial microparticles (EMPs) were found to be increased in active childhood vasculitis. The role of EMP in adult AAV and the relationship between EMP and CEC is unclear. PATIENTS AND METHODS: We studied 26 patients with AAV, 12 healthy volunteers and 10 patients with IgA nephropathy as disease control. Platelet-poor plasma was ultracentrifuged. MPs were identified and enumerated with flow cytometry, Annexin V, CD62E and CD105 antibodies. Leucocyte- and platelet-derived MPs were also measured. CEC were isolated and enumerated with CD146-driven immuno-magnetic isolation. RESULTS: EMPs are significantly elevated in patients with active vasculitis (CD62E: mean 248 MP/microl +/- 198 s.d.; CD105: 121 +/- 135/microl) compared with patients in remission/partial remission (CD62E: 55 +/- 30/microl, P = 0.001; CD105: 16 +/- 12/microl, P = 0.002) and healthy volunteers (CD62E: 66 +/- 33/microl, P = 0.002; CD105: 25 +/- 26/microl, P = 0.007). The MP count correlates with disease activity measured by the Birmingham Vasculitis Activity Score (BVAS) (CD62E: r = 0.703; CD105: r = 0.445, P < 0.023). CONCLUSION: EMPs are elevated in active adult AAV. EMP levels correlate with disease activity and might serve as a marker of endothelial activation and damage. Differential detection of endothelial, platelet- and leucocyte-derived MPs may provide more insight in to the pathogenesis of AAV.

Acute tubular injury in protocol biopsies of renal grafts: prevalence, associated factors and effect on long-term function.

Acute tubular injury (ATI) is commonly observed in renal allografts, especially early after transplantation. This study analyzes prevalence and associated clinical conditions of ATI in serial protocol biopsies (pBx) and indication biopsies (iBx), and its impact on long-term graft function. 612 pBx from 204 patients taken at 6 weeks, 3 and 6 months, and 151 iBx performed within the first year of transplantation were evaluated. Prevalence of ATI in pBx was 40% (6 weeks), 34% (3 months) and 37% (6 months), and 46% in iBx. ATI was associated with delayed graft function and prolonged cold ischemia time in pBx, and with acute rejections in iBx. The GFR at 1 and 2 years after transplantation correlated inversely with the frequency of ATI in both pBx and iBx (p < 0.001). Prevalence of chronic changes at 6 months was not significantly related to ATI (patients without ATI: 36%, patients with multiple ATI findings: 54%). ATI is linked to inferior long-term graft function. While this suggests lack of recovery from ATI with permanent allograft damage, the underlying molecular mechanisms need yet to be uncovered. Prevention of the potential pathogenetic factors identified in this study might be the key point to attain good long-term graft function.

Isolation and enumeration of circulating endothelial cells by immunomagnetic isolation: proposal of a definition and a consensus protocol.

BACKGROUND: Circulating endothelial cells (CECs) have been identified as markers of vascular damage in a variety of disorders, such as myocardial infarction, vasculitis, and transplantation. CD146-driven immunomagnetic isolation has gained widespread use, but the technique is hampered by the lack of a definition of CECs and the absence of a consensus for their enumeration. AIM: To evaluate several variables influencing immunomagnetic isolation of CECs, formulate a definition for CECs and propose a consensus protocol for their enumeration. METHODS: We devised a protocol based on CD146-driven immunomagnetic isolation and a subsequent confirmatory step with Ulex-Europaeus-Lectin-1 staining. In a multi-center effort, we evaluated the preanalytical and analytical phases of this protocol. We evaluated the effects of storage, anticoagulation and density centrifugation, and compiled previous experience with this technique. RESULTS: Our protocol permitted unequivocal identification of CECs with acceptable reproducibility. There was an effect of storage time in that median cell numbers declined to only 87.5% of their baseline values during 24 h of storage at 4 degrees C. Recovery was lower with citrate than with ethylene-diamine tetra-acetic acid after 4 h of storage; density centrifugation was also associated with lower recovery. We provide a comprehensive list of technical recommendations and potential pitfalls. Finally, based on our experience with this protocol and a recent consensus workshop, we formulated a working definition for CECs. CONCLUSION: Our work represents an important step toward consensus regarding the CECs. Our recommendations represent the experience of three major centers and should now be scrutinized by others in the field.

Circulating endothelial cells in relapse and limited granulomatous disease due to ANCA associated vasculitis.

OBJECTIVES: To evaluate numbers of circulating endothelial cells (CECs) in ANCA associated vasculitis and compare vasculitic relapse with limited granulomatous disease. METHODS: Sixteen patients with vasculitic relapse of ANCA associated vasculitis and 12 patients with limited granulomatous disease due to Wegener's granulomatosis (WG) were studied. Six patients with newly diagnosed vasculitic disease and six patients with vasculitis with infectious complications were also studied. Twenty two patients in remission were studied, as were 20 healthy controls. Counting of CECs was performed with anti-CD146 driven immunomagnetic isolation and staining with Ulex Europaeus lectin 1(UEA-1). RESULTS: Patients with vasculitic relapse had markedly increased numbers of circulating endothelial cells (12-800 cells/ml, median 88 cells/ml) as did patients with newly diagnosed systemic vasculitis (20-216 cells/ml, median 56 cells/ml). Patients with limited granulomatous disease due to WG had only slightly increased cell numbers (4-44 cells/ml, median 20 cells/ml), which were similar to those of patients in remission (4-36 cells/ml, median 16 cells/ml). Numbers of CECs in patients with granulomatous disease were significantly lower than in those patients with relapse or new onset vasculitis (p<0.001). Cell numbers in patients with relapse and new onset vasculitis declined with immunosuppressive treatment. Patients with infection had 4-36 cells/ml (median 10 cells/ml). A cut off value of 20 cells/ml for a positive result yielded 64% specificity and 95% sensitivity for active systemic vasculitis; the positive predictive value was 63% and the negative predictive value 95%. CONCLUSION: Markedly increased numbers of CECs discriminate active vasculitis from limited granulomatous disease and remission. These findings add further proof to the concept of CECs as a marker of ANCA associated small vessel vasculitis.

Subclinical cardiotoxic effects of anthracyclines as assessed by magnetic resonance imaging-a pilot study.

BACKGROUND: Anthracyclines are potent chemotherapeutics burdened by their cardiotoxicity. So far no marker to detect early cardiac damage exists. We tested the ability of magnetic resonance imaging (MRI) to show early changes in myocardial signal and cardiac function after anthracycline therapy. METHODS: Twenty-two patients with normal cardiac function were investigated by MRI before and 3 and 28 days after anthracycline chemotherapy. Contrast enhanced fast spin echo images were obtained to characterize myocardial enhancement. Left ventricular ejection fraction was measured by MRI in contiguous short-axis planes. RESULTS: All patients remained clinically stable. Ejection fraction decreased from 67.8% +/- 1.4% to 58.9% +/- 1.9% after 28 days (P < .05). The relative myocardial contrast enhancement increased from 3.8 +/- 0.4 to 6.9 +/- 1.1 (P < .01). An increase of the enhancement of >5 on day 3 compared with baseline predicted a significant loss of ejection fraction at 28 days (67.5% +/- 2.8% to 51.4% +/- 5.6%, mean difference 16.1% +/- 6.6%; P < .05), whereas an increase of +5 was not associated with a significant loss of ejection fraction (67.6% +/- 1.7% to 62.5% +/- 1.4%, mean difference 4.1% +/- 2.6%; P not significant). CONCLUSIONS: MRI detects early changes in myocardial contrast and slightly deteriorating cardiac function in patients receiving anthracyclines. Larger patient cohorts and longer follow-up are needed to evaluate MRI as a predictor for anthracycline cardiotoxicity.