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.

Serum leptin and ghrelin correlate with disease activity in ANCA-associated vasculitis.

OBJECTIVES: To study serum levels of leptin and ghrelin in ANCA-associated vasculitis (AAV). METHODS: Thirty-seven patients with AAV (21 patients with active AAV at initial presentation and during follow-up, 16 patients with AAV in long-term remission) and 21 matched healthy controls were included. Serum levels of leptin and ghrelin were measured at 0, 6 and 12 months by radioimmunoassay. Disease activity was gauged by Birmingham Vasculitis Activity Score (BVAS), CRP and circulating endothelial cells (CECs). RESULTS: Leptin levels were significantly lower in patients than in healthy controls (9.1 +/- 6.1 vs 22.3 +/- 22.4 ng/ml; P < 0.05). The difference persisted when corrected for BMI. Leptin levels increased significantly after 6 (27.8 +/- 21.9 ng/ml; P < 0.001) and 12 months (24.6 +/- 21.0 ng/ml; P < 0.001). Ghrelin levels were significantly elevated in patients compared with controls (402.6 +/- 112.9 vs 294.8 +/- 70.9 pmol/l; P < 0.005) and declined to normal values at 12 months (306.4 +/- 36.2 pmol/l). There was a significant positive correlation between ghrelin levels and disease activity, whereas leptin levels were negatively correlated with disease activity (CRP, BVAS and CECs). Accordingly, correlations between the ghrelin/leptin ratio and markers of disease activity reached the highest level of significance (all P < 0.001). CONCLUSIONS: Active AAV is characterized by decreased serum leptin and increased serum ghrelin, both of which return to normal with successful therapy. The role of leptin and ghrelin during the pathogenesis of AAV and the effects of these peptides on endothelial cells warrant further study.

Vascular endothelial damage and repair in antineutrophil cytoplasmic antibody-associated vasculitis.

OBJECTIVE: Antineutrophil cytoplasmic antibody-associated vasculitis (AAV) is characterized by necrotizing vessel wall inflammation, paralleled by the detachment of endothelial cells. The repair of such endothelial defects is crucial for the maintenance of regular structure and function of the injured vessels. Bone marrow-derived endothelial progenitor cells (EPCs) are thought to play a pivotal role in the regeneration of damaged endothelium. The aim of this study was to investigate whether EPCs are involved in vascular repair in AAV. METHODS: We assessed disease activity, CD34+ hematopoietic progenitor cells (HPCs) using flow cytometry, EPCs using an in vitro assay, and circulating endothelial cells (CECs) by immunomagnetic isolation from the peripheral blood of 31 patients with active AAV at 1, 3, and 6 months after the initiation of immunosuppressive therapy. RESULTS: In patients with untreated active disease, HPC and EPC numbers were comparable with those in healthy control subjects (n = 64). With the induction of remission, the number of HPCs and EPCs increased significantly, from a median of 1.5 cells/microl (range 0.0-7.0) to a median of 3.2 cells/microl (range 0.76-9.2) (P < 0.001) and from a median of 261 cells/high-power field (range 171-643) to a median of 470 cells/high-power field (range 168-996) (P < 0.021), respectively. In contrast, the initially elevated number of CECs decreased significantly (P < 0.001). We observed no correlation between the number of HPCs or EPCs and the leukocyte count, the thrombocyte count, or kidney function. CONCLUSION: In patients with AAV, the numbers of circulating CD34+ HPCs and EPCs increased significantly after the institution of immunosuppressive therapy and disease remission. This finding points to a role of circulating CD34+ HPCs and EPCs in endothelial repair in vasculitis. Targeted stimulation of these cells might represent a new possibility of improving vascular healing in AAV.

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.

Smoking habits in patients diagnosed with ANCA associated small vessel vasculitis.

OBJECTIVES: To study the prevalence of smoking at onset of symptoms in patients with small vessel vasculitis associated with antineutrophil cytoplasmic antibodies (ANCA). METHODS: A retrospective study, in 197 patients with ANCA associated vasculitis, of the history of cigarette smoking at onset of symptoms. Prevalence of smoking in patients with ANCA associated vasculitis was compared with age-specific values for the general population in Germany. RESULTS: 27 (14%) patients were smokers at the time of first disease manifestation (p < 0.001, compared with the entire population); 54 (27%) had smoked previously with 1-110 pack-years (median 18) but had stopped > or = 2 years before onset of vasculitis; 116 (59%) patients were lifelong non-smokers. At onset of symptoms, active smokers were younger (median age 42 years) than patients with vasculitis (median 54 years, p < 0.01, Mann-Whitney U test) with a lower percentage of women (15%, p < 0.005, Fisher's exact test) than in the entire group (47%). Smokers, non-smokers, or ex-smokers did not differ in organ manifestation, mortality, and development of end stage renal disease and relapse rate. CONCLUSIONS: The proportion of active smokers in the group of patients with ANCA associated vasculitis is significantly lower than in the entire population. Cigarette smoking may be associated with a reduced risk of ANCA associated vasculitis.

Acute liver failure: a message found under the skin.

Acute liver failure is a rare syndrome with rapid progression and high mortality. It is characterised by the onset of coma and coagulopathy usually within six weeks but can occur up to six months after the onset of illness. Viral hepatitis, idiosyncratic drug induced liver injury, and acetaminophen ingestion are common causes. This report describes the case of a 35 year old man who presented with acute liver failure shortly after binge drinking. Repeated history taking disclosed a gluteal disulfiram implant that the patient had received to treat his alcohol dependence. The patient recovered with maximum supportive care after surgical removal but without liver transplantation. This case illustrates that only meticulous history taking will disclose the sometimes bewildering causes of acute liver failure.

Counting the cost: markers of endothelial damage in hematopoietic stem cell transplantation.

During hematopoietic stem cell transplantation (HSCT), endothelial damage is the pathological hallmark of veno-occlusive disease of the liver, thrombotic microangiopathy, capillary leak syndrome and graft-versus-host disease. Events prior to conditioning, the conditioning regimen itself as well as calcineurin inhibitors may all induce endothelial damage. Unfortunately, the relative importance of these factors and their interactions, the time frame of endothelial damage and individual susceptibility remain unknown. Moreover, it is conceivable that conditioning regimens differ markedly in their propensity to initiate endothelial damage. Monitoring endothelial damage and response to treatment is hampered by the current lack of suitable markers. In this regard, an ideal marker should be sensitive and specific and indicate the development of an endothelial disorder prior to the onset of symptoms and organ dysfunction. Soluble markers, such as thrombomodulin, are easily amenable with immunoassays; yet, the interpretation of their levels is hampered by the influence of comorbidity. Evaluation of circulating endothelial cells in HSCT demonstrated a marked and dose-dependent increase in cell numbers after conditioning. The challenge ahead is to establish and evaluate novel markers of endothelial damage to permit early detection of disease, monitor response to treatment and evaluate different conditioning regimens.

A novel multimedia tool to improve bedside teaching of cardiac auscultation.

Training in cardiac auscultation is a core element of undergraduate teaching but recent studies have documented a remarkable decline in auscultatory skills. Therefore there is an interest in new ways to teach cardiac auscultation. In analogy to phonocardiography, an electronic system for simultaneous auscultation and visualisation of murmurs was sought. For this purpose, an electronic stethoscope was linked to a laptop computer and software created to visualise auscultatory findings. In a preliminary trial in undergraduate students, this approach greatly facilitated teaching. Amalgamating traditional phonocardiography with a multimedia approach, this system represents a novel tool for bedside teaching of cardiac auscultation.

An improved assay for enumeration of circulating endothelial cells.

Circulating endothelial cells have been established as markers of vascular disease, such as small vessel vasculitis, acute vascular rejection in renal transplant recipients, and cyclosporine-induced endothelial damage. Enumeration of these cells by immunomagnetic isolation and acridine staining remains the gold standard but necessitates considerable experience and expenditure. A simpler test would therefore be of great utility. Hence, our aim was to develop an improved simple assay to enumerate endothelial cells in peripheral blood. We had already used various surface markers to corroborate the endothelial origin of cells. Here, we studied the enumeration of cell numbers with immunomagnetic isolation and a variety of subsequent stains, such as CD31, von Willebrand's factor (vWF) immunocytochemistry, and Ulex europaeus lectin-1 (UEA-1). Eventually, we devised a simple protocol for enumeration using immunomagnetic isolation and a subsequent UEA-1 lectin stain. We evaluated the use of this protocol in parallel to immunomagnetic isolation and acridine counting alone in 92 renal transplant recipients who underwent renal biopsy. Recovery of various concentrations of human umbilical vein endothelial cells from blood was also studied. Immunomagnetic isolation and subsequent UEA-1 staining permits easier enumeration of circulating endothelial cells in peripheral blood. The assay is simple and easy to use, thus allowing for a more widespread use of circulating endothelial cells as a marker of vascular damage.

Efficiency of high-flux hemodialysis in the treatment of valproic acid intoxication.

Recently, highly efficient (i.e., high volume) dialysis systems have been successfully introduced for the treatment of critically ill patients in the intensive care unit. They also can be a safer, more effective, and less costly alternative to traditional extracorporal techniques in the treatment of severe intoxication. This holds true even if the substance to be eliminated is believed to be a poor candidate for dialysis treatment. We report a case of successful treatment of potentially life-threatening intoxication, with valproic acid (VPA) using a GENIUS batch dialysis system for combined standard and extended high-volume hemodialysis therapy. Concentration of VPA in the total collected dialysate were measured.

Vertebral osteomyelitis and endocarditis of a pacemaker lead due to Granulicatella (Abiotrophia) adiacens.

Systemic infection due to Granulicatella (formerly Abiotrophia), a species of nutrition-deficient gram-positive cocci, is rare. We present the case of a 68-year-old diabetic male who presented with back pain and a history of fever and chills. Imaging studies revealed vertebral osteomyelitis of the Th 10/11 region. Transesophageal echocardiography disclosed a vegetation adjacent to the pacemaker lead and blood cultures grew Granulicatella adiacens. A diagnosis of vertebral osteomyelitis and endocarditis due to G. adiacens was made and the patient improved with bed rest and medical treatment alone. Granulicatella ssp. should always be part of the differential diagnosis of fastidious bacteria in vertebral osteomyelitis and endocarditis.