Marion Haubitz

Joint European League Against Rheumatism and European Renal Association-European Dialysis and Transplant Association (EULAR/ERA-EDTA) recommendations for the management of adult and paediatric lupus nephritis

Objectives To develop recommendations for the management of adult and paediatric lupus nephritis (LN). Methods The available evidence was systematically reviewed using the PubMed database. A modified Delphi method was used to compile questions, elicit expert opinions and reach consensus. Results Immunosuppressive treatment should be guided by renal biopsy, and aiming for complete renal response (proteinuria <0.5 g/24 h with normal or near-normal renal function). Hydroxychloroquine is recommended for all patients with LN. Because of a more favourable efficacy/toxicity ratio, as initial treatment for patients with class III–IVA or A/C (±V) LN according to the International Society of Nephrology/Renal Pathology Society 2003 classification, mycophenolic acid (MPA) or low-dose intravenous cyclophosphamide (CY) in combination with glucocorticoids is recommended. In patients with adverse clinical or histological features, CY can be prescribed at higher doses, while azathioprine is an alternative for milder cases. For pure class V LN with nephrotic-range proteinuria, MPA in combination with oral glucocorticoids is recommended as initial treatment. In patients improving after initial treatment, subsequent immunosuppression with MPA or azathioprine is recommended for at least 3 years; in such cases, initial treatment with MPA should be followed by MPA. For MPA or CY failures, switching to the other agent, or to rituximab, is the suggested course of action. In anticipation of pregnancy, patients should be switched to appropriate medications without reducing the intensity of treatment. There is no evidence to suggest that management of LN should differ in children versus adults. Conclusions Recommendations for the management of LN were developed using an evidence-based approach followed by expert consensus.

Circulating endothelial cells Biomarker of vascular disease

Recent research has recognised new populations of non-hematopoietic cells in the blood. One of these, circulating endothelial cells (CECs), often defined by the expression of membrane glycoprotein CD146, are rarely found in the blood in health, but raised numbers are present in a wide variety of human conditions, including inflammatory, immune, infectious, neoplastic and cardiovascular disease, and seem likely to be evidence of profound vascular insult. An additional population are endothelial progenitor cells, defined by the co-expression of endothelial and immaturity cell surface molecules and also by the ability to form colonies in vitro. Although increased numbers of CECs correlate with other markers of vascular disease, questions remain regarding the precise definition, cell biology and origin of CECs. For example, they may be damaged, necrotic or apopototic, or alive, and could possess procoagulant and/or proinflammatory properties. However, since these cells seem to be representative of in situ endothelium, their phenotype may provide useful information. Indeed, whatever their phenotype, there is growing evidence that CECs may well be a novel biomarker, the measurement of which will have utility in various clinical settings related to vascular injury. Despite this promise, progress is impeded by the diversity of methodologies used to detect these cells. Accordingly, results are sometimes inconclusive and even conflicting. Nevertheless, increased CECs predict adverse cardiovascular events in acute coronary syndromes, suggesting they may move from being simply a research index to having a role in the clinic. The objective of the present communication is to condense existing data on CECs, briefly compare them with progenitor cells, and summarise possible mechanism(s) by which they may contribute to vascular pathology.

Circulating endothelial cells as markers for ANCA-associated small-vessel vasculitis

BACKGROUND Histological findings in small-vessel vasculitis associated with antineutrophil cytoplasmic antibodies (ANCAs) suggest that damaged endothelial cells undergo necrosis and detachment from the basement membrane. We postulated that isolation of these cells from peripheral blood might provide a novel marker of the disease and elucidate pathogenetic events. METHODS 18 patients with active ANCA-associated vasculitis, 20 patients in remission, 20 healthy controls, 12 patients with infection, and 12 patients with glomerular disease not associated with ANCA were studied. Endothelial cells were isolated from peripheral blood by use of Dynabeads coated with antibodies against CD146, and were stained for von Willebrand factor (vWF), CD31, and Ulex Europaeus lectin 1 (UEA-1). Tissue-factor immunocytochemistry and assays for markers of apoptosis and necrosis were also done. FINDINGS Few circulating endothelial cells were seen in healthy controls (0-20 cells/mL, median 5 cells/mL), patients with infection (0-16 cells/mL, median 8 cells/mL), and patients with non-ANCA glomerulonephritis (0-21 cells/mL, median 4 cells/mL). By contrast, large numbers of circulating endothelial cells were detected in patients with active vasculitis (20-5700 cells/mL, median 136 cells/mL, p<0.0001 when compared with healthy controls). Cell numbers fell substantially during 6 months of successful immunosuppressive treatment among those with active disease. Patients in remission had moderately raised cell numbers (0-60 cells/mL, median 16 cells/mL). 84% of cells obtained from patients with active disease stained positive for annexin/propidium iodide and 86% stained tissue factor positive, indicating a necrotic and procoagulant phenotype. INTERPRETATION Circulating endothelial cells are a novel marker of active ANCA-associated small-vessel vasculitis. The clinical use of this tool and the pathogenic mechanisms leading to these findings require further investigation.

Naturally occurring human urinary peptides for use in diagnosis of chronic kidney disease

Because of its availability, ease of collection, and correlation with physiology and pathology, urine is an attractive source for clinical proteomics/peptidomics. However, the lack of comparable data sets from large cohorts has greatly hindered the development of clinical proteomics. Here, we report the establishment of a reproducible, high resolution method for peptidome analysis of naturally occurring human urinary peptides and proteins, ranging from 800 to 17,000 Da, using samples from 3,600 individuals analyzed by capillary electrophoresis coupled to MS. All processed data were deposited in an Structured Query Language (SQL) database. This database currently contains 5,010 relevant unique urinary peptides that serve as a pool of potential classifiers for diagnosis and monitoring of various diseases. As an example, by using this source of information, we were able to define urinary peptide biomarkers for chronic kidney diseases, allowing diagnosis of these diseases with high accuracy. Application of the chronic kidney disease-specific biomarker set to an independent test cohort in the subsequent replication phase resulted in 85.5% sensitivity and 100% specificity. These results indicate the potential usefulness of capillary electrophoresis coupled to MS for clinical applications in the analysis of naturally occurring urinary peptides.

Intravenous pulse administration of cyclophosphamide versus daily oral treatment in patients with antineutrophil cytoplasmic antibody-associated vasculitis and renal involvement: a prospective, randomized study.

OBJECTIVE There is growing concern about the toxic side effects of daily oral cyclophosphamide (CYC) treatment. Intravenous (i.v.) pulse administration of CYC has been shown to be effective in patients with systemic lupus erythematosus, but contradictory results have been reported in patients with antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis. METHODS The efficacy and toxicity of i.v. pulse administration of CYC (0.75 gm/m2) versus daily oral CYC treatment (2 mg/kg body weight) were investigated in a prospective, randomized, multicenter study in patients with ANCA-associated vasculitis and renal involvement. RESULTS The cumulative CYC dose was reduced by 57% in patients with i.v. pulse treatment (n = 22) compared with patients treated with daily oral therapy (n = 25). Patient survival, remission rate, time of remission, relapse rate, and outcome of renal function were not different between the 2 treatment groups. However, the rate of leukopenia (P < 0.01) and severe infections (P < 0.05 by 1-tailed test) was significantly reduced in the i.v. pulse group compared with the group receiving daily oral treatment. Moreover, gonadal toxicity was reduced in the i.v. pulse group, as indicated by significantly lower levels of follicle-stimulating hormone. CONCLUSION This randomized study shows that i.v. CYC administration is an effective therapeutic tool with low toxicity in patients with ANCA-associated vasculitis and renal involvement.

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

Summary.  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 °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.

15-Deoxyspergualin in Patients with Refractory ANCA-Associated Systemic Vasculitis: A Six-Month Open-Label Trial to Evaluate Safety and Efficacy

The combination of cyclophosphamide (CYC) and oral corticosteroids is effective in the majority of patients with antineutrophil cytoplasmic antibody-associated vasculitis (AASV), but it carries substantial risk of drug-related morbidity and mortality. New regimens are desired, especially in refractory cases. The immunosuppressant 15-deoxyspergualin (DSG) is effective in experimental autoimmune disease and transplantation as well as in acute kidney transplant rejection in humans. To assess the efficacy and safety of DSG, an open label multicenter trial was conducted in patients with AASV who were either unresponsive or had contraindications for standard immunosuppressants. Included were 19 cases of Wegener granulomatosis and one case of microscopic polyangiitis. Nine of them had received CYC shortly before study entry without apparent therapeutic success. DSG (0.5 mg/kg per d) was given for 2 to 3 wk until the WBC count dropped to 3000/ micro l followed by a rest until at least a WBC of 4000/ micro l was reached again. This was repeated up to six cycles. During the study, no other immunosuppressants besides steroids were allowed. Disease improvement during treatment with DSG was achieved in 70% of cases (six cases of complete remission; eight cases of partial remission). Leucopenia occurred in each patient in a regular pattern during the cycles and was transient without exception. No mortality or septicemia was observed. Mild to moderate infections mainly in the respiratory tract were observed but resolved under adequate treatment without sequel. It is concluded that treatment with DSG is successful in patients with refractory Wegener granulomatosis under careful monitoring of WBC count.

Detection of Acute Tubulointerstitial Rejection by Proteomic Analysis of Urinary Samples in Renal Transplant Recipients

This study investigates proteomic analysis of urinary samples as a non‐invasive method to detect acute rejection of renal allografts.

Detection of circulating microparticles by flow cytometry: influence of centrifugation, filtration of buffer, and freezing

The clinical importance of microparticles resulting from vesiculation of platelets and other blood cells is increasingly recognized, although no standardized method exists for their measurement. Only a few studies have examined the analytical and preanalytical steps and variables affecting microparticle detection. We focused our analysis on microparticle detection by flow cytometry. The goal of our study was to analyze the effects of different centrifugation protocols looking at different durations of high and low centrifugation speeds. We also analyzed the effect of filtration of buffer and long-term freezing on microparticle quantification, as well as the role of Annexin V in the detection of microparticles. Absolute and platelet-derived microparticles were 10- to 15-fold higher using initial lower centrifugation speeds at 1500 × g compared with protocols using centrifugation speeds at 5000 × g (P < 0.01). A clear separation between true events and background noise was only achieved using higher centrifugation speeds. Filtration of buffer with a 0.2 μm filter reduced a significant amount of background noise. Storing samples for microparticle detection at −80°C decreased microparticle levels at days 28, 42, and 56 (P < 0.05 for all comparisons with fresh samples). We believe that staining with Annexin V is necessary to distinguish true events from cell debris or precipitates. Buffers should be filtered and fresh samples should be analyzed, or storage periods will have to be standardized. Higher centrifugation speeds should be used to minimize contamination by smaller size platelets.

Diagnosis and Prediction of CKD Progression by Assessment of Urinary Peptides

Progressive CKD is generally detected at a late stage by a sustained decline in eGFR and/or the presence of significant albuminuria. With the aim of early and improved risk stratification of patients with CKD, we studied urinary peptides in a large cross-sectional multicenter cohort of 1990 individuals, including 522 with follow-up data, using proteome analysis. We validated that a previously established multipeptide urinary biomarker classifier performed significantly better in detecting and predicting progression of CKD than the current clinical standard, urinary albumin. The classifier was also more sensitive for identifying patients with rapidly progressing CKD. Compared with the combination of baseline eGFR and albuminuria (area under the curve [AUC]=0.758), the addition of the multipeptide biomarker classifier significantly improved CKD risk prediction (AUC=0.831) as assessed by the net reclassification index (0.303±-0.065; P<0.001) and integrated discrimination improvement (0.058±0.014; P<0.001). Correlation of individual urinary peptides with CKD stage and progression showed that the peptides that associated with CKD, irrespective of CKD stage or CKD progression, were either fragments of the major circulating proteins, suggesting failure of the glomerular filtration barrier sieving properties, or different collagen fragments, suggesting accumulation of intrarenal extracellular matrix. Furthermore, protein fragments associated with progression of CKD originated mostly from proteins related to inflammation and tissue repair. Results of this study suggest that urinary proteome analysis might significantly improve the current state of the art of CKD detection and outcome prediction and that identification of the urinary peptides allows insight into various ongoing pathophysiologic processes in CKD.