Y.K. Onno Teng

Induction of long-term B-cell depletion in refractory rheumatoid arthritis patients preferentially affects autoreactive more than protective humoral immunity

IntroductionB-cell depletion has become a common treatment strategy in anti-TNF-refractory rheumatoid arthritis (RA). Although the exact mechanism of how B-cell depletion leads to clinical amelioration in RA remains to be elucidated, repetitive treatment with B-cell-depleting agents leading to long-term B-cell depletion has been reported to be beneficial. The latter has led to the hypothesis that the beneficial effects of B-cell depletion might act through their influence on pathogenic autoreactive plasma cells.MethodsIn this study, we investigated the effects of a fixed retreatment regimen with anti-CD20 mAbs on the humoral (auto)immune system in a cohort of therapy-refractory RA patients.ResultsFixed retreatment led to long-term B-cell depletion in peripheral blood, bone marrow and, to a lesser extent, synovium. Also, pathologic autoantibody secretion (that is, anticitrullinated peptide antibodies (ACPAs)) was more profoundly affected by long-term depletion than by physiological protective antibody secretion (that is, against measles, mumps and rubella). This was further illustrated by a significantly shorter estimated life span of ACPA-IgG secretion compared to total IgG secretion as well as protective antibody secretion.ConclusionBy studying plasma cell function during an extensive 2-year period of B-cell depletion, autoantibody secretion was significantly shorter-lived than physiologically protective antibody secretion. This suggests that the longevity of autoreactive plasma cells is different from protective long-lived plasma cells and might indicate a therapeutic window for therapies that target plasma cells.

A novel method for high-throughput detection and quantification of neutrophil extracellular traps reveals ROS-independent NET release with immune complexes

A newly-described first-line immune defence mechanism of neutrophils is the release of neutrophil extracellular traps (NETs). Immune complexes (ICxs) induce low level NET release. As such, the in vitro quantification of NETs is challenging with current methodologies. In order to investigate the role of NET release in ICx-mediated autoimmune diseases, we developed a highly sensitive and automated method for quantification of NETs. After labelling human neutrophils with PKH26 and extracellular DNA with Sytox green, cells are fixed and automatically imaged with 3-dimensional confocal laser scanning microscopy (3D-CLSM). NET release is then quantified with digital image analysis whereby the NET amount (Sytox green area) is corrected for the number of imaged neutrophils (PKH26 area). A high sensitivity of the assay is achieved by a) significantly augmenting the area of the well imaged (11%) as compared to conventional assays (0.5%) and b) using a 3D imaging technique for optimal capture of NETs, which are topologically superimposed on neutrophils. In this assay, we confirmed low levels of NET release upon human ICx stimulation which were positive for citrullinated histones and neutrophil elastase. In contrast to PMA-induced NET release, ICx-induced NET release was unchanged when co-incubated with diphenyleneiodonium (DPI). We were able to quantify NET release upon stimulation with serum from RA and SLE patients, which was not observed with normal human serum. To our knowledge, this is the first semi-automated assay capable of sensitive detection and quantification of NET release at a low threshold by using 3D CLSM. The assay is applicable in a high-throughput manner and allows the in vitro analysis of NET release in ICx-mediated autoimmune diseases.

Differential responsiveness to immunoablative therapy in refractory rheumatoid arthritis is associated with level and avidity of anti-cyclic citrullinated protein autoantibodies: a case study

In order to identify pathogenic correlates of refractory rheumatoid arthritis (RA), antibodies against anti-cyclic citrullinated protein (ACPAs) were investigated in RA patients in whom the dysregulated immune system had been ablated by high-dose chemotherapy (HDC) and autologous haematopoietic stem cell transplantation (HSCT). Six patients with refractory RA were extensively characterized in terms of levels of total immunoglobulins, RA-specific autoantibodies (ACPAs and rheumatoid factor) and antibodies against rubella, tetanus toxoid (TT) and phosphorylcholine before and after HDC plus HSCT. Additionally, the avidity of ACPAs was measured before and after treatment and compared with the avidity of TT antibodies following repeated immunizations. Synovial biopsies were obtained by arthroscopy before HDC plus HSCT, and analyzed by immunohistochemistry. In the three patients with clinically long-lasting responses to HDC plus HSCT (median 423 days), significant reductions in ACPA-IgG levels after therapy were observed (median level dropped from 215 to 34 arbitrary units/ml; P = 0.05). In contrast, stable ACPA-IgG levels were observed in three patients who relapsed shortly after HDC plus HSCT (median of 67 days). Clinical responders had ACPA-IgG of lower avidity (r = 0.75; P = 0.08) and higher degree of inflammation histologically (r = 0.73; P = 0.09). Relapse (after 38 to 530 days) in all patients was preceded by rising levels of low avidity ACPA-IgG (after 30 to 388 days), in contrast to the stable titres of high avidity TT antibodies. In conclusion, humoral autoimmune responses were differentially modulated by immunoablative therapy in patients with synovial inflammation and low avidity ACPA-IgG autoantibodies as compared with patients with high levels of high avidity ACPA-IgG. The distinct clinical disease course after immunoablative therapy based on levels and avidity of ACPA-IgG indicates that refractory RA is not a single disease entity.

Clinical and Histopathologic Characteristics Associated with Renal Outcomes in Lupus Nephritis

BACKGROUND AND OBJECTIVES The prognostic significance of histopathologic (sub)classes in the current classification of lupus nephritis (LN) is controversial. We analyzed clinical and histopathologic predictors of renal outcome in LN outside the framework of the classification. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS Variables (50 histopathologic and ten clinical) were tested in mixed, linear, and Cox regression models for their association with renal flare, ESRD, and eGFR during follow-up (1, 5, and 10 years) in 105 patients with LN who underwent biopsy from 1987 to 2011. The Cockcroft-Gault (normalized to a body surface area of 1.73 m2) and Schwartz formulas were used to calculate eGFR for adults and children, respectively. RESULTS During median follow-up of 9.9 years (25th-75th percentile, 5.9-13.8), 47 patients experienced a renal flare and 21 progressed to ESRD. Renal flare was predicted by fibrinoid necrosis (hazard ratio [HR], 1.04 per %; 95% confidence interval [95% CI], 1.00 to 1.07) and nonwhite race (HR, 2.23; 95% CI, 1.23 to 4.04). ESRD was predicted by fibrinoid necrosis (HR, 1.08 per %; 95% CI, 1.02 to 1.13), fibrous crescents (HR, 1.09 per %; 95% CI, 1.02 to 1.17), interstitial fibrosis/tubular atrophy (IF/TA) ≥25% (HR, 3.89; 95% CI, 1.25 to 12.14), eGFR at baseline (HR, 0.98 per ml/min per 1.73 m2; 95% CI, 0.97 to 1.00), and nonwhite race (HR, 7.16; 95% CI, 2.34 to 21.91). A higher mean eGFR during follow-up was associated with normal glomeruli (+0.2 ml/min per 1.73 m2 per %; 95% CI, 0.1 to 0.4). Like ESRD, a lower eGFR during follow-up was associated with fibrous crescents, IF/TA≥25%, and nonwhite race, as well as with cellular/fibrocellular crescents (-0.4 ml/min per 1.73 m2 per %; 95% CI, -0.6 to -0.2) and age (-0.8 ml/min per 1.73 m2 per year; 95% CI, -1.2 to -0.4). CONCLUSION The LN classification should include an index of evidence-based prognosticators. Awaiting validation of a formal index, we suggest that at least fibrinoid necrosis, fibrous crescents, and IF/TA warrant explicit independent scoring to assess the risk of progressive renal dysfunction in conjunction with clinical findings.

TAC-TIC use of tacrolimus-based regimens in lupus nephritis

Current guidelines do not mention tacrolimus (TAC) as a treatment option and no consensus has been reported on the role of TAC in lupus nephritis (LN). The present study aimed to guide clinical judgement on the use of TAC in patients with LN. A meta-analysis was performed for clinical studies investigating TAC regimens in LN on the basis of treatment target (induction or maintenance), concomitant immunosuppression and quality of the data. 23 clinical studies performed in patients with LN were identified: 6 case series, 9 cohort studies, 2 case-control studies and 6 randomised controlled trials (RCTs). Of the 6 RCTs, 5 RCTs investigated TAC regimens as induction treatment and 1 RCT as maintenance treatment. Five RCTs investigated TAC in combination with steroids and 2 TAC with mycophenolate plus steroids. All RCTs were performed in patients of Asian ethnicity. In a meta-analysis, TAC regimens achieved a significantly higher total response (relative risk (RR) 1.23, 95% CI 1.12 to 1.34, p<0.05) and significantly higher complete response (RR 1.48, 95% CI 1.23 to 1.77, p<0.05). The positive outcome was predominantly defined by the largest RCT investigating TAC with mycophenolate plus steroids. Regarding safety, the occurrence of leucopoenia was significantly lower, while the occurrence of increased creatine was higher. Clinical studies on TAC regimens for LN are limited to patients of Asian ethnicity and hampered by significant heterogeneity. The positive results on clinical efficacy of TAC as induction treatment in LN cannot be extrapolated beyond Asian patients with LN. Therefore, further confirmation in multiethnic, randomised trials is mandatory. Until then, TAC can be considered in selected patients with LN.

An evidence-based approach to pre-pregnancy counselling for patients with systemic lupus erythematosus

Patients with SLE are often young females of childbearing age and a pregnancy wish in this patient group is common. However, SLE patients are at high risk for adverse pregnancy outcomes that require adequate guidance. It is widely acknowledged that pre-pregnancy counselling is the pivotal first step in the management of SLE patients with a wish to become pregnant. Next, management of these patients is usually multidisciplinary and often requires specific expertise from the different physicians involved. Very recently a EULAR recommendation was published emphasizing the need for adequate preconception counselling and risk stratification. Therefore the present review specifically addresses the issue of pre-pregnancy counselling for SLE patients with an evidence-based approach. The review summarizes data retrieved from recently published, high-quality cohort studies that have contributed to a better understanding and estimation of pregnancy-related risks for SLE patients. The present review categorizes risks from a patient-oriented point of view, that is, the influence of pregnancy on SLE, of SLE on pregnancy, of SLE on the foetus/neonate and of SLE-related medication. Lastly, pre-pregnancy counselling of SLE patients with additional secondary APS is reviewed. Collectively these data can guide clinicians to formulate appropriate preventive strategies and patient-tailored monitoring plans during pre-pregnancy counselling of SLE patients.