Catherine Fournier

Characterization and Functional Consequences of Underexpression of Clusterin in Rheumatoid Arthritis

We previously compared by microarray analysis gene expression in rheumatoid arthritis (RA) and osteoarthritis (OA) tissues. Among the set of genes identified as a molecular signature of RA, clusterin (clu) was one of the most differentially expressed. In the present study we sought to assess the expression and the role of CLU (mRNA and protein) in the affected joints and in cultured fibroblast-like synoviocytes (FLS) and to determine its functional role. Quantitative RT-PCR, Northern blot, in situ hybridization, immunohistochemistry, and Western blot were used to specify and quantify the expression of CLU in ex vivo synovial tissue. In synovial tissue, the protein was predominantly expressed by synoviocytes and it was detected in synovial fluids. Both full-length and spliced isoform CLU mRNA levels of expression were lower in RA tissues compared with OA and healthy synovium. In synovium and in cultured FLS, the overexpression of CLU concerned all protein isoforms in OA whereas in RA, the intracellular forms of the protein were barely detectable. Transgenic overexpression of CLU in RA FLS promoted apoptosis within 24 h. We observed that CLU knockdown with small interfering RNA promoted IL-6 and IL-8 production. CLU interacted with phosphorylated IκBα. Differential expression of CLU by OA and RA FLS appeared to be an intrinsic property of the cells. Expression of intracellular isoforms of CLU is differentially regulated between OA and RA. We propose that in RA joints, high levels of extracellular CLU and low expression of intracellular CLU may enhance NF-κB activation and survival of the synoviocytes.

Syngeneic fibroblasts transfected with a plasmid encoding interleukin-4 as non-viral vectors for anti-inflammatory gene therapy in collagen-induced arthritis.

No effective long‐term treatment is available for rheumatoid arthritis. Recent advances in gene therapy and cell therapy have demonstrated efficiency in collagen‐induced arthritis (CIA). Interleukin‐4 (IL‐4) is already known to be efficient in CIA in systemic injection or administered by gene therapy. This study was designed to evaluate the effect of a non‐viral gene therapy of CIA, involving injection of syngeneic fibroblasts transfected with a plasmid encoding for IL‐4.

B cell apoptosis accelerates the onset of murine lupus.

To investigate whether the increased rate of lymphocyte apoptosis in systemic lupus erythematosus is involved in the onset of the disease, apoptotic or necrotic T or B lymphocytes from variouscell lines were injected intraperitoneally into pre‐autoimmune (NZB×NZW)F1 mice (BW) and non‐autoimmune BALB/c mice. The intraperitoneal production of cytokines and chemokines, the specific T cell response in the spleen, and the production of anti‐histone and anti‐dsDNA Ab were investigated. The onset of the disease was characterized by creatinine levels and evaluation of glomerular IgG deposits. In BW, but not in BALB/c mice, injection of apoptotic and not necrotic cells up‐regulated IL‐6 and IL‐10 in resident macrophages. Administration of apoptotic cells augmented the number of Th2 and B lymphocytes recruited in the peritoneal cavity. Only the treatment with apoptotic B cells promoted a systemic Th2 autoimmune response to H2 histones, associated with earlier occurrence of high levels of anti‐dsDNA autoantibodies, higher creatinine levels and more numerous glomerular IgG deposits than in BW controls not injected with apoptotic B cells. In genetically susceptible mice exposure to apoptotic of B, but not T, lymphocytes can elicit a Th2 response to H2 histones that helps B cell production of anti‐dsDNA Ab and finally triggers the onset of lupus.

Kidney function following nephrectomy: Similitude and discrepancies between kidney cancer and living donation

OBJECTIVESnThe reported long-term safety of kidney donation is inconsistent with the impairment of kidney function observed following nephrectomy for renal cell cancer. We aimed to investigate if indication for nephrectomy (kidney cancer vs. living donation) was an independent risk factor for kidney function deterioration.nnnMATERIALS AND METHODSnBetween 1985 and 2008, 124 patients with localized renal cell carcinoma who meet the criteria used for living donation, underwent radical nephrectomy (group 1) at our institution. Group 1 was retrospectively compared with 124 consecutive living donor nephrectomies (group 2) performed from 2004 to 2008. Kidney function evaluation was performed preoperatively and at 1, 2, 3, and 4 years postoperatively with calculation of estimated glomerular filtration rate through the Modification of Diet in Renal Disease (MDRD-eGFR) and the adjusted Cockroft and Gault (CG-eGFR) formula. Multivariate logistic regression included patients characteristics and indication for nephrectomy as predictors of kidney function deterioration.nnnRESULTSnMean decrease in MDRD-eGFR was 30.4% and 32.4% in groups 1 and 2 (P = 0.30). Prevalence of chronic kidney disease (CKD), defined by MDRD-eGFR < 60 mL/min/m(2), varied from 42.3% to 71% in group 1 and from 41.6% to 56% in group 2 at different time points (P = 0.073). Prevalence of CKD at 4 years defined by MDRD-eGFR < 45 mL/min/m(2) was significantly increased in group 1 compared with group 2 (16.2% and 5.3%, P < 0.005, respectively). Linear regression analysis showed only baseline kidney function and patient age predicted a significant decrease in postoperative kidney function (P < 0.001 and P = 0.04).nnnCONCLUSIONSnRenal cell carcinoma is not an independent risk factor for kidney function impairment following nephrectomy. Selected kidney cancer patients with few morbidities face the same deterioration of meanly 30% of kidney function compared with living donors, but their lower baseline function results in an increased risk for CKD.

Synthesis of Glycopeptides from Type II Collagen-Incorporating Galactosylated Hydroxylysine Mimetics and Their Use in Studying the Fine Specificity of Arthritogenic T Cells

Five analogues of the bovine type II collagen (bCII) immunodominant glycopeptide [β‐D‐Gal‐(5R)‐5‐Hyl264]CII(256–270) (1) carrying diverse modifications at the critical hydroxylysine (Hyl) 264 side chain were designed and synthesised, to explore the fine specificity of bCII‐reactive T cells involved in the initiation and/or regulation of collagen‐induced arthritis (CIA), a mouse model for rheumatoid arthritis (RA). β‐D‐Galactosyl‐(5R)‐5‐hydroxy‐L‐lysine (19) and corresponding mimetics (22–25), conveniently protected for solid‐phase synthesis, were all obtained by a divergent route involving enantiopure 5‐hydroxylated 6‐oxo‐1,2‐piperidinedicarboxylates as the key intermediates. All three bCII‐specific T hybridomas used, as well as a recurrent pathogenic CD4+ T‐cell clone isolated from bCII‐immunised DBA/1 mice, recognised the galactosylated form 1 of the immunodominant bCII (256–270) epitope. These cells were extremely sensitive to changes at the ε‐amino group of Hyl264, but differed in their pattern of recognition of analogues with a Hyl264 side chain modified at C‐5 (i.e. inversion of stereochemistry, methylation). These data further document the importance of collagen post‐translational modifications in autoimmunity and in the CIA model in particular, and provide a new insight into the molecular interaction between glycopeptide 1 and the TCR of pathogenic T cells.

Polyarthritis in MRL-1pr/1pr Mice: Mouse Type II Collagen is Antigenic but not Arthritogenic

In addition to a lupus-like syndrome and massive T cell proliferation, MRL-lpr/lpr(MRL/l) mice develop an arthritic process very similar serologically and histologically to human rheumatoid arthritis (RA). Recently, we have developed in DBA/1 mice an experimental model of autoimmune arthritis (EAA) which shares clinical features with RA, by injecting homologous type II collagen (CII). In order to investigate the possible relationship between the spontaneous polyarthritis of MRL/l mice and collagen induced EAA, we immunized MRL/l mice with mouse (M) CII. Our findings revealed that the injection of 100 micrograms M-CII in young or old MRL/l mice did not modify the articular pathology which spontaneously develops in non-injected mice. Circulating autoantibodies to native M-CII were found in the sera of immunized young mice but were not detected in non injected or immunized old mice. Conversely, denatured alpha 1 (II) chains or CB peptides derived from M-CII were recognized by most of the MRL/l sera whether mice had been immunized or not. The incidence of positive sera as well as the intensity of the response evaluated by Western blot analysis increased with the age of the mice. Taken together, our data suggest that, even if the injection of homologous CII in MRL/l mice may accelerate the onset of joint pathology, the spontaneous disease arises independently of an autoimmune response against native CII.

Use of computed tomography assessed kidney length to predict split renal GFR in living kidney donors.

AbstractObjectivesScreening of living kidney donors may require scintigraphy to split glomerular filtration rate (GFR). To determine the usefulness of computed tomography (CT) to split GFR, we compared scintigraphy-split GFR to CT-split GFR. We evaluated CT-split GFR as a screening test to detect scintigraphy-split GFR lower than 40xa0mL/min/1.73xa0m2/kidney.MethodsThis was a monocentric retrospective study on 346 potential living donors who had GFR measurement, renal scintigraphy, and CT. We predicted GFR for each kidney by splitting GFR using the following formula: Volume-split GFR for a given kidneyu2009=u2009measured GFR*[volume of this kidney/(volume of this kidneyu2009+u2009volume of the opposite kidney)]. The same formula was used for length-split GFR. We compared length- and volume-split GFR to scintigraphy-split GFR at donation and with a 4-year follow-up.ResultsA better correlation was observed between length-split GFR and scintigraphy-split GFR (ru2009=u20090.92) than between volume-split GFR and scintigraphy-split GFR (ru2009=u20090.89). A length-split GFR threshold of 45xa0mL/min/1.73xa0m2/kidney had a sensitivity of 100xa0% and a specificity of 75xa0% to detect scintigraphy-split GFR less than 40xa0mL/min/1.73xa0m2/kidney. Both techniques with their respective thresholds detected living donors with similar eGFR evolution during follow-up.ConclusionLength-split GFR can be used to detect patients requiring scintigraphy.Key points• Excellent correlation between kidney length and scintigraphy predicted GFRn • Kidney length screening detects all donors with GFR lower than 40xa0mL/min/1.73xa0m2n • Kidney length screening can replace scintigraphy screening.

Association of mGFR of the Remaining Kidney Divided by Its Volume before Donation with Functional Gain in mGFR among Living Kidney Donors

BACKGROUND AND OBJECTIVESnThe predictors of long-term renal function in living kidney donors are currently discussed. Our objectives were to describe the predictors of functional gain of the remaining kidney after kidney donation. We hypothesized that GFR of the remaining kidney divided by volume of this kidney (rk-GFR/vol) would reflect the density of functional nephrons and be inversely associated with functional gain of the remaining kidney.nnnDESIGN, SETTING, PARTICIPANTS, & MEASUREMENTSnWe conducted a prospective monocentric study including 63 living donors (26 men; 50.3±11.8 years old) who had been evaluated for (51)Cr-EDTA and measured GFR, split renal function by scintigraphy before donation (between 2004 and 2009), and measured GFR at 5.7±0.5 years after donation. For 52 donors, volume of the remaining kidney (measured and estimated with the ellipsoid formula using renal computed tomography scannography) was determined before donation. We tested our hypothesis in an external validation cohort of 39 living donors (13 men; 51.0±9.4 years old) from another single center during the same time period.nnnRESULTSnFor the main cohort, the mean measured GFR was 97.6±13.0 ml/min per 1.73 m(2) before donation and 63.8±9.4 ml/min per 1.73 m(2) at 5 years. Functional gain averaged 16.2±7.2 ml/min per 1.73 m(2) (+35.3%±16.7%). Multivariate analysis showed that age, body mass index, and rk-GFR/vol at donation were negatively correlated with functional gain and had strong predictive power of the 5-year functional gain (adjusted 5-year functional gain for age: -0.4 [95% confidence interval (95% CI), -0.5 to -0.1]; body mass index: -0.3 [95% CI, -0.6 to -0.1]; rk-GFR/vol: -55.1 [95% CI, -92.3 to -17.9]). We tested this model in the external validation cohort (adjusted 5-year functional gain for age: -0.1 [95% CI, -0.5 to 0.3]; body mass index: -0.9 [95% CI, -1.8 to -0.1]; rk-GFR/vol: -97.6 [95% CI, -137.5 to -57.6]) and confirmed that rk-GFR/vol was inversely associated with 5-year functional gain.nnnCONCLUSIONSnFor given age and body mass index, the long-term functional gain of the remaining kidney is inversely associated with the new variable rk-GFR/vol at donation.

Insights into spatial configuration of a galactosylated epitope required to trigger arthritogenic T-cell receptors specific for the sugar moiety

The immunodominant epitope of bovine type II collagen (CII256–270) in Aq mice carries a hydroxylysine-264 linked galactose (Gal-Hyl264), the recognition of which is central to the development of collagen-induced arthritis. This study explores the molecular interactions involved in the engagement of T-cell receptors (TCRs) with such epitopes. Responses of three anti-CII T-cell hybridomas and clone A9.2 (all sharing close TCR sequences) to a panel of CII256–270 analogues incorporating Gal-Hyl264 with a modified side chain were determined. Recognition of naturally occurring CII256–270 peptides by either group of T cells depended strictly upon the presence of the carbohydrate and, more precisely, its intact HO-4 group. Modifications of primary amino group on the hydroxylysine side chain eliminated T-cell reactivity, notwithstanding the presence of the galactosyl moiety. Moderate stereochemical changes, such as altered sugar orientation and methylation at the galactose anchor position, were still permissive. Conversely, robust transformations affecting the relative positions of the key elements were detrimental to TCR recognition. To conclude, these data provide strong new experimental evidence that integrity of both galactose HO-4 and hydroxylysine side chain primary amino groups are mandatory for activation of anti-Gal-Hyl264 TCRs. They also indicate that there is a certain degree of TCR plasticity in peptide-TCR interactions.