Azathioprine versus mycophenolate mofetil for long-term immunosuppression in lupus nephritis: results from the MAINTAIN Nephritis Trial

Background: Long-term immunosuppressive treatment does not efficiently prevent relapses of lupus nephritis (LN). This investigator-initiated randomised trial tested whether mycophenolate mofetil (MMF) was superior to azathioprine (AZA) as maintenance treatment. Methods: A total of 105 patients with lupus with proliferative LN were included. All received three daily intravenous pulses of 750 mg methylprednisolone, followed by oral glucocorticoids and six fortnightly cyclophosphamide intravenous pulses of 500 mg. Based on randomisation performed at baseline, AZA (target dose: 2 mg/kg/day) or MMF (target dose: 2 g/day) was given at week 12. Analyses were by intent to treat. Time to renal flare was the primary end point. Mean (SD) follow-up of the intent-to-treat population was 48 (14) months. Results: The baseline clinical, biological and pathological characteristics of patients allocated to AZA or MMF did not differ. Renal flares were observed in 13 (25%) AZA-treated and 10 (19%) MMF-treated patients. Time to renal flare, to severe systemic flare, to benign flare and to renal remission did not statistically differ. Over a 3-year period, 24 h proteinuria, serum creatinine, serum albumin, serum C3, haemoglobin and global disease activity scores improved similarly in both groups. Doubling of serum creatinine occurred in four AZA-treated and three MMF-treated patients. Adverse events did not differ between the groups except for haematological cytopenias, which were statistically more frequent in the AZA group (p=0.03) but led only one patient to drop out. Conclusions: Fewer renal flares were observed in patients receiving MMF but the difference did not reach statistical significance.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Long-term follow-up of the MAINTAIN Nephritis Trial, comparing azathioprine and mycophenolate mofetil as maintenance therapy of lupus nephritis

OBJECTIVE: To report the 10-year follow-up of the MAINTAIN Nephritis Trial comparing azathioprine (AZA) and mycophenolate mofetil (MMF) as maintenance therapy of proliferative lupus nephritis, and to test different definitions of early response as predictors of long-term renal outcome. METHODS: In 2014, data on survival, kidney function, 24 h proteinuria, renal flares and other outcomes were collected for the 105 patients randomised between 2002 and 2006, except in 13 lost to follow-up. RESULTS: Death (2 and 3 in the AZA and MMF groups, respectively) and end-stage renal disease (1 and 3, respectively) were rare events. Time to renal flare (22 and 19 flares in AZA and MMF groups, respectively) did not differ between AZA and MMF patients. Patients with good long-term renal outcome had a much more stringent early decrease of 24 h proteinuria compared with patients with poor outcome. The positive predictive value of a 24 h proteinuria <0.5 g/day at 3 months, 6 months and 12 months for a good long-term renal outcome was excellent (between 89% and 92%). Inclusion of renal function and urinalysis in the early response criteria did not impact the value of early proteinuria decrease as long-term prognostic marker. CONCLUSIONS: The long-term follow-up data of the MAINTAIN Nephritis Trial do not indicate that MMF is superior to AZA as maintenance therapy in a Caucasian population suffering from proliferative lupus nephritis. Moreover, we confirm the excellent positive predictive value of an early proteinuria decrease for long-term renal outcome

Combination of an optical parametric oscillator and quantum-dots 655 to improve imaging depth of vasculature by intravital multicolor two- photon microscopy References and links

International audienceSimultaneous imaging of different cell types and structures in the mouse central nervous system (CNS) by intravital two-photon microscopy requires the characterization of fluorophores and advances in approaches to visualize them. We describe the use of a two-photon infrared illumination generated by an optical parametric oscillator (OPO) on quantum-dots 655 (QD655) nanocrystals to improve resolution of the vasculature deeper in the mouse brain both in healthy and pathological conditions. Moreover, QD655 signal can be unmixed from the DsRed2, CFP, EGFP and EYFP fluorescent proteins, which enhances the panel of multi-parametric correlative investigations both in the cortex and the spinal cord

Phenotypic dynamics of microglial and monocyte-derived cells in glioblastoma-bearing mice

International audienceInflammatory cells, an integral component of tumor evolution, are present in Glioblastomas multiforme (GBM). To address the cellular basis and dynamics of the inflammatory microenvironment in GBM, we established an orthotopic syngenic model by grafting GL261-DsRed cells in immunocompetent transgenic LysM-EGFP//CD11c-EYFP reporter mice. We combined dynamic spectral two-photon imaging with multiparametric cytometry and multicolor immunostaining to characterize spatio-temporal distribution, morphology and activity of microglia and blood-derived infiltrating myeloid cells in live mice. Early stages of tumor development were dominated by microglial EYFP + cells invading the tumor, followed by massive recruitment of circulating LysM-EGFP + cells. Fluorescent invading cells were conventional XCR1 + and monocyte-derived dendritic cells distributed in subpopulations of different maturation stages, located in different areas relative to the tumor core. The lethal stage of the disease was characterized by the progressive accumulation of EGFP + /EYFP + monocyte-derived dendritic cells. This local phenotypic regulation of monocyte subtypes marked a transition in the immune response

Dynamics of Bev treatment effects on tumor development and blood vessel density.

<p>(<b>A–B</b>) Example of 3D projections (over 200 µm) of high-resolution images of the microvascularization observed at the margin (yellow line) between tumor and healthy brain in an untreated mouse (<b>A</b>) and a mouse treated with Bev for 1 week (<b>B</b>). Tumor (star) exhibits a high density of tortuous vessels in the untreated compared to less dense and straighter vessels in the Bev-treated mouse. (<b>C–D</b>) Evolutions of the mean blood vessel density (<b>C</b>) and the mean tumor cell density (<b>D</b>) in corresponding brain volumes of control (n = 7) and Bev-treated (n = 7) mice (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072655#s2" target="_blank">methods</a>). Tumor induced changes in blood vessel density are sustainably blocked by Bev (<b>C</b>) and mean tumor cell density is significantly reduced at day 21 (<b>D</b>) (*, p<0.05). (<b>E</b>) % reduction of blood vessel density relative to mean control values as a function of corresponding % reduction of tumor cell density. The absence of correlation (R²∼0) indicates that effect of Bev on tumor development is not mediated by its effect on blood vessel density. (<b>F</b>) Evolution of the increase rate of tumor cell densities in control and in mice treated with Bev for 3 weeks. Increase rate is expressed as the average of the ratios of fluorescence intensities recorded at one week intervals in the same areas. Increase rate is significantly reduced during the first 2 weeks but recovers the control increase rate on the third week despite a sustained blockade of vascular changes. (<b>G–I</b>) Immunostaining of vessels (anti-laminin, red) and BrdU positive cells (green) on D9 in untreated (<b>G</b>) or Bev-treated (<b>H</b>) tumors. Quantifications of BrdU(+) cells and blood vessels (<b>I</b>) in sections covering the whole tumor indicate that proliferation is not correlated with the amount of blood vessel density. Scale bars, 200 µm. Error bars represent SEM; (*, p<0.05).</p

Contribution of the micro­environment on tumor development.

<p>(<b>A</b>) Evolution of the tumor volume in mice pretreated with i.v. injections every 2 days of PBS (n = 8) or Bev (n = 5) for 1 week before tumor implantation. Tumor volume was normalized on the first day of visualization (day 0) and interpolated linearly between imaging sessions to take into account day of visualization mismatches between the two groups of mice. Interpolated data are indicated with squares linked together with dashed lines. A significant reduction of tumor volume was observed on D14 until it reached 58% on D28 similar to what observed upon continuous Bev treatment. (<b>B–C</b>) Tumor development and blood vessel density under AMD3100 treatment to prevent signaling by Stromal Derived Factor 1 alpha. Evolutions of mean tumor cell density (<b>B</b>) and mean blood vessel density (<b>C</b>) in control (n = 7) or AMD3100-treated (n = 6) mice (*, p<0.05). The average evolution under Bev is reported in gray for comparison. Blood vessel density was not significantly affected by AMD3100 treatment and remained significantly larger than the one observed under Bev on D21. (<b>D</b>) Evolution of the average tumor increase rate in control conditions and under AMD3100 treatment. Ratios between fluorescence intensities recorded in the same areas at one week interval have been averaged and evolution of this average was followed over the 3 weeks of treatment. Bev data are reported in gray for comparison. Reduction of tumor size is almost twice larger under AMD3100 than under Bev.</p

Imaging blood vessel network and interactions with tumor cells.

<p>(<b>A</b>) Mean projection image of a tumor and associated vascularization acquired through a 20× Water Immersion objective (N.A. = 1.0). One field of view corresponds to the dotted line box; 49 such fields were acquired to cover the full tumor extent. (<b>B</b>) For quantitative pharmacological experiments, a 10× dry objective (N.A. = 0.45) was used to increase data throughput by a factor 4. An overlap of 50 µm between fields of view ensured qualitative image stitching performed with ImageJ 3D Stitching macro, “pcm3D”. (<b>C</b>) Upper part, 3D projection of the vascular network over a depth of 150 µm of the tumor (right of the plain line) and of surrounding healthy brain (left of the plain line). Lower part: zooms of the outlined ROIs. Thin and straight normal brain vessels (left); tortuous and swollen tumor vessels (right). (<b>D</b>) Example of bicolor images showing at a 10 day interval the average vascular density (red) and tumor cell density (green) in a 25 µm thick horizontal section of the tumor located 200 µm below the brain surface. Note that the lower vascular density observed after 10 days is not associated with a reduction of tumor cell density. (<b>E,F</b>) Horizontal mean projection images over 100 µm subdural layers 15–19 days post implantation showing perivascular invasion (arrow heads). (<b>G</b>) Sagittal 3D rendering projection showing superficial tumor cells (arrow heads) migrating toward deeper brain regions along a large vertical vessel. (<b>H</b>) Mean projection of the volume of interest scanned for vascular changes occurring at a 1 day interval. All the changes (white dots) were found inside or within 150 µm off the tumor margin but none in the healthy brain region. Insets show examples of microvascular changes (arrows) at several locations. Scale bars, A&B: 1 mm; C&D: 300 µm, F&G: 50 µm,; H:300 µm, inset 25 µm.</p