Nonskeletal and skeletal effects of high doses versus low doses of vitamin D3 in renal transplant recipients: Results of the VITALE (VITamin D supplementation in renAL transplant recipients) study, a randomized clinical trial.

Vitamin D sufficiency is associated with a reduced risk of fractures, diabetes mellitus, cardiovascular events, and cancers, which are frequent complications after renal transplantation. The VITALE (VITamin D supplementation in renAL transplant recipients) study is a multicenter double-blind randomized trial, including nondiabetic adult renal transplant recipients with serum 25-hydroxy vitamin D (25(OH) vitamin D) levels of <30 ng/mL, which is randomized 12 to 48 months after transplantation to receive high (100 000 IU) or low doses (12 000 IU) of cholecalciferol every 2 weeks for 2 months and then monthly for 22 months. The primary outcome was a composite endpoint, including diabetes mellitus, major cardiovascular events, cancer, and death. Of 536 inclusions (50.8 [13.7] years, 335 men), 269 and 267 inclusions were in the high-dose and low-dose groups, respectively. The serum 25(OH) vitamin D levels increased by 23 versus 6 ng/mL in the high-dose and low-dose groups, respectively (P < .0001). In the intent-to-treat analysis, 15% versus 16% of the patients in the high-dose and low-dose groups, respectively, experienced a first event of the composite endpoint (hazard ratio, 0.94 [0.60-1.48]; P = .78), whereas 1% and 4% of patients in the high-dose and low-dose groups, respectively, experienced an incident symptomatic fracture (odds ratio, 0.24 [0.07-0.86], P = .03). The incidence of adverse events was similar between the groups. After renal transplantation, high doses of cholecalciferol are safe but do not reduce extraskeletal complications (trial registration: ClinicalTrials.gov; identifier: NCT01431430).

The sexual dimorphism of kidney growth in mice and humans.

Kidney mass and function are sexually determined, but the cellular events and the molecular mechanisms involved in this dimorphism are poorly characterized. By combining female and male mice with castration/replacement experiments, we showed that male mice exhibited kidney overgrowth from five weeks of age. This effect was organ specific, since liver and heart weight were comparable between males and females, regardless of age. Consistently, the androgen receptor was found to be expressed in the kidneys of males, but not in the liver. In growing mice, androgens led to kidney overgrowth by first inducing a burst of cell proliferation and then an increase of cell size. Remarkably, androgens were also required to maintain cell size in adults. In fact, orchiectomy resulted in smaller kidneys in a matter of few weeks. These changes paralleled the changes of the expression of ornithine decarboxylase and cyclin D1, two known mediators of kidney growth, whereas, unexpectedly, mTORC1 and Hippo pathways did not seem to be involved. Androgens also enhanced kidney autophagy, very likely by increasing transcription factor EB nuclear translocation. Functionally, the increase of tubular mass resulted in increased sodium/phosphate transport. These findings were relevant to humans. Remarkably, by studying living gender-paired kidney donors-recipients, we showed that tubular cell size increased three months after transplantation in men as compared to women, regardless of the donor gender. Thus, our results identify novel signaling pathways that may be involved in androgen-induced kidney growth and homeostasis and suggest that androgens determine kidney size after transplantation.

Three-dimensional architecture of nephrons in the normal and cystic kidney.

Kidney function is crucially dependent on the complex three-dimensional structure of nephrons. Any distortion of their shape may lead to kidney dysfunction. Traditional histological methods present major limitations for three-dimensional tissue reconstruction. Here, we combined tissue clearing, multi-photon microscopy and digital tracing for the reconstruction of single nephrons under physiological and pathological conditions. Sets of nephrons differing in location, shape and size according to their function were identified. Interestingly, nephrons tend to lie in planes. When this technique was applied to a model of cystic kidney disease, cysts were found to develop only in specific nephron segments. Along the same segment, cysts are contiguous within normal non-dilated tubules. Moreover, the shapes of cysts varied according to the nephron segment. Thus, our findings provide a valuable strategy for visualizing the complex structure of kidneys at the single nephron level and, more importantly, provide a basis for understanding pathological processes such as cystogenesis.

Is sexual harassment and psychological abuse among medical students a fatality? A 2-year study in the Paris Descartes School of Medicine.

INTRODUCTION: An observatory of sexual harassment and psychological abuse was set up at one of France's largest schools of medicine to both quantify and reduce sexual harassment or psychological abuse of medical students. METHODS: Over a 2-year period, we described the evolution of sexual harassment and psychological abuse and explored for associated factors. Moreover, a qualitative analysis using an inductive approach was performed from students' verbatim. RESULTS: 2795 responses were collected. Sexual harassment was reported in 7% and psychological abuse in 15%, at baseline, and decreased after the observatory was set up. Women had higher odds of being a victim of sexual harassment. Older students reported less often psychological abuse and being a witness of sexual harassment. Surgery departments were associated with up to 5.7-fold increased odds of sexual harassment. Surgery and pediatrics departments were associated with a 2-fold increased odds of psychological abuse. Qualitative analysis revealed four categories: humiliation, the feeling of inferiority, sexual harassment, and manifestations of violence. CONCLUSION: During clerkships, factors associated with higher odds of sexual harassment and psychological abuse were female gender, younger age, and departments of surgery. Setting up such an observatory may contribute to reduce this burden and provide a useful tool to raise awareness.

Fibroblast growth factor 23 decreases PDE4 expression in heart increasing the risk of cardiac arrhythmia; Klotho opposes these effects.

The concentration of fibroblast growth factor 23 (FGF23) rises progressively in renal failure (RF). High FGF23 concentrations have been consistently associated with adverse cardiovascular outcomes or death, in chronic kidney disease (CKD), heart failure or liver cirrhosis. We identified the mechanisms whereby high concentrations of FGF23 can increase the risk of death of cardiovascular origin. We studied the effects of FGF23 and Klotho in adult rat ventricular cardiomyocytes (ARVMs) and on the heart of mice with CKD. We show that FGF23 increases the frequency of spontaneous calcium waves (SCWs), a marker of cardiomyocyte arrhythmogenicity, in ARVMs. FGF23 increased sarcoplasmic reticulum Ca2+ leakage, basal phosphorylation of Ca2+-cycling proteins including phospholamban and ryanodine receptor type 2. These effects are secondary to a decrease in phosphodiesterase 4B (PDE4B) in ARVMs and in heart of mice with RF. Soluble Klotho, a circulating form of the FGF23 receptor, prevents FGF23 effects on ARVMs by increasing PDE3A and PDE3B expression. Our results suggest that the combination of high FGF23 and low sKlotho concentrations decreases PDE activity in ARVMs, which favors the occurrence of ventricular arrhythmias and may participate in the high death rate observed in patients with CKD.

MITF - A controls branching morphogenesis and nephron endowment.

Congenital nephron number varies widely in the human population and individuals with low nephron number are at risk of developing hypertension and chronic kidney disease. The development of the kidney occurs via an orchestrated morphogenetic process where metanephric mesenchyme and ureteric bud reciprocally interact to induce nephron formation. The genetic networks that modulate the extent of this process and set the final nephron number are mostly unknown. Here, we identified a specific isoform of MITF (MITF-A), a bHLH-Zip transcription factor, as a novel regulator of the final nephron number. We showed that overexpression of MITF-A leads to a substantial increase of nephron number and bigger kidneys, whereas Mitfa deficiency results in reduced nephron number. Furthermore, we demonstrated that MITF-A triggers ureteric bud branching, a phenotype that is associated with increased ureteric bud cell proliferation. Molecular studies associated with an in silico analyses revealed that amongst the putative MITF-A targets, Ret was significantly modulated by MITF-A. Consistent with the key role of this network in kidney morphogenesis, Ret heterozygosis prevented the increase of nephron number in mice overexpressing MITF-A. Collectively, these results uncover a novel transcriptional network that controls branching morphogenesis during kidney development and identifies one of the first modifier genes of nephron endowment.

Adverse events associated with currently used medical treatments for cystinuria and treatment goals: results from a series of 442 patients in France.

OBJECTIVE: To evaluate medical treatments, in terms of adverse events (AEs) and therapeutic goals, in a large series of patients with cystinuria. PATIENTS AND METHODS: Data from 442 patients with cystinuria were recorded retrospectively. Crystalluria was studied in 89 patients. A mixed-effects logistic regression model was used to estimate how urine pH, specific gravity and cysteine-binding thiols (CBT) correlate with risk of cystine crystalluria. RESULTS: Alkalizing agents and CBT agents were given to 88.8% (n = 381) and 55.3% (n = 238) of patients, respectively. Gastrointestinal AEs were reported in 12.3%, 10.4% and 2.6% of patients treated with potassium bicarbonate, potassium citrate and sodium bicarbonate, respectively (P = 0.008). The percentages of patients who experienced at least one AE with tiopronin (24.6%) and with D-penicillamine (29.5%) were similar (P = 0.45). Increasing urine pH and decreasing urine specific gravity significantly reduced the risk of cystine crystalluria, whereas D-penicillamine and tiopronin treatments did not reduce this risk (odds ratio [OR] 1 for pH ≤6.5; OR 0.52 [95% confidence interval {95% CI} 0.28-0.95] for 7.0 8.0, P <0.001). CONCLUSION: Adverse events were frequent with D-penicillamine and tiopronin. Alkaline hyperdiuresis was well tolerated and reduced cystine crystalluria. Urine specific gravity ≤1.005 and urine pH >7.5, while warning about calcium-phosphate crystallization, should be the goals of medical therapy.

Inhibition of the mTORC pathway in the antiphospholipid syndrome.

BACKGROUND: Although thrombosis is considered the cardinal feature of the antiphospholipid syndrome, chronic vascular lesions are common, particularly in patients with life-threatening complications. In patients who require transplantation, vascular lesions often recur. The molecular pathways involved in the vasculopathy of the antiphospholipid syndrome are unknown, and adequate therapies are lacking. METHODS: We used double immunostaining to evaluate pathway activation in the mammalian target of rapamycin complex (mTORC) and the nature of cell proliferation in the vessels of patients with primary or secondary antiphospholipid syndrome nephropathy. We also evaluated autopsy specimens from persons who had catastrophic antiphospholipid syndrome. The molecular pathways through which antiphospholipid antibodies modulate the mTORC pathway were evaluated in vitro, and potential pharmacologic inhibitors were also tested in vitro. Finally, we studied the effect of sirolimus in kidney-transplant recipients with the antiphospholipid syndrome. RESULTS: The vascular endothelium of proliferating intrarenal vessels from patients with antiphospholipid syndrome nephropathy showed indications of activation of the mTORC pathway. In cultured vascular endothelial cells, IgG antibodies from patients with the antiphospholipid syndrome stimulated mTORC through the phosphatidylinositol 3-kinase (PI3K)-AKT pathway. Patients with antiphospholipid syndrome nephropathy who required transplantation and were receiving sirolimus had no recurrence of vascular lesions and had decreased vascular proliferation on biopsy as compared with patients with antiphospholipid antibodies who were not receiving sirolimus. Among 10 patients treated with sirolimus, 7 (70%) had a functioning renal allograft 144 months after transplantation versus 3 of 27 untreated patients (11%). Activation of mTORC was also found in the vessels of autopsy specimens from patients with catastrophic antiphospholipid syndrome. CONCLUSIONS: Our results suggest that the mTORC pathway is involved in the vascular lesions associated with the antiphospholipid syndrome. (Funded by INSERM and others.).

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

OBJECTIVES: Screening 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 40 mL/min/1.73 m2/kidney. METHODS: This 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 kidney = measured GFR*[volume of this kidney/(volume of this kidney + volume 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. RESULTS: A better correlation was observed between length-split GFR and scintigraphy-split GFR (r = 0.92) than between volume-split GFR and scintigraphy-split GFR (r = 0.89). A length-split GFR threshold of 45 mL/min/1.73 m2/kidney had a sensitivity of 100 % and a specificity of 75 % to detect scintigraphy-split GFR less than 40 mL/min/1.73 m2/kidney. Both techniques with their respective thresholds detected living donors with similar eGFR evolution during follow-up. CONCLUSION: Length-split GFR can be used to detect patients requiring scintigraphy. KEY POINTS: • Excellent correlation between kidney length and scintigraphy predicted GFR • Kidney length screening detects all donors with GFR lower than 40 mL/min/1.73 m 2 • Kidney length screening can replace scintigraphy screening.

What is the significance of end-stage renal disease risk estimation in living kidney donors?

Two end-stage renal disease (ESRD) risk calculators were recently developed by Grams et al., and Ibrahim et al. to calculate ESRD risk before donation among living kidney donors. However, those calculators have never been studied among potential donors for whom donation was refused due to medical contraindications and compared to a group of donors. We compared 15-year and lifetime ESRD risk of donors and nondonors due to medical cause as estimated by those two calculators. Nondonors due to medical cause (n = 27) had a significantly higher 15-year ESRD risk compared to donors (n = 288) with both calculators (0.25 vs. 0.14, P < 0.001 for that developed by Grams et al. and 2.21 vs. 1.43, P = 0.002 for that developed by Ibrahim et al.). On the contrary, lifetime ESRD risk was not significantly different between the two groups. At both times (15 years and lifetime), we observed a significant overlap of ESRD risk between the two groups. ESRD risk calculators could be complementary to standard screening strategy but cannot be used alone to accept or decline donation.

Stat3 Controls Tubulointerstitial Communication during CKD.

In CKD, tubular cells may be involved in the induction of interstitial fibrosis, which in turn, leads to loss of renal function. However, the molecular mechanisms that link tubular cells to the interstitial compartment are not clear. Activation of the Stat3 transcription factor has been reported in tubular cells after renal damage, and Stat3 has been implicated in CKD progression. Here, we combined an experimental model of nephron reduction in mice from different genetic backgrounds and genetically modified animals with in silico and in vitro experiments to determine whether the selective activation of Stat3 in tubular cells is involved in the development of interstitial fibrosis. Nephron reduction caused Stat3 phosphorylation in tubular cells of lesion-prone mice but not in resistant mice. Furthermore, specific deletion of Stat3 in tubular cells significantly reduced the extent of interstitial fibrosis, which correlated with reduced fibroblast proliferation and matrix synthesis, after nephron reduction. Mechanistically, in vitro tubular Stat3 activation triggered the expression of a specific subset of paracrine profibrotic factors, including Lcn2, Pdgfb, and Timp1. Together, our results provide a molecular link between tubular and interstitial cells during CKD progression and identify Stat3 as a central regulator of this link and a promising therapeutic target.

Anti-inflammatory properties of Lipidosterolic extract of Serenoa repens (Permixon®) in a mouse model of prostate hyperplasia.

BACKGROUND: Permixon®, the hexanic lipidosterolic extract of saw palmetto Serenoa repens (LSESr), has shown properties that highlight its benefit in the management of benign prostate hyperplasia (BPH). To address its actual anti-inflammatory potency, we used a unique pro-inflammatory mouse model of prostate hyperplasia involving prostate-specific over-expression of prolactin transgene (Pb-Prl). METHODS: Six month-old Pb-Prl males were administered with Permixon® per os at the daily dose of 100 mg/kg for 28 days. Body and prostate weights were measured weekly and at sacrifice, respectively. Prostate histology was carefully assessed by a pathologist and detailed quantifications of epithelial and stromal compartments were performed using image analysis software. Luminal cell proliferation index was determined using Ki-67 immunostaining, and apoptosis using Bax/Bcl2 mRNA ratio. Tissue inflammation and fibrosis were assessed by histological analyses then quantified using CD45 immunostaining and picrosirius staining, respectively. Expression profiling of selected pro-inflammatory cytokines, chemokines, and chemokine receptors was performed by quantitative RT-PCR. RESULTS: In this model, Permixon® significantly decreased tissue weight and proliferation index specifically in the ventral lobe. Although treatment had no noticeable effect on epithelial histology of any lobe, it markedly reduced the histological hallmarks of inflammation in all lobes. This was confirmed by the global down-regulation of prostate pro-inflammatory cytokine profile, with significant reduction of CCR7, CXCL6, IL-6, and IL-17 expression. CONCLUSIONS: In this mouse model of prostate hyperplasia, Permixon® exerted potent anti-inflammatory properties in the whole prostate while anti-androgenic effects were lobe-specific, suggesting that distinct LSESr components may be involved in these effects. Our results support the beneficial role of Permixon® treatment for BPH. The relevance of CCR7, CXCL6, IL-6, and IL-17 as potential biomarkers to follow up BPH inflammatory status needs to be assessed.

EKLF-driven PIT1 expression is critical for mouse erythroid maturation in vivo and in vitro.

The PIT1/SLC20A1 protein, a well-described sodium/phosphate cotransporter and retrovirus receptor, has been identified recently as a modular of proliferation and apoptosis in vitro. The targeted deletion of the PIT1 gene in mice revealed a lethal phenotype due to severe anemia attributed to defects in liver development. However, the presence of immature erythroid cells associated with impaired maturation of the globin switch led us to investigate the role of PIT1 in hematopoietic development. In the present study, specific deletion of PIT1 in the hematopoietic system and fetal liver transplantation experiments demonstrated that anemia was associated with an erythroid cell- autonomous defect. Moreover, anemia was not due to RBC destruction but rather to maturation defects. Because Erythroid Krüppel-like Factor (EKLF)-knockout mice showed similar maturation defects, we investigated the functional link between PIT1 and EKLF. We demonstrated that EKLF increases PIT1 expression during RBC maturation by binding to its promoter in vivo and that shRNA-driven depletion of either PIT1 or EKLF impairs erythroid maturation of G1E cells in vitro, whereas reexpression of PIT1 in EKLF-depleted G1E cells partially restores erythroid maturation. This is the first demonstration of a physiologic involvement of PIT1 in erythroid maturation in vivo.

Assessment of hydration status in a large population.

Both acute and chronic dehydration can have important implications for human behaviour and health. Young children, non-autonomous individuals and the elderly are at a greater risk of dehydration. Mild hypertonic dehydration could be related to less efficient cognitive and physical performance and has been reported to be associated with frequently occurring pathological conditions, especially nephrolithiasis. The assessment of hydration status in a large sample appears to be of interest for conducting epidemiological and large clinical studies aimed at improving preventive and curative care. Especially in large-population studies, methods that are used have to be accurate, cheap, quick and require no technical expertise. Body weight change is widely used to determine acute hydration changes, but seems to be insufficiently accurate in longitudinal studies. Bioimpedance analysis methods enable the assessment of total body water content, but their use is still under debate. Because plasma osmolality directly reflects intracellular osmolality, it constitutes a good marker to assess acute hydration changes, but not chronic hydration status because it changes constantly. Moreover, venepuncture is considered to be invasive and is not suitable for a large-sample study, especially in children. Urinary markers appear to be good alternatives for assessing hydration status in large populations. Collection of urine samples is non-invasive and cheap. High technical expertise is not required to perform urinary marker measurements and these measurements can be carried out quickly. Thus, methods based on urinary markers are very well suited for field studies. Urine colour is probably the least sensitive marker despite its high specificity. Urine osmolality and especially urine specific gravity could be easily used for determining hydration status in large-sample studies.

The kidney as a reservoir for HIV-1 after renal transplantation.

Since the recent publication of data showing favorable outcomes for patients with HIV-1 and ESRD, kidney transplantation has become a therapeutic option in this population. However, reports have documented unexplained reduced allograft survival in these patients. We hypothesized that the unrecognized infection of the transplanted kidney by HIV-1 can compromise long-term allograft function. Using electron microscopy and molecular biology, we examined protocol renal transplant biopsies from 19 recipients with HIV-1 who did not have detectable levels of plasma HIV-1 RNA at transplantation. We found that HIV-1 infected the kidney allograft in 68% of these patients. Notably, HIV-1 infection was detected in either podocytes predominately (38% of recipients) or tubular cells only (62% of recipients). Podocyte infection associated with podocyte apoptosis and loss of differentiation markers as well as a faster decline in allograft function compared with tubular cell infection. In allografts with tubular cell infection, epithelial cells of the proximal convoluted tubules frequently contained abnormal mitochondria, and both patients who developed features of subclinical acute cellular rejection had allografts with tubular cell infection. Finally, we provide a novel noninvasive test for determining HIV-1 infection of the kidney allograft by measuring HIV-1 DNA and RNA levels in patients' urine. In conclusion, HIV-1 can infect kidney allografts after transplantation despite undetectable viremia, and this infection might influence graft outcome.

Determination of optimal vitamin D3 dosing regimens in HIV-infected paediatric patients using a population pharmacokinetic approach.

AIMS: To investigate 25-hydroxycholecalciferol [25(OH)D] population pharmacokinetics in children and adolescents, to establish factors that influence 25(OH)D pharmacokinetics and to assess different vitamin D3 dosing schemes to reach sufficient 25(OH)D concentrations (>30 ng ml(-1) ). METHODS: This monocentric prospective study included 91 young HIV-infected patients aged 3 to 24 years. Patients received a 100 000 IU vitamin D3 supplementation. A total of 171 25(OH)D concentrations were used to perform a population pharmacokinetic analysis. RESULTS: At baseline 28% of patients had 25(OH)D concentrations below 10 ng ml(-1) , 69% between 10 and 30 ng ml(-1) and 3% above 30 ng ml(-1) . 25(OH)D pharmacokinetics were best described by a one compartment model with an additional production parameter reflecting the input from diet and sun exposure. The effects of skin phototype and bodyweight were significant on 25(OH)D production before any supplementation. The basal level was 27% lower in non-white skin phototype patients and was slightly decreased with bodyweight. No significant differences in 25(OH)D concentrations were related to antiretroviral drugs. To obtain concentrations between 30 and 80 ng ml(-1) , patients with baseline concentrations between 10 and 30 ng ml(-1) should receive 100 000 IU per 3 months. However, vitamin D deficient patients (<10 ng ml(-1) ) would need an intensive phase of 100 000 IU per 2 weeks (two times) followed 2 weeks later by a maintenance phase of 100 000 IU per 3 months. CONCLUSIONS: Skin phototype and bodyweight had an influence on the basal production of 25(OH)D. According to 25(OH)D baseline concentrations, dosing schemes to reach sufficient concentrations are proposed.

Vitamin D status and outcomes after renal transplantation.

Kidney transplant recipients usually have low vitamin D levels, especially in the early posttransplantation period, but the association between vitamin D status with renal outcomes is not well described in this population. Here, we studied a prospective cohort of 634 kidney recipients who underwent transplantation at a single institution between January 2005 and June 2010. In this cohort, low 25-hydroxyvitamin D concentrations 3 months after transplantation did not predict early death or graft loss but were independently associated with lower measured GFR at 12 months (P=0.001) and higher risk for interstitial fibrosis and tubular atrophy (P=0.01). In contrast, levels of calcium, phosphorus, calcitriol, parathyroid hormone, or fibroblast growth factor-23 were not consistently associated with any of the studied outcomes. In conclusion, low 25-hydroxyvitamin D concentration measured 3 months after transplantation is an independent risk factor for interstitial fibrosis progression and is associated with a lower GFR 1 year after transplantation.

Slc20a1 and Slc20a2 regulate neuronal plasticity and cognition independently of their phosphate transport ability.

In recent years, primary familial brain calcification (PFBC), a rare neurological disease characterized by a wide spectrum of cognitive disorders, has been associated to mutations in the sodium (Na)-Phosphate (Pi) co-transporter SLC20A2. However, the functional roles of the Na-Pi co-transporters in the brain remain still largely elusive. Here we show that Slc20a1 (PiT-1) and Slc20a2 (PiT-2) are the most abundant Na-Pi co-transporters expressed in the brain and are involved in the control of hippocampal-dependent learning and memory. We reveal that Slc20a1 and Slc20a2 are differentially distributed in the hippocampus and associated with independent gene clusters, suggesting that they influence cognition by different mechanisms. Accordingly, using a combination of molecular, electrophysiological and behavioral analyses, we show that while PiT-2 favors hippocampal neuronal branching and survival, PiT-1 promotes synaptic plasticity. The latter relies on a likely Otoferlin-dependent regulation of synaptic vesicle trafficking, which impacts the GABAergic system. These results provide the first demonstration that Na-Pi co-transporters play key albeit distinct roles in the hippocampus pertaining to the control of neuronal plasticity and cognition. These findings could provide the foundation for the development of novel effective therapies for PFBC and cognitive disorders.

Usefulness and analytical performances of complement multiplex assay for measuring complement biomarkers in plasma.

INTRODUCTION: The complement system is involved in numerous diseases, through diverse mechanisms and degree of activation. With the emergence of complement targeting therapeutic, simple and accessible tools to evaluate the extent of complement activation are strongly needed. METHODS: We evaluated two multiplex panels, measuring complement activation fragments (C4a, C3a, C5a, Bb, Ba, sC5b9) and intact components or regulators (C1q, C2, C3, C4, C5, FD, FP, FH, FI). The specificity of each measurement was assessed by using complement proteins depleted sera and plasma collected from patients with complement deficiencies. Normal values distribution was estimated using 124 plasma samples from healthy donors and complement activation profile was assessed in plasma collected from 31 patients with various complement-mediated disorders. RESULTS: We observed good inter-assay variation. All tested protein deficiencies were accurately detected. We established assay-specific reference values for each analyte. Except for C3, C4 and C4a, the majority of the measurements were in good agreement with references methods or published data. CONCLUSION: Our study substantiates the utility of the Complement Multiplex assay as a tool for measuring complement activation and deficiencies. Quantifying complement cleavage fragments in patients exhibiting classical or alternative pathway activation allowed evaluating the activation state of the whole cascade.