Bénédicte Puissant-Lubrano

Hepatitis E Virus–Induced Cryoglobulinemic Glomerulonephritis in a Nonimmunocompromised Person

Hepatitis E virus (HEV)-related kidney disease and symptomatic cryoglobulinemia have been observed in solid-organ transplant recipients. However, HEV RNA in the cryoprecipitate has not yet been assessed. We report what to our knowledge is the first documented case of autochthonous HEV-induced cryoglobulinemic crescentic and membranoproliferative glomerulonephritis in an immunocompetent man with no notable medical history. He presented with edema, hypertension, increased serum creatinine level, and nephrotic syndrome. Type II cryoglobulinemia with monoclonal immunoglobulin G (IgG) κ light chain was detected. Anti-HEV IgG and IgM, as well as HEV RNA, were detected in serum and cryoprecipitate. Histologic analysis of a kidney biopsy specimen revealed features of crescentic and membranoproliferative glomerulonephritis. After HEV clearance, kidney and liver parameters improved and HEV RNA and cryoglobulinemia were undetectable. Hence, we conclude that HEV can cause severe kidney disease and should be considered in cases of unexplained glomerular disease.

Thymic output and peripheral T lymphocyte subsets in relapsing–remitting multiple sclerosis patients treated or not by IFN-β

We explored the parameters of central and peripheral tolerance in patients with stable relapsing-remitting multiple sclerosis, treated or not with IFN-beta. TREC-positive T cells were lower in patients compared with controls, mainly in CD4+ subset, compatible with a thymus dysfunction or an expansion of peripheral lymphocytes. Compared to controls, the frequency of activated CD4+CD25+ T cells was higher in patients without modification of the CD4+CD25(high) T cell proportion. The IFN-beta-treatment did not modify the TREC-positive cell frequency nor the naive/memory T cell subset percentage but was associated with lower blood lymphocyte count and a lower frequency of CD4+CD45RC(high) subset.

Impact of MHC class II polymorphism on blood counts of CD4+ T lymphocytes in macaque.

While the number of peripheral blood T lymphocytes and of their two main subsets (CD4+CD8− and CD4−CD8+) varies little in a given healthy individual, substantial variation is observed between individuals. It was proposed that these counts could be influenced by MHC polymorphisms because of the well-established role of MHC molecules in thymic T lymphocyte maturation and presentation of antigenic peptides to peripheral T lymphocytes. To test this hypothesis, we have chosen the crab-eating macaque (Macaca fascicularis), an animal model phylogenetically close to man. We selected the Philippine macaque population because of a restriction of the MHC polymorphism in this islander population. Peripheral blood lymphocytes were counted with an automated analyzer and T lymphocyte subsets were assessed by immunolabeling and flow cytometry. The MHC polymorphism was investigated in 200 unrelated subjects using 14 microsatellites markers distributed across the MHC and the DRB locus that was genotyped by denaturing gradient gel electrophoresis and sequencing. All markers were in Hardy–Weinberg equilibrium. Allelic associations were tested with the UNPHASED software. We revealed a significant influence of the MHC class II region on CD4+ T lymphocyte blood count with the largest effect associated with a two-locus haplotypes combining the DRACA allele 274 and the DRB haplotype #8a (p < 8 × 10−7). Our data should stimulate a similar association study of the CD4+ T cell counts in humans.

Influence of age, sex and HCMV-serostatus on blood lymphocyte subpopulations in healthy adults

Using a standardized immunophenotyping procedure we studied thirty-eight distinct subpopulations of T, B and NK lymphocytes in 253 healthy blood donors aged from 19 to 67. We analysed the influence of age, sex and HCMV seropositivity on each lymphocyte subpopulations and established reference ranges. We observed that aging influences the largest number of lymphocyte subpopulations with a slow increase of CD8+ EMRA T lymphocytes and of the numbers of circulating Tregs. The proportion of HLA-DR+ cells among Tregs increased with age and was correlated to the proportion of HLA-DR+ cells among effector T CD4+ lymphocytes. Sex had a major impact on absolute counts of CD4+ T cells which were higher in females. HCMV-seropositivity was associated with higher frequencies of CD8+ EMRA memory T lymphocytes while a high frequency of terminally differentiated EMRA CD4+ T cells was observed in 80% of HCMV-positive individuals and in none of the HCMV seronegative individuals.

Immunoglobulin IgA, IgD, IgG, IgM and IgG subclass reference values in adults

*Corresponding author: Pr. Antoine Blancher, Laboratoire d’Immunologie du CHU de Toulouse, Hôpital Rangueil, TSA 50032, 31059 Toulouse Cedex 9, France, Phone: +33 5 61 323432, Fax: +33 5 61 323424, E-mail: blancher.a@chu-toulouse.fr; and Laboratoire d’Immunogénétique Moléculaire, EA 3034, Université Paul Sabatier, Toulouse III, France Bénédicte Puissant-Lubrano, Pol-André Apoil and Nicolas CongyJolivet: Laboratoire d’Immunogénétique Moléculaire, EA 3034, Université Paul Sabatier, Toulouse III, France; and Laboratoire d’Immunologie, CHU de Toulouse, France Michael Peres: Laboratoire d’Immunologie, CHU de Toulouse, France Francis Roubinet: EFS Pyrénées-Méditerranée, Toulouse, France Letter to the Editor

Alternative complement pathway hemolytic assays reveal incomplete complement blockade in patients treated with eculizumab

Eculizumab is a monoclonal anti-C5 antibody used in the treatment of atypical hemolytic uremic syndrome (aHUS). We monitored complement inhibition in 16 eculizumab-treated patients suffering from HUS or transplant rejection (not aHUS patients). Blood samples were obtained one to four weeks after the last eculizumab injection. We observed that eculizumab efficiently blocked the terminal pathway (TP) through classical pathway (CP) activation measured by kinetic hemolytic assay (HA) (<10%) but incompletely blocked the TP through alternative pathway (AP) activation measured by rabbit (APH50>23%) or chicken erythrocytes HA (AP100>15%). Conversely, functional ELISA revealed a complete blockade of TP through AP activation in all patients (<10%). C5a and sC5b9 levels were not correlated with residual APH50 or AP100. Similar results were obtained after in vitro addition of increasing amounts of eculizumab to a control serum (in vitro APH50>60% and AP100>20%). We also showed that ELISA was less sensitive than HA.

Analytical performances of SPAPLUS® turbidimeter for the quantification of albumin and IgG in serum and cerebrospinal fluid.

OBJECTIVES The intrathecal production of IgG is part of the diagnosis criteria for Multiple Sclerosis. Its assessment requires both quantitative (quantification of IgG and albumin in serum and cerebrospinal fluid (CSF)) and qualitative assays (isoelectric focusing). We have evaluated the analytical performances of the SPAPLUS® immunoturbidimeter (The Binding Site®) for the quantification of IgG and albumin in serum and CSF. DESIGN AND METHODS Within-day and between-day precision, linearity and carry-over were assessed. Results obtained with SPAPLUS® were compared to those obtained with the nephelometer IMMAGE® 800, including albumin quotient and CSF IgG index. Isoelectric focusing was performed and considered as the gold standard for assessment of intrathecal production of IgG. RESULTS The within-day and the between-day precisions were obtained at two concentration levels and were below the recommendations of the manufacturer and of the French Society of Clinical Biology. Our evaluation confirmed the linearity of the assays and the absence of contamination. An agreement above 94% was observed between the results obtained with SPAPLUS® and those obtained with IMMAGE® 800. The use of the new reference material DA470k did not significantly modify IgG and albumin values. The confrontation of CSF IgG index and isoelectric focusing results led to a sensitivity of 79% and a specificity of 97% of CSF IgG index quantified on SPAPLUS® for the presence of oligoclonal bands at IEF. The sensitivity of intrathecal IgG calculated with the Reibers hyperbolic formula was 47.4% and specificity was 97% for the presence of oligoclonal bands at IEF. Automatic rerun managed by the device for concentrations outside the measuring range was satisfactory. CONCLUSION The SPAPLUS® immunoturbidimeter displays good analytical performances for the parameters evaluated in this work.

Distinct effect of age, sex, and CMV seropositivity on dendritic cells and monocytes in human blood

We analyzed the impact of age, sex, and CMV on blood monocyte and dendritic cell (DC) subpopulations in 256 healthy individuals aged from 19 to 96 years. Flow cytometry was performed on whole blood within the 4 h following blood drawing. Myeloid (mDC) and plasmacytoid DC (pDC), classical, intermediate, and nonclassical monocytes were enumerated by means of TruCount tubes (BD Biosciences). We provided reference values for mDC, pDC and the three monocyte subpopulations. The numbers of classical, intermediate, and nonclassical monocytes slightly increased with age while the numbers of mDC and pDC did not vary significantly. The level of expression of CD64 and CD163 on monocytes significantly increased with age while HLA‐DR expression did not vary significantly. More precisely, CD163 expression level on intermediate monocyte slightly increased with age in women only (Spearman P = 0.019) while CD64 expression increased on monocytes in CMV‐positive individuals only. We observed that sex had almost no impact on the numbers of monocytes and DC and on their expression level of CD64 and HLA‐DR. We observed a significant decrease in the numbers of pDC with age in CMV‐positive individuals, but not in CMV negative individuals. This suggests that the lifelong subclinical infection by CMV could influence the number of circulating DC of lymphoid origin. In contrast, CMV serostatus had no significant impact on absolute numbers of mDC and monocytes.

Reference values for T, B and NK human lymphocyte subpopulations in adults

The data presented in this paper are reference ranges for frequencies of thirty-eight subpopulations of T, B and NK lymphocytes, established from a cohort of 253 healthy blood donors aged from 19 to 67. When relevant, the influence of age or sex was taken into account to calculate these reference values. This article is related to the research article entitled “Influence of age, sex and HCMV-serostatus on blood lymphocyte subpopulations in healthy adults” (Apoil et al., 2017) [1]. Immunophenotyping data obtained from each individual is made publicly available for extended analyses.

A microplate assay to measure classical and alternative complement activity

Abstract Background: We developed and validated a kinetic microplate hemolytic assay (HA) to quantify classical and alternative complement activity in a single dilution of human plasma or serum. Methods: The assay is based on monitoring hemolysis of sensitized sheep (or uncoated rabbit) red blood cells by means of a 96-well microplate reader. The activity of the calibrator was evaluated by reference to 200 healthy adults. The conversion of 50% hemolysis time into a percentage of activity was obtained using a calibration curve plotted daily. Results: The linearity of the assay as well as interference (by hemolysis, bilrubinemia and lipemia) was assessed for classical pathway (CP). The within-day and the between-day precision was satisfactory regarding the performance of commercially available liposome immunoassay (LIA) and ELISA. Patients with hereditary or acquired complement deficiencies were detected (activity was measured <30%). We also provided a reference range obtained from 200 blood donors. The agreement of CP evaluated on samples from 48 patients was 94% with LIA and 87.5% with ELISA. The sensitivity of our assay was better than that of LIA, and the cost was lower than either LIA or ELISA. In addition, this assay was less time consuming than previously reported HAs. Conclusions: This assay allows the simultaneous measurement of 36 samples in duplicate per run of a 96-well plate. The use of a daily calibration curve allows standardization of the method and leads to good reproducibility. The same technique was also adapted for the quantification of alternative pathway (AP) activity.