Bénédicte Puissant

Immunomodulatory effect of human adipose tissue-derived adult stem cells: comparison with bone marrow mesenchymal stem cells.

Like mesenchymal stem cells from bone marrow (BM‐MSCs), adipose tissue‐derived adult stem cells (ADAS cells) can differentiate into several lineages and present therapeutical potential for repairing damaged tissues. The use of allogenic stem cells can enlarge their therapeutical interest, provided that the grafted cells could be tolerated. We investigate here, for the first time, the immunosuppressive properties of ADAS cells compared with the well‐characterized immunosuppressive properties of BM‐MSCs. ADAS cells did not provoke in vitro alloreactivity of incompatible lymphocytes and, moreover, suppressed mixed lymphocyte reaction (MLR) and lymphocyte proliferative response to mitogens. The impairment of inhibition when ADAS cells and BM‐MSCs were separated from lymphocytes by a permeable membrane suggests that cell contact is required for a full inhibitory effect. Hepatocyte growth factor is secreted by both stem cells but, similar to interleukin‐10 and transforming growth factor‐β (TGF‐β), the levels of which were undetectable in supernatants of MLR inhibited by ADAS cells or BM‐MSCs, it did not seem implicated in the stem cell suppressive effect. These findings support that ADAS cells share immunosuppressive properties with BM‐MSCs. Therefore, ADAS cell‐based reconstructive therapy could employ allogenic cells and because of their immunosuppressive properties, ADAS cells could be an alternative source to BM‐MSCs to treat allogenic conflicts.

FOXP3 mRNA levels are decreased in peripheral blood CD4+ lymphocytes from HIV-positive patients.

The impact of HIV infection on regulatory CD4+CD25high (Treg) lymphocyte subpopulations was evaluated by FOXP3 quantitative reverse transcriptase polymerase chain reaction and by flow cytometry. FOXP3 mRNA was quantified in peripheral blood mononuclear cells or purified CD4+ lymphocytes from HIV+ lymphopenic patients. Patients were distributed among clinical stages A, B, and C and received highly active antiretroviral therapy. The frequency of CD4+CD25high lymphocytes, measured by flow cytometry, was decreased in HIV+ patients (n = 38) compared with the group of uninfected subjects (n = 39). FOXP3 mRNA levels were found decreased in HIV+ patients (n = 25) compared with controls (n = 17) when expression of CD3γ or β-actin but not that of TATA box binding protein 1 was used for data normalization. Our results are compatible with a decrease of the Treg lymphocytes during HIV infection. The consequences of a Treg decrease are discussed in the context of immunologic anomalies observed during HIV infection.

Blood Concentrations of Hydroxychloroquine and Its Desethyl Derivative Correlate Negatively with the Percentage of CD45RO+ Cells among CD4+ Lymphocytes in Hydroxychloroquine-Treated Lupus Patients

Abstract:  The objective of the study was to investigate the influence of the blood concentrations of hydroxychloroquine ([HCQ]) and its derivative desethylhydroxychloroquine ([DHCQ]) on lymphocyte activation or differentiation in HCQ‐treated lupus patients. We studied the correlations between [HCQ], [DHCQ], and the frequency of various lymphocyte subsets in 58 HCQ‐treated lupus patients (mean HCQ dose: 4.93 ± 1.58 mg/kg/day; mean duration of the disease: 122 ± 64 months). [HCQ] and [DHCQ] were determined by high‐performance liquid chromatography (HPLC). Lymphocyte markers were studied by flow cytometry using monoclonal anti‐CD3, ‐CD4, ‐CD8, ‐CD25, ‐DR, ‐CD45RA,‐CD45RO, ‐CD19, ‐CD38, and ‐CD86 antibodies. sIL2‐R serum concentrations were measured by enzyme‐linked immunosorbent assay (ELISA). [HCQ] and [DHCQ] were 599.9 ng/mL (median: 529.5; range: 55–1935) and 353.43 (median: 286 ng/mL; range: 118–1090). In a multiple regression analysis, [HCQ] and [DHCQ] were associated with the HCQ prescribed dose in mg/kg/day (P= 0.0002 and P= 0.03) and with compliance to the treatment (P= 0.004 and P= 0.03). We found a negative correlation between [HCQ], [DHCQ], and the CD45RO+ cell frequency among CD3+CD4+ cells (P= 0.03 and P= 0.007, respectively). Other lymphocyte subset markers (LSMs) and sIL2‐R concentrations were not significantly associated with [HCQ] or [DHCQ]. In the multiple regression analysis, CD45RO+ expression was negatively influenced by [HCQ] (P= 0.005), and positively influenced by smoking habits (P= 0.005) and age (P= 0.005). Similar results were found in the multivariate model including [DHCQ]. Disease activity and taking more than 10 mg/day of corticosteroids or an immunosuppressive drug did not influence CD45RO+ expression. Lupus patients had less CD3+CD4+CD45RO+ cells than controls (P= 0.03). In lupus patients, HCQ and DHCQ may alter the generation or the blood circulation of CD4+CD45RO+ lymphocytes in a concentration‐dependent pattern.

Polymorphism of human and primate RANTES, CX3CR1, CCR2 and CXCR4 genes with regard to HIV/SIV infection

Among genes that influence human susceptibility to HIV (human immunodeficiency virus) infection or AIDS (acquired immunodeficiency syndrome) progression, chemokine-receptor and chemokine genes were extensively studied because of their role as HIV co-receptors or co-receptor competitors, respectively. We have studied in non-human primates (chimpanzee, gorilla, gibbon, orang-utan, crab-eating and rhesus macaque, baboon and marmoset) the RANTES, CCR2 and CX3CR1 gene sequences in regions surrounding human mutations that were associated with susceptibility to HIV or AIDS progression: RANTES G−403A and C−28G, CCR2 V64I, CX3CR1 V249I and CX3CR1 T280M. Among these five dimorphisms, only RANTES G−403A is observed in one of the eight primate species studied here (gibbon). This suggests that these mutations appeared recently in humans and probably do not account for variable HIV/SIV disease progression in primates. It is noteworthy that chimpanzees, which are naturally resistant to HIV-1- and HIV-2-induced AIDS, do not have the human mutations associated with delayed disease progression. Inter-species and intra-species polymorphic positions are observed in primates and we discuss the potential impact of these mutations on HIV/SIV disease progression. Particularly, we identified polymorphisms in old-world monkey (OWM) genes, and it could be of great importance to analyse the possible association between these polymorphisms and disease progression in OWM species that are currently used in research for HIV vaccine and therapy.

Decrease of lewis frequency in HIV-infected patients : possible competition of fucosylated antigens with HIV for binding to DC-SIGN

We explored the impact of human ABO glycosyltransferase and Lewis and secretor fucosyltransferase polymorphisms in HIV infection. We found that, compared with healthy blood donors, HIV-infected patients display a significant decrease in Lea−b+ phenotype frequencies. We showed that HIV binding on DC-SIGN-transduced Jurkat cells was inhibited by fucosyl bovine serum albumin. Our results suggest a slight protective effect of Lewis b antigen on HIV infection, possibly by the competition of Lewis antigens with HIV for binding to DC-SIGN.

Cryofibrinogénémie : étude monocentrique au CHU de Toulouse

PURPOSE Cryofibrinogenemia is an unknown disorder and studies dedicated to it are limited. The aim of our study was to report on the incidence, clinical manifestations and associated diseases in patients with isolated cryofibrinogenemia. METHODS This is a retrospective single-center study. Patients included in this study had a positive and isolated detection of cryofibrinogen between January 1st, 2011 and December 31st, 2012. Identification was possible through the database of the laboratory of immunology. RESULTS Two hundred and eighty-one consecutive orders of cryofibrinogenemia were identified. Seventy-three patients had a positive detection of cryofibrinogenemia. Among them, 12 had an isolated cryofibrinogenemia and sixty-one patients (84%) had concomitant cryofibrinogenemia and cryoglobulinemia. The mean age was 59±19years. Seven patients were female (58%). Cutaneous manifestations were present in half case. Peripheral nerve involvement was present in 5 cases (42%) and rheumatic manifestations in 4 patients (33%). A thrombotic event was reported in 7 patients (58%). Renal impairment was present in 7 patients. The median cryofibrinogen concentration was 254±304mg/L. Five patients had a secondary cryofibrinogenemia. The most often prescribed treatment was corticosteroids. CONCLUSION Cryofibrinogenemia is an unknown disorder. Testing for cryoglobulinemia is more frequent than for cryofibrinogenemia whereas clinical manifestations are similar. Detection of cryofibrinogen is positive in most of the cases, with an important prevalence of thrombotic events in this population. This study confirms the importance of conducting prospective studies on cryofibrinogenemia.

Cryofibrinogenemia and risk of cancer in cryoglobulinemic patients without vasculitis criteria

Cryofibrinogen is a cryoprotein that forms only in plasma and not in serum. In patients screened for a cryopathy, prevalence of cryofibrinogenemia varies from 12% to 51% [1–3]. Most studies concerning essential and secondary cryofibrinogenemia excluded patients with concomitant cryoglobulinemia [1]. In cryoglobulinemic patients, the presence of cryofibrinogenemia has been shown to be associated with cryoglobulinemia vasculitis [4,5]. Moreover, we found that patients with both cryoproteins had more frequently a cancer than patients with isolated cryoglobulinemia [5]. In routine practice, about half of the patients with a positive cryglobulinemia do not fulfil cryoglobulinemia vasculitis criteria. The present study aimed to determine the characteristics associated with the presence of cryofibrinogenemia in cryoglobulinemic patients without cryoglobulinemia vasculitis. We retrospectively included all cryoglobulinemic patients who were also tested for cryofibrinogenemia at the Immunology Laboratory of Toulouse University Hospital between January 2011 and December 2012. Toulouse University Hospital Research Ethics Committee approval was obtained for anonymous retrospective medical chart review. Patients who fulfilled cryoglobulinemia vasculitis criteria previously used in the CryoVas Cohort were excluded [6]. Medical files were reviewed. Baseline clinical and laboratory findings at the time of cryoprotein testing were recorded. Renal involvement was defined by a creatininemia N130 μmol/L or a glomerular filtration rate b60 ml/min/1.73 m (Modification of Diet in Renal Disease (MDRD) Study equation) or hematuria or proteinuria N500 mg/24 h. Blood was collected in citrated tubes for cryofibrinogenemia and in anticoagulant-free tubes for cryoglobulinemia. Both sera and plasma were kept at 37°C until centrifugation, which was performed within one hour for plasma and within 12 h for sera. Samples were kept at 4°C for 72 h for sera and 8 days for plasma and were then tested for the presence or absence of precipitate. Both cryoprecipitates were quantified by optic density absorbance at 280 nm. Presence of cryofibrinogen and of cryoglobulin was confirmed by western blotting, as previously described [2,5]. The study included 75 patients, 43 being cryoglobulin-positive/ cryofibrinogen-positive (CF-positive group) patients and 32 being cryoglobulin-positive/cryofibrinogen-negative (CF-negative group).