Estelle Seilles

Intravenous apoptotic spleen cell infusion induces a TGF-beta-dependent regulatory T-cell expansion.

Apoptotic leukocytes are endowed with immunomodulatory properties that can be used to enhance hematopoietic engraftment and prevent graft-versus-host disease (GvHD). This apoptotic cell-induced tolerogenic effect is mediated by host macrophages and not recipient dendritic cells or donor phagocytes present in the bone marrow graft as evidenced by selective cell depletion and trafficking experiments. Furthermore, apoptotic cell infusion is associated with TGF-β-dependent donor CD4+CD25+ T-cell expansion. Such cells have a regulatory phenotype (CD62Lhigh and intracellular CTLA-4+), express high levels of forkhead-box transcription factor p3 (Foxp3) mRNA and exert ex vivo suppressive activity through a cell-to-cell contact mechanism. In vivo CD25 depletion after apoptotic cell infusion prevents the apoptotic cell-induced beneficial effects on engraftment and GvHD occurrence. This highlights the role of regulatory T cells in the tolerogenic effect of apoptotic cell infusion. This novel association between apoptosis and regulatory T-cell expansion may also contribute to preventing deleterious autoimmune responses during normal turnover.

Genital human papillomavirus infection among women recruited for routine cervical cancer screening or for colposcopy determined by Hybrid Capture II and polymerase chain reaction.

The purpose of this study was to evaluate the clinical use of the Hybrid Capture (HC)-II system for the detection of human papillomavirus (HPV) DNA to identify women at risk of progression to high grade squamous intraepithelial lesions (HGSIL) and carcinomas by differentiating low risk (LR) HPV types (6, 11, 42, 43, 44) and high/intermediate risk (HR) HPV types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68). Five hundred and ninety-six women were enrolled in the study. Among them, 466 attended the hospital for routine cytologic screening and 130 were referred for colposcopy because of an abnormal Pap smear. The presence of HPV DNA was tested in cervical samples collected with the Digene Cervical Sampler in Digene Specimen Transport Medium (Digene Corporation, Silver Spring, MD, U.S.A.) using the HC-II assay. Results were compared with those obtained by polymerase chain reaction (PCR) using the MY09-MY11 primers followed by several hybridizations with specific probes. The overall HPV positivity was 32.9% by HC-II and 37.8% by PCR. Among cytologically normal smears, 19.5% were positive by HC-II (14.3% HR) and 25.1% by PCR. Of the atypical squamous cells of undetermined significance samples, 52.9% were positive by HC-II (41.1% HR) and 55.9% by PCR. Of the low grade SIL, 64.5% were positive by HC-II (59.4% HR) and 68.7% by PCR. The HPV positivity rate was found identical by both techniques in high grade smears (81.6%) and squamous cervical carcinomas (100%). By using PCR as the reference method, the sensitivity of HC-II was higher among women with abnormal cytology than with normal cytology (87.3% vs. 70%). Specificity was 80.8% and 97.5%, respectively. In summary, these results indicate that the HC-II method and MY-PCR identified nearly equivalent prevalences of HPV in cervical smear specimens.

Extended diagnostic criteria for plasmacytoid dendritic cell leukaemia.

The diagnosis of plasmacytoid dendritic cell leukaemia (pDCL) is based on the immunophenotypic profile: CD4+ CD56+ lineageneg CD45RA+/ROneg CD11cneg CD116low CD123+ CD34neg CD36+ HLA‐DR+. Several studies have reported pDCL cases that do not express this exact profile or expressing some lineage antigens that could thus be misdiagnosed. This study aimed to validate pDCL‐specific markers for diagnosis by flow‐cytometry or quantitative reverse transcription polymerase chain reaction on bone marrow samples. Expression of markers previously found in normal pDC was analysed in 16 pDCL, four pDCL presenting an atypical phenotype (apDCL) and 113 non‐pDC – lymphoid or myeloid – acute leukaemia. CD123 was expressed at significantly higher levels in pDCL and apDCL. BDCA‐2 was expressed on 12/16 pDCL and on 2/4 apDCL, but was never detected in the 113 non‐pDC acute leukaemia cases. BDCA‐4 expression was found on 13/16 pDCL, but also in 12% of non‐pDC acute leukaemia. High levels of LILRA4 and TCL1A transcripts distinguished pDCL and apDCL from all other acute leukaemia (except B‐cell acute lymphoblastic leukaemia for TCL1A). We thus propose a diagnosis strategy, scoring first the CD4+ CD56+/− MPOneg cCD3neg cCD79aneg CD11cneg profile and then the CD123high, BDCA‐2 and BDCA‐4 expression. Atypical pDCL can be also identified this way and non‐pDC acute leukaemia excluded: this scoring strategy is useful for diagnosing pDCL and apDCL.

Endothelial cell-derived microparticles induce plasmacytoid dendritic cell maturation: potential implications in inflammatory diseases.

Microparticles derived from vascular endothelium are thought to play a role in common inflammatory disorders. In this study, the authors show that microparticles derived from vascular endothelium specifically induce the maturation of plasmacytoid dendritic cells and production of inflammatory cytokines, suggesting that endothelial microparticles may serve as therapeutic targets for immune modulation. Background Increased circulating endothelial microparticles, resulting from vascular endothelium dysfunction, and plasmacytoid dendritic cell activation are both encountered in common inflammatory disorders. The aim of our study was to determine whether interactions between endothelial microparticles and plasmacytoid dendritic cells could contribute to such pathologies. Design and Methods Microparticles generated from endothelial cell lines, platelets or activated T cells were incubated with human plasmacytoid dendritic cells sorted from healthy donor blood or with monocyte-derived dendritic cells. Dendritic cell maturation was evaluated by flow cytometry, cytokine secretion as well as naive T-cell activation and polarization. Labeled microparticles were also used to study cellular interactions. Results Endothelial microparticles induced plasmacytoid dendritic cell maturation. In contrast, conventional dendritic cells were resistant to endothelial microparticle-induced maturation. In addition to upregulation of co-stimulatory molecules, endothelial microparticle-matured plasmacytoid dendritic cells secreted inflammatory cytokines (interleukins 6 and 8, but no interferon-α) and also induced allogeneic naive CD4+ T cells to proliferate and to produce type 1 cytokines such as interferon-γ and tumor necrosis factor-α. Endothelial microparticle endocytosis by plasmacytoid dendritic cells appeared to be required for plasmacytoid dendritic cell maturation. Importantly, the ability of endothelial microparticles to induce plasmacytoid dendritic cells to mature was specific as microparticles derived from activated T cells or platelets (the major source of circulating microparticules in healthy subjects) did not induce such plasmacytoid dendritic cell maturation. Conclusions Our data show that endothelial microparticles specifically induce plasmacytoid dendritic cell maturation and production of inflammatory cytokines. This novel activation pathway may be implicated in various inflammatory disorders and endothelial microparticles could be an important immunmodulatory therapeutic target.

Triazinic herbicide determination by gas chromatography-mass spectrometry in breast milk

A solid-phase extraction procedure using a graphitized carbon black cartridge for extraction and cleaning of a series of five triazines (atrazine, deethylatrazine, deisopropylatrazine, ametryne and prometryne) from breast milk samples was developed. Using a chemometric methodology, the optimisation of both the analysis time and the triazinic herbicide separation by gas chromatography-mass spectrometry (GC-MS) was then carried out with only 18 experiments. Detection and quantification limits for 1ml breast milk sample were, respectively, 0.3 and 1 ppb for each studied compound. The variation coefficients were less than 5% over the concentration range from 1 to 100 ppb. The accuracy was between 98.63 and 104.62% for each triazinic herbicide. The recovery was between 58.64 and 63.22% for the concentration range from 1 to 100 ppb for each triazinic herbicide. The assay was successfully applied to the analysis of several breast milk samples.

Humoral and cellular immunity in patients with hepatic alveolar echinococcosis. A 2 year follow-up with and without flubendazole treatment

Summary Parameters of humoral and cellular immunity were assessed in 12 patients with alveolar echinococcosis (AE) of the liver before, during and after discontinuation of treatment with flubendazole (FZ). In infected patients, before any medical treatment values of serum IgG, IgA, total haemolytic complement and C4 were significantly higher than those observed in control subjects; IgA levels were higher in jaundiced patients. Specific antibodies assayed by indirect haemagglutination and immunoelectrophoresis were present only in infected patients and were shown to decrease by the sixth month of treatment; however, similar fluctuations were observed without treatment. The percentage and absolute number of B lymphocytes, and total circulating lymphocytes, were significantly lower in patients with AE. An impairment of functional activity of T cells assayed by the leucocyte migration test, with PPD and Candidin as antigens, was demonstrated despite a normal percentage of SRBC rosettes. The ‘score’ of migration index still decreased during FZ treatment and returned to initial values after the year of follow‐up without treatment. These results suggest that human AE is associated with important immunological disturbances. Changes in humoral immunity can be unequivocally considered to be a consequence of the parasite infection. The primary or secondary nature of the impairment of cellular immune responses and its mechanisms remain to be elucidated. Flubendazole could be responsible for an increase of cellular immune alterations in these patients.

Increased Levels of Circulating Microparticles Are Associated with Increased Procoagulant Activity in Patients with Cutaneous Malignant Melanoma

Microparticles (MPs) are known to be increased in various malignancies and are involved in tumor invasion, angiogenesis, coagulation, and metastasis. We investigated the plasma levels of annexin-V MPs (AV(+)MPs), platelet-derived MPs (PMPs), and endothelial-derived MPs (EMPs) in patients with melanoma (n=129) and in healthy controls (n=49). A functional coagulation test STA Procoag-PPL measuring the clotting time was performed on samples containing MPs to evaluate their procoagulant potential. The plasma levels of PMPs, EMPs, and AV(+)MPs were significantly higher, and the clotting time-PPL was significantly lower in melanoma patients than in healthy controls. The plasma levels of PMPs, EMPs, and AV(+)MPs were higher in stage IV than in the other stages of melanoma, but with no significant difference. In addition, we observed an inverse correlation between PMPs, AV(+)MPs, and clotting times. Our data suggest that MPs are involved in the progression of melanoma and may be associated to melanoma-associated thrombogenesis.

Phosphatidylserine-expressing cell by-products in transfusion: A pro-inflammatory or an anti-inflammatory effect?

Labile blood products contain phosphatidylserine-expressing cell dusts, including apoptotic cells and microparticles. These cell by-products are produced during blood product process or storage and derived from the cells of interest that exert a therapeutic effect (red blood cells or platelets). Alternatively, phosphatidylserine-expressing cell dusts may also derived from contaminating cells, such as leukocytes, or may be already present in plasma, such as platelet-derived microparticles. These cell by-products present in labile blood products can be responsible for transfusion-induced immunomodulation leading to either transfusion-related acute lung injury (TRALI) or increased occurrence of post-transfusion infections or cancer relapse. In this review, we report data from the literature and our laboratory dealing with interactions between antigen-presenting cells and phosphatidylserine-expressing cell dusts, including apoptotic leukocytes and blood cell-derived microparticles. Then, we discuss how these phosphatidylserine-expressing cell by-products may influence transfusion.

Serum secretory IgA and secretory component in patients with non-cirrhotic alcoholic liver diseases

Elevated levels of secretory IgA in serum have been demonstrated in several liver dysfunctions such as hepatic cytolysis and cholestasis. However, these possible alterations at an early stage of liver diseases have not yet been investigated. We studied a cohort of chronic alcoholic patients without cirrhosis in order to assess the changes in serum secretory IgA and other forms of secretory component, the split product of the polymeric Ig-receptor of epithelial cells. The possible diagnostic value of these measurements in the assessment of alcoholic disease was compared to that of serum gamma-glutamyl transpeptidase activity. Serum levels of secretory IgA and IgM and free secretory component, were quantified by an enzyme-linked immunosorbent assay in 71 patients with chronic alcoholic liver disease without cirrhosis and in 45 healthy controls. Patients were divided into two groups according to the severity of the liver abnormalities. In addition, the reversibility of serum secretory IgA, IgM and free secretory component abnormalities after alcohol withdrawal was evaluated in 15 patients. Serum levels of the three molecular forms of secretory component were significantly higher than those measured in control subjects, both in the whole population of patients and in the two groups of alcoholic patients without cirrhosis. In all groups, serum secretory IgA levels were correlated to free secretory component but not to total IgA levels. Serum secretory IgA levels were as discriminative as gammaglutamyl transferase activity in distinguishing between chronic alcoholic patients without cirrhosis and non-alcoholic subjects. The abnormalities of serum secretory IgA concentrations were reversible after alcohol withdrawal.(ABSTRACT TRUNCATED AT 250 WORDS)

TP53 polymorphism at exon 4 in caucasian women from eastern France: lack of correlation with HPV status and grade of cervical precancerous lesions

OBJECTIVE To investigate the codon 72 TP53 polymorphism in women from eastern France with normal or abnormal cervical cytology. STUDY DESIGN We analyzed the TP53 allele distribution by denaturant gradient gel electrophoresis assay and the human papillomaviruses (HPV) infection in 138 cervical smears: 50 normal, 20 atypical squamous cells of undetermined significance, 40 low grade squamous intraepithelial lesions, 28 high grade squamous intraepithelial lesions. RESULTS The viral DNA prevalence increased with cytological abnormalities. The rates of arginine (Arg) and proline (Pro) homozygosity and Arg/Pro heterozygosity were 49, 0.72, and 51%, respectively. No association was found between HPV status and TP53 polymorphism. No differences were observed in the frequency of the TP53 genotypes according to cytology. CONCLUSION The TP53 Arg/Arg genotype does not appear to represent a risk factor in the progression of HPV associated cervical lesions. We were not able to confirm that the TP53 genotype increases the susceptibility to be infected by HPV or to develop HGSIL, and a fortiori invasive carcinoma of the cervix.