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HLA-DRB1*04:326 differs from HLA-DRB1*04:01:01 by one nucleotide substitution in codon 23 in exon 2., (© 2021 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

HLA-C*01:214 differs from HLA-C*01:02:01:01 by one nucleotide substitution in codon -10 in exon 1., (© 2021 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Histocompatibility laboratories perform the biological analyses linked related to organ transplant, hematopoietic stem cells transplant, some immune dysfunction diseases and immuno-allergy after therapeutic treatment. Most of these analyses are prospectively or retrospectively performed on sera and DNA. The Société Francophone d'Histocompatibilité et d'Immunogénétique (SFHI) has made some recommendations in order to define storage conditions and storage lifetime of the samples required in a histocompatibility laboratory. These recommendations have been drawn up by a working group of ten biologists. They have been established on literature review and data from method validation, which has been already performed within French laboratories (collected through a national questionnaire sent to participant laboratories). The recommendations made by the SFHI for the storage of samples for immunogenetics analyses facilitate the harmonization of practices among histocompatibility laboratories.

Identification of anti-human leukocyte antigen (HLA) antibodies (Abs) is based on Luminex™ technology. We used bioinformatics to (i) study the correlations of mean fluorescence intensities (MFIs) for all the possible allele pairs, and (ii) determine the degree of epitope homology between HLA antigens. Using MFI data on anti-HLA Abs from 6000 Luminex™ assays, we provide an updated overview of class I and II HLA antigen cross-reactivity in which each node corresponded to an allele and each link corresponded to a strong correlation between two alleles (Spearman's ρ > 0.8). We compared these correlations with the serological groups and the results of an epitope analysis. The strongest correlations concerned allele-specific Abs directed against the same antigen. For the HLA-A locus, the highest values of Spearman's ρ reflected broad specificity. For the HLA-B locus, graphs defined the HLA-Bw4 public epitope, and correlations between HLA-A and -B alleles were only present for beads with the same Bw4 public epitope. For the HLA-C locus, we identified two groups that differed with regard to their KIR ligand subclassification. Lastly, the HLA-DRB1 subgroups were part of a network. In the epitope analysis, Spearman's ρ was related to the number of matched epitopes within pairs of alleles. The combination of Spearman's ρ with simple, undirected graphing constitutes an effective tool for understanding routinely encountered cross-reactivity profiles. Based on this model, we have implemented an online data visualization tool available at http://cusureau.pythonanywhere.com/., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

A hematopoietic chimerism assay is the laboratory test for monitoring engraftment and quantifying the proportions of donor and recipient cells after hematopoietic stem cell transplantation recipients. Flow cytometry is the reference method for determining the purity of CD3 + cells on the chimerism of selected CD3 + cells. In the present study, we developed a single-step procedure that combines the CD3 + purity assay (using the PCR-based Non-T Genomic Detection Kit from Accumol, Calgary, Canada) and the qPCR chimerism monitoring assay (the QTRACE qPCR assay from Jeta Molecular, Utrecht, the Netherlands). First, for the CD3 + purity assay, we used a PCR-friendly protocol by changing the composition of the ready-to-use reaction tubes (buffer and taq polymerase) and obtained a satisfactory calibration plot (R 2 = 0.8924) with a DNA reference scale of 2 ng/μl. Next, 29 samples (before and after CD3 positive selection) were analyzed, the mean cell purity was, respectively, 19.6% ± 6.45 and 98.9% ± 1.07 in the flow cytometry assay; 26.8% ± 7.63 and 98.5% ± 1.79 in the PCR-based non-T genomic detection assay. Our results showed that the CD3 + purity assay using a qPCR kit is a robust alternative to the flow cytometry assay and is associated with time savings when combined with a qPCR chimerism assay., (© 2020 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Introduction: The appearance of de novo donor-specific anti-human leukocyte antigen antibodies (dnDSAs) after kidney transplantation is independently associated with poor long-term allograft outcomes. The objective of the present study was to evaluate the predictive value of a flow cytometry crossmatching (FC-XM) assay after the appearance of dnDSAs related to antibody-mediated allograft rejection (ABMR) after kidney transplantation., Materials and Methods: A total of 89 recipients with dnDSAs after transplantation were included. The crossmatching results were compared with the dnDSA profile (the mean fluorescence intensity (MFI), the complement-binding activity, and the IgG subclass profile) and the biopsy's morphological features., Results: Of the 89 patients, 59 (66%) were positive in an FC-XM assay, 17 (19%) had complement-binding DSAs, 55 (62%) were positive for IgG1 and/or IgG3 in a solid phase assay, and 45 (51%) had morphological biopsy features linked to ABMR., Conclusion: An FC-XM assay was unable to discriminate between cases with or without ABMR on biopsy findings; it had a low positive predictive value (<70%) and a low negative positive predictive value (<42.9%), taking into account the sensitivity of our assay (limit of detection: DSAs with an MFI >3000). In this context, the height of the MFI of the dnDSAs might be enough for a high positive predictive value for ABMR and additional testing for complement binding activity can remain optional., (Copyright © 2020 Elsevier B.V. All rights reserved.)

We report a discordance between complement-dependent cytotoxicity and next-generation sequencing molecular typing revealing HLA-B*15:47:01 allele with undefined serological equivalent confirmed by high-level immunization against the B15 serotype. Due to the high-level immunization against HLA-B15 and B70 antigens, we considered the HLA-B*15:47:01 allele to be B Blank and not as B15 or B70 serological specificity., (© 2019 John Wiley & Sons Ltd.)

The major histocompatibility complex class I polypeptide-related sequence A (MICA) glycoprotein mediates the activation of the natural killer group 2D receptor (NKG2D) expressed on NK and CD8+ T cells. A methionine or valine at position 129 in exon 3 results in strong (MICA129 met) or weak (MICA129 val) binding to NKG2D. The MICA A5.1 allele causes a premature stop codon. Various NKG2D polymorphisms are associated with low (NKC3 C/C and NKC4 C/C) or high (NKC3 G/G and NKC4 T/T) levels of NK cell cytotoxic activity. In 162 patients with spondyloarthritis (115 with ankylosing spondyloarthritis, 46 with psoriatic arthritis and 1 with reactive arthritis) compared to 124 healthy controls, MICA-129 with methionine allele was more frequent in patients with spondyloarthritis (odds ratio (OR) (95% confidence interval) = 4.84 (2.75‒8.67)), whereas MICA-129 val/val, MICA A5.1 and NKC3 C/C variants were less frequent (OR = 0.20 (0.11‒0.37), 0.15 (0.06‒0.36) and 0.24 (0.13‒0.44), respectively). After adjustment for HLA-B*27 status, only NKC3 C/C remained linked to spondyloarthritis (adjusted OR = 0.14 (0.06‒0.33)). Homozygosity for MICA A5.1 is linked to ankylosing spondyloarthritis, and NKC3 C/C and MICA-129 val/val to psoriatic arthritis. MICA and NKC3 polymorphisms (related to a low NK cell cytotoxic activity) constituted a genetic association with spondyloarthritis.

Flow cytometry crossmatching (FC-XM) assay is the most sensitive cell-based method for detecting donor-specific antibodies (DSAs). However, the use of FC-XM remains limited by methodological and clinical variations. This basic assay cannot discriminate between complement-fixing and noncomplement-fixing antibodies. FC-XM also detects patient all antibodies bound to donor cells and not only DSAs against to HLA molecules. Pretest factors associated with a donor's medical care can affect test results by changing the number, viability and target on lymphocytes (such as rituximab on CD20 + B-cells). Assay adjustment can be performed to improve the sensitivity and specificity of FC-XM. Pronase treatment (0.5-1 mg/mL) prevents false-positive B-cell FC-XM due to nonspecific immunoglobulin binding by Fc receptors and binding of surface immunoglobulins onto the surface of B-cells. Pronase treatment (2 mg/mL) or a serum incubation step with an anti-rituximab monoclonal antibody (Ab) prevents the interference induced by rituximab therapy. The use of 7 aminoactinomycin-D (7-AAD) or fluorochrome-conjugated C4d Ab, after complement incubation, allows complement-fixing antibodies to be distinguished from noncomplement-fixing antibodies. The use of donor endothelial precursor cells as target cells allows the detection of nonmajor histocompatibility complex Ab-binding endothelial cells. However, lymphocyte crossmatches still had some limits in specificity and sensitivity. This implies that this assay must be interpreted with the virtual crossmatch., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: The purpose of this article is to clarify and define medical qigong and to identify an appropriate study design and methodology for a large-scale study looking at the effects of qigong in patients with type 2 diabetes mellitus (T2DM), specifically subject enrollment criteria, selection of the control group and study duration. Methods: A comprehensive literature review of English databases was used to locate articles from 1980-May 2017 involving qigong and T2DM. Control groups, subject criteria and the results of major diabetic markers were reviewed and compared within each study. Definitions of qigong and its differentiation from physical exercise were also considered. Results: After a thorough review, it was found that qigong shows positive effects on T2DM; however, there were inconsistencies in control groups, research subjects and diabetic markers analyzed. It was also discovered that there is a large variation in styles and definitions of qigong. Conclusions: Qigong exercise has shown promising results in clinical experience and in randomized, controlled pilot studies for affecting aspects of T2DM including blood glucose, triglycerides, total cholesterol, weight, BMI and insulin resistance. Due to the inconsistencies in study design and methods and the lack of large-scale studies, further well-designed randomized control trials (RCT) are needed to evaluate the 'vital energy' or qi aspect of internal medical qigong in people who have been diagnosed with T2DM., Competing Interests: The authors are certified Yi Ren Qigong instructors and have no conflicts of interest.

Preformed donor-specific antibodies against human leukocyte antigen can induce antibody-mediated rejection after organ transplant. Hence, future transplant recipients undergo pretransplant screening for preformed antibodies (ie, virtual crossmatch). Subsequently, prospective (analytic) crossmatching is performed using conventional, complement-dependent cytotoxicity assays and/or flow cytometry-based methods. The present article reviews factors that must be considered when unexpected, positive, prospective crossmatches are observed. First, the prozone effect caused by the interference of complement or immunoglobulin M must be abrogated by treating the serum with moderate heat, dilution, hypotonic dialysis, EDTA, or dithiothreitol. Second, the physician must check for the presence of potentially interfering autoantibodies (in a context of autoimmune disease or human immunodeficiency virus infection) or therapeutic antibodies (such as rituximab and antithymocyte globulin). In conclusion, knowledge of each assay's technical characteristics will enable the physician to reliably interpret any discrepancies. The reasons for an unexpected, positive, prospective crossmatch must be elucidated before transplant to ensure efficient organ allocation and optimize patient outcomes.

BK virus is a common opportunistic post-transplantation viral infection. Although some risk factors have been studied in this context, the contribution of NK cells has not been assessed in detail. In a group of kidney transplant recipients, we studied the association between (i) the likelihood of BK virus replication during the two-year period after kidney transplantation and (ii) the genotypes of the killer cell immunoglobulin-like receptor (KIR) repertoire and their human leukocyte antigen (HLA) ligands. Other clinical factors (such as defective organ recovery and immunosuppressive treatment) were also assessed. BK virus replication was observed in 43 of the 103 recipients (41%). Patients with BK virus replication in the plasma were more likely to display defective organ recovery in the first seven days post-transplantation. BK virus replication was not associated with Missing KIR ligands. However, BK virus replication was more frequent in patients with responsive NK cells (i.e. when a ligand for activating KIRs was not homozygous in the recipient and present in the donor). Our results suggest that defective organ recovery and the recipient's activating KIR repertoire may be related (depending on HLA ligands present in the couple recipient / donor) to the reactivation of BK virus replication after kidney transplantation., (© 2016 Steunstichting ESOT.)

Introduction: Articular involvement is the most common extraintestinal manifestation associated with inflammatory bowel diseases (IBDs). Manifestations are 'paradoxical' when they occur during treatment, notably with anti-tumor necrosis factor (anti-TNF) drugs, which are expected to prevent or treat them. The aim of this study was to assess the frequency, characteristics, and associated factors of paradoxical articular manifestations in patients with IBD treated with anti-TNF., Patients and Methods: In this prospective single-center study, an examination by a rheumatologist was systematically offered to all patients with IBD treated with infliximab (IFX) to assess the prevalence of articular manifestations and distinguish between those related to treatment and those associated with intestinal disease. Paradoxical manifestations were defined as the occurrence of articular manifestations (excluding induced lupus and hypersensitivity reactions) during anti-TNF therapy in patients with intestinal remission. Measures of biological inflammatory, immunological markers, HLA-B27 allele, IFX trough levels, and anti-IFX antibody (Ab) were performed for all patients., Results: Between May 2013 and April 2014, 65 patients with Crohn's disease and 15 with patients ulcerative colitis treated with IFX were included. The median duration of anti-TNF therapy was 66 months [quartile (Q)1=23 months-Q3=81 months]. Articular manifestations were observed in 50 (62%) patients treated with IFX. Eleven percent (n=9) were considered to be associated with IBD and 16% (n=13) to be associated with anti-TNF therapy. Among articular manifestations associated with anti-TNF therapy, nine (11%) patients were considered paradoxical, two (2%) as drug-induced lupus, and two (2%) as a hypersensitivity reaction. Among the nine patients with paradoxical manifestations, all had Crohn's disease in clinical remission, three patients presented a spondyloarthropathy, and three developed associated paradoxical psoriasis. No patient discontinued anti-TNF because of the articular manifestations. Methotrexate was effective on articular symptoms in two of the three treated patients with paradoxical manifestations. No clinical or biological factors, including IFX trough levels, were associated with the occurrence of paradoxical manifestations., Conclusion: Paradoxical articular manifestations in IBD patients treated by anti-TNF are common, affecting more than 10% of patients. These events are generally mild and do not need discontinuation of anti-TNF therapy.

Crossmatching is essential prior to kidney transplantation to confirm compatibility between the donor and the recipient, particularly to prevent acute antibody-mediated rejection. An unexpected positive crossmatch may be obtained in recipients with an autoimmune disease or preexisting antibodies not detected by single-antigen bead array due to complement interference or who have been previously treated by desensitization protocols such as rituximab, antithymocyte globulin, or intravenous immunoglobulins. We report donor and recipient investigations that revealed unexpected positive B-cells crossmatch, probably due to donor cells, as the donor had received rituximab therapy shortly before organ harvesting, in a context of severe idiopathic thrombocytopenic purpura. We consequently detected unexpected Class II IgG complement-dependent cytotoxicity for all sera tested. Other laboratory investigations failed to elucidate the reasons for this recipient-related positivity.

Changes in classical and non-classical human leukocyte antigen expression by tumor cells can play a critical role in the generation of tumor antigen-specific immune responses and can modulate the interactions of natural killer cells and T cell subpopulations with target cells. Recently, several studies have investigated the relations between HLA molecules and prognosis in B-CLL, suggesting a potential clinical relevance of tumor escape mechanisms. In this paper, we will summarize conflicting information about the role of HLA-related prognostic factors in B-CLL, such as downregulation of HLA class I antigen expression, interactions between natural killer cell receptor and certain ligands and the role of HLA-G expression., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Objectives: Mastocytosis is a heterogeneous group of clonal mast cell disorders in which bone manifestations are frequently seen, but poorly understood. In this study, we analyzed correlation of clinical findings in mastocytosis patients with bone mineral density and bone turnover markers., Methods: Serum levels of bone turnover markers were measured in mastocytosis patients and healthy volunteers. Bone disease was evaluated using radiographic imaging, and measurement of bone mineral density., Results: Of 45 adult mastocytosis patients, bone abnormalities were detected in 34 (75%). Bone lesions were documented on radiographic imaging in 16 patients (36%), and bone mineral density in 24 patients (53%), of which 9 patients (20%) had osteoporosis and 15 (33%) had osteopenia. Serum levels of bone turnover markers that evaluate bone resorption (C-telopeptide, deoxypyridinoline), bone formation (bone-specific alkaline phosphatase), and bone remodeling (osteoprotegerin) were significantly higher in the patient population than in the control population (n=28). Levels of C-telopeptide and osteoprotegerin were higher in patients with advanced systemic mastocytosis than in patients with cutaneous or indolent systemic mastocytosis. Moreover, C-telopeptide and osteoprotegerin levels were significantly correlated with those of serum tryptase, a diagnostic marker of mastocytosis., Conclusion: The observed bone turnover markers variations indicate a complex process of bone turnover in mastocytosis-related bone manifestations. The highly significant correlation between serum tryptase and serum bone turnover markers levels, and the positive correlation of levels of bone turnover markers with advanced disease, support the existence of a link between bone remodeling and mast cell burden., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Accumulating evidence has suggested that ω3-polyunsaturated fatty acids (ω3-PUFAs) may have beneficial effects in the prevention/treatment of cardiovascular diseases, while controversies still remain regarding their anti-arrhythmic potential. It is not clear yet whether ω-3-PUFAs can suppress early afterdepolarizations (EADs) induced by oxidative stress. In the present study, we recorded action potentials using the patch-clamp technique in ventricular myocytes isolated from rabbit hearts. The treatment of myocytes with H(2)O(2) (200 μM) prolonged AP durations and induced EADs, which were significantly suppressed by docosahexaenoic acid (DHA, 10 or 25 μM; n = 8). To reveal the ionic mechanisms, we examined the effects of DHA on L-type calcium currents (I(Ca.L)), late sodium (I(Na)), and transient outward potassium currents (I(to)) in ventricular myocytes pretreated with H(2)O(2). H(2)O(2) (200 μM) increased I(Ca.L) by 46.4% from control (-8.4 ± 1.4 pA/pF) to a peak level (-12.3 ± 1.8 pA/pF, n = 6, p < 0.01) after 6 min of H(2)O(2) perfusion. H(2)O(2)-enhanced I(Ca.L) was significantly reduced by DHA (25 μM; -7.1 ± 0.9 pA/pF, n = 6, p < 0.01). Similarly, H(2)O(2)-increased the late I(Na) (-3.2 ± 0.3 pC) from control level (-0.7 ± 0.1 pC). DHA (25 μM) completely reversed the H(2)O(2)-induced increase in late I(Na) (to -0.8 ± 0.2 pC, n = 5). H(2)O(2) also increased the peak amplitude of and the steady state I(to) from 8.9 ± 1.0 and 2.16 ± 0.25 pA/pF to 12.8 ± 1.21 and 3.13 ± 0.47 pA/pF respectively (n = 6, p < 0.01, however, treatment with DHA (25 μM) did not produce significant effects on current amplitudes and dynamics of I(to) altered by H(2)O(2). In addition, DHA (25 μM) did not affect the increase of intracellular reactive oxygen species (ROS) levels induced by H(2)O(2) in rabbit ventricular myocytes. These findings demonstrate that DHA suppresses exogenous H(2)O(2)-induced EADs mainly by modulating membrane ion channel functions, while its direct effect on ROS may play a less prominent role.

Biphenotypic acute leukaemia (BAL) represents about 5% of adult acute leukaemia. Based on a previously described scoring system, the European Group for Immunologic Classification of Leukaemia (EGIL) proposed a set of diagnostic criteria for BAL. This scoring system is based on the number and degree of the specificity of several markers for myeloid or T/B lymphoid blasts. Here, we report the case of a BAL with Burkitt-like cytology, corresponding to "the acute lymphoblastic leukaemia, Burkitt type" L3 for the FAB classification. By flow cytometry, the blasts showed a positivity for B lymphoid cytoplasmic (CD79a and mu) and membrane (CD19, CD22, CD24, IgM) markers AND a positivity for the myeloid (CD13, CD33, CD65, CD15) markers.

Whereas many studies have considered the role of attention in prospective timing, fewer have established relations between movement complexity and prospective timing. The present study aims at assessing to what extent motion complexity interferes with prospective timing and at delineating a neuropsychophysical plausible model. We have thus designed a visual paradigm presenting stimuli in sequential pairs (reference comparison interval). Stimuli are motionless or moving according to different complexities, and stimulus complexities are intermixed within each pair. To prevent a possible attention-sharing effect, no concurrent task was required. Our study suggests that movement complexity is a key component of duration perception, and that the relative judgement of durations depends on spatio-temporal features of stimuli. In particular, it shows that movement complexity can bias subjects' perception and performance, and that subjects detect that comparison intervals are longer than reference before their end. In the discussion, we advocate that the classical internal clock model cannot easily account for our results. Consequently, we propose a model for time perception, based on a parallel processing between comparison interval perception and the reconstruction of the reference duration.

ZAP-70 and CD38 expression can identify B-cell chronic lymphocytic leukemia with an inferior clinical outcome. Many groups have investigated the meaning of the expression of these two proteins and the correlation with the bad prognosis in B-CLL. But nobody has investigated the relation between the multidrug resistance mediated by Pgp overexpression (MDR1) and ZAP-70/CD38 coexpression. Forty-one untreated and stage A patients, either ZAP-70(+)CD38(+) or ZAP-70(-)CD38(-), were tested to determine the MDR1 status. MDR1 was observed in 41% of CLL ZAP-70(+)CD38(+) and in 37% of CLL ZAP-70(-)CD38(-). The difference was not significant (p = 0.745). Patients with ZAP-70 and CD38 positive CLL can not be candidates for MDR1 antagonists.

Background and Objectives: Zeta-associated protein 70 (ZAP-70), a member of the Syk family of protein tyrosine kinases, is normally expressed in T and NK cells. While little is known about ZAP-70 expression in normal human B cells, it has been reported that ZAP-70 is expressed in a subset of patients with chronic lymphocytic leukemia (CLL) with a poor prognosis. In this study, we examined the expression and phosphorylation status of ZAP-70 in B-lineage acute lymphoblastic leukemia (Blin-ALL)., Design and Methods: First, ZAP-70 protein expression was assessed by Western blotting and flow cytometry and ZAP-70 mRNA transcripts were analyzed by reverse transcription polymerase chain reaction (RT-PCR) on human precursor B cell lines. Experiments were then carried out on cells obtained from 18 patients with Blin-ALL and from normal human bone marrow., Results: ZAP-70 was constitutively expressed and phosphorylated on tyr319 in human precursor Blin-ALL cell lines as well as in primary B leukemic cells from all examined Blin-ALL patients with pro-B, pre-B and B phenotypes, but not in malignant myeloid cells. Importantly, analysis of normal human bone marrow revealed expression of ZAP-70 transcripts only in the CD34+ cell fraction (either CD19-CD10- or CD19+CD10+) but not in the CD34- cell fraction (CD19+sIgM- pre-B cells or CD19+sIgM+ immature B cells)., Interpretation and Conclusions: ZAP-70 was found to be expressed in the CD34+ normal bone marrow compartment including earlier B-cell progenitors, but not in CD34- pre-B and immature B cells. By contrast, ZAP-70 was consistently expressed and phosphorylated in Blin-ALL cells. Further studies are required to determine whether ZAP-70 may play a pathophysiological role in Blin-ALL.

Background: The disappearance of anti-HCV antibodies over time, after a self-limited infection, also referenced seroreversion, has been observed. The frequency of this phenomenon remains controversial, especially in immunocompetent subjects. However, it has important implications in the context of transfusion inquiries, in particular in case of a blood donor suspected to have transmitted HCV through a past blood donation., Study Design and Methods: Our findings are presented of a longitudinal study, including 16 patients from a cohort of 78 immunocompetent, multitransfused individuals who were positive for anti-HCV (EIA and confirmatory assay [RIBA]) and followed over a long period of time without having received any antiviral therapy. The aim was to establish whether a past and self-resolved HCV infection could evolve toward a negative serology., Results: The 16 patients were classified in three groups: 1) 12 patients who remained anti-HCV positive with no evolution in their RIBA pattern after a mean follow-up of 7.6 years; 2) one patient who presented a complete seroreversion 6 years after enrollment; and 3) three patients with a partial seroreversion over a mean follow-up of 16 years., Conclusion: HCV infection is not always characterized by a persistent antibody response, even in immunocompetent individuals. This should be taken into consideration when transfusion inquiries are conducted.

Background: The main objective of the implementation of NAT for the screening of blood-borne viruses was to compensate for the failure of serologic assays during the window period. Because this new screening procedure theoretically covers the entire period of infectivity, the necessity for maintaining serologic assays in blood screening strategy could become questionable., Study Design and Methods: To investigate this issue, a panel of 35 samples has been studied by NAT. These samples had been collected from HIV-1 antibody-positive individuals presenting a persistently low viral RNA load (<400 copies/mL) in the absence of antiviral therapy. All samples were analyzed with the minipool (x8) NAT routinely used in blood bank setting (HIV-1 and HCV assay based on transcription-mediated amplification) and with single-donation testing., Results: The minipool NAT failed to detect the presence of HIV RNA in 15 of the 35 samples (11 remained negative when retested). Single-donation testing gave negative results in 4 samples (3 remained negative when retested). Fourteen of the 18 samples with a viral load greater than 50 copies per mL were positive by minipool NAT versus 6 of the 17 samples with fewer than 50 copies per mL (p = 0.02)., Conclusion: The results clearly demonstrate that anti-HIV screening should not be withdrawn from biologic qualification procedures of blood donations, even when single NAT is performed.

Chlamydia trachomatis heat shock protein 10 (Chsp10) is associated with chronic genital tract infection with C. trachomatis. Chsp10 is homologous to human chaperonin 10 (Cpn10) and early pregnancy factor (EPF), a form of human Cpn10 that is specifically secreted at the start of pregnancy. We investigated cross-reactions between serum anti-Chsp10 antibodies and anti-EPF antibodies in pregnant and nonpregnant patients. Pregnancy was found to be associated with the presence of anti-EPF antibodies, which are specifically induced in pregnant women with a history of C. trachomatis infection, and with the presence of serum anti-Chsp10 antibodies. We also found that infertility was associated with the presence of anti-Chsp10 and anti-EPF antibodies. The HLA class II haplotype DR8 DQ4 was associated with the presence of anti-Chsp10 antibodies but not of anti-EPF antibodies.

The objective of this study was to describe the cytological and immunophenotypical parameters evocative of B-cell Chronic Lymphocytic Leukaemia (B-CLL) and their ability to participate to the differential diagnosis of other B-chronic lymphoproliferatives disorders with blood dissemination (B-CLD). Two groups of pathology included 92 patients, 79 patients had a B-CLL and the 13 other had a B-CLD (1 Prolymphocytic Leukaemia, 12 non- Hodgkin's Lymphoma in which 4 Splenic Lymphoma with Villous Lymphocytes or SLVL). The lymphoid morphology was studied on blood smear stained with May Gr nwald Giemsa and the immunophenotypical analysis was performed by flow cytometry. The 72 patients with B-CLL were characterized by a predominance of small mature lymphocytes with a Matutes's CLL score 3 (generally CD5+, CD23+, SmIg poor expression). 4 out of B-CLL with cleaved lymphocytes 5 % showed the same immunological characteristics than the typical B-CLL cases. 3 cases of B-CLL with prolymphocytes between 5 and 55 % showed in 2 cases an immunophenotyping compatible with the diagnosis of B-CLD. The presence of shadow cells of Gumprecht was highly evocative of B-CLL. In conclusion, the cytological analysis remains at the root of any diagnosis and can be sufficient in most cases of typical CLL with the presence of shadow cells of Gumprecht on the blood smear. In case of presence of cleaved lymphocytes, the immunophenotyping becomes essential to confirme the diagnosis of B-CLL. In prolymphocytic cases, the differential diagnosis between mixed CLL and B-CLD (especially Mantle Cell Lymphoma and Marginal Zone B-Cell Lymphoma without villous lymphocytes) needs a multidisciplinary approach (clinical, cytogenetical and histological).

Cathepsin L is a cysteine protease whose overexpression in human melanoma cells increases their tumorigenicity and switches their phenotype from non-metastatic to highly metastatic. Regulation of the transcription of the gene encoding human cathepsin L has not been yet studied and only preliminary data exist on the promoter regulation of the gene encoding rodent cathepsin L. In the present study we identified molecular elements involved in the transcriptional regulation of human cathepsin L in melanoma cells. The sequence of the 5'-flanking region of the gene encoding human cathepsin L was determined up to 3263 bp upstream of the translation start site. The major transcription intiation site was located. Three mRNA splice variants, differing in their 5' untranslated ends, were identified. Regulatory regions crucial for cathepsin L promoter activity were characterized between -1489 and -1646 bp. In this region, two GC boxes (-1590/-1595 and -1545/-1550) and a CCAAT motif (-1571/-1575) were involved in specific DNA-protein interactions. An electrophoretic mobility-shift assay demonstrated that Sp1 and Sp3 transcription factors bound to these GC boxes, and only the transcription factor nuclear factor Y (NF-Y) bound to the CCAAT motif. Mutagenesis studies demonstrated that these binding sites contributed at least 85% of cathepsin L promoter activity. Thus structural and functional analysis demonstrated that binding sites for NF-Y, Sp1 and Sp3 are essential for transcription of the gene encoding human cathepsin L in melanoma cells.

We previously demonstrated that highly metastatic human melanoma cells secrete a 41 kDa proteinase that cleaves C3, the third component of complement, and shares antigenic determinants with procathepsin-L. Thus, we herein transfected the nonmetastatic DX-3 melanoma cells with the procathepsin-L cDNA. Three clones expressing and secreting high levels of procathepsin-L were selected. Conditioned medium and whole cell extracts from these clones, but not from control cells, carried a high C3-cleaving activity. The transfected clones displayed up to 60% resistance to complement-mediated lysis. Overexpression of procathepsin-L in melanoma cells increased their tumorigenicity and switched their phenotype from nonmetastatic to highly metastatic cells. This is the first report that demonstrates that enforced expression of procathepsin-L by human melanoma cells arms them with the ability to inactivate complement-mediated lysis and contributes to tumor growth and metastasis.

The functional significance of the extracellular amino-terminal region and of three highly conserved aromatic residues present in the fifth (TM-V) and sixth (TM-VI) transmembrane domains of the rat cholecystokinin (CCK)B receptor, transfected in Cos-7 cells, was investigated by site-directed mutagenesis. The amino-terminal region of the CCKB receptor seemed to be weakly involved in CCK binding in that the affinities of CCK8 and selective agonists and antagonists were not modified by truncation of this region. Substitution of Phe347 in TM-VI with alanine produced a mutant receptor that displays the same affinity and selectivity as the wild-type receptor for agonists, but a slightly increased affinity for the selective CCKB antagonist L-365,260. However, the addition of saturating CCK8 concentrations to cells expressing this mutant did not result in the production of inositol phosphates, demonstrating the critical role of Phe347 in CCKB receptor to G protein coupling. Substitution of Phe227 with alanine was without effect on the affinities of CCKB ligands and on phosphoinositide turnover but modified the affinity of the CCKA antagonist L-364,718. Residue Trp351 located within the CCKB receptor TM-VI is involved in the binding of CCK8 and CCK4 and of the CCK4-based antagonist PD-134,308, as illustrated by the decreased affinities of these ligands in W351A mutant. The lower affinity for CCK8 observed with this mutated CCKB receptor accounts for the higher EC50 value for phosphotidylinositol hydrolysis. This study suggests that at least part of the binding site for the agonist is located inside the transmembrane domain of the CCKB receptor, partially overlapping that of antagonists, and gives new insights into the regions involved in the transduction process.

The study of various social parameters as risk factors for coronary diseases gives contradictory results. The inadequacy of social status and the consequent subjective misconcept could reconcile these apparent oppositions. The present research seems to confirm the fact that misconcepts induced by inadequacity of social status are more frequent and more intensive in patients with coronary troubles.