Search

Background Endothelial damage has been described in antiphospholipid antibody (aPL)-positive patients. However, it is uncertain whether circulating endothelial cells (CECs)—which are released when endothelial injury occurs—can be a marker of patients at high risk for thrombosis. Methods Ninety-seven patients with aPL and/or systemic lupus erythematosus (SLE) were included. CECs were determined by an automated CellSearch system. We also assayed plasma levels of tissue factor-bearing extracellular vesicles (TF+/EVs) and soluble triggering receptor expressed on myeloid cells 1 (sTREM-1) as markers of endothelial dysfunction/damage. Results Patients' mean age was 46.1 ± 13.9 years, 77 were women. Thirty-seven had SLE and 75 patients were suffering from antiphospholipid syndrome. Thirty-seven percent of patients presented a medical history of arterial thrombosis and 46% a history of venous thromboembolism (VTE). Thirteen patients had increased levels of CECs (>20/mL), with a mean CEC level of 48.3 ± 21.3 per mL. In univariate analysis, patients with obesity or medical history of myocardial infarction (MI), VTE, or nephropathy had a significant increased CEC level. In multivariate analysis, obesity (odds ratio [OR] = 6.07, 95% confidence interval [CI]: 1.42–25.94), VTE (OR = 7.59 [95% CI: 1.38–41.66]), and MI (OR = 5.5 [95% CI: 1.1–26.6)] were independently and significantly associated with elevated CECs. We also identified significant correlations between CECs and other markers of endothelial dysfunction: sTREM-1 and TF+/EVs. Conclusion This study demonstrated that endothelial injury assessed by the levels of CECs was associated with thromboembolic events in patients with aPL and/or autoimmune diseases.

Several techniques have been developed to detect circulating tumor cells (CTC) in patients with head and neck squamous cell carcinoma (HNSCC), but their diagnostic and prognostic value are not yet fully established. A computerized retrieval of literatures was conducted without time restrictions using the electronic database in December 2014. Diagnostic accuracy variables were pooled and analyzed by the Meta-DiSc software. Engauge Digitizer and Stata software were used for pooled survival analysis. Twenty-two retrieved studies were eligible for systematic review, of which 9 conformed for the diagnostic test meta-analysis and 5 for the prognostic analysis. Subgroup analysis showed 24.6% pooled sensitivity and 100% pooled specificity of detections by using positive selection strategy, which moreover presented low heterogeneity. The presence of CTC was significantly associated with shorter disease free survival (DFS, HR 4.62, 95% CI 2.51–8.52). In conclusion, current evidence identifies the CTC detection assay as an extremely specific, but low sensitive test in HNSCC. Also, the presence of CTC indicates a worse DFS.

Objectives The diagnosis of solid cancer leptomeningeal metastasis (LM) relies on the cytology of cerebrospinal fluid (CSF) and/or imaging evidence of neuraxis, yet both lack sufficient sensitivity. The utility of the CellSearch ® , an FDA -approved technology, in assessing CSF tumor cell (CSFTC) was evaluated here in the diagnosis and treatment of patients with lung cancer-related LM. Materials and methods In 18 patients with magnetic resonance imaging (MRI) confirmed LM due to lung cancer, 5mL of CSF were collected in CellSave ® preservative tubes, which allow performing the assay within 96h after sampling. Using a previously adapted CellSearch ® method, we detected, visualized and enumerated CSFTCs and compared the results with conventional cytology. In 3 patients, tumor cells were evaluated sequentially to explore the predictive role of CSFTCs enumeration in the treatment response monitoring. Results CSFTCs were disclosed in 14 of 18 MRI confirmed LM samples (median 785CSFTCs/5mL CSF, range 1 to >20,000), yielding a sensitivity of 77.8%, compared with 44.4% for conventional cytology. CSFTC clusters were observed in 12 patients, similar to those previously described in blood as circulating tumor microemboli (CTM), and enumerated sequentially with reproducible results, which did not necessarily correlate with response to treatment. Conclusion The CellSearch ® technology, applied to limited sample volumes and allowing delayed processing, could be of great interest in the diagnosis of LM in lung cancer patients.

The triggering receptor expressed on myeloid cell type 1 (TREM-1) is a cell surface molecule that has been identified on both human and murine polymorphonuclear neutrophils and mature monocytes. The activation of TREM-1 in the presence of microbial components amplifies the inflammatory response and may be responsible for the hyperresponsiveness observed during the initial stage of sepsis. To investigate the effect of the modulation of the TREM-1 pathway during experimental murine sepsis, we used analogue synthetic peptides derived from the extracellular moiety of TREM-1. The TREM-1 ligand was expressed on both peritoneal and peripheral neutrophils during experimental peritonitis in mice. The TREM-1 peptides inhibited the recognition by TREM-1 of its ligand and protected endotoxinic mice from death. In septic rats, the TREM-1 peptides improved the hemodynamic status, attenuated the development of lactic acidosis, modulated the production of such proinflammatory cytokines as tumor necrosis factor alpha and interleukin-1β, and improved survival. The protective effect of these peptides on arterial pressure could partly be explained by a decreased production of nitric oxide. These data suggest that in vivo modulation of TREM-1 might be a suitable therapeutic tool for the treatment of sepsis. Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Background After undergoing heart transplantation and the subsequent compulsive immunosuppressive treatments, patients are at risk of rejection episodes, infectious complications or cancer development. Thus, it is probable that the various subsets of peripheral cytotoxic lymphocytes are modulated in such patients. This area of study can now be investigated by examining the numerous recently described natural killer (NK)-cell–related surface receptors. Methods A prospective cohort of 60 heart transplant recipients and 60 controls was studied. The partitioning of lymphocyte subsets, especially NK (CD3 − /CD56 + ), T (CD3 + /CD56 − ) and NKT-like (CD3 + /CD56 + ) cells, was compared in both groups using multi-parametric flow cytometry. Moreover, expression of a series of seven NK-related receptors was compared on the three subsets defined by CD56 expression. Results A significant increase in NK-cell levels was observed in transplanted patients, as compared with controls, whereas T and NKT-like cells were in similar proportions in both groups. Two NK-related receptors showed significantly different levels of expression in heart transplant recipients: the cytotoxic effector, CD244, which was in a significantly increased proportion on T and NKT-like cells; and the activating receptor, CD161, which was expressed significantly less on NK and NKT-like cells, but more on T cells. Conclusions These findings indicate that cytotoxic NK-related cells, increased in proportion, also display increased levels of activity-associated markers in heart transplant recipients. Viral infection or the immunosuppressive regimen could be responsible for the modulation of regulatory receptors on NK and NKT-like cells in heart transplant recipients.

Resume Les chloramines, sous-produits de desinfection de l'eau des piscines, ont ete incriminees dans des manifestations d'irritation des muqueuses chez les maitres nageurs et nageurs de haut niveau. Par ailleurs, l'entrainement sportif est susceptible d'avoir des repercussions sur le systeme immunitaire. La conjugaison de l'exposition aux chloramines et d'un entrainement intensif pourrait donc augmenter les risques d'anomalies immunologiques chez ces sportifs. Nous avons donc realise une etude en eau neuve et en eau usagee, avant et apres une periode d'entrainement chez 19 nageurs. Des prelevements sanguins ont ete realises afin d'evaluer les sous-populations lymphocytaires et leur etat d'activation. Les chloramines ayant ete impliquees dans des lesions de l'ADN, nous avons evalue l'apoptose des cellules circulantes par l'etude du cycle cellulaire. Pour apprecier l'impact sur le systeme immunitaire muqueux, des prelevements salivaires ont ete recueillis pour mesurer le taux d'IgA et les IgA specifiques de quatre germes frequemment responsables d'infections ORL. L'entrainement en eau neuve n'engendre aucune variation significative des parametres evalues. L'exposition a l'eau usagee entraine une diminution significative des proportions de lymphocytes B actives dans le sang peripherique. L'effet combine de l'entrainement et de l'exposition se traduit par une augmentation des lymphocytes T CD4+ et CD8+. En revanche, aucune anomalie du cycle cellulaire n'a ete constatee, en particulier il n'a pas ete detecte d'augmentation du nombre de cellule apoptotique. Ce travail indique que l'exposition aux chloramines a des effets essentiellement muqueux se traduisant non pas par des variations des IgA secretoires mais par des modifications de la recirculation des cellules impliquees dans les reponses immunitaires muqueuses.

To analyze the pattern of cell-surface expression of the triggering receptor expressed on myeloid cells (TREM) 1 during septic shock.Prospective clinical study in an adult 16-bed medical ICU.25 septic shock patients, 15 patients with shock of noninfectious origin and 7 healthy volunteers. Arterial blood was drawn within 12 h of admission and subjected to flow cytometry analysis after staining with anti-TREM-1 and anti-CD14 antibodies. Repeated sampling was performed on days 2, 3, 5, 7, and 14 in septic shock patients.Monocytic TREM-1 expression was significantly higher in septic shock patients (mean fluorescence intensity 2.3+/-0.2) than in nonseptic patients (1.0+/-0.1), and healthy volunteers (1.0+/-0.1). There was no difference in monocytic TREM-1 expression between nonseptic patients and healthy volunteers or between any of the three groups with respect to TREM-1 expression on neutrophils. The time course of TREM-1 expression on monocytes diverged significantly by day 3 between survivors and ns.The specificity of TREM-1 regulation by infection is highlighted. Moreover, surface TREM-1 expression on monocytes may prove useful in allowing the follow-up of septic patients during the course of the disease.

Background Quality assessment in flow cytometry cannot obey the same rules as those applicable to the measurement of chemical analytes. However, regular follow-up of known patients may provide a robust in-house control of cell subsets evaluation. Methods Sequential blood samples assessed for 32 HIV patients over several years and showing good stability were retrospectively assessed to establish coefficient of variations of the percentages of CD3+, CD4+, CD8+ cells, and CD4+ absolute counts (ACs). Results Mean relative standard variations for the whole cohort were of 0.04, 0.14, 0.08, and 0.18 for CD3%, CD4%, CD8%, and CD4 ACs, respectively. Discussion In-house follow-up of regularly checked compliant patients is a good alternative to traditional and costly repeatability and reproducibility studies for the validation of routine flow cytometry. © 2013 International Clinical Cytometry Society

Background Tetraspans are ubiquitous integral transmembrane molecules associated on the cell surface with such adhesion molecules as integrins. Their expression has been shown to vary in tumors, but has seldom been described on lung tumoral epithelial cells, and the conditions required for a proper association of tetraspans and integrins have not yet been fully explored. Methods We investigated the expression of 10 tetraspans and six β1 integrins on the tumoral lung epithelial cell line A549. Cells were examined both in quantitative flow cytometry and as monolayers, under normal or chelated conditions, in order to determine the cation dependency of their expression. Results Five tetraspans and four β1 integrins are expressed on the membrane of A549 cells. Both quantitative and qualitative surface and cytoplasmic modifications of this pattern were induced in chelating conditions, suggesting the importance of divalent cations for the expression of these molecules. Conclusions These data indicate that a specific pattern of tetraspans and integrins, relying strongly on the availability of divalent cations in the microenvironment, is expressed by tumoral epithelial cells. © 2004 Wiley-Liss, Inc.

Resume Les pathologies associees a l'usage du tabac sont nombreuses et pour certaines bien connues des medecins et du public, en particulier les cancers des interfaces epitheliales bronchiques, ORL et vesicales, mais d'autres pathologies a composante immunitaire lui sont aussi reliees. Les mecanismes physiopathologiques reliant exposition tabagique et pathologies sont paradoxalement encore mal connus, en particulier ceux concernant le systeme immunitaire et plus encore ceux mettant en jeu le systeme immunitaire muqueux. Cet article fait une revue des connaissances actuelles sur ce sujet sensible et de grande importance en Sante publique. Au niveau de l'immunite systemique, l'exposition tabagique induit un deficit immunitaire acquis, au moins partiel, qui peut favoriser le developpement de phenomenes d'hypersensibilite et de pathologies auto-immunes. En ce qui concerne le systeme immunitaire muqueux, l'exposition a la fumee de tabac induit des anomalies acquises de l'immunite innee aussi bien que de l'immunite adaptative, auxquelles s'ajoutent des phenomenes inflammatoires locaux.

A nephelometric immunoassay was developed to quantify immunoglobulin A (IgA) in children's stools. This method enables IgA in faecal protein extracts to be measured over a large range of concentrations (1.61–51.50 mg/L) with good accuracy (linear recovery in dilution‐overloading assay) and precision (within‐ and between‐run coefficients of variation (CVs) of 1–6%). An excellent recovery (105%) was obtained in stool samples overloaded by purified colostral IgA, demonstrating that the method used for faecal IgA extraction is adapted, not to induce significant IgA degradation, and probably allow a complete extraction of IgA. The amount of faecal IgA, as determined in stool samples from 125 children (6–24 months old), was an average of 14 mg per 100 g of stools (about 10% of the total protein stool content), with large individual variation (3–30 mg per 100 g of stools). No correlation was observed between faecal IgA amounts and the children's age or sex. Such an immunoassay could enable exhaustive noninvasive investigations of the maturation of the intestinal immune system, as well as accurate studies of the effect of oral dietary supplementation on IgA regulation. J. Clin. Lab. Anal. 18:195–199, 2004. © 2004 Wiley‐Liss, Inc.

The triggering receptor expressed on myeloid cells (TREM)-1 is a recently discovered receptor expressed on the surface of neutrophils and a subset of monocytes. Engagement of TREM-1 has been reported to trigger the synthesis of proinflammatory cytokines in the presence of microbial products. Previously, we have identified a soluble form of TREM-1 (sTREM-1) and observed significant levels in serum samples from septic shock patients but not controls. Here, we investigated its putative role in the modulation of inflammation during sepsis. We observed that sTREM-1 was secreted by monocytes activated in vitro by LPS and in the serum of animals involved in an experimental model of septic shock. Both in vitro and in vivo, a synthetic peptide mimicking a short highly conserved domain of sTREM-1 appeared to attenuate cytokine production by human monocytes and protect septic animals from hyper-responsiveness and death. This peptide seemed to be efficient not only in preventing but also in down-modulating the deleterious effects of proinflammatory cytokines. These data suggest that in vivo modulation of TREM-1 by sTREM peptide might be a suitable therapeutic tool for the treatment of sepsis.

Resume Les anticorps monoclonaux, decouverts par Kohler et Milstein en 1975, ont trouve de nombreuses et utiles applications. La plus recente est leur utilisation en therapeutique, dans des pathologies pour lesquelles des mecanismes physiopathologiques immunologiques sont etablis ou suspectes. Bloquer la synthese de cytokines pour limiter une reaction inflammatoire, diminuer voire arreter la proliferation de cellules malignes ou les eliminer specifiquement, inhiber l'agregation plaquettaire, neutraliser une toxine, prevenir une invasion virale, eviter la degranulation mastocytaire sont autant d'applications deja largement engagees, mais beaucoup restent encore a developper. Cet article constitue une revue des anticorps monoclonaux beneficiant d'une autorisation de mise sur le marche et des perspectives therapeutiques en cours d'etude.

The ovary can be the target of an autoimmune disease involving many different autoantigens. The clinical feature of this disease often results in premature ovarian failure or infertility and may be either isolated or associated with other autoimmune pathologies, especially with adrenal autoimmunity. The diagnosis of an autoimmune mechanism relies on the presence of anti-ovarian antibodies, whose prevalence is quite variable according to the different methods used to detect them, and to the different stages of the disease. In addition, their clinical significance is not always clear, as to their pathologic or epiphenomenal nature. However, the study of these autoantibodies has led to the identification of some of their antigenic targets which have to be known for a better understanding of the pathologic mechanisms involved. This paper reviews anti-steroid producing cells, anti-gonadotrophin receptor, anti-gonadotrophin, anti-corpus luteum, anti-zona pellucida and anti-oocyte antibodies.

Immunophenotyping is a major tool to assign acute leukemia blast cells to the myeloid lineage. However, because of the large heterogeneity of myeloid-related lineages, no clinically relevant immunological classification of acute myeloblastic leukemia (AML) has been devised so far. To attempt at formulating such a classification, we analyzed the pattern of expression of selected antigens, on blast cells collected at AML diagnosis. Patients were eligible if they had a first diagnosis of de novo AML and a sufficient number of blast cells for proper immunophenotyping. The relative expression of CD7, CD13, CD14, CD15, CD33, CD34, CD35, CD36, CD65, CD117, and HLA-DR were analyzed by cytometry in a test series of 176 consecutive AML cases. Statistical tools of clusterization allowed to remove antigens with overlapping distribution, leading us to propose an AML classification that was validated in a second AML cohort of 733 patients. We identified five AML subsets (MA to ME) based on the expression of seven antigens within four groups (CD13/CD33/DC117, CD7, CD3!5/CD36, CD15).-MA and MB-AML have exclusively myeloid features with seldom extramedullary disease and rare expression of lymphoid antigens. No cases of acute promyelocytic leukemia (APL) were observed within MB AML. MC AML have either myeloid or erythroblastic features. MD AML have more frequently high WBC counts than other subsets, which were related to the expression of CD35/CD36 and CD14 and to monoblastic differentiation. ME AML lack CD13, CD33, and CD117 but display signs of terminal myeloid differentiation. Specific independent prognostic factors were related to poor overall survival in each immunological subset: CD34+ (P

Immunoglobulin A (IgA), immunoglobulin A1 (IgA1) and immunoglobulin A2 (IgA2) subclasses, κ and λ immunoglobulin light chains, C3 and C4 complement fractions, and Mannan Binding Lectin were assayed using nephelometric immunoassays in milk samples collected from 79 mothers during the first 12 weeks of lactation. The data obtained provide a precise description of the dynamics of immune proteins in human milk evolution over time and allow to depict four periods. In the early post-partum period (days 1–4), the high colostrum concentration of all immune proteins rapidly decreases. The IgA2 subclass, λ light chain, and C3 are more concentrated than IgA1, κ light chain and C4, respectively. The relative concentrations of C3 and C4 complement fractions are reversed during the following period (days 5–12). In a third stage extending from day 13 to day 44 post-partum, the levels of total IgA, IgA1, IgA2, κ, λ, C3, C4 and Mannan binding lectin (MBL) still slightly decrease or remain stable, but the respective ratios of immunoglobulin light chains and of IgA subclasses reverse consecutively. During the days 45–84 further and last period, an increase of IgA, of the IgA1/IgA2 ratio and of immunoglobulin light chains is noted, contrasting with stability or slight persistent decrease of C3, C4, and MBL concentrations. These data suggest that the concentrations of immune components in human milk are finely tuned during lactation, in a highly specific fashion, likely to best suit the newborn's need and properly protect the lactating mammary gland.

Choroid plexus tissues are intraventricular structures composed of villi covered by a single layer of ciliated, cuboid epithelium. The plexuses secrete cerebrospinal fluid (CSF), synthesize numerous molecules, carry nutrients from the blood to CSF, reabsorb brain metabolism by-products and participate in brain immunosurveillance. During ageing, atrophy of epithelium occurs along with thickening of basement membranes. Enzymatic activities of epithelial cells decrease significantly. CSF secretion decreases as much as 50%. These modifications are concurrent with subnormal brain activity. In Alzheimer's disease, epithelial atrophy, thickening of basement membrane and stroma fibrosis are even more prominent. Ig and C1q deposition along the basement membrane can be frequently detected, suggesting immunological processes. Synthesis, secretory, and transportation functions are significantly altered resulting in decreased CSF turnover, reduced beta-amyloid clearance, and increased glycation phenomena as well as oxidative stress. Such modifications may favour fibrillary transformation of beta-amyloid protein and tau protein polymerisation.

The mannan binding lectin (MBL) activates the complement system by the lectin pathway after the recognition of some structural motifs (saccharides) present on the surface of microorganisms. MBL has been mostly identified and quantified in human serum by ELISA or microparticle immunonephelometry assays. This article reports the MBL levels as assessed by a microparticle immunonephelometric assay in 76 human milk samples. Immunonephelometry was performed using skim-milk samples diluted 20 times over a calibration range of 0.07–4.82 mg/L. MBL is indeed present in human milk and its concentration decreases significantly during development from colostrum (0.55±0.09 mg/L) to transitional (0.18±0.02 mg/L) and mature milk (0.17±0.02 mg/L). This innate molecule may be involved in the primary defenses of the mammary gland and the neonate, whose immune system is immature. The high levels observed during the first days of lactation support the hypothesis that this molecule plays a key role in limiting the colonization of the newborn gut by pathogens. J. Clin. Lab. Anal. 16:304–307, 2002. © 2002 Wiley-Liss, Inc.

The purpose of this study was to identify autoantigens contained in human ovary extracts. Serum samples from 36 infertile women with anti-ovary antibodies as detected with an ELISA technique were tested in Western blot against human ovary extracts. A reactive protein with a molecular mass matching that of the FSH was detected in 34 cases. These serum samples also reacted strongly in Western blot and ELISA with purified FSH and, in immunofluorescence, with pituitary cells. Using the Pepscan approach, with overlapping peptides matching the amino acid sequence of the human FSH beta-chain, several immunoreactive regions were evidenced. The 78-93 amino acid sequence of the human FSH beta-chain appeared as one of the major epitopes. Synthetic peptides of this region were prepared and demonstrated to react with human serum samples from women with anti-ovary antibodies. These data demonstrate that FSH can be an autoantigen, recognized by autoantibodies associated with infertility.

Resume Les interfaces epitheliales muqueuses, indispensables aux relations de l'organisme avec son environnement digestif, respiratoire, reproductif, sont defendues par un systeme immunitaire apparaissant de plus en plus elabore et controle. Il comporte une participation efficace des cellules et des molecules de l'immunite innee, ensemble physiologique de connaissance recente, mais phylogenetiquement tres ancien. Les muqueuses sont d'autre part colonisees par des cellules immunitaires (cellules presentatrices d'antigene, lignees lymphocytaires T, B), mobiles entre un site inducteur principal, les plaques de Peyer, et des sites effecteurs diversifies et produisant des molecules effectrices et regulatrices originales (IgA secretoires, cytokines, chemokines). Cet ensemble complexe est physiologiquement particulierement controle et reste habituellement silencieux. Il peut neanmoins etre le siege de pathologies specifiques et commence a pouvoir etre explore biologiquement. Le progres des connaissances fait esperer des modalites therapeutiques nouvelles a impact muqueux.

IgA, IgA1, and IgA2 concentrations were determined in 81 defatted human milk samples: colostrum (days 1-5, n = 42), transitional milk (days 6-14, n = 18) and mature milk (days 15-75, n = 21) by immunonephelometry. Correlations were found between total IgA levels and the concentrations of both IgA subclasses (P < 0.0001). The levels of the three molecules decreased over lactation with significant differences (P < 0.05) between colostrum and transitional milk levels and between colostrum and mature milk. Colostral IgA1 and IgA2 mean concentrations dropped respectively from 10.89 +/- 2.12 g/L, and 15.41 +/- 2.10 g/L to 1.83 +/- 0.73 g/L and 3.40 +/- 1.25 g/L in transitional milk reaching finally to 0.36 +/- 0.07 g/L and 0.27 +/- 0.06 g/L in mature milk. IgA2 concentrations were higher than those of IgA1 when the total IgA level was high. The IgA2 levels in colostrum could be an adaptation resistance of IgA to potentially harmful pathogens able to secrete IgA proteases and also a way to regulate colonization of the microflora in the newborn.

Resume Objectifs – Comprendre les mecanismes conduisant a la survenue tres frequente d'infections ORL chez les sportifs de haut niveau. Actualites – La survenue d'infections ORL suggere une baisse des defenses immunitaires locales, voire systemiques. Une telle immunosuppression peut resulter d'une part de l'entrainement physique lui-meme, mais egalement du stress lie a la competition. Cette revue de la litterature rappelle les moyens de defense immunitaire physiologiques de la sphere ORL, puis envisage les repercussions du stress et du sport respectivement sur le systeme immunitaire. Perspectives – Une meilleure connaissance des modifications du systeme immunitaire induites par la pratique de sports de haut niveau peut permettre une amelioration des conditions d'entrainement et de competition, visant a pallier les episodes d'immunosuppression accroissant la susceptibilite des sportifs aux infections.

Objective.Screening for latent tuberculosis infection (LTBI) is mandatory before initiating biologics in patients with chronic inflammatory arthritis (CIA). However, few studies have evaluated the discrepancies between the results of tuberculin skin test (TST) and interferon-γ release assays (IGRA) in these patients. The purpose of our study was to investigate factors associated with TST and IGRA results in a large cohort of patients with CIA before the introduction of biologics.Methods.A total of 563 consecutive patients with CIA (293 rheumatoid arthritis, 270 spondyloarthritis) and eligible for biologics were prospectively enrolled. Demographic, clinical, and biological data were recorded. Risk factors for LTBI were assessed. All patients underwent a TST, a chest radiograph, and an IGRA test (T-SPOT.TB).Results.Agreement between the 2 tests was low (κ = 0.16). The bacillus Calmette-Guerin (BCG) status was significantly associated with discordance between the 2 tests (p = 0.004). The TST positivity rate was 34.8%. Factors associated with a negative TST were female sex (p = 0.02) and immunosuppressive treatment (p = 0.003). The only LTBI risk factor associated with TST positivity was an abnormal chest radiograph (p = 0.02). T-SPOT.TB was positive in 21.7% of patients and indeterminate in 15.6%. Previous active TB and chest radiograph abnormalities were associated with IGRA positivity (p = 0.008 and p = 3.9 × 10−5, respectively). The BCG vaccination was associated with negative IGRA (p = 3 × 10−4). Indeterminate IGRA results were associated with age, C-reactive protein, and immunosuppressive treatment (p = 0.005, 0.007, and 0.004, respectively).Conclusion.Our data support the combined use of T-SPOT.TB and TST in patients with CIA before biologics introduction. However, despite these good diagnostic values, indeterminate results may complicate the use of IGRA.

The diagnosis of pulmonary sarcoidosis relies in part on the observation of alveolar CD4+ lymphocytosis. However, this criterion is not fully discriminative because this anomaly is also found in other types of lung diseases. Among other possible distinctive criteria, we investigated the expression of lymphocyte-addressing molecules, which could differ according to the pathophysiology of lung diseases. We investigated CD103 (alpha(E)beta7 integrin, CD103-beta7), reported to be both expressed on intra-epithelial lymphocytes in mucosal areas, including bronchi, and possibly involved in the recruitment of alveolar lymphocytes. The expression of CD103 was examined on bronchoalveolar lavage lymphocytes from 93 consecutive patients, including 34 patients with CD4+ lymphocytosis. For all patients, the expression of CD19, CD3, CD4, CD8, CD57, LFA1, DR, and CD103 was assessed by flow cytometry. Sarcoidosis seemed remarkably characterized by the lack of CD103 expression on the predominant CD4+ subset. Statistically significant differences were found between patients with sarcoidosis, with other types of CD4+ lymphocytosis, and with other lung disorders in the CD103+ cell levels and in the CD103/CD4 ratio. Combined use of the CD4/CD8 ratio (> 2.5) and the CD103/CD4 ratio (< 0.31) to assess bronchoalveolar lavage lymphocytes is a promising new tool for the diagnosis of sarcoidosis.

Microparticle-enhanced nephelometric immunoassays were specifically developed for the quantification ofα -lactalbumin, β -casein, serum albumin, lactoferrin, and lysozyme in human milk. These components, immunoglobulin A and total proteins were assayed in 780 samples collected from 79 mothers during the first 12 weeks of lactation. The data obtained provide a precise description of the dynamics of human milk evolution over time and allow to depict six periods. In the early post-partum period (days 1–4), colostrum contains principally immunological components. The relative concentrations of immunologically and nutritionally available proteins are reversed during the following short transition period (days 5–8). Milk then secreted (days 9–18 and 19–28) appears to have principally a nutritious function. During the two following periods (days 28–49 and 50–84), milk appears well balanced in its nutritional and immunological components, supplying breast-fed infants with appropriate food intake for growth and development and passively protecting them by reincreasing concentrations of immunological components.

The levels of complement fractions C3 and C4 were assayed in human milk in a classic nephelometric assay adapted to this secretion. Concentrations of these molecules were measured in 667 milk samples obtained sequentially from 76 volunteer lactating mothers during the first 12 weeks of lactation. Immunonephelometry was performed using skimmed milk samples diluted 10 times and yielded reproducible (coefficients of variation in within- and between-run precision lower than 9% for C3 and than 14% for C4) and accurate (linear recovery in dilution-overloading assay) data. High concentrations (mean ± SE) were found for C3 (199.32 ± 16.35 mg/L) and C4 (113.42 ± 11.16 mg/L) in colostrum samples (n = 159; days 1–5). A significant (P < 0.001) and rapid decrease was observed in transitional milk samples (n = 198; days 6–14), containing 57.71 ± 5.18 and 72.39 ± 4.98 mg/L of C3 and C4, respectively. Stable lower levels were noted in mature milk samples (n = 310; days 15–84) at 30.36 ± 1.57 mg/L for C3 (P < 0.001) and 53.38 ± 3.61 mg/L for C4 (P < 0.05). The decrease rate was different for C3 and C4, yielding a reversal of the C3/C4 ratio between colostrum and more mature milk.

The enumeration of lymphocyte subsets in absolute counts has long relied on different methods applied separately to whole blood cell count, lymphocyte differential appreciation, and flow cytometric evaluation of lymphocyte subsets percentages. The development of multicolor labeling methods inflow cytometry now allows a more homogeneous appreciation of several cell subsets among gated lymphocytes. The use of internal calibrators, such as microbead suspensions, also permits a direct appreciation of subsets in absolute counts in a single-platform method. These methods were compared with a traditional multiplatform method of assessing absolute counts of lymphocyte subsets in a pilot study in which all manipulations were performed by 1 person and in a full-scale larger study performed in the normal working conditions of a hospital laboratory. Microspheres seem to be a reliable tool to perform absolute count enumeration inflow cytometry, but several precautions in the sample preparation and flow cytometric analysis are required.

Quantitation of lysozyme in human milk was performed by a microparticle-enhanced nephelometric immunoassay based on the measurement of the light scattered during the competitive immunoagglutination of a microparticle–lysozyme conjugate with an anti-lysozyme antiserum. This immunoassay has a detection limit of 8 μg/L of reaction mixture and can be performed using diluted milk (1:6000, in reaction mixture), excluding sample pretreatment. Human milk lysozyme can be quantified over the concentration range 0.09–1.50 g/L, with within- and between-run coefficients of variation

Little is known of the maturation of the mucosae-associated lymphoid tissue (MALT) in man, because, for ethical reasons, tissues from newborns are not easy to obtain. We used the opportunity provided by autopsies systematically performed in infants who died of Sudden Infant Death Syndrome (SIDS) to study the maturation of the MALT after birth. Gut and bronchus samples of 90 infants from postpartum to 90 months and who died from SIDS were collected and studied by histological and immunofluorescence examination. Plasma cells, absent at birth, appeared within a few hours after birth and initially were of the IgM isotype. IgA plasma cells appeared at 12 days. These cells were first observed in gut and later in bronchi, indicating that maturation of the gut precedes that of bronchi. The number of plasma cells increased rapidly over time and IgA plasma cells became predominant after 3 weeks in the gut and 6 weeks in bronchi. At birth, only small IgM bearing B-cell foci were seen and organized germinal centers appeared to develop over a few days, first in the gut and only later in bronchi. These results confirm that, in man, the MALT organization at birth is still in its fetal form and that maturation depends on intestinal challenges and evolves over several weeks before IgA becomes the predominant isotype secreted.

This study prospectively analysed the relationships between immunophenotypic and cytogenetic features of blast cells in 432 acute non-lymphoblastic leukemias (ANLL) at presentation. An abnormal karyotype was detected in 232 cases (54%). These abnormalities were related to immunophenotypic markers as detected using a consensual panel of monoclonal antibodies allowing lineage assignment and investigation of myeloid marker expression on blast cells. In univariate analysis, CD9, CD10, CD15, CD34 and TdT expression appeared significantly associated with chromosomal anomalies. Multivariate analysis identified CD34 and CD9 expression as independently predictive of the presence of at least one cytogenetic abnormality (P < 10(-4) and P < 0.03, respectively). Significant associations between immunophenotypic and karyotypic features were observed both within individual FAB subgroups and independently from morphological criteria. Specific features were seen in five ANLL entities: M0 or M1/B lineage antigen positivity/t(9;22) or del(11)(q23); M2/CD13-/t(8;21); M4/CD13+, CD34+, CD36+/inv(16); M4 or M5/lack of B lineage antigen/del(11)(q23) or t(9;11). More practically, and although the relationships demonstrated only represent a fraction of homogeneous immunophenotypic subgroups, identification of such immunophenotypic features should prompt careful karyotypic examination, eventually using molecular biology analysis on non-growing cells.

The pathognomonic presence of IgA in the glomerular mesangium of patients with IgA nephropathy (IgAN) suggests an abnormal control of IgA metabolism in this disease that could be modified in transplanted IgAN patients. Alterations of IgA production have been demonstrated in both bone marrow samples and mucosae-associated tissues from IgAN patients, but the analysis of IgA production by peripheral B lymphocytes in culture has yielded conflicting results, some investigators showing an enhanced secretion of IgA and others reporting similar data as with control samples. Little is known about the endogenous activation state of B cells in IgAN, which can be approached by analyzing peripheral Ig-producing cells, a number of which are recirculating between lymphoid organs. In the present study, two methods were applied to appreciate the numbers of spontaneously activated peripheral B cells. The ELISPOT method provided information about short-term secretion of Igs. Intracytoplasmic immunofluorescence for IgA allowed to identify IgA-containing cells, either as short-rimmed immunocytes or as brightly stained immunoblasts with an enlarged cytoplasm. These cells were enumerated in IgAN patients (n = 31), transplanted IgAN patients (n = 27), control patients with other biopsy-proven renal diseases (nontransplanted, n = 48; transplanted, n = 38), and in healthy individuals (n = 18). The number of IgA spot-forming cells obtained in the control groups (1,251 +/- 95 cells/10(6) lymphocytes [mean +/- SE]) was consistent with those in similar previously reported studies, but differed significantly (P = 0.01) from those observed in nontransplanted IgAN patients, who had a surprisingly lower number of such cells (699 +/- 97 cells/10(6) lymphocytes); the number of IgA spot-forming cells in the transplanted IgAN patients (1,355 +/- 182 cells/10(6) lymphocytes) did not differ from that in the control groups. The same pattern was seen for IgA-containing immunoblasts. There was no difference in IgG (overall, 214 +/- 13 cells/10(6) lymphocytes) and IgM (overall, 61 +/- 10 cells/10(6) lymphocytes) spot-forming cell numbers between the five groups of individuals tested, suggesting that the anomaly noted in IgAN patients was not related to technical problems and that this could be a new feature of this renal disease. The normal levels of spontaneously activated peripheral IgA-producing cells found in transplanted IgAN patients suggest that immunosuppressive treatments could interfere with the anomalies of IgA metabolism in this disease.

Activating mutations of TSH-R have been described in toxic nodules and more recently in familial nonautoimmune thyrotoxicosis. This last entity is still confused with familial Graves' disease and the aim of this study is to define its phenotype. Based on 49 patients coming from our first family and on the 4 other kindreds secondarily described in the literature, the phenotypic expression is: a high incidence of hyperthyroidism, an early onset of disease, a higher men/women ratio (17/32) than in Graves, disease, the absence of ophthalmopathy and of circulating and intrathyroid signs of immunity, a pathology similar to toxic nodule, the need for a total destruction of thyroid tissue to cure the patients. The total analogy with toxic nodule leads us to name this new entity "toxic hyperplasia'. Among 92 successive diffuse nonfamilial thyrotoxicosis cases (initially considered as Graves) we isolated 5 cases without extra- and intrathyroidal autoimmunity, raising the question of the existence of an apparent "sporadic' form of toxic hyperplasia (neomutation?).

Background. Quality assessment in flow cytometry cannot obey the same rules as those applicable to the measurement of chemical analytes. However, regular follow-up of known patients may provide a robust in-house control of cell subsets evaluation. Methods. Sequential blood samples assessed for 32 HIV patients over several years and showing good stability were retrospectively assessed to establish coefficient of variations of the percentages of CD3+, CD4+, CD8+ cells and CD4+ absolute counts. Results. Mean relative standard variations for the whole cohort were of 0.04, 0.14, 0.08 and 0.18 for CD3%, CD4% CD8% and CD4 absolute counts respectively. Discussion. In-house follow up of regularly checked compliant patients is a good alternative to traditional and costly repeatability and reproducibility studies for the validation of routine flow cytometry. © 2013 Clinical Cytometry Society.

The progress of information and communication technologies has strongly influenced changes in healthcare and laboratory medicine. E-learning, the learning or teaching through electronic means, contributes to the effective knowledge translation in medicine and healthcare, which is an essential element of a modern healthcare system and for the improvement of patient care. E-learning also represents a great vector for the transfer knowledge into laboratory practice, stimulate multidisciplinary interactions, enhance continuing professional development and promote laboratory medicine. The European Federation of Laboratory Medicine (EFLM) has initiated a distance learning program and the development of a collaborative network for e-learning. The EFLM dedicated working group encourages the organization of distance education programs and e-learning courses as well as critically evaluate information from courses, lectures and documents including electronic learning tools. The objectives of the present paper are to provide some specifications for distance learning and be compatible with laboratory medicine practices.

SummaryA microparticle-enhanced nephelometric immunoassay has been developed for the determination of caseinomacropeptide (CMP) in bovine milk. It is based on the nephelometric quantification of the competitive immunoagglutination of a microparticle–CMP conjugate with an anti-κ-casein (κ-CN) antiserum. This one step immunoassay was sensitive (detection limit in reaction mixture, 16μg/l), accurate (linear recovery of CMP in dilution overloading) and reproducible (CV 7–14% for within and between run precision). Because of the specificity of the polyclonal antiserum used, it was necessary to separate CMP from κ-CN by ultrafiltration before the quantification of bovine milk CMP. Under the conditions of milk ultrafiltration used, κ-CN was entirely retained (> 99·5%) but the concentration of CMP measured in milk ultrafiltrates was underestimated (by ∼25%) compared with its concentration in whole milk. Microparticle-enhanced nephelometric immunoassay of CMP, with a calibration range from 0·32 to 20 mg/1 for 20- fold diluted milk ultrafiltrate, allowed contamination of bovine milk by rennet whey as low as 5 ml/1 to be detected. Applied to ultrafiltrates from milk stored at 4 °C, this immunoassay also detected proteolysis of κ-CN not revealed by measurement of κ-CN concentration in milk. A statistical lower limit of 3·21 mg/1 was determined as the increase in CMP concentration in milk ultrafiltrates that indicated probable κ-CN proteolysis in the milk sample. Previously demonstrated to be an easy to perform method for assaying the main proteins of bovine milk, microparticle-enhanced nephelometric immunoassay thus also appeared to be appropriate to quantify CMP so as to detect slight contamination of milk by whey and to indicate the proteolysis of κ-CN during milk storage at low temperature.

A microparticle-enhanced nephelometric immunoassay was developed for plasminogen quantitation in human plasma. It is based on the nephelometric measurement of the light scattered by microparticle clusters formed during a sandwich reaction between plasminogen, microparticle—anti-plasminogen conjugate, and the free antibodies of anti-plasminogen rabbit antiserum. This immunoassay was sensitive (detection limit in reaction mixture, 34 μg/L) and could be performed in 500-fold diluted human plasma, excluding any interference or sample pretreatment. It allowed the quantitation of plasminogen on a large range of concentrations (17–550 mg/L), with a security in antigen excess reaching 1,100 mg/L, with accuracy (linear recovery in dilution-overloading assay and correlation with conventional immunonephelometry), and precision (within- and between-run coefficients of variation lower than 8%). A normal reference range from 54 to 148 mg/L (mean ± 2 SD) was calculated from plasminogen concentration in plasma from 130 adults. Easy to perform (no washing or phase separation) and rapid (two steps of 30 minutes then 1 hour of incubation at room temperature), this microparticle-enhanced nephelometric immunoassay could be an interesting alternative method for human plasminogen quantitation. © 1996 Wiley-Liss, Inc.

Bacterial ribosomes, composed of proteins and RNA, have been used for nearly 30 years as immunostimulators. Preparations from about 30 different species of bacteria, as well as from fungi or parasites, have been used. Ribosomes have been tested in a number of animal models, and demonstrated to induce both specific immunity and protection. Paradoxically, although only a small part of these studies have dealt with micro-organisms involved in respiratory infections, most of the clinical trials involving ribosomes have used preparations obtained from such bacteria. Ribosomal preparations have been shown to reduce and prevent recurrent infections of the upper respiratory tract in humans.

The different degrees of lymphocytic infiltration observed in Graves' disease, Hashimoto's disease, and De Quervain thyroiditis suggest that the regulation of adhesion molecules expressed on endothelial cells could be different in these autoimmune disorders of the thyroid. Using immunohistological techniques, we observed that thyroid samples from patients with Graves' disease displayed a characteristic pattern of capillary proliferation, with CD62P, CD62E, and CD31 expression on endothelial cells. This was different from the pattern and size of endothelial cells expressing adhesion molecules in the two other types of thyroiditis where larger vessels and high endothelial venules were stained. Almost no signs of endothelial cell activation could be seen in a comparative series of non-autoimmune disorders.

Les caseines α, β et κ ont ete dosees par immunonephelemetrie microparticulaire, dans 1300 echantillons de lait, collectes bimensuellement pendant 13 mois (octobre 1992 a octobre 1993), aupres de 50 troupeaux de Normandie. Les concentrations de ces 3 caseines sont comparees a celles d'autres parametres de composition, mesures sur les memes laits (matieres proteiques, matieres grasses, lactose et extrait sec total). Les principaux facteurs, lies a l'exploitation, au troupeau, a sa conduite et a son alimentation, qui determinent les parametres de composition du lait a la production sont ensuite analyses. Ces memes parametres de composition biochimique et des parametres de coagulation a la presure (temps de floculation, indice de raffermissement K15, indice de fermete A30) sont mesures, sur la meme periode, dans des laits crus et standardises en matiere grasse, utilises dans une fabrication fromagere industrielle. Realise dans un cadre de temps et d'espace limite, l'ensemble du travail envisage l'etude du lait depuis sa production jusqu'a sa valorisation par transformation fromagere. Il permet de retrouver des notions bien connues et abondamment publiees, tant au niveau biochimique (liens entre les parametres de composition, variation et variabilite de ceux-ci), que zootechnique (influence de la race, des potentialites pedologiques du sol, de la repartition des velages, de l'alimentation, des conditions de transition entre stabulation et pâturage) et technologique (influence relative des parametres de composition sur la coagulabilite a la presure et les rendements de fabrication fromagere). L'immunonephelemetrie microparticulaire, qui permet un dosage specifique et precis des differentes caseines du lait, apparait comme une technique originale d'immunoanalyse, completant les moyens traditionnels d'evaluation de la qualite du lait.

IgA nephropathy (IgAN) is characterized by the presence of IgA and C3 deposits in the mesangium. Mesangial IgA is mostly dimeric IgA1 with a J chain, lacking a secretory component. In view of the recent demonstration of elevated serum levels of secretory component and secretory IgA in liver disease and HIV infection associated with hyper-IgA, we measured the serum secretory component in 50 IgAN patients and 45 controls. Free secretory component and secretory IgA and IgM levels were determined by enzyme-linked immunosorbent assay. Normal or low levels were found patients. These data support previous work suggesting that in IgAN circulating and probably mesangial IgA do not originate from the epithelial compartment of the mucosal immunoglobulins as it is the case in other hyper-IgA diseases.

Recurrent unexplained abortions are defined as at least two successive abortions during the first trimester of pregnancy. Implication of autoimmunity processes is now widely recognized. The list of biological assays proposed in recurrent abortions is inspired from immunological exploration of systemic lupus erythematosus in which pregnancy usually induces high risk for the conception product. Other assays derive from immunological hypotheses explaining the tolerance paradox of pregnancy, but many have not yet been established and will still require clinical research protocols.

Background The diagnosis of leptomeningeal metastasis (LM) in patients with solid tumors remains difficult. The usual diagnostic methods of cytomorphological assessment of cerebro-spinal fluid (CSF) and gadolinium enhanced MRI of the entire neuraxis lack both specificity and sensitivity. The Veridex CellSearch® technology has been designed for the detection of circulating tumor cells (CTC) in blood from cancer patients and validated for the follow-up and prognosis of breast, prostate, colorectal, and lung cancer. Our aim was to adapt this technology for the detection and the enumeration of tumor cells in the CSF of breast cancer patients presenting with LM. Methods On the occasion of a randomized phase III study evaluating the role of the intrathecal treatment in LM from breast cancer (DEPOSEIN, EudraCT N°: 2010-023134-23), the CellSearch® technology was adapted to direct enrichment, enumeration and visualization of tumor cells in 5 mL CSF samples, collected on CellSave® Preservative Tubes and analyzed within 3 days after CSF sampling. Results Sixteen CSF of 8 patients with primary breast cancer presenting with LM were studied. EpCAM+/cytokeratin + cells with typical morphology could be observed and enumerated sequentially with reproducible results in low or elevated numbers in 8 patients. Conclusion This methodology, established on a limited volume of sample and allowing delayed processing, could prove of great interest in the diagnosis and follow-up of cancer patients with LM, especially to appreciate the efficacy of chemotherapy.