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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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Bacterial ribosomes have been shown to induce effective humoral and cellular immunological responses to whole microorganisms. In this study, the numbers of specific antibody producing cells directed towards Klebsiella pneumoniae, Streptococcus pneumoniae, Streptococcus pyogenes and Haemophilus influenzae ribosomes or whole bacteria sonicates were compared in the peripheral blood and tonsils of 7 children, and in the tonsils, mesenteric and cervical lymph nodes of 10 sheep. No significant difference was noted between the two types of antigens, confirming that ribosomal preparations are able to mimic the immunogenicity of whole bacteria in the mucosae-associated lymphoid tissue.

Wheat flour is a complex organic dust likely to induce immune responses when inhaled in work environment conditions. We compared the humoral status of 159 exposed workers from 11 flour mills and one industrial bakery with that of 41 workers from a salt factory. IgG, IgA, and IgM levels of antibodies to whole flour and to gliadin were assayed using ELISA tests in serum and saliva samples. Serum levels of IgG and IgA to both antigens were significantly higher (p < 0.0001) in occupationally exposed workers. Exposed workers had significantly higher levels of salivary IgG (p = 0.005) and IgA (p < 0.0001) to whole flour and of salivary IgG (p = 0.0005) to gliadin. In both groups, similar levels of anti-gliadin salivary IgA antibodies were observed. These data suggest that occupational exposure to wheat flour triggers specific immune responses, most likely through stimulation of the mucosal immune system. The presence of significant levels of serum antibodies, however, indicates that a systemic immunologic response is also present among exposed individuals.

Crude thyroid peroxidase extracted from human thyroid microsomes was covalently bound onto polyacrylic and polyfunctional copolymerized microparticles. We observed agglutination of the thyroid peroxidase-microparticle conjugate with 13 monoclonal antibodies (mAbs) specific for epitopes on four different antigenic domains of human thyroid peroxidase (TPO; EC 1.11.1.7), after addition of anti-mouse immunoglobulins. We quantified agglutination by measuring with a specially designed nephelometer the light scattered by the conjugates. This allowed us to develop a microparticle-enhanced nephelometric immunoassay for human anti-TPO autoantibodies (aAbs) with defined epitopic specificity, based on the ability of aAbs to inhibit mAb-induced agglutination. Applied to patients with autoimmune thyroid diseases, this assay confirmed the polyclonality of anti-TPO aAbs and their preferential reactivity toward epitopes located on the A and B antigenic domains of the TPO molecule. The same specificities seem to be present in patients with Hashimoto thyroiditis or Graves disease.

Immunoglobulin kappa light chains are predominant in normal human serum and a kappa/lambda ratio of 1.7 to 2 has been reported. However, little is known of the partition of light chains in secretions. The levels of IgA, kappa and lambda were assayed by nephelometry in a series of secretions and in normal human serum. Although kappa chains remained predominant, the different secretions studied could be classified according to their kappa/lambda ratio. In saliva (P < 0.001), nasal fluid (P = 0.01) and tears (P = 0.04) the kappa/lambda ratio was significantly lower than in serum. Conversely, higher kappa/lambda ratios were obtained in gastric juice (P = 0.005) and hepatic bile (P = 0.004) and no significant difference was noted between serum and gall bladder bile (P = 0.62). The lower ratios were all observed in fluids produced by glands surrounded by lymphoid tissue included in the mucosae-associated lymphoid tissue. These data strengthen previous observations that suggest a preferential production of lambda chains in human mucosae.

Tonsillar abnormalities have previously been evidenced in IgA nephropathy (IgAN). We report the results of a systematic quantitative analysis of 303 tonsils obtained in 70 IgAN patients and 142 controls. IgG- and IgA-producing plasma cells, stained by immunofluorescence, were enumerated on serial sections of all samples. Both the percentage and number of IgA+ cells were significantly increased in IgAN patients. This anomaly was also observed in age- or sex-matched subgroups of patients. Performed on a large series of individuals this study confirms the dysregulation of IgA production the upper respiratory tract of IgAN patients.

An immunohistomorphometric study was performed on human samples of duodenum (10) and tonsil (25) to assess the numbers of plasma cells producing kappa or lambda chains. Different reagents were used and carefully assayed for specificity and absence of cross-reactivity. Kappa chains were found predominantly with these antibodies in 46 bone marrow-derived B-cell proliferations used as reagents' control samples. In contrast, plasma cells producing lambda chains were found to be more numerous in the samples of mucosal tissues. The kappa/lambda ratio observed was 0.53. This finding could be another feature reflecting the autonomy of the immune system of mucosae (MALT) in humans.

The expression of several epitopes of CD4, a molecule usually restricted to a subset of T-lymphocytes, was observed after immunofluorescent labeling of frozen-cut sections of human parathyroid. Seven samples obtained from three subjects presenting adenomas and from seven renal insufficiency patients with secondary or tertiary hyperplasia were found to express this molecule. Dot enzyme-linked immunosorbent assay, polyacrylamide gel electrophoresis, and Western blotting were used to characterize this peptide in cytosol and membrane fractions of these glands. These studies confirmed, in the parathyroids found positive in immunofluorescence, the presence of a protein with a mol wt similar to that of lymphocyte-derived CD4.

Thirty subjects who had received an oral ribosomal vaccine to common bacteria of upper respiratory tract infections, and ten controls were tonsillectomized for recurrent infections or rhonchologic pathology. A three-step indirect immunofluorescence technique was used to identify and enumerate specific antibody forming cells (SAFC) in their tonsils. Plasma-cells specific of the four strains being constituents of the oral vaccine ( H. influenzae, K. pneumonieae, S. pyogenes and S. pneumoniae ) were observed in all samples. The numbers of SAFC were significantly higher in the subjects who had received the oral vaccine. These results support the efficiency or oral immunization in increasing local defenses, even at distance from the sensitized gut-associated lymphoid tissue. These data provide evidence for the homing phenomenon in human mucosae associated lymphoid tissue (MALT).

This study reports that B-chronic lymphocytic leukemia (B-CLL) cells display the same pattern of toll-like receptors (TLRs) proteins expression as normal B-cells, yet with overexpression of TLR9. Furthermore, TLR7 and TLR9 appear to be functional and liable to respond to specific ligands, respectively imidazoquinolines and CpG-ODN thus potentially opening new therapeutic approaches.

Objective To further define the immunological tissular modifications in premature ovarian failure (POF). Method The patient was followed up for premature ovarian failure and mild endometriosis associated with serum antiovarian antibodies. A laparoscopic ovarian biopsy was decided on to analyze the tissue and support the onset of immunosuppressive therapy. Immunohistochemistry was performed using monoclonal antibodies directed against T cell membrane markers, as well as activation molecules, to define the composition of the cellular infiltrate and the consequences on ovarian tissue. Result(s) A dense infiltration of activated T lymphocytes was observed in close contact with follicular epithelium expressing HLA-DR and CD40. Conclusion(s) This observation supports the role of cellular immunity in ovarian autoimmunity with features very similar to those reported in murine models and other human autoimmune endocrine pathologies.

Nephelometric immunoassays were developed for human IgG, IgA, and IgM quantitation in B‐lymphocytes culture media. They allowed measurement of immunoglobulin (Ig) levels over a broad range of concentrations with good accuracy and precision. The kinetics of Ig production in B‐lymphocyte cultures was followed and the mean amount of each Ig was determined in six different samples after three days of culture. The nephelometric immunoassays reported here could be used to study, in vitro, the influence of various molecules (inhibitory or amplifying effect) on B‐lymphocytes' functional capacities.

While the involvement of anti-ovarian antibodies (AOAs) is highly likely, yet still controversial, in patients with patent premature ovarian failure (POF), it is even more difficult--for several reasons--to ascertain the clinical significance of these antibodies in patients without obvious ovarian failure. First, AOAs form a heterogeneous group of antibodies recognizing several different antigenic targets such as granulosa and thecal cells, zona pellucida, oocyte cytoplasm, corpus luteum, as well as gonadotrophins and their receptors. Second, the detection of AOAs in various clinical situations does not readily imply a causal relationship between these antibodies and impaired ovarian function. Third, diagnostic tools for detecting AOAs and their molecular targets have to be improved to yield more reliable data and allow a better comprehension of the pathophysiology of AOAs. Preliminary results with immunosuppressive therapy in selected AOA patients have been encouraging, but randomized trials have to be performed.

To describe the course of plasma sTREM (soluble triggering receptor expressed on myeloid cells)-1, procalcitonin (PCT), and C-reactive protein (CRP) concentrations during sepsis and their clinical informative value in predicting outcome.Prospective, noninterventional study.Medical adult intensive care unit at a university hospital in France.Sixty-three critically ill patients with sepsis, severe sepsis, or septic shock.None.Soluble TREM-1 concentrations were significantly lower at admission in nonsurvivors (n = 21) than in survivors (n = 42) (94 [30-258] vs. 154 [52-435] pg/mL, p = .02), whereas PCT levels were higher among nonsurvivors (19.2 [0.3-179] vs. 2.4 (0-254) pg/mL, p = .001). CRP levels did not differ between the two groups of patients. Plasma PCT and CRP decreased during the 14-day period of study in both survivors and nonsurvivors. Conversely, sTREM-1 plasma concentrations remained stable or even increased in nonsurviving patients and decreased in survivors. An elevated baseline sTREM-1 level was found to be an independent protective factor with an odds of dying of 0.1 (95% confidence interval, 0.1-0.8).A progressive decline of plasma sTREM-1 concentration indicates a favorable clinical evolution during the recovery phase of sepsis. In addition, baseline sTREM-1 level may prove useful in predicting outcome of septic patients.

In lung cancer as in other malignancies, tumor formation induces the development of local and systemic antitumoral immune responses. The tumor itself becomes surrounded by a local stroma reaction containing inflammatory cells, a large part of which being tumor infiltrating T-lymphocytes. This study was designed to investigate the potential clonality of these T-cells in non-small cell lung cancer. Two complementary methods where used: exploration of the Vbeta TCR repertoire usage in flow cytometry and analysis of the Vgamma TCR repertoire in multiplex PCR and gradient gel electrophoresis. These techniques were applied respectively to eluted fresh lymphocytes and extracted DNA from healthy lung tissue, tumor and lymph nodes from 44 patients. There was a good correlation between the two techniques used. An oligoclonal repertoire restriction was noted in most of the cases and in the three types of tissues studied suggesting the presence of tumor-specific clones. Moreover, Vbeta14 appeared to be the most frequent specificity used whatever the tissue considered, while Vbeta13.1 appeared to be selectively used in the stroma reaction of epidermoid lung carcinomas. A restricted TCRgamma band was also present in these tumors, and two more bands of TCRgamma where selectively present in adenocarcinomas. The demonstration of both alpha-beta and gamma-delta TCR restriction suggests both the recruitment of specific T-cells and their local proliferation within the tumoral tissue. The same feature in healthy lung tissue indicates that it might already be the site of specific anti-tumoral T-cell reactivity. In conclusion, this study reports on the presence of oligoclonal T-cell responses in most cases of non-small cell lung cancer. The comparison of tumor, healthy tissue and lymph nodes showed some degree of patient-dependent similarities suggestive of tumor specificity.

Humoral auto-immunity to the myelin oligodendrocyte glycoprotein (MOG) is likely involved in the pathogenesis of multiple sclerosis (MS). In 44 MS patients and 30 controls, Ig-producing B cells were identified by their isotype and as MOG-specific spot-forming cells (SFC). Peripheral anti-MOG antibodies were assayed in ELISA as well as anti-butyrophilin antibodies to investigate for molecular mimicry. MS patients had significantly higher levels of IgA- and MOG-SFC than controls, as well as significantly higher antibody responses to MOG and butyrophilin. These data provide added support for the implication of anti-MOG humoral immunity in the pathophysiology of MS, and suggest a balance of systemic (anti-self) and mucosal (environment-modulated) immune reactions in an attempt at regulating the pathogenic specific immune response.

It has been suggested that locally produced immunoglobulin (Ig)A could be more protective than IgG and that there could be a relationship between crevicular fluid-specific IgA levels and the onset of periodontal disease. This study was designed to investigate this hypothesis regarding specific immune responses towards 4 plaque anaerobes in gingival crevicular fluid and saliva from patients with periodontopathies and controls.Gingival crevicular fluid (GCF) and whole saliva were collected from 35 adults with periodontitis and 24 periodontally healthy adults (controls). Antigens were extracted from Actinomyces actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, and Fusobacterium nucleatum and used to set up specific enzyme-linked immunosorbent assay (ELISA) tests to assess IgA and IgG levels to these microorganisms in the fluids collected.The crevicular fluid of periodontitis patients contained significantly higher levels of IgG to the 4 microorganisms tested than that of controls (P10(-6) for all comparisons). IgA levels to the 4 bacteria were statistically significantly much higher in control crevicular fluid (P10(-7) for all comparisons). Controls also had statistically significantly higher levels of specific salivary IgA than patients (P0.02 for all comparisons).These data support the potentially protective role of specific IgA directed to oral microorganisms involved in the onset and development of periodontal disease.

• Proliferative sites within the overgrowing epithelium of cholesteatoma are still ill defined. In this study, we used the monoclonal antibody Ki-67 on frozen cut sections from 23 cholesteatoma samples obtained at surgery from 20 patients. This reagent has been reported to stain the nucleus of proliferating tumor cells and the cytoplasm of growing keratinocytes. In this series of samples, flat superficial layers of epithelium were consistently negative for Ki-67 or displayed a faint staining of the basal layer. By contrast, the deepest areas of epithelial recesses appeared brightly stained with Ki-67. The latter were often in the close vicinity of inflammatory cells present in the underlying mucosa. This observation suggests that cholesteatoma growth is initiated within the deep epithelial folds of overgrown tympanic skin, and that it might be triggered or sustained by inflammatory cytokines. ( Arch Otolaryngol Head Neck Surg. 1993;119:656-658)

Choroid plexuses form an interface between peripheral blood and cerebrospinal fluid. Dendritic-like cells have been reported in a few studies of choroid plexuses in man. Here we used electron microscopy and immunophenotyping to precise the morphologic features and phenotype of these cells. Examination of 10 human choroid plexuses evidenced intra-epithelial dendritic cells with a clear cytoplasm, reniform nucleus and long expansions. These cells express MHC Class II, CD11b, CD14, CD32, CD68 and IL-10, but not CD40, CD80 or CD86, suggesting an immunosuppressive role for these dendritic cells. Their sentinel position could make them participate to the immunological silence of the brain.

Anomalies of the cerebrospinal fluid flow rate and composition that have been reported in patients suffering from Alzheimer's disease (AD) could be related to alterations of the choroid plexuses (CD). Here we report a photonic and electron morphometric study in which we compared the height of CP epithelial cells and the thickness of their basement membrane on post-mortem samples from AD patients, age-matched controls and two newborns. Ageing appeared associated with epithelial atrophy and basement membrane thickening, but these features were significantly accentuated in AD. These data suggest that a dramatic alteration of the secretion and filtration could be involved in the multiparametric pathogenesis of late-onset AD.

Background: Helicobacter pylori is now recognised to be the major cause of antral gastritis and a risk factor for further development of gastric cancer. This infection results in local inflammation and a modification of gastric mucosal epithelial cell characteristics. Systematic investigation for the expression of the secretory component by gastric epithelium in a personal historical series of biopsy specimens showed the expression of this activation marker in 38% of the cases, 19% also showing clear signs of H. pylori infection. Aims: To further appreciate the activation of epithelial cells in the chronic gastric inflammation associated with H. pylori infection and other types of gastritis without consideration of the grade of gastritis. Methods: Punch gastric biopsies from 36 patients (10 patients with confirmed H. pylori gastritis, 16 patients with non-H. pylori gastritis and 10 controls) were tested for the expression of ICAM-1/CD54, HLA-DP, -DQ, -DR, secretory component and bcl-2 by immunofluorescence. Results: Up-regulation of most markers was observed both in H. pylori and non-H. pylori gastritis, although secretory component, CD54 and DP expression was more closely associated with H. pylori gastritis. Conclusion: H. pylori infection appears to activate gastric epithelial cells more strongly than other types of gastritis. This suggests an active relationship between this bacterium and epithelial cells. Investigation of the up-regulationship between this bacterium and epithelial cells. Investigation of the up-regulation of secretory component, ICAM-1/CD54 or DP in gastric biopsies may serve as extensive markers in search of H. pylori gastric infections.

Introduction Background. The therapeutic eYcacy of horse antilymphocyte globulins (ALG ) or of rabbit antithymo- Antilymphocyte and antithymocyte globulins (ALG cyte globulins (ATG ), used for both the prevention and ATG) are used in immunosuppression protocols and treatment of allograft rejection has been well for the treatment of aplastic anaemia, graft-versusdocumented. However, clinical use of these heterolog- host disease and organ transplantation [1‐3 ]. ALG ous antibodies can result in the production of antibod- and ATG are purified preparations of animal immunoies against horse or rabbit proteins and in the globulins specific for leukocyte surface molecules. development of serum sickness via circulating immune Among those are antibodies to diVerentiation antigens complexes. of human T cells such as CD2, CD3, CD4, CD8 etc., Methods. We studied the production of human IgG, or B cells such as CD21, CD19, CD40. These preparaIgA, and IgM anti-rabbit and anti-horse globulins, in tions also contain immunoglobulins directed to access240 serum samples from 111 kidney transplant recipi- ory or adhesion molecules, i.e. CD6, CD25, CD44, ents, of whom 89 were treated with ALG or ATG CD45, the integrin LFA1 and HLA DR [4‐7 ]. The (M erieux-France) as prophylaxis. antibody reactivity to these molecules varies between Results. Up to 8.9% of the patients had anti-ALG preparations of horse ALG and rabbit ATG and is and/or -ATG antibodies before the first transplanta- concentration-dependent. In vivo, the recognition of tion. This proportion increased significantly after. Pre- lymphocyte markers is followed by complementimmunization did not appear to be predictive of the dependent lysis or by phagocytosis of opsonized circuoccurrence of clinical serum sickness, yet sensitization lating lymphocytes [8]. ALG can also block the prolifincreased, after transplantation, in up to 71% of the eration of T lymphocytes by post-transcriptional subjects who developed this disorder (P=0.02). In inhibition of CD25. ALG used at an antibody concenpatients receiving a second transplant, pretransplanta- tration which can induce T cell proliferation has also tion antibody levels were not modified by the immuno- been shown to inhibit the proliferation of B cells and suppressive therapy applied. No relationship was found B cell lines, and to induce apoptosis of human B cells between early rejection and antiglobulin antibodies. [9‐11]. Conclusions. Serum anti-rabbit and/or -horse antibod- Before cyclosporin A became widely used, the clinies were demonstrated in a significant proportion of ical use of ALG and ATG provided a significant kidney recipients, even before transplantation, possibly improvement of graft survival and a lower incidence due to environmental exposure. A classical pattern of of graft rejection in organ transplantation [12 ]. One IgM increase was observed when the patients developed of the drawbacks of this treatment resides in the an immune response to ALG or ATG, and an IgA potential induction of immune-complex-mediated response after ALG. These results suggest that patients hypersensitivity reactions, as some patients treated with receiving ALG/ATG should be monitored for the ALG or ATG produce antibodies directed to horse or production of anti-ALG/ATG immunoglobulins. rabbit globulins [13,14]. Amemiya et al. [15 ], have

In Graves' disease, thyroid epithelial cells abnormally express HLA-DR Class II molecules in the vicinity of lymphocyte infiltrates, suggesting that lymphocyte proliferation is sustained by the appropriate presentation of antigenic material. The ability of thyroid epithelial cells to provide the necessary second signals has however not been documented. The expression of HLA-DR, CD40, CD40L, CD80, and CD28 was investigated on thyroid samples from 30 patients (Graves' disease, n = 16; benign toxic adenoma, n = 8; multinodular goiter, n = 6). Apoptotic cells were searched for using the TUNEL method. CD40 appeared to be coexpressed with HLA-DR in Graves' disease patients samples and in two control samples also containing lymphoid infiltrates. Almost no apoptotic cells were found. These data suggest that thyroid epithelial cells from Graves' disease patients have the ability to successfully present autoantigens. The near absence of apoptotic cells in surrounding lymphoid infiltrates is in keeping with this efficient provision of a rescue second signal.

To approach the physiopathology of the mucosal immune response in flour-mill and bakery workers, the authors developed a mouse model, which allowed them to study immune responses induced in the deep lung by acute inhalation of flour dust. In situ quantification of T lymphocytes (Thy1-2) and T-cell subsets (CD4 and CD8), macrophages, B lymphocytes, and immunoglobulin A plasma cells in the lungs of all animals revealed significant modifications of these cell compartments as early as 3 d after exposure; within 10 d of exposure, levels returned to baseline. The data suggest that the lung could be a sensitive target for inhaled xenobiotics, which might generate rapid immune local modifications that result in the maintenance of homeostasis.

The kinetics of specific IgA mucosal responses was assessed in 12 healthy volunteers over 3 weeks of treatment by oral administration of an immunostimulant, Ribomunyl, composed of ribosomes from the four bacteria Streptococcus pyogenes, Streptococcus pneumoniae, Klebsiella pneumoniae and Haemophilus influenzae. The levels of IgA specific for these four bacteria increased after each immunization and, after the third week of treatment, were significantly higher than baseline day 0 values. This study demonstrates that oral ribosomal immunostimulation results in the production of specific salivary antibodies liable to recognize whole bacteria antigens, and therefore likely to confer protection. The kinetic analysis performed also demonstrates the rapidity of specific mucosal immune responses after oral stimulation in man, a feature still seldom explored.

Discrepancies in the literature on acute leukemia blast cell immunophenotypes are sometimes related to differences between the epitopes recognized by various monoclonal antibodies (MoAb) in the same cluster of differentiation. CD15 is one example of such a variation. CD15 expression has been reported in 1.6% to 39% of acute lymphoblastic leukemias (ALL). We studied the expression of CD15 using 10 different commercially available anti-CD15 MoAbs and we observed three different expression patterns using anti-CD15 MoAbs by flow cytometry in 158 cases of ALL: Smy15c was found in 70% of B lineage ALLs, Smy15a and FMC-13 in 30 to 40% of cases and all others in less than 9% of B-ALL cases (p < 0.0001). In T lineage ALLs, Smy15c, Smy15a and FMC-10 identified CD15 in 30% of the cases and all others in less than 8% of the cases. Logistic regression revealed that Smy15a, CD34 and CD14 correlated significantly with Smy15c expression. We conclude that CD15 MoAbs have to be chosen carefully when ALL immunophenotype and subsequent studies of prognostic significance are performed particularly in assessing multiphenotypic ALLs.

A microparticle-enhanced nephelometric immunoassay was developed for alpha-lactalbumin quantitation in human milk. It is based on the nephelometric measurement of the light scattered during the competitive immunoagglutination of a microparticle-alpha-lactalbumin conjugate with an anti-alpha-lactalbumin antiserum. This immunoassay is sensitive (detection limit in reaction mixture, 1.5 micrograms/L) and could be performed in high dilution of milk, excluding any interference or sample pretreatment. It allowed the quantification of alpha-lactalbumin on a large range of concentrations (0.5-16.9 g/L) with accuracy (linear recovery in dilution-overloading assay) and precision (within- and between-run coefficients of variation from 1 to 7%). Changes in the alpha-lactalbumin concentration of human milk during lactation were determined in 162 samples. The concentration and ratio of alpha-lactalbumin total protein were found to be significantly lower in colostrum (4.9 g/l, 27%) than in transitional milk (5.2 g/L, 40%), then decreased in mature milk (3.4 g/L, 31%).

A microparticle-enhanced nephelometric immunoassay was developed for lactoferrin quantitation in human milk. It is based on the nephelometric measurement of the light scattered during the competitive immunoagglutination of a microparticle-lactoferrin conjugate with an antilactoferrin antiserum. This immunoassay is sensitive (detection limit in reaction mixture, 0.2 mg/L) and can be performed in diluted milk (1/3,000 in reaction mixture), excluding any interference or sample pretreatment. It allowed the quantification of lactoferrin on a large range of concentrations (0.675–21.6 g/L) with accuracy (linear recovery in dilution-overloading assay) and precision (within- and between-run coefficients of variation from 3% to 6%). Changes in the lactoferrin concentration of human milk during lactation were determined in 190 samples. The concentration and ratio of lactoferrin vs. total protein were found to be significantly higher in colostrum (5.9 g/l, 29%) than in transitional milk (2.9 g/L, 22%) or mature milk (2.5 g/L, 24%). J. Clin. Lab. Anal. 11:239–243, 1997. © 1997 Wiley-Liss, Inc.

Chronic lymphocytic leukemia (CLL) is characterized by the accumulation in the peripheral blood, secondary lymphoid tissues and bone marrow of functionally defective clonal B lymphocytes with prolonged survival in vivo. Despite therapeutic achievements have been accomplished in the management of this disease, however CLL remains incurable partially because of the resistance to apoptosis of CLL B-cells and of the altered immune system of CLL patients. CLL is hetereogenous, but its evolution is mostly slow, probably linked to some level of immune control of the leukemic cells. Indeed, clinical observations have been reported of spontaneous remissions associated with the intense immunological activity following a viral infection. Clinical responses have also been observed after treatment by immunomodulating cytokines and long-term survival is described, without disease, after allogeneic stem cells transplantation. All these data suggest that immunotherapy could be useful in the treatment of CLL, possibly as an adjuvant therapy after classical immunochemotherapy schedules. Toll like receptors (TLR) are proteins of the innate immune system belonging to the family of Pattern Recognition receptors (PRRs). Recognition of their ligands by the TLR present on neutrophils, macrophages, dendritic cells and B-cells are important in the initiation of adaptive immune responses. TLR-7 and 9 are expressed by CLL B-cells (Grandjenette, Hematologica, 2007). Previous studies have shown that stimulation of TLR-9 by CpG ODN (oligodinucleotides) induces an activation of CLL B-cells while triggering TLR-7 increases in vitro apoptosis of these cells. Here we show that these effects of TLR-9 and TLR-7 stimulation differ depending on the clinical form (stable or aggressive) of the disease and on the mutational status of CLL B-cells. In vitro stimulation with three doses of Imiquimod R837 or ODN CpG M362 was carried out for three days on cells from 40 patients (22 stable, 18 aggressive, mutational status known for 35, 18 IgVH mutated, 17 unmutated). Flow cytometry was used to measure apoptosis, proliferation after carboxyfluorescein succinimidyl ester (CFSE) labeling and modulation of surface differentiation antigens. Signaling pathways after incubation were further studied by antibody arrays and western blot. Spontaneous apoptosis occurring in vitro was demonstrated to involve the mitochondrial pathway. CLL B-cells were also confirmed to proliferate strongly and produce large amounts of IL-6 and IL-8 upon triggering by phorbol myrsistate (positive control), this compound almost completely aborting in vitro apoptosis. Cells from patients with a stable disease were significantly more prone to rapid apoptosis after TLR-7 triggering with Imiquimod, compared to cells from patients with an aggressive disease which displayed only spontaneous apoptosis. This rapid apoptosis in stable patients involved the p38 MAP-Kinase pathway. It was concomitant to an important production of IL-8 and IL-6. Conversely, CpG ODN conferred a protection against apoptosis to CLL B-cells from patients with an aggressive disease. This was accompanied by the activation of numerous anti-apoptotic proteins in the cells. CpG ODN also significantly increased CLL B-cells proliferation, concomitantly to the phosphorylation of Erk and Akt proteins. ODN finally increased the expression of CD20 and CD19 on the cells’surface. The same differences in reactivity were observed comparing mutated (∼stable) and unmutated (∼aggressive) cases. These data indicate that CLL B-cells from patients with a stable or aggressive (mutated/unmutated) disease answer differently when triggered through their surface TLRs. This might have an incidence on the behavior of these cells in vivo, in answer to stimulations by microbial compounds naturally binding these structures. These properties could also be used to develop adjuvant immunotherapies by loading CpG ODN-activated CLL B-cells with autologous apoptotic fragments issued from stimulation of part of the same cells with Imiquimod. Disclosures: No relevant conflicts of interest to declare.

Peyer's patches play a major role in the initiation of mucosal immune responses since most environmental antigens gain access to the digestive tract and are therefore liable to achieve contact with their specialized dome epithelium. We investigated kinetics of immunophenotypic modifications induced in the cells from murine Peyer's patches in a model of intensive controlled exposure to flour dust. Three groups of mice were placed in an atmosphere enriched in wheat flour for 3, 6 or 10 consecutive days. Although Ig bearing B cell numbers did not vary, the numbers of IgA-containing plasma-cells increased significantly during the course of exposure. A significant and increasing imbalance of the CD4/CD8 ratio was noted, as early as after 3 days of exposure related both to increasing numbers of CD4 + and decreasing numbers of CD8 + cells. These data indicate a rapid kinetics of immune responses in Peyer's patches in an original model of controlled respiratory challenge.

Labial salivary glands biopsies (LSG) performed to support clinical anomalies suggestive of Sjogren's syndrome (SS) sometimes fail to confirm the diagnosis. Here we investigated whether epithelial activation markers could provide further information. Frozen cut sections of LSG from 40 patients, including 23 confirmed SS, were examined in immunofluorescence for the expression of HLA class II molecules, the protector of apoptosis bcl-2, the intercellular adhesion molecule 1 (ICAM-1) and secretory component (SC). Class II molecules were highly expressed on epithelial cells in SS patients (DR > DP > DQ). Bcl2 was expressed in infiltrating cells which were more numerous in the group of SS patients. ICAM-1 was present on endothelial and infiltrating cells of a few patients in both groups. Epithelial cells produced SC in 83% of SS patients samples vs four cases of non-SS patients (P= 0.0002). Investigation of the expression of SC on glandular epithelial cells could therefore be proposed as a marker of SS.