Immunophenotyping of acute leukemia and lymphoproliferative disorders: a consensus proposal of the European LeukemiaNet Work Package 10.

The European LeukemiaNet (ELN), workpackage 10 (WP10) was designed to deal with diagnosis matters using morphology and immunophenotyping. This group aimed at establishing a consensus on the required reagents for proper immunophenotyping of acute leukemia and lymphoproliferative disorders. Animated discussions within WP10, together with the application of the Delphi method of proposals circulation, quickly led to post-consensual immunophenotyping panels for disorders on the ELN website. In this report, we established a comprehensive description of these panels, both mandatory and complementary, for both types of clinical conditions. The reason for using each marker, sustained by relevant literature information, is provided in detail. With the constant development of immunophenotyping techniques in flow cytometry and related software, this work aims at providing useful guidelines to perform the most pertinent exploration at diagnosis and for follow-up, with the best cost benefit in diseases, the treatment of which has a strong impact on health systems.

Reduced levels of both circulating CD4+ CD25+ CD127(low/neg) and CD4+ CD8(neg) invariant natural killer regulatory T cells in stable heart transplant recipients.

A cross-regulation between two regulatory T cell (T(reg) ) subsets [CD4(+) CD25(+) and invariant natural killer (NK) T - iNK T] has been described to be important for allograft tolerance induction. However, few studies have evaluated these cellular subsets in stable recipients as correlates of favourable clinical outcome after heart transplantation. T(reg) and iNK T cell levels were assayed by flow cytometry in peripheral blood samples from 44 heart transplant recipients at a 2-year interval in 38 patients, and related to clinical outcome. Multi-parameter flow cytometry used CD4/CD25/CD127 labelling to best identify T(reg) , and a standard CD3/CD4/CD8/Vα24/Vß11 labelling strategy to appreciate the proportions of iNK T cells. Both subtypes of potentially tolerogenic cells were found to be decreased in stable heart transplant recipients, with similar or further decreased levels after 2 years. Interestingly, the patient who presented with several rejection-suggesting incidents over this period displayed a greater than twofold increase of both cell subsets. These results suggest that CD4(+) CD25(+) CD127(low/neg) T(reg) and iNK T cells could be involved in the local control of organ rejection, by modulating immune responses in situ, in clinically stable patients. The measurement of these cell subsets in peripheral blood could be useful for non-invasive monitoring of heart transplant recipients, especially in the growing context of tolerance-induction trials.

Alteration of the immunological synapse in lung cancer: a microenvironmental approach.

This study was designed to investigate the immunological properties of stroma reaction T cells and tumoral cells by comparison with non-tumoral lung tissue and local lymph nodes in order to explore interactions between tumour cells and the immune system. Immunodetection of major histocompatibility complex (MHC) molecules, CD3/T cell receptor (TCR) complex and T cell subsets markers was carried out in situ on frozen sections, and the semi-quantitative expression of CD3, CD4 and CD8 was examined in flow cytometry on lymphocytes of nodal, tumoral and healthy lung tissue from 62 patients with non-small cell lung cancer. This study showed alterations on lymphocytes and tumour cells in lung cancer, consistent with an impairment of T cell activation. CD3, TCR alpha beta and accessory molecules expression is down-modulated on peri- or intra-tumoral lymphocytes. MHC class I and class II molecules are down-modulated significantly on tumour cells. Other differences were noted, such as the reversed CD4/CD8 ratio of tumour infiltrating cells, compared to healthy lung tissues, consistent with the development of cytotoxic anti-tumoral responses. This study reports on the presence of a strong in vivo immunomodulating effect of tumour cells in human non-small cell lung cancer, likely to impair proper formation of the immunological synapse.

[Ovarian autoimmunity and ovarian pathologies: antigenic targets and diagnostic significance]. / Auto-immunité anti-ovarienne et pathologies ovariennes: cibles antigéniques et implications diagnostiques.

The involvement of serum anti-ovarian autoantibodies (AOA) in ovarian pathology still remains controversial. In some cases of clinically patent ovarian failure, there seems to be a causal relationship between AOA and the ovarian disease. In patients with various organ-specific or systemic autoimmune diseases, or with unexplained, repeated reproductive failure, but otherwise normal ovarian function, it is even more difficult to determine the significance of AOA for several reasons: i) AOA recognize many different antigenic targets in the ovary ii) the antiovarian response may be transient or variable with time iii) the presence of AOA does not imply their aetiopathogenic role in the disease. The present paper reviews the clinical significance of AOA based on their ovarian targets as far as they have been identified until now.

Back-pack mice as a model of renal mesangial IgA dimers deposition.

Mesangial IgA in IgA nephropathy are dimers with a J chain but no poly-Ig receptor. This molecular structure has led to the hypothesis that these IgA are issued from the lamina propria of mucosal areas, reaching the kidney by way of the peripheral blood. The availability of hybridomas producing IgA dimers provided an opportunity to test this hypothesis in a new experimental model of IgA nephropathy. Mice were injected subcutaneously (back-pack mice) or intraperitoneally with hybridoma cells secreting either monoclonal IgA dimers, or monoclonal IgA monomers. The influence of immune complex formation was also tested in both these models. Renal IgA deposition was investigated 12 days after the injection of hybridoma cells. Backpack mice developed highly vascularized subcutaneous tumors. Mesangial IgA deposits were observed only in dimeric IgA hybridoma back-pack animals. No significant staining was observed in glomeruli from animals injected with hybridoma cells producing monomeric IgA. None of the hybridomas induced mesangial deposition when injected intraperitoneally. This animal model demonstrates the capacity of circulating IgA dimers to spontaneously form mesangial deposits and contributes to confirm the involvement of abnormalities of mucosal immunity in the pathogenesis of IgA nephropathy.

Most humoral non-responders to hepatitis B vaccines develop HBV-specific cellular immune responses.

About 10% of health care professionals vaccinated against hepatitis B virus (HBV) fail to develop protective antibodies. We tested the capacity of peripheral blood lymphocytes from 121 health care professionals, including 76 non-responders, to proliferate to four HBV vaccines, examined the proliferating cells' subset, production of IFN-gamma, IL-4 and IL-10, and for 22 subjects, the cytokine production genotype. Specific proliferative responses to at least one HBV antigen were noted in 75% humoral non-responders. These cells differed from the CD4+ strongly proliferating cells of responders. Non-responders frequently displayed a genotype of high TGF-beta and intermediate IL-10 secretion. Most humoral non-responders to HBV thus develop specific cellular immune responses, eventually liable to protect them against viral infection.

Autoimmunity and antigenic targets in ovarian pathology.

The involvement of autoimmune mechanisms in premature ovarian failure has been put forward by numerous investigators. In various other ovarian pathologies, such as idiopathic infertility, polycystic ovary syndrome, or endometriosis, similar mechanisms have been suggested. However, the exact role of autoimmunity in the pathophysiology of these diseases still remains controversial. The diagnosis of autoimmune ovarian disease relies on several clinical, biological and histological findings, but special interest has been focused on antiovarian autoantibodies. The search for these antibodies has been undertaken by several authors and yielded somewhat conflicting results which might be conditioned by methodological differences and by the multiplicity of potential immune targets. These targets, which comprise various steroidogenic enzymes, gonadotrophins and their receptors, the corpus luteum, zona pellucida and oocyte, are reviewed. Further investigation of these targets is required to improve the diagnostic tools that will lead to a precocious and reliable diagnosis of autoimmune ovarian disease, an appropriate clinical surveillance as well as the selection of patients who may benefit from immune-modulating therapy and possibly recover ovarian function and fertility.

[Recurrent pregnancy loss: which immunological laboratory tests?]. / Bilan immunologique d'une maladie abortive: quels examens demander?

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.

Immunological classification of acute myeloblastic leukemias: relevance to patient outcome.

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/CD117, CD7, CD35/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<3 x 10(-4)) in MA AML, CD7+ in MB AML, non-APL cases (P<0.03) in MC AML, CD34+ (P<0.002) and CD14+ (P<0.03) in MD AML, CD14+ in ME AML (P<0.01). The inclusion of seven key markers in the immunophenotyping of AML allows a stratification into clinically relevant subsets with individual prognostic factors, which should be considered to define high-risk AML populations.

Anti-myelin oligodendrocyte glycoprotein B-cell responses in multiple sclerosis.

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.

Enzymatic activities of bovine peripheral blood leukocytes and milk polymorphonuclear neutrophils during intramammary inflammation caused by lipopolysaccharide.

Leukocytes are recruited from peripheral blood into milk as part of the inflammatory response to mastitis. However, excessive accumulation of inflammatory cells alters the quality of milk and the proteases produced by polymorphonuclear neutrophils (PMNs) and macrophages may lead to mammary tissue damage. To investigate PMN recruitment and the kinetics of their intracytoplasmic enzymes in inflammation, we generated mastitis in six cows by intramammary infusion of lipopolysaccharide (LPS). Clinical signs of acute mastitis were observed in all of the cows, and normal status was resumed by 316 h. Intracytoplasmic elastase, collagenase, and cathepsin activities were measured within live cells by flow cytometry in peripheral blood leukocytes and milk PMNs before and during the inflammatory process (at 10 time points between 4 and 316 h). The proportion of immature PMNs was appreciated by CD33 surface labeling measured in flow cytometry. Leukopenia was observed in the peripheral blood 4 h postinfusion, concomitant to an increase in somatic cell counts in milk. CD33(+) PMNs were preferentially recruited from the peripheral blood to milk. Enzymatic activities were detected in PMNs, lymphocytes, and monocytes at levels depending on the cell type, sample nature, and time of collection. Milk PMNs had lower enzymatic activities than peripheral blood PMNs. This study showed that milk PMNs recruited during LPS-induced experimental mastitis have an immature phenotype and significantly lower enzymatic activities than peripheral blood PMNs. This suggests that CD33, an adhesion molecule, may be involved in the egress from blood to milk and that the enzymatic contents of PMNs are partly used during this process.

An immunoreactive peptide of the FSH involved in autoimmune infertility.

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.

Choroid plexus and ageing in rats: a morphometric and ultrastructural study.

Choroid plexuses (CP) are intraventricular structures involved in the production of cerebrospinal fluid (CSF) and in the synthesis and transport of numerous CSF components. Age-related modifications of the CP structure are still ill defined. We performed an ultrastructural and morphometric study of ageing CP in nine Sprague-Dawley rats 6, 18 and 30 months of age. Epithelial cells of CP villi were cubic in shape at 6 months, more dome-like at 18 months, and significantly flattened at 30 months of age. Epithelial basement membranes were thin and regular at 6 months, significantly thicker at 18 months and thicker and irregular at 30 months. Intravillous stroma increased nonhomogeneously with age. The ageing of CP in rats is characterized morphologically by epithelial atrophy, irregular fibrosis of the stroma and a thickening of epithelial basement membranes. These modifications suggest an alteration of secretory and filtrating functions in ageing CP.

Longitudinal study of rheumatoid arthritis patients discloses sustained elevated serum levels of soluble CD106 (V-CAM).

OBJECTIVE: To appreciate the evolution of serum angiogenic and/or adhesion molecules levels during a long term follow-up of rheumatoid arthritis (RA) patients. METHODS: Serum levels of 5 soluble adhesion/angiogenesis glycoproteins (VEGF, CD31, CD54, CD62E, CD106) were measured in Elisa in samples collected over 6 years in a cohort of 43 RA patients with monitored clinical parameters of disease activity and severity. RESULTS: RA patients had significantly higher levels (p < 0.0001) of sCD106 (VCAM-1) than control subjects. Conversely, the levels of soluble VEGF, CD31, CD54 and CD62E were normal or lower than normal. No statistically significant time effect was noted. No effect either was noted as related to the therapeutic agents taken by the patients. CONCLUSION: The sustained elevated serum levels of sCD106 observed here imply that this molecule might be related to the chronicity and progression of RA.

CSF-folate levels are decreased in late-onset AD patients.

Folates are involved in the cerebral metabolism of cobalamine, methionine, L-tyrosine and acetylcholine. Remarkably CSF-folate levels are 3 to 4 times higher than blood-folate levels. To reach the brain, folates are actively transported by choroid plexus (CP) as well as vitamins B6, B12, C and E. Epithelial atrophy having been reported in aging and in Alzheimer's disease (AD), we measured the CSF folate-levels of 126 patients, including 30 AD consecutive patients to evaluate whether CP functions of folate-transport were impaired. CSF-folate concentrations did not vary with age (10.47 +/- 1.93ng/ml between 20 and 60 years; 9.96 +/- 2.01 ng/ml in elderly control patients older than 60 years of age, p > 0.05) while late-onset AD patients had significantly lower CSF-folate levels (8.26 +/- 1.82 ng/ml, p < 0.001). These data support a specific alteration of CP transport function in AD patients.

Polyisotypic antipeanut-specific humoral responses in peanut-allergic individuals.

BACKGROUND: Peanut-containing food products may induce severe clinical reactions in sensitized subjects, and high levels of antipeanut IgE have been reported in the literature. Immunotherapy, proposed for the prevention of severe accidents, is often ill-tolerated and only partly efficient. This could be due to the spontaneous development of polyisotypic antipeanut antibodies. OBJECTIVE: To appreciate the presence and reactivity of other isotypes other than IgE of peanut-specific antibodies in serum samples from peanut-sensitized subjects. METHODS: Serum samples were obtained from 20 non-sensitized subjects and 23 sensitized patients divided in three groups according to their response to peanut oral challenge (no response or response to high or low doses, respectively). Peanut-specific IgG, IgG subclasses, IgA and IgM were assayed using an ELISA, and their reactivity against peanut proteins tested using Western Blot. RESULTS: A large dispersion of antipeanut antibody levels was observed in the three groups of patients, high levels of IgG, IgG1, IgG4 and IgA usually correlating with highly positive radioallergosorbent test (RAST). Such high levels were observed at onset in four patients who underwent peanut immunotherapy who had side effects and poor efficiency. Western blotting demonstrated that the polyisotypic response observed was directed to several peanut antigens, including the major allergens, Ara h1 and Ara h2. CONCLUSION: Peanut-sensitized patients who spontaneously develop specific IgE, display polyisotypic-specific antibody responses, whatever their response to oral challenge. This might explain the poor efficiency of peanut rush immunotherapy attempts.

Specific IgA to lactic acid bacteria in feces of children consuming milk fermented by yoghurt symbiosis and Lactobacillus casei (Danone strain DN 114 001).

An immunoreactive role of lactic acid bacteria established in animals has seldom been investigated in humans. In a large-scale clinical study, children from day-care centers received either yoghurt (Y), milk fermented by yoghurt symbiosis and Lactobacillus casei (DN 114 001) (YC), or gelified milk (GM) as diet supplements during two 30-day supplementation periods separated by one 30-day period without supplementation. Feces samples were collected before, during, and after the 2nd supplementation period. Proteins were extracted in a buffer containing enzymatic inhibitors. IgA levels were assessed and adjusted to the weight of feces samples. Specific IgA to lactic acid bacteria strains (Streptococcus thermophilus 8901A, 8902A; Lactobacillus bulgaricus; Lactobacillus casei) present in Y and YC were assayed in ELISA and adjusted to individual IgA levels. Mean levels of fecal IgA were within reported ranges for pediatric populations of similar age. IgA levels decreased significantly but transiently in children receiving Y, and increased significantly in children receiving GM, but did not vary in the group of children who were given YC. Specific IgA to the 4 strains tested increased significantly during the supplementation period only in the group of children receiving GM, while it was transient and not significant in children receiving YC. No variation was noted in children given Y Specific IgA to lactic acid bacteria can be assayed in feces. Supplementation with fermented milks might induce a mucosal tolerance to environmental flora.

Sequential C3 and C4 levels in human milk in relation to prematurity and parity.

Similarly to many immune molecules of human milk, C3 and C4 levels decrease during lactation. We investigated the influence, over the first three weeks of lactation, of both prematurity and parity on the sequential evolution of these levels. Milk C3 and C4 concentrations were measured by immunonephelometry in 494 individual samples collected from 76 lactating mothers. C3 and C4 concentrations were higher in milk from preterm or primiparous mothers. The major differences were observed in milk from days 5-8 and 9-20, likely due to pronounced interindividual variations in levels of days 1-4 milk. Milk from mothers of precocious (33 weeks' gestation or less) preterm newborns presented higher concentrations and a slower decrease of C3 and C4 levels than that from mothers of late (33-37 weeks' gestation) preterm newborns, when compared to term mothers. Finally, the inversion of the C3/C4 ratio occurring over time, previously reported, appeared later in milk from mothers of preterm newborns. The influence of prematurity was even greater in primiparous than in multiparous mothers. Both C3 and C4 levels therefore appear to be influenced in human milk by the parity and prematurity of the delivery. Mothers from preterm newborns seem to provide higher levels of C3 for a longer period post delivery.

Measurement of nine human milk proteins by nephelometric immunoassays: application to the determination of mature milk protein profile.

OBJECTIVES: Microparticle-enhanced nephelometric immunoassays for six human milk proteins (beta-casein, kappa-casein, alpha-lactalbumin, serum albumin, lactoferrin, and lysozyme) and conventional immunonephelometry assays for immunoglobulin A, C3, and C4 complement proteins were developed and characterized. DESIGN AND METHODS: Microparticle-enhanced nephelometric immunoassays are competitive assays based on the nephelometric quantification of the inhibition of microparticle-protein conjugates immunoagglutination by the proteins to be assayed. RESULTS: High precision (CVs ranged from 1% to 14% in within- and between-assays) and recovery (linear recovery in dilution-overloading assay) ensure a reliable determination of the main human milk proteins by single-step homogeneous nephelometric immunoassays, accurate over wide ranges of concentration. These immunoassays were easily applied to a large number of mature human milk samples (between 373 and 503 according to the proteins tested). CONCLUSIONS: The immunoassays developed could be applied to the fast determination of human milk protein profile usable for nursery milk bank and fortification.