Comparison of different blood collection, sample matrix, and immunoassay methods in a prenatal screening setting.

We compared how measurements of pregnancy-associated plasma protein A (PAPP-A) and the free beta subunit of human chorionic gonadotropin (fß-hCG) in maternal blood are influenced by different methods for blood collection, sample matrix, and immunoassay platform. Serum and dried blood spots (DBS) were obtained by venipuncture and by finger prick of 19 pregnant women. PAPP-A and fß-hCG from serum and from DBS were measured by conventional indirect immunoassay on an AutoDELFIA platform and by antibody microarray. We compared methods based on the recoveries for both markers as well as marker levels correlations across samples. All method comparisons showed high correlations for both marker concentrations. Recovery levels of PAPP-A from DBS were 30% lower, while those of fß-hCG from DBS were 50% higher compared to conventional venipuncture serum. The recoveries were not affected by blood collection or immunoassay method. The high correlation coefficients for both markers indicate that DBS from finger prick can be used reliably in a prenatal screening setting, as a less costly and minimally invasive alternative for venipuncture serum, with great logistical advantages. Additionally, the use of antibody arrays will allow for extending the number of first trimester screening markers on maternal and fetal health.

Indirect targeting of IGF receptor signaling in vivo by substrate-selective inhibition of PAPP-A proteolytic activity.

The insulin-like growth factor (IGF) signaling pathway is involved in certain human cancers, and the feasibility of directly targeting the IGF receptor has been actively investigated. However, recent evidence from clinical trials suggests that this approach can be problematic. We have developed an alternative strategy to indirectly inhibit the IGF signaling by targeting the metalloproteinase, pregnancy-associated plasma protein-A (PAPP-A). PAPP-A associated with the cell surface cleaves IGF binding protein-4 (IGFBP-4), when IGF is bound to IGFBP-4, and thereby increases IGF bioavailability for receptor activation in an autocrine/paracrine manner. We hypothesized that inhibition of PAPP-A would suppress excessive local IGF signaling in tissues where this is caused by increased PAPP-A proteolytic activity. To test this hypothesis, we developed an inhibitory monoclonal antibody, mAb 1/41, which targets a unique substrate-binding exosite of PAPP-A. This inhibitor selectively and specifically inhibits proteolytic cleavage of IGFBP-4 with an inhibitory constant (Ki) of 135 pM. In addition, it inhibited intracellular signaling of the IGF receptor (AKT phosphorylation) in monolayers of A549 cells, an IGF-responsive lung cancer-derived cell line found to express high levels of PAPP-A. We further showed that mAb 1/41 is effective towards PAPP-A bound to cell surfaces, and that it is capable of inhibiting PAPP-A activity in vivo. Using a murine xenograft model of A549 cells, we demonstrated that mAb 1/41 administered intraperitoneally significantly inhibited tumor growth. Analysis of xenograft tumor tissue recovered from treated mice showed penetration of mAb 1/41, reduced IGFBP-4 proteolysis, and reduced AKT phosphorylation. Our study provides proof of concept that IGF signaling can be selectively reduced by targeting a regulatory proteinase that functions extracellularly, upstream of the IGF receptor. PAPP-A targeting thus represents an alternative therapeutic strategy for inhibiting IGF receptor signaling.

Inflammatory biomarkers and coronary heart disease: from bench to bedside and back.

Inflammation plays a pivotal role in all stages of atherosclerosis from endothelial dysfunction and plaque formation to plaque destabilization and disruption. Inflammatory biomarkers, originally studied to better understand the pathophysiology of atherosclerosis, have generated increasing interest among clinicians, because of their utility in the challenging problems of diagnosis and risk assessment of patients with suspected or proved coronary heart disease. Moreover, in fascinating perspective, they could be used as therapeutic target, counteracting initiation, progression, and development of complications of atherosclerosis. In this review, we will provide an overview of the more promising inflammatory biomarkers, focusing on their utility and limitations in the clinical setting.

Resistance to age-dependent thymic atrophy in long-lived mice that are deficient in pregnancy-associated plasma protein A.

Pregnancy-associated plasma protein A (PAPPA) is a metalloproteinase that controls the tissue availability of insulin-like growth factor (IGF). Homozygous deletion of PAPPA in mice leads to lifespan extension. Since immune function is an important determinant of individual fitness, we examined the natural immune ecology of PAPPA(-/-) mice and their wild-type littermates reared under specific pathogen-free condition with aging. Whereas wild-type mice exhibit classic age-dependent thymic atrophy, 18-month-old PAPPA(-/-) mice maintain discrete thymic cortex and medulla densely populated by CD4(+)CD8(+) thymocytes that are capable of differentiating into single-positive CD4 and CD8 T cells. Old PAPPA(-/-) mice have high levels of T cell receptor excision circles, and have bone marrows enriched for subsets of thymus-seeding progenitors. PAPPA(-/-) mice have an overall larger pool of naive T cells, and also exhibit an age-dependent accumulation of CD44(+)CD43(+) memory T cells similar to wild-type mice. However, CD43(+) T cell subsets of old PAPPA(-/-) mice have significantly lower prevalence of 1B11 and S7, glycosylation isoforms known to inhibit T cell activation with normal aging. In bioassays of cell activation, splenic T cells of old PAPPA(-/-) mice have high levels of activation antigens and cytokine production, and also elicit Ig production by autologous B cells at levels equivalent to young wild-type mice. These data suggest an IGF-immune axis of healthy longevity. Controlling the availability of IGF in the thymus by targeted manipulation of PAPPA could be a way to maintain immune homeostasis during postnatal development and aging.

Quantification of proteolytically active pregnancy-associated plasma protein-A with an assay based on quenched fluorescence.

BACKGROUND: Maternal serum concentrations of pregnancy-associated plasma protein-A (PAPP-A, pappalysin-1, EC 3.4.24.79) are used to predict the occurrence of Down syndrome. In pregnancy, PAPP-A primarily circulates as a covalent 2:2 complex with the proform of eosinophil major basic protein (proMBP), which inhibits the proteolytic activity of PAPP-A. At term, however, approximately 1% of PAPP-A exists as an active, uncomplexed dimer with proteolytic activity directed specifically toward insulin-like growth factor binding protein (IGFBP)-4 and IGFBP-5. No assays have been developed that allow quantification of PAPP-A proteolytic activity. METHODS: We developed a sensitive and specific immunocapture assay for PAPP-A activity based on intramolecular quenched fluorescence. We used a 26-residue synthetic peptide derived from IGFBP-4 in which specific positions on each side of the PAPP-A cleavage site were substituted with 3-nitrotyrosine and o-aminobenzoic acid. RESULTS: The assay detected the activity of recombinant PAPP-A as well as PAPP-A in serum samples from pregnant women. The limit of detection (mean signal of blank plus 3 SD) of the active PAPP-A subunit was 13 pmol/L, and the intra- and interassay CVs were <10% and <15%, respectively. Interestingly, the fraction of active PAPP-A decreased gradually from week 7 to week 19 of pregnancy. CONCLUSIONS: This method allows the measurement of PAPP-A enzymatic activity and because of its specificity it is relevant to the study of the biological function of PAPP-A. The method may also be useful in the diagnosis of pregnancy disorders.

Review: immunomodulatory activity of pregnancy-associated plasma protein-A.

This mini-review highlights the growing number of indications for the immunological importance of pregnancy-associated plasma protein-A (PAPP-A), which is a remote member of the alpha-macroglobulin plasma protein family. PAPP-A can bind a variety of cytokines and specifically cleave a binding protein for insulin-like growth factors, thereby serving as a modulator of cytokine activity. Important immune functions, such as lymphocyte proliferation response to alloantigens and lectins and expression of HLA-DR molecules are predominantly suppressed in vitro by PAPP-A. It is likely that the immunoregulatory properties of PAPP-A are very similar to that of alpha 2-macroglobulin. The experimental data allows us to suppose that PAPP-A serves to prevent the recognition of the fetus by the maternal immune system and to suppress locally the host's immune response to the tumour.

Production and characterization of monoclonal antibodies against pregnancy-associated plasma protein A.

Pregnancy-associated plasma protein A (PAPP-A) was found to be a good first trimester maternal serum marker, together with free beta-human chorionic gonadotrophin (HCG) subunits, for the biochemical screening of fetal trisomy 21 (Down's syndrome). We have raised monoclonal antibodies (mAbs) against PAPP-A purified from human pregnancy serum. The different antibodies were characterized biochemically by Western blot analysis and in terms of specificity (reaction with non-pregnant and male serum). Their performance in Down's syndrome screening was assessed in comparison with an existing enzyme-linked immunosorbent assay method after labelling of the different mAbs with biotin or horseradish peroxidase. A pair of mAbs was eventually chosen for a double-antibody sandwich protocol. The selected combination was found to have a significantly increased specificity (P = 0.0116) over the method using (purified) polyclonal antibodies, together with slightly increased sensitivity. In our limited number of Down's syndrome pregnancy samples (n = 17) and controls (n = 18), the medians as well as the multiples of the median values (for the affected cases) were comparable between the two methods described.

Dual-label time-resolved immunofluorometric assay for simultaneous determination of pregnancy-associated plasma protein A and free beta-subunit of human chorionic gonadotrophin.

Using time-resolved fluorometry, a simple one-step dual-label immunometric assay has been developed, which allows simultaneous determination of pregnancy-associated plasma protein A (PAPP-A) and free beta-subunit of human chorionic gonadotrophin (beta-hCG) in first-trimester maternal serum samples. Two monoclonal antibodies were biotinylated and immobilized onto the surface of streptavidin-coated microtitration plates, and used to capture PAPP-A and beta-hCG. respectively. Europium (Eu) and Samarium (Sm) chelates were conjugated to two additional monoclonal antibodies acting as detection antibodies for PAPP-A and beta-hCG. The assay was performed using a 4-h one-step format. The within-run precision with buffer-based calibrators was below 8% over the working range of PAPP-A (40-10000 mIU/l) and beta-hCG (7.3-525 micrograms/l) and no hook effect was observed. The intra- and inter-assay coefficients of variation were below 7.1% for serum samples. PAPP-A and beta-hCG concentrations measured by the dual assay in 39 first-trimester serum samples correlated excellently with those obtained by DELFIA single-label PAPP-A (r = 0.997) and the beta-hCG part (r = 0.993) of the DELFIA AFP/beta hCG dual-label assay.

Rheumatoid arthritis and pregnancy.

Although some 70% of patients with rheumatoid arthritis remit during pregnancy, the mechanism for this is not known. A variety of mechanisms have been invoked but none appear to be sufficiently established as an explanation. The most likely explanation is that a factor, or factors, cause the inhibition of release of lymphokines which thereby have a suppressive effect on rheumatoid inflammation.

Immunochemical and biochemical relationship between human pregnancy-associated secreted endometrial alpha 1- and alpha 2-globulins (alpha 1- and alpha 2-PEG) and the soluble placental proteins 12 and 14 (PP12 and PP14).

Two proteins, pregnancy-associated endometrial alpha 1- and alpha 2-globulins (alpha 1- and alpha 2-PEG), synthesized de novo and secreted by the human endometrium and decidua during pregnancy, have been demonstrated to be immunochemically related to the soluble placental proteins PP12 and PP14 isolated from term placenta. However, although these immunochemically similar endometrially- and placentally-derived proteins differ in their reported biochemical properties, in this report we have demonstrated that PP12 and PP14 are biochemically identical to alpha 1- and alpha 2-PEG with respect to subunit size. We conclude that these placental proteins are derived from the endometrium by de novo synthesis, and we suggest that the localization of these proteins to the placenta reflects either absorption, specific binding or processing by the trophoblast. The significance of clinical studies involving PP12 and PP14 measurement must therefore be reassessed in the light of their exclusive endometrial origin.

In vitro release of pregnancy-associated plasma protein-A (PAPP-A) by human endometrial cells.

Evidence has now accumulated to sustain the idea that pregnancy-associated plasma protein-A (PAPP-A) is not pregnancy specific: it can be localized immunohistochemically in nonpregnant human endometrium. Thus, it was of interest to see if a primary cell culture of human endometrial cells was capable of producing PAPP-A. Immunoreactive PAPP-A-like material was detected in the culture medium of glandular as well as stromal cells. The presence of PAPP-A in endometrial culture medium was specific and could not be attributed to a nonspecific protease effect on the tracer used in radioimmunoassay. The production rate of PAPP-A by stromal cells could be enhanced by the addition of medroxyprogesterone acetate (MPA). Estradiol (E2) alone or in combination with MPA did not modify the production rate of PAPP-A. The production rate of PAPP-A by glandular cells remained unchanged after treatment with MPA and/or E2. These results confirm that in nonpregnant women PAPP-A is a progesterone-dependent protein produced by the endometrium.

[Non-specific immunological depression in pregnancy]. / La dépression immunitaire non spécifique de la grossesse.

The inhibitory effect of maternal plasma on in vitro lymphocyte responses shows that non specific inhibitory factors appear in early pregnancy. During the last decade the surge of interest in pregnancy proteins accompagnied by the advancement in immunological methodology has led to the isolation and characterization of these inhibitory factors. These pregnancy associated proteins play an important role in survival of the foetal allograft. It is significant that low plasma levels of inhibitory factors in early pregnancy are detected in women in whom spontaneous abortion occur.

Histochemical localization of pregnancy-associated plasma protein A in fetal, infant, and adult organs and comparison between antisera.

Histochemical localization of pregnancy-associated plasma protein A (PAPP A) was performed on various genital and extragenital organs from fetuses, infants, and adults. Three types of PAPP A reactive cells were found: endocrine, epithelial, and hematopoietic cells. Positive endocrine organs included the decidua and the fetal adrenal and Leydig cells. Positive epithelial cells were found in actively proliferating tissues, such as fetal renal tubules, fetal intestinal mucosa, and adult proliferative endometrial glands. In bone marrow, red cell precursors were PAPP A positive. Comparison of a commercially available antiserum with one raised in our own laboratory revealed significant differences in histochemical staining. The biological relevance of these findings remains to be determined.

The effect of excess antigen on the enzyme-linked immunosorbent assay of onco-placental proteins.

The time course of the antigen-antibody reaction between human chorionic gonadotrophin, pregnancy specific beta 1 glycoprotein and pregnancy associated plasma protein A and their respective immobilised antibodies has been investigated. All 3 antigens showed enhanced binding with increasing length of incubation up to a maximum value, followed by a decrease. It is suggested that the diminution in binding at high antigen concentration is caused by the solubilisation of the immobilised antigen-antibody complex and that this is promoted by the high concentration of antigen.

Immunological studies of the human placenta: functional and morphological analysis of pregnancy-associated plasma protein A (PAPP-A).

A blood protein, pregnancy-associated plasma protein A (PAPP-A), has been isolated from the plasma of pregnant women. An antiserum to this protein has been prepared and used to localize the protein within the placenta by immunofluorescence. It was found to be inconstantly present in the syncytiotrophoblast and intervillous fibrin. Neither antiserum nor antigen exerts a cytotoxic effect on peripheral blood lymphocytes, and neither affected blastogenic responses of lymphocytes to mitogens or in mixed lymphocyte culture reactions.