Anti-placental Alkaline Phosphatase Research Articles

135 Purification and characterization of human placental-derived exosomes from maternal serum using size exclusion chromatography

Exosomes are extracellular vesicles that contain a diverse array of signaling molecules and have a role in intercellular signaling. Placental tissues release exosomes that have been identified in maternal blood. The purpose of this study is to evaluate a method of exosome isolation using size-exclusion chromatography (SEC) columns to purify exosomes in a reproducible, time-efficient manner, enabling analysis of the biological activity of placental-derived exosomes (PDE). Stored samples from a previous prospective longitudinal study of women with uncomplicated pregnancies were used. Exosomes were isolated from third trimester and postpartum (24–48h after delivery) serum samples using SEC (Fig.1). We then confirmed the presence of exosomes using Nanodrop spectrophotometry (Abs 280 nm), Nanoparticle Tracking Analysis (NTA), and Western blot analysis for protein quantification, size determination and presence of syncytiotrophoblast specific marker Anti-Placental Alkaline Phosphatase (PLAP), respectively. After SEC of maternal serum (n=10), NTA revealed particle size to be 60 and 70 nm, on average. NTA and Nanodrop A280 of SEC fractions revealed a distinct exosome peak in both particle concentration and total protein content, indicating the presence of exosomes that could be further processed and analyzed. Western blot analysis of the proteins identified presence of syncytiotrophoblast specific marker PLAP in all samples (Fig.1). PDEs were present in maternal serum 24–48 hours after delivery. Although there were significant differences in the concentrations of exosomes in maternal serum from third trimester versus postpartum within individual patients, there was no consistent correlation of concentration difference between individual patients (Fig.2). SEC is a simple and reproducible technique to purify placental-derived exosomes from maternal serum. Placental exosomes are present in the serum of recently postpartum patients. These findings will facilitate further studies in the biological functions of placental-derived exosomes.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

The N-terminal Amphipathic α-Helix of Viperin Mediates Localization to the Cytosolic Face of the Endoplasmic Reticulum and Inhibits Protein Secretion

Viperin is an evolutionarily conserved interferon-inducible protein that localizes to the endoplasmic reticulum (ER) and inhibits a number of DNA and RNA viruses. In this study, we report that viperin specifically localizes to the cytoplasmic face of the ER and that an amphipathic α-helix at its N terminus is necessary for the ER localization of viperin and sufficient to promote ER localization of a reporter protein, dsRed. Overexpression of intact viperin but not the amphipathic α-helix fused to dsRed induced crystalloid ER. Consistent with other proteins that induce crystalloid ER, viperin self-associates, and it does so independently of the amphipathic α-helix. Viperin expression also affected the transport of soluble but not membrane-associated proteins. Expression of intact viperin or an N-terminal α-helix-dsRed fusion protein significantly reduced secretion of soluble alkaline phosphatase and reduced its rate of ER-to-Golgi trafficking. Similarly, viperin expression inhibited bulk protein secretion and secretion of endogenous α1-antitrypsin and serum albumin from HepG2 cells. Converting hydrophobic residues in the N-terminal α-helix to acidic residues partially or completely restored normal transport of soluble alkaline phosphatase, suggesting that the extended amphipathic nature of the N-terminal α-helical domain is essential for inhibiting protein secretion.

Ultrastructural localization of glycoprotein IIIa (GPIIIa, β3 integrin) on placental syncytiotrophoblast microvilli: implications for platelet alloimmunization during pregnancy

Fetal and neonatal alloimmune thrombocytopenia due to anti-human platelet antigen (HPA)-1a more commonly occurs in first pregnancies, unlike hemolytic disease of the newborn. Anti-D is produced after D+ fetomaternal hemorrhage; this usually occurs at parturition. Anti-HPA-1a could develop during pregnancy if maternal immunization is stimulated by HPA-1a expressed not only on platelets but also on other fetal cells. An ultrastructural study of fetal placental chorionic villi was undertaken to determine the localization of glycoprotein (GP)IIIa carrying the HPA-1a/1b polymorphism. First trimester and term villi were incubated with a monoclonal antibody (MoAb) to GPIIIa or with positive control MoAbs (anti-placental alkaline phosphatase and ED822 MoAb) to villous syncytiotrophoblast (ST). Binding of MoAbs was detected with a gold-conjugated secondary antibody before processing the tissues and examination of ultrathin sections in an electron microscope. Gold particles were evident on microvilli on the apical surface of ST when labeled with anti-GPIIIa and the placenta-specific MoAbs but not with an isotype control antibody. Immunolabeling for anti-GPIIIa on first trimester ST was similar to that of term ST. The apical surface of the ST is bathed in maternal blood. During the natural regenerative process of human placenta, senescent parts of the ST are shed into maternal blood during pregnancy. This includes both apoptotic ST nuclei and microparticulate ST debris. The presence of GPIIIa on this circulating ST cellular material could be the source of HPA-1a alloantigen causing primary immunization of susceptible primigravidae early enough for anti-HPA-1a to cause fetal thrombocytopenia during a first pregnancy.

Vitamins C and E Inhibit Apoptosis of Cultured Human Term Placenta Trophoblast

Preeclampsia can be lethal to both mother and baby. The prominent symptoms of this syndrome are hypertension, proteinuria and oedema, resulting from an exaggerated aseptic systemic inflammatory response, triggered by placental factors shed into the maternal circulation. Syncytiotrophoblast microparticles (STBM) are one possible factor, shed when the placenta is exposed to stressors such as hypoxia/reperfusion. These can disrupt mitochondria, triggering apoptosis and necrosis, placental pathologies which are increased in preeclampsia. We tested the effects of antioxidant vitamins C (50 μM) and E (50 μM) on trophoblast in culture, using term villous cytotrophoblast preparations. Following Percoll gradient centrifugation and MHC class I expressing cell depletion of placenta digests, syncytial fragments were removed using anti-placental alkaline phosphatase antibody. This yielded cytotrophoblasts of consistently high purity. EGF (10 ng/ml) stimulated syncytialisation and hCG and progesterone production. However, mitochondrial induced apoptosis (MIA) was evident 96 h post-isolation, as mitochondrial membrane potential loss and caspase 9 and caspase 3 activation. ROCK-1 cleavage and syncytiotrophoblast particle shedding increased concurrently with apoptosis induction. Vitamins blocked MIA and syncytiotrophoblast particle shedding and significantly increased hCG ( p < 0.005) and progesterone ( p < 0.02) concentrations in culture supernatants, reflecting the increased survival rates. Although more cells survived in culture, syncytialisation rate (%) was significantly reduced ( p < 0.005). We conclude that vitamins C and E can significantly reduce mitochondrial damage generated following syncytialisation in vitro. However, further work is required to determine whether antioxidant vitamins interfere with normal fusion processes.

Tumor Radioimmunolocalization in Nude Mice by Mono- and Divalent- Single-Chain Fv Antiplacental Alkaline Phosphatase Antibodies

One single-chain Fv antibody fragment (scFv) and a new recombinant covalently linked dimeric scFv antibody (sc(Fv)(2)) against placental alkaline phosphatase (PLAP) were investigated for selective tumor targeting. The biological behavior of these new antibodies was compared to that of the original native antibody, H7 MAb. The sc(Fv)(2)) antibody displayed convincing tumor localization properties with a rapid excretion pattern comparable to the scFv, but with a longer retention time in the tumor, and higher tumor-to-nontumor ratio (27:1), compared to the scFv (15:1), at 48 hours. For the sc(Fv)(2) antibody, more than 50% of the remaining activity in the mouse was present in the tumor between 24 and 48 hours after the injection. With this antibody, scintigraphic visualization of the tumor was also possible 1 week after the injection. It is concluded that this sc(Fv)(2) antibody fragment, with two binding sites, displays properties suitable for in vivo targeting of PLAP expressing tumors.

The p14 Fusion-associated Small Transmembrane (FAST) Protein Effects Membrane Fusion from a Subset of Membrane Microdomains

The p14 Fusion-associated Small Transmembrane (FAST) Protein Effects Membrane Fusion from a Subset of Membrane Microdomains

Immunostaining for placental alkaline phosphatase on fine‐needle aspiration specimens to detect noninvasive testicular cancer: a prospective evaluation in cryptorchid men

To assess, in a 12-year prospective study, the potential for early detection of testicular carcinoma in situ (CIS) by immunocytochemistry, using anti-placental alkaline phosphatase (PLAP) monoclonal antibodies on testicular fine-needle aspiration cytology (FNAC) specimens taken from a group of formerly cryptorchid patients, as such men are at greater risk of developing testicular cancer. Sixty-eight men who had had orchidopexy at the Urological Department of the University of Padova between 1975 and 1983 were evaluated first in 1993, by a protocol including a history, physical examination, testicular ultrasonography and serum tumour markers, to eliminate the presence of testicular cancer. In 57 of the 68 men, specimens taken from bilateral testicular FNAC were stained immunocytochemically using anti-PLAP monoclonal antibodies. After 8 years, the same protocol was repeated on the 57 men, and the follow-up was prolonged until March 2005 for men with previous positive PLAP immunostaining. In 1993, six of the 57 men, (10.5%) had unilateral positive immunostaining for PLAP. By 2001, none of these men had developed testicular cancer, while of the other 51 men, only one developed a nonseminomatous tumour. The uninterrupted surveillance of PLAP-positive men showed no overt cancer until March 2005. The present findings do not seem to confirm the reliability of PLAP immunostaining of testicular specimens from FNAC for detecting CIS. These findings might depend on the geographical variability of both CIS and testicular cancer incidence, as well as on the variable relationship between CIS and successive occurrence of invasive testis cancer.

Palmitoylation and Intracellular Domain Interactions Both Contribute to Raft Targeting of Linker for Activation of T Cells

Some transmembrane proteins must associate with lipid rafts to function. However, even if acylated, transmembrane proteins should not pack well with ordered raft lipids, and raft targeting is puzzling. Acylation is necessary for raft targeting of linker for activation of T cells (LAT). To determine whether an acylated transmembrane domain is sufficient, we examined raft association of palmitoylated and nonpalmitoylated LAT transmembrane peptides in lipid vesicles by a fluorescence quenching assay, by microscopic examination, and by association with detergent-resistant membranes (DRMs). All three assays detected very low raft association of the nonacylated LAT peptide. DRM association was the same as a control random transmembrane peptide. Acylation did not measurably enhance raft association by the first two assays but slightly enhanced DRM association. The palmitoylated LAT peptide and a FLAG-tagged LAT transmembrane domain construct expressed in cells showed similar DRM association when both were reconstituted into mixed vesicles (containing cell-derived proteins and lipids and excess artificial raft-forming lipids) before detergent extraction. We conclude that the acylated LAT transmembrane domain has low inherent raft affinity. Full-length LAT in mixed vesicles associated better with DRMs than the peptide. However, cells appeared to contain two pools of LAT, with very different raft affinities. Since some LAT (but not the transmembrane domain construct) was isolated in a protein complex, and the Myc- and FLAG-tagged forms of LAT could be mutually co-immunoprecipitated, oligomerization or interactions with other proteins may enhance raft affinity of one pool of LAT. We conclude that both acylation and other factors, possibly protein-protein interactions, target LAT to rafts.

Antitumor effect of radioimmunotherapy in a mouse model of testicular tumor with micrometastases defined by polymerase chain reaction

The efficacy of radioimmunotherapy (RIT) was evaluated by using a mouse model of testicular tumor with macro- and micrometastases to various organs. RIT consisting of a single intravenous injection of 5.8 MBq of I-131-labeled anti-placental alkaline phosphatase (PLAP) MAb was conducted one day or 7 days after testicular implantation in SCID mice of HeLa Hep2 cells expressing PLAP. RIT antitumor effect was significant for primary tumors as well as micrometastases defined by polymerase chain reaction (PCR) assay. However, ablation of tumor cells could be achieved with this treatment only at the initial stage of tumor growth in the testis, when metastasis could also be prevented. Once micrometastasis had occurred, however, complete elimination of tumor cell(s) was difficult. These findings appear to have implications for the use of RIT in treatment for micrometastasis, especially when detectable only by PCR assay.

Immunolocalization of anti-placental alkaline phosphatase monoclonal antibody in mice with testicular tumors and lymph node metastasis.

To evaluate the ability of an anti-placental alkaline phosphatase (PLAP) monoclonal antibody (MAb) to localize to PLAP-expressing tumors, we established a model of testicular tumor with metastasis to lymph nodes and liver in severe combined immunodeficient (SCID) mice. 131I-labeled or 125I-labeled MAb was simultaneously administered via the intravenous or lymphatic route, respectively. Preferential accumulation of MAb in PLAP-expressing tumors at primary as well as metastatic sites was demonstrated. The percentage of the injected dose of MAb found in the tumor was generally higher when MAb was administered intravenously. Identical tumor/blood ratios were found with the two routes of administration. These data suggest that intravenous administration of a radiolabeled MAb is superior to lymphatic administration for tumor imaging and radioimmunotherapy.

The T Cell-directed CC Chemokine TARC Is a Highly Specific Biological Ligand for CC Chemokine Receptor 4

Thymus and activation-regulated chemokine (TARC) is a recently identified CC chemokine that is expressed constitutively in thymus and transiently in stimulated peripheral blood mononuclear cells. TARC functions as a selective chemoattractant for T cells that express a class of receptors binding TARC with high affinity and specificity. To identify the receptor for TARC, we produced TARC as a fusion protein with secreted alkaline phosphatase (SEAP) and used it for specific binding. By stably transfecting five orphan receptors and five known CC chemokine receptors (CCR1 to -5) into K562 cells, we found that TARC-SEAP bound selectively to cells expressing CCR4. TARC-SEAP also bound to K562 cells stably expressing CCR4 with a high affinity (Kd = 0.5 nM). Only TARC and not five other CC chemokines (MCP-1 (monocyte chemoattractant protein-1), RANTES (regulated upon activation, normal T cells expressed and secreted), MIP-1alpha (macrophage inflammatory protein-1alpha), MIP-1beta, and LARC (liver and activation-regulated chemokine)) competed with TARC-SEAP for binding to CCR4. TARC but not RANTES or MIP-1alpha induced migration and calcium mobilization in 293/EBNA-1 cells stably expressing CCR4. K562 cells stably expressing CCR4 also responded to TARC in a calcium mobilization assay. Northern blot analysis revealed that CCR4 mRNA was expressed strongly in human T cell lines and peripheral blood T cells but not in B cells, natural killer cells, monocytes, or granulocytes. Taken together, TARC is a specific functional ligand for CCR4, and CCR4 is the specific receptor for TARC selectively expressed on T cells.

Factors Contributing to Imaging of Xenografts Using Anti-Placental Alkaline Phosphatase Monoclonal Antibody

Factors Contributing to Imaging of Xenografts Using Anti-Placental Alkaline Phosphatase Monoclonal Antibody

Factors Contributing to Imaging of Xenografts Using Anti-Placental Alkaline Phosphatase Monoclonal Antibody

Purpose To investigate factors that influence the imaging of placental alkaline phosphatase (PLAP) producing xenografts using an anti-PLAP monoclonal antibody (MAb). Materials and Methods Three xenografts (human seminoma, HeLa Hep 2 cells, and KK-47 bladder cancer cells) each expressing PLAP to a different degree were used to immunolocalize an anti-PLAP MAb, HPMS-1. Results Although the highest PLAP level was found in seminoma xenografts, the MAb was not useful for the imaging of seminoma xenografts because of poor accumulation. Fragmentation of the MAb, such as F(ab')2, however, was shown to be efficient for imaging seminoma xenografts. A distribution study with T1-201 revealed the highest blood flow in HeLa cells and the lowest in seminoma. A difference in blood flow may partially explain the disparity between the amount of MAb accumulation and the level of antigen expression in these three xenografts. Conclusions Blood flow in targeted tumors was shown to be more critical than their level of antigen expression for the imaging of xenografts with anti-tumor antibody. In addition, fragmentation of the MAb enabled tumor imaging because of a rapid clearance of the fragment from the circulation.

Combinations of nonlabeled, 125I-Labeled, and Anti-Idiotypic Antiplacental Alkaline Phosphatase Monoclonal Antibodies at Experimental Radioimmunotargeting

Combinations of nonlabeled, 125I-Labeled, and Anti-Idiotypic Antiplacental Alkaline Phosphatase Monoclonal Antibodies at Experimental Radioimmunotargeting

Construction, expression and characterisation of a single chain anti-tumour antibody (scFv)-EL-2 fusion protein

We describe the production and preliminary characterisation of a fusion protein between interleukin-2 and a single-chain Fv version of the H17E2 anti-placental alkaline phosphatase (PLAP) antibody. This molecule could be used to target interleukin-2 to PLAP-expressing tumours.

Syngeneic anti-idiotypic antibodies eliminate excess radiolabeled idiotypes at experimental radioimmunolocalization.

Significant improvements in tumor/nontumor ratio can be achieved by injections of nonlabeled anti-idiotypic monoclonal antibodies (MAbs) during radioimmunolocalization and radioimmunotherapy using MAbs to target experimental tumors. The in vivo effects of an anti-idiotypic MAb (alpha H7) against a radioiodinated, high affinity, low dissociation rate, monoclonal antiplacental alkaline phosphatase antibody (H7) was investigated. Following in vivo injection of the anti-idiotypic MAb, the radioactivity in experimental tumors was found to decrease only 25% while the reduction of corresponding radioactivity in nontumor tissues amounted to 65-85%, compared to the group receiving no anti-idiotypic MAbs. These results indicate that it is possible to partially clear the circulation and nontumor tissues from excess of radiolabeled idiotypic antibody, without significant decrease in specific tumor localization, increasing the tumor/nontumor ratio three- to fourfold. Circulating nontumor targeting radiolabeled antibodies is one of the major limiting factors in radioimmunotherapy today. Injection of anti-idiotypic MAbs could selectively significantly reduce the radiation dose to radio-sensitive tissues, i.e., bone marrow and intestine, thus improving efficiency in radioimmunoscintigraphy and radioimmunotherapy.

Sertoli cells of intratubular germ cell neoplasia express beta 2 microglobulin.

The cells in intratubular germ cell neoplasia in the vicinity of 38 germ cell tumors of the testis, including 20 pure seminomas, were studied for the expression of beta 2-microglobulin (beta 2m), the constant component of all HLA class I molecules. Immunohistochemistry using antibodies towards beta 2m, vimentin, placental alkaline phosphatase, and ferritin was employed. Whereas the intratubular cells in normal testis are beta 2m negative, beta 2m positive cells were identified in intratubular germ cell neoplasia tubules in 55 per cent of all tumors and in 60 per cent of the seminomas. The tubules with beta 2m positive cells were located in areas with invasive tumor or in the vicinity of such areas. The beta 2m positive cells were identified as Sertoli cells by morphology and by their staining with anti-vimentin. Neoplastic germ cells, identified by morphology and staining with anti-placental alkaline phosphatase and anti-ferritin were beta 2-microglobulin negative. The most intensely beta 2m-stained Sertoli cells were found in tubules with high concentrations of neoplastic germ cells. Intensely stained Sertoli cells were also found in 'Sertoli cell only' tubules inside invasive tumors and in areas without lymphocytic infiltration. The cells in adjacent normal tubules were beta 2m negative.

Pagetoid spread of intratubular germ cell neoplasia into rete testis: A morphologic and histochemical study of 100 orchiectomy specimens with invasive germ cell tumors

Intratubular germ cell neoplasia (ITGCN) is now considered to be the preinvasive phase of testicular germ cell tumors with the exceptions of spermatocytic seminoma, pure yolk sac tumor, and mature teratoma. Pagetoid spread of ITGCN into rete testis is a common yet unpublished finding in these cases. We reviewed 100 cases of testicular germ cell tumors from the Surgical Pathology service of Parkland Memorial Hospital (Dallas, TX) to evaluate the frequency of this pattern of spread. Additional sections were obtained from selected cases and were stained with anti-placental alkaline phosphatase, anti-low molecular weight keratin (clone AE1), and various lectins to highlight the process. Pagetoid spread of ITGCN into rete testis was identified in 24 of 60 cases (40%) in which histologic sections contained both ITGCN and rete testis. The incidence of pagetoid ITGCN involvement of the rete testis was lower in pure seminoma (seven of 25 cases [28%]) than in testes containing nonseminomatous germ cell tumors (17 of 35 cases [49%]). AE1 stained the epithelial cells of the rete testis but not the cells of the ITGCN, whereas placental alkaline phosphatase stained the neoplastic cells but not the epithelial cells of the rete testis. These stains were useful in delineating two cases in which the pagetoid involvement was so extensive that they were misdiagnosed as invasive seminomas. Pagetoid spread of ITGCN is a relatively common finding in testicular germ cell tumors and rarely can be mistaken for invasive seminoma. Immunohistochemistry can be helpful in distinguishing florid pagetoid spread from invasive seminoma.

Correlation of beta-camera imaging and immunohistochemistry in radioimmunotherapy using90Y-labeled monoclonal antibodies in ovarian cancer animal models

Tumor stroma contains much fibrin and monoclonal antifibrin antibody targeting is possible in tumors. In this study, nude mouse human ovarian carcinoma xenograft specimens were investigated after treatment with 90Y-labeled monoclonal antifibrin antibody Fab fragment or with 90Y-labeled OC125-monoclonal antibody F(ab')2 fragments. The mice received the radioimmunotherapy activity either intratumorally, intraperitoneally, or intravenously. Beta-camera imaging (BCI) is a novel device for studying activity distribution in tissue specimens and, together with immunohistochemistry (IHC) with OC125, antifibrin, anticarcinoembryonic antigen, anti-cytokeratin, and anti-placental alkaline phosphatase antibodies, was used for correlation of activity distribution of tissue specimens. These results were in concordance: Antigen distribution measured with IHC and radioactivity distribution were similar with the same antibodies, antifibrin, and OC125: However, these antigens demonstrated rather different distribution. Tissue studies revealed that activity was concentrated also in the necrotic tumor tissue, indicating that cell death was also caused by radiation. Differences in the tumor cell morphology were observed using different routes of administration. With BCI, it is possible to quantitate activities in frozen sections (microdosimetry), and these results were in concordance with absolute activities as measured by tissue sampling and well-counting. Three-dimensional reconstruction of tissue slices combined with radioactivity distribution measured with BCI allows estimation of total absorbed radiation dose in tumor after an appropriate dose planning.

Diagnostic tools for differentiating pleural mesothelioma from lung adenocarcinoma in paraffin embedded tissue. II. Design of an expert system and its application to the diagnosis of mesothelioma.

A panel of 14 antibodies (panepithelial antibody Lu-5, anti-keratin-18, anti-keratin-7, Ber-EP4, anti-Leu-M1, HEA-125, anti-carcinoembryonic antigen, anti-blood group-related antigens A, B, H, B72.3, anti-placental alkaline phosphatase, anti-vimentin and BMA-120), which have been evaluated for use in differentiating mesothelioma from lung adenocarcinoma, was applied to a group of 24 suspected mesotheliomas. Using the established qualitative, descriptive criteria derived from monovariate statistical analysis of the tumour control groups (definite mesotheliomas, adenocarcinomas), a definitive allocation was possible in only 25% of suspected cases. We therefore constructed two "expert systems", based on multivariate discriminant analysis with either the ALLOC 80 program for ordinal data or a newly developed analysis program for binomial data. With these two systems diagnostic allocation of suspected mesotheliomas was improved to 75% and 79%. The use of binomial data ("positive" versus "negative") in conjunction with the probability-based test system is of particular interest because the primary data are easy to record and the test results have a higher statistical probability.