Intestinal alkaline phosphatase at the crossroad of intestinal health and disease - a putative role in type 1 diabetes.

BACKGROUND: Patients with type 1 diabetes have shown an increase in circulating cytokines, altered lipoprotein metabolism and signs of vascular dysfunction in response to high-fat meals. Intestinal alkaline phosphatase (IAP) regulates lipid transport and inflammatory responses in the gastrointestinal tract. We therefore hypothesized that changes in IAP activity could have profound effects on gut metabolic homeostasis in patients with type 1 diabetes. METHODS: Faecal samples of 41 nondiabetic controls and 46 patients with type 1 diabetes were analysed for IAP activity, calprotectin, immunoglobulins and short-chain fatty acids (SCFAs). The impact of oral IAP supplementation on intestinal immunoglobulin levels was evaluated in C57BL/6 mice exposed to high-fat diet for 11 weeks. RESULTS: Patients with type 1 diabetes exhibited signs of intestinal inflammation. Compared to controls, patients with diabetes had higher faecal calprotectin levels, lower faecal IAP activities accompanied by lower propionate and butyrate concentrations. Moreover, the amount of faecal IgA and the level of antibodies binding to oxidized LDL were decreased in patients with type 1 diabetes. In mice, oral IAP supplementation increased intestinal IgA levels markedly. CONCLUSION: Deprivation of protective intestinal factors may increase the risk of inflammation in the gut - a phenomenon that seems to be present already in patients with uncomplicated type 1 diabetes. Low levels of intestinal IgA and antibodies to oxidized lipid epitopes may predispose such patients to inflammation-driven complications such as cardiovascular disease and diabetic nephropathy. Importantly, oral IAP supplementation could have beneficial therapeutic effects on gut metabolic homeostasis, possibly through stimulation of intestinal IgA secretion.

Removal of domain D2 or D3 of the human urokinase receptor does not affect ligand affinity.

The main ligand-binding determinant of the human urokinase receptor (uPAR) is located in the amino terminal domain D1, but when isolated this domain presents a 1500 fold lower affinity than the intact three-domain uPAR (D1D2D3). uPAR mutants missing either domain 2 (D1HD3) or domain 3 (D1D2) were expressed in murine LB6 cells and showed to be properly GPI-anchored to the cell surface. Binding assays with [125I]ATF demonstrated that these mutants possessed a normal (D1D2) or slightly reduced (D1HD3) affinity, indicating that a high ligand-affinity may be achieved by a combination of D1 with domain D2 or D3.

Monoclonal antibodies against endometrial protein PP14 and their use for purification and radioimmunoassay of PP14.

Monoclonal antibodies against placental protein 14 (PP14) were generated in mice. One of these (code 105DH1F1) was used for the purification of PP14 from mid-trimester amniotic fluid. The method is a simple one-step procedure, whereby monoclonal antibodies are bound to a solid phase and used for affinity immunoadsorption. 1.2 mg of PP14 was recovered from 25 ml of amniotic fluid. Using this purified PP14 and another monoclonal antibody (code 105AH7G3) a highly specific radioimmunoassay was developed. When the urine of pregnant women (7-10 weeks gestation) was tested little PP14 could be found at the detection level of 10 micrograms/l. The serum patterns of chorionic gonadotropin and PP14 were very similar in pregnancy. This study suggests that, unlike hCG, little PP14 is secreted into maternal urine. The purification method is gentle and allows quick purification of large amounts of PP14 for studies of its biological action(s).

CA-125 and placental protein 14 concentrations in plasma and peritoneal fluid of women with deeply infiltrating pelvic endometriosis.

OBJECTIVE: To investigate the plasma and peritoneal fluid (PF) concentrations of CA-125 and placental protein (PP14) in women with deeply infiltrating endometriosis. DESIGN: Plasma and PF were collected during 384 consecutive laparoscopies for pelvic pain or infertility. MAIN OUTCOME MEASURE: The presence and extent of endometriosis were carefully assessed, including the area, depth of infiltration, and volume of subtle lesions, typical lesions, and endometriomas. The day of the menstrual cycle was ascertained by endometrial biopsy and/or basal body temperature charts. RESULTS: Peritoneal fluid concentrations were some 100 and 10 times higher than plasma concentrations for CA-125 and PP14, respectively. Cyclic variations of CA-125 concentrations were only found in women with endometriosis showing increased plasma concentrations at the end of the cycle and increased PF concentrations in the early follicular phase. Cyclic variations of PP14 concentrations were found in women with and without endometriosis both in plasma and PF showing increased concentrations in the late luteal and early follicular phases. In women with endometriosis the increased plasma concentrations of PP14 and CA-125 correlated with the presence and volume of endometriomas and of deeply infiltrating endometriosis. The increased concentrations in PF correlated only with the pelvic area of subtle endometriotic lesions. The diagnostic sensitivity and specificity of CA-125 for endometriosis were 25% and 87%, respectively, and for endometriomas and/or deeply infiltrating endometriosis 36% and 87%, respectively, for a cutoff concentration of 25 U/mL. CONCLUSION: Superficial pelvic endometriosis secretes PP14 and CA-125 mainly toward the PF, whereas endometriomas and deeply infiltrating endometriosis secrete mainly toward the plasma. The increased plasma concentrations of CA-125 are most pronounced during the late luteal phase, and endometriomas and/or deeply infiltrating endometriosis can be detected with a sensitivity of 36% and a specificity of 87%.

Purification and characterization of endometrial protein PP14 from mid-trimester amniotic fluid.

Two methods are described for the purification of placental protein 14 from human mid-trimester amniotic fluid. The first includes gel filtration, anion exchange chromatography, and reversed-phase high performance liquid chromatography. The second method employs octyl-Sepharose chromatography instead of high performance liquid chromatography, and it also includes an anti-hCG adsorption step in order to remove the remaining traces of hCG from the purified PP14. In the first method, 362 micrograms of PP14 was recovered from 26 ml amniotic fluid with a final recovery of 25%. In the second method, 745 micrograms of PP14 was recovered from 200 ml amniotic fluid with a final recovery of 9.8%. As a result of either method sodium dodecyl sulfate polyacrylamide gel electrophoresis of purified protein showed one band at 28 kDa. Polyclonal antibodies against placental PP14 reacted with this band in immunoblot analysis and radioimmunoassay. A single N-terminal amino acid sequence of M D I P Q T K Q D L E L P K L A G T W H S M A was obtained for the isolated protein. This sequence is identical to that previously reported for human placental PP14. Due to its high PP14 concentration amniotic fluid serves as an excellent starting material for purification of this protein.

Time-resolved immunofluorometric assay for placental protein 14.

A sandwich-type solid phase time-resolved immunofluorometric assay (IFMA) was developed for endometrial protein PP14 (placental protein 14). The assay utilizes affinity-purified polyclonal antibodies for coating the microtiter wells and for labelling with europium (III) chelate. Maintaining specificity, the 0.6 micrograms/l sensitivity of IFMA is over 25 times higher than that of RIA. The immunofluorometric method enables detection and accurate quantitation of PP14 in all those samples in which PP14 is undetectable by RIA. The method is suitable for quantitative measurement of low PP14 levels in serum of postmenopausal and fertile-aged women and men, as well as in follicular fluid. At 14-16 micrograms/l, which is the sensitivity of radioimmunoassay, the intra-assay variation of IFMA is 6.6% and inter-assay variation 11.4%. In postmenopausal women the PP14 levels are 12.7-56.7 micrograms/l, in fertile-aged women 13.7-113.4 micrograms/l, and in men 3.1-53.1 micrograms/l. The levels in preovulatory follicular fluid are 1.2-20.5 micrograms/l. It is concluded that PP14 IFMA is highly sensitive, accurate and suitable for measurement of protein levels undetectable by other currently available methods.

Serous ovarian cyst fluids contain high levels of endometrial placental protein 14.

This paper reports the detection and characterization of endometrial placental protein 14 (PP14) in human ovarian cyst fluids. PP14 from ovarian cyst fluid has the same molecular size as PP14 purified from human amniotic fluid. No clear difference was found in the PP14 concentrations between benign, borderline and malignant cyst fluids, but serous ovarian cyst fluids had significantly higher PP14 concentrations (median 253.3 micrograms/l) than mucinous cyst fluids (median 17.5 micrograms/l).

Endometrial proteins: a reappraisal.

Uterine factors influence reproduction at the macro-anatomy level, and the effects of hormonal steroids on endometrial morphology are well recognized in the histopathological diagnosis of dysfunctional bleeding and infertility. During the past decade, attention has been paid to endometrial protein synthesis and secretion with respect to endocrine stimuli and implantation, and to the paracrine/autocrine effects of endometrial peptide growth factors, their binding proteins and other factors. The emphasis of this presentation is on protein secretion of the secretory endometrium, in which progesterone plays a pivotal role. Insulin-like growth factors have receptors on the endometrium, and IGF-binding proteins, stimulated by progesterone, modulate the effects of IGFs locally. Also other protein products of the secretory endometrium have been reviewed in this communication, with special emphasis on studies of a progesterone-associated endometrial protein which has many names in the literature, such as PEP, PP14, alpha 2-PEG and AUP. Extensive studies are ongoing in many laboratories to elucidate the regulation, function, interplay at tissue and cellular levels, and clinical significance of these proteins.

Evaluation of iron status in anemic patients with rheumatoid arthritis using an automated immunoturbidimetric assay for transferrin receptor.

We have evaluated a newly introduced immunoturbidimetric transferrin receptor assay (IdeA TfR-IT, Orion Diagnostica, Finland) in healthy subjects and in a study population consisting of patients with rheumatoid arthritis and juvenile chronic arthritis. The IdeA TfR-IT assay was found to provide reproducible results which were in good agreement with the ELISA assays from Orion Diagnostica (IDeA-ELISA, correlation R2=0.8, n=102) and R&D systems (Quantikine TfR ELISA assay, correlation R2=0.95, n=39). The analysis of the patient samples suggested that, on the basis of serum transferrin receptor and ferritin concentrations, in approximately one third of patients with rheumatoid arthritis anemia is due to the depletion of iron stores. Apparently, in all patients with rheumatoid arthritis iron deficiency must be considered as a potential cause of the anemia. Now, that assays which are suitable for automated analyzers have become available for the measurement of serum transferrin receptor, this analyte has the potential to become a part of the routine evaluation of iron status.

Progesterone-associated endometrial protein--a constitutive marker of human erythroid precursors.

Progesterone-associated endometrial protein (PAEP/PP14) is a 28-kD glycoprotein with sequence homology to beta-lactoglobulins containing a retinol-binding motif. PAEP/PP14 is present at nanomolar concentrations in human serum. It is produced by secretory and decidualized endometrium in women and by seminal vesicle epithelium in men. We report here that PAEP mRNA is constitutively expressed in normal hematopoietic tissue. Western immunoblotting of bone marrow cells with rabbit antibodies to PAEP gave a band at 28 kD, and immunocytochemical staining with monoclonal antibodies localized PAEP into the cytoplasm of erythroid precursors representing different stages of the normoblast series. PAEP was not detected in mature red blood cells, platelets, or in cells of the myeloid lineage. Untreated K562 leukemia cells did not contain PAEP, whereas treatment of the cells with tetradecanoylphorbol acetate (TPA) induced strong expression of PAEP mRNA and synthesis of the intact protein that was found both in the cytoplasm of the differentiating cells and in the supernatant of TPA-treated cultures. These findings add a new member to the growing family of genes that are constitutively expressed both in the reproductive tract and in the hematopoietic system.

Expression of endometrial protein PP14 in pelvic and ovarian endometriotic implants.

Localization and immunohistochemical staining patterns of endometrial protein PP14 were studied in 55 patients providing 86 histologically confirmed pelvic and ovarian endometriotic implants. The cellular localization of PP14 in endometriotic implants is comparable with that in the endometrium: epithelial cells express PP14, whereas stromal cells are negative. Positive immunostaining is restricted to apical secretory-like granules of the epithelial cells. In endometriotic implants with positive staining, PP14 is expressed by some, but not all, glandular epithelial cells. Furthermore, positive staining for PP14 is observed most frequently in foci with in-situ secretory differentiation, whereas implants with proliferative or atrophic implants only rarely express PP14. Finally, PP14 can be localized in endometriotic foci at different depths of infiltration, although positive labelling is seen more often in superficial implants. These data demonstrate that PP14 can be expressed by endometriotic glandular epithelial cells with secretory cellular differentiation and that the histological appearance of ectopic implants sometimes only poorly reflects their function.

Placental protein 14 in human in-vitro fertilization early pregnancies.

Placental protein 14 (PP14) and human chorionic gonadotrophin (HCG) were analysed in patients participating in an in-vitro fertilization-embryo transfer programme which did not include any kind of luteal support. Women with normal pregnancies, spontaneous abortions, ectopic pregnancies, biochemical pregnancies and non-pregnant women were compared. A combination of HCG and PP14 analyses distinguished between normal and abnormal implantation as early as 15 days after oocyte retrieval. The product of HCG (IU/l) and PP14 (micrograms/l) concentrations differed significantly between normal pregnancy, spontaneous abortion and ectopic pregnancy (P = 0.0248). It is concluded that both endometrial (PP14) and trophoblastic (HCG) markers, when used in combination, exhibit changes in abnormal implantation which may be clinically useful.

Suppression of prolactin secretion during ovarian hyperstimulation is followed by elevated serum levels of endometrial protein PP14 in the late luteal phase.

A double-blind placebo-controlled study on bromocriptine administration during days 2-12 of ovarian hyperstimulation for in-vitro fertilization (IVF) showed that, in bromocriptine cycles, levels of the endometrial protein PP14 were higher in the late luteal phase. This was verified both by calculating forward from the day of human chorionic gonadotrophin (HCG) administration and backward from the onset of the next period. Bromocriptine had no effect on IVF performance. During bromocriptine treatment the serum prolactin levels declined and serum oestradiol levels were higher on day 9 of the cycle. There was a positive correlation (r = 0.55; P = 0.012) between the serum oestradiol levels on day 9 and the PP14 levels on days 22-23 of the cycle. No difference was found in the luteal phase progesterone levels between bromocriptine- and placebo-treated cycles. These results suggest that low prolactin and/or high oestradiol levels during the follicular phase have an influence on the subsequent secretory capacity of the endometrium as reflected by secretion of a specific endometrial protein.

Studies on the transcobalamin receptor in hog kidney.

The binding of the cobalamin-transcobalamin complex by its solubilized receptor from hog kidney membrane was studied. The receptor bound the complex in a system containing bivalent cations, and the affinity was dependent on the NaCl concentration but not on temperature. The binding of cobalamin-transcobalamin to the receptor had an association constant of approximately 4.6 x 10(9) liter/mol and it was saturable and highly specific as competition by other proteins was not observed. The receptor had higher affinity for the cobalamin-transcobalamin complex (holo-TC) than for transcobalamin (apo-TC). Basic amino compounds known to interfere with tubular reabsorption of proteins did not inhibit the binding. Studies on subcellular fractions supported the view that the receptor was located on the brush border membrane of the kidney.

Suppression by human placental protein 14 of natural killer cell activity.

Human decidua of early pregnancy contains considerable numbers of CD3-CD56+ natural killer (NK) cells. In this study, two major protein products of the decidua, placental protein 14 (PP14) and placental protein 12 (PP12), were tested for the ability to regulate human NK cell activity. In vitro overnight exposure to PP14 of blood lymphocytes or purified large granular lymphocytes (LGL) resulted in suppression of cytotoxicity against K562 target cells in a 4-h 51Cr release assay. The NK inhibition was dependent on concentrations of PP14, being detectable at 5 micrograms/ml and reaching maximum at 50 micrograms/ml. Manifestation of PP14-induced NK suppression required 18-h contact with NK cells. The suppression of NK activity by PP14 was not abolished by indomethacin. In a target binding assay the number of PP14-treated LGL binding to K562 was comparable to that of untreated ones. By contrast with PP14, PP12 produced no effects on NK cells. These results indicate that PP14 suppresses the function of NK cells, which might be involved in prevention of maternal immune rejection of fetus at the fetomaternal interface.

Hematopoietic placental protein 14. An immunosuppressive factor in cells of the megakaryocytic lineage.

Placental protein 14 (PP14), an immunosuppressive molecule previously known to be expressed in the female and male reproductive tracts only, was shown to be expressed by hematopoietic cells of the megakaryocytic lineage. Northern blot analysis confirmed the induction specificity of PP14 mRNA in phorbol ester-treated K562 cells. Potent immunosuppressive activity in conditioned medium from phorbol ester-treated K562 cells was attributed to hematopoietic PP14 by anti-PP14 antibody blocking. Immunoprecipitation with anti-PP14 antibodies from conditioned medium revealed two distinct PP14 protein isoforms, designated PP14.1 and PP14.2. Polymerase chain reaction cloning and analysis demonstrated the presence of distinct mRNA counterparts to PP14.1 and PP14.2 that had not been resolved by Northern blot analyses. Hematopoietic PP14.1 mRNA corresponds in size to endometrial PP14 mRNA, whereas the smaller hematopoietic PP14.2 mRNA displays an internal in-frame 66-nucleotide deletion that can be explained by alternative splicing and predicts a 22-amino-acid deletion in the encoded gene product. Both PP14 mRNA isoforms were additionally detected by reverse transcriptase polymerase chain reaction analyses in two human megakaryocytic cell lines and in normal human megakaryocytes and platelets. PP14 mRNA was not detected by reverse transcriptase polymerase chain reaction in a panel of nonhematopoietic, nonendometrial tissues examined. The finding of hematopoietic PP14 within the megakaryocytic lineage provides an additional regulatory link between the coagulation and immune systems in normal and pathological settings.

Structural studies, localization in tissue and clinical aspects of human endometrial proteins.

Two human endometrial proteins, PP12 and PP14, are abundant in human amniotic fluid which is an excellent source for purification. In SDS-PAGE, purified PP12 migrates as several immunoreactive bands from 17,000 to 34,000, all having the same N-terminal amino acid sequence of Ala-Pro-Trp-Gln-Cys-Ala-, and all of them binding IFG-I. PP14 migrates at 28,000, and its N-terminal sequence is Met-Asp-Ile-Pro-Gln-Thr-Lys-Gln-Asp-Leu-Gln-Leu-Pro-Lys-Leu-Ala-Gly-Thr- Trp-His-Ser-Met-. There is a 59% identity between this sequence and that of horse beta-lactoglobulin, and also between PP14 and beta-lactoglobulins of various other species. PP14 and human retinol-binding protein show a 23% sequence identity, and the amino acid residues -Gly-Thr-Trp- at positions 17-19 of PP14 are identical with the corresponding residues of human retinol-binding protein. This site is assumed to play a part in the binding of retinol. An additional sequence identity (32%) is reported here for PP14 and protein BG, a 182 amino acid protein deduced from a 700-base pair cDNA clone isolated from the olfactory neuroepithelium of the frog. Sequence homology is also reported here between PP14 and insecticyanin, a camouflage-associated biliprotein in insects. The sequence of PP14 is therefore homologous to members of a family of proteins that bind and transport biologically active small molecules. Clinical studies have indicated an increase of PP12/IGF-bp and PP14 in the endometrium with advancing secretory changes. PP12/IGF-bp is also found in preovulatory follicular fluid. In hyperstimulated cycles of infertile women undergoing in-vitro fertilization, the serum PP12/IGF-bp concentration rises as multiple follicles mature, and luteinized granulosa cells contain this protein. In non-pregnant women, elevated values have been found in patients with advanced ovarian cancer and primary liver cancer. During pregnancy the serum PP12/IGF-bp concentration rises above the level in non-pregnant women around Week 8 of gestation. Abnormally high levels are seen in patients with pre-eclampsia and, in the third trimester, there is an inverse correlation between the maternal serum PP12/IGF-bp level and fetal weight. From these studies it is likely that a relationship exists between PP12/IGF-bp, the metabolism of IGFs and fetal growth. In non-pregnant women, serum PP14 concentrations appear to reflect endometrial secretory function. This is indicated by cyclic changes in the PP14 concentration in endometrial tissue and by the rising PP14 values in the late luteal phase.(ABSTRACT TRUNCATED AT 400 WORDS)