Search

Nucleic acid aptamers are promising ligands for analytical and preparative-scale affinity chromatography applications. However, a full industrial exploitation requires that aptamer-grafted chromatography media provide a number of high technical standards that remained largely untested. Ideally, they should exhibit relatively high binding capacity associated to a very high degree of specificity. In addition, they must be highly resistant to harsh cleaning/sanitization conditions, as well as to prolonged and repeated exposure to biological environment. Here, we present practical examples of aptamer affinity chromatography for the purification of three human therapeutic proteins from various sources: Factor VII, Factor H and Factor IX. In a single chromatographic step, three DNA aptamer ligands enabled the efficient purification of their target protein, with an unprecedented degree of selectivity (from 0.5% to 98% of purity in one step). Furthermore, these aptamers demonstrated a high stability under harsh sanitization conditions (100h soaking in 1M NaOH). These results pave the way toward a wider adoption of aptamer-based affinity ligands in the industrial-scale purification of not only plasma-derived proteins but also of any other protein in general., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

Endometriosis is a common condition in women, whose main repercussions are painful symptoms. In addition, it was shown that endometriosis was a major cause of infertility and various obstetric complications could be related to this pathology. Uterine rupture is a rare but serious complication whose incidence tends to decrease with the screening of women at risk, however, its fetal, maternal morbidity and mortality causes remains important. We were confronted with a case of posterior uterine rupture in a patient of 36 years, primipare term exceeded in immediate postpartum period. The patient's primary antecedent of uterine surgery torus was responsible for infertility endometriosis. The outcome was favorable for the mother, after a surgical treatment by laparotomy, and for the child. In the literature, two cases have been reported of uterine rupture after endometriosis surgery, which is why we found it interesting to report this rare case. Given the increase in surgical management of this disease, it seems relevant to ask whether, in the future, we should be more vigilant in monitoring pregnancy for these women., (Copyright © 2016 Elsevier Masson SAS. All rights reserved.)

Objective of the Review: To identify predictors of preterm delivery in the context of threatened preterm labour., Main Points: Tobacco use and previous history of preterm delivery are the main anamnestic elements to predict preterm birth. High positive predictive value of vaginal examination is restricted to cases with strong cervical alterations like dilatation over 4 cm. In case of discrete cervical alterations, literature confirms the great interest for cervical length ultrasonographic measurement as it reduces false positive cases. Absence of fetal respiratory movements appears to be as sensitive as cervical length and could be more specific but its clinical use remains rare. Vaginal detection of fetal fibronectin is the most useful biomarker with high negative predictive value (>90%). Fibronectin quantitative test seems to enhance the positive predictive value. No other biomarker is currently used in clinical practice. Electromyography and elastography of the cervix appear to be promising approaches., (Copyright © 2015 Elsevier Masson SAS. All rights reserved.)

Human T-cell lymphotropic virus type 1 (HTLV-1) entry involves the interaction between the surface (SU) subunit of the Env proteins and cellular receptor(s). Previously, our laboratories demonstrated that heparan sulfate proteoglycans (HSPGs) and neuropilin-1 (NRP-1), a receptor of VEGF(165), are essential for HTLV-1 entry. Here we investigated whether, as when binding VEGF(165), HSPGs and NRP-1 work in concert during HTLV-1 entry. VEGF(165) binds to the b domain of NRP-1 through both HSPG-dependent and -independent interactions, the latter involving its exon 8. We show that VEGF(165) is a selective competitor of HTLV-1 entry and that HTLV-1 mimics VEGF(165) to recruit HSPGs and NRP-1: (1) the NRP-1 b domain is required for HTLV-1 binding; (2) SU binding to target cells is blocked by the HSPG-binding domain of VEGF(165); (3) the formation of Env/NRP-1 complexes is enhanced by HSPGs; and (4) the HTLV SU contains a motif homologous to VEGF(165) exon 8. This motif directly binds to NRP-1 and is essential for HTLV-1 binding to, internalization into, and infection of CD4(+) T cells and dendritic cells. These findings demonstrate that HSPGs and NRP-1 function as HTLV-1 receptors in a cooperative manner and reveal an unexpected mimicry mechanism that may have major implications in vivo.

Vascular endothelial growth factor (VEGF) is essential for breast cancer progression and is a relevant target in anti-angiogenesis. Although VEGF121 and VEGF165, the fully or partially secreted isoforms, respectively, have been the focus of intense studies, the role of the cell-associated VEGF189 isoform is not understood. To clarify the contribution of VEGF189 to human mammary carcinogenesis, we established several clones of MDA-MB-231 cells stably overexpressing VEGF189 (V189) and VEGF165 (V165). V189 and V165 clones increased tumor growth and angiogenesis in vivo. Remarkably, V165 induced the most rapid tumor uptake, whereas V189 increased vasodilation. In vitro overexpression of VEGF165 and VEGF189 increases the proliferation and chemokinesis of these cancer cells. Interestingly, overexpression of VEGF189 increased cell adhesion on fibronectin (1.9-fold) and vitronectin (1.6-fold), as compared to VEGF165, through alpha5beta1 and alphavbeta5 integrins. Using the BIACore system we demonstrated for the first time that VEGF189 binds directly to neuropilin-1, which is strongly expressed in MDA-MB-231 cells. In contrast, VEGF-R2 was not significantly expressed and VEGF-R1 was expressed at low level. Our in vitro results suggest an autocrine effect of VEGF189 on breast cancer cells, probably through neuropilin-1. In conclusion, our data indicate that VEGF189 participates in mammary tumor growth through both angiogenesis and nonangiogenic functions. Whether VEGF189 overexpression is correlated to prognosis in human breast tumors remains to be established.

Background/aims: The incidence of hepatocellular carcinoma is higher in males, presumably due to the influence of sex steroids. Therefore, to further assess the role of sex steroids in the occurrence of hepatocellular carcinoma, this study investigated the predictive value of different sex hormones and their binding protein., Methods: Among 101 male patients with cirrhosis included in a prospective screening study, 29 developed hepatocellular carcinoma. We assessed the predictive value of 17 clinico-biological and 4 serum hormonal variables collected at enrollment, by the log-rank test and the Cox model., Results: Age (p = 0.003), bilirubin (p = 0.04), sex-hormone-binding-globulin (p = 0.006) and albumin (p = 0.08) were predictive using the log-rank test, while estradiol and total and free testosterone were not. The Cox model showed age (p = 0.0003; relative risk = 7.52), sex-hormone-binding globulin (p = 0.001, relative risk = 3.37) and albumin (p = 0.02, relative risk = 2.94) as the most predictive parameters., Conclusion: We conclude that high serum sex-hormone-binding-globulin levels have an independent predictive value for the occurrence of hepatocellular carcinoma. Serum sex-hormone-binding-globulin could be used to define patients at high risk for hepatocellular carcinoma and could hypothetically play a role in hepatocarcinogenesis.

The bioavailability of rapid-acting insulin administered as a nasal spray was studied in 6 type 1 (insulin-dependent) diabetic patients. They received long-acting bovine insulin (Ultratardum 40 U/ml, Organon) as basal treatment at 8 a.m. Rapid-acting insulin was also administered at 8 a.m., then at noon and 6 p.m, subcutaneously on day 1 as a 100 U/ml solution and intranasally by aerosol spray as a 100 U/ml and 500 U/ml with 1% (w/v) 9 lauryl ether solution on day 2 and day 3 respectively. On days 2 and 3, the dose of insulin was at least nine times higher than the subcutaneous dose on day 1. Free and total plasma insulin concentrations were assayed after the noon insulin administration. The peaks of the free and total plasma insulin levels were reached earlier and the return to basal levels was obtained earlier after nasal insulin administration than after insulin injected subcutaneously. The bioavailability of nasal spray insulin versus subcutaneous insulin with a 100 U/ml insulin solution was similar to that with a 500 U/ml insulin solution: 5.14 +/- 0.38% (m +/- SEM) and 4.64 +/- 0.46% according to the total plasma insulin level. This study suggests that the bioavailability of nasal spray insulin is not increased by increasing insulin concentration in our experimental conditions.

Anti-thyroid hormone autoantibodies are occasionally found in Hashimoto's disease. They can interfere with thyroid hormone assays. The authors report the case of a 35-year-old hypothyroid (TSH between 29 and 46 microIU/ml) female patient with Hashimoto's disease and surprisingly high free and total levels of plasma T4 and T3 assayed with different solid phase radio-immunologic kits. Two immunoenzymologic assay kits gave a T4 level close to the one expected. The presence of circulating anti-T4, T3 and reverse T3 autoantibodies was demonstrated by precipitating the corresponding tracers with the patient's serum in the presence of polyethylene glycol without heterologous antibodies. The replacement treatment of 150 micrograms l-thyroxine allowed normalization of TSH at 3.4 microIU/ml, but the patient remained tired and anxious, with headaches and tachycardia. After discontinuing treatment for one month a series of four plasma exchanges was carried out in six days. After the first exchange the binding percentages were already much lower and went even lower after the other exchanges. After the fourth one, the free T4 and T3 levels were closer to the expected levels:7.3 pmol/l (N:10-30 pmol/l) and 4.3 pmol/l (N:3-9 pmol/l). Treatment by prednisone made it possible to maintain the percentages of binding to the three tracers for two weeks at the levels reached after the fourth plasma exchange. Two months after discontinuing corticosteroid treatment they returned to the initial levels and were again interfering with the free T4 and T3 assays. This finding suggests that usefulness of the thyroid hormone immuno-enzymologic assay, which apparently is not affected by the anti-thyroid hormone autoantibody interference.(ABSTRACT TRUNCATED AT 250 WORDS)

Young and old, human erythrocytes, separated in vitro according to their age in vivo, were radioactively labeled at the cell-surface D-galactosyl and 2-acetamido-2-deoxy-D-galactosyl residues by treatment with D-galactose oxidase, followed by reduction with sodium borotritide. The labeling was quantitatively determined for each type of erythrocyte by measuring the molar amounts of borohydride necessary for the complete reduction of the oxidized residues. The number of surface residues per blood-group A+ erythrocyte was found to be 37.6 +/- 1.8 x 10(6) (n = 8) for young, 21.8 +/- 4.9 x 10(6) (n = 8) for old, and 24.8 +/- 6.4 x 10(6) (n = 8) for middle-aged erythrocytes, indicating a significant decrease of the residues during ageing.

Young and old human erythrocytes, separated in vitro according to age, were labeled at the surface-sialic acid residues by sodium periodate and borotritide treatment. No qualitative difference was observed between the sialic acid derivatives of young- and old-erythrocyte membranes. The number of labeled residues was significantly decreased in the in vivo aged erythrocytes (12 +/- 2.8 X 10(6); n = 8) when compared to the young ones (19.4 +/- 2.8 X 10(6); n = 8). Analysis of the labeled glycoproteins by sodium dodecyl sulfate gel-electrophoresis showed that this decrease affects the PAS-1, PAS-4, PAS-2, and PAS-3 bands (glycophorins A and B) of the old-erythrocyte membranes.

A high-performance liquid chromatographic method for an effective determination of glafenine and its main metabolite, glafenic acid, is described. The assay involves separate extraction procedures for glafenine and for its metabolite, but the same internal standard (floctafenine) and the same chromatographic conditions (including a 5-micron C8 column, a quaternary solvent mixture of water-acetonitrile-diethylamine-acetic acid and an ultraviolet detector set at 360 nm). For 1 ml of plasma, the detection limit is 0.05 mg/l for glafenine and 0.25 mg/l for glafenic acid. Compared with previously described techniques, this assay uses a very low glafenine linearity range, which allows the true pharmacokinetics of this drug to be described for the first time.

Aminoglycosides and in particular gentamicin are still largely used in the treatment of spontaneous bacterial peritonitis in the decompensed alcoholic cirrhotic patient. For that matter, we decided to study the various passage of gentamicin in the ascites after its administration through IM injection (80 mg in 6 patients and 120 mg in 6 other patients) and through IV injection (120 mg in 6 other patients). What could possibly be the best protocol to replace the daily intraperitoneal injections? We found out that no matter what the style of injection is, the peritoneal levels are only exceptionnaly higher than 3 mg/g at the highest peak period. These results brought us to analyze the passage of gentamicin in the blood when injected intraperitonealy (160 mg in 6 patients), and then to propose a therapeutic protocol combining on the first day, to the peritoneal injection a loading dose of an IP injection of 160 mg, the relay been taken over later with a peritoneal injection of 80 or 120 mg repeated 2 or 3 times daily. For each of the protocol the pharmacokinetic coefficients of gentamicin were calculated.

Gentamicin diffusion in pulmonary parenchyma was studied in 10 adult patients with normal renal functions before undergoing thoracic surgery. This aminoside was given in a one time dose of 120 mg directly and by way of IV route in 10 min time; blood samples from peripheral and pulmonary sources were drawn at regular intervals between the 10 min period and the 8th hr; parenchymal samples were collected 1 and 2 hrs after drug administration. After separation of residual blood by centrifugation at high speed, the tissues are grinded and homogenized. The assay was performed with an immunological method using a fluorescent polarized detection system. Tissue levels were low in all the patients (0.5 to 2.75 micrograms/g of fresh tissue) and this in spite of the high level of blood collected throughout the administration protocol. As a rule, the relation between tissue/blood is lower than those discussed in literature concerning bronchial secretion. Beside this, the concentrations are similar in peripheral and pulmonary blood collected. Practical conclusions are drawn for therapeutic purpose.

The contents of fucose, mannose, galactose, glucose, 2-acetamido-2-deoxy-D-glucose and -D-galactose, and sialic acid, when the results were expressed as nmol per mg of membrane dry-weights, were found to be significantly lower in the membranes of old erythrocytes than in the membranes of young ones. No significant difference was found between young and old membranes when the compositions were expressed as residues per one hundred carbohydrate residues, suggesting that a homogeneous decrease of the carbohydrate moieties may occur during aging in vivo.