Adaptation of the fish juvenile growth test (OECD TG 215, 2000) to the marine species Dicentrarchus labrax.

The OECD TG 215 method (2000) (C.14 method of EC Regulation 440/2008) was developed on the rainbow trout (Oncorynchus mykiss) to assess chronic toxicity (28d) of chemicals on fish juveniles. It contemplates to use other well documented species identifying suitable conditions to evaluate their growth. OECD proposes the European sea bass (Dicentrarchus labrax, L. 1758) as Mediterranean species among vertebrates recommended in the OECD guidelines for the toxicity testing of chemicals. In this context, our study is aimed to proposing the adaptation of the growth test (OECD TG 215, 2000) to D. labrax. For this purpose toxicity tests were performed with sodium dodecyl sulfate, a reference toxicant commonly used in fish toxicity assays. The main aspects of the testing procedure were reviewed: fish size (weight), environmental conditions, dilution water type, experimental design, loading rate and stocking density, feeding (food type and ration), test validity criteria. The experience gained from growth tests with the sea bass allows to promote its inclusion among the species to be used for the C.14 method.

Fitness Evaluation of Ruditapes philippinarum Exposed to Ni.

In this study, long-term effects of Ni, a widespread heavy metal in the aquatic ecosystems, have been determined on growth and lethality of the clam Ruditapes philippinarum, a known bioindicator of the marine environment. Three/four-month-old bivalves have been exposed to different concentrations of Ni dissolved in synthetic seawater. Growth and lethality as endpoints after 28 days of treatment have been observed. Obtained results are the following: EC25 = 3.97 ± 0.94 and 9.45 ± 1.59 mg/L and NOEC = 1.56 and 6.25 mg/L for growth and mortality, respectively. Moreover, this study can be considered a new tool for the evaluation of fitness of bivalve clam, together with other biological responses following to the biological impacts of metal pollution.

Can Artemia Hatching Assay Be a (Sensitive) Alternative Tool to Acute Toxicity Test?

Artemia sp. is extensively used in ecotoxicity testing, despite criticisms inherent to both acute and long-term tests. Alternative endpoints and procedures should be considered to support the use of this biological model. The hatching process comprises several developmental steps and the cyst hatchability seems acceptable as endpoint criterion. In this study, we assessed the reliability of the hatching assay on A. franciscana by comparing with acute and long-term mortality tests, using two chemicals: Diethylene Glycol (DEG), Sodium Dodecyl Sulphate (SDS). Both DEG and SDS tests demonstrated a dose dependent hatching inhibition. The hatching test resulted more sensitive than acute mortality test and less sensitive than the long-term one. Results demonstrate the reliability and high sensitivity of this hatching assay on a short time lag and support its useful application in first-tier risk assessment procedures.

Interdependence of initial cell density, drug concentration and exposure time revealed by real-time impedance spectroscopic cytotoxicity assay.

We investigated the combined effect of the initial cell density (12,500, 35,000, 75,000, and 100,000 cells cm(-2)) and concentration of the anti-cancer drug doxorubicin on HeLa cells by performing time-dependent cytotoxicity assays using real-time electrochemical impedance spectroscopy. A correlation between the rate of cell death and the initial cell seeding density was found at 2.5 µM doxorubicin concentration, whereas this was not observed at 5 or 100 µM. By sensing the changes in the cell-substrate interaction using impedance spectroscopy under static conditions, the onset of cytotoxicity was observed 5 h earlier than when using a standard colorimetric end-point assay (MTS) which measures changes in the mitochondrial metabolism. Furthermore, with the MTS assay no cytotoxicity was observed after 15 h of incubation with 2.5 µM doxorubicin, whereas the impedance showed at this time point cell viability that was below 25%. These results indicate that impedance detection reveals cytotoxic events undetectable when using the MTS assay, highlighting the importance of combining impedance detection with traditional drug toxicity assays towards a more in depth understanding of the effect of anti-cancer drugs on in vitro assays. Moreover, the detection of doxorubicin induced toxicity determined with impedance under static conditions proved to be 6 times faster than in perfusion culture.

Ecotoxicity of diethylene glycol and risk assessment for marine environment.

Diethylene glycol (DEG) is a chemical compound used during offshore oil activities to prevent hydrate formation, and it may be released into the sea. A full ecotoxicological characterization is required according to European and Italian regulations for chemical substances. We have evaluated long-term toxic effects of DEG on indicator species of the marine environment as algae (Phaeodactylum tricornutum), crustaceans (Artemia franciscana), molluscs (Tapes philippinarum) and fish (Dicentrarchus labrax). A range of no observed effect concentrations (365-25,000 mg/L) has been identified. Based on the toxicity results and the ratio between predicted environmental concentration and predicted no-effect concentration, we have estimated the maximum allowable value of DEG in the marine environment.

Binding of heparin-dependent antibodies to PF4 modified by enoxaparin oligosaccharides: evaluation by surface plasmon resonance and serotonin release assay.

BACKGROUND: The minimal structural requirements of low-molecular-weight heparins that determine the risk of developing heparin-induced thrombocytopenia (HIT) are not fully defined. OBJECTIVES: The ability of enoxaparin-derived oligosaccharides (OS) to induce platelet activation and exposure of platelet-factor 4 (PF4) epitopes recognized by antibodies developed in HIT was studied by surface plasmon resonance (SPR) and serotonin release assay. RESULTS: Decasaccharides with ≥ 11 sulfate groups induced platelet activation in the presence of plasma from patients with confirmed HIT. Serotonin release of > 80% without full inhibition at 100 µg mL(-1) was achieved with decasaccharides containing 14 or 15 sulfate groups, 2 dodecasaccharides and 2 tetradecasaccharides. An SPR method was developed using purified PF4 immobilized on carboxymethylated dextran. Antibodies from all HIT samples bound to PF4/heparin in SPR assays with resonance units (RU) ratio of 109-173 with HIT plasma vs. 88-93 with control plasma. RU ratios > 100 were measured when PF4 was pre-incubated with OS with ≥ 10 saccharide units and one octasaccharide containing 10 sulfate groups. RU ratios > 140, similar to those measured when PF4 was pre-incubated with unfractionated heparin or enoxaparin, were obtained with purified dodeca- and tetradecasaccharides. RU values strongly correlated with the number of sulfate groups in the decasaccharides tested (r = 0.93, P = 0.02). CONCLUSIONS: LMWHs with fragments > 10 saccharides and a large number of sulfate groups are more likely to be associated with a higher risk of HIT. These structure-activity relationships were independent of the ability of the OS to bind antithrombin.

Evaluation of a peptide ELISA for the detection of rituximab in serum.

Pharmacokinetic studies of therapeutic monoclonal antibodies necessitate the measurement of their biologically active fraction. The aim of this work was to develop an enzyme-linked immunosorbent assay (ELISA) for rituximab, a chimeric anti-CD20 monoclonal antibody, based on its binding to a 20-mer peptide (P20) derived from the extracellular loop of human CD20 (residues 165-184). Derivatives of P20 were prepared by conjugation to bovine serum albumin (BSA-P20ACM) or biotin (Biot-P20ACM). Interactions of P20 and its derived peptides with rituximab were analyzed by surface plasmon resonance (SPR) and by ELISA. A monoclonal anti-idiotype antibody (MB2A4) was used as the reference in each case. SPR analysis showed that P20 (conjugated or unconjugated) had a lower affinity for rituximab than MB2A4. ELISA methods based on P20 or MB2A4 were both highly accurate and reproducible for rituximab measurement in spiked samples, but the MB2A4-based assay had a lower limit of quantification. Interestingly, discrepant results were obtained with the two ELISA methods when analyzing pharmacokinetic samples, with the rituximab concentrations obtained with the MB2A4-based method being systematically higher than those determined by the P20-based method. Possible interference of circulating CD20 with the P20-based method was supported by competition experiments. Rituximab aggregation in the bloodstream may also account for the bias observed in samples from healthy mice. The P20-based ELISA is far less sensitive than the MB2A-based ELISA, thus limiting its utility for pharmacokinetic studies. However, the discrepancy observed between two different approaches for rituximab measurement indicates that data from different studies should be interpreted with care.

Differential estradiol requirement for the induction of estrus behavior and the luteinizing hormone surge in two breeds of sheep.

For a better understanding of the mechanisms that lead to the preovulatory GnRH/LH surge and estrus behavior, the minimum estradiol (E) requirements (dose and duration) to induce each of these events were determined and compared between two breeds of ewes having either single (Ile de France) or multiple (Romanov) ovulations. The ewes were initially studied during a natural estrus cycle, and were then ovariectomized and run through successive artificial estrus cycles. For these artificial cycles the duration and amplitude of the follucular phase E increase were manipulated by E implants. Under all conditions, the onset of estrus behavior was similar in the two breeds, although its duration was longer in Romanov ewes. While a moderate E signal (6 cm for 12 h) induced an LH surge in 10/10 Ile de France ewes, a larger E signal (12 cm for 12 h) was minimally effective in Romanov ewes (4/10). Additional studies revealed that a small E signal (3 cm for 6 h) induced full estrus behavior in all Romanov ewes but was completely ineffective in Ile de France animals (0/10). Higher doses and mostly longer durations of the E signal (12 cm for 24 h) were required to induce a surge in all the Romanov ewes. These results demonstrate a clear difference in the E requirement for the induction of estrus behavior and the LH surge between breeds of ewes that have different ovulation rates. These data provide compelling evidence that, in one breed, the neuronal systems that regulate both events require different estrogen signals.

Passage of progesterone into the brain changes with photoperiod in the ewe.

In this study we tested the hypothesis that photoperiod can modulate steroid access to the brain in a seasonal breeder. To this goal, we compared the passage of exogenous progesterone to the brain of female sheep maintained under short (SD) or long (LD) daylengths. In the first experiment, we studied two groups of ovariectomized females maintained under SD or LD, for three artificial cycles, consisting of bearing a subcutaneous oestradiol implant (E2-treated) and an intravaginal device releasing progesterone (CIDR). During the third cycle, the concentrations of progesterone and of its metabolites 5alpha-dihydroprogesterone and 3alpha-hydroxy-5alpha-pregnan-20-one were measured in the preoptic area (POA). The levels of progesterone in the POA were higher in ewes under LD than under SD while the amounts of metabolites were unchanged. In the second experiment, we compared ovariectomized female sheep equipped with a cannula in the third ventricle to sample the cerebrospinal fluid (CSF) under LD vs. SD. After progesterone (1 mg and 10 mg) was injected into the carotid artery, it was only detectable in the cerebrospinal fluid in sheep under LD. In the third experiment, we compared progesterone concentration in plasma and CSF in two groups of SD vs. LD ovariectomized E2-treated ewes for 2 h under CIDR treatment. Despite similar progesterone plasma concentrations, concentration in the CSF was 2.5 times higher in SD than in LD. Our results suggest a physiological modulation of the passage of progesterone to the brain according to the photoperiod.

The xenotransplantation of goat and human hematopoietic cells to sheep fetuses.

Hematopoietic xenografts were carried out in three experiments using goat fetal liver (44-48 days, experiments I and II) or purified human CD 34+ cells (experiment III) as the donor cells. Recipients were sheep fetuses at 41-47 days of gestation. Goat fetal liver cells were either injected without any pretreatment or stimulated by preincubation in a culturing in goat phytohemagglutinin-stimulated lymphocyte supernatant. Human CD 34+ myeloid progenitor cells were purified from bone marrow by minimacs immunomagnetic purification and cultured in medium supplemented with stem cell factor, IL3, and IL6. Goat-sheep chimerism was assessed by flow cytometry analysis (FCA) of peripheral blood and bone marrow cells using a mouse anti-goat CD 45 monoclonal antibody and by karyotype analysis of peripheral blood from goat/sheep chimeras. Human cell engraftment was assessed by polymerase chain reaction amplification of the human DAX1 gene in blood and bone marrow DNA from sheep which had received human cells. In the three experiments, a mean of 76% (26 of 34) of injected fetuses were born alive without any clinical evidence of graft-versus-host disease. Three lambs were found to be goat/sheep chimeric after flow cytometry analysis (peripheral blood and bone marrow) and karyotype (peripheral blood) analysis. Both tissues continued to express goat cells at 6 or 12 months (last assessment) depending on the experiment. No human chimerism was detected using polymerase chain reaction amplification in peripheral blood and bone marrow of any of the six sheep grafted with human cells. These data and those also obtained on other species (human, pig/sheep) show that it is possible to carry out hematopoietic xenografts using the sheep fetus as recipient provided both donor and recipient fetal cells are processed during the period of tolerance to foreign antigens.

Congenital heart defect in sibs with discordant karyotypes.

Congenital heart defects (CHDs) are genetically heterogeneous, associated with a variety of genetic conditions. Familial aggregation of CHD in patients with and without Down syndrome is rare. We report on the occurrence of concordant CHD in three sets of sibs with discordant karyotypes. In the first family, atrioventricular canal (AVC) was diagnosed in a chromosomally normal child and in his brother with Down syndrome. In the second family, AVC was associated with trisomy 21 in one sib and with trisomy 18 in the other. In the third family, tetralogy of Fallot was present in one patient with Down syndrome and in his nonsyndromic sister. Although the genetic heterogeneity of Down and non-Down CHD is not disputed, a susceptibility to both euploid and aneuploid CHDs could exist, and common predisposing factors could play a role in both conditions.

Autologous lymphocytes prevent the death of monocytes in culture and promote, as do GM-CSF, IL-3 and M-CSF, their differentiation into macrophages.

Blood monocytes collected by apheresis from healthy donors were differentiated in vitro to macrophages which were subsequently activated with recombinant human interferon-gamma. 7 day cultures were established by seeding Ficoll-separated mononuclear cells or elutriation-purified monocytes under different culture conditions. The best macrophage yields required the seeding of mononuclear cells (instead of purified monocytes) in teflon bags with a high air-liquid surface interface. The effects of GM-CSF, IL-3 and M-CSF on the macrophage yield were then evaluated. GM-CSF increased the average yield by 3.6- and 2.3-fold when purified monocytes or total mononuclear cells were seeded respectively. The corresponding increases with IL-3 were 2.5- and 2.1-fold respectively and with M-CSF 1.2- and 1.4-fold respectively. Macrophages matured under these various conditions displayed similar CD14, CD64, CD71, HLA-DR and Max 1 antigen expression and similar in vitro anti-tumoral activity against U937 cells. Culturing in the presence of cytokines permits the large scale production of activated macrophages for adoptive immunotherapy trials.

A case of torsade de pointes occurring in a newborn with persistent 2:1 atrioventricular block.

A case of QT interval prolongation with ventricular tachycardia and torsade de pointes is reported. Arrhythmias occurred in a baby with persistent 2:1 atrioventricular block and long QT interval 2 days after birth and were self-limiting. No structural cardiac defect was present. Serum levels of sodium, potassium, magnesium and calcium were in the normal range. Finally, the pathogenetic mechanism of cardiac block is discussed.

Production of human macrophages with potent antitumor properties (MAK) by culture of monocytes in the presence of GM-CSF and 1,25-dihydroxy vitamin D3.

Antitumoral macrophages (MAK) were obtained by the culture of human mononuclear cells in hydrophobic bags. From one cytapheresis, up to 10(9) mature macrophages could be purified by elutriation after one week of culture in IMDM medium in the presence of 2% human AB serum. These MAK cells were used for adoptive treatment in metastatic cancer patient with no dose-limiting toxicity. The present study aimed to improve the average MAK yield by addition of GM-CSF and of dihydroxy-cholecalciferol. The differentiated macrophages obtained presented higher antitumoral functionality in response to rh-IFN gamma than in their absence. These MAK presented all the differentiation antigens of cytotoxic macrophages compared to MAK cells differentiated in standard medium. They killed human tumor targets effectively in vitro at a low (1/1) effector/tumor ratio; furthermore, the antitumoral activity reached by MAK cells after IFN gamma activation appeared to be stabilized for several days.

Adoptive immunotherapy with activated macrophages grown in vitro from blood monocytes in cancer patients: a pilot study.

Ninety-three collections of leucocytes by cytapheresis followed by separation of monocytes by centrifugal elutriation were undertaken in twelve metastatic cancer patients (four melanomas, six colon carcinomas, one ovarian carcinoma, and one lung cancer). The leucaphereses were performed aiming to collect a product, ready for introduction into the elutriation chamber, i.e., with low contamination by erythrocytes and granulocytes. The median collection of leucocytes was 7.3 x 10(9). After elutriation, purified monocytes (mean: 0.91 x 10(9)) were cultured with 3-5% autologous serum for 7 days in the presence of 250 IU/ml of recombinant human gamma-interferon (Rh-IFN gamma) for the last 18 h of culture. The median number of activated macrophages (MAK) available for reinfusion was 2.4 x 10(8) for each culture. The phenotypes and the antitumoral potentiality of MAK cells were documented. Reinfusions performed i.v. or i.p. were well tolerated with no major side effects. No complete tumor response was obtained. One partial response and two stabilizations of the disease were observed in one melanoma and two colon carcinomas.

[HLA B27 phenotyping using flow cytometry]. / Phénotypage HLA B27 par cytométrie de flux.

Results of typing for HLA B27 antigens of 106 patients in whole blood with a FITC monoclonal anti HLA B27 antibody and analysis by flow cytometry are compared to the results obtained by the classical microlymphocytoxicity test. By flow cytometry analysis, there are not false negative result, but, because of cross reactions of the used monoclonal antibody with other specificities of the CREG B7 group, false positive result may be encountered. The flow cytometry analysis for typing HLA B27 antigens using a monoclonal antibody can be a good screening technique: so it's rapid and moreover the negative results can be accepted. The positive results must be confirmed.

Serial study of T-lymphocyte subsets in women during very early pregnancy.

The aim of this serial study was to define variations in T-lymphocyte populations during the first weeks of pregnancy. Results obtained with 14 women pregnant after artificial insemination show that the CD4+ population decreased significantly as soon as the fourth week. This decrease was concomitant with the HCG peak and could be compatible with certain immunological mechanisms participating in tolerance to the fetal graft.