Comparison of haematopoietic stem cell engraftment through the retro-orbital venous sinus and the lateral vein: alternative routes for bone marrow transplantation in mice.

Bone marrow transplantation in mice is performed by intravenous administration of haematopoietic repopulating cells, usually via the lateral tail vein. This technique can be technically challenging to carry out and may cause distress to the mice. The retro-orbital sinus is a large area where there is a confluence of several vessels that provides an alternative route for intravenous access. Retro-orbital injection, although aesthetically unpleasant, can be performed rapidly without requiring mechanical restriction or heat-induced vasodilation. In addition, this technique can be easily learned by novice manipulators. This route of administration has been reported for use in bone marrow transplantation but there is no comparison of retro-orbital and tail vein injections reported for this specific purpose, although both routes have been compared for many other applications. Here, we provide for the first time a comprehensive comparison between tail vein and retro-orbital injections for two different bone marrow transplant scenarios in P3B and B6D2F1 mice. In both cases, no significant differences regarding donor engraftment were observed between mice transplanted using each of the techniques. Haematological counts and leukocyte subpopulation distribution were practically identical between both animal groups. Moreover, donor engraftment levels were less homogenous when cells were transplanted by tail vein injection, probably due to a higher risk of failure associated with this technique. All these data suggest that retro-orbital injection is a compelling alternative to conventional tail vein injection for bone marrow transplant in mice, providing similar and more homogenous haematopoietic reconstitution.

Generation of iPSCs from genetically corrected Brca2 hypomorphic cells: implications in cell reprogramming and stem cell therapy.

Fanconi anemia (FA) is a complex genetic disease associated with a defective DNA repair pathway known as the FA pathway. In contrast to many other FA proteins, BRCA2 participates downstream in this pathway and has a critical role in homology-directed recombination (HDR). In our current studies, we have observed an extremely low reprogramming efficiency in cells with a hypomorphic mutation in Brca2 (Brca2(Δ) (27/) (Δ27)), that was associated with increased apoptosis and defective generation of nuclear RAD51 foci during the reprogramming process. Gene complementation facilitated the generation of Brca2(Δ) (27/) (Δ27) induced pluripotent stem cells (iPSCs) with a disease-free FA phenotype. Karyotype analyses and comparative genome hybridization arrays of complemented Brca2(Δ) (27/) (Δ27) iPSCs showed, however, the presence of different genetic alterations in these cells, most of which were not evident in their parental Brca2(Δ) (27/) (Δ27) mouse embryonic fibroblasts. Gene-corrected Brca2(Δ) (27/) (Δ27) iPSCs could be differentiated in vitro toward the hematopoietic lineage, although with a more limited efficacy than WT iPSCs or mouse embryonic stem cells, and did not engraft in irradiated Brca2(Δ) (27/) (Δ27) recipients. Our results are consistent with previous studies proposing that HDR is critical for cell reprogramming and demonstrate that reprogramming defects characteristic of Brca2 mutant cells can be efficiently overcome by gene complementation. Finally, based on analysis of the phenotype, genetic stability, and hematopoietic differentiation potential of gene-corrected Brca2(Δ) (27/) (Δ) (27) iPSCs, achievements and limitations in the application of current reprogramming approaches in hematopoietic stem cell therapy are also discussed.

Correction of SCID-X1 using an enhancerless Vav promoter.

The efficacy of gene therapy for the treatment of inherited immunodeficiency has been highlighted in recent clinical trials, although in some cases complicated by insertional mutagenesis and silencing of vector genomes through methylation. To minimize these effects, we have evaluated the use of regulatory elements that confer reliability of gene expression, but also lack potent indiscriminate enhancer activity. The Vav1 proximal promoter is particularly attractive in this regard and may be useful in situations where high-level or complex regulation of gene expression is not necessary. X-linked severe combined immunodeficiency (SCID-X1) is a good candidate for such an approach, particularly as there may be additional disease-related intrinsic risks of leukemogenesis, and where safety is therefore a paramount concern. We have tested whether lentiviral vectors expressing the common cytokine receptor gamma chain under the control of the proximal Vav1 gene promoter are effective for correction of signaling defects and the disease phenotype. Despite low-level gene expression, we observed near-complete restoration of cytokine-mediated STAT5 phosphorylation in a model cell line. Furthermore, at low vector copy number, highly effective T- and B-lymphocyte reconstitution was achieved in vivo in a murine model of SCID-X1, in both primary and secondary graft recipients. This vector configuration deserves further evaluation and consideration for future clinical trials.

A comparative solid-phase extraction study for the simultaneous determination of fluoxetine, amitriptyline, nortriptyline, trimipramine, maprotiline, clomipramine, and trazodone in whole blood by capillary gas-liquid chromatography with nitrogen-phosphorus detection.

This paper reports the simultaneous detection of the seven antidepressants fluoxetine, amitriptyline, nortriptyline, trimipramine, maprotiline, clomipramine, and trazodone in whole blood at concentration levels of 100-2000 ng/mL by gas chromatography with a nitrogen-phosphorus detector (GC-NPD). A comparative and validation study using two solid-phase extraction (SPE) columns, Chem Elut and Bond Elut Certify, were developed regarding their recovery, precision, sensitivity, and matrix purification efficiency. The Chem Elut columns, a diatomaceous earth, are closely related to conventional liquid-liquid extraction. The Bond Elut Certify columns, more recently developed in the market, are a mixed SPE: reversed-phase and cation exchange sorbent. Recoveries of the compounds using Chem Elut columns at 500 ng/mL were in the range 30-50%, with intra- and interassay precisions of less than 9% and 17%, respectively. Limits of detection (LODs) and quantitation (LOQs) ranged from 13 to 146 ng/mL and from 44 to 485 ng/mL, respectively. Recoveries of the compounds using Bond Elut Certify columns at 500 ng/mL were in the range 59-84% with intra- and interassay precisions of less than 8% and 11%, respectively. LODs and LOQs ranged from 8 to 67 ng/mL and from 25 to 223 ng/mL, respectively. An excellent linearity was observed with both extraction procedures from the LOQs up to 2000 ng/mL. Higher recoveries, cleaner extracts, better sensitivity, better precision, and less solvent consumption and disposal were achieved for the screening of these antidepressants with the use of the mixed SPE Bond Elut Certify compared with Chem Elut columns.

Acute nitrobenzene poisoning with severe associated methemoglobinemia: identification in whole blood by GC-FID and GC-MS.

A rare fatal case of self-poisoning with nitrobenzene following oral ingestion is reported. On presentation to the hospital, severe methemoglobinemia (70%) was observed in an 82-year-old male who had ingested 250 mL of an unknown substance in the previous 24 h. Methylene blue and exchange transfusion were the therapeutic methods applied in the treatment of the methemoglobinemia. Forty-eight hours after ingestion, a blood sample was collected in ICU and sent to our laboratory. We detected that the blood contained 3.2 microg/mL of nitrobenzene. The determination of nitrobenzene was performed using the combination of GC-FID for screening analysis and quantitation and GC-MS for confirmation of the obtained results.

Simultaneous determination of viloxazine, venlafaxine, imipramine, desipramine, sertraline, and amoxapine in whole blood: comparison of two extraction/cleanup procedures for capillary gas chromatography with nitrogen-phosphorus detection.

A comparative study for the simultaneous gas chromatographic (GC) resolution and detection of the six antidepressants viloxazine, venlafaxine, imipramine, desipramine, sertraline, and amoxapine in whole blood at concentration levels of 100-2000 ng/mL was developed. Two extraction/cleanup analytical procedures were compared regarding their recovery, precision, sensitivity and matrix purification efficiency. The first procedure consists of the employment of Chem Elut columns (diatomaceous earth) and is based on the principle of liquid-solid absorption extraction that is closely related to conventional liquid-liquid extraction. The second focuses on the use of Bond Elut Certify columns and a mixed SPE, reversed-phase and cation-exchange sorbent, more recently developed for the market. Each procedure required 2.0 mL of whole blood extraction and injection into a capillary GC equipped with a nitrogen-phosphorus detector. Mepivacaine was used as the extraction standard (surrogate), and prazepam was used as the chromatographic standard. No interferences were found, and the time for the chromatographic analysis was 16 min for one sample. Recoveries of the compounds using Chem Elut columns at 500 ng/mL were in the range of 28-74% with intra-assay and interassay precisions of less than 7% and 19%, respectively. Limits of detection (LOD) and quantitation (LOQ) ranged from 39 to 153 ng/mL and from 128 to 504 ng/mL, respectively. Recoveries of the compounds using Bond Elut Certify columns at 500 ng/mL were in the range of 64-86% with intra-assay and interassay precisions of less than 4% and 10%, respectively. LODs and LOQs ranged from 21 to 100 ng/mL and from 70 to 330 ng/mL, respectively. An excellent linearity was observed with both procedures from the LOQs up to 2000 ng/mL. The use of the reversed-phase and cation-exchange sorbent Bond Elut Certify showed advantages compared with Chem Elut columns for the screening of these antidepressants such as higher recoveries, cleaner extracts, better sensitivity, better precision, and less solvent consumption and disposal.

Análisis químicos realizados en casos de intoxicaciones por detergentes y limpiadores / Toxicological analysis performed in cases of poisonings due to detergents and cleansers

En este trabajo se estudian 21 casos relacionados con intoxicaciones por productos de limpieza. Los casos estudiados son de etiología accidental, suicida y criminal y corresponden a casos forenses y clínicos cuyos análisis se han realizado en el Servicio de Química del Instituto de Toxicología del Departamento de Madrid desde el año 1998 hasta el primer trimestre del año 2001. Este tipo de intoxicaciones por productos de limpieza ya sean detergentes y/o limpiadores son poco frecuentes en la casuística general de este Laboratorio, aunque en el contexto general de intoxicaciones las producidas por medicamentos y productos de limpieza son las más frecuentes. En este tipo de casos, dada la naturaleza de los productos causantes de la intoxicación, se requiere un amplio análisis que comprende: medida de pH, análisis de iones sodio y potasio, detección de alcoholes y tensioactivos. En algunas ocasiones, cuando se sospecha que las personas afectadas pudieran estar bajo tratamiento psiquiátrico, también se realiza la investigación de psicofármacos y drogas de abuso. La mayoría de las intoxicaciones accidentales sucedieron en establecimientos de hostelería y se produjeron por confusión de bebidas con detergentes y limpiadores de uso industrial. Sin embargo, las intoxicaciones suicidas se produjeron en domicilios con detergentes de uso doméstico o limpiacristales. Las intoxicaciones criminales procedieron de denuncias judiciales y se trataron fundamentalmente de alimentos a los que se les había añadido algún producto cáustico o tensioactivo con un fin criminal (AU)