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J Barquinero

Publications and source records attributed to J Barquinero.

At least 19 recordsLinked to original sources
PubMed2005

Translocation yields in peripheral blood lymphocytes from control populations.

PURPOSE: To record the latest information on control levels of translocations in cultured human lymphocytes. MATERIALS AND METHODS: Control-level data from seven European laboratories that are using fluorescence in situ hybridization (FISH) techniques for retrospective biological dosimetry have been combined in a meta-analysis. After correction for the differing probe combinations used, tests of consistency are performed. The combined data have been used to test for individual variation, systematic variation with age, gender and smoking habits. RESULTS: There is a strong variation of translocation yield with age but no variation was detectable with gender or smoking habits. After correction for age, homogeneity tests showed that about 10% of individuals were outside the 95% confidence limits as opposed to 5% expected. From a total of 385, there is an excess of about 20 individuals most of whom have an unexpectedly high yield of translocations. CONCLUSIONS: For retrospective biological dosimetry purposes a generic age-dependent control level can be assumed. No other lifestyle factors such as smoking appear to have a significant effect on translocation yield.

Adolescent↗
PubMed2005

Review of translocations detected by FISH for retrospective biological dosimetry applications.

Several European laboratories have combined their research efforts to arrive at a consensus view on using fluorescence in situ hybridisation (FISH) for retrospective dosimetry. The aim of this review is to report these views and to highlight some areas where further work is needed. Translocations in the stable cells should be measured only in the cells that contain the full complement of the painted material. Two-way and one-way translocations should be combined with equal weight. The control level of translocations has a strong dependence on age, which has now been measured and the system has been calibrated. In conclusion, the technique works and a lifetime dose to the bone marrow from low-linear energy transfer radiation of 0.5 Gy above normal background levels can be measured for any individual. The main application is considered to provide an independent verification of lifetime doses to individuals who might form a part of an epidemiological study.

Calibration↗
PubMed2004

Retroviral vectors: new applications for an old tool.

Retroviral vectors (RVs) have been used for stable gene transfer into mammalian cells for more than 20 years. The most popular RVs are those derived from the Moloney murine leukaemia virus (MoMLV). One of their main limitations is their inability to transduce noncycling cells. However, they have a relatively simple genome and structure, are easy to use, and are relatively safe for in vivo applications. For the last two decades, the artificial evolution of RVs has paralleled evolution in their applications, which now include those as diverse as the generation of transgenic animals, the stable delivery of small interfering RNA (siRNA) and gene therapy clinical trials. Recent reports of two successful gene therapy clinical trials in patients with severe immunodeficiency disease in France and Italy, and the development of T-cell acute leukaemia in two of 10 children participating in one of these clinical trials, demonstrate the great potential of RVs, but also some potential risks which may be intrinsically associated with their use. Basic aspects of RVs and vector production were reviewed in detail in a previous supplement of this journal. This article will first summarize some general aspects of retroviruses and RVs. Thereafter, recent developments in gene therapy using RVs, novel applications such as stable RNA interference and some other recent issues related to retroviral integration, including clonality studies after haematopoietic stem cell transplantation, retroviral tagging and insertional oncogenesis will be discussed.

Animals↗
PubMed2002

Myeloablation enhances engraftment of transduced murine hematopoietic cells, but does not influence long-term expression of the transgene.

To investigate to what extent myeloablation, graft size, and ex vivo manipulation influence the engraftment and long-term survival of transduced murine hematopoietic cells, groups of C57BL/6J (CD45.2) mice receiving total body irradiation (TBI) (1-9 Gy) or no irradiation were transplanted with either transduced bone marrow (BM) cells, at two cell doses, or with fresh BM cells from B6/SJL (CD45.1) congenic mice. Short (40 days) and long-term (5 months) engraftment and transgene expression were measured by FACS analysis. No donor cells were detected in the hematopoietic tissues of non-myeloablated mice, whereas in the irradiated animals, levels of engraftment correlated well with the dose of TBI administered. Similar percentages of transgene-expressing cells were found in the grafted hematopoietic cells of all groups of mice, regardless of the dose of TBI administered or the level of engraftment achieved. This suggests that the engrafted animals could become tolerant to the transgene product (enhanced green fluorescent protein, EGFP). Our results indicate that TBI facilitates the engraftment of manipulated hematopoietic cells in a dose-dependent manner, that mice engrafted with EGFP(+) hematopoietic cells probably acquire tolerance to EGFP, and that increasing the graft size and reducing the ex vivo manipulation required for retroviral gene transfer of hematopoietic cells also enhances their engrafting potential.

Animals↗
PubMed2000

Efficient transduction of human hematopoietic repopulating cells generating stable engraftment of transgene-expressing cells in NOD/SCID mice.

In an attempt to develop efficient procedures of human hematopoietic gene therapy, retrovirally transduced CD34(+) cord blood cells were transplanted into NOD/SCID mice to evaluate the repopulating potential of transduced grafts. Samples were prestimulated on Retronectin-coated dishes and infected with gibbon ape leukemia virus (GALV)-pseudotyped FMEV vectors encoding the enhanced green fluorescent protein (EGFP). Periodic analyses of bone marrow (BM) from transplanted recipients revealed a sustained engraftment of human hematopoietic cells expressing the EGFP transgene. On average, 33.6% of human CD45(+) cells expressed the transgene 90 to 120 days after transplantation. Moreover, 11.9% of total NOD/SCID BM consisted of human CD45(+) cells expressing the EGFP transgene at this time. The transplantation of purified EGFP(+) cells increased the proportion of CD45(+) cells positive for EGFP expression to 57. 7% at 90 to 120 days after transplantation. At this time, 18.9% and 4.3% of NOD/SCID BM consisted of CD45(+)/EGFP(+) and CD34(+)/EGFP(+) cells, respectively. Interestingly, the transplantation of EGFP(-) cells purified at 24 hours after infection also generated a significant engraftment of CD45(+)/EGFP(+) and CD34(+)/EGFP(+) cells, suggesting that a number of transduced repopulating cells did not express the transgene at that time. Molecular analysis of NOD/SCID BM confirmed the high levels of engraftment of human transduced cells deduced from FACS analysis. Finally, the analysis of the provirus insertion sites by conventional Southern blotting indicated that the human hematopoiesis in the NOD/SCID BM was predominantly oligoclonal.

Animals↗
PubMed1999

Retroviral gene transfer into human hematopoietic cells: an in vitro kinetic study.

BACKGROUND AND OBJECTIVE: Successful gene therapy applications require optimized strategies to increase gene transfer efficiency into hematopietic progenitor cells (HPCs) with long-term repopulating ability. One of the issues that needs to be clarified is how hematopoietic cells proliferate, differentiate and express the transgene after each cycle of transduction. We investigated the kinetics of cell expansion, CD34 antigen expression and transduction efficiency of human hematopoietic cells in culture conditions commonly used in retroviral gene transfer protocols. DESIGN AND METHODS: Purified CD34+ cells from cord blood (n=5) or leukapheresis products (n=9) and a retroviral vector encoding an enhanced version of the green fluorescent protein (EGFP) were used. Target cells were exposed daily to vector-containing supernatants and a combination of interleukin 3 (IL-3), interleukin 6 (IL-6), stem cell factor (SCF) and Flt3-ligand (FL). Cell samples were harvested from the cultures and analyzed at 24 hour intervals for seven consecutive days. RESULTS: We found that CD34+ cells proliferated and differentiated under our culture conditions. The number of genetically modified cells increased after each cycle of transduction. Median numbers of cells positive for both CD34 and EGFP increased steadily over the culture period, but after day four most of the EGFP+ cells had a low CD34 expression. INTERPRETATION AND CONCLUSIONS: Culturing and transducing CD34+ cells for longer periods of time under these conditions might be detrimental for ex vivo gene transfer applications since the transduced cells are likely to have a decreased potential for long-term engraftment and repopulation in vivo.

Antigens, CD34↗
PubMed1998

Highly efficient transduction of the green fluorescent protein gene in human umbilical cord blood stem cells capable of cobblestone formation in long-term cultures and multilineage engraftment of immunodeficient mice.

Purified CD34(+) and CD34(+)CD38(-) human umbilical cord blood (UCB) cells were transduced with the recombinant variant of Moloney murine leukemia virus (MoMLV) MFG-EGFP or with SF-EGFP, in which EGFP expression is driven by a hybrid promoter of the spleen focus-forming virus (SFFV) and the murine embryonic stem cell virus (MESV). Infectious MFG-EGFP virus was produced by an amphotropic virus producer cell line (GP+envAm12). SF-EGFP was produced in the PG13 cell line pseudotyped for the gibbon ape leukemia virus (GaLV) envelope proteins. Using a 2-day growth factor prestimulation, followed by a 2-day, fibronectin fragment CH-296-supported transduction, CD34(+) and CD34(+)CD38(-) UCB subsets were efficiently transduced using either vector. The use of the SF-EGFP/PG13 retroviral packaging cell combination consistently resulted in twofold higher levels of EGFP-expressing cells than the MFG-EGFP/Am12 combination. Transplantation of 10(5) input equivalent transduced CD34(+) or 5 x 10(3) input equivalent CD34(+)CD38(-) UCB cells in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice resulted in median engraftment percentages of 8% and 5%, respectively, which showed that the in vivo repopulating ability of the cells had been retained. In addition, mice engrafted after transplantation of transduced CD34(+) cells using the MFG-EGFP/Am12 or the SF-EGFP/PG13 combination expressed EGFP with median values of 2% and 23% of human CD45(+) cells, respectively, which showed that the NOD/SCID repopulating cells were successfully transduced. EGFP+ cells were found in all human hematopoietic lineages produced in NOD/SCID mice including human progenitors with in vitro clonogenic ability. EGFP-expressing cells were also detected in the human cobblestone area-forming cell (CAFC) assay at 2 to 6 weeks of culture on the murine stromal cell line FBMD-1. During the transduction procedure the absolute numbers of CAFC week 6 increased 5- to 10-fold. The transduction efficiency of this progenitor cell subset was similar to the fraction of EGFP+ human cells in the bone marrow of the NOD/SCID mice transplanted with MFG-EGFP/Am12 or SF-EGFP/PG13 transduced CD34(+) cells, ie, 6% and 27%, respectively. The study thus shows that purified CD34(+) and highly purified CD34(+)CD38(-) UCB cells can be transduced efficiently with preservation of repopulating ability. The SF-EGFP/PG13 vector/packaging cell combination was much more effective in transducing repopulating cells than the MFG-EGFP/Am12 combination.

Animals↗
PubMed1997

High-titer retroviral vectors containing the enhanced green fluorescent protein gene for efficient expression in hematopoietic cells.

Retroviral vectors constitute the most efficient system to deliver and integrate foreign genes into mammalian cells. We have developed a producer cell line that yields high titers of amphotropic retroviral vectors carrying the enhanced green fluorescent protein (EGFP) gene, a codon humanized, red-shifted variant of the green fluorescent protein (GFP) gene, which can be used as a selectable marker. We have used a hybrid vector that has been shown to efficiently drive gene expression in hematopoietic cells. Virtually all murine and human cell lines and primary human hematopoietic cells tested were transduced with varying efficiency after incubation with vector-containing supernatants. Human CD34(+) cells obtained from cord blood or aphereses products were transduced using a protocol that involves daily addition of vector-containing supernatants for 6 consecutive days. At day 6, up to 16% of the cells expressed EGFP, as assessed by flow cytometry. Sorted EGFP-expressing cells were able to produce fluorescent hematopoietic colonies. EGFP's main advantages are its fast flow cytometry determination and the possibility of cell sorting and simultaneous evaluation of the transduction efficiency along with other phenotypic markers.

3T3 Cells↗
PubMed1995

Myelosuppressive conditioning improves autologous engraftment of genetically marked hematopoietic repopulating cells in dogs.

We have studied the role of different conditioning regimens for engraftment of genetically marked hematopoietic repopulating cells in dogs. Peripheral blood (PB) and/or marrow cells collected after treatment with recombinant canine stem cell factor (rcSCF) or cyclophosphamide were transduced in a vector-containing long-term culture system. Three different vector-producing cell lines with similar viral titers were used. In two of them, the neo-containing LN vector was packaged either in the PA317 cell line with an amphotropic murine retrovirus envelope or the PG13 cell line with the gibbon ape leukemia virus (GALV) envelope. The MFG/GC vector produced in PA317 cells contained the human glucocerebrosidase gene. Nineteen dogs received either no conditioning (group A, n = 5), irradiation to both humeri with 1,000 cGy (group B, n = 5), a sublethal dose of cyclophosphamide 40 mg/kg (group C, n = 4), a sublethal dose of 200 or 300 cGy total body irradiation (TBI) (group D, n = 3), or an otherwise lethal dose of 920 cGy TBI (group E, n = 3) before intravenous (groups A, C, D, E) or intramedullary (group B) infusion of the transduced autologous hematopoietic cells. Transduction efficiency of hematopoietic cells at the time of infusion into the animals was similar among the different conditioning groups. Dogs were observed for at least 6 months. PB granulocytes were obtained at least every 3 weeks after transplant and analyzed by polymerase chain reaction for the presence of the transduced genes. The percentages of positive results in dogs more than 4 weeks after transplantation were 0% without conditioning, 5% with local irradiation, 18% with sublethal cyclophosphamide, 33% with sublethal TBI, and 17% with otherwise lethal TBI. Analyzing the influence of conditioning regimens by a generalized estimating equation (GEE) technique, which considered the use of different retrovirus vectors and the number of mononuclear cells infused as potential confounding variables, we found that engraftment of genetically marked repopulating cells was significantly improved (P < .001) in dogs receiving systemic conditioning with either otherwise lethal TBI, sublethal TBI, or sublethal cyclophosphamide compared to dogs with local irradiation only or no conditioning. Within the limitation of the experimental design, these data suggest that myeloablative or myelosuppressive conditioning improves engraftment of genetically marked hematopoietic repopulating cells.

Animals↗
PubMed1995

Allogeneic marrow grafts from donors with congenital chromosomal abnormalities in marrow cells.

To determine whether siblings with chromosomal abnormalities in marrow cells which are associated with cellular defects (e.g. Down syndrome or heterozygosity for Fanconi syndrome) are suitable donors for allogeneic bone marrow transplants, we have reviewed the patient files at the Fred Hutchinson Cancer Research Center (FHCRC) and carried out a survey among member centres of the International Bone Marrow Transplant Registry (IBMTR). The 57 of 253 (23%) member centres which responded to the survey reported seven transplants from donors with the following conditions: Down syndrome (n = 2), suspected heterozygotes for Fanconi syndrome (n = 3), and 47,XXX syndrome (n = 2), among a total of 5,561 allogeneic transplants from HLA-identical siblings. Adding the three cases seen at the Fed Hutchinson Cancer Research Center among 2,927 HLA-identical sibling transplants during 1992 resulted in 10 transplants among 8,488 cases transplanted overall: four with Down syndrome, four suspected of being heterozygous for Fanconi syndrome, and two trisomy X. Three out of four grafts from siblings with Down syndrome had complications, including poor graft function (n = 2) and graft failure (n = 1). Two of four recipients of marrow from presumed Fanconi syndrome heterozygotes presented with poor graft function and a third recipient developed graft failure after initial evidence of engraftment. The two patients given marrow from siblings with 47,XXX syndrome engrafted uneventfully. The experience reported here shows a low frequency of encountering an HLA-identical sibling donor who has chromosomal abnormalities in marrow cells consistent with Down syndrome or heterozygosity for Fanconi syndrome, about one case among 1,000 transplants. The much higher than expected incidence of graft problems with marrow from such a donor would make it reasonable to look either for an alternative marrow donor or consider an autologous transplant, in case a sibling marrow donor with Down syndrome or heterozygosity for Fanconi syndrome is encountered, although a donor with trisomy X seems acceptable.

Adolescent↗
PubMed1994

Antibodies against platelet-activating factor in patients with antiphospholipid antibodies.

We have studied the specificity of antiphospholipid antibodies in 148 patients with autoimmune diseases, 120 patients with systemic lupus erythematosus and 28 with the primary antiphospholipid syndrome. In addition, 20 patients suffering from syphilis were studied. As a control group, 64 healthy volunteers were investigated. Patient and control serum samples were tested for binding to seven different phospholipid antigens by ELISA. Interestingly, 90% of the sera from syphilis patients and 6% of the autoimmune patients exhibited a significant binding to platelet-activating factor (PAF), a molecule similar to the structure of phosphatidylcholine. In addition, the IgG fraction from one of the lupus patients, which showed a high binding activity to PAF, was further affinity-purified using both liposomes and an affinity chromatography column. Preincubation of these antibodies with PAF inhibited subsequent binding to immobilized PAF. These observations might suggest a putative interaction of antiphospholipid autoantibodies with PAF 'in vivo', which may have, in some patients, important pathophysiological consequences.

Antibodies, Antiphospholipid↗
PubMed1994

Effect of human IgG antiphospholipid antibodies on an in vivo thrombosis model in mice.

High levels of IgG antiphospholipid antibodies (aPL) have been associated with clinical thrombosis. It is uncertain however whether these antibodies play a direct role in thrombosis or are merely epiphenomena. To investigate whether antiphospholipid antibodies might play a role in thrombosis, we utilized a novel mouse model in which the dynamics of in vivo thrombosis can be studied. CD1 mice (26-30 g) were passively immunized with 25 mg of human IgG from a patient with the Antiphospholipid Syndrome (IgG-APS) (n = 17), IgG from normal pooled sera (IgG-NHS) (n = 9), or saline solution (n = 12), followed by 40 mg of the same preparations at 48 h. At 72 h, levels of human aPL antibodies, detected using the anticardiolipin ELISA test (aCL ELISA test), in mice immunized with IgG-APS, were 50-100 GPL units. Each animal was anesthetized, femoral vein minimally mobilized and subjected to a standardized "pinch" injury to induce thrombosis. The vessel was transilluminated using acrylic optical fibers connected to a light source, and clot formation and dissolution were visualized by a standard surgical microscope equipped with a video camera, video recorder, and computer assisted analysis system. Results showed that average clot size was significantly larger in mice immunized with IgG-APS compared to those treated with saline (p < 0.037). In addition, the thrombus persisted longer in a significantly higher number of mice immunized with IgG-APS (10/17) compared to mice immunized with IgG-NHS (1/9) or saline (2/12) (p < 0.02).(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1992

Valvular heart disease in the primary antiphospholipid syndrome.

OBJECTIVE: To determine the prevalence of cardiac valvular involvement in patients with the primary antiphospholipid syndrome. DESIGN: Cross-sectional study with evaluation of case patients and control patients by Doppler echocardiography. The mean follow-up for case patients was 22 months. SETTING: University-based tertiary medical center. PATIENTS: Twenty-eight consecutive patients who were diagnosed with the primary antiphospholipid syndrome during a 10-year period; 28 age- and sex-matched healthy controls. MEASUREMENTS AND MAIN RESULTS: Ten patients (36%; 95% Cl, 19% to 56%) with the primary antiphospholipid syndrome had cardiac valvular involvement: Four patients had mitral valve involvement; four patients, aortic valve involvement; and two patients, both mitral and aortic valvular involvement; no patients had tricuspid or pulmonary valve disease. Eight of 10 patients had a regurgitant murmur. None of the control patients had valvular disease. The mean mitral valve thickness in patients with mitral valve involvement was 7.0 +/- 1.6 mm, compared with 2.7 +/- 0.8 mm in patients with normal valves and 3.2 +/- 0.9 mm in the control group. The mean aortic valve thickness in patients with aortic valve involvement was 3.8 +/- 0.5 mm compared with 1.4 +/- 0.3 mm in patients with normal valves and 1.4 +/- 0.5 mm in the control group. Stenotic lesions were not found. Regurgitation was severe in two patients (one required surgery), moderate in three patients, and mild in three patients. CONCLUSIONS: Valvular involvement is frequently found in patients with the primary antiphospholipid syndrome. The lesions are left-sided, causing regurgitation that may be clinically important.

Adult↗
PubMed1990

[Primary antiphospholipid syndrome: study of 27 patients].

The clinical and biological features of a series of 27 patients with the recently described primary antiphospholipid syndrome are reported. Most of them belonged to a cohort of 90 patients who were carriers of lupus anticoagulant, which had been detected in the systematic evaluation of prolonged activated partial thromboplastin times in our hospital. Since the diagnosis they underwent a prospective protocol of follow up, with a peak follow up period of 9 years. The mean age of the 27 patients was 40.8 years and there were virtually no differences between sexes. Venous thrombosis was the most common clinical finding (16 episodes in 14 of the 27 patients). The most prevalent laboratory findings were lupus anticoagulant and IgG anticardiolipin antibodies.

Abortion, Spontaneous↗