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Biomedical subjects

C Bueno

Publications and source records attributed to C Bueno.

At least 19 recordsLinked to original sources
PubMed2006

Human embryonic stem cells: A journey beyond cell replacement therapies.

Success in the derivation of human embryonic stem cell (hESC) lines has opened up a new area of research in biomedicine. Human ESC not only raise hope for cell replacement therapies but also provide a potential novel system to better understand early human normal development, model human abnormal development and disease, and perform drug-screening and toxicity studies. The realization of these potentials, however, depends on expanding our knowledge about the cellular and molecular mechanisms that regulate self-renewal and lineage specification. Here, we briefly highlight the potential applications of hESC and review how flow cytometry has contributed to the initial characterization of both undifferentiated hESC cultures and hematopoietic development arising from hESC. We envision that a combination of state-of-the-art technologies, including cytomics, proteomics and genomics, will be instrumental in moving the field forward, ultimately lending invaluable knowledge to research areas such as human embryology, oncology and immunology.

Animals↗
PubMed2006

[Intravenous immunoglobulin therapy: ELISA measurement of antidesmogleins 1 and 3 in three patients with pemphigus vulgaris].

Intravenous immunoglobulin therapy (IVIg) has been used in the treatment of autoimmune bullous diseases unresponsive to conventional therapy in recent years. The action mechanism, which is not well known, suggests a wide spectrum of immunoregulation. In the last five years, several studies on patients with unresponsive pemphigus vulgaris with a clinical and serological outcome after IVIg administration in 80%-90% of cases have been published. We report the case of 3 patients with pemphigus vulgaris in whom we measured autoantibody titers to desmoglein 3 and 1 during 8 months. In spite of the clinical improvement, no significant decrease in antibody concentration was observed. Therapy with IVIg, although it has clinical benefit, did not decrease antibody values in our patients and thus it may need to be combined with immunosuppressant that inhibit pathogen antibody production.

Adult↗
PubMed2005

Complement-fixing activity of anticardiolipin antibodies in patients with and without thrombosis.

We have analysed in vitro the complement-fixing activity of anticardiolipin antibodies (C-fix aCL) from patients with persistent and moderate/high titres IgG aCL antibodies: 21 with thrombosis and 11 without thrombosis. Titre and C-fix ability of aCL were measured by ELISA. APS and non-APS patients were similar with regard to mean levels of IgG aCL (46 +/- 24 versus 51 +/- 30 GPL, P = 0.7), frequency of IgM aCL (P = 0.7) and a comparable predominance of IgG2 aCL reactivity on ELISA (95% versus 100%, respectively, P = 1.0). Remarkably, a high frequency of C-fix aCL (71% versus 92%, P = 0.35) was observed in both groups. Similarly, no difference was observed in the mean level of C-fix aCL in APS and non-APS patients (7 +/- 6 versus 9 +/- 8 SDunits, P = 0.3). Analysis of 10 primary and 11 secondary APS also revealed a comparable IgG aCL mean titre (57 +/- 29 versus 37 +/- 11, P = 0.06), frequency of IgM aCL (P = 0.6) and of C-fix aCL (70% versus 73%, P = 0.99). Among APS patients six had exclusive arterial events and seven exclusive venous events. The IgG aCL mean titre (36 +/- 10 versus 36 +/- 11 GPL, P = 0.9) and the frequency of IgM aCL antibodies (P = 0.56) in these subgroups of patients were comparable. There was a trend of higher frequency of C-fix aCL in patients with exclusive venous events (100%) compared to 50% of those with exclusive arterial events (p = 0.07). Importantly, C-fix aCL titre was higher in the former group compared to the later one (8 +/- 5 SDunits versus 2 +/- 2 SDunits, P = 0.016). Our data support the notion of a high frequency of C-fix aCL in APS. Although it does not discriminate those patients without thrombotic events with persistent moderate/high levels of aCL, this property seems to be more relevant in venous events and may provide the basis for further understanding the distinct pathogenic mechanisms underlying arterial and venous occlusive disorders of APS.

Adult↗
PubMed2004

Quantitative analysis of bcl-2 expression in normal and leukemic human B-cell differentiation.

Lack of apoptosis has been linked to prolonged survival of malignant B cells expressing bcl-2. The aim of the present study was to analyze the amount of bcl-2 protein expressed along normal human B-cell maturation and to establish the frequency of aberrant bcl-2 expression in B-cell malignancies. In normal bone marrow (n=11), bcl-2 expression obtained by quantitative multiparametric flow cytometry was highly variable: very low in both CD34(+) and CD34(-) B-cell precursors, high in mature B-lymphocytes and very high in plasma cells. Bcl-2 expression of mature B-lymphocytes from peripheral blood (n=10), spleen (n=8) and lymph node (n=5) was significantly higher (P<0.02) in CD23(-) as compared to CD23(+) B cells, independent of the type of tissue analyzed. Upon comparison with normal human B-cell maturation, bcl-2 expression in neoplastic B cells from 144 patients was found to be aberrant in 66% of the cases, usually corresponding to bcl-2 overexpression (63%). Follicular lymphoma (FL) carrying t(14;18) and MALT lymphoma were the only diagnostic groups constantly showing overexpression of bcl-2. Bcl-2 overexpression was also frequently found in precursor B-acute lymphoblastic leukemia (84%), typical (77%) and atypical (75%) B-cell chronic lymphocytic leukemia, prolymphocytic leukemia (two of three cases), mantle cell lymphoma (55%), but not in t(14;18)(-) FL, splenic marginal zone lymphoma, Burkitt lymphoma and multiple myeloma.

Adult↗
PubMed2002

C-ANCA-positive IgG fraction from patients with Wegener's granulomatosis induces lung vasculitis in rats.

The aim of the present study was to analyse in rats the ability of C-ANCA-positive IgG fraction in triggering inflammatory response on pulmonary tissue. Wistar rats (n = 18) were injected via the the internal jugular vein with 20 mg of total C-ANCA-positive IgG fraction isolated from serum of three different Wegener's granulomatosis patients obtained before therapy. Similarly, control rats were treated with IgG fraction from two rheumatoid arthritis patients (n = 7), IgG from six normal human sera (n = 15) or saline (n = 18), respectively. Animals were sacrificed after 24h of injection for histological analysis of the lungs. Vasculitis and inflammatory infiltrate were consistently absent in rats injected with rheumatoid arthritis IgG or saline and in 14/15 of normal IgG treated animals. In contrast, marked vasculitis was observed in all 18 animals injected with C-ANCA-positive IgG fraction. The histological features were characterized by the presence of a perivascular pleomorphic cellular sheath, particularly around small vessels, endothelial adherence and diapedesis of polymorphonuclear leucocytes and presence of granuloma-like lesions. A dose-response relationship was observed between protein concentration of C-ANCA IgG sample and the intensity of the inflammatory response in the animals. In addition, IgG fraction with undetectable C-ANCA, obtained from one patient in remission after treatment, was not able to reproduce the pulmonary tissue alterations induced by its paired IgG that was positive for C-ANCA taken before therapy. The experimental model described herein may be useful to characterize more effectively the pathogenic mechanism of C-ANCA in Wegener's disease.

Adult↗
PubMed2002

The excited-state interaction of resazurin and resorufin with amines in aqueous solutions. Photophysics and photochemical reactions.

The photophysics and photochemical behavior of the phenoxazin-3-one dyes, resazurin and resorufin, have been studied in aqueous solutions. The irradiation of resazurin in the presence of amines leads to deoxygenation of the N-oxide group, giving resorufin. This photoreaction is highly dependent on the amine structure and is efficient only in the presence of tertiary aliphatic amines. The absorption and fluorescence properties of these dyes are dependent on pH. At pH above 7.5 both dyes are in their anionic form. For resorufin this form is highly fluorescent (phiF = 0.75). At lower pH the fluorescence is strongly reduced. The N-oxide dye presents a very weak fluorescence quantum yield (0.11), which also is reduced at low pH. Flash photolysis experiments allowed characterization of the triplet state and the transients formed after irradiation of these dyes in the absence and presence of amines. The triplet quantum yields are 0.08 for resazurin and 0.04 for resorufin. The photodeoxygenation of N-oxide in the presence of amines occurs from the triplet state.

Amines↗
PubMed2001

Flow cytometric analysis of cytokine production by normal human peripheral blood dendritic cells and monocytes: comparative analysis of different stimuli, secretion-blocking agents and incubation periods.

In this paper, we comparatively analyze the effects of the following different stimuli on the production and intracellular accumulation of the interleukin (IL)-1 beta, IL-6, IL-12, tumor necrosis factor-alpha (TNF-alpha), and IL-8 inflammatory cytokines in both normal human peripheral blood (PB) dendritic cell (DC) subsets and monocytes: lipopolysaccharide (LPS) versus Staphylococcus aureus cowan I (SAC) in the presence or absence of interferon-(IFN)-gamma-, cytokine secretion-blocking agents (brefeldin A alone versus brefeldin A plus monensin), and incubation periods (6, 12, and 24 h). For this purpose, a four-color multiple-staining direct immunofluorescence technique analyzed by flow cytometry was systematically used in all experiments (n = 19). Our results show that after stimulation, an important proportion of each of the two CD33(+) myeloid DC subsets as well as the monocytes produce significant amounts of all cytokines analyzed under each of the experimental conditions assayed. In contrast, CD33(-/+lo) lymphoplasmocytoid DC failed to produce detectable levels of any of the above-mentioned cytokines under the same stimulatory conditions. Upon comparing the different stimuli used, LPS was associated with higher percentages of cytokine-producing cells compared with SAC, especially within the CD33(hi) DC subset; interestingly, the addition of IFN-gamma enhanced the response of monocytes to both LPS and SAC. As regards the secretion-blocking agents, brefeldin A alone was superior to the combination of brefeldin A and monensin. This is because it was frequently associated with both a higher percentage of cytokine-positive cells and greater amounts of detectable cytokines per cell. Sequential analysis of cytokine production by PB DC and monocytes after 6, 12, and 24 h of cell culture showed that after 6 h, an increased cell death rate existed among DC, which became even undetectable at 24 h, in the absence of a significant increase in cytokine secretion. In summary, our results show that from the experimental conditions assayed in this paper, to induce cytokine production by normal human DC and monocytes, maximum response is obtained once PB samples are stimulated for 6 h with LPS (with or without IFN-gamma) in the presence of brefeldin A alone.

Adolescent↗
PubMed2001

Comparative analysis of the morphological, cytochemical, immunophenotypical, and functional characteristics of normal human peripheral blood lineage(-)/CD16(+)/HLA-DR(+)/CD14(-/lo) cells, CD14(+) monocytes, and CD16(-) dendritic cells.

Human peripheral blood (PB) CD14(lo)/HLA-DR(+) cells were initially described as a subset of mature monocytes. Recently, it has been suggested that these represent a part of a new subset of dendritic cells (DC), characterized by the coexpression of MDC-8/HLA-DR/CD16. The aim of the present paper was to analyze the morphological, cytochemical, phenotypical, and functional characteristics of PB CD16(+)/HLA-DR(+) cells compared to both PB CD14(+) monocytes and CD16(-) DC. In contrast to CD14(+) monocytes, purified CD16(+)/HLA-DR(+) cells displayed cytoplasmic veils and lacked cytoplasmic myeloperoxidase and alpha-naphthyl acetate esterase. Normal human PB CD16(+)/HLA-DR(+) cells also displayed phenotypic characteristics different from those of CD14(+) monocytes: they lacked the CD64 Fcgamma receptor, showed lower levels of CD32, and expressed higher amounts of CD16 compared to CD14(+) monocytes. They also displayed a different pattern of expression of other antigens, including CD14, HLA-DR, CD45RA, CD45RO, complement receptors and complement regulatory surface proteins, adhesion and costimulatory molecules, and cytokine receptors, among others. When compared to CD16(-) DC, CD16(+)/HLA-DR(+) cells showed reactivity for CD16, dim positivity for CD14, higher expression of both Ig- and complement-receptors and lower reactivity for HLA-DR, adhesion, and costimulatory molecules (with the exception of CD86). The CD16(+)/HLA-DR(+) cell subset displayed a higher Ig/complement-mediated phagocytic/oxidative activity than CD16(-) DC, although this activity was significantly lower than that of mature monocytes. Regarding cytokine production at the single cell level, LPS plus IFN-gamma-stimulated PB CD16(+)/HLA-DR(+) cells produced significant amounts of IL1beta, IL6, IL12, TNFalpha, and IL8; however, the percentage of cytokine-producing cells and the amount of cytokine/cell were lower in CD16(+)/HLA-DR(+) cells than in CD14(+) monocytes. In addition, upon comparing CD16(+)/HLA-DR(+) cells with CD33(+++)/CD16(-) DC, we found that the percentage of cytokine-producing cells and the amount of cytokine/cell were significantly different in both cell subsets. In summary, our results show that CD16(+)/HLA-DR(+) cells clearly display different morphologic, cytochemical, immunophenotypical, and functional characteristics compared to both mature monocytes and CD16(-) DC. Interestingly, these cells are more frequent than other DC in normal human adult PB and cord blood samples, while they are less represented in normal bone marrow.

Adult↗
PubMed2001

Serotonergic regulation of inhibitory avoidance and one-way escape in the rat elevated T-maze.

It has been proposed that distinct 5-HT pathways modulate different types of anxiety. Activation of the ascending dorsal raphe (DR)-5-HT pathway, innervating the amygdala and frontal cortex, would facilitate learned defensive behaviors. On the other hand, activation of the DR-periventricular 5-HT pathway, which innervates the dorsal periaqueductal gray matter (DPAG), would inhibit innate flight or fight reactions. Dysfunction of these pathways has been suggested to relate to generalized anxiety disorder (GAD) and panic disorder (PD) in humans, respectively. The elevated T-maze has been developed to separate conditioned (inhibitory avoidance) from unconditioned (escape) defensive responses in the same rat. Pharmacological validation of this model has shown that the GAD-effective serotonergic anxiolytic buspirone or the putative anxiolytic ritanserin selectively impaired inhibitory avoidance while leaving one-way escape unchanged. Chronic injection of the 5-HT/noradrenaline reuptake inhibitor imipramine impaired inhibitory avoidance and prolonged escape, an effect that may be related to the therapeutic action of this drug on both GAD and PD. Like imipramine, intra-DPAG injection of the 5-HT(1A) agonist 8-OH-DPAT impaired both inhibitory avoidance and one-way escape. Intra-DPAG administration of the 5-HT(2A/2C) agonist DOI prolonged escape, without affecting inhibitory avoidance. The reversible inactivation of the DRN by muscimol impaired inhibitory avoidance, while facilitating escape from the open arm. Taken together, these results suggest that 5-HT exerts differential control on inhibitory avoidance and escape response in the elevated T-maze, mobilizing different types of 5-HT receptors in key structures implicated in fear/anxiety.

Animals↗
PubMed2001

Human neural stem cells in vitro. A focus on their isolation and perpetuation.

Because of their ability to generate all the cell types in the nervous system, neural stem cells are promising candidates for the development of cellular and genetic therapies for nervous system disorders and, in particular, neurodegenerative diseases. In recent years, researchers have discovered ways of expanding and perpetuating these cells in culture, as well as different sources for these tissue-specific stem cells, ranging from embryonic to adult tissue, and also from human pluripotent stem cells. Current efforts are oriented to the understanding of the molecular mechanisms controlling their fate decisions, their genetic engineering, and how to harness their potential to make them useful from a therapeutic point of view.

Cells, Cultured↗
PubMed2001

Sequential analysis of CD34+ and CD34- cell subsets in peripheral blood and leukapheresis products from breast cancer patients mobilized with SCF plus G-CSF and cyclophosphamide.

Administration of stem cell factor (SCF) has been proven to enhance cytokine-induced mobilization of CD34+ hematopoietic progenitor cells (HPC) into the peripheral blood (PB). The aim of the present study was to explore in a homogeneous group of 22 uniformly treated breast cancer patients: (1) the kinetics of mobilization into PB of both CD34+ and CD34- cell subsets, including dendritic cells, in sequential samples obtained from day +7 up to day +12 after mobilization; and (2) the composition of the CD34+ and CD34- cell subsets present in the two leukapheresis products obtained for each patient. The following CD34+ and CD34- subsets were analyzed: early CD34+ HPC, erythroid-, myeloid- and B-lymphoid-committed CD34+ precursor cells, mature T, B and NK cells, monocytes, neutrophils, eosinophils, basophils, and dendritic cells (DC) including three subsets of lin-/HLADR+DC (CD16+, CD33high and CD123high). Our results show that the absolute number of PB CD34+ HPC progressively increases from day +7 onwards. As far as the CD34- PB leukocyte subsets are concerned, monocytes (CD14+) displayed the earliest recovery after mobilization predicting neutrophil recovery 1 day in advance. The number of CD34+ HPC collected in a single leukapheresis product was always > or = 1.4 x 10(6) cells/kg body weight. No significant changes were observed between the two leukapheresis sessions either as regards their composition in CD34+ HPC subsets or their CD34- leukocyte populations except for a higher ratio of both CD34+ erythroid/CD34+ myeloid HPC (0.35 +/- 0.13 vs 0.30 +/- 0.13; P = 0.04) and neutrophils/monocytes (1.58 +/- 2.1 vs 0.69 +/- 0.27; P = 0.009) found for the first leukapheresis. Interestingly, the overall number of dendritic cells (DC) was higher in the second leukapheresis (1.06 +/- 0.56 vs 1.9 +/- 0.46; P = 0.02) due to a selective increase of the CD16+ antigen-presenting cells. In summary, our results show that the combination of cyclophosphamide, G-CSF and SCF is highly effective for stem cell mobilization, with differences observed in the mobilization kinetics of the different hematopoietic cell subsets analyzed.

Adolescent↗
PubMed2001

Human neural stem and progenitor cells: in vitro and in vivo properties, and potential for gene therapy and cell replacement in the CNS.

The generation of unlimited quantities of neural stem and/or progenitor cells derived from the human brain holds great interest for basic and applied neuroscience. In this article we critically review the origins and recent developments of procedures developed for the expansion, perpetuation, identification, and isolation of human neural precursors, as well as their attributes. Factors influencing their in vitro properties, both under division and after differentiation conditions, are evaluated, with the aim of identifying properties common to the different culture systems reported. This analysis suggests that different culture procedures result in cells with different properties, or even in different cells being isolated. With respect to in vivo performance, present evidence obtained in rodents indicate that cultured human neural precursors, in general, are endowed with excellent integrative properties. Differentiation of the implanted cells, in particular in the case of adult recipients, seems not to be complete, and functionality still needs to be demonstrated. In relation to gene transfer and therapy, aspects currently underexplored, initial data support the view that human neural stem and progenitor cells may serve a role as a platform cell for the delivery of bioactive substances to the diseased CNS. Although a large deal of basic research remains to be done, available data illustrate the enormous potential that human neural precursors isolated, expanded, and characterized in vitro hold for therapeutic applications. In spite of this potential, maintaining a critical view on many unresolved questions will surely help to drive this research field to a good end, that is, the development of real therapies for diseases of the human nervous system.

Animals↗
PubMed2000

Genetically perpetuated human neural stem cells engraft and differentiate into the adult mammalian brain.

Human neural stem cells (HNSCs) may serve as a cellular vehicle for molecular therapies as well as for cell replacement in the human CNS. The survival, integration, and differentiation of HNSC.100, a multipotent cell line of HNSCs (A. Villa et al. (2000), Exp. Neurol. 161, 67-84), conditionally perpetuated by genetic and epigenetic means, was investigated after transplantation to the striatum and substantia nigra of the adult, intact rat brain. These are two key regions in the mammalian brain involved in the control of voluntary movement and motor coordination, among other functions. Soon after transplantation (1 week), the cells had already integrated in a nondisruptive manner into the surrounding tissue and migrated out of the implantation site to different distances depending on graft location (in the range of 0.5-2.5 mm). Cell migration was markedly more extensive in the striatum, where the cells colonized the whole extent of the caudate-putamen, than in the substantia nigra region. The engrafted cells completely downregulated the stem cell marker nestin and, due to their multipotential nature, differentiated and expressed mature neural markers. As expected from cells grafted into nonneurogenic regions of the intact brain, the majority of differentiated cells expressed GFAP (astroglia), but expression of other markers, like GalC (oligodendroglia) and MAP2, beta-tubulin III, NeuN, and NSE (for mature neurons) could also be detected. These results demonstrate that genetically perpetuated HNSCs, once transplanted, find residence in the host brain, where they differentiate, generating mature neural cells in the host, chimeric, adult mammalian brain. HNSCs cell lines may be a highly useful model for the development of humanized systems for cell replacement and/or gene transfer to the CNS, which will likely be strong candidates for future therapeutic application in human neurodegenerative conditions.

Animals↗
PubMed1999

Photochemical and pharmacokinetic properties of selected flavins.

Some photochemical and pharmacokinetic properties of riboflavin, lumiflavin and the 2',3',4',5' tetraacetyl, tetrapropionyl, tetrabutiryl and tetrapalmitoyl esters of riboflavin have been studied. The esters are considerably more photostable than riboflavin but less so than lumiflavin, and appear to be photosensitizers with a behaviour similar to that of riboflavin, promoting photoreactions of biological targets even in the absence of molecular oxygen. The various flavins display important differences in their pharmacokinetic behaviour. Riboflavin, lumiflavin and the short-chain esters (the acetyl and propionyl esters) are rapidly cleared from serum, and recovered in comparable amounts from the liver and kidneys. These results are in agreement with their hydrophilic or moderately hydrophobic character. In contrast, the longer-chain butiryl and palmitoyl esters exhibit a prolonged retention in serum and undergo a significantly larger accumulation in the liver as compared with the kidneys; they are also found in the spleen. In all cases the tissue uptake of these esters becomes appreciable only after 24 h. These results are coherent with the highly hydrophobic character of these esters, which induce a slow release from serum lipoproteins and have a preferential clearance via the bile-gut pathway, showing affinity for the components of the reticuloendothelial system. These long-chain riboflavin esters will probably have a greater and more persistent risk of photoinduced hepatotoxicity than riboflavin, lumiflavin and the short-chain esters.

Animals↗
PubMed1999

Extensive characterization of the immunophenotype and pattern of cytokine production by distinct subpopulations of normal human peripheral blood MHC II+/lineage- cells.

Dendritic cells (DC) represent the most powerful professional antigen-presenting cells (APC) in the immune system. The aim of the present study was to analyse, on a single-cell basis by multiparametric flow cytometry with simultaneous four-colour staining and a two-step acquisition procedure, the immunophenotypic profile and cytokine production of DC from 67 normal whole peripheral blood (PB) samples. Two clearly different subsets of HLA-II+/lineage- were identified on the basis of their distinct phenotypic characteristics: one DC subset was CD33strong+ and CD123dim+ (0.16 +/- 0.06% of the PB nucleated cells and 55.9 +/- 11. 9% of all PB DC) and the other, CD33dim+ and CD123strong+ (0.12 +/- 0.04% of PB nucleated cells and 44.53 +/- 11.5% of all PB DC). Moreover, the former DC subpopulation clearly showed higher expression of the CD13 myeloid-associated antigen, the CD29 and CD58 adhesion molecules, the CD2, CD5 and CD86 costimulatory molecules, the CD32 IgG receptor and the CD11c complement receptor. In addition, these cells showed stronger HLA-DR and HLA-DQ expression and a higher reactivity for the IL-6 receptor alpha-chain (CD126) and for CD38. In contrast, the CD123strong+/CD33dim+ DC showed a stronger reactivity for the CD4 and CD45RA molecules, whereas they did not express the CD58, CD5, CD11c and CD13 antigens. Regarding cytokine production, our results show that while the CD33strong+/CD123dim+ DC are able to produce significant amounts of inflammatory cytokines, such as IL-1beta (97 +/- 5% of positive cells), IL-6 (96 +/- 1.1% of positive cells), IL-12 (81.5 +/- 15.5% of positive cells) and tumour necrosis factor-alpha (TNF-alpha) (84 +/- 22.1% of positive cells) as well as chemokines such as IL-8 (99 +/- 1% of positive cells), the functional ability of the CD123strong+/CD33dim+ DC subset to produce cytokines under the same conditions was almost null. Our results therefore clearly show the presence of two distinct subsets of DC in normal human PB, which differ not only in their immunophenotype but also in their functionality, as regards cytokine production.

AC133 Antigen↗