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Biomedical subjects

G Juan

Publications and source records attributed to G Juan.

At least 19 recordsLinked to original sources
PubMed2006

Directly observed treatment for tuberculosis in pharmacies compared with self-administered therapy in Spain.

OBJECTIVES: To compare directly observed treatment (DOT) of tuberculosis through pharmacy offices with self-administered treatment (SAT) in patients at risk for non-adherence. METHODS: Prospective study for DOT (1999-2002) and retrospective study for SAT (1996-1998) in patients at risk for non-adherence (human immunodeficiency virus [HIV] infection, alcoholism, illicit drug use, immigrant or homeless status and/or previous failure to complete). Patients in the DOT programme received medication as out-patients twice a week in pharmacies that supervised adherence and provided socio-sanitary support to patients. RESULTS: There were 101 and 112 patients in the DOT and SAT groups, respectively. Demographic and clinical characteristics were similar in both groups. Differences were observed in risk factors for non-adherence (more immigrants and fewer intravenous drug users in the DOT vs. the SAT groups; P < 0.05). In the DOT group, 76 patients (75.2%) completed treatment and were cured compared to only 30 patients (26.7%) in the SAT group (P < 0.001). Implementation of DOT increased the cost of treatment by 400 Euro per patient compared to SAT. CONCLUSION: In patients at risk for non-adherence, DOT implemented through pharmacy offices was better than SAT; however, completion rates were still low.

Adolescent↗
PubMed2001

Neuroblastic and Schwannian stromal cells of neuroblastoma are derived from a tumoral progenitor cell.

The coexistence of neuroblastic and Schwannian stromal (SS) cells in differentiating neuroblastoma (NB), and derivation of Schwannian-like cells from neuroblastic clones in vitro, were accepted previously as evidence of a common pluripotent tumor stem line. This paradigm was challenged when SS cells were suggested to be reactive in nature. The advent of microdissection techniques, PCR-based allelic analysis, and in situ fluorescent cytometry made possible the analysis of pure cell populations in fresh surgical specimens, allowing unequivocal determination of clonal origins of various cell subtypes. To overcome the complexity and heterogeneity of three-dimensional tissue structure, we used: (a) Laser-Capture Microdissection to obtain histologically homogeneous cell subtype populations for allelotype analysis at chromosomes 1p36, 11q23, 14q32, and 17q and study of MYCN copy number; (b) multiparametric analysis by Laser-Scanning Cytometry of morphology, DNA content, and immunophenotype of intact cells from touch imprints; and (c) bicolor fluorescence in situ hybridization on touch imprints from manually microdissected neuroblast and stroma-rich areas. Histologically distinct SS and neuroblastic cells isolated by Laser-Capture Microdissection had the same genetic composition in 27 of 28 NB analyzed by allelic imbalance and gene copy number. In all 20 cases studied by Laser-Scanning Cytometry, SS cells identified by morphology and S-100 immunostaining had identical DNA content and GD2-staining pattern as their neuroblastic counterparts. In 7 cases, fluorescence in situ hybridization demonstrated the same chromosomal makeup for SS and neuroblastic cells. These results provide unequivocal evidence that neuroblastic and SS cells in NB are derived from genetically identical neoplastic cells and support the classical paradigm that NB arises from tumoral cells capable of development along multiple lineages.

Adolescent↗
PubMed2001

Endothelial apoptosis as the primary lesion initiating intestinal radiation damage in mice.

Gastrointestinal (GI) tract damage by chemotherapy or radiation limits their efficacy in cancer treatment. Radiation has been postulated to target epithelial stem cells within the crypts of Lieberkühn to initiate the lethal GI syndrome. Here, we show in mouse models that microvascular endothelial apoptosis is the primary lesion leading to stem cell dysfunction. Radiation-induced crypt damage, organ failure, and death from the GI syndrome were prevented when endothelial apoptosis was inhibited pharmacologically by intravenous basic fibroblast growth factor (bFGF) or genetically by deletion of the acid sphingomyelinase gene. Endothelial, but not crypt, cells express FGF receptor transcripts, suggesting that the endothelial lesion occurs before crypt stem cell damage in the evolution of the GI syndrome. This study provides a basis for new approaches to prevent radiation damage to the bowel.

Animals↗
PubMed2001

Intranuclear compartmentalization of cyclin E during the cell cycle: disruption of the nucleoplasm-nucleolar shuttling of cyclin E in bladder cancer.

Cyclin E/cyclin-dependent kinase 2 complexes are essential during the cell cycle for entrance into S phase. Cyclin E expression starts in mid-G1, reaches a maximum at S-phase entrance, and undergoes proteolysis mediated by the ubiquitin pathway as the cell progresses through S phase. Laser scanning cytometry, a microscope-based cytofluorometer combining the advantages of both flow and image analysis, allowed the determination of subcellular localization of cyclin E, p27, and retinoblastoma protein during cell cycle progression in normal human fibroblasts and nine bladder cancer cell lines. We observed that in normal fibroblasts and most tumor cell lines, cyclin E localizes in the nucleoplasm during mid-G1, and is translocated to the nucleolus during G1-S-phase transition, and its levels are undetectable in G2-M phase. Neither levels nor subcellular localization of p27 and retinoblastoma protein was cell cycle dependent in normal or tumor cells. However, four of nine bladder cancer cell lines continued to express cyclin E in all phases of the cycle, and image analysis revealed that it was localized to nucleoli. These observations suggest that the nucleolus mediates a cyclin E "shuttling" between the nucleus and the cytoplasm that is probably involved in its regulation and that this mechanism could be disrupted in bladder cancer.

Cell Cycle↗
PubMed2001

Use of V4R in patients who sustain blunt chest trauma.

OBJECTIVE: In blunt chest trauma, the right ventricle is more vulnerable than the left. The purpose of this study was to determine whether recording V4R in patients with blunt chest trauma would provide additional useful information to that already obtained from the standard 12-lead electrocardiogram (ECG). METHODS: Forty-five patients with blunt chest trauma and 40 unmatched control subjects without blunt chest trauma had standard 12-lead ECG and right precordial leads recorded. The ECGs were read blindly by three physicians. RESULTS: Patients with chest trauma were distinguishable from controls on the basis of the left-sided ECGs (odds ratio, 2.9; 95% confidence interval, 1.71-4.90). This was not the case using V4R (odds ratio, 1.23; 95% confidence interval, 0.59-2.0). CONCLUSION: Patients with a significant mechanism and physical findings of blunt chest trauma were more likely than controls to have an abnormal ECG. They were not more likely to have abnormalities in V4R. We recommend that a 12-lead ECG, but not V4R, be routinely obtained on these patients.

Adolescent↗
PubMed2001

Magnetic resonance imaging of the pharynx in OSA patients and healthy subjects.

Obstructive sleep apnoea (OSA) occurs because of recurrent narrowing and occlusion of the velopharynx (VP) during sleep. The specific cause of OSA is unknown. Cephalometric radiography, fibreoptic nasopharyngoscopy, acoustic reflection techniques, and computerized tomography have limitations (dynamic and tridimensional evaluation) in the mechanism of occlusion investigation. Static and dynamic examination of the soft tissue structures surrounding the upper airway during the respiratory cycle in wakefulness and sleep, can lead to a better understanding of the process. Ultrafast magnetic resonance imaging (one image per 0.8 s) was used to study the upper airway and surrounding soft tissue in 17 patients with OSA during wakefulness and sleep, and in eight healthy subjects whilst awake. The major findings of this investigation in the 25 subjects were as follows: 1) the VP was smaller in apnoeic patients, only during part of the respiratory cycle; 2) the variation in VP area during the respiratory cycle was greater in apnoeic patients than in controls, particularly during sleep, suggesting an increased compliance of the VP in these patients; 3) VP narrowing was similar in the lateral and anterior-posterior dimensions, both in controls and apnoeic patients while awake; apnoeic patients during sleep have a more circular VP upon reaching the minimum area; 4) there was an inverse relationship between dimensions of the lateral pharyngeal walls and airway area, probably indicating that lateral walls are passively compressed or stretched as a result of changes in the airway calibre; and 5) soft palate and parapharyngeal fatpads were larger in apnoeic patients, although their role in the genesis of OSA is uncertain. It was concluded that changes in the velopharynx area and diameter during the respiratory cycle are greater in apnoeic patients than in normal subjects, particularly during sleep. This suggests that apnoeic patients have a more collapsible velopharynx, this being the main mechanism of obstruction.

Adult↗
PubMed2000

Fenretinide-induced caspase 3 activity involves increased protein stability in a mechanism distinct from reactive oxygen species elevation.

Fenretinide (4-HPR) is a synthetic retinoid that displays a broad range of biological effects and has also demonstrated clinical efficacy as a chemopreventative agent. One cellular activity of 4-HPR is its ability to induce apoptosis. This effect has been proposed to relate to changes in intracellular reactive oxygen species. We show herein that a 1-h treatment of HL-60 cells with 4-HPR led to a dose-dependent increase in hydroperoxides. Pretreatment of cells with the antioxidant vitamin C abolished apoptosis, measured as the appearance of the sub-G1 peak, in 4-HPR-treated cells. The retinoid also elicited a 3.6-fold increase in caspase 3 activity; however, this increase was not affected by vitamin C treatment. Analysis of caspase 3 protein expression by Western blot analysis revealed that 4-HPR resulted in a significant increase in the appearance of the active p17 subunit without effecting a concomitant change in p32 procaspase 3 levels. Studies on de novo synthesis and stability of caspase 3 by pulse-chase and immunoprecipitation methods show that 4-HPR-treated samples had decreased incorporation of radioactive amino acid precursors into newly synthesized procaspase 3 but, during the chase (for up to 9 h), had more labeled caspase 3 remaining when compared with controls. These studies suggest that 4-HPR may effect changes in caspase 3 activity by modulating changes in zymogen stability by a mechanism distinct from the retinoid-elicited increase in reactive oxygen species.

Acetylcysteine↗
PubMed2000

Cell cycle arrest and apoptosis of melanoma cells by docosahexaenoic acid: association with decreased pRb phosphorylation.

The incidence of cutaneous malignant melanoma is undergoing a dramatic increase in persons with light-color skin in all parts of the world. The prognosis for individuals with advanced disease is dismal due to the lack of effective treatment options. Thus, there is a need for new approaches to control tumor progression. Epidemiological, experimental, and mechanistic data implicate omega-6 polyunsaturated fatty acids (PUFAs) as stimulators and long-chain omega-3 PUFAs as inhibitors of development and progression of a range of human cancers, including melanoma. The aim of this study was to assess the mechanisms by which docosahexaenoic acid (DHA), an omega-3 PUFA, affects human melanoma cells. Exponentially growing melanoma cell lines were exposed in vitro to DHA and then assessed for (a) inhibition of cell growth; (b) expression of cyclins and cyclin-dependent kinase inhibitors in individual cells by flow cytometry and immunocytochemistry using specific monoclonal antibodies to cyclin D1, cyclin E, p21WAF1/CIP1, or p27(KIP1); and (c) expression of total pRb(T) independent of phosphorylation state and hypophosphorylated pRb(P-) in fixed cells by flow cytometry and immunocytochemistry using specific monoclonal antibodies to pRb(T) or pRb(P-), respectively. After treatment with increasing concentrations of DHA, cell growth in a majority of melanoma cell lines (7 of 12) was inhibited, whereas in 5 of 12 cell lines, cell growth was minimally affected. Two melanoma cell lines were examined in detail, one resistant (SK-Mel-29) and one sensitive (SK-Mel-110) to the inhibitory activity of DHA. SK-Mel-29 cells were unaffected by treatment with up to 2 microg/ml DHA whether grown in the absence or presence of 1% fetal bovine serum (FBS). No appreciable change was observed in cell growth, cell cycle distribution, the status of pRb phosphorylation, cyclin D1 expression, or the levels of the cyclin-dependent kinase inhibitors p21 and p27. In contrast, SK-Mel-110 cell growth was inhibited by DHA with the cells accumulating either in G1 or S phase: 0% in SK-Mel-29 versus 13.3 or 41.2% in SK-Mel-110 in the absence or presence of FBS, respectively. In the absence of serum, considerable death occurred by apoptosis. In addition, DHA treatment resulted in increasing numbers of SK-Mel-110 cells (from 12 to >40%) expressing hypophosphorylated pRb, whereas the levels of cyclin D1 and p21 changed little. Expression of p27 in these cells increased >2.5 times when grown in the absence of FBS but not in the presence of 1% FBS. Thus, we show for the first time that DHA inhibits the growth of cultured metastatic melanoma cells. Furthermore, growth inhibition correlates with a quantitative increase in hypophosphorylated pRb in the representative sensitive melanoma cell line SK-Mel-110. Although multiple factors influence pRb phosphorylation, it appears that both cyclin D1 and p21 expression do not change in the presence of DHA, although p27 was strikingly increased in SK-Mel-110 cells in the absence of FBS. The fact that pRb became hypophosphorylated after exposure to DHA suggests a cross-talk mechanism between fatty acid metabolism and the pRb pathway. Determining the mechanism by which PUFAs can inhibit melanoma growth will be an important first step in the rational use of PUFAs as antitumor agents.

Animals↗
PubMed1999

Resveratrol increases nitric oxide synthase, induces accumulation of p53 and p21(WAF1/CIP1), and suppresses cultured bovine pulmonary artery endothelial cell proliferation by perturbing progression through S and G2.

Epidemiological studies have shown that the regular consumption of red wine may in part account for the apparent compatibility of a high fat diet with a low incidence of coronary atherosclerosis. This phenomenon, commonly referred to as the French paradox, may be associated with red wine constituents that exhibit tumor-preventive properties as well as inhibit reactions that increase the risk of coronary heart disease. Here we show that resveratrol, a polyphenol in red wine, induces nitric oxide synthase, the enzyme responsible for the biosynthesis of NO, in cultured pulmonary artery endothelial cells, suggesting that resveratrol could afford cardioprotection by affecting the expression of nitric oxide synthase. We also show that resveratrol inhibits the proliferation of pulmonary artery endothelial cells, which, based on flow cytometric analysis, correlates with the suppression of cell progression through S and G2 phases of the cell cycle. Western blot analysis and immunocytochemical protein detection combined with multiparameter flow cytometry further demonstrate that the perturbed progression through S and G2 phases is accompanied by an increase in the expression of tumor suppressor gene protein p53 and elevation of the level of cyclin-dependent kinase inhibitor p21(WAF1/CIP1). All of the observed effects of resveratrol, including induction of apoptosis at its higher concentration, are also compatible with its putative chemopreventive and/or antitumor activity.

Animals↗
PubMed1999

Histone H3 phosphorylation in human monocytes and during HL-60 cell differentiation.

Phosphorylation of the nucleosome core histone H3 (H3) on Ser-10 is thought to be a prerequisite for chromatin condensation at mitosis. Although during interphase, cell differentiation, or mitogenic activation of quiescent cells, changes in chromatin structure that involve local chromatin condensation/decondensation also occur, little is known about H3 phosphorylation during these transitions. Using the recently developed sensitive marker to monitor H3 phosphorylation, namely, the mAb that recognizes the phosphorylated epitope of H3 (anti-H3-P mAb), the status of H3 phosphorylation was assayed in individual human lymphocytes after their mitogenic stimulation (G0 to G1 transition) and in human leukemic HL-60 cells induced to differentiate by all-trans-retinoic acid (RA), 1,25-dihydroxyvitamin D3 (vit D3), dimethyl sulfoxide (DMSO), or phorbol myristate acetate (PMA). Multiparameter flow cytometry was used to correlate H3 phosphorylation with cell cycle position. The specificity of the anti-H3-P mAb was confirmed by the loss of its binding following cell treatment with alkaline phosphatase. The presence of phosphorylated H3 was detected during interphase in HL-60 cells and in normal lymphocytes at a level severalfold lower than during mitosis. No significant changes in H3 phosphorylation were observed during lymphocyte stimulation. Unexpectedly, the level of H3 phosphorylation was over fourfold higher in monocytes than in lymphocytes or granulocytes from peripheral blood. The punctate pattern of labeling with anti-H3-P mAb in monocyte nuclei suggests that H3 is phosphorylated in small clusters of adjacent nucleosomes. Differentiation of HL-60 cells was accompanied by a rise in H3 phosphorylation, which was higher after induction by RA, vit D3, and PMA (approx. threefold) than after DMSO (approximately 20%). The data indicate that in addition to being a critical event during chromatin condensation at mitosis, H3 phosphorylation plays a role during chromatin changes accompanying differentiation of HL-60 cells, in particular, along the monocytic lineage. The high level of H3 phosphorylation in monocytes may serve as a marker of these cells and is being explored as a possible diagnostic and prognostic tool in monocytic leukemias.

Antibodies, Monoclonal↗
PubMed1999

[Acute respiratory insufficiency as initial manifestation of brain stem lesions].

We describe three patients with different brainstem lesions (bulbar angioma, bulbar infarct, and Arnold-Chiari malformation) who debuted with acute respiratory insufficiency. Other neurological manifestations had gone unobserved in all three cases. Respiratory insufficiency worsened notably during sleep to the point that mechanical ventilation was required or death occurred (Ondine s curse). The patient with a bulbar angioma is stable with only assisted ventilation by a nasal route at night, with good quality of life. Our conclusions are that: a) central nervous system anomalies need to be investigated as possible causes of respiratory insufficiency when lungs are normal; b) the respiratory control of patients with brainstem lesions should be studied, particularly at night (polysomnography), and c) even when awake-state ventilation is adequate, nighttime assisted ventilation may be required.

Acute Disease↗
PubMed1998

Phosphorylation of retinoblastoma protein assayed in individual HL-60 cells during their proliferation and differentiation.

Expression of pRb and its state of phosphorylation were immunocytochemically assayed in individual HL-60 cells during their proliferation and after induction of differentiation, using mAb which detects hypophosphorylated pRb (pRbP-) combined with mAb which reacts with pRb regardless of its phosphorylation (total pRb; pRbT). Correlated measurements of pRbP-, pRbT, a ratio of pRbP-/pRbT, and cellular DNA content by flow cytometry revealed expression of total pRb and its phosphorylation state vis-à-vis the cell cycle position. Following mitosis (during the exponential phase of cell growth) a mixture of hypo- and hyperphosphorylated pRb was present within the cell for less than 2 h, i.e., early in G1; no hypophosphorylated pRb was detected throughout remainder of the cycle. Cellular pRb content was increasing primarily during G1 and the cell entrance to S was correlated with attainment of a distinct threshold level of pRb. No correlation was seen between the content of pRb per cell and its state of phosphorylation during G1. Cell differentiation whether induced by 1,25-dihydroxyvitamin D3, retinoic acid, or phorbol myristate acetate led to cell arrest primarily in G0/1. The G0/1 cells in these cultures, compared to G1 cells from the untreated cultures, had increased level of both pRbT and pRbP-. However, because the relative increase of pRbP- was disproportionally greater than of pRbT, the pRbP-/pRbT ratio of the differentiating cells was markedly elevated. The cells that still were in S and G2/M in the differentiating cultures also showed the presence of hypophosphorylated pRb. Our data suggest that the mechanism of irreversible cell cycle arrest during terminal differentiation involves both the increase in content of pRb and dephosphorylation of pRb already present within the cell. This provides a large pool of hypophosphorylated pRb that can effectively remove all free E2F, thereby precluding activation of the genes whose transcription is needed to pass the G1 restriction point. In contrast to terminal differentiation the transient quiescence (G0 state) manifests only by dephosphorylation of pRb, without a change in its cellular level.

Cell Differentiation↗
PubMed1998

Histone H3 phosphorylation and expression of cyclins A and B1 measured in individual cells during their progression through G2 and mitosis.

Phosphorylation of histone H3 (H3) on Ser-10 correlates with chromatin condensation at mitosis. A new monoclonal antibody (anti-H3-P) was developed that recognizes phosphorylated H3 (H3-P). This antibody was used in multiparameter flow cytometric analysis to relate H3 phosphorylation in individual human leukemic cells to the cells' position in the cycle as well as their expression of cyclins A and B1. Mitotic cells, from prophase to telophase, reacted with anti-H3-P; the binding of the antibody to chromatin of interphase cells was several times weaker. Cell growth in the presence of staurosporine, an inhibitor of the kinase(s) that phosphorylate H3, abolished the cells' reactivity with the antibody. The reactivity also was abolished by incubation of permeabilized mitotic cells with alkaline phosphatase. These data indicate that, within permeabilized cells, the antibody is indeed specific for H3-P and does not detect the unphosphorylated epitope. All cells reacting with anti-H3-P, with the exception of prophase and early prometaphase, were cyclin A negative; the expression of cyclin B1 in these cells was threefold higher than in G2 cells. The analysis of phosphorylation of H3 in individual cells when combined with multiparameter analysis of their cycle position and expression of other proteins offers new possibilities to study molecular mechanisms associated with the G2 to M transition and chromatin condensation. It also offers an assay to screen in vivo inhibitors of kinase(s) or phosphatase(s) involved in H3 phosphorylation or dephosphorylation, and it provides a valuable marker to identify mitotic cells by cytometry.

Alkaline Phosphatase↗