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PubMed2004

The duplicated B-class heterodimer model: whorl-specific effects and complex genetic interactions in Petunia hybrida flower development.

In both Antirrhinum (Antirrhinum majus) and Arabidopsis (Arabidopsis thaliana), the floral B-function, which specifies petal and stamen development, is embedded in a heterodimer consisting of one DEFICIENS (DEF)/APETALA3 (AP3)-like and one GLOBOSA (GLO)/PISTILLATA (PI)-like MADS box protein. Here, we demonstrate that gene duplications in both the DEF/AP3 and GLO/PI lineages in Petunia hybrida (petunia) have led to a functional diversification of their respective members, which is reflected by partner specificity and whorl-specific functions among these proteins. Previously, it has been shown that mutations in PhDEF (formerly known as GREEN PETALS) only affect petal development. We have isolated insertion alleles for PhGLO1 (FLORAL BINDING PROTEIN1) and PhGLO2 (PETUNIA MADS BOX GENE2) and demonstrate unique and redundant properties of PhDEF, PhGLO1, and PhGLO2. Besides a full homeotic conversion of petals to sepals and of stamens to carpels as observed in phglo1 phglo2 and phdef phglo2 flowers, we found that gene dosage effects for several mutant combinations cause qualitative and quantitative changes in whorl 2 and 3 meristem fate, and we show that the PHDEF/PHGLO1 heterodimer controls the fusion of the stamen filaments with the petal tube. Nevertheless, when the activity of PhDEF, PhGLO1, and PhGLO2 are considered jointly, they basically appear to function as DEF/GLO does in Antirrhinum and to a lesser extent as AP3/PI in Arabidopsis. By contrast, our data suggest that the function of the fourth B-class MADS box member, the paleoAP3-type PETUNIA HYBRIDA TM6 (PhTM6) gene, differs significantly from the known euAP3-type DEF/AP3-like proteins; PhTM6 is mainly expressed in the developing stamens and ovary of wild-type flowers, whereas its expression level is upregulated in whorls 1 and 2 of an A-function floral mutant; PhTM6 is most likely not involved in petal development. The latter is consistent with the hypothesis that the evolutionary origin of the higher eudicot petal structure coincided with the appearance of the euAP3-type MADS box genes.

Antirrhinum↗
PubMed2006

Does the rate of rewarming from profound hypothermic arrest influence the outcome in a swine model of lethal hemorrhage?

BACKGROUND: Rapid induction of profound hypothermic arrest (suspended animation) can provide valuable time for the repair of complex injuries and improve survival. The optimal rate for re-warming from a state of profound hypothermia is unknown. This experiment was designed to test the impact of different warming rates on outcome in a swine model of lethal hemorrhage from complex vascular injuries. METHODS: Uncontrolled lethal hemorrhage was induced in 40 swine (80-120 lbs) by creating an iliac artery and vein injury, followed 30 minutes later (simulating transport time) by laceration of the descending thoracic aorta. Through a thoracotomy approach, a catheter was placed in the aorta and hyperkalemic organ preservation solution was infused on cardiopulmonary bypass to rapidly (2 degrees C/min) induce profound (10 degrees C) hypothermia. Vascular injuries were repaired during 60 minutes of hypothermic arrest. The 4 groups (n = 10/group) included normothermic controls (NC) where core temperature was maintained between 36 to 37 degrees C, and re-warming from profound hypothermia at rates of: 0.25 degrees C/min (slow), 0.5 degrees C/min (medium), or 1 degrees C/min (fast). Hyperkalemia was reversed during the hypothermic arrest period, and blood was infused for resuscitation during re-warming. After discontinuation of cardiopulmonary bypass, the animals were recovered and monitored for 6 weeks for neurologic deficits, cognitive function (learning new skills), and organ dysfunction. Detailed examination of brains was performed at 6 weeks. RESULTS: All the normothermic animals died, whereas survival rates for slow, medium and fast re-warming from hypothermic arrest were 50, 90, and 30%, respectively (p < 0.05 slow and medium warming versus normothermic control, p < 0.05 medium versus fast re-warming). All the surviving animals were neurologically intact, displayed normal learning capacity, and had no long-term organ dysfunction. CONCLUSIONS: Rapid induction of hypothermic arrest maintains viability of brain during repair of lethal vascular injuries. Long-term survival is influenced by the rate of reversal of hypothermia.

Animals↗
PubMed2005

Refining the complex rheumatoid arthritis phenotype based on specificity of the HLA-DRB1 shared epitope for antibodies to citrullinated proteins.

OBJECTIVE: The main genetic risk factor for rheumatoid arthritis (RA), the HLA region, has been known for 25 years. Previous research has demonstrated, within the RA population, an association between HLA-DRB1 alleles carrying the shared epitope (SE) and antibodies directed against cyclic citrullinated peptides (anti-CCP antibodies). We undertook this study to make the first comparison of SE allele frequencies in the healthy population with those in RA patients who do or do not harbor anti-CCP antibodies. METHODS: HLA-DRB1 typing was performed in 408 RA patients from the Leiden Early Arthritis Clinic (the Leiden EAC; a Dutch population-based inception cohort in which disease course was followed up over time), in 423 healthy Dutch controls, and in 720 affected members of 341 US multiplex (sibpair) families of Caucasian origin from the North American RA Consortium (NARAC) with well-established disease and fulfilling the American College of Rheumatology classification criteria for RA. The presence of anti-CCP antibodies was determined by enzyme-linked immunosorbent assay. RESULTS: For the Leiden EAC, the odds ratio (OR) describing the association of 2 copies of the SE allele with anti-CCP positivity (using no copies of the SE allele in the healthy control group as the referent) was 11.79 (P < 0.0001), while the OR for 1 SE allele was 4.37 (P < 0.0001). No association with the SE was observed in the Dutch anti-CCP-negative RA patients. For the NARAC families, linkage and association analysis revealed the SE to be associated only with anti-CCP-positive disease and not with anti-CCP-negative disease. Stratified analyses indicated that anti-CCP antibodies primarily mediated association of the SE with joint damage or disease persistence. CONCLUSION: HLA-DRB1 alleles encoding the SE are specific for disease characterized by antibodies to citrullinated peptides, indicating that these alleles do not associate with RA as such, but rather with a particular phenotype.

Arthritis, Rheumatoid↗
PubMed2005

Conservation of pregnancy-specific glycoprotein (PSG) N domains following independent expansions of the gene families in rodents and primates.

BACKGROUND: Rodent and primate pregnancy-specific glycoprotein (PSG) gene families have expanded independently from a common ancestor and are expressed virtually exclusively in placental trophoblasts. However, within each species, it is unknown whether multiple paralogs have been selected for diversification of function, or for increased dosage of monofunctional PSG. We analysed the evolution of the mouse PSG sequences, and compared them to rat, human and baboon PSGs to attempt to understand the evolution of this complex gene family. RESULTS: Phylogenetic tree analyses indicate that the primate N domains and the rodent N1 domains exhibit a higher degree of conservation than that observed in a comparison of the mouse N1 and N2 domains, or mouse N1 and N3 domains. Compared to human and baboon PSG N domain exons, mouse and rat PSG N domain exons have undergone less sequence homogenisation. The high non-synonymous substitution rates observed in the CFG face of the mouse N1 domain, within a context of overall conservation, suggests divergence of function of mouse PSGs. The rat PSG family appears to have undergone less expansion than the mouse, exhibits lower divergence rates and increased sequence homogenisation in the CFG face of the N1 domain. In contrast to most primate PSG N domains, rodent PSG N1 domains do not contain an RGD tri-peptide motif, but do contain RGD-like sequences, which are not conserved in rodent N2 and N3 domains. CONCLUSION: Relative conservation of primate N domains and rodent N1 domains suggests that, despite independent gene family expansions and structural diversification, mouse and human PSGs retain conserved functions. Human PSG gene family expansion and homogenisation suggests that evolution occurred in a concerted manner that maintains similar functions of PSGs, whilst increasing gene dosage of the family as a whole. In the mouse, gene family expansion, coupled with local diversification of the CFG face, suggests selection both for increased gene dosage and diversification of function. Partial conservation of RGD and RGD-like tri-peptides in primate and rodent N and N1 domains, respectively, supports a role for these motifs in PSG function.

Amino Acid Motifs↗
PubMed2005

Immunochemical analysis of protein expression in breast epithelial cells transformed by estrogens and high linear energy transfer (LET) radiation.

Breast cancer is a complex disease involving numerous genetic aberrations. Immunochemical analysis of protein expression is presented in a human breast epithelial cell line neoplastically transformed by high linear energy transfer (LET) alpha particle radiation in the presence of 17beta estradiol (E) and in the parental human breast epithelial cell line (MCF-10F) which served as a non-tumorigenic control. The aim of this work was to determine the levels of mRNA and protein expression in control and transformed cells at various stages of the neoplastic process. The levels of mRNA and protein expression of PCNA, c-fos, JNK2 and Fra-1 were increased in the transformed cell line compared to the levels in non-tumorigenic control cells. The transforming factor Rho A was significantly increased only in the tumor cell line. Furthermore, the levels of mRNA and protein expression of ErbB2 were significantly increased in the transformed cell line and in tumor cells derived from the transformed cells after injecting them into nude mice. A decrease in RbA/p48 protein expression and mRNA levels was observed in cells treated with double doses of alpha particle radiation in the presence of estrogen, regardless of tumorigenicity. Such expression was lower than that in the control untreated MCF-10F cells. In summary, these studies show that estrogen and high LET-radiation induce changes in oncoprotein expression and mRNA levels of human breast cell lines. These changes are indicative of a cascade of events that characterize the process of cell transformation in breast cancer. These results provide evidence that multiple steps with consecutive changes are involved when normal cells become tumorigenic cells as a result of alpha particle irradiation and estrogen treatments.

Adult↗
PubMed2002

Subcellular redistribution of la/SSB autoantigen during physiologic apoptosis in the fetal mouse heart and conduction system: a clue to the pathogenesis of congenital heart block.

OBJECTIVE: In isolated congenital heart block, the mechanism by which maternal autoantibodies target the intracellular components of the Ro/La RNP complex is unclear. Previous studies have demonstrated that cultured fetal cardiac myocytes rendered apoptotic bind antibodies to 48-kd La/SSB. This study further investigated the subcellular distribution of the La antigen during apoptosis in the fetal mouse heart and conduction system. METHODS: The atrioventricular (AV) node, AV bundle, and sinoatrial (SA) node were identified in serial sections prepared from paraffin blocks of normal mouse fetuses on days 15, 17, and 19 of gestation. Apoptosis was detected by TUNEL assay. Under confocal microscopy, fluorescent labeling of fragmented DNA in apoptotic cells was assessed by TUNEL, and La protein localization was visualized simultaneously using a murine monoclonal antibody or affinity-purified human polyclonal anti-La antibodies. RESULTS: Apoptotic cells were detected in and at the periphery of the AV and SA nodes as well as in the fetal heart valve insertions and working myocardium. In contrast, no apoptosis was detected in the adult heart AV node or surrounding myocardium. As expected, the La antigen was predominantly immunolocalized to the nucleus in nonapoptotic cells. However, apoptotic cells showed a marked reduction of nuclear La and redistribution of La to the cytoplasm. High-resolution confocal microscopy revealed that in cells that had undergone apoptosis, La antigen asymmetrically clustered near the surface of TUNEL-positive nuclei and apoptotic bodies. CONCLUSION: These data provide the first in vivo demonstration of the subcellular translocation of La autoantigen during apoptosis in the fetal heart and the conduction system under physiologic conditions. This observation supports the hypothesis that subcellular redistribution of La in the normally developing heart facilitates the binding of cognate maternal antibodies and subsequent tissue damage.

Animals↗
PubMed2004

The search for genes contributing to the low level of response to alcohol: patterns of findings across studies.

BACKGROUND: Alcoholism is a complex genetically influenced disorder in which multiple phenotypes [e.g., disinhibition, alcohol-metabolizing patterns, and the low level of response (LR) to alcohol] contribute to the risk. A low LR to alcohol is one of the more thoroughly studied risk phenotypes; data indicate that LR relates to the risk status, predicts future alcoholism, and has a heritability as high as 60%. This article reviews data from animal and human studies regarding the LR to alcohol, searching for a convergence of results that might lead to the identification of relevant genes. METHODS: A literature search was performed regarding animal and human genetic studies focusing on genes that might affect the LR to alcohol as a risk factor for alcoholism. The goal was to synthesize these results and highlight potential patterns. RESULTS: Focusing on both genetic linkage and association studies, a number of chromosomal regions and genes potentially relevant to findings across two or more sources were identified. The genes of potential interest fell into several categories, including second-messenger systems (e.g., G proteins, adenylyl cyclase, and protein kinases); neurotransmitters or drug-related receptors (e.g., gamma-aminobutyric acid-A, glutamate, serotonin, and cannabinoid and opioid receptors); genes that affect alcohol metabolism; and genes that might relate to an overlap in the risk for alcoholism and some psychiatric conditions (e.g., catechol-O-methyltransferase regarding schizophrenia and bipolar disorder). CONCLUSIONS: The review identifies several genes that may contribute to a low LR to alcohol and, thus, to an increased risk for alcohol use disorders. The chromosomal regions and genes highlighted here may form the basis for more focused genetic studies of alcohol use disorders, with the goals of developing more specific and effective prevention and treatment approaches.

Alcohol Drinking↗
PubMed2005

Analysis of the interaction between human steroid 21-hydroxylase and various monoclonal antibodies using comparative structural modelling.

OBJECTIVE: To study the interaction between human steroid 21-hydroxylase (21-OH) and monoclonal antibodies (MAbs) to 21-OH directed to 3 different epitopes recognised by 21-OH autoantibodies characteristic of autoimmune Addison's disease. DESIGN: Build comparative structural models of 21-OH, 21-OH MAbs and complexes of 21-OH-21-OH MAbs and study the effects of 21-OH MAbs on 21-OH enzyme activity. Then, analyse the relationship between sites important for binding of 21-OH MAbs and 21-OH autoantibodies and sites important for 21-OH enzyme activity. METHODS: Variable (V) regions of 21-OH MAbs (M21-OH1, M21-OH3, M21-OH5) were sequenced and models of the MAbs built using structures of antibodies in the database as templates. A comparative model of 21-OH was built using the crystal structure of rabbit cytochrome p450 2c5/3LVdH as template. 21-OH enzyme activity was measured in terms of conversion of [3H]progesterone to deoxycorticosterone and the effect of purified MAb IgGs on 21-OH enzyme activity was assessed. RESULTS: M21-OH1, M21-OH3 and control MAb had no effect on 21-OH enzyme activity with 88.8% +/- 24% (n = 6), 86.7% +/- 7.6% (n = 6) and 86.5% +/- 10.6% (n = 6) of activity remaining in the presence of the respective IgGs. This was consistent with the epitopes for M21-OH1 and M21-OH3 being located distant from 21-OH enzyme active sites in our 21-OH model. The epitope for M21-OH5 which inhibited 21-OH enzyme activity (48.5 +/- 8.3% activity remaining; P < 0.001 compared with control MAb IgG) was found close to the redox protein binding site in our 21-OH model. CONCLUSIONS: A comparative model of 21-OH has been produced. Analysis of experimental data in the context of the model suggests that M21-OH5 inhibits 21-OH enzyme activity through interference with redox protein binding.

Animals↗
PubMed2005

Preclinical models of shock and sepsis: what can they tell us?

The goal of translational research is to transform biologic knowledge into new treatments for human disease. Although preclinical models replicate some of the features of the disease process modeled, they invariably fail to reproduce the complexity of human illness, and by their very experimental nature, they are readily manipulated to maximize evidence of efficacy. The result is that successful translation from preclinical models to clinically effective therapy is uncommon, and that clinical trials are often undertaken without a comprehensive and realistic preclinical portfolio of studies to optimize their design. The lethal and morbid human conditions of sepsis and shock are attractive targets for new therapies and enormously challenging processes to translate because they entail considerable clinical heterogeneity, require emergent effective intervention, and are shaped not only by the initial insult, but by approaches to subsequent resuscitation and support. A colloquium jointly sponsored by the Shock Society and the International Sepsis Forum in June 2004 addressed the challenges of translational research in shock and sepsis. Through a comprehensive review of a broad variety of model approaches, and vigorous debate about the merits of differing strategies, a series of common themes emerged. We concluded that there is no single ideal model of shock or sepsis, but rather a large number of complementary models that recapitulate some discrete features of the disorders while minimizing others. Consequently, successful preclinical investigation mandates the use of a panel of preclinical studies consciously designed to address specific questions of relevance to the clinical setting. A corollary of this conclusion is that preclinical studies can shape concepts of disease and can be used to refine decisions regarding optimal patient populations for therapeutic interventional trials. We further recognized that the design and reporting of preclinical studies is highly variable, thereby limiting effective data interpretation and integration between studies. Hence, greater model standardization would aid in interpreting data and in pooling results into systematic data syntheses: such efforts should be promoted and undertaken.

Animals↗
PubMed2006

An evaluation of the level of response to alcohol, externalizing symptoms, and depressive symptoms as predictors of alcoholism.

OBJECTIVE: The development of alcohol-use disorders (AUDs) reflects a complex relationship between genetic influences and environmental/cultural forces. Some genes operate through intermediate phenotypes, including a low level of response (LR) to alcohol, externalizing symptoms (EXT), and internalizing characteristics such as depressive syndromes (DEP). This article evaluates the impact of these three intermediate phenotypes and additional domains in a structural equation model (SEM). METHOD: Data were available from baseline at approximately age 20 for LR, as well as from additional domains at the 10- and 15-year follow-up periods for 393 men from the San Diego Prospective Study. Correlational analyses and an AMOS-based SEM were used to evaluate the development of alcohol problems, including AUDs, with the hypothetical model based on results from prior studies evaluating each key intermediate phenotype separately. RESULTS: The SEM explained 51% of the variance of the 15-year outcome, and had good fit characteristics. The family history ofAUDs (FHalc) was linked, directly or indirectly, to all three key domains. The combination of LR and EXT mediated the relationship between FHalc and 15-year alcohol outcomes, with a trend (p = .07) for LR to mediate between FHalc and the 10-year outcome. DEP, by itself, did not mediate FHalc to alcoholism. The LR predicted the 15-year outcome both through alcohol problems at 10 years and via drinking to cope (COPE), with each of these domains functioning as mediators. The relationship of EXT to outcome was mediated by alcohol expectations (EXPECT) and by COPE. DEP added to the model in the context of an FH of independent depressions, stress, and lower social supports, subsequently affecting COPE. CONCLUSIONS: The results indicate that the development of AUDs reflects several genetically influenced endophenotypes in the context of multiple additional domains. Both EXPECT and COPE represented important pathways through which the phenotypes influenced the AUD risk.

Adaptation, Psychological↗
PubMed2004

3 dimensional modelling of early human brain development using optical projection tomography.

BACKGROUND: As development proceeds the human embryo attains an ever more complex three dimensional (3D) structure. Analyzing the gene expression patterns that underlie these changes and interpreting their significance depends on identifying the anatomical structures to which they map and following these patterns in developing 3D structures over time. The difficulty of this task greatly increases as more gene expression patterns are added, particularly in organs with complex 3D structures such as the brain. Optical Projection Tomography (OPT) is a new technology which has been developed for rapidly generating digital 3D models of intact specimens. We have assessed the resolution of unstained neuronal structures within a Carnegie Stage (CS)17 OPT model and tested its use as a framework onto which anatomical structures can be defined and gene expression data mapped. RESULTS: Resolution of the OPT models was assessed by comparison of digital sections with physical sections stained, either with haematoxylin and eosin (H&E) or by immunocytochemistry for GAP43 or PAX6, to identify specific anatomical features. Despite the 3D models being of unstained tissue, peripheral nervous system structures from the trigeminal ganglion (approximately 300 microm by approximately 150 microm) to the rootlets of cranial nerve XII (approximately 20 microm in diameter) were clearly identifiable, as were structures in the developing neural tube such as the zona limitans intrathalamica (core is approximately 30 microm thick). Fourteen anatomical domains have been identified and visualised within the CS17 model. Two 3D gene expression domains, known to be defined by Pax6 expression in the mouse, were clearly visible when PAX6 data from 2D sections were mapped to the CS17 model. The feasibility of applying the OPT technology to all stages from CS12 to CS23, which encompasses the major period of organogenesis for the human developing central nervous system, was successfully demonstrated. CONCLUSION: In the CS17 model considerable detail is visible within the developing nervous system at a minimum resolution of approximately 20 microm and 3D anatomical and gene expression domains can be defined and visualised successfully. The OPT models and accompanying technologies for manipulating them provide a powerful approach to visualising and analysing gene expression and morphology during early human brain development.

Brain↗
PubMed2004

Complexities of the pathogenesis of Mannheimia haemolytica and Haemophilus somnus infections: challenges and potential opportunities for prevention?

Progress in producing improved vaccines against bacterial diseases of cattle is limited by an incomplete understanding of the pathogenesis of these agents. Our group has been involved in investigations of two members of the family Pasteurellaceae, Mannheimia haemolytica and Haemophilus somnus, which illustrate some of the complexities that must be confronted. Susceptibility to M. haemolytica is greatly increased during active viral respiratory infection, resulting in rapid onset of a severe and even lethal pleuropneumonia. Despite years of investigation, understanding of the mechanisms underlying this viral-bacterial synergism is incomplete. We have investigated the hypothesis that active viral infection increases the susceptibility of bovine leukocytes to the M. haemolytica leukotoxin by increasing the expression of or activating the beta2 integrin CD11a/CD18 (LFA-1) on the leukocyte surface. In vitro exposure to proinflammatory cytokines (i.e. interleukin-1beta, tumor necrosis factor-alpha and interferon-gamma) increases LFA-1 expression on bovine leukocytes, which in turn correlates with increased binding and responsiveness to the leukotoxin. Alveolar macrophages and peripheral blood leukocytes from cattle with active bovine herpesvirus-1 (BVH-1) infection are more susceptible to the lethal effects of the leukotoxin ex vivo than leukocytes from uninfected cattle. Likewise, in vitro incubation of bovine leukocytes with bovine herpesvirus 1 (BHV-1) potentiates LFA-1 expression and makes the cells more responsive to leukotoxin. A striking characteristic of H. somnus infection is its propensity to cause vasculitis. We have shown that H. somnus and its lipo-oligosaccharide (LOS) trigger caspase activation and apoptosis in bovine endothelial cells in vitro. This effect is associated with the production of reactive oxygen and nitrogen intermediates, and is amplified in the presence of platelets. The adverse effects of H. somnus LOS are mediated in part by activation of endothelial cell purinergic receptors such as P2X7. Further dissection of the pathways that lead to endothelial cell damage in response to H. somnus might help in the development of new preventive or therapeutic regimens. A more thorough understanding of M. haemolytica and H. somnus virulence factors and their interactions with the host might identify new targets for prevention of bovine respiratory disease.

Animals↗
PubMed2002

Simultaneous flow cytometric analysis of two cell surface markers, telomere length, and DNA content.

BACKGROUND: Various protocols for estimation of telomere length in individual cells by flow cytometry using fluorescence in situ hybridization of fluorescently labeled peptide nucleic acid (PNA) probes (Flow-FISH) have been described. Combined analysis of telomere length and cell phenotype, however, remains difficult because few fluorochromes with suitable emission spectra tolerate the harsh conditions needed for DNA denaturation during hybridization of the telomere-specific PNA probe. We overcame these problems and developed a method for measuring telomere length in cell subsets characterized by the expression of two surface antigens. METHODS: Alexa Fluor 488 and Alexa Fluor 546 were used for cell surface staining. Antigen-antibody complexes were covalently cross-linked onto the cell membrane before Flow-FISH. Cells were hybridized with a PNA probe conjugated to cyanine 5 (Cy5). Hoechst 33342 (HO342) was added for determination of cellular DNA content. For assay standardization, we added an aliquot of a single batch of 1,301 cells to each sample as an internal control before hybridization with the PNA probe. Samples were prepared in duplicate and analyzed on a standard three-laser BD LSR flow cytometer. For assay validation, the same samples were analyzed in parallel to correlate the percentage of telomere length of the sample versus 1,301 control cells to the mean size of terminal restriction fragments (TRFs) of DNA as determined by Southern gel analysis. RESULTS: The method permitted clear identification of lymphocyte subsets in samples hybridized for Flow-FISH, with subset frequencies comparable to those of untreated samples. At a concentration of 10 nM, the Cy5-labeled telomere-specific PNA probe produced a bright fluorescence signal well separated from background. Addition of HO342 in low concentration did not interfere with Cy5 telomere fluorescence, produced adequate DNA histograms, and permitted clear identification of cell phenotype. The probe concentration of 10 nM also proved optimal for inclusion of 1,301 control cells for assay standardization. Telomere length estimations by the current method correlated highly with TRF calculations by Southern gel hybridization (r(2)= 0.9, P = 0.0003). Application of our protocol to the analysis of human CD8CD28 lymphocyte subsets showed that CD8(+bright)CD28(-) lymphocytes generally exhibit shorter telomeres than CD8(+bright)CD28(+) cells. These data concurred with previous results of telomere shortening in CD8(+)CD28(-) T cells that were obtained by using different techniques. CONCLUSIONS: The multiparameter Flow-FISH protocol permitted rapid determination of differences in telomere length in subpopulations characterized by two surface markers without prior cell separation.

Antigens, CD↗
PubMed2006

In vitro and in vivo activity of R547: a potent and selective cyclin-dependent kinase inhibitor currently in phase I clinical trials.

The cyclin-dependent protein kinases are key regulators of cell cycle progression. Aberrant expression or altered activity of distinct cyclin-dependent kinase (CDK) complexes results in escape of cells from cell cycle control, leading to unrestricted cell proliferation. CDK inhibitors have the potential to induce cell cycle arrest and apoptosis in cancer cells, and identifying small-molecule CDK inhibitors has been a major focus in cancer research. Several CDK inhibitors are entering the clinic, the most recent being selective CDK2 and CDK4 inhibitors. We have identified a diaminopyrimidine compound, R547, which is a potent and selective ATP-competitive CDK inhibitor. In cell-free assays, R547 effectively inhibited CDK1/cyclin B, CDK2/cyclin E, and CDK4/cyclin D1 (K(i) = 1-3 nmol/L) and was inactive (K(i) > 5,000 nmol/L) against a panel of >120 unrelated kinases. In vitro, R547 effectively inhibited the proliferation of tumor cell lines independent of multidrug resistant status, histologic type, retinoblastoma protein, or p53 status, with IC(50)s </= 0.60 mumol/L. The growth-inhibitory activity is characterized by a cell cycle block at G(1) and G(2) phases and induction of apoptosis. R547 reduced phosphorylation of the cellular retinoblastoma protein at specific CDK phosphorylation sites at the same concentrations that induced cell cycle arrest, suggesting a potential pharmacodynamic marker for clinical use. In vivo, R547 showed antitumor activity in all of the models tested to date, including six human tumor xenografts and an orthotopic syngeneic rat model. R547 was efficacious with daily oral dosing as well as with once weekly i.v. dosing in established human tumor models and at the targeted efficacious exposures inhibited phosphorylation of the retinoblastoma protein in the tumors. The selective kinase inhibition profile and the preclinical antitumor activity of R547 suggest that it may be promising for development for use in the treatment of solid tumors. R547 is currently being evaluated in phase I clinical trials.

Animals↗
PubMed2003

Endocrinologic and immunologic factors associated with recovery of growth in children with human immunodeficiency virus type 1 infection treated with protease inhibitors.

INTRODUCTION: Growth failure is a common presenting sign in children with HIV disease and is a sensitive indicator of disease progression in children with AIDS. Highly active antiretroviral therapy (HAART) is associated with a significant decrease in viral load and a subsequent rise in CD4+ T cell counts in HIV-1-infected children and also with increased height and weight. The underlying mechanisms of catch-up growth during HAART are yet unknown. METHODS: Height and weight measurements, blood sample analyses for HIV-1 RNA and peripheral blood CD4+ T cell counts were obtained twice within 1 month before the start of HAART and after 12, 24, 36 and 48 weeks of treatment. Serum concentrations of insulin-like growth factor I (IGF-1), IGFs complexed to specific, structurally homologous binding proteins (IGFBP-3), cortisol, free thyroxine and tumor necrosis factor alpha (TNF-alpha) were measured before the start of therapy and after 24 weeks. In addition serum IGF-1 and IGFBP-3 values were determined after 48 weeks. RESULTS: Twenty-seven HIV-1-infected children with a median age of 5.5 years (range, 0.3 to 14.9 years) were included. Overall no significant changes in height and body mass index (BMI) z scores were observed. The median baseline plasma viral load of 68,800 copies/ml decreased to less than the detection limit of 500 copies/ml in 80% of the children after 48 weeks. TNF-alpha values were elevated (44 pg/ml) at baseline and decreased significantly to 37 pg/ml after 24 weeks. At baseline elevated TNF-alpha was observed in 78%, which decreased to 55% after 24 weeks. Baseline free thyroxine and cortisol values were normal and did not change during therapy. Baseline serum of IGF-1 and IGFBP-3 concentrations were normal, but IGF-1 tended to be lower than IGFBP-3. Both values increased significantly after the initiation of therapy. IGFBP-3 decreased after 48 weeks whereas IGF-1 stabilized. The increase in IGF-1 was significantly higher in children in whom the BMI and length (after correction for age and sex) increased the most. CONCLUSION: Hypothyroidism and adrenal axis abnormalities are not associated with restoration of growth after the initiation of antiretroviral therapy in HIV-1-infected children. The combination of relatively high serum IGFBP-3 concentration and relatively lower serum IGF-1 suggests the presence of a growth hormone-resistant state. During treatment with a protease inhibitor-containing regimen, decreased serum IGFBP-3 and stabilization of IGF-1 after a significant initial increase suggest restoration of normal sensitivity to growth hormone and recovery to an anabolic condition.

Antiretroviral Therapy, Highly Active↗
PubMed2002

A quantitative evaluation of IMRT dose distributions: refinement and clinical assessment of the gamma evaluation.

BACKGROUND AND PURPOSE: Although intensity modulated radiotherapy (IMRT) is a step forward in comparison to conventional, static beam delivery, quality assurance is more complex and labour intensive, demanding detailed two-dimensional dosimetric verification. Regardless of the technique used for measuring the dose distribution, what is essential to the implementation of routine verification of IMRT fields is the efficient and accurate comparison of the measured versus desired dose distribution. In order to achieve a fast, yet accurate quantitative measure of the correspondence between measured and calculated dose, the theoretical concept of the gamma evaluation method presented by Low et al. (Med. Phys., 25 (1998) 656) was converted into a calculation algorithm, taking into account practical considerations related to the discrete nature of the data. MATERIALS AND METHODS: A filter cascade of multiple levels was designed to obtain fast and accurate comparison of the two dose distributions under evaluation. The actual comparison consists of classification into accepted or rejected datapoints with respect to user-defined acceptance criteria (dose difference and distance to agreement). The presented algorithm was tested on dosimetric images calculated and/or acquired by means of a liquid filled portal imaging device during the course of intensity modulated treatments of prostate cancer, including pre-treatment verification as well as verification during treatment. To assess its ability to intercept possible errors in dose delivery, clinically relevant errors were deliberately introduced into the dose distributions. RESULTS: The developed gamma filter method proves successful in the efficient comparison of calculated versus measured IMRT dose distribution. Secondly, intercomparison of dosimetric images acquired during different treatment sessions illustrate its potential to highlight variations in the dosimetric images. The simulated errors were unmistakably intercepted. CONCLUSIONS: The readily obtained gamma evaluation images are an easy tool for quality control of IMRT fields. To reduce the artefacts related to the discrete nature and limited resolution of the data, a fast and accurate filter cascade was developed, offering the possibility to use the gamma method for day to day evaluation of patient dosimetric portal images with or without comparison to a predicted portal dose distribution.

Algorithms↗
PubMed2026

Management of Soft Tissue and Visceral Leiomyosarcomas.

IMPORTANCE: Leiomyosarcoma is a rare and heterogeneous malignant mesenchymal neoplasm associated with substantial morbidity and mortality. Given recent advances in biologic understanding and the complexity of leiomyosarcoma, a consensus-driven approach is needed to harmonize management and address remaining clinical and research gaps. OBJECTIVE: To provide an evidence-based synthesis of current diagnostic and therapeutic approaches for leiomyosarcoma by an international panel of physicians, researchers, and patient advocates, focusing on site-specific management, systemic therapy strategies, and key areas of clinical uncertainty, while identifying unmet needs and research priorities. EVIDENCE REVIEW: This review is based on a comprehensive evaluation of the literature, including clinical trials, observational studies, and international consensus guidelines. Sources were identified through MEDLINE (via PubMed) and Embase database searches and reference screening, then supplemented by multidisciplinary expert consensus. Emphasis was placed on studies informing diagnosis, surgical management, radiotherapy, and systemic therapy in leiomyosarcoma. FINDINGS: The rarity and heterogeneity of leiomyosarcoma poses substantial challenges in its management. In localized disease, complete surgical resection remains the cornerstone of treatment, with evidence supporting the use of site-specific perioperative treatment strategies. Prospective data supporting neoadjuvant or adjuvant chemotherapy are lacking, and the role of radiotherapy differs across anatomic disease sites and institutions. In advanced disease, multiple systemic therapies demonstrate activity, including anthracycline-based and gemcitabine-based combinations, trabectedin, and tyrosine kinase inhibitors, although optimal sequencing after first-line therapy remains undefined. Emerging data suggest potential benefit from treatment continuation strategies and selected use of local therapies in oligometastatic settings. Molecular heterogeneity is increasingly recognized but has not yet translated into routine clinical implementation, and integration of molecular profiling into diagnostic pathways for predictive and therapeutic insights remains an unmet need. CONCLUSIONS AND RELEVANCE: This international consensus addresses the diagnosis and management of leiomyosarcoma. Management requires a multidisciplinary, site-specific approach informed by limited but evolving evidence. Key uncertainties persist, particularly regarding perioperative therapy, optimal sequencing and combination of systemic treatments, and integration of molecular data. Continued international collaboration and leiomyosarcoma-specific clinical trials are needed to refine treatment strategies and improve patient outcomes.

Journal Article↗
PubMed2004

Conservation of the microstructure of genome segments in Brassica napus and its diploid relatives.

The cultivated Brassica species are the group of crops most closely related to Arabidopsis thaliana (Arabidopsis). They represent models for the application in crops of genomic information gained in Arabidopsis and provide an opportunity for the investigation of polyploid genome formation and evolution. The scientific literature contains contradictory evidence for the dynamics of the evolution of polyploid genomes. We aimed at overcoming the inherent complexity of Brassica genomes and clarify the effects of polyploidy on the evolution of genome microstructure in specific segments of the genome. To do this, we have constructed bacterial artificial chromosome (BAC) libraries from genomic DNA of B. rapa subspecies trilocularis (JBr) and B. napus var Tapidor (JBnB) to supplement an existing BAC library from B. oleracea. These allowed us to analyse both recent polyploidization (under 10,000 years in B. napus) and more ancient polyploidization events (ca. 20 Myr for B. rapa and B. oleracea relative to Arabidopsis), with an analysis of the events occurring on an intermediate time scale (over the ca. 4 Myr since the divergence of the B. rapa and B. oleracea lineages). Using the Arabidopsis genome sequence and clones from the JBr library, we have analysed aspects of gene conservation and microsynteny between six regions of the genome of B. rapa with the homoeologous regions of the genomes of B. oleracea and Arabidopsis. Extensive divergence of gene content was observed between the B. rapa paralogous segments and their homoeologous segments within the genome of Arabidopsis. A pattern of interspersed gene loss was identified that is similar, but not identical, to that observed in B. oleracea. The conserved genes show highly conserved collinearity with their orthologues across genomes, but a small number of species-specific rearrangements were identified. Thus the evolution of genome microstructure is an ongoing process. Brassica napus is a recently formed polyploid resulting from the hybridization of B. rapa (containing the Brassica A genome) and B. oleracea (containing the Brassica C genome). Using clones from the JBnB library, we have analysed the microstructure of the corresponding segments of the B. napus genome. The results show that there has been little or no change to the microstructure of the analysed segments of the Brassica A and C genomes as a consequence of the hybridization event forming natural B. napus. The observations indicate that, upon polyploid formation, these segments of the genome did not undergo a burst of evolution discernible at the scale of microstructure.

Brassica↗