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Biomedical subjects

David Harris

Publications and source records attributed to David Harris.

At least 19 recordsLinked to original sources
PubMed2006

Studying the right cell in acute myelogenous leukemia: dynamic changes of apoptosis and signal transduction pathway protein expression in chemotherapy resistant ex-vivo selected "survivor cells".

We hypothesized that studying protein expression in cells surviving in vitro chemotherapy ("survivor cells", SV), could provide more important insight into the biology of drug-resistant AML cells than analysis of the bulk population of leukemic cells. Leukemia-enriched samples from 79 patients with new or relapsed AML were cultured for four days +/- cytarabine (5-10 microM). Early apoptotic cells were removed to yield purified SV. Expression of BCL2, bax, PKC alpha, ERK2 and pERK2 proteins was measured using laser scanning cytometry. The SV population was enriched for CD34+ stem cells. Protein expression patterns in SV differed considerably from those in controls; culture and reanalysis of protein expression revealed stability, reversion, or new patterns of change. Patterns of pairs or triads of proteins were nonrandomly distributed and appeared at statistically unlikely frequencies, suggesting preferential adoption of certain patterns. The patterns of change were highly predictive of remission attainment, relapse, and survival in univariate and multivariate analysis. We conclude that in vitro SV cells have protein expression patterns distinct from those of the bulk population of leukemic cells and that these patterns are predictive of outcome. Analysis of SV cells may be more informative than analysis of the bulk population of leukemia cells.

Adult↗
PubMed2006

Common inheritance of chromosome Ia associated with clonal expansion of Toxoplasma gondii.

Toxoplasma gondii is a globally distributed protozoan parasite that can infect virtually all warm-blooded animals and humans. Despite the existence of a sexual phase in the life cycle, T. gondii has an unusual population structure dominated by three clonal lineages that predominate in North America and Europe, (Types I, II, and III). These lineages were founded by common ancestors approximately10,000 yr ago. The recent origin and widespread distribution of the clonal lineages is attributed to the circumvention of the sexual cycle by a new mode of transmission-asexual transmission between intermediate hosts. Asexual transmission appears to be multigenic and although the specific genes mediating this trait are unknown, it is predicted that all members of the clonal lineages should share the same alleles. Genetic mapping studies suggested that chromosome Ia was unusually monomorphic compared with the rest of the genome. To investigate this further, we sequenced chromosome Ia and chromosome Ib in the Type I strain, RH, and the Type II strain, ME49. Comparative genome analyses of the two chromosomal sequences revealed that the same copy of chromosome Ia was inherited in each lineage, whereas chromosome Ib maintained the same high frequency of between-strain polymorphism as the rest of the genome. Sampling of chromosome Ia sequence in seven additional representative strains from the three clonal lineages supports a monomorphic inheritance, which is unique within the genome. Taken together, our observations implicate a specific combination of alleles on chromosome Ia in the recent origin and widespread success of the clonal lineages of T. gondii.

Animals↗
PubMed2006

Atiprimod blocks phosphorylation of JAK-STAT and inhibits proliferation of acute myeloid leukemia (AML) cells.

In studies of multiple myeloma cells, atiprimod was shown to block Stat3 activation and inhibited colony-forming cell proliferation. We hypothesized that atiprimod may also inhibit activation of intracellular signaling pathways in AML cells resulting in apoptosis and growth inhibition. We demonstrate that atiprimod inhibited clonogenic growth of AML cell lines and fresh AML marrow cells whereas it did not significantly affect growth of normal hematopoietic progenitors from marrow samples of healthy controls. Atiprimod decreased phosphorylation of Stat3 and Stat5, and protein levels of Jak2, whereas gene expression of Jak2 was not affected. Atiprimod further induced apoptosis by cleavage of caspase 3 and PARP. In summary, our data suggest that atiprimod has a significant antiproliferative and proapoptotic effect on AML cells. This effect may be facilitated by inhibition of the Jak-Stat signaling pathway. Further evaluation of atiprimod in clinical trials of AML should be considered.

Acute Disease↗
PubMed2006

Simultaneous inhibition of PDK1/AKT and Fms-like tyrosine kinase 3 signaling by a small-molecule KP372-1 induces mitochondrial dysfunction and apoptosis in acute myelogenous leukemia.

Phosphoinositol-3-kinase (PI3K)/protein kinase B (AKT) and Fms-like tyrosine kinase 3 (FLT3) signaling are aberrantly activated in acute myelogenous leukemia (AML) cells. Constitutively activated AKT and FLT3 regulate leukemia cell survival and resistance to chemotherapy. In this study, we investigated the effects of the novel multiple kinase inhibitor KP372-1 on the survival of AML cell lines and primary AML samples. KP372-1 directly inhibited the kinase activity of AKT, PDK1, and FLT3 in a concentration-dependent manner. Western blot analysis indicated that KP372-1 decreased the phosphorylation of AKT on both Ser(473) and Thr(308); abrogated the phosphorylation of p70S6 kinase, BAD, and Foxo3a via PI3K/AKT signaling; and down-regulated expression of PIM-1 through direct inhibition of FLT3. Treatment of AML cell lines with KP372-1 resulted in rapid generation of reactive oxygen species and stimulation of oxygen consumption, followed by mitochondrial depolarization, caspase activation, and phosphatidylserine externalization. KP372-1 induced pronounced apoptosis in AML cell lines and primary samples irrespective of their FLT3 status, but not in normal CD34(+) cells. Moreover, KP372-1 markedly decreased the colony-forming ability of primary AML samples (IC(50) < 200 nmol/L) with minimal cytotoxic effects on normal progenitor cells. Taken together, our results show that the simultaneous inhibition of critical prosurvival kinases by KP372-1 leads to mitochondrial dysfunction and apoptosis of AML but not normal hematopoietic progenitor cells.

3-Phosphoinositide-Dependent Protein Kinases↗
PubMed2006

Triptolide induces caspase-dependent cell death mediated via the mitochondrial pathway in leukemic cells.

Triptolide, a diterpenoid isolated from the Chinese herb Tripterygium wilfordii Hook.f, has shown antitumor activities in a broad range of solid tumors. Here, we examined its effects on leukemic cells and found that, at 100 nM or less, it potently induced apoptosis in various leukemic cell lines and primary acute myeloid leukemia (AML) blasts. We then attempted to identify its mechanisms of action. Triptolide induced caspase-dependent cell death accompanied by a significant decrease in XIAP levels. Forced XIAP overexpression attenuated triptolide-induced cell death. Triptolide also decreased Mcl-1 but not Bcl-2 and Bcl-X(L) levels. Bcl-2 overexpression suppressed triptolide-induced apoptosis. Further, triptolide induced loss of the mitochondrial membrane potential and cytochrome C release. Caspase-9 knock-out cells were resistant, while caspase-8-deficient cells were sensitive to triptolide, suggesting criticality of the mitochondrial but not the death receptor pathway for triptolide-induced apoptosis. Triptolide also enhanced cell death induced by other anticancer agents. Collectively, our results demonstrate that triptolide decreases XIAP and potently induces caspase-dependent apoptosis in leukemic cells mediated through the mitochondrial pathway at low nanomolar concentrations. The potent antileukemic activity of triptolide in vitro warrants further investigation of this compound for the treatment of leukemias and other malignancies.

Antineoplastic Agents, Alkylating↗
PubMed2006

Genetic analysis of the capsular biosynthetic locus from all 90 pneumococcal serotypes.

Several major invasive bacterial pathogens are encapsulated. Expression of a polysaccharide capsule is essential for survival in the blood, and thus for virulence, but also is a target for host antibodies and the basis for effective vaccines. Encapsulated species typically exhibit antigenic variation and express one of a number of immunochemically distinct capsular polysaccharides that define serotypes. We provide the sequences of the capsular biosynthetic genes of all 90 serotypes of Streptococcus pneumoniae and relate these to the known polysaccharide structures and patterns of immunological reactivity of typing sera, thereby providing the most complete understanding of the genetics and origins of bacterial polysaccharide diversity, laying the foundations for molecular serotyping. This is the first time, to our knowledge, that a complete repertoire of capsular biosynthetic genes has been available, enabling a holistic analysis of a bacterial polysaccharide biosynthesis system. Remarkably, the total size of alternative coding DNA at this one locus exceeds 1.8 Mbp, almost equivalent to the entire S. pneumoniae chromosomal complement.

Bacterial Capsules↗
PubMed2006

Mechanisms of apoptosis sensitivity and resistance to the BH3 mimetic ABT-737 in acute myeloid leukemia.

BCL-2 proteins are critical for cell survival and are overexpressed in many tumors. ABT-737 is a small-molecule BH3 mimetic that exhibits single-agent activity against lymphoma and small-cell lung cancer in preclinical studies. We here report that ABT-737 effectively kills acute myeloid leukemia blast, progenitor, and stem cells without affecting normal hematopoietic cells. ABT-737 induced the disruption of the BCL-2/BAX complex and BAK-dependent but BIM-independent activation of the intrinsic apoptotic pathway. In cells with phosphorylated BCL-2 or increased MCL-1, ABT-737 was inactive. Inhibition of BCL-2 phosphorylation and reduction of MCL-1 expression restored sensitivity to ABT-737. These data suggest that ABT-737 could be a highly effective antileukemia agent when the mechanisms of resistance identified here are considered.

Animals↗
PubMed2006

Comparison of the genome sequence of the poultry pathogen Bordetella avium with those of B. bronchiseptica, B. pertussis, and B. parapertussis reveals extensive diversity in surface structures associated with host interaction.

Bordetella avium is a pathogen of poultry and is phylogenetically distinct from Bordetella bronchiseptica, Bordetella pertussis, and Bordetella parapertussis, which are other species in the Bordetella genus that infect mammals. In order to understand the evolutionary relatedness of Bordetella species and further the understanding of pathogenesis, we obtained the complete genome sequence of B. avium strain 197N, a pathogenic strain that has been extensively studied. With 3,732,255 base pairs of DNA and 3,417 predicted coding sequences, it has the smallest genome and gene complement of the sequenced bordetellae. In this study, the presence or absence of previously reported virulence factors from B. avium was confirmed, and the genetic bases for growth characteristics were elucidated. Over 1,100 genes present in B. avium but not in B. bronchiseptica were identified, and most were predicted to encode surface or secreted proteins that are likely to define an organism adapted to the avian rather than the mammalian respiratory tracts. These include genes coding for the synthesis of a polysaccharide capsule, hemagglutinins, a type I secretion system adjacent to two very large genes for secreted proteins, and unique genes for both lipopolysaccharide and fimbrial biogenesis. Three apparently complete prophages are also present. The BvgAS virulence regulatory system appears to have polymorphisms at a poly(C) tract that is involved in phase variation in other bordetellae. A number of putative iron-regulated outer membrane proteins were predicted from the sequence, and this regulation was confirmed experimentally for five of these.

Animals↗
PubMed2006

Potential role of activated platelets in homing of human endothelial progenitor cells to subendothelial matrix.

Endothelial progenitor cells (EPCs) mobilize from the bone marrow in response to tissue injury and participate in vascular repair. However, there is limited data about the homing mechanisms of EPCs to vascular injury sites. Recently animal experiments indicated that platelets play a role in recruitment of EPCs to injury sites. However, data on the possible interaction between platelets and EPCs within the human system are limited. We, therefore, examined in-vitro human platelet-EPC interaction under static and flow conditions. Human EPCs were isolated from donated buffy coats by magnetic microbeads and flow cytometry cell sorting using CD133 and VEGFR-2, respectively, as markers. Platelets were tested in the form of washed platelets, platelet rich plasma or whole blood. EPCs formed heterotypic aggregates with resting platelets under static conditions, an interaction that was greatly enhanced when platelets were activated by collagen, ADP or thrombin-activation peptide. The platelet-EPC interaction was inhibited by antibodies to P-selectin or P-selectin glycoprotein ligand-1 (PSGL-1), but not by antibodies to glycoproteins Ib-IX-V or IIb/IIIa. When perfused over activated platelets under shear stress of 2.5 dyn/cm(2), EPCs tethered to platelayers and either adhered immediately or rolled a short distance before adhering. In addition, platelets promoted the colonization of adherent EPCs in culture conditions. Consistent with recent animal studies, these findings demonstrate that human EPCs interact in vitro with activated platelets under static and flow conditions, mediated through P-selectin-PSGL-1 interaction. This interaction may be a central mechanism for homing of EPCs to vascular injury sites.

AC133 Antigen↗
PubMed2005

Genomic sequence of the pathogenic and allergenic filamentous fungus Aspergillus fumigatus.

Aspergillus fumigatus is exceptional among microorganisms in being both a primary and opportunistic pathogen as well as a major allergen. Its conidia production is prolific, and so human respiratory tract exposure is almost constant. A. fumigatus is isolated from human habitats and vegetable compost heaps. In immunocompromised individuals, the incidence of invasive infection can be as high as 50% and the mortality rate is often about 50% (ref. 2). The interaction of A. fumigatus and other airborne fungi with the immune system is increasingly linked to severe asthma and sinusitis. Although the burden of invasive disease caused by A. fumigatus is substantial, the basic biology of the organism is mostly obscure. Here we show the complete 29.4-megabase genome sequence of the clinical isolate Af293, which consists of eight chromosomes containing 9,926 predicted genes. Microarray analysis revealed temperature-dependent expression of distinct sets of genes, as well as 700 A. fumigatus genes not present or significantly diverged in the closely related sexual species Neosartorya fischeri, many of which may have roles in the pathogenicity phenotype. The Af293 genome sequence provides an unparalleled resource for the future understanding of this remarkable fungus.

Allergens↗
PubMed2005

Response shift masks the treatment impact on patient reported outcomes (PROs): the example of individual quality of life in edentulous patients.

BACKGROUND: Quality of life (QoL) is now established as an important outcome for evaluating the impact of disease, and for assessing the efficacy of treatments. However, individuals change with time and the basis on which they make a QoL judgement may also change, a phenomenon increasingly referred to as response shift. Here, the individual may change his or her internal standards, values, and/or conceptualization on the target construct as a result of external factors such as a treatment or a change in health status. This has important implications for assessing the effects of treatments as a change in QoL may reflect a response shift, a treatment effect, or a complex combination of both. In this study, we used an individualised quality of life (IQoL) measure, the SEIQoL, together with a then-test to determine whether response shift would influence the measurement of treatment efficacy in edentulous patients. METHODS: Data are reported here for the first phase of a randomised controlled clinical trial designed to assess the impact, on IQoL, of implant supported dentures compared with high quality conventional dentures. IQoL was measured using the SEIQoL-DW in 117 patients (mean age 64.8; 32% male) at baseline (T1) and 3 months (T2) after receiving high quality conventional dentures. The work was carried out in dental teaching hospitals in Dublin and Belfast. RESULTS: Unadjusted SEIQoL index scores revealed no significant impact of treatment at three months (baseline: 75.0; 3 months: 73.2, p = .33, n.s.). However, the then-test at 3 months revealed that patients retrospectively rated their baseline IQoL as significantly lower (P < .001) than they had rated it at the time (then-test baseline: 69.2). Comparison of the 3 month scores with this readjusted baseline indicated a significant treatment effect (then-test baseline: 69.2; 3 months: 73.2, p = 0.016). 81% of patients nominated at least one different IQoL domain at 3 months. CONCLUSION: The positive impact of denture treatment for edentulous patients on IQoL was seen only when response shifts were taken into consideration. The nature of the response shifts was highly complex but the data indicated a degree of re-conceptualization and reprioritisation. Assessment of the impact of treatments using patient-generated reports must take account of the adaptive nature of patients.

Adaptation, Psychological↗
PubMed2005

Circulating endothelial progenitor cells and coronary collaterals in patients with non-ST segment elevation myocardial infarction.

BACKGROUND: Endothelial progenitor cells (EPCs) are bone marrow-derived cells that are augmented in response to ischemia and incorporated into neovascularization sites. We sought to determine whether circulating EPCs are related to collateral formation following non-ST segment elevation myocardial infarction (NSTEMI). METHODS: Twenty patients who underwent percutaneous coronary intervention (PCI) within a week of NSTEMI were divided into two groups: patients without collaterals (coll-, n=10) and patients with Rentrop grade 3--4 collaterals (coll+, n=10). Blood samples were drawn before PCI and 24+/- 2 h after PCI. EPC colonies were grown from peripheral blood mononuclear cells, characterized, and counted. Using flow cytometry the percentage of cells co expressing vascular endothelial growth factor receptor-2 and CD 133 was determined. RESULTS: The coll+ group had higher degree of culprit vessel stenosis and lower initial thrombolysis in myocardial infarction flow grade. The relative number of EPCs before PCI was significantly higher in the coll+ group than in the coll- group (1.49 +/- 0.9% vs. 0.77+/- 0.4%, p= 0.045). There were no significant intergroup differences in the number of EPC colony-forming cells. The number of EPC colonies increased in the coll- group after PCI (9.5 +/- 4.8 to 14.0 +/- 5.9/10(6) cells, p=0.01). CONCLUSIONS: This study supports an association between circulating EPC levels and collateral formation in patients with an NSTEMI.

AC133 Antigen↗
PubMed2005

The genome of the kinetoplastid parasite, Leishmania major.

Leishmania species cause a spectrum of human diseases in tropical and subtropical regions of the world. We have sequenced the 36 chromosomes of the 32.8-megabase haploid genome of Leishmania major (Friedlin strain) and predict 911 RNA genes, 39 pseudogenes, and 8272 protein-coding genes, of which 36% can be ascribed a putative function. These include genes involved in host-pathogen interactions, such as proteolytic enzymes, and extensive machinery for synthesis of complex surface glycoconjugates. The organization of protein-coding genes into long, strand-specific, polycistronic clusters and lack of general transcription factors in the L. major, Trypanosoma brucei, and Trypanosoma cruzi (Tritryp) genomes suggest that the mechanisms regulating RNA polymerase II-directed transcription are distinct from those operating in other eukaryotes, although the trypanosomatids appear capable of chromatin remodeling. Abundant RNA-binding proteins are encoded in the Tritryp genomes, consistent with active posttranscriptional regulation of gene expression.

Animals↗
PubMed2005

Gene arrays at Pneumocystis carinii telomeres.

In the fungus Pneumocystis carinii, at least three gene families (PRT1, MSR, and MSG) have the potential to generate high-frequency antigenic variation, which is likely to be a strategy by which this parasitic fungus is able to prolong its survival in the rat lung. Members of these gene families are clustered at chromosome termini, a location that fosters recombination, which has been implicated in selective expression of MSG genes. To gain insight into the architecture, evolution, and regulation of these gene clusters, six telomeric segments of the genome were sequenced. Each of the segments began with one or more unique genes, after which were members of different gene families, arranged in a head-to-tail array. The three-gene repeat PRT1-MSR-MSG was common, suggesting that duplications of these repeats have contributed to expansion of all three families. However, members of a gene family in an array were no more similar to one another than to members in other arrays, indicating rapid divergence after duplication. The intergenic spacers were more conserved than the genes and contained sequence motifs also present in subtelomeres, which in other species have been implicated in gene expression and recombination. Long mononucleotide tracts were present in some MSR genes. These unstable sequences can be expected to suffer frequent frameshift mutations, providing P. carinii with another mechanism to generate antigen variation.

Amino Acid Sequence↗
PubMed2005

A novel ring-substituted diindolylmethane,1,1-bis[3'-(5-methoxyindolyl)]-1-(p-t-butylphenyl) methane, inhibits extracellular signal-regulated kinase activation and induces apoptosis in acute myelogenous leukemia.

We investigated the antileukemic activity and molecular mechanisms of action of a newly synthesized ring-substituted diindolylmethane derivative, 1,1-bis[3'-(5-methoxyindolyl)]-1-(p-t-butylphenyl) methane (DIM #34), in acute myelogenous leukemia (AML) cells. DIM #34 inhibited AML cell growth via the induction of apoptosis and abrogated clonogenic growth of primary AML samples. Exposure to DIM #34 induced loss of mitochondrial inner transmembrane potential, release of cytochrome c into the cytosol, and caspase activation. Bcl-2-overexpressing, Bax knockout, and caspase-9-deficient cells were partially resistant to cell death, suggesting the involvement of the intrinsic apoptotic pathway. Furthermore, DIM #34 transiently inhibited the phosphorylation and activity of the extracellular signal-regulated kinase and abrogated Bcl-2 phosphorylation. Because other methylene-substituted diindolylmethane analogues have been shown to transactivate the nuclear receptor peroxisome proliferator-activated receptor gamma (PPARgamma), we studied the role of PPARgamma in apoptosis induction. Cotreatment of cells with a selective PPARgamma antagonist or with retinoid X receptor and retinoic acid receptor ligands partially modulated apoptosis when combined with DIM #34, suggesting PPARgamma receptor-dependent and receptor-independent cell death. Together, these findings suggest that diindolylmethanes are a new class of compounds that selectively induce apoptosis in AML cells through the modulation of the extracellular signal-regulated kinase and PPARgamma signaling pathways.

Acute Disease↗
PubMed2005

The genome of the protist parasite Entamoeba histolytica.

Entamoeba histolytica is an intestinal parasite and the causative agent of amoebiasis, which is a significant source of morbidity and mortality in developing countries. Here we present the genome of E. histolytica, which reveals a variety of metabolic adaptations shared with two other amitochondrial protist pathogens: Giardia lamblia and Trichomonas vaginalis. These adaptations include reduction or elimination of most mitochondrial metabolic pathways and the use of oxidative stress enzymes generally associated with anaerobic prokaryotes. Phylogenomic analysis identifies evidence for lateral gene transfer of bacterial genes into the E. histolytica genome, the effects of which centre on expanding aspects of E. histolytica's metabolic repertoire. The presence of these genes and the potential for novel metabolic pathways in E. histolytica may allow for the development of new chemotherapeutic agents. The genome encodes a large number of novel receptor kinases and contains expansions of a variety of gene families, including those associated with virulence. Additional genome features include an abundance of tandemly repeated transfer-RNA-containing arrays, which may have a structural function in the genome. Analysis of the genome provides new insights into the workings and genome evolution of a major human pathogen.

Animals↗
PubMed2005

A comprehensive survey of the Plasmodium life cycle by genomic, transcriptomic, and proteomic analyses.

Plasmodium berghei and Plasmodium chabaudi are widely used model malaria species. Comparison of their genomes, integrated with proteomic and microarray data, with the genomes of Plasmodium falciparum and Plasmodium yoelii revealed a conserved core of 4500 Plasmodium genes in the central regions of the 14 chromosomes and highlighted genes evolving rapidly because of stage-specific selective pressures. Four strategies for gene expression are apparent during the parasites' life cycle: (i) housekeeping; (ii) host-related; (iii) strategy-specific related to invasion, asexual replication, and sexual development; and (iv) stage-specific. We observed posttranscriptional gene silencing through translational repression of messenger RNA during sexual development, and a 47-base 3' untranslated region motif is implicated in this process.

3' Untranslated Regions↗