Growth inhibition of human promonocytic leukaemic U937 cells by interferon gamma is irreversible and not cell cycle phase-specific.

Growth of human promonocytic leukaemic U937 cells was found arrested within 24 h upon exposure to interferon gamma (IFN-gamma). Removal of the interferon did not result in the resumption of growth, as is evident from the absence of doubling of viable cell count and(3)H-thymidine incorporation. 5-Bromo-2'-deoxyuridine-based flow cytometric analysis of the growth-arrested cells, 24 h subsequent to the removal of IFN-gamma, showed absence of DNA synthesis, confirming the irreversible nature of the growth inhibition. Propidium iodide-based flow cytometric analysis of the growth-arrested cells showed a distribution which is typical of a growth inhibition without resulting in the accumulation of cells in any specific phase of the cell cycle. These results indicated that IFN-gamma arrested growth of U937 cells in an irreversible and cell cycle phase-independent manner. These observations were in contrast to our earlier report on the reversible and cell cycle phase-specific growth inhibition of human amniotic (fetal epithelial) WISH cells by the interferon. Copyright 1999 Academic Press.

Genome-wide association study of prostate cancer-specific survival

BACKGROUND: Unnecessary intervention and overtreatment of indolent disease are common challenges in clinical management of prostate cancer. Improved tools to distinguish lethal from indolent disease are critical. METHODS: We performed a genome-wide survival analysis of cause-specific death in 24,023 prostate cancer patients (3,513 disease-specific deaths) from the PRACTICAL and BPC3 consortia. Top findings were assessed for replication in a Norwegian cohort (CONOR). RESULTS: We observed no significant association between genetic variants and prostate cancer survival. CONCLUSIONS: Common genetic variants with large impact on prostate cancer survival were not observed in this study. IMPACT: Future studies should be designed for identification of rare variants with large effect sizes or common variants with small effect sizes.

Development of Novel Assays for Measuring Different Molecular Forms of Prostate Specific Antigen

Prostate cancer (PCa) is the most commonly diagnosed non-skin cancer and second leading cause of cancer-related death of men in developed countries. Measurement of prostate specific antigen (PSA) is a very sensitive method for diagnosing and monitoring of prostate cancer (PCa), but the specificity needs improvement. Measurements of different molecular forms of PSA have been shown to improve differentiation between PCa and benign prostatic diseases. However, accurate measurement of some isoforms has not been achieved in previous assays. The aim of the present study was to develop new assays that reliably measure enzymatically active PSA, PSA-α1-chymotryposin (PSA-ACT) and PSA-α1-protease inhibitor (PSA-API), and to evaluate their diagnostic value. Double-label immunofluorometric assays using a novel monoclonal antibody (MAb) and another antibody to either free PSA (fPSA) or total PSA (tPSA) were developed and used to measure PSA-ACT and fPSA or tPSA at the same time. These assays provide enough sensitivity for measurement of PSA-ACT in sera with low PSA levels. The results obtained confirmed that proportion of PSA-ACT to tPSA (%PSA-ACT) was as useful as proportion of fPSA to tPSA (%fPSA) for discrimination between PCa and benign prostatic hyperplasia (BPH). We developed an immunoassay for detection of PSA-API based on proximity ligation, which improved assay sensitivity 10-fold compared with conventional assays. Our results confirmed previous findings that the PSA-API level is somewhat lower in men with than without PCa, and the combination of %fPSA and proportion of PSA-API to tPSA (%PSA-API) provides diagnostic improvement compared with either method alone. Assays based on this principle should be applicable to other immunoassays in which the nonspecific background is a problem. An immunopeptidometric sandwich assay (IPMA) was developed to measure the enzymatically active PSA. This assay showed high specificity, but sensitivity was not good enough for measurement of PSA concentrations in the gray zone, 2-10 µg/L, in which tPSA does not efficiently differentiate between PCa and BPH. We further developed a solid-phase proximity ligation immunoassay, which provided a 10-fold improvement in sensitivity. This proof of concept study shows that peptides reacting with proteins are potentially useful for sensitive and specific measurement of protein variants for which specific MAbs cannot be obtained.

A CD8(+) T cell clone specific for antigen also recognizes peptidomimics present in anti-idiotypic antibody: Implications for T cell memory

The relay hypothesis [R. Nayak, S. Mitra-Kaushik, M.S. Shaila, Perpetuation of immunological memory: a relay hypothesis, Immunology 102 (2001) 387-395] was earlier proposed to explain perpetuation of immunological memory without requiring long lived memory cells or persisting antigen. This hypothesis envisaged cycles of interaction and proliferation of complementary idiotypic B cells (Burnet cells) and anti-idiotypic B cells (Jerne cells) as the primary reason for perpetuation of immunological memory. The presence of pepti-domimics of antigen in anti-idiotypic antibody and their presentation to antigen specific T cells was postulated to be primary reason for perpetuation of T cell memory. Using a viral hemagglutinin as a model, in this work, we demonstrate the presence of peptidomimics in the variable region of ail anti-idiotypic antibody capable of functionally mimicking the antigen derived peptides. A CD8(+) CTL clone was generated against the hemagglutinin protein which specifically responds to either peptidomimic synthesizing cells or peptidomimic pulsed antigen presenting cells. Thus, it appears reasonable that a population of activated antigen specific T cells is maintained in the body by presentation of peptidomimic through Jerne cells and other antigen presenting cells long after immunization. (C) 2007 Elsevier Inc. All rights reserved.

Specific language impairment in pre-adolescence, adolescence, and adulthood with special emphasis on health-related quality of life

This clinical study focused on effects of childhood specific language impairment (SLI) on daily functioning in late life. SLI is a neurobiological disorder with genetic predisposition and manifests as poor language production or comprehension or both in a child with age-level non-verbal intelligence and no other known cause for deficient language development. The prevalence rate of around 7% puts it among the most prevalent developmental disorders in childhood. Negative long-term effects, such as problems in learning and behavior, are frequent. In follow-up studies the focus has seldom been on self-perception of daily functioning and participation, which are considered important in the International Classification of Functioning, Disability, and Health (ICF). To investigate the self-perceived aspects of everyday functioning in individuals with childhood receptive SLI compared with age- and gender-matched control populations, the 15D, 16D, and 17D health-related quality of life (HRQoL) questionnaires were applied. These generic questionnaires include 15, 16, and 17 dimensions, respectively, and give both a single index score and a profile with values on each dimension. Information on different life domains (rehabilitation, education, employment etc.) from each age-group was collected with separate questionnaires. The study groups comprised adults, adolescents (12-16 years), and pre-adolescents (8-11 years) who had received a diagnosis of receptive SLI and had been examined, usually before school age, at the Department of Phoniatrics of Helsinki University Central Hospital, where children with language deficits caused by various etiologies are examined and treated by a multidisciplinary team. The adult respondents included 33 subjects with a mean age of 34 years. Measured with 15D, the subjects perceived their HRQoL to be nearly as good as that of their controls, but on the dimensions of speech, usual activities, mental functioning, and distress they were significantly worse off. They significantly more often lived with their parents (19%) or were pensioned (26%) than the adult Finnish population on average. Adults with self-perceived problems in finding words and in remembering instructions, manifestations of persistent language impairment, showed inferior every day functioning to the rest of the study group. Of the adolescents and pre-adolescents, 48 and 51, respectively, responded. The majority in both groups had received special education or extra educational support at school. They all had attended speech therapy at some point; at the time of the study only one adolescent, but every third pre-adolescent still received speech therapy. The 16D score of the adolescent or the 17D score of the pre-adolescents did not differ from that of their controls. The 16D profiles differed on some dimensions; subjects were significantly worse off on the dimension of mental functioning, but better off on the dimension of vitality than controls. Of the 17D dimensions, the study group was significantly worse off on speech, whereas the control group reported significantly more problems in sleeping. Of the childhood performance measures investigated, low verbal intelligence quotient (VIQ), which is often considered to reflect receptive language impairment, was in adults subjects significantly associated with some of the self-perceived problems, such as problems in usual activities and mental functioning. The 15D, 16D, and 17D questionnaires served well in measuring self-perceived HRQoL. Such standardized measures with population values are especially important in confirming with the ICF guidelines. In the future these questionnaires could perhaps be used on a more individual level in follow-up of children in clinics, and even in special schools and classes, to detect those children at greatest risk of negative long-term effects and perhaps diminished well-being regarding daily functioning and participation.

Clinical application of biomarkers in prostate cancer in men with metabolic syndrome project: Prostate Specific Membrane Antigen (PSMA) Positron Emission Tomography (PET) in diagnosing localised prostate cancer

Localised prostate cancer is a heterogenous disease and a multi-modal approach is required to accurately diagnose and stage the disease. Whilst the use of magnetic resonance imaging (MRI) has become more common, small volume and multi-focal disease are oft en diffi cult to characterise. Prostate specifi c membrane antigen is a cell surface protein, which is expressed in nearly all prostate cancer cells. Its expression is signifi cantly higher in high grade prostate cancer cells. In this study, we compare multi-parametric magnetic resonance imaging and 68-Gallinium-PSMA PET with whole-mount pathology of the prostate to evaluate the applicability of multiparameteric (MP) MRI and 68Ga-PSMA PET in detecting and locating tumour foci in patients with localised prostate cancer.

Virus-Specific Cytolytic Antibodies to Nonstructural Protein 1 of Japanese Encephalitis Virus Effect Reduction of Virus Output from Infected Cells

We demonstrate the presence of nonstructural protein 1 (NS1)-specific antibodies in a significant proportion of convalescent-phase human serum samples obtained from a cohort in an area where Japanese encephalitis virus (JEV) is endemic. Sera containing antibodies to NS1 but not those with antibodies to other JEV proteins, such as envelope, brought about complement-mediated lysis of JEV-infected BHK-21 cells. Target cells infected with a recombinant poxvirus expressing JEV NS1 on the cell surface confirmed the NS1 specificity of cytolytic antibodies. Mouse anti-NS1 cytolytic sera caused a complement-dependent reduction in virus output from infected human cells, demonstrating their important role in viral control. Antibodies elicited by JEV NS1 did not cross lyse West Nile virus- or dengue virus-infected cells despite immunoprecipitating the NS1 proteins of these related flaviviruses. Additionally, JEV NS1 failed to bind complement factor H, in contrast to NS1 of West Nile virus, suggesting that the NS1 proteins of different flaviviruses have distinctly different mechanisms for interacting with the host. Our results also point to an important role for JEV NS1-specific human immune responses in protection against JE and provide a strong case for inclusion of the NS1 protein in next generation of JEV vaccines.

Molecular basis for the development of diagnostic methods and specific treatment modalities for idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is an interstitial lung disease with unknown aetiology and poor prognosis. IPF is characterized by alveolar epithelial damage that leads tissue remodelling and ultimately to the loss of normal lung architecture and function. Treatment has been focused on anti-inflammatory therapies, but due to their poor efficacy new therapeutic modalities are being sought. There is a need for early diagnosis and also for differential diagnostic markers for IPF and other interstitial lung diseases. The study utilized patient material obtained from bronchoalveolar lavage (BAL), diagnostic biopsies or lung transplantation. Human pulmonary fibroblast cell cultures were propagated and asbestos-induced pulmonary fibrosis in mice was used as an experimental animal model of IPF. The possible markers for IPF were scanned by immunohistochemistry, RT-PCR, ELISA and western blot. Matrix metalloproteinases (MMPs) are proteolytic enzymes that participate in tissue remodelling. Microarray studies have introduced potential markers that could serve as additional tools for the assessment of IPF and one of the most promising was MMP 7. MMP-7 protein levels were measured in the BAL fluid of patients with idiopathic interstitial lung diseases or idiopathic cough. MMP-7 was however similarly elevated in the BAL fluid of all these disorders and thus cannot be used as a differential diagnostic marker for IPF. Activation of transforming growth factor (TGF)-ß is considered to be a key element in the progression of IPF. Bone morphogenetic proteins (BMP) are negative regulators of intracellular TGF-ß signalling and BMP-4 signalling is in turn negatively regulated by gremlin. Gremlin was found to be highly upregulated in the IPF lungs and IPF fibroblasts. Gremlin was detected in the thickened IPF parenchyma and endothelium of small capillaries, whereas in non-specific interstitial pneumonia it localized predominantly in the alveolar epithelium. Parenchymal gremlin immunoreactivity might indicate IPF-type interstitial pneumonia. Gremlin mRNA levels were higher in patients with end-stage fibrosis suggesting that gremlin might be a marker for more advanced disease. Characterization of the fibroblastic foci in the IPF lungs showed that immunoreactivity to platelet-derived growth factor (PDGF) receptor-α and PDGF receptor-β was elevated in IPF parenchyma, but the fibroblastic foci showed only minor immunoreactivity to the PDGF receptors or the antioxidant peroxiredoxin II. Ki67 positive cells were also observed predominantly outside the fibroblastic foci, suggesting that the fibroblastic foci may not be composed of actively proliferating cells. When inhibition of profibrotic PDGF-signalling by imatinib mesylate was assessed, imatinib mesylate reduced asbestos-induced pulmonary fibrosis in mice as well as human pulmonary fibroblast migration in vitro but it had no effect on the lung inflammation.

Enhanced Tetrahedral Ordering of Water Molecules in Minor Grooves of DNA: Relative Role of DNA Rigidity, Nanoconfinement, and Surface Specific Interactions

Confinement and Surface specific interactions call induce Structures otherwise unstable at that temperature and pressure. Here we Study the groove specific water dynamics ill the nucleic acid sequences, poly-AT and poly-GC, in long B-DNA duplex chains by large scale atomistic molecular dynamics simulations, accompanied by thermodynamic analysis. While water dynamics in the major groove remains insensitive to the sequence differences, exactly the opposite is true for the minor groove water. Much slower water dynamics observed in the minor grooves (especially in the AT minor) call be attributed to all enhanced tetrahedral ordering (< t(h)>) of water. The largest value of < t(h)> in the AT minor groove is related to the spine of hydration found in X-ray Structure. The calculated configurational entropy (S-C) of the water molecules is found to be correlated with the self-diffusion coefficient of water in different region via Adam-Gibbs relation D = A exp(-B/TSC), and also with < t(h)>.

Specific Small-Molecule Activator of Aurora Kinase A Induces Autophosphorylation in a Cell-Free System

Aurora kinases are essential for chromosomal segregation and cell division and thereby important for maintaining the proper genomic integrity. There are three classes of aurora kinases in humans: A, B, and C. Aurora kinase A is frequently overexpressed in various cancers. The link of the overexpression and tumorigenesis is yet to be understood. By employing virtual screening, we have found that anacardic acid, a pentadecane aliphatic chain containing hydroxylcarboxylic acid, from cashew nut shell liquid could be docked in Aurora kinases A and B. Remarkably, we found that anacardic acid could potently activate the Aurora kinase A mediated phosphorylation of histone H3, but at a similar concentration the activity of aurora kinase B remained unaffected in vitro. Mechanistically, anacardic acid induces the structural changes and also the autophosphorylation of the aurora kinase A to enhance the enzyme activity. This data thus indicate anacardic acid as the first small-molecule activator of Aurora kinase, which could be highly useful for probing the function of hyperactive (overexpressed) Aurora kinase A.

The GAF Domain of the cGMP-Binding, cGMP-Specific Phosphodiesterase (PDE5) Is a Sensor and a Sink for cGMP

We describe here a novel sensor for cGMP based on the GAF domain of the cGMP-binding, cGMP-specific phosphodiesterase 5 (PDE5) using bioluminescence resonance energy transfer (BRET). The wild type GAFa domain, capable of binding cGMP with high affinity, and a mutant (GAFaF163A) unable to bind cGMP were cloned as fusions between GFP and Rluc for BRET2 assays. BRET2 ratios of the wild type GAFa fusion protein, but not GAFaF163A, increased in the presence of cGMP but not cAMP. Higher basal BRET2 ratios were observed in cells expressing the wild type GAFa domain than in cells expressing GAFaF163A. This was correlated with elevated basal intracellular levels of cGMP, indicating that the GAF domain could act as a sink for cGMP. The tandem GAF domains in full length PDE5 could also sequester cGMP when the catalytic activity of PDE5 was inhibited. Therefore, these results describe a cGMP sensor utilizing BRET2 technology and experimentally demonstrate the reservoir of cGMP that can be present in cells that express cGMP-binding GAF domain-containing proteins. PDE5 is the target for the anti-impotence drug sildenafil citrate; therefore, this GAF-BRET2 sensor could be used for the identification of novel compounds that inhibit cGMP binding to the GAF domain, thereby regulating PDE5 catalytic activity.