The psychological impact of Stevens-Johnson syndrome and toxic epidermal necrolysis on patients' lives: a Critically Appraised Topic.

CLINICAL SCENARIO: A 65-year-old man presented with a 12-h history of deteriorating rash. Two weeks previously he had completed a course of neoadjuvant chemotherapy for ductal carcinoma of the breast. On examination there were bullae, widespread atypical targetoid lesions and 15% epidermal detachment. There was no mucosal involvement on presentation, but subsequently it did evolve. Skin biopsy showed subepidermal blistering with epidermal necrosis. This confirmed our clinical diagnosis of overlap Stevens-Johnson syndrome (SJS)/toxic epidermal necrolysis (TEN). On transfer to intensive care he was anxious and fearful. MANAGEMENT QUESTION: What are the psychological impacts of SJS/TEN on this man's life? BACKGROUND: SJS and TEN have devastating outcomes for those affected. OBJECTIVES: To conduct a Critically Appraised Topic to (i) analyse existing research related to the psychological impact of SJS and TEN and (ii) apply the results to the clinical scenario. METHODS: Seven electronic databases were searched for publications focusing on the psychological impact of SJS/TEN on adults over 18 years of age. RESULTS: Six studies met the inclusion criteria. Healthcare practitioners' (HCPs') lack of information around the disorder was highlighted. Patients experienced undue stress and fear. Some patients had symptoms aligned to post-traumatic stress disorder (PTSD), anxiety and depression. DISCUSSION AND RECOMMENDATION: The evidence suggests that SJS and TEN impact psychologically on patients' lives. Education of HCPs, to address their lack of awareness and information on SJS/TEN, should facilitate their capacity to provide information and support to patients, thereby reducing patient anxiety. On discharge, a follow-up appointment with relevant HCPs to reduce the possibility of PTSD occurring should be considered.

Dicarboxylic acids as pH sensors for hyperpolarized 13C magnetic resonance spectroscopic imaging.

Imaging tumoral pH may help to characterize aggressiveness, metastasis, and therapeutic response. We report the development of hyperpolarized [2-13C,D10]diethylmalonic acid, which exhibits a large pH-dependent 13C chemical shift over the physiological range. We demonstrate that co-polarization with [1-13C,D9]tert-butanol accurately measures pH via13C NMR and magnetic resonance spectroscopic imaging in phantoms.

International study of the place of death of people with cancer: a population-level comparison of 14 countries across 4 continents using death certificate data.

BACKGROUND: Where people die can influence a number of indicators of the quality of dying. We aimed to describe the place of death of people with cancer and its associations with clinical, socio-demographic and healthcare supply characteristics in 14 countries. METHODS: Cross-sectional study using death certificate data for all deaths from cancer (ICD-10 codes C00-C97) in 2008 in Belgium, Canada, Czech Republic, England, France, Hungary, Italy, Mexico, the Netherlands, New Zealand, South Korea, Spain (2010), USA (2007) and Wales (N=1,355,910). Multivariable logistic regression analyses evaluated factors associated with home death within countries and differences across countries. RESULTS: Between 12% (South Korea) and 57% (Mexico) of cancer deaths occurred at home; between 26% (Netherlands, New Zealand) and 87% (South Korea) occurred in hospital. The large between-country differences in home or hospital deaths were partly explained by differences in availability of hospital- and long-term care beds and general practitioners. Haematologic rather than solid cancer (odds ratios (ORs) 1.29-3.17) and being married rather than divorced (ORs 1.17-2.54) were most consistently associated with home death across countries. CONCLUSIONS: A large country variation in the place of death can partly be explained by countries' healthcare resources. Country-specific choices regarding the organisation of end-of-life cancer care likely explain an additional part. These findings indicate the further challenge to evaluate how different specific policies can influence place of death patterns.

An orbital period of 0.94 days for the hot-Jupiter planet WASP-18b.

The 'hot Jupiters' that abound in lists of known extrasolar planets are thought to have formed far from their host stars, but migrate inwards through interactions with the proto-planetary disk from which they were born, or by an alternative mechanism such as planet-planet scattering. The hot Jupiters closest to their parent stars, at orbital distances of only approximately 0.02 astronomical units, have strong tidal interactions, and systems such as OGLE-TR-56 have been suggested as tests of tidal dissipation theory. Here we report the discovery of planet WASP-18b with an orbital period of 0.94 days and a mass of ten Jupiter masses (10 M(Jup)), resulting in a tidal interaction an order of magnitude stronger than that of planet OGLE-TR-56b. Under the assumption that the tidal-dissipation parameter Q of the host star is of the order of 10(6), as measured for Solar System bodies and binary stars and as often applied to extrasolar planets, WASP-18b will be spiralling inwards on a timescale less than a thousandth that of the lifetime of its host star. Therefore either WASP-18 is in a rare, exceptionally short-lived state, or the tidal dissipation in this system (and possibly other hot-Jupiter systems) must be much weaker than in the Solar System.

Development and evaluation of human AP endonuclease inhibitors in melanoma and glioma cell lines.

AIMS: Modulation of DNA base excision repair (BER) has the potential to enhance response to chemotherapy and improve outcomes in tumours such as melanoma and glioma. APE1, a critical protein in BER that processes potentially cytotoxic abasic sites (AP sites), is a promising new target in cancer. In the current study, we aimed to develop small molecule inhibitors of APE1 for cancer therapy. METHODS: An industry-standard high throughput virtual screening strategy was adopted. The Sybyl8.0 (Tripos, St Louis, MO, USA) molecular modelling software suite was used to build inhibitor templates. Similarity searching strategies were then applied using ROCS 2.3 (Open Eye Scientific, Santa Fe, NM, USA) to extract pharmacophorically related subsets of compounds from a chemically diverse database of 2.6 million compounds. The compounds in these subsets were subjected to docking against the active site of the APE1 model, using the genetic algorithm-based programme GOLD2.7 (CCDC, Cambridge, UK). Predicted ligand poses were ranked on the basis of several scoring functions. The top virtual hits with promising pharmaceutical properties underwent detailed in vitro analyses using fluorescence-based APE1 cleavage assays and counter screened using endonuclease IV cleavage assays, fluorescence quenching assays and radiolabelled oligonucleotide assays. Biochemical APE1 inhibitors were then subjected to detailed cytotoxicity analyses. RESULTS: Several specific APE1 inhibitors were isolated by this approach. The IC(50) for APE1 inhibition ranged between 30 nM and 50 µM. We demonstrated that APE1 inhibitors lead to accumulation of AP sites in genomic DNA and potentiated the cytotoxicity of alkylating agents in melanoma and glioma cell lines. CONCLUSIONS: Our study provides evidence that APE1 is an emerging drug target and could have therapeutic application in patients with melanoma and glioma.

'I am part of the community but...' The changing context of rural living for persons with advanced cancer and their families.

INTRODUCTION: Older rural persons who are receiving palliative care experience multiple co-existing transitions that can be distressing. These transitions do not occur in a vacuum, but occur in a context that reflects the uniqueness of rural living and the complexities of end of life in rural settings. The context or situation (geographical, physical, and social) in which an experience occurs influences the way people view and interpret the world around them; this contextual perspective contributes greatly to perceptions held by rural residents. PURPOSE: The purpose of this study was to explore the context in which older rural patients receiving palliative care and their families experience transitions. Following a study of the transition experiences of older rural palliative patients, an in-depth interpretive description analysis was conducted specific to the context in which the participants' transitions occurred. METHODS: Twenty-seven open-ended, individual, audio-taped, qualitative interviews were conducted and 4 focus group discussions were held to gather data. Individual audio-taped interviews were conducted with six older rural persons with advanced cancer and 10 bereaved (post-death) family caregivers. Four focus groups were conducted with 12 palliative care healthcare professionals. Participants were recruited from 3 rural health regions in a western Canadian province classified as one of the most 'rural' Canadian provinces. All interviews were transcribed verbatim, coded, and analyzed using Thorne's interpretive description qualitative approach. RESULTS: From the data analysis four themes emerged: (1) community connectedness/isolation; (2) lack of accessibility to care; (3) communication and information issues; and (4) independence/dependence. Participants described feelings of being connected to the community at the same time as they also reported feeling isolated. They described their value of independence at the same time as finding themselves becoming increasingly dependent on others. At times this value of independence interfered with their seeking and accessing needed health or supportive care. They perceived their lack of access to health care resulted in little or no choice in where they die. CONCLUSIONS: These findings reveal that the rural context has a major impact on the types of community support and healthcare services needed by older persons with advanced disease and their families. With advanced disease, the participants' sense of solitude became one of isolation, and with increasing dependence on others, they needed more connection and support from others. The findings reflected a more complex view of rural aging and dying than has been cited in the literature to date. This study suggests there is a need to renegotiate community supports and the independence available to persons with advanced disease as they undergo multiple transitions near the end of life.

Participation of DNA repair in the response to 5-fluorouracil.

The anti-metabolite 5-fluorouracil (5-FU) is employed clinically to manage solid tumors including colorectal and breast cancer. Intracellular metabolites of 5-FU can exert cytotoxic effects via inhibition of thymidylate synthetase, or through incorporation into RNA and DNA, events that ultimately activate apoptosis. In this review, we cover the current data implicating DNA repair processes in cellular responsiveness to 5-FU treatment. Evidence points to roles for base excision repair (BER) and mismatch repair (MMR). However, mechanistic details remain unexplained, and other pathways have not been exhaustively interrogated. Homologous recombination is of particular interest, because it resolves unrepaired DNA intermediates not properly dealt with by BER or MMR. Furthermore, crosstalk among DNA repair pathways and S-phase checkpoint signaling has not been examined. Ongoing efforts aim to design approaches and reagents that (i) approximate repair capacity and (ii) mediate strategic regulation of DNA repair in order to improve the efficacy of current anticancer treatments.

Characterization of Mg2+ binding to the DNA repair protein apurinic/apyrimidic endonuclease 1 via solid-state 25Mg NMR spectroscopy.

Apurinic/apyrimidinic endonuclease 1 (APE1), a member of the divalent cation-dependent phosphoesterase superfamily of proteins that retain the conserved four-layered alpha/beta-sandwich structural core, is an essential protein that functions as part of base excision repair to remove mutagenic and cytotoxic abasic sites from DNA. Using low-temperature solid-state (25)Mg NMR spectroscopy and various mutants of APE1, we demonstrate that Mg(2+) binds to APE1 and a functional APE1-substrate DNA complex with an overall stoichiometry of one Mg(2+) per mole of APE1 as predicted by the X-ray work of Tainer and co-workers (Mol, C. D.; Kuo, C. F.; Thayer, M. M.; Cunningham, R. P.; Tainer, J. A. Nature 1995, 374 , 381-386). However, the NMR spectra show that the single Mg(2+) site is disordered. We discuss the probable reasons for the disorder at the Mg(2+) binding site. The most likely source of this disorder is arrangement of the protein-ligands about the Mg(2+) (cis and trans isomers). The existence of these isomers reinforces the notion of the plasticity of the metal binding site within APE1.

CD8 positive T cells influence antigen-specific immune responses through the expression of chemokines.

The potential roles of CD8(+) T-cell-induced chemokines in the expansion of immune responses were examined using DNA immunogen constructs as model antigens. We coimmunized cDNA expression cassettes encoding the alpha-chemokines IL-8 and SDF-1alpha and the beta-chemokines MIP-1alpha, RANTES, and MCP-1 along with DNA immunogens and analyzed the resulting antigen-specific immune responses. In a manner more similar to the traditional immune modulatory role of CD4(+) T cells via the expression of Th1 or Th2 cytokines, CD8(+) T cells appeared to play an important role in immune expansion and effector function by producing chemokines. For instance, IL-8 was a strong inducer of CD4(+) T cells, indicated by strong T helper proliferative responses as well as an enhancement of antibody responses. MIP-1alpha had a dramatic effect on antibody responses and modulated the shift of immune responses to a Th2-type response. RANTES coimmunization enhanced the levels of antigen-specific Th1 and cytotoxic T lymphocyte (CTL) responses. Among the chemokines examined, MCP-1 was the most potent activator of CD8(+) CTL activity. The enhanced CTL results are supported by the increased expression of Th1 cytokines IFN-gamma and TNF-alpha and the reduction of IgG1/IgG2a ratio. Our results support that CD8(+) T cells may expand both humoral and cellular responses in vivo through the elaboration of specific chemokines at the peripheral site of infection during the effector stage of the immune response.

Base excision repair and the central nervous system.

Reactive oxygen species generated during normal cellular metabolism react with lipids, proteins, and nucleic acid. Evidence indicates that the accumulation of oxidative damage results in cellular dysfunction or deterioration. In particular, oxidative DNA damage can induce mutagenic replicative outcomes, leading to altered cellular function and/or cellular transformation. Additionally, oxidative DNA modifications can block essential biological processes, namely replication and transcription, triggering cell death responses. The major pathway responsible for removing oxidative DNA damage and restoring the integrity of the genome is base excision repair (BER). We highlight herein what is known about BER protein function(s) in the CNS, which in cooperation with the peripheral nervous system operates to control physical responses, motor coordination, and brain operation. Moreover, we describe evidence indicating that defective BER processing can promote post-mitotic (i.e. non-dividing) neuronal cell death and neurodegenerative disease. The focus of the review is on the core mammalian BER participants, i.e. the DNA glycosylases, AP endonuclease 1, DNA polymerase beta, X-ray cross-complementing 1, and the DNA ligases.

Targeted antigen delivery to antigen-presenting cells including dendritic cells by engineered Fas-mediated apoptosis.

Immunity to tumors as well as to viral and bacterial pathogens is often mediated by cytotoxic T lymphocytes (CTLs). Thus, the ability to induce a strong cell-mediated immune response is an important requirement of novel immunotherapies. Antigen-presenting cells (APCs), including dendritic cells (DCs), are specialized in initiating T-cell immunity. Harnessing this innate ability of these cells to acquire and present antigens, we sought to improve antigen presentation by targeting antigens directly to DCs in vivo through apoptosis. We engineered Fas-mediated apoptotic death of antigen-bearing cells in vivo by co-expressing the immunogen and Fas in the same cell. We then observed that the death of antigen-bearing cells results in increased antigen acquisition by APCs including DCs. This in vivo strategy led to enhanced antigen-specific CTLs, and the elaboration of T helper-1 (Th1) type cytokines and chemokines. This adjuvant approach has important implications for viral and nonviral delivery strategies for vaccines or gene therapies.

Engineering of in vivo immune responses to DNA immunization via codelivery of costimulatory molecule genes.

Nucleic acid immunization is a novel vaccination technique to induce antigen-specific immune responses. We have developed expression cassettes for cell surface markers CD80 and CD86, two functionally related costimulatory molecules that play an important role in the induction of T cell-mediated immune responses. Coimmunization of these expression plasmids, along with plasmid DNA encoding for HIV-1 antigens, did not result in any significant change in the humoral response; however, we observed a dramatic increase in cytotoxic T-lymphocyte (CTL) induction as well as T-helper cell proliferation after the coadministration of CD86 genes. In contrast, coimmunization with a CD80 expression cassette resulted in a minor, but positive increase in T-helper cell or CTL responses. This strategy may be of value for the generation of rationally designed vaccines and immune therapeutics.

DNA Polymerase Beta Participates in Mitochondrial DNA Repair.

We have detected DNA polymerase beta (Polß), known as a key nuclear base excision repair (BER) protein, in mitochondrial protein extracts derived from mammalian tissue and cells. Manipulation of the N-terminal sequence affected the amount of Polß in the mitochondria. Using Polß fragments, mitochondrion-specific protein partners were identified, with the interactors functioning mainly in DNA maintenance and mitochondrial import. Of particular interest was the identification of the proteins TWINKLE, SSBP1, and TFAM, all of which are mitochondrion-specific DNA effectors and are known to function in the nucleoid. Polß directly interacted functionally with the mitochondrial helicase TWINKLE. Human kidney cells with Polß knockout (KO) had higher endogenous mitochondrial DNA (mtDNA) damage. Mitochondrial extracts derived from heterozygous Polß mouse tissue and KO cells had lower nucleotide incorporation activity. Mouse-derived Polß null fibroblasts had severely affected metabolic parameters. Indeed, gene knockout of Polß caused mitochondrial dysfunction, including reduced membrane potential and mitochondrial content. We show that Polß is a mitochondrial polymerase involved in mtDNA maintenance and is required for mitochondrial homeostasis.

New insights into the structure of abasic DNA from molecular dynamics simulations.

Abasic (AP) sites constitute a common form of DNA damage, arising from the spontaneous or enzymatic breakage of the N-glycosyl bond and the loss of a nucleotide base. To examine the effects of such damage on DNA structure, especially in the vicinity of the abasic sugar, four 1.5 ns molecular dynamics simulations of double-helical DNA dodecamers with and without a single abasic (tetrahydrofuran, X) lesion in a 5'-d(CXT) context have been performed and analyzed. The results indicate that the abasic site does not maintain a hole or gap in the DNA, but instead perturbs the canonical structure and induces additional flexibility close to the abasic site. In the apurinic simulations (i.e., when a pyrimidine is opposite the AP site), the abasic sugar flipped in and out of the minor groove, and the gap was water filled, except during the occurrence of a novel non-Watson-Crick C-T base pair across the abasic site. The apyrimidinic gap was not penetrated by water until the abasic sugar flipped out and remained extrahelical. Both AP helices showed kinks of 20-30 degrees at the abasic site. The Watson-Crick hydrogen bonds are more transient throughout the DNA double helices containing an abasic site. The abasic sugar displayed an unusually broad range of sugar puckers centered around the northern pucker. The increased motion of the bases and backbone near the abasic site appear to correlate with sequence-dependent helical stability. The data indicate that abasic DNA contorts more easily and in specific ways relative to unmodified DNA, an aspect likely to be important in abasic site recognition and hydrolysis.

Expression specificity of the mouse exonuclease 1 (mExo1) gene.

Genetic recombination involves either the homo-logous exchange of nearly identical chromosome regions or the direct alignment, annealing and ligation of processed DNA ends. These mechanisms are involved in repairing potentially lethal or mutagenic DNA damage and generating genetic diversity within the meiotic cell population and antibody repertoire. We report here the identification of a mouse gene, termed mExo1 for mouse exonuclease 1, which encodes a approximately 92 kDa protein that shares homology to proteins of the RAD2 nuclease family, most notably human 5' to 3' exonuclease Hex1/hExo1, yeast exonuclease 1 (Exo1) proteins and Drosophila melanogaster Tosca. The mExo1 gene maps to distal chromosome 1, consistent with the recent mapping of the orthologous HEX1 / hEXO1 gene to chromosome 1q42-q43. mExo1 is expressed prominently in testis, an area of active homologous recombination, and spleen, a prominent lymphoid tissue. An increased level of mExo1 mRNA was observed during a stage of testis development where cells that are actively involved in meiotic recombination arise first and represent a significant proportion of the germ cell population. Comparative evaluation of the expression patterns of the human and mouse genes, combined with previous biochemical and yeast genetic studies, indicate that the Exo1-like proteins are important contributors to chromosome processing during mammalian DNA repair and recombination.

Functional characterization of Ape1 variants identified in the human population.

Apurinic/apyrimidinic (AP) sites are common mutagenic and cytotoxic DNA lesions. Ape1 is the major human repair enzyme for abasic sites and incises the phosphodiester backbone 5' to the lesion to initiate a cascade of events aimed at removing the AP moiety and maintaining genetic integrity. Through resequencing of genomic DNA from 128 unrelated individuals, and searching published reports and sequence databases, seven amino acid substitution variants were identified in the repair domain of human Ape1. Functional characterization revealed that three of the variants, L104R, E126D and R237A, exhibited approximately 40-60% reductions in specific incision activity. A fourth variant, D283G, is similar to the previously characterized mutant D283A found to exhibit approximately 10% repair capacity. The most common substitution (D148E; observed at an allele frequency of 0.38) had no impact on endonuclease and DNA binding activities, nor did a G306A substitution. A G241R variant showed slightly enhanced endonuclease activity relative to wild-type. In total, four of seven substitutions in the repair domain of Ape1 imparted reduced function. These reduced function variants may represent low penetrance human polymorphisms that associate with increased disease susceptibility.

DNA base excision repair activities and pathway function in mitochondrial and cellular lysates from cells lacking mitochondrial DNA.

Mitochondrial DNA (mtDNA) contains higher steady-state levels of oxidative damage and mutates at rates significantly greater than nuclear DNA. Oxidative lesions in mtDNA are removed by a base excision repair (BER) pathway. All mtDNA repair proteins are nuclear encoded and imported. Most mtDNA repair proteins so far discovered are either identical to nuclear DNA repair proteins or isoforms of nuclear proteins arising from differential splicing. Regulation of mitochondrial BER is therefore not expected to be independent of nuclear BER, though the extent to which mitochondrial BER is regulated with respect to mtDNA amount or damage is largely unknown. Here we have measured DNA BER activities in lysates of mitochondria isolated from human 143B TK(-) osteosarcoma cells that had been depleted of mtDNA (rho(0)) or not (wt). Despite the total absence of mtDNA in the rho(0) cells, a complete mitochondrial BER pathway was present, as demonstrated using an in vitro assay with synthetic oligonucleotides. Measurement of individual BER protein activities in mitochondrial lysates indicated that some BER activities are insensitive to the lack of mtDNA. Uracil and 8-oxoguanine DNA glycosylase activities were relatively insensitive to the absence of mtDNA, only about 25% reduced in rho(0) relative to wt cells. Apurinic/apyrimidinic (AP) endonuclease and polymerase gamma activities were more affected, 65 and 45% lower, respectively, in rho(0) mitochondria. Overall BER activity in lysates was also about 65% reduced in rho(0) mitochondria. To identify the limiting deficiencies in BER of rho(0) mitochondria we supplemented the BER assay of mitochondrial lysates with pure uracil DNA glycosylase, AP endonuclease and/or the catalytic subunit of polymerase gamma. BER activity was stimulated by addition of uracil DNA glycosylase and polymerase gamma. However, no addition or combination of additions stimulated BER activity to wt levels. This suggests that an unknown activity, factor or interaction important in BER is deficient in rho(0) mitochondria. While nuclear BER protein levels and activities were generally not altered in rho(0) cells, AP endonuclease activity was substantially reduced in nuclear and in whole cell extracts. This appeared to be due to reduced endogenous reactive oxygen species (ROS) production in rho(0) cells, and not a general dysfunction of rho(0) cells, as exposure of cells to ROS rapidly stimulated increases in AP endonuclease activities and APE1 protein levels.

Malignant transformation of human fibroblasts by a transfected N-ras oncogene.

The only ras oncogene as yet identified in cells from human fibrosarcomas is N-ras, but the relationship between N-ras oncogene expression and the malignant state of these cell lines is not known. To determine if expression of an N-ras oncogene causes human cells to become malignant, we transfected the N-ras oncogene from human leukemia cell line 8402, cloned into a high expression vector pSV N-ras, into MSU-1.1 cells, a nontumorigenic, infinite life span fibroblast cell strain with a normal morphology and a stable near-diploid karyotype. The transformants formed distinct foci composed of morphologically transformed cells. Cells from such foci expressed higher than normal levels of N-ras protein, exhibited growth factor independence, and formed large colonies in soft agar at a high frequency. Injection of progeny of these focus-derived cells s.c. into athymic mice resulted in progressively growing, invasive malignant tumors (round cell, spindle cell, or giant cell sarcomas) which reached a diameter of 6 mm in 3 to 4 weeks. Injection of focus-derived or tumor-derived cells i.v. resulted in tumors in various organs of the mice. The focus-derived cell strain tested, as well as the majority of the cells derived from the tumor it produced, exhibited the same near-diploid karyotype as the parental MSU-1.1 cells. Cells transfected with an N-ras oncogene that was expressed at a normal level formed only a single, indistinct focus, and cells from that focus were not malignant.

Dynamic nuclear polarization of biocompatible (13)C-enriched carbonates for in vivo pH imaging.

A hyperpolarization technique using carbonate precursors of biocompatible molecules was found to yield high concentrations of hyperpolarized (13)C bicarbonate in solution. This approach enabled large signal gains for low-toxicity hyperpolarized (13)C pH imaging in a phantom and in vivo in a murine model of prostate cancer.