Novel DNA methylation signatures of tobacco smoking with trans-ethnic effects.

BACKGROUND: Smoking remains one of the leading preventable causes of death. Smoking leaves a strong signature on the blood methylome as shown in multiple studies using the Infinium HumanMethylation450 BeadChip. Here, we explore novel blood methylation smoking signals on the Illumina MethylationEPIC BeadChip (EPIC) array, which also targets novel CpG-sites in enhancers. METHOD: A smoking-methylation meta-analysis was carried out using EPIC DNA methylation profiles in 1407 blood samples from four UK population-based cohorts, including the MRC National Survey for Health and Development (NSHD) or 1946 British birth cohort, the National Child Development Study (NCDS) or 1958 birth cohort, the 1970 British Cohort Study (BCS70), and the TwinsUK cohort (TwinsUK). The overall discovery sample included 269 current, 497 former, and 643 never smokers. Replication was pursued in 3425 trans-ethnic samples, including 2325 American Indian individuals participating in the Strong Heart Study (SHS) in 1989-1991 and 1100 African-American participants in the Genetic Epidemiology Network of Arteriopathy Study (GENOA). RESULTS: Altogether 952 CpG-sites in 500 genes were differentially methylated between smokers and never smokers after Bonferroni correction. There were 526 novel smoking-associated CpG-sites only profiled by the EPIC array, of which 486 (92%) replicated in a meta-analysis of the American Indian and African-American samples. Novel CpG sites mapped both to genes containing previously identified smoking-methylation signals and to 80 novel genes not previously linked to smoking, with the strongest novel signal in SLAMF7. Comparison of former versus never smokers identified that 37 of these sites were persistently differentially methylated after cessation, where 16 represented novel signals only profiled by the EPIC array. We observed a depletion of smoking-associated signals in CpG islands and an enrichment in enhancer regions, consistent with previous results. CONCLUSION: This study identified novel smoking-associated signals as possible biomarkers of exposure to smoking and may help improve our understanding of smoking-related disease risk.

Síndrome de Nicolau: A propósito de un caso / Nicolau syndrome: About a case

Se evaluó paciente femenino de 51 años, con antecedentes de diabetes mellitus tipo II e infecciones urinarias a repetición, procedente de la comunidad Mario Briceño Iragorry, quien acude al ambulatorio centro de especialidades "Los Grillitos", de la Corporación de Salud del Estado de Aragua - Venezuela, presentando lesión ulcerada a nivel de hemicadera izquierda, posterior a la colocación vía intramuscular de diclofenac sódico el 28/03/17, el que fue indicado por facultativo para tratar dolor por infección urinaria alta. Posteriormente, a las 24 horas presenta en la zona de la inyección una coloración violácea, induración y mucho dolor, motivo por el que acude nuevamente al facultativo. Se le realizó ecosonograma de partes blandas en región glútea izquierda, reportando imagen heterogénea a predominio hipoecoico con ecos internos de mediana intensidad, que genera reforzamiento posterior de 49x39 mm, con aproximadamente 30 cc de líquido, que correspondería a un proceso inflamatorio e infeccioso (absceso). Posteriormente a la evaluación, se indica antibiótico terapia a base de oxacilina 1 g cada 8 horas vía endovenosa por siete días, además 100 mg cada 12 horas vía oral de nitrofurantoína para el proceso infeccioso urinario, que el urocultivo indica ser sensible a la E. Coli. Al tercer día de la enfermedad actual, se le drena absceso con material purulento aproximadamente 20 cc y cura sucesiva por cinco días, evolucionando satisfactoriamente del proceso infeccioso de partes blandas e infección urinaria.

A 51-year-old female patient with a history of type II diabetes mellitus and recurrent urinary tract infections from the Mario Briceño Iragorry community, attended the outpatient clinic "Los Grillitos" of the health corporation of Aragua Venezuela, with an ulcerated lesion at the left hemi-hip level, following the intramuscular placement of diclofenac sodium on 03/28/17, which was indicated by facultative to treat pain due to high urinary tract infection, afterwards approximately 24 hours presents at the injection site a violet coloration, induration and a lot of pain, which is why she returned to facultative, she performed a soft-tissue echosonogram in the left gluteal region reporting a heterogeneous image to hypoechoic predominance with internal echoes of medium intensity that generates posterior reinforcement which measures 49x39 mm with approximately 30 cc of liquid that could correspond to an inflammatory and infectious process (abscess), post-evaluation is indicated antibiotic therapy based on oxacillin 1 g every 8 hours intravenous route for seven days plus 100 mg every 12 hours oral route of nitrofurantoin for the urinary infectious process that the urine culture indicates to be sensitive E. coli, the third day of illness is drained abscess purulent material approximately 20 cc and successive cure for five days. Satisfactory evolution of the infectious process of soft tissue and urinary tract infection.

Cadmium body burden and increased blood pressure in middle-aged American Indians: the Strong Heart Study.

Cadmium (Cd) is an environmental pollutant that has been associated with cardiovascular disease in populations, but the relationship of Cd with hypertension has been inconsistent. We studied the association between urinary Cd concentrations, a measure of total body burden, and blood pressure in American Indians, a US population with above national average Cd burden. Urinary Cd was measured using inductively coupled plasma mass spectrometry, and adjusted for urinary creatinine concentration. Among 3714 middle-aged American Indian participants of the Strong Heart Study (mean age 56 years, 41% male, 67% ever-smokers, 23% taking antihypertensive medications), urinary Cd ranged from 0.01 to 78.48 µg g-1 creatinine (geometric mean=0.94 µg g-1) and it was correlated with smoking pack-year among ever-smokers (r2=0.16, P<0.0001). Participants who were smokers were on average light-smokers (mean 10.8 pack-years), and urinary Cd was similarly elevated in light- and never-smokers (geometric means of 0.88 µg g-1 creatinine for both categories). Log-transformed urinary Cd was significantly associated with higher systolic blood pressure in models adjusted for age, sex, geographic area, body mass index, smoking (ever vs never, and cumulative pack-years) and kidney function (mean blood pressure difference by lnCd concentration (ß)=1.64, P=0.002). These associations were present among light- and never-smokers (ß=2.03, P=0.002, n=2627), although not significant among never-smokers (ß=1.22, P=0.18, n=1260). Cd was also associated with diastolic blood pressure among light- and never-smokers (ß=0.94, P=0.004). These findings suggest that there is a relationship between Cd body burden and increased blood pressure in American Indians, a population with increased cardiovascular disease risk.

Genome-wide linkage scan for plasma high density lipoprotein cholesterol, apolipoprotein A-1 and triglyceride variation among American Indian populations: the Strong Heart Family Study.

BACKGROUND: Recent studies have identified chromosomal regions linked to variation in high density lipoprotein cholesterol (HDL-C), apolipoprotein A-1 (apo A-1) and triglyceride (TG), although results have been inconsistent and previous studies of American Indian populations are limited. OBJECTIVE: In an attempt to localise quantitative trait loci (QTLs) influencing HDL-C, apo A-1 and TG, we conducted genome-wide linkage scans of subjects of the Strong Heart Family Study. METHODS: We implemented analyses in 3484 men and women aged 18 years or older, at three study centres. RESULTS: With adjustment for age, sex and centre, we detected a QTL influencing both HDL-C (logarithm of odds (LOD) = 4.4, genome-wide p = 0.001) and apo A-1 (LOD = 3.2, genome-wide p = 0.020) nearest marker D6S289 at 6p23 in the Arizona sample. Another QTL influencing apo A-1 was found nearest marker D9S287 at 9q22.2 (LOD = 3.0, genome-wide p = 0.033) in the North and South Dakotas. We detected a QTL influencing TG nearest marker D15S153 at 15q22.31 (LOD = 4.5 in the overall sample and LOD = 3.8 in the Dakotas sample, genome-wide p = 0.0044) and when additionally adjusted for waist, current smoking, current alcohol, current oestrogen, lipid treatment, impaired fasting glucose, and diabetes, nearest marker D10S217 at 10q26.2 (LOD = 3.7, genome-wide p = 0.0058) in the Arizona population. CONCLUSIONS: The replication of QTLs in regions of the genome that harbour well known candidate genes suggest that chromosomes 6p, 9q and 15q warrant further investigation with fine mapping for causative polymorphisms in American Indians.

Detection of MetastaticTumor Cells in Blood by Reverse Transcriptase-Polymerase Chain Reaction.

In oncology, the correct assessment of tumor stage is crucial information, as it relates to disease-free interval and prognosis. Routine staging modalities use histopathology to evaluate the presence or absence of metastasis mainly in tissues such as lymph nodes, as described in Chapter 1 by Roskell and Buley. Retrospective analysis using fine sectioning techniques has shown that approx 30% of tumor patients may be understaged at the time of diagnosis (1) Since the early 1980s, immunohistochemical and -cytochemical methods have been devised to increase the sensitivity in detecting small tumor loads or single tumor cells (2) Clinical studies using monoclonal antibodies against cytokeratins, intermediate filament proteins characterizing epithelial cells and thus carcinomas, have shown that the presence of such cells in the bone marrow is an independent prognostic marker of disease-free and overall survival (3-5). The methodology for this approach is given in Chapter 5 by Braun and Pantel. With the advent of extremely powerful nucleic acid amplification and detection methods a significant increase in analytical and diagnostic sensitivity of tumor cell detection has been achieved. Such micrometastasis assays detect messenger RNA (mRNA) molecules transcribed from genes active in the cancer cell, but not the non-malignant cellular components otherwise present in the sample material. In this technique, a first strand cDNA copy is first produced from the RNA molecules using a reverse transcriptase (RT). The RT step is then followed by an enzymatic exponential amplification reaction, most commonly performed using the polymerase chain reaction (PCR).

Induction of protective immunity against syngeneic rat cancer cells by expression of the cytosine deaminase suicide gene.

The use of the cytosine deaminase (CD)/5-fluorocytosine suicide system as a cancer gene therapy approach enables selective killing of CD-modified cells as well as the ablation of non-modified tumor cells due to a bystander effect that has been suggested to involve the immune system in vivo. Using a stable CD transfectant of the tumorigenic rat adenocarcinoma cell line AS (AS/CD), an antitumoral response against the CD expressing cell line as well as the parental cell line could be induced by stepwise vaccinations in syngeneic animals. AS/CD tumor regression occurred independently of 5-fluorocytosine treatment and was sufficient to protect 37% of the animals against subsequent challenge with tumorigenic doses of the parental AS cell line. Immune rats contained lymphocytes able to specifically lyse CD modified as well as unmodified AS tumor cells in vitro, most likely contributing to the in vivo antitumoral reaction. Thus, the CD suicide system seems to be suitable not only for a local tumor gene therapy but also for the application as therapy of metastatic tumors and minimal residual disease.

Fractionated radiation therapy in combination with adenoviral delivery of the cytosine deaminase gene and 5-fluorocytosine enhances cytotoxic and antitumor effects in human colorectal and cholangiocarcinoma models.

Radiosensitization of human gastrointestinal tumors by 5-fluorouracil (5-FU) has been studied in vitro and clinically in human cancer therapy trials. The bacterial enzyme cytosine deaminase (CD) converts the nontoxic prodrug 5-fluorocytosine (5-FC) into 5-FU. Human colon cancer cells stably expressing CD have been shown by other investigators to be sensitized to radiation following treatment with 5-FC. We previously used an adenoviral vector under control of the cytomegalovirus promoter (AdCMVCD) encoding the CD gene in combination with 5-FC and a single fraction of radiation exposure to enhance cytotoxicity to human cholangiocarcinoma cells in vitro and in vivo. The purpose of this study was to determine whether AdCMVCD infection and 5-FC with multiple fraction low-dose radiotherapy results in enhanced cytotoxicity. In the present study, we utilized AdCMVCD and 5-FC with single fraction radiotherapy to demonstrate enhanced cytotoxicity to WiDr human colon carcinoma cells in vitro. Additionally, we tested this gene therapy/prodrug treatment strategy employing a fractionated radiation dosing schema in animal models of WiDr colon carcinoma and SK-ChA-1 cholangiocarcinoma. A prolonged WiDr tumor regrowth delay was obtained with AdCMVCD infection in combination with systemic delivery of 5-FC and fractionated external beam radiation therapy compared with control animals treated without radiation, without 5-FC, or without AdCMVCD. The results of treatment with AdCMVCD + 5-FC + radiation therapy to cholangiocarcinoma xenografts were equivalent to those obtained with systemic 5-FU administration + radiation. Thus, the use of AdCMVCD can be effectively combined with clinically relevant 5-FC and radiation administration schemes to achieve enhanced tumor cell killing and increased control of established tumors of human gastrointestinal malignancies.

Double suicide gene (cytosine deaminase and herpes simplex virus thymidine kinase) but not single gene transfer allows reliable elimination of tumor cells in vivo.

Suicide genes such as cytosine deaminase (CD) and herpes simplex virus thymidine kinase (TK) encode products that convert nontoxic substances (prodrugs) into toxic metabolites. Suicide gene transfer is currently being used in cancer therapy or can be used as a safety modality. To analyze the reliability of suicide genes as a safety modality for a vaccination study with viable cytokine/B7 gene-modified tumor cells, the individual and combined efficacy of the two suicide genes was compared for in vitro and in vivo cell killing of a murine mammary adenocarcinoma cell line (TS/A). To adapt the system to an in vivo gene delivery situation, bulk cultures cotransfected with the CD and TK gene were used instead of selected clones. In vitro, both CD and TK conferred sensitivity to the respective prodrug but the combined cytotoxic effects of both gene products were always superior. For in vivo analysis BALB/c mice were injected subcutaneously with CD- and TK-modified TS/A cells, treated with prodrugs, and tumor size was evaluated for a period of 100 days. In the in vivo situation the combination of both enzyme/prodrug systems was again most effective. The highest single concentration of 5-FC (500 mg/kg) or GCV (100 mg/kg) was not able to fully protect the animals from developing tumors, whereas a combination of 5-FC (250 mg/kg) and GCV (50 mg/kg) resulted in complete tumor eradication. In nude mice treated in the same way, most CD/TK tumors could not be eliminated. Furthermore, BALB/c mice cured of TS/A-CD/TK tumors developed a systemic tumor immunity against challenge with parental TS/A cells. These findings indicate that reliable tumor elimination by the suicide genes depends on T cells. The cooperative effect of both suicide genes was confirmed in vitro with the human renal cell carcinoma line RCC26. We conclude that TK and CD together, but neither gene alone, act as a safety mechanism for the elimination of tumor cells in a reliable fashion and suggest that a rapid and quantitative antigen release by effective TK- and CD-mediated tumor destruction is necessary for T cell immunity to develop.

Detection of cytosine deaminase in genetically modified tumor cells by specific antibodies.

Bacterial cytosine deaminase (CD) converts the non-toxic prodrug 5-fluorocytosine (5-FC) into 5-fluorouracil (5-FU), which is toxic for mammalian cells. Therefore, the CD gene is used in cancer gene therapy to achieve high local concentration of a toxic metabolite without significant systemic toxicity. To allow the detection of CD expression at the protein level, we raised both polyclonal rabbit antisera and a monoclonal antibody (mAb) against a histidine-tagged CD fusion protein. The specificity of the polyclonal antisera and the mAb was confirmed by immunohistochemistry, immunoblot analysis, and immunoprecipitation using CD-expressing tumor cell lines. Furthermore, the antibodies can be used for ELISA assays and flow cytometry. Finally, the CD protein could be demonstrated in frozen tissue sections of CD-modified tumors in a rat tumor model using the anti-CD serum. With these antibodies, CD expression can now be monitored throughout in vitro and in vivo gene transfer studies, including clinical protocols relying on the CD suicide gene strategy.

Adenovirus-assisted lipofection: efficient in vitro gene transfer of luciferase and cytosine deaminase to human smooth muscle cells.

Smooth muscle cells (SMC) are a central cell type involved in multiple processes of coronary artery diseases including restenosis and therefore are major target cells for different aspects of gene transfer. Previous attempts to transfect primary arterial cells using different techniques like liposomes, CaPO4 and electroporation resulted in only low transfection efficiency. The development of recombinant adenoviruses dramatically improved the delivery of foreign genes into different cell types including SMC. However, cloning and identification of recombinants remain difficult and time-consuming techniques. The present study demonstrates that a complex consisting of reporter plasmid encoding firefly luciferase (pLUC), polycationic liposomes and replication-deficient adenovirus was able to yield very high in vitro transfection of primary human smooth muscle cells under optimized conditions. The technique of adenovirus-assisted lipofection (AAL) increases transfer and expression of plasmid DNA in human smooth muscle cells in vitro up to 1000-fold compared to lipofection. To verify the applicability of AAL for gene transfer into human smooth muscle cells we studied a gene therapy approach to suppress proliferation of SMC in vitro, using the prokaryotic cytosine deaminase gene (CD) which enables transfected mammalian cells to deaminate 5-fluorocytosine (5-FC) to the highly toxic 5-fluorouracil (5-FU). The effect of a transient CD expression on RNA synthesis was investigated by means of a cotransfection with a RSV-CD expression plasmid and the luciferase reporter plasmid. Western blot analysis demonstrated high expression of CD protein in transfected SMC. Cotransfected SMC demonstrated two-fold less luciferase activity in the presence of 5-FC (5 mmol/l) after 48 h compared to cells transfected with a non-CD coding plasmid. The data demonstrate that a transient expression of CD could be sufficient to reduce the capacity of protein synthesis in human SMC. This simple and effective in vitro transfection method may also be applicable to in vivo delivery of target genes to the vascular wall to inhibit SMC proliferation.

Monitoring gene therapy with cytosine deaminase: in vitro studies using tritiated-5-fluorocytosine.

UNLABELLED: Genetically modified mammalian cells that express the cytosine deaminase (CD) gene are able to convert the nontoxic prodrug 5-fluorocytosine (5-FC) to the toxic metabolite 5-fluorouracil (5-FU). PET with 18F-5-FC may be used for in vivo measurement of CD activity in genetically modified tumors. METHODS: A human glioblastoma cell line was stably transfected with the Escherichia coli CD gene. After incubation of lysates of CD-expressing cells and control cells with 3H-5-FC high-performance liquid chromatography (HPLC) was performed. The uptake of 5-FC was measured after various incubation times using therapeutic amounts of 5-FC. In addition, saturation and competition experiments with 5-FC and 5-FU were performed. Finally, the efflux was measured. RESULTS: We found that 3H-5-FU was produced in CD-expressing cells, whereas in the control cells only 3H-5-FC was detected. Moreover, significant amounts of 5-FU were found in the medium of cultured cells, which may account for the bystander effect observed in previous experiments. However, uptake studies revealed a moderate and nonsaturable accumulation of radioactivity in the tumor cells, suggesting that 5-FC enters the cells only through diffusion. Although a significant difference in 5-FC uptake was seen between CD-positive and control cells after 48 hr of incubation, no difference was observed after 2 hr of incubation. Furthermore, a rapid efflux could be demonstrated. CONCLUSION: 5-Fluorocytosine transport may be a limiting factor for this therapeutic procedure. Quantitation with PET has to rely more on dynamic studies and modeling, including HPLC analysis of the plasma, than on nonmodeling approaches.