Prostate cancer is the second leading cause of male death in the United States. The incidence increases most rapidly with age, and multiple genetic and epigenetic factors have been implicated in the initiation, progression, and metastasis of the cancer. Nevertheless, scientific knowledge of the molecular mechanisms underlying the disease is still limited; and hence treatment has only been partially successful. The objective of the current studies was to examine the role of caspase 3 (CPP32) and NAD(P)H:quinone oxidoreductase (NQO1) in the signaling of genistein-and β-lapachone (bLap)-induced apoptosis in human prostate carcinoma cells PC3.Both genistein and bLap produced dose-dependent growth inhibition and treatment-induced apoptosis in PC3. Treatment with caspase 3 inhibitor, DEVD-fmk before exposure to genistein, significantly inhibited caspase 3 expression and treatment-induced apoptosis; implicating CPP32 as the main target in genistein-induced apoptosis in PC3. Contrary to this observation, inhibition of CPP32 did not significantly influence bLap-induced apoptosis; implying that the major target of bLap-induced apoptosis may not be the caspase. Treatment with NQO1 inhibitor, dicoumarol (50 μM), prior to exposure of PC3 to bLap led to significant decrease in bLap toxicity concurrent with significant decrease in treatment-induced apoptosis; thus implicating NQO1 as the major target in β-lapachone-induced apoptosis in PC3. In addition, the data demonstrated that NQO1 is the major target in bLap-genistein (combination)-induced apoptosis. On the contrary, blocking NQO1 activity did not significantly affect genistein-induced apoptosis; implying that NQO1 pathway may not be the main target for genistein-induced apoptosis in PC3 cells. Furthermore, blocking NQO1 and CPP32 did not confer 100% protection against genistein-induced or bLap-induced apoptosis.The data thus demonstrate that both genistein-and bLap-induced apoptosis are mostly but not completely dependent on CPP32 and NQO1 respectively. Other minor alternate death pathways may be involved. This suggests that some death receptor signals do not utilize the caspase CPP32 and/or the NQO1 death pathways in PC3. The demonstrated synergism between genistein and bLap justifies consideration of these phytochemicals in chemotherapeutic strategic planning. Prostate cancer is the most common non-skin malignancy and the second leading cause of male death in the United States . The incThe problem often faced with the clinical management of prostate cancer is derived not only from the fact that no single gene or molecule can serve as a reliable marker ,3, but aThe molecular mechanism(s) and intracellular mediators of both spontaneous-and treatment-induced apoptosis are not fully elucidated. However, evidence from several research investigations seem to indicate that a variety of stimuli, including physiological, pathologic, environmental or cytotoxic, can trigger the process of apoptosis in many mammalian cells ,6, and t2-M stage [Genistein isoflavone [4',5',]-trihydroxyisoflavone) is a metabolite of soy  and has -M stage ; inhibit-M stage ; competi-M stage ,18; stim-M stage ,20.The other phytochemical of potential therapeutic significance is β-lapachone [3, 4-dihydro-2, 2-dimethyl-2H-naphtol (1,2-b) pyran-5,6-dione], a simple plant product with a chemical structure different from currently used anti-cancer drugs. It has been previously demonstrated that the primary mode of cytotoxicity of β-Lapachone is through the induction of apoptosis ,22. Struin vitro effects of β-lapachone and genistein have been described, knowledge of the key intracellular targets of β-lapachone and genistein is limited. Recent reports have suggested that by β-lapachone-induced apoptosis is non-caspase mediated in breast [1 and/or S phase followed by apoptotic and/or necrotic cell death [While a number of n breast  and prosn breast ,34, and n breast ,27. B-Lan breast ,29. It dn breast -32 and ill death . The apoll death , and hasll death  and caspll death ,33.The objective of this study was to determine the potential chemosensitivity of human prostate adenocarcinoma, PC3 cells to β-lapachone and Genistein and the role of caspase 3 (CPP32) and NAD(P)H:Quinone oxidoreductase enzyme (NQO1) in the signaling of β-Lapachone and genistein-induced apoptosis in PC3 cells. The hypothesis is that combination treatment with the two phytochemicals will be strongly preventive and/or interceptive against prostate cancer by modulating epigenetic events (apoptosis) associated with the progression of active and latent cancer cells to clinical malignancy.in vitro using Trypan blue exclusion, LDH and MTS bioassays. In single and combination treatments, both genistein and β-lapachone inhibited cell growth and decreased cell survival through induction of cell death [Figures. Human prostate carcinoma cells PC3, was used to determine the chemosensitivity of prostate cancer to genistein isoflavone and β-lapachone Figures. ,2,3. TheExtensive cell death was observed in proliferating human prostate cancer cells after treatment with β-lapachone and genistein isoflavone. To determine if the treatment-induced cell occurred through cytotoxic necrosis and/or apoptosis, cells were harvested and assayed for apoptosis induction with Annexin V-FITC and TUNEL apoptosis assays to detect early and late apoptosis respectively. Aliquots of cells were also stained with acridine orange/ethidium bromide nuclear stain to distinguish between apoptotic and necrotic cells. The results revealed that in both single and combination treatments, cell death was mostly through apoptosis in a dose-dependent manner Figures ,5. With To determine the potential role of the enzyme NAD(P)H:quinone oxidoreductase (NQO1) in β-lapachone (bLap)-and genistein (Gn)-induced apoptosis in PC3, the cells were exposed to Gn and bLap in the presence or absence of dicoumarol in single and combination treatments; and then assayed for apoptosis by the Annexin V-FITC and TUNEL assays. Dicoumarol is a specific inhibitor of NQO1. The results revealed that blocking NQO1 activity with dicoumarol (50 μM) significantly reduced bLap-induced apoptosis Figure ; indicatTo determine if apoptosis induced by β-lapachone and/or genistein involved activation of caspase 3 protease (CPP32), the PC3 cells, were subjected to treatments with Gn and/or bLap co-administered with or without CPP32 inhibitor (DEVD-fmk), and then cultured as previously described. Post-treatment apoptosis was determined as previously described. As shown in Figures In this study, we determined the role of caspase 3 (CPP32) and the enzyme NAD(P)H:quinone oxidoreductase (NQO1) in the signaling of β-lapachone (bLap)-and genistein (Gn)-induced apoptosis in human prostate adenocarcinoma, PC3 cells. Data from this study demonstrate significant inhibition of cell growth and proliferation in PC3 cells, with significant difference in chemosensitivity of PC3 to genistein and β-lapachone (P < 0.01). Furthermore, growth inhibition of PC3 cells strongly correlated with the MTS and LDH assay results. The pattern of response and percent post-treatment live cells was consistent with previous results ,37-39. TThe present data also implicates caspase-3 protease, CPP32, in the molecular pathway of genistein-induced apoptosis in prostate PC3 cancer cells, consistent with previous investigations ,11,39. UThe present data indicate a possible alternate CPP32 pathway in bLap-induced apoptosis in PC3. However, unlike the observation in genistein-induced apoptosis, blocking the CPP32 activity with the specific caspase inhibitor, DEVD-fmk, did not significantly change the percentage of bLap-induced apoptosis in PC3 cells; indicating that CPP32 many not be the main death pathway of bLap-induced apoptosis in PC3 cells. Activation of the caspase 3 in bLap-induced apoptosis has been reported in previous studies .The potential role of NAD(P)H:quinone oxydoreductase (NQO1) activity in genistein-and bLap-induced apoptosis in PC3 was investigated. Co-culture of PC3 cells with dicoumarol, a specific inhibitor of NQO1 activity, significantly reduced the cytotoxicity of β-Lapachone in PC3 cells, as reflected in the significant reduction in the percentage of treatment-induced apoptosis. Dicoumarol increased cell survival. These results implicate NQO1 as the main target in bLap-induced apoptosis in PC3, consistent with previous observations ,34,40. HContrary to the observation in bLap-induced apoptosis, blocking NQO1 activity did not significantly influence genistein-induced apoptosis in PC3 cells; implying NQO1 may not be the major target in genistein-induced apoptosis in PC3 cells. However, the overall data indicate a synergistic effect of genistein-bLap combination treatment of PC3 and, that the major target in the combination treatment-induced apoptosis in PC3 cells is NQO1. Investigation into genistein/bLap synergism in a number human cancer cells is on-going.It is concluded from the data obtained that: i) both genistein and bLap exert growth inhibition effects in PC3 cells, with significant differences in chemosensitivity of PC3 to the two agents; ii) there is a manifestation of synergism between genistein-bLap combination treatment; iii) both genistein and bLap induce apoptosis in PC3 cells; iv) the major target in genistein-induced apoptosis in PC3 cells seems to be CPP32; v) the major molecular target in bLap-induced apoptosis in PC3 cells appear to be NQO1; vi) the major target in the genistein-bLap combination treatment-induced apoptosis appears to be NQO1; and vii) combination treatment appears significantly more efficacious than single treatments. More extensive studies are ongoing to delineate and clarify the molecular mechanisms underlying the combination effects.10-70) and frozen at -37°C until used. β-lapachone (Sigma Scientific (St. Louis, MO, USA) was constituted in DMSO solvent as 1, 2, 3, 5 and 8 μM/ml solutions (bL1-8) and stored at -37°C. Dicoumarol (Sigma Scientific St. Louis, MO, USA) was constituted in DMSO as 50 μM/ml and frozen at -37°C until used. The caspase 3 inhibitor, DEVD-fmk and substrate DEVD-afc were purchased from Biovision . Culture media (RPMI 1640), antibiotics, trypsin-EDTA, and other reagents were purchased from Sigma scientific (St. Louis, MO, USA).Genistein isoflavone  was constituted in DMSO (dimethylsulfoxide) solvent as 10, 20, 30, 50 and 70 μg/ml solutions (GHuman prostate adenocarcinoma (PC3) was a generous donation from Rambaugh-Goodwin Cancer Research Institute, Plantation FL.2 at 37°C for 48 hrs to reach 80–90% confluence. All cells were grown and maintained as monolayers in 25 m2 tissue culture flasks (Sigma Scientific, St. louis, MO, USA) in RPMI 1640 containing 15 mM HEPES, and supplemented with 0.45% glucose (w/w), 5.0% FBS and 100 U. mL-1 penicillin + 100 mg. ml-1 streptomycin. The cells were then harvested by gentle scraping with a cell scraper. The cell suspensions were then grown at a density of 2.5 × 103 cells/well in 24-well microtiter plates (MTP) for 36 hr to allow adherence. The supernatants were discarded and the agents (Gn or bLap) were added over a range of 5 cytotoxic concentrations in single and combination treatments. In preliminary studies with bL1-8, the IC50 ranged between 1.8–3 μM for a number of cells. Therefore in the present studies, 2.0 μM (bL2) was used in the combination studies with varying concentrations of genistein. All treatments were in triplicates. Dicoumarol was added to the cells and incubated for 4 hr before treatment with either genistein or β-lapachone alone or in combination. All plates were then incubated at 37°C in a humidified atmosphere of 5% CO2 in air for a maximum of 72 hr. At 12, 24 and 36 hr of incubation, 100 μl of the supernatant from each well was gently aspirated and replenished with 100 μl of fresh media. The supernatants were stored at -37°C until assayed for lactate dehydrogenase (LDH) enzyme activity. At 36 hr incubation, the cells were harvested by trypsinization with trypsin-EDTA, and processed for post-treatment metabolic activity using cell viability and apoptotic assays as described.To assess the chemosensitivity of human prostate cancer cells to single and combination treatment with genistein (Gn) and β-lapachone (bLap), cells were sub-cultured under 5% CO2 and 37°C, the purple formazan product (indicative of reduction of MTS) was visible. The absorbance was read on Multiskan biochromatic automated microplate reader (Multiskan, DC) at 490 nm. The signal generated (color intensity) is directly proportional to the number of viable  cells in the wells. Relative cell numbers can therefore be determined based on the optical absorbance  of the sample. The blank values were subtracted from each well of the treated cells and controls; and the mean and standard error for each treatment (singles and combination) were calculated relative to the control:MTS assay depends on mitochondrial enzyme reduction of MTS solution to detect and determine cell viability. The MTS cell proliferation assay is a colorimetric method for determining the number of viable cells in proliferation. It is composed of solutions of a tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS] and an electron coupling reagent . MTS is bioreduced by the cells into formazan product that is soluble in cell culture medium. Following cell culture as described above, 100 μL of cells were harvested from each treatment group and added to a 96 MTP followed by addition of 20 μl of MTS  stock solution to each well. After 2 hr incubation under standard conditions of 5% COC = absorbance of the control : AT = absorbance of the treated cells where AB = absorbance of the blank AFor the Trypan blue exclusion test, cells were treated and cultured as described. They were harvested and Trypan blue dye solution was added to the cell suspensions. Total cell counts and viable cell number  were determined by a standard hemocytometer procedure. Live-viable cells were seen as colorless (impermeable to the dye due to intact cell membrane) and dead cells were seen as blue (permeable to dye due to disruption of cell membrane):Lactase dehydrogenase activity was measured by a non-radioactive protocol using the LDH cytotox kit (Cat. #. 1644 793: Boehringer-Mannheim GmbG, Bochemica). The previously frozen supernatants were thawed for LDH determination. Briefly, 100 μL/well of each cell-free supernatant was transferred in triplicate into wells in a 96-well microtiter plate (MTP) and 100 μL of LDH-assay reaction mixture  added to each well. After 90 min incubation under standard conditions the absorbance of the color generated was read on Multiskan biochromatic automatic microplate reader at 490 nm. The mean absorbance/optical density (OD) for each treatment group was calculated. The blank values were subtracted from each well and the mean percent treatment-induced cytotoxicity for each cell line and treatment type (single and combination) was calculated as:Where:expt = mean absorbance from the treated cells: ABSlow = mean absorbance from controls (untreated cells)(spontaneous release of LDH)ABShi = mean absorbance from Triton X-100 treated cells (standard/maximum LDH release)(positive control).ABSTreatment-induced apoptosis was assessed by two independent assays, Annexin V-FITC assay and the DNA Fragmentation (TUNEL) assay. PC3 cells were treated and co-cultured with the test agents as previously described in this study; and the subjected to the apoptosis determination assays as below:Apoptosis-associated translocation of phosphatidylserine from the inner to the outer leaflet of the plasma membrane in GC27 and K833 cells was assessed with the use of FITC-labeled Annexin V, a calcium-dependent phospholipid-binding protein with a high affinity for phosphatidylserine; using AnnexinV-FITC Staining Kit (Boehringer Mannheim). Briefly, 100 μl aliquots of the previously prepared cell suspensions were centrifuged, and the cell pellets re-suspended in Annexin binding buffer, incubated with AnnexinV-FITC substrate; then cells were smeared onto microscope slides and either evaluated immediately with fluorescence microscope, or smears were fixed with 4% depolymerized paraformaldehyde and stored at -40°C for later examination as previously described . PercentR kit (Boehringer Mannheim Co, Indianapolis, IN) as previously described [in situ by specific end labeling and detection of DNA fragments produced by the apoptotic process. To perform the TUNEL assay, slides of the PBS suspended cells were fixed with 4% paraformaldehyde for 30 minutes. The cells (slides) were then permeabilized with Triton X-100 at 4°C for 2 min; then flooded with TdT enzyme and digoxigenin-dUTP reaction buffer (TUNEL) reagent for 60 min in a humidity chamber at 37°C, washed with distilled water, incubated for 10 minutes with streptavidin-horseradish peroxidase complex. The stained mounted cells were examined at 100×, 200× and 400× magnification of the microscope (Olympus BH-2). Cell death was quantified by counting 150 cells in 5–7 separate fields of view per slide, and noting the percentage of apoptotic cells based on morphological appearance, as previously described [The presence of apoptosis was determined by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL), using the ApopTagescribed . The kitescribed .The potential involvement of caspase-3 protease (CPP32) and/or the enzyme NQO1 [NAD(P)H:quinone oxidoreductase] in the molecular pathways of β-lapachone-and/or genistein-induced growth inhibition and apoptosis in PC3 cells were determined, after treatment of the cells as already described.In order to determine the potential role of caspase-3 proteases (CPP32) in the common pathways of β-lapachone and genistein-induced growth inhibition and apoptosis, human prostate cancer cell lines were treated as previously described above. The activity of caspase 3 was determined using a the fluorometric substrate DEVD-afc and caspase 3 inhibitor DEVD-fmk according to the protocol of the Caspase Activity Assay kit.Briefly, PC3 cells were treated and incubated as previously described. At 24, 48 and 72 hr cells were scrapped into suspension and centrifuged at 10,000 rpm for 10 min. The pellet was resuspended in 100 μl of lysis buffer and incubated at 4°C for 10 min, followed by centrifugation at 10,000 rpm for 10 min. Fifty μl aliquots of the supernatants were removed and placed in a 96-well microtiter plate (MTP) containing reaction buffer. The DEVD-afc substrate was added and the MTP was incubated at 37°C for 30 min. Activity was monitored with the linear cleavage and release of the afc side chain; and compared with a linear standard curve generated by the controls on the same MTP.In order to determine the potential role of enzyme NQO1 [NAD(P)H:quinone oxidoreductase] in the molecular pathways of β-lapachone-and genistein-induced growth inhibition and apoptosis in human prostate cancer, PC3 cell lines were treated as previously described. Dicoumarol (3-3'-methylene-bis (4-hydroxycou-marin) is a commonly used inhibitor of NQO1, which competes with NADH or NADPH for binding to the oxidized form of NQO1. Dicoumarol thereby prevents reduction and activation of various target quinines like β-lapachone. The cells, cultured as previously described, were treated concomitantly in single and combination treatments of varying concentrations β-lapachone (bLap), genistein (gen), and bLap-Gn combination with and without 50 μM dicoumarol as previously described. The treated cells were harvested and tested for treatment-induced apoptosis by the methods previously described in this study.JKD contributed 50% in all aspects of the research. All other authors contributed equally-50%All authors read and approved the final manuscript.


The purpose of the present study was to further elucidate the anti-viral spectrum of PAP by examining its effects on the survival of mice challenged with lymphocytic choriomeningitis virus (LCMV).Others and we have previously described the potent We examined the therapeutic effect of PAP in CBA mice inoculated with intracerebral injections of the WE54 strain of LCMV at a 1000 PFU dose level that is lethal to 100% of mice within 7–9 days. Mice were treated either with vehicle or PAP administered intraperitoneally 24 hours prior to, 1 hour prior to and 24 hours, 48 hours 72 hours and 96 hours after virus inoculation.in vivo anti- LCMV activity in mice challenged intracerebrally with an otherwise invariably fatal dose of LCMV. At non-toxic dose levels, PAP significantly prolonged survival in the absence of the majority of disease-associated symptoms. The median survival time of PAP-treated mice was >21 days as opposed to 7 days median survival for the control (p = 0.0069).PAP exhibits significant Our results presented herein provide unprecedented experimental evidence that PAP exhibits antiviral activity in the CNS of LCMV-infected mice. The broad-spectrum anti-viral agent PAP   is a welThe purpose of the present study was to further elucidate the anti-viral spectrum of PAP by examining its effects on survival of mice challenged with intracerebral inoculations of lymphocytic choriomeningitis virus (LCMV). LCMV is a rodent-borne arena virus that can result in persistent neuronal infection on mice ,44. AlphIn the present study, we describe the significant efficacy of the highly stable, potent and broad-spectrum anti-viral agent PAP in a murine model of LCMV. Our results presented herein provide unprecedented experimental evidence that PAP exhibits antiviral activity in the CNS of LCMV-infected mice.P. americanca, in four steps [IIIB with an IC50 p24 of 14 ± 2 nM.PAP was purified from spring leaves of the pokeweed plant, ur steps ,38. Brie50 dose. Each group of animals was placed in a separate cage.Animal infection was performed in an appropriate Animal BioSafety Level-3 Laboratory (ABL-3) at BRIEM (Research Institute for Epidemiology and Microbiology, MINSK, Belarus) with the technician wearing appropriate facility clothing. The culture was thawed in a water bath at 37°C and then diluted in normal saline to achieve the required concentration. In this study, all mice were challenged with 1000 PFU which is 100-times higher than the LDth and 7th days by presenting: weight loss, immobility, disheveled hair, convulsions, severe decubitus paralysis and death. All subjective measurement of decreased mobility and scruffy fur were done in a blinded fashion as not to influence the results. The protective properties of the experimental anti-viral drugs were assessed by using the following treatment-preventative regimen: Mice were treated either with vehicle (n = 20) or PAP (n = 10) (0.25 mg/kg) administered intraperitoneally 24 hours prior to, 1 hour prior to, and 24 hours, 48 hours, 72 hours, and 96 hours after virus inoculation. Mice were then observed for 21-days post infection. The protective effect of the experimental anti-viral drugs was evaluated according to the rise of the survival rate and prolongation of mean life of the experimental animals as compared with the control animals.Three week old CBA mice were intracerebrally infected with 1000 PFU of WE54 strain of LCMV that resulted in lethality of 100% of control (non-treated) animals in 6–8 days after infection. Control animals were given physiologic salt solution as a placebo instead of the compound. In general, for non-treated animals, clinical signs of the disease manifested on the 5Statistical significance was determined using the Kaplan Meier Log-Rank test.In order to evaluate the anti-LCMV activity of PAP, mice were inoculated with intracerebral injections of LCMV at a dose level that is lethal to 100% of mice within 6–8 days. Mice were treated either with vehicle or PAP as described in the methods section. Of the 20 control mice, 3 died on day 1 immediately after intracerebral injection due to accidental brain injury and are excluded from the data analysis. All 17 of the remaining control mice developed clinical signs of LCMV infection between day 4 and 6, including weight loss, disheveled hair, decreased mobility and paralysis  , 2 died on day 1 immediately after intracerebral injection due to accidental brain injury and are invaluable. Five of the remaining 8 mice (62.5%) treated with PAP remained alive > 21 days post-LCMV inoculation In the present study, we describe the significant efficacy of the highly stable, potent and broad-spectrum anti-viral agent PAP in a murine model of LCMV. Our results presented herein provide unprecedented experimental evidence that PAP exhibits antiviral activity in the CNS of LCMV-infected mice. Future studies will examine the therapeutic activity of PAP against LCMV in post-challenge settings. As PAP is well described as an HIV agent, this observation is also relevant as HIV-1 infects the central nervous system (CNS) and it has been feared that the CNS may be a sanctuary site where HIV-1 could hide and continue to replicate despite otherwise effective antiretroviral treatment ,49. AntiCases of LCMV infections have been reported in Europe, the Americas, Australia, and Japan. According to the Center for Disease Control, currently there is no specific drug therapy for LCMV. Although the anti-viral agent ribavirin is effective against LCMV in vitro, there is no established evidence to support its use for treatment of LCMV in humans .The anti-viral activity of PAP has been described against numerous pathogenic viruses, which included poliovirus, HIV-1, herpes simplex virus, cytomegalovirus, influenza virus and now, the negative-strand RNA virus Lymphocytic choriomeningitis virus. The ability of PAP to inhibit viral protein synthesis and depurinate viral RNA and DNA ,16 as weTreatment of CBA mice with the broad-spectrum anti-viral agent, PAP significantly improved the probability of survival following the LCMV challenge and decreased overall LCMV-related symptoms. The ability of PAP to exhibit anti-viral activity within the central nervous system is also encouraging within the framework of potential HIV-treatment. We have recently described the rational design and engineering of several recombinant PAP mutants with superior anti-HIV activity ,17. In oLCMV: Lymphocytic choriomeningitis virusPAP: Pokeweed anti-viral proteinHIV and FIV: Human and Feline immunodeficiency virus, respectivelyFMU is listed as an inventor on a number of PAP patents which are owned by PHI. FMU, AV and HET are salaried employees at PHI. PHI is a non-profit public charity.FMU designed the research project. LR, AP & LT conducted the Lassa experiments. FMU & AV provided PAP and coordinated the Lassa experiments with LR, AP and LT. FMU and HT wrote the manuscript. All authors read and approved manuscript.The pre-publication history for this paper can be accessed here:


TGFBR1*6A, a common hypomorphic variant of the type I Transforming Growth Factor Beta receptor, is emerging as a tumor susceptibility allele that predisposes to the development of breast, colon and ovarian cancer. The association with prostate cancer has not yet been explored. A total of 907 cases and controls from New York City were genotyped to test the hypothesis that TGFBR1*6A may contribute to the development of prostate cancer. TGFBR1*6A allelic frequency among cases (0.086) was slightly higher than among controls (0.080) but the differences in TGFBR1*6A genotype distribution between cases and controls did not reach statistical significance (p = 0.67). Our data suggest that TGFBR1*6A does not contribute to the development of prostate cancer.Prostate cancer is the most commonly diagnosed cancer in men and one of the leading causes of cancer deaths. There is strong genetic evidence indicating that a large proportion of prostate cancers are caused by heritable factors but the search for prostate cancer susceptibility genes has thus far remained elusive.  TGFBR1*6A is a common variant of the type I TGF-ß receptor, TGFBR1 [TGFBR1*6A (*6A) has a deletion of three GCG triplets coding for alanine within a nine alanine (9A) repeat sequence of TGFBR1 (*9A) exon 1, resulting in a six alanine (6A) repeat sequence. The 9-bp deletion that differentiates *6A from *9A is located within the predicted signal sequence cleavage region. In vitro studies have demonstrated that TGFBR1*6A responds less effectively than TGFBR1 to TGF-ß growth inhibitory signals [TGFBR1*6A heterozygotes and homozygotes among patients with a diagnosis of cancer as compared with the general population led us to postulate that TGFBR1*6A might act as a tumor susceptibility allele [TGFBR1*6A carriers may have an increased risk of breast, colon and ovarian cancer [TGFBR1*6A may contribute to the development of prostate cancer, we conducted a case control study of patients with biopsy verified prostate cancer cases and geographically and ethnic-status matched controls.Transforming Growth Factor Beta (TGF-ß) is one of the most potent inhibitor of cell growth . Almost , TGFBR1 . TGFBR1* signals ,5. The an cancer . To testTGFBR1*6A. The mean age of cases was significantly higher than controls (p < 0.01) but there were no differences in ethnic status between the two groups. There were 59 TGFBR1*6A heterozygotes and three TGFBR1*6A homozygotes among cases, 62 TGFBR1*6A heterozygotes and 1 TGFBR1*6A homozygote among controls. TGFBR1*6A allelic frequency among cases (0.086) was slightly higher than among controls (0.080) but the differences in TGFBR1*6A genotype distribution between cases and controls did not reach statistical significance (p = 0.67)  but was not significant after adjustment for race and age strata within groups (OR 2.11, 95% CI 0.98–4.57) (Table TGFBR1*6A was significant (p = 0.01).A total of 907 cases and controls were genotyped for BRCA2 gene have an increased prostate cancer risk [BRCA2 mutations in the general population, it is unlikely to account for a significant proportion of prostate cancer cases. Approximately 14% of the general population carries at least one copy of TGFBR1*6A, which makes it the most common candidate tumor susceptibility allele reported to date. While there is growing evidence that TGFBR1*6A predisposes to the development of breast, colon and ovarian cancer, our data do not suggest that it predisposes to the development of prostate cancer. We have previously shown that TGFBR1*6A homozygotes have an O.R of 2.69 and 2.02 for ovarian and colon cancer, respectively. The present study has the power to detect an O.R. for prostate cancer of 1.70 or higher and therefore rules out a major association between TGFBR1*6A and prostate cancer. However, it does not exclude a smaller O.R., which might have clinical relevance given the high TGFBR1*6A allelic frequency in the general population. It is possible that age differences in cases and controls affected the allele frequencies observed. If the TGFBR1*6A allele predisposes to a lethal malignancy such as prostate cancer, however, its frequency could be higher, not lower, in a younger cohort. Thus, the younger mean age of controls could result in a bias toward the null hypothesis, resulting in a stronger association than that observed. The intriguing findings of a high TGFBR1*6A allelic frequency among prostate cancer cases diagnosed before the age of 55 have to be cautiously interpreted given the fact that this group only included 46 patients. We have previously shown that TGFBR1*6A is not associated with an increased risk of bladder cancer [TGFBR1*6A and prostate cancer is at best very weak but further studies are needed to formally exclude an association with early onset prostate cancer.Prostate cancer is the most common cancer and the second most common cause of cancer death among U.S. men . A similcer risk . HoweverDNA was extracted from lymphocytes of blood specimens from 465 consecutive individuals diagnosed with adenocarcinoma of the prostate who received care at the outpatient urology clinic at Memorial Sloan-Kettering Cancer Center from April 2000 to September 2002. The blood samples were collected following completion of diagnostic studies. They were unselected for age or family history. Clinical and pathological records were reviewed to confirm the diagnosis of prostate cancer in all subjects. Once pathological diagnosis of prostate cancer was confirmed, the age of diagnosis was recorded, and all other identifying links were destroyed. The study design and anonymization method were approved by the Memorial Sloan-Kettering Cancer Center Institutional Review Board.A population of 465 healthy male controls aged 20 to 87 years with well-defined ethnic background who had donated blood for various reasons  constituted the control group. Controls were matched to the cases on ethnicity and were from the same geographic locations as the prostate cancer cases. None of the controls had any personal history of cancer at the time of blood donation. This was ascertained by a questionnaire completed by each control. Exact age information was not available for 205 controls since it was not collected prospectively but the age range (20 to 40) was known. All personal identifiers were permanently removed from both cases and controls.® GC rich kit . The PCR reaction mixture included 20 ng of genomic DNA in a 10-µL reaction volume and the following concentration of other reagents: primers (0.25 µM each), 1X GC genomic PCR reaction buffer, 1.625 mM Mg2+, 0.2 mM dNTPs and 0.16 µL of Advantage-GC genomic polymerase mix. Polymerase chain reaction cycling conditions consisted of an initial denaturation period of 3 minutes at 94°C, then 35 cycles of denaturation for 30 seconds at 94°C and annealing/extension for 2 minutes at 72°C, followed by a final extension step of 5 minutes at 72°C. Quality controls were run on a 2% agarose gel. The ABI Prism 310 Genetic Analyzer (Applied Biosystems, Foster City, CA) was used for data acquisition. A peak at 115 base pairs corresponded to TGFBR1 allele, whereas a peak at 107 base pairs corresponded to the TGFBR1*6A variant. The rare equivocal results were confirmed by cloning of the PCR product followed by automated sequencing. Samples were read by two independent investigators unaware of the case /control status. Ten percent of samples were randomly selected and run for quality assurance. Concordance rate was 100%.DNA was extracted by standard technique using the Qiagen DNA extraction kit. The PCR primers used were 5'-CCA CAG GCG GTG GCG GCG CGA TG-3' in the forward direction and 5'-CGT CGC CCC CGG GAF CAG CGC CGC-3' in the reverse direction. A standard solution was prepared using the Clontech AdvantageTGFBR1 genotypes, age, and ethnicity were compared between cases and controls using Fisher's exact tests. To test the hypothesis that the hypomorphic TGFBR1*6A gene is related to an increased prostate cancer risk, adjusted odds ratios of prostate cancer were estimated using both conditional and unconditional logistic regression models. Both models were run since the matched controls of cases with missing genotypes had to be excluded in the conditional models but could be included in the unconditional models. Adjusted odds ratios of prostate cancer were estimated comparing carriers of TGFBR1*6A versus non-carriers under dominant models. Potential confounders such as age (in four strata) and ethnicity were controlled in the analysis. Whether the effects of TGFBR1*6A on prostate cancer differ by age was evaluated by stratified analysis and tests for multiplicative interaction. A small p value indicates that interaction of age and gene is statistically significant on the multiplicative level. For the unconditional models, sensitivity analysis was conducted to evaluate the impact of the fact that the exact age of some controls with age 20–40 years is unknown (N = 126). With 442 cases and 465 controls, the power to detect an OR of 1.7 and 2 in the present study was 0.86 and 0.98, respectively, based on a two-tailed test at the 0.05 significance level.Distributions of All authors made substantial contributions to this paper, including conceiving of the ideas, discussion and writing. All authors read and approved the final manuscript.


The finite element method (FEM) is a powerful mathematical tool to simulate and visualize the mechanical deformation of tissues and organs during medical examinations or interventions. It is yet a challenge to build up an FEM mesh directly from a volumetric image partially because the regions (or structures) of interest (ROIs) may be irregular and fuzzy.A software package, ImageParser, is developed to generate an FEM mesh from 3-D tomographic medical images. This software uses a semi-automatic method to detect ROIs from the context of image including neighboring tissues and organs, completes segmentation of different tissues, and meshes the organ into elements.The ImageParser is shown to build up an FEM model for simulating the mechanical responses of the breast based on 3-D CT images. The breast is compressed by two plate paddles under an overall displacement as large as 20% of the initial distance between the paddles. The strain and tangential Young's modulus distributions are specified for the biomechanical analysis of breast tissues.The ImageParser can successfully exact the geometry of ROIs from a complex medical image and generate the FEM mesh with customer-defined segmentation information. Diagnostic imaging devices such as CT, MRI and PET scanners are able to produce three-dimensional 3-D) descriptions of various features such as tissues and organs. In a computer, these images are some data to describe the intensity at each spatial point of a volume. The interpretation of the dataset requires special training and depends on the experience. Researchers have introduced a variety of algorithms to visualize 3-D medical images, and to extract the geometric information of objects from volumetric image data -3.-D descriIn recent years, the finite element method (FEM) has widely been used to simulate the mechanical deformation of tissues and organs during examinations or interventions -6. To buThe purpose of this work is to establish an FEM model to simulate the deformation of a woman breast based on mammography compression. A patient's breast may include three kinds of tissues: fatty, parenchyma, and cancerous tissues . During In this paper, a software package called the ImageParser is developed to generate an FEM model from 3-D medical images. While aiming at the imaging segmentation, mesh generation, and deformation simulation of heterogeneous breast tissues, the method is applicable to the many biomedical imaging and biomechanical analysis of soft/hard tissues such as mammography and cardiovascular imaging. This software uses a semi-automatic method to detect the objective constituents from the context of an image including neighboring tissues and organs. It segments an image based on customer-defined grayscale ranges, and meshes tissues into elements with a customer-defined size. Inputting the generated FEM mesh into an FEM program, we can calculate the mechanical deformation under specific boundary conditions and mechanical loadings. The ImageParser is written in Microsoft Visual C# .NET (Microsoft Development Environment 2003 Version 7.1), and can be integrated into a high-level image analysis environment with a good extendibility and scalability.The ImageParser provides a window style GUI as shown in Figure This software provides a semi-automatic interface to detect the borderline of the breast as shown in Figure Figure While we are able to directly output the segmentation information based on the voxels, it cannot be effectively used by any FEM software since the whole breast includes more than ten million voxels. We therefore mesh the breast into larger elements based on the specific requirements of precision and computation capability. Figure After the mesh of the breast is generated, we can implement it into an FEM software package to simulate the mechanical deformation of the breast with given material constants of all the tissues and appropriate boundary conditions.A medical image typically includes many kinds of organs and tissues. However, biomedical engineers may only be interested in a small number of regions in a complex medical image. While certain algorithms have been developed to automatically detect the surface of the 3-D image -3,11, thx0, y0) is selected close to the borderline, a function is then used to search the most possible border point in the square region with the left-top point (x0 - s, y0 - s) and the right-bottom point (x0 + s, y0 + s). Here s is a customer-defined parameter with a default value as 3 pixels. In this region, the gradient of each point Δ(x, y) is defined by a Laplace operator [1. We first focus on one slice. When the first point (operator so thatf(x, y) denotes the intensity at (x, y). The detected point is the one with the different color from the background and with the maximum value of Δ(x, y)/[(x - x0)2 + (y - y0)2 + ε] where ε is a customer-defined parameter with a default value as 0.1 to prevent the singularity on the point (x0, y0). The detected point is denoted as (x1, y1). It is noted that the Laplacian normalized by the distance of the point to the selected seed point is to make the neighboring points have a higher priority to be detected. At certain regions, the borderline may not be clear or two borderlines are close enough, in which cases the program will not get lost.where x1, y1), the software can adjust the location of the point and detect the second point (x2, y2) on the borderline. As seen in Figure x1, y1) and (x2, y2). Two squares with edge length 2s are marked by the dark color in a big square having a diagonal line from (x1, y1) to (x2, y2). We find the most possible border point in the two dark squares by using the same method in the first step. If this new border point is in the right-top square, we replace (x2, y2) by this point. Otherwise, we replace (x1, y1) by the new border point. Once (x1, y1) or (x2, y2) is updated, we continue to find the next border point in the same way. Repeat this procedure until the distance between the two points is less than 2s. Connecting all points in such an orderly way, we obtain the borderline. It is noted that this method is convergent because the distance between two working points becomes smaller and smaller in this procedure.2. We start to select the next point of the borderline. After we manually select one point visually close to the borderline following the method of detecting  plane  in the z direction.ANSYS 7.0  is the cTo simulate the nonlinearly elastic deformation, we divide this compression process into 20 incremental steps. At each step the displacement, strain, and stress fields can be calculated. Figure σε and εe are the von Mises stress and strain, respectively. It is noted that, for linear elastic material, E is always a material constant. Because the material properties of skin, normal entity and tumor are all nonlinear, E should change during the process of compression. Figure 8(a) shows the von Mises strain for the sections at z = 69,78,87 mm. The strain around the thorax is quite significant due to the boundary constraint, whereas the strain close to skin is small because of the free boundary condition. In the region of tumor, it is shown that the strain is much smaller than that in the neighboring region because the tumor is much harder than other tissues. Figure 8(b) demonstrates that the tangential Young's modulus is no longer uniform even in the same tissue because its strain field is not uniform.where The ImageParser system has been developed to create FEM mesh models from 3-D medical images. A semi-automatic method has been proposed to detect the ROIs from the context of complex image structures. The ROIs can be meshed into cuboidal elements and segmented based on the grayscale of the voxels. It has been demonstrated that, through a 3-D CT image volume of the woman breast, the ImageParser can effectively mesh the breast into cuboidal elements, and simulate the realistic nonlinear deformation responses of the breast tissues upon compression.LZS, GW, and MWV conceived and planned this research project. HMY and LZS designed and developed this software. TY prepared the CT image volume of the breast. TY, JW, and GW analyzed the CT images. HMY, LZS, and GW wrote the manuscript.


Ten potential reference genes were compared for their use in experiments investigating cellular mRNA expression of virus infected cells. Human cell lines were infected with Cytomegalovirus, Human Herpesvirus-6, Camelpox virus, SARS coronavirus or Yellow fever virus. The expression levels of these genes and the viral replication were determined by real-time PCR. Genes were ranked by the BestKeeper tool, the GeNorm tool and by criteria we reported previously. Ranking lists of the genes tested were tool dependent. However, over all, β-actin is an unsuitable as reference gene, whereas TATA-Box binding protein and peptidyl-prolyl-isomerase A are stable reference genes for expression studies in virus infected cells. Quantitative real-time PCR (QPCR) has become the favoured tool in mRNA expression analysis and also in virus diagnostics . Real-tiAn efficient infection could be evidenced by a significant increase of viral RNA or DNA for all 5 viruses over time table . DespiteT values should be expected for useful reference genes and a high SD for genes that are susceptible to virus replication. Corresponding to the recent estimation the SD of the CT value was highest for Act in 4 of 5 viruses, indicating that Act is no reliable reference gene in this setting. In contrast, TBP and PPI displayed the highest expressional stability for 4 of 5 viruses. To find a general conclusion, the total of all SD values from all virus experiments (sumV) was calculated for each reference gene. As shown in table v = 2.29 for both), followed by GAP (sumv = 3.49) and β2M (sumv = 3.96). All other genes showed moderate total SD values (sumv > 4.58), except Act (sumv = 11.28), confirming to be the most inappropriate reference gene. It is remarkable that the obtained BestKeeper index values are low, despite the inclusion of Act in the calculation. Calculating BestKeeper vs. each reference gene using Pearson correlation displayed very inconsistent results (table HRG), it seems that CAMP infection caused the highest variations in reference gene expression.The experimentally obtained data for each virus and each gene were analysed using three different methods. The reference gene evaluation of the BestKeeper tool is shown in table P sumv = .49 and βV, representing the SD of a reference gene over all viruses, was lowest for PPI (sumV = 6.08) confirming the results obtained by the Bestkeeper tool. However, β2M (sumV = 6.11), GAP (sumV = 6.19) and TBP (sumV = 6.29) turned out to be comparably reliable as reference genes. Second, also the GeNorm tool showed that Act is by far the worst reference gene (sumV = 14.20).Analysing the expression data with the GeNorm tool showed slightly deviant results table . First, T values . The two genes with the lowest ΔΔCT value were TBP (sumV = 9.82) and PPI (sumV = 10.04), corresponding to the results of the Bestkeeper and the GeNorm tool.Applying the calculation mode presented previously , that ises table , Act wasT method, and their results were compared.To date, it is generally accepted, that the selection of the ideal reference gene in gene expression analysis has to be done for each individual experimental setting by evaluating several genes and using the best two or three of these genes as reference. Obviously there is no "one good gene for all experiments" recommendation. However, it is helpful to find putative candidates that can be shortlisted when setting up a new experimental design. Therefore, we determined the expression of previously tested reference genes in a setting of virus infected human cell lines. Capable reference genes were evaluated using three independent methods: Bestkeeper, GeNorm and the ΔΔCAll three tools ranked actin at the last position, indicating that it is an unsuitable reference gene in virus infected cells. The actin gene shows significant variations with increasing degree of infection.The best genes obtained from all three calculation tools were TBP and PPI. TBP seems to be a relative stable expressed gene during the course of virus replication of different viruses in different cells. However, as previously shown  TBP is nInterestingly, classical reference genes like β2M and GAP were also acceptable regarding to a stable expression in virus infected cells. All other genes showed moderate expression stability.Pearson correlation. As described by Pfaffl et al. its use is limited to groups without heterogeneous variances, but the tested reference genes have very different expression levels resulting in significant variances. Paffl et al. also described that new versions of Bestkeeper should circumvent these problems by use of Sperman and Kendall Tau correlation. However, one problem still remains to be solved; both tools, the BestKeeper and the GeNorm, can not compare paired probes. This is the great advantage of the ΔΔCT method, or any other method which directly compares paired samples. From this point of view the use of a method like the ΔΔCT should be applied first before considering additional tools for further elucidation of the acquired data.The analysis of our data set according to the Bestkeeper tool revealed very good BestKeeper indices; even actin was included into our gene panel. These findings demonstrate the usefulness of analysing a wide variety of reference gene candidates. The inconsistent data regarding to the Bestkeeper calculation of the coefficient of correlation and the corresponding p-values may be a result of the In summary, TBP and PPI turned out to be the best reference genes in virus infected cells. These genes are a good point to start reference gene selection in gene expression studies in virus infection experiments.Camelpox strain CP-19, CMV strain AD169, HHV-6 strain U1102, SARS coronavirus strain 6109 and YFV strain 17D were propagated according to standard procedures -10.The respective MOI and time of cell culture are shown in table 6 cells was prepared using the QIAamp RNA Blood Mini Kit and RNase-free DNase set (Qiagen, Hilden, Germany) according to the manufacturer's recommendations for cultured cells. RNA solution was treated with DNA-free (Ambion, Huntingdon, United Kingdom).Total RNA from 1 × 1020 primed cDNA-synthesis. cDNA synthesis was performed using 1 μg of RNA, at 50°C. Finally, cDNA was diluted 1:5 before use in QPCR.cDNA was produced using the Superscript III RT-PCR System (Invitrogen, Karlsruhe, Germany) according to the manufacturer's recommendations for oligo(dT)Primers, TaqMan probes and QPCR conditions for reference gene analysis were used as previously described . PCR wasT value was calculated as follows: First the ΔCT for each time point of probe assessment between virus and Mock infected cells was calculated. In a second step the maximal differences between the time points were calculated as ΔΔCT.Analysis was performed with the BestKeeper  and GeNoThe author(s) declare that they have no competing interests.AR conceived the study, carried out the HHV-6 experiments and real-time PCR assays and drafted the manuscript. ST carried out the CMV experiments. HB carried out the YF experiments. MM carried out the SARS experiments. WS participated in the design of the study. AN carried out the CAMP experiments, participated in design and coordination of the study and helped to draft the manuscript. All authors read and approved the final manuscript.


In response to the Blackburn and Rowley essay on the President's Council on Bioethics, several thought-provoking opinions on ethical challenges in biomedical research are expressed by prominent stakeholders PLoS Biology, Elizabeth In their essay in the April 2004 issue of Stem cell research and its applications have the potential to revolutionize human health care. Recent polls show support for embryonic stem cell research, even with conservative voters. The public, as the major benefactor of biomedical research and the target population of beneficial clinical advances, has the right to a fact-based discussion of the science regarding stem cells. It is therefore time that the debate on stem cell research, with its risks and benefits, be taken to the public. A debate on stem cell research restricted to the President's Council on Bioethics is a disservice to the public.Nearly three decades ago, the advent of recombinant DNA technology and in vitro fertilization (IVF) techniques, raised similar concerns regarding research. Contrary to apprehensive expectations, recombinant DNA technology has boosted enormous advances in the health care and pharmaceutical industry. IVF evolved to be a widely accepted, safe medical procedure, with over one million healthy babies born by IVF and related treatments. Similarly, once stem cells are successfully used in the clinic, most of today's political and ethical issues will evaporate.The International Society for Stem Cell Research (ISSCR), a society whose membership encompasses the bulk of the stem cell research brain trust, holds the position that research on both adult and embryonic stem cells will guarantee the fastest progress in scientific discovery and clinical advances. The ISSCR also strongly opposes reproductive cloning and supports the National Academy of Science's proposal to develop voluntary guidelines to encourage responsible practices in human embryonic stem cell research.One of the original recommendations of the President's Council on Bioethics was a four-year moratorium on stem cell research. The purpose of this moratorium was theoretically to open a large, national discourse on the topic of stem cell research, a debate intended to bring all sides into thoughtful reflection on the issue. To that end, the ISSCR has repeatedly and consistently offered an open forum for all sides in the debate at our conferences, and has carefully offered invitations to join our society and to speak at our annual meeting to members of the President's Council, including colleagues whose opposition to stem cell research has been clear. None have accepted. Dr. Kass, in particular, has received several direct appeals but has turned down every such opportunity to make his case to the researchers who arguably are his discourse partners, from whom he could learn much, and whom he should be actively engaged in teaching. It is tragic that voices of dissent and debate are stilled, for it is this very quality of open debate that is at the heart of both the scientific method and an ethically directed American democracy—surely a goal that we all share.


Nocardia brasiliensis is a rare human pathogen usually associated with localized cutaneous infections.Nocardia brasiliensis infection developed after a bone fracture of the left hand of an otherwise healthy 32-year-old man. Treatment with trimethoprim-sulfamethoxazole given for a total of three months combined with surgical debridement resulted in complete resolution of the infection.We report a case of primary lymphocutaneous Nocardiosis should be part of the differential diagnosis in patients with sporotrichoid infection, particularly those with a history of outdoor injury. Culture of the affected tissue and antimicrobial susceptibility testing of the isolate should be performed for diagnosis and treatment. Nocardia. This genus consists of strictly aerobic, Gram-positive, variably acid-fast, filamentous bacteria with a tendency to fragment into bacillary and coccoid forms [N. asteroides, N. farcinica, N. nova (included in the N. asteroides complex) and N. brasiliensis are the species most often involved in human disease [N. brasiliensis has been recovered from the soil in many tropical and subtropical areas but rarely in temperate areas. Traumatic inoculation of N. brasiliensis into the skin is the most typical mode of acquisition of the infection due to this organism [Nocardiosis is a rare localized or systemic infection caused by several species of the genus id forms . N. aste disease ,2. N. brorganism ,2.N. brasiliensis.Herein, we describe a case of a man, who had an accident at work and 1 week later developed lymphocutaneous infection caused by A previously healthy 32-year-old man was referred to the emergency department of orthopedics with traumatic injuries of the index, middle and ring fingers of the left hand. The injury happened at work while he was operating a machine of cotton elaboration.3), while red blood cell count, haemoglobin, chemistry and urine analysis were within normal limits. Radiography of the bones of his left hand revealed fractures of the nail bones of the middle and the ring finger. Surgical debridement of the damaged soft tissue was undertaken with amputation of these two nail bones. The patient was hospitalized and intravenous therapy with ceforanide (1 g/12 h), ofloxacin (200 mg/12 h) and metronidazole (500 mg/8 h) was initiated.On admission, routine laboratory investigations showed only elevated white blood cell count  without any other systemic symptoms.Physical examination revealed multiple erythematous subcutaneous nodules along the lymphatics extending up the patient's left forearm. These nodules were tender and painful. There was no regional lymphadenopathy. Debridement of the lesions was performed and the tissue was submitted for bacterial and fungal cultures. The Gram-stained smear showed polymorphonuclear leucocytes and Gram-positive fine, branching filaments, partially acid-fast, with a tendency to fragment into coccoid and bacillary forms.3), absolute neutrophil count, 12,780/mm3, erythrocyte sedimentation rate, 75 mm/h; and C-reactive protein, 14.2 mg/dl .Laboratory tests showed: white blood cell count (18,000/mmNocardia brasiliensis (Table After 5 days of incubation cultures of the pus and the tissue on Columbia blood agar grew white colonies adherent to the agar, rough with a velvety surface, having a characteristic mouldy odor Figure . Coloniais Table . SusceptN. brasiliensis accounts for only about 7–14% of the reported cases [N. brasiliensis lymphocutaneous syndrome has been previously described in the same geographic area [In the United States, there are an estimated 500–1,000 new cases of nocardiosis each year . On the ed cases . The inchic area .N. brasiliensis although rarely implicated in pulmonary and disseminated infections in immunocompromised patients, has been most commonly associated with cutaneous infections [Nocardia enters the skin after traumatic inoculation injuries, varying from contaminated abrasions and puncture wounds to insect and animal bites. The most common resulting skin lesions are on the upper and lower extremities [fections . Nocardiremities . CutaneoNocardia and entered the wound after the accident.The present case is consistent with the classical presentation of lymphocutaneous infection with a primary lesion at the site of injury on the hand and an ascending lymphangitis involving the forearm. The inoculation probably occurred from the cotton that had been contaminated by Sporothrix schenkii and Nocardia brasiliensis, include Mycobacterium marinum and Leishmania species. Less common causes are Coccidioides immitis, Cryptococcus neoformans, Histoplasma capsulatum, Blastomyces dermatitidis, Pseudoallescheria boydii, other species of Mycobacterium, Streptococcus pyogenes, Staphylococcus aureus and viruses as cowpox virus and herpes simplex [The lymphocutaneous syndrome can be caused by a wide variety of microorganisms. The most common causative agents of the syndrome in addition to  simplex . A histo simplex ,9.N. brasiliensis after only 2 to 3 weeks of therapy. In patients with sulfa intolerance or those who fail therapy with trimethoprim-sulfamethoxazole, alternative therapy must be based on sensitivity testing. Minocycline, tetracycline, amikacin and amoxicillin-clavulanic acid have been successfully used [Trimethoprim-sulfamethoxazole combination is recognized the drug of choice for nocardiosis . Primarylly used .Although rare, lymphocutaneous nocardiosis must be considered, diagnosed with appropriate cultures and adequately treated, in order to prevent progression to dissemination of the primary skin disease.


The rates of Sexually transmitted diseases (STDs) in England have been rising steadily since the mid 1990s, making them a major public health concern. In 2003, 672,718 people were diagnosed with an STD in England, and around one third of those cases were diagnosed in London.. These maps are in Scalable Vector Graphics (SVG) format and require a freely available Adobe SVG browser plug-in to be displayed. They are based on data obtained from the House of Commons Hansard Written Answers for 15 October 2004. They show steadily rising rates of STDs in London over the covered seven-year period. Also, one can clearly see on the maps that PCTs located in central London had the highest numbers of STD diagnoses throughout the mapped seven years. A companion bar chart allows users to instantly compare the STD figure of a given PCT for a given year against the average figure for all 25 mapped PCTs for the same year, and also compare those figures across all seven years. The maps offer users a rich set of useful features and functions, including the ability to change the classification method in use, the number of ranges in the map, and the colour theme, among others.Using GeoReveal v1.1 for Windows, we produced Web-based interactive choropleth maps of diagnoses of STDs by Primary Care Trust (PCT) in London for the years from 1997 to 2003 Wizard-driven tools like GeoReveal have made it very easy to transform complex raw data into valuable decision support information products (interactive Web maps) in very little time and without requiring much expertise. The resultant interactive maps have the potential of further supporting health planners and decision makers in their planning and management tasks by allowing them to graphically interrogate data, instantly spot trends, and make quick and effective visual comparisons of geographically differentiated phenomena between different geographical areas and over time.® Flash and bitmap graphics.SVG makes an ideal format for such maps. SVG is a World Wide Web Consortium non-proprietary, XML-based vector graphics format, and is an extremely powerful alternative to Macromedia Sexually transmitted diseases (STDs) have become a major public health concern in the UK during recent years. The rates of STDs in England have been rising steadily since the mid 1990s. In 2003, the number of STDs in England rose by 4% compared to 2002. Overall, 672,718 people were diagnosed with an STD in England in 2003, and around one third of those cases were diagnosed in the London area alone [The House of Commons Hansard Written Answers for 15 October 2004 included an answer by Melanie Johnson MP, Minister for Public Health at the Department of Health, to a question by Sarah Teather MP on "how many cases of diagnosed STDs there were in each Primary Care Trust (PCT) in London in each year since 1997". The answer was provided in the form of a long table showing the figures for 25 PCTs in London  clinic data sets, are crucial for the decision maker wanting, for example, to:- improve access to GUM clinics and make decisions regarding the expansion or closure of existing clinics, or the creation of new ones;- channel resources and target STD prevention programmes to areas with the most need, or scale such programmes according to the magnitude of the problem in different areas ; and/or- monitor the impact of such programmes in a given area over time.In this paper, we describe a much better way of presenting the same Hansard table data in the form of interactive Web maps in Scalable Vector Graphics (SVG) format to further support health planners and decision makers in their planning and management tasks. – see 'Methods' section below), we produced Web-based interactive choropleth maps of diagnoses of STDs by PCT in London for the years from 1997 to 2003, which readers can browse at   for all 25 mapped PCTs . Using the 'Select Map Topic' list, users can select the topic that is shown on the main map (a year from 1997 to 2003). Finally, users can select from the 'Colour of Theme' list the colour theme that is used in the main map (orange, green, or blue).Map zooming, panning, MapTips (displaying PCT names), and a dynamic legend are available. An overview map shows a miniature version of the full extent map. When the user zooms in, a rectangle on the overview map highlights the area that is currently being displayed in the main map window. The user can click and drag this rectangle to change the view in the main map window. After zooming into the main map, users can use the 'Reset' button to return to the full extent of the main map.An 'Info' button is also available. Clicking this button will open a pop-up window with extra information about the maps, links to the Web sites of London PCTs, and detailed help about the map interface Figure .Turning raw tabular data into much more useful and accessible visual information in the form of interactive Web maps is much needed to support and empower decision makers, and even members of the general public. Such maps help us understand the relationships, patterns and trends buried in the original data sets and also enable instant visual comparisons to be made between different geographical areas and over time (when data sets for successive periods of time are available) . to see the difference for themselves and appreciate the value of interactive maps.We believe this transformation of raw data into valuable decision support information is very evident in the London STD example described in this paper. Readers only have to compare the original Hansard table Table  with the). It is rapidly becoming a popular choice for delivering interactive Web maps, being designed to work effectively across platforms, output resolutions, colour spaces, and a range of available bandwidths. It offers a rich modern graphics format providing the ability for better map display, and advanced graphical features such as transparency, arbitrary geometry, filter effects (shadows, lighting effects, etc.), scripting, and animation [SVG is a non-proprietary language for describing rich, stylable two-dimensional graphics and graphical applications in XML (eXtensible Markup Language). SVG is fully endorsed by the W3C (World Wide Web Consortium – nimation .® Flash format [® Shockwave/Flash File) formats, will keep their sharp character when enlarged, while raster-based images (storing information about each and every pixel in the image), such as those saved in GIF (Graphics Interchange Format) or JPEG (Joint Photographic Experts Group) formats, will show jagged edges.All these features have made SVG a direct competitor to the proprietary Macromediah format . Vector-), in the same way the free Adobe Reader software is available for rendering PDF (Portable Document Format) files.A free SVG Web browser plug-in is available from Adobe for different platforms , other SVG/Flash mapping tools available today for publishing maps created in desktop GIS (Geographic Information Systems) include GéoClip , SVGMapMaker , MapViewSVG , and SVGMapper . The latter two tools (MapViewSVG and SVGMapper) are specific to ESRI ArcView GIS. Table Besides GeoReveal . These maps are in SVG format and require a freely available Adobe SVG browser plug-in to be displayed. They are based on data obtained from the House of Commons Hansard Written Answers for 15 October 2004. They show steadily rising rates of STDs in London over the covered seven-year period. Also, one can clearly see on the maps that PCTs located in central London had the highest numbers of STD diagnoses throughout the mapped seven years. A companion bar chart allows users to instantly compare the STD figure of a given PCT for a given year against the average figure for all 25 mapped PCTs for the same year, and also compare those figures across all seven years. The maps offer users a rich set of useful features and functions, including the ability to change the classification method in use, the number of ranges in the map, and the colour theme, among others.Using GeoReveal v1.1 for Windows, we produced Web-based interactive choropleth maps of diagnoses of STDs by PCT in London for the years from 1997 to 2003 We also presented a quick review of some of the tools available today for creating interactive vector graphics maps from desktop GIS projects.Wizard-driven tools like GeoReveal have made it very easy to transform complex raw data into valuable decision support information products (interactive Web maps) in very little time and without requiring much expertise. The resultant interactive maps have the potential of further supporting health planners and decision makers in their planning and management tasks by allowing them to graphically interrogate data, instantly spot trends, and make quick and effective visual comparisons of geographically differentiated phenomena between different geographical areas and over time.® Flash and bitmap graphics.SVG makes an ideal format for such maps. SVG is a W3C non-proprietary, XML-based vector graphics format, and is an extremely powerful alternative to Macromedia® .NET framework v1.1 to be installed on the production machine.We used GeoReveal v1.1 for Windows to create the interactive SVG maps described in this paper. GeoReveal runs under Windows 98/NT/2000/XP and requires Microsoft, and is freely available in both ArcView and MapInfo formats to the UK academic community from EDINA UKBORDERS service with the support of the ESRC and JISC . We also prepared a spreadsheet containing data about the number of STDs recorded in each London PCT between 1997 and 2003 using data from [, creating a MapInfo .TAB file. This file was exported to MID/MIF format, the format that files need to be in, in order to be used by GeoReveal.We started by extracting London PCT boundaries from a larger data set of all England (2001 Census PCT – post April 2002 change), which is the copyright of the Crown/Ordnance Survey We created our presentation (the interactive SVG Web maps) using the GeoReveal Wizard; an eight-step process that allows users to create a fully interactive SVG page.The first Wizard dialog that must be completed is the 'Introduction' dialog, which is used to specify the directory to which the final GeoReveal output files will be saved Figure .To select the output directory, we clicked the 'Browse' button and in the resulting file browse dialog, we selected the required directory and clicked 'OK'. To move to the next step, we clicked 'Next'.The first Wizard step is the 'Choose Template' dialog Figure . This diWe selected the template to be used. Ten templates are provided; four that include a bar chart, four that include a pie chart and two that have no chart. With our London PCT STD data, the most appropriate is a bar chart template.We selected a logo image to be added to the top-left corner of our GeoReveal page. We clicked the 'Browse' button and selected the required file – in this instance, a University of Bath logo has been used..We then specified the title of the GeoReveal page. This title is placed next to the logo at the top of the output page  is the piece of text that is displayed when the mouse cursor is hovered over a region in the map. In this instance, the PCT Name field has been selected.Users can also choose to turn the information box on or off. When it is turned on, a user can click a region in the map to display a dialog containing all information held within the MID/MIF file about that region Figure .Finally, we selected the background colour and highlight colour for the map. The highlight colour is the colour a map region  will be displayed in when the mouse cursor hovers over it. To move to the next step, we clicked 'Next'.The third step is the 'Select Overview Map Data' dialog Figure . This diWe selected the MID/MIF file to be used for the overview map by clicking the 'Browse' button and selecting the required file. It is possible to use the same MID/MIF file for both the main and overview maps.We selected the overview map colour and the overview rectangle colour (this rectangle shows a user where they are currently zoomed in on the main map). Additionally, users can select the background colour and border colour for the overview map panel. To move to the next step, we clicked 'Next'.The fourth step is the 'Select Options Controls' dialog Figure . This diFirst, we selected the background map image that is to be used. We ticked the top 'Checkbox Visible' box to activate the background map option and then clicked the top 'Browse' button to select the file that will be used. This file must be a JPEG or GIF image and information is needed about its width and height in real terms, and also the bounding coordinates.Once the required file is selected, the 'Background Image Settings' dialog is displayed Figure . In thisIn the 'Select Options Controls' dialog, it is also possible to:- enable a vector map option; and- select the text colour, border colour, background colour and control colour (the colour in which the fields are rendered) for the 'Options Panel'.To move to the next step, we clicked 'Next'.The fifth step is the 'Set Bar Chart' dialog Figure . This di- specify the bar colour, grid colour (if the grid is enabled), text colour, background colour and border colour;- add or remove a % sign to the figures that are shown at the end of each bar. As the data being used in this presentation are absolute, the 'Show %' box should be unchecked; and- specify the opacity of the bars in the bar chart.After specifying our settings for the bar chart, we clicked 'Next' to move to the next step.The sixth step is the 'Set Legend' dialog Figure . This di- specify a title and subtitle for the legend. Rather than specifying a main title, we checked the 'Use Dynamic Legend' box. When this box is checked, the title will be determined by the topic that the GeoReveal map is based on (a year from 1997 to 2003 in our case);- enable or disable the 'Highlight Range' option. If this option is enabled (as is the case in our presentation), when the mouse is hovered over a map region (PCT), the legend range that this region falls into will be highlighted; and- specify the text colour, background colour and border colour for the legend panel.After specifying our settings for the legend, we clicked 'Next' to move to the next step.The seventh step is the 'Set Information Panel' dialog Figure . This di- enable or disable each of the information panels and select the fields from the MID/MIF file that will be used to populate each panel. A title can also be entered for each panel (in our instance, Panel 1 was enabled, has been given the title 'PCT Name' and will display the PCT Name field from the MID/MIF file); and- specify the text colour, background colour and border colour for the 'Information Panel'.We then clicked 'Next' to move to the next step.The eighth step is the 'Set Navigation Panel' dialog Figure . This diWe then clicked 'Next' to move to the final step.Lastly, we saved the settings for our presentation and generated an initial presentation page Figure . The 'Wi- save the settings they have just made by clicking 'Save Settings', then in the resulting dialog, browsing to the required directory and clicking 'Save'. The saved settings file enables users to restore their settings and edit the presentation at a later date; and- generate the presentation by clicking 'Finish'. A message is displayed to confirm that the presentation has been successfully generated Figure . ClickinWhen the presentation is created, the 'Advanced View' window is also opened. This can be used to add the finishing touches to the presentation. It contains all the options that the wizard does, along with a few advanced options.The 'Legend' tab can be used to select the legend colour schemes that will be available Figure . In thisThe 'Bar Chart' tab can be used to edit the bar chart Figure . It is p- we ticked the 'Show Average Value Bars' box to add average value bars to the bar chart; and- set the average bar colour and specified the opacity of the bars.Finally, we used the 'Navigation Toolbar' tab to edit the 'Navigation Panel' Figure . In this- we enabled the help button by ticking the 'Display Help Button' box;- entered the text that will be shown on the button. In this instance, it will show 'Info'; and- selected the help file that will be used by clicking the 'Browse' button and selecting the required file. In this instance, an HTML document was created that contains hyperlinks to the PCT Web sites, and information about using the presentation , the company that produces GeoReveal.All three authors contributed equally to this paper.


The 6 minutes walk test (6MWT) is a useful assessment instrument for the exercise capacity of elderly persons. The impact of the health status on the 6MWT-distance in elderly, however, remains unclear, reducing its value in clinical settings. The objective of this study was to investigate to what extent the 6MWT-distance in community dwelling elderly is determined by health conditions.A  to D (signs of active disease at the moment of examination).One hundred and fifty-six community dwelling elderly people  were assessed for health status and performed the 6MWT. After clinical evaluation, electrocardiography and laboratory examination participants were categorized into a stratified six-level classification system according to their health status, going from The mean 6MWT-distance was 603 m (SD = 178). The 6MWT-distance decreased significantly with increasing age (ANOVA p = 0.0001) and with worsening health status (ANCOVA, corrected for age p < 0.001).A multiple linear regression model with health status, age and gender as independent variables explained 31% of the 6MWT-distance variability. Anthropometrical measures (stature, weight and BMI) did not significantly improve the prediction model. A significant relationship between 6MWT-distance and stature was only present in category A .Significant differences in 6MWT-distance are observed according to health status in community-dwelling elderly persons. The proposed health categorizing system for elderly people is able to distinguish persons with lower physical exercise capacity and can be useful when advising physical trainers for seniors. Aging results in an important decrease of muscle power and exercise capacity. TherefoThe 6MWT has first been introduced as a functional exercise test by Lipkin in 1986 . Its res2; ankle-arm blood pressure index <0.9; 1-second forced expiratory volume and absence of stroke history, use of diuretics and smoking). They report that gender-specific reference equations based upon age, height and weight explained 40% of the variance in 6MWT-distance, which was significantly less for men and women who were older and heavier, and for shorter men. Also, Rikli et al. [Several authors studied the determining factors of the 6MWT-distance in healthy adults and propose either reference equations or normative data for the 6MWT-outcome -16. Trooi et al. , to the i et al.  and to ti et al. ,19,20. SAll members of a large Belgian Health Insurance Organization (BHIO) who registered for a health-conditioning week for seniors, organized by the BHIO, were invited by advertisement to participate in our study. The program of the health-conditioning week included general instructions for a healthy life-style and physical exercise classes. One hundred and fifty-six subjects  volunteered to participate in the study and gave their informed consent. All participants were living in the community and belonged to the A-category according to Katz et al.  and thusAll participants underwent extensive health screening by medical doctors. First, blood & urine samples were collected after overnight fasting for determination of erythrocyte sedimentation rate, mean red blood cell corpuscular volume, leukocyte count (with differentiation), and concentration of haemoglobin, urea, alkaline phosphatase, glucose, Aspartate Aminotransferase (ASAT), Alanine Aminotransferase (ALAT), protein and electrophoresis for the blood samples and determination of protein, glucose and sediment for the urine samples (according to the SENIEUR protocol ). SecondThe classification system was originally developed in order to grade the participants according to the risk for dangerous complications during physical exercise and to allow physical therapists to adapt the scheduled program of the health-conditioning week . Therefore, cardiovascular abnormalities were considered to present a higher risk than non-cardiovascular conditions.A were completely healthy and were considered as presenting no particular risk for any kind of physical exercise. An additional distinction can be made between those using no medication (A1) and those using preventive medication only (A2). This subdivision might be important in specific clinical contexts (e.g. assessment of Vitamin D levels in elderly); in the context of our study the distinction between health categories A1 and A2 is less relevant and therefore these participants will be considered together in all statistical analysis. Category B consisted of participants who were functioning normally, presented no major medical restrictions, but could be in need of special instructions for exercising due to their health status. Category B1 was accorded to participants having a disease that was non-cardiovascular and stable. Category B2 was given to participants using medication having cardiovascular effects. Subjects in category C had cardiovascular pathology or a history thereof; they were considered as having an increased risk of cardiovascular complications during exercise. Those belonging to category D were found to present signs of acute disease or exacerbation of chronic disease. If combinations of health conditions existed, subjects were classified in the worst health category.We distinguished four categories of decreasing health escribed . All parStatistical analysis was performed using the SPSS (for Windows, release 11.5.1) software package. All data subsets were assessed for the presence of a normal distribution (Kolmogorov-Smirnov Goodness of Fit Test p > 0.05) before using parametric analysis. Correlations between data subsets with a normal distribution were performed using Pearson's Correlation Coefficient; non-normally distributed datasets were analyzed for correlation using Kendall's Correlation Coefficient. Differences between data subsets were analyzed using Analysis of Variance (ANOVA), Analysis of Co-Variance (ANCOVA) and Students t-test. Bonferroni post-hoc test was performed to detect significant differences between subgroups. A multiple linear regression model was designed in order to explain the variability of the 6MWT-distance. Significance level was set at p < 0.05 .The mean 6MWT-distance was 603 m (SD = 178) for the whole group (N = 156). Overall, male participants were significantly heavier (p < 0.001), taller (p < 0.001) and covered a significantly longer distance in six minutes p = 0.022) than female participants. There was no significant difference in age or BMI between male and female participants  participants (44 in group A1 and 14 in group A2) could be considered as completely healthy. No significant difference was found regarding age or 6MWT-distance between participants of group A1 and A2 (mean age respectively 62.0 years SD = 6.6 and 62.7 years SD = 10.0, mean 6MWT-distance respectively 696.4 m SD = 150.7 and 686.9 m SD = 118.4 for A1 and A2). ANOVA testing  revealed a significantly lower 6MWT-distance (p < 0.001) with worsening health category (A → C), even after correction for age (ANCOVA p < 0.001). Participants with poorer health status were significantly older (p = 0.0001) than those in better health , A &C (p < 0.01), and B1 &C (p < 0.05). Subjects in health-category A were significantly (p < 0.01) younger than those in category B2 and C; those in health-category B1 were significantly (p < 0.01) younger than category B2.Post-hoc tests allowed detecting significant differences in the 6MWT-distance between health-categories A , and C , but not within category B1  or B2  (table A).There was a negative correlation between the 6MWT-distance and the participant's age for the whole group (r = -0.42, p < 0.001), and for both males (r = -0.32, p = 0.019) and females (r = -0.47, p < 0.001). A correlation between 6MWT-distance and age was present within health-categories When the 156 participants were divided in age categories, again a significant (p < 0.001) decrease in 6MWT-distance was observed with increasing age figure . Subject2 = 0.33) revealed that neither weight nor height was a significant coefficient, contributing to the model (t = -1.4, p = 0.16 and t = -0.98, p = 0.33 for weight and height respectively). Therefore, a new multiple linear regression model was computed without weight and height as independent variables explaining 31% of the variability of the 6MWT-distance (R2 = 0.31). In figure In a first step a multiple linear regression model was computed with 6MWT-distance as dependent variable and with age, gender, weight, height and health status as independent variables. Analysis of this regression model R2 = 0.3 reveale6MWT-distance predicted (m) = 1192 - (6 × age) -  - (69 × gender)A, 2 for health-category B1, 3 for health-category B2, 4 for health-category C. The standard error of the estimate was 147 m.with age expressed in years; 0 for male and 1 for female; 1 for health-category 2 = 0.18) in a first step, in which we introduced in a second step the entry of health status as supplementary independent variable. Using this method we obtained the partial correlation coefficient  between health status and 6MWT-distance.In order to determine the impact of health status in the regression model, we have computed a model with 6MWT-distance as dependent variable and with age and gender as independent variables (RA) and to differentiate them from the "apparently healthy". The latter group was functioning normally, but could not be considered as completely healthy since participants either presented stabilized non-cardiovascular conditions (group B1) or were using cardiovascular medication, however, without any sign of active cardiovascular disease other than normalized hypertension and without significant abnormalities on ECG (group B2). When a history or signs of cardiovascular disease, other than hypertension, were noticed, participants were considered as belonging to group C. When evaluating large numbers of participants there is always the possibility of encountering individuals who are acutely ill; since they will not be able to fully participate in an exercise program, we choose to classify them separately (group D). The classification system was primarily designed to allow the establishment of recommendations concerning the exercise schedule (type, duration and intensity) of elderly persons in the absence of direct medical supervision. Therefore, the classification system is rather conservative and it is easy for an individual to be considered at risk for complications. Roughly, participants in category A will have no particular limitations for exercising; for those in category B1 the instructions will vary with the nature of the health problem; those in category B2 will only exercise at higher intensity  when guided by an instructor qualified for training elderly persons; those in category C will only be allowed to exercise under supervision of an instructor and with medical guidance of the training program; those in category D will not exercise unless cleared by a physician. Since any exercise schedule will depend upon its objective, we do not go into further details here for these recommendations.The 6MWT presents several interesting advantages for the evaluation of the exercise capacity in elderly people. Different authors have described reference equations and tables to predict the 6MWT-distance in healthy elderly subjects. Gender, age, weight and height of these subjects appear to explain a large proportion of the variability in the 6MWT-distance -16. AdvaA1 = 44 of 156) or 37% (Groups A1 and A2 = 58 of 156) of the participants were completely healthy, depending on whether the use of preventive medication was taken into account. However, 79% (groups A, B1 and B2 = 123 of 156) were functioning normally in the community and had no overt history of cardiovascular disease. Rikli et al. (1999) [A, B1 and B2. Our study, based upon a much smaller population sample, demonstrates that normative data based upon the population of the aforementioned authors is not representative for completely healthy elderly. Indeed, the mean 6MWT-distance of completely healthy participants (category A, mean age 62 years (SD = 7); mean 6MWT-distance 764 m (SD = 162) and 657 m (SD = 118) for male and female respectively) is much higher than the norm reported by Rikli et al. [It is clear that elderly persons often termed as 'apparently healthy' do not correspond to criteria for being completely healthy. In our study, only 28% (group . (1999)  studied 4 m SD = 2 and 6574 m SD = 2 and 657i et al.  or Troosi et al. . Our stui et al.  and the i et al. . CurrentA). This relationship is attenuated or disappears in subjects presenting chronic pathology even without apparent functional limitations. Our study demonstrates that there exists a significant decrease in 6MWT-distance with increasing health problems. Since the main objective of our classification system was to stratify for the risk for cardiovascular or metabolic (e.g. hypoglycemia) complications during exercise, we did not expect that the performance on the 6MWT would vary with the health categories. It is probable that the relationship between health status and 6MWT-distance reflects the influence of the status of the cardiovascular system on the general fitness. Inversely, however, it can also be explained by the negative prognostic value of lack of fitness for the ulterior development of cardiovascular disease. It has, indeed, repeatedly been reported for healthy middle-aged adults that this risk is greater for the least fit individuals [A minor familiarization effect has been described for the 6MWT with better values obtained at a second testing . Since rividuals ,29. Sinc2-value. This is different from other studies that describe regression equations for the 6MWT-distance in healthy elderly [2-value  can be due to the more heterogeneous composition in health status and the repartition of the population in several health categories in our study. The remaining unexplained variability in 6MWT-distance might be found in the differences in skeletal muscle strength, the training levels and the physical activity levels of the participants. However, these parameters were not measured in our study.In a multiple linear regression model age, gender and health status explained 31% of the variability of the 6MWT-distance in the population of our study. All three independent variables had a highly significant contribution to the model . The addition of weight, height and BMI to the prediction model did not significantly improve the R elderly ,15, and r female ) than ouWe propose to incorporate the present health classification for elderly people as outcome of the medical screenings preceding the start of a physical exercise program. As shown in our study, significant differences can be found in physical exercise capacity according to the health status. We used the 6MWT-distance, which is an established exercise test for elderly persons, to document the physical exercise capacity of our participants. Especially in the elderly, which show increased prevalence of co-morbidities (most frequently (cardio)vascular, metabolic or rheumatological), physical exercise programs should be established according to the individual exercise capacity. Following the health classification of the elderly participant, the training instructor can adapt the modalities of the training program in order to obtain the best results at the most appropriate training intensity. This should finally lead to concrete training guidelines for elderly people in relation to their health status, diminishing the risk for injuries or complications during physical exercise.None declared.TM conceived and coordinated the study, participated in the evaluation of the health condition of the participants, the analysis and the redaction. IB performed the statistical analysis and participated in the design and the redaction. ML participated in the evaluation of the health condition of the participants, the analysis and the redaction. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


In response to the Blackburn and Rowley essay on the President's Council on Bioethics, several thought-provoking opinions on ethical challenges in biomedical research are expressed by prominent stakeholders PLoS Biology by Elizabeth Blackburn and Janet Rowley, two of the scientific members of President Bush's Council on Bioethics. Invited by the President to serve on this Council, they say that it was ‘a difficult invitation to accept’. Maybe, but that they did accept the invitation is to be applauded. As the Council's report ‘Monitoring Stem Cell Research’ states, ‘fairness in ethical evaluation and judgment depends on … fair and accurate description of the relevant facts of the case at hand’. In other (fewer) words, sound ethics requires a solid base in sound science. It is crucial that any bioethics committee or council made up of ten to twenty members should include at least two or three scientists broadly acquainted with the field in general, and with recent published findings. I was only sorry to read that Elizabeth Blackburn  had her Council term terminated by White House directive on February 27, 2004.I read with interest the article in a recent issue of Of course, any bioethics committee or council  is likely also to include philosophers, lawyers, theologians, sociologists, and probably ‘lay’ people of appropriate interests. The scientists may well find that other members of the group have ‘strong opposing views’ on ethical issues, as well as on the costs and benefits of technologies arising from biomedical research. Elizabeth Blackburn and Janet Rowley were assured, both by Leon Kass, the chairman of the Council, and by President Bush himself, that their voices would be heard and integrated into the Council statements. It is therefore disappointing to learn that, in the ‘Beyond Therapy’ report (which I have not yet read), their requests for revision of certain aspects was declined. Were they not offered the option of a brief minority report? It would be expected in such circumstances that dissenting opinions would be recorded (as was done, for example, in the 1984 UK report by the The ‘Monitoring Stem Cells Research’ report (which I have read) contains no recommendations, but includes a rather comprehensive survey of the various ethical positions relating to human embryonic stem cell research, a historical account of the development up to the present time of federal law and policy, and a chapter on recent  developments in human stem cell research and therapy. The scientists must have contributed substantially to this section of the report. Emphasis is put on the need for research on both adult and embryonic stem cells, since at present there is no way to assess which approach has the more promising therapeutic potential for which diseases. Some funding figures are given: on human embryonic stem cell research the US National Institutes of Health spent $10.7 million in 2002 and $17 million in 2003, with an estimated total spent by US companies of $70 million, while in the same two years the National Institutes of Health spent $170 million in 2002 and $181.5 million in 2003 on adult stem cell research. However, it is not obvious that there are any US scientists wanting to work on human embryonic stem cells within the constraints of US federal funding who are prevented from doing so by lack of money.To my mind, the major deficiency in the ‘Monitoring Stem Cells Research’ report is the almost complete lack of reference to what Elizabeth Blackburn and Janet Rowley correctly call ‘years of rigorous and careful research in animal models’. Some mention is made of experiments with human embryonic stem cells in immunologically handicapped mice, but in any such model both the stem cells and the mice are difficult to work with. Much of the science-based optimism that human embryonic stem cells may eventually prove of therapeutic value springs from the results of experiments with mouse embryonic stem cells in intact mice. Curiously, only a single such experiment is cited: an impressive but somewhat recondite piece of work from Jaenisch's laboratory , using c