There is mounting evidence for associations between sedentary behaviours and adverse health outcomes, although the data on occupational sitting and mortality risk remain equivocal. The aim of this study was to determine the association between occupational sitting and cardiovascular, cancer and all-cause mortality in a pooled sample of seven British general population cohorts.The sample comprised 5380 women and 5788 men in employment who were drawn from five Health Survey for England and two Scottish Health Survey cohorts. Participants were classified as reporting standing, walking or sitting in their work time and followed up over 12.9 years for mortality. Data were modelled using Cox proportional hazard regression adjusted for age, waist circumference, self-reported general health, frequency of alcohol intake, cigarette smoking, non-occupational physical activity, prevalent cardiovascular disease and cancer at baseline, psychological health, social class, and education.In total there were 754 all-cause deaths. In women, a standing/walking occupation was associated with lower risk of all-cause  and cancer  mortality, compared to sitting occupations. There were no associations in men. In analyses with combined occupational type and leisure-time physical activity, the risk of all-cause mortality was lowest in participants with non-sitting occupations and high leisure-time activity.Sitting occupations are linked to increased risk for all-cause and cancer mortality in women only, but no such associations exist for cardiovascular mortality in men or women. The health benefits of leisure-time physical activity are well defined through several decades of epidemiological and clinical studies Studies employing objective measures have reported a very high prevalence of daily sedentary time Total sedentary time is comprised of at least four domains: (a) occupational, (b) leisure-time, (c) transport-related, and (d) domestic. Although there is still no strong supporting evidence, the results of previous studies indicate that different domains of sedentary behaviour might show specific relationships with health Further research on the relationship of occupational sitting and health risks is particularly important because the large majority adults aged 15–64 years are employed Therefore, the aim of this study was to determine the impact of occupational sitting on cardiovascular, cancer and all-cause mortality in a pooled sample of seven British population cohorts. The main hypothesis was that people in sitting jobs have a higher risk for all-cause, cancer and cardiovascular mortality than those in occupations that involve mostly standing or walking. A secondary aim was to examine the combined effect of non-occupational physical activity and occupational sitting on mortality risk.Details of the sample design and selection can be found elsewhere These analyses used secondary data from the Health Survey for England (HSE) and the Scottish Health Survey (SHS) in multiple survey years. Ethical approval was granted for all aspects of these studies by the following Ethics Committees prior to each survey year data collection: HSE 1994 was approved by the Medical Ethics Committee of the British Medical Association; HSE 1998/99 were approved by North Thames Multi Centre Research Ethics Committee; HSE 2003/2004 were approved by the London Multi-Centre Research Ethics Committee; SHS 1998 was approved by the Research Ethics Committees for All Health Boards for Scotland; SHS 2003 was approved by the Multi Research Ethics Committee for Scotland. Each sampled address for the HSE and SHS was sent an advance letter which introduces the survey and states that an interviewer would be calling to seek permission to interview. A leaflet was also enclosed providing general information about the survey and some of the findings from previous surveys. Individual interviews were conducted with adults who give verbal informed consent. At the end of individual interviews, participants were asked for agreement to a follow-up visit by a trained nurse. There was no formal record that participants have given verbal consent to the individual interview or gave physical measurements that are not biological samples . It was made clear in the advance letters and information leaflets that participation in the survey is entirely voluntary, and that participants may decline to answer individual questions, withdraw or stop at any time, or refuse any particular measurement if they wish to do so. The procedures used in the HSE to obtain informed consent were very closely scrutinised by a National Health Service (NHS) and the Scottish Executive ethics committee each year. Information leaflets and both the content and wording of questionnaires were also reviewed by the ethics committees.Participants in this study were aged 40 years and over at study induction. In the present analyses we included cohort members with complete data on all required variables who consented to their death being flagged by the National Registry.Computer-assisted personal interviewing modules assessed respondents’ demographics, self-reported general health and history of disease , health behaviours , and socioeconomic characteristics . Psychological health was evaluated using the 12-item version of General Health Questionnaire (GHQ) When you’re at work are you mainly sitting down, standing up or walking about?” The  physical activity questionnaire used in these cohorts has been described in detail elsewhere Main activity at work  was assessed with the following question: “Non-occupational physical activity was converted into tertiles of MET-hours/week using standard physical activity intensity tables International Classification of Diseases, Ninth (ICD-9) and Tenth (ICD-10) Revisions. Codes corresponding to cardiovascular disease mortality were 390–459 for ICD-9 and I01–I99 for ICD-10. Codes corresponding to cancer mortality were 1400–2399 for ICD-9, and C000–D489 for ICD-10.Participants were flagged by the British National Health Service (NHS) Central Registry, who notified us of the date and cause of death where applicable. Diagnoses for primary (underlying) cause of death was based on the st of December 2009 (SHS) or 15th of February 2011 (HSE). We tested for interactions of occupational activity type with sex and non-occupational physical activity by entering an interaction term in the corresponding age-adjusted Cox models for all three outcomes. Due to evidence for sex interactions for all-cause and cancer mortality (both p<0.001) all analyses were stratified by sex. Due to the low number of events in women we repeated a non-sex specific Cox regression with cardiovascular mortality as the outcome and sex entered as a confounder.Due to evidence of violation of the proportional hazards assumption because of the low number of events in the walking-based occupations group, we developed Cox regression models  with the standing and walking occupational activity groups combined and mainly sitting work as the reference. Surviving participants were censored at 31All Cox regression models were adjusted for age (model 1), waist circumference, self-reported general health, frequency of alcohol intake, cigarette smoking, psychological health, MET-hours/week of non-occupational physical activity, prevalent cardiovascular disease (angina/stroke/ischaemic heart disease) at baseline, and prevalent cancer at baseline (model 2). To specifically examine the influence of socioeconomic position in the last stage (model 3) we also adjusted for occupational social class , of which 379 (160 in women) were attributed to cancer and 177 (42 in women) to cardiovascular disease. The observed (unadjusted) all-cause, cancer, and cardiovascular mortality are presented in We restricted the main analyses among those 5180 women  and 5552 men  who were followed up for three years or more. The direction and magnitude of the observed associations were very similar to those in the main analyses  and 3, fWe also repeated the above Cox analyses among 2634 women 2160 men who reported ‘never being a regular smoker’. The direction and magnitude of the observed associations were very similar to those shown in In women, we found evidence for an interaction between occupational activity type and non-occupational physical activity in terms of all-cause mortality (p = 0.011), but not for cancer (p = 0.130) and cardiovascular mortality (p = 0.087). Nevertheless, in the stratified analyses by physical activity level (using sex-specific median as the cut-off point) we found that for both all-cause and cancer mortality, sitting occupations were linked to an increased risk in those women who were in the high non-occupational physical activity group , but not in the low non-occupational physical activity group . These results were virtually unchanged when we restricted the stratified analyses to those women who reported no cancer and no cardiovascular disease at baseline, e.g. the all-cause mortality multivariable-adjusted HR for the high non-occupational activity group was 0.45 . There were no differences in the association of main activity at work and mortality by non-occupational physical activity level groups in men.In the analyses with the combined occupational activity and non-occupational physical activity variable as the exposure  and 2, tThe main aim of this study was to examine the association of occupational sitting with cardiovascular, cancer and all-cause mortality risk. We found partial support for our hypothesis that people with jobs involving mainly sitting would have higher risk of all-cause, cancer- and cardiovascular disease mortality compared to people with jobs involving mostly standing or walking.We found that women with standing/walking occupations had lower risk of dying from all-causes and cancer , but not from cardiovascular disease, relative to women with sitting occupations, after adjusting for multiple covariates. In men, we found no differences in mortality risk from all-causes, cancer or cardiovascular disease after adjusting for multiple covariates when comparing those in standing/walking occupations with those in sitting occupations.The findings of the present analyses provide an important contribution to the currently equivocal literature about associations between occupational sitting and mortality risk The sub-analysis with non-cancer death as outcome suggested that the associations observed between occupational sitting and all-cause mortality in women were driven by cancer-related deaths, while the lack of associations observed between occupational sitting with cardiovascular mortality was likely explained by the relatively small number of cardiovascular disease-related deaths that occurred over the follow-up period. There is fairly consistent evidence that sedentary behaviour A secondary aim of this study was to examine the combined effect of non-occupational physical activity and occupational sitting on mortality risk. We found that in men and women with high non-occupational physical activity and non-sitting occupations , the risk of all-cause mortality was 26% and 53% lower, respectively, compared to those with low non-occupational physical activity and sitting occupations . Cancer-related mortality risk was significantly lower (by 58%) in women with high non-occupational physical activity and non-sitting occupations relative to those with low non-occupational physical activity and sitting occupations, but no association was observed for men. These results demonstrate the benefits of physical activity in non-occupational domains combined with occupational activity, which have been examined together infrequently. Studies that have examined occupational and non-occupational activity and their associations with mortality separately have yielded similar results The mechanism through which sitting occupations are potentially detrimental to health is unclear. The rodent model-based hypothesis that prolonged sitting causes dramatic reduction of lipoprotein lipase activity (by 80–90%) compared with standing up or ambulating was put forward more than a decade ago The main strengths of this study were the large study population pooled from seven independent cohort studies, the prospective design of the analyses with mean follow-up of approximately 13 years, the multiple analytical measures we took to minimise the chances of reverse causality, and the linkage of data with the national death registry with cause-specific details. Our analyses were adjusted for a range of demographic and behavioural variables, although residual confounding from unmeasured factors remains a possibility.One limitation was that despite the relatively large sample size there was a low number of cardiovascular death events that limits our ability to draw conclusions from the corresponding analyses. The use of self-reported measures of occupational and non-occupational physical activity raises the potential of bias or measurement error. However, the non-occupational physical activity measure has demonstrated sufficient validity against accelerometers The findings of this study have implications against a backdrop of declining trends in daily activity and the increasingly sedentary nature of work. For working populations, occupational time makes up a large part of their day Figure S1Observed all-cause mortality rates by main activity while at work. Whiskers denote 95% confidence intervals for percentages. P-values (group-to-group comparison) were calculated using two proportions z-test.(TIF)Click here for additional data file.Figure S2Observed cancer mortality rates by main activity while at work. Whiskers denote 95% confidence intervals for percentages. P-values (group-to-group comparison) were calculated using two proportions z-test.(TIF)Click here for additional data file.Figure S3Observed CVD mortality rates by main activity while at work. Whiskers denote 95% confidence intervals for percentages. P-values (group-to-group comparison) were calculated using two proportions z-test.(TIF)Click here for additional data file.Table S1Cox regression models for main activity while at work and all-cause/cancer/cardiovascular mortality in women aged ≥40 years who were in employment and reported no cancer or cardiovascular disease (angina/stroke/ischaemic heart disease) at baseline (n = 5027).†Model 1: adjusted for age; Model 2; also adjusted for waist circumference, self-reported general health, psychological health, frequency of alcohol intake, cigarette smoking, MET-hours/week of non-occupational physical activity; Model 3: also adjusted for occupational social class  and age finished educations . ‡p-values in brackets correspond to the trend in the Cox models when the main activity at work variable is entered in its original form with 3-categories .(DOCX)Click here for additional data file.Table S2Cox regression models for main activity while at work and all-cause/cancer/cardiovascular mortality in men aged ≥40 years who were in employment and reported no cancer or cardiovascular disease (angina/stroke/ischaemic heart disease) at baseline (n = 5329).†Model 1: adjusted for age; Model 2; also adjusted for waist circumference, self-reported general health, psychological health, frequency of alcohol intake, cigarette smoking, MET-hours/week of non-occupational physical activity; Model 3: also adjusted for occupational social class  and age finished educations . ‡p-values in brackets correspond to the trend in the Cox models when the main activity at work variable is entered in its original form with 3-categories .(DOCX)Click here for additional data file.


Analysis of gene expression from different species is a powerful way to identify evolutionarily conserved transcriptional responses. However, due to evolutionary events such as gene duplication, there is no one-to-one correspondence between genes from different species which makes comparison of their expression profiles complex.In this paper we describe a new method for cross-species meta-analysis of gene expression. The method takes the homology structure between compared species into account and can therefore compare expression data from genes with any number of orthologs and paralogs. A simulation study shows that the proposed method results in a substantial increase in statistical power compared to previously suggested procedures. As a proof of concept, we analyzed microarray data from heat stress experiments performed in eight species and identified several well-known evolutionarily conserved transcriptional responses. The method was also applied to gene expression profiles from five studies of estrogen exposed fish and both known and potentially novel responses were identified.http://bioinformatics.math.chalmers.se/Xspecies/.The method described in this paper will further increase the potential and reliability of meta-analysis of gene expression profiles from evolutionarily distant species. The method has been implemented in R and is freely available at Gene expression microarray and RNA-seq provide fast and cost-efficient measurement of mRNA abundance for thousands of genes simultaneously. The amount of gene expression data generated by these techniques is constantly increasing and public repositories such as Gene Expression Omnibus and ArrayExpress contains today a large body of information from a wide range of species and experimental conditions,2. LargeDrosophila melanogaster and the worm Caenorhabditis elegans). The latter compensates for the number of paralogs in order to avoid bias from large homology groups. The weights wj are arbitrarily and can be set to weigh individual experiments up and down. This is for example useful when multiple experiments are performed in a single organism (see Estrogen exposure below for an example). However, more sophisticated weighting strategies are also possible, such as weights based on the evolutionary distance between the included species (e.g. evolutionary distinctiveness score[ss score).Si can be calculated explicitly depending on the weightsThe density function of  becomesSi using a Gamma distribution with the same expectation value and variance. Approximating a weighted sum of exponentially distributed variables with a Gamma distribution has previously shown to accurate enough for our purpose[Si becomes  Analogously, density functions for the cases when two or more weights are equal can also be derived. However, evaluating the cumulative density function (CDF) requires numerical integration which is computationally expensive. We therefore approximate the distribution of  purpose. The expα and β should be  Hence, the shape and scale parameters Si with the null distribution of Si. The hypothesis in 1 can now be tested and a corresponding p-value calculated by comparing the observed value Saccharomyces cerevisiae (4932), Schizosaccharomyces pombe (4896), Arabidopsis thaliana (3702), Oryza sativa (4530), Drosophila melanogaster (7227), Danio rerio (7955) Mus musculus (10090), Homo sapiens (9606) (NCBI Taxonomy IDs are given in parenthesis). Each gene was assumed to be measured in two different groups, one control and one treated, with three independent observations from each. Data was simulated from a Gaussian distribution with mean value 0 and variance 1 and p-value calculated using a two-population t-test assuming equal variance. For differentially expressed orthologous groups  an effect ranging from 0 to 10 was added to the treated group . For groups and species with in-paralogous genes the effect was added to one single in-paralog (randomly selected). The weights wij in Si were set to be uniform. For the combined method all observations from in-paralogs treated as independent replicated observations for one single gene (homology group). For the average method, an average was taken over all observations from in-paralogs generating one single observation for each observation. For the random method one of the in-paralogs was randomly selected and other discarded. For these three methods the cross-species p-value was calculated by Fisher’s combined probability test[i was estimated by calculating the proportion of false positives among the i most significant groups.Simulations were performed on homology groups from Homologene for the species ity test. The falIntensity data from Affymetrix type of microarrays was pre-processed using RMA while inftp://ftp.geneontology.org/pub/go/gene-associations/). Only terms with an experimental evidence code  were considered. Functional enrichment was inferred using the topGO R package[Gene expression data from eight experiments investigating the effects of heat stress in eight species were fetched from Gene Express Omnibus and ArrayExpress Table. Homolog package.Oncorhynchus mykiss the arbitrary weight component was set to 0.25, 0.25, 0.25, 0.125 and 0.125  multiple in-paralogs with similar expression profile (2) multiple in-paralogs with divergent expression profile, (3) noise with thick tails (t-distribution with five degrees of freedom), (4) errors in homology structure, error rate=0.1 and (5) errors in the homology structure, error rate=0.5.Click here for fileList of analyzed homology groups from the meta-analysis of heat stress experiments. Results for each homology group based on heat stress experiments performed in eight different species. The list contains combined p-value and false discovery rate as well as individual p-values from each experiment.Click here for fileResults of the functional enrichment of Gene Ontology terms. Results from the Gene Ontology (GO) term enrichment analysis of the significant homology groups from the heat stress analysis. The file contains results from the biological process (BP), cellular component (CC) and molecular function (MF) ontologies.Click here for fileList of analyzed homology groups from the meta-analysis of aquatic vertebrates exposed to estrogens. Results for each homology group based on estrogen exposure experiments performed in four aquatic vertebrates. The list contains combined p-value and false discovery rate as well as individual p-values from each experiment.Click here for file


Xenotropic murine leukemia virus related virus (XMRV) has been the subject of intense investigation since its discovery and initial characterization in human prostate carcinoma. Notwithstanding the important and ongoing controversies surrounding detection methods, seroprevalence, disease association, or viral origin, we sought to determine whether XMRV gene expression was sufficient to promote prostate pathology in a transgenic mouse model. Using the probasin promoter to drive prostate specific expression of XMRV genes in a transgenic mouse (PRO-XMRV) in vivo, we compared prostates of age matched male littermates over time. Using immunohistochemistry to stain envelope proteins we have been able to detect XMRV gene expression in the prostate of 3 of 3, 11-15 month old mice with the highest levels found in the lateral lobes. The lateral lobes also exhibit thickened layers of glandular epithelia that contain elevated numbers of bromodeoxyuridine retaining cells. While this model produces wild-type particles that can be detected in the urine by RT-PCR, infection is not thought to be occurring due to the absence of the receptor, XPR-1, in mouse cells. To determine if viral infection and integration is required for XMRV-related prostate pathology, we have also created a transgenic strain in which XPR-1 is expressed in the mouse prostate (PRO-XPR1). Breeding PRO-XMRV mice with PRO-XPR1 mice will allow us to test whether XMRV integration or gene expression can cause more advanced prostate pathology in vivo. With these XMRV mouse models we seek to address the question that remains unanswered to date as to whether XMRV is capable of causing prostate dysplasia or cancer in vivo.


This cross-sectional study aimed to investigate associations between a marker of cardiac strain, the N-terminal prohormone B-type natriuretic peptide (NT-proBNP), and inflammation as reflected by either a conventional or novel inflammatory marker in a bi-ethnic South African cohort.We measured NT-proBNP, C-reactive protein (CRP) and plasma-soluble urokinase plasminogen activator receptor (suPAR) levels along with conventional biomarkers in black (n = 117) and white (n = 116) men.NT-proBNP, CRP and suPAR levels were higher in black compared to white men. NT-proBNP was significantly associated with both CRP  and suPAR  in black men only. After full adjustment in multiple regression analyses, the above associations of NT-proBNP with CRP  and suPAR  were confirmed in black men.These results suggest that a low-grade inflammatory state as reflected by both a conventional and novel marker of inflammation may contribute to higher cardiovascular risk as reflected by the associations obtained with a marker of cardiac strain in black South African men. High morbidity and mortality among the black population in South Africa is a major concern due to the high prevalence of communicable and non-communicable diseases, especially in communities of low socio-economic standing with limited healthcare facilities CRP, which reflects systemic inflammation, is elevated in acute heart failure and N-terminal prohormone B-type natriuretic peptide (NT-proBNP), reflecting cardiac strain, are proposed reliable risk markers of cardiovascular disease as well as good predictors of cardiovascular morbidity and mortality The associations of NT-proBNP with markers of inflammation have been studied in part in white populations of American and European descent The SAfrEIC study was approved (06M01) by the Ethics Review Board of the North-West University and the study protocol conformed to the ethical guidelines of the Declaration of Helsinki (2008) for investigation of human participants. This cross-sectional study formed part of a larger South African investigation on the role of Sex, Age and Ethnicity on Insulin sensitivity and Cardiovascular function (SAfrEIC) study that involved 382 black and 372 white participants from the North West province of Southern Africa. This sub-study included 117 black and 116 white men after excluding all patients who were using antihypertensive or anti-inflammatory medication (n = 41) and those infected with HIV (n = 55).Approximately 10 to 20 recruited participants visited the Metabolic Unit facility daily at the Potchefstroom campus of the North-West University over a period of seven weeks. Each participant gave written informed consent to take part in the study after all the procedures were comprehensively explained. A participant sheet guided the participants through the different research stations where various measurements were performed. Basic health and demographic questionnaires were completed during the morning. An informative description regarding the results of the health assessment was given to each participant at the end of the study. In the event where abnormalities became evident (e.g. hypertension or diabetes), the participant was advised to visit their local clinic, hospital or physician.Duplicate office blood pressure measurements were taken with the Omron HEM-757  apparatus. The first blood pressure measurement was taken after an initial 10 minute resting period and a second measurement was taken 5 minutes after the first. Pulse pressure was subsequently calculated. Participants with a systolic (SBP) ≥140 mmHg and/or diastolic blood pressure (DBP) ≥90 mmHg were considered hypertensive Body height was measured to the nearest 1.0 cm by using the Invicta Stadiometer  and body weight to the nearest 0.1 kg . Subsequently, the body mass index (BMI) was calculated for each participant. The waist circumference was measured at the midpoint between the lowest rib and the top of the iliac crest with a Holtain non-stretchable, flexible metal measuring tape Participants were requested to fast for a minimum of eight hours. Fasting lipids , high-density lipoprotein cholesterol (HDL), and triglycerides (TG)), serum glucose, γ-glutamyl transferase, creatinine and high-sensitivity serum CRP were determined with the Konelab autoanalyzer . Low density lipoprotein cholesterol (LDL) was determined with the Friedewald formula: LDL = TC – HDL – TG/2.17. Insulin was determined with the ST AIA-PACK IRI kit  using a two-site immuno-enzymometric assay. Serum cotinine was determined with the IMMULITE 2000 nicotine metabolite assay . The Elecsys proBNP sandwich immunoassay was used on an Elecsys 2010  to determine the serum NT-proBNP concentration of each participant. Plasma (EDTA) suPAR levels were measured using the suPARnostic® ELISA kit . Human immunodeficiency virus status was determined directly after blood sampling with rapid tests according to the protocol of the National Department of Health of South Africa. Serum was used for testing with the First Response Test  and was repeated with the Pareeshak test (BHAT Bio-tech India) to confirm the results.2) were used to compare proportions and independent t-tests to compare the means of continuous variables. We plotted unadjusted associations between NT-proBNP and markers of inflammation in single regression analysis. We divided CRP and suPAR values into quartiles to explore associations with NT-proBNP levels, while adjusting for SBP and arterial compliance in analysis of covariance (ANCOVA). A standard multiple regression analyses were performed to investigate independent associations between NT-proBNP and inflammatory markers. Several covariates were considered for entry into the regression model including age, BMI, SBP, arterial compliance, serum creatinine, γ-glutamyl transferase, fasting glucose, total cholesterol to high density lipoprotein cholesterol ratio and cotinine. Probability values of α≤0.05 were considered statistically significant.Statistica software v10.0  was used for database management and statistical analyses. The normal distribution of the variables was tested prior to any further statistical analyses. Variables that deviated from normality  were logarithmically transformed. Associations of NT-proBNP with markers of inflammation were tested for interaction with ethnicity by performing the appropriate interaction terms. Chi-square tests , and suPAR . It is also noteworthy to mention that the mean blood pressures of the black men were high normal compared to white men with optimal pressures. By performing ANCOVA with adjustments for BMI, SBP, cotinine and γ-glutamyl transferase the initial difference in CRP levels between black and white men disappeared . None of these variables seem to alter the difference in mean suPAR levels in black or white men .et al. that suggested a positive relationship between NT-proBNP and inflammatory markers including CRP, orosomucoid, haptoglobulin and alpha1-antitrypsin in European (Swedish) men and woman et al. investigated the potential of suPAR as a marker of adverse cardiovascular outcome in over 2500 Danish men and women. From those results, suPAR associated with an increased risk of cardiovascular disease, cancer, type 2 diabetes and mortality, more strongly in men than in women This study investigated the associations of NT-proBNP with CRP and suPAR in black and white South African men. NT-proBNP associated positively with both inflammatory markers in black men. However, this relationship was absent in the white group. It is known that CRP reflects metabolic inflammation with its relation to BMI and lipids Increased levels of the cardiac hemodynamic volume load biomarker, NT-proBNP, reflect the risk of developing left ventricular dysfunction and congestive heart failure The underlying and combined mechanisms relating NT-proBNP to CRP and suPAR in cardiovascular disease are not well understood and we can therefore only speculate to explain our findings. A cascade of contributing factors is involved in the poor cardiovascular state of the black South African population. Among these factors are adverse lifestyle choices which include alcohol misuse, insufficient dietary intake and excessive smoking. In turn, these factors contribute to alterations in hemodynamic and metabolic processes after which systemic inflammation becomes unavoidable Normally, the natriuretic peptide clearance receptor type C and the kidneys maintain the concentrations of BNP and its amino fragment to restore the equilibrium of the natriuretic peptide system and its effects Our study has certain limitations. It was cross-sectional and therefore causality cannot be inferred due to confounding variables or unknown factors that were associated with both NT-proBNP and inflammation. The black men in this study were from a low socio-economic class , which could have rendered these subjects as high risk for developing disease, due to limited healthcare provision. However, no interactions were observed with socio-economic standing. NT-proBNP is known as a stable and sensitive marker of cardiac function, including right ventricular dysfunction In conclusion, NT-proBNP persistently associated with CRP and suPAR in black men, but not in white men. Our result suggests that a low-grade inflammatory state may potentiate adverse changes in hemodynamic strain on the heart. This is in line with the higher prevalence of hypertension and early onset hemodynamic alterations known to this group of black men. However, these findings need confirmation in larger prospective and experimental studies to identify potential mechanisms of early onset cardiovascular changes.


A common goal of persons post-stroke is to regain community ambulation. The plantar flexor muscles play an important role in propulsion generation and swing initiation as previous musculoskeletal simulations have shown. The purpose of this study was to demonstrate that simulation results quantifying changes in plantar flexor activation and function in individuals post-stroke were consistent with (1) the purpose of an intervention designed to enhance plantar flexor function and (2) expected muscle function during gait based on previous literature.Three-dimensional, forward dynamic simulations were created to determine the changes in model activation and function of the paretic ankle plantar flexor muscles for eight patients post-stroke after a 12-weeks FastFES gait retraining program.An median increase of 0.07  was seen in simulated activation averaged across all plantar flexors during the double support phase of gait from pre- to post-intervention. A concurrent increase in walking speed and plantar flexor induced forward center of mass acceleration by the plantar flexors was seen post-intervention for seven of the eight subject simulations. Additionally, post-training, the plantar flexors had an simulated increase in contribution to knee flexion acceleration during double support.For the first time, muscle-actuated musculoskeletal models were used to simulate the effect of a gait retraining intervention on post-stroke muscle model predicted activation and function. The simulations showed a new pattern of simulated activation for the plantar flexor muscles after training, suggesting that the subjects activated these muscles with more appropriate timing following the intervention. Functionally, simulations calculated that the plantar flexors provided greater contribution to knee flexion acceleration after training, which is important for increasing swing phase knee flexion and foot clearance. FurtherComputer simulation studies have previously been used to demonstrate the function of individual muscles in healthy -13 and pDue to the role of plantar flexors and leg extension  in generThis study used subject-specific musculoskeletal models to simulate the changes in activation and function of the ankle plantar flexor muscles in individuals post-stroke after a FastFES gait retraining program, as well as to identify relationships between simulation results and clinical gait variables. We developed forward dynamic gait simulations using gait data collected before and after 12-weeks of FastFES gait training. The purpose of this study was to demonstrate that simulation results were consistent with (1) the purpose of the intervention and (2) expected muscle function during gait based on previous literature. Additionally, we evaluated the ability of the model results to predict intervention outcomes by demonstrating correlations between our simulation results and clinically relevant outcome measures.° plantar flexion with the knee flexed, and presence of deficits in walking function. Exclusion criteria were defined as: severe aphasia, substantial cognitive deficits, cerebellar involvement, or preexisting conditions affecting walking function [Twelve individuals post-stroke  were recruited to participate in a 12-week FES gait retraining intervention, involving both plantar and dorsiflexor stimulation . Inclusifunction .Training consisted of four six minute bouts of treadmill walking at the subjects’ fastest possible speed with FES delivered to the paretic limb during the first, third, and fifth minutes of each bout. A fifth bout consisted of three minutes of treadmill walking with FES followed by three minutes of overground walking without FES. During the overground walking subjects were instructed to walk with the same pattern as practiced with the FES. This fifth bout is designed to help transfer the training to a more natural overground setting. FES was delivered to the ankle dorsiflexor muscles during the paretic swing phase and to the ankle plantar flexor muscles during the paretic pre-swing phase of gait . Timing Kinematic and kinetic gait data were collected using an 8-camera motion capture system  as subjects walked at their self-selected walking speed on an instrumented split belt treadmill  pre- and post- intervention. The self-selected walking speed was determined during over ground walking using the average of three trials of the 6-meter walk test. Subjects walked without a brace or assistive device and no FES was delivered during these evaluation sessions. Subjects wore an overhead support harness with no body weight support and held on to a handrail (if needed) for safety.Three-dimensional, subject-specific forward dynamic simulations were created from motion capture walking trials using OpenSim . The modBased on the muscles targeted by the intervention, the paretic medial gastrocnemius (MG), soleus (SOL), and tibialis posterior (TP) muscles were analyzed in this study. All analysis in this manuscript is focused on the paretic limb. While the authors acknowledge the importance of all muscles, both ipsilateral and contralateral, in contributing to the variables of interest in this study, the plantar flexor muscles are being analyzed here due to the targeted nature of the intervention. Of the three plantar flexor muscles, the MG and SOL muscles were directly stimulated by FES. The TP was also studied, as changes in the TP would reflect changes in a muscle that serves a similar function but was not exposed to FES. For each muscle, the simulated activation ranged from 0 to 1, where 0 indicates no muscle activation and 1 indicates full muscle activation. We calculated the average simulated activation over a period of time, where 1.0 would be the maximum average activation, for the complete gait cycle and during double support. Double support in this study is defined as the period of double support on the paretic limb during pre-swing. Muscle force perturbations were used to determine individual muscle contributions to knee joint and center of mass (COM) accelerations . For thiDifferences between simulated activation for each muscle pre- and post- intervention were evaluated using the Wilcoxon signed-rank test. Non-parametric statistics per performed due to low sample size and high variability between subjects. Changes in the average knee joint and COM accelerations from pre- to post-intervention were assessed on an individual basis. Linear regressions were performed to assess the following relationships: (1) Changes in simulated activation of the PF muscles versus changes in peak knee flexion during swing phase of gait and walking speed. (2) Changes in simulated plantar flexion activation and trailing limb angle versus changes in knee flexion acceleration. (3) Forward COM acceleration produced by the PF muscles versus walking speed.P < 0.01)) than pre-intervention . Medial gastrocnemius experienced the largest median change across subjects, at 0.12  (P = 0.04), and a 0.04  median increase (P = 0.01) was seen in the tibialis posterior muscle. Although not significant, the soleus muscle showed a median increase of 0.05  with training (P = 0.20), with 5 of 8 subjects increasing predicted activation and two subjects showing no change (< 0.01). No significant change with training was seen for the average of these muscles over the full gait cycle )  was seen in simulated activation averaged across all plantar flexor muscles over double support after 12-weeks of gait training  wa2 ) were not statistically significant for the total or individual muscles across subjects, two additional subjects  achieved forward COM acceleration from the PF muscles post-intervention. Overall, the sum of the PF muscles for seven of the eight subjects either improved acceleration of the COM or decelerated the COM less after training.Prior to training, the sum of the PF muscles for seven of the eight subjects decelerated the COM in the direction of walking during double support; only one subject’s PF showed the normal pattern of accelerating the COM forward during double support Figure . Althoug2 ) (P = 0.04) post-intervention  with increases in peak knee flexion during the swing phase of gait . Neither change in simulated PF activation nor trailing limb angle alone were significant predictors of change in knee flexion acceleration induced by the plantar flexors. Increased simulated plantar flexion activation with training  was related to increases in self-selected walking speed. Greater self-selected walking speed post-intervention exhibited a trend for a relationship with greater forward COM acceleration by the plantar flexors during double support .Increases from pre- to post-training in model predicted PF activation during pre-swing showed a significant positive relationship  the purpose of the intervention and (2) expected muscle function during gait based on previous literature. In addition, we identified correlations between simulation results and clinical measures of walking function. Sixteen subject-specific models were created to represent pre- and post-training gait for 8 individuals post-stroke. To our knowledge, this is the first study to use musculoskeletal simulations of persons with chronic hemiparesis to simulate changes in muscle activation and function before and after participation in a gait retraining intervention.Significant increases were seen in simulated plantar flexor activation during double support with no changes in total predicted activation over the entire gait cycle, suggesting selective increases in predicted plantar flexion activation during the phase of gait targeted by the intervention, i.e. double support. This new simulated activation pattern post-intervention is consistent with the timing of PF FES used during the intervention, which stimulated PF muscles only during pre-swing. This post-intervention activation pattern also agrees with healthy PF muscle coordination where the plantar flexors are activated most during late single leg stance and pre-swing. Thus, this new simulated pattern of activation for the plantar flexor muscles suggests that the subjects activated these muscles with more appropriate timing following the intervention.The soleus and gastrocnemius play key roles in forward progression and swing initiation, respectively, primarily during the double support phase of gait . Post-stIn hemiparetic individuals, enhancing the contribution of the soleus towards forward COM acceleration has been identified as an important mechanism for increasing walking speed . InteresNeptune and colleagues have shown that the gastrocnemius muscle is critical for swing initiation during pre-swing, increasing swing phase knee flexion and enabling foot clearance during swing by accelerating the knee into flexion . For sevIn a previous experimental study investigating immediate effects of FES, peak knee flexion during swing was hypothesized to increase with greater forward propulsive forces as a result of increases in simulated PF activation during pre-swing . In our This study examined changes in simulated post-stroke muscle activation and function at self-selected speed after a gait retraining intervention. Due to the limited number of subjects and the high variability in individuals post-stroke, it is not known if the findings of this study apply to the population of individuals post-stroke as a whole. Also, all subjects walked at a faster self-selected walking speed post-intervention. While the use of greater walking speeds post-intervention may have confounding effects on variables such as forward COM acceleration, we believe this analysis is important because it allows us to determine what changes in model predicted muscle activation and function were necessary to achieve the increases seen in walking speed post-intervention, a common goal of gait is retraining. In fact, this the first study to show concurrent improvements in speed, propulsion, and predicted PF activation after a targeted training intervention with individuals post-stroke. Due to the absence of a control group, it is not clear if the changes seen from pre- to post-intervention are a specific result of either FES or fast treadmill walking. Future work will include groups trained with FES or at fast walking speeds to assess the individual components of the intervention.The analysis of muscle function is somewhat limited since activation was simulated without the use of EMG for this study. However, our modeling does account for the effect of limb posture on muscle function, which is not accounted for by EMG. For some of the muscles included in this model, surface EMG cannot be obtained. When used longitudinally with an intervention, EMG magnitude can be insensitive to hypertrophy of muscle, which makes comparisons difficult over time. Additionally, relying on EMG signal amplitudes can be difficult, as signal magnitude can vary based on electrode placement and tissue conductivity . HoweverSubjects were allowed to use handrails during walking trials for safety purposes. Although the subjects were instructed to use the handrails with a ‘light touch,’ some subjects may have applied larger forces to the handrails. While the forces applied to the handrails were not accounted for explicitly, residual forces were calculated during the simulation and applied to the COM to account for kinetic imbalances due to external forces (i.e. handrail force). These residual forces were found to agree closely with the forces applied on the instrumented handrails during data collection.For the first time, muscle-actuated simulations were used to detect the effect of a gait retraining intervention on post-stroke modeled muscle activation and function. Improvements predicted by the simulations were consistent with improvements in kinematic measures of gait performance, demonstrating that musculoskeletal simulations can provide insight into clinically meaningful results. The simulations showed that a new pattern of predicted activation for the plantar flexor muscles emerged after training, suggesting that the subjects activated these muscles with more appropriate timing following the intervention. Functionally, after training, the plantar flexors provided greater contribution to knee flexion acceleration, which is important for increasing swing phase knee flexion and foot clearance. Improvement in trailing limb angle combined with increased predicted activation of the plantar flexors was shown to predict improvements in forward COM acceleration produced by the plantar flexors pre-swing. Also, greater contribution to forward COM acceleration by the plantar flexors after training was predictive of greater self-selected walking speed post-training. The correlations seen in this study are noteworthy, as they demonstrate a connection between musculoskeletal model predictions and clinically relevant gait variables measured pre- and post- interventionFES: Functional electrical stimulation; PF: Plantar flexors; COM: Center of mass; MG: Medial gastrocnemius; SOL: Soleus; TP: Tibialis posterior; EMG: Electromyography.The authors declare that they have no competing interests.BK created the musculoskeletal models, analyzed the data, and drafted the manuscript. TK participated in the design of the study and coordination and helped to draft the manuscript. DR conceived of the study, and participated in its design and coordination and helped to draft the manuscript. SBM conceived of the study, and participated in its design and coordination and helped to draft the manuscript. JS conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.


In HIV-1-infected patients a long lasting CD4+ cell decline influences the host-EBV balance and thereby increases the risk for EBV related malignancies. In spite of a world-wide access to combination antiretroviral therapy (cART) there are still a considerable number of HIV-1-infected patients who will develop severe immunodeficiency. These undiagnosed HIV-1 infected patients, so called late testers, demonstrate an increased lymphoma risk, compared to patients diagnosed early. Consecutive individual screening for EBV DNA-load in late testers might be a useful predictor of emerging EBV-malignancy.in vitro and further analyzed.Patient biopsies and ascites were analyzed morphologically, by immuncyto-histochemistry and in-situ hybridization. Viral DNA and RNA load were quantified by PCR. Cell lines from primary tumor and from ascites, were established 6/L. The lymphoma was CD45-negative and weakly CD22- and CD30-positive. The patient had a history of Kaposi sarcoma and HHV-8 seropositivity. The lymphoma biopsies, and three cell lines derived on different occasions from the tumor cell effusion, were all EBV-positive but HHV-8 negative.We here report on a case of EBV-positive lymphoma in an AIDS patient, first presenting with pleural effusion and ascites and was thus initially considered a primary effusion lymphoma (PEL) but was later diagnosed as a plasmablastic lymphoma (PBL). The patient had responded to cART with undetectable HIV-RNA and increased CD4 cell count one year prior to lymphoma presentation. At the time of lymphoma diagnosis the HIV-RNA values were <50 RNA-copies per mL blood (undetectable) and the CD4-positive cell count 170 ×10A noticeable EBV-DNA load decline was observed during the remission of the lymphoma following CHOP-therapy. The EBV-DNA load increased dramatically at the time of recurrence.EBV DNA load might be useful in monitoring the effect of lymphoma treatment as well as in estimating the risk of EBV-associated lymphoma in HIV-1 infected patients with pronounced immunosuppression. EBV infects the vast majority of humans and resides latently in B-cells. The virus carries genes that can induce and maintain mature B cell growth. In immunocompromised post-transplant and HIV-infected patients the virus may cause lymphoproliferative or malignant diseases. Although EBV negative lymphoid malignancies have been described in HIV-infected persons EBV has a strong association with a wide range of B-cell lymphomas in HIV-carriers including Burkitt’s lymphoma (BL), Hodgkin lymphoma and Diffuse Large B-cell lymphomas .Plasmablastic lymphoma (PBL) is a rare aggressive subtype of non-Hodgkin lymphoma (NHL) most commonly localized to mucosal tissues such as the oral cavity, but may also be manifest in other organs such as liver, breast or bone . MorpholFurthermore, PBL is CD45-negative, while PEL is CD45-positive. Both PBL and PEL display markers associated with plasma cell differentiation such as CD38 and CD138 and usually express cytoplasmic immunoglobulin and may be variably positive for CD30. Although most cases of PBL occur in AIDS-patients with profound immunodeficiency, HIV-1 negative cases have also been reported [In the era of modern combination antiretroviral therapy (cART) there has been an impressive reduction of AIDS-related opportunistic infections and lymphomas. However, HIV-1 patients still suffer an increased risk for NHL  and the 6/L. In a malignancy assessment the same year endoscopic biopsies were taken from the gut mucosa. By routine histopathological analysis the biopsies showed signs of chronic inflammation and macrophage infiltration, but no lymphoma. Bone marrow aspiration was also performed with May-Grunewald stained smears showing immature cells of the myeloid series and many lymphoid cells (44%), including cells of lymphoplasmocytoid/plasmacell type. Staining for kappa and lambda light chains gave no evidence for clonal B-cell proliferation. The findings were suggested to represent a polyclonal B-cell hyperplasia without suspicion of lymphoma.When the patient was diagnosed with HIV-1 the CD4+ cell count was 170 × 10Oral hairy leukoplakia was diagnosed early the year after HIV-diagnosis and the same year the patient developed hemolytic anaemia Figure . The ana6/L, and CMV-retinitis was diagnosed and treated with altering foscarnet and ganciclovir. During the fourth year HIV-RNA values increased to 65 000/mL prompting a change in the anti-retroviral treatment to AZT and didanosine. Second half of fourth year cART was introduced . After cART introduction, including a protease inhibitor, HIV-RNA was <500/mL, and remained undetectable during the following two years. The patients CD4 cell count went from 30 × 106/L in second half of year two after HIV-1 diagnosis to 190 × 106/L after introduction of cART year four. The suppressive CMV-treatment was withdrawn the same year (year 4).In December the second year antiretroviral treatment with azidothymidine (AZT) was initiated, and shortly thereafter a conjunctival Kaposi lesion was removed. In February the following year , and one month later ascites. CT scan revealed a mass in the gastric wall and pleural effusion. An initial cytological analysis of the effusion suggested a primary effusion lymphoma (PEL). A biopsy from the tumor in the gastric wall confirmed the presence of an EBV-positive Non-Hodgkin lymphoma with immunoblastic features considered to be a manifestation of PEL.After diagnosis of the lymphoma cytostatic treatment with cyclophosphamide, hydroxydaunorubicin (Doxorubicin), vincristine (Oncovin), prednisolone (CHOP), and intrathecal methotrexate was initiated and the ascites disappeared within four days. At the time of lymphoma treatment Rituximab was not yet approved and was therefore not a treatment option. The patient continued with cART and suppressive toxoplasmosis treatment. The CHOP treatment was repeated every 3rd week and the patient completed six treatment cycles in total. Before every CHOP treatment blood was drawn for EBV-DNA analysis. EBV-load was also measured on three occasions the month after completing CHOP treatment. The remission of the tumor mass of the gastric wall was confirmed with a CT-scan and the patient did clinically well. Then, one month after the final CHOP course, ascites reappeared together with pleural effusion. Reintroduced cytostatic treatment now only had marginal effect on the lymphoma. Pleural effusions were evacuated twice. The patient died four and a half years after HIV-1 diagnosis due to progression of the lymphoma.At autopsy lymphoma manifestations were observed in spleen, liver, lymphoid organs and lymphomatous dissemination to pleura and peritoneal cavity. A small cerebral mass in globus pallidus was also found, but this was not further analyzed to rule out lymphomatous spread to the central nervous system lymphoma or a lesion related to the patient’s earlier diagnosed cerebral toxoplasmosis.Retrospective in situ hybridization of the patient’s early Kaposi manifestation did show EBER-expression in some (probably lymphatic) cells.When the patient presented with a gastric tumor and ascitic and pleural effusions, cytological examination of the ascitic fluid showed a population of dispersed large blastic cells with a high frequency of mitoses and partly vacuolated dark blue cytoplasm. The tumor cells were morphologically highly reminiscent of blastic lymphoid cells, with features of immunoblastic plasmablastic differentiation Figure . By immuA trans-abdominal core biopsy was subsequently obtained from the extra nodal tumor in the gastric wall, which was prepared for flow cytometry and histopathology and molecular analysis. The routine hematoxylin-eosin (H-E) staining showed an infiltration of a large cell malignant lymphoma with immunoblastic/plasmablastic features, but without any mucosal component or lymph node structures in the specimen Figure . The morAdditional analysis was performed on autopsy material. Immunohistochemical analysis on sections was positive for CD43 and CD138  than SPIBL-II and III (one fragment at 87–92 kD and one at 108 kD), which both have the same clonal origin.The clinical features of this patient’s lymphoma manifestations were at the time of diagnosis consistent with primary effusion lymphoma (PEL), with ascitic and pleural effusions. Gene expression profile analysis of PEL has suggested a plasmablastic derivation in common with PBL . NeverthHIV-associated PBL is considered as a very aggressive lymphoma, with a poor prognosis also in the era of cART .As far as could be analyzed on the autopsy material this lymphoma had histopathological and phenotypical features that were similar at first diagnosis and after recurrence. At relapse the lymphoma was spread both to extra-nodal sites and reappearing in effusions.The CD4 expression on the PBL cells was unexpected, but has been seen in other cases, as has recently been reviewed . MoreoveThe cell lines derived from the biopsies taken before and after recurrence were distinctly different. Cell lines from the early lymphoma had a lymphoblastoid cell line feature, i.e. similar to immunoblastic lymphoma. The cell lines derived from pleural effusion at recurrence were BL like, but did not have the t translocation typical for BL. They expressed EBV-latency program I/II, which altogether would be compatible with the plasmablastic phenotype, also suggested by other markers on the cell lines. The cell lines were also different from reported HHV-8-positive PEL lines, which regularly do not express any immunoglobulin .From clonal analysis of immunoglobulin-rearrangement we can conclude that the cell lines seem to share one marker of rearrangement of the heavy chain locus, while another marker is seen in addition to the BL like/PBL-cell lines. This might indicate that they represent clonal progression from the original lymphoma precursor cell, although it is more likely that they are derived from different clones. SPIBL-I could also be a spontaneous lymphoblastoid cell line derived from latently EBV infected cells not related to the tumor, although its origin from the ascitic effusion would be compatible with a tumor origin.At the time of progression the HIV-RNA was non-detectable and CD4+ cell count had increased, although not to an acceptable level. A persevering decreased CD4+ cell count can be reflected in the lack of EBV control . A long Our finding of an EBV-DNA decline and persistence of low EBV-DNA loads clearly reflects the cytostatic treatment effect on the lymphoma. The subsequent, pronounced EBV-DNA increase preceding the lymphoma relapse suggests a value of EBV monitoring in treatment as well as follow up of EBV-positive lymphomas. This indicates the use of EBV-DNA load as a marker for recurrence as well as a treatment effect marker. The use of EBV-DNA load has also been clearly shown in the assessment of risk for EBV-positive PTLD in transplanted patients -25.In HIV-1-infected patients a long lasting decline of CD4+ cells will disrupt the host-EBV balance and increase the risk for EBV related malignancy. In spite of the worldwide access to cART and awareness of HIV-1 there are still a considerable number of undiagnosed HIV-1-infected individuals, who will have developed severe immune deficiency at the time of HIV diagnosis. When initiating cART treatment at the time of HIV-diagnosis in this patient group , repeated analysis of EBV- could be of value in estimating the lymphoma risk.In this rare PBL with effusion our finding of EBV-DNA decline and persistence of low EBV-DNA load reflects the positive effect of cytostatic treatment on the lymphoma. This finding together with a pronounced EBV-DNA increase preceding the lymphoma relapse demonstrates the value of EBV monitoring in treatment of EBV-positive lymphomas in AIDS-patients. The result indicates the usefulness of EBV-DNA load as a marker for recurrence and treatment effects. EBV DNA-load might also be useful as an indicator of lymphoma risk in HIV-infected patients presenting late in HIV-1 infection with severe immunodeficiency.6/L, antiretroviral treatment was initiated and in year three the patient began with cART. In the middle of year four the patient was diagnosed with a lymphoma. The lymphoma went into remission after cytostatic treatment, but recurred three months later with massive pleural effusion. The patient died in middle of year five. Biopsies were taken from the gut the first year, four years prior to the lymphoma diagnosis and from the developed lymphoma on three occasions in year five.A 36 year old homosexual man was diagnosed with HIV-1 in 1993. Two years later a Kaposi conjunctival lesion was removed . For immunocytochemistry an alkaline phosphatase chromogenic substrate was used, whereas for immunohistochemistry a peroxidase-based detection system was employed according to the manual . Pre-treatment protocols for antigen demasking on paraffin sections were used according to standard procedure for each antibody.Flow cytometry was performed with a Becton-Dickinson analyser (Becton-Dickinson).Tissue biopsy materials, aspiration from ascitic and pleural effusions, were collected upon surgical excision or aspiration from the patient, stored in sterile 0.9% NaCl and were set up within 4 hours for tissue culture. Biopsy material was minced with scissors and forceps and passed through a metal wire mesh 3–4 times. The cell suspension thus generated was set up in 12-well microplates with RPMI 1640 and antibiotics. The cultures were followed regularly by microscopical examination and fed by replacing some half the culture medium once every 4–7 days in the beginning. Cells from the first biopsy occasion were transferred to microplates with irradiated fibroblast feeder layer. After one-two weeks clumps or loose aggregates of cells were detected and they were explanted in five mL Falcon plastic tubes on feeder layers after 2–3 months, and subsequently upon expansion of the cells to 25 mL Falcon bottles . The second and third attempts to establish cell lines (SPIBL II and III) from the relapsed pleura effusions were very different and cells grew out much faster than on the first attempt, already 1–2 weeks after in vitro explanation. The established cell lines were used for characterization and also stored away by freezing.The cell lines were analyzed by standard light microscopy.Chromosomes in the cell lines were analyzed by making metaphase plates and stained for Giemsa-banding, according to Zech et al. .Cellular DNA was analyzed using a PCR method .EBV-PCR from blood analysis and statistics were performed as previously reported by Friis et al. . EBV wasEBV: Epstein-Barr virus; cART: Combined anti-retroviral treatment; HIV-1: Human immunodeficiency virus 1; HHV-8: Human herpes virus number 8; PEL: Primary effusion lymphoma; PBL: Plasma blastic lymphoma; SPIBL: Spontaneous immunoblastoid cell line.No commercial relationship or potential conflict of interest related to the submission exists. No non-financial competing interests exist.AF participated in the design of the study, carried out EBV laboratory work and wrote manuscript. BÅ participated in the design of the study, recruited patient and wrote manuscript. KG participated in the design and coordination of the study, recruited patient and wrote manuscript. AA carried out sample preparation and cell line related laboratory work. IE participated in the design of the study, wrote manuscript. BC made pathology evaluations and wrote manuscript. JZZ carried out HHV-8 laboratory work. All authors read and approved the final manuscript.


In an analysis of 26 oligonucleotide drugs recently evaluated in late-stage clinical trials we found that to date at least half have demonstrated suppression of the target mRNA and/or protein levels in the relevant cell types in man, including those present in liver, muscle, bone marrow, lung, blood and solid tumors. Overall, this strongly implies that the drugs are being delivered to the appropriate disease tissues. Strikingly we also found that the majority of the drug targets of the oligonucleotides lie outside of the drugable genome and represent new mechanisms of action not previously investigated in a clinical setting. Despite the high risk of failure of novel mechanisms of action in the clinic, a subset of the targets has been validated by the drugs. While not wishing to downplay the technical challenges of oligonucleotide delivery in vivo, here we demonstrate that target selection and validation are of equal importance for the success of this field.Oligonucleotide delivery  While not wishing to downplay the technical challenges of oligonucleotide delivery in vivo, to our view it plays equal fiddle to a parameter that is rarely discussed, that of target validation.Successful drug development is a precarious combination of the right target, the right drug, the right disease and the right patient population. When one of these parameters is ill-chosen the drug fails. As a handful of oligonucleotide drugs inch toward regulatory approval, consensus according to recent reviews and perspectives is that the ‘delivery problem’ is the pivotal factor which will see this new therapeutic paradigm stand or fall ,2. HowevThe pharmaceutical industry considers the optimal drug target to have a well-understood role in the pathophysiology of the disease, to be disease-modifying when modulated with drugs, to be non-essential in unaffected tissues, to have its expression restricted to the disease tissue, to be assayable with a biomarker to monitor therapeutic efficacy and to have a favorable competitive situation . Drug tavia RNase H-dependent mechanisms. The remainder modulates splicing, inhibits translation or blocks RNA function . Several are extracellular proteins  and therefore potentially drugable with therapeutic antibodies or proteins. The majority of the targets was clinically unvalidated at the outset of the trials and therefore represents a new mechanism of action for their respective diseases. Three of the targets—APOB, TTR and DMD—are the products of genes which are disregulated or non-functional because of changes in their DNA sequence  and which have been proven to cause disease. Hence, they are ‘genetically validated’.The targets of these oligonucleotides come from a range of protein families which is notably under-represented in the classical drugable families, i.e. enzymes and receptors. Of the targets, five are enzymes and therefore possibly drugable with small molecules  mir-122. In these 14 cases it can be safely assumed that the oligonucleotide drug is being delivered to its cellular target, although it cannot be assumed that the magnitude of target downregulation is sufficient to mediate a therapeutic effect.We highlight below 14 drugs for which inhibition of the molecular target at either mRNA or protein level in patient samples has been reported. They are: ApoB/mipomersen for familial hypercholesterolemia (FH) [1]. The liver-derived apoliprotein B-100 (ApoB) protein plays a fundamental role in cholesterol homeostasis as a structural component of very low-density lipoprotein (VLDL) and as a ligand for LDL receptor-mediated endocytosis of LDL by liver cells. Its over-production and decreased clearance are seen in cardiovascular-associated diseases, as well as inherited diseases such as familial hypercholesterolemia (FH: OMIM 107730). Familial defective ApoB hypercholesterolemia is a result of non-sense or frame-shift mutations within the LDL-receptor binding domain of ApoB and causes severe hypercholesterolemia and premature cardiovascular disease. Statin drugs are unable to reduce cholesterol sufficiently for these patients. ApoB is an ideal target for an mRNA-targeting oligonucleotide in FH (reviewed in (O-methoxyethyl (MOE)-ribonucleotides. It induces ApoB mRNA degradation. It has been tested in numerous clinical trials including phase 3 studies in which efficacy endpoints were met (iewed in ). It is were met ,10. The were met . In recewere met ) constitwere met . The druwere met . It seemDystrophin/eteplirsen, drisapersen for Duchenne muscular dystrophy (DMD) . Dystrophin is essential for the normal function of skeletal and cardiac muscle. Its gene is prone to mutations that result in muscular dystrophy (muscle weakening) (OMIM: 300377). DMD is mostly due to DNA deletions which disrupt the reading frame and produce a dysfunctional protein. High affinity, chemically modified ASOs can be designed to bind sequence-specifically to pre-mRNA and induce ‘skipping’ of exons containing out-of-frame deletions leading to accumulation of a shortened but nevertheless partially functional protein. This concept was first shown in cells and has been validated in numerous examples in vivo (O-methyl-phosphorothioate RNA (drisapersen) oligonucleotides which induce exon skipping in the dystrophin mRNA. In pioneering clinical trials, treatments of DMD patients with eteplirsen and drisapersen have led to restoration of the functional target protein in high percentages of muscle fibers thereby validating the target . In a phase 1-2a study of drisapersen treatment of young patients, weekly subcutaneous injections of 0.5–6.0 mg/kg of drug led to new dystrophin expression through exon-51 skipping in all patients receiving the two highest doses. All trial participants then received the highest dose of drug in a 12-week extension phase resulting in a mean improvement of 35 m in the functional walk test /oblimersen for cancer [4]. Several late-stage oligonucleotide drugs have been or are being investigated for treatment of cancers. Three of the targets—clusterin, survivin, Bcl-2—have well-characterized roles in mechanisms which protect cancer cells from apoptosis induced by cytotoxic drugs. The pro-survival factor Bcl-2 inhibits apoptosis and enhances cell survival by inhibiting the release of cytochrome c during apoptosis. Bcl-2 overexpression in chronic lymphocytic leukemia (CLL) is associated with aggressive disease and resistance to chemotherapy. Bcl-2 family members heterodimerize with pro-apoptotic regulators and efforts to identify small-molecule drugs to inhibit these protein–protein interactions have been largely unsuccessful. One exception is the Bcl-2 antagonist obatoclax mesylate. The drug is in clinical trials however the results to date have not shown strong activity . In oneactivity . In anotactivity . Target activity . ClinicaSurvivin/LY2181308 for cancer [5]. Survivin is a member of the Inhibitors of apoptosis (IAP) family. In cells, it functions as a stress-induced cytoprotective chaperone exerting its anti-apoptotic effects through binding to the second mitochondrial activator of caspases (SMAC) (reviewed in (O-(MOE) ribonucleotides which targets the translation initiation codon of survivin. An account of a first in-human dose study described how the drug was delivered intravenously as a saline solution initially daily for 3 days as a loading dose and then up to 750 mg weekly into cancer patients. Survivin mRNA and protein in a wide range of tumor tissues were both reduced by ∼20% and the apoptosis marker CC3 indicated restored apoptosis, thereby validating the clinical rationale (iewed in ). It is ationale . Positroationale . No objeClusterin/custirsen for cancer [6]. Clusterin is a stress-induced, cytoprotective small heat-shock chaperone protein. It is expressed in virtually all tissues and is secreted. In cancer clusterin inhibits apoptosis through its suppression of proapoptotic BAX and activates the PI3K/AKT cell survival pathway (reviewed in (iewed in ). Clusteiewed in . Treatmeiewed in . This driewed in . These tiewed in  or time Hsp27/OGX-427 for cancer [7]. Heat Shock 27 kDa Protein 1 (Hsp27) is another small heat shock protein under clinical investigation as a target for cancer treatment using a MOE-modified ASO. It is highly expressed in several types of cancer: in castration-resistant prostate cancer (CRPC) its expression is induced by androgen removal and/or chemotherapy. It inhibits the activation of caspases as well as activating STAT3 and AKT oncogenic pathways. It is therefore an attractive target in cancer however it is undrugable with small molecules. OGX-427 is a 20-mer MOE ASO which has completed single agent and combination phase 1 trials to determine dose in prostate, bladder, as well as breast and lung cancer. Reduction of tumor markers in prostate (prostate-specific antigen (PSA): >40% reduction in 3 from 16 patients) and ovarian (cancer-antigen 125: >20% reduction in 3 from 5 patients) cancers was reportedly observed in patients, as well as decreased numbers of circulating tumor cells and lowered Hsp27 expression measured by immunofluorescence  is often upregulated in tumors (n tumors . A 20-men tumors . The ASORNR/GTI-2040 for cancer [9]. Overexpression of ribonucleotide reductase (RNR), an enzyme composed of R1 and R2 subunits which converts ribonucleosides to deoxyribonucleosides, is commonly observed in cancer cells. It increases levels of deoxynucleosides, thereby competing with nucleoside cytotoxics and providing a mechanism of chemoresistance. RNR has been validated as a target in cancer using small-molecule drugs however selectivity issues with such compounds have been reported . GTI-20reported . A stronreported . Levels reported : continuc chain/TPI ASM8 for asthmaCCR3, β [10]. Eosinophils play an important pathogenic role in allergic asthma. The late asthmatic response (LAR) to inhaled allergen challenge induces the proliferation and migration of eosinophils. This depends upon chemokine signaling involving C-C chemokine receptor type 3 (CCR3) and the stimulation of receptors for the cytokines IL-3, IL-5 and granulocyte-macrophage colony-stimulating factor (GM-CSF) comprising a common beta chain (βc). Indeed, IL-5 has been proposed as an important drug target for allergy and associated disorders  and at r 4 days . A signi results ,49. DrugICAM1/alicaforsen for pouchitis [11]. Intercellular adhesion molecule-1 (ICAM1) is a transmembrane glycoprotein expressed in endothelial cells and leukocytes. It facilitates leukocyte migration from blood to sites of inflammation and is involved in inflammatory bowel pathophysiology. Inflammatory bowel disease (IBD) encompasses an array of disorders including Crohn’s disease (CD) and ulcerative colitis (UC). ICAM1 was selected as a target for IBD because of its role in cell trafficking, because it is present during intestinal inflammation and because increasing levels of circulating protein are observed in patients with CD, UC and pouchitis. Alicaforsen is a 20-mer phosphorothioate oligodeoxynucleotide targeted to ICAM1. Several clinical trials of alicaforsen have been completed (reviewed in (iewed in ). Cliniciewed in . It has iewed in . Alicafoiewed in , but theiewed in . The resiewed in ). IntereC-Reactive protein/ISIS-CRPRx for cardiovascular disorder [12]. C-reactive protein (CRP) is produced in the liver and increased levels of the protein are seen in a variety of disorders including cardiovascular disease, diabetes and various inflammatory conditions. ISIS-CRPRx is a MOE ASO which recently completed a phase 1 clinical trial in 80 subjects at single and multiple doses from 50–600 mg/week . An average reduction of more than 70% in circulating CRP was reported. The drug sponsors have announced that this first CRP inhibitor will be employed as an early proof of concept for the target in a variety of diseases, including multiple myeloma and rheumatoid arthritis.TTR/ALN-TTR02 for amyloidosis [13]. We identified five siRNAs in phase 2 clinical trials . From these, target suppression has only been reported for one—ALN-TTR02, though a working RNAi mechanism in man was shown for a formulated siRNA whose development has since been discontinued  is caused by mutations in the transthyretin gene (TTR), which is expressed predominantly in the liver, and results in the accumulation of pathogenic deposits of mutant and wild-type TTR protein in multiple extra-hepatic tissues, including the peripheral nervous system and heart. The intravenously administered, lipid nanoparticle (LNP)-formulated chemically unmodified siRNA ALN-TTR02 targets the mutant TTR protein responsible for TTR-mediated amyloidosis. Recently this siRNA has progressed into phase 2 trials following promising phase 1 results in which the drug was well tolerated and demonstrated an up to 94% reduction in serum TTR levels in healthy volunteers after a single dose (0.01–0.50 mg/kg) . Furthermore, TTR downregulation was reportedly sustained at up to 80% 1 month after treatment ceased.ontinued . The herMiscellaneous drug/target combinations not reporting target inhibition. Many of the oligonucleotide drugs in advanced clinical trials reportedly have positive effects in patients but, for a variety of possible reasons, the PD data describing inhibition of target by the drug has not been reported (14–26]). Miravirsen (SPC-3649: (PC-3649: . Circula15]) is a 20-mer first generation phosphorothioate oligodeoxynucleotide targeting TGF-β2 mRNA in phase 3 clinical trials. The drug has generated promising data in numerous clinical trials for the treatment of malignant brain cancers after local delivery directly into tumors (reviewed in (Trabedersen (AP12009:  which targets the nucleocapsid protein of respiratory syncytial virus (RSV), a structural protein responsible for encapsidation of the viral genome. This siRNA is an unmodified 19-mer RNA phosphorodiester duplex with 3′-overhangs of thymidine deoxynucleotides. In phase 1 trials it proved to be safe and well tolerated in healthy subjects  and in eProperly designed oligonucleotides are specific inhibitors of gene expression. They are well suited to the modern paradigm of pharmaceutical research which begins with the gene coding for a disease-relevant target. Where a target represents a new mechanism of action and is being investigated for the first time in patients there is a steep learning curve and a high probability of failure . We examIE2 gene of human cytomegalovirus which was administered by intravitreal injection . Aftiewed in . HoweverTarget selection is a critical phase in drug discovery, particularly for oligonucleotide drugs. Decreasing productivity in pharma R&D has cast doubts on the modern paradigm of pharmaceutical research that begins with a target gene: many successful drugs originally discovered by compound profiling modulate multiple targets and it is recognized that drug targets often have redundancy pathways , limitinAPOB, DMD, TTR) are genetically validated: mutations in the DNA sequence of the gene cause the disease. For APOB and DMD, second generation ASOs  have shown the expected effects on the target protein in patients in multiple clinical trials. In turn, this has yielded improvements in disease thereby providing clinical validation of the targets. A subset of the targets  is being investigated for cancers in combination with approved cytotoxic drugs. Varying degrees of target mRNA and protein inhibition by oligonucleotides have been observed in tumor tissues in patients and in many cases this has been associated with anti-tumor activity, However, the general lack of robust effects on disease outcome seen so far leads some investigators to question whether it is insufficient target inhibition due to sub-optimal drug delivery or an invalid target which is to blame  have alOligonucleotide drugs still represent a promising new paradigm in drug discovery/development. Emerging data have confirmed that they are delivered to many organs/tissues at concentrations sufficient to inhibit their RNA targets and see therapeutic effects in man. New robust methods of, for example, LNP-formulated delivery to currently inaccessible tissues may expand their application further, once associated issues of toxicity, manufacturing complexity and cost have been solved. Although in principle, oligonucleotides can be targeted to any gene of the genome, in reality, they will likely be most successful when targeting genes in new mechanisms outside of the drugable genome and in monogenic diseases.Novartis Foundation (formerly the Ciba Geigy Jubilee Foundation) (to H.L.L.). Funding for open access charge: ETH Zürich, Zürich.Conflict of interest statement. None declared.


ESRRB) gene is the underlying cause of autosomal recessive nonsyndromic hearing impairment (ARNSHI) due to the DFNB35 locus which maps to 14q24.3. A genome scan of a large consanguineous Pakistani pedigree with ARNSHI established linkage with a maximum multipoint LOD score of 4.2 to the 14q24 region and the region of homozygosity contained the ESRRB gene. Sequencing of the ESRRB gene using DNA samples from hearing-impaired family members uncovered a novel three-nucleotide deletion c.1018_1020delGAG (p.Glu340del). The deletion segregates with hearing impairment in the pedigree and was not observed in 500 control chromosomes. The deletion of glutamic acid residue occurs in the ligand-binding domain of ESRRB protein. It is expected that the deletion affects the ligand-binding activity of the domain in ESRRB, which leads to the ARNSHI.Mutations in the estrogen-related receptor beta ( ESRRB gene . ESRRB belongs to a subfamily of orphan nuclear receptors that are structurally related to estrogen receptors (ER) but do not directly bind estrogen. The ESRR proteins have DNA-binding and ligand-binding domains, which are both required for transcriptional regulation of ER target genes. The ligand-binding domain (LBD) provides structural stability and influences the binding affinity of the ESRRB protein [ESRRB gene which is responsible for ARNSHI. Hearing impairment (HI) has been associated with estrogen physiology for decades. Turner's syndrome in females and Noonan's syndrome in males are both marked by hypoestrinism and include the HI phenotype. Variations in hearing function have also been found according to gender, menstrual cycle or menopausal state, estrogen therapy, and oral contraceptive use, which implicate estrogen as a primary requirement for hearing preservation . Recentlne MIM 60167; 2. EThe study was approved by the Institutional Review Boards of the Quaid-I-Azam University and the Baylor College of Medicine and Affiliated Hospitals prior to initiation. Informed consent was obtained from all members from family 4243 who participated in the study. Family 4243  is a conGenomic DNA was extracted from venous blood which was provided by five hearing and four HI family members . The ninESRRB (RefSeq NM_004452.3) gene were designed using Primer3 software [Exon primers for software . PCR-ampESRRB-related HI [The audiogram of HI individual IV-4 displays bilateral, severe-to-profound HI affecting all frequencies . This islated HI . θ = 0) was observed at marker rs935340  in exon 8 which segregates with ARNSHI . The glutamic acid residue 340 is located in the ligand-binding domain of the ESRRB protein. The identification of the deletion mutation of ESRRB gene will expand our understanding of hearing impairment due to mutations in the estrogen-related gene ESRRB.A genome-wide linkage scan performed using DNA samples from a large consanguineous ARNSHI pedigree and the subsequent sequencing of the


Patients presenting with suspected deep vein thrombosis (DVT) are common in the emergency department. Evidence based strategies for the diagnostic evaluation of these patients exists. The diagnosis is based on three criteria: a clinical pre-test probability score (Wells score), D-dimer testing, and ultrasound examination of the deep veins. The aim of our study was to evaluate the adherence to these guidelines in our emergency department.In a retrospective design 252 patients admitted to our department for the suspicion of DVT in 2010 were identified. All patient records were reviewed for the documentation of a pre-test clinical probability score, the result of D-dimer testing and the result of any ultrasound testing. The diagnostic work-up vas categorised as either: appropriate, inappropriate, or indeterminable due to lack of data.Wells score was documented in 92 (37%) cases. D-dimer was measured in 252 (100%) cases and 175 (69%) patients underwent an ultrasound examination. Ultrasound examination was repeated in 18 cases.The diagnostic work-up could be considered appropriate in 106 (42%) cases. In 53 (21%) cases the diagnostic work-up was inappropriate, in 48 (91%) cases because an ultrasound examination was not performed although indicated by the guideline and in five (9%) cases because an ultrasound examination was performed although not indicated. In 93 (37%) cases the diagnostic work-up could not be classified as appropriate or inappropriate, in all cases due to lack of documentation of Wells score.We identified three cases were the patient was readmitted and diagnosed with DVT (n=2) or pulmonary embolism (n=1) less than three months after DVT had been ruled-out. In all three cases the appropriateness of the initial diagnostic work-up could not be determined due to lack of documentation of Wells score.We found poor adherence to diagnostic guidelines in our department. Documentation of the pre-test clinical probability is often missing and we found that under-utilisation of ultrasound is much more common than over-utilisation. Further more we found some indication that cases of DVT might have been misdiagnosed because Wells score was not applied in the diagnostic work-up.


Photodynamic procedures are based on the light-induced, photosensitizer-mediated overproduction of reactive oxygen species for removal of harmful or unwanted cells/pathogens. With approvals for various applications by health agencies in most industrial countries, Photodynamic Therapy (PDT) represents the method of choice for treatment of age-related macular degeneration and is appreciated as minimally invasive therapeutic procedure to treat skin, esophageal, head and neck, lung, and bladder cancers with high cure rates, low side effects, and excellent cosmetic outcome. Being motivated by the success of PDT in management of various human diseases, new and very promising applications of this procedure are being identified and explored by the PDT research community. So, for example, Photodynamic Inactivation of microorganisms (PDI) has the potential to avert the severe threat of increasing antimicrobial resistance. This special issue presents a collection of papers dealing with novel photosensitizers with improved photochemical and photophysical properties, alternatives to (single) photon excitation of the photoactive drug, new clinical targets of photodynamic procedures, and innovative reviews on crucial aspects, which substantiate the success of Photodynamic Therapy and Photodynamic Inactivation.Hydrogen bond acceptors and additional cationic charges in methylene blue derivatives: photophysics and antimicrobial efficiency,” A. Felgenträger et al. report on the synthesis and photodynamic efficiency of six novel methylene blue derivatives with highly polar and/or hydrophilic groups. Based on a systematic analysis of singlet oxygen formation in combination with the absorbed light energy and on investigations of the phototoxicity towards Staphylococcus aureus and Escherichia coli, a structure-activity relationship has been drawn from a chemical point of view. The data presented in this study demonstrate that hydrogen acceptor bond moieties and additional tertiary charges in the substituent have a positive influence on the overall antimicrobial efficacy of methylene blue derivatives, resulting in a convincing inactivation of viable Gram (+) or Gram (−) bacteria up to 7 log units.In a research paper entitled “N-methyl-D-glucamine gold nanoparticles and the respective photodynamic activity is described by B. Lkhagvadulam et al.'s “Size-dependent photodynamic activity of gold nanoparticles conjugate of water soluble purpurin-18-N-methyl-D-glucamine”. The authors demonstrate that the PDT efficiency of photosensitizer-nanoparticle conjugates against A549 lung cancer cells is higher when compared to the unbound photosensitizer because of the increased internalization of the PS into the target cells using the size effect. The highest photodynamic activity is reported to be associated with rather bigger conjugates with a diameter of about 60 nm.Conjugates of nanoparticles and photosensitizers represent a very promising new generation of photoactive substances. The correlation of the size of purpurin-18-Photo-induced antitumor effect of 3,6-Bis(1-methyl-4-vinylpyridinium) carbazole diiodide” evaluated the photo-induced antitumor effect of 3,6-bis(1-methyl-4-vinylpyridinium) carbazole diiodide (BMVC) in a lung tumor cell line (TC-1) and the corresponding in vivo murine model (TC-1 cell tumors in C57BL/6 mice). BMVC is able to induce a phototoxic effect in TC-1 cells at light fluences greater than 40 J/cm² and significantly reduced the tumor growth rate in vivo following fine-needle interstitial light illumination of the photosensitizer. Immunohistochemistry studies showed that the microvascular density was lower not only in the PDT group but also in animals, which received light only (without photosensitizer). Therefore, the antivascular effect might be partially attributed to mild hyperthermia induced by the laser illumination. Y-.S. Chou et al.'s “Photosensitizer adhered to cell culture microplates induces phototoxicity in carcinoma cells” discusses the phototoxic effect of adherence of photosensitizers on the surfaces of cell culture microplates. Using two rather lipophilic and two hydrophilic substances currently employed in PDT, the authors show that lipophilic photosensitizer molecules adhered to microplates are able to induce cytotoxicity in A431 human epidermoid carcinoma cells upon illumination, independently of whether the photoactive molecule relocalizes from the surface into cells or not. This effect is negligible for hydrophilic photosensitizers. The ability of plastic materials to (reversibly) store photosensitizers might, according to V. Ziegler et al. represent a new approach for photosensitizer delivery or development of antimicrobial coatings.The study of V. Ziegler et al. entitled “Sonodynamic excitation of rose bengal for eradication of Gram-positive and Gram-negative bacteria” excited the photosensitizer Rose Bengal with 28 kHz ultrasound. The results presented in the study demonstrate for the first time that the novel “sonodynamic” activation of Rose Bengal is able to reduce the number of viable Staphylococcus aureus and Escherichia coli by up to 4 log units. Sonodynamic treatment represents a promising approach for curing internal infections and sterilization.Inactivation of microorganisms within skin or tissue based on photosensitizers activated by illumination may be complicated by the relatively low penetration depth of visible light. As an alternative to light illumination, F. Nakonechny et al.'s “Innovative strategies to overcome biofilm resistance” by A. Taraszkiewicz et al. strategies to combat bacterial microfilms are discussed. If organized in biofilms , bacteria are much more resistant to antimicrobial therapies, including Photodynamic Inactivation. The authors suggest two innovative approaches to increase the bactericidal effect of PDI, namely, the employment of enzymes that affect the biofilm or a combination of PDI with antibiotics, plant extracts, or other biofilm disrupting substances. Application of PDI together with enzymes specific for microbial structures might furthermore increase the selectivity of phototoxicity and/or allow for the usage of lower photosensitizer concentrations. In a comprehensive review article entitled “In vitro analysis of photosensitizer accumulation for assessment of applicability of fluorescence diagnosis of squamous cell carcinoma of epidermolysis bullosa patients” investigated the applicability of fluorescence diagnosis of hypericin and endogenous protoporphyrin IX using an in vitro model system, consisting of different (EB) skin cell lines exposed to normal and proinflammatory conditions. A proinflammatory milieu simulated by addition of TNF-alpha appeared not to influence photosensitizer accumulation in skin cells. Carcinoma cells of recessive dystrophic EB showed lower hypericin or PpIX-induced fluorescence than nonmalignant recessive dystrophic EB cells. Further experiments will be necessary to evaluate the possible application of fluorescence diagnosis for early cancer detection of EB patients.Patients suffering from the genetically induced skin disorder epidermolysis bullosa (EB) are exposed to an increased skin tumor risk. P. Larisch et al.'s “Two-photon photodynamic therapy by water-soluble self-assembled conjugated porphyrins” review a set of studies on two-photon PDT with water-soluble and self-assembling conjugated porphyrins. Based on data of two-photon cross section values obtained by open-aperture Z-scan measurements, singlet oxygen production and phototoxicity towards cancer cells in vitro, the conjugated porphyrins are suggested as candidates for two-photon PDT agents.Two-photon absorption is a nonlinear optical process, by which two photons are absorbed simultaneously, thus allowing for doubling the wavelength of excitation of a given photosensitizer in PDT, thereby increasing the penetration depth of the PDT illumination light in tissue and allowing for treatment of thicker malignancies. In this issue, K. Ogawa and Y. Kobuke through their paper “Killing effect of Ad5/F35-APE1 siRNA recombinant adenovirus in combination with hematoporphrphyrin derivative-mediated photodynamic therapy on human nonsmall cell lung cancer,” L. Xia and coworkers report on the PDT efficiency and the molecular mechanism of cytotoxicity of the Ad5/F35-APE1 siRNA recombinant adenovirus in combination with hematoporphyrin derivative (HpD) in A549 human lung adenocarcinoma cells. Infection of cancer cells with the Ad5/F35-APE1 siRNA recombinant adenovirus significantly reduced the HpD-PDT-induced expression of the APE1 protein and could therefore enhance the photokilling of HpD-mediated PDT.In the paper entitled “Effective combination of photodynamic therapy and imiquimod 5% cream in the treatment of actinic keratoses: three cases” of L. Held et al. evaluate the therapeutic effect of combining PDT using 5-aminolevulinic-acid-(ALA-) induced PPIX as photosensitizer with imiquimod 5% cream for the clinical treatment of actinic keratoses. Imiquimod is known to regulate the level of cytokines and thereby to influence the immune response and was applied to the treatment area two weeks after PDT. Although a rather small population of patients was part of the pilot study, the results are promising and should be studied in subsequent larger clinical trials: PDT followed by imiquimod treatment was well tolerated and combination of both improved the reduction of actinic keratoses, as demonstrated by the authors. Three papers discuss the interconnection between PDT and the host's immune response. The methodology report entitled “Use of photodynamic therapy for treatment of actinic keratoses in organ transplant recipients”. It is well known that long-term immunosuppressive medication performed on organ transplant recipients increases the risk to develop skin cancers and/or their precursors. The authors present PDT as efficient, safe, and well-tolerated treatment option for these malignancies in immunosuppressed patients but point out that early and numerous treatments on a regular basis are necessary to obtain an optimal response and to prevent development of new actinic keratoses.Photodynamic treatment of actinic keratoses and nonmelanoma skin cancer in solid organ transplant recipients as well as alternatives to PDT are reviewed by C. Wlodek et al.'s “Immunogenic cell death: can it be exploited in photodynamic therapy for cancer?” review the undoubted key role of the immune response triggered by PDT for the control of cancer cells revival after an antitumor treatment. By elucidating the role of damage associated molecular patterns (DAMPs) at the crossroad between cancer cell death and immunogenicity in PDT, the convincing advantage of PDT over several other cancer treatment options, which is to potentially elicit a specific antitumor immunity, is discussed.E. Panzarini et al. in their paper “A comprehensive tutorial on in vitro characterization of new photosensitizers for photodynamic antitumor therapy and photodynamic inactivation of microorganisms” present a comprehensive tutorial on the initial experimental procedures in vitro, which help to identify possible applications of newly synthesized photosensitizers in the frame of PDT/PDI. The article covers a detailed description of the experimental approach to the characterization of the photochemical and photophysical properties of a photosensitizer, its uptake kinetics into cells, intracellular localization, and photodynamic action in both, tumor cells and microorganisms. The authors hope that the special issue on PDT in general and the tutorial article in particular will help to stimulate the efforts to expand the convincing benefits of photodynamic procedures within both established and new fields of applications.The process of scientific development of new applications of PDT is still highly active and viable. The finalizing contribution of this special issue is therefore an invitation to other researchers to join the successful photodynamic community. T. Kiesslich and coworkers in the paper entitled “