In the present study, we have investigated whether CB1IR was assessed in formalin-fixed, paraffin-embedded specimens collected with a consecutive intent during primary tumour surgical resection from a series of cases diagnosed with colorectal cancer. Tumour centre (n = 483) and invasive front (n = 486) CB1IR was scored from 0 (absent) to 3 (intense staining) and the data was analysed as a median split i.e. CB1IR <2 and ≥2. In microsatellite stable, but not microsatellite instable tumours , there was a significant positive association of the tumour grade with the CB1IR intensity. The difference between the microsatellite stable and instable tumours for this association of CB1IR was related to the CpG island methylation status of the cases. Cox proportional hazards regression analyses indicated a significant contribution of CB1IR to disease-specific survival in the microsatellite stable tumours when adjusting for tumour stage. For the cases with stage II microsatellite stable tumours, there was a significant effect of both tumour centre and front CB1IR upon disease specific survival. The 5 year probabilities of event-free survival were: 85±5 and 66±8%; tumour interior, 86±4% and 63±8% for the CB1IR<2 and CB1IR≥2 groups, respectively.CB1 receptor expression in colorectal cancer is associated with the tumour grade in a manner dependent upon the degree of CpG hypermethylation. A high CB1IR is indicative of a poorer prognosis in stage II microsatellite stable tumour patients.The level of CB However, CB1 receptors and their endogenous ligands anandamide (arachidonoylethanolamide) and 2-arachidonoylglycerol mediate a multitude of effects in the body including the regulation of pain, bone formation, energy homeostasis and gastrointestinal function 1 receptors are found in the crypt epithelium as well as in subepithelial inflammatory cells, in smooth muscle of blood vessels and in submucosal plexus The G-protein coupled cannabinoid1−/− mice than in their wild type littermates, whilst treatment with a CB receptor agonist, or with compounds blocking the cellular removal and metabolism of endocannabinoids, alleviates the inflammation 1 receptor polymorphism (1359 G/A) is associated with a reduced susceptibility to ulcerative colitis in man 1 receptor inverse agonist In addition to the functions described above, the endocannabinoid system acts as a “damage limiting” system to mitigate the effects of pathological situations. This appears to be particularly true for the gastrointestinal endocannabinoid system. Thus, inflammation induced by agents such as 2,4-dinitrobenzene sulfonic acid, trinitrobenzene sulfonic acid, mustard oil or dextran sulfate sodium is more pronounced in CB1-dependent and -independent effects of endogenous and/or synthetic cannabinoids upon cell viability have been reported Min/+ mice), animals lacking the CB1 receptor showed a greater number of small intestinal and colonic polyps than the corresponding CB1+/+ mice 1 receptor polymorphism show a shorter survival time than the G/G wild-type patients 1 receptor mRNA and protein have been reported in colorectal cancer Cannabinoids and endocannabinoids produce potentially useful effects upon cancer cell proliferation, motility and invasive behaviour  approved the handling of tissue samples and patient data in the present study, including the procedure whereby patients verbally gave their informed consent. This consent was documented in each patient record, and this was considered by the ethical committee to be sufficient. In the database used for the analyses here, the tissue samples were given a case number and year, and the patient names were not indicated in the database.1 receptor immunoreactivity (see below). The clinical information in the database for these patients was as follows: median age 71 years ; cancer location in right colon 31.1%, left colon 31.1%, rectum 37.8% (n = 482); disease stage I 15.5%, II 39.3%, III 21.0%, IV 24.2% (n = 476); tumour grade well/well-moderately differentiated 48.75%, moderate-poor/poorly differentiated 51.25% (n = 480); microsatellite stable 85.0%, microsatellite instable 15.0%; (n = 473); CIMP status negative 50.4%, low 37.2%, high 12.4% (n = 484). Further, 82.4% (of 483 cases) did not receive preoperative radiotherapy (either 5×5 Gy or 25×2 Gy); 75.0% (of 476 cases) had radical surgery; and 13.7% (of 475 cases) received adjuvant chemotherapy.The formalin-fixed, paraffin-embedded samples used in the present study were obtained from tissue collected during primary tumour surgical resection of colorectal cancer ). The samples were collected with a consecutive intent at the Department of Surgery, Umeå University Hospital, Sweden, during the period 1995–2003 and where possible the patients were followed for up to 113 months 1 receptor antibody  and the secondary components  were then added. The antibody, a rabbit polyclonal raised to an peptide corresponding to C terminal amino acids 461–472 of the human CB1 receptor and which cross-reacts with the human, mouse and rat CB1 receptor according to data from the manufacturers, has been shown previously by researchers in Umeå (including the corresponding author) to produce the appropriate pattern of staining in human cerebellum, but not to produce immunoreactivity in forebrains from CB1−/− mice http://expasy.org/tools/blast/) of the peptide sequence gave CB1 and its two splice variants as the only hits in man. In several other species, CB1 receptors were again identified, the only non-CB1 hits being from proteins termed “Uncharacterized protein [Gene: CNR1]”  as well as “Putative uncharacterized protein [Gene: PANDA_015085] - Ailuropoda melanoleuca (Giant panda)” and “Chromosome 14 SCAF15003, whole genome shotgun sequence fragment” in Tetraodon nigroviridis (Green pufferfish).The paraffin-embedded tissue sections were deparaffinized and rehydrated before antigen retrieval in a pressure cooker  in Diva Decloaker . Samples were subsequently placed in a Ventana semiautomated immunostaining machine . The CB1IR was assessed by one investigator (SBG) who was blinded to the clinical status of the patients. The samples were graded on the basis of the dominant CB1 receptor immunoreactive intensity (CB1IR) in the tumour interiors and in the tumour invasive fronts and scored from 0 (absent) to 3 (intense) for the cells. When all samples had been scored, the investigator repeated the procedure (without access to the previous scores) and then compared the scores on the two runs. Cases where the scores were divergent were then assessed a third time, again without access to the previous scores. For the tumour front samples scored, for example, there were 53 occasions where the first and second scores disagreed, due mainly to erroneous scores from the initial stage of the first run. The final scores were then entered by another investigator (CJF) into the database for analysis.CB2 tests were undertaken using the statistical package built into the GraphPad Prism 5 computer programme for the Macintosh . Cox proportional-hazards and binary logistic regression analyses were conducted using IBM SPSS statistics 19 software for the Macintosh . For survival analyses, disease-specific events were defined as death with known disseminated or recurrent disease (“†ca”). Death from other causes was censored, as were the cases where the patient was still alive at the date of last follow-up. The duration of event-free survival is defined as the time from diagnosis until either the date of colorectal cancer death, death of other causes, or if no death occurred, until the date of last follow-up.Kaplan-Meier survival analyses, Fisher's exact test and χ1IR was found in the epithelial cells of the crypts, with scattered positivity in subepithelial inflammatory cells , is not sufficient to remove antibody-bound positive control peptide adhering non-specifically to glass and/or to zwitterionic lipids.A common way of testing for antibody specificity is to investigate preadsorbtion of the antibody with the immunising peptide, although the usefulness of this measure has been questioned 1IR intensity and entered into the database. Both plasma membrane and cytoplasmic CB1IR was scored, so the values represent the total pool of CB1 receptors. Nuclear staining of CB1IR (found in 42 cases) was not scored. The frequency distributions of the CB1IR for the tumour centre and fronts were similar, with a score of 0 being returned for 77 and 60 cases; a score of 1 for 196 and 185 cases; a score of 2 for 140 and 156 cases; and a score of 3 for 70 and 85 cases . There was no significant difference in the distribution pattern of the two sets of scores . It was noted that the scores for the two regions were not always the same for a given case. Indeed, for the 482 cases scored for both tumour centre and front, the scores were the same in only 319 (66%) of the cases, being higher in the tumour front in 110 (23%) cases and higher in the tumour centre in 53 (11%) cases. In consequence, throughout this study, both tumour centre and tumour front scores have been analysed separately. Non-malignant tissue was not scored, but in general its level of immunoreactivity appeared to be lower rather than higher than seen for the tumour tissue.A total of 483 (tumour interior) and 486 (tumour front) cases were scored for CB1IR, the analyses were conducted using a simple median split, i.e. CB1IR<2 and ≥2. Using the entire data set to search for variables, a binary logistic regression with parameters gender, site , radiotherapy (prior to surgery), disease stage, tumour histological grade (i.e. differentiation), tumour type (i.e. mucinous or non-mucinous), microsatellite instability screening status [stable (MSS) or instable (MSI)], amount of lymphocytes at the tumour front and the frequency of tumour buds . These effects can simply be visualised by dividing the dataset into four groups on the basis of the two significant parameters , tumour grade, whether the tumours were mucinous or non-mucinous, incidence of lymphocytes at the tumour front or CIMP status . For gender, the p values for the CB1IR distributions were 0.069 and 0.18 for tumour centre and front, respectively (Fisher's exact test), and for the incidence of buds at the tumour front, the p values (χ2 test) were 0.086 and 0.073, respectively (data not shown).In view of the frequency distribution of the CBrameters . The majSS cases . Further1IR, the CIMP distributions were: MSS (n = 396), negative 225 (57%), low 153 (39%) and high 18 (4.5%); MSI (n = 71), negative 15 (21%), low 17 (24%) and high 39 (55%) . When the CIMP status of the samples was added into the binary logistic regression of the whole dataset with the parameters described above, the significant effect of the tumour histological grade was retained (p<0.005) for both tumour centre and front CB1IR, but the effect of the microsatellite instability screening status was lost for the tumour centres (p>0.1) but not for the tumour fronts (p<0.05) (data not shown). In order to investigate this further, the data for the samples was divided into the three CIMP groups: negative, low and high regardless of microsatellite instability screening status or whether the patients had received radiotherapy prior to surgery. There was a clear influence of CIMP status on the results, where the effect of the tumour histological grade was seen in the cases with a CIMP-negative score but not in the cases with a CIMP-high score, the CIMP-low cases being somewhere in the middle  0.60 [95% CI 0.40–0.91] p<0.05) where the number of cases not receiving/receiving radiotherapy was 441/102. The Cox proportional hazards regression analysis is a test used to determine the contribution of the parameter(s) under investigation upon disease-specific survival without making assumptions as to the nature of the survival curve. The measure Exp(B), sometimes called the hazards ratio, indicates the change in risk as the parameter under investigation is changed from the default parameter (here no radiotherapy) to the test parameter (here radiotherapy). In this case, the radiotherapy reduced the risk of death due to the disease since the Exp(B) value was significantly lower than unity. In order to remove the influence of this parameter, the CB1IR data was analysed only for the cases that did not receive radiotherapy. In addition, the MSS and MSI cases were analysed separately.The cases in the database had been followed for up to 113 months 1IR upon disease-specific survival. However, when a bivariate analysis was undertaken with disease stage as the second parameter, a highly significant contribution of CB1IR was seen . Upon further subdivision of the stage II cases according to cancer site, a significant contribution of both tumour centre and front CB1IR upon disease-specific survival was seen for the rectal cancers, but not for the colon cancers, although the direction  was the same. It is important to note, however, that interpretation of results with these subgroups are limited by a low power and are not supported by the Cox analyses (see below).For the MSS cases, a univariate Cox proportional hazards regression analysis failed to show a significant effect of either tumour interior or front CBwas seen . This waeen , the influence of the tumour histological grade upon disease outcome was reduced, whereas the influence of both tumour center and front CB1IR was significant  1.77  p<0.05).The fact that in the MSS patients the CBanalyses , where inificant . These d1IR impacted upon disease-specific survival in stage II cases, simply because of the 32 cases that fell into this category (after exclusion of cases receiving radiotherapy prior to surgery), only one died of the cancer during the follow-up period.For the MSI cases, no conclusions could be drawn as to whether CB1 receptors were scored. CB1 receptors are often regarded as cell surface receptors, but it is well established in many cells and tissues that they have been found intracellularly 1 receptors with Gα subunits 1 receptors are highly lipophilic, and thus the plasma membrane is not a barrier to their cellular penetration. Extracellular signal-regulated kinase is an important signalling molecule, and has been implicated in antiproliferative effects of cannabinoids in a number of different cancer cell lines 1 receptors. There are three main results in from the study, and these are discussed in turn.The present study was motivated by data from both cultured cells and patient samples suggesting that a dysfunctional endocannabinoid signalling system is involved either in the pathogenesis and/or as a consequence of colorectal cancer 1IR scores from absent (0) to pronounced (3) were seen. Although there was no significant difference between the CB1IR distribution patterns for the two regions, one third of the cases had a score for the tumour invasive front that was different from that for the corresponding tumour centre CB1IR. One possible explanation for this difference is that the tumour front is an area of intense immunological and inflammatory activity 1 receptors in this region. One potential candidate in this respect is the cytokine interleukin-4 (IL-4), given that it can increase CB1 receptor expression 1 receptor transcription.For both tumour centres and invasive fronts, a gamut of CB1IR<2∶≥2 among the cases of approximately 2∶1, whilst the ratio is close to 1∶1 for the cases with moderate-poor/poorly differentiated tumours at surgery. In the MSI cases, no such difference is seen and the cases have a ratio near 1∶1. MSI cases are characterised by a high incidence of mutated DNA microsatellite markers as a result of a loss of DNA mismatch repair, and differ from MSS cases not only in terms of tumour characteristics and gene expression profiles 1IR distributions. However, a major difference between MSS and MSI cases are the relative incidences of CIMP-negative, low and high 1IR. It is notable that for all cases with a negative CIMP status and moderate-poorly differentiated tumours, the relative proportion of cases with CB1IR≥2 is similar to that seen for well/well-moderately and for moderate-poor/poorly differentiated CIMP-high tumours. This would suggest that the shift in CB1IR distribution seen with histological grade is brought about along the same pathway as the shift seen with CIMP-high, so that the effects are not additive.For MSS cases, well/well-moderately differentiated tumours distribute with the ratio of CB1 receptor expression, Wang et al. Cnr1 gene (the gene responsible for the CB1 receptor). These authors found hypermethylation of these sites in the colorectal tumours, ranging from cases with a single action at position +108 to cases with hypermethylation in ≥15 sites. They reported that the hypermethylation resulted in Cnr1 gene silencing Cnr1 gene. It is possible that one or several genes that are commonly inactivated by hypermethylation in CIMP-high tumours have downstream effects upon the transcription and/or turnover of the CB1 receptor, and this produces the results seen here, or alternatively that the pattern of Cnr1 hypermethylation in colorectal cancer is rather different from that picked up in the CIMP screen. In the latter situation, a case with a low level of CB1 promotor methylation but a high CIMP score (or vice versa) would be classified differently in the two studies, so it is not surprising that the results are divergent. In future studies, it would clearly be of interest to investigate Cnr1 hypermethylation in our tissue material, to be able to distinguish between these alternatives.With respect to the effects of DNA hypermethylation upon CB1 tumour receptor expression would be beneficial to the patients, whereas the opposite was found to be the case, at least for the patients with stage II MSS tumours at surgery. To our knowledge, only three studies have been undertaken to investigate the prognostic value of CB1IR in solid tumours. In hepatocellular carcinoma, the 35 cases with an undetectable or faint CB1IR showed a significantly poorer disease-free survival than the 29 cases with a moderate or intense CB1IR 1IR was also associated with histological grade, with 20/34 of the cases with well/well-moderately differentiated tumours showing a high CB1IR while only 9/30 cases with moderate-poor/poorly differentiated tumours showed a high CB1IR 1 receptor mRNA expression with quantitative RT-PCR. In the prostate cancer study , a composite score was again used, and the disease-specific survival was significantly poorer for the 192 cases with a CB1IR equal to or above the median  than for the 77 cases with a CB1IR below the median  From the introduction, it might have been expected that a high CB1IR score is going to be the same regardless of the cancer in question, but the present study would suggest that hepatocellular carcinoma, rather than colorectal cancer, is the “odd cancer out”. The question nevertheless remains as to why a high, rather than a low, CB1 expression should be associated with a poorer disease-specific survival. One possible explanation has been furnished by a recent study using cultured astrocytoma cells transfected with CB1 receptors 1 receptor expression levels and found that at a low receptor expression, the receptors coupled primarily to extracellular signal-regulated kinases, and that activation of the CB1 receptors led to apoptosis. In contrast, at a high level of CB1 receptor expression, activation of the receptors led additionally to the activation of the Akt survival pathway, and cannabinoids only produced apoptosis when this pathway was inhibited 1 receptor expression results in the switch from a pro-apoptotic to a predominantly pro-survival pathway would mean that the local endocannabinoid tone no longer acts to limit the damaging influence of the tumour but rather to exacerbate it and thereby result in a poorer prognosis for the patient. The hepatocellular carcinoma data 1IR do not have a sufficiently high level of expression to trigger the switch in these cells.It is of course naïve to assume that the influence of the CB1IR as a prognostic marker to aid treatment decisions in cancer. In prostate cancer, CB1IR looks to be a very promising marker that provides additive prognostic information to that supplied by other variables such as the Gleason score and the tumour stage 1IR as a prognostic marker to aid such treatment decisions in stage II MSS colorectal cancer.A final note concerns the potential of CBFigure S11 receptor immunoreactivity in non-malignant and adenocarcinoma samples.CB Panel A, non-malignant tissue; Panel B adenocarcinoma tissue, both stained using the antibody batch used in (TIF)Click here for additional data file.Figure S21 receptor immunoreactivity in forebrain samples from wild-type and CB1 receptor knockout mice.CB Panels A and B show the immunoreactivity from wild-type and CB1 receptor knockout mice, respectively. Objective magnification is 1.25×. The tiff image from the selected areas was imported into Adobe Photoshop (version CS4 for the Macintosh) and the colour histograms were captured. Panels C (wild-type) and D (CB1 receptor knockout) show immunoreactivity from different forebrain tissue slides to those in Panels A and B, at a higher objective magnification (20×). The colour histograms are for the whole images. The paraffin embedded, formalin-fixed mouse tissue was kindly provided by Drs. Beat Lutz and Giacomo Mancini, Department of Physiological Chemistry, Johannes Gutenberg-University Mainz, Germany.(TIF)Click here for additional data file.Figure S31IR scores according to tumour grade, CIMP status and microsatellite stability screening status.Division of CB Panel A, tumour centre; Panel B, tumour front. The data are grouped according to tumour grade  and microsatellite stability  and the CIMP status. P values were determined using Fisher's exact test. The total (i.e. CB1IR<2 and ≥2) number of cases is shown enclosed within each bar.(TIF)Click here for additional data file.


There was information omitted from the Funding statement. The correct version of the Funding statement is available below:This work was supported by a service development grant from the Jewish Federation of New York and a research grant MH071651 from the National Institute of Mental Health. Dr. YIS received an investigator-initiated grant from Lundbeck Pharmaceuticals for this study and for a collaborative study  entitled “Prevention of PTSD by Escitalopram.” The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials.


In the present study, we have investigated the prognostic usefulness of phosphorylated Akt immunoreactivity (pAkt-IR) in prostate cancer using a well-characterised tissue microarray from men who had undergone transurethral resection due to lower urinary tract symptoms.473) antibody. Immunoreactive intensity was determined for 282 (tumour) and 240 (non-mlignant tissue) cases. Tumour pAkt-IR scores correlated with Gleason score, tumour Ki67-IR (a marker of cell proliferation) and tumour phosphorylated epidermal growth factor receptor (pEGFR)-IR. For cases followed with expectancy, a high tumour pAkt-IR was associated with a poor disease-specific survival, and the prognostic information provided by this biomarker was additive to that provided by either (but not both) tumour pEFGR-IR or Ki67-IR. Upon division of the cases with respect to their Gleason scores, the prognostic value of pAkt-IR was seen for patients with Gleason score 8–10, but not for patients with Gleason score 6–7.pAkt-IR in prostate epithelial and tumour cells was assessed using a monoclonal anti-pAkt (SerTumour pAkt-IR is associated with both disease severity and disease-specific survival. However, its clinical use as a biomarker is limited, since it does not provide prognostic information in patients with Gleason scores 6–7. According to recent statistics, the global incidence of prostate cancer (Pca) in 2008 was approximately 900 000 cases The serine-threonine kinase Akt is an important regulator of cell proliferation and apoptosis. Downstream effects of Akt activation include the phosphorylation and thereby inactivation of the protein BAD, a pro-apoptotic protein Epidermal growth factor receptor (EGFR) is a cell surface receptor tyrosine kinase responsive to a number of growth factors such as epidermal growth factor and amphiregulin. Phosphorylation of EGFRs leads to activation of a number of different intracellular signalling pathways, in turn resulting in cell growth and survival In the present study, we investigated pAkt immunoreactivity in a well-characterised Pca tissue array The formalin-fixed, paraffin-embedded samples used in the present study were collected between 1975 and 1991 at the Central Hospital, Västerås, Sweden, from a total of 412 patients diagnosed with prostate cancer at transurethral resection for lower urinary tract symtoms The material was collected according to Swedish Regulations at a time when informed consent was not required. The research ethical committee at Umeå university hospital  approved of the study and waived the need for informed consent. In the database used for the analyses, the tissue samples were given a case number and year, and the patient names were not indicated in the database.The presence of metastases was determined by bone scans shortly after the transurethral resection, and the patients were followed until death or until 2003. The Gleason scores, the percentage of the specimen that contained tumour and the disease stage were assessed in each sample. Cause of death was determined by evaluation of medical records. Tissue microarrays using cores with a diameter of 0.6 mm were constructed using a Beecher Instrument . Each tissue microarray slide  contained up to eight  samples of tumour tissue (which included both primary and secondary Gleason grade areas) and up to four samples of non-malignant tissue from each patient 473) monoclonal antibody  and the secondary system . Ki67 was analysed either as the number of positive stained cells crossing 11 horizontal lines across the core, or as the percentage of cells positive for this marker , after which they were exposed to the primary  who did not access the clinical data at the time of evaluation. The scanned scores were analysed on the basis of intensity  and distribution . The composite score for each core was then determined. Thus, for example, a core with 25% intensity 3 and 75% intensity 2 would be given a score of 0.25×3+0.75×2 = 2.25. Examples of staining intensities 1–4 are shown in With the exception of the intraclass correlation coefficient and Cox proportional-hazards regression analyses, which were conducted using SPSS software , all statistical calculations were undertaken using the statistical package built into the GraphPad Prism 5 computer programme for the Macintosh . Partial correlation coefficients were calculated on an Excel spreadsheet from the Spearman correlation coefficients for matched samples . For survival analyses, an event was defined as death due to prostate cancer and entered into the database as “event = 1″, thereby allowing us to determine disease-specific survival. Death from other causes was censored, as were cases where the patient was alive at the date of last follow-up. Three cases where the disease outcome was not known were excluded from the survival analyses.Cores from 282 (tumour samples) and 240  cases were scored for pAkt-IR. Consistent with previous studies Correlation coefficients for the tumour and non-malignant pAkt-IR scores vs. clinical and biochemical parameters scored for the same samples are summarised in ab is the Spearman correlation coefficient for the interaction between parameters a and b, and Γab.c refers to the first order partial coefficient between parameters a and b when parameter c is taken into consideration. The equation represents the general formula for partial correlation derived in An important question to be investigated is whether the associations between non-malignant pAkt-IR and the clinical markers of disease severity are true correlations, or simply reflect the correlation between tumour and non-malignant pAkt-IR. This can be assessed using the formula:pAkt-IR (T) was significantly correlated to pEGFR-IR scores for both tumour and non-malignant samples . In contThe samples were grouped into four groups on the basis of their tumour pAkt-IR and pEGFR-IR scores and their Gleason scores, incidence of metastases at diagnosis and tumour Ki67-IR scores were compared . The disFor the 282 cases where tumour pAkt-immunoreactivity (pAkt-IR) could be scored, 207 were followed by expectancy, as was the standard approach at the time in Sweden. The other patients received radiotherapy, hormonal treatment or radical prostatectomy. Three cases where outcome was not known were excluded, giving a total of 204 cases available for survival analysis. The corresponding number of cases for the non-malignant pAkt-IR was 194. These samples allow determination as to whether pAkt-IR has prognostic value. For the 204 tumour cases, 101, 20 and 83 cases were below, equal to and above the median value (2.75), respectively.In the search of potential prognostic markers, the choice of cut-off is important. In order to investigate this, Cox proportional-hazards regression analyses were undertaken for both tumour and non-malignant pAkt-IR and over a wide range of cut-off values. The data are shown in In contrast to the situation for tumour pAkt-IR, non-malignant pAkt-IR scores had a narrow band where significant prognostic value was seen , and theCox regression data using a wide range of cut-offs were also constructed for the tumor Ki67-IR and pEGFR-IR data available in the database Kaplan-Meier plots of the influence of tumour pAkt-IR upon survival for the entire data set and subdivided on the basis of either the pEGFR-IR or the Ki67-IR cut-offs are shown in The curves shown in In the present study, pAkt-IR was assessed in a well characterised Pca tissue microarray Previous studies have reported that pAkt-IR scores are higher in tumour tissue than in non-malignant tissue In their large cohort, Ayala et al. We For a useful prognostic marker, there should be a relatively wide range of cut-off values that provide a significant discrimination of cases with good/poor prognoses. This criteria is simply to allow for application of the marker in clinical praxis, and analysis of the previously published data for tumour pEGFR-IR and Ki67-IR scores per se in Gleason score cases 8–10 is related to the proliferative capacity of the cells with high pAkt-IR expression.Although at first sight the wide window of significant prognostic information is promising, the clinical usefulness of tumour pAkt-IR is limited, since it provides no prognostic information at all at Gleason scores 6–7, i.e. those cases where treatment decisions are the most difficult. The finding, however, that pAkt-IR does provide prognostic information at Gleason scores 8–10 is mechanistically interesting, since it would suggest that activation of this survival pathway adds to the negative prognosis seen in poorly differentiated tumours. Cases with a Gleason score 8–10 in the database have a significantly higher median Ki-67 index than those with lower scores In the present study we found that there was a high correlation between tumour pAkt-IR and pEGFR-IR scores, confirming the study of Koumakpayi et al. A final note concerns the lack of correlation between pAkt-IR and PDGFRß in either the tumour or non-metastatic samples. PDGFRß is a tyrosine kinase implicated in a number of cancers, including prostate cancer In conclusion, the present study has demonstrated that although tumour pAkt-IR is associated with both disease severity and disease-specific survival, it is of limited clinical usefulness as a biomarker, since it does not provide prognostic information in Gleason 6–7 cases. The association with tumour pEGFR-IR is consistent with the hypothesis that a high activity along the EGFR-Akt signalling pathway facilitates epithelial-mesenchymal transition and thereby an adverse clinical outcome.Figure S1Distribution of pAkt-IR scores in tumour (T) and non-malignant (N) samples. Shown are the scores for 282 (tumour) and 240  cases, using bin widths of 0.5 IR units to group the samples. The median (with 25% and 75% percentile in brackets) scores were: tumour 2.75 (2.25–3.125) and 2.0625 (1.578–2.609). For the 189 cases scored for both tumour and non-malignant pAkt-IR, the Spearman’s rho value was 0.3105 (P<0.0001), and the median values were significantly different .(TIF)Click here for additional data file.Figure S2Exp(B) values for tumour pEGFR-IR (Panels A and B) and Ki67-IR (Panels C and D) for patients followed by expectancy; influence of Gleason score. Sample sizes are: pEGFR-IR, Gleason scores 6–7 (A), n = 141; pEGFR-IR, Gleason scores 8–10 (B), n = 59; Ki67-IR, Gleason scores 6–7 (C), n = 140; Ki67-IR, Gleason scores 8–10 (D), n = 61. For explanation of the symbols, see legend to (TIF)Click here for additional data file.Table S1Non-parametric partial coefficients for pairwise comparisons of pAkt-IR vs. clinical parameters with a single controlling factor.(DOCX)Click here for additional data file.Table S2Non-parametric partial coefficients for pairwise comparisons of pAkt-IR vs. clinical parameters with a single controlling factor.(DOCX)Click here for additional data file.Table S3COX proportional-hazards regression analyses for tumour pAkt-IR and Ki-67-IR for patients with Gleason scores 8–10 at diagnosis and followed by expectancy(DOCX)Click here for additional data file.


The onset of double diffusive convection in a viscoelastic fluid-saturated porous layer is studied when the fluid and solid phase are not in local thermal equilibrium. The modified Darcy model is used for the momentum equation and a two-field model is used for energy equation each representing the fluid and solid phases separately. The effect of thermal non-equilibrium on the onset of double diffusive convection is discussed. The critical Rayleigh number and the corresponding wave number for the exchange of stability and over-stability are obtained, and the onset criterion for stationary and oscillatory convection is derived analytically and discussed numerically. The problem of double diffusive convection in porous media has attracted considerable interest during the past few decades because of its wide range of applications, including the disposal of the waste material, high quality crystal production, liquid gas storage and others.Early studies on the phenomena of double diffusive convection in porous media are mainly concerned with problem of convective instability in a horizontal layer heated and salted from below. The double-diffusive convection instabilities in a horizontal porous layer was studied primarily by Nield On the other hand, viscoelastic fluid flow in porous media is of interest for many engineering fields. Unfortunately, the convective instability problem for a binary viscoelastic fluid in the porous media has not been given much attention. Wang and Tan In present research, we perform the linear stability of double diffusive convection in a viscoelastic fluid-saturated porous layer, with the assumption that the fluid and solid phases are not in local thermal equilibrium (LTE). The effects of parameters of the system on the onset of convection are discussed analytically and numerically. The critical Rayleigh number, wave number and frequency for exchange of stability are determined.We consider an infinite horizontal porous layer of depth Assuming slow flows in porous media, the momentum balance equation can be linearized asFor general viscoelastic fluids, the constitutive relations between stress tensor We assume that the diffusion of temperature obeys the following equations, which is a non-equilibrium model between the solid and fluid phases, suggested by The onset of double diffusive convection can be studied under the Boussinesq approximation and an assumption that the fluid The basic state is assumed to be quiescent and we superimpose a small perturbation on it. We eliminate the pressure from the momentum transport equation(4) and define stream function Then the following dimensionless variables are defined asHere the symbol “Hence the boundary conditions areIn this section, we discuss the linear stability of the system. According to the normal mode analysis, the Eqs.(10)–(13) is solved using the time dependent periodic disturbances in a horizontal plane. We assume that the amplitudes are small enough, so the perturbed quantities can be expressed as followsWhere Since The steady onset corresponds to This result is obtained by Banu and Rees Further Eq.(20) can be written asIn the absence of the solute effect, Eq.(21) reduces toThe value of Rayleigh number Case 1: For very small values of When the value of H is very small, the critical value of the Rayleigh number To minimize We also expand Substituting Eq.(26) into the Eq.(25), and rearranging the terms and then equating the coefficients of same powers of H will allow us to obtain the Substituting these values of Case 2: For very large values of For the very large values of Letting Similarly, we expand Substituting Eq.(30) into the Eq.(29), we getThen, substituting these values of For oscillatory onset Effect of different values of conductivity ratio The variation of conductivity ratio on the critical Rayleigh number for stationary mode with the heat transfer coefficient for different values of conductivity ratio is shown in The effect of relaxation time on the neutral curves is shown in From The stationary Rayleigh number increases with an increase in the value of heat transfer coefficient In The effect of Lewis number The onset of double diffusive convection in a binary Maxwell fluid, which is heated and salted from below, is studied analytically using using a thermal non-equilibrium model. Based on the normal mode technique, the linear stability has been studied analytically. The effects of relaxation time, heat transfer coefficient, normalized porosity parameter and other parameters on the stationary and oscillatory convection are discussed and shown graphically. It is found that the increasing relaxation time increases the elasticity of a viscoelastic fluid thus causing instability. The asymptotic solutions for both small and large values of


The microenvironment within solid tumours can influence the metastatic dissemination of tumour cells, and recent evidence suggests that poorly oxygenated (hypoxic) cells in primary tumours can also affect the survival and proliferation of metastatic tumour cells in distant organs. Hypoxic tumour cells have been historically targeted during radiation therapy in attempts to improve loco-regional control rates of primary tumours since hypoxic cells are known to be resistant to ionizing radiation-induced DNA damage. There are, therefore, a number of therapeutic strategies to directly target hypoxic cells in primary (and metastatic) tumours, and several compounds are becoming available to functionally inhibit hypoxia-induced proteins that are known to promote metastasis. This mini-review summarizes several established and emerging experimental strategies to target hypoxic cells in primary tumours with potential clinical application to the treatment of patients with tumour metastases or patients at high risk of developing metastatic disease. Targeting hypoxic tumour cells to reduce metastatic disease represents an important advance in the way scientists and clinicians view the influence of tumour hypoxia on therapeutic outcome. While reduced oxygen tensions can be lethal for some cells, many tumour cells are able to survive under poorly oxygenated (hypoxic) conditions. It is well-established that hypoxic tumour cells are resistant to radiation therapy, but the clinical impact of hypoxic tumour cells extends beyond the treatment of localized primary tumours with ionizing radiation. Hypoxic tumour cells promote tumour progression and metastasis through a variety of direct and indirect mechanisms, and hypoxic tumour cells, therefore, represent a significant impediment to successful cancer therapy.The cellular environment within solid tumours is increasingly being appreciated as an important limitation to current cancer therapy. The vasculature within most solid tumours consists of abnormally formed, poorly functional blood vessels that are incapable of delivering sufficient oxygen and nutrients to properly support the growing tumour mass . AvailabPatient survival rates are closely associated with the development of distant metastatic disease -4, with in vivo is inherently difficult and the tumour-specificity of HIF-1α inhibition is not clear. Thus, the inhibition of metastasis-associated proteins induced by hypoxia may provide more specific effects on metastatic tumour cell dissemination, metastatic tumour cell homing to distant organs, and metastatic tumour growth compared to HIF-1α inhibition, and several intriguing targets have been identified that hold promise for treating metastatic disease.Hypoxic tumour cells are known to up-regulate a number of genes that promote metastasis. Consequently, the therapeutic inhibition or functional targeting of hypoxia-induced proteins holds promise as a potential strategy to decrease metastases in patients with hypoxic tumours. There are a number of small molecule inhibitors of HIF-1α (the hypoxia-responsive α-subunit of HIF-1) that have been identified ,21 incluCarbonic anhydrase-9 (CAIX) is a hypoxia-induced cell surface protein involved in the regulation of intracellular pH. Therapeutic inhibition of CAIX has recently been shown to decrease primary tumour growth and metastasis in pre-clinical breast tumour models , partly LOX is a hypoxia-induced secreted enzyme that cross-links collagens and elastin in the extracellular matrix ,29. LOX C-X-C chemokine receptor type-4 (CXCR4) is expressed on a range of normal cell types and is involved in several physiological processes, including hematopoiesis, angiogenesis, leukocyte trafficking, and leukocyte homing . CXCR4 iThe established role of several hypoxia-induced genes in promoting metastatic tumour cell dissemination and growth of metastatic tumour foci underscore the therapeutic potential of inhibiting the activity of hypoxia-induced proteins to reduce tumour metastases. Inhibition of HIF-1α, CAIX, LOX, or CXCR4 to reduce the development and growth of tumour metastases represent rational therapeutic strategies to disrupt the metastatic process. A complimentary strategy is to utilize compounds that have been designed to specifically kill cells at low oxygen tensions, and there are several hypoxia-activated cytotoxins with exciting potential to treat tumour metastases.3 can be hypoxic [2 in diameter [18F-EF5 assessed by positron emission tomography (PET) [Hypoxic tumour cells can be directly targeted using pro-drugs that are metabolically reduced to cytotoxic agents in cells at low oxygen tensions ,40. The  hypoxic -46, whildiameter . Clinicahy (PET) . Taken tTirapazamine (TPZ)  is a hypAnother hypoxia-activated cytotoxin with encouraging activity is the alkylaminoanthraquinone N-oxide AQ4N ,57. AQ4NThe 3,5-dinitrobenzamide-2-mustard PR-104 is an emerging hypoxia-activated pre-prodrug with intriguing therapeutic potential ,65. PR-12 probes inserted into tumours, to evaluating the expression levels of hypoxia-induced proteins, to administering compounds that bind in hypoxic tumour cells  tumour must be used to identify patients most likely to benefit from therapies designed to target or inhibit hypoxic tumour cells to treat metastases. There are several methods used to detect and quantify hypoxia in solid tumours ranging from physical pOnidazole  or EF5 [nidazole ) that ar[18F-EF5 ). In addIn patients without obvious (detectable) tumour metastases at the time of presentation, the presence of hypoxic cells in their primary tumour would suggest the patient is more likely to have undetected disseminated tumour cells in their system and/or may be at higher risk for developing metastatic disease after treatment of their primary tumour -4. TheseRational design of therapeutic strategies to overcome metastatic disease based on targeting hypoxic tumour cells and/or inhibiting the hypoxia-induced proteins that influence tumour metastasis holds great promise for improving the treatment of metastatic cancer. Pre-clinical and clinical therapeutic strategies to overcome metastatic disease based on the presence of hypoxic tumour cells in primary and metastatic tumours should be pursued to address this important issue.2: partial pressure of oxygen; SDF-1α: stromal cell-derived factor-1α; TPZ: tirapazamine.βAPN: β-aminoproprionitrile; CAIX: carbonic anhydrase-9; CXCR4: C-X-C chemokine receptor type-4; HIF: hypoxia-inducible factor; LOX: lysyl oxidase; PET: positron emission tomography; pOThe authors declare that they have no competing interests.KLB and SD drafted and approved the final version of the manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/11/504/prepub


Fluid status was determined by bioimpedance spectroscopy method, Body Composition Monitor. The study population was further classified into four groups according to the median of relative hydration status (△HS =fluid overload/extracellular water) and the presence or absence of diabetes. The median level of relative hydration status was 7%. Among all patients, 207(43.9 %) were diabetic. 71 (15.0%) subjects had commencing dialysis, and 187 (39.6%) subjects presented rapid renal function decline during a median 17.3-month follow-up. Patients with fluid overload had a significantly increased risk for commencing dialysis and renal function decline independent of the presence or absence of diabetes. No significantly increased risk for renal progression was found between diabetes and non-diabetes in late CKD without fluid overload. In conclusion, fluid overload has a higher predictive value of an elevated risk for renal progression than diabetes in late CKD.Fluid overload is one of the major presentations in patients with late stage chronic kidney disease (CKD). Diabetes is the leading cause of renal failure, and progression of diabetic nephropathy has been associated with changes in extracellular fluid volume. The aim of the study was to assess the association of fluid overload and diabetes in commencing dialysis and rapid renal function decline (the slope of estimated glomerular filtration rate (eGFR) less than -3 ml/min per 1.73 m Chronic kidney disease (CKD) has become a global public health problem. Among many traditional risk factors of renal progression, diabetes is the leading cause of CKD in the developed world, and patients with diabetes and CKD have a greater increased risk of end-stage renal disease, and all-cause and cardiovascular mortality than those without diabetes . ProgresFluid overload is one of the major presentations in patients with late CKD. The abnormal fluid status has been correlated with arterial hypertension, left ventricular hypertrophy, and other adverse cardiovascular sequelae ,4. AccumSince both diabetes and fluid overload are significantly associated with renal progression and diabetes has been closely correlated with fluid overload, there might be a complex interaction between diabetes, fluid overload, and renal progression. Many mechanisms have been reported in the causes of renal function decline in diabetes patients . HoweverAll 612 of CKD stages 4-5 patients were invited to participate in the study from January 2011 to December 2011 at one hospital in Southern Taiwan. All patients had been enrolled in our integrated CKD program for more than 3 months (30.9±27.0 months). CKD was staged according to K/DOQI definitions and the estimated glomerular filtration rate (eGFR) was calculated using the equation of the 4-variable Modification of Diet in Renal Disease (MDRD) Study [The study protocol was approved by the Institutional Review Board of the Kaohsiung Medical University Hospital (KMUH-IRB-990125). Informed consents were obtained in written form from patients and all clinical investigations were conducted according to the principles expressed in the Declaration of Helsinki. The patients gave consent for the publication of the clinical details.th percentile of ΔHS was 7%. Accordingly, when ΔHS was greater than 7%, it was classified as “fluid overload” [Fluid status was measured once by a bioimpedance spectroscopy method, Body Composition Monitor  at enrollment. Based on the difference of impedance in each tissue, BCM provides the information of extracellular fluid overload, normohydrated lean tissue, and normohydrated adipose tissue in whole body. Normal extracellular water (ECW) and intracellular water (ICW) can be determined for a given weight and body composition. Fluid overload can be calculated from the difference between the normal expected and measured ECW . BCM hasverload” . In thisThe values of PWV were measured by an ankle brachial index-form device, which automatically and simultaneously measured blood pressures in both arms and ankles using an oscillometric method . After oDemographic and clinical data were obtained from medical records and interviews with patients at enrollment. Diabetes was defined as those with a medical history through chart review. Cardiovascular disease was defined as a history of heart failure, acute or chronic ischemic heart disease, and myocardial infarction. Cerebrovascular disease was defined as a history of cerebral infarction or hemorrhage. Information regarding patient medications including diuretics, calcium channel blockers, β-blocker, and angiotensin converting enzyme inhibitors (ACEI), and angiotensin II receptor blockers (ARB) within 3 months before enrollment was obtained from medical records. The participant was asked to fast for at least 12 hours before blood sample collection for the biochemistry study and protein in urine was measured using an immediate semiquantitative urine protein dipstick test and graded as negative, trace, 1+, 2+, 3+, or 4+. The severity of leg edema was also measured and graded individually on a 4-point system . Blood pCommencing dialysis was confirmed by reviewing medical charts or catastrophic illness certificate  and defined as requiring commencing hemodialysis or peritoneal dialysis. The timing for commencing dialysis was considered according to the regulations of the Bureau of the National Health Insurance of Taiwan regarding the laboratory data, eGFR, uremic status, and nutritional status . 2 per year. All eGFR values available from ascertainment of fluid status to the end of the observation period were included for calculation. At least three eGFR values were required to estimate the eGFR slope. Rapid renal progression was defined as the lowest quartile  [Patients are followed at nephrologic outpatient clinics at three-month intervals to ascertain the renal and vital status. Every patient received eGFR measurement at least every three month. The decline in kidney function was assessed by the eGFR slope, defined as the regression coefficient between eGFR and time in units of ml/min/1.73 mR slope) . th, 75th percentile), as appropriate, and categorical variables were expressed as percentages. Skewed distribution continuous variables were log-transformed to attain normal distribution. The significance of differences in continuous variables between groups was tested using the one-way analysis of variance (ANOVA) followed by the post hoc test adjusted with a Bonferroni correction or the Kruskal-Wallis H test, as appropriate. The difference in the distribution of categorical variables was tested using the Chi-square test. Time-to-event survival analysis by Kaplan-Meier survival curve was used to test fluid overload or diabetes as a predictor of the risk of commencing dialysis. Cox regression models were utilized to evaluate the relationship between fluid overload and diabetes in commencing dialysis. Multivariable logistic regression models were also utilized to examine the association of renal function decline with fluid overload and diabetes. Age, gender, medication (diuretics and ACEI/ARB), and all the variables in 2/dl2, proteinuria (urine protein above 1+), and systolic blood pressure cut at 140mmHg were selected in multivariate analysis. Statistical analyses were conducted using SPSS 18.0 for Windows . Statistical significance was set at a two-sided p-value of less than 0.05.The study population was further classified into four groups according to △HS of 7% and the presence or absence of diabetes. Continuous variables were expressed as mean±SD or median (252) among all patients. Diabetic patients with fluid overload received more diuretics, calcium channel blocker, and β-blocker treatment. TBW, ECW and ECW/TBW were higher in patients with both diabetes and fluid overload than those with only diabetes or fluid overload. Blood urea nitrogen, serum glycated hemoglobin and uric acid levels, and the severity of urine protein were the highest and serum albumin was the lowest in patients with diabetes and fluid overload than others. The median levels of fluid overload and fluid overload/ECW (△HS) of the entire cohort were 0.9 L and 7%, respectively. The comparison of clinical characteristics between groups based on △HS divided at 7% and the presence or absence of diabetes is shown in Seventy-one patients (15.0%) progressed to commencing dialysis, 65 of hemodialysis and 6 of peritoneal dialysis during a median follow-up of 17.3  months . No more2/year respectively) was significantly faster than those without fluid overload  and -1.2 mL/min/1.73 m2/year respectively) independent of diabetes. △HS was significantly associated with high risk for renal function decline 〔every 1 unit increase odds ratio (OR): 1.06, 95% CI:1.02-1.10), P=0.001〕. The unadjusted and adjusted odds ratio (OR) for renal function decline in non-diabetic patients with fluid overload compared with those without fluid overload was 3.00  and 4.26  in diabetic nephropathy was also associated with changes in extracellular fluid volume . IncreasThough diabetic patients have usually been associated with fluid overload , a substEvaluation of volume status is an important issue in CKD patients in clinical practice. Essig et al. indicated that only 8% increase in ECW might easily be underestimated by clinical examination . PhysicaThis study has some limitations that must be mentioned. Fluid status and the other clinical variables were measured only once at enrollment. The effect of time-varying fluid status and the other clinical variables on renal progression might be underestimated. The BCM has been validated in the dialysis population, but not in CKD populations. This is indeed a limitation of the reality of the parameters of fluid status measured by BCM in CKD populations. Besides, we recorded use of ACEI/ARB and diuretics as categorical variables within 3 months before enrollment, not time-averaged defined daily dose of these drugs. The effect of time-varying dose of medication might be underestimated. In conclusion, our study demonstrates that fluid overload leads to an increased risk for commencing dialysis and renal function decline in stages 4-5 CKD patients in the presence or absence of diabetes. Fluid overload has a higher predictive value of an elevated risk for renal progression than diabetes in late CKD. Future intervention studies will be necessary to demonstrate whether objective identification and relief of fluid overload can slow down the renal function decline in CKD patients with and without diabetes.


In the Netherlands, infection with varicella-zoster virus (VZV) is considered a benign common childhood illness and routine vaccination against VZV is not done. In 1995 it was estimated that 98-100% of the adult Dutch general population is immune, yet the estimate is based on a database in which a relative small number of people of non-Dutch ethnic origin were represented. As the city of Amsterdam has large immigrant communities originating from various subtropical and tropical countries, such as Morocco, Surinam, and Turkey with probably lower VZV transmission, this study aimed to estimate the seroprevalence of VZV IgG antibodies (anti-VZV) among various ethnic groups in Amsterdam, and identify factors associated with seronegative VZV status.The study was a cross-sectional survey of the Amsterdam population (2004), and the study sample was stratified by age and ethnicity, with deliberate oversampling of minority ethnic groups. Serum samples obtained from 1,341 residents in 2004 were tested for antibodies to VZV. Basic demographic data  were also available.The anti-VZV seroprevalence in the overall Amsterdam population was estimated to be 94% . Regarding ethnic origin, first generation immigrants , especially those that migrated after the age of 11 years, were more likely to be anti-VZV seronegative compared to those arriving at an earlier age or those born in the Netherlands (97–98%). Both ethnic origin and generation of immigration were positive predictors for IgG seronegativity to VZV (p<0.015). No other predictors for seronegativity were found.The results of this study imply that about 4–8% of the general adult Amsterdam population is still susceptible to infection with VZV, and that susceptibility is even higher in some immigrant groups. When assessing the risk of infection after VZV exposure alertness is needed for vulnerable persons like pregnant women, patients with hematological malignancies or organ transplants in particular among first-generation immigrants. In the Netherlands, primary varicella infection (chicken pox) is considered a benign common childhood illness. After a first infection with varicella-zoster virus (VZV), immunity is regarded as life-long . In lateVZV circulates widely in the Dutch population, and exposure to the virus is relatively frequent. The risk for VZV infection is highest in pre-school children aged 1–5 years, and by the age of 5, at least 93% of the children have VZV antibodies. According to a national population-based VZV seroprevalence study 1995–1996), almost all Dutch adults (98–100%) have antibodies against VZV, yet that study included relatively few residents of non-Dutch origin , almost . As the The data used for this study were obtained from a cross-sectional population-based health survey . The survey was carried out in 2004 by the Amsterdam Public Health Service (GGD) in collaboration with the National Institute for Public Health and the Environment (RIVM). Amsterdam consists of thirteen districts and the sample for the survey was drawn from five of them. These five contain a population that was representative concerning socioeconomic status and ethnicity for the total population of Amsterdam. The survey was approved by the Medical Ethics Committee of the Academic Medical Centre. The sample was stratified by ethnic background  and five age groups . Within each stratum a random sample was drawn. The Turkish and Moroccan ethnic groups were oversampled to ensure sufficient numbers of individuals from these groups. Data were weighted to correct for oversampling by ethnic groups. After weighting the data for age, gender and ethnicity, respondents appeared to have an annual income and unemployment rate that was comparable to that of the total population of Amsterdam in 2004. The overall response rate among ethnic Dutch, Turkish and Moroccan subjects was 45%. More details on the survey are described elsewhere .Respondents were invited for an interview and medical examination in a community health centre. All interviews were conducted in the language of choice of the respondent . From all the issues that were addressed the following variables were considered pertinent to the current study: sex, age, country of birth of the participant and his/her parents, age at the time of migration, and the number and age of children living with the participant currently and/or in the past. Participants were classified into five ethnic groups , according to the self-reported country of birth and the country of birth of the respondent's mother or father. Furthermore, participants of non-Dutch ethnic origin were classified as first- or second-generation immigrants depending on their own country of birth. Those born in the Netherlands were considered as second-generation and all others as first-generation immigrants.Participants were requested to provide a blood sample. These were collected, after obtaining written informed consent, and were stored at 7°C within 30 minutes, then centrifuged and frozen at −80°C within 48 hr. Seventy-nine per cent of the participants  donated a blood sample for the serum repository.Plasma samples were tested for IgG-class antibodies to VZV by means of quantitative enzyme immunoassays. The assays were performed in the Public Health Laboratory in Amsterdam according to the instructions of the manufacturer. The serological test was a microplate enzyme-linked immunosorbent assay system that uses purified antigen  to detect VZV IgG .In estimating anti-VZV seroprevalence, only samples with a positive result were considered immune. All negative and equivocal test results  were considered as not immune. We thereby increased the specificity and reduced the number of false positive results. As the purpose of this study was to establish factors associated with VZV susceptibility, reduced assay sensitivity is preferable to reduced specificity [In order to obtain results representative for the adult population in Amsterdam, prevalences and confidence intervals of 95% (95%CI) corrected for stratification were calculated using the complex samples modules of SPSS, version 17 . In these analyses data were weighted for age, sex and ethnic origin, using a weighing method which corrected the oversampling by ethnic groups as described elsewhere ,11,12. PFor this study, 97.5%  of the collected blood samples were available for laboratory analysis. Table Table The results of the univariable analysis for IgG seronegativity to VZV are shown in Table Our study shows a high seroprevalence (94%) of VZV IgG antibodies in the overall adult Amsterdam population (95%CI 92–96%), which is in line with other seroprevalence estimates in adults living in temperate zones ,5,13-19.One explanation for this difference may be the ethnic diversity present in urban populations. The city of Amsterdam has large migrant communities, with people originating from various subtropical and tropical countries, which are known to have less VZV transmission. In temperate regions, VZV causes annual epidemics among susceptible household members, in day care centers, and in schools, resulting in high seroprevalence. In warmer climates, VZV infection is less frequent and as many as 50% of young adults in tropical countries may never have had a primary VZV infection ,5,7,22. Other studies have described that anti-VZV seroprevalence may be related to household composition (≥ 4 persons) and school attendance by a household member ,6,28-30 In this study, we increased the specificity of the test by considering those with equivocal test results as non-immune. This may have led to an underestimate of the true anti-VZV seroprevalence and thus the immunity in the Amsterdam population, and overestimated the VZV susceptibility. However, as the aim of this study was to identify factors associated with VZV susceptibility, and in order to reduce the number of false positives, this approach seems justified. A subsidiary analysis in which the equivocal test results were considered as false negative showed similar, although less significant outcomes (data not shown).The relatively low response rate of the AHM (45%) and its sampling methods may be considered as potential sources of bias, which may have affected the results of this study. However, the oversampling and non-response bias by ethnic groups were addressed by weighing the data by sex, age, and ethnicity. A non-responders survey showed that the sample appeared to be representative of the population on most health determinants . FurtherThe introduction of a two-dose universal childhood VZV vaccination programme in the Netherlands is being considered. In terms of health policies and the cost-effectiveness of the introduction of a universal vaccination programme for VZV, the finding of 4–8% of adult susceptibles in Amsterdam should be taken into account. One of several unresolved questions is the impact of a VZV vaccination programme on the incidence of herpes zoster. Another issue related to cost-effectiveness is the uncertainty of the burden of disease of VZV in children . CompareIn conclusion, the results of this study imply that about 4-8% of the general adult Amsterdam population is still susceptible to infection with VZV, and that susceptibility is even higher in some immigrant groups. When assessing the risk of infection after VZV exposure alertness is needed for vulnerable persons like pregnant women, patients with hematological malignancies or organ transplants in particular among first-generation immigrants. .The authors declare that they have no competing interests.GGCvR performed the data analysis and wrote the first draft of the manuscript. MD advised and supervised the carrying out of the immunoassays. MSvdL contributed to the statistical analysis. GBS and AvdH made substantial changes to the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/12/140/prepub


To review the measurement methods of radiological ‘tumour response' in two clinical trials.Neo-tAnGo and ARTemis are national 800-patient breast cancer trials assessing neo-adjuvant chemotherapy regimens, both with a secondary endpoint of radiological tumour response, assessing treatment effect in terms of change in tumour size. Both trials record radiological sizes at baseline, mid-way through and at the end of the chemotherapy regimens.Neo-tAnGo (randomising Jan'05 to Sept'07) recorded mammograms and/or ultrasounds and/or MRI scans, identifying up to two breast and one axillary lesion as ‘target' lesions. At each of the 3 time-points and for each target lesion, the scan types and largest diameters observed (maximum of two) were reported, along with a clinical judgement of response.ARTemis (randomising May'09 to Jan'13) recorded ultrasounds only, recording the longest single diameter of each tumour (including axillary tumours) along with total ‘tumour bulk' in each breast. At mid- and end-chemotherapy, a clinical judgement of response per breast is also recorded.In neo-tAnGo, the type of radiological scan used and number of recorded dimensions varied, not only across hospitals but also within-hospitals across patients and across assessment times, creating challenges regarding tumour comparisons. In ARTemis, difficulties existed surrounding the clinical response categorisation and interpretation of ‘tumour bulk', and non-adherence to the specified radiological technique raised methodological issues.Optimally recording radiological ‘tumour response' and making meaningful inter-patient, inter-tumour or inter-time-point comparisons is challenging. Specifications regarding the authorised radiological scanning techniques and reporting of findings are imperative in a trial protocol.


Dose and time intensifications of chemotherapy improved the outcome of lymphoma therapy. However, recent study results show that too intense therapies can result in inferior tumour control. We hypothesise that the immune system plays a key role in controlling residual tumour cells after treatment. More intense therapies result in a stronger depletion of immune cells allowing an early re-growth of the tumour.We propose a differential equations model of the dynamics and interactions of tumour and immune cells under chemotherapy. Major model features are an exponential tumour growth, a modulation of the production of effector cells by the presence of the tumour (immunogenicity), and mutual destruction of tumour and immune cells. Chemotherapy causes damage to both, immune and tumour cells. Growth rate, chemosensitivity, immunogenicity, and initial size of the tumour are assumed to be patient-specific, resulting in heterogeneity regarding therapy outcome. Maximum-entropy distributions of these parameters were estimated on the basis of clinical survival data. The resulting model can explain the outcome of five different chemotherapeutic regimens and corresponding hazard-ratios.We conclude that our model explains observed paradox effects in lymphoma therapy by the simple assumption of a relevant anti-tumour effect of the immune system. Heterogeneity of therapy outcomes can be explained by distributions of model parameters, which can be estimated on the basis of clinical survival data. We demonstrate how the model can be used to make predictions regarding yet untested therapy options. High-grade non-Hodgkin lymphoma (NHL) is a haematologic malignancy, which is curable by multi-drug and multi-cycle cytotoxic chemotherapy in a substantial proportion of cases .There is a loss of effector cells due to degradation, consumption by fighting the tumour, and chemotherapy.Higher chemotherapy doses result in both higher tumour and effector cell depletion. The model will be linked to clinical survival data derived from randomised studies, with the aim to predict and explain data of chemotherapy outcomes. More precisely, we claim that the model fulfils the following requirements: heterogeneity of patient outcomes can be traced back to plausible heterogeneity of certain model parameters,the estimated parameters and parameter distributions are biologically plausible,the time scale and variance of time to relapse is consistent with reported event-free survival curves,extent of tumour at diagnosis (e.g. stage) emerges as prognostic factor,observed treatment difference of several clinical trials comparing CHOP and CHOEP variants are quantitatively reproduced.We propose an ordinary differential equations (ODE) model of the interaction of a growing tumour, an anti-tumour immune response, and the effect of chemotherapy on both the tumour and immune system. The system is based on a model proposed by Kuznetsov et al. . To keepIn particular, we are interested in exhibiting the possibility of “paradoxic” treatment effects namely that a more intense chemotherapy has not superior, but even inferior results. This may happen if chemotherapy is not able to completely eradicate the tumour but the immune system is weakened to such an extent that it cannot control residual tumour cells.As basic model of an immunogenic tumour we adapt a model of Kuznetsov et al.  originalAdditionally, the effect of chemotherapy is added as transient depletion of cell counts following a first order kinetic (i.e. a fixed percentage of cells survive) both for tumour and for immune effector cells. Corresponding parameters require estimation, also Sect. .1-mice: Firstly, tumour growth stimulates the production of new cytotoxic T-lymphocytes; secondly immune cells destroy tumour cells. Depending on parameter settings, possible stable outcomes of the models are extinction or (saturated) permanent growth of the tumour or persistence of a residual tumour controlled by the immune system. This model meets all of our assumptions and requirements made in Sect. The ODE-model introduced by Kuznetsov is based on a system of two ordinary differential equations describing the kinetics of growth and regression of an immunogenic tumour and the interaction with tumour-specific effector cells of the immune system. The model exhibits two major phenomena that were observed in experiments with BCLT be the number of tumour cells and E be the number of tumour-specific effector cells. The system of differential equations of the model in Kuznetsov et al. ,the number of tumour cells where the stimulation of effector cells is half-maximal T=0, (B)T≈4.6⋅108, E≈1.6⋅105 (large tumour).(C)T≈8⋅106, E≈1.6⋅106 (“dormant” tumour).(D)T≈2.5⋅108, E≈8⋅105.Kuznetsov estimated the parameters of the model on the basis of experimental data in mice  is a saddle node on a curve, the separatrix, which divides the phase space into two domains: Initial conditions at one domain result in uncontrolled tumour growth (stable steady state (B)) while at the second domain the tumour is eventually controlled by the immune system (stable steady state (C)). Steady state (A) is an unstable fixed point and expresses a tumour-free effector cell population. The number of fixed points as well as their stability depend on the parameter setting.Kuznetsov’s model is designed to express the interactions of tumour cells and immune cells in leukaemia for mice. We adapt this ODE-system to model tumour growth and therapy in human diffuse large B-cell lymphoma (DLBCL).T2/3 by the following considerations: We treat the tumour as a sphere. Then the number of cells T is proportional to its volume: T∝r3 where r denotes the radius of the tumour. The tumour surface is proportional to r2∝T2/3. As B-cell-lymphomas grow in multiple tumour nodes, we set the exponent slightly larger than c and set c=0.75 /(tumour doubling time in days)). The immunogenicity ρ is assumed as 0.1245 day−1 in Kuznetsov et al. :=trace(J)2−4⋅det(J) determines whether the point is a node or a spiral. The Jacobian matrix for the linearised system =−νcETc−1+o(Tc−1) and det(J)=δνcETc−1−o(Tc−1) where o is the Landau order symbol. This leads to With v et al.  the stabT from the second equation of the system -axis at log(α/ν). An increase of α shifts this straight line in log(E)-direction.Furthermore, we obtain the nullcline of e system  4\documE is The nullcline of entclass1pt{minimaT: c=0.75 we obtain a polynomial of degree 7: α and immunogenicity ρ. Steady states were determined numerically using the statistical software package R . The function is based on the algorithm given in Brent . Precise information about the number of distinct positive roots is given by Sturm’s method .To illustrate model behaviour, we chose v et al. . The quaod Stoer . There aα=0.18,ρ=0.1245: A: saddle node,   B: unstable node or focus,   C: saddle node.We get three numerical solutions for the steady states of Eq.  with α=0The system exhibits three steady states. Table Tumour overgrows immune system:An initially small tumour population stimulates the immune system by increasing the effector cell population. By reaching a size large enough to weaken the immune system, the tumour depletes the effector cell population to 0.Tumour eventually becomes extinct:There are two distinct dynamics here: Either a small tumour population becomes extinct immediately. Or, after an initial growth of both tumour cells and effector cells, the tumour is eliminated by the effector cells. We can study the characteristics of the system by examining the nullclines ))Tumour oα and the immunogenicity ρ on the system behaviour. Decreasing α respectively increasing ρ enlarges the domain where the tumour eventually is eliminated. Additionally the quantity of immunogenicity ρ determines the steepness of the effector cell curve: a higher ρ implies a steeper peak. Note that similar phase diagrams can be obtained by either increasing tumour growth velocity α or decreasing immunogenicity ρ. In the first case, the immune system has not enough time to react against the fast growing tumour, while in the second case the immune system is insufficiently stimulated. Figure α, immunogenicity of the tumour ρ, size of the tumour Tdiag, and chemosensitivity of tumour kT as well as effector cells kE. We aim to estimate this multi-dimensional distribution from survival data of randomised chemotherapy trials.In this section, we describe how the model is linked to clinical data in order to estimate unknown parameters of the model  study effect by introducing a parameter Patient-specific:These parameters differ between patients. A population distribution is assumed for these kind of parameters.Therapy-specific:The parameter value depends on the applied chemotherapeutic drugs and drug doses.Study-specific:The parameter value varies for different studies with similar patient populations.Some parameters are considered as patient-, therapy-, or study-specific  or whether it is suppressed by the immune system before it is clinically apparent. The parameter distribution to be estimated is restricted to this admissible set. We start with an initial condition of T=104 and E=σ/δ=2⋅105, which describes the immune equilibrium state. The exact choice of T(0) is unimportant for model behaviour as long as one starts with a sufficiently small tumour, i.e. as long as the loss of effector cells due to presence of tumour represented by parameter μ can be neglected compared to the natural degradation of effector cells represented by parameter δ. In case of a growing tumour, we determine the effector cell population Ediag if T=Tdiag is reached for the first time. In the second step, we simulate the therapy by starting at the initial condition  for each parameter combination . Therapy regimes vary in the number of cycles (6 or 8), time between dose application (e.g. 2 or 3 weeks), and chemotherapy dose expressed by the dose-dependence of kT and kE . Altogether we consider 51(α)⋅35(ρ)⋅7(Tdiag)⋅41(kT)⋅31(kE) grid points in the five-dimensional space. For instance, we used the seven grid points {10i/210+,i=0,…,6} as possible tumour sizes at diagnose Tdiag. Density of the grid is chosen in dependence on the corresponding parameter sensitivity of the fitness function. If small changes of a parameter results in large differences of the fitness function, the grid was chosen finer to achieve a better approximation.We discretise the ranges of the parameters by introducing grid points. Therapy simulation is performed on the grid for each parameter combination  and 0.216 (doubling time 3.2 days). The immunogenicity ρ has an expected value of 0.078. The expected tumour load Tdiag is 11.31 on log10-scale. The expected chemosensitivity kT is 2.36, which implies a tumour kill of 90.6 % per shot. 90 % of values are between 1.86 (84.3 % kill per shot) and 3.00 (95.0 % per shot). The effector toxicity kE is 0.48 resulting in a kill of 37.9 % effector cells per shot. These values indicate that the grid is chosen adequately as the values are not located on the edge of the grid. The toxicity exponent for intensified therapies  and predicting corresponding estimates of 2-years survival , CD8+ cytotoxic T-cells and other lymphocytes . At this volume, the growth is far below saturation, and thus, our simplification is reasonable .Chemotherapy is introduced by a transient first-order loss of both tumour and immune cells for the duration of one day after chemotherapy application. The dependence of corresponding toxicity parameters on the drug or drug concentration was modelled by a simple power function of the effective dose of a drug introduced in Hasenclever et al. . AlternaSince Kuznetsov’s model was parameterised for the murine system, it was necessary to re-parameterise the model for patients. We retrieved parameter estimates or parameter ranges from the literature or set parameters to biologically or clinically plausible values.α, immunogenicity of tumour ρ, chemosensitivity of tumour kT, and volume at diagnosis Tdiag can vary within a certain parameter range. We estimated the distribution of these patient specific parameters by fitting the predictions of our model to clinical survival data. We developed an innovative algorithm to solve this task: At first, we simulated a discrete grid of parameter values. Then we determined the maximum entropy distribution parameterised by certain moment constrains on the grid, which induces a survival curve. The agreement of survival curve and clinical data is optimised by evolutionary strategies. Kirkby et al.  and Michaelis–Menten term (η) have a relatively small impact on model behaviour since they are only relevant for small tumour sizes. Similarly, for large tumour sizes, the second term of the first model equation is linear in E so that the fourth term (containing parameter δ) simply implies a shift of the distribution of immunogenicity (ρ). Rather than the single values, the quotient of the mutual destruction rates μ and ν is relevant. It has a larger impact on the quantitative model behaviour but the same results can be achieved by alternative distributions of the other model parameters (results not shown). Better data are required to remove the uncertainty of model parameters in the future. For example, time series data of different immune cell fractions and surrogate markers of tumour load would be helpful.Only a few parameters with large impact on survival curves were determined by fitting model predictions to clinical data, namely expectation and variance of immunogenicity ρ imply large values of tumour growth velocity α  correlation between parameters. However, due to the restriction to admissible parameter sets  normal and greater than normal, complete range of the International Prognostic Index is covered). The tumour stimulation rate of effector cells ρ=0.078 is difficult to interpret as it comprises many aspects of the immune system. However, it is in the same order of magnitude compared to Kuznetsov’s estimate of 0.1245 for mice. The expected tumour volume at diagnose Tdiag is 1011.31. This fits well to our understanding because in this magnitude the turnover of effector cells is also high, i.e. the tumour becomes symptomatic.The mean tumour doubling time was estimated as about 6 days with 90 % of values between 3 and 18 days. It is well known that aggressive non-Hodgkin’s lymphoma are highly proliferating with a tumour doubling time between 24 hours and about 30 days in dependence on the histologic subtype (Frolund et al. Within the time-frame used for model fitting, parameter estimates resulted in a good agreement of model and survival data of the two randomised clinical trials NHL-B2 and RICOVER-60 comprising five different chemotherapy regimens (Pfreundschuh et al. To demonstrate practical use of our model, we performed simulations of modified CHOP regimens by varying therapy intervals and number of cycles. We predicted for example that six cycles of CHOP-14 are necessary for reasonable cure rates. There is only limited potential to improve therapy by further time-intensifications and increasing number of cycles. Results are robust against the uncertainty of our parameter estimates.α and that stage of disease is a surrogate marker of tumour size Tdiag. We also have to acknowledge that we do not have a surrogate marker for immunogenicity since detailed immune statuses of patients are hardly available. A model extension to account for immunotherapy based on the CD20+ antibody Rituximab is also a work in progress.Many more predictions are possible, especially with respect to stratified therapies in dependence on patient characteristics. We aim to elaborate this issue and possible clinical consequences in the near future. However, translation of model insights into clinical practice is challenging since there are no well established correlates of model parameters with clinical data. One can only speculate at the moment whether for example LDH is a surrogate marker of tumour growth


Prior to the antiretroviral (ARV) drug roll out in 2004, people living with HIV (PLHIV) in South Africa received disability grants when they were defined as "AIDS-sick". In the absence of available and effective medication, a diagnosis of AIDS portended disability. The disability grant is a critical component of South Africa's social security system, and plays an important role in addressing poverty among PLHIV. Given the prevalence of unemployment and poverty, disability grants ensure access to essential resources, like food, for PLHIV. Following the ARV roll out in South Africa, PLHIV experienced improved health that, in turn, affected their grant eligibility. Our aim is to explore whether PLHIV reduced or stopped treatment to remain eligible for the disability grant from the perspectives of both PLHIV and their doctors.A mixed-methods design with concurrent triangulation was applied. We conducted: (1) in-depth semi-structured interviews with 29 PLHIV; (2) in-depth semi-structured interviews with eight medical doctors working in the public sector throughout the Cape Peninsula; (3) three focus group discussions with programme managers, stakeholders and community workers; and (4) a panel survey of 216 PLHIV receiving ARVs.Unemployment and poverty were the primary concerns for PLHIV and the disability grant was viewed as a temporary way out of this vicious cycle. Although loss of the disability grant significantly affected the well-being of PLHIV, they did not discontinue ARVs. However, in a number of subtle ways, PLHIV "tipped the scales" to lower the CD4 count without stopping ARVs completely. Grant criteria were deemed ad hoc, and doctors struggled to balance economic and physical welfare when assessing eligibility.It is crucial to provide sustainable economic support in conjunction with ARVs in order to make "positive living" a reality for PLHIV. A chronic illness grant, a basic income grant or an unemployment grant could provide viable alternatives when the PLHIV are no longer eligible for a disability grant. The estimated 5.7 million South Africans living with HIV in 2010 make this the largest HIV epidemic in the world .3 received ARV therapy  disability grant. In cases where there is no income through employment, I offer [a] disability grant. I have offered disability grant[s] to all those who start ARVs regardless of their CD4 count. I have even prescribed ARVs for patients whose CD4 count is above 200. I base my assessment for initiation of ARVs on the general health condition of the patient." Another doctor, however, was more reluctant to incorporate social dimensions into her assessment of an individual's disability grant eligibility:"Sometimes they bring their kids and you can see that they are hungry. But then again, the guidelines state that you cannot give this person a disability grant. You know what is going on at home and that there are no social workers to take care of the person. You know that you can help, but then at the same time you do not want to be seen as the "fraudulent" doctor. I guard against that because one day when you give that disability grant and SASSA decided to do an audit - then you may be seen as fraudulent. But honestly speaking, I have been "fraudulent", two or even three times, not a lot; I mean I am very careful. They (HIV-positive patients) think I have empathy and understanding, because where they come from is probably where I come from." The participants in the in-depth interviews were unclear about the way the doctors decided on grant eligibility and the way the social services managed the grants. Some of the participants described their frustration with corruption in the welfare system, referring to officials working in the social security agency who demanded the first month of the disability grant payment as payment for them assisting in the disability grant application procedure. Some PLHIV clearly indicated that the application procedure was difficult and suggested that it would be a useful to have professional people assisting them in the process.The narratives included accounts of people who had received a disability grant in the 1990s and were still accessing it even though their health was good due to ARV treatment. They reported that these people on "long-term grants" had received the grant from a friendly doctor at a time when some doctors interpreted the rules to mean that PLHIV without access to treatment would qualify for permanent grants. This was before the roll out of ARVs.In most cases, grant recipients were not aware of how long they had the grant, and a few of the participants seemed to wonder why they still received the grant. One recurrent story was linked to people who got the grant for six months. We were told that they could not apply for a new grant period before the previous one had ended, and then it took some months before they got a new grant. As a result, the story of a person receiving a grant might sound like this: six months with a disability grant, then a period without a grant, and then another six months with a grant again, and so on. One of the in-depth participants, an HIV-positive 44-year-old man, stated, "The grant is coming and going."South Africa's high level of unemployment complicates the development of a comprehensive welfare system based on income tax revenues ,30. We fThe two key narratives that emerged from the interviews suggested that unemployment affects PLHIV in two ways: HIV illness may prevent PLHIV from seeking work; and even if PLHIV are able to seek work, they are unlikely to find work given South Africa's high unemployment levels. The participants struggled with the dual impact of the loss of their disability grants as their health improved, and the general lack of employment opportunities. Similarly, Coetzee found that ARVs enabled PLHIV to look for work in the labour market, but given the high levels of unemployment, ARVs and resumed health did not translate directly into employment .The number of grants awarded to HIV-positive individuals rose from 27% in 2001 to 41% in 2003 ,34. BetwSustained adherence to ARVs is crucial to ensure the effectiveness of treatment and to slow the development of AIDS and the spread of drug-resistant viral strains. People who were taking ARVs had less functional impairment, fewer HIV-related symptoms, and a greater capacity to work than those who had not yet started ARV treatment .Our findings indicate that the disability grant played an important role in minimizing the negative consequences of unemployment among PLHIV. The disability grants played a crucial role in maintaining their financial, emotional and physical well-being. However, we found a number of conflicting views as to whether disability grants were a disincentive to adherence. Despite the reported reliance on disability grants among PLHIV in the context of high unemployment levels, this study did not find that PLHIV taking ARVs sought to compromise their health in order to remain eligible for the grant. However, a few participants mentioned their own and other people's strategies for reducing their CD4 count prior to their tests through alcohol consumption and short-term discontinuation of their ARVs.Even though the PLHIV and the doctors described the adherence to medication in quite different ways, they seemed to agree about the consequences of receiving or losing the grant. These findings suggest nuanced approaches to balancing health and welfare where health usually takes precedence. Alcohol abuse, insufficient financial resources and lack of food were also found to act as barriers to ARV adherence by Nachega and colleagues . A systeEven though we did not find strong evidence that low adherence, coupled with the aim of re-qualifying for a disability grant, was a widespread phenomenon, we found that some PLHIV practised a form of "circumstantial" non-adherence. It was also clear that the grant application system puts PLHIV under pressure because of the uncertainty related to the renewal and loss of the disability grant. This in turn could result in a vicious cycle where PLHIV experience a restoration of health but have difficulty maintaining a nutritionally adequate diet after the loss of disability grant income when no other sources of income are available. A secure source of income is necessary to ensure a good diet for PLHIV, and this in turn is pivotal for adherence to the medication.The most common reason for non-adherence given in the interviews and survey was hunger. This problem is linked to the fact that in some cases, the disability grant was the only source of economic relief for PLHIV. Coetzee and Nattrass noted that as ARVs restored the health of PLHIV, the unemployment rate increased because despite their improved health, many individuals were still unable to find employment .In contrast to studies that present health and welfare as an oppositional dyad, this study offers scope for engaging with the subtleties of negotiating health and welfare in a country struggling with high rates of HIV prevalence and unemployment ,40.Disability grants are not a sufficient response to intransigent poverty. This puts pressure on both the state and local decision makers, like doctors, to find alternative means of supporting individuals and families affected by HIV and unemployment. Citing Fakir, Jones summarizes the challenges of providing social services in South Africa; these include institutional weaknesses, gaps in quality, and a failure to adequately focus on the norms, values and attitudes of public officials with regard to HIV ,42. In aSimilarly, Macgregor discussed the ways in which the notion of health as a human right engenders expectations of the state to provide the means to ensure health . She desDoctors are deemed "fit" to make medical assessments for the grant-issuing authority (SASSA) so they play a critical role in the administration of the grants. Their role of assessing grant eligibility in the public health system is based on their integration into the public services. Given that the South African social welfare system places the responsibility for determining disability grant eligibility on doctors, our findings indicate that doctors experience considerable pressure when recommending eligibility, so that they use different kinds of criteria to determine eligibility.Through their role in determining eligibility, doctors have a strong influence on each individual's quality of life. As long as the disability grant aims at compensating for a clinical condition or a disability, the doctors' role will remain pre-eminent. If the disability grant primarily becomes a means for poverty relief, the doctors' role becomes problematic. The crucial role of doctors in approving the grant applications combined with weak institutional structures and the needs of the PLHIV revealed in our study, may explain why the doctors feel that they are under such pressure. It is likely that the institutional weaknesses of the social services increase the pressure on the doctors by further enhancing the importance of their role in the system. The general lack of clarity with regards to the guidelines and the variation in the ways in which the grant is approved also contributes to the doctor's increased importance, and perhaps to the participants' perception of "inequity" in grant eligibility and administration. It seems that the pressure on the doctors comes from both higher administrative levels through weaknesses in the system and from lower levels through the social conditions of the individuals and families affected by HIV.Access to financial resources is necessary if PLHIV are to maintain their health by taking ARVs; PLHIV also need to have a set of structures in place to support them when they are on treatment.The disability grant plays a critical role in supporting PLHIV who are unable to find employment. However, there seems to be some unfairness in the social grant system. We did not find evidence that PLHIV stopped taking ARVs in order to qualify or re-qualify for a disability grant. However, the disability grant is crucial to many PLHIV because it ensures them some degree of economic stability. Given the importance of the public sector ARV roll out, it is decisive for the state to provide these resources.The traditional understanding of disability implies that the disabled cannot work at all. Being HIV positive and on ARVs represents a position that challenges this kind of understanding. It is possible to work, but the kind of work that PLHIV can do is affected by both HIV and ARVs. This offers scope for further research on the contingencies of living with HIV, on ARVs.A sound welfare policy could take these more diffuse kinds of living conditions into account, and develop means that are either targeted towards groups with special needs or universal in the sense that all groups in need are covered. Alternatives to disability grants could be a chronic illness grant or a basic income grant, and should be considered by policymakers given the role that social assistance plays in making "positive living" a reality for indigent and unemployed PLHIV.Furthermore, incentives to create more jobs are pivotal. Given that unemployment is a central concern for many people with HIV, an unemployment grant would also be an important improvement. It is therefore important to ensure a stable economy for unemployed individuals and households affected by HIV through strategic and multi-layered support.The authors declare that they have no competing interests, neither financial nor non-financial.MMDP participated in the data collection, the data analysis and the writing of the paper. She has taken the main lead in the revision of the paper. EAM participated in the data collection, the data analysis and the writing of the paper. ABG participated in the data collection, the data analysis and the writing of the paper. All authors have read and approved the final version of this manuscript.