Lives saved have become a standard metric to express health benefits across interventions and diseases. Recent estimates of malaria-attributable under-five deaths prevented using the Lives Saved tool (LiST), extrapolating effectiveness estimates from community-randomized trials of scale-up of insecticide-treated nets (ITNs) in the 1990s, confirm the substantial impact and good cost-effectiveness that ITNs have achieved in high-endemic sub-Saharan Africa. An even higher cost-effectiveness would likely have been found if the modelling had included the additional indirect mortality impact of ITNs on preventing deaths from other common child illnesses, to which malaria contributes as a risk factor.As conventional ITNs are being replaced by long-lasting insecticidal nets and scale-up is expanded to target universal coverage for full, all-age populations at risk, enhanced transmission reduction may--above certain thresholds--enhance the mortality impact beyond that observed in the trials of the 1990s. On the other hand, lives saved by ITNs might fall if improved malaria case management with artemisinin-based combination therapy averts the deaths that ITNs would otherwise prevent.Validation and updating of LiST's simple assumption of a universal, fixed coverage-to-mortality-reduction ratio will require enhanced national programme and impact monitoring and evaluation. Key indicators for time trend analysis include malaria-related mortality from population-based surveys and vital registration, vector control and treatment coverage from surveys, and parasitologically-confirmed malaria cases and deaths recorded in health facilities. Indispensable is triangulation with dynamic transmission models, fitted to long-term trend data on vector, parasite and human populations over successive phases of malaria control and elimination.Sound, locally optimized budget allocation including on monitoring and evaluation priorities will benefit much if policy makers and programme planners use planning tools such as LiST - even when predictions are less certain than often understood. The ultimate success of LiST for supporting malaria prevention may be to prove its linear predictions less and less relevant. Ongoing scale-up of malaria control in an era of leveling funds for health requires that investments are justified in terms of their impact and value for money . The RolLives saved have become a standard metric to express these benefits across interventions and diseases -8. The sThe Lives Saved Tool (a.k.a. LiST) used by Eisele and colleagues is a suitable mortality projection model, that has the advantage of considering the impact of malaria interventions in the context of multiple key child health interventions, and the full envelope of child deaths from all causes -12.The authors qualify their malaria impact estimates as conservative, which is probably correct despite several uncertainties; in any case the presented cost-effectiveness ratios are somewhat below those published earlier ,14.First, lives saved were evaluated within an envelope of malaria-attributable deaths, estimated at 17% of all-cause deaths in children 1-59 months in the year 2000 . In the malaria-attributable deaths in the same age group [Second, the ITN trials, reaching a near-universal 80-100%) household possession of ITNs, estimated effectiveness at a stable 17% reduction in all-cause mortality among children 1-59 months, or 5.5 per 1,000 child-years . Re-analge group . However0% househge group -23.A third uncertainty is how ITN effectiveness plays out at coverage levels different from the near-universal household possession achieved in the trials. Across the 36 countries now analysed, household ownership ranged from 3.5% to 94% in 2010 . ConversFurther re-analysis of data from the 1990s trials will not improve certainty here. More up-to-date reassurance comes from a recent ecological analysis of household surveys across 22 countries. At ITN ownership levels ranging from 1% to 54% (over 2001-2009), ITN household ownership reduced all-cause post-neonatal under-five mortality by 24% , more than the 17% in the cluster-randomized trials . While tEnhanced validation of ITN/LLIN effectiveness assumptions would require model fitting to individual settings that scaled-up ITN coverage and measured all-cause and malaria-attributable mortality trends, in the absence of change in other child health interventions. This situation is--fortunately--becoming rarer, as countries scale-up vector control alongside improved malaria treatment ,27, and Validation against recent programme scale-up and mortality trends will furthermore help to monitor whether mortality patterns as of 2000 (adjusted only for population growth) remain the best counterfactual. Even if ITN usage stopped altogether, malaria-related mortality may not resurge to levels as high as in 2000, since child deaths from other major--interacting--causes such as measles, pneumonia and HIV have fallen since ,30. Alsointerim approach [Next ambitions should focus on enhancing mortality measurement, and on better understanding the impact of vector control in settings of varying endemicity, mortality and anti-malarial treatment patterns. This requires better population-based data on the effective coverage of ACT-based treatment  and IRS,approach ,36.Synthesizing new insights from improved impact data requires a very different type of models, simulating interactions between vector, parasite and human populations over successive phases of malaria control and elimination ,37. DynaMeanwhile, LiST remains useful to guide short-term strategic planning and M&E priorities in endemic countries, notably reinforcing international recommendations to measure child service coverage and impact indicators through nationally representative household surveys. The pending incorporation of ACT-based treatment  will impMalaria elimination is becoming a tempting goal even for several currently endemic African countries. But recent gains remain fragile in view of evolving drug and insecticide resistance, malaria's epidemic nature, and the challenge to sustain programme funding despite many competing development priorities that are all (very) cost-effective . Sound, My views do not necessarily represent the decisions, policy or views of the Global Fund to Fight AIDS, Tuberculosis and Malaria. I declare that I have no conflicts of interest.


The aim of the present study was to comprehensively evaluate systemic and local inflammation as well as progression of vascular inflammation in normal and mechanically injured vessels in a large animal model of mild hypercholesterolemia. Our aim was also to test the effect of high-dose statin treatment on these processes.Pigs were kept for 120 days on a standard diet , high-cholesterol diet  or high-cholesterol diet with Atorvastatin starting after 50 days . Left carotid artery balloon injury was conducted in all groups after 60 days of diet treatment. Biochemical analysis together with evaluation of blood and tissue markers of vascular injury and inflammation were performed in all groups at the end of experiment.HCD compared to SD induced systemic inflammation demonstrated by increased number of circulating monocytes and lymphocytes. HCD compared to SD induced also local inflammation demonstrated by adipocyte hypertrophy and infiltration of T-lymphocytes in abdominal white adipose tissue, activation of hepatic stellate cells with infiltration of T- and B-lymphocytes and macrophages in the liver and increased macrophage content in lung parenchyma. These changes were accompanied by increased Intima/Media thickness, stenosis, matrix deposition and activated T-cell infiltrates in injured but not in uninjured contralateral carotid artery as we previously reported. The treatment with high-dose statin attenuated all aspects of systemic and local inflammation as well as pathological changes in injured carotid artery.Diet related mild hypercholesterolemia induce systemic and local inflammation in the liver, lung and adipose tissue that coincide with enhanced inflammation of injured vessel but is without deleterious effect on uninjured vessels. High dose statin attenuated systemic and local inflammation and protected injured vessels. However, finding exact role of reduced systemic and remote inflammation in vascular protection requires further studies. The long-term consequences of high-fat and/or high-cholesterol diet consumption are associated with an increased risk of cardiovascular disease, fatty liver disease, obesity and type 2 diabetes .The fattMetabolic cells that are exposed to excess of nutrients and energy respond triggering a chronic inflammation not only in vasculature but also in other organs. The architecture of liver, white adipose tissue (WAT) and lung is characterized by a close interaction between metabolic and immune cells. In obese individuals, cells of the metabolic tissues (such as adipose tissue and liver) can eventually initiate the pro-inflammatory signaling cascade causing activation of leukocytes, thus leading to tissue-specific inflammation . Recent evidence in mice suggested that dietary cholesterol exacerbates inflammatory changes due to an increased recruitment of leukocytes in WAT, particularly macrophages and T-lymphocytes –7. Over Heme oxygenase 1 (HO-1) is a stress-induced protein that is expressed in response to a variety of stimuli. HO-1 expression is implicated in protection against atherosclerosis. Previous studies have shown that the increase in HO-1 expression and activity by statins may have an anti-atherosclerotic effect in humans and in animal models of atherosclerosis ,17.. HO-Statins, by inhibiting 3-hydroxy-3-methylglutaryl-coenzyme A (HMGCoA) reductase, decrease endogenous cholesterol synthesis. Most data indicate that statins exert pleiotropic effects in addition to the lowering of serum cholesterol . These iMouse models have been widely used to evaluate the effects of high-fat feeding, however many of the evidences obtained are biased by differences in anatomy, lipid metabolism and lipoprotein profile. Unlike humans, even an excess of blood cholesterol in mice is carried by high-density lipoprotein (HDL) and not by low-density lipoprotein (LDL), this could hamper the translation of experimental results obtained in mice to humans . On the In the present study we evaluated for the first time in a porcine model of mild-hypercholesterolemia whether high-cholesterol diet, besides vascular changes, is sufficient to induce a systemic and tissue-specific inflammation. We addressed the effects of high-dose atorvastatin treatment on entire spectrum of vascular, systemic and tissue specific inflammation to identify key factors involved in pathology. Large White female pigs (n=21) were purchased from a commercial breeder. Experiments involving animals were carried out according to a protocol approved by the Animal Care Committee of the Italian Minister of Public Health. The investigation conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health .Pigs were allocated into three groups: SD (n=7), pigs fed a standard porcine diet (3.6% lipid content); HCD (n=7), pigs fed a high-cholesterol diet ; STATIN (n=7), high-cholesterol fed pigs (the same diet as for HCD group) treated orally with high-dose atorvastatin  starting after 50 days of high-cholesterol diet (80 mg/day/55Kg pig) until the end of the experimental procedure . HCD-fed pigs received nearly 1100 Kcal extra/day compared to SD-fed pigs. The diet had an overall duration of 120 days.After 60 days, the vascular injury in porcine carotid was induced as previously described .Blood samples were collected from pigs at the beginning of diet administration and at sacrifice. The plasma obtained was kept at -80°C until the measurements. Aspartate aminotransferase (AST), Alanine aminotransferase (ALT), total, LDL and HDL cholesterol and glucose were determined with colorimetric/enzymatic assays . All biochemical assays were carried out on an automated analyzer (Olympus AU 400). To determine counts of red blood cells, hemoglobin and hematocrit levels, WBCs, monocytes, lymphocytes, neutrophils, basophils, eosinophils, and platelets, complete blood counts were performed on EDTA anticoagulated blood samples using a Cell-Dyn 3500 system (Abbott Diagnostics). Hemograms were performed using a Cell-Dyn 3500 system (Abbott Diagnostics). Blood smears were May-Grunwald-Giemsa stained and visually evaluated for white blood cell differential counts. Analyses of inflammatory cytokines were performed using the SearchLight Chemiluminescent Array Kit specific for IL-1β, IL-6, interferon-gamma (IFNγ) and TNFα (Pierce Biotechnology). Assays were performed according to the manufacturer’s instructions. Balloon-injured left carotid arteries and contralateral carotid arteries (as negative control) as well as liver, lung and subcutaneous abdominal WAT were harvested at sacrifice, fixed in paraformaldehyde 4% and embedded in paraffin blocks. Serial sections (3 µm thickness) were cut and either stained with hematoxylin&eosin or Masson’s Trichrome staining (Bio-Optica).Immunohistochemical staining was performed on paraffin-embedded tissue sections of carotid arteries, liver, lung and subcutaneous abdominal WAT from each pig. To test antibody specificity, for each primary antibody used, control sections were incubated a) without the primary antibody or b) with the antibody, which had been previously incubated in presence of the peptide antigen . Antigen retrieval was achieved by boiling in citrate buffer (pH 6.0). Tissue sections were incubated with primary antibodies for staining the following antigens: mouse anti-human CD45RO (M0742) DAKO, dilution 1:200; mouse anti-human MAC387 (sc-66204), Santa Cruz, dilution 1:200; mouse anti-human CD20 (18-0088), Zymed, dilution 1:300; mouse anti-human CD15 (MY1), Histo-Line Laboratories, dilution 1:100; mouse anti-human CD138 (MI15), DAKO, dilution 1:100; mouse anti-human smooth muscle actin (MS-113-P), Neomarkers, dilution 1:3000; mouse anti-human type I collagen (sc-59772), Santa Cruz, dilution 1:400; rabbit anti-porcine TGF-β1 (sc-146), Santa Cruz, dilution 1:250; rabbit anti-human TGFβRII (sc-400), Santa Cruz, dilution 1:50; rabbit anti-human HO-1 (spa-895), Stressgen, dilution 1:1000; rabbit anti-human iNOS (sc-651), Santa Cruz, dilution 1:200. VSMCs in carotid arteries have been detected using an antibody specific for the alpha smooth muscle actin . Proliferation index was evaluated by immunohistochemistry with a mouse monoclonal anti-Ki67 antibody . Apoptosis index was evaluated by immunohistochemistry with a rabbit polyclonal antibody specific for the cleaved form of caspase-3 . A biotinylated secondary antibody was used for streptavidine-biotin-complex peroxidase staining . DAB was used as chromogen (Sigma-Aldrich), and sections were counterstained with hematoxylin . At least three slides per tissue per pig were analyzed for each staining. The Aperio ScanScope GL Slide Scanner  system was used to capture digital images with a 40x scanning magnification (20x with 2x magnification changer). The ScanScope console was equipped with a Nikon 20x/0.75 Plan Apochromat objective producing a 0.25 µm/pixel scanning resolution. The Aperio ImageScope software (version 8.2.5.1263) was used to acquire and process digital images. The carotid arteries were harvested 60 days after the balloon-injury procedure. The carotid arteries were fixed in paraformaldeyde 4% and embedded in paraffin blocks. Serial sections (3 um thickness) were cut and stained with hematoxylin and eosin. Two independent operators (MB and AF) blindly evaluated all the sections obtained for each carotid artery by microscopic analysis. The ten sections showing the worst damage were selected for a full morphometric analysis, including: Intima-Media Area ratio, Intima-Media Thickness ratio, Intima-Media Thickness, degree of stenosis, intimal thickness, medial thickness, internal elastic lamina perimeter, external elastic lamina perimeter, internal elastic lamina area, intimal area, external elastic lamina area, medial area. The computer assisted analysis has been performed using specific software . The degree of stenosis has been evaluated using the following formula: /*100. The results reported for each animal for Intima/Media (I/M) Area ratio, I/M Thickness ratio, I/M Thickness and degree of stenosis represent the average value obtained by considering the three sections showing the worst damage. p<0.05. The statistical analyses were done using SPSS 17.0.Data are presented as the mean ±S.E.M. Datasets from each experiment were tested for normal distribution using SigmaPlot software. For studies involving two groups, comparisons were made using two-tailed t-tests. For multiple group comparisons were made using one-way ANOVA followed by Tuckey post-hoc test. Correlations were determined using Spearman’s rank order analysis. Differences were considered significant when We already reported that after 120 days the high-cholesterol diet significantly increased serum total cholesterol, LDL-cholesterol [Blood analysis performed after 120 days showed a significantly increased number of total WBCs in HCD-fed pigs compared to SD-fed pigs . Blood sSubcutaneous abdominal WAT from HCD-fed pigs showed adipocyte hypertrophy. The mean cross-sectional area of adipocytes from HCD-fed pigs was significantly increased compared to the adipocytes from SD-fed pigs. An immunohistochemical staining specific for activated T lymphocytes showed that high-cholesterol diet induced a significant increase in the amount of infiltrating T-lymphocytes into the abdominal WAT compared to SD. Treatment with atorvastatin effectively reduced both the adipocyte mean cross-sectional area and the amount of infiltrating T-lymphocytes compared to HCD . The immImmunohistochemical liver analysis revealed increased leukocyte infiltration in the hepatic parenchyma of HCD-fed pigs compared with SD-fed pigs. The majority of leukocytes infiltrating the liver were macrophages . A signiIn HCD-fed pigs the expression of the stress-inducible genes HO-1 and iNOS was significantly increased compared to SD-fed pigs . AtorvasLung sections from HCD-fed pigs showed a significantly increased recruitment of macrophages compared to the amount of macrophages detected in SD-fed pigs . The treAtorvastatin significantly reduced I/M Area and I/M Thickness ratio compared to HCD . The degThe present study demonstrates for the first time that statin treatment reduces generalized inflammation in spite of a minimal effect on lipids in a model of vascular injury in pigs fed high cholesterol diet. In fact diet is sufficient to cause a systemic inflammation by increasing the number of circulating WBCs, and to promote tissue-specific inflammation, by raising the amount of infiltrating leukocytes in WAT, liver and lung. These changes could contribute to increased vascular injury in hypercholesterolemic pigs. However statin treatment reversed not only enhanced vascular injury in hypercholesterolemic pigs but also attenuated most of systemic and localized inflammatory responses. Previous studies have indicated that the pig could represent an ideal preclinical model providing insight into lipoprotein metabolism for several reasons. In particular, pigs have omnivorous habits and a lipoprotein distribution similar to that found in humans; high-cholesterol diets are able to induce human-like changes in the plasma lipoprotein profile of pigs, with ~ 60% of plasma cholesterol distributed in LDL particles ,26.In the present study we set up a model of mild-hypercholesterolemic pig, with total and LDL cholesterol concentrations 20% higher than in normocholesterolemic pigs. These supra-physiological concentrations are similar to those of the vast majority of patients, very different from other models showing an increase of cholesterol up to 700%. In fact, nearly 50% of Western Countries adults have total cholesterol concentrations at the level that the National Cholesterol Education Program (NCEP) expert panel considers “borderline-high risk” [Epidemiological studies have reported that leukocyte counts rise in atherosclerotic patients and there is a positive correlation between increased amounts of circulating leukocytes and coronary artery disease ,28. TranIn this regard, blood analysis performed at sacrifice, after 16 weeks of hypercholesterolemic diet, displayed a significant increase in total WBCs, monocytes and lymphocytes in HCD-fed pigs compared to SD-fed pigs. This is in accordance with previous findings from our research group in which pigs fed a high-cholesterol diet showed an increased number of circulating WBCs, particularly monocytes . The strPrevious studies in hypercholesterolemic mice have shown that macrophage and lymphocyte infiltration is of critical importance in WAT inflammation. T-lymphocytes may be enrolled during early adipose tissue inflammation preceding the appearance of macrophages, and the recruitment of these leukocytes to sites of inflammation is usually mediated by chemokines released from preadipocytes and adipocytes . In addiThe occurrence of WAT inflammation in response to the excess of lipids leads to a low-level induction of inflammatory cytokines such as TNF-α, IL-1β and IL-6: this low-grade inflammation induces the further recruitment and activation of many professional immune cells . In our Metabolic disorders related to lipid metabolism such as the increase of serum triglycerides, total cholesterol and LDL-cholesterol, are well known causes of liver injury characterized by inflammation, steatosis and fibrosis . HSCs plThe administration of a high-cholesterol diet in our preclinical model did not induce either steatosis or fibrosis, HSCs activation notwithstanding. The evaluation of ECM deposition by trichrome staining and the immunohistochemical analysis specific for type I collagen, TGF-β1 and TGFβRII expression did not show significant differences on liver sections from HCD- and SD-fed pigs. Interestingly, HSCs activation and leukocyte infiltration of hepatic tissue did not alter hepatic function. In particular, HCD-fed pigs did not display increased levels of ALT and AST after 16 weeks on a high-cholesterol diet compared to SD-fed pigs. It is widely demonstrated that the administration of large amounts of dietary cholesterol causes hepatic inflammation in both mice and rabbits and it has been recently showed that high-fat feeding in mice increases the hepatic recruitment of T-lymphocytes and macrophages ,37,38. IIn our model, the inflammation extended beyond the WAT and the liver to the lung parenchyma, with a significantly increased amount of infiltrating macrophages in HCD-fed pigs compared to SD-fed pigs. This enrichment in lung macrophages was associated with neither parenchyma remodeling nor fibrosis. Moreover, no signs of increased type I collagen deposition as well as significant differences in the expression of TGF-β1 and TGFβRII were are observed. Our results are in contrast with a recent work from Naura et al. in which it has been shown that a high-fat diet induces the persistence of lung inflammation associated with tissue remodeling in apoE-KO mice .To test whether reducing total cholesterolemia could determine a significant decrease of circulating leukocytes, a subset of HCD-fed pigs received high-dose atorvastatin (80mg/die) for 8 weeks. Statins, the inhibitors of HMG-CoA reductase, are extensively used in medical practice for their cholesterol-lowering effect. Large clinical trials have demonstrated that this class of drugs greatly reduces cardiovascular-related morbidity and mortality in patients with and without coronary disease. Several works have established that statins, in addition to their lipid-lowering effects, exert immuno-modulatory actions being able to decrease the expression of adhesion molecules, chemokines and chemokine receptors on both leukocytes and endothelial cells, ultimately limiting the recruitment of leukocytes across the vessel wall ,40. The Consistent with above considerations, we determined that treatment of animals with high-dose atorvastatin resulted in a significant reduction of serum total cholesterol and LDL-cholesterol concentration. However, no significant reduction in triglycerides was noted.Compared to the findings in HCD-fed pigs, treatment with atorvastatin markedly lowered the number of circulating WBCs, monocytes and lymphocytes. The immuno-modulatory effects of atorvastatin went beyond the reduction of circulating WBCs. Our findings demonstrated that high-dose atorvastatin treatment leads to a substantial decrease of the hypercholesterolemia-induced inflammatory infiltrates in WAT, liver and lung. In particular, treatment with atorvastatin in HCD-fed pigs prevented adipocyte hypertrophy limiting the number of infiltrating T-lymphocytes. Furthermore, statin-treatment considerably reduced the amount of circulating pro-inflammatory cytokines IFN-γ and TNF-α. In the liver, atorvastatin caused a significant reduction of activated HSCs and of the amount of infiltrating leukocytes, with a concomitant reduction of the stress-inducible gene HO-1 but without affecting the hepatic expression of iNOS. Last, atorvastatin treatment in HCD-fed pigs was effective in reducing the macrophage content in lung parenchyma.Inflammatory response in liver, lung and adipose tissue correlated with enhanced intimal hyperplasia in carotid artery. Interestingly, no response was observed in contralateral uninjured artery. This highlights the importance of local injury and systemic/remote inflammation in the development of vascular pathology as we previously identified . AtorvasIn conclusion, our study demonstrates for the first time in a clinically relevant porcine model of diet induced hypercholesterolemia that even a moderate increase in plasma cholesterol levels can induce a significant increase in the amount of circulating WBCs. This effect extends beyond systemic inflammation to tissue-specific inflammation in WAT, liver and lung. We also establish that a mild-hypercholesterolemia is sufficient to induce an augmented leukocyte infiltrates in these organs. HCD-fed pigs treated with high-dose atorvastatin results in a reduction of total and LDL cholesterol and in markedly decreased systemic and tissue-specific inflammatory markers by preventing the development of an inflammatory milieu and the accumulation of infiltrating leukocytes.


Moreover, database screening revealed overall more intracellular protein interaction partners in pre-mating than in post-mating sperm proteins. Finally, post-mating sperm proteins evolved at significantly higher evolutionary rates than pre-mating sperm and non-reproductive proteins on the branches to multi-male breeding species, while no such increase was observed on the branches to unimale and monogamous species. We conclude that less protein–protein interactions of post-mating sperm proteins account for lowered functional constraint, allowing for stronger impact of post-mating sexual selection, while the opposite holds true for pre-mating sperm proteins. This pattern is particularly strong in multi-male breeding species showing high female promiscuity.To assess the relative impact of functional constraint and post-mating sexual selection on sequence evolution of reproductive proteins, we examined 169 primate sperm proteins. In order to recognize potential genome-wide trends, we additionally analysed a sample of altogether 318 non-reproductive (brain and postsynaptic) proteins. Based on cDNAs of eight primate species (Anthropoidea), we observed that pre-mating sperm proteins engaged in sperm composition and assembly show significantly lower incidence of site-specific positive selection and overall lower non-synonymous to synonymous substitution rates ( But against expectations, many sperm proteins are evolutionarily conserved ,18. Thisproteins ,20. Stilzona pellucida- and egg-binding, gamete recognition, sperm–egg interaction, egg-activation and gamete fusion). The expectation is that species-specific levels of sexual selection may have a stronger impact on evolutionary rates of post-mating proteins, whereas functional constraint may particularly restrict sequence evolution of pre-mating sperm proteins.This study aims at assessing the relative impact of both functional constraint and sexual selection on evolutionary rates of functionally distinct sperm proteins. Present analyses are based on 169 human sperm proteins with increased expression in testis or prostate and a clear assignment to one of the following categories: (i) pre-mating sperm proteins that are engaged in sperm composition  or sperm assembly  and (ii) post-mating sperm proteins that prepare  or actively participate in fertilization . Assuming neutral evolution of synonymous exchanges, dN/dS ratios > 1 stand for selection for more amino acid exchanges than expected under neutrality . In turn, dN/dS values < 1 can be taken as evidence for negative selection, and hence selection against amino acid exchanges. We quantified levels of functional constraint on the basis of direct and indirect protein interaction partners. The impact of post-mating sexual selection was evaluated by comparing sequence evolution between primate species with higher and lower levels of female promiscuity. Potential associations between dN/dS, protein interactions and mating system variation may provide new insights into the mechanisms involved in the evolution of sperm proteins. Furthermore, they may open up new perspectives regarding genes/proteins as targets for diagnosis and treatment of impaired male fertility, development of non-hormonal contraceptives and identification of fertility markers in animal husbandry. In order to verify whether high numbers of direct and indirect protein interactions reflect levels of functional constraint, we compiled and analysed an additional, non-reproductive dataset comprising a total of 318 brain and postsynaptic proteins. The sample of non-reproductive proteins additionally enabled us to control for species-specific differences in demographic history that should affect entire genomes.Evolutionary rates of primate (anthropoid) sperm proteins were assessed at the cDNA level using the ratio of non-synonymous to synonymous substitution rates (2.(a)et al. [et al. [et al. [et al. [http://www.ebi.ac.uk/gxa/) for human microarray data and excluded all proteins without consistent information concerning their upregulation in at least one of the search items under consideration . We also excluded proteins, for which no alignment could be compiled containing the aspired species set of eight primates (see below) owing to missing entries, insecure annotation and/or insufficient sequence quality. Based on UniProt gene ontology annotations and original literature, we assigned each of these sperm proteins to one of the following two functional categories:— Pre-mating sperm proteins : the 110 proteins falling into this category are constituents of structural components, such as cytoskeleton , axoneme and outer dense fibres. Furthermore, these proteins participate in gene regulation, spermatogenesis or sperm maturation, finally leading to mature spermatozoa ready for ejaculation. The included motor proteins of the dynein complex are involved in the bending of the sperm tail.— Post-mating sperm proteins : the 59 proteins of this category are either involved in post-mating processes increasing sperm motility and priming spermatozoa for sperm–egg interaction  or contribute immediately to gamete recognition, sperm–egg interaction, egg-activation and gamete fusion via interaction with female molecules.Our sample of 169 sperm proteins was based on three proteomic studies carried out by Ficarro et al. , Martíne [et al.  and Part [et al. . The fir [et al. , we only(b)Homo sapiens, Hsa), common chimpanzee , western lowland gorilla , Sumatran orang-utan , northern white-cheeked gibbon , Rhesus monkey , olive baboon  and white-tufted-ear marmoset . Coding DNAs were retrieved from ENSEMBL and NCBI databases . Subsequent analyses of sequence evolution were conducted in the maximum-likelihood (ML) framework implemented in the PAML v. 4.4  package [For each of the 169 sperm proteins, we generated a codon-based alignment using the MUSCLE algorithm implemented in the GUIDANCE web-server . Alignme package . The loa(i)dN/dS interval . However, while the alternative version (M8) allows for an extra site class under positive selection (dN/dS ≥ 1), dN/dS of this extra site class is fixed at 1 in the null version (M8A). To ensure convergence at global optima, M8 analyses were run thrice with different initial dN/dS values . For LRT, 2Δl was compared with critical values following a 50 : 50 mixture of a point mass at zero and a χ2 distribution with degrees of freedom (d.f.) equal to the difference in the number of free parameters between M8A and M8 (=1). To reduce the number of false positives, we applied a 1% level of significance  .Each of the 169 alignments was tested for the presence of positively selected codon sites employing a likelihood ratio test (LRT) that compares the fit of two beta model versions implemented in Codeml . Both moχ2-test. Additionally, levels of dN/dS across sites (M8A) were compared between both groups by employing Mann–Whitney U-test (two sided). All tests were conducted with SPSS v. 20.0 applying a 5% level of significance and sequential Bonferroni correction for multiple comparisons. We computed 95% confidence intervals (CIs) for each proportion and median on the basis of 100 000 bootstrap replicates using in-house PERL scripts.Subsequently, we analysed if the distribution of proteins with/without candidate sites of positive selection differed between pre- and post-mating sperm proteins using the (ii)Effects of post-mating sexual selection on sequence evolution of pre-mating and post-mating sperm proteins were investigated across protein groups considering variant mating systems in the sampled species . In orddN/dS to vary across branches, on each of the cDNA alignments and compared the estimates for terminal branches to northern white-cheeked gibbon and common chimpanzee. These two species were chosen because they represent the two extremes in the range of mating systems covered by our sample: while common chimpanzees are multi-male breeders with an extraordinarily high number of periovulatory mating partners, northern white-cheeked gibbons are monogamous and extra-pair matings have not been reported , which allows eported  and higher levels of post-mating sexual selection (foreground 2: chimpanzee and Rhesus monkey). The branches representing white-tufted-ear marmoset and Sumatran orang-utan were sampled into the background instead of foreground 1 owing to frequent extra-pair and -group matings in both species that impair predictions regarding levels of post-mating sexual selection [Papio hamadryas [We additionally ran a branch model that inferred election –32. Furtamadryas ,34.dN/dS estimates across pre-mating sperm, post-mating sperm and non-reproductive proteins using Kruskal–Wallis rank-sum test . If Kruskal–Wallis test rejected equality of distribution, we conducted post hoc Mann–Whitney U-test on pairs of protein groups (two sided). Kruskal–Wallis and Mann–Whitney U-tests were conducted with SPSS v. 20.0 applying a 5% level of significance and sequential Bonferroni correction. Ninety-five per cent CIs of medians were inferred using an in-house PERL script. Whenever short branches impaired the inference of dN/dS estimates for at least one of the terminal branches or foregrounds compared, a protein was excluded from downstream analyses. This procedure led to inclusion of 29 pre-mating sperm, 25 post-mating sperm and 102 non-reproductive proteins when focusing on the branches to common chimpanzee and northern white-cheeked gibbon . Comparisons of foregrounds 1 and 2 were based on 44 pre-mating sperm, 43 post-mating sperm and 177 non-reproductive proteins .Subsequently, we tested gibbon branch, chimpanzee branch, foreground 1 and foreground 2 for different distributions of (c)We assessed levels of functional constraint for each of the 169 sperm proteins based on numbers of direct and indirect protein–protein interaction partners (PIP) as taken from 17 out of 25 databases available through PSICQUIC , using human protein IDs as search items  and employing the PSICQUIC clustering feature that provides a non-redundant list of interactants. The GeneMANIA, iRefIndex, Interoporc and STRING databases were opted out in order to avoid that assumed (instead of proven) interactions biased our results. For the same reason, we ignored search results with the attributes ‘unspecified method’, ‘predictive text mining’ and/or ‘inferred by curator’ (quotation marks highlight PSICQUIC terminology). Moreover, results without information about the underlying methodology or referring to interactions between human proteins and proteins of other species including pathogens were excluded. Therefore, we also ignored hits from the MPIDB and VirHostNet databases, which focus on interactions with microbes and viruses. Additionally, we avoided to record interactions between proteins and drug-like molecules, and thus excluded the BindingDB and ChEMBL databases. In the electronic supplementary material, tables S5 and S6, we list numbers of interactions per sampled pre-mating and post-mating sperm protein.nPIP) and dN/dS across sites (M8A), employing Spearman's rank correlation. Additionally, we investigated whether levels of dN/dS across sites (M8A) differed between 71 sperm proteins with nPIP < 10 and 16 sperm proteins with nPIP > 100 using Mann–Whitney U-test. Finally, we examined whether levels of nPIP differed between pre- and post-mating sperm proteins, employing Mann–Whitney U-test again. Ninety-five per cent CIs of medians  were inferred from 100 000 bootstrap replicates, each. All analyses were carried out with SPSS v. 20.0 applying a 5% level of significance and sequential Bonferroni correction for multiple testing. An analogous procedure was applied to a set of 318 non-reproductive proteins (see below), thereof 136 with nPIP < 10 and 29 with nPIP > 100 .We checked for a correlation between the number of direct and indirect protein interaction partners per protein ((d)nPIP and sequence evolution, we compiled a sample of non-reproductive proteins, adopting a previous approach [et al. [et al. [nPIP are reported in the electronic supplementary material, tables S9 and S10.In order to (i) recognize potential effects of demography on sequence evolution of sperm proteins and (ii) validate a potential relationship between approach . This sa [et al.  and Bayé [et al.  that sho3.(a)a; electronic supplementary material, table S11). Additionally, median dN/dS values (M8A) pointed to overall enhanced non-synonymous/synonymous substitution rate ratios of post-mating proteins (=0.233) versus pre-mating sperm proteins (=0.077) (figure 1b). In line with this, χ2 and Mann–Whitney U-tests provided highly significant support for increased incidence of site-specific positive selection and overall higher dN/dS values (M8A) in post-mating relative to pre-mating sperm proteins .At the 1% level of significance, LRT statistics supported the presence of positively selected codon sites for 33 out of 169 cDNA alignments , each representing a constant set of eight primate (anthropoid) species. The proportion of alignments including candidate sites of positive selection was markedly higher in post-mating sperm proteins preparing or actively participating in fertilization (36%) than in pre-mating sperm proteins engaged in sperm composition or sperm assembly (11%) (figure 1(b)dN/dS estimates across pre- and post-mating sperm and non-reproductive proteins for the branch to multi-male breeding common chimpanzee (p < 0.05), but not for the branch to monogamous northern white-cheeked gibbon . Indeed, 95% CIs of median dN/dS estimates were highly overlapping with respect to the gibbon branch. On the contrary, 95% CI of post-mating sperm proteins ranged above the respective intervals of pre-mating and non-reproductive proteins with regard to the chimpanzee branch (a). As far as the chimpanzee branch was concerned, levels of dN/dS estimates were more than twofold higher in post-mating sperm proteins (median dN/dS = 0.428) than in pre-mating sperm (median dN/dS = 0.190) and non-reproductive proteins (median dN/dS = 0.211) . Accordingly, post hoc analyses of the chimpanzee branch provided significant support for different dN/dS levels in post-mating sperm relative to pre-mating sperm proteins (p < 0.05) and non-reproductive proteins . However, no such support was provided when comparing dN/dS estimates of pre-mating sperm and non-reproductive proteins for the chimpanzee branch .Kruskal–Wallis test rejected equal distribution of e branch a. As farp < 0.05), but not for the branches to monogamous gibbon and human and unimale breeding gorilla . Moreover, Mann–Whitney U-test confirmed significantly higher dN/dS estimates for post-mating sperm proteins (median dN/dS = 0.339) as compared with pre-mating sperm proteins  and non-reproductive proteins  for the branches to chimpanzee and Rhesus monkey. However, focusing on the same branches, Mann–Whitney U-test did not support different levels of dN/dS in pre-mating sperm and non-reproductive proteins .These findings could be reproduced when expanding analyses from chimpanzee and gibbon to our species samples representing higher and lower levels of post-mating sexual selection. Thus, test results suggested unequal evolutionary rates of the three distinguished protein classes for the terminal branches to multi-male breeding chimpanzee and Rhesus monkey dN/dS values across sites (M8A) and nPIP per human orthologue were negatively correlated in non-reproductive proteins . In our sample of sperm proteins, the negative correlation was even more pronounced . In line with this general trend, levels of dN/dS across sites (M8A) were consistently higher in sperm and non-reproductive proteins with nPIP < 10  than in their counterparts having nPIP > 100 . Thus, overall higher numbers of protein interactants in pre-mating (median nPIP = 22) than in post-mating sperm proteins  probably reflect increased levels of functional constraint in the former relative to the latter group . Taken together, our data suggest that post-mating sperm proteins are less functionally constrained and more subjected to some form of post-mating sexual selection than are pre-mating sperm proteins. This pattern is more obvious in multi-male breeding species than in monogamous and unimale breeding species.Spearman's correlation indicated with high significance that 4.(a)dN/dS values across sites in post-mating versus pre-mating sperm proteins confirms a general association between protein function and evolutionary rate nPIP < 10 versus nPIP > 100) made the negative association between numbers of interactants and evolutionary rates even more obvious in our sperm and non-reproductive protein samples (a).Protein–protein interactions mediate diverse intra- and intercellular processes and are pivotal for the functionality of cells and organisms. As impaired functioning of one protein affects all or at least some of its direct and indirect interactions, proteins having more interactants are more likely crucial for cell functioning than proteins with less interactants  most likely reflect their peripheral role in the sperm interactome and not a bias from potentially less comprehensive data on postcopulatory interactions between male and female proteins. Consequently, we ascribe less incidence of site-specific positive selection and lower dN/dS values across sites (M8A) in pre-mating versus post-mating sperm proteins to overall higher numbers of intracellular interactants in the former relative to the latter group .(c)dN/dS values in post-mating relative to pre-mating sperm and non-reproductive proteins for the branch to common chimpanzee and for foreground 2 comprising chimpanzee and Rhesus branches. On the contrary, post-mating sperm, pre-mating sperm and non-reproductive proteins evolved at similar rates on the gibbon branch and across foreground 1 which merges gibbon, human and gorilla branches . The models employed (free-ratio model and branch model) might also have had an impact on the respective dN/dS estimates. However, the most probable explanation for this observation is that levels of post-mating sexual selection are lower in Rhesus monkey  than common chimpanzee  and in a multi-species approach (foreground 1 versus foreground 2), the increase of evolutionary rates of post-mating sperm proteins appeared not as strong in the combined analysis of chimpanzee and Rhesus branches as in the isolated analysis of the chimpanzee branch  despiteIt is further noteworthy that post-mating sperm proteins might also evolve at slightly increased evolutionary rates in northern white-cheeked gibbon, western lowland gorilla and human (see medians in 5.Our data suggest that less functional constraint and more effective post-mating sexual selection explain overall increased non-synonymous/synonymous substitution rate ratios in post-mating relative to pre-mating sperm proteins. Present analyses further illustrate that the accelerating effect of post-mating sexual selection on sequence evolution is particularly effective on post-mating sperm proteins. But despite an apparent effect of functional constraint and post-mating sexual selection, other factors might affect sequence evolution of sperm proteins too. In particular, immune evasion, which describes the evolutionary escape of male reproductive proteins from female immune system, functional redundancy and defence against transposable elements through the piRNA pathway –57 may a


The STING signaling pathway has emerged as a central TLR-independent mediator of host innate defense in response to sensing cytosolic nucleic acids, either through direct binding of CDNs secreted by intracellular bacteria, or through binding of a structurally distinct CDN produced by a host cell receptor, cyclic GMP-AMP (cGAMP) synthase (cGAS), in response to binding cytosolic double-stranded DNA. Binding of CDNs to STING initiates a signaling cascade through the TBK-1/IRF-3 axis to induce type I interferon (IFN) and other co-regulated genes. Discovered recently, CDNs synthesized by cGAS and bacteria are structurally distinct. The cGAS product, termed non-canonical CDN, has a phosphate bridge with both 2’-5’ and 3’-5’ linkages that reportedly increases its affinity for STING by 200-fold. Here we show in a panel of human donors that the non-canonical linkage confers significantly increased activity of CDNs to activate PBMCs, as measured by expression of IFN and NF-κB dependent cytokines, as compared to CDNs with canonical 3’-5’ phosphate linkages. By conducting Immunogenicity-Structure Activity Relationship studies in vitro and in various animal models of infection and cancer, we selected synthetic derivatives of native CDNs produced by bacteria or eukaryotic cells with increased potency. CDN derivatives with sulfur atoms at non-bridging oxygens of the internucleotide phosphate bridge were resistant to digestion with phosphodiesterase. Surprisingly, R,R di-thio CDN diastereomers induced higher levels of IFN in vitro, and induced a higher magnitude of peak and memory antigen-specific CD4 and CD8 T cell responses correlated with protective immunity, as compared to either the R,S di-thio CDN diastereomer, or di-oxo CDN. CDNs based on adenosine nucleotides were comparatively independent of host cell permeabilization to activate STING signaling. We applied this finding to enhance the activity of immunotherapy regimens utilizing irradiated GM-CSF expressing tumor cell vaccines. Co-formulation with R,R dithio-c-di-AMP significantly inhibited tumor growth in several models, correlated with increased mobilization and activation of dendritic cells, increased tumor lymphocyte infiltration, and T cell immunity. Immunologic potency of CDNs was essentially lost in mice encoding a non-functional STING allele. Collectively, CDNs have high translation potential for diverse applications in clinical oncology.


Endogenous retroviruses (ERVs) are integrated as DNA proviruses in the genomes of all mammalian species. Several ERVs are replication-competent and produced as fully infectious viruses from host cell. Thus, live-attenuated vaccines and biological substances have been prepared using the cell lines which may produce ERV. Indeed, we recently reported that several commercial live-attenuated vaccines for pets were contaminated with the infectious feline endogenous retrovirus, RD-114. In this study, to establish a cell line for vaccine manufacture with reduced risk of ERVs, we generated a cell line stably expressing human tetherin (Teth-CRFK cells). The release of infectious ERV from Teth-CRFK cells was suppressed to undetectable levels, while the production of parvovirus in Teth-CRFK cells was similar to that in parental CRFK cells. These observations suggest that Teth-CRFK cells will be useful as a cell line for the manufacture of live-attenuated vaccines or biological substances with reduced risk of ERV. Endogenous retroviruses (ERVs) are integrated as DNA proviruses in the genomes of all mammalian species. Several ERVs are replication-competent and produced as fully infectious viruses from host cells, although most are inactivated by deletions and mutations with stop codons Recently, we reported that several commercial live-attenuated vaccines for pets were contaminated with the infectious feline endogenous gammaretrovirus, RD-114 RD-114 is a polytropic virus Tetherin  was originally identified as a cellular restriction factor that blocks the release of HIV-1 in the absence of the viral accessory protein, Vpu In this study, we attempted to establish the CRFK cell line with reduced risk of endogenous RD-114 by suppressing the release of infectious RD-114 from cells by the antiviral action of tetherin.2 incubator in Dulbecco's modified Eagle's medium  supplemented with 10% fetal bovine serum and penicillin/streptomycin. CRFK cells were transfected with an expression plasmid for human tetherin containing a FLAG-tag at the N-terminus, pTeth-FL, using Trans-IT LT-1  CRFK cells (ATCC CCL-94) were maintained at 37°C in a 5% CO6 cells) were cultured for 3 days. The culture media from each cell line were collected and then centrifuged to sediment RD-114 virions CRFK and Teth-CRFK cells (1×10LacZ marker rescue assay established previously lacZ (FeLV-B) virus 6 cells), CRFK (LacZ) and Teth-CRFK (LacZ) cells, were cultured for 3 days. We confirmed by X-gal staining that the transduction efficiency of lacZ gene into CRFK (LacZ) and Teth-CRFK (LacZ) cells were 100%. After changing the culture media, cells were further incubated for 4 days. The culture supernatants were collected and then filtrated through 0.45-µm filters. The filtrated culture supernatants were inoculated into TE671 cells. After 2 days, cells were stained with X-gal and then foci were counted.The infectious titer of RD-114 virus produced from CRFK cells and Teth-CRFK cells was quantified using the method modified the 5 cells/well into 96-well plates and then cultured for 2 days. The assay was performed according to the manufacturer's instructions. The spectrophotometric absorbance of the sample was measured at 595 nm wavelength using a TriStar LB941 .The cell viabilities of CRFK cells and Teth-CRFK cells were measured by MTT assay using a Cell Proliferation Kit I (MTT) . CRFK and Teth-CRFK cells were seeded at 1×1050 using FL74 cells (feline lymphoblastoid cell line) as described previously In this study, we used two parvovirus strains, TU1 strain The CRFK cell line stably expressing human tetherin, Teth-CRFK cell line, was established as described in 6 cells) for 3 days and measured the copy number of viral RNA derived from RD-114 virion released from each cell line into the culture media by real-time RT-PCR. As shown in CRFK cells are known to be constitutively produce infectious RD-114 LacZ marker rescue assay. The infectious titer of RD-114 virus produced from CRFK cells was 46.7 focus-forming units (f.f.u.)/ml, while that produced from Teth-CRFK cells was below the detection limit (<1 f.f.u./ml)  . These rWe next examined the cell viability of Teth-CRFK cells by MTT assay, since the constitutive expression of tetherin may affect the proliferation of CRFK cells. As shown in Feline cell lines, including CRFK cells, have been widely used to manufacture vaccines for parvoviruses such as feline panleukopenia virus (FPLV) and canine parvoviruses (CPVs). Indeed, there have been some reports regarding contamination of commercial live-attenuated vaccines for these viruses with infectious RD-114 50 using FL74 cells. As shown in To examine the effects of tetherin expression on the growth of parvoviruses, we next examined the growth of parvoviruses in Teth-CRFK cells. CRFK and Teth-CRFK cells were infected with TU1 strain of FPLV or V220 strain of CPV-2a and then incubated for 2 days. The titers of parvoviruses produced in CRFK or Teth-CRFK cells were measured in TCIDLacZ marker rescue assay showed that infectious RD-114 was not detected in the samples from Teth-CRFK cells infected with FPLV and CPV (data not shown). These were consistent with the results from We also measured the titer of RD-114 in the parvovirus preparations from both CRFK cells and Teth-CRFK cells by real-time RT-PCR. As shown in We previously demonstrated that RD-114 proviral DNA was never detected in PBMC from the dogs inoculated with 1,200 FFU of RD-114 . Thus, we estimate that parvovirus vaccines prepared from the Teth-CRFK cell line should not establish RD-114 infection in vivo even after concentrating the supernatants >1,200-foldTaken together, these observations suggested that Teth-CRFK cells would be useful for the manufacture of live-attenuated vaccines of nonenveloped viruses or biological substances with reduced risk of contamination with infectious RD-114, although the use for enveloped viruses which could be inhibited by tetherin might be limited. Furthermore, similar strategies using tetherin would be applicable to establish various cell lines with reduced risk of endogenous retrovirus contamination.


The acute effects of HCE on blood pressure were studied in anaesthetised normotensive male Wistar rats challenged with intravenous hypotonic saline infusion after a 3.5-hour equilibration for four hours of one-hour control, 1.5-hour treatment and 1.5-hour recovery periods. HCE was added to the infusate during the treatment period. Sub-chronic hypotensive effects of HCE were examined in weanling Dahl salt-sensitive (DSS) genetically hypertensive rats, which progressively develop hypertension with age, treated with HCE (80 mg/kg) every third consecutive day for seven weeks. Isolated atrial muscle strips, portal veins and descending thoracic aortic rings of healthy normotensive Wistar rats were used to investigate the vascular effects of HCE.The aim of this study was to examine some in vivo and p < 0.05) fall in blood pressure in the normotensive anaesthetised Wistar rats. DSS hypertensive rats treated with HCE displayed low arterial blood pressure and heart rate values from weeks five to seven. HCE produced concentration-dependent negative inotropic and chronotropic effects on rat isolated electrically driven left, and spontaneously beating right atrial muscle preparations, respectively. HCE also evoked concentration-dependent relaxation responses of endothelium-intact aortic rings and portal veins isolated from healthy normotensive Wistar rats. The vasorelaxant effects of HCE in intact aortic rings were significantly reduced, but not completely abolished by adding endothelial-derived factor (EDRF) inhibitor, L-NAME, suggesting that the vasorelaxant effect of the extract is mediated via EDRF-dependent and independent mechanisms.Acute HCE administration caused a significant (The results of the study suggest that the hypotensive action of HCE is elicited, in part, directly by decreasing myocardial contractile performance and total peripheral vascular resistance due to its negative inotropic and chronotropic effects on rat isolated atrial muscle strips; and vasorelaxant effects on isolated vascular smooth muscles. The observed cardiovascular effects of HCE partly support the basis for its use in the management of high blood pressure in folkloric medicine. Prevailing economic conditions and poor health services in many developing populations necessitate the development of alternative low-cost therapies to manage various human ailments. Plant extracts present another option of easily accessible sources of new drugs. Indeed, some plant extracts have been reported to be effective in the management of hypertension, including complications such as coronary heart disease, angina, arrhythmias and congestive heart failure.4Helichrysum  possess anti-inflammatory and anti-allergic properties.Helichrysum ceres S Moore [Asteraceae] to treat kidney and cardiorespiratory disorders.H ceres leaf extract in anaesthetised male Sprague-Dawley rats can, in part, be attributed to the extract’s natriuretic and diuretic properties.H ceres leaf extract may avert the progression of high blood pressure in the Dahl salt-sensitive rats (DSS), a genetic model of salt-sensitivity hypertension.9Ethnomedical evidence indicates that extracts of the genus The main aim of the present study was, therefore, to assess the effects of H ceres leaf extract on mean arterial blood pressure in normotensive Wistar and hypertensive DSS rats. We also evaluated the effects of the leaf extract on the myocardial contractile performance of rat isolated atrial muscle strips, and the vasodilatory effects on isolated portal veins and thoracic aortic rings of healthy normal Wistar rats. We envisaged that establishment of the mechanism(s) of the cardiovascular effects of the leaf extract would provide scientific evidence for the development of an inexpensive and accessible source of novel drugs for the treatment of hypertension and certain cardiac disorders in underprivileged, developing populations.Methoxamine hydrochloride (ME), acetylcholine chloride (ACh), indomethacin, NG-nitro-L-arginine-methyl-ester (LNAME), methylene blue, atropine sulphate (ATR), glibenclamide, (±)-propranolol hydrochloride, (-)-noradrenaline hydrochloride (NA), and nifedipine were obtained from Sigma . All other chemicals were of the analytical grade and supplied by Merck chemicals (Pty) Ltd, . Indomethacin was dissolved in 0.5% sodium bicarbonate. Glibenclamide was dissolved in dimethyl sulphoxide (DMSO). All other drugs, including Krebs’ solution were freshly prepared in deionised water.Helichrysum ceres (S Moore) [Asteraceae] identified by Professor H Baijnath, the former chief taxonomist/curator of the University of KwaZulu-Natal’s Department of Botany, were collected around Durban, South Africa, between January and June 2005. A voucher specimen of the plant has been deposited in the University’s Botany Herbarium.Leaves from H ceres leaf extract was prepared as previously described.Ethanolic ad libitum. Procedures involving animals and their care were conducted in conformity with institutional guidelines of the University of KwaZulu-Natal.Male Wistar (250–300 g) and weanling Dahl salt-sensitive rats (100–150 g), bred and housed at the Biomedical Research Unit, University of KwaZulu-Natal were used in this study. The rats were maintained on a 12-h light/12-h dark regime, and given both food  and water in vivo in the Wistar and DSS rats, respectively (DSS rats develop hypertension as they age). The effect of HCE on myocardial contractile performance was evaluated on isolated rat atrial muscle strips, while the vasodilatory effects of the extract were examined on isolated thoracic aortic rings and portal veins of healthy Wistar rats.The acute and sub-chronic effects of HCE on mean arterial blood pressure (MAP) were examined Wistar normotensive rats were prepared for acute blood pressure studies using a method previously described in detail.A heparinised cannula inserted into the carotid artery directly measured blood pressure and heart rate at 30-min intervals via a pressure transducer  compatible with the PowerLab System ML410/W . The urinary bladder of each rat was also cannulated, for urine voidance, with a similar calibre polythene tubing via an incision in the abdominal wall.Following a 3- to 5-hour equilibration period, measurements were recorded over the four-hour post-equilibration period of one-hour control, 1.5-hour treatment and 1.5-hour recovery periods. In those animals in which the effects of the extract were studied, HCE was added to the infusate at 360 μg/h for 1.5 hours (treatment period), resulting in a total dose of 1.8 mg/kg (for a 300-g rat), before the animals were returned to infusate alone for the last 1.5 hours (recovery period). The depth of anaesthesia was monitored throughout the experiment and additional intravenous bolus doses of inactin (0.05 g/kg body weight) were administered when necessary. The administration rates of the extracts were chosen from previous experience.7n = 8, per group) were used. Mean arterial blood pressure was measured every third consecutive day for seven weeks at 09:00 in both the untreated control group and the HCE-treated  DSS rats. Control rats were similarly treated with deionised water (3 ml/kg). A tail-cuff computerised blood pressure monitor  was used to assess the cardiovascular effects of HCE. The method was standardised and used routinely in our laboratory and is described elsewhere in detail.10In order to evaluate the long-term effects of HCE on blood pressure, separate groups comprising control and extract-treated groups of DSS rats  containing Krebs-Henseleit solution (KHS) of the following composition in mM: NaCl, 118; KCl, 4.7; NaHTo evaluate the contractile force of the extract (inotropic), the electrically driven left atrium was impaled on a thin platinum wire electrode and stimulated (5–10 mV) with square wave pulses of 5-ms duration at a frequency of 3 Hz via an SRI stimulator . To examine the extract’s effect on atrial pacemaker activity (chronotropic), spontaneously beating right atria were set up under the same physiological experimental conditions. Two isolated electrically driven left atrial muscle strips, and two isolated spontaneously beating right atrial muscle preparations were always set up at a time  to allow for changes in the atrial muscle sensitivity.n = 8 preparations for each concentration).Concentration–response curves for HCE (0–160 mg/ml) and/or reference agonist drugs were obtained. Control atrial muscle strips were treated with volumes of deionised water equivalent to the volumes of bath-applied HCE solution (0.1–0.6 ml). The electrically provoked and spontaneous contractions of the atrial muscles, as well as the HCE (and reference agonist drug)-induced responses of the atrial muscle preparations were recorded isometrically by means of Ugo Basile force–displacement transducers and pen-writing Gemini recorders (model 7070). The effects of HCE/drugs were expressed as a percentage of the baseline values . Satisfactory and functional endothelium was checked by at least 10% and 70% relaxation, respectively, to 10-6 M ACh. After a sustained stable tonic contraction was achieved, concentration–response curves for HCE (0–160 mg/ml) and/or reference agonist drugs were obtained.Aorta pieces (4–5 mm long) suspended under a resting tension of 1 g in a 30-ml Ugo-Basile organ bath containing KHS were left to equilibrate for 45–60 minutes. In some experiments, the endothelium was denuded mechanically G-nitro-L-arginine methyl ester, L-NAME, 100 μM, a nitric oxide synthase inhibitor; methylene blue, 10 μM, a guanylate cyclase inhibitor; and indomethacin, 10 μM, a non-selective cyclooxygenase inhibitor).The involvement of endothelium-derived factors in HCE-induced relaxation was examined in intact aortic rings pre-treated with appropriate antagonists  in the presence glibenclamide, and high potassium , respectively, as previously described.14To assess the role of potassium or calcium in the vasorelaxant effect of the extract, concentration–response curves of HCE were constructed in endothelium-intact aortic rings pre-contracted with potassium  suspended under a resting tension of 0.5 g in a 30-ml Ugo-Basile organ bath containing KHS was allowed to equilibrate for 60 minutes, following which, graded concentrations of HCE  were added to the organ bath in separate preparations to establish its effects on the amplitude of myogenic contractions.To investigate whether the effects of HCE were mediated through modulation of alpha-1 adrenergic receptors or voltage-operated calcium channels, some of the portal vein preparations were pre-treated with an alpha-1 adrenergic receptor blocker, prazosin (1 μM), and an L-type voltage-operated calcium channel blocker, nifedipine (1 μM), respectively, five minutes before re-establishing the cumulative concentration–dependent responses. Control venous muscle strips were treated with deionised water equivalent to the volumes of bath-applied HCE solution. Two isolated venous tissue preparations (one control and the other HCE- or reference drug-treated test) were always set up in order to make allowances for changes in the venous tissue sensitivity.n = 8 preparations for each concentration).The HCE- and/or drug-induced responses of the venous smooth muscle preparations recorded isometrically by means of Ugo Basile force–displacement transducers and pen-writing Gemini recorders were calculated as a percentage of the baseline values .All results are expressed throughout as means ± SEM. Data were analysed by one-way analysis of variances (ANOVA) followed by Dunnett multiple-comparison tests. A value of in vivo in normotensive Wistar and DSS hypertensive rats. The MAP and heart rate of Wistar rats challenged with continuous jugular infusion of 0.077 M NaCl at 9 ml/h did not change during the four-hour experiment with HCE To evaluate the long-term effects of HCE on mean arterial blood pressure and heart rate, DSS rats were treated every third consecutive day for seven weeks at 09:00 with HCE . By comparison with values in week one and untreated animals after four weeks, HCE treatment of DSS rats prevented the expected age-related increase in blood pressure and slightly reduced the heart rate p < 0.05) and concentration-dependently decreased the force of myocardial contraction (negative inotropy) of the rat isolated electrically driven left atria Pre-treatment of the atrial preparations with ATR elicited a complete abolition of the negative chronotropic effect of HCE 2 (0–8 mM) were established in isolated electrically driven left atria in the absence or presence of HCE (40 or 80 mg/ml). Both noradrenaline and calcium induced concentration–response curves (positive inotropy) To determine whether HCE influenced the noradrenaline- or calcium-induced inotropic effects, cumulative concentration–response relationships for noradrenaline (0–1.5 ml) or CaCl1-adrenoceptor agonist) evoked concentration-dependent relaxation responses of endothelium-intact aortic rings Addition of cumulative concentrations of HCE (0–160 mg/ml) to aortic rings pre-contracted with 10 μM ME  were constructed in separate preparations of ME pre-contracted endothelium-intact aortic rings in the absence or presence of EDRF inhibitors: nitric oxide synthase inhibitor (L-NAME), guanylate cyclase inhibitor, (methylene blue) and cyclooxygenase inhibitor, (indomethacin). The vasorelaxant effects of HCE were significantly reduced by the antagonists +, 20 mM) or a high potassium concentration  to evaluate the role of potassium and calcium channels, respectively, on the vasorelaxant effects of the extract. In order to assess the involvement of KATP-sensitive channels, the rings were pre-treated with 3 μM glibenclamide (ATP-sensitive potassium channel blocker) before pre-contracting with K+ (20 mM). Vasorelaxation of aortic ring preparations pre-contracted with low potassium (K+ < 30 mM) concentration occurs via opening of the potassium channels.+ did not cause any change in the vasorelaxant effects of HCE Concentration–response curves of HCE 0–160 mg/ml) were established in endothelium-intact aortic rings pre-contracted with a low . HCE (0–160 mg/ml) induced a significant vasorelaxation in the endothelium-intact aortic rings pre-contracted with high K+2+ through L-type voltage-sensitive channels (VOCs).18To evaluate the role of calcium channels in the vasorelaxant effects of HCE, endothelium-intact aortic rings were precontracted with high concentrations of potassium (Kp > 0.05) in endothelium-intact aortic rings contracted with low  or high  potassium concentrations The vasorelaxant effects of graded concentrations of HCE (10–160 mg/ml) were not statistically different  on the rhythmic, myogenic spontaneous contractions were investigated in rat isolated spontaneously contracting portal veins. HCE induced a significant and concentration-dependent vasorelaxation in these portal vein preparations Helichrysum ceres leaf extract reduces blood pressure.in vivo and exhibited vasorelaxant effects in vascular tissue preparations, suggesting that the extract decreases cardiac output and the total peripheral resistance of blood vessels.The experimental evidence presented in this study advances our previous report that Sub-chronic HCE treatment of DSS rats prevented the development of hypertension, presumably via the polyphenols, tannins, triterpenes and saponins present in the extract.1-adrenergic receptors and calcium channels, as evidenced by the extract’s reduction of the noradrenaline or calcium concentration-dependent positive inotropic effects Pre-treatment of atrial preparations with atropine completely abolished the negative chronotropic and inotropic effects of lower concentrations of HCE, but partial negative inotropic effects persisted at higher concentrations (160 mg/ml). We speculate that HCE’s cardio-inhibitory effects involve inhibition of β1-adrenoceptor agonist) Removal of the aortic endothelium did not completely abolish the HCE-evoked vasorelaxant effects in rings pre-contracted with ME (α+ (< 30 mM) occurs via the opening of the potassium channels.+ (> 35 mM).HCE exhibited concentration-dependent vasorelaxation in endothelium-intact aortic rings pre-contracted with potassium concentrations of 20 mM. Vasorelaxation of aortic preparations pre-contracted with low KATP channels.14The vasorelaxant effect of HCE endothelium-intact aortic rings was, however, not significantly affected by pre-treatment with glibenclamide, a selective blocker of K2+-activated Kca channelsWe suggest that the vasorelaxant effects of HCE in KCl-contracted aortic rings were mediated by endothelium-derived hyperpolarising factor (EDRF), which directly opens arterial smooth muscle Ca+ (80 mM) concentrations + in KCl-depolarised muscles are due to the influx of extracellular Ca2+ through L-type voltage-sensitive channels (VOCs).2+ influx. Taken together, the opening of potassium and calcium channels also appears to be a possible mechanism for the HCE-induced vasorelaxation.HCE elicited concentration-dependent vasorelaxations in the intact aortic rings pre-contracted with high KHCE elicited concentration-dependent vasorelaxation in myogenic spontaneously contracting isolated portal vein preparations 21H ceres has been reported to contain polyphenols, tannins, triterpenes and saponins with a large variety of pharmacological actions.Although the bioactive constituents of HCE were not established in this study, The observed vascular effects of HCE provide, in part, not only mechanisms of hypotensive action of the extract, but also the basis for its use in the management of high blood pressure in folkloric medicine.


To apply deconvolution algorithms to voxel-wise analysis of first-pass myocardial perfusion MR data and to determine how noise affects perfusion estimation using different quantification methods.One of the main advantages of cardiac first-pass perfusion MR is its high spatial resolution. Though several methods have been used to quantify myocardial perfusion rate, no previous work has been done on voxel-wise analysis and published methods were applied to standard myocardial segments. Signal-to-noise ratio (SNR) might negatively affect the accuracy of the measurements obtained with methods developed for segmental analysis.Fermi function modelling, auto-regressive moving-average model (ARMA), model-independent approach using B-spline-basis and exponential-basis deconvolution were used to quantify myocardial perfusion rate in 6 patients. Perfusion data were acquired on a 3 Tesla Philips CMR scanner in three LV short axis slices with a saturation recovery gradient echo method  during adenosine-induced hyperaemia (140µg/kg/min) using 0.05mmol/kg Gd-DTPA  at 4ml/minute followed by a 20 ml saline flush. Voxel-wise perfusion estimates were compared to the results obtained from segmental analysis, for different levels of spatial averaging (10 voxels - 1 segment - whole slice). The data shown in Figure ARMA method analysis resulted in the lowest model curve-fit error for different level of noise in both rest and stress condition. Exponential deconvolution had a lower error when compared to Fermi function modelling and model independent analysis Figure . The oveThe curve-fit error increased when analyzing rest perfusion images, most likely as a result of the increased baseline signal intensity values or of a lower perfusion rate in the rest images, acquired after stress.This study confirms the importance of adequate SNR in first-pass perfusion images. Before voxel-wise analysis can be used in clinical practice, more studies will be needed to define the best algorithms to deal with reduced SNR typical for voxel-wise tissue data. ARMA approach and Exponential basis deconvolution were the least sensitive methods to noise.


Anaplastic lymphoma kinase-positive non-small cell lung carcinoma patients are generally highly responsive to the dual anaplastic lymphoma kinase and MET tyrosine kinase inhibitor crizotinib. However, they eventually acquire resistance to this drug, preventing the anaplastic lymphoma kinase inhibitors from having a prolonged beneficial effect. The molecular mechanisms responsible for crizotinib resistance are beginning to emerge, e.g., in some anaplastic lymphoma kinase-positive non-small cell lung carcinomas the development of secondary mutations in this gene has been described. However, the events behind crizotinib-resistance currently remain largely uncharacterized. Thus, we report on an anaplastic lymphoma kinase-positive non-small cell lung carcinoma patient with concomitant occurrence of epidermal growth factor receptor and V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog mutations upon development of crizotinib-resistance.epidermal growth factor receptor and V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog mutations concomitant with the original anaplastic lymphoma kinase gene rearrangement and without signs of anaplastic lymphoma kinase fusion gene amplification or secondary anaplastic lymphoma kinase mutations.A 61-year-old Caucasian never-smoking male was diagnosed with anaplastic lymphoma kinase -positive pulmonary adenocarcinoma, stage T4N3M1b. Treatment with crizotinib initially resulted in complete objective response in the thorax and partial response in the abdomen, but after 8 months of therapy the patient acquired resistance and progressed. Biopsies from new metastases revealed development of anaplastic lymphoma kinase-positive pulmonary adenocarcinoma, which upon emergence of crizotinib resistance acquired 2 new somatic mutations in the epidermal growth factor receptor and V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog genes, respectively, concomitant with the original anaplastic lymphoma kinase rearrangement. Thus, these 3 driver mutations, usually considered mutually exclusive, may coexist in advanced non-small cell lung carcinoma that becomes resistant to crizotinib, presumably because heterogeneous tumor clones utilize epidermal growth factor receptor and/or V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog signaling to circumvent the inhibition of anaplastic lymphoma kinase-mediated signaling by crizotinib. The identification of new targetable somatic mutations by tumor re-biopsy may help clarify the mechanism behind the development of the acquired crizotinib resistance and pave the way for combined strategies involving multiple targeted therapies.To our knowledge, this is the first report of an  EGFR  and ALK (anaplastic lymphoma kinase) genes can obtain remarkable benefit from therapies targeting these oncogenic drivers [ALK gene fusions were identified in a distinct subpopulation (approximately 5%) of NSCLC patients [EGFR- or KRAS  genes or amplification of MET  gene [ALK-positive NSCLC patients have been shown to be highly responsive to the oral small-molecule triple ALK, ROS1 and MET tyrosine kinase inhibitor (TKI) crizotinib  [ALK-rearranged NSCLC, crizotinib was approved as second-line treatment for this subset of patients by the US Food and Drug Administration in August 2011 [ALK-positive NSCLC [ALK rearrangements typically consist of a small inversion on chromosome 2p23-21, resulting in the fusion between exon 20–29 of the ALK gene (encoding the kinase-domain) and exons 1–13 (different variants due to different breakpoints) in the N-terminal portion of the Echinoderm Microtubule-associated protein-Like 4 (EML4) gene. Rarer ALK translocation partners, such as Kinesin Family member 5B (KIF5B) and TRK-fused gene (TFG) have also been reported in NSCLC. Cases of atypical translocation with partial loss of chromosomal material, resulting in so called single-red signals by fluorescence in-situ hybridization (FISH) analysis of ALK gene rearrangements in tumor cell nuclei, may also occur [ALK rearrangements result in the expression of stabilized chimeric ALK fusion-proteins with constitutive kinase activity and oncogenic properties [Approximately 85% of newly diagnosed lung cancers are non-small-cell lung carcinoma (NSCLC). Despite surgery, radiotherapy and intensive chemotherapy, the median 5-year survival rate remains around 10% [ drivers . Oncogenpatients  typicallon) gene . ALK-pos Pfizer) . Based ove NSCLC . ALK reaALK-positive NSCLC patients initially show rapid and effective response to crizotinib [ALK-positive NSCLC patients the development of secondary point mutations in the ALK kinase domain or ALK fusion gene amplification have been shown to be responsible for the acquired resistance to crizotinib [Although most of izotinib ,6, they izotinib ,12,13. Aizotinib ,12,14,1518F-FDG). Furthermore, bilateral pleural effusion and direct tumor invasion into mediastinum and pericardium was observed. All together, the TNM stage was T4N3M1b.A 61-year-old Caucasian never-smoking male was referred to hospital in June 2011 due to respiratory complains and weight loss. The initial diagnosis was pneumonia but fused imaging positron emission tomography (PET) and computerized tomography (CT) of the chest revealed an irregular tumor infiltrate of 5 × 5 cm and possible lymphangitis carcinomatosa in the left lung’s upper lobe, enlargement of bilateral mediastinal and right cervical lymph nodes, and enlargement of both adrenals glands. All these lesions displayed increased uptake of 18F-fluoro-2-deoxy-D-glucose , revealed intense positive staining in the metastatic tumor tissue . The cell nuclei with rearrangement appeared with one normal fusion signal and an abnormal single red signal for ALK, characteristic for ALK gene fusion with partial deletion of genetic material [A small transbronchial biopsy of the pulmonary infiltrate showed a histology and immunohistochemistry (IHC) profile  of primary adenocarcinoma (data not shown). Because of insufficient biopsy material for molecular tests and quickly deteriorating patient performance, first-line chemotherapy including carboplatin, vinorelbine, and bevacizumab was started. Despite an initial partial response to this treatment, the disease further progressed with massive enlargement of mediastinal, retroperitoneal, and inguinal lymph nodes  IHC-staining for ALK protein in the tumor tissue   were detected in the relapsed metastasis , tumor clones bearing the L862R mutation could also be potentially responsive to EGFR-TKI. Meanwhile, because of rapid worsening while waiting for these new molecular tests the patient received third-line pemetrexed, showing further progression in abdomen and pelvis, but still no sign of relapse in the thorax. Unfortunately, the patient rapidly deteriorated and died before being able to attempt a palliative radiation of the lymph nodes in pelvis and a fourth-line treatment with erlotinib.However, after 8 months of treatment the patient displayed massive disease progression in the retroperitoneal, pelvic and inguinal lymph nodes, while there was still complete response in the thorax Figure . In ordee Figure C-2D. Impsistance ,12,13. H) Figure  and in eALK-positive, EGFR and KRAS wild-type, pulmonary adenocarcinoma, who after 8 months of response to crizotinib treatment acquired resistance to this drug and progressed rapidly. Re-biopsy of relapsed metastatic tumor tissue showed newly emerged somatic mutations in both the KRAS and the EGFR gene, while the tumor cells still carried the original ALK rearrangement but without signs of ALK fusion gene amplification or secondary ALK mutations. These results indicated that the observed tumor progression was not due to one of the known mechanisms of acquired crizotinib resistance, i.e. loss of ALK rearrangement, amplification of ALK fusion gene, or secondary ALK mutations [EGFR and KRAS mutations. To our knowledge, this is the first report of a concomitant presence of ALK gene rearrangement, EGFR mutation, and KRAS mutation in the same NSCLC patient. Although the vast majority of ALK-positive pulmonary adenocarcinomas lack concomitant EGFR- or KRAS-mutations, suggesting that these mutations are mutually exclusive [EML4-ALK fusion gene and EGFR-mutations have been reported [ALK rearrangement and wild-type EGFR and KRAS, while re-biopsy from a new metastasis at progression after 8 months of crizotinib treatment revealed the appearance of EGFR and KRAS mutations in addition to an unchanged ALK rearrangement. This suggests that these 3 mutations, typically considered mutually exclusive [in vivo, presumably because the tumor cells or heterogeneous tumor clones utilize EGFR and/or KRAS signaling to circumvent the inhibition of ALK-mediated signaling by crizotinib. The emergence of either EGFR or KRAS mutations have been reported in single NSCLC cases acquiring resistance to crizotinib, both with and without concomitant loss of ALK gene rearrangement [Multiple molecular mechanisms are emerging as causes of acquired resistance to treatment with TKI like crizotinib, posing new challenges for targeted therapy of NSCLC. We have presented a patient with advanced, utations ,13,15, bxclusive ,12,16, sreported -20. Thes18F-FDG PET/CT scan, showed that 6% of them had metastasis to abdominal lymph nodes and only 0.5% to external iliac lymph nodes [Metastatic spreading of NSCLC to subdiaphragmatic lymph nodes, as observed in our case, is relatively uncommon compared to other metastatic sites, such as the liver. Indeed, a recent study of 1,191 NSCLC patients consecutively staged by ph nodes . HoweverKRAS mutation could render the patient resistant to an EGFR-targeting therapy. This could however not be verified since the patient died before a new targeted treatment could be initiated. Nevertheless, the possibility of treating the patient with the EGFR-TKI erlotinib was considered. Indeed, even if the co-existence of KRAS and EGFR mutations could be interpreted as double mutation occurring in the crizotinib-resistant tumor cells, the possibility of different tumor clones with activation of EGFR and KRAS signaling as different mechanisms of crizotinib-resistance appeared to be an alternative, perhaps more likely explanation. Furthermore, the identified L862R EGFR mutation, although not reported before, was located near mutations known to be sensitive to EGFR-TKI, such as L858R and L861X. Therefore, the tumor clone bearing this EGFR mutation could have been potentially responsive to EGR-TKI therapy. However, a previously described single case of L862V EGFR mutation was associated with lack of response to gefitinib and the same could have been the scenario with the L862R EGFR mutation. Functional characterization of this and other rare EGFR mutations is needed in order to elucidate their impact on treatment with TKI [Together with previous reports, our case clearly underlines the need for re-biopsy in patients who develop acquired resistance to a TKI such as crizotinib. This should be done primarily to investigate the mechanisms behind the acquired resistance, and hopefully identify new somatic mutations that potentially could be targeted. The appearance of new targetable somatic mutations could pave the way for combinatorial therapeutic strategies involving multiple targeted therapies. In this case, however, the presence of the G13D with TKI .EGFR and KRAS mutations could indicate a shift in the oncogenic survival and proliferation dependency from the ALK signaling pathway to EGFR and/or KRAS pathways in the ALK-TKI resistant cells. Activation of HER-family proteins has been associated with sustained down-stream signaling in the presence of ALK-TKI, indicative of the shift in survival dependency from the ALK signaling pathway to HER-family-driven pathways in the ALK-TKI resistant cells [KIT amplification, auto-phosphorylation of EGFR, and aberrant expression of other receptor tyrosine kinases have also been postulated by in vitro experiments to cause resistance to crizotinib in ALK-rearranged cancer cells [The appearance of new nt cells . Cell lint cells , suggester cells ,13,15.EGFR and KRAS mutations can arise completely independently, they share a common progenitor that acquires these drivers independently as later events, or both drivers coexist within the same cell, and then one or other is lost in subclonal evolution is still unclear. Regardless of which evolutionary clonal process takes place, the potential role for combinatorial therapeutics in overcoming crizotinib resistance in the clinic appears of increasing importance [However, the mechanisms behind crizotinib resistance in NSCLC patients are still poorly understood. NSCLC is increasingly recognized as heterogeneous set of diseases at the morphological and molecular level and these differences may drive therapeutic decision making. Tumor heterogeneity, the invariable development of resistance to targeted monotherapy with TKI, and the occurrence of multiple driver mutations in TKI-resistant tumor cells, appear to be problems strictly related to one another and capable of undermining effective treatment. Whether separate oncogenic driver subclones with portance ,15.ALK-rearranged pulmonary adenocarcinoma. Moreover, our case indicates that ALK-rearranged NSCLC may possibly acquire resistance to crizotinib through the emergence of concurrent somatic mutations in both EGFR and KRAS genes. The concomitant occurrence of EGFR and KRAS mutations with ALK rearrangement upon crizotinib resistance suggests that the most effective therapeutic strategy for ALK-positive lung cancers may ultimately require combined strategies targeting not only ALK gene fusions and ALK resistance mutations, but also other driver mutations that activate alternative TKI-resistance pathways.Our case demonstrates the utility of tumor re-biopsies in order to better understand the mechanisms of crizotinib resistance in patients with Written informed consent was obtained from the wife of the deceased patient for publication of this Case report and any accompanying images. A copy of the written consent is available for review by the Editor of this journal.Eric Santoni-Rugiu has received speaker fees from Pfizer and Roche as well as research grants from Pfizer. Jens Benn Sørensen has received speaker fees and research grants from Pfizer.Edyta Maria Urbanska has received speaker fees from Pfizer.The other authors declare that they have no competing interests.


O-methyloxymorphone, which may represent novel therapeutic molecules for IBD. These 14-alkoxymorphinans were pharmacologically and immunologically characterized.Of the three opioid receptors, μ (MOP), δ (DOP) and κ (KOP), the MOP type is the most involved in the action of opioids in the gut. Experimental studies on models of intestinal inflammation and inflammatory bowel disease (IBD) support an anti-inflammatory role of peripheral MOP receptors in the gut, besides their involvement in pain control  and gastrointestinal motility . Research focuses increasingly on exploring the therapeutic potential of peripheral MOP receptors aiming for identification of peripheral ligands as improved treatment for debilitating conditions associated with bowel functions. One strategy to increase peripheral selectivity includes chemical modifications that enhance hydrophilicity ,2. Our win vitro. Antinociceptive activities were assessed using acetic acid-induced writhing and tail-flick tests. Physicochemical properties (logP and logD) were determined using the MarvinSketch software.Synthesis of novel zwitterionic 14-alkoxymorphinans was accomplished by multi-step syntheses. Binding, functional and immunomodulatory activities were determined In vitro, the new 6-amino-acid-substituted 14-alkoxymorphinans bound with high affinity and showed agonist activity towards the MOP receptor. They significantly inhibited the nuclear transcription factor kappaB (NF-κB) activation in tumor-necrosis factor-α (TNF-α) and lipopolysaccharide (LPS)-stimulated human monocytic THP-1 cells. In vivo, they produced dose-dependent antinociceptive effects in mice after subcutaneous administration, being several-fold more potent than morphine. Based on the calculated logP and logD values, an increase of hydrophilicity, and thus peripheral selectivity can be achieved by attachment of amino acid residues to the morphinan skeleton.Novel MOP agonists acting in the periphery with combined immunosuppressive and analgesic properties may provide a new approach for the treatment of IBD.


Infection of human cells by Human T-cell leukaemia virus (HTLV-1) is mediated by the viral envelope glycoproteins. The gp46 surface glycoprotein binds to a cell surface receptors, including heparan sulphate proteoglycans, neuropilin-1, and Glut-1, allowing the transmembrane glycoprotein to initiate fusion of the viral and cellular membranes. The envelope glycoproteins are recognised by neutralising antibodies and cytotoxic T lymphocytes following a protective immune response, and therefore represent attractive components for an HTLV-1 vaccine. Here we begin to explore the immunological properties of recombinant SU as a potential subunit vaccine candidate. We have used a soluble recombinant gp46 fused to the Fc-region of human IgG (sRgp46-Fc) as an immunogen to vaccinate mice and generate monoclonal antibodies (mAbs) targeting SU. The recombinant SU protein is highly immunogenic, inducing high titre antibody responses to the immunogen and facilitating selection of hybridomas that secrete anti-SU mAbs. Many of these mAbs recognise envelope expressed by HTLV-1 infected cells and these antibodies strongly stimulate neutrophil-mediated cytotoxic responses. Moreover, several of the mAbs directly and robustly antagonise envelope-mediated fusion and neutralize pseudovirus infectivity. While antibodies targeting either the receptor-binding domain or C-terminal domain of SU exhibit anti-viral activity, the most potently neutralizing mAbs tend to recognise epitopes that cluster in the N-terminal receptor-binding domain of SU. Thus, our data demonstrate that recombinant forms of SU possess immunological features that are of direct utility to subunit vaccine design.


Fast, accurate, and reliable strain-typing is a prerequisite in managing MRSA epidemics. In this study, we compared three different strain-typing methods.The usefulness of spa-typing, pulse-field gel electrophoresis (PFGE) and automated repetitive extragenic palindromic sequence-based (rep-PCR) method  for typing Finnish MRSA strains was studied. A total of 110 clinical MRSA isolates collected from well-defined outbreaks in Ostrobothnia region and 96 isolates from several epidemics or sporadic sources all over Finland were analysed.Among all studied isolates 11 different major rep-PCR groups, 20 different PFGE and 10 different spa types were formed. Geographically close strains from separate local epidemics in Ostrobothia caused difficulties in the grouping and discrepancies among the typing methods. From this materialthree minor rep-PCR groups could be formed by DiversiLab software’s overlay analysis and 4 different PFGE groups. All of these isolates were, however, undistinguishable by spa-typing.PFGE method had the highest discriminatory power, but it is a non-automated and labour intensive method. Spa-typing was unable to differentiate close strains that belong to separate epidemics. Automated rep-PCR typing represents an attractive option for accurate and rapid molecular typing of MRSA. In addition, a rep-PCR strain library can be created that aids laboratories to classify isolates and hospitals to monitor the occurrence of different types.None declared.


Non ST-segment acute coronary syndromes (NSTE-ACS) comprise 70% of all ACS. Difficulty in recognizing which patients have myocardium at risk contributes to heterogeneity in NSTE-ACS management, leading to poor outcomes compared to ST-elevation myocardial infarction (STEMI). To date, lack of suitable animal models has impeded progress in optimizing care. We developed a novel canine model to mimic NSTE-ACS, and evaluated ex vivo myocardial characteristics in the context of in vivo T2-CMR that may identify at-risk and salvageable regions.Five mongrel dogs underwent thoracotomy to create a left circumflex coronary artery stenosis without complete occlusion and to place epicardial pacing electrodes, and two dogs underwent sham thoracotomy. Pacing the animals with circumflex stenosis produced ST depression that resolved upon termination of pacing. Pre- and post-pacing CMR was performed in anesthetized, intubated animals on a 1.5 Tesla scanner with T2 mapping and late gadolinium enhancement (LGE) scans.T2 increased significantly  in the circumflex-supplied lateral wall post-pacing in animals with stenosis compared to their remote myocardium  without producing evident LGE-positivity; no such T2 increase occurred in sham-operated animals. Ex vivo measurements of cellular respiration revealed decreased respiration in ischemic vs. remote regions . Electron microscopy showed mitochondrial swelling in the ischemic region, but no membrane breaks or other signs of cellular necrosis.in vivo T2-CMR can identify regions of that heart with potentially reversible myocardial and mitochondrial injury in a novel canine model of NSTE-ACS.These preliminary studies suggest that DHLRI, AHA


Endogenous retroviruses (ERVs), remnants of ancient retroviral infections, have invaded the germ line of every vertebrate species including both non-human primates and humans. Most ERVs are functionally crippled by extensive deletions, mutations and hypermethylation, leading to the view that they are inert genomic fossils. However, some ERVs retain the potential for biological activity and can produce mRNA transcripts, functional viral proteins, and even non-infectious virus particles during certain developmental and pathological processes. Although ERV gene products are implicated in the etiology of disease, their impact during host infection with exogenous retroviruses such as human and simian immunodeficiency virus (HIV/SIV) remains unknown. Here, we identify the homologue of the biologically active human ERV-K, macaque ERV-K(macERV-K), which retainsintact open reading frames in the macaque genome. Indeed, we isolated a fully-spliced mRNA product encoding macERV-K Env, which contains all the structural features of a canonical retroviral Envelope protein. Furthermore, we identified robust CD8+ and CD4+ T cell responses directed at macERV-K Env in one SIV-infected Indian rhesus macaque. The macERV-K Env-specific CD4+ T cell response in this animal was larger than the entire 5lV-specific CD4+ T cell response and had a uniform effector memory phenotype, indicating continual low-level antigen exposure. In support of this, we also identified antibody responses targeting macERV-K in this animal. Although SIV infection did not appear to trigger ERV Env-specific T cell responses, these data suggest that Env proteins from ERVs can be active during infections with exogenous retroviruses. The interaction of ERV Env proteins expressed in HIV-infected cells may have major implications for the pathogenicity of HIV and warrants further investigation.