CPCA produced concentration-dependent relaxation of FA, which was notably, but not completely, reduced after endothelial denudation. DPCPX, A1 receptor antagonist, had no significant effect, while SCH 58261 (A2A receptor antagonist) notably reduced CPCA-evoked effect. Pharmacological inhibition of nitric oxide synthase or cyclooxygenase comparably reduced CPCA-evoked action, still in a lesser degree than after denudation. In the presence of buffer with high K+ (100 mM), CPCA-produced relaxations were almost abolished. TEA (nonselective KCa blocker), glibenclamide (KATP blocker), Ba++ (KIR blocker), or ouabain (Na+/K+-ATPase inhibitor) did not change CPCA-induced relaxation. Concentration-response curve for CPCA was significantly shifted to the right after the incubation of apamin (SK channel blocker). CPCA produced concentration-dependent relaxation of FA that was partly dependent on endothelial cells. Endothelium-related portion of CPCA-elicited effect was mediated by combined action of endothelial NO, prostacyclin, and EDHF after activation of endothelial A2A receptors. Small conductance KCa channels were involved in this action.We examined the contribution of endothelial relaxing factors and potassium channels in actions of CPCA, potent adenosine A Vasodilatations in response to adenosine are predominantly due to activation of adenosine A2 receptors located on vascular smooth muscle or endothelial cells -1,2,4-triazolo pyrimidine SCH 58261, L-phenylephrine, ouabain, glibenclamide ; acetylcholine iodide, tetraethylammonium bromide (TEA) ; NμM) produced concentration-dependent relaxation of intact rat femoral artery. After the endothelial denudation control relaxations induced by CPCA were significantly reduced  significantly reduced CPCA-evoked relaxation . CPCA (0.1–100 μM) and indomethacin  produced significant and comparable inhibitions of intact rat femoral artery response to CPCA (P < 0.05). Individual incubations of L-NOARG , glibenclamide , Ba++ , or ouabain  did not change control relaxations induced by CPCA on examined blood vessel  CPCA-induced relaxation on intact vascular rings was strongly inhibited , an adenosine A2 receptor agonist, was not antagonized by DPCPX [2A receptors located on endothelial cells. Furthermore, it is reasonable to suggest that the endothelium-independent part of the CPCA-produced effect was probably associated with activation of another adenosine A2 receptor subtype, most likely A2B, which is located on smooth muscle cells. This hypothesis needs further evaluation.In order to characterize the subtype of adenosine receptors associated with CPCA action, we used DPCPX and SCH 58261, adenosine Aby DPCPX . On the 2 receptor agonist, obtained in another investigation after the incubation of L-NNA  [2Areceptors [In the next part of our study, it was of interest to evaluate possible contribution of endothelial nitric oxide and prostacyclin in vascular response to CPCA. Thus, CPCA-evoked relaxation was notably reduced by L-NOARG. This was in accordance with partial inhibition of rat femoral artery response to 2-octynyladenosine (YT-146), adenosine Ahibitor) . Moreoveeceptors . However+ (100 mM) resulting in strong inhibition of the CPCA-induced relaxation. Thus, it can be proposed that EDHF notably contributed to the endothelium-dependent fraction of CPCA action. However, if we take into account that the inhibition in the presence of high potassium buffer was more pronounced than the one recorded after endothelial denudation, it can be assumed that, apart from the indirect action mediated via EDHF, a significant part of CPCA effect was actually a result of direct action on smooth muscle cells and subsequent increase of cellular membrane potassium conductance. Although this suggestion requires secondary experimental assessment, in our recent investigation on femoral artery we obtained that adenosine-induced relaxation was endothelium-independent and fully inhibited in the presence of a buffer with high K+ [The corresponding analysis of previous results showed that endothelial nitric oxide and prostacyclin equally contributed to CPCA-induced relaxation of investigated blood vessel. Moreover, as mentioned earlier, the obtained reduction in the presence of L-NOARG or indomethacin was significantly lower than that after endothelial denudation. This was indicative for the assumption that separate endothelium-dependent component of CPCA-evoked effect was not completely mediated by nitric oxide or prostacyclin. Thus, we hypothesized that some other endothelial relaxing factor, presumably endothelium-derived hyperpolarizing factor (EDHF), may be involved in CPCA-induced effect. This assumption was confirmed in our experiments in which potassium conductance was blocked by buffer with high K high K+ .+/K+-ATPase or different potassium channels [Ca), glibenclamide (ATP-sensitive potassium channel blocker—KATP), Ba++ (inward-rectifying potassium channel blocker—KIR), or ouabain (Na+/K+-ATPase inhibitor) did not change control relaxations induced by CPCA on examined blood vessel. This excluded the possible role of blocked pharmacological targets in femoral artery relaxant response to CPCA. On the other hand, the control pEC50 value for CPCA-produced concentration-response curve was significantly reduced after the incubation of apamin, which suggests certain role of small conductance calcium-activated potassium channels (SK) in studied vasorelaxant process.In general, EDHF-mediated vascular effects can be exerted through activation of Nachannels –23. In o2A receptors. Small conductance KCa channels were involved in transduction mechanism of CPCA-induced vascular response on examined blood vessel. It appears that endothelium-independent portion of CPCA-evoked relaxation of rat femoral artery did not involve adenosine A2A receptors. This direct effect of CPCA on smooth muscle cells was most likely dependent upon activation of adenosine A2B receptors and subsequent increase of cellular membrane potassium conductance. However, these assumptions need further clarification. In conclusion, CPCA produced concentration-dependent relaxation of isolated rat femoral artery that was partly dependent on endothelial cells. Endothelium-related part of the CPCA-elicited effect was mediated by combined action of endothelial nitric oxide, prostacyclin, and EDHF after predominant activation of endothelial adenosine A


Understanding the organization of the nervous system requires methods for dissecting the contributions of each component cell type to circuit function. One widely used approach combines genetic targeting of Cre recombinase to specific cell populations with infection of recombinant adeno-associated viruses (rAAVs) whose transgene expression is activated by Cre (“Cre-On”). Distinguishing how the Cre-expressing neurons differ functionally from neighboring Cre-negative neurons requires rAAVs that are inactivated by Cre (“Cre-Off”) and can be used in tandem with Cre-On viruses. Here we introduce two rAAV vectors that are inactivated by Cre and carry different fluorophore and optogenetic constructs. We demonstrate single and dual rAAV systems to achieve Cre-On and Cre-Off expression in spatially-intermingled cell populations of the striatum. Using these systems, we uncovered cryptic genomic interactions that occur between multiple Cre-sensitive rAAVs or between Cre-sensitive rAAVs and somatic Cre-conditional alleles and devised methods to avoid these interactions. Our data highlight both important experimental caveats associated with Cre-dependent rAAV use as well as opportunities for the development of improved rAAVs for gene delivery. One such method combines transgenic animals in which site-specific recombinases (SSRs) expressed in specific cell populations trigger transgene expression by activating DNA-based recombinant adeno-associated virus (rAAV) vectors  Cre-On and Double-floxed Orientation (DO)/FAS Cre-Off vectors was based on the pAAV-Ef1á-DIO-hChR2(H134R)-mCherry-WPRE-pA vector from the Deisseroth Lab (http://www.stanford.edu/group/dlab/optogenetics/sequence_info.html). Cloning and sequence confirmation was done either in house or outsourced (Genscript). DIO and DO vectors were cloned using the Asc1 and Nhe1 restriction sites, introduced at the ends of transgenes through transgene-specific primers and PCR amplification. All transgenes were amplified from existing vectors except for ChETA-TdTomato, which was custom synthesized (Genscript). For DIO cloning, the Asc1 site was N-Terminal and the Nhe1 site C-Terminal with respect to the transgene; for DO cloning, the sites were reversed. All N-Terminal sites included a Kozak sequence (GCCACC) directly preceding the start codon. To generate FAS vectors, a custom insert was synthesized to replace the FLEx insert and sub-cloned into the original pAAV-Ef1α-DIO-hChR2(H134R)-mCherry-WPRE-pA backbone using BamHI and EcoRI sites. The FAS insert preserves the original multiple cloning site of the FLEx insert and contains Asc1 and Nhe1 sites within the FAS sites which were used to subclone the other FAS transgenes. Full maps and clones are available on Addgene (http://www.addgene.org/).Cloning of All vectors were amplified with recombination deficient bacteria  and tested functionally for Cre-On and Cre-Off expression by calcium phosphate transfection (Invitrogen) into HEK 293 cells, HEK 293 cells constitutively expressing Cre . Striatum was targeted by directing the needle approximately 0.9mm anterior and 2.4mm lateral from Bregma and 2.1mm from the pial surface. Cortical injections were targeted using the same anterior–posterior and medial-lateral coordinates, but the needle was directed 0.4mm from the pial surface. Coordinates were adjusted slightly by age and 300–1000nl were injected bilaterally. Following injections and wound closure, mice received ketoprofen (10 mg/kg) for analgesia and were returned to home cages for 3–4 weeks. All surgical procedures, animal handling, and euthanasia were carried out in accordance with federal guidelines and were approved by the Harvard Medical School Institutional Animal Care and Use Committee.Isoflorane anesthetized mice were transcardially perfused with 4% paraformaldehyde and the brains were post-fixed for 1–7 days. Brains were sectioned coronally or sagitally at 40–50 μm thickness using a Vibratome. No immuno-enhancement was used to increase the signal of native fluorophores. For Cre immunohistochemistry, slices were incubated overnight at 4°C with mouse anti-Cre antibody  in 1x PBS containing 5% normal goat serum (GIBCO) and 0.3% TritonX-100. The following day, tissue was rinsed in PBS, reacted with goat anti-mouse Alexa 647  for 1 h at room temperature in the same blocking buffer as for the primary antibody reaction. Slices were rinsed, mounted onto superfrost slides, dried and coverslipped under ProLong antifade reagent with DAPI (Molecular Probes). Whole-slide images were acquired with a VS110 slice scanning microscope (Olympus). To determine fluorophore co-expression, confocal stacks were taken with a Zeiss LSM 150 or Olympus FV1000 and analyzed using ImageJ.http://www.addgene.org/).We used the DIO Cre-On vector pAAV-Ef1α-DIO-hChR2(H134R)-mCherry-WPRE-pA  of striatal neurons are medium spiny neurons (MSNs), which provide the only striatal output. MSNs come in two types which can be grouped by axonal projection pattern and dopamine receptor expression   neurons in the striatum of D2-Cre mice infected with DO-GFP were identified with an antibody against Cre. Confocal imaging of the infection zone and quantification of the number of cells positive for Cre and/or GFP revealed that of 692 Cre+ and 496 GFP+ cells (N = 4 infections) only one cell was double-positive . Thus, either constitutive expression of Cre from a BAC or simultaneous viral introduction of Cre efficiently turns off expression of DO rAAVs.To quantify the efficiency with which stable, transgenic Cre expression turns off DO rAAV expression, Cre-expressing   Figure .+ projections were observed in the SNr, while Cre-Off TdTomato+ projections were visible in the GP   Figure . Since T+ arborizations to the SNr but only minor arborizations in GP . Thus stable transgenic Cre expression efficiently eliminates FAS rAAV transgene expression, leading to undetectable fluorophore expression within 3–4 weeks.The efficiency of stable transgenic Cre expression in turning off FAS expression was quantified in fixed slices of striatum of D2-Cre mice infected with FAS-TdTomato and immunostained for Cre Figure . Of 477 + and 650 TdTomato+ cells, 40 were double-positive (N = 4 infections). Thus viral introduction of Cre efficiently turns off expression of simultaneously infected FAS rAAVs.To quantify the efficiency of virally introduced Cre to turn off FAS expression, we infected the striata of wild-type mice simultaneously with rAAVs carrying Cre-GFP and FAS-TdTomato at titers where approximately equal numbers of cells expressed Cre-mCherry and GFP Figure . We used+ and TdTomato+ cells were intermingled in the striatum and did not exhibit interference . Thus co-infection of FAS and DIO rAAVs targets efficient Cre-Off and Cre-On expression without spatial interference.To test whether Cre-Off FAS and Cre-On DIO rAAVs could be co-infected without spatial interference, FAS-TdTomato and DIO-GFP were injected into the striatum of D2-Cre mice Figure . GFP+ ane Figure . GFP+ bue Figure     Figure . Of 177 ) Figure . ExclusiDue to the limited packaging capacity of rAAVs, infecting multiple Cre-On or Cre-Off rAAVs may be an effective way to introduce more than one transgene in the same cell populations. We tested whether multiple Cre-On or Cre-Off rAAVs could be co-infected without spatial interference of transgene expression by infecting the striatum and quantifying GFP and mCherry/TdTomato fluorescence. Co-infection of the striatum of D2-Cre mice with DIO-mCherry and DIO-GFP resulted in similar levels of mCherry and GFP expression Figure  without We designed rAAV expression vectors that allow different transgenes to be simultaneously targeted to spatially intermingled cell populations with and without Cre. Although the FLEx system is widely used to achieve Cre-On (DIO) expression, Cre-Off rAAVs are not readily available. Therefore, we used the FLEx system to generate Cre-Off rAAVs (DO) and confirmed the efficacy of this design using striatal injections. High efficiency Cre-On and Cre-Off expression when each system is used in isolation was confirmed both for constitutive genomic Cre expression as well as for acute virally-induced Cre expression.Whereas DIO and DO rAAVs achieve the expected Cre-On and Cre-Off expression when injected individually, when co-infected, DIO expression was absent in areas with DO expression. This data suggests that many cells within the infection volume were transduced by both DIO and DO rAAV particles, yet expression failed to occur only in Cre expressing cells. However, the presence of Cre and multiple FLEx rAAVs is not sufficient for interference, since DIO/DIO rAAV co-infection led to robust expression from both vectors. The absence of DO expression at the periphery of the example infection was not present in all DIO and DO combinations and thus likely represents a lack of DO rAAV spread and infection rather than Cre-independent interference. Therefore the interference we describe requires Cre and FLEx rAAVs with oppositely oriented transgenes.Although the exact nature of the destructive, Cre-dependent interference occurring between DIO and DO rAAVs present in the same cell is unknown, we hypothesize that it resulted from intermolecular recombination involving loxP and lox2272 sites. Intermolecular recombination between rAAVs is well documented  might be sufficient to interfere with transcription and be considered, in effect, insertion of an exogenous rAAV-based stop cassette. Since loxFAS sites do not efficiently recombine with loxP, this putative integration would be inefficient in FAS rAAVs (Siegel et al., AAV integration into somatic genomes is well documented Smith,  and Cre-The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be constructed as a potential conflict of interest.


The purpose of this paper is to describe imaging techniques and findings of T2-weighted magnetic resonance imaging (MRI) of edema in myocardial diseases. T2-weighted cardiac MRI is acquired by combining acceleration techniques with motion and signal suppression techniques. The MRI findings should be interpreted based on coronary artery supply, intramural distribution, and comparison with delayed-enhancement MRI. In acute myocardial diseases, such as acute myocardial infarction and myocarditis, the edema is larger than myocardial scarring, whereas the edema can be smaller than the scarring in some types of nonischemic cardiomyopathy, including hypertrophic cardiomyopathy. T2-weighted MRI of edema identifies myocardial edema associated with ischemia, inflammation, vasculitis, or intervention in the myocardium and provides information complementary to delayed-enhancement MRI. Delayed-enhancement magnetic resonance imaging (MRI) is valuable for the diagnosis and assessment of the severity of both ischemic and nonischemic myocardial diseases –8. HowevT2-weighted cardiac MRI of edema is acquired by combining acceleration techniques with motion suppression and prepulse techniques. These MRI techniques freeze the cardiac and respiratory motion effectively with giving high contrast between the blood, fat, normal myocardium, and myocardial edema. Turbo spin-echo imaging with multiple refocusing pulses has replaced spin-echo imaging in T2-weighted cardiac MRI because the scan time is reduced by a factor of 10–12 . A paralAn ECG-gating technique is usually used for cardiac MRI. This technique allows for data acquisition at the end diastole when the myocardium is static. A breath-hold technique suppresses respiratory artifacts. Alternative methods to the breath-holding technique are navigator-gating and respiratory-gating techniques . The black-blood prepulse technique, consisting of two inversion-recovery pulses combined with ECG-gating, is applied to T2-weighted cardiac MRI . By usinMyocardial edema is quantified with T2-weighted MRI with T2-prepared or multiecho acquisition . ZagroseImage interpretation of T2-weighted cardiac MRI is based on comparison with coronary artery supply, intramural distribution  and morphology  of the edema, and comparison with delayed-enhancement MRI.The location of the myocardial edema should be compared with the coronary artery supply. Myocardial edema or ischemia associated with acute myocardial infarction distribute to the coronary artery supply and often show transmural involvement . The myoAt the acute stage of ischemic or inflammatory cardiomyopathy, myocardial edema is larger than myocardial scarring seen in delayed-enhancement MRI, because the edema may surround inflammatory or dying tissues , 11. WheThe intraventricular flow close to the hypokinetic myocardium is not nullified sufficiently . ArrhythMyocardial edema distributes to the coronary artery supply in acute myocardial infarction , 11. T2-In acute myocarditis, myocardial edema is usually observed in the lateral wall  11, 19], 1911, 1 In eosinophilic myocarditis, myocardial edema appears patchy or diffuse . Churg-S18FDG-PET (Patchy myocardial edema is occasionally observed in cardiac sarcoidosis . The myo8FDG-PET . Scarred8FDG-PET . Patchy mesocardial edema is often observed in hypertrophic cardiomyopathy . The myo Takotsubo cardiomyopathy is a reversible cardiomyopathy that occurs following a stressful event. This disease affects postmenopausal women, and the clinical and ECG findings are similar to those of myocardial infarction. However, takotsubo cardiomyopathy does not show myocardial scarring on delayed-enhancement MRI . T2-weigT2-weighted MRI is useful for the detection of myocardial edema after ablation therapy for ventricular arrhythmia associated with some cardiomyopathies. These patients tend to receive repeated ablations for arrhythmogenic myocardial scarring. T2-weighted MRI distinguishes the recently ablated region with edema from the myocardial scarring or previously ablated region .T2-weighted cardiac MRI of edema is acquired in combination with acceleration, motion suppression, and other techniques. T2-weighted cardiac MRI visualizes myocardial edema that corresponds to ischemia, active inflammation, vasculitis, or recently performed intervention in the myocardium and provides information complementary to delayed-enhancement MRI.


The second study, with a GM-CSF-expressing herpes simplex virus type 1 , was presented at the recent American Society of Clinical Oncology meeting. Four hundred thirty-six patients were randomized (2:1) to single-agent T-Vec or subcutaneous GM-CSF, resulting in a significantly greater durable response rate and median time to treatment failure for the virotherapy arm (http://chicago2013.asco.org/immunotherapies-offer-bright-prospects-advanced-melanoma). Again, both oncolytic and loco-regional/systemic immunotherapeutic activity was documented. In light of these studies, it is important to recognize that Pexa-Vec and T-Vec merely represent two regiments of a larger army of targeted biological agents that includes reovirus , various adenoviruses, coxsackie virus and other herpes and VV vectors. In that vein, VV appear to be particularly promising candidate anti-cancer agents. As we have shown, elucidation of intracellular mechanisms may uncover other avenues of attack, through which combination therapies can be exploited for greater anti-tumour efficacy. Further therapeutic alliances might be sought by genetic manipulation of the viral genome itself to deliver a therapeutic payload and form a ‘coalition of killing’. The potential of targeted, multi-modality therapy centred on the biology of viral oncolytics is an emerging, yet exciting, field of investigation and highlights the crucial role of understanding viral biology. In the words of Sun Tzu (“The Art of War”): “We cannot enter into alliances with neighbouring princes until we are acquainted with their designs.”In a recent study of combined vaccinia virus (VV) and radiation therapy (RT) in BRAF-mutant melanomas, we dissected the precise events by which RT enhances viral cytotoxicity. Our studies revealed that the complex interplay between RT, virus and cell signaling pathways caused a completely novel synthetically lethal form of tumor cell death. We initially sought to assess the effect of combining a VV GLV-1h68) and RT in melanoma cell lines with different genetic backgrounds . We hypothesised that RT would induce stress responses in all tumor genotypes and activate host survival pathways to create an environment favorable to viral replication and subsequent oncolysis. Contrary to our expectations, enhanced tumor cell kill with the combination of virus and RT only occurred in BRAF-mutant melanoma cells. In mechanistic studies, it was clear that VV triggered activation of the c-Jun N-terminal Kinase (JNK) pathway and tumor necrosis factor-α (TNF-α) signaling in all genotypes. In BRAF-mutant cells, virus-induced TNF-α production was associated with anti-apoptotic/pro-survival effects, as previously described with MEK inhibitor-induced cytotoxicity h68 and R and Akt


Aminoacyl-tRNA synthetases (ARSs) are essential and ubiquitous ‘house-keeping’ enzymes responsible for charging amino acids to their cognate tRNAs and providing the substrates for global protein synthesis. Recent studies have revealed a role of multiple ARSs in pathology, and their potential use as pharmacological targets and therapeutic reagents. The ongoing discovery of genetic mutations in human ARSs is increasing exponentially and can be considered an important determinant of disease etiology. Several chemical compounds target bacterial, fungal and human ARSs as antibiotics or disease-targeting medicines. Remarkably, ongoing exploration of noncanonical functions of ARSs has shown important contributions to control of angiogenesis, inflammation, tumourigenesis and other important physiopathological processes. Here, we summarize the roles of ARSs in human diseases and medicine, focusing on the most recent and exciting discoveries. Aminoacyl-tRNA synthetases (ARSs) comprise an ancient ubiquitous family of enzymes in all cells from three major kingdoms of life. They catalyse the esterification reactions that link amino acids with cognate tRNAs bearing the correct anticodon triplet to ensure the accurate transfer of information directed by the genetic code , 18 mitochondrial ARSs, and 2 dual-localized ARSs present in both cytoplasm and mitochondria (GARS and KARS). In human and other mammalian cells, a large tRNA multi-synthetase complex (MSC) organizes 9 cytoplasmic ARSs and 3 non-enzyme factors, namely MSC p43, p38 and p18 .GARS (glycyl-tRNA synthetase)  and related neuropathies . In contYARS (tyrosyl-tRNA synthetase) are associated with DI-CMT type C (DI-CMTC)  is characterized by slow progressive neuropathy, intermediate nerve conduction velocities, axonal degeneration, and demyelination of peripheral motor and sensory neurons. Three dominant mutations  in KARS (lysyl-tRNA synthetase) is the third ARS gene associated with CMT disease. Compound heterozygous mutations in the KARS gene were identified in a patient with severe neurological symptoms including peripheral neuropathy  in AARS  in a mouse model of ataxia was reported in 2006  was investigated  and play a key role in autoimmune diseases.Unique sequence at the N-terminal part of human cytosolic seryl-tRNA synthetase.GARS, YARS, KARS and AARS associated with CMT and related disorders have been discovered and investigated  showed that increased dosage of disease-causing alleles led to more severe neurological phenotypes due to gain-of-function effects that could not be rescued by over-expression of wild-type GARS, arguing against a loss-of-function defect   or in peripheral nerves of excluded , as wellexcluded . ConsistGARS secreted from human macrophages exhibits anti-tumourigenic activity  and RARS2 , have been implicated in leukoencephalopathy with brain stem and spinal cord involvement and lactate elevation (LBSL)  has been identified  in the HARS2  at two highly conserved amino acids (p.L200V and p.V368L), and an alternative splice-derived deletion of 12 in-frame codons, were shown to be causative  in the enzymatic active site of SARS2  was identified   are fatal disorders occurring during the neonatal period or first year after birth. The exome of a hypertrophic mitochondrial CMP infant with cardiac respiratory chain deficiency, who died at 10 months of age, was sequenced. A homozygous missense mutation (p.R592W) was identified in To date, all mitochondrial ARSs bearing genetic mutations causing human diseases exhibit compromised aminoacylation activity, and effects on noncanonical functions unrelated to translation have not been demonstrated. Thus, the etiology underlying human mitochondrial disorders might be restricted to defective enzymatic activities. These observations raise an important question: Given that genetic mutations from mitochondrial ARSs affect fundamental mitochondrial functions, for example, translational repression of mitochondrial proteins and consequent respiratory chain defects, why do they cause a diversity of disease phenotypes in distinct affected tissues? Possibly, the threshold amount of an ARS required for optimal enzymatic activity differs for each individual ARS and tissue combination. Thus, a change in the activity of a mutant ARS might be tolerated in some tissues but not in others. Alternatively, there might exist tissue-specific proteins that bind the mutant ARS, leading to disease phenotypes by inhibiting the enzymatic activity, or by diminishing potential noncanonical functions.Ala, EARS2-mtT-DNAGlu, HARS2-mtT-DNAHis, RARS2-mtT-DNAArg and YARS2-mtT-DNATyr) cause the same or similar pathological phenotypes, possibly due to a common loss-of-activity mechanism . Mutations at distinct sites in the same mtT-DNA gene sometimes lead to multiple disease types, possibly due to diverse effects on tRNA synthesis, maturation, accepting activity, or stability. In contrast, mutations in mitochondrial ARSs impact aminoacylation activity without affecting production, processing or turnover of cognate tRNAs, possibly accounting for the phenotype mismatch between mutations of ARS-tRNA pairs.Human mitochondrial DNA encodes 22 mitochondrial tRNAs  high evolutionary divergence of prokaryotic and eukaryotic ARSs; (ii) the full complement of bacterial ARSs provides 20 distinct targets; (iii) a wealth of structural information on both bacterial and eukaryotic ARSs and (iv) an abundance of known natural inhibitors that can be used for starting points for drug development. Mupirocin (pseudomonic acid) is currently the only ARS inhibitor commercially used as an antibiotic. It is a natural product isolated from Pseudomonas fluorescens that inhibits eubacterial and archaeal IARS catalytic activity, and is topically effective against gram-positive pathogens . The high specificity of AN2690 for the fungal enzyme is possibly due to inefficient penetration of human cell plasma membranes or inaccessibility of LARS in the MSC.AN2690  is a broad-spectrum, anti-fungal therapeutic agent specifically developed for topical treatment of onychomycosis  cells, a T helper cell sub-class characterized by robust interleukin 17 (IL-17) production, have a key role in autoimmune diseases , vascular endothelial growth factor-A (VEGF-A), and several chemokine receptors and ligands among others  to form an inactive pre-GAIT complex. Ser999 phosphorylation directs the formation of the active GAIT complex that binds initiation factor eIF4G and blocks recruitment of the pre-initiation complex. Unexpectedly, a C-terminus truncated form of EPRS, termed EPRSN1, was found to bind GAIT target mRNAs and prevent binding of the functional GAIT complex, thereby acting as a dominant-negative inhibitor of GAIT activity. This imposes a ‘translational trickle’ of target protein expression at basal levels required for tissue well-being  complex suppresses translation of a ‘regulon’ of inflammation-related mRNAs, including ceruloplasmin , releasing tumour suppressor MSC p18 for nuclear re-localization. Phosphorylated MARS exhibits significantly reduced catalytic activity due to diminished tRNAiMet binding, and a consequent repression of global translation. Interestingly, UV-stress-induced MARS phosphorylation, and subsequent global translational repression, mirrors IFN-γ-induced EPRS phosphorylation and transcript-selective translational silencing program, revealing a noncanonical theme common to these and possibly other synthetases.Human MARS has an especially critical role in translation initiation by transferring Met to initiator tRNAsars gene were found that affect vascular development and maintenance, including aortic arch vessel dilatation, aberrant hindbrain capillary patterning, abnormal intersomitic vessels, or disorganized vessels with abnormal branching of established intersegmental vessels  domain, appended to SARS at the C-termini and containing nuclear localization signal (NLS), was identified as an essential domain for shuttling the enzyme into nucleus and repressing VEGF-A mRNA transcription, thereby permitting normal vascular development  controls various critical cellular processes including cell growth, autophagy, protein synthesis and metabolism. Activation of the mTOR signalling pathway contributes to multiple human pathologies including cancer and tissue hypertrophy. Pharmacological inhibitors of mTOR are used clinically to prevent graft rejection and restenosis after angioplasty. mTOR complex 1 (mTORC1) is activated by metabolic inputs such as elevated Leu and other amino acids . KARS inhibits ubiquitin-dependent degradation of 67LR, thereby promoting laminin-induced cell migration. This pro-migratory function of KARS suggests a possible role in tumour cell metastasis.Human KARS exhibits regulatable cellular localization and multiple functions beyond translation including transcription control upon nuclear import , might provide a new set of physiologic extracellular or intracellular pathways as basis for developing novel therapeutics with minimal side-effects.ARSs have a triple-faceted relationship with human health and disease. First, the loss-of-function of an ARS resulting from genetic mutations can cause a broad spectrum of diseases. Human LARS can correct mitochondrial dysfunctions caused by tRNAClarification of molecular mechanisms underlying ARS mutation-derived CMT disease.Development of ARS-based therapeutic reagents to treat CMT and mitochondrial ARS-defective diseases.Systematic exploration of the physiopathological relevance of noncanonical function of human ARSs.Development of therapeutic strategies based on secretory ARSs, endogenous ARSs, or ARS antibodies.


Endothelial dysfunction in resistance arteries alters end organ perfusion in type 2 diabetes. Superoxides and cyclooxygenase-2 (COX-2) derivatives have been shown separately to alter endothelium-mediated relaxation in aging and diabetes but their role in the alteration of vascular tone in old diabetic subjects is not clear, especially in resistance arteries. Consequently, we investigated the role of superoxide and COX-2-derivatives on endothelium-dependent relaxation in 3 and 12 month-old Zucker diabetic fatty (ZDF) and lean (LZ) rats. Mesenteric resistance arteries were isolated and vascular tone was investigated using wire-myography. Endothelium (acetylcholine)-dependent relaxation was lower in ZDF than in LZ rats . Blocking NO production with L-NAME was less efficient in old than in young rats. L-NAME had no effect in old ZDF rats although eNOS expression level in old ZDF rats was similar to that in old LZ rats. Superoxide level and NADPH-oxidase subunits (p67phox and gp91phox) expression level were greater in ZDF than in LZ rats and were further increased by aging in ZDF rats. In young ZDF rats reducing superoxide level with tempol restored acetylcholine-dependent relaxation to the level of LZ rats. In old ZDF rats tempol improved acetylcholine-dependent relaxation without increasing it to the level of LZ rats. COX-2 (immunolabelling and Western-blot) was present in arteries of ZDF rats and absent in LZ rats. In old ZDF rats arterial COX-2 level was higher than in young ZDF rats. COX-2 blockade with NS398 restored in part acetylcholine-dependent relaxation in arteries of old ZDF rats and the combination of tempol and NS398 fully restored relaxation in control (LZ rats) level. Accordingly, superoxide production and COX-2 derivatives together reduced endothelium-dependent relaxation in old ZDF rats whereas superoxides alone attenuated relaxation in young ZDF or old LZ rats. Type 2 diabetes is the most frequently encountered metabolic disorder, currently affecting 5% to 10% of most populations, and the incidence continues to grow among developing countries Type 2 diabetes is associated with an increased reactive oxygen species (ROS) production Using Zucker Diabetic Fatty (ZDF) rats, we found that ROS and COX-2 vasoconstrictor derivatives additionally reduced endothelium-dependent relaxation in old diabetic rats whereas ROS alone are involved in the decrease in endothelium-dependent relaxation in young ZDF or in old LZ rats.Adult male Zucker fatty diabetic (ZDF) and lean Zucker (LZ) rats, 12–14 and 50–52 week-old, were purchased from Charles River . Rats were anesthetized  and the right femoral artery was catheterized for blood pressure measurement In a separated series of experiments 12-month-old male LZ and ZDF rats were treated with the COX2 inhibitor Celecoxib  or with the combination of Celecoxib (25 mg/kg/day) plus 1-Oxyl-2, 2, 6, 6-tetramethyl-4-hydroxypiperidine  The procedure followed in the care and euthanasia of the study animals complied with the European Community Standards on the Care and Use of Laboratory Animals . The protocol was approved by the Committee on the Ethics of Animal Experiments of the “Pays de la Loire” Region .Segments of mesenteric arteries (2 mm long) were mounted on a wire-myograph  Other segments of arteries were homogenized and proteins  were separated by SDS-PAGE using a 4% stacking gel followed by a 10% running gel. Proteins were detected with specific antibodies . COX-2 polyclonal antibodies (1∶1000) were obtained from Santa Cruz Biotechnology. Protein expression was visualized using the ECL-Plus Chemiluminescence kit (Amersham) Other arterial segments were embedded vertically in Tissue-tek (Sakura) and frozen in isopentane. ROS detection was performed on transverse cross sections 7 µm thick incubated with dihydroethydine (DHE) as previously described Immunostaining of COX-2 was performed as previously described Results are expressed as means ± SEM. Significance of the difference between arteries was determined by ANOVA . Means were compared by paired t-test or by the Bonferroni test for multigroup comparisons. Values of p<0.05 were considered to be significant.Rat body weight, greater in young ZDF rats than in young LZ rats decreased with age in ZDF rats , in agreAcetylcholine-dependent relaxation was reduced in ZDF rats compared to lean rats . FurtherIn young LZ rats, L-NAME suppressed acetylcholine-mediated relaxation whereas it was inhibited, but not suppressed, in old LZ rats . In ZDF As excessive oxidative stress has been shown to reduce endothelium-mediated dilation in diabetes, we investigated the ROS level in mesenteric arteries was analyzed using dihydroethidin (DHE) microfluorography. We found that DHE staining was higher in ZDF than in LZ rats whatever the age. In addition, in older animals the ROS level was higher than in younger animals in both LZ and ZDF rats . The incCOX-2 was expressed in ZDF rats and not in LZ animals . In addiFinally, in old rats phenylephrine-mediated contraction was greater than in young rats . The conIn old ZDF rats treated chronically with the COX-2 inhibitor celecoxib, acetylcholine-induced relaxation was improved whereas celecoxib did not affect relaxation in old LZ rats . NeverthIn this study we found that endothelium-dependent relaxation was reduced in young rats suffering from type 2 diabetes due to excessive ROS production, whereas in one year-old diabetic rats COX-2 derivatives in association with an excessive ROS production reduced endothelium dependent relaxation.First, we found that endothelium-mediated dilation, evidenced by acetylcholine-dependent relaxation was reduced in ZDF rats compared to lean rats. This finding is in agreement with previous studies showing that type 2 diabetes is associated with endothelial dysfunction in rats As endothelium-mediated relaxation in mesenteric arteries relies greatly on the production of NO As excessive oxidative stress has a major role in the endothelium dysfunction observed in diabetes, we investigated the role of superoxide in acetylcholine-mediated relaxation. Certainly, oxidative stress has been shown to play a major role in the reduction in endothelium-mediated relaxation in diabetes in most vascular territories 2α has an major role in mediating endothelial dysfunction In order to better understand this endothelial dysfunction found in old ZDF rats, we investigated the involvement of COX-2 in acetylcholine-induced relaxation. Indeed, COX-2 is expressed in resistance arteries Finally, in old rats phenylephrine-mediated contraction was greater than in young rats. Furthermore, phenylephrine-mediated contraction was also higher in ZDF than in LZ rats. These observations are in agreement with previous studies, which have shown that both aging We found that long-term exposure to diabetes increased endothelium damages and further reduced endothelium-mediated relaxation in mesenteric resistance arteries. In these arteries COX-2 derivatives in addition to ROS contributed to severely reduce endothelium-dependent dilation. This finding is of importance as it shows that deleterious pathways may add one to each other when diabetes evolves over time. Certainly, the present experimental conditions most probably represent a more common pathological condition as diabetes is usually diagnosed several months or years after the onset of the metabolic disorders leading to hyperglycemia. Consequently, the present study reinforces the assumption that not only oxidative stress but also inflammation contribute, most probably synergistically, to the vascular disorders occurring in type 2 diabetes.


Yersinia pestis, Y. pseudo-tuberculosis, and Y. enterocolitica) are all psychrotropic bacteria capable of growth at 4°C; however, cold growth is dependent on the presence of an exoribonuclease, polynucleotide phosphorylase (PNPase). PNPase has also been implicated as a virulence factor in several notable pathogens including the salmonellae, Helicobacter pylori, and the yersiniae [where it typically influences the type three secretion system (TTSS)]. Further, PNPase has been shown to associate with ribonuclease E (endoribonuclease), RhlB (RNA helicase), and enolase (glycolytic enzyme) in several Gram-negative bacteria forming a large, multi-protein complex known as the RNA degradosome. This review will highlight studies demonstrating the influence of PNPase on the virulence potentials and stress responses of various bacterial pathogens as well as focusing on the degradosome-dependent and -independent roles played by PNPase in yersiniae stress responses.Microbes are incessantly challenged by both biotic and abiotic stressors threatening their existence. Therefore, bacterial pathogens must possess mechanisms to successfully subvert host immune defenses as well as overcome the stress associated with host-cell encounters. To achieve this, bacterial pathogens typically experience a genetic re-programming whereby anti-host/stress factors become expressed and eventually translated into effector proteins. In that vein, the bacterial host-cell induced stress-response is similar to any other abiotic stress to which bacteria respond by up-regulating specific stress-responsive genes. Following the stress encounter, bacteria must degrade unnecessary stress responsive transcripts through RNA decay mechanisms. The three pathogenic yersiniae ( Escherichia coli, with limited (but a growing number of) studies performed in Gram-positive organisms   is the predominant endoribonuclease in  Condon, . The fir Condon, . SpecialYersinia pseudotuberculosis degradosome, comprised of RNase E, PNPase, and RhlB helicase associates  is a slightly smaller enzyme (~78 kDa) but shares 89% sequence similarity with that of its E. coli counterpart. Additionally, PNPase acts as a polymerase when the concentration of nucleotide di-phosphates is greater than inorganic phosphate, as shown in E. coli  involved in regulating gene expression, the entire paradigm associated with “what constitutes a gene” was shifted. sRNAs became increasingly studied when they were found to influence bacterial physiology and even virulence  will bind to sRNAs either promoting their stabilization or degradation  during cold shock .Bacterial responses to low temperature stress are generally referred to as “cold shock.” PNPase confers cold growth capabilities to a number of bacteria  including: E. coli, cold inducible genes, including six cold shock protein genes (csp's), become upregulated  during stationary phase growth of E. coli  and other cold-inducible transcripts in both E. coli and Yersinia enterocolitica  in csp transcripts , PNPase was found to negatively regulate virulence/twitching motility, primarily through its S1 RNA-binding domain . However, in a seemingly counter-intuitive observation, PNPase works to negatively regulate macrophage invasion and expression of type TTSS genes involved in macrophage invasion  transcript levels by acting through the spv activator/regulator spvR, thereby reducing intracellular survival of S. enterica  has caused three major human pandemics as well as the great plagues of London in the 1660s   might play a facilitating role in binding RNA while residues F67 and K112 actually contact the RNA molecule , which proved necessary for not just TTSS function (as measured by HeLa cell rounding) but also cold growth  and sda (encoding an anti-neutrophil DNase) degradation during late-exponential growth phase , containing only its amino terminal catalytic and S1 domains, was ectopically expressed (Briegel et al., Y. pseudotuberculosis (Yang et al., Using bacterial two-hybrid analysis as well as co-immunoprecipitation techniques, the Y. pseudotuberculosis proliferation was significantly reduced when the degradosome was unable to assemble (Yang et al., pnp deletion mutants observed earlier (Rosenzweig et al., pnp deletion mutants (Rosenzweig et al., To determine whether degradosome assembly was required for yersiniae virulence, both a proliferation assay in co-culture with macrophage-like cells as well as a TTSS functional evaluation were carried out. Interestingly, Y. pseudotuberculosis PNPase confers cold-growth capability in a degradosome-independent manner while PNPase responds to oxidative stress, in the form of H2O2 exposure, in a degradosome-dependent manner (Henry et al., Yersinia oxidative stress response also requires PNPase in a degradosome-dependent manner (Henry et al., Was the PNPase contribution to yersiniae cold-growth degradosome-dependent or -independent, and did PNPase contribute to the oxidative stress response? To address this, the dominant-negative, truncated RNase E variant, residues 1–465 (Yang et al., E. coli during oxidative stress was degradosome-independent since an RNase E truncated variant devoid of the degradosome scaffolding region did not demonstrate compromised growth when exposed to H2O2 (Wu et al., E. coli, PNPase independently prevented the accumulation of oxidatively damaged RNA (Wu et al., E. coli and Y. pseudotuberculosis (Wu et al., In sharp contrast, the PNPase requirement of S. enterica and D. nodosus also require PNPase for optimal virulence, while PNPase works to enhance survival of C. jejuni, S. aureus, S. pyogenes, and S. mutans during unfavorable conditions (Table Throughout the literature, many examples of how ribonucleases impact bacterial virulence are apparent (Lawal et al., ns Table . With rens Table .Despite our furthering the aforementioned study, to determine that yersiniae degradosome assembly was also necessary for oxidative stress responses but not cold stress responses (Henry et al., The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Cardiovascular disease (CVD) risk in type 2 diabetes (T2DM) is only partially reduced by intensive glycemic control. Diabetic dyslipidemia is suggested to be an additional important contributor to CVD risk in T2DM. Multiple lipid lowering medications effectively reduce fasting LDL cholesterol and triglycerides concentrations and several of them routinely reduce CVD risk. However, in contemporary Western societies the vasculature is commonly exposed to prolonged postprandial hyperlipidemia. Metabolism of these postprandial carbohydrates and lipids yields multiple proatherogenic products. Even a transient increase in these factors may worsen vascular function and induces impaired endothelial dependent vasodilatation, a predictor of atherosclerosis and future cardiovascular events. There is a recent increased appreciation for the role of gut-derived incretin hormones in controlling the postprandial metabolic milieu. Incretin-based medications have been developed and are now used to control postprandial hyperglycemia in T2DM. Recent data indicate that these medications may also have profound effects on postprandial lipid metabolism and may favorably influence several cardiovascular functions. This review discusses (1) the postprandial state with special emphasis on postprandial lipid metabolism and its role in endothelial dysfunction and cardiovascular risk, (2) the ability of incretins to modulate postprandial hyperlipidemia and (3) the potential of incretin-based therapeutic strategies to improve vascular function and reduce CVD risk. The prevalence of type 2 diabetes mellitus (T2DM) is increasing worldwide at alarming rates. In the next 20 years the prevalence of T2DM is expected to be more than 350 million people worldwide . CardiovAlthough the risk of major cardiovascular events in diabetes is closely related to glycemic control in observational studies, therapeutic targeting of glycated hemoglobin levels has not been effective in decreasing cardiovascular risk outcomes -12. FurtHistorically, the associations between metabolic abnormalities and cardiovascular disease have been studied largely during fasting conditions. However, the important contribution of postprandial state to cardiovascular disease is increasingly being recognized, particularly in conditions of insulin resistance and T2DM. The mechanisms of postprandial hyperglycemia, as well as its clinical importance (including cardiovascular) risk have been addressed in several excellent review articles -20 and wMultiple lipid lowering medications have been developed that effectively reduce fasting concentrations of LDL cholesterol and triglycerides (TG). Although several of these medications, particularly the statins, routinely reduce CVD risk by 25-35%, there remains substantial residual and absolute risk in higher CVD risk populations, such as in T2DM. This may be in part explained by postprandial elevation in lipids. In fact, in contemporary post-industrialized societies most individuals spend the majority of non-sleeping hours in the postprandial state. For example, as the typical American diet consists of 3 or more meals per day and it takes more than 8 hours for triglyceride concentrations to return to fasting levels after a meal, postprandial triglyceride concentrations often remain elevated throughout the day. Importantly postprandial triglyceride concentrations may in fact be a better predictor of cardiovascular events than fasting triglycerides. The adverse effect of postprandial triglycerides is thought to be mediated by proatherogenic lipolysis products of nascent triglyceride-rich lipoproteins, such as remnant lipoproteins and fatty acids, and even a transient increase in these factors may worsen vascular function.Several large observational studies have assessed the association between non-fasting lipid concentrations and cardiovascular risk Table . In the The Physicians' Health Study, a prospective nested case control study was conducted in 14,916 men aged 40 to 84 years, 85% of whom had baseline blood samples taken under non-fasting conditions . The priNon-fasting TG were also associated with myocardial infarction (MI) and ischemic heart disease and death after 26 years of follow-up in a prospective cohort of almost 14,000 women and men in the Copenhagen study . The assSince none of the above studies included measurement of fasting lipids, it is not clear whether similar relationships with CVD would be observed with fasting TG values. However, the combination of a screening visit without requirements to be in fasting condition along with a protocol for strictly fasting enrollment visits in 2,809 asymptomatic men who participated in the Multiple Risk Factor Intervention Trial provided an opportunity to compare the effects of both fasting and non-fasting TG levels on non-fatal and fatal coronary heart disease . The anaIn contrast, based on the time after the last meal for baseline blood draws, 26,509 initially healthy women in Women's Health Study  were strTriglycerides of dietary origin enter the lymph and then systemic circulation as chylomicrons packaged together with apolipoprotein B-48 (apoB-48). Following a fat enriched meal, chylomicrons rapidly increase and lead to pronounced triglyceride elevations peaking approximately 4 hours post-meal. In humans, the presence of apoB-48 distinguishes chylomicrons from smaller VLDL particles carrying triglycerides of hepatic origin that contain apolipoprotein B-100 (apoB-100). Although increases in particles carrying apoB-48 explain about 80% of the postprandial triglyceride increase, much of the increase in the particle count is represented by particles containing apoB-100 . Both chin vitro and in vivo experimental data showed that apoCIII may directly induce endothelial dysfunction via inhibition of insulin-induced activation of Akt resulting in reduced nitric oxide (NO) release and subsequent impaired vasodilation [RLPs after lipolysis become enriched in cholesterol, deposit 5- to 20-fold more cholesterol into the vessel wall per particle, and are preferentially retained in the vessel wall where they are avidly taken up by macrophages -31. RLPsdilation . Possibldilation ,41.Fatty acids, an additional product of postprandial TG hydrolysis, may also contribute to increased cardiovascular risk. Increased non-esterified fatty acids (NEFA) concentrations are prospectively associated with both cardiovascular morbidity and mortality ,43. NEFAEndothelial dysfunction is believed an important link between the postprandial state, atherosclerosis and CVD. It is characterized by impaired endothelium-dependent vasodilation and increased pro-coagulant and pro-inflammatory activity . CoronarThe incretin hormones, glucagon-like peptide-1 (GLP-1) produced by L-cells in the distal gut, and glucose-dependent insulinotropic polypeptide (GIP) produced by duodenal K-cells in response to ingested nutrients, are important regulators of glucose homeostasis . IncretiNotably, the incretin effect appears reduced or lost in individuals with impaired glucose tolerance and T2DM ,80. ThisIn vitro, the active form of GLP-1 (7-36) induced endothelium-dependent vasodilation in preconstricted pulmonary arteries [In vitro, GLP-1 inhibited tumor necrosis factor alpha (TNF-α) induced plasminogen activator inhibitor 1 (PAI-1) gene and protein expression in endothelial cells [In vivo, administration of GLP-1 improved endothelial function in salt-sensitive hypertensive rats [Recent studies indicate that GLP-1 may also exert beneficial effects on the cardiovascular system independent of its effects on glucose, lipid or energy metabolism . In vitrarteries . In vitral cells . In vivoive rats . Of greaive rats ,88 and hive rats . In addiive rats , improveive rats  and atteive rats .Although GLP-1 is highly effective in lowering blood glucose and has promising cardiovascular effects, its therapeutic potential is severely limited because of rapid degradation by dipeptidyl peptidase 4 (DPP-4) to GLP-1 (9-36) which does not stimulate GLP-1 receptor and therefore does not exert the metabolic effects of active GLP-1 . TherefoExenatide (exendin-4), the first FDA approved GLP-1 mimetic, has only 53% homology to the human GLP-1 amino acid sequence; as such, it is relatively more resistant to DPP-4, reaching maximum levels approximately 2 hours following subcutaneous injection . Exenati2), fat-enriched breakfast meal . Blood was collected for lipid assays over an 8 hour postprandial period. The single dose of exenatide strongly suppressed postprandial elevation of TG, apoB-48, RLP-TG and RLP-cholesterol (Figure To assess the effects of typical clinical dosing of exenatide on postprandial lipid and lipoprotein excursions, we conducted a double-blinded, randomized, placebo-controlled, crossover study in participants with IGT or with recent onset T2DM in good control with diet alone . The intWe also tested whether the metabolic effects of exenatide would translate to improved postprandial endothelial dysfunction . Twenty in vitro and in vivo in rodents was profoundly reduced by both exendin-4 and sitagliptin [Clinical studies with DPP-4 inhibitors provide additional support for incretin-based therapies as an effective tool in ameliorating postprandial dyslipidemia. These compounds only moderately increase endogenous GLP-1 levels and as such they do not have a detectable effect on gastric emptying rate ,106. Desagliptin . A summaSince more intensive glycemic control with standard diabetes medications has been largely ineffective in reducing the high residual cardiovascular risk in individuals with T2DM, aggressive treatment of other cardiovascular risk factors appears to be the logical next step in the effort to decrease cardiovascular risk in this group. In fact, the results of the STENO-2 study show that a target-driven, long-term, intensified intervention aimed at multiple risk factors in patients with type 2 diabetes and microalbuminuria, may reduce the risk of cardiovascular events by about 50 percent . An incrAlthough this review highlights clinical and experimental data that provide evidence for favorable cardiovascular effects of incretins, the lack of outcome studies and the short history of clinical use of these agents limit our knowledge about their clinical cardiovascular efficacy. Large multi-center longitudinal studies designed not just to prove CVD safety as shown in two recent meta-analyses of short-term randomized clinical trials of exenatide  or sitagapoB-100: apolipoprotein B-100; apoB-48: apolipoprotein B-48; apoCIII: apolipoprotein CIII; BMI: body mass index; CHD: coronary heart disease; CVD: cardiovascular disease; CRP: C-reactive protein; DPP-4: dipeptidyl peptidase 4; GIP: glucose-dependent insulinotropic polypeptide; GLP-1: glucagon-like peptide-1; IGT: impaired glucose tolerance; MCP-1: monocytes chemoattracting protein 1; MI: myocardial infarction; NEFA: non-esterified fatty acids; NO: nitric oxide; PAI-1: plasminogen activator inhibitor 1; PAT: peripheral arterial tonometry; RLPs: remnant lipoprotein particles; T2DM: type 2 diabetes mellitus; TNF-α: tumor necrosis factor alpha.JK and PDR are recipients of unrestricted research grants from Amylin/Lilly. SA declares no competing interests.SA and JK drafted the manuscript. PDR conceived of the concept, initiated and helped drafting, and reviewed the final version of the manuscript. All authors read and approved the final manuscript.


CD4+ T cells augment the antigen-presenting capacity of dendritic cells and promote the expansion of tumor-reactive CTLs. Antibodies mediate their antitumor effects via antibody-dependent cellular cytotoxicity, activation of the complement system, improving the uptake of coated tumor cells by phagocytes, and the functional interference of biological pathways essential for tumor growth. Consequently, several tumor-associated antigens (TAAs) have been identified that represent promising targets for T cell- or antibody-based immunotherapy. These TAAs comprise proteins preferentially expressed in normal and malignant prostate tissues and molecules which are not predominantly restricted to the prostate, but are overexpressed in various tumor entities including PCa. Clinical trials provide evidence that specific immunotherapeutic strategies using such TAAs represent safe and feasible concepts for the induction of immunological and clinical responses in PCa patients. However, further improvement of the current approaches is required which may be achieved by combining T cell- and/or antibody-based strategies with radio-, hormone-, chemo- or antiangiogenic therapy. Prostate cancer (PCa) is the most common noncutaneous cancer diagnosis and the second leading cause of cancer-related deaths among men in the United States. Effective treatment modalities for advanced metastatic PCa are limited. Immunotherapeutic strategies based on T cells and antibodies represent interesting approaches to prevent progression from localized to advanced PCa and to improve survival outcomes for patients with advanced disease. CD8 Prostate cancer (PCa) represents the most common noncutaneous cancer and the second leading cause of cancer mortality among men in the United States, with an estimated incidence of 240,890 cases and an estimated number of 33,720 deaths in 2011 ,6. There+ cytotoxic T cells (CTLs) can efficiently recognize and destroy tumor cells which expose peptides derived from intracellular tumor-associated antigens (TAAs) in complex with human leukocyte antigen (HLA) class I molecules on the surface [+ T cells recognizing peptides in the context of HLA class II molecules on the cell surface also play an important role in antitumor immunity [+ T cells improve the capacity of dendritic cells (DCs) to induce CTLs by the interaction between CD40 on DCs and CD40 ligand on activated CD4+ T cells. Furthermore, CD4+ T cells provide help for the maintenance and expansion of CTLs by secreting cytokines, and can eradicate tumor cells directly. Clinical studies focusing on the adoptive transfer of naturally occuring human T cells demonstrated tumor regression in cancer patients [Specific immunotherapy emerged as a promising treatment modality for advanced PCa ,8. T cel surface . CD4+ T immunity . CD4+ T patients ,12. Follpatients ,14. Humapatients ,16. + CTLs and CD4+ T cells. Owing to their extraordinary capacity to induce and expand tumor-reactive T cells, DCs emerged as promising candidates for cancer vaccination therapy [DCs are professional antigen presenting cells (APCs) which display a unique capacity to induce, sustain and regulate T-cell responses ,18. In t therapy ,20.+ CTLs and CD4+ T cells. Furthermore, they can interfere with biological pathways essential for tumor growth. Immunotherapeutic strategies based on the administration of mAbs have been successfully introduced into clinic [Monoclonal antibodies (mAbs) recognize target structures on the tumor cell surface and mediate their antitumor effects by various mechanisms of action . They aro clinic . More reo clinic ,24. Consequently, much attention has been paid to the identification of TAAs that represent attractive targets for T cell- or antibody-based immunotherapy. The group of molecules preferentially expressed in normal and malignant prostate tissues comprises prostate-specific antigen (PSA), prostate-specific membrane antigen (PSMA), prostatic acid phosphatase (PAP), prostate stem cell antigen (PSCA), T cell receptor gamma alternate reading frame protein (TARP), transient receptor potential (trp)-p8 and six-transmembrane epithelial antigen of the prostate 1 (STEAP1).b-transgenic mice [PSA, a kallikrein-like serin-protease, is almost exclusively expressed by prostate epithelial cells, can be detected in the majority of PCa tissues, and represents the most widely used serum marker for diagnosis and monitoring of PCa ,26,27,28nic mice . Furthernic mice ,38,39. in vitro when compared to protein-pulsed DCs [et al. [+ T cell tolerance to a prostate-restricted model antigen by androgen ablation indicating that specific immunotherapy of PCa may be more efficacious when administered after androgen ablation [Consequently, much attention has been paid to the optimization of delivery strategies for an active immunotherapy such as the transduction of DCs with an adeno-associated virus-based vector which more effectively stimulated PSA-specific CTLs lsed DCs . The cap [et al.  showed tablation .The integral membrane glycoprotein PSMA represents a marker for normal prostate cells and can be detected in the majority of prostate tumors, particularly in undifferentiated, metastatic HRPC ,44. Althin vitro [in vitro which efficiently induce T cell responses in human HLA class II transgenic mice [in vitro and in vivo studies for optimized antigen capability to stimulate T cell responses. In this context, co-transduction of genes encoding the extracellular domain of PSMA and a costimulatory protein using an adenoviral vector has been proven to effectively activate specific T cell responses in vitro and to elicit protective and therapeutic anti-tumor immunity in a murine tumor model [HLA-A2-restricted PSMA-derived peptides were shown to induce antitumoral CTL responses in vitro ,49,50. Iin vitro ,52 and tin vitro  have beenic mice ,54. PSMAor model .213Bi-J591, humanized 90Y-chelate-J591 and 131I-J591 have also been shown to markedly reduce the tumor volume in nude mice bearing LNCaP xenografts [131I or 111In showed preferential accumulation in areas of the viable tumor in xenograft models which is a prerequisite for effective radioimmunotherapy [in vitro and in vivo [et al. [Pseudomonas Exotoxin A with PSMA-specificity and therapeutic efficacy in a mouse xenograft model.Based on the surface expression on PCa cells, PSMA also represents a promising target molecule for antibody therapy. Evaluation of different anti-PSMA mAbs coupled to ricin A and the bismuth-conjugated mab J591 binding to the extracellular PSMA portion revealed target-specific cytotoxicity against PSMA-expressing PCa cells ,57. 213Bnografts ,58. The  in vivo . Wolf et [et al.  develope+ and CD4+ T cells and to recruit them to the tumor site revealed efficient inhibition of tumor growth in a xenograft model [The use of an anti-PSMA x anti-CD3 bispecific diabody to selectively activate PSMA-specific CD8ft model ,63. Anotft model .The expression of PAP which represents one of the major proteins secreted by prostate epithelial cells is mainly restricted to benign and malignant prostate tissue . Studiesin vivo [+ T cells in the blood of PCa patients and healthy donors [in vitro screening for CD4+ T cell reactivity directed to PAP sequence motifs of an overlapping 20-mer peptide library, CD4+ T cell epitopes for several HLA-DR allelic variants were identified [Naturally generated, HLA-A2-binding, immunogenic peptides were identified ,70 and rin vivo ,72. Addiy donors . Furthery donors ,74. A pey donors . Such prentified ,77. in vitro to PA2024, a fusion protein consisting of human granulocyte-macrophage colony-stimulating factor and PAP [The immunotherapeutical potential of vaccination strategies using PAP as target antigen have been demonstrated in mouse models and clinical trials. In a recent study, a DNA vaccine encoding murine PAP induced PAP-specific CTL responses and effectively suppressed tumor growth in a Transgenic Adenocarcinoma of the Mouse Prostate (TRAMP) model . Moreove and PAP . PatientPSCA is a glycosylphosphatidylinositol-anchored cell surface glycoprotein that is expressed in basal and secretory epithelial cells of the prostate and was originally identified by its upregulation in a human PCa xenograft model ,81. On tin vitro [+ T cells recognizing two of these peptides in the blood of PCa patients [We and others identified HLA-A2-restricted PSCA peptides capable of generating tumor-reactive CTL responses in vitro ,86,87. Wpatients . Several studies focusing on the immunotherapeutical potential of PSCA were conducted in the TRAMP mouse model displaying PSCA expression pattern in the spontaneously developing PCa. When vaccinated with a viral vector encoding PSCA after priming with PSCA cDNA, TRAMP mice with prostate intraepithelial neoplasia developed antigen-specific CTL responses and displayed a significantly increased survival rate when compared to the control group . Anotherin vitro and in vivo and revealed Fc-independent induction of cell death requiring target cross-linking [in vivo [PSCA has also been evaluated as target for antibody-based immunotherapy. Anti-PSCA mAbs conjugated to the toxin maytansinoid were effectively internalized by PCa cells resulting in cytotoxicity and regression of xenografts in mice . Further-linking . Several[in vivo ,95. Furt[in vivo . In anot[in vivo .The transmembrane protein prostein is typically expressed in normal and malignant prostate tissues and is potentially involved in PCa cell migration and invasion ,99,100. in vitro-stimulation of CD8+ T lymphocytes with peptide-loaded DCs we identified an autochthonously generated HLA-A*0201-presented, prostein-derived peptide which was able to stimulate tumor-reactive CTLs [By ive CTLs . Immunogive CTLs . TARP is derived from a unique androgen-regulated transcript of a portion of the non-rearranged T cell receptor gamma-chain locus ,105. In in vitro were identified [et al. defined two TARP-derived HLA class II-binding peptides which were shown to elicit effective CD4+ T cell responses [Several naturally generated HLA-A*0201-restricted TARP-peptides which stimulated PCa and breast cancer cell-reactive CTLs entified ,108. In esponses .Pseudomonas Exotoxin A. TARP was subject of evaluation of a novel targeting approach using antibodies specifically binding individual HLA class I-peptide complexes on the surface of tumor cells for diagnostic and therapeutic purposes . In thistrp-p8 encodes a seven-span transmembrane protein with significant homology to a family of Ca2+ channel proteins [in vitro [The gene proteins . Trp-p8 proteins . Furtherproteins . We idenin vitro . STEAP1 is a transmembrane protein originally identified by its overexpression in a PCa xenograft model that mimics an advanced disease stage . It is pin vitro and in vivo as well as three promiscuous CD4+ T cell epitopes have been identified [Several naturally processed HLA-A2-restricted peptides capable of inducing CTLs entified ,118,119.Some vaccination strategies using recombinant cDNA or viral vectors encoding mouse STEAP1 were successful in the induction of specific T cell responses, reduction of tumor growth and increase of survival in mouse models ,120,121.Recent data suggest that STEAP1 additionally represents an attractive target for antibody-based immunotherapy as two STEAP-specific mAbs significantly inhibited the growth of PCa xenografts in mice .Several potential target structures for specific immunotherapy are not predominantly restricted to the prostate, but are overexpressed in different tumors of epithelial and/or hematopoietic origin including PCa. This group comprises parathyroid hormone-related protein (PTHrP), human telomerase reverse transcriptase (hTERT), survivin, members of the epidermal growth factor receptor family, N-cadherin, erythropoietin-producing hepatocellular receptor tyrosine kinase class A2 (EphA2) and synovial sarcoma X chromosome break point (SSX) proteins.PTHrP is a factor that binds receptors on osteoblasts and induces bone formation. It is highly overexpressed in PCa and other cancers of epithelial origin including gastric, breast, lung, colon, cervical and renal cancer and is considered to be involved in the development of bone metastases ,123. Thein vitro, and two of them were additionally described to induce antitumoral CTL responses in vivo [in vitro [Four HLA-A2-restricted PTHrP peptides were shown to elicit tumor-reactive CTLs  in vivo ,125. Furin vitro . Due to its role in metastasis formation and bone destruction in malignant diseases, neutralizing anti- PTHrP antibodies may provide an interesting immunotherapeutic tool. Promising results have been obtained in a mouse model where neutralizing antibodies inhibited the formation of osteolytic bone metastases of lung cancer cells .in vitro and in vivo [Another potential target for specific immunotherapy is hTERT. This molecule is undetectable in most non-transformed somatic cells but is expressed in more than 85% of human tumors including PCa . Several in vivo ,131,132. in vivo ,136,137. in vivo ,139. Survivin, an inhibitor of apoptosis and promoter of proliferation, is expressed in many tissues during fetal development whereas expression is almost absent in differentiated healthy adult tissues. However, expression can be detected in cells undergoing self renewal as hematopoietic precursor cells, keratinocytes, lymphocytes, activated endothelial cells and epithelial cells of the uterine cervix . Surviviin vitro [+ T cells reactive against one of the previously defined survivin peptides and a peptide modified at an anchor aminoacid were found in the blood of tumor patients [+ T cell epitopes restricted to other HLA class I molecules were defined by analyzing the peptide specificity of spontaneous CTL responses in cancer patients [in vitro stimulation of PBMCs [We and others identified two naturally generated HLA-A*0201-restricted peptides which induced specific CTL responses in vitro ,145. Furpatients . A numbepatients ,148 and of PBMCs . Recentlof PBMCs ,151.In pre-clinical models, immunotherapeutical targeting of survivin has been shown to effectively induce T cell responses and to exert anti-tumor effects for various solid tumors ,153.Several cell surface proteins of the ErbB receptor tyrosine kinase family such as c-erbB-1 , c-erbB-2 (HER-2/neu) and c-erbB-4 (HER-4) are overexpressed in different tumors including PCa  and may The tyrosine kinase receptor HER-2/neu is the target structure for the humanized mAb trastuzumab which has been successfully used for the treatment of breast and gastric cancer ,155. RecIn vivo studies which were based on the administration of the EGFR-specific mAbs cetuximab and panitumumab have shown inhibition of tumor growth in various PCa xenograft models [EGFR is overexpressed in a significant percentage of PCa and was found to be involved in PCa progression to androgen independence ,163. In t models ,165. Altt models ,167. in vitro and in a mouse xenograft model [Recently, c-erbB-4 also emerged as potential target molecule due to its frequent overexpression in PCa . C-erbB-ft model ,170. In N-cadherin is involved in the interaction between cells and extracellular matrix components. Upregulation of N-cadherin is a characteristic feature of tumor progression in the context of epithelial-mesenchymal transition, thereby promoting cell motility, invasiveness and metastasis formation including pelvic lymph node infiltration and bone metastasis in PCa ,172. In EphA2 is a cell-membrane bound receptor tyrosine kinase which is expressed in a wide panel of normal tissues , but is in vivo models. Such antibodies can induce internalization and degradation of the receptor as demonstrated by an effective down-regulation of EphA2 on the cell surface [The immunotherapeutical potential of mAbs with agonistic activity has been intensively evaluated in pre-clinical  surface . In addi surface ,183. Enh surface . Due to  surface ,186,187.SSX proteins represent a superfamily of highly homologous cancer-testis antigens with nuclear localization, restricted expression in HLA class I-deficient testis or ovary germline cells and frequent overexpression in tumors of various histological origins, especially in advanced stage cancer ,189. Due+ CTLs efficiently recognize and destroy tumor cells. CD4+ T cells augment the antigen-presenting capacity of DCs and promote the expansion of tumor-reactive CTLs. Antibodies directed against tumor surface antigens can mediate their antitumor effects by engaging cytotoxic effector cells such as natural killer cells, complement activation, improving the uptake of coated tumor cells by phagocytes, and the functional interference of biological pathways essential for tumor growth. Clinical trials which aimed at the in vivo-activation of CD8+ CTLs and CD4+ T cells by the administration of peptides, proteins, DNA, or TAA-loaded DCs revealed that these approaches were safe and feasible. Furthermore, immunological and clinical responses were induced in PCa patients. Clinical studies including mAbs directed against prostate cancer surface antigens were also conducted and resulted in some clinical responses. However, further improvement of current immunotherapeutic treatment modalities for advanced PCa is required which may be achieved by combining T cell- and/or antibody-based strategies with radio-, hormone-, chemo- or antiangiogenic therapy. In recent years, various TAAs have been identified that represent attractive target structures for specific immunotherapy of prostate cancer. These TAAs comprise proteins preferentially expressed in normal and malignant prostate tissues and molecules which are not predominantly restricted to the prostate, but are overexpressed in various tumor entities including PCa. The identification of TAAs and derived HLA class I and class II-restricted T cell epitopes paved the way for the design of novel T cell- or antibody-based immunotherapeutic strategies. CD8


Exosomes are endosome-derived, 30–100 nm small membrane vesicles released by most cell types including tumor cells. They are enriched in a selective repertoire of proteins and nucleic acids from parental cells and are thought to be actively involved in conferring intercellular signals. Tumor-derived exosomes have been viewed as a source of tumor antigens that can be used to induce antitumor immune responses. However, tumor-derived exosomes also have been found to possess immunosuppressive properties and are able to facilitate tumor growth, metastasis, and the development of drug resistance. These different effects of tumor-derived exosomes contribute to the pathogenesis of cancer. This review will discuss the roles of tumor-derived exosomes in cancer pathogenesis, therapy, and diagnostics. Membranous vesicle shedding from live cells was first observed in the early 1980s and was proposed to be a mechanism through which cells discard inert debris . Differeβ [Exosomes contain cytosolic and membrane proteins derived from the parental cells. The protein content largely depends on their cellular origin and are generally enriched for certain molecules, including targeting/adhesion molecules , membrane trafficking molecules , cytoskeleton molecules , proteins involved in MVB formation , chaperones , signal transduction proteins  and cytoplasmic enzymes  , 8, 24. β . In addiβ –29. in vitro studies, including (1) binding of vesicles to the surface of a recipient cell through exosomal adhesion molecules, or phosphatidylserine (PS)/lysophosphatidylcholine and cellular receptors ; (2) direct fusion of vesicles with recipient plasma membrane after adhesion; or (3) internalization of vesicles into endocytic compartments through receptor-mediated endocytosis or phagocytosis [Exosomes are now recognized as important mediators of cell-to-cell communication . Howeverocytosis . Also, tocytosis . The interaction between exosomes and target cells can lead to direct stimulation of target cells via surface-expressed growth factors or bioactive lipids, transfer of membrane receptors, or delivery of proteins to target cells. Also, the presence of mRNA and microRNA, termed “exosomal shuttle RNA,” in exosomes suggests that genetic material exchange could be an additional level of exosome-mediated communication between cells .There is still some confusion in describing different types of vesicles secreted by cells. The terms “exosomes,” “microvesicles,” and “membrane particles” are sometimes used interchangeably. Generally, the term “microvesicles” refers to vesicles shed from the plasma membrane, have a relatively larger size (100–1000 nm) than exosomes and can be sedimented at 10,000 ×g. The term “membrane particles” refers to vesicles that also originate from plasma membrane, but have a small size similar to exosomes . In thisex vivo model system, DCs pulsed with exosomes derived from malignant effusions expressing tumor antigens cross-present the antigens to antigen-specific cytotoxic T lymphocytes (CTLs) [The protein composition of exosomes largely reflects that of their parental cells and thus shows cell-type specificity. In particular, tumor-derived exosomes contain tumor-specific antigens expressed in the parental tumor cells. Enrichment of tumor antigens such as melan-A , Silv 33, carcins (CTLs) . Recentls (CTLs) . in vitro and in vivo in both autologous and allogeneic murine models, suggesting that these exosomes can stimulate antitumor immunity in an MHC-independent manner [Direct application of tumor-derived exosomes for the enhancement of antitumor immunity also has been investigated. It was reported that tumor-derived exosomes could induce specific antitumor responses when the parental tumor cells were genetically modified to express pro-inflammatory cytokines such as IL-18, IL-12, and IL-2 –39 or wht manner . Moreovet manner . Heat-stt manner . Furthert manner  or chickt manner  resultedThe promising results obtained in animal tumor models led to several phase I clinical trials using tumor-derived exosomes or exosome-pulsed DCs as cancer vaccines , 47. Howβ (GSK-3β) activation and decreased pyruvate dehydrogenase activity in treated cells, sequestered β-catenin-dependent survival pathway, and counteracted the constitutively activated phosphatidylinositol 3-kinase/Akt survival pathway to drive tumor cells toward apoptosis [In addition to the potential immunostimularoty effects, a proapoptotic function of tumor-derived exosomes directly on tumor cells was also reported. Exosome-like vesicles produced by human pancreatic tumor cells were reported to increase Bax and decrease Bcl-2 expression, inducing tumor cells toward mitochondria apoptotic pathway. These exosomes also induced phosphatase and tensin homolog (PTEN) and glycogen synthase kinase-3poptosis . The intpoptosis . in vivo. Notably, in cancer patients with advanced disease, tumor-derived exosomes are produced abundantly in the tumor microenvironment, however effective immunostimulatory or antitumor effects of these vesicles are rarely observed. In fact, there is substantial evidence supporting a role of tumor-derived exosomes in preventing antitumor immune responses and promoting tumorigenesis.Despite the potential antitumor effects of tumor-derived exosomes, it is still unclear whether the constant production of exosomes by tumor cells is beneficial or harmful for their own survival ζ chain expression and impair TCR signaling [in vivo by suppressing NK cell function [The observation that membrane vesicles shed from murine melanoma cell lines inhibited the expression of the immune response region-associated antigen by macrophages provided early evidence that tumor-derived membrane vesicles is a possible mechanism whereby tumor-bearing hosts become immunocompromised . More reignaling , suggestignaling , 56. Simfunction . Taken tβ1 mediated suppressive activity on T cells in vitro. Interestingly, significant expansion of MDSC-like CD14+HLA-DR-/low and TGF-β-secreting cells was also found in the peripheral blood of late-stage melanoma patients and high levels of MDSCs is usually associated with poor responses to tumor vaccines [β molecules present on tumor-derived exosomes [Tumor-derived exosomes can also target myeloid cells to modulate their differentiation and function. Exosomes derived from human melanoma cell lines and colorectal carcinoma cell lines were shown to skew monocyte differentiation into DCs toward the generation of myeloid-derived suppressor cells (MDSCs) and exert TGF-vaccines . Similarvaccines . These mexosomes . MyD88 aexosomes . Similarexosomes , althougexosomes , 64.The effect of tumor-derived exosome on BM cells is thought to be a coevolutionary strategy of the primary tumor and the tumor microenvironment . Alteratβ-dependent mechanism [β- and IL-10-dependent mechanism [In addition, tumor-derived exosomes can also support the function of regulatory T (Treg) cells. For example, human tumor-derived exosomes were found to selectively impair the IL-2 response to cytotoxic effector cells while supporting Treg cell activities through a TGF-echanism . Tumor-dechanism . A similechanism .Given that tumor-derived exosomes are capable of altering APC function and enhancing regulatory cell activity while at the same time are a source of tumor antigen, it is tempting to speculate that tumor-derived exosomes may also have the ability to promote tolerance to tumor-specific antigens. Indeed, we have demonstrated that tumor-derived exosomes bearing a model tumor antigen were able to induce antigen-specific immunosuppression in a murine delayed-type hypersensitivity model. We proposed a mechanism that tumor-derived exosomes provide tumor antigens to DCs as well as condition DCs toward a suppressive/tolerogenic phenotype, resulting in the downregulation of antigen-specific immune responses .In addition to attenuating different branches of the antitumor immunity to help tumor cells survive immunosurvelliance, tumor-derived exosomes have also been implicated in facilitating tumor invasion and metastasis. By stimulating angiogenesis, modulating stromal cells, and remodeling extracellular matrix, tumor-derived exosomes have been found to contribute to the establishment of a premetastatic niche, generating a suitable microenvironment in distant metastatic sites . Early proteomic analysis of mesothelioma cell-derived exosomes detected the presence of strong angiogenic factors that can increase vascular development in the neighborhood of tumor . Melanomin vitro and angiogenesis in vivo in a rat model [in vitro and an increase in branching in vivo  [Tetraspanins, which are constitutively enriched in exosomes, have been found to contribute to exosome-mediated angiogenesis. It was reported that exosomes derived from a pancreatic tumor line overexpressing D6.1A, a tetraspanin associated with poor prognosis in patients with gastriointestinal cancer, strongly induced endothelial cell branching at model . Tumor-dat model . Tumor-din vivo  .β on their surface in association with betaglycan and can trigger SMAD-dependent signaling. Exosomal delivery of TGF-β is capable of driving the differentiation of fibroblasts into myofibroblasts, whose enrichment in solid tumor represents an altered stroma that usually supports tumor growth, vascularization, and metastasis. Exosomal TGF-β delivery is also qualitatively different from soluble TGF-β in that they induce a more significant elevation of fibroblast FGF2 production [β in addition to their immunosuppressive functions. However, it was also noted that TGF-β is not universally present on exosomes derived from all cancer cells. Another pronounced effect of tumor-derived exosomes is their ability to modulate the function of stromal cells such as fibroblasts. It was recently shown that exosomes produced by a certain type of cancer cells contain TGF-oduction . These oFurthermore, exosomes shed by gynecologic neoplasias, including ovarian cancer and breast cancer cells, were found to contain metalloproteinases that have proteolytic activity. These exosomes can increase extracellular matrix degradation and augment tumor invasion into the stroma –80. It wInterestingly, tumor-derived microvesicles, which are mostly shed from tumor plasma membrane, were found to have certain effects similar to exosomes, such as stimulating angiogenesis , 83, modlet-7 genes target oncogenes including RAS and HMGA2 and generally play a tumor-suppressor role, the release of let-7 microRNA via exosomes could deliver oncogenic signals and promote metastasis [The intercellular exchange of proteins and genetic materials via exosomes is a potentially effective approach for cell-to-cell communication within the tumor microenvironment . In parttastasis . Moreovetastasis . Similartastasis .Tumor-derived exosomes may also transport apoptosis-inhibitory proteins induced under stress conditions to promote tumor survival. For example, survivin, a member of the inhibitor of apoptosis protein family, can be absorbed by cancer cells from extracellular media and inhibit their apoptosis following genotoxic stress as well as increase their replicative and metastatic ability . It was in vitro and in vivo. The exosome-antibody interactions inhibit the overall effect of Trastuzumab on the proliferation of cancer cells by reducing antibody binding to cancer cells [The protumorigenic role of tumor-derived exosomes is also reflected by their active participation in drug resistance through several mechanisms. One mechanism is by drug exportation via the exosome pathway. In human ovarian carcinoma cells that stably acquired resistance to the cancer chemotherapy drug cisplatin, the lysosome compartment, where the drug usually accumulates, was reduced with more exosomes released compared to cisplatin-sensitive cells. Moreover, when the cells were loaded with cisplatin, exosomes released from cisplatin-resistant cells contained 2.6-fold more platinum than those released from cisplatin-sensitive cells, suggesting that exosome secretion can be utilized by cancer cell to export anticancer drugs . A similer cells . Such aner cells .Taken together, tumor-derived exosomes exert pro-tumorigenic effects via pleiotropic mechanisms . HoweverAs discussed above, tumor-derived exosome-pulsed DCs, tumor-derived exosomes, and exosomes isolated from malignant ascites all have been investigated for their ability to elicit antitumor immune response in patients. However, although these clinical approaches appear to be safe, there has been a lack of clinical efficacy of exosome-based vaccines in contrast to the promising results obtained in many animal tumor models. Because of their potential immunosuppressive properties, direct administration of tumor-derived exosomes may actually result in promoted tumor growth. Therefore, clinical studies have focused on the use of tumor-derived exosome-loaded mature DCs  or ascitThe protumorigenic potential of tumor-derived exosomes in cancer patients is supported by the observations that in patients with breast or ovarian cancer, the level of circulating exosomes and exosomes with tumor markers is much higher than nonmalignant individuals and increases with tumor progression , 105, anOn the other hand, tumor-derived exosomes containing tumor-specific protein and microRNA profiles have been proposed to be cancer diagnostic markers. Early detection of cancer could be easily performed using exosomes isolated from body fluids such as blood plasma, serum, and urine. Evidence supporting this approach include: (1) ovarian cancer-associated expression of claudin proteins can be detected in the circulating vesicles of a majority of ovarian cancer patients , (2) in Increasing evidence suggests that tumor-derived exosomes can confer either antitumorigenic or protumorigenic effects. These seemingly controversial effects can be the results of complex interactions between exosomes, responding cells, and environmental factors. In cancer patients, the immunostimulatory or immuosuppressive effects of tumor-derived exosomes may also depend on the stage of cancer progression as well as the immune status. Notably, as close replicas of their parental cells, tumor-derived exosomes are well positioned to transmit the detrimental effects of tumor cells onto the immune system to facilitate their survival, growth, and metastasis. Therefore, a better understanding of the roles of tumor-derived exosomes in cancer pathogenesis is needed to further improve anti-cancer therapeutics as well as exosome-based cancer diagnostics.


Pim3 gene. Accordingly, lymphomas arising in Myc-transgenic mice and Burkitt lymphoma cell lines exhibit elevated levels of Pim-3. Interestingly, inhibition of Pim kinases by a novel pan-Pim kinase inhibitor, Pimi, in Myc-induced lymphoma results in cell death that appears independent of caspases. The data indicate that Pim kinase inhibition could be a viable treatment strategy in certain human lymphomas that rely on Pim-3 kinase expression.The Pim kinases are weak oncogenes. However, when co-expressed with a strong oncogene, such as c-Myc, Pim kinases potentiate the oncogenic effect resulting in an acceleration of tumorigenesis. In this study we show that the least studied Pim kinase, Pim-3, is encoded by a gene directly regulated by c-Myc via binding to one of the conserved E-boxes within the  Therefore, Myc expression is often selected for during various steps in the genesis of many types of human cancer. To avoid transformation, normal cells can respond to Myc overexpression by undergoing apoptosis or senescence thienopyrimidin-4-one), and the non-active control substance, Pim-ci, were obtained from Abbott Laboratories.-Myc or Eμ-Myc transgenic mice -thymidine (Amersham)/ml media was added and the cells were harvested by a semiautomatic cell harvester (Inotech CH-5605 Dottikon). [3H]-thymidine incorporation was measured using a liquid scintillation counter (PerkinElmer).Cells were cultured in 96-well plates for 48 h in the presence of either DMSO or various concentrations of Pimi. The last 2 h of incubation, 20 μCi of [


To investigate the effectiveness of a pilot program using a CAM multi-therapy treatment program focusing on self-care behaviors for the treatment of Generalized Anxiety Disorder (GAD).This is a quasi-experimental one-group pretest-posttest design using a convenience sample of volunteers at a military treatment facility in the Pacific Northwest. Participants (N=37) were enrolled if they had a documented history of GAD or met screening criteria for GAD using the GAD-7, with 68% completing the program (N=25). Participants received acupuncture treatments one time/week for six weeks and were asked to engage in yogic breathing exercises, self and/or partner assisted massage therapy using scented oils, episodic journaling, nutrition counseling, and exercise.Significant reductions were identified on pre and post GAD-7, Depression-Anxiety-Stress Scale-21, UCLA Loneliness Scale, and a significant increase was noted in the Rosenberg Self-esteem Scale. In addition patient behavior (participation in study therapies) remained consistently high and a secondary outcome was reduction in anti-anxiety medication use.The findings in this pilot study suggest multimodal interventions to facilitate self-care is feasible and that a multi-therapy treatment program using CAM therapy, focusing on self-care behaviors may be an effective adjunct therapy for the treatment of Generalized Anxiety Disorder.


Immune inhibitory molecules on immune cells as well as on tumor cells appear to play a key role in modulating the immunogenicity of tumors and the efficacy of immunotherapies. One mediator is represented by the non-classical HLA-G antigen, which is often overexpressed in human tumors when compared to corresponding normal tissues thereby inhibiting both innate as well as adaptive immune responses. Since discordant HLA-G transcript and protein expression levels were often found in tumors of distinct origin posttranscriptional control mechanisms have been recently suggested. Indeed, different HLA-G-specific miRs have been identified, which were able to downregulate HLA-G surface expression. The miR-mediated inhibition of HLA-G enhanced the NK cell recognition. These miRs were also differentially expressed in renal cell carcinoma (RCC) versus normal kidney epithelium. Immunohistochemical analysis demonstrated of a high frequency HLA-G expression in RCC lesions, which was associated with disease progression and inversely correlated with the expression of HLA-G-specific miRs. These data postulate that HLA-G-specific miRs might be used as prognostic markers as well as potential therapeutics for targeting HLA-G expressing RCC.


Occlusion of femoro-popliteal bypass graft represents a limb and life-threatening condition requiring emergency intervention.A 66-year-old man was admitted to our hospital on November 11, 2012. He had a painful, pulseless, and cold below-knee in right lower extremity. He had previous right cross-over composite sequential femoro-popliteal bypass. Angiography showed acute occlusion of cross-over composite femoro-popliteal bypass graft and stent graft used in treatment of pseudoaneurysm developing in popliteal anastomosis region of this graft. Proximal EPTFE graft was occluded with thrombus in arteriography.Thrombectomy was performed with the 5F Fogarty balloon catheter and excessive thrombus was extracted. A thrombo-embolectomy was performed to the distal of popliteal artery, but thrombus was not detected in this region. The patient was performed a new interposition saphenous vein graft between the proximal EPTFE graft and distal popliteal artery. The patient received low-molecular weight heparin in postoperative period. He received warfarin and acetylsalicylic acid in follow-up. Right lower extremity pulses were palpable in postoperative period. Right lower extremity had good function in follow-up at 6 months.We showed importance of saphenous vein graft bypass and thrombectomy in a case of having acute occlusion of cross-over composite sequential femoro-popliteal bypass.