Many functional neuroimaging studies have reported differential patterns of spontaneous brain activity in posttraumatic stress disorder (PTSD), but the findings are inconsistent and have not so far been quantitatively reviewed. The present study set out to determine consistent, specific regional brain activity alterations in PTSD, using the Effect Size Signed Differential Mapping technique to conduct a quantitative meta-analysis of resting-state functional neuroimaging studies of PTSD that used either a non-trauma (NTC) or a trauma-exposed (TEC) comparison control group. Fifteen functional neuroimaging studies were included, comparing 286 PTSDs, 203 TECs and 155 NTCs. Compared with NTC, PTSD patients showed hyperactivity in the right anterior insula and bilateral cerebellum, and hypoactivity in the dorsal medial prefrontal cortex (mPFC); compared with TEC, PTSD showed hyperactivity in the ventral mPFC. The pooled meta-analysis showed hypoactivity in the posterior insula, superior temporal, and Heschl’s gyrus in PTSD. Additionally, subgroup meta-analysis (non-medicated subjects vs. NTC) identified abnormal activation in the prefrontal-limbic system. In meta-regression analyses, mean illness duration was positively associated with activity in the right cerebellum (PTSD vs. NTC), and illness severity was negatively associated with activity in the right lingual gyrus (PTSD vs. TEC). Posttraumatic stress disorder (PTSD) is a psychiatric illness caused by traumatic events, characterized by traumatic event re-experiencing (e.g. flashbacks), avoidance of trauma-related events, hyperarousal (e.g. hypervigilance), and negative cognitions and mood2454Multiple neuroimaging modalities such as functional magnetic resonance imaging (fMRI), single-photon emission computed tomography (SPECT), and positron emission tomography (PET) have been employed to investigate the aforementioned altered brain activities in PTSD. In general terms, fMRI makes use of the blood oxygen level-dependent (BOLD) signal to show patterns of activity in the brain4141516Resting-state neuroimaging, which evaluates regional interactions that occur when a subject is not performing an explicit task, has proved an informative and reliable research tool1219202122232425262727293124Although many task-related neuroimaging meta-analyses, synthesizing a variety of symptom provocation and cognitive-emotional studies, have been performed to elucidate the neural underpinnings of PTSD141516171617In the present study, we used a voxel-based meta-analytic technique, Effect Size Signed Differential Mapping (ES-SDM), to identify consistent functional brain alterations in PTSD by integrating the full range of studies reporting resting regional brain activity. We performed two individual pooled meta-analyses, comparing PTSD with TEC and with NTC respectively, to explore the different pathophysiological implications in PTSD. In addition, we used subgroup meta-analyses to control for comorbidity and medication. Finally, we performed meta-regression analysis to examine the potential effects of age, illness severity and illness duration of PTSD patients.The search strategy identified a total of 138 papers , of whic121920212223242627282930313233353637232930283132373219122133353621241229Finally, our total sample comprised 286 patients with PTSD, 203 TECs and 155 NTCs. In the PTSD vs. TEC group were 7 studies comprising 178 PTSD and 203 TEC; controlling for comorbidity and medication there were 4 studies comprising 132 PTSD and 150 TEC, and 5 studies comprising 112 PTSD and 132 TEC. In the PTSD vs. NTC group were 11 studies comprising 149 PTSD and 155 NTC; controlling for comorbidity and medication there were 6 studies comprising 74 PTSD and 82 NTC, and 8 studies comprising 106 PTSD and 111 NTC, respectively. In no study was there any significant difference in age or sex between the PTSD and control groups.In the pooled whole-brain meta-analysis, in PTSD compared to non-trauma controls, resting-state activity was increased in the bilateral cerebellum, right insula (anterior part) and inferior frontal gyrus (IFG), and decreased in the dorsal medial prefrontal cortex (dmPFC)  and anterior cingulate gyrus ), left insula  and adjacent left auditory cortex  and Heschl’s gyrus ) . The resIn the pooled whole-brain meta-analysis, in PTSD compared to trauma-exposed controls, resting-state activity was increased in the ventral mPFC  and bilateral ACC), left SMG, and middle frontal gyrus (MFG), and decreased in the right posterior insula and adjacent right auditory cortex (including the right HG and STG) and right visual cortex  and calcarine fissure and surrounding cortex ) . In the In a whole-brain jack-knife sensitivity analysis of PTSD vs. NTC , the finWhole-brain jack-knife sensitivity analysis of PTSD vs. TEC  showed tVariables explored by regression were age and the duration and severity of illness . In metaWe used quantitative ES-SDM meta-analytic methods to synthesize findings from 15 resting-state functional neuroimaging studies of PTSD, in which patients with PTSD were compared to either TEC or NTC. The result confirmed a subset of regional differences that have frequently been reported in previous PTSD studies, including hyperactive anterior insula and hypoactive dmPFC in PTSD patients compared with NTC, and hyperactive vmPFC in PTSD compared with TEC. In addition differences were found in other regions, such as the auditory and visual cortex and cerebellum, that have hitherto been neglected in the modeling of trauma symptoms. Results were the same in subgroup meta-analysis of ‘non-comorbidity’ studies compared with NTC, and additional hyperactivity of the amygdala and hippocampus in the PTSD patients were identified in the subgroup meta-analysis of ‘non-medication’ studies compared with NTC.In the meta-analyses, PTSD patients had increased resting-state brain activity in vmPFC compared with TEC, and decreased activity in dmPFC when compared with NTC. Alterations in mPFC have often been reported in PTSD studies using various imaging modalities including structural MRI7We found hyperactive right anterior insula in PTSD patients compared with NTC at rest. Hyperactivity in the anterior insula has been reported in resting-state functional neuroimaging studies of PTSD2746505151Our finding of cerebellar hyperactivity in PTSD is consistent with reports of elevated cerebellum rCBF activity in PTSD at rest205860In the subgroup meta-analysis of non-medication studies, increased activity in the left amygdala, parahippocampal gyrus and hippocampus was observed in PTSD compared to NTC which was not identified in the pooled meta-analysis. The amygdala participates in the enhancement of startle7Meta-regression analyses of PTSD vs. NTC studies showed that the mean illness duration was positively associated with resting-state activity in the right cerebellum. This is the first study to our knowledge to report this positive relationship. Partially consistent with this, a longitudinal fMRI study found increased activation in cerebellum in acute PTSD patients and decreased cerebellar activation in symptoms-improved PTSD patients after 2 years of follow-up, which suggested the cerebellum may reflect symptom improvementWe also found that PTSD symptom severity was negatively associated with activity of the right LG in the meta-regression analyses of PTSD vs. TEC studies. A previous voxel-based morphometry analysis demonstrated reduced gray matter volume in the lingual gyrus in the chronic PTSD group compared with the symptoms-improved groupThis meta-analysis has some limitations. One constraint was the availability of studies that met our criteria for inclusion. The exclusion of studies that did not report stereotaxic coordinates or used functional connectivity approaches likely reduced our power to detect less-robust activations. Further, the small number of studies precluded separate meta-analyses for some moderator variables, such as the characteristics of patients , imaging method , and analysis method . Although we conducted subgroup meta-analyses of ‘non-comorbidity’ and ‘non-medication’ studies in PTSD compared with TEC, these included only 4 studies and 5 studies, respectively, and had limited power; further investigation will needed to determine their relative contributions to PTSD pathology.In addition, like all coordinate-based methods, ES-SDM assumes that effect sizes originate from homogeneous t-value contrasts; in fact they might originate from different covariate models or from different raw statistics, and this limitation could be controlled by SDM covariate analyses, if relevant. Finally, all neuroimaging data are highly sensitive to common artifacts such as head motion and breathing effects that may influence the results69The present meta-analyses provide a unique opportunity to assess altered intrinsic brain activities across individual PTSD studies during rest. The results confirmed a subset of consistent regional differences often reported in previous PTSD studies, including the vmPFC, insula, and limbic regions . Additional regions were found, such as the auditory and visual cortex and cerebellum, that have received less attention. It is noteworthy that different parts of mPFC and insula may have different pathophysiological implications in PTSD, and future PTSD studies should subdivide these regions, especially in functional connectivity analysis. Differential brain regions and activities found in two individual pooled meta-analyses revealed differential pathophysiological implications in two different comparisons. Further studies are needed to determine whether the findings reported here are disease-related or stress-related. Subgroup analyses also suggested an influence of medication and co-morbidity on the pathophysiology of PTSD, which will need to be verified by further study.Finally, it must be acknowledged that differences observed between PTSD and controls during resting-state fMRI are still not easily interpreted, because diverse interpersonal differences  may influence the neuroimaging result. Transcending this limitation will require innovative methodological approaches. This is a developing field and our results should be considered provisional.A systematic search strategy was used to select studies published between January 1995 and April 2015. A combination search strategy of Mesh terms and text words was conducted in PubMed, Web of Knowledge, Embase and Science Direct, China National Knowledge Infrastructure (CNKI), National Technical Information Service, and System for Information on Grey Literature. The terms used were as follows: ALFF <or> ReHo <or> rCBF <or> rCMRglu <or> ASL <or> amplitude of low frequency fluctuations <or> low frequency fluctuations <or> regional homogeneity <or> regional cerebral perfusion <or> regional cerebral metabolic <or> arterial spin labeling; PTSD <or> posttraumatic stress disorder; baseline <or> resting-state <or> rest <or> resting. We assessed all search results for potential suitability. The abstracts were all in English; articles in Chinese were translated into English for assessment. Studies were selected according to the following inclusion criteria: 1) the study had used at least one of the functional imaging techniques of fMRI, PET, or SPECT to analyze altered spontaneous brain activity in patients with PTSD; 2) the study included comparison of PTSD patients with NTC or TEC; 3) 3-dimensional coordinates in stereotactic space of the activation areas were clearly reported. Studies were excluded if they were case reports, reported only region of interest (ROI) findings or used seed-voxel-based analysis procedures, if the participants were not classified using current diagnostic criteria for PTSD, if the data contributed to another publication (in which case the publication with the largest group size was selected), or if the data, when added in, tipped the balance into significant heterogeneity (p < 0.005). The reference lists of the identified articles were searched for additional studies. Two authors (T.W and J.L) independently conducted the literature search. The results were compared, any inconsistent result was discussed, and a consensus decision was reached.Individual study quality was assessed using a 10-point checklist, which focused on the clinical and demographic aspects of the study samples and the imaging methodology see . The che71http://www.sdmproject.com/software)7374www.sdmproject.com/utilities/?show=Statistics). For studies not reporting any measure related to effect size , we write a “p” for positive peaks (i.e. patients > controls) and an “n” for negative peaks (i.e. patients < controls), according to the SDM tutorials. Second, peak coordinates and their t-values were used to recreate a standard Montreal Neurological Institute (MNI) map of the differences for each study by means of a non-normalized Gaussian kernel, which assigns higher effect sizes to the voxels more correlated with peaks. In the assignment, a relatively large full-width at half-maximum  was used to control false positive resultsPapers were divided into two based upon the nature of the control group, TEC or NTC . Two individual meta-analyses were performed comparing PTSD with TEC and with NTC. Additional subgroup meta-analyses were performed to control for comorbidity and medication. The meta-analyses were performed using ES-SDM  in the observed range of values of the variable. The main output for each variable was a map of the regression slopeHow to cite this article: Wang, T. et al. Altered resting-state functional activity in posttraumatic stress disorder: A quantitative meta-analysis. Sci. Rep.6, 27131; doi: 10.1038/srep27131 (2016).


The small interfering RNA (siRNA) is suggested to offer a novel means of treating atopic dermatitis (AD) because it allows the specific silencing of genes related to AD pathogenesis. In our previous study, we found that siRNA targeted against RelA, an important nuclear factor-kappa B (NF-κB) subdomain, with functional peptides, showed therapeutic effects in a mouse model of AD. In the present study, to develop a topical skin application against AD, we prepared a hydrogel containing anti-RelA siRNA and functional peptides and determined the intradermal permeation and the anti-AD effects in an AD mouse model. We selected the silk protein, sericin (SC), which is a versatile biocompatible biomaterial to prepare hydrogel as an aqueous gel base. We found that the siRNA was more widely delivered to the site of application in AD-induced ear skin of mice after topical application via the hydrogel containing functional peptides than via the preparation without functional peptides. In addition, the ear thickness and clinical skin severity of the AD-induced mice treated with hydrogel containing anti-RelA siRNA with functional peptides improved more than that of mice treated with the preparation formulated with negative siRNA. Atopic dermatitis (AD) is a chronic inflammatory disease of the skin with a complex pathogenesis that is associated with both genetic and environmental factors . In its The use of topical corticosteroids in combination with aggressive moisturizing is the most common and effective treatment for AD. However, the use of high-strength corticosteroids combined with an occlusive dressing can cause side effects, such as an increase in the risk of skin thinning. In addition, the long-term use of steroids can result in complications such as weight gain, reduced bone density, and suppression of the immune system .Nuclear factor-kappa B (NF-κB) plays a critical role in the immunological disturbances observed in AD, and NF-κB decoy oligodeoxynucleotides (ODNs) have been reported to ameliorate atopic skin lesions in NC/Nga mice with fewer side effects than corticosteroids ,4,5.Small interfering RNA (siRNA) can specifically silence genes related to AD pathogenesis through a process called RNA interference (RNAi). Therefore, it is also expected to offer a potential means of treating AD without causing side effects. In our previous study, we reported several findings on the effects of treating AD with topical siRNAs targeted against RelA, which is the most important NF-κB subdomain involved in the production of inflammatory cytokines, as shown in the AD-induced mouse model . We alsoTopical applications of therapeutic peptides, proteins, and nucleic acids, such as plasmid DNA and siRNA, have been increasingly studied because of the medical importance of treating skin diseases, topical vaccination, and improving skin properties ,9. HowevIn the present study, we develop a hydrogel formulated with sericin (SC), which contained anti-RelA siRNAs and functional peptides, as a topical application for use against AD. We previously reported the silk protein SC as a useful biocompatible biomaterial for the formation of hydrogels without cross-linkages, as an aqueous gel base ,26. TherThe 6-carboxyfluorescein-aminohexyl (FAM)-siRNA, which served as a fluorescence-labeled siRNA, , anti-RelA siRNA , and negative control siRNA  were obtained from Cosmo Bio Co., Ltd., . The TAT peptide , and AT1002 peptide  were obtained from BEX Co., Ltd., .ad libitum. All experiments with animals were carried out in accordance with a protocol approved by the Animal Care and Ethics Committee of the Tokyo University of Pharmacy and Life Sciences .Six-week-old female ICR mice were purchased from SLC  and 6-week-old male NC/Nga mice were purchased from Charles River Laboratories Japan, Inc. . The mice were housed under controlled conditions of 22–24 °C temperature, 40%–60% humidity, and 12-h light/dark cycle with the light period starting at 8:00 a.m. Food and water were supplied g for 5 min to remove the insoluble material, and then cooled to room temperature. The 1% SC solution was kept at 5 °C for 1 day. Then, GL was added to the SC hydrogel as a plasticizer (v/v = 1/1), to improve its tensibility.The siRNA/Tat complexes with a nitrogen to phosphorous (N/P) ratio of 10 were prepared by mixing both solutions for 30 min at room temperature. The siRNA/Tat + AT1002 were prepared by mixing the AT1002 and siRNA/Tat complex solutions before use. SC Hope  was used as the source of intact SC. Glycerin  was used as the plasticizer in the SC hydrogel. To prepare the SC hydrogel, 1% SC was dissolved in purified water at 100 °C for 20 min. Because the SC solutions contain some insoluble SC and other particulates, each solution was centrifuged at 1000 2 cross-sections of skin tissue at the application site. The skin tissue samples were soaked in Tissue Mount  at 4 °C overnight in the dark, mounted with Tissue Mount in cold acetone, and 20-μm frozen sections were cut using a Cryostat HM550  and fixed in cold acetone. Slides were washed three times with PBS, and then the tissue sections were with Fluoroment-G. To examine the permeability of the siRNAs in the mouse skin, FAM-siRNA were observed using confocal laser microscopy .The 6-week-old male ICR mice used in this study were anesthetized with intraperitoneal pentobarbital (50 mg/kg), and their backs were shaved clean using an electric clipper followed by a cream-based hair remover . Then, the backs of the mice were tape-stripped 20 times with surgical tape  and 40 μL of FAM-siRNA containing SC hydrogels  were applied to the backs of the mice. After 10 h, the mice were euthanized, and then the dermal tissue was washed with phosphate-buffered saline (PBS) and resected in 1-cmAD-like skin was induced in the left ears of 6-week-old NC/Nga mice. First, the mice were sensitized by topically applying 2,4-dinitrofluorobenzene  dissolved in acetone to their footpads and shaved backs on day 0 and 3. After the first sensitization exposure, DNFB dissolved in acetone/olive oil was applied to their left ears on day 7 and 10.The AD-induced mice were used in this experiment on day 9 after the first sensitization. Then, the ears were not tape-stripped. All the mice were anesthetized with intraperitoneal pentobarbital (50 mg/kg). The FAM-siRNA-containing SC hydrogels (40 μL of SC gel containing naked FAM-siRNA or FAM-siRNA/Tat + AT1002) were applied to both sides of the left ear lobes of the mice. After 10 h, the mice were euthanized, and then the ear lobes were washed with PBS and resected. The tissue samples were then processed identically to the tape-stripped mouse skin in n = 4 per group) were treated topically with 50 μL of siRelA/Tat + AT1002 without SC hydrogel or SC hydrogels containing naked siRelA, siControl/Tat + AT1002, or siRelA/Tat + AT1002  three times a week for 2 weeks. The left ears were covered with olive oil after every application while olive oil alone was applied to the untreated control mice ears. The ear thickness and clinical skin severity score were periodically evaluated throughout the study. The mice were not evaluated blindly. Ear thickness was measured using a digital thickness gauge  and skin lesions were examined macroscopically. The clinical score was determined by assessing the individual clinical severity of the five symptoms  and assigning scores from 0 to 3 (Maximum score: 15). Then, on day 15, 20-μm frozen sections of left ear lobe tissue samples of the AD mice were prepared and stained with hematoxylin and eosin (H&E) or toluidine blue (TB) and stained sections were observed using microscopy.The AD-like left ears of mice . The statistical analysis of the data was performed using analysis of variance (ANOVA) followed by Dunnett’s test. Statistical significance was defined as The distribution of the FAM-siRNA in the mouse epidermis following the application of SC hydrogels containing naked FAM-siRNA, FAM-siRNA/Tat, or FAM-siRNA/Tat + AT1002 was determined. As shown in We also examined whether siRNA could penetrate the AD-like ear skin following topical application of the FAM-siRNA/Tat+AT1002 SC hydrogel on day 9 after the first sensitization . No fluoWe examined whether SC hydrogels containing anti-RelA siRNA (siRelA) had a therapeutic effect on the clinical symptoms of AD. Several siRelA formulations were applied three times a week for two weeks from day 3 of the sensitization. The ear thickness and total clinical score of dermatitis in the AD-induced NC/Nga mice are shown in Finally, the tissue sections of left earlobes of NC/Nga mice were examined histologically with H&E and TB staining on day 15 . H&E staAs shown In the present study, we found that the siRNA was more widely delivered to the site of application in AD-induced ear skin of mice after topical application via the hydrogel with functional peptides than via the preparation without functional peptides. In addition, anti-AD effects in the AD-induced mice treated with hydrogel containing siRelA combined with functional peptides strongly improved by the additive effects of silencing effect by the siRelA/Tat + AT1002, and moisturizing, antioxidant, and UV-resistant effects of the SC hydrogel. In general, the systemic side effects with weakened immune systems by the long-term reduction of NF-κB reduction are important issue. However, the systemic side effects could not be occurred in this study, because siRelA was administered at the just topical inflammatory site. These findings indicate that SC hydrogel containing siRNA/Tat + AT1002 has the potential for application as a versatile and efficacious topical hydrogel formulation, not only for use in AD, but also numerous other skin diseases.


Kaposi’s sarcoma (KS)-associated herpesvirus (KSHV) is an oncogenic human herpesvirus. KSHV is associated with three cancers in the human population: KS, primary effusion lymphoma (PEL), and multicentric Castleman’s disease (MCD). KS is the leading cause of cancer in HIV-infected individuals. In this review, we discuss the most recent discoveries behind the mechanisms of KSHV latency maintenance and lytic replication. We also review current therapies for KSHV-associated cancers. Subsequent studies further identified KSHV as the etiologic agent of primary effusion lymphoma (PEL)2 and the B-cell hyperplasia known as multicentric Castleman’s disease (MCD)3. KSHV is also linked to two under-studied inflammatory syndromes. One KSHV inflammatory disease recognized, immune reconstitution inflammatory syndrome-KS (IRIS-KS), is the paradoxical rapid development of KS after the start of highly active antiretroviral therapy (HAART) for HIV and during the rebound of CD4+ T-cells5. Uldricket al. discovered another inflammatory disease, termed KSHV inflammatory cytokine syndrome (KICS), in patients infected with both HIV and KSHV with high levels of viral interleukin-6 (vIL-6), human IL-6 (hIL-6), and KSHV viral loads6. Subsequent to this initial report, KICS has also been found to affect non-HIV-infected KSHV-positive individuals7.Kaposi’s sarcoma (KS)-associated herpesvirus (KSHV), also known as human herpesvirus 8 (HHV-8), is a linear double-stranded DNA virus and a member of the gammaherpesvirus subfamily. The virus was first isolated by Chang9. Following primary infection, both latent and lytic genes are expressed, but after several rounds of replication, lytic gene expression decreases and latency is established. Latency is the default program of the virus10. During latency establishment, the linear KSHV genome circularizes to become an episome. This latent form of KSHV expresses only a few proteins, including latency-associated nuclear antigen (LANA), viral FADD-like interleukin-1-β-converting enzyme (FLICE/caspase 8)-inhibitory protein (vFLIP), vCyclin, and multiple microRNAs11. Additional genes that are expressed at low levels during latency include K1, vIL-612, and K1513. The expression of LANA is sufficient and necessary to establish latency, as it plays a pivotal role in episome maintenance and latent replication. Two LANA proteins form a dimer and the N-termini bind to the host chromosomes while the C-termini interact with LANA-binding sites (LBSs) in the KSHV episome14. Recently, three labs have crystalized the C-terminus of LANA and found that the LANA dimers oligomerize, forming a higher order of organization that facilitates the binding of DNA18. It was also discovered that LANA contains positively charged patches opposite to the DNA-binding face. Mutations of these residues did not alter LANA’s DNA binding capabilities but diminished the interaction with cellular chromatin bromodomain (BRD) proteins, which play a role in latent replication and maintenance19.KSHV, like other herpesviruses, has a latent and lytic phase to its lifecycle20. As the transcription of IE genes does not require prior viral protein synthesis, IE genes are experimentally defined by their transcription in the presence of inhibitors of protein synthesis such as cycloheximide. DE gene expression can be inhibited by cycloheximide because they require proteins encoded by IE genes to transactivate their promoters but are also not dependent on DNA replication. Late genes are expressed subsequent to the start of viral DNA replication and encode for structural proteins required for assembling new virions as well as envelope glycoproteins. Viral replication inhibitors  can prevent the production of infectious progeny virions.Lytic replication is divided into three phases of gene expression: immediate early (IE), delayed early (DE), and late8. However, it has recently been proposed that KSHV can be reactivated and enter lytic replication in a RTA-independent manner22. In this pathway, KSHV reactivation is induced by cellular apoptosis and is dependent on the activation of caspase 3. It is interesting to note that the virions produced though this RTA-independent lytic pathway appear to be less infectious than virions produced through RTA-dependent lytic replication21. This observation needs to be furthered expanded upon in the future.Latent KSHV can be induced into lytic replication with the addition of chemicals such as 12-O-tetradecanoylphorbol-13-acetate (TPA), valproic acid (VPA), and sodium butyrate. These chemicals activate the expression of the IE gene replication and transcription activator (RTA), encoded by ORF50, which is the key regulator of KSHV lytic replication as its ectopic expression is sufficient to start the lytic cascadeKSHV is a pathogenic virus whose mechanism of disease is not fully understood. It is clear that both the latent and lytic phases of the KSHV lifecycle play a role in virus-related disease and a better understanding of these phases can help guide the development of treatments. This review covers recent advances in understanding the latent/lytic switch and discusses current and potential future therapeutic treatments for KSHV-related malignancies.23. The KSHV episome is packaged in chromatin and several labs have shown that in actively transcribed latency regions, the chromatin is in an open configuration, lacks nucleosomes, and exhibits active histone marks while lytic genes are packaged in closed chromatin  have been shown to repress TPA-induced reactivation through epigenetic changes32. Liet al. examined the effect of class III HDACs, known as sirtuins (SIRTs), and found that they also repress reactivation through epigenetic changes33. SIRT1 is able to inhibit lytic replication through its ability to bind to RTA and inhibit its transactivation activity33. In fact, inhibition of SIRT1 was sufficient to induce lytic replication33. Dillonet al. reported that the knockdown of another family of histone-modifying enzymes, the tousled-like kinases (TLKs), resulted in loss of latency and reactivation of the virus34. This was due to a decrease in inhibitory phospho-histone H3 associated with the RTA promoter.How latent KSHV reactivates and efficiently makes new progeny virus is a complex process that requires not only viral but also cellular proteins. To maintain latency, it is important that latent genes are expressed while lytic gene transcription is repressedromatin 24–30. 37. Caiet al. reported that this interaction is strengthened by poly-SUMO-2-ylation of KAP1 so it can bind to a LANA SUMO-2 interacting motif37. LANA and KAP1 form a complex with another cellular protein named nuclear factor E2-related factor 2 (Nrf2)38, which targets the RTA promoter and allows for LANA-KAP1 to inhibit RTA expression, thereby repressing lytic replication  is a chromatin remodeler, and several groups have shown that it also interacts with LANAication 35,36.39. Using the HSP90 inhibitors 17-dimethylaminoethylamino-17-demethoxygeldana-mycin (17-DMAG) and AUY922, Chenet al. disrupted this interaction and found it led to degradation of LANA39. It was also observed that these inhibitors along with a third HSP90 inhibitor (PU-H71) caused apoptosis of PEL cell lines. Another group also reported an increase in apoptosis of PEL cell lines treated with PU-H7140. K1 is another viral protein involved in preventing apoptosis that was found to interact with HSP9041. When cells expressing K1 were treated with HSP90 inhibitors, it was discovered that K1 expression was decreased and K1’s anti-apoptotic effect was diminished. These studies show the important role of HSP90 in maintaining latency through stability of LANA39 and inhibition of apoptosis41.Heat shock protein 90 (HSP90) is a cellular chaperone protein that interacts with the N-terminus of LANA42. Recently, it has been found that sustained activation of ERK-RSK leads to the phosphorylation and accumulation of c-Fos, which binds to KSHV promoters43. This accumulation of active c-Fos allows for efficient late lytic gene expression, as shown by a knockdown of c-Fos resulting in a decrease of ORF64 lytic gene expression and the fact that a non-functional form of c-Fos resulted in decreased virion production. Fuet al. also examined ORF45 activation of ERK-RSK signaling and discovered that amino acids 56–70 of ORF45 are critical for its interaction with RSK44. In fact, a single amino acid mutation of ORF45 at F66 can disrupt its ability to activate RSK, which leads to decreased late lytic gene expression and reduction of new virus. It was also shown that reactive oxygen species (ROS) can induce KSHV reactivation from latency45 and that induction of the KSHV lytic cycle further upregulates ROS, which can be targeted with N-acetyl-L-cysteine (NAC) to inhibit the development of KS46.Once KSHV is reactivated, it is important for efficient completion of the lytic cycle to make infectious viral progeny. Though RTA is the driver of reactivation, completion of the lytic cycle requires cellular proteins. The KSHV IE/DE protein ORF45 has been shown to activate cellular kinases in the ERK-RSK pathway, and inhibition of this leads to reduced lytic replicationet al. have demonstrated that upon reactivation, early lytic gene expression activates DDR kinases47, as does primary infection48. When inhibitors of ATM and ATR were added to cell culture, it was found that the virus could reactivate and enter lytic replication, but late gene expression was diminished, resulting in fewer infectious viral progeny being made47.Another pathway shown to be important for late lytic replication is the DNA damage response (DDR) pathway. Hollingworth9. Some researchers have hypothesized that the induction of lytic replication would be a good therapy for KSHV cancers if used in combination with a lytic inhibitor such as ganciclovir. By initiating reactivation but not allowing full lytic replication, more immunodominant targets could be produced that would be recognized by the immune system and provide more druggable targets to kill infected cells50.Most KSHV-infected cells harbor the latent form of the virus. In the case of KS and PEL, most tumor cells are latent with only a few cells exhibiting lytic gene expression. In MCD, a larger proportion of the tumor mass displays lytic gene expression. Lytic replication is thought to be required to promote the growth of KSHV-associated cancers and help spread the virus. In most cases, the high proportion of cells undergoing abortive lytic replication express lytic proteins involved in the activation of angiogenesis and signal transduction, and complete viral replication does not occur51. In this study, patients were treated with high-dose zidovudine along with valganciclovir. The KSHV kinases ORF36 and ORF21 phosphorylated these compounds, making them toxic to the cell. Overall, 86% of treated patients obtained a major clinical response and 50% obtained a major biochemical response as determined by improvements in clinical parameters such as hemoglobin, albumin, and C-reactive protein levels. The 5-year survival rate reported in this study was 86%51. Another report showed thatin vitro treatment of KSHV-infected cells with the HIV protease drug nelfinavir resulted in less infectious KSHV virus being produced52.In 2011, a pilot study was published in support of induction therapy in the treatment of KSHV-related MCDet al. screened 650 US Food and Drug Administration (FDA)-approved drugs in anin vitro assay53. This screen identified three topoisomerase II inhibitors  as strong inducers of viral reactivation and that daunorubicin was even more powerful than the classic inducer, sodium butyrate. These three drugs were able to cause apoptosis through DNA intercalation, but the virus produced was capable of infecting new cells. Hence, if these inducers were to be used in patients, it would require their use in combination with a viral replication inhibitor.In a search for effective inducers of lytic replication, Kang56. SIRT1 function is also linked to promoting increased fatty acid synthesis, and, as previously stated, inhibition of SIRT1 leads to increased lytic replication33. Bhattet al. showed that inhibition of fatty acid synthase (FASN) with a drug, C75, led to cellular apoptosis by activation of caspase 3 54. Moreover, KSHV-latent endothelial cells go through caspase 3/7-induced apoptosis when glutaminolysis is inhibited57. Daiet al. have also demonstrated that by inhibiting sphingosine kinase 2 and sphingolipid metabolism, PEL cells build up ceramides in the cell that result in lytic replication and apoptosis58. Furthermore, Leunget al. also demonstrated that clinically achievable amounts of the glucose metabolic analog 2-Deoxy-D-glucose (2-DG) induce endoplasmic reticulum stress and inhibit KSHV replication and reactivation from latency59. These data suggest that new therapeutics targeting metabolic pathways in KSHV cancer cells should also be explored.Several groups have shown that latency is linked to a dysregulated metabolic state of the cell with increased fatty acid synthesiset al. recently reported that latent KSHV cells have increased glutamate secretion and metabotropic glutamate receptor 1 expression60. Inhibitors of glutamate secretion and receptor expression in KS and PEL cells were found to decrease cellular proliferation. Another study has demonstrated the ability of the drug celecoxib to suppress RTA expression and viral production by blocking the activation of the p38 MAPK pathway61. Another key pathway that has been targeted is the PI3K/Akt/mTOR pathway. Sinet al. demonstrated that the use of the mTOR inhibitor rapamycin was capable of inhibiting PEL tumor growth by reducing cytokine secretion and autocrine signaling62. Since then, more reports have come out showing other inhibitors of this pathway have similar effects65. It is interesting to note that not only does rapamycin inhibit tumor growth but it is also capable of inhibiting viral reactivation by repressing RTA expression through transcriptional and post-transcriptional mechanisms66.Other modes of therapies for KSHV-associated cancers have also been reported. Valiya Veettil68 through the upregulation of membrane-type 1 matrix metalloproteinase (MT1-MMP)67 and the transcription factors Snail, Slug, Twist, ZEB1, and ZEB268. This EndMT event allows for the KSHV-infected cell to invade other tissue, an important aspect for the development of KS, and this knowledge of Notch signaling and KSHV provides new molecular targets for therapy.Another pathway, the Notch signaling pathway, which is activated by KSHV, has recently been reported to cause endothelial-to-mesenchymal transition (EndMT)KSHV is a double-stranded DNA oncogenic herpesvirus. After infection, the virus goes latent and expresses only a few proteins and microRNAs. This latent virus can be reactivated and enter the lytic cycle through either cellular stress or chemical induction that alters the epigenetics of the cell. During the complete lytic cycle, the virus expresses its genes in a temporal fashion and produces new, infectious virus particles that ultimately kill the cell.69. To inhibit late lytic replication, classical drugs such as valganciclovir can be used. Other possibilities include inducing RTA-independent lytic replication, possibly by targeting metabolic processes, where a significant decrease in viral progeny is observed. Continued advances in this field will provide additional insights into the biology and pathogenesis of KSHV infection as well as better treatments and cures for KSHV-related cancers.Even though the lytic cycle is important for pathogenesis, the vast majority of cells in KSHV malignancies harbor latent virus. Viral induction therapy is a promising method to treat KSHV-related diseases. It is important, however, to create a balance between efficient reactivation of latent cells and controlling the spread of infection through the use of combination therapies involving lytic replication inhibitors. This method of treatment has the potential to induce immunodominant viral proteins, cause apoptosis of the cell, and inhibit the production of structural proteins so new virions cannot be made. Future experiments should explore new combinations of KSHV-reactivating drugs and late lytic cycle inhibitors. Some potential inducers to be used in these experiments include the anthracyclines and HSP90 inhibitors described above as well as the growing number of SIRT1 inhibitorsDDR, DNA damage response; DE, delayed early; EndMT, endothelial-to-mesenchymal transition; HDAC, histone deacetylase; HSP90, Heat shock protein 90; IE, immediate early; KAP1, Krüppel-associated box domain-associated protein 1; KICS, KSHV inflammatory cytokine syndrome; KSHV, Kaposi’s sarcoma-associated herpesvirus; LANA, latency-associated nuclear antigen; MCD, multicentric Castleman’s disease; PEL, primary effusion lymphoma; RTA, replication and transcription activator; ROS, reactive oxygen species; SIRT, sirtuin; TLK, tousled-like kinase; TPA, 12-O-tetradecanoylphorbol-13-acetate; vIL-6, viral interleukin-6.


Trachomatous trichiasis (TT) needs to be managed to reduce the risk of vision loss. The long-term impact of epilation  in preventing visual impairment and corneal opacity from TT is unknown. We conducted a randomized controlled trial of epilation versus surgery for the management of minor TT (fewer than six lashes touching the eye) in Ethiopia. Here we report the four-year outcome and the effect on vision and corneal opacity.1300 individuals with minor TT were recruited and randomly assigned to quality trichiasis surgery or repeated epilation using high quality epilation forceps by a trained person with good near vision. Participants were examined six-monthly for two-years, and then at four-years after randomisation. At two-years all epilation arm participants were offered free surgery. At four-years 1151 (88.5%) were re-examined: 572 (88%) and 579 (89%) from epilation and surgery arms, respectively. At that time, 21.1% of the surgery arm participants had recurrent TT; 189/572 (33%) of the epilation arm had received surgery, while 383 (67%) declined surgery and had continued epilating (“epilation-only”). Among the epilation-only group, 207 (54.1%) fully controlled their TT, 166 (43.3%) had minor TT and 10 (2.6%) had major TT . There were no differences between participants in the epilation-only, epilation-to-surgery and surgery arm participants in changes in visual acuity and corneal opacity between baseline and four-years.Most minor TT participants randomised to the epilation arm continued epilating and controlled their TT. Change in vision and corneal opacity was comparable between surgery and epilation-only participants. This suggests that good quality epilation with regular follow-up is a reasonable second-line alternative to surgery for minor TT for individuals who either decline surgery or do not have immediate access to surgical treatment. Trachoma causes visual impairment through the effect of in-turned eyelashes (trichiasis) on the surface of the eye. Epilation is a common traditional practice of intermittent plucking of lashes touching the eye, however, its long-term effectiveness in preventing visual impairment is unknown. We conducted a randomized controlled trial of epilation versus eyelid surgery (the main treatment option) in 1300 people with mild trichiasis in Ethiopia. We defined mild trichiasis as fewer than six lashes touching the eye. We have previously reported results to two years and have now re-assessed these individuals at four years. Overall, we found no difference between the epilation and surgery groups in terms of change in vision and corneal opacity between baseline and four years. Most mild trichiasis participants randomised to the epilation arm continued epilating and controlled their trichiasis. This suggests that good quality epilation is a reasonable second-line alternative to surgery for mild trichiasis for individuals who either decline surgery or do not have immediate access to surgical treatment. Chlamydia trachomatis infection and inflammation in which the upper eyelid is distorted and rolled inwards (entropion) and the eyelashes turn towards the eye [Trachoma is the leading infectious cause of blindness worldwide . Trachom the eye . Trachom the eye –6. The c the eye . Some la the eye ,8. Globa the eye . UpdatedEyelid surgery is performed to correct the anatomical abnormality, in the expectation that this reduces the risk of sight loss ,9. The WDespite considerable efforts to scale-up surgery programmes only around 150,000 people per year have been reported as treated surgically in recent years worldwide . It has Many individuals with trichiasis, particularly those with mild disease, decline surgery, even when this is provided free and close to home –16. LackEpilation is a widespread traditional practice in many trachoma endemic societies, with up to 70% of people with trichiasis using this treatment strategy ,6,13,21.This study was approved by the National Health Research Ethics Review Committee (NHRERC) of the Ethiopian Ministry of Science and Technology, the London School of Hygiene and Tropical Medicine Ethics Committee and Emory University Institutional Review Board. Potential participants were provided with both written and oral information in Amharic about the trial. For those agreeing to participate, written informed consent in Amharic was required prior to enrolment. If the participant was unable to read and write, the information sheet and consent form were read to them and their consent recoded by witnessed thumbprint. The detailed Trial Protocol is described in S1 Text and the CONSORT statement in Text S2 of the report of the two-year results .The trial methods and results up to two-years have been published previously . BrieflyParticipants were followed-up at 6, 12, 18 and 24 months and re-assessed using the same protocol. Participants who showed evidence of disease progression during the follow-up period, defined as five or more lashes touching the eye or corneal changes related to observed lashes, were immediately offered primary surgery (epilation arm) or repeat surgery (surgery arm) to be performed by a senior surgeon. New epilating forceps were provided for epilation arm participants as required. Individuals with other ophthalmic pathology (e.g. cataract) were referred to the regional ophthalmic service in Bahirdar. At the end of the trial at two-years all epilation arm participants were offered free trichiasis surgery in the community. Some individuals accepted this, but the majority chose to continue epilating.About four years after enrolment participants were invited for a follow-up assessment (March to August 2012). They were notified by a letter sent out through the village administration teams, which explained the purpose, date and place of follow-up assessment. People not able to come to the health facilities for assessment were assessed in their homes. Reasons for loss to follow-up were identified and documented. Participants were interviewed in Amharic about their vision, ocular symptoms, epilation forceps retention and history of epilation and/or surgery since the two-year follow-up. Individuals were considered to be “frequent epilators” if they performed epilation at least once in two months. Participants enrolled into the epilation arm were asked about their views on epilation and epilation practices.Unaided and pinhole LogMAR visual acuities were measured at 4 metres. Ophthalmic examinations were conducted in the same manner as the previous follow-ups by a single observer (EH) who had also conducted the 6 and 18-month follow-ups. Grades of trichiasis, entropion, and corneal opacity were documented and the eyes were photographed. The examiner was masked to the intervention allocation. The treatment allocation code had been previously broken for the two year analysis. Recurrent trichiasis was defined as one or more lashes touching the eye or evidence of epilation or repeat surgery. Clinical evidence of epilation was identified by the presence of broken or newly growing lashes, or areas of absent lashes. Change in corneal opacity was assessed by direct comparison of the baseline and four-year cornea photographs. Photographs were viewed on a computer screen at about 10x magnification by a single masked ophthalmologist (MJB). These were graded as improved, no change, or worse.2 tests. The Wilcoxon rank-sum test was used to test for significant differences in number of lashes. Logistic regression was used to assess factors associated with trichiasis progression within the epilation-only group, to identify predictors of surgery uptake in all epilation arm participants and corneal opacity deterioration in all study participants. Ordinal logistic regression was used to assess factors associated with reduction in visual acuity by four-years in all study participants. Variables that were associated with the outcome on univariable analyses at a level of p<0.05 were retained in the multivariable logistic regression models.Patients initially randomised to epilation were sub-divided according to whether or not they had surgery during the four-year follow-up period. These are subsequently referred to as epilation-to-surgery and epilation-only groups, respectively. Baseline and four-year follow-up demographic and clinical characteristics, and the change in clinical phenotypes during follow-up were compared between the surgery-only, epilation-only and epilation-to-surgery participants using XAt baseline, 1300 individuals were recruited, of whom 650 were randomised to immediate surgery and 650 to epilation . The basThere were 149 participants who were not re-examined at four-years. The reasons for not being re-examined are shown in Amongst those re-examined at four-years, there were more female participants randomised to surgery compared to those randomisation to epilation (p = 0.03), however, there was no difference in age or literacy status . BaselinThe 383 epilation arm participants who were still only epilating at four-years (epilation-only) were slightly older than both the 579 surgery arm and 189 epilation-to-surgery participants  of the 579 participants randomised to surgery. Of these 122, 61 (50.0%) were practicing epilation at four years. Twenty-one (3.6%) had undergone repeat surgery during the four years. Among the 189 epilation-to-surgery participants, 42 (22.2%) had failed surgery (recurrent trichiasis), of whom 27 (64.2%) were epilating.At four-years, among the 383 epilation-only participants, 207 (54.1%) were successfully epilating (had no lashes touching the eye), 166 (43.3%) had minor trichiasis (<6 lashes) and 10 (2.6%) had major trichiasis (>5 lashes). Overall, the epilation-only group had more trichiasis, entropion and lid margin conjunctivalisation than either the surgery arm or the epilation-to-surgery group .Changes in clinical phenotype between baseline and four-years are shown in At four-years the surgery arm and epilation-to-surgery participants had slightly better LogMAR visual acuity than the epilation-only group . HoweverOverall, few individuals had a change in corneal opacification, determined by the comparison of baseline and four-year photographs . There wAmong the epilation-only group 259 (67.6%) were “frequent epilators” (at least once in two months) between the two and four-year follow-ups. They were asked about their experience: 185 (72%) reported “no problem”, 37 (14.3%) did not always find the trained epilators when needed, 17 (6.6%) had found epilation uncomfortable, the trained epilators of 9 (3.5%) reported difficulty epilating, and 7 (2.7%) had found people unwilling to epilate them. Among the 124 who were not frequently epilating, 119 (96%) did not have a specific reason for not epilating other than not needing to; the other five had nobody to perform epilation. Epilation frequency was not associated either with age (p = 0.31) or gender (p = 0.60). Compared to the “infrequent epilators”, the “frequent epilators” had a slightly higher lash burden at baseline , but lower lash burden at four-years .The epilation-only group were asked if they had tried to obtain trichiasis surgery at any time between the two and four-year follow-up: 352 (92%) replied “Never” and 325 (85%) reported that they were happy epilating. There was no statistically significant difference in the average lash burden at four-years between those who were happy epilating and those that were not . Participants who were not happy epilating were more likely to have tried to obtain surgical treatment for their trichiasis between two and four-year follow-ups .At the two-year follow-up, 589 / 603 (98%) of epilation arm participants still had their epilation forceps. At the four-year follow-up, 351 / 383 (92%) of the epilation-only group had retained at least one pair of epilation forceps. Females were more likely to have retained their forceps than males . At four-years, new forceps were provided to those who had lost their forceps, and did not want surgery.Univariate and multivariable associations with having surgery in epilation arm participants at any point during the four years of follow-up are shown in At the four-year follow-up, all participants with recurrent trichiasis in the surgery arm and all participants in the epilation arm who had not previously had surgery were offered free surgery: only 17 / 383 (4.4%) of the epilation only participants accepted surgery, the remaining 366 (95.6%) preferred to continue epilating.Trachomatous trichiasis has a wide disease spectrum, with many individuals having relatively few lashes touching the eye ,7. This In this study, surgery was better than epilation at correcting entropion and controlling trichiasis. However, it should be noted that at four-years 76.2% of the epilation-only participants had mild or no entropion, and 63.4% had no entropion progression. The epilation-only group generally controlled their trichiasis well by epilation, with only a few showing signs of significant progression. At four-years 2.6% of this group had major trichiasis (>5 lashes), which is low compared to the Gambian longitudinal study, in which 37% of the eyes progressed from minor to major trichiasis over four years . HoweverThe proportion of participants in the surgery arm with recurrent trichiasis at four-years was relatively low compared to other trials and longitudinal studies ,23,24. TThe epilation-only group had poorer baseline visual acuity compared to both the surgical arm participants and the epilation-to-surgery group. However, there was no difference in visual acuity change (baseline to four-years) between the epilating and surgery groups, which is similar to what we reported at two-years . SeveralThe epilation arm as a whole and the epilation-only sub-group had more baseline corneal opacity than the surgery arm participants. However, there was no significant difference in change of corneal opacity between the different groups at four-years. This result is consistent with our findings at two-years and a report from a longitudinal study in The Gambia, which compared change in corneal opacity in individuals with minor trichiasis who had undergone surgery with those who had declined surgery and practiced epilation . In farmMore than two-thirds of the epilation-only participants reported frequent epilation. Most reported no problems. Difficulty of getting the trained epilator when they needed help was cited as the main problem, encountered by 14%. However, this could be addressed by training more than one family member. The high retention rate of forceps in this study is encouraging, suggesting that the forceps are valued.This study has a number of limitations. This four-year follow-up and analysis was not pre-specified in the original trial protocol, which covered the period up to the two-year follow-up. The study ceased to have a fully randomised treatment allocation at two-years when all the epilation arm participants were offered free surgery, and hence we have adjusted follow-up outcomes for baseline imbalances. Those not examined at four-years were older and had worse baseline presenting visual acuity than those seen at four-years. This could have underestimated change in vision over time as older age is associated with greater visual impairment. However, this is unlikely to introduce bias in vision change between the surgery arm and epilation only group as those lost to follow-up were equally distributed between these groups.We found that surgery was more effective for controlling trichiasis than epilation; however there was no difference in change in visual acuity and corneal opacity. The progression of minor trichiasis can be effectively mitigated with frequent epilation. We found low rates of surgery uptake among people with mild disease, even with free community-based surgery. There is a need for clear guidelines on how programmes should manage patients with a few non-entropic lashes who refuse surgery. Trichiasis in general and particularly major trichiasis warrants surgical treatment. However, the results of this study and the reality of low surgical uptake in many regions, suggest that good quality epilation, in the context of regular follow-up by a service that can provide surgery if subsequently needed, is a reasonable second-line alternative to surgery for minor trichiasis for individuals who either decline surgery or do not have immediate access to surgical treatment.


The mortality rate of cerebellar hemorrhage (CH) is generally higher than other types of intracranial hemorrhage. Recently, the increased survey rate of CH has come from improved clinical imaging and earlier surgical intervention. Hence, the predictors of intermittent- (1 to 6 months) and long-term (> 6months) mortality are clinically practical use for educational and therapeutic decisions. Unfortunately, the factors predictive mortality within six-month had not yet been systematically investigated.Seventy-two patients with acute spontaneous CH were retrospectively analyzed. The patients were divided into the six-month mortality group  and survival group . The independent predictors of six-month mortality were investigated by multivariate Cox proportional hazards regression.The radiological brainstem compression  was independent predictor of mortality within six-month after CH onset. The median onset time of six-month mortality was 5 days in patients with brainstem compression (p < 0.001) and the hazard ratios for the onset time was 13.1 compared with those without brainstem compression .We report the first study that radiological brainstem compression predicted the mortality within six-month after onset of CH. Patients with radiological brainstem compression were about 23 times more likely to die within 6 months after CH than those without radiological brainstem compression. Cerebellar hemorrhage (CH) is the least common type of intracranial hemorrhage (ICH), accounting for 10% of all ICH cases in western countries . In receThis retrospective study was approved by the Institutional Review Board (IRB)/Ethics Committee of Tri-Service General Hospital, National Defense Medical Center, Taiwan (No. 100-05-104). Between August 2004 to May 2012, 72 consecutive patients, diagnosed of acute spontaneous CH without prior disability and admitted to our hospital within 48 hours following onset, were collected, reviewed, de-identified by the authors, and analyzed anonymously. The authors had access to the patients’ records prior to data anonymization, and the IRB/Ethics Committee waived the need for informed consent. None of the patients had a prior stroke or complications associated with another hemorrhagic lesion or hemorrhagic transformation of ischemic stroke.On admission, all patients underwent detailed physical examinations, routine laboratory tests, and imaging. The initial neurological state was evaluated by the GCS. The patients, blood sugar (BS), systolic blood pressure (SBP), diastolic blood pressure (DBP), heart rate (HR), and body temperature (BT) were measured immediately before they underwent computer tomography. Basic characteristics, including age, gender, date of death, and pre-existing or concomitant illnesses, were collected. Coagulopathy were defined by an international normalized ratio > 1.4, partial thromboplastin time > 35, and platelet count > 100,000 . Image fIn our center, patients with hematoma < 3cm in diameter, and no hydrocephalus or brainstem compression were candidates for conservative treatments. Otherwise, surgical decompression consist of craniotomy and evacuation of hematoma were performed immediately in patients with neurologic deterioration or brainstem compression and/or hydrocephalus from ventricular obstruction (except patients who had a large hemorrhage or coma status without brainstem reflexes due to its predictably poor outcome) . All theStatistical analyses were performed with SPSS 13.0 for Windows. To determine the independent predictors of mortality within six-month, univariate and multivariate Cox proportional hazards regression models were used. The cut-off points of continuous variables for predicting mortality within six-month were based on previously published cut-off values: age 65 , BS leveThe baseline characteristics of the study population and the potential factors affecting six-month mortality were summarized in Tables The results from the univariate and multivariate forward Cox regression analyses for six-month mortality were shown in Median onset time of six-month mortality was 5 days in the radiological brainstem compression group (p < 0.001) and the HRs for the onset time of six-month mortality was 13.1 compared with patients without brainstem compression  . The KapIdentifying effective predictors of intermediate-term mortality in patients with CH is important. This study showed that radiological brainstem compression (HRs = 23.5) was a strong independent predictor of mortality within six-month in patients with spontaneous CH. Patients with radiological brainstem compression were about 23 times more likely to die within 6 months after CH than those without radiological brainstem compression.Brainstem compression by cerebellar hematoma is a specific and severe complication of CH and may contribute to neurological deterioration, impaired consciousness, and death . Indeed,Moreover, radiological brainstem compression was associated with unfavorable long-term outcomes and mortality , 7. HoweOnly four studies have investigated predictive factors of intermediate- and long-term mortality or outcomes after CH onset –8 Table. Louis eA few published studies investigating patients with intracranial hemorrhage (ICH) mention predictors of intermediate- and long-term mortality can be used for reference. However no or very few cases of CH were included in these studies because of the relatively low prevalence. These studies were summarized in Reviewing the above mentioned studies, the increasing age was the most common predictor of long-term mortality of ICH. However, age 65 years was not predictive of six-month mortality in the current study. In addition to a higher prevalence of radiological brainstem compression in CH, the relatively younger mean age (65.2 ± 16.1 years) of the patients in this study might contribute to the difference in findings. In general, the condition of the patient and complications associated with increasing age may have larger effects on long-term mortality, especially in older patients.The results of this study must be viewed in light of its limitations. First, the case number is relatively small because of the low incidence of CH compared with other types of ICH despite an 8-year period collection. Second, it might be slightly subjective to define radiological brainstem compression on the basis of imaging findings alone and without brainstem-evoked potentials or electroencephalography; however, it is difficult to perform them in such an emergent condition. Third, no information was available regarding medications prescribed during the follow-up period, such as anticoagulant agents. The inability to exclude the effects of these differences may have affected outcome results. However, hypertension is the major cause of CH and is reported to account for 60–89% of all cases. The likelihood of anticoagulant medications being prescribed to patients with hemorrhagic stroke was very low compared to patients with ischemic stroke. Therefore, the confounding resulting from side effects of anticoagulant agents, such as recurrent hemorrhage, was unlikely in this study. Last, the bias caused by the single-center retrospective hospital-based analysis posed limitations in generalization. Although a prospective design would be better, it would take many years to collect an adequate number. Further prospective analysis is need in the future.Radiological brainstem compression was a strong predictive factor for mortality within six-month in patients with CH and patients with radiological brainstem compression were about 23 times more likely to die within 6 months after CH than those without radiological brainstem compression. These findings may facilitate the identification of predictive factors for intermediate-term mortality after CH, thereby helping clinicians and patients.


Response to cardiac resynchronization therapy (CRT) is often assessed six months after implantation. Our objective was to assess the number of patients changing from responder to non-responder between six and 14 months, so-called late non-responders, and compare them to patients who were responder both at six and 14 months, so-called stable responders. Furthermore, we assessed predictive values of six and 14-month response concerning clinical outcome.105 patients eligible for CRT were enrolled. Clinical, laboratory, ECG, and echocardiographic parameters and patient-reported health status (Kansas City Cardiomyopathy Questionnaire [KCCQ]) were assessed before, and six and 14 months after implantation. Response was defined as ≥15% LVESV decrease as compared to baseline. Major adverse cardiac events (MACE) were registered until 24 months after implantation. Predictive values of six and 14-month response for MACE were examined.In total, 75 (71%) patients were six-month responders of which 12 (16%) patients became late non-responder. At baseline, late non-responders more often had ischemic cardiomyopathy and atrial fibrillation, higher BNP and less dyssynchrony compared to stable responders. At six months, late non-responders showed significantly less LVESV decrease, and higher creatinine levels. Mean KCCQ scores of late non-responders were lower than those of stable responders at every time point, with the difference being significant at 14 months. The 14 months response was a better predictor of MACE than six months response.The assessment of treatment outcomes after six months of CRT could be premature and response rates beyond might better correlate to long-term clinical outcome. Cardiac resynchronization therapy (CRT) is an established treatment for patients with congestive heart failure (CHF) and a wide QRS complex. A commonAt present, many studies and clinicians evaluate CRT response within six months after device implantation and focus on pre-implantation factors predicting this response. However, limited data are available concerning the prevalence and predictors of long-term changes in response to CRT. Therefore, in the current study we assessed the number and characteristics of patients whose response at 14 months differed from their response at six months. Our main focus was on late non-responders as we hypothesize that these might have a worse prognosis than (late and stable) responders and should therefore be identified. Hence, we also examined the correlation of 14 months response with health outcomes, including patient-reported health status and major adverse cardiac events (MACE).This was a prospective, single center study designed to study the influence of PSYchological factors on health outcomes in HEART failure patients treated with CRT (PSYHEART-CRT). Patients eligible to CRT, according to applicable guidelines and evidence-based medicine at time of inclusion, were enrolled between January 2009 and August 2011 at the University Medical Center Utrecht (UMCU).The study was conducted in accordance with the Declaration of Helsinki, and the study protocol was approved by the local Medical Ethics Committee of the UMCU (protocol number 08–246) and patients signed informed consent. A more extensive description has been published previously.[Echocardiographic studies were performed prior to implantation (baseline), and six and 14 months after device implantation. Data were acquired using Philips IE 33  or Vivid 7  ultrasound machines. Apart from speckle tracking analysis, echocardiographic parameters were assessed offline using Xcelera software (R3.3L1). Speckle tracking was performed for studies on the Vivid 7 and analyzed using EchoPac software . Volumes and other measurements were assessed by one observer and in accordance with the guidelines of the American Society of Echocardiography (ASE) and European Association of Echocardiography (EAE).[Mitral regurgitation at baseline and after six months was visually assessed and extracted from echocardiographic records.LVESV was assessed by Simpsons’ biplane method. Volume changes were assessed between baseline and six months FU, baseline and 14 months FU and between six and 14 months FU.Response to CRT was defined as relative decrease in LVESV of ≥15%, which has been shown to predict clinical outcome up to five years after CRT implantation. Non-respDoppler flows over the pulmonary and aortic valve were recorded and time from Q to onset of flow was assessed for both valves. IntervenΔIVMD was assessed between baseline and six months FU.sept) was evaluated using speckle tracking by evaluation of longitudinal septal strain, as previously described.[sept was assessed between baseline and six months FU.Systolic rebound stretch of the septum  to assess CHF-specific health status. The KCCQMACE cases were defined as hospitalization due to heart failure, LVAD implantation, heart transplantation or death due to heart failure. Assessment took place for up to 24 months after CRT implantation.Statistical analysis was performed using SPSS version 20.0 . Continuous variables are presented as mean with standard deviation (SD) when normally distributed and as median with interquartile range (IQR) in case of non-normal distribution. Categorical variables are presented as numbers and percentages. Differences at baseline and at six months FU between stable responders and late non-responders were assessed. Categorical variables were compared using Pearson’s Chi-square and continuous variables were assessed using students T-tests or Mann-Whitney U, as appropriate. Related samples of continuous variables were assessed using students T-tests or Friedman’s two-way analysis of variance by ranks test. Related samples of categorical variables were assessed with McNemar. Furthermore, the correlation between response rates, at six and 14 months, and MACE was assessed and compared with Pearson’s Chi-square and net reclassification index (NRI). The NRI A sensitivity analysis was performed for a subsample thereby excluding patients with atrial fibrillation (AF) as this is associated with reduced CRT response. –15Of 139 patients that consented to participate in the study, 12 lacked a baseline echocardiographic study, 11 cases had insufficient image quality, nine were lost to follow-up, two died of non-cardiac cause.Six months after CRT implantation 71% (n = 75) patients were responders and 29% (n = 30) were non-responders. Of these responders 84% (n = 63) were stable responders and 16% (n = 12) became late non-responders, as shown in At baseline, six and 14 months FU, 15 patients demonstrated AF at least during one assessment. Of these, three had permanent AF, six had persistent AF (for which one a His-ablation was perfomed), and three had paroxysmal AF. Three patients had AF solely at baseline, and device interrogation did not show AF anymore during follow-up. If all patients with AF were neglected, crossover from response to late non-response still occurred in 12% of the six-month responders.Baseline characteristics of the 75 six-month responders, stratified by stable responders and late non-responders, are shown in sept, and higher B-type natriuretic peptide (BNP) levels compared to stable responders.As shown in If patients with AF at baseline were excluded from the analysis, baseline differences between late non-responders and stable responders did not change, except for RV peak systolic velocity which appeared to be significantly lower in non-responders.An overview of the six-month FU data of stable responders and late non-responders is provided in sept which did not show a significant difference anymore between late non-responders and stable responders.If patients with AF at six months were disregarded in the analyses, six-month results showed only minor changes. Differences between late non-responders and stable responders did not differ, except for pacing percentage, ΔLVEF and SRSIn total, 91% (68/75) of the six-month responders completed the KCCQ three times; at baseline, and at six and 14-month FU. At each assessment, the nine late non-responders reported a lower mean health status score than the 59 stable responders . This difference was statistically significant at 14 months FU only. In addition, the stable responders reported significantly increased KCCQ scores from baseline to six months FU (p<0.001), while this increase did not occur for the late non-responder group.Of the total population, 24% (26/105) suffered a MACE within two years after CRT implantation. For six-month responders, late non-responders and stable responders the prevalence of patients suffering a MACE were: 19% (14/75), 58% (7/12) and 11% (7/63), respectively. The main finding of this study was that 16% of the six-month responders turned into non-responders after 14 months of CRT. Furthermore, we found that 14-months response rates correlated significantly better with patient-reported health status and occurrence of MACE compared to six months response rates, indicating that the change from responder to non-responder has important consequences for prognosis.sept was used to define mechanical dyssynchrony. Our center previously demonstrated that this parameter is a good predictor of volumetric response to CRT as well as clinical outcome.[sept had important additional value for the identification of CRT responders. CRT aims for the correction of dyssynchrony, thereby improving ventricular functioning and reducing heart failure symptoms. Consequently, patients with underlying dyssynchrony are more likely to respond to CRT.[electrical dyssynchrony. Nevertheless, our study did not show significant differences concerning QRS duration or the presence of LBBB between stable responders and late non-responders; implicating that according to current guidelines they were equally suitable to receive a CRT device and a priori would have similar chances of becoming a responder. Finally, a relatively high share of late non-responders suffered from AF as compared with stable responders. AF is associated with reduced CRT response; however the mechanism remains unclear as AF could be the result of more advanced heart failure, whereas, at the same time, it can reduce biventricular capture.[Although late non-responders and stable responders were both eligible to CRT according to the guidelines, significant differences between these groups were already present prior to implantation. Pre-implantation BNP was lower in stable responders, whereas volumes did not differ significantly between late non-responders and stable responders. Lower BNP levels are associated with more reverse remodeling and better prognosis, as high BNP indicates high wall stress associated with dilated myocardium.,17 In ad outcome.,20 In ad outcome. recentlyd to CRT.,21 Howev capture. Both adv capture.,15Late non-responders demonstrated significantly lower volume reductions and BNP did not reduce during the first six months of CRT. This indicates that they had less benefit from CRT compared with stable responders. It has been demonstrated that less reverse remodeling is correlated with an increase in MACE. MoreoverLate non-responders showed larger average left atrial volumes, more often demonstrated AF and pacing percentages were significantly lower at six months, the three of which could very well be correlated. In general, patients with AF showed a significantly lower median pacing percentage; 90% vs 99%. At 14 months, late non-responders still more often demonstrated AF than stable responders , whereas median pacing percentages no longer differed significantly; 98% vs 99%, (p = 0.34). Consequently, difference in pacing percentages at six months probably may not contribute to the response conversion in late non-responders, as this improved thereafter. This might implicate a merely modest role for AF concerning the occurrence of late non-response. Especially considering the fact that crossover from response to non-response still occurred in 12% when AF patients were not taken into account. However, the difference in AF burden complicates the interpretation of the influence of AF on late non-response. In addition, the results of this subanalysis have to be interpreted with even more caution as without AF patients sample size is compressed even further.Moreover, despite lower volume reductions, a lack of BNP reduction, higher creatinine levels, higher frequency of AF, and lower pacing percentages, the late non-responders did show relevant reverse remodeling at six months.At baseline, and six and 14 months FU, late non-responders reported lower health status than stable responders, but the difference was significant at 14 months only. This finding suggests that the correlation between volume response and patient-reported health status increases after six months of CRT. At 14 moIn daily practice the effect of CRT is usually assessed six months after device implantation. However, the high number of late non-responders found in our study indicates that the long term effect of CRT is not yet visible after six months, leading to premature and possibly incorrect conclusions about patients’ response to treatment. Patients should be monitored closely after six months of CRT. Our recommendation would be to repeat ECG, echocardiography, laboratory measurements, and health status reports beyond the first year after CRT implantation, in order to be able to consider other interventions in case of deterioration, thereby improving prognosis and preventing early MACE.This is a single-center study in a real-world setting with its inherent limitations. Twelve patients lacked a baseline echocardiographic study and 11 patients had insufficient image quality. In addition, the study is underpowered for multivariable analysis; however, the main focus of this paper was to assess the prevalence of late non-responders. Nevertheless, we would encourage investigating these findings in a larger cohort, in order to confirm our results and to investigate the independent determinants of late (non)response. Moreover, the amount of biventricular pacing was estimated based on the pacing percentage provided by the device. Furthermore, during AF, biventricular pacing could be overestimated because of pseudo-fusion between intrinsic conduction and pacing. Finally, our follow-up period was 14 months, as this was the time point patients came into the clinic for their regular check-up. Yet, as previously addressed, it has been shown that reverse remodeling can continue even thereafter; until 24 months after CRT implantation. ThereforWe demonstrated that 16% of six-month volume responders changes into non-responders after more than 1 year of CRT. Furthermore, 14-month response had a stronger correlation with health outcomes  than six-month response, indicating that the crossover from responder to non-responder represents a relevant change in patients’ health. This knowledge is essential for daily clinical practice as well as for future research projects on CRT as it indicates that the assessment of treatment outcomes after six months of CRT might be premature.


P = 0.037), which suggests that ADAM-10 regulates immunoresponse in melanoma initiation and progression. No statistically significant correlations were found between ADAM-10 expression in primary tumor cells and nodal metastases and other histopathological parameters analyzed. Decreased immunoreactivity of ADAM-10 in cancer cells from regional lymph nodes was correlated with worse prognosis; however this correlation was statistically nonsignificant (P = 0.065). Review of the literature shows that our study is the first one ever to describe the significance of ADAM-10 expression in correlation with detailed histopathological parameters of the primary tumor and data on long-term survival of cutaneous melanoma patients.ADAM-10  is a protein belonging to a superfamily of metalloproteases, enzymes capable of degrading the extracellular matrix. ADAMs have also been shown to be primarily involved in ectodomain cleavage. The aim of the study was to assess the expression and intracellular location of ADAM-10 in 104 primary skin melanomas and 16 metastatic lesions from regional lymph nodes. Also, prognostic significance of ADAM-10 expression in primary tumor cells and metastatic lesion cells was evaluated during 5-year observation. It was revealed that high expression of ADAM-10 positive cells was strictly related with lower intensity of tumor-infiltrating lymphocytes ( ADAM-10  is a protein belonging to a superfamily of metalloproteases, enzymes capable of degrading the extracellular matrix. ADAMs have also been shown to be primarily involved in ectodomain cleavage [ADAM family proteins are membrane anchored glycoproteins which play a major role in a number of cytobiochemical processes such as proteolysis, intercellular adhesion, and activation of various signaling cascades , 3. The γ-secretase, intracellular domain (ICD) fragments are formed, which upon translocation to the nucleus modulate several signal transduction pathways and become either transcriptional activators or repressors [α and epidermal growth factor receptors [In a normal cell, catalytically active ADAM-10 protein functions as an enzyme shedding extracellular fragments of membrane-bound proteins (shedding), which leads to the release of active protein ectodomains. ADAM-10 also participates in the biochemical process referred to as regulated intramembrane proteolysis (RIP), during which, and with mediation by presenilin-dependent pressors , 6. A wieceptors .The aim of the study was to assess the expression and intracellular location of ADAM-10 in 104 primary skin melanomas and 16 metastatic lesions from regional lymph nodes. Also, prognostic significance of ADAM-10 expression in primary tumor cells and metastatic lesion cells was evaluated during 5-year observation. Correlations were also analyzed between ADAM-10 immunoreactivity parameters and detailed clinicopathological parameters such as Breslow thickness and Clark level, presence of nodal and distant metastases, recurrence of primary tumor, mitotic rate, ulceration, lymphocytic inflammatory infiltration, regression, and microsatellitosis.The study group consisted of 104 patients with CMM, who were diagnosed between 2005 and 2010 and treated in the Lower Silesian Oncology Center in Wroclaw, Poland. Additionally, tissue material obtained from 16 nodal metastatic foci was included in the study. The group was selected on the basis of tissue material (paraffin blocks and histopathology slides) and the availability of medical documentation. Comprehensive clinical data were obtained from archival medical records. The diagnostic and therapeutic procedures utilized were determined from medical records in the Oncology Outpatient Clinic of the Lower Silesian Oncology Center and data provided by the Lower Silesian Cancer Registry and Civil Register Office. The retrospective study was approved by the Ethical Committee of the Wroclaw Medical University, Poland.Patients enrolled in the study were treated by then-available methods. Excisional biopsy of the primary lesion was performed. Once cutaneous melanoma was diagnosed in histopathological examination, the primary procedure was extended by excising the scar with a margin of 5, 10, or 20 mm of unaffected skin depending on Breslow thickness and primary tumor location, if any. If nodal metastases (cN0) were not clinically manifested and Breslow thickness was above 1 mm (>pT1a), sentinel lymph node biopsy (SNLB) was performed. When metastases were observed in the regional lymph nodes , lymphadenectomy was performed.Clinicopathological profile of patients included the following parameters: age and gender, primary tumor location, tumor stratification according to AJCC (pT), presence or absence of nodal (pN) and distant (pM) metastases, information on disease recurrence, and sentinel lymph node biopsy (SLNB) procedures .2), presence of ulceration, lymphangio invasion, microsatellitosis, intensity of lymphocytic inflammatory infiltrate (TILs: tumor-infiltrating lymphocytes), and microscopic evidence of regression . The sections were mounted on superfrost slides , dewaxed with xylene, and gradually hydrated. The activity of endogenous peroxidase was blocked by 5-minute exposure to 3% H2O2. The sections were boiled for 15 minutes at 250 W in Antigen Retrieval Solution . Immunohistochemical reactions were then performed using polyclonal antibody detecting ADAM-10 , N-cadherin , and SPARC . The tested sections were incubated with antibodies for 1 hour at room temperature. The subsequent incubations involved biotinylated antibodies (15 minutes at room temperature) and streptavidin-biotinylated peroxidase complex (15 minutes at room temperature) . DAB  was used as a chromogen (10 minutes at room temperature). All sections were counterstained with Meyer's hematoxylin. In every case control reactions were performed, with the relevant antibody substituted by Primary Mouse Negative Control .Formalin-fixed, paraffin embedded tissue was freshly cut  and the reaction intensity. The final immunohistochemical reaction results are expressed according to the semiquantitative IRS (ImmunoReactive Score) scale devised by Remmele and Stegner . This scχ2 test, exact Fisher test in the case of 2 × 2 tables, and Spearman rank correlation were used to analyze associations between mitotic rate and the presence of ulceration and clinicopathological parameters. Differences between the means were tested with a nonparametric test ; the log-rank test was used to compare survival in two groups, the overall survival rate was estimated by the Kaplan-Meier method, and the influence of explanatory variables on death risk was analyzed by means of the Cox proportional hazard regression. P values < 0.05 were considered statistically significant.Statistical analysis was performed using the Statistica 10.0 and IBM SPSS 21 software packages. Overall survival (OS) was defined as the time between the primary surgical treatment and death, and OS was censored at last follow-up for patients who were still alive. A ADAM-10 expression defined as IRS > 0 was found in 82 primary tumors (78.8% of patients ). No ADAP = 0.037), which suggests that ADAM-10 regulates immunoresponse in melanoma initiation and progression. No statistically significant correlations were found between ADAM-10 expression in primary tumor cells and nodal metastases and other histopathological parameters analyzed . Interestingly, lower ADAM-10 expression in cancer cells from nodal metastases was also correlated with older age at diagnosis (P = 0.037)  .P = 0.088). Other clinical parameters such as pT, pN, recurrence, sex, and location of the primary tumor did not show any significant correlations with ADAM-10 expression . Other parameters of ADAM-10 expression in primary tumor and nodal metastasis cells did not have a statistically significant impact on the prognosis in cutaneous melanoma patients in the analyzed group of patients.Decreased immunoreactivity of ADAM-10 in cancer cells from regional lymph nodes was correlated with worse prognosis; however this correlation was statistically nonsignificant  revealed only two articles that describe immunohistochemical analysis of ADAM-10 expression in cutaneous melanoma , 9.Lee et al.  analyzedSimilarly to our results, Lee et al.  did not P = 0.04), which is not compatible with our results. Interestingly, our statistical analysis showed statistically nonsignificant trend of increased ADAM-10 immunoexpression in cancer cells from the primary tumor and the presence of distant metastasis (P = 0.088). Other clinical parameters such as pT, pN, sex, or location of the primary tumor were not shown to be significantly correlated with ADAM-10 expression.Lee et al.  showed tP = 0.065).It must be stressed that in our study it was only ADAM-10 immunoreactivity in metastases that had prognostic value. Analysis of Kaplan-Meier curves showed that decreased expression of ADAM-10 in cancer cells from regional lymph node metastases was correlated with worse prognosis; however this correlation was statistically nonsignificant . The possible meaning of this correlation is unclear and needs further studies, yet it suggests that ADAM-10 is involved in the regulation of immunological response in the initiation and progression of melanoma. Neither of the research teams described in the quoted papers analyzed or correlated ADAM-10 expression parameters with the details of the histopathology report [P = 0.029) was reduced with increasing age at diagnosis and that decreased expression of ADAM-10 in cancer cells from nodal metastases was also correlated with more advanced age at diagnosis (P = 0.037). Lee et al. [An innovative feature of our study was the finding that decreased intensity of tumor-infiltrating lymphocytes (TILs) was strictly correlated with the increased number of ADAM-10 positive cancer cells in the primary tumor (y report , 9. Furte et al.  and AndeTo sum up, it must be pointed out that, in the two papers referred to above by Lee et al.  and Ande