Dual induction of PKR with E2F-1 and IFN-alpha to enhance gene therapy against hepatocellular carcinoma

Overexpression of the transcription factor E2F-1 induces apoptosis in tumor cells. This apoptotic effect is partly mediated through the induction of the double-stranded RNA-activated protein kinase (PKR). Here, we investigate if agents that upregulate PKR could enhance the apoptotic effect of E2F-1 overexpression in liver tumors. In human hepatocellular carcinoma (HCC) cells (Hep3B, HepG2, Huh7), adenovirus-mediated overexpression of E2F-1 (AdCMV-E2F) transcriptionally increased PKR mRNA. The subsequent increase of total and phosphorylated PKR protein was followed by induction of apoptosis. When AdCMV-E2F was combined with the PKR modifier interferon alpha (IFNalpha), PKR was additionally upregulated and both PKR activation and apoptosis were increased. Subcutaneous xenograft tumors were selectively targeted using an adenoviral vector expressing E2F-1 under the control of the human telomerase reverse transcriptase (hTERT) promoter (AdhTERT-E2F). Weekly systemic administration of AdhTERT-E2F inhibited tumor growth. The tumor suppressive effect of AdhTERT-E2F therapy was further enhanced in combination with IFNalpha.Our results demonstrate that PKR activating agents enhance the anti-tumor effect of E2F-1 overexpression in HCC in-vitro and in-vivo. Hence, modulation of PKR is a potential strategy to increase the efficacy of PKR-dependent anti-tumor therapies.

Effective treatment of advanced colorectal cancer by rapamycin and 5-FU/oxaliplatin monitored by TIMP-1

AIM: The mTOR-inhibitor rapamycin has shown antitumor activity in various tumors. Bedside observations have suggested that rapamycin may be effective as a treatment for colorectal carcinomatosis. METHODS: We established an orthotopic syngenic model by transplanting CT26 peritoneal tumors in Balb/C mice and an orthotopic xenograft model by transplanting SW620 peritoneal tumors in nu/nu mice. Expression levels of tissue inhibitor of matrix-metalloproteinases 1 (TIMP-1) in the tumor and serum was determined by enzyme-linked immunosorbent assay. RESULTS: Rapamycin significantly suppressed growth of syngenic and xenografted peritoneal tumors. The effect was similar with intraperitoneal or oral rapamycin administration. Tumor suppression was further enhanced when rapamycin was combined with 5-fluorouracil and/or oxaliplatin. The combination treatment showed no acute toxicity. TIMP-1 serum levels correlated well (CC = 0.75; P < 0.01) with rapamycin treatment. CONCLUSIONS: Rapamycin suppressed advanced stage colorectal cancer, even with oral administration. Combining rapamycin with current chemotherapy regimens significantly increased antitumor efficacy without apparent toxicity. The treatment efficacy correlated with serum TIMP-1 levels, suggesting its potential as a surrogate marker in future clinical trials.

The stem-loop binding protein stimulates histone translation at an early step in the initiation pathway

Metazoan replication-dependent histone mRNAs do not have a poly(A) tail but end instead in a conserved stem-loop structure. Efficient translation of these mRNAs is dependent on the stem-loop binding protein (SLBP). Here we explore the mechanism by which SLBP stimulates translation in vertebrate cells, using the tethered function assay and analyzing protein-protein interactions. We show for the first time that translational stimulation by SLBP increases during oocyte maturation and that SLBP stimulates translation at the level of initiation. We demonstrate that SLBP can interact directly with subunit h of eIF3 and with Paip1; however, neither of these interactions is sufficient to mediate its effects on translation. We find that Xenopus SLBP1 functions primarily at an early stage in the cap-dependent initiation pathway, targeting small ribosomal subunit recruitment. Analysis of IRES-driven translation in Xenopus oocytes suggests that SLBP activity requires eIF4E. We propose a model in which a novel factor contacts eIF4E bound to the 5' cap and SLBP bound to the 3' end simultaneously, mediating formation of an alternative end-to-end complex.

Characterization and Clinical Relevance of ALDHbright Populations in Prostate Cancer

PURPOSE High aldehyde dehydrogenase (ALDH) has been suggested to selectively mark cells with high tumorigenic potential in established prostate cancer cell lines. However, the existence of cells with high ALDH activity (ALDH(bright)) in primary prostate cancer specimens has not been shown so far. We investigated the presence, phenotype, and clinical significance of ALDH(bright) populations in clinical prostate cancer specimens. EXPERIMENTAL DESIGN We used ALDEFLUOR technology and fluorescence-activated cell-sorting (FACS) staining to identify and characterize ALDH(bright) populations in cells freshly isolated from clinical prostate cancer specimens. Expression of genes encoding ALDH-specific isoforms was evaluated by quantitative real-time PCR in normal prostate, benign prostatic hyperplasia (BPH), and prostate cancer tissues. ALDH1A1-specific expression and prognostic significance were assessed by staining two tissue microarrays that included more than 500 samples of BPH, prostatic intraepithelial neoplasia (PIN), and multistage prostate cancer. RESULTS ALDH(bright) cells were detectable in freshly excised prostate cancer specimens (n = 39) and were mainly included within the EpCAM((+)) and Trop2((+)) cell populations. Although several ALDH isoforms were expressed to high extents in prostate cancer, only ALDH1A1 gene expression significantly correlated with ALDH activity (P < 0.01) and was increased in cancers with high Gleason scores (P = 0.03). Most importantly, ALDH1A1 protein was expressed significantly more frequently and at higher levels in advanced-stage than in low-stage prostate cancer and BPH. Notably, ALDH1A1 positivity was associated with poor survival (P = 0.02) in hormone-naïve patients. CONCLUSIONS Our data indicate that ALDH contributes to the identification of subsets of prostate cancer cells of potentially high clinical relevance.

Marked increase of the astrocytic marker S100B in the cerebrospinal fluid of HIV-infected patients on LPV/r-monotherapy

Objective: To determine changes of cerebrospinal fluid (CSF) biomarkers of patients on monotherapy with lopinavir/ritonavir. Design: The Monotherapy Switzerland/Thailand study (MOST) trial compared monotherapy with ritonavir-boosted lopinavir with continued therapy. The trial was prematurely stopped due to virological failure in six patients on monotherapy. It, thus, offers a unique opportunity to assess brain markers in the early stage of HIV virological escape. Methods: Sixty-five CSF samples (34 on continued therapy and 31 on monotherapy) from 49 HIV-positive patients enrolled in MOST. Using enzyme-linked immunosorbent assay, we determined the CSF concentration of S100B (astrocytosis), neopterin (inflammation), total Tau (tTau), phosphorylated Tau (pTau), and amyloid-β 1–42 (Aβ), the latter three indicating neuronal damage. Controls were CSF samples of 29 HIV-negative patients with Alzheimer dementia. Results: In the CSF of monotherapy, concentrations of S100B and neopterin were significantly higher than in continued therapy (P = 0.006 and P = 0.013, respectively) and Alzheimer dementia patients (P < 0.0001 and P = 0.0005, respectively). In Alzheimer dementia, concentration of Aβ was lower than in monotherapy (P = 0.005) and continued therapy (P = 0.016) and concentrations of tTau were higher than in monotherapy (P = 0.019) and continued therapy (P = 0.001). There was no difference in pTau among the three groups. After removal of the 16 CSF with detectable viral load in the blood and/or CSF, only S100B remained significantly higher in monotherapy than in the two other groups. Conclusion: Despite full viral load-suppression in blood and CSF, antiretroviral monotherapy with lopinavir/ritonavir can raise CSF levels of S100B, suggesting astrocytic damage.

Factors affecting screening for hepatocellular carcinoma

Background and aim. Hepatocellular carcinoma (HCC) is a frequent cancer. Its prognosis is highly dependent on early diagnosis. Patients at risk for developing HCC should be enrolled in a surveillance programme. Nevertheless, many patients at risk are not regularly screened. We aimed at exploring the characteristics that affect enrolment in a surveillance programme. Material and methods. The characteristics of the patients included in the prospective Bern HCC cohort between August 2010 and August 2011 were analysed according to their participation in a surveillance programme. Results. Among the 82 patients included in the cohort during this period of time, 48 were in a surveillance program before the diagnosis of HCC. Thirty five percent of cirrhotic patients were not screened. Age, sex, level of education, Child-Pugh status and MELD score were similar between the patients who were screened and those who were not screened. Patients with a private insurance and patients treated by a liver specialist were more frequently enrolled in a surveillance program. Sixty seven percent of the screened patients were eligible for curative treatment whereas only 15% of the non-screened patients were. Conclusions. In conclusion the surveillance of patients at risk for developing HCC increases their chances to be diagnosed at an early stage to allow curative treatment. More than one third of cirrhotic patients were not regularly screened. Patients with chronic liver disease should be referred to identify those at risk and enrol them in a surveillance program.

Shying away: An observer-based microprocess analysis of experiential avoidance and resource activation in outpatients with generalized anxiety spectrum disorder

Despite long-standing calls for patient-focused research on individuals with generalized anxiety spectrum disorder there is little systematized knowledge about the in-session behaviors of these patients. The primary objective of this study was to describe of in-session trajectories of the patients' level of explication (as an indicator of an elaborated exposure of negative emotionality) and the patients' focus on their own resources and how these trajectories are associated with post-treatment outcome. In respect to GAD patients, a high level of explication might be seen as an indicator of successful exposure of avoided negative emotionality during therapy sessions. Observers made minute-by-minute ratings of 1100 minutes of video of 20 patients-therapists dyads. The results indicated that a higher level of explication generally observed at a later stage during the therapy sessions and the patients' focus on competencies at an early stage was highly associated with positive therapy outcome at assessment at post treatment, independent of pretreatment distress, rapid response of well-being and symptom reduction, as well as the therapists' professional experience and therapy lengths. These results will be discussed under the perspective of emotion regulation of patients and therapist's counterregulation. It is assumed that GAD-Patients are especially skilled in masking difficult emotions. Explication level and emotion regulation are important variables for this patient group but there's relation to outcome is different.

Delivery of health care at the end of life in cancer patients of four swiss cantons a retrospective database study (SAKK 89/09)

Background The use of cancer related therapy in cancer patients at the end-of-life has increased over time in many countries. Given a lack of published Swiss data, the objective of this study was to describe delivery of health care during the last month before death of cancer patients. Methods Claims data were used to assess health care utilization of cancer patients (identified by cancer registry data of four participating cantons), deceased between 2006-2008. Primary endpoints were hospitalization rate and delivery of cancer related therapies during the last 30 days before death. Multivariate logistic regression assessed the explanatory value of patient and geographic characteristics. Results 3809 identified cancer patients were included. Hospitalization rate (mean 68.5%, 95%CI 67.0-69.9) and percentage of patients receiving anti-cancer drug therapies (ACDT, mean 14.5%, 95%CI 13.4-15.6) and radiotherapy (mean 7.7%, 95%CI 6.7-8.4) decreased with age. Canton of residence and insurance type status most significantly influenced the odds for hospitalization or receiving ACDT. Conclusions The intensity of cancer specific care showed substantial variation by age, cancer type, place of residence and insurance type status. This may be partially driven by cultural differences within Switzerland and the cantonal organization of the Swiss health care system.

Early versions in medieval textual traditions. Wolfram’s Parzival as a test case

Wolfram von Eschenbach’s novel Parzival is a courtly romance composed in German language shortly after 1200. In a project, based at the University of Bern, a new critical edition of the poem is prepared in electronic and printed form. It visualizes parallel textual versions, which, depending on particular circumstances of oral performance, have developed in the early stage of the poem’s transmission. Philological research as well as phylogenetic techniques common in the natural sciences, e.g. in molecular biology, have been used to demonstrate the existence of these early textual versions. The article shows how both methods work and how they are applied to the ongoing edition. Exemplary passages to be presented include the text of some rare fragments written in the first decades of the 13th century, which might even go back to the author’s lifetime and which allow to date the existence of the versions they belong to.

The cyclotron laboratory and the RFQ accelerator in Bern

Two proton accelerators have been recently put in operation in Bern: an 18 MeV cyclotron and a 2 MeV RFQ linac. The commercial IBA 18/18 cyclotron, equipped with a specifically conceived 6 m long external beam line ending in a separate bunker, will provide beams for routine 18-F and other PET radioisotope production as well as for novel detector, radiation biophysics, radioprotection, radiochemistry and radiopharmacy developments. The accelerator is embedded into a complex building hosting two physics laboratories and four Good Manufacturing Practice (GMP) laboratories. This project is the result of a successful collaboration between the Inselspital, the University of Bern and private investors, aiming at the constitution of a combined medical and research centre able to provide the most cutting-edge technologies in medical imaging and cancer radiation therapy. The cyclotron is complemented by the RFQ with the primary goals of elemental analysis via Particle Induced Gamma Emission (PIGE), and the detection of potentially dangerous materials with high nitrogen content using the Gamma-Resonant Nuclear Absorption (GRNA) technique. In this context, beam instrumentation devices have been developed, in particular an innovative beam profile monitor based on doped silica fibres and a setup for emittance measurements using the pepper-pot technique. On this basis, the establishment of a proton therapy centre on the campus of the Inselspital is in the phase of advanced study.

Oral syphilis: a series of 5 cases.

Syphilis is an infectious, usually sexually transmitted, disease caused by Treponema pallidum, subspecies pallidum. Because of the increasing prevalence in Europe during the past few years, dentists could be confronted with patients with oral manifestations of syphilis. Because oral lesions are highly contagious, it is vital to make the correct diagnosis quickly to initiate the proper therapy and to interrupt the chain of infection. We present the cases of 5 patients with syphilis-related oral lesions. These cases are representative because of their clinical presentation, age, and gender distribution and the diagnostic approach. The aim of the present report is to emphasize the importance of the dentist knowing and identifying syphilis in different stages to diagnose the disease and institute treatment at an early stage.

Transcriptional profiles of cytokine/chemokine factors of immune cell-homing to the parasitic lesions: a comprehensive one-year course study in the liver of E. multilocularis-infected mice.

Pathogenesis of chronically developing alveolar echinococcosis (AE) is characterized by a continuous, granulomatous, periparasitic infiltration of immune cells surrounding the metacestode of Echinococcus multilocularis (E.multilocularis) in the affected liver. A detailed cytokine and chemokine profile analysis of the periparasitic infiltrate in the liver has, however, not yet been carried out in a comprehensive way all along the whole course of infection in E. multilocularis intermediate hosts. We thus assessed the hepatic gene expression profiles of 18 selected cytokine and chemokine genes using qRT-PCR in the periparasitic immune reaction and the subsequent adjacent, not directly affected, liver tissue of mice from day 2 to day 360 post intra-hepatic injection of metacestode. DNA microarray analysis was also used to get a more complete picture of the transcriptional changes occurring in the liver surrounding the parasitic lesions. Profiles of mRNA expression levels in the hepatic parasitic lesions showed that a mixed Th1/Th2 immune response, characterized by the concomitant presence of IL-12α, IFN-γ and IL-4, was established very early in the development of E. multilocularis. Subsequently, the profile extended to a combined tolerogenic profile associating IL-5, IL-10 and TGF-β. IL-17 was permanently expressed in the liver, mostly in the periparasitic infiltrate; this was confirmed by the increased mRNA expression of both IL-17A and IL-17F from a very early stage, with a subsequent decrease of IL-17A after this first initial rise. All measured chemokines were significantly expressed at a given stage of infection; their expression paralleled that of the corresponding Th1, Th2 or Th17 cytokines. In addition to giving a comprehensive insight in the time course of cytokines and chemokines in E. multilocularis lesion, this study contributes to identify new targets for possible immune therapy to minimize E. multilocularis-related pathology and to complement the only parasitostatic effect of benzimidazoles in AE.

Impaired mTORC1-Dependent Expression of Homer-3 Influences SCA1 Pathophysiology.

Spinocerebellar ataxia type 1 (SCA1), due to the expansion of a polyglutamine repeat within the ubiquitously expressed Ataxin-1 protein, leads to the premature degeneration of Purkinje cells (PCs), the cause of which is poorly understood. Here, we identified the unique proteomic signature of Sca1(154Q/2Q) PCs at an early stage of disease, highlighting extensive alterations in proteins associated with synaptic functioning, maintenance, and transmission. Focusing on Homer-3, a PC-enriched scaffold protein regulating neuronal activity, revealed an early decline in its expression. Impaired climbing fiber-mediated synaptic transmission diminished mTORC1 signaling, paralleling Homer-3 reduction in Sca1(154Q/2Q) PCs. Ablating mTORC1 within PCs or pharmacological inhibition of mTORC1 identified Homer-3 as its downstream target. mTORC1 knockout in Sca1(154Q/2Q) PCs exacerbated and accelerated pathology. Reinstating Homer-3 expression in Sca1(154Q/2Q) PCs attenuated cellular dysfunctions and improved motor deficits. Our work reveals that impaired mTORC1-Homer-3 activity underlies PC susceptibility in SCA1 and presents a promising therapeutic target.

Non-surgical treatment of pain associated with posterior tibial tendon dysfunction: study protocol for a randomised clinical trial

BACKGROUND Symptoms associated with pes planovalgus or flatfeet occur frequently, even though some people with a flatfoot deformity remain asymptomatic. Pes planovalgus is proposed to be associated with foot/ankle pain and poor function. Concurrently, the multifactorial weakness of the tibialis posterior muscle and its tendon can lead to a flattening of the longitudinal arch of the foot. Those affected can experience functional impairment and pain. Less severe cases at an early stage are eligible for non-surgical treatment and foot orthoses are considered to be the first line approach. Furthermore, strengthening of arch and ankle stabilising muscles are thought to contribute to active compensation of the deformity leading to stress relief of soft tissue structures. There is only limited evidence concerning the numerous therapy approaches, and so far, no data are available showing functional benefits that accompany these interventions. METHODS After clinical diagnosis and clarification of inclusion criteria (e.g., age 40-70, current complaint of foot and ankle pain more than three months, posterior tibial tendon dysfunction stage I & II, longitudinal arch flattening verified by radiography), sixty participants with posterior tibial tendon dysfunction associated complaints will be included in the study and will be randomly assigned to one of three different intervention groups: (i) foot orthoses only (FOO), (ii) foot orthoses and eccentric exercise (FOE), or (iii) sham foot orthoses only (FOS). Participants in the FOO and FOE groups will be allocated individualised foot orthoses, the latter combined with eccentric exercise for ankle stabilisation and strengthening of the tibialis posterior muscle. Participants in the FOS group will be allocated sham foot orthoses only. During the intervention period of 12 weeks, all participants will be encouraged to follow an educational program for dosed foot load management (e.g., to stop activity if they experience increasing pain). Functional impairment will be evaluated pre- and post-intervention by the Foot Function Index. Further outcome measures include the Pain Disability Index, Visual Analogue Scale for pain, SF-12, kinematic data from 3D-movement analysis and neuromuscular activity during level and downstairs walking. Measuring outcomes pre- and post-intervention will allow the calculation of intervention effects by 3×3 Analysis of Variance (ANOVA) with repeated measures. DISCUSSION The purpose of this randomised trial is to evaluate the therapeutic benefit of three different non-surgical treatment regimens in participants with posterior tibial tendon dysfunction and accompanying pes planovalgus. Furthermore, the analysis of changes in gait mechanics and neuromuscular control will contribute to an enhanced understanding of functional changes and eventually optimise conservative management strategies for these patients. TRIAL REGISTRATION ClinicalTrials.gov Protocol Registration System: ClinicalTrials.gov ID NCT01839669.

How often parametrial involvement leads to post-operative adjuvant treatment in locally advanced cervical cancer after neoadjuvant chemotherapy and type C radical hysterectomy?

OBJECTIVE Parametrial involvement (PMI) is one of the most important factors influencing prognosis in locally advanced stage cervical cancer (LACC) patients. We aimed to evaluate PMI rate among LACC patients undergoing neoadjuvant chemotherapy (NACT), thus evaluating the utility of parametrectomy in tailor adjuvant treatments. METHODS Retrospective evaluation of consecutive 275 patients affected by LACC (IB2-IIB), undergoing NACT followed by type C/class III radical hysterectomy. Basic descriptive statistics, univariate and multivariate analyses were applied in order to identify factors predicting PMI. Survival outcomes were assessed using Kaplan-Meier and Cox models. RESULTS PMI was detected in 37 (13%) patients: it was associated with vaginal involvement, lymph node positivity and both in 10 (4%), 5 (2%) and 12 (4%) patients, respectively; while PMI alone was observed in only 10 (4%) patients. Among this latter group, adjuvant treatment was delivered in 3 (1%) patients on the basis of pure PMI; while the remaining patients had other characteristics driving adjuvant treatment. Considering factors predicting PMI we observed that only suboptimal pathological responses (OR: 1.11; 95% CI: 1.01, 1.22) and vaginal involvement (OR: 1.29 (95%) CI: 1.17, 1.44) were independently associated with PMI. PMI did not correlate with survival (HR: 2.0; 95% CI: 0.82, 4.89); while clinical response to NACT (HR: 3.35; 95% CI: 1.59, 7.04), vaginal involvement (HR: 2.38; 95% CI: 1.12, 5.02) and lymph nodes positivity (HR: 3.47; 95% CI: 1.62, 7.41), independently correlated with worse survival outcomes. CONCLUSIONS Our data suggest that PMI had a limited role on the choice to administer adjuvant treatment, thus supporting the potential embrace of less radical surgery in LACC patients undergoing NACT. Further prospective studies are warranted.