Neoadjuvant chemoradiotherapy with or without panitumumab in patients with wild-type KRAS, locally advanced rectal cancer (LARC): a randomized, multicenter, phase II trial SAKK 41/07.

BACKGROUND: We conducted a randomized, phase II, multicenter study to evaluate the anti-epidermal growth factor receptor (EGFR) mAb panitumumab (P) in combination with chemoradiotherapy (CRT) with standard-dose capecitabine as neoadjuvant treatment for wild-type KRAS locally advanced rectal cancer (LARC). PATIENTS AND METHODS: Patients with wild-type KRAS, T3-4 and/or N+ LARC were randomly assigned to receive CRT with or without P (6 mg/kg). The primary end-point was pathological near-complete or complete tumor response (pNC/CR), defined as grade 3 (pNCR) or 4 (pCR) histological regression by Dworak classification (DC). RESULTS: Forty of 68 patients were randomly assigned to P + CRT and 28 to CRT. pNC/CR was achieved in 21 patients (53%) treated with P + CRT [95% confidence interval (CI) 36%-69%] versus 9 patients (32%) treated with CRT alone (95% CI: 16%-52%). pCR was achieved in 4 (10%) and 5 (18%) patients, and pNCR in 17 (43%) and 4 (14%) patients. In immunohistochemical analysis, most DC 3 cells were not apoptotic. The most common grade ≥3 toxic effects in the P + CRT/CRT arm were diarrhea (10%/6%) and anastomotic leakage (15%/4%). CONCLUSIONS: The addition of panitumumab to neoadjuvant CRT in patients with KRAS wild-type LARC resulted in a high pNC/CR rate, mostly grade 3 DC. The results of both treatment arms exceeded prespecified thresholds. The addition of panitumumab increased toxicity.

Expression of the proto-oncogene c-fos in three-dimensional fetal brain cell cultures and the lack of correlation with maturation-inducing stimuli.

Previous work has shown that aggregating fetal brain cell cultures are able to attain a highly differentiated state, and that their development is greatly enhanced by growth and/or differentiation factors such as epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and the protein kinase C-activating tumor promoter mezerein. The present study shows that in these 3-dimensional cultures the peptide growth factors EGF and bFGF as well as mezerein are able to induce the expression of the proto-oncogene c-fos. This induction was rapid and transient, in good agreement with observations reported from a wide variety of cell types in vitro. The maximal levels of c-fos mRNA found after stimulation were low in immature cultures and increased greatly as maturation progressed. Of the three factors tested, mezerein was the most potent inducer of c-fos. In contrast to the peptide growth factors EGF and bFGF which were found to induce c-fos only in glial cells, mezerein was stimulatory in glial cells as well as in neurons. A similar cell type specificity has been observed previously for the maturation-enhancing response in immature aggregate cultures. However, in the present study no correlation was found between the degree of c-fos induction and the extent of the maturation-enhancing stimulation. Immature cultures known to be most sensitive and responsive to these maturation-enhancing agents required relatively high doses of peptide growth factors for the induction of c-fos, and the maximal levels of c-fos mRNA elicited were much lower than those in differentiated cultures which did not show any long-term response to these stimuli.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of glucagon secretion by glucose transporter type 2 (glut2) and astrocyte-dependent glucose sensors.

Ripglut1;glut2-/- mice have no endogenous glucose transporter type 2 (glut2) gene expression but rescue glucose-regulated insulin secretion. Control of glucagon plasma levels is, however, abnormal, with fed hyperglucagonemia and insensitivity to physiological hypo- or hyperglycemia, indicating that GLUT2-dependent sensors control glucagon secretion. Here, we evaluated whether these sensors were located centrally and whether GLUT2 was expressed in glial cells or in neurons. We showed that ripglut1;glut2-/- mice failed to increase plasma glucagon levels following glucoprivation induced either by i.p. or intracerebroventricular 2-deoxy-D-glucose injections. This was accompanied by failure of 2-deoxy-D-glucose injections to activate c-Fos-like immunoreactivity in the nucleus of the tractus solitarius and the dorsal motor nucleus of the vagus. When glut2 was expressed by transgenesis in glial cells but not in neurons of ripglut1;glut2-/- mice, stimulated glucagon secretion was restored as was c-Fos-like immunoreactive labeling in the brainstem. When ripglut1;glut2-/- mice were backcrossed into the C57BL/6 genetic background, fed plasma glucagon levels were also elevated due to abnormal autonomic input to the alpha cells; glucagon secretion was, however, stimulated by hypoglycemic stimuli to levels similar to those in control mice. These studies identify the existence of central glucose sensors requiring glut2 expression in glial cells and therefore functional coupling between glial cells and neurons. These sensors may be activated at different glycemic levels depending on the genetic background.

STRATEGIC-1: A multiple-lines, randomized, open-label GERCOR phase III study in patients with unresectable wild-type RAS metastatic colorectal cancer.

BACKGROUND: The management of unresectable metastatic colorectal cancer (mCRC) is a comprehensive treatment strategy involving several lines of therapy, maintenance, salvage surgery, and treatment-free intervals. Besides chemotherapy (fluoropyrimidine, oxaliplatin, irinotecan), molecular-targeted agents such as anti-angiogenic agents (bevacizumab, aflibercept, regorafenib) and anti-epidermal growth factor receptor agents (cetuximab, panitumumab) have become available. Ultimately, given the increasing cost of new active compounds, new strategy trials are needed to define the optimal use and the best sequencing of these agents. Such new clinical trials require alternative endpoints that can capture the effect of several treatment lines and be measured earlier than overall survival to help shorten the duration and reduce the size and cost of trials. METHODS/DESIGN: STRATEGIC-1 is an international, open-label, randomized, multicenter phase III trial designed to determine an optimally personalized treatment sequence of the available treatment modalities in patients with unresectable RAS wild-type mCRC. Two standard treatment strategies are compared: first-line FOLFIRI-cetuximab, followed by oxaliplatin-based second-line chemotherapy with bevacizumab (Arm A) vs. first-line OPTIMOX-bevacizumab, followed by irinotecan-based second-line chemotherapy with bevacizumab, and by an anti-epidermal growth factor receptor monoclonal antibody with or without irinotecan as third-line treatment (Arm B). The primary endpoint is duration of disease control. A total of 500 patients will be randomized in a 1:1 ratio to one of the two treatment strategies. DISCUSSION: The STRATEGIC-1 trial is designed to give global information on the therapeutic sequences in patients with unresectable RAS wild-type mCRC that in turn is likely to have a significant impact on the management of this patient population. The trial is open for inclusion since August 2013. TRIAL REGISTRATION: STRATEGIC-1 is registered at Clinicaltrials.gov: NCT01910610, 23 July, 2013. STRATEGIC-1 is registered at EudraCT-No.: 2013-001928-19, 25 April, 2013.

Comprehensive Expression Analyses of Neural Cell-Type-Specific miRNAs Identify New Determinants of the Specification and Maintenance of Neuronal Phenotypes.

MicroRNAs (miRNAs) have been shown to play important roles in both brain development and the regulation of adult neural cell functions. However, a systematic analysis of brain miRNA functions has been hindered by a lack of comprehensive information regarding the distribution of miRNAs in neuronal versus glial cells. To address this issue, we performed microarray analyses of miRNA expression in the four principal cell types of the CNS (neurons, astrocytes, oligodendrocytes, and microglia) using primary cultures from postnatal d 1 rat cortex. These analyses revealed that neural miRNA expression is highly cell-type specific, with 116 of the 351 miRNAs examined being differentially expressed fivefold or more across the four cell types. We also demonstrate that individual neuron-enriched or neuron-diminished RNAs had a significant impact on the specification of neuronal phenotype: overexpression of the neuron-enriched miRNAs miR-376a and miR-434 increased the differentiation of neural stem cells into neurons, whereas the opposite effect was observed for the glia-enriched miRNAs miR-223, miR-146a, miR-19, and miR-32. In addition, glia-enriched miRNAs were shown to inhibit aberrant glial expression of neuronal proteins and phenotypes, as exemplified by miR-146a, which inhibited neuroligin 1-dependent synaptogenesis. This study identifies new nervous system functions of specific miRNAs, reveals the global extent to which the brain may use differential miRNA expression to regulate neural cell-type-specific phenotypes, and provides an important data resource that defines the compartmentalization of brain miRNAs across different cell types.

Long-Term Trends of HIV Type 1 Drug Resistance Prevalence among Antiretroviral Treatment-Experienced Patients in Switzerland.

Background. Accurate quantification of the prevalence of human immunodeficiency virus type 1 (HIV-1) drug resistance in patients who are receiving antiretroviral therapy (ART) is difficult, and results from previous studies vary. We attempted to assess the prevalence and dynamics of resistance in a highly representative patient cohort from Switzerland. Methods. On the basis of genotypic resistance test results and clinical data, we grouped patients according to their risk of harboring resistant viruses. Estimates of resistance prevalence were calculated on the basis of either the proportion of individuals with a virologic failure or confirmed drug resistance (lower estimate) or the frequency-weighted average of risk group-specific probabilities for the presence of drug resistance mutations (upper estimate). Results. Lower and upper estimates of drug resistance prevalence in 8064 ART-exposed patients were 50% and 57% in 1999 and 37% and 45% in 2007, respectively. This decrease was driven by 2 mechanisms: loss to follow-up or death of high-risk patients exposed to mono- or dual-nucleoside reverse-transcriptase inhibitor therapy (lower estimates range from 72% to 75%) and continued enrollment of low-risk patients who were taking combination ART containing boosted protease inhibitors or nonnucleoside reverse-transcriptase inhibitors as first-line therapy (lower estimates range from 7% to 12%). A subset of 4184 participants (52%) had 1 study visit per year during 2002-2007. In this subset, lower and upper estimates increased from 45% to 49% and from 52% to 55%, respectively. Yearly increases in prevalence were becoming smaller in later years. Conclusions. Contrary to earlier predictions, in situations of free access to drugs, close monitoring, and rapid introduction of new potent therapies, the emergence of drug-resistant viruses can be minimized at the population level. Moreover, this study demonstrates the necessity of interpreting time trends in the context of evolving cohort populations.

Redirection of T cells by delivering a transgenic mouse-derived MDM2 tumor antigen-specific TCR and its humanized derivative is governed by the CD8 coreceptor and affects natural human TCR expression.

Retroviral transfer of T cell antigen receptor (TCR) genes selected by circumventing tolerance to broad tumor- and leukemia-associated antigens in human leukocyte antigen (HLA)-A*0201 (A2.1) transgenic (Tg) mice allows the therapeutic reprogramming of human T lymphocytes. Using a human CD8 x A2.1/Kb mouse derived TCR specific for natural peptide-A2.1 (pA2.1) complexes comprising residues 81-88 of the human homolog of the murine double-minute 2 oncoprotein, MDM2(81-88), we found that the heterodimeric CD8 alpha beta coreceptor, but not normally expressed homodimeric CD8 alpha alpha, is required for tetramer binding and functional redirection of TCR- transduced human T cells. CD8+T cells that received a humanized derivative of the MDM2 TCR bound pA2.1 tetramers only in the presence of an anti-human-CD8 anti-body and required more peptide than wild-type (WT) MDM2 TCR+T cells to mount equivalent cytotoxicity. They were, however, sufficiently effective in recognizing malignant targets including fresh leukemia cells. Most efficient expression of transduced TCR in human T lymphocytes was governed by mouse as compared to human constant (C) alphabeta domains, as demonstrated with partially humanized and murinized TCR of primary mouse and human origin, respectively. We further observed a reciprocal relationship between the level of Tg WT mouse relative to natural human TCR expression, resulting in T cells with decreased normal human cell surface TCR. In contrast, natural human TCR display remained unaffected after delivery of the humanized MDM2 TCR. These results provide important insights into the molecular basis of TCR gene therapy of malignant disease.

Nasal immunization of mice with human papillomavirus type 16 virus-like particles elicits neutralizing antibodies in mucosal secretions.

To specifically induce a mucosal antibody response to purified human papillomavirus type 16 (HPV16) virus-like particles (VLP), we immunized female BALB/c mice orally, intranasally, and/or parenterally and evaluated cholera toxin (CT) as a mucosal adjuvant. Anti-HPV16 VLP immunoglobulin G (IgG) and IgA titers in serum, saliva, and genital secretions were measured by enzyme-linked immunosorbent assay (ELISA). Systemic immunizations alone induced HPV16 VLP-specific IgG in serum and, to a lesser extent, in genital secretions but no secretory IgA. Oral immunization, even in the presence of CT, was inefficient. However, three nasal immunizations with 5 microgram of VLP given at weekly intervals to anesthetized mice induced high (>10(4)) and long-lasting (>15 weeks) titers of anti-HPV16 VLP antibodies in all samples, including IgA and IgG in saliva and genital secretions. CT enhanced the VLP-specific antibody response 10-fold in serum and to a lesser extent in saliva and genital secretions. Nasal immunization of conscious mice compared to anesthetized mice was inefficient and correlated with the absence of uptake of a marker into the lung. However, a 1-microgram VLP systemic priming followed by two 5-microgram VLP intranasal boosts in conscious mice induced both HPV16 VLP-specific IgG and IgA in secretions, although the titers were lower than in anesthetized mice given three intranasal immunizations. Antibodies in serum, saliva, and genital secretions of immunized mice were strongly neutralizing in vitro (50% neutralization with ELISA titers of 65 to 125). The mucosal and systemic/mucosal HPV16 VLP immunization protocols that induced significant titers of neutralizing IgG and secretory IgA in mucosal secretions in mice may be relevant to genital HPV VLP-based human vaccine trials.

NEUROPATHOPHYSIOLOGICAL MECHANISMS IN GLUTARIC ACIDURIA TYPE I: 3-HYDROXYGLUTARIC ACID LEADS TO AMMONIA INCREASE AND NON-APOPTOTIC CELL DEATH

A 3D in vitro model of rat organotypic brain cell cultures in aggregates was used to investigate neurotoxicity mechanisms in glutaric aciduria type I (GA-I). 1 mM glutarate (GA) or 3-hydroxyglutarate (3OHGA) were repeatedly added to the culture media at two different time points. In cultures treated with 3OHGA, we observed an increase in lactate in the medium, pointing to a possible inhibition of Krebs cycle and respiratory chain. We further observed that 3OHGA and to a lesser extend GA induced an increase in ammonia production with concomitant decrease of glutamine concentrations, which may suggest an inhibition of the astrocytic enzyme glutamine synthetase. These previously unreported findings may uncover a pathogenic mechanism in this disease which has deleterious effects on early stages of brain development. By immunohistochemistry we showed that 3OHGA increased non-apoptotic cell death. On the cellular level, 3OHGA and to a lesser extend GA led to cell swelling and loss of astrocytic fibers whereas a loss of oligodendrocytes was only observed for 3OHGA. We conclude that 3OHGAwas the most toxic metabolite in our model for GA-I. 3OHGA induced deleterious effects on glial cells, an increase of ammonia production, and resulted in accentuated cell death of non-apoptotic origin.

T-type Ca2+ channels, SK2 channels and SERCAs gate sleep-related oscillations in thalamic dendrites.

T-type Ca2+ channels (T channels) underlie rhythmic burst discharges during neuronal oscillations that are typical during sleep. However, the Ca2+-dependent effectors that are selectively regulated by T currents remain unknown. We found that, in dendrites of nucleus reticularis thalami (nRt), intracellular Ca2+ concentration increases were dominated by Ca2+ influx through T channels and shaped rhythmic bursting via competition between Ca2+-dependent small-conductance (SK)-type K+ channels and Ca2+ uptake pumps. Oscillatory bursting was initiated via selective activation of dendritically located SK2 channels, whereas Ca2+ sequestration by sarco/endoplasmic reticulum Ca2+-ATPases (SERCAs) and cumulative T channel inactivation dampened oscillations. Sk2-/- (also known as Kcnn2) mice lacked cellular oscillations, showed a greater than threefold reduction in low-frequency rhythms in the electroencephalogram of non-rapid-eye-movement sleep and had disrupted sleep. Thus, the interplay of T channels, SK2 channels and SERCAs in nRt dendrites comprises a specialized Ca2+ signaling triad to regulate oscillatory dynamics related to sleep.

Flagellin promotes myeloid differentiation factor 88-dependent development of Th2-type response.

Activation of dendritic cells (DC) by microbial products via Toll-like receptors (TLR) is instrumental in the induction of immunity. In particular, TLR signaling plays a major role in the instruction of Th1 responses. The development of Th2 responses has been proposed to be independent of the adapter molecule myeloid differentiation factor 88 (MyD88) involved in signal transduction by TLRs. In this study we show that flagellin, the bacterial stimulus for TLR5, drives MyD88-dependent Th2-type immunity in mice. Flagellin promotes the secretion of IL-4 and IL-13 by Ag-specific CD4(+) T cells as well as IgG1 responses. The Th2-biased responses are associated with the maturation of DCs, which are shown to express TLR5. Flagellin-mediated DC activation requires MyD88 and induces NF-kappaB-dependent transcription and the production of low levels of proinflammatory cytokines. In addition, the flagellin-specific response is characterized by the lack of secretion of the Th1-promoting cytokine IL-12 p70. In conclusion, this study suggests that flagellin and, more generally, TLR ligands can control Th2 responses in a MyD88-dependent manner.

Autofluorescence Findings Of A Novel Maculopathy In Patient With Spinocerebellar Ataxia Type 1 And Of A Cone - Rod Type Retinal Dysfunction In Three Generation Family With Spinocerebellar Ataxia Type 7

Purpose: To report a novel maculopathy in a patient with SCA1. To describe autofluorescence findings in family with SCA7 and associated cone-rod retinal dysfunction.Methods: 4 affected patients from two families were assessed to investigate a progressive loss of visual acuity (VA). Examinations included fundus photography, autofluorescence (AF) fundus fluorescein angiogragraphy (FFA) and optical coherence tomography. Electroretinogram (full-field) was performed in 2 affected patients. All patients had color vision testing using Ishihara pseudoisochromatic plates. Molecular analysis was performed in family 2.Results: The patient with known diagnosis of SCA1 had a visual acuity of 20/200 bilaterally and dyschromatopsia. He had saccadic pursuit. Fundus examination showed mild retinal pigment epithelium (RPE) changes at the macula. OCT showed bilateral macular serous detachment, which was not obvious at the FFA and explained his VA. AF imaging showed a central hyperfluorescence. The 45 year old proband from family 2 had a visual acuity of 200/20 and dyschromatopsia. ERG testing showed cone type dysfunction of photoreceptors. Her daughter affected at a younger age had the same ERGs findings. Fundus examination showed mild RPE changes in proband, normal findings in her daughter. AF imaging of both patients showed a ring of high density AF around the fovea. The ring was also obvious on near infrared AF. Later onset of gait imbalance led to the diagnosis of SCA7Conclusions: Within the group of spinocerebellar ataxias, only the type 7 is associated with retinal dysfunction. We present the first report of maculopathy associated with SCA1 causing severe vision loss. The ring of high density AF in SCA7 confirmed an early retinal photoreceptor dysfunction in patient with normal fundus.

Phosphodiesterase-5 inhibition mimics intermittent reoxygenation and improves cardioprotection in the hypoxic myocardium.

Although chronic hypoxia is a claimed myocardial risk factor reducing tolerance to ischemia/reperfusion (I/R), intermittent reoxygenation has beneficial effects and enhances heart tolerance to I/R. AIM OF THE STUDY: To test the hypothesis that, by mimicking intermittent reoxygenation, selective inhibition of phosphodiesterase-5 activity improves ischemia tolerance during hypoxia. Adult male Sprague-Dawley rats were exposed to hypoxia for 15 days (10% O₂) and treated with placebo, sildenafil (1.4 mg/kg/day, i. p.), intermittent reoxygenation (1 h/day exposure to room air) or both. Controls were normoxic hearts. To assess tolerance to I/R all hearts were subjected to 30-min regional ischemia by left anterior descending coronary artery ligation followed by 3 h-reperfusion. Whereas hypoxia depressed tolerance to I/R, both sildenafil and intermittent reoxygenation reduced the infarct size without exhibiting cumulative effects. The changes in myocardial cGMP, apoptosis (DNA fragmentation), caspase-3 activity (alternative marker for cardiomyocyte apoptosis), eNOS phosphorylation and Akt activity paralleled the changes in cardioprotection. However, the level of plasma nitrates and nitrites was higher in the sildenafil+intermittent reoxygenation than sildenafil and intermittent reoxygenation groups, whereas total eNOS and Akt proteins were unchanged throughout. CONCLUSIONS: Sildenafil administration has the potential to mimic the cardioprotective effects led by intermittent reoxygenation, thereby opening the possibility to treat patients unable to be reoxygenated through a pharmacological modulation of NO-dependent mechanisms.

Tightening of DNA knots by supercoiling facilitates their unknotting by type II DNA topoisomerases.

Using numerical simulations, we compare properties of knotted DNA molecules that are either torsionally relaxed or supercoiled. We observe that DNA supercoiling tightens knotted portions of DNA molecules and accentuates the difference in curvature between knotted and unknotted regions. The increased curvature of knotted regions is expected to make them preferential substrates of type IIA topoisomerases because various earlier experiments have concluded that type IIA DNA topoisomerases preferentially interact with highly curved DNA regions. The supercoiling-induced tightening of DNA knots observed here shows that torsional tension in DNA may serve to expose DNA knots to the unknotting action of type IIA topoisomerases, and thus explains how these topoisomerases could maintain a low knotting equilibrium in vivo, even for long DNA molecules.