GILZ-mimics as novel therapeutic agents for progressive multiple sclerosis

poster abstractMultiple sclerosis (MS), a leading cause of neurological disability is an inflammatory demyelinating disease of the central nervous system (CNS). The clinical course of MS is highly variable ranging from isolated neurologic episodes to frequently relapsing or progressive disease. Currently there are no effective treatments for progressive MS. The long-term goal of this project is to evaluate a novel therapeutic strategy for progressive MS. Under physiological conditions signaling via the transcription factor, nuclear factor-kappa B (NF-κB) and glucocorticoid (GC) stimulation pathways regulate the immuno-inflammatory responses of the CNS resident glial cells. While NF-κB induces transcriptional activation, signaling via GC receptor functions to suppress immune responses. Persistent activation of NF-κB in the glial cells precipitates neuronal degeneration and axonal loss characteristic of progressive MS. Interactome analysis between the GC and NF-κB pathways suggested a novel strategy to inhibit NF-κB. Glucocorticoid-induced leucine zipper (GILZ) is a GC inducible protein that binds p65, the functionally critical subunit of NF-κB, and prevent transactivation of pathological mediators. The sites of interaction are localized to the proline rich region of the GILZ protein and the p65 transactivation domain. A 23 residue GILZ peptide prevented nuclear translocation of p65 and suppressed disease in an animal model of MS. Structurally GILZ peptide adopted polyproline type II (PPII) helical conformation, a favorable feature for drug development. The objective of this study is to optimize the lead peptide and develop drug like analogs. Specific features of the GILZ-p65 interactions were adapted in the design of over 25 GILZ analogs such that each exhibit optimum PPII helix, bind p65 transactivation domain and potentially accommodate modified residues that enhance the binding specificity with the p65. The analogs were ranked after passing through the Lipinski filter to determine the drug like properties. The top ranked analogs will be evaluated for functional efficacy

Щоденники експедиційної роботи Григорія Дем'яна (Вступна стаття та підготовка текстів Василя Сокола)

The interaction between S(-II) and ferric oxides exerts a major control for the sulphur and iron cycle and in particular for the carbon and electron flow in many aquatic systems. It is regarded to be a key reaction leading ultimately to pyrite formation, the pathways still remaining unresolved. We have studied the reaction between lepidocrocite (γ-FeOOH, 21–42 mmol L−1) and dissolved S(-II) (3–9 mmol L−1) in batch experiments at pH 7 in a glove box using TEM, XRD, Mössbauer spectroscopy, and wet chemistry extraction to explore the nanocrystalline products forming at different time steps in close contact to the lepidocrocitesurface. S(0) and acid extractable Fe(II) (Fe(II)HCl) were the main products detected by wet chemistry extraction. The reaction could be divided into three steps: a rapid (<15 min) consumption of dissolved S(-II), formationof S(0) and the build-up of an Fe(II)HCl pool. Then in the absence of dissolved S(-II) concentrations of S(0) and Fe(II)HCl increased only slightly. TEM measurements revealed the occurrence of a mackinawite rim covering the lepidocrocite crystals and being separated from the lepidocrocitesurface by an interfacial magnetite layer that can be regarded as a steady state product of the interaction between lepidocrocite and mackinawite. A significant fraction of Fe(II) was formed in excess to FeS within the first 2 h. The amount of this fraction increased with decreasing ratio between dissolved S(-II) concentration and the concentration ofsurface sites, which we attributed to a kinetic decoupling of S(-II) oxidation and Fe(II) detachment from the lepidocrocitesurface. At low ratios, S(-II) seems to transfer electrons to lepidocrocite faster then stoichiometric amounts of FeS could. After 2 days Fe(II)HCl and S(0) started to decrease resulting in pyrite formation accompanied by traces of magnetite. TEM measurements indicated that mackinawite completely dissolved and precipitation of pyrite occurred dislocated from the lepidocrocitesurface. The absence of dissolved sulphide under these conditions suggest that excess Fe(II) is involved in the formationof polysulphides which are key precursors during pyrite formation. We propose that the occurrence of excess Fe(II) is a common phenomenon particularly in low sulphide – high iron environments attributing significant reactivity to ferric (hydr)oxide

Ano-genital Granulomatosis and Crohn's Disease: A Case Series of Males Presenting with Genital Lymphoedema.

Background and Aims: Ano-genital granulomatosis is a rare chronic granulomatous condition of the skin that causes lymphoedema of the external genitalia. There is a reported association with Crohn's disease. Mechanisms of disease and optimal methods of treatment are poorly understood. Methods: A retrospective casenote review of 25 male patients with ano-genital granulomatosis presenting with genital lymphoedema was performed to determine the clinical and histopathological features of this condition and its relationship to intestinal Crohn's disease. Results: A combination of penile and scrotal oedema was reported at presentation in 80% of patients; 40% of patients had associated intestinal Crohn's disease. The average time from symptom onset to diagnosis was 52.7 months. Half of cutaneous biopsies contained non-caseating granulomas and 14% contained intralymphatic granulomas. In all, 72% of patients responded to oral steroids initially but recurrence was common. Complete or partial response was achieved in 60% of patients treated with azathioprine. Three of six patients responded to anti-tumour necrosis factor [TNF] therapy. A small proportion of patients required circumcision or de-bulking surgery for more debilitating disease. Conclusions: Ano-genital granulomatosis is a rare condition that presents with genital lymphoedema, and there is frequently a protracted delay in diagnosis. There is a very strong association with intestinal Crohn's disease. Genital lymphoedema associated with gastrointestinal symptoms should prompt careful evaluation to exclude both ano-genital granulomatosis and Crohn's disease

Astabiotics: Antimicrobial Signal Transduction Activators

poster abstractThe increasing prevalence of multi-drug resistant Gram-negative bacteria is a major public health concern. All available antibiotics are inhibitors of targets essential for virulence or growth. CpxRA is a highly conserved bacterial signal transduction system that responds to extracytoplasmic membrane stress. CpxA is a sensor with kinase and phosphatase activity; upon activation, CpxA donates a phosphate group to CpxR, activating a transcriptional response. Activation of CpxRA reduces the flow of protein traffic through the cytoplasmic membrane, dramatically reducing the expression of virulence determinants. Activation of CpxRA abolishes the virulence of Salmonella Typhimurium in mice. We found that activation of CpxRA crippled the ability of Haemophilus ducreyi to cause disease in experimentally infected human volunteers. Using an Escherichia coli reporter strain, we developed a high throughput screen to detect compounds that activate CpxRA. In a pilot screen of 36,000 compounds, we identified 1 class of compounds that shifts the equilibrium of CpxA to kinase activity, activating CpxR. Based on its potency, the calculated effective dose of the lead compound (a nitroindole) was 10 mg/kg. Female mice tolerated 100 mg/kg of the nitroindole given twice a day for 3 days. A CpxRA activating mutant constructed in uropathogenic E. coli (UPEC) was severely impaired in a murine urinary tract infection model; thus, activation of CpxRA is a valid treatment strategy for UPEC. However, when female mice were challenged with UPEC and treated with 100 mg/kg of the nitroindole using the schedule above, there were no differences in the recovered CFU in the urine, bladder, and kidney of sham and compound-treated mice. Future studies will include medicinal optimization of the nitroindole and identification and optimization other leads that activate CpxRA. Although our pilot test of efficacy was negative, astabiotics have great potential as broad-spectrum, adjunctive therapies to existing antimicrobials for treatment of Gram-negative infections

Crystal growth patterns in solid solution systems:case studies on oscillatory zoning and mineral replacement reactions

Oszillierende Zonierung und Mineralverdrängungsreaktionen sind Beispiele für Kristallwachstumsmuster in Mischkristallsystemen. Beide Themenbereiche wurden mit hochauflösender Mikroanalytik und thermodynamischer Modellierung behandelt. Oszillierend zonierte Granate der Grossular-Andradit-Reihe wurden mit der analytischen Transmissions-Elektronenmikroskopie (ATEM) untersucht und der Maßstab und die Grenzflächen in der Bedeutung für die Wachstumsdynamik diskutiert. Mit Molekularsimulationen wurden die thermodynamischen Mischungseigenschaften und die Grenzflächenstabilität berechnet und in Bezug zu den natürlichen Proben gesetzt. Mineralverdrängungsmuster metamorpher Granate eines teilweise eklogitisierten Gesteins wurden mikroanalytisch mittels ATEM untersucht und in Bezug zu Reaktionsmechanismen gesetzt. Die Volumenänderung (Porosität) bei Verdrängungsreaktionen von Mischkristallen in wässriger Lösung wird als Modell vorgestellt und auf das System KBr-KCl-H2O angewandt

Bayesian Approach to Model CD137 Signaling in Human M.tuberculosis in vitro Responses

Abstract Immune responses are qualitatively and quantitatively influenced by a complex network of receptor-ligand interactions. Among them, the CD137:CD137L pathway is known to modulate innate and adaptive human responses against Mycobacterium tuberculosis. However, the underlying mechanisms of this regulation remain unclear. In this work, we developed a Bayesian Computational Model (BCM) of in vitro CD137 signaling, devised to fit previously gathered experimental data. The BCM is fed with the data and the prior distribution of the model parameters and it returns theirposterior distribution and the model evidence, which allows comparing alternative signaling mechanisms. The BCM uses a coupled system of non-linear differential equations to describe the dynamics of Antigen Presenting Cells, Natural Killer and T Cells together with the interpheron (IFN)-c and tumor necrosis factor (TNF)-a levels in the media culture. Fast and complete mixing of the media is assumed. The prior distribution of the parameters that describe the dynamics of the immunological response was obtained from the literature and theoretical considerations Our BCM applies successively the Levenberg-Marquardt algorithm to find the maximum a posteriori likelihood (MAP); the Metropolis Markov Chain Monte Carlo method to approximate the posterior distribution of the parameters and Thermodynamic Integration to calculate the evidence of alternative hypothesis. Bayes factors provided decisive evidence favoring direct CD137 signaling on T cells. Moreover, the posterior distribution of the parameters that describe the CD137 signaling showed that the regulation of IFNc levels is based more on T cells survival than on direct induction. Furthermore, the mechanisms that account for the effect of CD137 signaling on TNF-a production were based on a decrease of TNF-a production by APC and, perhaps, on the increase in APC apoptosis. BCM proved to be a useful tool to gain insight on the mechanisms of CD137 signaling during human response against Mycobacterium tuberculosis.Fil: Darío A Fernández Do Porto. UNIV.DE BUENOS AIRES. FAC.DE CS.EXACTAS Y NATURALES. UNIV.DE BUENOS AIRES. FAC.DE CS.EXACTAS Y NATURALES. INST QUIM FISICA D/L/MATERIALES MED AMB Y ENERG.Fil: Jerónimo Auzmendi. UNIV.DE BUENOS AIRES. FAC.DE CS.EXACTAS Y NATURALES. INST QUIM FISICA D/L/MATERIALES MED AMB Y ENERG.Fil: Delfina Peña. UNIV.DE BUENOS AIRES. FAC.DE CS.EXACTAS Y NATURALES. CONSEJO NAC.DE INVEST.CIENTIF.Y TECNICAS. OFICINA DE COORDINACION ADMINISTRATIVA CIUDAD UNIVERSITARIA. INSTITUTO DE QUIMICA BIOLOGICA DE LA FACULTAD DE CS. EXACTAS Y NATURALES. UNIV.DE BUENOS AIRES. FAC.DE CS.EXACTAS Y NATURALES. DTO.DE QUIMICA BIOLOGICA.Fil: Veronica E Garcia. CONSEJO NAC.DE INVEST.CIENTIF.Y TECNICAS. OFICINA DE COORDINACION ADMINISTRATIVA CIUDAD UNIVERSITARIA. INSTITUTO DE QUIMICA BIOLOGICA DE LA FACULTAD DE CS. EXACTAS Y NATURALES.Fil: Luciano Moffatt. UNIV.DE BUENOS AIRES. FAC.DE CS.EXACTAS Y NATURALES. INST QUIM FISICA D/L/MATERIALES MED AMB Y ENERG

Pitch Memory in Nonmusicians and Musicians: Revealing Functional Differences Using Transcranial Direct Current Stimulation

For music and language processing, memory for relative pitches is highly important. Functional imaging studies have shown activation of a complex neural system for pitch memory. One region that has been shown to be causally involved in the process for nonmusicians is the supramarginal gyrus (SMG). The present study aims at replicating this finding and at further examining the role of the SMG for pitch memory in musicians. Nonmusicians and musicians received cathodal transcranial direct current stimulation (tDCS) over the left SMG, right SMG, or sham stimulation, while completing a pitch recognition, pitch recall, and visual memory task. Cathodal tDCS over the left SMG led to a significant decrease in performance on both pitch memory tasks in nonmusicians. In musicians, cathodal stimulation over the left SMG had no effect, but stimulation over the right SMG impaired performance on the recognition task only. Furthermore, the results show a more pronounced deterioration effect for longer pitch sequences indicating that the SMG is involved in maintaining higher memory load. No stimulation effect was found in both groups on the visual control task. These findings provide evidence for a causal distinction of the left and right SMG function in musicians and nonmusician

Impairment of Auditory-Motor Timing and Compensatory Reorganization after Ventral Premotor Cortex Stimulation

Integrating auditory and motor information often requires precise timing as in speech and music. In humans, the position of the ventral premotor cortex (PMv) in the dorsal auditory stream renders this area a node for auditory-motor integration. Yet, it remains unknown whether the PMv is critical for auditory-motor timing and which activity increases help to preserve task performance following its disruption. 16 healthy volunteers participated in two sessions with fMRI measured at baseline and following rTMS (rTMS) of either the left PMv or a control region. Subjects synchronized left or right finger tapping to sub-second beat rates of auditory rhythms in the experimental task, and produced self-paced tapping during spectrally matched auditory stimuli in the control task. Left PMv rTMS impaired auditory-motor synchronization accuracy in the first sub-block following stimulation (p<0.01, Bonferroni corrected), but spared motor timing and attention to task. Task-related activity increased in the homologue right PMv, but did not predict the behavioral effect of rTMS. In contrast, anterior midline cerebellum revealed most pronounced activity increase in less impaired subjects. The present findings suggest a critical role of the left PMv in feed-forward computations enabling accurate auditory-motor timing, which can be compensated by activity modulations in the cerebellum, but not in the homologue region contralateral to stimulation

Potentiation of Carboplatin-Mediated DNA Damage by the Mdm2 Modulator Nutlin-3a in a Humanized Orthotopic Breast-to-Lung Metastatic Model

Triple-negative breast cancers (TNBC) are typically resistant to treatment, and strategies that build upon frontline therapy are needed. Targeting the murine double minute 2 (Mdm2) protein is an attractive approach, as Mdm2 levels are elevated in many therapy-refractive breast cancers. The Mdm2 protein-protein interaction inhibitor Nutlin-3a blocks the binding of Mdm2 to key signaling molecules such as p53 and p73α and can result in activation of cell death signaling pathways. In the present study, the therapeutic potential of carboplatin and Nutlin-3a to treat TNBC was investigated, as carboplatin is under evaluation in clinical trials for TNBC. In mutant p53 TMD231 TNBC cells, carboplatin and Nutlin-3a led to increased Mdm2 and was strongly synergistic in promoting cell death in vitro. Furthermore, sensitivity of TNBC cells to combination treatment was dependent on p73α. Following combination treatment, γH2AX increased and Mdm2 localized to a larger degree to chromatin compared with single-agent treatment, consistent with previous observations that Mdm2 binds to the Mre11/Rad50/Nbs1 complex associated with DNA and inhibits the DNA damage response. In vivo efficacy studies were conducted in the TMD231 orthotopic mammary fat pad model in NOD.Cg-Prkdc(scid)Il2rg(tm1Wjl)/SzJ (NSG) mice. Using an intermittent dosing schedule of combined carboplatin and Nutlin-3a, there was a significant reduction in primary tumor growth and lung metastases compared with vehicle and single-agent treatments. In addition, there was minimal toxicity to the bone marrow and normal tissues. These studies demonstrate that Mdm2 holds promise as a therapeutic target in combination with conventional therapy and may lead to new clinical therapies for TNBC

Defective antigen presentation by monocytes in ESRD patients not responding to hepatitis B vaccination: impaired HBsAg internalization and expression of ICAM-1 and HLA-DR/Ia molecules

This study was undertaken to evaluate the monocyte function of uraemic non-responders to hepatitis B vaccination. Therefore, some parameters concerning antigen processing by monocytes (Mo) as antigen presenting cells (APC) were analysed. It was found that in uraemic non-responders, (1) the internalization of HBsAg by monocytes was significantly decreasjed—HBsAg complexed with specific IgG or as immune complex isolated from patients is better internalized compared with free HBsAg; (2) during antigen presentation the expression of adhesion (ICAM-1) and accessory (HLA-DR/Ia) molecules was significantly decreased in uraemic patients, especially in non-responders; and (3) impaired internalization of HBsAg as well as a decrease in ICAM-1 and HLA-DR/Ia expression, correlated well with the blunted proliferation of CD4+ T cells stimulated by autologous monocytes induced by HBsAg