Sodium-specific foraging by leafcutter ant workers (Atta cephalotes, Hymenoptera: Formicidae)

1. Sodium is often a limiting nutrient for terrestrial animals, and may be especially sought by herbivores. Leafcutter ants are dominant herbivores in the Neotropics, and leafcutter foraging may be affected by nutritional demands of the colony and/or the demands of their symbiotic fungal mutualists. We hypothesized that leafcutter colonies are sodium limited, and that leafcutter ants will therefore forage specifically for sodium. 2. Previous studies demonstrated that leafcutter Atta cephalotes Linnaeus workers preferentially cut and remove paper baits treated with NaCl relative to water control baits. Atta cephalotes colonies in this study were presented with baits offering NaCl, Na2SO4, and KCl to test whether leafcutters forage specifically for sodium. Sucrose and water were used as positive and negative controls, respectively. 3. Atta foragers removed significantly more of the baits treated with NaCl and Na2SO4 than the KCl treatment, which did not differ from water. The NaCl and Na2SO4 treatments were collected at similar rates. We conclude A. cephalotes forage specifically for sodium rather than for anions (chloride) or solutes in general. This study supports the hypothesis that leafcutter ants are limited by, and preferentially forage for, sodium.

Enhancing spiral-wound ultrafiltration performance for direct drinking water treatment through operational procedures improvement: A feasible option for the Sao Paulo Metropolitan Region

This work aimed to evaluate the influence of specific operational conditions on the performance of a spiral-wound ultrafiltration pilot plant for direct drinking water treatment, installed at the Guarapiranga's reservoir, in the Sao Paulo Metropolitan Region. Results from operational tests showed that the volume of permeate produced in the combination of periodic relaxation with flushing and chlorine dosage procedures was 49% higher than the volume obtained when these procedures were not used. Two years of continuous operation demonstrated that the ultrafiltration pilot plant performed better during fall and winter seasons, higher permeate flow production and reduced chemical cleanings frequency. Observed behavior seems to be associated with the algae bloom events in the reservoir, which are more frequent during spring and summer seasons, confirmed by chlorophyll-a analysis results. Concentrate clarification using ferric chloride was quite effective in removing NOM and turbidity, allowing its recirculation to the ultrafiltration feed tank. This procedure made it possible to reach almost 99% water recovery considering a single 54-hour recirculation cycle. Water quality monitoring demonstrated that the ultrafiltration pilot plant was quite efficient, and that potential pathogenic organisms, Escherichia coil and total coliforms, turbidity and apparent color removals were 100%, 95.1%, and 91.5%, respectively. (C) 2012 Elsevier B.V. All rights reserved.

Efficacy of graded activity versus supervised exercises in patients with chronic non-specific low back pain: protocol of a randomised controlled trial

Abstract Background Low back pain is a relevant public health problem, being an important cause of work absenteeism worldwide, as well as affecting the quality of life of sufferers and their individual functional performances. Supervised active physical routines and of cognitive-behavioral therapies are recommended for the treatment of chronic Low back pain, although evidence to support the effectiveness of different techniques is missing. Accordingly, the aim of this study is to contrast the effectiveness of two types of exercises, graded activity or supervised, in decreasing symptoms of chronic low back pain. Methods/design Sample will consist of 66 patients, blindly allocated into one of two groups: 1) Graded activity which, based on an operant approach, will use time-contingent methods aiming to increase participants’ activity levels; 2) Supervised exercise, where participants will be trained for strengthening, stretching, and motor control targeting different muscle groups. Interventions will last one hour, and will happen twice a week for 6 weeks. Outcomes (pain, disability, quality of life, global perceived effect, return to work, physical activity, physical capacity, and kinesiophobia) will be assessed at baseline, at treatment end, and three and six months after treatment end. Data collection will be conducted by an investigator blinded to treatment allocation. Discussion This project describes the randomisation method that will be used to compare the effectiveness of two different treatments for chronic low back pain: graded activity and supervised exercises. Since optimal approach for patients with chronic back pain have yet not been defined based on evidence, good quality studies on the subject are necessary. Trial registration NCT01719276

Citrus aurantium L. essential oil exhibits anxiolytic-like activity mediated by 5-HT1A-receptors and reduces cholesterol after repeated oral treatment

Abstract Background The current treatments for anxiety disorders and depression have multiple adverse effects in addition to a delayed onset of action, which has prompted efforts to find new substances with potential activity in these disorders. Citrus aurantium was chosen based on ethnopharmacological data because traditional medicine refers to the Citrus genus as useful in diminishing the symptoms of anxiety or insomnia, and C. aurantium has more recently been proposed as an adjuvant for antidepressants. In the present work, we investigated the biological activity underlying the anxiolytic and antidepressant effects of C. aurantium essential oil (EO), the putative mechanism of the anxiolytic-like effect, and the neurochemical changes in specific brain structures of mice after acute treatment. We also monitored the mice for possible signs of toxicity after a 14-day treatment. Methods The anxiolytic-like activity of the EO was investigated in a light/dark box, and the antidepressant activity was investigated in a forced swim test. Flumazenil, a competitive antagonist of benzodiazepine binding, and the selective 5-HT1A receptor antagonist WAY100635 were used in the experimental procedures to determine the mechanism of action of the EO. To exclude false positive results due to motor impairment, the mice were submitted to the rotarod test. Results The data suggest that the anxiolytic-like activity observed in the light/dark box procedure after acute (5 mg/kg) or 14-day repeated (1 mg/kg/day) dosing was mediated by the serotonergic system (5-HT1A receptors). Acute treatment with the EO showed no activity in the forced swim test, which is sensitive to antidepressants. A neurochemical evaluation showed no alterations in neurotransmitter levels in the cortex, the striatum, the pons, and the hypothalamus. Furthermore, no locomotor impairment or signs of toxicity or biochemical changes, except a reduction in cholesterol levels, were observed after treatment with the EO. Conclusion This work contributes to a better understanding of the biological activity of C. aurantium EO by characterizing the mechanism of action underlying its anxiolytic-like activity.

Human Endogenous Retrovirus K(HML-2) Gag and Env specific T-cell responses are not detected in HTLV-I-infected subjects using standard peptide screening methods

Abstract Background An estimated 10–20 million individuals are infected with the retrovirus human T-cell leukemia virus type 1 (HTLV-1). While the majority of these individuals remain asymptomatic, 0.3-4% develop a neurodegenerative inflammatory disease, termed HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). HAM/TSP results in the progressive demyelination of the central nervous system and is a differential diagnosis of multiple sclerosis (MS). The etiology of HAM/TSP is unclear, but evidence points to a role for CNS-inflitrating T-cells in pathogenesis. Recently, the HTLV-1-Tax protein has been shown to induce transcription of the human endogenous retrovirus (HERV) families W, H and K. Intriguingly, numerous studies have implicated these same HERV families in MS, though this association remains controversial. Results Here, we explore the hypothesis that HTLV-1-infection results in the induction of HERV antigen expression and the elicitation of HERV-specific T-cells responses which, in turn, may be reactive against neurons and other tissues. PBMC from 15 HTLV-1-infected subjects, 5 of whom presented with HAM/TSP, were comprehensively screened for T-cell responses to overlapping peptides spanning HERV-K(HML-2) Gag and Env. In addition, we screened for responses to peptides derived from diverse HERV families, selected based on predicted binding to predicted optimal epitopes. We observed a lack of responses to each of these peptide sets. Conclusions Thus, although the limited scope of our screening prevents us from conclusively disproving our hypothesis, the current study does not provide data supporting a role for HERV-specific T-cell responses in HTLV-1 associated immunopathology.

Treatment of neuromyelitis optica: an evidence based review

Neuromyelitis optica (NMO) is an inflammatory disease of the central nervous system characterized by severe optic neuritis and transverse myelitis, usually with a relapsing course. Aquaporin-4 antibody is positive in a high percentage of NMO patients and it is directed against this water channel richly expressed on foot processes of astrocytes. Due to the severity of NMO attacks and the high risk for disability, treatment should be instituted as soon as the diagnosis is confirmed. There is increasing evidence that NMO patients respond differently from patients with multiple sclerosis (MS), and, therefore, treatments for MS may not be suitable for NMO. Acute NMO attacks usually are treated with high dose intravenous corticosteroid pulse and plasmapheresis. Maintenance therapy is also required to avoid further attacks and it is based on low-dose oral corticosteroids and non-specific immunosuppressant drugs, like azathioprine and mycophenolate mofetil. New therapy strategies using monoclonal antibodies like rituximab have been tested in NMO, with positive results in open label studies. However, there is no controlled randomized trial to confirm the safety and efficacy for the drugs currently used in NMO.

Standard surgical treatment for benign prostatic hyperplasia is safe for patients over 75 years: analysis of 100 cases from a high-volume urologic center

OBJECTIVES: In this study, we aimed to determine the complications of standard surgical treatments among patients over 75 years in a high-volume urologic center. METHODS: We analyzed 100 consecutive patients older than 75 years who had undergone transurethral prostatic resection of the prostate or open prostatectomy for treatment of benign prostatic hyperplasia from January 2008 to March 2010. We analyzed patient age, prostate volume, prostate-specific antigen level, international prostatic symptom score, quality of life score, urinary retention, co-morbidities, surgical technique and satisfaction with treatment. RESULTS: Median age was 79 years. Forty-eight patients had undergone transurethral prostatic resection of the prostate, and 52 had undergone open prostatectomy. The median International Prostatic Symptom Score was 20, the median prostate volume was 83 g, 51% were using an indwelling bladder catheter, and the median prostatespecific antigen level was 5.0 ng/ml. The most common comorbidities were hypertension, diabetes and coronary disease. After a median follow-up period of 17 months, most patients were satisfied. Complications were present in 20% of cases. The most common urological complication was urethral stenosis, followed by bladder neck sclerosis, urinary fistula, late macroscopic hematuria and persistent urinary incontinence. The most common clinical complication was myocardial infarction, followed by acute renal failure requiring dialysis. Incidental carcinoma of the prostate was present in 6% of cases. One case had urothelial bladder cancer. CONCLUSIONS: Standard surgical treatments for benign prostatic hyperplasia are safe and satisfactory among the elderly. Complications are infrequent, and urethral stenosis is the most common. No clinical variable is associated with the occurrence of complications.

Differential cell type-specific transcriptional regulation of the CYP1A1 gene

Cytochrome P450 1A1 (CYP1A1) monooxygenase plays an important role in the metabolism of environmental pollutants such as polycyclic aromatic hydrocarbons (PAHs) and halogenated polycyclic aromatic hydrocarbons (HAHs). Oxidation of these compounds converts them to the metabolites that subsequently can be conjugated to hydrophilic endogenous entities e.g. glutathione. Derivates generated in this way are water soluble and can be excreted in bile or urine, which is a defense mechanism. Besides detoxification, metabolism by CYP1A1 may lead to deleterious effects since the highly reactive intermediate metabolites are able to react with DNA and thus cause mutagenic effects, as it is in the case of benzo(a) pyrene (B[a]P). CYP1A1 is normally not expressed or expressed at a very low level in the cells but it is inducible by many PAHs and HAHs e.g. by B[a]P or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Transcriptional activation of the CYP1A1 gene is mediated by aryl hydrocarbon receptor (AHR), a basic-helix-loop-helix (bHLH) transcription factor. In the absence of a ligand AHR stays predominantly in the cytoplasm. Ligand binding causes translocation of AHR to the nuclear compartment, its heterodimerization with another bHLH protein, the aryl hydrocarbon nuclear translocator (ARNT) and binding of the AHR/ARNT heterodimer to a DNA motif designated dioxin responsive element (DRE). This process leads to the transcriptional activation of the responsive genes containing DREs in their regulatory regions, e.g. that coding for CYP1A1. TCDD is the most potent known agonist of AHR. Since it is not metabolized by the activated enzymes, exposure to this compound leads to a persisting activation of AHR resulting in diverse toxic effects in the organism. To enlighten the molecular mechanisms that mediate the toxicity of xenobiotics like TCDD and related compounds, the AHR-dependent regulation of the CYP1A1 gene was investigated in two cell lines: human cervix carcinoma (HeLa) and mouse hepatoma (Hepa). Study of AHR activation and its consequence concerning expression of the CYP1A1 enzyme confirmed the TCDD-dependent formation of the AHR/ARNT complex on DRE leading to an increase of the CYP1A1 transcription in Hepa cells. In contrast, in HeLa cells formation of the AHR/ARNT heterodimer and binding of a protein complex containing AHR and ARNT to DRE occurred naturally in the absence of TCDD. Moreover, treatment with TCDD did not affect the AHR/ARNT dimer formation and binding of these proteins to DRE in these cells. Even though the constitutive complex on DRE exists in HeLa, transcription of the CYP1A1 gene was not increased. Furthermore, the CYP1A1 level in HeLa cells remained unchanged in the presence of TCDD suggesting repressional mechanism of the AHR complex function which may hinder the TCDD-dependent mechanisms in these cells. Similar to the native, the mouse CYP1A1-driven reporter constructs containing different regulatory elements were not inducible by TCDD in HeLa cells, which supported a presence of cell type specific trans-acting factor in HeLa cells able to repress both the native CYP1A1 and CYP1A1-driven reporter genes rather than species specific differences between CYP1A1 genes of human and rodent origin. The different regulation of the AHR-mediated transcription of CYP1A1 gene in Hepa and HeLa cells was further explored in order to elucidate two aspects of the AHR function: (I) mechanism involved in the activation of AHR in the absence of exogenous ligand and (II) factor that repress function of the exogenous ligand-independent AHR/ARNT complex. Since preliminary studies revealed that the activation of PKA causes an activation of AHR in Hepa cells in the absence of TCDD, the PKA-dependent signalling pathway was the proposed endogenous mechanism leading to the TCDD-independent activation of AHR in HeLa cells. Activation of PKA by forskolin or db-cAMP as well as inhibition of the kinase by H89 in both HeLa and Hepa cells did not lead to alterations in the AHR interaction with ARNT in the absence of TCDD and had no effect on binding of these proteins to DRE. Moreover, the modulators of PKA did not influence the CYP1A1 activity in these cells in the presence and in the absence of TCDD. Thus, an involvement of PKA in the regulation of the CYP1A1 Gen in HeLa cells was not evaluated in the course of this study. Repression of genes by transcription factors bound to their responsive elements in the absence of ligands has been described for nuclear receptors. These receptors interact with protein complex containing histone deacetylase (HDAC), enzyme responsible for the repressional effect. Thus, a participation of histone deacetylase in the transcriptional modulation of CYP1A1 gene by the constitutively DNA-bound AHR/ARNT complex was supposed. Inhibition of the HDAC activity by trichostatin A (TSA) or sodium butyrate (NaBu) led to an increase of the CYP1A1 transcription in the presence but not in the absence of TCDD in Hepa and HeLa cells. Since amount of the AHR and ARNT proteins remained unchanged upon treatment of the cells with TSA or NaBu, the transcriptional upregulation of CYP1A1 gene was not due to an increased expression of the regulatory proteins. These findings strongly suggest an involvement of HDAC in the repression of the CYP1A1 gene. Similar to the native human CYP1A1 also the mouse CYP1A1-driven reporter gene transfected into HeLa cells was repressed by histone deacetylase since the presence of TSA or NaBu led to an increase in the reporter activity. Induction of reporter gene did not require a presence of the promoter or negative regulatory regions of the CYP1A1 gene. A promoter-distal fragment containing three DREs together with surrounding sequences was sufficient to mediate the effects of the HDAC inhibitors suggesting that the AHR/ARNT binding to its specific DNA recognition site may be important for the CYP1A1 repression. Histone deacetylase is recruited to the specific genes by corepressors, proteins that bind to the transcription factors and interact with other members of the HDAC complex. Western blot analyses revealed a presence of HDAC1 and the corepressors mSin3A (mammalian homolog of yeast Sin3) and SMRT (silencing mediator for retinoid and thyroid hormone receptor) in both cell types, while the corepressor NCoR (nuclear receptor corepressor) was expressed exclusively in HeLa cells. Thus the high inducibility of CYP1A1 in Hepa cells may be due to the absence of NCoR in these cells in contrast to the non-responsive HeLa cells, where the presence of NCoR would support repression of the gene by histone deacetylase. This hypothesis was verified in reporter gene experiments where expression constructs coding for the particular members of the HDAC complex were cotransfected in Hepa cells together with the TCDD-inducible reporter constructs containing the CYP1A1 regulatory sequences. An overexpression of NCoR however did not decrease but instead led to a slight increase of the reporter gene activity in the cells. The expected inhibition was observed solely in the case of SMRT that slightly reduced constitutive and TCDD-induced reporter gene activity. A simultaneous expression of NCoR and SMRT shown no further effects and coexpression of HDAC1 with the two corepressors did not alter this situation. Thus, additional factors that are likely involved in the repression of CYP1A1 gene by HDAC complex remained to be identified. Taking together, characterisation of an exogenous ligand independent AHR/ARNT complex on DRE in HeLa cells that repress transcription of the CYP1A1 gene creates a model system enabling investigation of endogenous processes involved in the regulation of AHR function. This study implicates HDAC-mediated repression of CYP1A1 gene that contributes to the xenobiotic-induced expression in a tissue specific manner. Elucidation of these processes gains an insight into mechanisms leading to deleterious effects of TCDD and related compounds.

Genetic engineering and preclinical evaluation of oncolytic herpes simplex viruses retargeted to cancer-specific receptors

Oncolytic virotherapy exploits the ability of viruses to infect and kill cells. It is suitable as treatment for tumors that are not accessible by surgery and/or respond poorly to the current therapeutic approach. HSV is a promising oncolytic agent. It has a large genome size able to accommodate large transgenes and some attenuated oncolytic HSVs (oHSV) are already in clinical trials phase I and II. The aim of this thesis was the generation of HSV-1 retargeted to tumor-specific receptors and detargeted from HSV natural receptors, HVEM and Nectin-1. The retargeting was achieved by inserting a specific single chain antibody (scFv) for the tumor receptor selected inside the HSV glycoprotein gD. In this research three tumor receptors were considered: epidermal growth factor receptor 2 (HER2) overexpressed in 25-30% of breast and ovarian cancers and gliomas, prostate specific membrane antigen (PSMA) expressed in prostate carcinomas and in neovascolature of solid tumors; and epidermal growth factor receptor variant III (EGFRvIII). In vivo studies on HER2 retargeted viruses R-LM113 and R-LM249 have demonstrated their high safety profile. For R-LM249 the antitumor efficacy has been highlighted by target-specific inhibition of the growth of human tumors in models of HER2-positive breast and ovarian cancer in nude mice. In a murine model of HER2-positive glioma in nude mice, R-LM113 was able to significantly increase the survival time of treated mice compared to control. Up to now, PSMA and EGFRvIII viruses (R-LM593 and R-LM613) are only characterized in vitro, confirming the specific retargeting to selected targets. This strategy has proved to be generally applicable to a broad spectrum of receptors for which a single chain antibody is available.

Flagellin:allergen fusion proteins as novel vaccines for the treatment of severe type I allergies

Over the last decades the prevalence of food allergies has continually increased on a world wide scale. While there are effective treatments available for bee and wasp venom allergic patients, there is currently no established therapy for the treatment of severe food allergies. Aim of the project was to genetically fuse different food allergens with the immune modulating Toll-like receptor 5 (TLR5)-ligand flagellin and to test these constructs for their immune modulatory capacities both in vitro and in vivo. Chicken ovalbumin (Ova) as model antigen, Pru p 3, and Ara h 2 the respective major allergens from peach and peanut were used as allergens. The potential vaccine candidates were characterized by protein biochemical methods (purity, folding, endotoxin contaminations). Moreover, their immune modulating effects on cell culture lines (TLR5-receptor activation) and primary mouse immune cells (myeloid and plasmacytoid dendritic cells) were investigated. Additionally, the prophylactic and therapeutic use of the flagellin Ova fusion protein (rflaA:Ova) were investigated in a mouse model of intestinal allergy. In myeloid dendritic cells (mDC) stimulation with the fusion proteins led to a strong cell activation and cytokine secretion. Here, the fusion proteins proved to be a much stronger stimulus than the equimolar amount of both proteins provided alone or as a mixture. Noteworthy, stimulation with rflaA:Ova induced the secretion of the anti-inflammatory cytokine IL-10 from mDC. In co-culture experiments this IL-10 secretion suppressed the Ova-induced secretion of Th1 and Th2 cytokines from Ova-specific CD4 T cells. Using MyD88-deficient mDC this repression of cytokine secretion was shown to be TLR-dependent. Finally, the potency of the rflaA:Ova construct was investigated in a mouse model of Ova-induced intestinal allergy. In a prophylactic vaccination approach rflaA:Ova was shown to prevent the establishment of the intestinal allergy and all associated symptoms (weight loss, temperature drop, soft faeces). This fusion protein-mediated protection was accompanied by a reduced T cell activation, and reduced Th2 cytokines in intestinal homogenates. These effects were paralleled by a strong induction of Ova-specific IgG2a antibodies in rflaA:Ova-vaccinated sera, while Ova-specific IgE antibody production was significantly reduced. Therapeutic vaccination with rflaA:Ova reduced allergic symptoms and T cell activation but did not influence weight loss and antibody production. In all in vivo experiments vaccination with both proteins either provided alone or as a mixture did not have comparable effects. Future experiments aim at elucidating the mechanism and further optimization of the therapeutic vaccination approach. The results presented in this thesis demonstrate, that fusion proteins containing flagellin have strong immune modulatory capacities both in vitro and in vivo. Therefore, such constructs are promising vaccine candidates for the therapy of type I allergies.

Study of PI3K/Akt signaling pathway as potential molecular target for T-cell acute lymphoblastic leukemia (T-ALL) treatment: pan-inhibition of PI3K catalitic isoforms as better therapeutic approach

Class I phosphatidylinositol 3-kinases (PI3Ks) are heterodimeric lipid kinases consisting of a regulatory subunit and one of four catalytic subunits (p110α, p110β, p110γ or p110δ). p110γ/p110δ PI3Ks are highly enriched in leukocytes. In general, PI3Ks regulate a variety of cellular processes including cell proliferation, survival and metabolism, by generating the second messenger phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P3). Their activity is tightly regulated by the phosphatase and tensin homolog (PTEN) lipid phosphatase. PI3Ks are widely implicated in human cancers, and in particular are upregulated in T-cell acute lymphoblastic leukemia (T-ALL), mainly due to loss of PTEN function. These observations lend compelling weight to the application of PI3K inhibitors in the therapy of T-ALL. At present different compounds which target single or multiple PI3K isoforms have entered clinical trials. In the present research, it has been analyzed the therapeutic potential of the pan-PI3K inhibitor BKM120, an orally bioavailable 2,6-dimorpholino pyrimidine derivative, which has entered clinical trials for solid tumors, on both T-ALL cell lines and patient samples. BKM120 treatment resulted in cell cycle arrest and apoptosis, being cytotoxic to a panel of T-ALL cell lines and patient T-lymphoblasts. Remarkably, BKM120 synergized with chemotherapeutic agents currently used for treating T-ALL patients. BKM120 efficacy was confirmed in in vivo studies to a subcutaneous xenotransplant model of human T-ALL. Because it is still unclear which agents among isoform-specific or pan inhibitors can achieve the greater efficacy, further analyses have been conducted to investigate the effects of PI3K inhibition, in order to elucidate the mechanisms responsible for the proliferative impairment of T-ALL. Overall, these results indicated that BKM120 may be an efficient treatment for T-ALLs that have aberrant up-regulation of the PI3K signaling pathway and strongly support clinical application of pan-class I PI3K rather than single-isoform inhibitors in T-ALL treatment.

Development of a highly potent bispecific antibody format targeting the novel tumor-specific antigen CLDN18.2

Due to multiple immune evasion mechanisms of cancer cells, novel therapy approaches are required to overcome the limitations of existing immunotherapies. Bispecific antibodies are potent anti-cancer drugs, which redirect effector T cells for specific tumor cell lysis, thus enabling the patient’s immune system to fight cancer cells. The antibody format used in this proof of concept study–bispecific ideal monoclonal antibodies termed BiMAB–is a tailor-made recombinant protein, which consists of two fused scFv antibodies recognizing different antigens. Both are arranged in tandem on a single peptide chain and the individual variable binding domains are separated by special non-immunogenic linkers. The format is comprised of a scFv targeting CLDN18.2–a gastric cancer tumor associated antigen (TAA) –while the second specificity binds the CD3 epsilon (CD3ε) subunit of the T cell receptor (TCR) on T cells. For the first time, we compared in our IMAB362-based BiMAB setting, four different anti-CD3-scFvs, respectively derived from the mAbs TR66, CLB-T3, as well as the humanized and the murine variant of UCHT1. In addition, we investigated the impact of an N- versus a C-terminal location of the IMAB362-derived scFv and the anti-CD3-scFvs. Thus, nine CLDN18.2 specific BiMAB proteins were generated, of which all showed a remarkably high cytotoxicity towards CLDN18.2-positive tumor cells. Because of its promising effectiveness, 1BiMAB emerged as the BiMAB prototype. The selectivity of 1BiMAB for its TAA and CD3ε, with affinities in the nanomolar range, has been confirmed by in vitro assays. Its dual binding depends on the design of an N-terminally positioned IMAB362 scFv and the consecutive C-terminally positioned TR66 scFv. 1BiMAB provoked a concentration and target cell dependent T cell activation, proliferation, and upregulation of the cytolytic protein Granzyme B, as well as the consequent elimination of target cells. Our results demonstrate that 1BiMAB is able to activate T cells independent of elements that are usually involved in the T cell recognition program, like antigen presentation, MHC restriction, and co-stimulatory effector molecules. In the first in vivo studies using a subcutaneous xenogeneic tumor mouse model in immune incompetent NSG mice, we could prove a significant therapeutic effect of 1BiMAB with partial or complete tumor elimination. The initial in vitro RIBOMAB experiments correspondingly showed encouraging results. The electroporation of 1BiMAB IVT-RNA into target or effector cells was feasible, while the functionality of translated 1BiMAB was proven by induced T cell activation and target cell lysis. Accordingly, we could show that the in vitro RIBOMAB approach was applicable for all nine BiMABs, which proves the RIBOMAB concept. Thus, the CLDN18.2-BiMAB strategy offers great potential for the treatment of cancer. In the future, administered either as protein or as IVT-RNA, the BiMAB format will contribute towards finding solutions to raise and sustain tumor-specific cellular responses elicited by engaged and activated endogenous T cells. This will potentially enable us to overcome immune evasion mechanisms of tumor cells, consequently supporting current solid gastric cancer therapies.

Implementation of enhanced biological phosphorus removal (ebpr) wastewater treatment processes enriched with different microbial communities

The EBPR (Enhanced Biological Phosphorus Removal) is a type of secondary treatment in WWTPs (WasteWater Treatment Plants), quite largely used in full-scale plants worldwide. The phosphorus occurring in aquatic systems in high amounts can cause eutrophication and consequently the death of fauna and flora. A specific biomass is used in order to remove the phosphorus, the so-called PAOs (Polyphosphate Accumulating Organisms) that accumulate the phosphorus in form of polyphosphate in their cells. Some of these organisms, the so-called DPAO (Denitrifying Polyphosphate Accumulating Organisms) use as electron acceptor the nitrate or nitrite, contributing in this way also to the removal of these compounds from the wastewater, but there could be side reactions leading to the formation of nitrous oxides. The aim of this project was to simulate in laboratory scale a EBPR, acclimatizing and enriching the specialized biomass. Two bioreactors were operated as Sequencing Batch Reactors, one enriched in Accumulibacter, the other in Tetrasphaera (both PAOs): Tetrasphaera microorganisms are able to uptake aminoacids as carbon source, Accumulibacter uptake organic carbon (volatile fatty acids, VFA). In order to measure the removal of COD, phosphorus and nitrogen-derivate compounds, different analysis were performed: spectrophotometric measure of phosphorus, nitrate, nitrite and ammonia concentrations, TOC (Total Organic Carbon, measuring the carbon consumption), VFA via HPLC (High Performance Liquid Chromatography), total and volatile suspended solids following standard methods APHA, qualitative microorganism population via FISH (Fluorescence In Situ Hybridization). Batch test were also performed to monitor the NOx production. Both specialized populations accumulated as a result of SBR operations; however, Accumulibacter were found to uptake phosphates at higher extents. Both populations were able to remove efficiently nitrates and organic compounds occurring in the feeding. The experimental work was carried out at FCT of Universidade Nova de Lisboa (FCT-UNL) from February to July 2014.

Lineage-specific transcription factor aberrations in AML

Transcription factors play a key role in the commitment of hematopoietic stem cells to differentiate into specific lineages [78]. This is particularly important in that a block in terminal differentiation is the key contributing factor in acute leukemias. This general theme of the role of transcription factors in differentiation may also extend to other tissues, both in terms of normal development and cancer. Consistent with the role of transcription factors in hematopoietic lineage commitment is the frequent finding of aberrations in transcription factors in AML patients. Here, we intend to review recent findings on aberrations in lineage-restricted transcription factors as observed in patients with acute myeloid leukemia (AML).

Organ-specific eosinophilic disorders of the skin, lung, and gastrointestinal tract

Eosinophils are multifunctional leukocytes that increase in various tissues in patients with a variety of disorders. Locally, they can be involved in the initiation and propagation of diverse inflammatory responses. In this review the clinical association of eosinophils with diseases of the skin, lung, and gastrointestinal tract is summarized. An approach to determining the causal role of eosinophils in these diseases is presented. Recent findings concerning molecular diagnosis, cause, and treatment are discussed.