The Development of Candidate Therapeutics for Transmissible Spongiform Encephalopathies

Previous studies have shown that addition of recombinant prion into a cell free prion replication assay – PMCA inhibits the formation of PrPSc. Previously naturally existing versions of ovine prion protein were tested: rARQ, rARR and rVRQ within this assay. Of these, rVRQ was the most potent inhibitor of amplification of different scrapie isolates (IC50 value 120 nM) and bovine BSE (IC50 – 171 nM). The main aim of this study was to produce additional molecular clones for expression of recombinant ovine prion protein where codon 136 had been mutated to code for different amino acids. These rPrPs were tested in dose response experiments in order to investigate whether the change at 136 position in ovine PrP could impact on the ability to inhibit or stop the prion protein misfolding compared to previously tested rVRQ. In order to produce rPrP mutants at codon 136, site-directed mutagenesis was used. All rPrPs were purified by metal affinity chromatography taking advantage of the metal binding properties of PrP molecule. All mutated rPrPs were added to protein misfolding cyclic amplification (PMCA) at different concentration and compared to rVRQ. After amplification, samples were digested with Proteinase K (100 µg/ml) and quantified on immunoblots. The best inhibitors were tested with different ovine scrapie (ARQ/VRQ, VRQ/VRQ, AHQ/VRQ), bovine BSE and ovine BSE isolates (ARQ/ARQ). The results showed that three of the recombinant prion proteins: rRRQ, rKRQ and rPRQ (with arginine, lysine and proline at 136 position, respectively) were found to inhibit the PrPSc misfolding significantly better than naturally occurred rVRQ. The structure of rPrP variants and amino acid substitution at 136 position were analysed and different length peptides containing the valine, arginine, lysine and proline at 136 position were designed. None of these peptides analysed in PMCA gave similar levels of inhibition to the equivalent full length recombinant prion protein response. Moreover, structural analysis showed that introduction of longer amino acids at position 136 did not alter the whole scaffold of prion protein. In addition, the longer side chains for arginine136 and lysine136 or pyrrolidine loop in proline could result in more interatomic bonding in comparison to valine136 and therefore could act to stabilize the whole PrP molecule. Furthermore, the presence of the longer side chains of arginine136 and lysine136 would not predict further structure changes because of the ‘structural’ pocket present on the opposite site of position 136 in ovine PrP. The Rov9 cell line could be persistently infected with processed (heated and sonicated) scrapie brain homogenate SSBP1 (VRQ/VRQ) and SSBP1 derived, NaPTA precipitated PrPSc. In both cases, PrPres was detected in cell lysates from induced with 1 µg/ml doxycycline and when 500 µg of total protein was digested with 20 µg/ml of PK followed by PrPres concentration by centrifugation. The best inhibitory rPrPs were used in experiments to prevent the infection with SSBP1 isolate or reduce the PrPres in persistently infected Rov9 cells. As a result, addition of 250 nM of rRRQ, rKRQ and rPRQ prevented the infection of Rov9 cells at culture passage 1. The rPrP variants showed more promising results than natural rVRQ. In contrast, no significant reduction of PrPres was observed when persistently infected Rov9 cells were treated with 250 nM of either variants or natural rPrPs for 4 days. Overall, this work demonstrated a novel therapeutic approach for prion diseases using recombinant prion proteins. The recombinant protein treatment was effective not only in scrapie model but also among other TSEs and therefore these rPrPs or analogous strategy could be applied as potential human TSE therapeutic

The Development of Candidate Therapeutics for Transmissible Spongiform Encephalopathies

Previous studies have shown that addition of recombinant prion into a cell free prion replication assay – PMCA inhibits the formation of PrPSc. Previously naturally existing versions of ovine prion protein were tested: rARQ, rARR and rVRQ within this assay. Of these, rVRQ was the most potent inhibitor of amplification of different scrapie isolates (IC50 value 120 nM) and bovine BSE (IC50 – 171 nM). The main aim of this study was to produce additional molecular clones for expression of recombinant ovine prion protein where codon 136 had been mutated to code for different amino acids. These rPrPs were tested in dose response experiments in order to investigate whether the change at 136 position in ovine PrP could impact on the ability to inhibit or stop the prion protein misfolding compared to previously tested rVRQ. In order to produce rPrP mutants at codon 136, site-directed mutagenesis was used. All rPrPs were purified by metal affinity chromatography taking advantage of the metal binding properties of PrP molecule. All mutated rPrPs were added to protein misfolding cyclic amplification (PMCA) at different concentration and compared to rVRQ. After amplification, samples were digested with Proteinase K (100 µg/ml) and quantified on immunoblots. The best inhibitors were tested with different ovine scrapie (ARQ/VRQ, VRQ/VRQ, AHQ/VRQ), bovine BSE and ovine BSE isolates (ARQ/ARQ). The results showed that three of the recombinant prion proteins: rRRQ, rKRQ and rPRQ (with arginine, lysine and proline at 136 position, respectively) were found to inhibit the PrPSc misfolding significantly better than naturally occurred rVRQ. The structure of rPrP variants and amino acid substitution at 136 position were analysed and different length peptides containing the valine, arginine, lysine and proline at 136 position were designed. None of these peptides analysed in PMCA gave similar levels of inhibition to the equivalent full length recombinant prion protein response. Moreover, structural analysis showed that introduction of longer amino acids at position 136 did not alter the whole scaffold of prion protein. In addition, the longer side chains for arginine136 and lysine136 or pyrrolidine loop in proline could result in more interatomic bonding in comparison to valine136 and therefore could act to stabilize the whole PrP molecule. Furthermore, the presence of the longer side chains of arginine136 and lysine136 would not predict further structure changes because of the ‘structural’ pocket present on the opposite site of position 136 in ovine PrP. The Rov9 cell line could be persistently infected with processed (heated and sonicated) scrapie brain homogenate SSBP1 (VRQ/VRQ) and SSBP1 derived, NaPTA precipitated PrPSc. In both cases, PrPres was detected in cell lysates from induced with 1 µg/ml doxycycline and when 500 µg of total protein was digested with 20 µg/ml of PK followed by PrPres concentration by centrifugation. The best inhibitory rPrPs were used in experiments to prevent the infection with SSBP1 isolate or reduce the PrPres in persistently infected Rov9 cells. As a result, addition of 250 nM of rRRQ, rKRQ and rPRQ prevented the infection of Rov9 cells at culture passage 1. The rPrP variants showed more promising results than natural rVRQ. In contrast, no significant reduction of PrPres was observed when persistently infected Rov9 cells were treated with 250 nM of either variants or natural rPrPs for 4 days. Overall, this work demonstrated a novel therapeutic approach for prion diseases using recombinant prion proteins. The recombinant protein treatment was effective not only in scrapie model but also among other TSEs and therefore these rPrPs or analogous strategy could be applied as potential human TSE therapeutic

In vitro antileukemic activity of novel adenosine derivatives bearing boron cluster modification

A series of adenosine derivatives bearing a boron cluster were synthesized and evaluated for their cytotoxicity against primary peripheral mononuclear cells from the blood of 17 patients with leukemias (16 CLL and 1 very rare PLL), as well as from 5 healthy donors used as a control. Among the tested agents, two, i.e., compounds 1 and 2, displayed high in vitro cytotoxicity and proapoptotic potential on leukemic cells, with only scarce activity being seen against control cells. Biological tests related to apoptosis revealed the activation of the main execution apoptotic enzyme, procaspase-3, in CLL and PLL cells exposed to compounds 1 and 2. Moreover, the above compounds indicated high activity in the proteolysis of the apoptotic markers PARP-1 and lamin B1, fragmentation of DNA, and the induction of some changes in the expression of the Mcl-1, protein apoptosis regulator in comparison with control cells

A trematode parasite derived growth factor binds and exerts influences on host immune functions via host cytokine receptor complexes

The trematode Fasciola hepatica is responsible for chronic zoonotic infection globally. Despite causing a potent T-helper 2 response, it is believed that potent immunomodulation is responsible for rendering this host reactive non-protective host response thereby allow- ing the parasite to remain long-lived. We have previously identified a growth factor, FhTLM, belonging to the TGF superfamily can have developmental effects on the parasite. Herein we demonstrate that FhTLM can exert influence over host immune functions in a host receptor specific fashion. FhTLM can bind to receptor members of the Transforming Growth Factor (TGF) superfamily, with a greater affinity for TGF-β RII. Upon ligation FhTLM initiates the Smad2/3 pathway resulting in phenotypic changes in both fibroblasts and macrophages. The formation of fibroblast CFUs is reduced when cells are cultured with FhTLM, as a result of TGF-β RI kinase activity. In parallel the wound closure response of fibroblasts is also delayed in the presence of FhTLM. When stimulated with FhTLM blood monocyte derived macrophages adopt an alternative or regulatory phenotype. They express high levels interleukin (IL)-10 and arginase-1 while displaying low levels of IL-12 and nitric oxide. Moreover they also undergo significant upregulation of the inhibitory recep- tor PD-L1 and the mannose receptor. Use of RNAi demonstrates that this effect is depen- dent on TGF-β RII and mRNA knock-down leads to a loss of IL-10 and PD-L1. Finally, we demonstrate that FhTLM aids newly excysted juveniles (NEJs) in their evasion of antibody- dependent cell cytotoxicity (ADCC) by reducing the NO response of macrophages—again dependent on TGF-β RI kinase. FhTLM displays restricted expression to the F. hepatica gut resident NEJ stages. The altered fibroblast responses would suggest a role for damp- ened tissue repair responses in facilitating parasite migration. Furthermore, the adoption of a regulatory macrophage phenotype would allow for a reduced effector response targetingjuvenile parasites which we demonstrate extends to an abrogation of the ADCC response. Thus suggesting that FhTLM is a stage specific evasion molecule that utilises host cytokine receptors. These findings are the first to clearly demonstrate the interaction of a helminth cytokine with a host receptor complex resulting in immune modifications that facilitate the non-protective chronic immune response which is characteristic of F. hepatica infection

Intrauterine contraceptive device embedded in the omentum – case report

Piotr Zolnierczyk, Krzysztof Cendrowski, Wlodzimierz Sawicki Department of Obstetrics, Gynecology and Oncology, 2nd Faculty of Medicine, Medical University of Warsaw, Warsaw, Poland Abstract: This report describes the case of a 29-year-old patient, female (nulliparous) who had an intrauterine device (IUD) inserted in 2010 and who has had no gynecological control since then (for 4 years). After this time, the asymptomatic patient had a gynecological appointment, during which a doctor did not find the strings of IUD in the speculum. Ultrasound examination did not reveal the presence of the IUD in the uterine cavity, which led to the suspicion of its presence outside the uterus. The patient was referred to a hospital, where she underwent ultrasound and X-ray examination of the pelvis that confirmed the presence of the IUD outside the uterus. Laparoscopy was performed during which the IUD was localized as being embedded in the omentum. It was removed by performing a resection of a part of the omentum with inflammatory infiltration. The patient was discharged home on the second postoperative day in a good condition. This case confirms the need for gynecological control and ultrasound examination shortly after insertion. An ultrasound or/and X-ray is mandatory in any case of absence of IUD strings previously visible in the vagina, if the patient did not observe its expulsion. Keywords: intrauterine device, myometrium, IUD threads, uterine cavity, ultrasound examinatio

Transfundal puncture of a large ovarian cyst with hysteroscopic and ultrasonographic guidance

Piotr Zolnierczyk, Krzysztof Cendrowski, Wlodzimierz Sawicki Department of Obstetrics, Gynecology and Oncology, 2nd Faculty of Medicine, Medical University of Warsaw, Warsaw, Poland Abstract: This paper describes the case of an 83-year-old patient with hypertension, diabetes, obesity (body mass index – 38), congestive heart failure, and history of cardiac surgery, who was referred for a diagnostic–therapeutic decompression of a large, symptomatic ovarian cyst. Due to anatomical conditions, the only safe way was a transfundal puncture under mini-hysteroscopic and ultrasound guidance. A puncture with aspiration of 300 mL of serous fluid from the cyst was performed without technical problems and complications. Cytology showed no cancer cells in the examined liquid. Relief from pain and compression discomfort was achieved in the patient. This case shows the possibility of combining ultrasound and minimally invasive diagnostic methods like hysteroscopy in selected clinical situations. Keywords: ovarian cyst puncture, hysteroscopy, ultrasound guided puncture, transfundal cyst puncture, vaginoscopy&nbsp

A User Study for the Evaluation of Adaptive Interaction Systems for Inclusive Industrial Workplaces

In recent years, production systems have become highly sophisticated and complex. As a result, while on the one hand, the least-skilled labor has been partially displaced by machines, high-skilled labor is more required to supervise and control advanced automation systems. In many cases, the complexity of machines implies an increased complexity of human-machine interfaces (HMIs), which are the main point of contact between the operator and the machine. To enable effective use of HMIs and to enable their usage by workers with different knowledge and capabilities, novel design approaches have been proposed. In particular, in this article, we consider the approach developed in the framework of the European research project INCLUSIVE, which aimed at designing industrial HMIs that adapt to the skills and capabilities of human operators. As a case study, we consider an adaptive interaction system for the woodworking industry and present an extensive evaluation carried out in real production environment with shopfloor workers. The effectiveness of the INCLUSIVE approach has been assessed with subjective and objective measurements and compared to that of interaction systems customarily used in industry. Results have shown that users appreciated the INCLUSIVE system and largely preferred it over the customary system. Moreover, with regard to objective performance-related measurements, they performed better when using the INCLUSIVE system since they received tailored guidance during the considered working tasks