Epitope mapping of a PrP(Sc)-specific monoclonal antibody: identification of a novel C-terminally truncated prion fragment

Monoclonal antibodies (mAbs) against prion proteins (PrPs) are indispensable in research and diagnosis of prion diseases, however the majority of these bind both the cellular (PrP^C) and the disease-associated (PrP^Sc) isoforms. According to the widely accepted protein-only hypothesis the two isoforms share the same sequence, but differ in their conformation. In the present study we set to determine the critical binding residues of our PrP^Sc-specific mAbs with the view of discerning which residues play a key role in the conformational transition between PrP^C and PrP^Sc. Focussing on the V5B2 mAb that provided differential labelling of prion-affected tissue from individuals positive for transmissible spongiform encephalopathies, we performed alanine scanning and phage-display epitope mapping to elucidate the antigenic determinants of this mAb and gain insight into its specificity on a molecular level. We observed that instead of discriminating between the two prion protein isoforms based on conformational differences, V5B2 binds a previously uncharacterized C-terminally truncated form of PrP^Sc that ends with the residue Y226, which we named PrP226*. The addition of a single C-terminal amino-acid residue completely abolished V5B2 binding, while Western blots using recombinant full-length PrPs and PrPs terminating at Y226 confirmed that the V5B2 mAb discriminates between the two based on their difference in length.     

Osteoblastic bone metastases from renal cell carcinoma

Background

RCC accounts for only 2–3% of all cancers. Due to its’ non-specific symptoms disease is often diagnosed in advanced stage. Disseminated RCC frequently produces bone metastases that are almost always highly destructive, hyper vascularized and purely osteolytic.

Case report.

In this article we describe a case of a 71-year old male patient with disseminated osteoblastic bone metastases from renal cell carcinoma (RCC), and present a short review of published literature reporting cases of osteoblastic bone metastases from RCC. Our patient presented with thoracic pain aggravated by movement. He was diagnosed with predominantly osteoblastic bone metastases in the skeleton of thoracic and lumbar vertebra along with metastases in iliac bones, ribs, humerus and clavicles. Initially, origin of bone metastases was unknown, but later a small tumor in patient’s right kidney was identified. Microscopic evaluation of the open bone biopsy showed clear cell RCC with sarcomatoid differentiation.

Conclusions

Although, due to its’ rarity, RCC is not included in the primary differential diagnosis in patients with osteoblastic metastases, such rare cases suggest that RCC may be considered in the diagnosis when there no other primary tumor is found.     

Obstacles and pitfalls in the PEGylation of therapeutic proteins

In recent years, PEGylation has become widely used as a post-production modification methodology for improving the biomedical efficacy and physicochemical properties of therapeutic proteins. Several marketed drugs that have already been in use for more than a decade have proved the applicability and safety of this technology and, with the successes already achieved, it is expected that PEGylation will be applied to other potential therapeutic proteins. The non-biodegradable nature of PEG, however, may become a limiting factor for the next generation of protein pharmaceuticals, for which use in high concentrations and in the long term is the aim, especially considering the trend for the use of branched and high molecular mass PEGs. This review addresses various obstacles and pitfalls in the production of PEGylated biopharmaceuticals, in particular, the specificity of PEGylation reactions, separation and purification issues, the analysis of inherently polydisperse PEG reagents and PEGylated products, the consistency of products and processes, and the accurate determination of pharmacokinetic properties.     

Cone-beam Computed Tomography Guided Nusinersen Administrations in Adult Spinal Muscular Atrophy Patients with Challenging Access: A Single- Center Experience

BackgroundThe challenging anatomic predispositions in adult patients with spinal muscular atrophy (SMA) preclude the conventional lumbar punctures. Consequently, an introduction of alternative method for intrathecal delivery of nusinersen is required. Cone-beam CT (CBCT) allows volumetric display of the area of interest, pre-procedural planning and real time needle guidance which results in accurate anatomic navigation. The aim of the study was to evaluate technical success, safety, and feasibility of CBCT lumbar intrathecal delivery of nusinersen in the adult SMA patients with challenging anatomical access.Patients and methodsThirty-eight adult SMA patients were treated in our institution. Patients with challenging access were selected by multidisciplinary board for image guided administration of nusinersen either due to implantation of the posterior fusion instrumentation, severe scoliosis defined as Cobb’s angle > 40º or body mass index over 35. Technical success, radiation exposure and occurrence of adverse events were assessed.ResultsTwenty patients were selected, and 108 CBCT-guided procedures were performed. Each patient underwent at least 4 administrations. Transforaminal approach was performed in 82% of patients. The technical success was 100%, with primary success of 93.5%. The median radiation effective dose of the administrations was 5 mSv, the mean value equalled 10 mSv. Only mild adverse events were reported in the study.ConclusionsCBCT-guided lumbar intrathecal administrations of nusinersen in an adult SMA population with challenging access was feasible and safe image guided method.Key words: nusinersen, cone-beam CT, lumbar puncture     

Chimeric flagellin as the self-adjuvanting antigen for the activation of immune response against Helicobacter pylori

Helicobacter pylori infection can cause gastritis, peptic ulcer and can lead to gastric cancer. Lengthy antibiotic therapy does not protect the host against reinfection. H. pylori evolved to evade the recognition of the immune response by modifying several of its components whose orthologous proteins from other bacteria activate the innate immune response. Flagella are essential for the H. pylori effective colonization of human duodenum and stomach. TLR5, a member of the Toll-like receptor family, recognizes flagellin of most bacteria, such as Escherichia coli, but does not recognize the flagellin FlaA of H. pylori. We restored the ability of FlaA for the recognition by TLR5 by engineering a chimeric flagellin, in which both terminal segments of H. pylori flagellin were replaced by the corresponding segments from TLR5-activating E. coli flagellin. Recombinant chimeric flagellin folded correctly and was able to activate TLR5. Significantly increased serum IgG and IgA antibody responses were determined in mice vaccinated with chimeric flagellin in comparison to mice vaccinated with a control protein (FlaA) or negative control. Antibody titers remained high even 8 months after the last immunization. Antibodies were able to bind native flagellin from H. pylori lysate. Vaccination with chimeric flagellin provided mice with significant protection against H. pylori. The approach of chimeric flagellin can therefore generate effective immunogens that enable activation of innate and adaptive immune response and can be used to construct efficient vaccines against H. pylori or other flagellated bacteria that evade TLR5 recognition.     

Development and characterization of a novel mAb against bilitranslocase - a new biomarker of renal carcinoma

Background

Bilitranslocase (TC 2.A.65.1.1) is a bilirubin-specific membrane transporter, found on absorptive (stomach and intestine) and excretory (kidney and liver) epithelia and in vascular endothelium. Polyclonal antibodies have been raised in rabbits in the past, using a synthetic peptide corresponding to AA65-77 of rat liver bilitranslocase, as an antigen. Affinity-purified antibodies from immune sera have been found to inhibit various membrane transport functions, including the bilirubin uptake into human hepatocytes and the uptake of some flavonoids into human vascular endothelial cells. It was described by means of immunohistochemistry using polyclonal antibodies that bilitranslocase expression is severely down-regulated in clear cell renal carcinoma. The aim of our work was development and characterization of high-affinity, specific mAbs against bilitranslocase, which can be used as a potential diagnostic tool in renal cell carcinoma as well as in a wide variety of biological assays on different human tissues.

Materials and methods

Mice were immunized with a multi-antigen peptide corresponding to segment 65–75 of predicted primary structure of the bilitranslocase protein. By a sequence of cloning, immune- and functional tests, we aimed at obtaining a specific monoclonal antibody which recognizes a 37 kDa membrane protein, and influences the transport activity of bilitranslocase.

Results

On the basis of previous results, specific IgM monoclonal antibodies were produced in BALB/c mice, in order to further improve and extend the immunological approach to the study of bilitranslocase in renal cancer cells as well as to develop its potential diagnostics use.

Conclusions

In this article we show an immunological approach, based on newly developed monoclonal antibodies, to a detailed biochemical and functional characterization of a protein whose gene and protein structure is still unknown. We were able to demonstrate our novel mAb as a tumor marker candidate of renal cell carcinoma, which may prove useful in the diagnostic procedures.     

Nurr1 haplotypes are associated with femoropopliteal restenosis/re-occlusion after percutaneous transluminal angioplasty

Restenosis/re-occlusion remains a frequent complication in the first year after percutaneous transluminal angioplasty (PTA). In this study, association of nuclear receptor related 1 protein (Nurr1) haplotypes to the restenosis/re-occlusion rate after femoropopliteal PTA was investigated. Patients (n = 142) with disabling claudication or critical limb ischaemia, who had undergone technically successful femoropopliteal PTA, were prospectively followed up by vascular ultrasound imaging 12 months after the procedure. Nurr1 haplotypes 2 and 3 were associated significantly with the restenosis/re-occlusion rate (adjusted odds ratio 1.6, 95% confidence interval (CI) 1.1–2.3 and 2.0, 1.3–2.8, respectively) on univariate analysis.     

Novel ASAP1‐USP6, FAT1‐USP6, SAR1A‐USP6, and TNC‐USP6 fusions in primary aneurysmal bone cyst

Aneurysmal bone cyst (ABC) is a benign but locally aggressive neoplasm, with a tendency for local recurrence. In contrast to other bone tumors with secondary cystic change, ABC is characterized by USP6 gene rearrangement. There is a growing list of known USP6 fusion partners, characterization of which has been enabled with the advent of next‐generation sequencing (NGS). The list of known fusion partners includes CDH11, CNBP, COL1A1, CTNNB1, EIF1, FOSL2, OMD, PAFAH1B1, RUNX2, SEC31A, SPARC, STAT3, THRAP3, and USP9X. Using NGS, we analyzed a series of 11 consecutive ABCs and identified USP6 fusions in all cases, providing further evidence that USP6 fusions are universally present in primary ABCs. We identified four novel fusion partners in five ABCs and confirmed them by RT‐PCR and Sanger sequencing, ASAP1, FAT1, SAR1A, and TNC (in two cases). Because of high sensitivity and specificity, detection of a USP6 fusion by NGS may assist in differentiating between ABC and its mimics, especially in small biopsy samples when a definite diagnosis cannot be achieved on morphological grounds alone. Further studies with a large number of cases and follow‐up are needed to determine whether different fusion partners are associated with specific clinical and pathologic features of ABCs.