Localization and RNAi-driven inhibition of a Brugia malayi encoded Interleukin-5 Receptor Binding protein

A molecule termed BmIL5Rbp (aka Bm8757) was identified from Brugia malayi filarial worms and found to competitively inhibit human IL-5 binding to its human receptor. After the expression and purification of a recombinant BmIL5Rbp and generation of BmIL5Rbp-specific rabbit antibody, we localized the molecule on B. malayi worms through immunohistochemistry and immunoelectron microscopy. RNA interference was used to inhibit BmIL5Rbp mRNA and protein production. BmIL5Rbp was shown to localize to the cuticle of Brugia malayi and to be released in their excretory/secretory products. RNAi inhibited BmIL5Rbp mRNA production by 33% and reduced the surface protein expression by ~50% and suppressed the release of BmIL5Rbp in the excretory/secretory products. RNAi has been used successfully to knock down the mRNA and protein expression of BmIL5Rbp in the early larval stages of B. malayi and provided a proof-of-principle for the local inhibition of the human IL5 receptor. These findings provide evidence that a parasite encoded IL5R antagonist could be utilized therapeutically.

Development of a Multiplex PCR-Based Assay for Rapid Serotyping of Erysipelothrix Species

ABSTRACT The Gram-positive bacterium Erysipelothrix rhusiopathiae is a zoonotic pathogen that causes erysipelas in a wide range of mammalian and avian species. Historically, E. rhusiopathiae has been differentiated from other Erysipelothrix species by serotyping. Among 28 serovars of Erysipelothrix species, specific serovars, namely, 1a, 1b, and 2 of E. rhusiopathiae, are associated mainly with the disease in pigs, poultry, and humans; however, other serovar strains are often simultaneously isolated from diseased and healthy animals, indicating the importance of isolate serotyping for epidemiology. The traditional serotyping protocol, which uses heat-stable peptidoglycan antigens and type-specific rabbit antisera in an agar-gel precipitation test, is time-consuming and labor-intensive. To develop a rapid serotyping scheme, we analyzed sequences of the 12- to 22-kb chromosomal region, which corresponds to the genetic region responsible for virulence of serovar 1a and 2 strains of E. rhusiopathiae, of the 28 serovars of Erysipelothrix species. We confirmed that the serovar 13 strain lacks the genomic region and that some serovar strains possess very similar or the same genetic structure, prohibiting differentiation of the serovars. We created 4 multiplex PCR sets allowing the simultaneous detection and differentiation of the majority of Erysipelothrix serovars. Together with a previously reported multiplex PCR that can differentiate serovars 1a, 1b, 2, and 5, the multiplex PCR-based assay developed in this study covers all but one (serovar 13) of the reported serovars of Erysipelothrix species and should be a valuable tool for etiological as well as epidemiological studies of Erysipelothrix infections.

In Vitro Analyses Reveal the Effect of Synthetic Cytokinin Forchlorfenuron (FCF) on a Septin-Like Protein of Taeniid Cysticerci

Cytokinin forchlorfenuron (FCF), a synthetic cytokinin, has been used specifically for the characterization of septins. In spite of genomic evidence of their existence, nothing is known about septin filaments in taeniid cestodes. The aim of this work was to determine the presence of a septin-like protein in cysticerci of Taenia crassiceps and Taenia solium using the deduced amino acid sequence of T. solium septin 4 (SEPT4_Tsm), to design and synthesize a derived immunogenic peptide (residues 88 to 103), to prepare a specific rabbit polyclonal antibody, and to examine the effects of FCF at different concentrations and exposure times on an in vitro culture of T. crassiceps cysticerci. In vitro, FCF altered the morphology and motility of T. crassiceps cysticerci, and its effects were reversible under specific concentrations. In addition, we observed by ultrastructural observation that FCF alters the cellular subunit of the protonephridial system of cestodes, where disruption of the axoneme pattern of flame cells was observed. The rabbit polyclonal antibody prepared against the synthetic peptide recognized a major band of 41 kDa in both parasites. Our results establish the importance of SEPT4_Tsm in the dynamics and survival of taeniid cysticerci, as well as their susceptibility to FCF. This is also the first report that a septin is present in the cytoskeleton of taeniids.

Identification of the Chromosomal Region Essential for Serovar-Specific Antigen and Virulence of Serovar 1 and 2 Strains ofErysipelothrix rhusiopathiae

ABSTRACTErysipelothrix rhusiopathiaecauses swine erysipelas, an infection characterized by acute septicemia or chronic endocarditis and polyarthritis. Among 17E. rhusiopathiaeserovars, determined based on heat-stable peptidoglycan antigens, serovars 1 and 2 are most commonly associated with the disease; however, the molecular basis for the association between these serovars and virulence is unknown. To search for the genetic region defining serovar 1a (Fujisawa) strain antigenicity, we examined the 15-kb chromosomal region encompassing a putative pathway for polysaccharide biosynthesis, which was previously identified in theE. rhusiopathiaeFujisawa strain. Six transposon mutants of Fujisawa strain possessing a mutation in this region lost antigenic reactivity with serovar 1a-specific rabbit serum. Sequence analysis of this region in wild-type strains of serovars 1a, 1b, and 2 and serovar N, which lacks serovar-specific antigens, revealed that gene organization was similar among the strains and that serovar 2 strains showed variation. Serovar N strains displayed the same gene organization as the serovar 1a, 1b, or 2 strain and possessed certain mutations in this region. In two of the analyzed serovar N strains, restoration of the mutations via complementation with sequences derived from serovar 1a and 2 strains recovered antigenic reactivity with 1a- and 2-specific rabbit serum, respectively. Several gene mutations in this region resulted in altered capsule expression and attenuation of virulence in mice. These results indicate a functional connection between the biosynthetic pathways for the capsular polysaccharide and peptidoglycan antigens used for serotyping, which may explain variation in virulence among strains of different serovars.

Corrigendum

McElroy M and Keshmiri A, Impact of using conventional inlet/outlet boundary conditions on haemodynamic metrics in a subject-specific rabbit aorta, Proc IMechE, Part H: Journal of Engineering in Medicine, first published on March 25, 2017, DOI: 10.1177/0954411917699237 Following OnlineFirst publication of the article, the authors informed SAGE of an error in the transient velocity inlet profile which had been defined inaccurately due to a human error in the interpretation of clinical data in the literature. As a result of this error in boundary conditions, some of the results of transient flow computations were incorrect. A watermarked version of the first publication of the article (as first published on March 25, 2017) is attached for reference in the PDF version of this corrigendum. The authors have revised and corrected their article. The revised version of the article has been accepted following peer review and replaces the article first published on March 25, 2017. Date received: 8 August 2017; accepted: 30 November 2017 (Revised version) Date received: 29 March 2016; accepted: 21 February 2017 (Original version) The correct and citable version of the article is accessible at the following DOI: 10.1177/0954411917699237

A rational approach to obtaining high-specific polyclonal antibodies against recombinant alpha-synuclein

The approach for the quick and efficient production ofpolyclonal antibodies tothe target antigen alpha-synuclein has been proposed. Two methods have been employed to purify specific rabbit polyclonal antibodies against recombinant human alpha-synuclein, produced by subcutaneous immunization with complete Freund's adjuvant. It was shown that purification on CNBr-activated Sepharose with immobilized alpha-synuclein resulted in antibody preparation with rabbit serum histidine-rich glycoprotein as a contaminant. Two-stage antibody purification procedure first on Sepharose with immobilized protein G, and then on alpha-synuclein immobilized column helps to avoid contamination and to obtain homogenous antibody preparation. Antibodies recognize different conformations of alpha-synuclein and can be used in a variety of immunochemical approaches, including immunocytochemistry.

Impact of Using Conventional Inlet/Outlet Boundary Conditions on Haemodynamic Metrics in a Subject-Specific Rabbit Aorta

Computational fluid dynamics is a tool capable of accurately measuring metrics currently used to predict the behaviour of cardiovascular diseases. This study quantifies the impact various commonly used inlet and outlet boundary conditions have on various shear rate–based haemodynamic metrics currently used for predicting the localisation of cardiovascular diseases. Simulations are conducted on an accurately represented rabbit aorta configuration and comparison has been made against available in vivo data. The boundary conditions studied include two different inlet profiles, three outlet boundary conditions, and steady-state versus pulsatile flow cases. Large variations were found in the results, particularly when using different outlet boundary conditions, and the discrepancies were evaluated both quantitatively and qualitatively. The results clearly highlight the importance of the type of boundary condition used when simulating complex cardiovascular models. By restricting the attention to the flow within the aorta and the intercostal branches, the results suggest that prescribing transient simulation and fully developed flow at the inlet are not required. Furthermore, assuming the widely accepted low wall shear stress theory of Caro, it was found that Murray’s law–based outlet boundary condition returns the most physiologically accurate results when compared to in vivo data.

A New Dosing Scheme of ATG-F Prevents Rejection and Maintains Immune Recovery in Haploidentical T and B Cell Depleted Stem Cell Transplantation

Abstract 4154 T and B cell depletion of haploidentical peripheral stem cells with CD3/CD19 coated magnetic microbeads prevents GvHD and allows to coinfuse large numbers of donor NK cells and other accessory cells. The anti CD3 specific OKT3 antibody was routinely used as rejection prophylaxis without affecting CD3 negative cells. However, due to its restricted availability, the substance had to be substituted by polyclonal ATG preparations with a longer half life period and comprising a broader variety of antigen, with the CD56 antigen in particular. We present data with reduced ATG doses given at the beginning of the conditioning regimen in order not to impair cotransfused NK cells and immune recovery. A total of 27 pediatric patients (ALL/AML n=9, relapsed solid tumors n=13, nonmalignant diseases n=5) received either 3×5 mg/kg (n=7) or 3×10 mg/kg (n=20) ATG-F (Fresenius) starting at day -12, followed by fludarabine (160 mg/m2, d -8 to -5) thiotepa (10 mg/m2 d -4) and melphalan (140 mg/m2 d −3 to −2). A median number of 14.4×106 CD34/kg and 62×106 NK cells/kg with 37×103 T cells/kg were infused. Median time to ANC>500 was 9 days in both groups. Graft rejection occurred in 3/7 patients with 15 mg ATG (42%) and in 2/20 patients with 30 mg ATG (10%). After reconditioning with a TLI based regimen, final engraftment was achieved in all patients. Acute GvHD grade II-IV was observed in 1/4 (15 mg) and 1/18 (6%, 30 mg) patients without rejection. Extensive chronic GvHD occurred in 1/4 (15 mg group) and 1/18 (30 mg group) patients. Immune recovery was monitored in the 30 mg group and compared with a historical group receiving OKT3 and the same chemotherapy (n=34). Recovery of CD56+ NK cells was fast with a mean number of 473 vs. 230 cells/μl at day +14, 299 vs. 281 cells/μl at day +30 and 245 vs. 236 cells/μl at day +90. CD3+ T cells reached 12 vs. 16/μl at day +30 and 138 vs. 217/μl at day +90 (30 mg group vs. OKT3 group; no significant differences for all data pairs). ATG serum levels were measured in 9 patients by flow cytometry (amount of ATG binding to the Jurkat cell line, defined as T cell specific rabbit IgG). Median peak levels of 10.5 μg/ml (15 mg group) and 15.0 μg/ml (30 mg group) specific rabbit IgG were reached between day -8 and -6 and dropped to 1.2 and 2.6 μg/ml at day 0. In vitro incubation of NK cells from healthy donors with a comparable dosage of ATG-F (1 or 10 μg/ml) resulted in 26 (41)% apoptosis and 0.2 (0.2)% necrosis (70 (52)% vital cells) after 24 hours. Conclusions: Our aim was to substitute OKT3 by ATG in patients who receive CD3/19 depleted haploidentical peripheral stem cells without hampering donor NK cells infused on day 0 and subsequent immune recovery. Administration of 15 or 30 mg/kg ATG-F was started at an early time point (day -12) of the regimen. Both doses resulted in low serum levels of specific ATG at day 0 and in a fast NK cell recovery. In vitro results suggested, that the majority of NK cells will not be damaged herewith. However, 15 mg/kg seemed to be not effective in preventing graft rejection and use of 30 mg/kg ATG-F has to be recommended. Immune recovery of T and NK cells was comparable to that of a historical control group who received OKT3. This approach will be also of interest for other transplantation strategies in which various components of the grafts and additionally given Tregs or specific T cells have to be preserved. Disclosures: Martinius: Fresenius Biotech: Employment.