Direct Intracranial Injection of AAVrh8 Encoding Monkey ß-N-Acetylhexosaminidase Causes Neurotoxicity in the Primate Brain.

GM2 gangliosidoses, including Tay-Sachs disease and Sandhoff disease, are lysosomal storage disorders caused by deficiencies in ß-N-acetylhexosaminidase (Hex). Patients are afflicted primarily with progressive central nervous system (CNS) dysfunction. Studies in mice, cats, and sheep have indicated safety and widespread distribution of Hex in the CNS after intracranial vector infusion of AAVrh8 vectors encoding species-specific Hex α- or ß-subunits at a 1:1 ratio. Here, a safety study was conducted in cynomolgus macaques (cm), modeling previous animal studies, with bilateral infusion in the thalamus as well as in left lateral ventricle of AAVrh8 vectors encoding cm Hex α- and ß-subunits. Three doses (3.2 × 1012 vg [n = 3]; 3.2 × 1011 vg [n = 2]; or 1.1 × 1011 vg [n = 2]) were tested, with controls infused with vehicle (n = 1) or transgene empty AAVrh8 vector at the highest dose (n = 2). Most monkeys receiving AAVrh8-cmHexα/ß developed dyskinesias, ataxia, and loss of dexterity, with higher dose animals eventually becoming apathetic. Time to onset of symptoms was dose dependent, with the highest-dose cohort producing symptoms within a month of infusion. One monkey in the lowest-dose cohort was behaviorally asymptomatic but had magnetic resonance imaging abnormalities in the thalami. Histopathology was similar in all monkeys injected with AAVrh8-cmHexα/ß, showing severe white and gray matter necrosis along the injection track, reactive vasculature, and the presence of neurons with granular eosinophilic material. Lesions were minimal to absent in both control cohorts. Despite cellular loss, a dramatic increase in Hex activity was measured in the thalamus, and none of the animals presented with antibody titers against Hex. The high overexpression of Hex protein is likely to blame for this negative outcome, and this study demonstrates the variations in safety profiles of AAVrh8-Hexα/ß intracranial injection among different species, despite encoding for self-proteins.

Cell junction remodeling in gingival tissue exposed to a microbial toxin.

UNLABELLED: The gingival epithelium plays a key role in protecting the supporting structures of the teeth from bacteria and their products. In ex vivo experiments, we recently showed that the cytolethal distending toxin (Cdt) from the periodontal pathogen Aggregatibacter actinomycetemcomitans causes extensive damage to gingival tissue. Morphological changes included detachment of the keratinized outer layer, distention of spinous and basal cells in the oral epithelium, disruption of rete pegs, and apparent dissolution of cell junctions. Adherens junctions (zonula adherens) are essential for maintaining barrier function and integrity of gingival epithelium. Therefore, immunohistochemical and RT-PCR analyses of human gingival explants (HGX) and human gingival epithelial cells (HGEC) were utilized for a closer examination of the effects of the Cdt on E-cadherin, the key membrane component of adherens junctions. Although there was some variability among tissue donors, exposure of gingival tissue or isolated epithelial cells to the toxin generally resulted in a pronounced increase in the expression and cytosolic distribution of E-cadherin, accompanied by an increase in levels of the intracellular scaffolding proteins ß-catenin and ß-actin. These results indicate that the Cdt induced substantial remodeling of adherens junctions, with a potential impact on the barrier function of gingival epithelium. ABBREVIATIONS: cytolethal distending toxin (Cdt), 4',6-diamidino-2-phenylindole (DAPI), human gingival epithelial cells (HGEC), human gingival explants (HGX), human gingival fibroblasts (HGF), transepithelial resistance (TER).

Epitopes from two soybean glycinin subunits are antigenic in pigs.

BACKGROUND: Glycinin is a seed storage protein in soybean (Glycine max) that is allergenic in pigs. Glycinin is a hexamer composed of subunits consisting of basic and acidic portions joined by disulfide bridges. There are five glycinin subunit isoforms designated Gy1-Gy5. The purpose of this study is to identify epitopes from selected glycinin subunits that are antigenic in pigs. RESULTS: Twenty-seven out of 30 pigs had antibodies against glycinin in their sera. Ten of these sera had immunoglobulin G (IgG) against the Gy4 (A5A4B3) or Gy1 (A1aBx) subunit. Three sera recognised overlapping regions between the two subunits tested, though no serum stained both A5A4B3 and A1aBx. Two sera stained a highly conserved region between A5A4B3 and A1aBx, though again neither serum stained both peptides. The basic part of the A1aBx subunit was not recognised by any of the sera tested even though immunoblot data indicated that the basic and acidic subunits of glycinin are nearly equally antigenic. CONCLUSION: Two antigenic regions of A5A4B3 and A1aBx were identified that bound antibodies in half of the sera that reacted with these two proteins. Half of the sera reacted with unique regions of A5A4B3 and A1aBx. The failure of the basic portion of A1aBx to bind pig antibodies may indicate that it is less antigenic than the basic portion of A5A4B3 and other glycinin subunits.

Characterization and preparation of Aspergillus niger naringinase for debittering citrus juice.

UNLABELLED: Naringinase from Aspergillus niger was prepared and characterized to evaluate its effectiveness in debittering citrus juice. The enzyme was purified to homogeneity by sulfate fractionation and chromatographies on Q-Sepharose, Sephacryl S-200, and S-100 HR columns, and estimated by gel filtration chromatography (GFC) to have a molecular weight (MW) of 131 kDa, of which its subunit was measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be around 65.5 kDa. The enzyme showed active and stable pH ranges both within 4.5 to 5.0. Its optimal temperature was in the range of 45 to 55 °C. Freeze drying provided an estimated enzymatic recovery of 95.9%, greater than spray drying with the recovery at 55.6%. The freeze-drying powder could retain its enzymatic activity stably at 4 °C for 6 mo. Also, the enzyme in 0.220 U/mL citrus juice could sufficiently remove the naringin for the bitterness. Oral acute toxicity study revealed the maximum tolerated dose (MTD) of the naringinase powder was >10 g/kg in mice. The contents of arsenic (As), lead (Pb), mercury (Hg), the aerobic plate count, and coliform number in the enzyme powder all met the criteria for food use. These characteristics suggest that the naringinase from A. niger is efficient and suitable for debittering the citrus juice, and the process consisting of fermentation, salt precipitation, ion exchange, ultrafiltration, and freeze drying is a promising means to prepare the naringinase for food industry, setting up a strong base to enzymatically debitter citrus juice. PRACTICAL APPLICATION: This study focused on characterization, preparation, and validation of naringinase from A. niger, which provided useful information on how to prepare, store, and use the naringinase. In addition, this naringinase met the safety standards for food use and showed strong ability to remove the bitter taste from citrus juice, which provided useful information for interested readers, and the food industry.

Inhibited proliferation of human periodontal ligament cells and gingival fibroblasts by Actinobacillus actinomycetemcomitans: involvement of the cytolethal distending toxin.

Actinobacillus actinomycetemcomitans can inhibit fibroblast proliferation. The objective of this study was to characterize the early proliferative responses of human periodontal ligament cells (PDLC) and gingival fibroblasts (GF) to A. actinomycetemcomitans components and to investigate the possible involvement of the cytolethal distending toxin (cdt) produced by this bacterium. The PDLC and GF were challenged with surface components of A. actinomycetemcomitans. Both DNA and protein synthesis as well as cell lysis or apoptosis were assayed for a 6-h period after addition of the bacterial extract. Unlike the controls, inhibition of DNA synthesis had already occurred in the challenged cells at the end of the initial 3- to 6-h period. No lysis or apoptosis was detected, and the total protein synthesis remained unaffected. The persistence of the effect on cell growth was confirmed after a 72-h period of challenge, during which the cells remained viable but exhibited an elongated and distended cell body. No significant differences were observed between PDLC and GF. When a cdt-knockout strain of A. actinomycetemcomitans was used almost no inhibitory effect on cell proliferation was observed. It was concluded that A. actinomycetemcomitans causes a non-lethal inhibition of proliferation in PDLC and GF as a result of an early arrest of DNA synthesis. Cytolethal distending toxin is responsible for most of this effect. This bacterial property may compromise tissue homeostasis in the periodontium.