Poster Session: Retinal Changes Associated with Football-Related Concussions and Head Impacts.

This pilot study explored the sensitivity of retinal markers to CNS sequelae of concussive and subconcussive head hits. Three groups of college athletes were assessed at pre-season, post-season and 4-months later: Football players with a concussion history (FB+C) (n = 9), players without a concussion history (FB-C) (n = 11), and non-contact athletes (swimmers, track & field; Non-FB) (n = 12). Measures included optical coherence tomography (OCT), OCT angiography, electroretinography, and visual acuity testing. Head impacts during the season were tracked with in-helmet accelerometers. At pre-season, FB+C demonstrated thicker macular central subfields (CSF) (Hedge's g (effect size) = 1.05, p = 0.02) and retinal nerve fiber layers (RNFL) (g = 0.81, p = 0.08), relative to other athletes. Differences in CSF thickness were also observed at post-season and follow-up (gs > 1.00, ps < 0.04), reflecting their non-short-term nature. RNFL was thicker in FB+C at post-season (g = 0.93, p = 0.06) but not later. Total head impacts during the season correlated with increases in CSF thickness from baseline to follow-up only (r = -0.53, p = 0.02). High intensity head impacts in particular correlated with increases in cup-to-disc ratio at post-season and follow-up (rs > 0.53, ps < 0.03). These data suggest that concussion history is associated with retinal changes that are not short-term, and that severe head impacts are associated with acute changes whose duration is not yet known.

Efficacy of non-toxic deletion mutants of protective antigen from Bacillus anthracis.

Current human anthrax vaccines available in the United States and Europe consist of alum-precipitated supernatant material from cultures of a toxigenic, nonencapsulated strain of Bacillus anthracis. The major component of human anthrax vaccine that confers protection is protective antigen (PA). A second-generation human vaccine using the recombinant PA (rPA) is being developed. In this study, to prevent the toxicity and the degradation of the native rPA by proteases, we constructed two PA variants, delPA (163-168) and delPA (313-314), that lack trypsin (S(163)-R(164)-K(165)-K(166)-R(167)-S(168)) or chymotrypsin cleavage sequence (F(313)-F(314)), respectively. These proteins were expressed in Bacillus brevis 47-5Q. The delPAs were fractionated from the culture supernatant of B. brevis by ammonium sulfate at 70% saturation, followed by anion exchange chromatography on a Hitrap Q, Hiload 16/60 superdex 200 gel filtration column and phenyl sepharose hydrophobic interaction column. In accordance with previous reports, both delPA proteins combined with lethal factor protein did not show any cytotoxicity on J774A.1 cells. The delPA (163-168) and delPA (313-314) formulated either in Rehydragel HPA or MPL-TDM-CWS (Ribi-Trimix), elicited a comparable amount of anti-PA and neutralizing antibodies to those of native rPA in guinea pigs, and confers full protection of guinea pigs from 50xLD50 of fully virulent B. anthracis spore challenges. Ribi-Trimix was significantly more effective in inducing anti-PA and neutralizing antibodies than Rehydragel HPA. These results indicate the possibility of delPA (163-168) and delPA (313-314) proteins being developed into nontoxic, effective and stable recombinant vaccine candidates.

Expression and secretion of the protective antigen of Bacillus anthracis in Bacillus brevis.

We used the Bacillus brevis-pNU212 system to develop a mass production system for the protective antigen (PA) of Bacillus anthracis. A moderately efficient expression-secretion system for PA was constructed by fusing the PA gene from B. anthracis with the B. brevis cell-wall protein signal-peptide encoding region of pNU212, and by introducing the recombinant plasmid, pNU212-mPA, into B. brevis 47-5Q. The clone producing PA secreted about 300 microg of recombinant PA (rPA) per ml of 5PY-erythromycin medium after 4 days incubation at 30 degrees C. The rPA was fractionated from the culture supernatant of B. brevis 47-5Q carrying pNU212-mPA using ammonium sulfate at 70% saturation followed by anion exchange chromatography on a Hitrap Q, a Hiload 16/60 Superdex 200 gel filtration column and a phenyl sepharose hydrophobic interaction column, yielding 70 mg rPA per liter of culture. The N-terminal sequence of the purified rPA was identical to that of native PA from B. anthracis. The purified rPA exhibited cytotoxicity towards J774A.1 cells when combined with lethal factor. The rPA formulated in either Rehydragel HPA or MPL-TDM-CWS adjuvant (Ribi-Trimix) elicited the expression of a large amount of anti-PA and neutralizing antibodies in guinea pigs and completely protected them against a 100 LD50 challenge with fully virulent B. anthracis spores.