Faculty Bibliography

Introduction: The Ichtchenko laboratory has developed methods that enable facile production of purified recombinant derivatives of botulinum neurotoxins (BoNTs) that retain the structural and trafficking properties of wt BoNTs. Surprisingly, and unlike previously described recombinant BoNT derivatives, they remain physiologically active despite inactivating mutations to the light chain (LC) protease. As such, they are referred to as atoxic derivatives rather than nontoxic derivatives. In this study, we compared the biological activities of BoNT/A ad0 (E224 > A, Y336 > A) with more extensively modified BoNT/A ad derivatives. Methods: Recombinant BoNT/A derivatives and primary neuronal cultures derived from E19 rat hippocampus were prepared as previously described, and their toxicity determined using a modified murine LD50 assay. Mouse embryonic stem cell–derived neuron (ESN) cultures were tested at 34 d after differentiation. After 24 and 48 hours exposure to 25 nM BoNT/A derivative, monosynaptic miniature postsynaptic currents were measured. Intrinsic electrical characteristics of treated ESNs were measured to evaluate any potential cytotoxic activity. Results: BoNT/A ad0 is internalized into hippocampal neurons. It co-localizes with and cleaves SNAP-25. In ESN cultures, BoNT/A ad0 reduced synaptic activity by 88% at 24 hours and by 99% at 48 hours. The introduction of more extensive amino acid substitutions disabled in vitro detection of BoNT/A ad derivatives, such that they had no effect on synaptic activity in ESN cultures for up to 72 hours. None of the BoNT/A ad derivatives tested evoked evidence of cytotoxicity. Despite the effect of amino acid substitutions on physiological activities measured in vitro, all BoNT ad derivatives tested retained some level of toxicity in vivo, with LD50 values 100,000-fold to 500,00-fold greater than wt BoNT/A. Conclusion: To design atoxic BoNT derivatives as molecular vehicles for delivering drugs to the neuronal cytoplasm, metalloprotease and substrate-binding activity need to be carefully balanced.

OBJECTIVE: We investigated the relationship between the molecular weight (MW) distribution of hyaluronan (HA) in synovial fluid (SF) and risk of knee osteoarthritis (OA) progression. METHODS: HA MW was analyzed for 65 baseline knee SFs. At 3-year follow-up, knees were scored for change in joint space narrowing (JSN), osteophyte (OST) progression, or occurrence of total knee arthroplasty (TKA). HA MW distribution was analyzed using agarose gel electrophoresis (AGE), and its relationship to OA progression was evaluated using logistic regression. The association between HA MW and self-reported baseline knee pain was analyzed using Pearson's correlation coefficients. RESULTS: Knee OA was categorized as non-progressing (OST-/JSN-, 26 knees, 40%), or progressing based on OST (OST+/JSN-, 24 knees, 37%), OST and JSN (OST+/JSN+, 7 knees, 11%) or total knee arthroplasty (TKA, 8 knees, 12%). The MW distribution of HA in baseline SFs was significantly associated with the odds of OA progression, particularly for index knees. After adjusting for age, gender, BMI, baseline X-ray grade and pain, each increase of one percentage point in %HA below 1 million significantly increased the odds of JSN (odds ratios (OR) = 1.45, 95% CI 1.02-2.07), TKA or JSN (OR = 1.24, 95%CI 1.01-1.53) and the odds of any progression (OR = 1.16, 95% CI 1.01-1.32). HA MW distribution significantly correlated with pain. CONCLUSION: These data suggest that the odds of knee OA progression increases as HA MW distribution shifts lower and highlight the value of reporting MW distribution rather than just average MW values for HA.

OBJECTIVE: To establish whether there is an association between TSG-6 activity and osteoarthritis progression. DESIGN: TSG-6 activity was determined in 132 synovial fluids from patients with OA of the knee, using a novel quantitative TSG-6 activity assay. The association between TSG-6 activities at baseline and four distinct disease progression states, determined at 3-year follow-up, was analyzed using logistic regression. RESULTS: There was a statistically significant relationship between TSG-6 activity at baseline and all OA progression states over a 3-year period. Patient knees with TSG-6 activities in the top tenth percentile, compared to the median activity, had an odds ratio (OR) of at least 7.86 (confidence interval (CI) [3.2, 20.5]) for total knee arthroplasty (TKA) within 3 years, and of at least 5.20 (CI [1.8, 13.9]) after adjustment for confounding factors. Receiver operating characteristic (ROC) analysis for knee arthroplasty yielded a cut-off point of 13.3 TSG-6 activity units/ml with the following parameters: area under the curve 0.90 (CI [0.804, 0.996]), sensitivity 0.91 (CI [0.59, 0.99]), specificity 0.82 (CI [0.74, 0.88]) and a negative predictive value (NPV) of 0.99 (CI [0.934, 0.994]). CONCLUSION: The TSG-6 activity is a promising independent biomarker for OA progression. Given the high NPV, this assay may be particularly suitable for identifying patients at low risk of rapid disease progression and to assist in the timing of arthroplasty.

We have previously described genetic constructs and expression systems that enable facile production of recombinant derivatives of botulinum neurotoxins (BoNTs) that retain the structural and trafficking properties of wt BoNTs. In this report we describe the properties of one such derivative, BoNT/A ad, which was rendered atoxic by introducing two amino acid mutations to the light chain (LC) of wt BoNT/A, and which is being developed as a molecular vehicle for delivering drugs to the neuronal cytoplasm. The neuronal binding, internalization, and intracellular trafficking of BoNT/A ad in primary hippocampal cultures was evaluated using three complimentary techniques: flow cytometry, immunohistochemistry, and Western blotting. Neuronal binding of BoNT ad was significantly increased when neurons were incubated in depolarizing medium. Flow cytometry demonstrated that BoNT/A ad internalized into neurons but not glia. After 24 hours, the majority of the neuron-bound BoNT/A ad became internalized, as determined by its resistance to pronase E-induced proteolytic degradation of proteins associated with the plasma membrane of intact cells. Significant amounts of the atoxic LC accumulated in a Triton X-100-extractable fraction of the neurons, and persisted as such for at least 11 days with no evidence of degradation. Immunocytochemical analysis demonstrated that the LC of BoNT/A ad was translocated to the neuronal cytoplasm after uptake and was specifically targeted to SNARE proteins. The atoxic LC consistently co-localized with synaptic markers SNAP-25 and VAMP-2, but was rarely co-localized with markers for early or late endosomes. These data demonstrate that BoNT/A ad mimics the trafficking properties of wt BoNT/A, confirming that our platform for designing and expressing BoNT derivatives provides an accessible system for elucidating the molecular details of BoNT trafficking, and can potentially be used to address multiple medical and biodefense needs.

Researchers and clinicians operate in an increasingly complex clinical and regulatory environment in which understanding the principles governing human research is essential. However, most orthopaedic surgeons have not received in-depth training in regulatory requirements and scientific research methods. Ensuring that research is conducted in accordance with state and federal laws and ethical principles is essential to guard compromising patient information and avoid severe penalties for noncompliance. The researcher must understand the regulations for compliance and proper data management, including the requirements of the Health Insurance Portability and Accountability Act, proper application of informed consent, use of the Institutional Review Board, and data protection guidelines. Tools such as a regulatory binder can assist investigators in complying with requirements, maintaining regulatory standards, and ensuring a robust study design and conduct.