Ongoing decision-making dilemma for treatment of de novo spinal infections: a comparison of the Spinal Infection Treatment Evaluation Score with the Spinal Instability Spondylodiscitis Score and Spine Instability Neoplastic Score.

OBJECTIVE: De novo spinal infections are an increasing medical problem. The decision-making for surgical or nonsurgical treatment for de novo spinal infections is often a non-evidence-based process and commonly a case-by-case decision by single physicians. A scoring system based on the latest evidence might help improve the decision-making process compared with other purely radiology-based scoring systems or the judgment of a single senior physician. METHODS: Patients older than 18 years with an infection of the spine who underwent nonsurgical or surgical treatment between 2019 and 2021 were identified. Clinical data for neurological status, pain, and existing comorbidities were gathered and transferred to an anonymous spreadsheet. Patients without an MR image and a CT scan of the affected spine region were excluded from the investigation. A multidisciplinary expert panel used the Spine Instability Neoplastic Score (SINS), Spinal Instability Spondylodiscitis Score (SISS), and Spinal Infection Treatment Evaluation Score (SITE Score), previously developed by the authors' group, on every clinical case. Each physician of the expert panel gave an individual treatment recommendation for surgical or nonsurgical treatment for each patient. Treatment recommendations formed the expert panel opinion, which was used to calculate predictive validities for each score. RESULTS: A total of 263 patients with spinal infections were identified. After the exclusion of doubled patients, patients without de novo infections, or those without CT and MRI scans, 123 patients remained for the investigation. Overall, 70.70% of patients were treated surgically and 29.30% were treated nonoperatively. Intraclass correlation coefficients (ICCs) for the SITE Score, SINS, and SISS were 0.94 (95% CI 0.91-0.95, p < 0.01), 0.65 (95% CI 0.91-0.83, p < 0.01), and 0.80 (95% CI 0.91-0.89, p < 0.01). In comparison with the expert panel decision, the SITE Score reached a sensitivity of 96.97% and a specificity of 81.90% for all included patients. For potentially unstable and unstable lesions, the SISS and the SINS yielded sensitivities of 84.42% and 64.07%, respectively, and specificities of 31.16% and 56.52%, respectively. The SITE Score showed higher overall sensitivity with 97.53% and a higher specificity for patients with epidural abscesses (75.00%) compared with potentially unstable and unstable lesions for the SINS and the SISS. The SITE Score showed a significantly higher agreement for the definitive treatment decision regarding the expert panel decision, compared with the decision by a single physician for patients with spondylodiscitis, discitis, or spinal osteomyelitis. CONCLUSIONS: The SITE Score shows high sensitivity and specificity regarding the treatment recommendation by a multidisciplinary expert panel. The SITE Score shows higher predictive validity compared with radiology-based scoring systems or a single physician and demonstrates a high validity for patients with epidural abscesses.

Cage subsidence-A multifactorial matter!

STUDY DESIGN: Retrospective cohort study OBJECTIVE: Wider cages are associated with improved decompression and reduced subsidence, but variation in cage physical properties limits consistent outcome analysis after thoracolumbar interbody fusion. This study investigated cage subsidence and its relationship to lateral and posterior approaches with a focus on the hypothesis that the larger surface area of lateral cages results in lower subsidence rates. METHODS: This study retrospectively reviewed 194 patients who underwent interbody fusion between 2016 and 2019 with a primary outcome of cage subsidence. Secondary outcomes were cage distribution (patients, approaches, expandability), cage dimensions, t­scores, length of hospital stay, blood loss, surgical time, and pelvic incidence-lumbar lordosis (PI-LL) mismatch. RESULTS: Medical records were reviewed for 194 patients receiving 387 cages at 379 disc levels. Subsidence was identified in 35.1% of lateral cages, 40.9% of posterior cages, and 36.3% of all cages. Lower surface area (p = 0.008) and cage expandability were associated with subsidence risk. Lower anteroposterior cage length proved to be a significant factor in the subsidence of posteriorly placed cages (p = 0.007). Osteopenic and osteoporotic patients experienced cage subsidence 36.8% of the time compared to 3.5% of patients with normal t­scores (p = 0.001). Cage subsidence correlated with postoperative deterioration of the PI-LL mismatch (p = 0.03). Patients receiving fusion augmentation with bone morphogenic protein experienced higher fusion rates (p < 0.01). CONCLUSION: Cage subsidence is a common complication that can significantly impact operative outcomes following thoracolumbar interbody fusion. Low t­scores, smaller surface area, cage expandability, and lower cage length in posterior approaches contribute significantly to cage subsidence.

A novel scoring system concept for de novo spinal infection treatment, the Spinal Infection Treatment Evaluation Score (SITE Score): a proof-of-concept study.

OBJECTIVE: De novo infections of the spine are an increasing healthcare problem. The decision for nonsurgical or surgical treatment is often made case by case on the basis of physician experience, specialty, or practice affiliation rather than evidence-based medicine. To create a more systematic foundation for surgical assessments of de novo spinal infections, the authors applied a formal validation process toward developing a spinal infection scoring system using principles gained from other spine severity scoring systems like the Spine Instability Neoplastic Score, Thoracolumbar Injury Classification and Severity Score, and AO Spine classification of thoracolumbar injuries. They utilized an expert panel and literature reviews to develop a severity scale called the "Spinal Infection Treatment Evaluation Score" (SITE Score). METHODS: The authors conducted an evidence-based process of combining literature reviews, extracting key elements from previous scoring systems, and obtaining iterative expert panel input while following a formal Delphi process. The resulting basic SITE scoring system was tested on selected de novo spinal infection cases and serially refined by an international multidisciplinary expert panel. Intra- and interobserver reliabilities were calculated using the intraclass correlation coefficient (ICC) and Fleiss' and Cohen's kappa, respectively. A receiver operating characteristic analysis was performed for cutoff value analysis. The predictive validity was assessed through cross-tabulation analysis. RESULTS: The conceptual SITE scoring system combines the key variables of neurological symptoms, infection location, radiological variables for instability and impingement of neural elements, pain, and patient comorbidities. Ten patients formed the first cohort of de novo spinal infections, which was used to validate the conceptual scoring system. A second cohort of 30 patients with de novo spinal infections, including the 10 patients from the first cohort, was utilized to validate the SITE Score. Mean scores of 6.73 ± 1.5 and 6.90 ± 3.61 were found in the first and second cohorts, respectively. The ICCs for the total score were 0.989 (95% CI 0.975-0.997, p < 0.01) in the first round of scoring system validation, 0.992 (95% CI 0.981-0.998, p < 0.01) in the second round, and 0.961 (95% CI 0.929-0.980, p < 0.01) in the third round. The mean intraobserver reliability was 0.851 ± 0.089 in the third validation round. The SITE Score yielded a sensitivity of 97.77% ± 3.87% and a specificity of 95.53% ± 3.87% in the last validation round for the panel treatment decision. CONCLUSIONS: The SITE scoring concept showed statistically meaningful reliability parameters. Hopefully, this effort will provide a foundation for a future evidence-based decision aid for treating de novo spinal infections. The SITE Score showed promising inter- and intraobserver reliability. It could serve as a helpful tool to guide physicians' therapeutic decisions in managing de novo spinal infections and help in comparison studies to better understand disease severity and outcomes.

An Assessment of the Safety of Surgery and Hardware Placement in de-novo Spinal Infections. A Systematic Review and Meta-Analysis of the Literature.

OBJECTIVES: Primary objectives were outcomes comparison of instrumented surgery used for de-novo spinal infections in terms of infection recurrence, reoperations, primary failure, mortality, and length of stay relative to non-instrumented surgery. Secondary objectives were outcomes for surgical and non-surgical treatment of de-novo spinal infections regarding recurrence of infection, mortality, quality of life, and length-of-stay. METHODS: A systematic literature review was performed using the PubMed database. Studies comparing outcome variables of patients with de-novo spinal infections (DNSI) treated with and without instrumentation and surgical versus non-surgical treatment were included. Studies primarily focusing on epidural abscesses or non-de-novo infections were excluded. A meta-analysis was performed for infection recurrence, reoperation, primary treatment failure, mortality, and quality-of-life parameters. RESULTS: A total of 17 retrospective studies with 2.069 patients met the inclusion criteria. 1.378 patients received surgical treatment with or without instrumentation; 676 patients were treated non-surgically. For the comparison of instrumented to non-instrumented surgery Odds-Ratios were .98 (P = .95) for infection recurrence, .83 (P = .92) for primary failure, .53 (P = .02) for mortality and .32 (P = .05) for reoperation. For the comparison of non-surgical to surgical treatment, Odds-Ratios were .98 (P = .95) for infection recurrence, and 1.05 (P = .89) for mortality. CONCLUSION: Available data support that instrumented surgery can be performed safely without higher rates of infection recurrence or primary failure and lower reoperation and mortality rates compared to nonsurgical treatment for DNSI. Furthermore, spine surgical treatment may generally be performed without higher risk of infection recurrence and mortality and better quality-of-life outcomes compared to generic non-surgical treatment.

Engaging cervical spinal circuitry with non-invasive spinal stimulation and buspirone to restore hand function in chronic motor complete patients.

The combined effects of cervical electrical stimulation alone or in combination with the monoaminergic agonist buspirone on upper limb motor function were determined in six subjects with motor complete (AIS B) injury at C5 or above and more than one year from time of injury. Voluntary upper limb function was evaluated through measures of controlled hand contraction, handgrip force production, dexterity measures, and validated clinical assessment batteries. Repeated measure analysis of variance was used to evaluate functional metrics, EMG amplitude, and changes in mean grip strength. In aggregate, mean hand strength increased by greater than 300% with transcutaneous electrical stimulation and buspirone while a corresponding clinically significant improvement was observed in upper extremity motor scores and the action research arm test. Some functional improvements persisted for an extended period after the study interventions were discontinued. We demonstrate that, with these novel interventions, cervical spinal circuitry can be neuromodulated to improve volitional control of hand function in tetraplegic subjects. The potential impact of these findings on individuals with upper limb paralysis could be dramatic functionally, psychologically, and economically.

In vivo cleavage of transgene donors promotes nuclease-mediated targeted integration.

Targeted DNA integration is commonly used to eliminate position effects on transgene expression. Integration can be targeted to specific sites in the genome via both homology-based and homology-independent processes. Both pathways start the integration process with a site-specific break in the chromosome, typically from a zinc-finger nuclease (ZFN). We previously described an efficient homology-independent targeted integration technique that captures short (<100 bp) pieces of DNA at chromosomal breaks created by ZFNs. We show here that inclusion of a nuclease target site on the donor plasmid followed by in vivo nuclease cleavage of both the donor and the chromosome results in efficient integration of large, transgene-sized DNA molecules into the chromosomal double-strand break. Successful targeted integration via in vivo donor linearization is demonstrated at five distinct loci in two mammalian cell types, highlighting the generality of the approach. Finally, we show that CHO cells, a cell type recalcitrant to homology-based integration, are proficient at capture of in vivo-linearized transgene donors. Moreover, we demonstrate knockout of the hamster FUT8 gene via the simultaneous ZFN- or TALE nuclease-mediated integration of an antibody cassette. Our results enable efficient targeted transgene addition to cells and organisms that fare poorly with traditional homology-driven approaches.

Highly efficient deletion of FUT8 in CHO cell lines using zinc-finger nucleases yields cells that produce completely nonfucosylated antibodies.

IgG1 antibodies produced in Chinese hamster ovary (CHO) cells are heavily alpha1,6-fucosylated, a modification that reduces antibody-dependent cellular cytotoxicity (ADCC) and can inhibit therapeutic antibody function in vivo. Addition of fucose is catalyzed by Fut8, a alpha1,6-fucosyltransferase. FUT8(-/-) CHO cell lines produce completely nonfucosylated antibodies, but the difficulty of recapitulating the knockout in protein-production cell lines has prevented the widespread adoption of FUT8(-/-) cells as hosts for antibody production. We have created zinc-finger nucleases (ZFNs) that cleave the FUT8 gene in a region encoding the catalytic core of the enzyme, allowing the functional disruption of FUT8 in any CHO cell line. These reagents produce FUT8(-/-) CHO cells in 3 weeks at a frequency of 5% in the absence of any selection. Alternately, populations of ZFN-treated cells can be directly selected to give FUT8(-/-) cell pools in as few as 3 days. To demonstrate the utility of this method in bioprocess, FUT8 was disrupted in a CHO cell line used for stable protein production. ZFN-derived FUT8(-/-) cell lines were as transfectable as wild-type, had similar or better growth profiles, and produced equivalent amounts of antibody during transient transfection. Antibodies made in these lines completely lacked core fucosylation but had an otherwise normal glycosylation pattern. Cell lines stably expressing a model antibody were made from wild-type and ZFN-generated FUT8(-/-) cells. Clones from both lines had equivalent titer, specific productivity distributions, and integrated viable cell counts. Antibody titer in the best ZFN-generated FUT8(-/-) cell lines was fourfold higher than in the best-producing clones of FUT8(-/-) cells made by standard homologous recombination in a different CHO subtype. These data demonstrate the straightforward, ZFN-mediated transfer of the Fut8- phenotype to a production CHO cell line without adverse phenotypic effects. This process will speed the production of highly active, completely nonfucosylated therapeutic antibodies.

Zinc-finger nuclease-driven targeted integration into mammalian genomes using donors with limited chromosomal homology.

We previously demonstrated high-frequency, targeted DNA addition mediated by the homology-directed DNA repair pathway. This method uses a zinc-finger nuclease (ZFN) to create a site-specific double-strand break (DSB) that facilitates copying of genetic information into the chromosome from an exogenous donor molecule. Such donors typically contain two approximately 750 bp regions of chromosomal sequence required for homology-directed DNA repair. Here, we demonstrate that easily-generated linear donors with extremely short (50 bp) homology regions drive transgene integration into 5-10% of chromosomes. Moreover, we measure the overhangs produced by ZFN cleavage and find that oligonucleotide donors with single-stranded 5' overhangs complementary to those made by ZFNs are efficiently ligated in vivo to the DSB. Greater than 10% of all chromosomes directly incorporate this exogenous DNA via a process that is dependent upon and guided by complementary 5' overhangs on the donor DNA. Finally, we extend this non-homologous end-joining (NHEJ)-based technique by directly inserting donor DNA comprising recombinase sites into large deletions created by the simultaneous action of two separate ZFN pairs. Up to 50% of deletions contained a donor insertion. Targeted DNA addition via NHEJ complements our homology-directed targeted integration approaches, adding versatility to the manipulation of mammalian genomes.

BAK and BAX deletion using zinc-finger nucleases yields apoptosis-resistant CHO cells.

Anoxic and metabolic stresses in large-scale cell culture during biopharmaceutical production can induce apoptosis. Strategies designed to ameliorate the problem of apoptosis in cell culture have focused on mRNA knockdown of pro-apoptotic proteins and over-expression of anti-apoptotic ones. Apoptosis in cell culture involves mitochondrial permeabilization by the pro-apoptotic Bak and Bax proteins; activity of either protein is sufficient to permit apoptosis. We demonstrate here the complete and permanent elimination of both the Bak and Bax proteins in combination in Chinese hamster ovary (CHO) cells using zinc-finger nuclease-mediated gene disruption. Zinc-finger nuclease cleavage of BAX and BAK followed by inaccurate DNA repair resulted in knockout of both genes. Cells lacking Bax and Bak grow normally but fail to activate caspases in response to apoptotic stimuli. When grown using scale-down systems under conditions that mimic growth in large-scale bioreactors they are significantly more resistant to apoptosis induced by starvation, staurosporine, and sodium butyrate. When grown under starvation conditions, BAX- and BAK-deleted cells produce two- to fivefold more IgG than wild-type CHO cells. Under normal growth conditions in suspension culture in shake flasks, double-knockout cultures achieve equal or higher cell densities than unmodified wild-type cultures and reach viable cell densities relevant for large-scale industrial protein production.

Knockout rats via embryo microinjection of zinc-finger nucleases.

The toolbox of rat genetics currently lacks the ability to introduce site-directed, heritable mutations into the genome to create knockout animals. By using engineered zinc-finger nucleases (ZFNs) designed to target an integrated reporter and two endogenous rat genes, Immunoglobulin M (IgM) and Rab38, we demonstrate that a single injection of DNA or messenger RNA encoding ZFNs into the one-cell rat embryo leads to a high frequency of animals carrying 25 to 100% disruption at the target locus. These mutations are faithfully and efficiently transmitted through the germline. Our data demonstrate the feasibility of targeted gene disruption in multiple rat strains within 4 months time, paving the way to a humanized monoclonal antibody platform and additional human disease models.