A Direct Comparison of rAAV5 Variants Derived from the Baculovirus Expression System Using LC-MS Workflows Demonstrates Key Differences in Overall Production Yield, Product Quality and Vector Efficiency.

Gene therapy holds great promise for the treatment of severe diseases, and adeno-associated virus (AAV) vectors have emerged as valuable tools in this field. However, challenges such as immunogenicity and high production costs complicate the commercial viability of AAV-based therapies. To overcome these barriers, improvements in production yield, driven through the availability of robust and sensitive characterization techniques that allow for the monitoring of critical quality attributes to deepen product and process understanding are crucial. Among the main attributes affecting viral production and performance, the ratio between empty and full capsids along with capsid protein stoichiometry are emerging as potential parameters affecting product quality and safety. This study focused on the production of AAV vectors using the baculovirus expression vector system (BEVS) in Sf9 cells and the complete characterization of AAV5 variants using novel liquid chromatography and mass spectrometry techniques (LC-MS) that, up to this point, had only been applied to reference commercially produced virions. When comparing virions produced using ATG, CTG or ACG start codons of the cap gene, we determined that although ACG was the most productive in terms of virus yield, it was also the least effective in transducing mammalian cells. This correlated with a low VP1/VP2 ratio and a higher percentage of empty capsids. Overall, this study provides insights into the impact of translational start codon modifications during rAAV5 production using the BEVS, the associated relationship with capsid packaging, capsid protein stoichiometry and potency. The developed characterization workflow using LC-MS offers a comprehensive and transferable analysis of AAV-based gene therapies, with the potential to aid in process optimization and facilitate the large-scale commercial manufacturing of these promising treatments.

Degradation behavior and osseointegration of Mg-Zn-Ca screws in different bone regions of growing sheep: a pilot study.

Magnesium (Mg)-based implants are highly attractive for the orthopedic field and may replace titanium (Ti) as support for fracture healing. To determine the implant-bone interaction in different bony regions, we implanted Mg-based alloy ZX00 (Mg < 0.5 Zn < 0.5 Ca, in wt%) and Ti-screws into the distal epiphysis and distal metaphysis of sheep tibiae. The implant degradation and osseointegration were assessed in vivo and ex vivo after 4, 6 and 12 weeks, using a combination of clinical computed tomography, medium-resolution micro computed tomography (µCT) and high-resolution synchrotron radiation µCT (SRµCT). Implant volume loss, gas formation and bone growth were evaluated for both implantation sites and each bone region independently. Additionally, histological analysis of bone growth was performed on embedded hard-tissue samples. We demonstrate that in all cases, the degradation rate of ZX00-implants ranges between 0.23 and 0.75 mm/year. The highest degradation rates were found in the epiphysis. Bone-to-implant contact varied between the time points and bone types for both materials. Mostly, bone-volume-to-total-volume was higher around Ti-implants. However, we found an increased cortical thickness around the ZX00-screws when compared with the Ti-screws. Our results showed the suitability of ZX00-screws for implantation into the distal meta- and epiphysis.

Proteomic Landscape of Adeno-Associated Virus (AAV)-Producing HEK293 Cells.

Adeno-associated viral (AAV) vectors are widely used for gene therapy, providing treatment for diseases caused by absent or defective genes. Despite the success of gene therapy, AAV manufacturing is still challenging, with production yields being limited. With increased patient demand, improvements in host cell productivity through various engineering strategies will be necessary. Here, we study the host cell proteome of AAV5-producing HEK293 cells using reversed phase nano-liquid chromatography and tandem mass spectrometry (RPLC-MS/MS). Relative label-free quantitation (LFQ) was performed, allowing a comparison of transfected vs. untransfected cells. Gene ontology enrichment and pathway analysis revealed differential expression of proteins involved in fundamental cellular processes such as metabolism, proliferation, and cell death. Furthermore, changes in expression of proteins involved in endocytosis and lysosomal degradation were observed. Our data provides highly valuable insights into cellular mechanisms involved during recombinant AAV production by HEK293 cells, thus potentially enabling further improvements of gene therapy product manufacturing.

A Rare Case of Spinal Cord Injury Following Thoracic Radiofrequency Ablation.

Medial branch radiofrequency ablation (RFA) has become a common treatment for facet-related back pain. While this procedure is often performed in the lumbar and cervical spinal segments, it can also be applied to the thoracic spine. Complications of spinal RFA at any level are scarce in the literature and are often mild. The patient is a 37-year-old male with a family history of multiple sclerosis in his siblings who underwent thoracic RFA at the T2-T5 nerve root levels. Within one week of the procedure, the patient experienced paresthesias below the nipple line and progressive lower-extremity weakness. He was also found to exhibit urinary retention on presentation to our facility one month later. MRI showed focal cord short T1 inversion recovery (STIR) signal abnormality at the T3-T4 level, favored to represent myelomalacia. An extensive laboratory and imaging workup was otherwise unrevealing. The patient was treated with neuromodulators and a short course of inpatient rehabilitation. One year later, he used knee-ankle-foot orthoses for ambulating short distances and a manual wheelchair for longer distances, and he no longer required intermittent catheterization for bladder management. This case presents a rare and unusual timeline of symptom evolution, laboratory findings, and imaging results that do not unveil a clear pathophysiological mechanism, which led to the patient's spinal cord injury. The clinical level of injury based on the patient's symptoms and location of myelomalacia on MRI, however, strongly support a causative contribution by the thoracic RFA procedure.

Cervical Myelopathy and Peripheral Polyneuropathy in a 26-Year-Old Female Following Bariatric Surgery.

The frequency, clinical course, and prognosis of the neurological sequelae following bariatric surgery remain obscure and continue to be a subject of medical research. We present the case of a 26-year-old female who underwent sleeve gastrectomy for the treatment of obesity and demonstrated progressive neurological deficits within months of her procedure. Extensive testing revealed very low thiamine and copper levels, peripheral polyneuropathy, and spinal cord lesions on imaging. She was treated with intravenous copper and thiamine and was admitted to the rehabilitation unit. Eighteen months following her admission, she achieved complete recovery. Previous studies have reported neurological complications following bariatric surgery in 1.1-8.6% of cases. Some of the most common nutritional deficiencies involve copper, iron, calcium, magnesium, and vitamins B1, B12, D, and E. Patients may experience central and peripheral neurological deficits following bariatric surgery. Fortunately, a path to recovery exists and it involves both pharmacological and rehabilitative treatment.

Pain relief following genicular nerve radiofrequency ablation: does knee compartment matter?

Aim: To investigate the effect of knee osteoarthritis (OA) compartment location on pain relief following genicular radiofrequency ablation. Materials & methods: A retrospective chart review was performed on 62 patients. Visual analog scale scores at 3 and 6 months post procedure were compared with baseline and between compartment groups. Results: Pain significantly improved for all patients at 3 and 6 months (p < 0.001 and p = 0.005, respectively). Medial compartment OA was a significant predictor of improvement at 3 months (p = 0.042). Patellofemoral compartment OA was a significant predictor for a higher visual analog scale at 3 months (p = 0.018). Conclusion: Compartmental location of knee OA impacts pain relief following genicular radiofrequency ablation. Future protocols could target nerves based on which compartments are more affected on imaging.

Lay abstract Aim: To investigate the effect of knee arthritis location on pain relief following planned nerve disruption using radiofrequency ablation (RFA). Materials & methods: This study analyzed existing records of 62 cases of patients who underwent an ablation procedure to the sensory nerves of the knee. On a scale from one to ten, pain after RFA at 3 and 6 months was compared with baseline and compared between arthritis location groups. Results: Pain decreased for all patients at 3 and 6 months. Inner knee arthritis was a predictor of pain improvement at 3 months. Arthritis of the kneecap was a predictor for worse pain at 3 months. Conclusion: Location of knee arthritis impacts pain relief following nerve disruption using RFA. Future protocols could target nerves based on arthritis location.

A rationally designed oral vaccine induces immunoglobulin A in the murine gut that directs the evolution of attenuated Salmonella variants.

The ability of gut bacterial pathogens to escape immunity by antigenic variation-particularly via changes to surface-exposed antigens-is a major barrier to immune clearance1. However, not all variants are equally fit in all environments2,3. It should therefore be possible to exploit such immune escape mechanisms to direct an evolutionary trade-off. Here, we demonstrate this phenomenon using Salmonella enterica subspecies enterica serovar Typhimurium (S.Tm). A dominant surface antigen of S.Tm is its O-antigen: a long, repetitive glycan that can be rapidly varied by mutations in biosynthetic pathways or by phase variation4,5. We quantified the selective advantage of O-antigen variants in the presence and absence of O-antigen-specific immunoglobulin A and identified a set of evolutionary trajectories allowing immune escape without an associated fitness cost in naive mice. Through the use of rationally designed oral vaccines, we induced immunoglobulin A responses blocking all of these trajectories. This selected for Salmonella mutants carrying deletions of the O-antigen polymerase gene wzyB. Due to their short O-antigen, these evolved mutants were more susceptible to environmental stressors (detergents or complement) and predation (bacteriophages) and were impaired in gut colonization and virulence in mice. Therefore, a rationally induced cocktail of intestinal antibodies can direct an evolutionary trade-off in S.Tm. This lays the foundations for the exploration of mucosal vaccines capable of setting evolutionary traps as a prophylactic strategy.

A Rare Case of Nontraumatic Thoracic Compression Fracture in a 28-Year-Old Man.

ABSTRACT: A 28-yr-old African American man with a history of synovitis, acne, pustulosis, hyperostosis, and osteitis syndrome, tobacco use, and sickle cell trait was referred to a physiatrist at a multidisciplinary spine center with a 1-yr history of nontraumatic thoracic back pain that had significantly worsened over the previous 4 wks. In the context of recurrent infections requiring hospital admissions and the patient's immunosuppressed status, magnetic resonance imaging of his thoracic spine was obtained, showing an acute or subacute anterior compression deformity of the T7 vertebral body. He was subsequently provided with a hyperextension brace, physical therapy referral, and a trial of intranasal calcitonin. The patient reported significant improvement in pain at his 3-mo follow-up appointment and continued to show pain and functional improvement in physical therapy sessions up to 6 mos later.

A lean magnesium-zinc-calcium alloy ZX00 used for bone fracture stabilization in a large growing-animal model.

Over the last decade, demand has increased for developing new, alternative materials in pediatric trauma care to overcome the disadvantages associated with conventional implant materials. Magnesium (Mg)-based alloys seem to adequately fulfill the vision of a homogeneously resorbable, biocompatible, load-bearing and functionally supportive implant. The aim of the present study is to introduce the high-strength, lean alloy Mg‒0.45Zn‒0.45Ca, in wt% (ZX00), and for the first time investigate the clinical applicability of screw osteosynthesis using this alloy that contains no rare-earth elements. The alloy was applied in a growing sheep model with osteotomized bone (simulating a fracture) and compared to a non-osteotomy control group regarding degradation behavior and fracture healing. The alloy exhibits an ultimate tensile strength of 285.7 ± 3.1 MPa, an elongation at fracture of 18.2 ± 2.1%, and a reduced in vitro degradation rate compared to alloys containing higher amounts of Zn. In vivo, no significant difference between the osteotomized bone and the control group was found regarding the change in screw volume over implantation time. Therefore, it can be concluded that the fracture healing process, including its effects on the surrounding area, has no significant influence on degradation behavior. There was also no negative influence from hydrogen-gas formation on fracture healing. Despite the proximal and distal screws showing chronologically different gas release, the osteotomy showed complete consolidation. STATEMENT OF SIGNIFICANCE: Conventional implants involve several disadvantages in pediatric trauma care. Magnesium-based alloys seem to overcome these issues as discussed in the recent literature. This study evaluates the clinical applicability of high-strength lean Mg‒0.45Zn‒0.45Ca (ZX00) screws in a growing-sheep model. Two groups, one including a simulated fracture and one group without fracture, underwent implantation of the alloy and were compared to each other. No significant difference regarding screw volume was observed between the groups. There was no negative influence of hydrogen-gas formation on fracture healing and a complete fracture consolidation was found after 12 weeks for all animals investigated.

Comparing pain relief and functional improvement between methylprednisolone and dexamethasone lumbosacral transforaminal epidural steroid injections: a self-controlled study.

BACKGROUND: Previous studies have shown varying results between lumbosacral transforaminal epidural steroid injections (TFESIs) performed with particulate versus non-particulate corticosteroids. The purpose of this study was to investigate the difference in pain relief and functional improvement between particulate and non-particulate lumbosacral TFESIs in patients who had undergone both injections, sequentially. METHODS: This was a self-controlled, retrospective study of 20 patients who underwent both a methylprednisolone and a dexamethasone TFESI to the same vertebral level and side. Primary outcomes included pain relief according to the visual analogue scale (VAS) and functional improvement determined by a yes/no answer to questions regarding mobility and the activities of daily living. Post-injection data was recorded at 2, 3, and 6 months. RESULTS: A decrease in VAS scores of -3.4 ± 3.0 (mean ± standard deviation), -3.1 ± 3.1, and -2.8 ± 3.4 was seen for the methylprednisolone group at 2, 3, and 6 months, respectively. Similar decreases of -3.9 ± 3.5, -3.4 ± 2.8, and -2.3 ± 3.4 were seen in the dexamethasone group. There was no significant difference in pain relief at any point between the two medications. The percentage of subjects who reported improved function at 2, 3, and 6 months was 65%, 51%, and 41%, respectively, for the methylprednisolone group and 75%, 53%, and 42% for the dexamethasone group. CONCLUSIONS: These findings support the use of non-particulate corticosteroids for lumbosacral TFESIs in the context of documented safety concerns with particulate corticosteroids.

CoCrMo surface modifications affect biocompatibility, adhesion, and inflammation in human osteoblasts.

In this study, different surface modifications were performed on a Cobalt-Chrome-Molybdenum (CoCrMo) alloy and the effects on cell viability and cytotoxicity as well as the adhesion potential of human osteoblasts (hFOB) and their inflammation reaction were investigated in vitro. CoCrMo discs were coated with TiN, with polished and porous coated surfaces, or with pure titanum (cpTi) surfaces and examined by Scanning Electron Microscopy to evaluate surface modifications. In vitro cell viability, adhesion behaviour, and expression of inflammation markers of hFOB human osteoblasts were measured via CellTiter-Glo, CytoTox, ELISA, and RT-PCR respectively. All results were compared to CoCrMo without surface modifications. The biocompatibility data showed high compatibility for the TiN hard coatings. Likewise, the porous surface coating increased cell viability significantly, compared to an untreated CoCrMo alloy. None of the investigated materials influenced cytotoxicity. Different surface modifications did not influence expression of fibronectin, although TiN, porous surface coatings and polished surfaces showed highly significant reductions in integrin subunit expression. In addition to the regulation of adhesion potential these three surfaces stimulated an anti-inflammatory response by osteocytes. Improved biocompatibility and adhesion properties may contribute to better osteointegration of prosthetics.

Resorbable implants in pediatric fracture treatment.

Pediatric osteosynthesis has developed over the last 20 years, thereby reducing medical and economic burden, including long and expensive hospitalization. Currently, conventional and rigid alloying systems such as titanium are used for stabilization of bone fractures in children. In many cases, implants must be removed, as otherwise growth would be impeded. Biodegradable implant materials exhibit beneficial properties and would make a second removal surgery unnecessary. In the following article, we will give an overview of implant materials that are currently used in pediatric traumatology with a focus on Mg-based implants. Furthermore, we will discuss current scientific knowledge on resorbable implants, including results from pre-clinics and clinics.

Histone deacetylase inhibitors vorinostat and panobinostat induce G1 cell cycle arrest and apoptosis in multidrug resistant sarcoma cell lines.

Synovial sarcoma and high grade chondrosarcoma are characterized by their lack of response to conventional cytotoxic chemotherapy, the tendency to develop lung metastases, and low survival rates. Research within the field prioritizes the development and expansion of new treatment options for dealing with unresectable or metastatic diseases. Numerous clinical trials using histone deacetylases inhibitors (HDACi) have shown specific efficacy as an active antitumor agent for treating a variety of solid tumors. However, as of yet the effect of different HDACi on synovial- and chondrosarcoma cells has not been investigated. In this study, vorinostat (SAHA), panobinostat (LBH-589), and belinostat (PXD101) decreased cell viability of synovial sarcoma (SW-982) and chondrosarcoma (SW-1353) cells in a time- and dose dependent manner and arrested SW-982 cells in the G1/S phase. Western blot analysis determined the responsible cell cycle regulator proteins. In addition, we found apoptotic induction by caspase 3/7 activity, caspase 3 cleavage, and PARP cleavage. In SW-1353 cells only SAHA showed comparable effects. Noteworthy, all HDACi tested had synergistic effects with the topoisomerase II inhibitor doxorubicin in SW-1353 chondrosarcoma cells making the cells more sensitive to the chemotherapeutic drug. Our results show for the first time that SAHA and LBH-589 reduced viability of sarcoma cells and arrested them at the G1/S checkpoint, while also inducing apoptosis and enhancing chemotherapeutic sensitivity, especially in chondrosarcoma cells. These data demonstrate the exciting potential of HDACi for use in sarcoma treatment.

Pre-operative corticosteroid injections improve functional outcomes in patients undergoing arthroscopic repair of high-grade partial-thickness rotator cuff tears.

BACKGROUND: Subacromial corticosteroid injections (CSI's) are a common non-surgical treatment for rotator cuff tears. Few studies have assessed the effects of pre-operative CSI's on postoperative functional outcomes. METHODS: A retrospective analysis was conducted of 132 patients with high-grade, partial-thickness rotator cuff tears (PTRCT's). The subjects were divided into two groups based on whether they received a CSI or not. The CSI group was further divided into three subgroups based on when they received a pre-operative injection: 0-3 months, 3-6 months, >6 months before surgery. The Visual Analog Scores (VAS), American Shoulder and Elbow Surgeon scores (ASES), and Constant scores were recorded prior to surgery and at a one-year post-operative follow-up appointment for each subject. RESULTS: Patients who received a pre-operative CSI (n=92) improved significantly more than the non-injection group (n=40) in all outcome measures. The 0-3 months injection subgroup experienced a significant increase in ASES and Constant score (p=0.019 and 0.014, respectively) compared to the other two subgroups, but the VAS score decrease only trended toward significance (p=0.091). The sample as a whole experienced significant improvement in all three outcome measures. CONCLUSION: Patients undergoing arthroscopic repair of a high-grade PTRCT may benefit from a pre-operative CSI 0-3 months before surgery. LEVEL OF EVIDENCE: IIb.

Salmonella enterica Serovar Typhi Lipopolysaccharide O-Antigen Modification Impact on Serum Resistance and Antibody Recognition.

Salmonella enterica serovar Typhi is a human-restricted Gram-negative bacterial pathogen responsible for causing an estimated 27 million cases of typhoid fever annually, leading to 217,000 deaths, and current vaccines do not offer full protection. The O-antigen side chain of the lipopolysaccharide is an immunodominant antigen, can define host-pathogen interactions, and is under consideration as a vaccine target for some Gram-negative species. The composition of the O-antigen can be modified by the activity of glycosyltransferase (gtr) operons acquired by horizontal gene transfer. Here we investigate the role of two gtr operons that we identified in the S Typhi genome. Strains were engineered to express specific gtr operons. Full chemical analysis of the O-antigens of these strains identified gtr-dependent glucosylation and acetylation. The glucosylated form of the O-antigen mediated enhanced survival in human serum and decreased complement binding. A single nucleotide deviation from an epigenetic phase variation signature sequence rendered the expression of this glucosylating gtr operon uniform in the population. In contrast, the expression of the acetylating gtrC gene is controlled by epigenetic phase variation. Acetylation did not affect serum survival, but phase variation can be an immune evasion mechanism, and thus, this modification may contribute to persistence in a host. In murine immunization studies, both O-antigen modifications were generally immunodominant. Our results emphasize that natural O-antigen modifications should be taken into consideration when assessing responses to vaccines, especially O-antigen-based vaccines, and that the Salmonellagtr repertoire may confound the protective efficacy of broad-ranging Salmonella lipopolysaccharide conjugate vaccines.

High-Grade Articular, Bursal, and Intratendinous Partial-Thickness Rotator Cuff Tears: A Retrospective Study Comparing Functional Outcomes After Completion and Repair.

We conducted a study to assess the impact of tear location on functional outcomes in high-grade partial-thickness rotator cuff tears (PTRCTs) after arthroscopic completion and repair. Retrospectively, we evaluated the preoperative and postoperative findings of 60 patients who underwent arthroscopic completion and repair of Ellman grade 3 partial-thickness tears of the supraspinatus. The 60 patients were grouped by tear subtype (20 articular, 20 bursal, 20 intratendinous) as identified by preoperative imaging and confirmed at time of surgery. After surgery, the 3 subtypes showed similar significant (P < .001) improvements in American Shoulder and Elbow Surgeons scores (articular, 46.9, 85.1; bursal, 44.3, 80.3; intratendinous, 43.6, 86.1), Constant scores (articular, 54.3, 79.4; bursal, 49.9, 75.0; intratendinous, 56.8, 80.9), and visual analog scale scores (articular, 5.1, 1.2; bursal, 5.8, 1.6; intratendinous, 6.0, 1.2). Our study findings validate use of the current algorithm for Ellman grade 3 PTRCTs of the supraspinatus and advocate their completion and repair, regardless of tear location.