Comparison of ligamentization potential between anterior cruciate ligament-derived cells and adipose-derived mesenchymal stem cells reseeded to acellularized tendon allograft.

AIMS: To test the hypothesis that reseeded anterior cruciate ligament (ACL)-derived cells have a better ability to survive and integrate into tendon extracellular matrix (ECM) and accelerate the ligamentization process, compared to adipose-derived mesenchymal stem cells (ADMSCs). METHODS: Acellularized tibialis allograft tendons were used. Tendons were randomly reseeded with ACL-derived cells or ADMSCs. ACL-derived cells were harvested and isolated from remnants of ruptured ACLs during reconstruction surgery and cultured at passage three. Cell suspensions (200 µl) containing 2 × 106 ACL-derived cells or ADMSCs were prepared for the purpose of reseeding. At days 1, 3, and 7 post-reseeding, graft composites were assessed for repopulation with histological and immunohistochemical analysis. Matrix protein contents and gene expression levels were analyzed. RESULTS: In the graft reseeded with ACL-derived cells, a large number of elongated cells that integrated into the matrix were evident at day 3 and day 7. However, in the graft reseeded with ADMSCs, only a small number of elongated cells were found integrated into the matrix. Immunofluorescence for Ki-67 and type I collagen confirmed the pronounced production of type I collagen by Ki-67-positive ACL-derived cells integrated into the ECM. A messenger RNA (mRNA) expression assay demonstrated significantly higher gene expression levels of types I (p = 0.013) and III (p = 0.050) collagen in the composites reseeded with ACL-derived cells than ADMSCs. CONCLUSION: ACL-derived cells, when reseeded to acellularized tendon graft, demonstrated earlier better survival and integration in the tendon ECM and resulted in higher gene expression levels of collagen, which may be essential to the normal ligamentization process compared to ADMSCs.Cite this article: Bone Joint Res 2022;11(11):777-786.

Characterization of nebulized liposomal amikacin (Arikace) as a function of droplet size.

The stress of nebulization has been shown to alter the properties of liposomal drugs. What has not been demonstrated is whether nebulized liposomes differ as a function of droplet size. Because droplet size influences lung deposition, liposomes with different properties could be deposited in different areas of the lung (e.g., central vs. peripheral). In this report, a liposomal amikacin formulation (Arikace, a registered trademark of Transave, Inc., Monmouth Junction, NJ) that is being developed as an inhaled treatment for gram negative infections was aerosolized with an eFlow (registered trademark of PARI, GmbH, Munich, Germany) nebulizer, reclaimed from the various stages of an Andersen cascade impactor (ACI) and analyzed for lipid-to-drug (L/D) (w/w) ratio, amikacin retention, and liposome size. For the nebulized solution, 99.7% of the total deposited drug was found on ACI stages 0 through 5, which have cutoff diameters of 9, 5.8, 4.7, 3.3, 2.1, and 1.1 microm, respectively. Properties were found to differ for drug reclaimed on stage 0 compared stages 1-5, which were not different from one another. For drug found on stages 1-5 (97% of total drug), the averages (n = 3) for L/D, percent encapsulated amikacin, and liposome mean diameter ranged from 0.59 to 0.68 (w/w), 71% to 75%, 248 to 282 nm, respectively. Drug found on stage 0 (2.8% of total drug) had an average L/D ratio of 0.51 and average liposome mean diameter of 375 nm. Examination of another batch of liposomal amikacin revealed no statistically significant differences between drug reclaimed on stages 0-5. Although a droplet size dependence was noted for one batch of Arikace aerosolized with the eFlow, the effect was considered to be inconsequential because the fraction in doubt represented nonrespirable particles >9 microm and accounted for <3% of the total deposited dose. The methodology applied here appears useful in evaluating aerosolized liposome systems. However, our results should not be assumed to apply to other liposome/drug compositions and nebulizers.

Functional interaction between paramyxovirus fusion and attachment proteins.

Paramyxovirinae envelope glycoproteins constitute a premier model to dissect how specific and dynamic interactions in multisubunit membrane protein complexes can control deep-seated conformational rearrangements. However, individual residues that determine reciprocal specificity of the viral attachment and fusion (F) proteins have not been identified. We have developed an assay based on a pair of canine distemper virus (CDV) F proteins (strains Onderstepoort (ODP) and Lederle) that share approximately 95% identity but differ in their ability to form functional complexes with the measles virus (MV) attachment protein (H). Characterization of CDV F chimeras and mutagenesis reveals four residues in CDV F-ODP (positions 164, 219, 233, and 317) required for productive interaction with MV H. Mutating these residues to the Lederle type disrupts triggering of F-ODP by MV H without affecting functionality when co-expressed with CDV H. Co-immunoprecipitation shows a stronger physical interaction of F-ODP than F-Lederle with MV H. Mutagenesis of MV F highlights the MV residues homologous to CDV F residues 233 and 317 as determinants for physical glycoprotein interaction and fusion activity under homotypic conditions. In assay reversal, the introduction of sections of the CDV H stalk into MV H shows a five-residue fragment (residues 110-114) to mediate specificity for CDV F-Lederle. All of the MV H stalk chimeras are surface-expressed, show hemadsorption activity, and trigger MV F. Combining the five-residue H chimera with the CDV F-ODP quadruple mutant partially restores activity, indicating that the residues identified in either glycoprotein contribute interdependently to the formation of functional complexes. Their localization in structural models of F and H suggests that placement in particular of F residue 233 in close proximity to the 110-114 region of H is structurally conceivable.

Reversible inhibition of the fusion activity of measles virus F protein by an engineered intersubunit disulfide bridge.

In search of target sites for the development of paramyxovirus inhibitors, we have engineered disulfide bridges to introduce covalent links into the prefusion F protein trimer of measles virus. F-Edm-452C/460C, predicted to bridge head and stalk domains of different F monomers, shows a high degree of proteolytic maturation and surface expression, predominantly as stable, dithiothreitol-sensitive trimers, but no fusion activity. Reduction of disulfide bridges partially restores activity. These findings underscore the importance of reversible intersubunit interactions between the stalk and head domains for F activity. Noncovalent small molecules mimicking this behavior may constitute a potent strategy for preventing paramyxovirus entry.

Nonnucleoside inhibitor of measles virus RNA-dependent RNA polymerase complex activity.

Paramyxoviruses comprise several major human pathogens. Although a live-attenuated vaccine protects against measles virus (MV), a member of the paramyxovirus family, the virus remains a principal cause of worldwide mortality and accounts for approximately 21 million cases and 300,000 to 400,000 deaths annually. The development of novel antivirals that allow improved case management of severe measles and silence viral outbreaks is thus highly desirable. We have previously described the development of novel MV fusion inhibitors. The potential for preexisting or emerging resistance in the field constitutes the rationale for the identification of additional MV inhibitors with a diverse target spectrum. Here, we report the development and implementation of a cell-based assay for high-throughput screening of MV antivirals, which has yielded several hit candidates. Following confirmation by secondary assays and chemical synthesis, the most potent hit was found to act as a target-specific inhibitor of MV replication with desirable drug-like properties. The compound proved highly active against multiple primary isolates of diverse MV genotypes currently circulating worldwide, showing active concentrations of 35 to 145 nM. Significantly, it does not interfere with viral entry and lacks cross-resistance with the MV fusion inhibitor class. Mechanistic characterization on a subinfection level revealed that the compound represents a first-in-class nonnucleoside inhibitor of MV RNA-dependent RNA polymerase complex activity. Singly or in combination with the fusion inhibitors, this novel compound class has high developmental potential as a potent therapeutic against MV and will likely further the mechanistic characterization of the viral polymerase complex.

Assessing a system to capture stray aerosol during inhalation of nebulized liposomal cisplatin.

The aim of this study was to determine the efficacy of using a high-efficiency particulate air (HEPA) filter air cleaning system, a demistifier, to reduce the potential risk of fugitive aerosol contact in health care personnel working with patients inhaling nebulized liposomal encapsulated SLIT (Sustained-release Lipid Inhalation Targeting) Cisplatin. Filters were used to sample platinum in the air outside the tent and from the tent's exhaust stream. Air collection was performed under three conditions: (1) during patient dosing (14 h of air collection); (2) immediately after the patient has left the demistifier tent (4 h of air collection); and (3) when 7 mL of drug product was nebulized to dryness in the tent without a patient being present. Filters were collected, and placed in an extraction solvent. Subsequently, the solvent was assayed for platinum content by inductively coupled plasma-mass spectrometry (ICP-MS). Platinum levels in the extraction solvent were indistinguishable from the blank controls for all conditions. Measured levels were below workplace exposure limits established for cisplatin by the Occupational Safety and Health Administration (i.e., 2 ng . (L(1)). In addition, the demistifier was able to effectively capture aerosolized SLIT Cisplatin following nebulization of 7 mL of drug product to dryness in the tent. The demistifier tent is effective at containing any nebulized liposomal encapsulated cisplatin during patient treatment. Importantly, because the tent's HEPA filtration system is effective at removing any nebulized liposomal cisplatin, the exhausted air, which is free of platinum, can be returned into the room with no additional ventilation precautions.