Mechanisms of aberrant organization of growth plates in conditional transgenic mouse model of spondyloepiphyseal dysplasia associated with the R992C substitution in collagen II.

Mutations in collagen II, a main structural protein of cartilage, are associated with various forms of spondyloepiphyseal dysplasia (SED), whose main features include aberrations of linear growth. Here, we analyzed the pathomechanisms responsible for growth alterations in transgenic mice with conditional expression of the R992C collagen II mutation. Specifically, we studied the alterations of the growth plates of mutant mice in which chondrocytes lacked their typical columnar arrangement. Our studies demonstrated that chondrocytes expressing the thermolabile R992C mutant collagen II molecules endured endoplasmic reticulum stress, had atypical polarization, and had reduced proliferation. Moreover, we demonstrated aberrant organization and morphology of primary cilia. Analyses of the extracellular collagenous deposits in mice expressing the R992C mutant collagen II molecules indicated their poor formation and distribution. By contrast, transgenic mice expressing wild-type collagen II and mice in which the expression of the transgene encoding the R992C collagen II was switched off were characterized by normal growth, and the morphology of their growth plates was correct. Our study with the use of a conditional mouse SED model not only indicates a direct relation between the observed aberration of skeletal tissues and the presence of mutant collagen II, but also identifies cellular and matrix elements of the pathomechanism of SED.

Testing the anti-fibrotic potential of the single-chain Fv antibody against the α2 C-terminal telopeptide of collagen I.

Abstract This study focuses on the single-chain fragment variable (scFv) variant of the original IgA-type antibody, recognizing the α2 C-terminal telopeptide (α2Ct) of human collagen I, designed to inhibit post-traumatic localized fibrosis via blocking the formation of collagen-rich deposits. We have demonstrated that the scFv construct expressed in yeast cells was able to fold into an immunoglobulin-like conformation, but it was prone to forming soluble aggregates. Functional assays, however, indicate that the scFv construct specifically binds to the α2Ct epitope and inhibits collagen fibril formation both in vitro and in a cell culture model representing tissues that undergo post-traumatic fibrosis. Thus, the presented study demonstrates the potential of the scFv variant to serve as an inhibitor of the excessive formation of collagen-rich fibrotic deposits, and it reveals certain limitations associated with the current stage of development of this antibody construct.

Engineering and characterization of the chimeric antibody that targets the C-terminal telopeptide of the α2 chain of human collagen I: a next step in the quest to reduce localized fibrosis.

Inhibition of the extracellular process of collagen fibril formation represents a new approach to limiting posttraumatic or postsurgical localized fibrosis. It has been demonstrated that employing a monoclonal antibody that targets the C-terminal telopeptide of the α2 chain of collagen I blocks critical collagen I-collagen I interaction, thereby reducing the amount of collagen deposits in vitro and in animal models. Here, we developed a chimeric variant of a prototypic inhibitory antibody of mouse origin. The structure of this novel antibody was analyzed by biochemical and biophysical methods. Moreover, detailed biochemical and biological studies were employed to test its antigen-binding characteristics. The ability of the chimeric variant to block formation of collagen fibrils was tested in vitro and in high-density cultures representing fibrotic processes occurring in the skin, tendon, joint capsule, and gingiva. The potential toxicity of the novel chimeric antibody was analyzed through its impact on the viability and proliferation of various cells and by testing its tissue cross-reactivity in sets of arrays of human and mouse tissues. Results of the presented studies indicate that engineered antibody-based blocker of localized fibrosis is characterized by the following: (1) a correct IgG-like structure, (2) high affinity and high specificity for a defined epitope, (3) a great potential to limit the accumulation of collagen-rich deposits, and (4) a lack of cytotoxicity and nonspecific tissue reactivity. Together, the presented study shows the great potential of the novel chimeric antibody to limit localized fibrosis, thereby setting ground for critical preclinical tests in a relevant animal model.

"Learning From Disaster: What Past Events Can Teach Radiology Departments about Planning for a Mass Casualty Incident".

The increased frequency of mass shootings, terror attacks, and natural disasters in recent years have presented challenges to provision of quality medical care in both short and long-term stressful situations. While emergency departments and trauma surgeons are usually the face of the response to mass casualty incidents (MCI), other departments such as radiology are often active participants in caring for these patients but may not be as well prepared. In this article, we review nine papers describing the experiences of various radiology departments with specific MCIs and the lessons they learned from those experiences. By analysis of common themes raised in these papers, we hope to enable departments to incorporate these lessons into their disaster plans to enhance their preparedness for such events.

Differential cellular composition of human palatine and pharyngeal tonsils.

OBJECTIVE: The goal of this study was to gain a better understanding of the potential functional specialization of palatine and pharyngeal tonsils, by comparing their cellular composition in paired specimens from a large cohort of adenotonsillectomy patients. DESIGN: Resident B cell, T cell, dendritic cell, and stromal cell subsets were characterized using multicolor flow cytometry in palatine and pharyngeal tonsil specimens from 27 patients, age 2-34 years. RESULTS: Paired comparisons showed highly significant intra-individual differences in resident cell subsets of palatine and pharyngeal tonsils. Palatine tonsils harbored higher fractions of germinal center B cells/plasmablasts and IgD- CD27- double-negative B cells, and conversely lower fractions of IgD + CD38- resting naïve B cells compared to pharyngeal tonsils. Palatine tonsils also showed lower fractions of plasmacytoid dendritic cells, and higher percentages of two subsets of stromal cells - fibroblastic reticular cells and lymphatic endothelial cells - compared to pharyngeal tonsils from the same individual. CONCLUSIONS: Despite their physical proximity and histological similarities, palatine and pharyngeal tonsils display marked intra-individual differences in their cellular composition with regard to functionally important immune and stromal subsets. These differences are likely to have immunologic, pathologic, and physiologic significance.

Prospects and limitations of improving skeletal growth in a mouse model of spondyloepiphyseal dysplasia caused by R992C (p.R1192C) substitution in collagen II.

Skeletal dysplasias form a group of skeletal disorders caused by mutations in macromolecules of cartilage and bone. The severity of skeletal dysplasias ranges from precocious arthropathy to perinatal lethality. Although the pathomechanisms of these disorders are generally well defined, the feasibility of repairing established aberrant skeletal tissues that developed in the presence of mutant molecules is currently unknown. Here, we employed a validated mouse model of spondyloepiphyseal dysplasia (SED) that enables temporal control of the production of the R992C (p.R1192C) collagen II mutant that causes this disease. Although in our earlier studies we determined that blocking the expression of this mutant at the early prenatal stages prevents a SED phenotype, the utility of blocking the R992C collagen II at the postnatal stages is not known. Here, by switching off the expression of R992C collagen II at various postnatal stages of skeletal development, we determined that significant improvements of cartilage and bone morphology were achieved only when blocking the production of the mutant molecules was initiated in newborn mice. Our study indicates that future therapies of skeletal dysplasias may require defining a specific time window when interventions should be applied to be successful.

Blocking collagen fibril formation in injured knees reduces flexion contracture in a rabbit model.

Post-traumatic joint contracture is a frequent orthopaedic complication that limits the movement of injured joints, thereby severely impairing affected patients. Non-surgical and surgical treatments for joint contracture often fail to improve the range of motion. In this study, we tested a hypothesis that limiting the formation of collagen-rich tissue in the capsules of injured joints would reduce the consequences of the fibrotic response and improve joint mobility. We targeted the formation of collagen fibrils, the main component of fibrotic deposits formed within the tissues of injured joints, by employing a relevant rabbit model to test the utility of a custom-engineered antibody. The antibody was delivered directly to the cavities of injured knees in order to block the formation of collagen fibrils produced in response to injury. In comparison to the non-treated control, mechanical tests of the antibody-treated knees demonstrated a significant reduction of flexion contracture. Detailed microscopic and biochemical studies verified that this reduction resulted from the antibody-mediated blocking of the assembly of collagen fibrils. These findings indicate that extracellular processes associated with excessive formation of fibrotic tissue represent a valid target for limiting post-traumatic joint stiffness. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1038-1046, 2017.

Auxiliary proteins that facilitate formation of collagen-rich deposits in the posterior knee capsule in a rabbit-based joint contracture model.

Post-traumatic joint contracture is a debilitating consequence of trauma or surgical procedures. It is associated with fibrosis that develops regardless of the nature of initial trauma and results from complex biological processes associated with inflammation and cell activation. These processes accelerate production of structural elements of the extracellular matrix, particularly collagen fibrils. Although the increased production of collagenous proteins has been demonstrated in tissues of contracted joints, researchers have not yet determined the complex protein machinery needed for the biosynthesis of collagen molecules and for their assembly into fibrils. Consequently, the purpose of our study was to investigate key enzymes and protein chaperones needed to produce collagen-rich deposits. Using a rabbit model of joint contracture, our biochemical and histological assays indicated changes in the expression patterns of heat shock protein 47 and the α-subunit of prolyl 4-hydroxylase, key proteins in processing nascent collagen chains. Moreover, our study shows that the abnormal organization of collagen fibrils in the posterior capsules of injured knees, rather than excessive formation of fibril-stabilizing cross-links, may be a key reason for observed changes in the mechanical characteristics of injured joints. This result sheds new light on pathomechanisms of joint contraction, and identifies potentially attractive anti-fibrotic targets.