Active immunisation targeting nerve growth factor attenuates chronic pain behaviour in murine osteoarthritis.

OBJECTIVES: Nerve growth factor (NGF) has emerged as a key driver of pain in osteoarthritis (OA) and antibodies to NGF are potent analgesics in human disease. Here, we validate a novel vaccine strategy to generate anti-NGF antibodies for reversal of pain behaviour in a surgical model of OA. METHODS: Virus-like particles were derived from the cucumber mosaic virus (CuMV) and coupled to expressed recombinant NGF to create the vaccine. 10-week-old male mice underwent partial meniscectomy to induce OA or sham-surgery. Spontaneous pain behaviour was measured by Linton incapacitance and OA severity was quantified using OARSI histological scoring. Mice (experimental and a sentinel cohort) were inoculated with CuMVttNGF (Vax) or CuMVttctrl (Mock) either before surgery or once pain was established. Efficacy of anti-NGF from the plasma of sentinel vaccinated mice was measured in vitro using a neurite outgrowth assay in PC12 cells. RESULTS: Anti-NGF titres were readily detectable in the vaccinated but not mock vaccinated mice. Regular boosting with fresh vaccine was required to maintain anti-NGF titres as measured in the sentinel cohort. Both prophylactic and therapeutic vaccination demonstrated a reversal of pain behaviour by incapacitance testing, and a meta-analysis of the two studies showing analgesia at peak anti-NGF titres was highly statistically significant. Serum anti-NGF was able to inhibit neurite outgrowth equivalent to around 150 ug/mL of recombinant monoclonal antibody. CONCLUSIONS: This study demonstrates therapeutic efficacy of a novel NGF vaccine strategy that reversibly alleviates spontaneous pain behaviour in surgically induced murine OA.

Modeling and Assessing Osteoarthritis in Mice by Destabilization of the Medial Meniscus (DMM).

In this chapter, we describe an induced model of osteoarthritis in mice, frequently employed in the study of this disease. We outline in detail the surgical induction of disease and preparation of samples for histological assessment of disease.

Ciliary IFT88 Protects Coordinated Adolescent Growth Plate Ossification From Disruptive Physiological Mechanical Forces.

Compared with our understanding of endochondral ossification, much less is known about the coordinated arrest of growth defined by the narrowing and fusion of the cartilaginous growth plate. Throughout the musculoskeletal system, appropriate cell and tissue responses to mechanical force delineate morphogenesis and ensure lifelong health. It remains unclear how mechanical cues are integrated into many biological programs, including those coordinating the ossification of the adolescent growth plate at the cessation of growth. Primary cilia are microtubule-based organelles tuning a range of cell activities, including signaling cascades activated or modulated by extracellular biophysical cues. Cilia have been proposed to directly facilitate cell mechanotransduction. To explore the influence of primary cilia in the mouse adolescent limb, we conditionally targeted the ciliary gene Intraflagellar transport protein 88 (Ift88fl/fl ) in the juvenile and adolescent skeleton using a cartilage-specific, inducible Cre (AggrecanCreERT2 Ift88fl/fl ). Deletion of IFT88 in cartilage, which reduced ciliation in the growth plate, disrupted chondrocyte differentiation, cartilage resorption, and mineralization. These effects were largely restricted to peripheral tibial regions beneath the load-bearing compartments of the knee. These regions were typified by an enlarged population of hypertrophic chondrocytes. Although normal patterns of hedgehog signaling were maintained, targeting IFT88 inhibited hypertrophic chondrocyte VEGF expression and downstream vascular recruitment, osteoclastic activity, and the replacement of cartilage with bone. In control mice, increases to physiological loading also impair ossification in the peripheral growth plate, mimicking the effects of IFT88 deletion. Limb immobilization inhibited changes to VEGF expression and epiphyseal morphology in Ift88cKO mice, indicating the effects of depletion of IFT88 in the adolescent growth plate are mechano-dependent. We propose that during this pivotal phase in adolescent skeletal maturation, ciliary IFT88 protects uniform, coordinated ossification of the growth plate from an otherwise disruptive heterogeneity of physiological mechanical forces. © 2022 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Role of Ciliary Protein Intraflagellar Transport Protein 88 in the Regulation of Cartilage Thickness and Osteoarthritis Development in Mice.

OBJECTIVE: Mechanical and biologic cues drive cellular signaling in cartilage development, health, and disease. Primary cilia proteins, which are implicated in the transduction of biologic and physiochemical signals, control cartilage formation during skeletal development. This study was undertaken to assess the influence of the ciliary protein intraflagellar transport protein 88 (IFT88) on postnatal cartilage from mice with conditional knockout of the Ift88 gene (Ift88-KO). METHODS: Ift88fl/fl and aggrecanCreERT2 mice were crossed to create a strain of cartilage-specific Ift88-KO mice (aggrecanCreERT2 ;Ift88fl/fl ). In these Ift88-KO mice and Ift88fl/fl control mice, tibial articular cartilage thickness was assessed by histomorphometry, and the integrity of the cartilage was assessed using Osteoarthritis Research Society International (OARSI) damage scores, from adolescence through adulthood. In situ mechanisms of cartilage damage were investigated in the microdissected cartilage sections using immunohistochemistry, RNAScope analysis, and quantitative polymerase chain reaction. Osteoarthritis (OA) was induced in aggrecanCreERT2 ;Ift88fl/fl mice and Ift88fl/fl control mice using surgical destabilization of the medial meniscus (DMM). Following tamoxifen injection and DMM surgery, the mice were given free access to exercise on a wheel. RESULTS: Deletion of Ift88 resulted in progressive reduction in the thickness of the medial tibial cartilage in adolescent mice, as well as marked atrophy of the cartilage in mice during adulthood. In aggrecanCreERT2 ;Ift88fl/fl mice at age 34 weeks, the median thickness of the medial tibial cartilage was 89.42 µm (95% confidence interval [95% CI] 84.00-93.49), whereas in Ift88fl/fl controls at the same age, the median cartilage thickness was 104.00 µm (95% CI 100.30-110.50; P < 0.0001). At all time points, the median thickness of the calcified cartilage was reduced. In some mice, atrophy of the medial tibial cartilage was associated with complete, spontaneous degradation of the cartilage. Following DMM, aggrecanCreERT2 ;Ift88fl/fl mice were found to have increased OARSI scores of cartilage damage. In articular cartilage from maturing mice, atrophy was not associated with obvious increases in aggrecanase-mediated destruction or chondrocyte hypertrophy. Of the 44 candidate genes analyzed, only Tcf7l2 expression levels correlated with Ift88 expression levels in the microdissected cartilage. However, RNAScope analysis revealed that increased hedgehog (Hh) signaling (as indicated by increased expression of Gli1) was associated with the reductions in Ift88 expression in the tibial cartilage from Ift88-deficient mice. Wheel exercise restored both the articular cartilage thickness and levels of Hh signaling in these mice. CONCLUSION: Our results in a mouse model of OA demonstrate that IFT88 performs a chondroprotective role in articular cartilage by controlling the calcification of cartilage via maintenance of a threshold of Hh signaling during physiologic loading.

TSG-6 Is Weakly Chondroprotective in Murine OA but Does not Account for FGF2-Mediated Joint Protection.

OBJECTIVE: Tumor necrosis factor α-stimulated gene 6 (TSG-6) is an anti-inflammatory protein highly expressed in osteoarthritis (OA), but its influence on the course of OA is unknown. METHODS: Cartilage injury was assessed by murine hip avulsion or by recutting rested explants. Forty-two previously validated injury genes were quantified by real-time polymerase chain reaction in whole joints following destabilization of the medial meniscus (DMM) (6 hours and 7 days). Joint pathology was assessed at 8 and 12 weeks following DMM in 10-week-old male and female fibroblast growth factor 2 (FGF2)-/- , TSG-6-/- , TSG-6tg (overexpressing), FGF2-/- ;TSG-6tg (8 weeks only) mice, as well as strain-matched, wild-type controls. In vivo cartilage repair was assessed 8 weeks following focal cartilage injury in TSG-6tg and control mice. FGF2 release following cartilage injury was measured by enzyme-linked immunosorbent assay. RESULTS: TSG-6 messenger RNA upregulation was strongly FGF2-dependent upon injury in vitro and in vivo. Fifteeen inflammatory genes were significantly increased in TSG-6-/- joints, including IL1α, Ccl2, and Adamts5 compared with wild type. Six genes were significantly suppressed in TSG-6-/- joints including Timp1, Inhibin ßA, and podoplanin (known FGF2 target genes). FGF2 release upon cartilage injury was not influenced by levels of TSG-6. Cartilage degradation was significantly increased at 12 weeks post-DMM in male TSG-6-/- mice, with a nonsignificant 30% reduction in disease seen in TSG-6tg mice. No differences were observed in cartilage repair between genotypes. TSG-6 overexpression was unable to prevent accelerated OA in FGF2-/- mice. CONCLUSION: TSG-6 influences early gene regulation in the destabilized joint and exerts a modest late chondroprotective effect. Although strongly FGF2 dependent, TSG-6 does not explain the strong chondroprotective effect of FGF2.

Studying Osteoarthritis Pathogenesis in Mice.

With the increasing availability and complexity of mouse models of disease, either spontaneous or induced, there is a concomitant increase in their use in the analysis of pathogenesis. Among such diseases is osteoarthritis, a debilitating disease with few treatment options. While advances in our understanding of the pathogenesis of osteoarthritis has advanced through clinical investigations and genome-wide association studies, there is still a large gap in our knowledge, hindering advances in therapy. Patient samples are available ex vivo, but these are generally in the very late stages of disease. However, with mice, we are able to induce disease at a defined time and track the progression in vivo and ex vivo, from inception to end stage, to delineate the processes involved in disease development. © 2018 by John Wiley & Sons, Inc.

Regulation of Merkel cell development by Pax6.

Merkel cells are mechanoreceptors widely distributed in the vertebrate skin. In rodents, Merkel cells within the whisker pads are innervated by free sensory nerve endings derived from the maxillary branch of the trigeminal nerve. This study identified expression of the transcription factor Pax6 in Merkel cells and investigated its role. Immunohistochemistry and Western blot for Pax6 and Merkel cell markers, cytokeratin-8 (K8) and cytokeratin-20 (K20) were performed in wild-type and Pax6 (-/-) fetuses. The subcellular localisation of Pax6 in Merkel cells in vitro was manipulated using hydrogen peroxide. Pax6 was primarily localised within the cytoplasm of the Merkel cells at birth, but postnatally was also detected within the nuclei. In vitro, after 4 days in culture Pax6 protein was completely relocated to the nuclei of fetal-derived Merkel cells, mimicking the in vivo situation, suggesting that Pax6 acts as an active nucleo-cytoplasmic shuttling protein in common with many other homeodomain transcription factors. The subcellular localisation of Pax6 could be modulated in vitro by changing the redox potential of the culture medium for Merkel cells. Differentiation of cultured Pax6 (-/-) Merkel cells was shown to be inhibited. At perinatal stages, it was found that Pax6 is required for maintaining cytokeratin-8 expression, an early Merkel cell marker, whereas cytokeratin-20 was retained by the Pax6 (-/-) mutant cells. Pax6 is expressed in developing Merkel cells as a nucleo-cytoplasmic shuttling protein and its activity is required for normal differentiation, possibly through regulating cell maturation.

PAX6 dosage effects on corneal development, growth, and wound healing.

The requirement for correct dosage of the transcription factor Pax6 during corneal growth and development was investigated using the Pax6-overexpressing (PAX77) transgenic mouse. Transgenics had a microcornea phenotype due to failure of postnatal growth, associated with reduction in the number of cells layers in the corneal epithelium. Cell cycle progression was monitored using bromodeoxyuridine, p63, cyclin E, and phosphohistone-3 labeling: proliferation rates were higher in PAX77+ than wild-type, without a concomitant increase in apoptosis. Hence, failure of proliferation did not underlie microcornea. PAX77+ corneal epithelia had reduced levels of cytokeratin-12, and exhibited severe wound healing delay that, in contrast to Pax6+/- mice, could not be modulated by exogenous growth factors. PAX77+ lenses showed partial failure of lens fiber differentiation. The data demonstrate that anterior eye development is very sensitive to Pax6 dosage. Although there are similarities between the eye phenotype of Pax6 heterozygotes and overexpressing mice, there are also striking differences. Developmental

Brachytelephalangic chondrodysplasia punctata with severe spinal cord compression: report of four new cases.

Brachytelephalangic chondrodysplasia punctata (CDPX1, OMIM: #302950) is a rare congenital skeletal dysplasia caused by arylsulfatase E deficiency (OMIM: #300180). Although the symptoms are usually mild, severe spinal cord compression by dysplastic vertebras may develop. We report four new cases with severe cervical spinal canal narrowing documented by radiography, magnetic resonance imaging (MRI), and autopsy. In all, nine cases of CDPX1 with severe cervical spinal cord compression have now been described. Because these cases account for a large proportion of all reported CDPX1 cases, we believe that an antenatal suspicion of CDPX1 should lead to genetic counseling and to investigations for spinal cord compression. After birth, this complication must be routinely anticipated, and we suggest spinal MRI in all CDPX1 infants. Unless spinal cord compression is confidently ruled out, we recommend that these newborns receive the same care as trauma patients suspected of craniocervical junction disruption.

Clinical phenotype of lathosterolosis.

Lathosterolosis (LS) is a defect of cholesterol biosynthesis due to the deficiency of the enzyme sterol-C5-desaturase. Only two patients have been described to date, both presenting with multiple malformations, mental retardation, and liver involvement. In addition in one of them pathological examination revealed mucolipidosis-like inclusions on optic microscopy analysis, and peculiar lysosomal lamellar bodies on electron microscopy analysis. This study is focused on a better characterization of the clinical phenotype of LS. We describe a further case in a fetus, sibling of the first patient reported, presenting with neural tube defect, craniofacial and limb anomalies, and prenatal liver involvement. The fetal phenotype suggests the possible occurrence of significant intrafamilial variability in LS, and expands the phenotypic spectrum of the disease. Histological examination of autopsy samples from the fetus and skin fibroblasts from the living sibling suggested that the mucolipidosis-like picture previously reported is not a constant feature of LS, being possibly associated with the most severe biochemical defects, but confirmed the ultrastructural finding of lamellar inclusions. The LS phenotype appears to be characterized by the distinctive association of a recognizable pattern of congenital anomalies, involving axial and appendicular skeleton, liver, central nervous and urogenital systems, and lysosomal storage. This condition partially overlaps with other defects of sterol metabolism, suggesting intriguing pathogenic links among these conditions.