Primary cilia attenuate hedgehog signalling in neoplastic chondrocytes.

Primary cilia can act as either a negative or positive regulator of the hedgehog (Hh) signaling pathway. Many cartilage tumors are characterized by abnormal activation of the Hh pathway. Here, we report that the presence of primary cilia occurs at a low frequency (12.4%) in neoplastic chondrocytes from malignant human chondrosarcomas, compared with chondrocytes from normal articular cartilage (67.7%). To determine the function of primary cilia in cartilaginous neoplasia, we studied benign cartilage tumors that are formed in mice by chondrocyte-specific overexpression of Gli2, a downstream transcriptional activator of the Hh pathway. Col2A1-Gli2 mice were crossed with Ift88+/- mice, which display a partial loss of ciliogenesis. Surprisingly, cartilage tumors developed in Ift88+/- mice that were phenotypically similar to those that arise in Col2A1-Gli2 mice. Further activation of the Hh pathway was observed in Col2A1-Gli2; Ift88+/- mice compared with either Col2A1-Gli2 or Ift88+/- mice, which was associated with an increased incidence of cartilage tumors. Chondrosarcomas were established in explant cultures, and treated with choral hydrate, which disrupts the functional primary cilia. Thus, treatment resulted in hyperactivity of the Hh signaling pathway, as well as cellular changes that could promote tumor growth. Primary cilia functions to inhibit Hh signaling in neoplastic chondrocytes. The activation of Hh signaling is sufficient to induce benign cartilage tumors without another oncogenic initiating event. Moreover, as primary cilia suppress Hh pathway activation in chondrosarcoma, cellular mechanisms inhibiting proper cilia function may be important in maintaining the neoplastic phenotype.

The first successful lower extremity transplantation: 6-year follow-up and implications for cortical plasticity.

Vascularized composite allotransplantation as a viable reconstructive option is gaining recognition and new cases are being reported with increasing frequency including hand, face and laryngeal transplantation. However, only one successful complete lower limb transplantation has been reported to date, in which a functioning limb from one ischiopagus twin with a lethal cardiac anomaly was transplanted to the other. Six years later, the patient is mobilizing well and engaging in sporting activities with her peers in a mainstream school. Clinical evaluation of motor and sensory modalities demonstrated a good functional result. Quality of life was assessed using the short form-36 health survey and lower extremity functional scale disclosing a high level of social and physical capacity. Functional magnetic resonance imaging was performed and showed cortical integration of the limb; the implications of cortical plasticity and vascularized composite allotransplantation for the correction of congenital limb anomalies are presented.

Testosterone regulates cell proliferation in aggressive fibromatosis (desmoid tumour).

BACKGROUND: Aggressive fibromatosis (desmoid tumour) is a locally invasive tumour caused by mutations resulting in ß-catenin protein stabilisation. Apc1638N mice are predisposed to developing aggressive fibromatosis tumours, and male mice develop greater numbers of tumours than female mice, suggesting a role for androgens in this tumour type. METHODS: Human aggressive fibromatosis tumours were examined for the expression of the androgen receptor, and primary human tumour cell cultures were treated with testosterone. Orchidectomised Apc1638N mice were investigated for the development of tumours, and were treated with testosterone to study the effect of tumour formation and the level of ß-catenin. RESULTS: Androgen receptors are universally expressed in human aggressive fibromatosis tumours. Testosterone increased the proliferation rate and ß-catenin protein level in a dose-dependent manner in human aggressive fibromatosis tumours. Orchiectomy reduced the number and size of tumours that formed in male Apc1638N mice to a similar level as observed in female mice. Testosterone treatment increased the number of tumours that formed in orchidectomised male mice, and resulted in a marked increase in ß-catenin protein levels. CONCLUSION: Testosterone regulates ß-catenin protein level and proliferation rate in this mesenchymal tumour. This work identifies the therapeutic use of testosterone blockade in aggressive fibromatosis as an area for further investigation.

Allograft airway fibrosis in the pulmonary milieu: a disorder of tissue remodeling.

Obliterative bronchiolitis (OB) is thought to be a form of chronic allograft rejection. However, immunosuppressive therapy is not effective once fibrosis has developed. We hypothesize that disordered tissue remodeling is a mechanism for the pathogenesis of OB. We examined allograft airway fibrosis in an intrapulmonary tracheal transplant model of OB. Allograft airways were completely obliterated at day 21 by fibrotic tissue; however, tissue remodeling continued thereafter, as demonstrated by the change of collagen deposition density, shift from type I to type III collagen, shift from fibroblasts to myofibroblasts and shift of expression profiles and activities of matrix metalloproteinases (MMPs). We then used a broad-spectrum MMP inhibitor, SC080, to attempt to manipulate tissue remodeling. Administration of the MMP inhibitor from day 0 to day 28 reduced airway obliteration, without inhibiting T-cell activation. MMP inhibition from day 14 to day 28 showed similar effects on airway obliteration. MMP inhibition from day 21 to day 35 did not reverse the airway obliteration, but significantly reduced the collagen deposition, type III collagen and myofibroblasts in the lumen. We conclude that tissue remodeling plays a critical role in the development and maintenance of fibrosis after transplantation.

Skeletal dysplasias and the growth plate.

Skeletal dysplasias are disorders in which there is derangement in the growth or shape of the skeleton. Long bone grows from cartilage that persists near the ends until skeletal maturity as the growth plate. Developmental biology work has identified the major regulatory proteins in growth plate chondroyte function. There are hundreds of skeletal dysplasias, and the molecular genetic etiology of many was defined in the past decade and a half. Now that the causative genes for these disorders have been identified, they can be broadly classified by the function of the protein that these genes encode for into disorders caused by extracellular structural proteins, proteins that regulate normal growth plate chondrocyte differentiation and patterning, and enzymes that process these proteins. There are clinical similarities within each group, and the phenotype can be predicted based on the role of the mutated protein in normal growth plate function. As such, this framework to classify the skeletal dysplasias has practical clinical implications.

Eosinophilic granuloma. A different behaviour in children than in adults.

Localised Langerhans-cell histiocytosis of bone (eosinophilic granuloma) is a benign tumour-like condition with a variable clinical course. Different forms of treatment have been reported to give satisfactory results. However, previous series all contain patients with a wide age range. Our aim was to investigate the effect of skeletal maturity on the rate of recurrence of isolated eosinophilic granuloma of bone excluding those arising in the spine. We followed up 32 patients with an isolated eosinophilic granuloma for a mean of five years; 17 were skeletally immature. No recurrences were noted in the skeletally immature group even after biopsy alone. By contrast, four of 13 skeletally mature patients had a recurrence and required further surgery. This suggests that eosinophilic granuloma has a low rate of recurrence in skeletally immature patients.

Tcf-3 expression and beta-catenin mediated transcriptional activation in aggressive fibromatosis (desmoid tumour).

Aggressive fibromatosis harbours mutations resulting in beta-catenin protein stabilization. Primary cell cultures demonstrate constitutive tcf activation in aggressive fibromatosis. Expression and co-immunoprecipitation studies suggest that beta-catenin binds and activates tcf-3 in this tumour. This is the first demonstration of tcf-3 activation by beta-catenin stabilization in a human neoplastic process.

Potential treatment for clubfeet based on growth factor blockade.

Select soft tissues in clubfeet are contracted, resulting in stiffness. These contracted tissues share ultrastructural characteristics with palmar fibromatosis (Dupuytren contracture), in which the growth factors transforming growth factor-beta (TGF-beta) and platelet-derived growth factor (PDGF) are expressed and play a role in regulating cell behavior. More contracted tissue (medial side of the foot) and less contracted tissue (lateral side of the foot) from 20 clubfeet were studied using reverse transcription-polymerase chain reaction and western analysis for expression of TGF-beta and PDGF (along with collagen type I and type III). Cell cultures were established from the more contracted tissues to determine the effect of blockade of these factors with neutralizing antibodies on proliferation, chemotaxis, and collagen expression. Both growth factors were expressed at higher levels by the contracted tissues, and blockade led to decreased collagen expression, proliferation, and chemotaxis. Growth factor blockade has the potential to change the behavior of these cells in a way that would lessen the severity of the contractures, perhaps improving the outcome of clubfoot treatment.

Cyclooxygenase-two (COX-2) modulates proliferation in aggressive fibromatosis (desmoid tumor).

Aggressive fibromatosis is a locally invasive soft tissue lesion. Seventy-five per cent of cases harbor a somatic mutation in either the APC or beta-catenin genes, resulting in beta-catenin protein stabilization. Cyclooxygenase-2 (COX-2) is an enzyme involved in prostaglandin synthesis that modulates the formation of colonic neoplasia, especially in cases due to mutations resulting in beta-catenin stabilization. Human aggressive fibromatoses and lesions from the Apc+/Apc1638N mouse (a murine model for Apc-driven fibromatosis) demonstrated elevated COX-2 levels. COX-2 blockade either by the selective agent DFU or by non-selective COX blocking agents results in reduced proliferation in human tumor cell cultures. Breeding mice with Cox-2-/- mice resulted in no difference in number of aggressive fibromatoses formed, but in a smaller tumor size, while there was a decrease in number of GI lesions by 50%. Mice fed various COX blocking agents also showed a decline in tumor size. COX-2 expression was regulated by tcf-dependent transcription in this lesion. COX-2 partially regulates proliferation due to beta-catenin stabilization in aggressive fibromatosis. Although COX blockade alone does not cause tumor regression, this data suggests that it may have a role as an adjuvant therapy to slow tumor growth in this lesion.

Local complications after limb-salvage surgery for pediatric bone tumours: a pictorial essay.

The prognosis for patients with bone sarcoma treated with LSS has improved considerably over the past 2 decades, but this has also lead to an increase in the number of complications requiring treatment. Imaging plays an important role, not only in assessing the primary tumour, but also in identifying postsurgical complications. Plain radiography demonstrates the majority of the complications associated with LSS and remains the mainstay of follow-up imaging. Complications such as fractures are common and warrant frequent plain film follow-up. Imaging with scintigraphy, MRI and CT should be tailored to the patient's clinical history, type of surgery and suspected complications. A baseline postoperative bone scan examination can be helpful for comparisons with subsequent scans for the detection of complications. Sonography should be considered if infection is suspected. Finally, tumour recurrence may be frequent enough to consider more extensive use of MRI.

Prominent expression of mRNA for proinflammatory cytokines in synovium in patients with juvenile rheumatoid arthritis or chronic Lyme arthritis.

OBJECTIVE: To examine the cytokine profiles in synovium of patients with juvenile rheumatoid arthritis (JRA) or Lyme arthritis, 2 chronic inflammatory arthritides that affect children. METHODS: We used in situ hybridization with specific riboprobes to determine the expression of mRNA for proinflammatory or antiinflammatory cytokines in synovial samples from 5 patients with early, untreated JRA, 15 patients with late, treated JRA, and 9 patients with chronic Lyme arthritis. For comparison, synovia were examined from 6 patients with rheumatoid or psoriatic arthritis, and from 9 patients with various orthopedic conditions. RESULTS: Among the children with early, untreated JRA, a median of 3 to 8% of inflammatory cells in synovial samples expressed mRNA for the proinflammatory cytokines interleukin 1beta (IL-1beta), tumor necrosis factor-alpha(TNF-alpha), or interferon-gamma (IFN-gamma). Although a median of 3.9% of the cells expressed mRNA for the antiinflammatory cytokine IL-10, none had IL-4 mRNA. Most of the patients with late, treated JRA, chronic Lyme arthritis, rheumatoid, or psoriatic arthritis had mRNA for each of these proinflammatory cytokines in about 1% of the cells, whereas mRNA for the antiinflammatory cytokines was less frequent. The inflammatory cell density was much less in the synovium of patients with various orthopedic conditions, but about 1% of the infiltrating cells expressed mRNA for at least one of the proinflammatory cytokines. CONCLUSION: Patients with early or late JRA or chronic Lyme arthritis have expression of mRNA in synovial tissue primarily for proinflammatory cytokines, with less expression of antiinflammatory cytokines.

Predominance of beta-catenin mutations and beta-catenin dysregulation in sporadic aggressive fibromatosis (desmoid tumor).

Aggressive fibromatosis (also called desmoid tumor) occurs as a sporadic lesion or as part of Familial Adenomatous Polyposis, which is caused by germ line mutations in the Adenomatous polyposis Coli (APC) gene. APC is involved in the regulation of the cellular level of beta-catenin, which is a mediator in Wnt signaling. Mutational analysis of the beta-catenin and APC genes was performed in 42 sporadic aggressive fibromatoses. Nine tumors had mutations in APC, and 22 had a point mutation in beta-catenin at either codon 45 or codon 41 (producing a stabilized beta-catenin protein product). Immunohistochemistry showed an elevated beta-catenin protein level in all tumors, regardless of mutational status. Beta-catenin localized to the nucleus, and was not tyrosine phosphorylated in the six tumors in which this was tested. The demonstration of mutations in two mediators in the Wnt-APC-beta-catenin pathway implicates beta-catenin stabilization as the key factor in the pathogenesis of aggressive fibromatosis. This is the first demonstration of somatic beta-catenin mutations in a locally invasive, but non metastatic lesion composed of spindle cells, illustrating the importance of beta-catenin stabilization in a variety of cell types and neoplastic processes. Moreover, this tumor has one of the highest reported frequencies of beta-catenin mutations of any tumor type.

Expression of osteocalcin and its transcriptional regulators core-binding factor alpha 1 and MSX2 in osteoid-forming tumours.

Osteosarcoma, fibrous dysplasia, and myositis ossificans contain osteoid-producing cells that are not necessarily morphologically typical osteoblasts. Nevertheless, these pathologic cells may share differentiation steps with osteoblasts at the molecular level. Osteocalcin, a bone-specific extracellular matrix protein, is a marker of mature osteoblasts. Osteocalcin is upregulated by the transcription factor core-binding factor alpha 1, which is responsible for commitment to the osteoblastic lineage, and is downregulated by MSX2, a homeobox-containing transcription factor expressed during the early proliferative phase of osteoblast differentiation. Semiquantitative reverse transcription-polymerase chain reaction was used to compare expression levels of osteocalcin, core-binding factor alpha 1, and MSX2 in 34 osteosarcoma, five fibrous dysplasia, and five myositis ossificans specimens, as well as in seven normal cortical bone samples. Despite normal or elevated levels of core-binding factor alpha-1 expression in most specimens, osteocalcin expression was low or undetectable in most cases of osteosarcoma (25 of 34) and myositis ossificans (4 of 5). Single-strand conformation polymorphism and sequencing did not identify any mutations in the DNA-binding domain of core-binding factor alpha 1. However, a high level of MSX2 expression was demonstrated in these lesions, which may inhibit osteocalcin transcription. The presence of moderate levels of osteocalcin in fibrous dysplasia may contribute to the characteristic disconnected appearance of trabeculae in that entity because osteocalcin is a negative regulator of bone formation.

Pelvic obliquity after fusion of the spine in Duchenne muscular dystrophy.

Spinal fusion, ending caudally at L5 rather than at the sacrum, is recommended for selected patients with scoliosis due to Duchenne muscular dystrophy. We present a retrospective review of 48 patients operated on for this condition. Patients having spinal curvature with a Cobb angle of less than 40 degrees and with less than 10 degrees between a line tangential to the superior margins of both iliac crests and a line perpendicular to the spinous processes of L4 and L5, were fused to L5 (38 patients); patients not meeting these criteria were fused to the sacrum (10 patients). Spinal and sitting obliquity increased in patients fused to L5, rather than to the sacrum, but the severity of the worsening obliquity was significantly greater in patients in whom the apex of the curve was below L1. Two of the ten latter patients required revision procedures for worsening obliquity when their pulmonary function deteriorated to less than 25% of predicted values. We recommend fusion to the sacrum for scoliosis in Duchenne muscular dystrophy, especially for patients with an apex to their curve below L1.

Adenomatous polyposis coli gene mutation alters proliferation through its beta-catenin-regulatory function in aggressive fibromatosis (desmoid tumor).

Aggressive fibromatosis is a monoclonal proliferation of spindle (fibroblast-like) cells. A subset of lesions contain somatic truncating adenomatous polyposis coli (APC) gene mutations, and all of the lesions contain an elevated beta-catenin protein level. A major function of APC is to regulate beta-catenin protein level. Beta-catenin has a dual function in the cell: it is a member of the adherens junction, and it binds transcription factors in the tcf-lef family, transactivating transcription. Cell cultures from aggressive fibromatoses containing an APC mutation were studied. Transient transfection of the full-length APC gene caused decreased proliferation and beta-catenin protein level in these cultures. To determine whether beta-catenin protein level was responsible for the change in proliferation rate, stable transfections of deltaN89beta-catenin (a stabilized form that is not degraded by APC, but retains all other functions) were achieved in half of the cultures derived from each tumor, whereas the other half were transfected with an empty vector. Transfection of the full-length APC gene in cultures that were stably transfected with deltaN89beta-catenin did not result in a change in proliferation. The type I promotor of p56lck contains an HMG consensus region, to which members of the tcf-lef family can bind. p56lck was expressed in cultures not transfected with the full-length APC gene and in cultures transfected with the full-length APC gene and deltaN89beta-catenin, but not in cultures transfected with only the full-length APC gene. These data show that APC truncating mutations give aggressive fibromatosis cells a proliferative advantage through beta-catenin and suggest that beta-catenin acts to transactivate transcription.

Increased beta-catenin protein and somatic APC mutations in sporadic aggressive fibromatoses (desmoid tumors).

Sporadic aggressive fibromatosis (also called desmoid tumor) is a monoclonal proliferation of spindle (fibrocyte-like) cells that is locally invasive but does not metastasize. A similarity to abdominal fibromatoses (desmoids) in familial adenomatous polyposis and a cytogenetic study showing partial deletion of 5q in a subset of aggressive fibromatoses suggests that the adenomatous polyposis coli (APC) gene plays a role in its pathogenesis. APC helps regulate the cellular level of beta-catenin, which is a downstream mediator in Wnt (Wingless) signaling. beta-Catenin has a nuclear function (binds transcription factors) and a cell membrane function (is a component of epithelial cell adherens junctions). Six cases of aggressive fibromatosis of the extremities from patients without familial adenomatous polyposis, or a family history of colon cancer, were studied. Immunohistochemistry, using carboxy and amino terminus antibodies to APC, and DNA sequencing showed that three of the six contained an APC-truncating mutation, whereas normal tissues did not contain a mutation. Western blot and Northern dot blot showed that all six tumors had a higher level of beta-catenin protein than surrounding normal tissues, despite containing similar levels of beta-catenin mRNA. Immunohistochemistry localized beta-catenin throughout the cell in tumor tissues, although it localized more to the periphery in cells from normal tissues. Reverse transcription polymerase chain reaction showed that the tumors expressed N-cadherin but not E-cadherin (a pattern of expression of proteins making up adherens junctions similar to fibrocytes), suggesting that the specific adherens junctions present in epithelial cells are not necessary for beta-catenin function. Increased beta-catenin may cause the growth advantage of cells in this tumor through a nuclear mechanism. The increased protein level, relative to the RNA level, suggests that beta-catenin is degraded at a lower rate compared with normal tissues. In some cases, this is caused by a somatic mutation resulting in a truncated APC protein.

Differential collagen I gene expression in fetal fibroblasts.

PURPOSE: Fetal wound healing is characterized by the regeneration of normal dermis and the absence of scar. Transforming growth factor beta-1 (TGF-beta1) is a ubiquitous cytokine with potent fibrogenic effects in both postnatal and fetal wounds. Supplementing fetal wounds with TGF-beta1 results in increased fibrosis consisting primarily of collagen I. We hypothesized that the lack of scar formation in fetal wounds may be caused by differential collagen I gene (COL1A1) expression. The authors examined basal collagen Ia gene expression in human fetal, newborn, and adult dermal fibroblasts after stimulation with exogenous TGF-beta1. METHODS: Subconfluent human dermal fibroblasts from fetal, newborn, and adult cell lines were incubated for 24 hours, then stimulated by incubation for 4 hours with 1 ng/mL of human recombinant TGF-beta1, or with media alone for basal collagen gene expression, and then placed in guanidium isothyocyanate buffer. To quantitate COL1A1 gene expression, total cellular RNA was extracted and subjected to northern and slot blot hybridization analysis with Dig-labeled COL1A1 probes. The membrane was exposed to x-ray film for 15 minutes and developed. RESULTS: Scant COL1A1 gene transcript was detected in control fetal fibroblasts. Brief stimulation with of TGF-beta1 upregulated the COL1A1 gene transcription in fetal fibroblasts. Gene expression for COL1A1 in both postnatal cell lines appeared similar in treated and untreated cells. Housekeeping control (GAPDH) confirmed no difference in total amount of RNA at the start or end of the experiment. CONCLUSION: COL1A1 gene expression is notably absent in unstimulated fetal fibroblasts, but is upregulated by TGF-beta1. In contrast, postnatal fibroblasts demonstrate significant constitutive COL1A1 gene expression at baseline and unchanged after TGF-beta1 stimulation. This differential regulation may contribute to the ability of fetal wounds to regenerate without scar and explain the effect of exogenous TGF-beta1 to increase fibroplasia in fetal dermal incisional wounds.

Aggressive fibromatosis (desmoid tumor) is a monoclonal disorder.

Aggressive fibromatosis (also called deep fibromatosis or desmoid tumor) is a proliferation of cytologically benign-appearing fibrocytes, often resulting in significant functional loss. The nature of the lesion is controversial: some evidence suggests that it is a reactive process, whereas other evidence supports a neoplastic etiology. The pattern of X chromosome inactivation, using a technique based on polymerase chain reaction (PCR) amplification of a hypervariable CAG repeat region flanking Hhal restriction sites of the human androgen receptor gene, was determined in four cases in which cryopreserved tumor and adjacent normal tissue were available. All four tumors demonstrated a monoclonal pattern, while the adjacent normal tissues demonstrated a polyclonal pattern. This demonstrates that aggressive fibromatosis is proliferation of cells derived from a single clone with a growth advantage, and thus is likely a neoplastic process.

Molecular genetic and immunohistochemical analysis of the tumor suppressor genes Rb and p53 in palmar and aggressive fibromatosis.

This pilot project analyzed the tumor suppressor genes p53 and Rb in 13 cases of aggressive fibromatoses and six cases of palmar fibromatoses (Dupuytren contracture). Immunohistochemistry, reverse transcription polymerase chain reaction, polymerase chain reaction followed by single-strand confirmation polymorphism analysis, and Southern blot to detect gene rearrangements were used. No abnormalities were detected in p53. The aggressive fibromatoses demonstrated a lack of Rb immunohistochemical staining and decreased mRNA for Rb. No structural mutation in the coding sequence of the Rb gene was detected. The decreased level of Rb gene expression, despite a normal coding sequence, may indicate increased proliferation and may suggest potential treatment schemes.