Activation of cytotoxic lymphocytes through CD6 enhances killing of cancer cells.

Immune checkpoint inhibitors (ICIs) have demonstrated efficacy and improved survival in a growing number of cancers. Despite their success, ICIs are associated with immune-related adverse events that can interfere with their use. Therefore, safer approaches are needed. CD6, expressed by T-lymphocytes and human NK cells, engages in cell-cell interactions by binding to its ligands CD166 (ALCAM) and CD318 (CDCP1). CD6 is a target protein for regulating immune responses and is required for the development of several mouse models of autoimmunity. Interestingly, CD6 is exclusively expressed on immune cells while CD318 is strongly expressed on most cancers. Here we demonstrate that disrupting the CD6-CD318 axis with UMCD6, an anti-CD6 monoclonal antibody, prolongs survival of mice in xenograft mouse models of human breast and prostate cancer, treated with infusions of human lymphocytes. Analysis of tumor-infiltrating immune cells showed that augmentation of lymphocyte cytotoxicity by UMCD6 is due to effects of this antibody on NK, NKT and CD8 + T cells. In particular, tumor-infiltrating cytotoxic lymphocytes from UMCD6-treated mice expressed higher levels of perforin and were found in higher proportions than those from IgG-treated mice. Moreover, RNA-seq analysis of human NK-92 cells treated with UMCD6 revealed that UMCD6 up-regulates the NKG2D-DAP10 receptor complex, important in NK cell activation, as well as its downstream target PI3K. Our results now describe the phenotypic changes that occur on immune cells upon treatment with UMCD6 and further confirm that the CD6-CD318 axis can regulate the activation state of cytotoxic lymphocytes and their positioning within the tumor microenvironment.

Effects of placebo administration on immune mechanisms and relationships with central endogenous opioid neurotransmission.

Behavioral conditioning and expectation can have profound impact on animal and human physiology. Placebo, administered under positive expectation in clinical trials, can have potent effects on disease pathology, obscuring active medications. Emerging evidence suggests placebo-responsive neurotransmitter systems (e.g., endogenous opioid) regulate immune function by manipulating inflammatory proteins including IL-18, a potent pro-inflammatory, nociceptive cytokine implicated in pathophysiology of various diseases. Validation that neuroimmune interactions involving brain µ-opioid receptor (MOR) activity and plasma IL-18 underlie placebo analgesic expectation could have widespread clinical applications. Unfortunately, current lack of mechanistic clarity obfuscates clinical translation. To elucidate neuroimmune interactions underlying placebo analgesia, we exposed 37 healthy human volunteers to a standardized pain challenge on each of 2 days within a Positron Emission Tomography (PET) neuroimaging paradigm using the MOR selective radiotracer, 11C-Carfentanil (CFN). Each day volunteers received an intervention (placebo under analgesic expectation or no treatment), completed PET scanning, and rated their pain experience. MOR BPND parametric maps were generated from PET scans using standard methods. Results showed placebo reduced plasma IL-18 during pain (W74 = -3.7, p < 0.001), the extent correlating with reduction in pain scores. Placebo reduction in IL-18 covaried with placebo-induced endogenous opioid release in the left nucleus accumbens (T148 = 3.33; puncorr < 0.001) and left amygdala (T148 = 3.30; puncorr < 0.001). These findings are consistent with a modulating effect of placebo (under analgesic expectation in humans) on a potent nociceptive, pro-inflammatory cytokine (IL-18) and underlying relationships with endogenous opioid activity, a neurotransmitter system critically involved in pain, stress, and mood regulation.

Mammalian Glycosylation Patterns Protect Citrullinated Chemokine MCP-1/CCL2 from Partial Degradation.

Monocyte chemoattractant protein-1 (MCP-1/CCL2) is a potent chemotactic agent for monocytes, primarily produced by macrophages and endothelial cells. Significantly elevated levels of MCP-1/CCL2 were found in synovial fluids of patients with rheumatoid arthritis (RA), compared to osteoarthritis or other arthritis patients. Several studies suggested an important role for MCP-1 in the massive inflammation at the damaged joint, in part due to its chemotactic and angiogenic effects. It is a known fact that the post-translational modifications (PTMs) of proteins have a significant impact on their properties. In mammals, arginine residues within proteins can be converted into citrulline by peptidylarginine deiminase (PAD) enzymes. Anti-citrullinated protein antibodies (ACPA), recognizing these PTMs, have become a hallmark for rheumatoid arthritis (RA) and other autoimmune diseases and are important in diagnostics and prognosis. In previous studies, we found that citrullination converts the neutrophil attracting chemokine neutrophil-activating peptide 78 (ENA-78) into a potent macrophage chemoattractant. Here we report that both commercially available and recombinant bacterially produced MCP-1/CCL2 are rapidly (partially) degraded upon in vitro citrullination. However, properly glycosylated MCP-1/CCL2 produced by mammalian cells is protected against degradation during efficient citrullination. Site-directed mutagenesis of the potential glycosylation site at the asparagine-14 residue within human MCP-1 revealed lower expression levels in mammalian expression systems. The glycosylation-mediated recombinant chemokine stabilization allows the production of citrullinated MCP-1/CCL2, which can be effectively used to calibrate crucial assays, such as modified ELISAs.

Inhibition of EZH2 prevents fibrosis and restores normal angiogenesis in scleroderma.

Scleroderma (SSc) is a complex disease that involves activation of the immune system, vascular complications, and tissue fibrosis. The histone methyltransferase enhancer of zeste homolog 2 (EZH2) mediates trimethylation of lysine 27 of histone 3 (H3K27me3), which acts as a repressive epigenetic mark. Both EZH2 and H3K27me3 were elevated in SSc dermal fibroblasts and endothelial cells compared with healthy controls. EZH2 inhibitor DZNep halted fibrosis both in vitro and in vivo. In SSc fibroblasts, DZNep dose-dependently reduced the expression of profibrotic genes and inhibited migratory activity of SSc fibroblasts. We show that epigenetic dysregulation and overexpression of LRRC16A explains EZH2-mediated fibroblast migration in SSc. In endothelial cells, inhibition of EZH2 restored normal angiogenesis in SSc via activating the Notch pathway, specifically by up-regulating the Notch ligand DLL4. Our results demonstrate that overexpression of EZH2 in SSc fibroblasts and endothelial cells is profibrotic and antiangiogenic. Targeting EZH2 or EZH2-regulated genes might be of therapeutic potential in SSc.

Angiogenic and Arthritogenic Properties of the Soluble Form of CD13.

Aminopeptidase N/CD13 is expressed by fibroblast-like synoviocytes (FLS) and monocytes (MNs) in inflamed human synovial tissue (ST). This study examined the role of soluble CD13 (sCD13) in angiogenesis, MN migration, phosphorylation of signaling molecules, and induction of arthritis. The contribution of sCD13 was examined in angiogenesis and MN migration using sCD13 and CD13-depleted rheumatoid arthritis (RA) synovial fluids (SFs). An enzymatically inactive mutant CD13 and intact wild-type (WT) CD13 were used to determine whether its enzymatic activity contributes to the arthritis-related functions. CD13-induced phosphorylation of signaling molecules was determined by Western blotting. The effect of sCD13 on cytokine secretion from RA ST and RA FLS was evaluated. sCD13 was injected into C57BL/6 mouse knees to assess its arthritogenicity. sCD13 induced angiogenesis and was a potent chemoattractant for MNs and U937 cells. Inhibitors of Erk1/2, Src, NF-κB, Jnk, and pertussis toxin, a G protein-coupled receptor inhibitor, decreased sCD13-stimulated chemotaxis. CD13-depleted RA SF induced significantly less MN migration than sham-depleted SF, and addition of mutant or WT CD13 to CD13-depleted RA SF equally restored MN migration. sCD13 and recombinant WT or mutant CD13 had similar effects on signaling molecule phosphorylation, indicating that the enzymatic activity of CD13 had no role in these functions. CD13 increased the expression of proinflammatory cytokines by RA FLS, and a CD13 neutralizing Ab inhibited cytokine secretion from RA ST organ culture. Mouse knee joints injected with CD13 exhibited increased circumference and proinflammatory mediator expression. These data support the concept that sCD13 plays a pivotal role in RA and acute inflammatory arthritis.

Keys to Class II correction: A comparison of 2 extraction protocols.

INTRODUCTION: The purpose of this study was to evaluate the effects of 2 extraction patterns on incisor and molar movements in patients with growing Class II Division 1. METHODS: The sample included 54 patients 10-17 years of age treated by 2 private practice orthodontists using Tweed directional force mechanics, 4 premolar extractions, J-hook headgears, and Class II elastics or Saif springs. The sample was divided on the basis of having maxillary and mandibular first premolars (4/4) or maxillary first and mandibular second premolars (4/5) extracted. Each group included 27 patients. Treatment lasted 2.8 ± 0.60 years and 2.6 ± 0.54 years for the 4/4 and 4/5 groups, respectively. Pretreatment (T1) and posttreatment lateral cephalograms and dental casts were evaluated. Cranial base, mandibular, and maxillary superimpositions were performed to quantify tooth movements and displacements. RESULTS: There were no statistically significant T1 between-group differences in crowding or in the SNA, SNB, ANB, and MPA angles. Analyses of covariance, controlling for statistically significant (P <0.05) differences in T1 mandibular incisor position, showed that mandibular first premolars extractions produced greater (1.6 mm) mandibular incisor retraction than second premolar extractions. The mandibular first molars were protracted significantly more (0.7 mm) after the second premolar than the first premolar extractions. Within-group changes of the MPA, between-group differences in the changes in MPA, and the amount of vertical eruption of the maxillary and mandibular molars were not significantly different between the 2 extraction patterns. CONCLUSIONS: Extraction of mandibular second premolars enhances Class II molar correction, with greater mesial first molar movement and less distal incisor movement. Neither extraction pattern has an effect on the MPA or the vertical dimension (ie, there was no "wedge effect").

Tazemetostat for patients with relapsed or refractory follicular lymphoma: an open-label, single-arm, multicentre, phase 2 trial.

BACKGROUND: Activating mutations of EZH2, an epigenetic regulator, are present in approximately 20% of patients with follicular lymphoma. We investigated the activity and safety of tazemetostat, a first-in-class, oral EZH2 inhibitor, in patients with follicular lymphoma. METHODS: This study was an open-label, single-arm, phase 2 trial done at 38 clinics or hospitals in France, the UK, Australia, Canada, Poland, Italy, Ukraine, Germany, and the USA. Eligible patients were adults (≥18 years) with histologically confirmed follicular lymphoma (grade 1, 2, 3a, or 3b) that had relapsed or was refractory to two or more systemic therapies, had an Eastern Cooperative Oncology Group performance status of 0-2, and had sufficient tumour tissue for central testing of EZH2 mutation status. Patients were categorised by EZH2 status: mutant (EZH2mut) or wild-type (EZH2WT). Patients received 800 mg of tazemetostat orally twice per day in continuous 28-day cycles. The primary endpoint was objective response rate based on the 2007 International Working Group criteria for non-Hodgkin lymphoma, assessed by an independent radiology committee. Activity and safety analyses were done in patients who received one dose or more of tazemetostat. This study is registered with ClinicalTrials.gov, NCT01897571, and follow-up is ongoing. FINDINGS: Between July 9, 2015, and May 24, 2019, 99 patients (45 in the EZH2mut cohort and 54 in the EZH2WT cohort) were enrolled in the study. At data cutoff for the analysis (Aug 9, 2019), the median follow-up was 22·0 months (IQR 12·0-26·7) for the EZH2mut cohort and 35·9 months (24·9-40·5) for the EZH2WT cohort. The objective response rate was 69% (95% CI 53-82; 31 of 45 patients) in the EZH2mut cohort and 35% (23-49; 19 of 54 patients) in the EZH2WT cohort. Median duration of response was 10·9 months (95% CI 7·2-not estimable [NE]) in the EZH2mut cohort and 13·0 months (5·6-NE) in the EZH2WT cohort; median progression-free survival was 13·8 months (10·7-22·0) and 11·1 months (3·7-14·6). Among all 99 patients, treatment-related grade 3 or worse adverse events included thrombocytopenia (three [3%]), neutropenia (three [3%]), and anaemia (two [2%]). Serious treatment-related adverse events were reported in four (4%) of 99 patients. There were no treatment-related deaths. INTERPRETATION: Tazemetostat monotherapy showed clinically meaningful, durable responses and was generally well tolerated in heavily pretreated patients with relapsed or refractory follicular lymphoma. Tazemetostat is a novel treatment for patients with follicular lymphoma. FUNDING: Epizyme.

Effects of transverse bodily movements of maxillary premolars on the surrounding hard tissue.

INTRODUCTION: This experimental study was designed to (1) produce buccal translation of maxillary premolars and (2) evaluate the effects on the buccal alveolar bone. METHODS: A randomized split-mouth study was designed based on 7 adult male beagle dogs. The experimental side received a custom cantilever appliance fabricated to produce a translatory force through the maxillary second premolar's center of resistance. The contralateral second premolar received no appliance and served as the control. The premolars underwent 6-7 weeks of buccal translation, followed by 3 weeks of fixed retention. Biweekly tooth movements were evaluated using intraoral and radiographic measurements. Pretreatment and posttreatment models were measured to assess tipping. Three-dimensional microscopic tomography was used to quantify the amount and density of buccal bone. Bone formation and turnover were assessed using fluorescent labeling, hematoxylin and eosin staining, tartrate-resistant acid phosphatase staining, and bone sialoprotein immunostaining. RESULTS: The applied force (100 g of force) translated (1.4 mm) and minimally tipped (4°) the experimental teeth. Lateral translation produced dehiscences at the mesial and distal roots, with 2.0 mm and 2.2 mm loss of vertical bone height, respectively. Bone thickness decreased significantly (P < 0.05) at the apical (∼0.4 mm), midroot (∼0.4 mm), and coronal (∼0.2 mm) levels. Fluorescent imaging, hematoxylin and eosin staining, and immunostaining for bone sialoprotein all showed new bone formation extending along the entire periosteal surface of the second premolar's buccal plate. Tartrate-resistant acid phosphatase staining demonstrated greater osteoclastic activity on the experimental than that of control sections. CONCLUSIONS: New buccal bone forms on the periosteal surface during and after tooth translation, but the amount of bone that forms is less than the amount of bone loss, resulting in a net decrease in buccal bone thickness and a loss of crestal bone.

Effects of micro-osteoperforations on tooth movement and bone in the beagle maxilla.

PURPOSE: The purpose of this study was to determine how micro-osteoperforations (MOPs) affect tooth movements, bone turnover, bone density, and bone volume. METHODS: A split-mouth experimental design with 7 beagle dogs was used to evaluate bone surrounding maxillary second premolars that had been retracted for 7 weeks. One month after the maxillary third premolars were extracted, 8 MOPs (1.5 mm wide and 7 mm deep) were created without flaps with the use of the Propel device (6 were placed 3 mm distal to the second premolar and 2 were placed in the premolar furcation) on one randomly chosen side. The maxillary second premolars were retracted bilaterally with the use of 200 g nickel-titanium closed coil springs. Tooth movements were measured intraorally and radiographically. Microscopic computed tomography was used to evaluate the material density and volume fraction of bone distal to the premolars. Hematoxylin and eosin-stained and fluorescent sections were used to examine the bone remodeling. RESULTS: Neither the intraoral (P = 0.866) nor radiographic (P = 0.528) measures showed statistically significant side differences in tooth movements. There also were no statistically significant differences in the density (P = 0.237) or volume fraction (P = 0.398) of bone through which the premolars were being moved. Fluorescent and histologic evaluations showed no apparent differences in osteoblasts, osteoclasts, or mineralization of bone near the teeth being moved. Bone healing was evident in and near the MOP sites, which had nearly but not completely healed after 7 weeks. Regions of acellular bone were evident extending ∼0.8 mm from the MOP sites. CONCLUSIONS: MOPs placed 3 mm away from teeth do not increase tooth movements and have limited and transitory effect on bone.

Localizing the osseous boundaries of micro-osteoperforations.

INTRODUCTION: The aim of this work was to determine how far the effects of micro-osteoperforations (MOPs) extend within bone by quantifying the damage caused and the short-term bony adaptations that occur in and around the injury site. METHODS: With the use of a split-mouth design, 34 MOPs (Propel) were randomly placed in the mandibular furcal bone of 13 beagle dogs either 2 or 4 weeks before killing them. The control side received no treatment. Vickers hardness microindentation, microscopic computed tomography, and histologic analyses were performed to evaluate the bone surrounding the MOPs. RESULTS: Microfractures produced during insertion extended ∼0.6 mm from the MOP sites. Cortical and trabecular bone were significantly less dense on the experimental than on the control side up to 4.2 mm from the edge of the MOP, but side differences were small (<5%) beyond 1.5 mm from the MOP. Experimental cortical bone was significantly softer than the control bone up to 0.8 mm from the MOP after 2 weeks of healing, and up to 0.5 mm from the MOP after 4 weeks of healing. Hematoxylin and eosin stained sections of cortical and trabecular bone showed small areas of woven bone within the MOP sites after 2 weeks, and acellular areas of bone extending ∼0.5 mm from the MOP. After 4 weeks of healing, there were greater amounts of woven bone, as well as early signs of lamellar bone, in and around the MOP sites. Markedly increased TRAP activity extending up to 2.5 mm from the MOP was evident after 2 weeks, but not after 4 weeks. Vital fluorescence staining showed diffuse bone deposition on the experimental side up to 1.5 mm from the MOP margin. CONCLUSIONS: When MOPs are performed in beagle dogs, demineralization is transient and healing of the injured area, as well as remineralization of bone affected by MOP placement, begins during the first 2 weeks. Although the transient effects extend farther, the principal effects extend only ∼1.5 mm from the MOP site.

Pharmacokinetic optimitzation of CCG-203971: Novel inhibitors of the Rho/MRTF/SRF transcriptional pathway as potential antifibrotic therapeutics for systemic scleroderma.

We recently reported the development of a novel inhibitor of Rho-mediated gene transcription (1, CCG-203971) that is efficacious in multiple animal models of acute fibrosis, including scleroderma, when given intraperitoneally. The modest in vivo potency and poor pharmacokinetics (PK) of this lead, however, make it unsuitable for long term efficacy studies. We therefore undertook a systematic medicinal chemistry effort to improve both the metabolic stability and the solubility of 1, resulting in the identification of two analogs achieving over 10-fold increases in plasma exposures in mice. We subsequently showed that one of these analogs (8f, CCG-232601) could inhibit the development of bleomycin-induced dermal fibrosis in mice when administered orally at 50mg/kg, an effect that was comparable to what we had observed earlier with 1 at a 4-fold higher IP dose.

Effects of bone grafting, performed with corticotomies and buccal tooth movements, on dehiscence formation in dogs.

INTRODUCTION: A randomized split-mouth experiment was performed in dogs to determine the effects of bone grafting, together with corticotomies and buccal tooth movements, on dehiscence formation. METHODS: Bilateral full-thickness mucoperiosteal buccal flaps were raised, and corticotomies were performed with a piezosurgery unit adjacent to the maxillary second premolars in 7 dogs. The experimental (graft+) side received a demineralized freeze-dried allograph and a resorbable collagen membrane. The second premolars were expanded with archwires for 9 weeks, followed by 3 weeks of consolidation. Soft tissue measurements included probing depths, attachment loss, and recession. Tooth movements were monitored using intraoral, radiographic, and model measurements. Bone surrounding the second premolars was evaluated with microcomputed tomography. New bone formation was analyzed histologically using calcein and alizarin fluorescent labels, and hematoxylin and eosin stains. RESULTS: Postsurgical healing progressed normally with no signs of infection. The graft+ and control (graft-) second premolars underwent similar amounts of expansion (about 2.5 mm intraorally; about 1.7 mm radiographically) and tipping, with no statistically significant side differences. The soft tissue periodontium was not affected on either side. There were bony dehiscences on both the graft+ and graft- sides, with slightly but significantly (P = 0.038) more bone loss over the mesial root on the graft- side. Bone material density was significantly (P = 0.028) greater on the graft+ side. Buccal bone apposition was evident surrounding graft particles, and mineralized particulate graft material was present at the apical aspect of the roots on the graft+ side. CONCLUSIONS: Bone grafting does not prevent dehiscence formation because only a limited amount of new bone is formed, primarily at the more apical aspects of the tooth's roots.

Prevalence of gingival recession after orthodontic tooth movements.

INTRODUCTION: This study was designed to evaluate the long-term prevalence of gingival recession after orthodontic tooth movements, focusing on the effects of mandibular incisor proclination and expansion of maxillary posterior teeth. METHODS: Records of 205 patients (162 female, 43 male) were obtained from 2 private practice orthodontists. Using pretreatment (age, 14.0 ± 5.9 years) and posttreatment (age, 16.5 ± 6.0 years) lateral cephalograms and dental models, mandibular incisor proclination and maxillary arch widths were measured. Gingival recession was measured based on posttreatment and postretention (age, 32.3 ± 8.5 years) intraoral photographs and models. Associations between tooth movements and gingival recession were evaluated statistically. RESULTS: Only 5.8% of teeth exhibited recession at the end of orthodontic treatment (only 0.6% had recession >1 mm). After retention, 41.7% of the teeth showed recession, but the severity was limited (only 7.0% >1 mm). There was no relationship between mandibular incisor proclination during treatment and posttreatment gingival recession. Incisors that finished treatment angulated (IMPA) at 95° or greater did not show significantly more recession than did those that finished less than 95°. There were weak positive correlations (r = 0.17-0.41) between maxillary arch width increases during treatment and posttreatment recession. CONCLUSIONS: Orthodontic treatment is not a major risk factor for the development of gingival recession. Although greater amounts of maxillary expansion during treatment increase the risks of posttreatment recession, the effects are minimal.

Elevation of a full-thickness mucoperiosteal flap alone accelerates orthodontic tooth movement.

INTRODUCTION: Our objective was to determine whether the elevation of a full-thickness mucoperiosteal flap alone, without cortical cuts, decreases the amount of bone around teeth and accelerates mesial tooth movements. METHODS: The mandibular second premolars of 7 beagle dogs were extracted, and on a randomly selected side, a full-thickness mucoperiosteal buccal flap extending from the distal aspect of the third premolar to the mesial aspect of the first premolar was elevated. The other side did not receive flap surgery. The mandibular third premolars were protracted with orthodontic appliances. Tooth movements were analyzed biweekly over an 8-week period with calipers and radiographs. The amount and density of bone were analyzed using microcomputed tomography; bone remodeling was evaluated with histologic sections. RESULTS: Experimental tooth movements measured intraorally between cusp tips were significantly greater (25.3%) than control tooth movements. The approximate center of resistance measured radiographically also moved significantly more (about 31%) on the experimental than on the control side. The experimental premolar tipped more than the control premolar (10.5° vs 8.7°), but the difference was not statistically significant. The medullary bone volume fraction mesial to the third premolar was significantly less (9.1%) and the bone was significantly less dense (9%) on the experimental side than on the control side. Histology showed no apparent side differences in the numbers of osteoclasts and osteoblasts evident in the medullary bone. CONCLUSIONS: Elevation of a full-thickness mucoperiosteal flap alone (ie, without injury to bone) decreases the amount and density of medullary bone surrounding the tooth and accelerates tooth movement. Due to its limited effects, elevation of a flap alone to increase tooth movements may not be justified.

Scleroderma dermal microvascular endothelial cells exhibit defective response to pro-angiogenic chemokines.

OBJECTIVES: Angiogenesis plays a critical role in SSc (scleroderma). The aim of this study was to examine the expression of growth-regulated protein-γ (Gro-γ/CXCL3), granulocyte chemotactic protein 2 (GCP-2/CXCL6) and their receptor CXCR2 in endothelial cells (ECs) isolated from SSc skin and determine whether these cells mount an angiogenic response towards pro-angiogenic chemokines. The downstream signalling pathways as well as the pro-angiogenic transcription factor inhibitor of DNA-binding protein 1 (Id-1) were also examined. METHODS: Skin biopsies were obtained from patients with dcSSc. ECs were isolated via magnetic positive selection. Angiogenesis was measured by EC chemotaxis assay. RESULTS: Gro-γ/CXCL3 and GCP-2/CXCL6 were minimally expressed in both skin types but elevated in SSc serum. Pro-angiogenic chemokine mRNA was greater in SSc ECs than in normal ECs. SSc ECs did not migrate to vascular endothelial growth factor (VEGF), Gro-γ/CXCL3, GCP-2/CXCL6 or CXCL16. The signalling pathways stimulated by these chemokines were also dysregulated. Id-1 mRNA in SSc ECs was lower compared with normal ECs, and overexpression of Id-1 in SSc ECs increased their ability to migrate towards VEGF and CXCL16. CONCLUSION: Our results show that SSc ECs are unable to respond to pro-angiogenic chemokines despite their increased expression in serum and ECs. This might be due to the differences in the signalling pathways activated by these chemokines in normal vs SSc ECs. In addition, the lower expression of Id-1 also decreases the angiogenic response. The inability of pro-angiogenic chemokines to promote EC migration provides an additional mechanism for the impaired angiogenesis that characterizes SSc.

Miniscrew-assisted slow expansion of mature rabbit sutures.

INTRODUCTION: In this study, we experimentally evaluated whether complex, mature sutures can be separated using skeletal anchorage and light, continuous forces. METHODS: Twelve adult, 8- to 9-month-old female New Zealand white rabbits were randomly assigned to 1 control group and 2 experimental groups. Open-coil nickel-titanium springs delivered constant forces of 100 g across the sagittal suture to miniscrew implants placed bilaterally in the frontal bone. Sutural separation was measured biweekly. Separation was also measured with microcomputed tomography. Bone formation (mineral apposition) was measured with fluorescent labels. Qualitative histologic analyses of the suture tissues were performed using hematoxylin and eosin staining; osteoclasts were evaluated with tartrate resistant acid phosphatase staining. RESULTS: All 24 miniscrew implants remained stable throughout the experiment. There was no statistically significant sutural separation in the control group. In the experimental groups, sutural separation was significant (P <0.05) at all time points after the initial records were taken. The rate of separation was linear during the first 42 days. There were moderate correlations (R = 0.59-0.89; P <0.05) between miniscrew implant separation and bone marker separation. Mineral apposition rate, which was not measureable in the control group, was significant in the experimental group. The mineral apposition rate was greater between 14 and 28 days than between 28 and 38 days, and it was greater on the ectocranial than on the endocranial surface. Based on the microcomputed tomography analysis, 3-dimensional sutural volume of the experimental group increased significantly (P = 0.02), but surface area did not (P = 0.26). CONCLUSIONS: It is possible to separate the sagittal suture of mature rabbits. Sutural separation is limited, indicating involvement of other articulations.

Effect of longitudinal flutes on miniscrew implant stability and 3-dimensional bone formation.

INTRODUCTION: The purpose of this study was to evaluate the effects of longitudinal flutes on miniscrew implant (MSI) stability and bone healing. METHODS: Using 11 skeletally mature New Zealand white rabbits, we placed 31 longitudinally fluted and 31 nonfluted, 3-mm-long MSIs in standardized positions in their calvaria and immediately loaded them with 100 g using nickel-titanium coil springs. Insertion torque values were obtained for each MSI placed; removal torque values were obtained for 28 MSIs that had been in place for 6 weeks and 20 MSIs that had been in place for 2 weeks. The bone volume fractions at 6 to 24, 24 to 42, and 42 to 60 µm from the MSI surfaces were evaluated using microcomputed tomography with an isotropic resolution of 6 µm. RESULTS: The success rate was 97% for both the fluted and nonfluted MSIs. The difference in insertion torque between the fluted and nonfluted MSIs was not statistically significant (P = 0.930). After 2 weeks, there was no statistically significant (P = 0.702) difference in removal torque between the fluted and nonfluted MSIs. After 6 weeks, removal torque values were significantly (P = 0.008) higher for the fluted (3.42 ± 0.26 N.cm) than the nonfluted (2.49 ± 0.20 N.cm) MSIs. Bone volume fractions of the 6-to-24-, 24-to-42-, and 42-to-60-µm layers were significantly (P <0.05) greater for the nonfluted than the fluted MSIs. CONCLUSIONS: Loaded 3-mm-long MSIs with and without flutes have high success rates. Longitudinal flutes placed in 3-mm MSIs increased their removal torque by 37% and decreased the amount of bone immediately surrounding them.

A key role for Fut1-regulated angiogenesis and ICAM-1 expression in K/BxN arthritis.

OBJECTIVES: Angiogenesis contributes to the pathogenesis of rheumatoid arthritis. Fucosyltransferases (Futs) are involved in angiogenesis and tumour growth. Here, we examined the role of Fut1 in angiogenesis and K/BxN serum transfer arthritis. METHODS: We examined Fut1 expression in human dermal microvascular endothelial cells (HMVECs) by quantitative PCR. We performed a number of angiogenesis assays to determine the role of Fut1 using HMVECs, Fut1 null (Fut1(-/-)), and wild type (wt) endothelial cells (ECs) and mice. K/BxN serum transfer arthritis was performed to determine the contribution of Fut1-mediated angiogenesis in Fut1(-/-) and wt mice. A static adhesion assay was implemented with RAW264.7 (mouse macrophage cell line) and mouse ECs. Quantitative PCR, immunofluorescence and flow cytometry were performed with Fut1(-/-) and wt ECs for adhesion molecule expression. RESULTS: Tumour necrosis factor-α induced Fut1 mRNA and protein expression in HMVECs. HMVECs transfected with Fut1 antisense oligodeoxynucleotide and Fut1(-/-) ECs formed significantly fewer tubes on Matrigel. Fut1(-/-) mice had reduced angiogenesis in Matrigel plug and sponge granuloma angiogenesis assays compared with wt mice. Fut1(-/-) mice were resistant to K/BxN serum transfer arthritis and had decreased angiogenesis and leucocyte ingress into inflamed joints. Adhesion of RAW264.7 cells to wt mouse ECs was significantly reduced when Fut1 was lacking. Fut1(-/-) ECs had decreased intercellular adhesion molecule-1 (ICAM-1) expression at mRNA and protein levels compared with wt ECs. ICAM-1 was also decreased in Fut1(-/-) arthritic ankle cryosections compared with wt ankles. CONCLUSIONS: Fut1 plays an important role in regulating angiogenesis and ICAM-1 expression in inflammatory arthritis.

Flapless cortical bone damage has no effect on medullary bone mesial to teeth being moved.

INTRODUCTION: In this study, we evaluated the effects of bone awl-induced damage to bone surrounding a tooth that was moved. METHODS: A randomized split-mouth design with 7 foxhounds was performed to evaluate protraction of the mandibular third premolars for 56 days with 200 g of orthodontic force. Before initiating tooth movements, a bone awl was used on the experimental side to create 60 buccal and lingual microfracture injuries to the cortical bone without a periosteal flap. Tooth movements were performed on the control and experimental sides. Microcomputed tomography and histology were used to assess bone morphology and modeling. Radiographic and caliper measures were used to assess tooth movements. RESULTS: The awl-induced injuries produced significant damage and microfractures (95 mm(3)). Buccal and lingual cortical bone volume fractions and densities were significantly less and cortical modeling was significantly greater on the experimental sides than on the control sides. Bone volume fractions and densities mesial to the third premolars were the same on the experimental and control sides. Experimental side tooth movements (1.40 ± 0.25 mm) were statistically the same as the control side tooth movements (1.57 ± 0.45 mm). CONCLUSIONS: The effects of flapless, bone awl-induced damage were limited to the cortical bone. Because there was no effect on the medullary bone mesial to the tooth being moved, no differences in tooth movements were produced.

Targeting the myofibroblast genetic switch: inhibitors of myocardin-related transcription factor/serum response factor-regulated gene transcription prevent fibrosis in a murine model of skin injury.

Systemic sclerosis (SSc), or scleroderma, similar to many fibrotic disorders, lacks effective therapies. Current trials focus on anti-inflammatory drugs or targeted approaches aimed at one of the many receptor mechanisms initiating fibrosis. In light of evidence that a myocardin-related transcription factor (MRTF)-and serum response factor (SRF)-regulated gene transcriptional program induced by Rho GTPases is essential for myofibroblast activation, we explored the hypothesis that inhibitors of this pathway may represent novel antifibrotics. MRTF/SRF-regulated genes show spontaneously increased expression in primary dermal fibroblasts from patients with diffuse cutaneous SSc. A novel small-molecule inhibitor of MRTF/SRF-regulated transcription (CCG-203971) inhibits expression of connective tissue growth factor (CTGF), α-smooth muscle actin (α-SMA), and collagen 1 (COL1A2) in both SSc fibroblasts and in lysophosphatidic acid (LPA)-and transforming growth factor ß (TGFß)-stimulated fibroblasts. In vivo treatment with CCG-203971 also prevented bleomycin-induced skin thickening and collagen deposition. Thus, targeting the MRTF/SRF gene transcription pathway could provide an efficacious new approach to therapy for SSc and other fibrotic disorders.