Nicholas A. Dewyer

Targeted Deletion of CCR2 Impairs Deep Vein Thombosis Resolution in a Mouse Model

CCR2 is required for monocyte recruitment in many inflammatory processes, as well as conferring Th1 lymphokine responses. Deep vein thrombosis (DVT) resolution represents a specific inflammatory response whereby the thrombus must be dissolved for restoration of blood flow. Using a stasis model of DVT in the mouse, we investigated the role of CCR2 on DVT resolution. Genetic deletion of CCR2 (CCR2−/−) was associated with larger thrombi at early and later time points, increased thrombus collagen, fewer thrombus monocytes (F4/80), and significantly impaired neovascularization. IL-2 and IFN-γ were significantly reduced in early CCR2−/− thrombi, whereas MCP-1 was significantly increased, and Th2 lymphokines were unaffected. Supplementation of CCR2−/− mice with IFN-γ normalized early thrombus resolution without increasing monocyte influx. Neither Ab depletion of IFN-γ nor genetic deletion of IFN-γ impaired early DVT resolution. Early fibrinolysis was not impaired in CCR2−/− mice, but a significant reduction in both matrix metalloproteinase (MMP)-2 and MMP-9 activity was observed. However, only MMP-9 activity was restored with administration of IFN-γ. We conclude that an early CCR2-dependent Th1 lymphokine response predominates in normal DVT resolution, mediates this in part by MMP-9 activation, but is not solely dependent on IFN-γ.

Fibrotic injury after experimental deep vein thrombosis is determined by the mechanism of thrombogenesis

Vessel wall matrix changes occur after injury, although this has not been well studied in the venous system. This study tested the hypothesis that the thrombus dictates the vein wall response and vein wall damage is directly related to the duration of thrombus contact. To determine the injury response over time, rats underwent inferior vena cava (IVC) ligation to produce a stasis thrombus, with harvest at various time points to 28 days (d). Significant vein wall matrix changes occurred with biomechanical injury (stiffness) peaking at 7-14 d, with concurrent early reduction in total collagen, an increase in early matrix metalloproteinase (MMP)-9 and late MMP-2, and concomitant increase in tumor necrosis factor (TNF)alpha, monocyte chemoattractant(MCP)-1 and tumor growth factor (TGF)beta (all P<0.05). To isolate the effect of the thrombus and its mechanism of genesis, rats underwent 7 d or limited stasis (24 hours), non-stasis thrombosis, or non-thrombotic IVC occlusion (Silicone plug). Vein wall stiffness was increased seven-fold, with a five-fold reduction in collagen, and 5.5- to seven-fold increase in TNFalpha, MCP-1, and TGFbeta with 7 d stasis as compared with controls (all P<0.05). By Picosirus red staining analysis, collagenolysis was significantly greater with 7 d stasis injury (P=0.01) but neither MMP-9 nor MMP-2 activity correlated with injury mechanism. In addition, vein wall cellular proliferation and uPA gene expression paralled the stasis thrombotic injury. Limited stasis, non-stasis thrombosis and non-thrombotic IVC occlusion showed a lesser inflammatory response. These data suggest both a static component and the thrombus directs vein wall injury via multiple mechanisms.

Neutrophils modulate post-thrombotic vein wall remodeling but not thrombus neovascularization

Early deep venous thrombosis (DVT) resolution is associated with neutrophil (PMN) influx. This study examined the role of PMNs in thrombus neovascularization and vein wall injury after DVT. A rat model of DVT by inferior vena cava (IVC) ligation was performed with control serum or rabbit anti-rat PMN serum administered perioperatively with sacrifice at 2 and 7 days. At 2 days, neutropenic rats had 1.6-fold larger thrombi (P = .04) and 1.4-fold higher femoral venous pressures by water manometry (P = .008) but no difference in thrombus neovascularization was observed. By 7 days, DVT sizes were similar, but vein wall injury persisted in the neutropenic rats with a 2.0-fold increase in vein wall stiffness by microtensiometry (P < .05), as well as a 1.2-fold increased thickness (P = .04). Collagen and profibrotic growth factors were significantly increased in neutropenic IVC at 7 days (all P < .05). Vein wall and intrathrombus uPA byWestern immunoblotting, and intrathrombus MMP-9 gelatinase activity were significantly less in neutropenic rats than controls (P < .001). Conversely, MMP-2 was significantly elevated in neutropenic IVC at 2 days after DVT. However, neutropenia induced 24 hours after DVT formation resulted in no significant increase in vein wall stiffness or collagen levels at 7 days, despite 1.4-fold larger thrombi (P < .05). These data suggest a critical early role for PMN in post DVT vein wall remodeling.

The role of urokinase plasminogen activator and plasmin activator inhibitor-1 on vein wall remodeling in experimental deep vein thrombosis

OBJECTIVE Deep vein thrombosis (DVT) resolution instigates an inflammatory response, resulting in vessel wall damage and scarring. Urokinase-plasminogen activator (uPA) and its inhibitor, plasminogen activator inhibitor-1 (PAI-1), are integral components of the fibrinolytic system, essential for venous thrombosis (VT) resolution. This study determined the vein wall response when exposed to increased and decreased plasmin activity. METHODS A mouse inferior vena cava (IVC) ligation model in uPA -/- or PAI-1 -/- and their genetic wild types (B6/SvEv and C57/BL6, respectively) was used to create stasis thrombi, with tissue harvest at either 8 or 21 days. Tissue analysis included gene expression of vascular smooth muscle cells (alpha smooth muscle actin [αSMA], SM22) and endothelial marker (CD31), by real-time polymerase chain reaction, enzyme-linked immunosorbent assay, matrix metalloproteinase (MMP)-2 and -9 activity by zymography, and vein wall collagen by picro-Sirius red histologic analysis. A P < .05 was considered significant. RESULTS Thrombi were significantly larger in both 8-day and 21-day uPA -/- as compared with wild type (WT) and were significantly smaller in both 8-day and 21-day PAI-1 -/- as compared with WT. Correspondingly, 8-day plasmin levels were reduced in half in uPA -/- and increased three-fold in PAI-1 -/- when compared with respective WT thrombi (P < .05; n = 5-6). The endothelial marker CD31 was elevated two-fold in PAI-1 -/- mice at 8 days, but reduced 2.5-fold at 21 days in uPA -/- as compared with WT (P = .02; n = 5-6), suggesting less endothelial preservation. Vein wall vascular smooth muscle cell (VSMC) gene expression showed that 8-day and 21-day PAI-1 -/- mice had 2.3- and 3.8-fold more SM22 and 1.8- and 2.3-fold more αSMA expression than respective WT (P < .05; n = 5-7), as well as 1.8-fold increased αSMA (+) cells (P ≤ .05; n = 3-5). No significant difference in MMP-2 or -9 activity was found in the PAI-1 -/- mice compared with WT, while 5.4-fold more MMP-9 was present in 21-day WT than 21-day uPA -/- (P = .03; n = 5). Lastly, collagen was ∼two-fold greater at 8 days in PAI-1 -/- IVC as compared with WT (P = .03; n = 6) with no differences observed in uPA -/- mice. CONCLUSIONS In stasis DVT, plasmin activity is critical for thrombus resolution. Divergent vein wall responses occur with gain or loss of plasmin activity, and despite smaller VT, greater vein wall fibrosis was associated with lack of PAI-1.

Divergent effects of Tlr9 deletion in experimental late venous thrombosis resolution and vein wall injury

Deep-vein thrombosis (DVT) resolves via a sterile inflammatory response. Defining the inflammatory response of DVT may allow for new therapies that do not involve anticoagulation. Previously, we have shown that Toll-like receptor 9 (Tlr9) gene deleted mice had impaired venous thrombosis (VT) resolution. Here, we further characterise the role of Tlr9 signalling and sterile inflammation in chronic VT and vein wall responses. First, we found a human precedent exists with Tlr9+ cells present in chronic post thrombotic intraluminal tissue. Second, in a stasis VT mouse model, endogenous danger signal mediators of uric acid, HMGB-1, and neutrophil extracellular traps marker of citrullinated histone-3 (and extracellular DNA) were greater in Tlr9-/- thrombi as compared with wild-type (WT), corresponding with larger VT at 8 and 21 days. Fewer M1 type (CCR2+) monocyte/macrophages (MØ) were present in Tlr9-/- thrombi than WT controls at 8 days, suggesting an impaired inflammatory cell influx. Using bone marrow-derived monocyte (BMMØ) cell culture, we found decreased fibrinolytic gene expression with exposure to several endogenous danger signals. Next, adoptive transfer of cultured Tlr9+/+ BMMØ to Tlr9-/- mice normalised VT resolution at 8 days. Lastly, although the VT size was larger at 21 days in Tlr9-/- mice and correlated with decreased endothelial antigen markers, no difference in fibrosis was found. These data suggest that Tlr9 signalling in MØ is critical for later VT resolution, is associated with necrosis clearance, but does not affect later vein wall fibrosis. These findings provide insight into the Tlr9 MØ mechanisms of sterile inflammation in this disease process.

Circulating CD4-positive lymphocyte levels as predictor of response to induction chemotherapy in patients with advanced laryngeal cancer

Background. Tumor regression after induction chemotherapy (ICT) identifies laryngeal cancers that are responsive to chemoradiation. Patient immune parameters have recently been associated with response to chemotherapy and may identify responding patients. A retrospective analysis was performed to determine if pretreatment, circulating T lymphocyte levels predicted ICT response in patients with advanced laryngeal cancer.

Stapedectomy Effects on Tinnitus Relationship of Change in Loudness to Change in Severity

Objective To relate poststapedectomy change in tinnitus loudness to change in tinnitus severity. Study Design Prospective, within-subjects. Setting A single otology and neurotology subspecialty referral practice. Subjects and Methods Forty-nine subjects undergoing stapedectomy completed the study between January 2012 and October 2013. Tinnitus instruments, audiometric data, and demographic information were collected prior to and 1 and 6 months after surgery. Tinnitus loudness was assessed using an 11-point (0 = none; 5 = conversation level; 10 = jet engine) visual analog scale, and severity was measured using the validated Tinnitus Functional Index. The relationship between change in tinnitus loudness and change in tinnitus severity was evaluated using linear regression and receiver operating characteristic (ROC) analyses. Results A linear regression model of change in tinnitus loudness averaged for both ears on a visual analog scale (ΔVASavg) versus change in Tinnitus Functional Index score (ΔTFI) showed a strong correlation (ΔTFI = 9.35 ×ΔVASavg; R = 0.64; P < .001). An ROC analysis identified ΔVASavg between 1.5 and 2.0 as the optimal threshold for predicting a clinically significant change in tinnitus severity (ΔTFI ≥ 13), with sensitivity and specificity of approximately 0.62 and a positive predictive value (PPV) of 0.64. Conclusion For poststapedectomy patients, a VAS loudness change by 1.5 to 2.0 points averaged for both ears in bilateral tinnitus or ~3 points in unilateral tinnitus has a PPV ~0.64 for a clinically significant change in tinnitus severity.

A Novel Approach to Oropharyngeal Foreign Body Removal

Grill wire brush bristle foreign bodies most commonly embed in the oropharynx. Often these bristles can be removed in the clinic; however, on occasion, the patient requires general anesthesia for retrieval because of the gag reflex and difficulty with access and visualization. We report here on 2 cases of patients who underwent successful transoral robotic surgical retrieval of wire bristles from the base of tongue after unsuccessful direct laryngoscopy. Otolaryngologists should be aware of the use of robotic assistance for oropharyngeal foreign body retrieval.

Automated smartphone audiometry: Validation of a word recognition test app

Develop and validate an automated smartphone word recognition test.

Pathology quiz case 2. Keratocystic odontogenic tumor (KCOT) of the maxilla.

A N OTHERWISE HEALTHY 15-YEAR-OLD GIRL presented with an 8-month history of gradually progressive right superior globe dystopia and right nasal obstruction without diplopia, eye pain, or pressure. Ophthalmologic examination showed 2 mm of superior displacement of the right globe, with 3 mm of orbital proptosis and 2 mm of inferior scleral show. There was a palpable mass extending from the inferior orbital rim to the gingivobuccal sulcus. Anterior rhinoscopy revealed medial displacement of the inferior turbinate and lateral nasal wall. A computed tomogram demonstrated a 7 6 5-cm cystic expansile mass centered in the right maxillary sinus, with expansion into the soft tissues of the cheek, the nasal cavity, and the ethmoid region (Figure1). There was secondary obstruction of the frontal, ethmoid, and sphenoid sinuses, with substantial bone thinning along the floor of the orbit. An unerupted/ displaced tooth was also seen in the superomedial portion of the cyst (Figure 2). The patient was taken to the operating room for biopsy. Intraoperative findings included a large amount of keratinaceous debris within the cyst, and the cyst wall was adherent to the orbit. After frozen-section diagnosis, the cystic lesion was widely marsupialized by excision of the inferior turbinate and medial cyst wall along with the attached tooth. Pathologic analysis revealed uniform squamous epithelium with basal palisading and a thin refractile parakeratinized lining (Figure 3). What is your diagnosis?