Preclinical development of AMG 139, a human antibody specifically targeting IL-23.

BACKGROUND AND PURPOSE: AMG 139 is a human anti-IL-23 antibody currently in a phase II trial for treating Crohn's disease. To support its clinical development in humans, in vitro assays and in vivo studies were conducted in cynomolgus monkeys to determine the pharmacology, preclinical characteristics and safety of this monoclonal antibody. EXPERIMENTAL APPROACH: The in vitro pharmacology, pharmacokinetics (PK), pharmacodynamics and toxicology of AMG 139, after single or weekly i.v. or s.c. administration for up to 26 weeks, were evaluated in cynomolgus monkeys. KEY RESULTS: AMG 139 bound with high affinity to both human and cynomolgus monkey IL-23 and specifically neutralized the biological activity of IL-23 without binding or blocking IL-12. After a single dose, linear PK with s.c. bioavailability of 81% and mean half-life of 8.4-13 days were observed. After weekly s.c. dosing for 3 or 6 months, AMG 139 exposure increased approximately dose-proportionally from 30 to 300 mg·kg(-1) and mean accumulation between the first and last dose ranged from 2- to 3.5-fold. Peripheral blood immunophenotyping, T-cell-dependent antigen responses and bone formation markers were not different between AMG 139 and vehicle treatment. No adverse clinical signs, effects on body weight, vital signs, ophthalmic parameters, clinical pathology, ECG, organ weights or histopathology were observed in the monkeys with the highest dose of AMG 139 tested (300 mg·kg(-1) s.c. or i.v.). CONCLUSIONS AND IMPLICATIONS: The in vitro pharmacology, PK, immunogenicity and safety characteristics of AMG 139 in cynomolgus monkeys support its continued clinical development for the treatment of various inflammatory diseases.

IL-36 in psoriasis.

Psoriasis is a common but severe skin disease with significant health consequences, both physical and psychological. Evidence has emerged during the past several years pointing to a key role for IL-36 in psoriasis. Overexpression of IL-36 in mouse skin leads to a disease quite similar to human plaque psoriasis, and inhibition of IL-36 in human psoriatic skin ameliorates the inflammation. Loss of the natural antagonist of IL-36, IL-36Ra, results in a different, more severe skin disease known as pustular psoriasis. These effects are likely a consequence of the actions of IL-36 both on cells of the immune system as well as on components of skin including fibroblasts and keratinocytes.

Are Th17 cells in the gut pathogenic or protective?

Th17 cells are abundant in multiple chronic inflammatory and autoimmune diseases. Clinical trials with antibodies to interleukin (IL)-17A, one of the Th17-cell signature cytokines, have recently reported therapeutic benefit in multiple patient populations; however, in Crohn's disease the role of Th17 cells and IL-17A appears to be more complicated. The development of different subsets of Th17 cells and their relative pathogenic activities with a focus on the gut environment will be discussed.

11 IL-1 family members in inflammatory skin disease.

The cytokines IL-1alpha and IL-1beta have long been known to play a profound role in inflammation, and in the past decade another cytokine, IL-18 (originally known as IGIF), has also been realized to be an IL-1 family member and to possess significant inflammatory activity. Half a dozen additional members of the IL-1 family have been identified in recent years, and given their relatedness to IL-I and IL-18, it is tempting to speculate that they too might possess inflammatory potential. We have demonstrated that certain of these cytokines can activate MAP kinases and the pathway leading to NFkappaB, via known IL-1R family members. Moreover, when overexpressed in skin, they are capable of causing an inflammatory skin condition resembling that seen in human disease.

Aquaporin 5-deficient mouse lungs are hyperresponsive to cholinergic stimulation.

Although aquaporin 5 (AQP5) is the major water channel expressed in alveolar type I cells in the lung, its actual role in the lung is a matter of considerable speculation. By using immunohistochemical staining, we show that AQP5 expression in mouse lung is not restricted to type I cells, but is also detected in alveolar type II cells, and in tracheal and bronchial epithelium. Aqp5 knockout (Aqp5(-/-)) mice were used to analyze AQP5 function in pulmonary physiology. Compared with Aqp5(+/+) mice, Aqp5(-/-) mice show a significantly increased concentration-dependent bronchoconstriction to intravenously administered Ach, as shown by an increase in total lung resistance and a decrease in dynamic lung compliance (P < 0.05). Likewise, Penh, a measure of bronchoconstriction, was significantly enhanced in Aqp5(-/-) mice challenged with aerosolized methacholine (P < 0.05). The hyperreactivity to bronchoconstriction observed in the Aqp5(-/-) mice was not due to differences in tracheal smooth muscle contractility in isolated preparations or to altered levels of surfactant protein B. These data suggest a novel pathway by which AQP5 influences bronchoconstriction. This observation is of special interest because studies to identify genetic loci involved in airway hyperresponsiveness associated with asthma bracket genetic intervals on human chromosome 12q and mouse chromosome 15, which contain the Aqp5 gene.

PECAM-1/IgG attenuates peroxynitrite-mediated extremity reperfusion injury.

OBJECTIVE: Neutrophil transendothelial migration, a key feature of skeletal muscle ischemia and reperfusion (I/R) injury, is mediated by the platelet endothelial cell adhesion molecule-1 (PECAM-1). Peroxynitrite anion, a toxic product of neutrophil superoxide anion and nitric oxide, contributes to oxidative skeletal muscle injury and can be quantified by measurement of protein tyrosine nitration after I/R. This study hypothesizes that administration of the PECAM-1/IgG antibody chimera will inhibit peroxynitrite-mediated injury after I/R. METHODS: The study was composed of five groups: an I/R group (n = 4), a sham treatment group anesthetic control (n = 3), a treatment group receiving the PECAM-1/immunoglobulin G (IgG) antibody chimera with I/R (n = 9), a treatment group receiving human IgG with I/R as an antibody control (n = 6), and a treatment group receiving normal saline solution with I/R as a vehicle control (n = 5). The right hind limb in male New Zealand white rabbits was rendered ischemic by occluding the iliac and femoral arteries for 3 hours, followed by 2 hours of reperfusion (I/R). Sham-treated rabbits underwent arterial dissection without arterial occlusion. PECAM-1/IgG-treated rabbits and IgG-treated rabbits received an infusion of 1 mg/kg in normal saline solution 20 mL via an ear vein catheter during the last 5 minutes of ischemia and the first 15 minutes of reperfusion. Saline solution-treated rabbits similarly received normal saline solution 20 mL. The anterior tibialis muscle was harvested after reperfusion. Immunohistochemical staining for nitrotyrosine was performed with monoclonal antinitrotyrosine antibodies and fluorescently labeled secondary antibodies. Computed morphometric study was performed to calculate relative fluorescence scores for each histologic section. Averaged fluorescence scores were analyzed by one-way analysis of variance with Bonferroni post hoc comparison. RESULTS: The averaged fluorescence scores (mean +/- SEM) for the sham-treated (2.88 +/- 0.78) and PECAM-1/IgG-treated (6.16 +/- 0.43) groups demonstrated a significant reduction in quantitative fluorescence compared with the IgG- (15.17 +/- 2.01) and saline solution-treated (17.46 +/- 3.71) control groups, and the I/R-treated (18.52 +/- 3.00) group, (P <.05). CONCLUSIONS: These results suggest that PECAM-1/IgG diminishes peroxynitrite-mediated oxidative skeletal muscle injury by inhibiting neutrophil transendothelial migration and may therefore prove a useful therapeutic agent in the treatment of reperfusion injury.

Lessons learned in adopting endovascular techniques for treating abdominal aortic aneurysm.

HYPOTHESIS: Endovascular exclusion of abdominal aortic and common iliac aneurysms can be performed safely, and in the short term represents a feasible alternative to traditional, open aneurysm repair. PATIENTS AND METHODS: Forty-one patients were treated with endovascular grafts for 39 abdominal aortic and 2 common iliac artery aneurysms. RESULTS: All devices were successfully deployed. The size of the abdominal aortic aneurysms varied from 4.9 to 11.9 cm (average, 6.13 cm). The median procedure time was 195 minutes. There was one iliac artery rupture, which required celiotomy for repair. The hospital stay varied from 2 to 39 days (average, 6.7 days). The perioperative mortality rate was 2.4%. Sixteen patients (39%) had groin wound complications. Ten patients (24%) had evidence of contrast (endoleak) within the aneurysm sac on completion of the procedure. There were no obvious direct leaks from either the point of proximal or distal fixation. Seven of these endoleaks have resolved spontaneously. Two patients required additional procedures in the postoperative period to treat endoleak. The final patient has evidence of persistent endoleak on 3-month surveillance computed tomography scan. Major late problems occurred in 3 patients. CONCLUSION: Patients with large abdominal aortic aneurysms and considerable cardiac comorbidity can safely undergo endovascular aneurysm repair. Femoral groin wound complications resulting in prolonged hospitalization remain the major cause of perioperative morbidity. In contradistinction to open aneurysm repair, long-term surveillance is essential to detect migration of the device and identify flow within the residual aneurysm sac-complications that could lead to aneurysm rupture following endovascular repair.

Salivary acinar cells from aquaporin 5-deficient mice have decreased membrane water permeability and altered cell volume regulation.

Aquaporins (AQPs) are channel proteins that regulate the movement of water through the plasma membrane of secretory and absorptive cells in response to osmotic gradients. In the salivary gland, AQP5 is the major aquaporin expressed on the apical membrane of acinar cells. Previous studies have shown that the volume of saliva secreted by AQP5-deficient mice is decreased, indicating a role for AQP5 in saliva secretion; however, the mechanism by which AQP5 regulates water transport in salivary acinar cells remains to be determined. Here we show that the decreased salivary flow rate and increased tonicity of the saliva secreted by Aqp5(-)/- mice in response to pilocarpine stimulation are not caused by changes in whole body fluid homeostasis, indicated by similar blood gas and electrolyte concentrations in urine and blood in wild-type and AQP5-deficient mice. In contrast, the water permeability in parotid and sublingual acinar cells isolated from Aqp5(-)/- mice is decreased significantly. Water permeability decreased by 65% in parotid and 77% in sublingual acinar cells from Aqp5(-)/- mice in response to hypertonicity-induced cell shrinkage and hypotonicity-induced cell swelling. These data show that AQP5 is the major pathway for regulating the water permeability in acinar cells, a critical property of the plasma membrane which determines the flow rate and ionic composition of secreted saliva.

Tumor necrosis factor-alpha inhibits aquaporin 5 expression in mouse lung epithelial cells.

Aquaporin 5 (AQP5), the major water channel expressed in alveolar, tracheal, and upper bronchial epithelium, is significantly down-regulated during pulmonary inflammation and edema. The mechanisms that underlie this decrease in AQP5 levels are therefore of considerable interest. Here we show that AQP5 expression in cultured lung epithelial cells is decreased 2-fold at the mRNA level and 10-fold at the protein level by the proinflammatory cytokine tumor necrosis factor alpha (TNF-alpha). Treatment of murine lung epithelial cells (MLE-12) with TNF-alpha results in a concentration- and time-dependent decrease in AQP5 mRNA and protein expression. Activation of the p55 TNF-alpha receptor (TNFR1) with an agonist antibody is sufficient to cause decreased AQP5 expression, demonstrating that the TNF-alpha effect is mediated through TNFR1. Inhibition of nuclear factor kappaB (NF-kappaB) translocation to the nucleus blocks the effect of TNF-alpha on AQP5 expression, indicating that activation of NF-kappaB is required, whereas inhibition of extracellular signal-regulated or p38 mitogen-activated protein kinases showed no effect. These data show that TNF-alpha decreases AQP5 mRNA and protein expression and that the molecular pathway for this effect involves TNFR1 and activated NF-kappaB. The ability of inflammatory cytokines to decrease aquaporin expression may help explain the connection between inflammation and edema.

Determinates of functional disability after complex upper extremity trauma.

This is a retrospective chart review of 71 patients who were operated on for presumed upper extremity arterial trauma between June 1992 and June 1998. Penetrating trauma occurred in 50 (70%) patients, and blunt trauma in 21 (30%). There were 2 innominate, 6 subclavian, 13 axillary, 26 brachial, 5 radial, 6 ulnar, and 6 multiple arterial injuries. There were 7 negative explorations (4 venous injuries, 2 false-positive angiograms, and 1 branch artery injury). In addition to the vascular injury, 44 patients (69%) had another injury in the extremity, including 8 (12.5%) orthopedic injuries, 12 (19%) nerve injuries, and 24 (37.5%) combination nerve and orthopedic injuries. There were three arterial thromboses, one arterial disruption, and four amputations, resulting in a patency rate and limb salvage rate of 94%. Persistent disability was more common in those patients with blunt injury (p = 0.02) and in those patients with associated neurologic and orthopedic injuries (p < 0.05). Full functional recovery was seen in 21 (33%) patients, while some form of disability was noted in the remaining 67%. The magnitude of the concomitant neurologic injury was the major determinate of functional outcome in this patient population.

Shear force regulates matrix metalloproteinase activity in human saphenous vein organ culture.

BACKGROUND: Development of vein graft intimal hyperplasia has been related both to shear force and to the activity of matrix metalloproteinases (MMPs). Little data are available regarding the effects of shear on MMP expression and activity. The aim of this study was to examine the relationship among shear force, metalloproteinase activity, and intimal thickening in human saphenous vein segments maintained in organ culture. MATERIALS AND METHODS: Segments of human saphenous vein were cultured under static conditions, or perfused under low-flow and high-flow conditions in a perfusion apparatus for 7 days. Metalloproteinase levels and activities were measured using ELISA and substrate gel zymography, respectively. Intimal thickening was determined by morphometric analysis. Results were compared with control vein tissue, which was not subjected to organ culture, using a one-way ANOVA. RESULTS: A 13% increase in proteolytic activity was noted on substrate gel zymography at 68-72 kDa in high-flow vein tissue. The protein content of MMP-2, MMP-9, tissue inhibitor of metalloproteinase-1 (TIMP-1), and TIMP-2 was increased in high-flow vein tissue by 21%, 126%, more than 100-fold, and 86%, respectively. In culture media bathing the outside of the vein, TIMP-2 was increased in high-flow specimens, while TIMP-1 was inversely related to flow rate. Intimal thickening was directly related to flow rates, and was progressively increased in the low-flow and high-flow groups by 3-fold and 4-fold, respectively. CONCLUSIONS: Metalloproteinase levels in human saphenous vein cultures are related to shear force. MMP levels and activity correlate with the degree of intimal thickening. This model may provide a valuable tool for the analysis of physical forces and their influence on intimal thickening in human saphenous vein.

Valuing educational scholarship at the Medical College of Wisconsin.

Since the late 1980s faculty and staff at the Medical College of Wisconsin (MCW) have actively sought to align their school's academic culture and promotional process with its mission of educational excellence and innovation. As one of the top 50 medical schools receiving NIH funds, MCW has well-established mechanisms to evaluate and recognize the scholarship of discovery. Understanding, evaluating, and recognizing the value of individuals engaged in the scholarship of teaching, however, required changes in individuals' beliefs and in the MCW's promotion processes and organizational infrastructure. Building on the successful introduction of the MCW's Educator's PortfolioCopyright, a tool for documenting educational scholarship, a multifaceted change strategy was implemented to influence underlying beliefs and values about clinician-educators. Retrospectively, this strategy was consistent with John Kotter's eight-step change model, which the authors apply as an organizing framework for this case report of educational evolution at the MCW. Through creating a guiding coalition, developing vision and strategy, generating short-term wins, and anchoring new approaches in the MCW's culture, the MCW has made substantive progress in recognizing and rewarding educational scholarship. Changing academic cultures to value education is itself an educational process, requiring persistence and the ability to teach others about educational scholarship and its associated criteria.

All-trans-retinoic acid decreases cell proliferation and increases apoptosis in an animal model of vein bypass grafting.

BACKGROUND: We have previously demonstrated a decrease in intimal hyperplasia in vein bypass grafts from animals treated with all-trans-retinoic acid (atRA). The purpose of this study was to examine the effect of atRA on proliferation and apoptosis rates in healing vein bypass grafts. METHODS: Interposition jugular vein bypass grafts were placed in the carotid artery of 30 New Zealand white rabbits. Animals received either atRA (10 mg/kg/d) or vehicle (corn oil) for a period of 2 weeks. Animals were killed at 3, 7, or 28 days after graft placement after having received 3 doses of 5-bromo-2'-¿Deoxyuridine (BRDU, 35 Mg/KG). Animals Were Perfusion Fixed, And Vein Grafts Were Prepared For Immunohistochemistry By Using Antibodies To Brdu, Proliferating Cell Nuclear Antigen, And Bcl-XL. Apoptosis Was Measured By Using The Tunel Assay. Histologic Sections Were Analyzed By A Pathologist Blinded To The Study, And An Index Of Positively Stained Cells Was Generated For Each Layer Of The Vein Graft Wall. RESULTS: All-trans-retinoic acid reduced the proliferation index in the neointima of vein grafts during the first week after surgery. Apoptotic rates were higher in the intima of vein grafts from animals treated with atRA, which could not be explained by changes in bcl-xl expression. No differences were noted in the media or adventitia between the groups. CONCLUSIONS: atRA decreased cell proliferation and increased apoptosis in the intima of healing vein bypass grafts. These effects contribute to decreased intimal hyperplasia, which has been previously noted.

All-trans-retinoic acid decreases vein graft intimal hyperplasia and matrix metalloproteinase activity in vivo.

BACKGROUND: Development of vein graft intimal hyperplasia has been associated with increased activity of matrix metalloproteinases (MMPs). All-trans-retinoic acid (atRA) decreases expression and activity of MMPs in tissue culture and has decreased intimal hyperplasia following arterial balloon catheter injury. We examined the effect of oral administration of atRA on intimal hyperplasia and MMP expression in an animal model of vein bypass grafting. MATERIALS AND METHODS: Interposition jugular vein bypass grafts were placed in the carotid artery of New Zealand white rabbits. Animals received either atRA (10 mg/kg/day) or vehicle (corn oil) for a period of 2 weeks. Retinoic acid serum levels were determined by HPLC. Intimal and medial areas were measured using morphometric analysis of perfusion-fixed vein graft specimens, and intimal thickness was calculated using circumferential measurements. Expression of MMP-2, MMP-9, and TIMP-1 in vein grafts and unoperated control veins was determined using Northern analysis, and proteolytic activity was determined using substrate gel zymography. RESULTS: Animals treated with atRA had significantly elevated serum levels of this compound and its metabolites. A decrease in intimal to medial ratio was noted after 28 days in vein grafts from treated animals (0.63 vs 0.88, P < 0.01), and a decrease in calculated intimal thickness was noted at 7 and 28 days. Expression of MMP-2 was decreased in treated animals 7 days following surgery, and expression of both MMP-2 and MMP-9 was decreased at 28 days. A decrease in proteolytic activity was noted on zymography at 68 kDa, 7 and 28 days following surgery in vein grafts from animals treated with atRA, corresponding with a decrease in the active form of MMP-2. Increased expression of TIMP-1 was noted in vein grafts from both the treated and the control groups, 7 and 28 days following graft placement. CONCLUSIONS: Oral administration of all-trans-retinoic acid resulted in decreased intimal hyperplasia in an animal model of vein bypass grafting. This was associated with decreased expression and activity of MMP-2 in treated animals.

Intraoperative carotid evaluation.

HYPOTHESIS: Intraoperative duplex scanning can identify technical defects and increase the quality of carotid artery repair. DESIGN: We evaluated 100 consecutive carotid operations in 96 patients (60 men and 36 women) from 1995 to 1998. Spectral-derived peak systolic flow velocities (PSV) were graded (PSV < 100 cm/s, normal laminar flow; PSV 100-150 cm/s, mild or moderate flow disturbance; PSV > 150 cm/s, severe flow disturbance). Prospective criteria for intraoperative revision included PSV greater than 150 cm/s, spectral broadening, and B-mode imaging of intimal flaps or intraluminal debris. Preoperative, intraoperative, and 6-week follow-up duplex scan results were analyzed. SETTING: All patients were evaluated and treated at a single academic institution. INTERVENTIONS: All procedures were performed with the patient under general endotracheal anesthesia; 86% underwent shunting and 70% underwent patching. MAIN OUTCOME MEASURE: Number and type of revisions, patency of repair, residual and recurrent stenosis, and ipsilateral neurologic events. RESULTS: There were 33 intraoperative duplex studies with abnormal findings. Seven involved the common carotid artery and resulted in intraoperative revision of 5 intimal flaps at the site of the proximal clamp. In 11 patients, incomplete eversion endarterectomy resulted in elevated distal intimal flaps in the external carotid artery that were repaired through a separate arteriotomy. There were 15 abnormalities in the internal carotid artery prompting 5 revisions. Five studies with PSV of 100 to 150 cm/s had no defects on B-mode imaging and were observed without treatment. Five false-positive studies were attributed to increased flow velocity due to contralateral occlusive discase. At 6 weeks' follow-up, 4 of 5 repaired common carotid arteries were normal on duplex scan and 1 had a mild residual stenosis. Ten of the 11 external carotid repairs were patent and 1 was occluded. Four of the 5 internal carotid artery repairs were normal on postoperative evaluation and 1 had a mild residual stenosis. Of the 10 abnormal internal carotid arteries that were observed, 9 were normal on postoperative duplex and 1 had a mild residual stenosis. One perioperative stroke occurred in a patient with a normal, patent carotid repair. CONCLUSIONS: Intraoperative duplex evaluation of carotid reconstruction is an efficient, sensitive tool that can detect technical lesions that will jeopardize surgical reconstruction. Interpretive judgment is required because all flow disturbances do not dictate surgical intervention. This technique enables the surgeon to maximize the quality of the arterial reconstruction during carotid artery surgery.