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Restoration is frequently aimed at the recovery of target species, but also influences the larger food web in which these species participate. Effects of restoration on this broader network of organisms can influence target species both directly and indirectly via changes in energy flow through food webs. To help incorporate these complexities into river restoration planning, we constructed a model that links river food web dynamics to in-stream physical habitat and riparian vegetation conditions. We present an application of the model to the Methow River, Washington, USA, a location of on-going restoration aimed at recovering salmon. Three restoration strategies were simulated: riparian vegetation restoration, nutrient augmentation via salmon carcass addition, and side channel reconnection. We also added populations of nonnative aquatic snails and fish to the modeled food web to explore how changes in food web structure mediate responses to restoration. Simulations suggest that side channel reconnection may be a better strategy than carcass addition and vegetation planting for improving conditions for salmon in this river segment. However, modeled responses were strongly sensitive to changes in the structure of the food web. The addition of nonnative snails and fish modified pathways of energy through the food web, which negated restoration improvements. This finding illustrates that forecasting responses to restoration may require accounting for the structure of food webs, and that changes in this structure, as might be expected with the spread of invasive species, could compromise restoration outcomes. Unlike habitat-based approaches to restoration assessment that focus on the direct effects of physical habitat conditions on single species of interest, our approach dynamically links the success of target organisms to the success of competitors, predators, and prey. By elucidating the direct and indirect pathways by which restoration affects target species, dynamic food web models can improve restoration planning by fostering a deeper understanding of system connectedness and dynamics., (© 2017 by the Ecological Society of America.)

Objectives: We hypothesized that the method of stress external rotation more accurately reproduces the mechanism of injury, and therefore this diagnostic method more likely detects ankle instability than the fibular stress examination., Design: Prospective cohort comparison study., Setting: Level 1 trauma center., Patients: Twenty-eight consecutive patients with unstable ankle fractures presenting within 7 days from the time of injury. Previous ankle surgical history or age younger than 18 years was excluded., Intervention: Stress external rotation and lateral fibular stress examination was performed intraoperatively., Main Outcome Measure: Radiographic measurement of the tibiofibular clear space, tibiofibular overlap, and medial clear space were recorded., Results: After normalization of the fluoroscopic measurements, there was no difference in detecting changes in tibiofibular clear space or tibiofibular overlap. However, there was a significant difference in detecting medial clear space widening with stress external rotation. Compared with lateral fibular stress, stress external rotation demonstrated a 35% increase (P < 0.05) in medial clear space widening. This difference correlates with the 1-2-mm difference of additional widening with stress external rotation., Conclusions: Untreated instability impacts patient outcomes. The difference in widening with stress external rotation was significantly greater than lateral fibular stress and appreciable on standard fluoroscopic views. Stress external rotation radiographs are a more reliable indicator of mortise instability than traditional lateral fibular stress., Level of Evidence: Diagnostic Level IV. See Instructions for Authors for a complete description of levels of evidence.

Objectives: Administration of early and appropriate antibiotic in treating patients with open fractures is an important early factor in preventing infection and optimizing outcomes. The purpose of this study is to evaluate the effects of an orthopaedic trauma performance improvement program directed at early antibiotic administration for open fracture patients at our trauma center., Design: Retrospective comparative cohort study of patients treated for an open fracture by before and after implementation of our performance improvement program specifically designed to address early open fracture care., Setting: Single metropolitan level 2 regional trauma center., Patients: Patients with open fractures treated by orthopaedic surgery (hand and spine excluded) at our institution between January 2012 and December 2013 were included. Patients transferred from another facility were excluded., Intervention: Patients were divided into one of the following 2 groups. Group 1 included patients treated before our open fracture performance improvement program (January 2012-December 2012) and group 2 comprised those treated after the program was instituted (January 2013-December 2013)., Main Outcome Measurements: Patient demographics, injury factors, and performance measures relating to early open fracture care [eg, the characteristics of early antibiotic administration in their treatment course, including timeliness of prophylactic intravenous (IV) antibiotic therapy and reasons for delay or omission of these treatments] were evaluated., Results: Group 1 was comprised of 127 patients with a total of 142 open fractures, whereas group 2 included 132 patients with a total of 156 open fractures. Patient and injury factors were not significantly different between the 2 groups. Group 1 received IV antibiotics at an average of 70.5 minutes after arrival at our institution compared with group 2 who received antibiotics at an average of 32.4 minutes (P < 0.001). The average times from emergency department arrival to physician evaluation improved from 6.5 to 4.5 minutes (P = 0.02) and antibiotic order to antibiotic delivery improved from 37 to 13 minutes (P < 0.001) for group 1 compared with group 2, respectively. The average time between physician evaluation and antibiotic showed a trend toward improvement (12.7-8.0 minutes, P = 0.57). Fifty percent of patients in group 1 (63/127) had antibiotics initiated within 1 hour of hospital arrival, whereas 78% (100/132) in group 2 had antibiotics initiated within 1 hour (P < 0.001). Eighty-five percent (112/127) of patients in group 1 had antibiotics initiated within 3 hours of hospital arrival, whereas 95% (125/132) in group 2 had antibiotics initiated within 3 hours (P = 0.03). Of those patients receiving standard antibiotics (cephalosporin), 79% (85/107) in group 1 and 91% (104/114) in group 2 received the recommended dose of IV antibiotic for their body weight (e.g., 2 g cefazolin for patients of >80 kg) (P < 0.006)., Conclusions: Optimal treatment of open fracture patients with early and appropriate antibiotic prophylaxis was lacking for many patients at our trauma center. A multifaceted performance improvement program specifically concentrating on education, accountability, and antibiotic availability aimed at this aspect of orthopaedic trauma care was very effective in improving our early treatment of these patients., Level of Evidence: Therapeutic level III. See instructions for authors for a complete description of levels of evidence.

Background: TLR-2 is expressed on the surface of leucocytes, lung and liver tissue and initiates the activation of immune response after interaction with components of the bacterial cell wall. In this experiment we investigated whether immunostimulation with TLR-2 agonists under conditions of sterile inflammation (hemorrhagic shock (HS)) may affect the immune response and remote organ inflammation., Methods: Male C57/BL6 mice were subjected to standardized pressure-controlled HS (MAP of 35 mmHg for 90 minutes). The TLR-2 agonist macrophage-activated lipopeptide-2 (MALP-2) was administered (i.p.) either 12 hours prior to the induction of HS (Group MALP PT) or after the hypotensive period (90 minutes) (Group MALP T). After six hours, plasma cytokine levels (IL-6, KC, IL-10, and MCP-1) and lung and liver MPO activity were assessed., Results: Pre-treatment with MALP-2 resulted in a significant attenuation of the systemic pro-inflammatory (IL-6) response (MALP PT: 0.83±0.2 ng/ml vs. MALP T: 1.7±0.09 ng/ml) (p<0.05). In comparison to the liver MPO activity, lung MPO levels in in group MALP PT did not show differences to levels measured in MALP T mice (1.200±200 ng/mg vs. 1.800±200 ng/mg)., Conclusions: After initial inflammation, MALP-2 pre-treatment was associated with attenuated systemic immune response after sterile stimulus. The TLR-2 agonist appears to affect sterile inflammation pathways. The exact mechanisms should be studied further to better understand these affects.

Introduction: Bilateral femoral shaft fractures have been reported to be an independent risk factor for morbidity and mortality; however, the value of these studies is limited due to small sample sizes and the timing of these studies before the establishment of damage control orthopaedics. The objective of this study was to compare the incidence of morbidity and mortality in patients with bilateral vs. unilateral femoral shaft fractures in the era of damage control orthopaedics., Methods: Retrospective analysis of the TraumaRegister DGU from 2002 to 2005. Inclusion criteria were uni- or bilateral femoral shaft fractures and complete demographic data documentation. Univariate data analysis and logistic regression analysis were performed with SPSS., Results: Between 2002 and 2005, 776 patients with unilateral and 118 patients with bilateral femoral shaft fractures were identified. Patients with bilateral femoral shaft fractures had a significantly higher Injury Severity Score (ISS) (29.5 vs. 25.7 points), a significantly higher incidence of pulmonary (34.7% vs. 20.6%) and multiple organ failure (25.0% vs. 14.6%) as well as a significantly higher mortality rate (16.9% vs. 9.4%). In the overall patient population, early total care (ETC) was significantly more often performed in patients with unilateral femoral shaft fractures (50.9% vs. 33.6%). Logistic regression analysis revealed no significant association between bilateral femoral shaft fractures and multiple organ failure or mortality; however, bilateral femoral shaft fractures are an independent risk factor for pulmonary failure. Subgroup analysis revealed that the impact of the bilateral femoral shaft fracture was especially pronounced in patients with an ISS<25 points., Discussion: Bilateral femoral shaft fractures are an independent risk factor for pulmonary failure but not for multiple organ failure or mortality. The impact of the additional femoral shaft fracture for pulmonary failure appears to be especially pronounced in the less severely injured patients, whose injuries are often underestimated when stratified with the ISS. Patients with bilateral femoral shaft fractures have significantly more often severe abdominal injuries as well as severe blood loss which may account for the increased mortality rate. Therefore, the presence of bilateral femoral shaft fractures should be recognised as an increased risk for systemic complications., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Purpose of Review: There is still an ongoing debate whether damage control orthopedics (DCO) or other treatment strategies should be favored in the treatment of multiply injured patients. This review gives an overview of the current literature concerning this important question in the treatment of severely injured patients., Recent Findings: Several studies could show that DCO can reduce the inflammatory burden due to surgery (second hit). The only randomized study showed a benefit for borderline patients treated by DCO in comparison to early total care. Other studies showed advantages for early care treatment in similar patients., Summary: In severely injured patients, DCO should be considered. On the other hand, there is still a lack of randomized studies for a more precise characterization of the patients who benefit from DCO treatment.

We present a novel technique of intramedullary fixation of the fibula using a humeral guide wire as an adjunct to tibial fixation, in the setting of tibial shaft fracture. Not only does this technique aid in determining length, alignment, and rotation of the tibial fracture, but it may also help the support of the lower extremity as whole by stabilizing the lateral column. In addition, this technique can be used to help maintain reduction of the fibula when there is concern for the soft tissues of the lower extremity secondary to swelling or injury. Our clinical case series demonstrates this safe, effective, and cost-sensitive technique to be used in the treatment of select concurrent fractures of the tibia and fibula.

Background and Purpose: Thoracoscopic-assisted ventral stabilisation for thoracolumbar fractures has been shown to be associated with decreased recovery time and less morbidity when compared with open procedures. However, there are a limited number of studies evaluating late clinical and radiological results after thoracoscopic spinal surgery., Methods: We performed an analysis of the late outcomes of thoracolumbar fractures after minimally invasive thoracoscopic ventral instrumentation. Between August 2003 and December 2008, 70 patients with thoracolumbar fractures (T5-L2) underwent ventral thoracoscopic stabilisation. Tricortical bone grafts, anterior plating systems (MACS-System), and cage implants were used for stabilisation. Outcomes measured include radiologic images (superior inferior endplate angle), Visual Analogue Scale (VAS), VAS Spine Score, quality of life scores SF-36 and Oswestry Disability Index (ODI)., Results: Forty seven patients (67%, 47 out of 70) were recruited for the follow up evaluation (2.2 ± 1.5 years). Lower VAS Spine scores were calculated in patients with intra- or postoperative complications (44.7 (± 16.7) vs. 65.8 (± 24.5), p=0.0447). There was no difference in outcome between patients treated with bone graft vs. cage implants. Loss of correction was observed in both bone graft and titanium cage groups., Interpretation: The present study demonstrates diminished long-term quality of life in patients treated with thoracoscopic ventral spine when compared with the outcome of german reference population. In contrast to the other patients, those patients without intra-operative or post-operative complications were associated with improved outcome. The stabilisation method (bone graft versus spinal cage) did not affect the long-term clinical or radiographic results in this series.

Interleukin-10 is known to modulate the systemic inflammatory response after trauma. This study investigates differences in the systemic and end-organ inflammation in animals treated with either inhalative or systemic IL-10 after experimental hemorrhagic shock (HS). Pressure controlled HS was performed in C57/BL6 mice for 1.5h (6 animals per group). Inhalative or systemic recombinant mouse IL-10 (50 μg/kg dissolved in 50 μl PBS) was administered after resuscitation. Animals were sacrificed after 4.5 or 22.5h of recovery. Serum levels of IL-6, IL-10, KC, MCP-1, and LBP were determined by ELISA. Pulmonary and liver inflammation was analyzed by standardized Myeloperoxidase (MPO) kits. Systemic and inhalative IL-10 administration affected the systemic inflammatory response as well as end-organ inflammation differently. Differences were obvious in the early (6h) but not later (24h) inflammatory phase. Systemic IL-10 application was associated with a decreased systemic inflammatory response as well as hepatic inflammation, whereas nebulized IL-10 solely reduced the pulmonary inflammation. Our study demonstrates that systemic and nebulized IL-10 administration differentially influenced the systemic cytokine response and end-organ inflammation. Early pulmonary but not hepatic protection appears to be possible by inhalative IL-10 application. Further studies are necessary to assess exact pathways., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The global response to the 12 January 2010 earthquake in Haiti revealed the ability to mobilise medical teams quickly and effectively when academic medical centres partner non-governmental organisations (NGO) that already have a presence in a zone of devastation. Most established NGOs based in a certain region are accustomed to managing the medical conditions that are common to that area and will need additional and specialised support to treat the flux of myriad injured persons. Furthermore, an NGO with an established presence in a region prior to a disaster appears better positioned to provide sustained recovery and rehabilitation relief. Academic medical centres can supply these essential specialised resources for a prolonged time. This relationship between NGOs and academic medical centres should be further developed prior to another disaster response. This model has great potential with regard to the rapid preparation and worldwide deployment of skilled medical and surgical teams when needed following a disaster, as well as to the subsequent critical recovery phase., (© 2012 The Author(s). Journal compilation © Overseas Development Institute, 2012.)

The tibia is the most commonly fractured long bone. Although the goals of fracture management are straightforward, methods for achieving anatomical alignment and stable fixation are limited. Type of management depends on fracture pattern, local soft-tissue involvement, and systemic patient factors. Tibial shaft fractures with concomitant fibula fractures, particularly those at the same level, may be difficult to manage because of their inherent instability. Typically, management of lower extremity fractures is focused on the tibia fixation, and the associated fibula fracture is managed without fixation. In this article, we describe a novel technique for intramedullary fixation of the fibula, using a humeral guide wire as an adjunct to tibia fixation in the setting of tibial shaft fracture. This technique aids in determining length, alignment, and rotation of the tibia fracture and may help support the lower extremity as whole by stabilizing the lateral column. In addition, this technique can be used to help maintain reduction of the fibula when there is concern about the soft tissues of the lower extremity secondary to swelling or injury. Our clinical case series demonstrates the safety, effectiveness, and cost-sensitivity of this technique in managing select concurrent fractures of the tibia and fibula.

Background: The earthquake that struck Haiti on 10 January 2010, killed 200,000 persons and injured thousands more. Working with Partners in Health, a non-governmental organization already present in Haiti, Dartmouth College, and the University of Pennsylvania sent multidisciplinary surgical teams to hospitals in the villages of Hinche and Cange. The purpose of this report is to describe the injuries seen and evolution of treatments rendered at these two outlying regional hospitals during the first month following the earthquake., Methods: A retrospective review of the database maintained by each team was performed. In addition to a list of equipment taken to Haiti, information collected included patient age, American Society of Anesthesiology (ASA) physical status, injuries sustained, procedures performed, wound management strategy, antibiotic therapy, and early outcomes., Results: A total of 113 surgical procedures were performed in 15 days by both teams. The average patient age was 25 years and average ASA score was 1.4. The majority of injuries involved large soft tissue wounds and closed fractures, although 21-40% of the patients at each hospital had either an open fracture or amputation wound. Initially, wound debridement was the most common procedure performed, but after two weeks, skin grafting, fracture fixation, and amputation revision were the more commonly needed operations., Conclusions: Academic surgical teams can ameliorate the morbidity and mortality following disasters caused by natural hazards by partnering with organizations that already have a presence in the affected region. A multidisciplinary team of surgeons and nurses can improve both mortality and morbidity following a disaster.

Purpose: To compare standard of care nuclear SPECT imaging with cardiac magnetic resonance imaging (MRI) for emergency room (ER) patients with chest pain and intermediate probability for coronary artery disease., Materials and Methods: Thirty-one patients with chest pain, negative electrocardiogram (ECG), and negative cardiac enzymes who underwent cardiac single photon emission tomography (SPECT) within 24 h of ER admission were enrolled. Patients underwent a comprehensive cardiac MRI exam including gated cine imaging, adenosine stress and rest perfusion imaging and delayed enhancement imaging. Patients were followed for 14 +/- 4.7 months., Results: Of 27 patients, 8 (30%) showed subendocardial hypoperfusion on MRI that was not detected on SPECT. These patients had a higher rate of diabetes (P = 0.01) and hypertension (P = 0.01) and a lower global myocardial perfusion reserve (P = 0.01) compared with patients with a normal cardiac MRI (n = 10). Patients with subendocardial hypoperfusion had more risk factors for cardiovascular disease (mean 4.4) compared with patients with a normal MRI (mean 2.5; P = 0.005). During the follow-up period, patients with subendocardial hypoperfusion on stress MRI were more likely to return to the ER with chest pain compared with patients who had a normal cardiac MRI (P = 0.02). Four patients did not finish the MR exam due to claustrophobia., Conclusion: In patients with chest pain, diabetes and hypertension, cardiac stress perfusion MRI identified diffuse subendocardial hypoperfusion defects in the ER setting not seen on cardiac SPECT, which is suspected to reflect microvascular disease., ((c) 2009 Wiley-Liss, Inc.)

Objectives: This study sought to investigate the prevalence and mechanisms underlying right ventricular (RV) dyssynchrony in arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C) using tissue Doppler echocardiography (TDE)., Background: An ARVD/C is characterized by fibrofatty replacement of RV myocardium and RV dilation. These pathologic changes may result in electromechanical dyssynchrony., Methods: Echocardiography, both conventional and TDE, was performed in 52 ARVD/C patients fulfilling Task Force criteria and 25 control subjects. The RV end-diastolic and -systolic areas, right ventricular fractional area change (RVFAC), and left ventricular (LV) volumes and function were assessed. Mechanical synchrony was assessed by measuring differences in time-to-peak systolic velocity (T(SV)) between the RV free wall, ventricular septum, and LV lateral wall. An RV dyssynchrony was defined as the difference in T(SV) between the RV free wall and the ventricular septum, >2 SD above the mean value for control subjects., Results: The mean difference in RV T(SV) was higher in ARVD/C compared with control subjects (55 +/- 34 ms vs. 26 +/- 15 ms, p < 0.001). Significant RV dyssynchrony was not noted in any of the control subjects. Based on a cutoff value of 56 ms, significant RV dyssynchrony was present in 26 ARVD/C patients (50%). Patients with RV dyssynchrony had a larger RV end-diastolic area (22 +/- 5 cm(2) vs. 19 +/- 4 cm(2), p = 0.02), and lower RVFAC (29 +/- 8% vs. 34 +/- 8%, p = 0.03) compared with ARVD/C patients without RV dyssynchrony. No differences in QRS duration, LV volumes, or function were present between the 2 groups., Conclusions: An RV dyssynchrony may occur in up to 50% of ARVD/C patients, and is associated with RV remodeling. This finding may have therapeutic and prognostic implications in ARVD/C.

Despite recent advances in cartilage regeneration and restoration procedures, isolated, large, full-thickness cartilage lesions in young patients continue to pose significant challenges to patients and orthopedic surgeons. Treatment options for this difficult problem have traditionally included arthrodesis, osteotomy, osteochondral allograft, and prosthetic reconstruction. We present a case of an adolescent patient with isolated idiopathic lateral tibial chondrolysis treated with a custom ceramic hemi-unicondylar hemiarthroplasty. Preoperatively, a 3-dimensional computed tomography scan of the patient's knee was obtained to begin manufacturing a conforming custom ceramic insert that would articulate between the tibial base plate and the patient's native lateral femoral cartilage. Through a lateral parapatellar approach, the tibial preparation was carried out using the Zimmer M/G unicompartmental knee system (Warsaw, Indiana), and the tibial base plate was cemented into position in the standard fashion. A custom, conforming, prefabricated ceramic insert (CeramTec, Memphis, Tennessee) was then inserted onto the tibial base plate. At 5-year follow-up, this salvage procedure was successful in relieving pain and restoring function in this young patient. There were no signs of implant loosening or lysis. Magnetic resonance imaging of the knee at last follow-up revealed that the cartilage thickness of the patient's lateral femoral condyle remained unchanged. Unicondylar hemiarthroplasty performed in patients with large unipolar lesions in the knee can provide durable and reliable pain relief. Ceramic is a viable material that can be considered for articulation with native cartilage.

Background: Accurate preoperative assessment of biliary anatomy in live donor hepatectomy may be helpful to assess the suitability of a graft and to stratify risk of biliary complications., Methods: A retrospective review of existing data among donor and recipients of 36 living donor transplants was performed to assess role of preoperative magnetic resonance cholangiography (MRC) for defining biliary anatomy and to stratify risk of biliary complications., Results: Thirty-six living liver donors underwent MRC, and subsequently right lobectomy. Intraoperative cholangiography and biliary exploration revealed that 24 donors (66.6%) had conventional and 12 (33.3%) had aberrant biliary anatomy. Intraoperative cholangiography demonstrated a strong correlation with MRC (P=0.001) and intraoperative findings (P=0.001). MRC had specificity and positive predictive value of 100%. The risk of developing biliary complication was 5.9 times higher if the biliary anatomy was of any type other than A (P=0.03, CI 1.06-32.9) after controlling for donor age, recipient age, and type of anastomosis., Conclusion: MRC reliably identified variant biliary anatomy. The preoperative MRC demonstrated congruence with the intraoperative cholangiogram and with the intraoperative findings. MRC is helpful in predicting risk of biliary complications in recipients, and identifies donors who would otherwise be excluded intraoperatively by cholangiography, thus limiting the risk of an unnecessary operation.

The Tec family kinase Itk is an important regulator of Ca(2+) mobilization and is required for in vivo responses to Th2-inducing agents. Recent data also implicate Itk in TCR-induced regulation of the actin cytoskeleton. We have evaluated the requirements for Itk function in TCR-induced actin polarization. Reduction of Itk expression via small interfering RNA treatment of the Jurkat human T lymphoma cell line or human peripheral blood T cells disrupted TCR-induced actin polarization, a defect that correlated with decreased recruitment of the Vav guanine nucleotide exchange factor to the site of Ag contact. Vav localization and actin polarization could be rescued by re-expression of either wild-type or kinase-inactive murine Itk but not by Itk containing mutations affecting the pleckstrin homology or Src homology 2 domains. Additionally, we find that Itk is constitutively associated with Vav. Loss of Itk expression did not alter gross patterns of Vav tyrosine phosphorylation but appeared to disrupt the interactions of Vav with SLP-76. Expression of membrane-targeted Vav, Vav-CAAX, can rescue the small interfering RNA to Itk-induced phenotype, implicating the alteration in Vav localization as directly contributing to the actin polarization defect. These data suggest a kinase-independent scaffolding function for Itk in the regulation of Vav localization and TCR-induced actin polarization.

XLP is caused by mutations affecting SAP, an adaptor that recruits Fyn to SLAM family receptors. SAP-deficient mice recapitulate features of XLP, including increased T cell activation and decreased humoral responses post-infection. SAP-deficient T cells also show increased TCR-induced IFN-gamma and decreased T(H)2 cytokine production. We demonstrate that the defect in IL-4 secretion in SAP-deficient T cells is independent of increased IFN-gamma production. SAP-deficient cells respond normally to polarizing cytokines, yet show impaired TCR-mediated induction of GATA-3 and IL-4. Examination of TCR signaling revealed normal Ca(2+) mobilization and ERK activation in SAP-deficient cells, but decreased PKC-theta recruitment, Bcl-10 phosphorylation, IkappaB-alpha degradation, and nuclear NF-kappaB1/p50 levels. Similar defects were observed in Fyn-deficient cells. SLAM engagement amplified PKC-theta recruitment in wt but not SAP- or Fyn-deficient cells, arguing that a SAP/Fyn-mediated pathway enhances PKC-theta/NF-kappaB1 activation and suggesting a role for this pathway in T(H)2 regulation.

Cell polarization and migration in response to chemokines is essential for proper development of the immune system and activation of immune responses. Recent studies of chemokine signaling have revealed a critical role for PI3-Kinase, which is required for polarized membrane association of pleckstrin homology (PH) domain-containing proteins and activation of Rho family GTPases that are essential for cell polarization and actin reorganization. Additional data argue that tyrosine kinases are also important for chemokine-induced Rac activation. However, how and which kinases participate in these pathways remain unclear. We demonstrate here that the Tec kinases Itk and Rlk play an important role in chemokine signaling in T lymphocytes. Chemokine stimulation induced transient membrane association of Itk and phosphorylation of both Itk and Rlk, and purified T cells from Rlk(-/-)Itk(-/-) mice exhibited defective migration to multiple chemokines in vitro and decreased homing to lymph nodes upon transfer to wt mice. Expression of a dominant-negative Itk impaired SDF-1alpha-induced migration, cell polarization, and activation of Rac and Cdc42. Thus, Tec kinases are critical components of signaling pathways required for actin polarization downstream from both antigen and chemokine receptors in T cells.

Beta-2-glycoprotein I (beta2GP1) has been implicated as the primary antigenic target in antiphospholipid syndrome. To study the role beta2GP1 antibodies play in thrombosis associated with this syndrome, the clearance and binding of phosphatidylserine (PS)-containing target membranes were monitored in beta2GP1-immune mice. Clearance in immune mice (T(1/2)4.8 min) was faster than in normal mice (T(1/2)11.0 min). Analysis of PS vesicles recovered from immune mice by sequencing and Western blotting showed the presence of bound beta2GP1 and autologous antibody, respectively. Bleeding times in immune mice were approximately 30% shorter than in control mice. In vitro clotting times, however, were the same in both populations. To determine if the in vivo results could be attributed to the interaction of autoantibodies with the vascular endothelium, the binding of PS-containing target membranes to normal and apoptotic endothelial cells was studied. While endothelial cells bound PS vesicles, beta2GP1 reduced uptake by approximately 50% in both normal and apoptotic endothelium. In the presence of beta2GP1 antibodies, however, uptake in apoptotic cells, but not normal cells, increased by more than two-fold. These results suggest that thrombosis in antiphospholipid syndrome could, in part, be due to antibody-dependent cross-linking of beta2GP1 bound to PS-expressing cells and the vascular endothelium., (Copyright 2000 Academic Press.)

The invasive ability of C. jejuni/coli strains isolated from children with diarrhea was studied using an in vitro HEp-2 cell invasion assay. The ratio between the number of intracellular bacteria and the number of bacteria in the inoculum was determined (invasion index). It was found that under anaerobic conditions, there was a significant decrease in the invasion index as compared to standard conditions (5% CO2). Of 11 strains tested, seven were determined as invasive on the basis of invasion indexes within the range of 0.0002-0.01. In a previous study [D. Schwartz et al., Zbl. Bakt. 280, 338-347 (1994)], it was found, that most of the C. jejuni/coli isolates tested produced an outer membrane protein when grown under conditions of iron depletion (IRP). The IRP were detected in eight of the nine strains tested in the present study (five invasive and three non-invasive strains). In one non-invasive strain, IRP was not detected. When kept under conditions of iron depletion, one of the invasive strains exhibited a significant increase in invasive capacity. The results suggest that iron depletion seems to stimulate the invasion capacity of C. jejuni/coli in vitro.

Human pancreatic alpha-amylase has been isolated using a glycogen affinity precipitation procedure and crystallized in a form suitable for high resolution three-dimensional X-ray crystallographic analyses. Crystals are of the orthorhombic space group P2(1)2(1)2(1), with unit cell dimensions of a = 53.04 A, b = 74.80 A and c = 137.34 A, and contain only one protein molecule per asymmetric unit. Diffraction data have been collected and found to extend to 1.6 A resolution. These studies form the basis for elucidating the full atomic structure of human pancreatic alpha-amylase and thereby providing insight into the catalytic mechanism of this enzyme.

In order to determine if prostaglandin values correlate with gestational age, birth weight, postnatal age, or respiratory distress syndrome (RDS), we determined plasma prostaglandin F2 alpha (PGF2 alpha) and prostaglandin E2 (PGE2) by specific radioimmunoassay in 34 samples from 27 preterm infants. Neither prostaglandin correlated with gestational age or with birth weight. PGF2 alpha decreased (p less than 0.3) with postnatal age. Values for PGF2 alpha and PGE2 in each sample varied together (p less than .01) but only PGF2 alpha increased (p less than .09) in infants with RDS. The highest PGF2 alpha values occurred in infants with severe RDS, including four infants with patent ductus arteriosus (PDA). In contrast, plasma PGE2 was not elevated in infants with RDS or PDA.

Ethidium fluorescence assays were adapted for the rapid and sensitive detection of precA; in addition, fluorescence measurements on binding precA to linear, OC and CCC PM2 DNAs have enabled the stoichiometry of precA binding as well as the precA-induced unwinding angle of DNA to be determined. The stoichiometry of binding was independently confirmed by sedimentation analysis to be one precA molecule per 3 bp. The unwinding angle was also independently confirmed by measurements of fluorescence changes induced by the binding of precA to CCC DNA which was relaxed by topoisomerase to give a precA-induced unwinding angle of 51 degrees. Electron microscopy of OC DNA molecules which bound nonsaturating amounts of precA revealed that the length increase in DNA due to precA was approximately 55%. Finally, examination of negatively stained precA complexes with a variety of linear DNAs showed that the minor groove is the primary site of interaction for this protein.

The stereochemical courses of the hydrolyses catalysed by three glycosidases have been determined directly by 1H nmr. The anomeric configuration of the initially formed product was ascertained in each case by observation of the chemical shift and coupling constant of the anomeric proton at the new hemiacetal centre. Two of the enzymes investigated, an endo-glucanase and an exo-glucanase are components of the cellulase complex of Cellulomonas fimi. The third enzyme is the beta-glucosidase from almond emulsin. Two of these enzymes, the exo-glucanase and the almond beta-glucosidase catalysed hydrolysis with retention of anomeric configuration, in agreement with previous observations on the almond enzyme. The endo-glucanase catalysed hydrolysis with inversion of configuration, this result being confirmed by optical rotation measurements. This 1H nmr approach has several advantages over other techniques in that it is applicable to a wide variety of glycosidases and substrates and it is non-destructive, allowing recovery of the enzyme.

Fab fragments from hybridoma HEd 10 [Lee, J. S., Lewis, J.R., Morgan, A.R., Mosmann, T.R., & Singh, B. (1981) Nucleic Acids Res. 9, 1707-1721] were prepared in large amounts by papain digestion of the immunoglobulin G (IgG) fraction from ascites fluid. Binding data were generated by a fluorescence quenching technique, and binding constants [K(0)] were estimated from Scatchard plots. The Fab fragments bound tightly to poly(dT) [K(0) = 12.7 X 10(6) M-1], and analysis of binding constants for the series p(dT)2 to p(dT)17 showed that the recognition sequence consisted of four consecutive residues. The effect of ionic strength on the interaction suggested that only two phosphates were involved. Binding constants for poly(dU), poly[d(brU)], poly[d(brC)], and poly(rU) were 1.0 X 10(6) M-1, 18.8 X 10(6) M-1, 0.5 X 10(6) M-1, and less than 0.5 X 10(6) M-1, respectively, implicating the involvement of the 3, 4, and 5 positions of the pyrimidine ring as well as the deoxyribose sugar in the recognition process. At high ionic strength (0.5 M) K(0) for whole IgG binding to poly(dT) was 75 X 10(6) M-1 compared to a value of 1.1 X 10(6) M-1 for the Fab fragment. These results may have implications for the tissue damage caused by DNA-containing immune complexes in systemic lupus erythematosus.