Inter-rater agreement on assessment of outcome within a trauma registry.

INTRODUCTION: To better evaluate the degree of ongoing disability in trauma patients, it has been recommended that trauma registries introduce routine long-term outcome measurement. One of the measures recommended for use is the Extended Glasgow Outcome Scale (GOS-E). However, few registries have adopted this measure and further research is required to determine its reliability with trauma populations. This study aimed to evaluate the inter-rater agreement of GOS-E scoring between an expert rater and trauma registry follow-up staff with a sample of detailed trauma case scenarios. METHODS: Sixteen trauma registry telephone interviewers participated in the study. They were provided with a written summary of 15 theoretical adult trauma cases covering a spectrum of disability and asked to rate each case using the structured GOS-E interview. Their ratings were compared with those of an expert rater in order to calculate the inter-rater agreement for each individual rater-expert rater pair. Agreement was reported as the percentage of agreement, the kappa statistic, and weighted kappa. A multi-rater kappa value was also calculated for agreement between the 16 raters. RESULTS: Across the 15 cases, the percentage of agreement between individual raters and the expert ranged from 63% to 100%. Across the 16 raters, the percentage of agreement with the expert rater ranged from 73-100% (mean=90%). Kappa values ranged from 0.65 to 1.00 across raters (mean=0.86) and weighted kappa values ranged from 0.73 to 1.00 (mean=0.89) The multi-rater kappa value was 0.78 (95% CI: 0.66, 0.89). CONCLUSIONS: Sixteen follow-up staff achieved 'substantial' to 'almost perfect' agreement with an expert rater using the GOS-E outcome measure to score 15 sample trauma cases. The results of this study lend support to the use of the GOS-E within trauma populations and highlight the importance of ongoing training where multiple raters are involved to ensure reliable outcome reporting. It is also recommended that the structured GOS-E interview guide be used to achieve better agreement between raters. Ensuring the reliability of trauma outcome scores will enable more accurate evaluation of patient outcomes, and ultimately, more targeted trauma care.

Chronic inhibition of mammalian target of rapamycin by rapamycin modulates cognitive and non-cognitive components of behavior throughout lifespan in mice.

Aging is, by far, the greatest risk factor for most neurodegenerative diseases. In non-diseased conditions, normal aging can also be associated with declines in cognitive function that significantly affect quality of life in the elderly. It was recently shown that inhibition of Mammalian TOR (mTOR) activity in mice by chronic rapamycin treatment extends lifespan, possibly by delaying aging {Harrison, 2009 #4}{Miller, 2011 #168}. To explore the effect of chronic rapamycin treatment on normal brain aging we determined cognitive and non-cognitive components of behavior throughout lifespan in male and female C57BL/6 mice that were fed control- or rapamycin-supplemented chow. Our studies show that rapamycin enhances cognitive function in young adult mice and blocks age-associated cognitive decline in older animals. In addition, mice fed with rapamycin-supplemented chow showed decreased anxiety and depressive-like behavior at all ages tested. Levels of three major monoamines (norepinephrine, dopamine and 5-hydroxytryptamine) and their metabolites (3,4-dihydroxyphenylacetic acid, homovanillic acid, and 5-hydroxyindolacetic acid) were significantly augmented in midbrain of rapamycin-treated mice compared to controls. Our results suggest that chronic, partial inhibition of mTOR by oral rapamycin enhances learning and memory in young adults, maintains memory in old C57BL/6J mice, and has concomitant anxiolytic and antidepressant-like effects, possibly by stimulating major monoamine pathways in brain.

Characterisation of orthopaedic trauma admitted to adult level 1 trauma centres.

BACKGROUND: Despite the vast number of traumatic injuries that are orthopaedic in nature, comprehensive epidemiological data that characterise orthopaedic trauma are limited. The aim of this study was to investigate the nature of orthopaedic trauma admitted to adult Level 1 Trauma Centres. METHODS: Data were obtained from the Victorian Orthopaedic Trauma Outcomes Registry (VOTOR), which includes all patients with orthopaedic trauma admitted to the two adult Level 1 Trauma Centres in Victoria (Australia). Information was collected from the medical record and hospital databases on patients' demographics and injury event, diagnoses and management. RESULTS: Data were analysed on 784 patients recruited between August 2003 and March 2004. Patients were mainly young (<65 years) (70.7%), male (59.1%) and injured in a transport collision (51.3%). Fractures of the femur (23.7%) and spine (23.5%) were the most common injuries and were predominately managed with operative (87.6%) and conservative (78.8%) methods, respectively. Differences in most parameters were evident between younger (<65 years) and older (> or =65 years) patients. CONCLUSIONS: This study presents epidemiological data on patients with orthopaedic trauma who were admitted to adult Level 1 Trauma Centres. This information is critical for the future monitoring and evaluation of the outcomes of orthopaedic trauma.

A prospective trial of computed tomography and ultrasonography for diagnosing appendicitis in the atypical patient.

BACKGROUND: The surgical diagnosis of acute appendicitis is customarily made on clinical grounds alone using history, physical examination, and white blood cell count. In the atypical patient, ie, the patient with prolonged symptoms, inconsistent history, or misleading physical examination, diagnostic studies should be helpful in establishing the appropriate diagnosis. Computed tomography (CT) scan and ultrasonography (US) have demonstrated utility in diagnosing appendicitis but have not been studied in the atypical patient population. METHODS: A retrospective review of 500 consecutive appendectomy patients, coupled with a review of the literature, resulted in the development of an algorithm to help the medical physician to differentiate the atypical patient requiring an imaging study from those requiring immediate surgical consultation. This patient population was entered into a prospective, randomized study of CT scan or US examination. RESULTS: Between May 1997 and May 1999, 106 patients were enrolled in the study; 17 were later excluded from the study because of typical presentation and direct admission to surgery without diagnostic imaging. Forty-nine patients were randomly assigned to CT scan and 40 to US examination. CT scan was 100% specific and 97% sensitive (P = 0.018). US was 90% specific and 76% sensitive. Based on the results of these imaging studies, 70 of the study patients underwent exploration for suspected appendicitis or other acute inflammatory process. CONCLUSION: A subset of patients presenting with possible acute appendicitis has been identified that should benefit from imaging of the appendix prior to surgical consultation. For this group, CT scan appears superior to abdominal and pelvic US in terms of diagnostic accuracy and reliability. With this approach, a low incidence of negative laparotomies was achieved.

Modulation of HIV-1 replication by a novel RhoA effector activity.

The RhoA GTPase is involved in regulating actin cytoskeletal organization, gene expression, cell proliferation, and survival. We report here that p115-RhoGEF, a specific guanine nucleotide exchange factor (GEF) and activator of RhoA, modulates HIV-1 replication. Ectopic expression of p115-RhoGEF or Galpha13, which activates p115-RhoGEF activity, leads to inhibition of HIV-1 replication. RhoA activation is required and the inhibition affects HIV-1 gene expression. The RhoA effector activity in inhibiting HIV-1 replication is genetically separable from its activities in transformation of NIH3T3 cells, activation of serum response factor, and actin stress fiber formation. These findings reveal that the RhoA signal transduction pathway regulates HIV-1 replication and suggest that RhoA inhibits HIV-1 replication via a novel effector activity.

Functional interaction between the cytoplasmic leucine-zipper domain of HIV-1 gp41 and p115-RhoGEF.

The long cytoplasmic tail of the human immunodeficiency virus (HIV)-1 transmembrane protein gp41 (gp41C) is implicated in the replication and cytopathicity of HIV-1 [1]. Little is known about the specific functions of gp41C, however. HIV-1 or simian immunodeficiency virus (SIV) mutants with defective gp41C have cell-type- or species-dependent phenotypes [2] [3] [4] [5] [6]. Thus, host factors are implicated in mediating the functions of gp41C. We report here that gp41C interacted with the carboxy-terminal regulatory domain of p115-RhoGEF [7], a specific guanine nucleotide exchange factor (GEF) and activator of the RhoA GTPase, which regulates actin stress fiber formation, activation of serum response factor (SRF) and cell proliferation [8] [9]. We demonstrate that gp41C inhibited p115-mediated actin stress fiber formation and activation of SRF. An amphipathic helix region with a leucine-zipper motif in gp41C is involved in its interaction with p115. Mutations in gp41C leading to loss of interaction with p115 impaired HIV-1 replication in human T cells. These findings suggest that an important function of gp41C is to modulate the activity of p115-RhoGEF and they thus reveal a new potential anti-HIV-1 target.

Importance of surgical resection in the successful management of soft tissue sarcoma.

HYPOTHESIS: A systemic disease-free state necessitates a local disease-free state. This cannot be accomplished without a properly performed resection by an experienced surgical team. Successful local management of soft tissue sarcoma (STS) may lead to improved disease-free survival. An STS treatment protocol using wide local excision followed by radiation therapy is effective in achieving local tumor control and survival similar to that of multiple-modality regimens, but with lower morbidity. DESIGN: Retrospective cohort review (August 1, 1987, to May 6, 1998). SETTING: Referral to a single musculoskeletal oncologic surgeon, with surgery performed at a tertiary care medical center in a large urban area. PATIENTS: Ninety patients with STS of the trunk or extremities. INTERVENTIONS: Preoperative evaluation included surveillance computed tomographic scan of the chest, magnetic resonance imaging of primary site to assess tumor extent and to plan the surgical approach, and angiography if vascular bypass was proposed. Wide local excision of tumor was performed, with concomitant vascular bypass and/or complex plastic reconstruction as needed. Postoperative radiation therapy was given in most patients. Adjuvant chemotherapy was used selectively. MAIN OUTCOME MEASURES: Morbidity, local recurrence rates, and survival. RESULTS: Histologically negative margins were obtained in 89 (99%) of 90 patients; 86 (96%) remained free of local disease at follow-up. Five patients died of systemic metastatic disease. CONCLUSION: Excellent local control obtained with aggressive, appropriate surgery followed by radiation therapy in most patients and chemotherapy in only selective high-risk patients leads to excellent survival, with low morbidity and good functional outcome.

Prospective evaluation of formalin therapy for radiation proctitis.

BACKGROUND: Radiation proctitis is a troublesome complication of radiation therapy for as many as 75% of patients after pelvic irradiation. Five percent progress to chronic radiation proctitis complicated by telangiectasias and hemorrhage. The utility of formalin rectal instillation for treatment of bleeding is prospectively evaluated in this study. METHODS: Eleven patients (9 male, 2 female) with rectal bleeding after pelvic irradiation were treated with formalin therapy. In a single treatment, 4% formalin was instilled into the rectum in four separate 20-cc aliquots with total mucosal contact time of approximately 15 minutes. Patients were initially evaluated at 7 to 10 days and 1 month postoperatively and assessed for bleeding. RESULTS: All patients presented with rectal bleeding. Twenty-seven percent required transfusion. Thirty-six percent had failed other previous therapy. In follow-up of 3 to 64 months, 100% had initial success with cessation of bleeding. Three patients had recurrent bleeding; none required transfusion. One patient required repeat formalin instillation, with no further bleeding at 3 months follow-up. CONCLUSION: Local rectal instillation of 4% formalin is an efficacious therapy for treatment of radiation-induced lower gastrointestinal bleeding.

In vitro release kinetics of gentamycin from a sodium hyaluronate gel delivery system suitable for the treatment of peripheral vestibular disease.

For certain patients who experience intense vertigo arising from unilateral vestibular lesions, the primary therapy is a vestibular nerve section, an intracranial surgical procedure. One alternative to this treatment is therapeutic ablation of vestibular function on the unaffected side using an ototoxic agent. We prepared a biodegradable sustained-release gel delivery system using sodium hyaluronate that can be administered into the middle ear using only a local anesthetic. The gel contains gentamycin sulfate, the ototoxic agent of choice for treatment of unilateral vestibulopathy, and it exhibits diffusion-controlled release of the drug over a period of hours. The released gentamycin could then diffuse into the inner ear through the round membrane. This represents an important advance over previous formulations, which used only gentamycin sulfate solutions, in that it should allow more careful control of the dose, it should reduce loss of the drug from the middle ear site, and it should maintain intimate contact with the round membrane. By carefully controlling the dose, it should be possible to inhibit vestibular function while minimizing hearing loss. Herein we describe the in vitro release kinetics of gentamycin sulfate from sodium hyaluronate gels and find that the system obeys Fickian behavior.

p115 RhoGEF, a GTPase activating protein for Galpha12 and Galpha13.

Members of the regulators of G protein signaling (RGS) family stimulate the intrinsic guanosine triphosphatase (GTPase) activity of the alpha subunits of certain heterotrimeric guanine nucleotide-binding proteins (G proteins). The guanine nucleotide exchange factor (GEF) for Rho, p115 RhoGEF, has an amino-terminal region with similarity to RGS proteins. Recombinant p115 RhoGEF and a fusion protein containing the amino terminus of p115 had specific activity as GTPase activating proteins toward the alpha subunits of the G proteins G12 and G13, but not toward members of the Gs, Gi, or Gq subfamilies of Galpha proteins. This GEF may act as an intermediary in the regulation of Rho proteins by G13 and G12.

Direct stimulation of the guanine nucleotide exchange activity of p115 RhoGEF by Galpha13.

Signaling pathways that link extracellular factors to activation of the monomeric guanosine triphosphatase (GTPase) Rho control cytoskeletal rearrangements and cell growth. Heterotrimeric guanine nucleotide-binding proteins (G proteins) participate in several of these pathways, although their mechanisms are unclear. The GTPase activities of two G protein alpha subunits, Galpha12 and Galpha13, are stimulated by the Rho guanine nucleotide exchange factor p115 RhoGEF. Activated Galpha13 bound tightly to p115 RhoGEF and stimulated its capacity to catalyze nucleotide exchange on Rho. In contrast, activated Galpha12 inhibited stimulation by Galpha13. Thus, p115 RhoGEF can directly link heterotrimeric G protein alpha subunits to regulation of Rho.

Downregulation of beta-catenin by human Axin and its association with the APC tumor suppressor, beta-catenin and GSK3 beta.

BACKGROUND: Inactivation of the adenomatous polyposis coli (APC) tumor suppressor protein is responsible for both inherited and sporadic forms of colon cancer. Growth control by APC may relate to its ability to downregulate beta-catenin post-translationally. In cancer, mutations in APC ablate its ability to regulate beta-catenin, and mutations in beta-catenin prevent its downregulation by wild-type APC. Moreover, signaling by the protein product of the wnt-1 proto-oncogene upregulates beta-catenin and promotes tumorigenesis in mice. In a Xenopus developmental system, Wnt-1 signaling was inhibited by Axin, the product of the murine fused gene. This suggests a possible link between Axin, the Wnt-1 signaling components beta-catenin and glycogen synthase kinase 3 beta (GSK3 beta), and APC. RESULTS: Human Axin (hAxin) binds directly to beta-catenin, GSK3 beta, and APC in vitro, and the endogenous proteins are found in a complex in cells. Binding sites for Axin were mapped to a region of APC that is typically deleted due to cancer-associated mutations in the APC gene. Overexpression of hAxin strongly promoted the downregulation of wild-type beta-catenin in colon cancer cells, whereas mutant oncogenic beta-catenin was unaffected. The downregulation was increased by deletion of the APC-binding domain from Axin, suggesting that APC may function to derepress Axin activity. In addition, hAxin dramatically facilitated the phosphorylation of APC and beta-catenin by GSK3 beta in vitro. CONCLUSIONS: Axin acts as a scaffold upon which APC, beta-catenin and GSK3 beta assemble to coordinate the regulation of beta-catenin signaling.

Evidence for distinct mechanisms of transition state stabilization of GTPases by fluoride.

GTPase-activating proteins (GAPs) function by stabilizing the GTPase transition state. This has been most clearly demonstrated by the formation of a high-affinity complex between various GAPs and GDP-bound GTPases in the presence of aluminum tetrafluoride, which can mimic the gamma-phosphate of GTP. Herein, we report that p190 RhoGAP forms a high-affinity complex with Rho GTPases in the presence of fluoride ions, suggesting that p190 also functions to stabilize the GTPase transition state. However, this Rho-p190 complex does not require aluminum ions or even guanine nucleotide, indicating a distinct role for fluoride that is not consistent with the gamma-phosphate-mimicking hypothesis. These results indicate that it is necessary to reconsider the assumed role of fluoride in stabilizing a variety of other GTPase-GAP interactions where the requirement for aluminum or guanine nucleotide has not yet been addressed.

Identification of an actin cytoskeletal complex that includes IQGAP and the Cdc42 GTPase.

The Rho subfamily of low molecular weight GTPases have been implicated in a variety of cellular functions that include reorganization of the actin cytoskeleton and stress-induced activation of the c-Jun kinase. The downstream targets that mediate the effects of Cdc42 on the actin cytoskeleton have yet to be fully identified. We have used the transient transfection of COS-7 cells with epitope-tagged Cdc42 to identify candidate signaling partners for this GTPase and identified the IQGAP protein as a major in vivo target for activated Cdc42. Epidermal growth factor stimulation of serum-starved COS-7 cells promoted the formation of a Cdc42-IQGAP complex, indicating that growth factors can increase the pool of activated Cdc42. Activated HA-Cdc42 co-localized with IQGAP or F-actin in vivo, whereas cells transfected with dominant-negative forms of Cdc42 (Cdc42(T17N)) showed predominantly dispersed distributions for both HA-Cdc42 and endogenous IQGAP. In detergent lysates from COS-7 cells transiently transfected with different forms of Cdc42, or from stably transfected CHO cells, the induction of actin polymerization by phalloidin resulted in the incorporation of both IQGAP and Cdc42 into actin-containing complexes. Taken together, these findings are consistent with a model whereby IQGAP serves as a target for GTP-bound Cdc42 providing a direct link between the activated GTPase and the actin cytoskeleton.

Calmodulin modulates the interaction between IQGAP1 and Cdc42. Identification of IQGAP1 by nanoelectrospray tandem mass spectrometry.

Calmodulin regulates numerous fundamental metabolic pathways by binding to and modulating diverse target proteins. In this study, calmodulin-binding proteins were isolated from normal (Hs578Bst) and malignant (MCF-7) human breast cell lines with calmodulin-Sepharose and analyzed by SDS-polyacrylamide gel electrophoresis. A protein that migrated at approximately 190 kDa bound to calmodulin in the presence of Ca2+ and was the only calmodulin-binding protein detected in the absence of Ca2+. This 190-kDa protein was identified as IQGAP1 by nanoelectrospray mass spectrometry and collision-induced dissociation tandem mass spectrometry. IQGAP1 coimmunoprecipitated with calmodulin from lysates of MCF-7 cells. Moreover, overlay with 125I-calmodulin confirmed that IQGAP1 binds directly to calmodulin. Analysis of the functional effects of the interaction revealed that Ca2+/calmodulin disrupted the binding of purified IQGAP1 to the Ras-related protein Cdc42 in a concentration-dependent manner. These data clearly identify IQGAP1 as the predominant calmodulin-binding protein in Ca2+-free breast cell lysates and reveal that calmodulin modulates the interaction between IQGAP1 and Cdc42.

IQGAP1, a Rac- and Cdc42-binding protein, directly binds and cross-links microfilaments.

Activated forms of the GTPases, Rac and Cdc42, are known to stimulate formation of microfilament-rich lamellipodia and filopodia, respectively, but the underlying mechanisms have remained obscure. We now report the purification and characterization of a protein, IQGAP1, which is likely to mediate effects of these GTPases on microfilaments. Native IQGAP1 purified from bovine adrenal comprises two approximately 190-kD subunits per molecule plus substoichiometric calmodulin. Purified IQGAP1 bound directly to F-actin and cross-linked the actin filaments into irregular, interconnected bundles that exhibited gel-like properties. Exogenous calmodulin partially inhibited binding of IQGAP1 to F-actin, and was more effective in the absence, than in the presence of calcium. Immunofluorescence microscopy demonstrated cytochalasin D-sensitive colocalization of IQGAP1 with cortical microfilaments. These results, in conjunction with prior evidence that IQGAP1 binds directly to activated Rac and Cdc42, suggest that IQGAP1 serves as a direct molecular link between these GTPases and the actin cytoskeleton, and that the actin-binding activity of IQGAP1 is regulated by calmodulin.

Radioimmunoguided surgery for colorectal cancer.

BACKGROUND: Improving the survival rate of surgical treatments of cancer depends upon accurate staging of disease and the subsequent ability to completely resect the lesions. Radioimmunoguided surgery (RIGS) has been employed in the treatment of colon and rectal cancer, and a multicenter trial is under way. METHODS: Twenty-eight patients with either primary or recurrent colorectal cancer were injected with the monoclonal antibody CC49 labeled with iodine 125. A hand-held gamma-detecting probe was used intraoperatively to detect radiolabeled antibody. At surgery the patients underwent both traditional and RIGS explorations. RESULTS: RIGS detected radiolabeled antibody in 73% (14 of 19) of patients with primary colorectal cancer and in 100% (7 of 7) of patients with recurrent disease. Based on RIGS, 26% (5 of 19) of patients with primary cancer were upstaged from stage I/II to stage III/IV. CONCLUSIONS: Although the final results of this phase III trial are incomplete, RIGS appears to provide immediate, valuable information that is not available from more conventional methods.

Identification of a novel guanine nucleotide exchange factor for the Rho GTPase.

The Rho GTPase promotes proliferation and cytoskeletal rearrangements in mammalian cells. To understand the regulation of Rho, it is important to characterize guanine nucleotide exchange factors (GEFs), which stimulate the dissociation of GDP and subsequent binding of GTP. Using Rho as an affinity ligand, we have isolated a 115-kDa protein (p115-RhoGEF) that binds specifically to the nucleotide-depleted state. A full-length cDNA encoding p115-RhoGEF was isolated, and its protein product, which exhibited sequence homology to Dbl and Lbc, catalyzed the exchange of GDP for GTP specifically on Rho and not on the Rac, Cdc42, or Ras GTPases. p115-RhoGEF is capable of regulating cell proliferation, as determined by its ability to induce the transformation of NIH 3T3 cells. Northern and Western analysis suggests that p115-RhoGEF is ubiquitously expressed. These results indicate that p115-RhoGEF may be a general regulator of Rho and its associated cellular phenotypes.

IQGAP1, a calmodulin-binding protein with a rasGAP-related domain, is a potential effector for cdc42Hs.

Proteins that associate with the GTP-bound forms of the Ras superfamily of proteins are potential effector targets for these molecular switches. A 195 kDa protein was purified from cell lysates by affinity chromatography on immobilized cdc42Hs-GTP and a corresponding cDNA was isolated. Sequence analysis revealed localized identities to calponin, the WW domain, unconventional myosins and to the rasGAP-related domain (GRD) contained in IRA, NF-1, SAR1 and rasGAP. p195 was found to be identical to IQGAP1, a protein previously reported to bind ras. Purified recombinant p195/IQGAP1 bound to and inhibited the GTPase activity of cdc42Hs and rac whereas no interaction with ras was detected. The C-terminal half of IQGAP1 containing the GRD bound to cdc42 and rac in a GRD-dependent fashion, but a smaller fragment containing only the GRD did not. Cdc42 was also co-immunoprecipitated from cell lysates with antibody specific to p195/IQGAP1. Calmodulin also co-immunoprecipitated with p195/IQGAP1 and was found to associate with fragments containing the IQ domain. Expression of a cDNA fragment encoding the GRD inhibited the CDC24/CDC42 pathway in yeast, but no effect on ras was observed. In mammalian cells, both endogenous and ectopically expressed p195/IQGAP1 were localized to lamellipodia and ruffling cell membranes, where co-localization with actin was apparent. These results suggest that IQGAP1 is an effector target for cdc42Hs and may mediate the effects of this GTPase on cell morphology.