The prevalence and impact of respiratory symptoms in asthmatics and nonasthmatics during deployment

OBJECTIVE: The purpose of this study was to compare the prevalence, severity, and impact of respiratory symptoms in asthmatics and nonasthmatics during Operation Enduring Freedom and Operation Iraqi Freedom. METHODS: A survey was given to 1,250 active duty soldiers and Department of Defense contractors returning from Operation Enduring Freedom/Operation Iraqi Freedom. Subjects were asked about demographics, smoking habits, respiratory symptoms, and impact on job performance before and during deployment. Patients with a history of asthma were asked method of diagnosis, current symptoms, and asthma therapy. RESULTS: A total of 1,193 subjects returned the completed questionnaire (95% response rate). Mean age of respondents was 38 +/- 11 years, 83% (n = 977) were male, and 31% (n = 375) were past or present smokers. Sixty-one subjects (5%) reported a previous diagnosis of asthma. Both asthmatics and nonasthmatics had increased respiratory symptoms of wheezing, cough, sputum production, chest pain/tightness, and allergy symptoms during deployment compared to predeployment (p < 0.05 for all). When compared to nonasthmatics, asthmatic subjects reported more wheezing, sputum production, and chest pain/tightness during deployment (p < 0.0001, 0.05, 0.05 respectively), had more difficulty with military duties (p < 0.05), and were more likely to seek medical attention and receive duty restrictions (p < 0.0001). Twenty-six percent (n = 16) of asthmatics reported poor baseline symptom control, and this group had significantly increased symptoms, functional limitations, and health care utilization when compared to asthmatics who were symptom-controlled at baseline. CONCLUSIONS: Respiratory symptoms were common among both asthmatics and nonasthmatics during deployment. Differences in symptoms and health care utilization in this group of asthmatics were primarily due to subjects with poor baseline control.     

Dislocation of the proximal tibiofibular joint in association with a tibial shaft fracture: two case reports and a literature review

Dislocation of the proximal tibiofibular joint (PTFJ) in association with a displaced tibial shaft fracture and an intact fibula is an exceedingly rare injury. We present 2 cases of tibia fractures associated with an intact fibula and a PTFJ dislocation. The first case involves a man who sustained a closed spiral fracture of the distal tibial shaft, with an intact fibula, an anterolaterally dislocated PTFJ, and a partial tear of the lateral collateral ligament. The tibia was percutaneously plated, and the PTFJ was reduced and then stabilized with temporary screw fixation. The second case involves a woman who sustained a closed fracture of the tibia in association with a PTFJ dislocation. The tibia was fixed with an intramedullary nail, and the PTFJ was similarly reduced and fixed with a temporary screw. We also provide a brief literature review focusing on classification of PTFJ dislocations, mechanism of injury, associated injuries, and treatment options.     

Multiplexed antibody arrays for the discovery and validation of glycosylated protein biomarkers

Protein glycosylation, the enzymatic linkage of mono- and poly-saccharides to proteins, is a critical determinant of protein function; however, there is a lack of tools for studying the glycosylation of specific proteins in complex samples. A new type of antibody-lectin sandwich assay enables the measurement of the glycosylation of specific proteins that have been captured from complex samples using antibody arrays combined with lectin-based detection probes. Antibody-lectin sandwich arrays have the potential to expand our understanding of the role of glycans and protein glycosylation in disease and to identify and investigate new biomarkers for early detection, disease prognosis and therapeutic response prediction. While antibody-lectin sandwich arrays yield less-detailed structural information regarding protein glycosylation than other available methods, they do provide a simple and reproducible method for investigating changes in protein abundance and glycosylation of multiple proteins and can be easily applied to large or small sample sets. By profiling protein and glycan variations, new disease-associated glycan alterations can be identified and validated for use as biomarkers.     

Coumarin-based inhibitors of human NAD(P)H:quinone oxidoreductase-1. Identification, structure-activity, off-target effects and in vitro human pancreatic cancer toxicity

The enzyme human NAD(P)H quinone oxidoreductase-1 (NQO1), which is overexpressed in several types of tumor cell, is considered a design target for cancer therapeutics. We identify new coumarin-based competitive inhibitors of NQO1, one of which is nanomolar. Using computational docking and molecular dynamics, we obtain insights into the structural basis of inhibition. Selected inhibitors were then assessed for off-target effects associated with dicoumarol and were found to have differing effects on superoxide formation and mitochondrial respiration. A comparison of NQO1 inhibition and off-target effects for dicoumarol and its derivatives suggests that the ability of dicoumarol to kill cancer cells is independent of NQO1 inhibition, that cellular superoxide production by dicoumarol does not seem linked to NQO1 inhibition but may be related to mitochondrial decoupling, and that superoxide does not appear to be a major determinant of cytotoxicity. Implications are discussed for NQO1 inhibition as an anticancer drug design target and superoxide generation as the dicoumarol-mediated mechanism of cytotoxicity.     

Chemotherapy for colorectal cancer prior to liver resection for colorectal cancer hepatic metastases does not adversely affect peri-operative outcomes

BACKGROUND: Systemic chemotherapy is being used increasingly in patients with colorectal cancer. The effects of prior systemic adjuvant or palliative chemotherapy on morbidity following hepatic resection for metastases are not well defined. OBJECTIVES: To assess the peri-operative impact of systemic chemotherapy on liver resection for colorectal cancer hepatic metastases. METHODS: Ninety-six resections for colorectal cancer hepatic metastases performed from July 2001 to July 2003 (93% > or =2 segments) were reviewed. Pre-operative demographics, peri-operative features, and post-operative outcomes were collected prospectively. Type of chemotherapy and the temporal relationship of chemotherapy to the liver resection were analyzed. RESULTS: Fifty-three of 96 patients (55%) received a mean of 5.7 cycles (6.1 months) of systemic chemotherapy prior to hepatic resection, with a median interval of 12 months from end of chemotherapy to liver resection (range 1-75 months). Thirty-five received 5-fluorouracil/leucovorin (5-FU/LV) alone, nine had irinotecan (CPT-11) in addition to 5-FU/LV, and nine were not specified. Pre-operative age, sex, co-morbidities, ASA score, biochemical and liver enzyme profiles, tumor number, and extent and technique of hepatic resection were the same in the chemotherapy and non-chemotherapy cohorts. Mean tumor size was smaller (4.5 cm vs. 5.8 cm) and synchronous metastases were half as common (25% vs. 49%) in the chemotherapy group. Liver resection operative time was equivalent (270 min) in the two groups. Higher estimated blood loss (EBL) (1,000 ml vs. 850 ml), but fewer transfusions (23% vs. 15%) were associated with the chemotherapy group. Although not statistically significant, post-operative liver enzyme peaks were higher in the chemotherapy group (AST = 402 U/L vs. 302 U/L, P = 0.09 and ALT = 433 U/L vs. 312 U/L, P = 0.1). Peak changes in INR and serum bilirubin did not differ. Complications and length of stay (LOS) did not differ between the groups. The only post-operative death was in the non-chemotherapy group. Interestingly, hepatic steatosis was present in 28% of the non-chemotherapy cases and 57% of the chemotherapy resection specimens (P = 0.005) and was marked (>30%) in 7% and 10%, respectively. Further analysis of the chemotherapy group based on the interval between completion of chemotherapy and the hepatic resection (<6 months, 7-12 months, 1-2 years, and >2 years) revealed a trend towards worse outcomes in most categories for those in the >2 years cohort. When comparing the 5-FU/LV alone, to the CPT-11 group there were no significant differences except higher intra-operative blood loss in the group receiving 5-FU/LV alone (1,295 ml vs. 756 ml, P = 0.01). CONCLUSION: Despite variations in biochemical function and hepatic steatosis, short-term clinical outcomes are not affected by the administration of chemotherapy prior to hepatic resection. Furthermore, there is no detrimental effect of close timing of chemotherapy prior to resection, and there are no appreciable differences between irinotecan containing regimes and more traditional 5-FU-only based therapies, although the subset sample sizes were small in this study.     

Rapid identification of heterozygous mutations in Drosophila melanogaster using genomic capture sequencing

One of the key advantages of using Drosophila melanogaster as a genetic model organism is the ability to conduct saturation mutagenesis screens to identify genes and pathways underlying a given phenotype. Despite the large number of genetic tools developed to facilitate downstream cloning of mutations obtained from such screens, the current procedure remains labor intensive, time consuming, and costly. To address this issue, we designed an efficient strategy for rapid identification of heterozygous mutations in the fly genome by combining rough genetic mapping, targeted DNA capture, and second generation sequencing technology. We first tested this method on heterozygous flies carrying either a previously characterized dac⁵ or sens^E2 mutation. Targeted amplification of genomic regions near these two loci was used to enrich DNA for sequencing, and both point mutations were successfully identified. When this method was applied to uncharacterized twr mutant flies, the underlying mutation was identified as a single-base mutation in the gene Spase18-21. This targeted-genome-sequencing method reduces time and effort required for mutation cloning by up to 80% compared with the current approach and lowers the cost to <$1000 for each mutant. Introduction of this and other sequencing-based methods for mutation cloning will enable broader usage of forward genetics screens and have significant impacts in the field of model organisms such as Drosophila.The availability of genetic tools in Drosophila to generate, screen, and characterize mutations with phenotypes of interest makes it one of the most powerful genetic model organisms. One of the most commonly used mutagens, ethyl methane sulfonate (EMS), has been widely used in genetic screens, and mutations identified in more than 3000 genes have been recorded in FlyBase (http://flybase.org/). In order to clone an EMS or other chemical mutagen-induced novel mutation, it is often necessary to reduce the candidate locus to a small interval, typically of ∼20 kb or less in size, by fine genetic mapping (H Bellen, pers. comm.). However, due to the large number of flies that must be scored in order to achieve such resolution, the fine mapping step is quite labor intensive and costly. Additionally, for some genomic regions, it is not always possible to map a mutation to a small interval due to limitations such as low recombination rates and lack of mapping stocks.Several molecular methods have been proposed for cloning EMS mutations without the need for genetic mapping. One method, named TILLING, is based on Cel-I-mediated heteroduplex DNA cleavage and can be used to identify changes in genes of interest between the wild-type and mutant flies (Winkler et al. 2005). It is ideal for isolating alleles of given genes from a large collection of EMS-induced mutants; however, this method is not well suited for identifying mutations obtained from forward genetic screens, since candidate genes need to be predefined for testing using TILLING. Another approach of mutation cloning is to take advantage of the recent development of second-generation sequencing technology. Rapid progress in sequencing technologies has dramatically increased the throughput and reduced the cost of DNA sequencing (Mardis 2008a,b; Shendure and Ji 2008; Ansorge 2009). Recently, identification of homozygous mutations by direct whole-genome sequencing has been reported (Sarin et al. 2008; Smith et al. 2008; Srivatsan et al. 2008; Blumenstiel et al. 2009). Mutations in genes that are important for development, however, are often homozygous lethal, and it is necessary to detect heterozygous mutations. The detection of heterozygous mutations requires much more sequencing coverage (Bentley et al. 2008; Ley et al. 2008; Wheeler et al. 2008). We estimate that about 30× sequencing coverage is necessary to detect heterozygous mutations with high sensitivity (>95%) and accuracy (error rate <10⁻⁶) (Supplemental Fig. 1). At the current cost of about $1500 for every 10× sequencing coverage of the Drosophila genome, the direct whole-genome sequencing approach is still too expensive for broad usage. This approach is further limited, as parental fly strains and multiple alleles often need to be sequenced in parallel to distinguish single nucleotide polymorphisms (SNPs) and other changes from causative mutations.When a cloning by sequencing approach is used, rough genetic mapping of the mutation is often highly valuable, as it can greatly reduce downstream data analysis efforts. Since approximately one base mutation is induced per 400 kb when flies are treated with 25 mM EMS, a commonly used experimental condition, a total of about 400 base changes exist in each mutant fly (Cooper et al. 2008; Blumenstiel et al. 2009). Mapping of the mutation can greatly reduce the number of candidate changes that need to be analyzed. Although fine genetic mapping is quite labor intensive, intermediate levels of genetic mapping where a mutation is mapped to within a few megabases (Mb) can be achieved efficiently using methods such as P element or SNP mapping in Drosophila (Zhai et al. 2003; Chen et al. 2008). Once a mutation is mapped to an interval of several megabases or less, it is then necessary to sequence only the candidate region (often <1% of the genome) instead of the entire genome, resulting in significant cost reduction. Recently, several DNA enrichment methods have been developed that enable enrichment of DNA from targeted regions (Albert et al. 2007; Dahl et al. 2007; Porreca et al. 2007; Gnirke et al. 2009; Ng et al. 2009; Okou et al. 2009). With optimized probe design and capture conditions, the vast majority of a targeted region (>95%) can be efficiently enriched (H Wang and R Chen, unpubl.). Among the numerous methods for targeted DNA enrichment, padlock capture, which relies on a combination of oligonucleotide hybridization and enzymatic activity, shows the greatest specificity (Bau et al. 2009; Li et al. 2009b). As shown in Figure 1A, a pool of probes that contain target-specific capturing arms can be annealed to the target regions. DNA synthesis using the target regions as templates will generate circular DNA, which is selectively amplified by PCR using common sequences flanking the capture arms as primers. A key feature of padlock probes is that they can be regenerated by PCR, thereby greatly reducing costs when multiple enrichments are needed, as in the case of sequencing complementing mutations of the same locus.Open in a separate windowFigure 1.Two known mutations were detected using padlock capture sequencing. (A) Flowchart of mutation detection by padlock capture and next-generation sequencing technologies. Padlock capture technology requires probes that contain two target-specific capturing arms (green) connected by a common linker (red). The unique targeting arms of individual targeting oligonucleotides are designed to hybridize immediately upstream and downstream from each exon (purple bars) of interest. Hybridization to genomic DNA is followed by an enzymatic gap filling and ligation step, such that a copy of the sequence of interest is incorporated into a circle (purple dashed line). Then the enriched DNA is PCR amplified and is used to prepare libraries for the next-generation platform sequencing. (B) sens^E2 and (C) dac⁵ mutation detection. Reads alignment at the mutation is shown between the vertical lines. Each read is drawn as a gray line, and bases that are different from the reference are colored. Sanger sequencing is conducted to confirm the mutation with the heterozygous mutated base indicated by the arrow.To test the feasibility of identifying heterozygous mutations by targeted genome sequencing, we first applied the proposed method to two known mutations, dac⁵ and sens^E2. Exons within 0.5 Mb flanking the two known mutations were enriched and sequenced using both the 454 Life Sciences (Roche) and the Illumina GAII systems. We found that the causative point mutations could be reliably identified by either platform using only a small fraction of each platform''s capacity. Next, we applied this method to uncover the mutation in a previously uncharacterized mutant, twr¹, and identified a missense mutation in the Spase18-21 gene. Two lines of evidence support that the identified mutation in Spase18-21 is indeed the causative mutation for the twr phenotype. First, we identified mutations in Spase18-21 in two additional twr mutant alleles, twr² and twr¹¹. Second, a P-element insertion that fails to complement the twr¹ mutant allele maps to the 5′ end of the Spase18-21 gene. In summary, we conclude that combining rough genetic mapping, DNA targeted capture, and second generation sequencing is highly cost effective and immediately applicable to mutation cloning in Drosophila, which will enable broader usage of the forward genetic screen.     

Fibrinogen Regulates the Cytotoxicity of Mycobacterial Trehalose Dimycolate but Is Not Required for Cell Recruitment,Cytokine Response,or Control of Mycobacterial Infection

During inflammatory responses and wound healing, the conversion of soluble fibrinogen to fibrin, an insoluble extracellular matrix, long has been assumed to create a scaffold for the migration of leukocytes and fibroblasts. Previous studies concluded that fibrinogen is a necessary cofactor for mycobacterial trehalose 6,6′-dimycolate-induced responses, because trehalose dimycolate-coated beads, to which fibrinogen was adsorbed, were more inflammatory than those to which other plasma proteins were adsorbed. Herein, we investigate roles for fibrin(ogen) in an in vivo model of mycobacterial granuloma formation and in infection with Mycobacterium tuberculosis, the causative agent of tuberculosis. In wild-type mice, the subcutaneous injection of trehalose dimycolate-coated polystyrene microspheres, suspended within Matrigel, elicited a pyogranulomatous response during the course of 12 days. In fibrinogen-deficient mice, neutrophils were recruited but a more suppurative lesion developed, with the marked degradation and disintegration of the matrix. Compared to that in wild-type mice, the early formation of granulation tissue in fibrinogen-deficient mice was edematous, hypocellular, and disorganized. These deficiencies were complemented by the addition of exogenous fibrinogen. The absence of fibrinogen had no effect on cell recruitment or cytokine production in response to trehalose dimycolate, nor was there a difference in lung histopathology or overall bacterial burden in mice infected with Mycobacterium tuberculosis. In this model, fibrin(ogen) was not required for cell recruitment, cytokine response, or response to infection, but it promoted granulation tissue formation and suppressed leukocyte necrosis.Fibrinogen is an approximately 340-kDa circulating glycoprotein, a heterotrimer of Aα, Bβ, and γ chains (35). This plasma protein is produced primarily in the liver by hepatocytes, with strong upregulation in response to proinflammatory agents (16, 20, 35). The role of fibrinogen in the coagulation cascade is well characterized, with the cleavage of fibrinogen by thrombin to form fibrin, which then is covalently cross-linked by activated Factor XIII to form the rigid, hemostatic fibrin clot. Less well-known are the other functions of fibrin(ogen), such as the mediation of platelet spreading, the promotion of angiogenesis, and the stimulation of fibroblast proliferation (3, 7, 14, 22, 34). Fibrin(ogen) also is thought to provide a scaffold along which leukocytes can migrate, and stimulated monocytes and neutrophils express a high-affinity receptor for fibrin(ogen), the integrin α_Mβ₂ (Mac-1) (1).Mycobacterium tuberculosis is the leading cause of death due to bacterial infection and currently is estimated to infect a third of the world population (10, 12). In the context of tuberculosis, fibrinogen has been shown to be upregulated during experimental tuberculosis in mouse models and in natural human infections, as occurs in other infectious diseases, resulting in a hypercoagulable state (15, 30). Fibrinogen also has been identified as a cofactor for the pathological effects of mycobacterial trehalose 6,6′-dimycolate (TDM). Retzinger et al. demonstrated that TDM adsorbed fibrinogen preferentially to the exclusion of other plasma proteins, which increased the pyogranulomatous response to TDM (25). TDM is a predominant mycobacterial cell wall component and an important virulence factor for M. tuberculosis, in that it is granulomagenic, can delay phagosome maturation, and is an active component of complete Freund''s Adjuvant (2, 17, 18, 32). The presentation of TDM as a higher-order polymer, as a monolayer either in oil-water emulsions or on polystyrene particles, is required for maximal bioactivity (13, 26). We use a bead-based delivery model using 90-μm polystyrene microspheres suspended within Matrigel, a commercially available mixture of extracellular matrix components, to study the contributions of individual lipids to the inflammatory response to mycobacteria (13, 27).The development of a fibrinogen Aα chain-deficient mouse (Fib knockout [KO]) has facilitated the direct study of the roles of fibrinogen in inflammation (36). The use of this mouse strain has revealed the roles of fibrinogen in the limitation of Listeria monocytogenes growth in vivo (21), in the exacerbation of crescentic glomerulonephritis (9), in the organization of wound healing and wound stability (8), in tumor metastasis (23), and in cell adhesion to biomaterials (6). On the other hand, a study of bleomycin-induced pulmonary fibrosis in Fib KO mice showed that fibrosis developed independently of fibrin(ogen), and the absence of fibrin(ogen) increased the presence of neutrophils (40).In this study, we use a subcutaneous granuloma model in Fib KO mice to determine whether fibrinogen is necessary for the inflammatory response to TDM. Our results show that while fibrinogen is important for the organized formation of granulation tissue, fibrinogen deficiency has no effect on leukocyte recruitment to TDM-coated beads or proinflammatory cytokine production by the recruited cells. Fib KO mice also show no differences in pulmonary histopathology and only a transient difference in pulmonary bacterial burden in response to intravenous infection with M. tuberculosis. However, Fib KO mice show a suppurative response to TDM, resulting from the exacerbation of neutrophil necrosis and matrix degradation, whereas wild-type (WT) mice develop a granulomatous response with less cell degeneration, necrosis, and matrix degradation. These results show that fibrinogen is required neither as a cofactor for initiating an inflammatory response to TDM nor for controlling infection with M. tuberculosis, but rather suppresses the cytotoxic effects of TDM on recruited neutrophils while promoting the formation of granulation tissue.     

Particularities of the vasculature can promote the organ specificity of autoimmune attack

How certain autoimmune diseases target specific organs remains obscure. In the 'K/BxN' arthritis model, autoantibodies to a ubiquitous antigen elicit joint-restricted pathology. Here we have used intravital imaging to demonstrate that transfer of arthritogenic antibodies caused macromolecular vasopermeability localized to sites destined to develop arthritis, augmenting its severity. Vasopermeability depended on mast cells, neutrophils and FcgammaRIII but not complement, tumor necrosis factor or interleukin 1. Unexpectedly, radioresistant FcRgamma-expressing cells in an organ distant from the joint were required. Histamine and serotonin were critical, and systemic administration of these vasoactive amines recapitulated the joint localization of immune complex-triggered vasopermeability. We propose that regionally distinct vascular properties 'interface' with immune effector pathways to foster organ-specific autoimmune damage, perhaps explaining why arthritis accompanies many human infectious and autoimmune disorders.