Pseudofracture: An Acute Peripheral Tissue Trauma Model

Following trauma there is an early hyper-reactive inflammatory response that can lead to multiple organ dysfunction and high mortality in trauma patients; this response is often accompanied by a delayed immunosuppression that adds the clinical complications of infection and can also increase mortality.^1-9 Many studies have begun to assess these changes in the reactivity of the immune system following trauma.^10-15 Immunologic studies are greatly supported through the wide variety of transgenic and knockout mice available for in vivo modeling; these strains aid in detailed investigations to assess the molecular pathways involved in the immunologic responses.^16-21The challenge in experimental murine trauma modeling is long term investigation, as fracture fixation techniques in mice, can be complex and not easily reproducible.^22-30This pseudofracture model, an easily reproduced trauma model, overcomes these difficulties by immunologically mimicking an extremity fracture environment, while allowing freedom of movement in the animals and long term survival without the continual, prolonged use of anaesthesia. The intent is to recreate the features of long bone fracture; injured muscle and soft tissue are exposed to damaged bone and bone marrow without breaking the native bone.The pseudofracture model consists of two parts: a bilateral muscle crush injury to the hindlimbs, followed by injection of a bone solution into these injured muscles. The bone solution is prepared by harvesting the long bones from both hindlimbs of an age- and weight-matched syngeneic donor. These bones are then crushed and resuspended in phosphate buffered saline to create the bone solution.Bilateral femur fracture is a commonly used and well-established model of extremity trauma, and was the comparative model during the development of the pseudofracture model. Among the variety of available fracture models, we chose to use a closed method of fracture with soft tissue injury as our comparison to the pseudofracture, as we wanted a sterile yet proportionally severe peripheral tissue trauma model. ³¹Hemorrhagic shock is a common finding in the setting of severe trauma, and the global hypoperfusion adds a very relevant element to a trauma model. ^32-36 The pseudofracture model can be easily combined with a hemorrhagic shock model for a multiple trauma model of high severity. ³⁷Download video file.^{(62M, mov)}     

Stoichiometry in an ecological context: testing for links between <Emphasis Type="Italic">Daphnia</Emphasis> P-content,growth rate and habitat preference

We used laboratory experiments with ten Daphnia taxa to test for links between Daphnia P-content, growth rate and habitat preference. The taxa represent a wide range of body sizes and most show distinct preferences for one of three habitats: shallow lakes, deep, stratified lakes or fishless ponds. Previous studies show that taxa from shallow lakes and fishless ponds experience high predation risk and rich food resources, whereas taxa from deep lakes experience low predation risk, strong food limitation and potentially P-deficient resources. Thus, we predicted higher P-content and higher maximal growth rates in taxa from ponds and shallow lakes and lower P-content, lower maximal growth but reduced sensitivity to P-limitation in taxa preferring stratified lakes. In each of 25 experiments, a clonal Daphnia cohort was cultured for 4 days on a P-sufficient (molar C:P ratio 70) or a P-deficient (C:P 1,000) diet of a green alga at a high concentration (1 mg C l^–1). The P-content of adult Daphnia fed the P-sufficient diet ranged from 1.52 to 1.22% mass. Small-bodied taxa from shallow lakes had higher P-content than larger-bodied taxa from deep lakes or fishless ponds. However, we found a nonsignificant negative correlation between P-content and growth on the P-sufficient diet, rather than the positive relationship predicted by the growth rate hypothesis. The P-deficient diet resulted in declines in both growth rate and P-content compared with the P-sufficient controls and the extent of the declines differed between taxa. Taxa from ponds showed a marginally greater decline in growth with the P-deficient diet compared with taxa from shallow or deep lakes. However, contrary to stoichiometric theory, no relationship was found between a species P-content and growth depression on the P-deficient diet. Although we found evidence for habitat adaptations, our results show that factors other than Daphnia P-content are important in determining differences between Daphnia species in both maximal growth rate and sensitivity to P-limited growth.     

Effects of hypothermia and re-warming on the inflammatory response in a murine multiple hit model of trauma

INTRODUCTION: Although, hypothermia is a frequent event after trauma, it is unclear whether its beneficial or detrimental effects are more important. This study aims to quantify the effects of hypothermia and re-warming on the inflammatory response after fracture/hemorrhage and subsequent fracture stabilization with resuscitation. MATERIALS AND METHODS: Eighty-one male C57Bl/6 mice (aged 8-10 weeks, weighing 22.0+/-3.0 g) underwent femoral fracture and hemorrhage followed by resuscitation and splint fixation of the fracture. Animals were sacrificed 3h after induction of hemorrhage and fracture. Besides a sham group (n=6), four experimental groups were created: A: normothermia (n=12), B: hypothermia after trauma (n=21), C: re-warming after resuscitation and before stabilization (n=21), and D: hypothermia before trauma (n=21). Groups B-D were further subdivided into three subgroups according to the degree of hypothermia (subgroup 1: 35-33 degrees C, subgroup 2: 32.9-30.0 degrees C, and subgroup 3: 29.9-27.0 degrees C). Plasma cytokine (TNF-alpha, IL-6, and IL-10) and chemokine (MCP-1) concentrations were determined by ELISA, pulmonary permeability changes were quantified, and histological analysis of lung and liver tissues was performed. RESULTS: Normothermia resulted in a significantly increased early mortality rate. A significantly increased pro-inflammatory and decreased anti-inflammatory responses were also observed in normothermia as compared to hypothermia. The extent of these changes was most pronounced in the severe hypothermic group. Re-warming after mild hypothermia resulted in a pro-inflammatory response comparable to normothermia. CONCLUSION: Hypothermia has a beneficial effect on early survival after trauma, which appears to be independent of the level of hypothermia and re-warming. Re-warming, however, enhanced the pro-inflammatory response. Further studies with a longer posttraumatic observation period are required to investigate the long term effects of the hypothermia and re-warming-induced changes on the pro- and anti-inflammatory responses.     

The cell-cell adhesion molecule EpCAM interacts directly with the tight junction protein claudin-7

We recently described that in the metastasizing rat pancreatic carcinoma line BSp73ASML the cell-cell adhesion molecule EpCAM, CD44 variant isoforms and the tetraspanins D6.1A and CD9 form a complex that is located in glycolipid-enriched membrane microdomains. This complex contains, in addition, an undefined 20 kDa protein. As such complex formation influenced cell-cell adhesion and apoptosis resistance, it became of interest to identify the 20 kDa polypeptide. This 20 kDa protein, which co-precipitated with EpCAM in BSp73ASML lysates, was identified as the tight junction protein claudin-7. Correspondingly, an association between EpCAM and claudin-7 was noted in rat and human tumors and in non-transformed tissues of the gastrointestinal tract. Co-localization of the two molecules was most pronounced at basolateral membranes, but was also observed in tight junctions. Evidence for direct protein-protein interactions between EpCAM and claudin-7 was obtained by co-immunoprecipitation after treatment of tumor cells with a membrane-permeable chemical cross-linker. The complex, which is located in glycolipid-enriched membrane microdomains, is not disrupted by partial cholesterol depletion, but claudin-7 phosphorylation is restricted to the localization in glycolipid-enriched membrane microdomains. This is the first report on an association between EpCAM and claudins in both non-transformed tissues and metastasizing tumor cell lines.     

Accurate prediction of the functional significance of single nucleotide polymorphisms and mutations in the ABCA1 gene

The human genome contains an estimated 100,000 to 300,000 DNA variants that alter an amino acid in an encoded protein. However, our ability to predict which of these variants are functionally significant is limited. We used a bioinformatics approach to define the functional significance of genetic variation in the ABCA1 gene, a cholesterol transporter crucial for the metabolism of high density lipoprotein cholesterol. To predict the functional consequence of each coding single nucleotide polymorphism and mutation in this gene, we calculated a substitution position-specific evolutionary conservation score for each variant, which considers site-specific variation among evolutionarily related proteins. To test the bioinformatics predictions experimentally, we evaluated the biochemical consequence of these sequence variants by examining the ability of cell lines stably transfected with the ABCA1 alleles to elicit cholesterol efflux. Our bioinformatics approach correctly predicted the functional impact of greater than 94% of the naturally occurring variants we assessed. The bioinformatics predictions were significantly correlated with the degree of functional impairment of ABCA1 mutations (r² = 0.62, p = 0.0008). These results have allowed us to define the impact of genetic variation on ABCA1 function and to suggest that the in silico evolutionary approach we used may be a useful tool in general for predicting the effects of DNA variation on gene function. In addition, our data suggest that considering patterns of positive selection, along with patterns of negative selection such as evolutionary conservation, may improve our ability to predict the functional effects of amino acid variation.     

High-density polymerase-mediated incorporation of fluorochrome-labeled nucleotides

DNA-polymerase-mediated incorporation of different fluorochrome-labeled nucleotides (FdNTPs) was investigated with the goals of optimizing the high-density labeling of probes and exploring DNA sequencing strategies that rely on the controlled, sequential addition of such compounds. By systematically evaluating variables--including polymerase type, buffer conditions, and fluorochrome chemistries--a rational strategy for the sequential addition of labeled nucleotides to a DNA template was demonstrated. A simple structural model of the polymerase-DNA template complex that considered the fluorochrome moiety of the FdNTPs and the linker length also guided this strategy. Complementary results that portend the use of simple photobleaching to enable the reliable quantitation of consecutive additions are presented.     

Inhibition of hepatitis C virus-like particle binding to target cells by antiviral antibodies in acute and chronic hepatitis C

Hepatitis C virus (HCV) is a leading cause of chronic viral hepatitis worldwide. The study of antibody-mediated virus neutralization has been hampered by the lack of an efficient and high-throughput cell culture system for the study of virus neutralization. The HCV structural proteins have been shown to assemble into noninfectious HCV-like particles (HCV-LPs). Similar to serum-derived virions, HCV-LPs bind and enter human hepatocytes and hepatoma cell lines. In this study, we developed an HCV-LP-based model system for a systematic functional analysis of antiviral antibodies from patients with acute or chronic hepatitis C. We demonstrate that cellular HCV-LP binding was specifically inhibited by antiviral antibodies from patients with acute or chronic hepatitis C in a dose-dependent manner. Using a library of homologous overlapping envelope peptides covering the entire HCV envelope, we identified an epitope in the N-terminal E2 region (SQKIQLVNTNGSWHI; amino acid positions 408 to 422) as one target of human antiviral antibodies inhibiting cellular particle binding. Using a large panel of serum samples from patients with acute and chronic hepatitis C, we demonstrated that the presence of antibodies with inhibition of binding activity was not associated with viral clearance. In conclusion, antibody-mediated inhibition of cellular HCV-LP binding represents a convenient system for the functional characterization of human anti-HCV antibodies, allowing the mapping of envelope neutralization epitopes targeted by naturally occurring antiviral antibodies.     

Genes and neurons: molecular insights to fear and anxiety

Experimental animal models provide an important tool for the identification of inheritable components of fear and anxiety. 'Pavlovian' fear conditioning has been tremendously successful to characterize the neuronal circuitry and cellular mechanisms of the formation, consolidation and extinction of fear memories. Here we summarize recent progress that has led to the identification of gene products contributing to such experience-dependent changes in fear and anxiety and may guide the search for genetic factors involved in the development and treatment of human anxiety disorders.     

Cardiac mitochondrial preconditioning by Big Ca2+-sensitive K+ channel opening requires superoxide radical generation

ATP-sensitive K+ channel opening in inner mitochondrial membranes protects hearts from ischemia-reperfusion (I/R) injury. Opening of the Big conductance Ca2+-sensitive K+ channel (BK(Ca)) is now also known to elicit cardiac preconditioning. We investigated the role of the pharmacological opening of the BK(Ca) channel on inducing mitochondrial preconditioning during I/R and the role of O2-derived free radicals in modulating protection by putative mitochondrial (m)BK(Ca) channel opening. Left ventricular (LV) pressure (LVP) was measured with a balloon and transducer in guinea pig hearts isolated and perfused at constant pressure. NADH, reactive oxygen species (ROS), principally superoxide (O2(-*)), and m[Ca2+] were measured spectrophotofluorometrically at the LV free wall using autofluorescence and fluorescent dyes dihydroethidium and indo 1, respectively. BK(Ca) channel opener 1-(2'-hydroxy-5'-trifluoromethylphenyl)-5-trifluoromethyl-2(3H)benzimid-axolone (NS; NS-1619) was given for 15 min, ending 25 min before 30 min of global I/R. Either Mn(III)tetrakis(4-benzoic acid)porphyrin (TB; MnTBAP), a synthetic dismutator of O2(-*), or an antagonist of the BK(Ca) channel paxilline (PX) was given alone or for 5 min before, during, and 5 min after NS. NS pretreatment resulted in a 2.5-fold increase in developed LVP and a 2.5-fold decrease in infarct size. This was accompanied by less O2(-*) generation, decreased m[Ca2+], and more normalized NADH during early ischemia and throughout reperfusion. Both TB and PX antagonized each preconditioning effect. This indicates that 1) NS induces a mitochondrial-preconditioned state, evident during early ischemia, presumably on mBK(Ca) channels; 2) NS effects are blocked by BK(Ca) antagonist PX; and 3) NS-induced preconditioning is dependent on the production of ROS. Thus NS may induce mitochondrial ROS release to initiate preconditioning.