Validation of a model to predict adverse outcomes in patients with pulmonary embolism.

AIMS: To validate a model for quantifying the prognosis of patients with pulmonary embolism (PE). The model was previously derived from 10 534 US patients. METHODS AND RESULTS: We validated the model in 367 patients prospectively diagnosed with PE at 117 European emergency departments. We used baseline data for the model's 11 prognostic variables to stratify patients into five risk classes (I-V). We compared 90-day mortality within each risk class and the area under the receiver operating characteristic curve between the validation and the original derivation samples. We also assessed the rate of recurrent venous thrombo-embolism and major bleeding within each risk class. Mortality was 0% in Risk Class I, 1.0% in Class II, 3.1% in Class III, 10.4% in Class IV, and 24.4% in Class V and did not differ between the validation and the original derivation samples. The area under the curve was larger in the validation sample (0.87 vs. 0.78, P=0.01). No patients in Classes I and II developed recurrent thrombo-embolism or major bleeding. CONCLUSION: The model accurately stratifies patients with PE into categories of increasing risk of mortality and other relevant complications. Patients in Risk Classes I and II are at low risk of adverse outcomes and are potential candidates for outpatient treatment.     

Parathyroidectomy: Overview of the Anatomic Basis and Surgical Strategies for Parathyroid Operations

It is imperative for the surgeon who performs parathyroidectomy to have a thorough understanding of the anatomy and embryology of the parathyroid glands in order to optimize the cure rate for patients with hyperparathyroidism (HPT). Furthermore, all clinicians caring for patients with hyperparathyroidism should be aware of the advancements in preoperative parathyroid localization, intraoperative PTH monitoring and surgical strategies for treatment of hyperparathyroidism. In this chapter, the anatomy and embryology of the parathyroid glands will be reviewed. The available surgical options for treatment of patients with hyperparathyroidism will be addressed, including “focused” parathyroidectomy, bilateral neck exploration, radioguided parathyroidectomy, and endoscopic and video-assisted parathyroidectomy. The unique challenge associated with reoperative parathyroidectomy for persistent or recurrent hyperparathyroidism will be outlined. Finally, insight into how to locate a qualified surgeon will be provided and recommendations will be made on what constitutes an appropriate choice of operation for specific patients with primary hyperparathyroidism.     

Exposure to magnetic resonance imaging does not produce taste aversion in rats

A taste aversion test was used to evaluate possible toxic effects of magnetic resonance imaging (MRI). Thirty male Sprague-Dawley rats were randomly assigned to four groups: Group One (n = 10) received 30 minutes exposure inside the MRI scanner; Group Two (n = 10) received a sham exposure to the MRI scanner; Group Three (n = 5) was injected with 0.15 M lithium chloride; and Group Four (n = 5) was injected with vehicle. All groups were given 10 minutes access to a 0.1% saccharin solution immediately prior to their respective treatment. The rats treated with lithium chloride displayed a taste aversion to the saccharin solution upon subsequent testing over an eight day period. The two control groups (Two and Four) and the rats exposed to MRI did not display any aversion to the saccharin solution. These results are compared to other studies that have shown that magnetic fields can influence biological systems.     

Double-Negative T Cell Levels Correlate with Chronic Graft-versus-Host Disease Severity

Chronic graft-versus-host disease (cGVHD) is a major complication, affecting 50% to 80% of long-term survivors of allogeneic hematopoietic stem cell transplantation. Current cGVHD therapies are neither specific nor curative, and patients are typically maintained for several months to years under immunosuppressive regimens that are associated with important side effects and increased susceptibility to life-threatening infections. As a result, continued investigation into the pathology of the disease and the search for novel diagnostic and therapeutic strategies to treat cGVHD remains a high priority. We report that the cellular dynamics of various immune cell subsets are related to cGVHD onset and severity in a cohort of allogeneic hematopoietic stem cell transplantation recipients. We document a decrease in the proportion of CD45RO⁺ CD4^?CD8^? (double-negative [DN]) T cells at the onset of cGVHD, a time at which serum levels of B cell activating factor and B cells are increased. We also find that DN T cell levels are correlated with cGVHD severity. Our present findings are in line with the view that activated DN T cells exhibit their immunoregulatory potential by eliminating B cells in vivo. Taken together, these findings suggest that maintaining elevated DN T cell numbers before the onset of cGVHD may prevent pathological B cell responses.     

Hematopoietic plakophilin‐3 regulates acute tissue‐specific and systemic inflammation in mice

Plakophilin‐3 (PKP3) is a member of the armadillo protein family, which is important in cell?cell contacts and signaling during development and tumorigenesis. In conventional facilities, PKP3‐deficient mice (PKP3^?/?) develop spontaneous dermatitis, indicating a possible involvement of PKP3 in inflammatory responses. Here, we show that PKP3 deficiency sensitizes mice to irritant contact dermatitis induced by phorbol myristate acetate (PMA). This sensitization occurred in mice with PKP3 deficiency in the hematopoietic system (PKP3^?/?hem), but not if the deficiency was specific to skin keratinocytes (PKP3^?/?ker). In a model of dextran sulfate sodium induced colitis, ubiquitous PKP3 deletion, but not intestinal epithelial PKP3 deficiency (PKP3^?/?IEC), impaired survival from disease. Interestingly, PKP3^?/?hem mice also displayed increased sensitivity to dextran sulfate sodium induced colitis. Finally, PKP3^?/? mice were more sensitive to the lethality of lipopolysaccharide (LPS) injection than wild‐type (WT) mice, and this phenotype was associated with increased intestinal permeability. PKP3^?/?IEC mice did not reproduce the enhanced endotoxin reactivity of PKP3^?/? mice, in contrast to PKP3^?/?hem mice. Finally, in vitro stimulation of WT neutrophils with LPS or PMA increased Pkp3 expression. In conclusion, our data highlight a novel role for hematopoietic PKP3 in the regulation of both locally and systemically induced immune responses. Nonetheless, further research is needed to unravel the underlying mechanism.     

Histidine-Rich Glycoprotein Modulates the Anti-Angiogenic Effects of Vasculostatin

Brain angiogenesis inhibitor 1 (BAI1) is a transmembrane protein expressed on glial cells within the brain. Its expression is dramatically down-regulated in many glioblastomas, consistent with its functional ability to inhibit angiogenesis and tumor growth in vivo. We have shown that the soluble anti-angiogenic domain of BAI1 (termed Vstat120) requires CD36, a cell surface glycoprotein expressed on microvascular endothelial cells (MVECs), for it to elicit an anti-angiogenic response. We now report that Vstat120 binding to CD36 on MVECs activates a caspase-mediated pro-apoptotic pathway, and this effect is abrogated by histidine-rich glycoprotein (HRGP). HRGP is a circulating glycoprotein previously shown to function as a CD36 decoy to promote angiogenesis in the presence of thrombospondin-1 or −2. Data here show that Vstat120 specifically binds HRGP. Under favorable MVEC growth conditions this interaction allows chemotactic-directed migration as well as endothelial tube formation to persist in in vitro cellular systems, and increased tumor growth in vivo as demonstrated in both subcutaneous and orthotopic brain tumor models, concomitant with an increase in tumor vascularity. Finally, we show that HRGP expression is increased in human brain cancers, with the protein heavily localized to the basement membrane of the tumors. These data help define a novel angiogenic axis that could be exploited for the treatment of human cancers and other diseases where excess angiogenesis occurs.Angiogenesis is the process through which new blood vessels are formed from pre-existing capillaries. In adulthood the vasculature is normally quiescent, a homeostatic state that is maintained by a precise balance of pro-angiogenic inducers such as vascular endothelial growth factor and anti-angiogenic inhibitors such as thrombospondin (TSP)−1 and −2. This angiostatic balance can be physiologically regulated in favor of new blood vessel formation during processes such as menstruation and wound healing allowing normal tissue regeneration; while pathological disruption of this balance is associated with many disease states, including diabetic retinopathy, atherosclerosis-induced tissue ischemia, chronic inflammation, tumor growth and metastasis, obesity, asthma, and several autoimmune diseases.¹ Therefore, it is extremely important to identify and dissect the pathways involved in vessel growth in both normal and aberrant conditions.Neovascularization is a major component of malignant tumor growth and many therapeutic strategies have been developed to inhibit tumor angiogenesis, including antibodies or decoys that bind and neutralize vascular endothelial growth factor,^2,3 small molecule inhibitors of growth factor signaling pathways,⁴ and peptides based on the anti-angiogenic type I repeat domains (TSR) of TSP-1.^5,6,7 Studies on the mechanisms of TSP-mediated anti-angiogenesis revealed that the TSR domains play an essential role⁸ and that the type B scavenger receptor CD36 functions as the critical endothelial cell surface receptor.^9,10 Using mouse corneal pocket angiogenesis assays we recently demonstrated that CD36 also functions as the receptor for a 120 kDa anti-angiogenic fragment derived from an unrelated TSR-containing protein, Brain Angiogenesis Inhibitor 1 (BAI1). This fragment, known as vasculostatin or Vstat120, suppressed neovessel formation in corneas from wild-type mice yet no effect was observed in CD36 null animals, showing for the first time that a TSR-containing protein distinct from TSP-1 and −2 mediates its anti-angiogenic functions through interactions with CD36.¹¹BAI1 is a 1584-aa brain-specific protein predicted to have seven transmembrane segments and a large extracellular domain. The extracellular domain contains an RGD integrin recognition motif, a putative hormone receptor (HomR) domain, and five TSR domains. Its expression is down-regulated in glioblastomas¹² and inversely correlated with vascularity and metastasis in colorectal cancer,¹³ consistent with an anti-angiogenic role. Kaur et al¹⁴ have shown that the TSR-containing fragment, Vstat120, was released from the cell membrane via proteolytic cleavage at a G protein–coupled receptor cleavage site and that this fragment inhibited microvascular endothelial cell (MVEC) proliferation, migration, and tube formation equivalent to that seen with full-length BAI1. Strikingly, restoration of Vstat120 expression in human glioma cells suppressed tumorigenicity and vascularity and enhanced animal survival in subcutaneous and orthotopic tumor implantation models in nude mice.¹¹The binding site on CD36 for the TSR domains of TSP-1 and −2 and Vstat120 has been localized to amino acids 93 to 120^11,15,16 within a highly conserved region termed the CLESH domain (CD36, LIMP2, EMP structural homology – see Figure 1A). CLESH domains are also found in proteins genetically distinct from the CD36 family,¹⁷ including some, such as HIV gp120 and histidine-rich glycoprotein (HRGP) which are known to bind TSP-1. HRGP, a 75-kDa glycoprotein synthesized in the liver, circulates in the blood at moderately high concentrations (approximately 100 to 150 μg/ml), and is secreted from activated platelets. In addition to TSP-1 and −2, it binds a wide range of ligands including divalent metal cations and several components of the provisional matrix laid down by healing wounds and tumors where angiogenesis is prevalent (for review see¹⁸). It has been implicated in the coagulation and fibrinolytic systems, and high levels have been associated with thrombotic disorders.^19,20 Work in our lab has shown that HRGP acts as a CD36 decoy to promote angiogenesis by binding TSP-1 and −2 and thereby preventing them from binding to CD36 on the endothelial cell surface.^21,22 Because both HRGP and CD36 compete for the identical binding region in TSP-1 and −2, we hypothesized that the relative concentrations of ligands, receptor, and decoy within a given microenvironment would ultimately determine the degree of angiogenesis. Within tumors and other areas of chronic inflammation, HRGP can be released from activated platelets as well as deposited from plasma that gained access via poorly organized hyperpermeable vessels. The angiogenic balance would then be tipped to favor angiogenesis by HRGP binding to TSPs as well as potentially other TSR containing anti-angiogenic proteins.Open in a separate windowFigure 1HRGP CLESH domain fusion protein specifically precipitates Vstat120 from tumor cell postculture media. A: HRGP/GST fusion proteins. Three recombinant GST fusion proteins were designed using fragments of the HRGP protein spanning amino acids 155 to 213 (within the second cystatin domain), amino acids 330 to 389 (the HRR), and amino acids 428 to 502 (CLESH domain). The linear structure of CD36 with the CLESH domain highlighted is shown below for reference. B: Coomassie stained gel (top panel) showing the purified GST-HRGP protein fragments after glutathione sepharose chromatography. The bottom panel shows Western blot analysis of each fusion protein probed with anti-GST antibody. C: Western blot analysis of a GST pull-down assay. The three GST-HRGP fusion proteins and GST alone were bound to glutathione sepharose beads, and CM from LN229V120 glioma cells were added to each sample. Each precipitate was then probed using the Vstat120/BAI1-specific antibody. Vstat120 was only detected when the CLESH domain (HRGP 428 to 502) was used in the pull-down and not the cystatin (HRGP 155 to 213) or the HRR (HRGP 330 to 389) domains. HRGP 428 to 502 only precipitated Vstat120 when CM from Vstat120-transfected LN229 cells (+ lanes) was used. No protein was detected when CM from the nontransfected parent cells (− lane) was used. This is a representative image from n = 3 experiments.In this article we show that the CLESH domain of HRGP binds Vstat120 and suppresses its anti-angiogenic activity by reversing inhibition of endothelial cell migration and tube formation. Furthermore, we show in both subcutaneous and orthotopic brain tumor models that HRGP exacerbates glioblastoma tumor growth and enhances tumor vascularity. We also show that the amount of HRGP present in human brain is increased in patients with primary tumors with the protein localized predominantly within the basement membrane. Finally, we offer some insight into the mechanism of action of Vstat120 by showing caspase-3 activation and endothelial cell apoptosis on Vstat120 addition. Together these results suggest that deposition of HRGP into angiogenic microenvironments, perhaps as the result of the inherent “leakiness” of the neo-vasculature and/or of platelet granule release, can modulate the anti-angiogenic processes mediated by the general family of TSR-containing proteins, and may shed light on the mechanism of angiogenesis regulation in the brain.     

Electrophysiological mechanisms of delayed excitotoxicity: positive feedback loop between NMDA receptor current and depolarization-mediated glutamate release

Delayed excitotoxic neuronal death after insult from exposure to high glutamate concentrations appears important in several CNS disorders. Although delayed excitotoxicity is known to depend on NMDA receptor (NMDAR) activity and Ca(2+) elevation, the electrophysiological mechanisms underlying postinsult persistence of NMDAR activation are not well understood. Membrane depolarization and nonspecific cationic current in the postinsult period were reported previously, but were not sensitive to NMDAR antagonists. Here, we analyzed mechanisms of the postinsult period using parallel current- and voltage-clamp recording and Ca(2+) imaging in primary hippocampal cultured neurons. We also compared more vulnerable older neurons [about 22 days in vitro (DIV)] to more resistant younger (about 15 DIV) neurons, to identify processes selectively associated with cell death in older neurons. During exposure to a modest glutamate insult (20 microM, 5 min), similar degrees of Ca(2+) elevation, membrane depolarization, action potential block, and increased inward current occurred in younger and older neurons. However, after glutamate withdrawal, these processes recovered rapidly in younger but not in older neurons. The latter also exhibited a concurrent postinsult increase in spontaneous miniature excitatory postsynaptic currents, reflecting glutamate release. Importantly, postinsult NMDAR antagonist administration reversed all of these persisting responses in older cells. Conversely, repolarization of the membrane by voltage clamp immediately after glutamate exposure reversed the NMDAR-dependent Ca(2+) elevation. Together, these data suggest that, in vulnerable neurons, excitotoxic insult induces a sustained positive feedback loop between NMDAR-dependent current and depolarization-mediated glutamate release, which persists after withdrawal of exogenous glutamate and drives Ca(2+) elevation and delayed excitotoxicity.     

Adrenal function in Smith-Lemli-Opitz syndrome

Smith-Lemli-Opitz syndrome (SLOS) is a multiple malformation syndrome due to mutations of the 7-dehydrocholesterol reductase gene (DHCR7), which leads to a deficiency of cholesterol synthesis and an accumulation of 7-dehydrocholesterol. The SLOS clinical spectrum ranges from multiple major malformations to a mild phenotype with minor anomalies and intellectual disability. Several children with SLOS and adrenal insufficiency have been described. We performed ovine corticotropin (oCRH) testing in 35 SLOS patients and 16 age- and gender-matched controls. We reviewed prior adrenocorticotropin (ACTH) stimulation tests of our SLOS patients (19 of 35 available) and reviewed results of ACTH stimulation tests from 10 additional SLOS patients. Results from oCRH testing showed that patients with SLOS had significantly higher ACTH baseline values than healthy controls (24.8 ± 15.3 pg/ml vs. 17.8 ± 7.5 pg/ml, P = 0.034). However, no statistically significant differences were noted for peak ACTH values (74.4 ± 35.0 pg/ml vs. 64.0 ± 24.9 pg/ml, P = 0.303) and for baseline (14.2 ± 7.8 mcg/dl vs. 14.2 ± 6.3 mcg/dl, P = 0.992) and peak cortisol values (28.2 ± 7.9 mcg/dl vs. 24.8 ± 8.1 mcg/dl, P = 0.156). The area-under-the-curve (AUC) was not significantly different in SLOS patients compared to controls for both ACTH (250.1 ± 118.7 pg/ml vs. 195.3 ± 96.6 pg/ml, P = 0.121) as well as cortisol secretion (83.1 ± 26.1 mcg/dl vs. 77.8 ± 25.9 mcg/dl, P = 0.499). ACTH stimulation test results were normal in 28 of 29 tests. The individual with the abnormal test results had subsequent normal oCRH tests. The slightly increased baseline ACTH level seen during oCRH testing may be due to compensated adrenocortical insufficiency. However, we were able to show that our patients with SLOS had an adequate glucocorticoid response, and thus, in mild to moderate cases of SLOS stress steroid coverage may not be warranted.     

Development of a catadioptric endoscope objective with forward and side views

Autofluorescence endoscopy is a promising functional imaging technique to improve screening of pre-cancerous or early cancer lesions in the gastrointestinal (GI) tract. Tissue autofluorescence signal is weak compared to white light reflectance imaging. Conventional forward-viewing endoscopes are inefficient in the collection of light from objects of interest along on the GI luminal wall. A key component of a complete autofluorescence endoscope is the light collection module. In this paper, we report the design, optimization, prototype development, and testing of an endoscope objective that is capable of acquiring simultaneous forward and radial views. The radial-view optical design was optimized for a balance between image quality and light collection. Modulation transfer function (MTF), entrance pupil radius, manufacturability, and field-of-view were parameters used in the lens optimization. In comparison with the typical forward-viewing endoscopes, our nonsequential ray trace simulations suggest the proposed radial-view design is more practical in the light collection. To validate the proposed simulation methods, a 3:1 scaled-up prototype was fabricated. Contrast measurements were taken with the prototype, and then compared with the simulated MTF.     

An Inhibitor of Arachidonate 5-Lipoxygenase, Nordy, Induces Differentiation and Inhibits Self-Renewal of Glioma Stem-Like Cells

Recent progress in cancer biology indicates that eradication of cancer stem cells (CSCs) is essential for more effective cancer therapy. Unfortunately, cancer stem cells such as glioma stem-like cells (GSLCs) are often resistant to either radio- or chemotherapy. Therefore, screening and development for novel therapeutic modalities against CSCs has been an important emerging field in cancer research. In this study, we report that a synthetic dl-nordihydroguaiaretic acid compound (dl-NDGA or “Nordy”), inhibited self-renewal and induced differentiation of GSLCs in vitro and in vivo. We found that Nordy inhibited an enzyme known to be involved in leukemia stem cell and leukemia progression, Alox-5, and attenuated the growth of GSLCs in vitro. Nordy reduced the GSLC pool through a decrease in the CD133⁺ population and abrogated clonogenicity. Nordy appeared to exert its effect via astrocytic differentiation by up-regulation of GFAP and down-regulation of stemness related genes, rather than by inducing apoptosis of GSLCs. The growth inhibition of xenografted glioma by Nordy was more long-lasting compared with that of the akylating agent BCNU, which exhibited significant relapse on drug discontinuation resulting from an enrichment of GSLCs. Meanwhile, transient exposure to Nordy reduced tumorigenecity of GSLCs and induced differentiation of the xenografts. Taken together, we have identified Alox-5 as a novel target in GSLCs and its inhibition with Nordy exhibits therapeutic implications through inducing GSLC differentiation.