SESSION TITLE: Disorders of the Mediastinum 1 SESSION TYPE: Med Student/Res Case Rep Postr PRESENTED ON: 10/09/2018 01:15 pm - 02:15 pm INTRODUCTION: Lynch syndrome is an autosomal dominant condition caused by genetic defects in one or more DNA mismatch repair (MMR) genes, including MLH1, MSH2, MSH6, and PMS2. Patients with such mutations have an elevated risk of developing multiple malignancies - the colon, rectum, endometrium, ovary, stomach etc. Thymoma has been reported in patients with Lynch syndrome but not thymic carcinoma (1). We hereby present a case of thymic carcinoma in a patient with Lynch syndrome due to an MLH1 mutation. CASE PRESENTATION: A 45-year-old female with a familial and personal history of Lynch syndrome (MLH1 heterozygous mutation) presented with dyspnea, chest pain, and dysphagia for 2 weeks. A chest CT showed an anterior, diffuse mediastinal mass measuring 5.7 x 8.7 x 8.9 cm with bilateral pulmonary nodules, a pericardial effusion (fig1). A pericardial window was performed. Biopsy of a lung nodule demonstrated a poorly differentiated carcinoma with squamous features with extensive necrosis, favoring thymic origin. Immunohistochemistry showed that the tumor was positive for Keratin AE1/AE3, p63, CD117, and negative for CK7, CK20, synaptophysin, chromogranin, CD5, and PAX-8. Genomic studies on the tumor revealed deficient MMR status with a two-copy deletion of MLH1 at 3p22.2 and c-kit mutation. The patient received two cycles of carboplatin and paclitaxel, with initial clinical improvement, but then disease progressed. A chest CT showed the progression of malignancy, compression of the superior vena cava, a severe compression of the left atrium due to the tumor (fig2). A drug to target c-kit was initiated; however, she died shortly thereafter, and within 3 months after diagnosis due to the progression of the disease. DISCUSSION: Defects in the MMR genes underlie Lynch syndrome. Thymic cancer has usually been associated with mutations of TP53 and CDKN2A and others including ALK, ATM, ERBB4, FGFR3, KIT and NRAS(2). There is literature on the association of thymic carcinoma with an MLH1 mutation. A case report by Yufeng Jiang et al reports finding MLH1 and MSH2 mutation more in thymic carcinoma than Type AB thymoma(3), and an article by Yisong Wang et al reports multiple genetic mutations in thymic carcinomas which were probably due to the presence of mutations of MLH1 and XRCC1. There is a study by Yulan Qing et al which suggests that MSH2-/- Hematopoietic Stem Cells are Pre-Leukemic Stem Cells and can transform into lymphoma/leukemia-initiating cells in the thymic environment. Role of MLH1 mutations in the pathogenesis of thymic cancer is not very clear. Our patient had Lynch syndrome due to heterozygous MLH1 mutation. Her thymic carcinoma had a two-copy deletion of MLH1. It could be that this lethal malignancy is associated with MLH 1 Mutations. CONCLUSIONS: Thymic carcinoma may be one of the extracolonic malignancies associated with Lynch syndrome and MLH1 gene mutation may have a role in pathogenesis. Reference #1: Stage IB Malignant Thymoma in a Lynch Syndrome Patient with Multiple Cancers: Response to Incidental Administration of Oxaliplatin and 5-Fluorouracil M. Tampellini, I. Alabiso, C.M. Sculli, M. Barberis, D. Giachino, A. Berruti & L. Dogliotti, Journal of Chemotherapy Vol. 18, Iss. 4, 2006 Reference #2: F, Pichlhöfer B, Zaharie AT, et al. Molecular Profiling of Thymoma and Thymic Carcinoma: Genetic Differences and Potential Novel Therapeutic Targets. Pathology Oncology Research. 2017;23(3):551-564. https://doi.org/10.1007/s12253-016-0144-8. Reference #3: Jiang Y, Liu Y, Shi X, Mao X, Zhao Y, Fan C. An unusual combined thymic carcinoma composed of squamous cell carcinoma and type AB thymoma: a rare case report. Diagnostic Pathology. 2017;12:9. https://doi.org/10.1186/s13000-016-0590-3. DISCLOSURES: No relevant relationships by Deepali Pandey, source=Web Response No relevant relationships by david shepro, source=Web Response

Congestive Heart FailureVolume 12, Issue 4 p. 235-235 Free Access Microvascular Research: Biology and Pathology First published: 29 January 2007 https://doi.org/10.1111/j.1527-5299.2006.05461.xAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article. Volume12, Issue4July/August 2006Pages 235-235 RelatedInformation

Ischemia and reperfusion of the lower torso lead to leukotrieneand neutrophil (PMN)-dependent lung injury characterized by lung PMN sequestration, increased permeability, and noncardiogenic edema. It is thought that PMNs require adhesion to endothelium to alter barrier function. This study tests the role of CD 18, the PMN adherence receptor, in mediating lung permeabilily after lower torso ischemia and reperfusion. Anesthetized rabbits (n=9) underwent 3 hours of bilateral hind limb ischemia. Ten minutes after the release of the tourniquets, plasma leukotriene B4 levels increased to 395±85 pglml, higher than 129±35 pg/ml in controls (n=9,p <0.01). At this time there was a reduction in circulating white blood cells (x103), 3.56±0.49/mm3 relative to 6.07±0.61/mm3 in controls (p<0.01). PMNs were sequestered in the hind limbs, indicated by increased myeloperoxidase activity of 1.06±0.19 units/g compared with 0.56±0.09 units/g in controls (p<0.05). Four hours after tourniquet release, PMNs were sequestered in the lungs, 52±4 PMNs per 10 high-power fields, a value higher than 31.5±3 PMNs per 10 high-power fields in controls; bronchoalveolar lavage fluid protein content increased to 554±90 Lg/ml relative to 277±46 tg/ml in controls; and there was lung edema, measured by increased wet weight-to-dry weight ratios of 5.19±0.10, higher than 4.29±0.21 in controls (all p<0.01). The wet/dry ratios of the heart, liver, and kidney were unchanged. Pretreatment of other rabbits (n=8) with a purified anti-CD 18 monoclonal antibody (R 15.7, 1 mg/kg) 10 minutes before tourniquet release did not affect the rise in leukotriene B4 but was effective in preventing leukopenia (7.29±+1.05/mm3, p<0.01) and sequestration of PMNs in the hind limbs (myeloperoxidase activity, 0.46±0.12 units/g, p<0.05). Further, the anti-CD 18 monoclonal antibody prevented lung sequestration of PMNs (34±3 PMNs per 10 high-power fields, p<0.01) and reduced permeability, shown by a fall in bronchoalveolar lavage fluid protein to 324±41 ,tg/ml and a fall in wet/dry weight ratio to 4.61±0.10 (both p<0.05). These data suggest that CD 18 is important in PMN-dependent lung injury after remote ischemia. (Circulation Research 1992;71:82-86)

The thalidomide analogue and immunomodulatory drug (IMiD®) lenalidomide (CC-5013, REVLIMID™) is emerging as a useful treatment for a number of cancers and has recently entered phase III trials for multiple myeloma. It has been suggested that the anti-tumor effect of lenalidomide is related to its anti-angiogenic potency. In this regard, we have previously shown that lenalidomide inhibits angiogenesis in both rat and human in vitro models but does not affect endothelial cell proliferation. We now show that oral administration of lenalidomide attenuates growth factor-induced angiogenesis in vivo; the rat mesenteric window assay was utilized to show that lenalidomide significantly inhibits vascularization in a dose-dependent manner. We also found that lenalidomide significantly inhibits growth factor-induced endothelial cell migration. This correlates with the inhibitory effect of lenalidomide on growth factor-induced Akt phosphorylation, thereby providing a potential mechanism for its anti-migratory and subsequent anti-angiogenic effects. These data further support the use of lenalidomide as an orally administered drug for the effective treatment of angiogenesis-dependent conditions, including cancer, and suggest a potential mechanism of action.

The inflammation-induced subcellular redistribution of key cytoskeletal and junctional proteins in cultured human lung microvessel endothelial cells is investigated as part of a study on the posttranslational regulation of paracellular permeability. Inflammatory agonist-stimulated cells are detergent fractionated into three subcellular compartments followed by quantitative immunoblot analysis. Actin, γ-catenin, and VE-cadherin increasingly associate with the cytoskeletal fraction upon thrombin stimulation. Concomitantly, actin is reduced in the cytosol fraction, whereas γ-catenin and VE-cadherin are reduced in the membrane fraction. α- and β-catenin show baseline distributions similar to those of VE-cadherin and γ-catenin, but do not significantly redistribute. Additionally, vimentin is found exclusively in the cytoskeletal fraction and also does not significantly redistribute following thrombin treatment. The VE-cadherin response is independent of the presence of F-actin or actin redistribution. Immunofluorescence microscopy reveals that membrane and cytoskeletal VE-cadherin is present in alternating patches along the cell junctions. Furthermore, VE-cadherin is lost from zones of interendothelial cell pore formation. A model is formulated describing these membrane-associated VE-cadherin patches as predetermined zones of potential intercellular gap formation. During inflammation, VE-cadherin is lost from these zones and sequestered at the remaining cell–cell contact sites, anchored to the cytoskeleton in an actin-independent fashion.

This study examined changes in the biomechanical properties of cultured pulmonary microvascular endothelial cells (ECs) and neutrophils induced by adhesion of neutrophils to these ECs. The biomechanical properties of cells were evaluated using magnetic twisting cytometry, which measures the angular rotation of ferromagnetic beads bound to cells through antibody ligation on application of a specified magnetic torque. Adhesion of neutrophils to 24-hour tumor necrosis factor-alpha (TNF-alpha)-treated ECs, but not to untreated ECs, induced an increase in EC stiffness within 2 minutes, which was accompanied by an increase and a reorganization of F-actin in ECs. A cell-permeant, phosphoinositide-binding peptide attenuated the EC stiffening response, suggesting that intracellular phosphoinositides are required. The stiffening response was not inhibited by ML-7, a myosin light-chain kinase inhibitor, or BAPTA, an intracellular Ca2+ chelator. Moreover, the phosphorylation pattern of the regulatory myosin light chains was unaltered within 15 minutes of neutrophil adherence. These data suggested that the EC stiffening response appeared not to be mediated by myosin light-chain-dependent mechanisms. Concomitantly, neutrophil adhesion to 24-hour TNF-alpha-treated ECs also induced changes in the biomechanical properties of neutrophils compared to neutrophils bound to untreated ECs. Taken together, these results demonstrated that neutrophil adhesion to TNF-alpha-treated ECs induces changes in the biomechanical properties of both cell types through actin cytoskeletal remodeling. These changes may modulate neutrophil transmigration across the endothelium during inflammation.

By using streptozotocin-induced diabetic rats as a studied model, our previous experimental results have indicated that daily oral feeding of garlic extract (100 mg/kg BW) could increase the cardiovascular functions in streptozotocin (STZ) rats; the abnormality of lipid profile was prevented; and garlic extract could increase fibrinolitic activities with the decrease of platelet aggregation. Moreover, the plasma insulin level was Increased concomitantly with the decrease of plasma glucose level, However, due to the high incidence of atherosclerosis in diabetes, the present study has been continued for further investigation of the effect of garlic extract on the coronary vascular ultrastructual changes, In addition, to identify the possible mechanism(s) of garlic's therapeutic effects, the cyclooxygenase Inhibitor, aspirin, has been included in this present study,By using transmission electron microscopic studies, 16 weeks of daily oral feeding of garlic extract (100 mg/kg BW) caused as an antiatherosclerotic agent at the coronary arteriolar (15-30 mu m) wall in STZ-rats, Interestingly, the thickening of coronary capillary (5-10 mu m) basement membrane also was significantly attenuated within the group of STZ-rats treated with garlic extract, However, the possible direct action of garlic through the cyclooxygenase pathway has not been confirmed by the results of aspirin: The daily oral feeding of aspirin (10 mg/kg BW) in 16-week STZ-rats has not showed reduced arteriolar vascular wall abnormalities. The irregular patterns of fiber matrix, arranging the basement membrane at the arteriolar walls, were still recognized in the same manner as in STZ-rats, Interestingly, the thickening of the capillary basement membrane occurred in 16-week STZ-rats seems to be attenuated by the aspirin received, At present, garlic extract may open the new era in the medicinal use of garlic to prevent diabetic cardiovascular complications.

Protein distribution profiles may be used to characterize both physiological and pathophysiological cellular changes, but rigorous biochemical assays for measuring such movements are lacking. This paper reports on a protein redistribution assay that combines reversible metal chelate-based total protein detection with a four-fraction subcellular detergent fractionation procedure. TNF-α stimulated cultured human omental microvessel endothelial cells are fractionated into cytosol, membrane/organelle, nuclear (envelope and associated), and cytoskeletal/DNA compartments. Protein fractions are separated electrophoretically and electroblotted or slot-blotted onto PVDF membranes without electrophoretic separation. A key feature is that total protein is measured and analyzed directly on the resultant PVDF membrane, using a Ferrozine/ferrous metal-chelate stain, without the added step of a prior solution-phase protein assay. As a result, factors that may adversely affect NFκB quantification, such as saturation of the solid-support membrane, are rigorously evaluated and controlled. Following removal of the Ferrozine/ferrous total protein stain, NFκB distribution is determined via standard immunodetection procedures. This assay reveals a new level of complexity regarding NFκB distribution and translocation. NFκB is shown to translocate from the cytosol to the membrane/organelle and cytoskeletal/DNA fractions, whereas trace levels of NFκB are observed in the nuclear (envelope and associated) fraction. Dose-curve analysis reveals that the response is initiated at 10 U/ml of TNF-α, plateaus at approximately 1000 U/ml, and remains essentially constant up to 2000 U/ml. Time-course analysis demonstrates a measurable response as early as 5 min and a peak response at approximately 30 min, after which the distribution begins to return to baseline. The assay should provide a valuable tool for rapid evaluation and mechanistic studies of NFκB redistribution.

P-selectin is an adhesion molecule expressed on activated endothelial and platelet surfaces. The function of the short consensus repeats (SCRs) of P-selectin, homologous with the SCRs of complement regulatory proteins is largely unknown. In a model of murine hindlimb ischemia where local reperfusion injury is partly mediated by IgM natural antibody and classical complement pathway activation, we hypothesized that human soluble P-selectin (sP-sel) would moderate the complement component of the inflammatory response. Infusion of sP-sel supernatant or purified (p) sP-sel prepared from activated human platelets, reduced ischemic muscle vascular permeability by 48% and 43%, respectively, following reperfusion. Hindlimb immunohistochemistry demonstrated negligible C3 staining colocalized with IgM in these groups compared with intense staining in the untreated injured mice. In vitro studies of mouse serum complement hemolytic activity showed that psP-sel inhibited the classical but not alternative complement pathway. Flow cytometry demonstrated that psP-sel inhibited C1q adherence to sensitized red blood cells. From these data we conclude that sP-sel moderates skeletal muscle reperfusion injury by inhibition of the classical complement pathway.

Electrophoretically separated proteins may be visualized using organic dyes, such as Ponceau red, Amido black, fast green, or most commonly Coomassie Brilliant Blue (,). Alternatively, sensitive detection methods have been devised using metal ions and colloids of gold, silver, copper, carbon, or iron (, , , , , , , , , ). Metal chelates form a third class of stains, consisting of organometallic complexes that bind avidly to proteins resolved in polyacrylamide gels or immobilized on solid-phase supports (). The metal chelate staining procedures are simple, requiring reagents that are easily prepared, are stable at room temperature, and can be reused several times without loss of sensitivity. The staining procedures are relatively inexpensive, since they do not utilize precious metals, such as gold or silver. Like Ponceau red stain, the metal chelate stains are readily reversible. Complexes form at acidic pH and elute on increasing the pH to 7.0–10.0. Metal chelates can be used to detect proteins on nitrocellulose, PVDF, and nylon membranes as well as in polyacrylamide gels. The metal complexes do not modify proteins, and are compatible with immunoblotting, lectin blotting, mass spectrometry, and Edman-based protein sequencing (, , , , ). Metal chelate stains are suitable for routine protein measurement in solid-phase assays owing to the quantitative stoichiometry of complex formation with proteins and peptides (,). Such solid-phase protein assays are more sensitive and resistant to chemical interference than their solution-based counterparts ().

Prevalent methods for visualizing proteins resolved by two-dimensional (2-D) gel electrophoresis include autoradiography, silver staining, and Coomassie brilliant blue staining (1,2. The organic dye Coomassie brilliant blue is capable of detecting as little as 100 ng of protein, but this is considerably less sensitive than silver staining or autoradiography (1,2. Silver staining allows detection of low-nanogram amounts of protein, but detection sensitivity of autoradiography can be considerably better, since it depends principally on the specific activity of the radiolabeling method. Other staining techniques are valuable for specific applications in 2-D gel electrophoresis, such as Edman-based protein sequencing, mass spectrometry, and immunoblotting. Many of these applications require stains that do not covalently modify proteins, are relatively sensitive, and are easily reversible (3). Additionally, immunoblotting protocols are often based upon the enzymatic formation of a colored formazan product or the chemiluminescent production of light (4). Thus, image acquisition requires detection of diverse chromogenic, radioactive, and luminescent signals.

Vascular pathologies induced by ischemia/reperfusion involve the production of reactive oxygen species (ROS) that in part cause tissue injury. The production of ROS that occurs upon reperfusion activates specific second messenger pathways. In diabetic retinopathy there is a characteristic loss of the microvascular pericyte. Pericytes are more sensitive than endothelial cells to low concentrations of ROS, such as hydrogen peroxide (H2O2) when tested in vitro. Whether the pericyte loss is due to toxic cell death triggered by the noxious H2O2 or apoptosis, due to activation of specific second messenger pathways, is unknown. During apoptosis, a cell's nucleus and cytoplasm condense, the cell becomes fragmented, and ultimately forms apoptotic bodies. It is generally assumed that apoptosis depends on nuclear signaling, but cytoplasmic morphological processes are not well described. We find that exposing cultured retinal pericytes to 100 μM H2O2 for 30 min leads to myosin heavy chain translocation from the cytosol to the cytoskeleton and a significant decrease in cell surface area. Pericyte death follows within 60–120 min. Exposing cells to 150 mJ/cm2 ultraviolet radiation, an alternate free radical generating system, also causes pericyte myosin translocation and apoptosis. Proteolytic cleavage of actin is not observed in pericyte apoptosis. 3‐aminobenzamide, a pharmacological inhibitor of the cleavage and activation of the DNA‐repairing enzyme poly (ADP‐ribose) polymerase (PARP) inhibits pericyte apoptosis, and prevents myosin translocation. Deferoxamine, an iron chelator known to interfere with free radical generation, also inhibits pericyte myosin translocation, contractility, and cell death. Myosin translocation to the cytoskeleton may be an early step in assembly of a competent contractile apparatus, which is involved in apoptotic cell condensation. These results suggest that pericyte loss associated with increased free radical production in diabetic retina may be by an apoptotic phenomenon. J. Cell. Biochem. 75:118–129, 1999. © 1999 Wiley‐Liss, Inc.

Endothelial cell (EC) propagation has been simplified by developing cell-specific selection criteria. Methods commonly used for selectively isolating EC include: (i) differential sieving of disaggregated tissue, (ii) differential plating of cells on extracellular matrices, (iii) lectin affinity isolation of cell populations and (iv) fluorescence-activated cell sorting of cells labeled with a carbocyanine dye of acetylated low-density lipoprotein (DiI-Ac-LDL). Few criteria for selectively propagating pericytes (PC) are currently available. Nonspecific esterases exhibit a high degree of multiplicity when compared with other mammalian isozymes and may be suitable for the identification and selective propagation of cells of the microvasculature. Evaluation of esterase isotype expression in PC and EC by zymography indicates PC contain alpha-naphthyl acetate and alpha-naphthyl butyrate hydrolyzing esterases as well as dipeptidyl peptidase I, while EC only contain alpha-naphthyl acetate esterase. The cytotoxic response of PC and EC to various amino acid esters is assessed by monitoring vital dye uptake and by light microscopy. Several amino acid esters are cytotoxic to both cell types, whereas 50 mM L-leucine methyl ester (L-Leu OMe) is toxic to EC but not to PC. This amino acid ester is also toxic to mesothelial and retinal pigmented epithelial cells, other common contaminants of PC cultures. Analysis of protein composition by two-dimensional gel electrophoresis indicates that L-Leu OMe does not stimulate expression of stress response proteins in PC. Thus, L-Leu OMe can be utilized to cultivate PC selectively from mixed cell populations.

Two principal forms of the actin binding protein, filamin, are expressed in mammalian cells: nonmuscle and muscle isotypes (FLN-1 and FLN-2). A protein that copurifies with an alpha-naphthyl acetate hydrolyzing esterase from human omentum microvessel endothelial cells (EC) is isolated by nondenaturing electrophoresis, sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and electroblotting. The purified protein is subjected to in situ trypsin cleavage, reversed-phase high performance liquid chromatography (HPLC) and automated Edman degradation. Six peptide fragments from the protein are identified to have 60-66% identity with nonmuscle filamin (ABP-280). Two of these peptides are 100% identical to a previously sequenced human muscle filamin fragment. Polyclonal antibody is produced using a 16-residue synthetic peptide corresponding to a structural beta-sheet region of muscle filamin. Compared with a variety of vascular cells evaluated, retinal pericytes express an abundance of both muscle and non-muscle filamin isotypes. Pericytes contain at least 10 times more muscle filamin than human umbilical vein EC and at least three times the amount expressed in human omentum microvessel and bovine pulmonary artery EC. Differential detergent fractionation indicates that both filamin isotypes are primarily localized in the cytosol and membrane/organelle fractions of pericytes. Another actin crosslinking protein, alpha-actinin, is primarily found in the cytosol and cytoskeletal fractions. The dynamic regulation of actin microfilament organization in pericytes may be controlled in part by the two filamin isotypes, which in turn may contribute to pericyte contractility.

Nonspecific vasodilatation during iontophoresis is an important confounding factor in experimental pharmacology. In this investigation, we studied the involvement of sensory nerves and histamine-related reactions in causing nonspecific vasodilatation in a model of anodal and cathodal iontophoresis of sodium chloride. Firstly, we applied a mixture of local anesthetic (EMLA) cream to confirm its suppressive effect on nonspecific vasodilatation and to measure its efficacy in three different dosages (duration: 1, 2, and 3 h). We then investigated the role of histamine in nonspecific vasodilatation by giving an oral antihistamine drug (cetirizine) to subjects who had and had not been given EMLA. We found substantial suppression of the nonspecific vasodilatation in all EMLA-treated groups (all dosages) compared with untreated controls (with suppression rates of 60–65%). Dosage had no significant effect. A further suppression of nonspecific vasodilatation was seen after oral cetirizine during anodal and cathodal iontophoresis in both EMLA-treated and untreated groups. The antihistamine effect was most pronounced during anodal iontophoresis. These results suggest a histaminergic increase in perfusion that may be independent of neurogenic mechanisms and depend on polarity (anode or cathode). Local nerve blocks (EMLA) together with cetirizine may therefore be used to reduce nonspecific vasodilatation in both anodal and cathodal iontophoresis.