Induction of apoptosis and chemosensitization of mesothelioma cells by Bcl-2 and Bcl-xL antisense treatment

Our study was designed to investigate the role of the anti-apoptotic proteins Bcl-2 and Bcl-xL in the chemoresistance of cells derived from malignant pleural mesothelioma. First, we determined the basal expression levels of Bcl-2 and Bcl-xL in mesothelioma cells and examined the effect of their downregulation by antisense oligonucleotides. Bcl-xL mRNA and protein could be readily detected in mesothelioma cell lines, whereas only low levels of Bcl-2 mRNA and protein were found. Preferential downregulation of either Bcl-xL alone or of Bcl-xL and Bcl-2 simultaneously was achieved by treatment with antisense oligonucleotides 4259 and 4625, respectively, whereas the expression of other apoptosis-relevant genes remained unaffected. Treatment with oligonucleotides 4259 or 4625 lowered the apoptosis threshold in ZL34 mesothelioma cells, as indicated by an increase in cell death accompanied by increased caspase-3-like activity, a decrease of the mitochondrial transmembrane potential and the cleavage of procaspase-7 and ICAD. In addition to the direct induction of apoptosis, antisense treatment sensitized ZL34 cells to the cytostatic effect of cisplatin and gemcitabine, with the combination of 4625 and cisplatin being the most effective. Our results demonstrate that Bcl-2 and Bcl-xL antisense treatment facilitates apoptosis in mesothelioma cells and suggest the use of Bcl-2/Bcl-xL bispecific antisense treatment in combination with cisplatin or gemcitabine for therapy of malignant pleural mesothelioma.     

Perinatal human hypoxia-ischemia vulnerability correlates with brain calcification

Deregulation of intracellular calcium homeostasis is widely considered as one of the underlying pathophysiological mechanisms of hypoxic-ischemic brain injury. Whether this alteration can result in cerebral calcification was investigated in basal ganglia, cerebral cortex, and hippocampus of human premature and term neonates together with glial reaction. In all samples nonarteriosclerotic calcifications were observed, their number and size were area-specific and increased in term neonates. Basal ganglia always presented the highest degree of calcification and hippocampus the lowest, located mainly in the CA1 subfield. In all cases, neuronal damage was associated with astroglial reaction and calcium precipitates, with microglial reaction only in basal ganglia and cerebral cortex, and argues for the participation of excitatory amino acid receptors in hypoxia-ischemia damage. These data correlate with hypoxia-ischemia vulnerability in the perinatal period. The clinical relevance of these precipitates and the neuroprotective interest of non-NMDA receptor manipulation are discussed in the light of our results.     

In vitro function and phagocytosis of galactosylated platelet concentrates after long-term refrigeration

BACKGROUND: Short-term refrigeration of platelets (PLTs) in the absence of plasma results in their rapid clearance after transfusion. Blocking beta-N-acetylglucosamine (beta-GlcNAc) residues of glycoprotein Ibalpha (GPIbalpha) with galactose prevents binding of refrigerated human and mouse PLTs to macrophages and prolongs the circulation times of refrigerated mouse PLTs. PLT-associated galactosyltransferase efficiently galactosylates chilled PLTs in the presence of its substrate UDP-galactose is added to PLT-rich plasma. STUDY DESIGN AND METHODS: To characterize the hemostatic function of refrigerated and galactosylated human PLTs processed in the blood bank, PLT aggregation was studied in vitro under static and flow conditions and expression of integrin beta3 (CD61), CD62P (P-selectin), GPIbalpha (CD42b), annexin V binding, and integrin alphaIIbeta3 activation with flow cytometry. Affinity of macrophages for galactosylated refrigerated PLTs was evaluated with THP-1 cells, which recognize and phagocytize refrigerated PLTs. RESULTS: PLTs refrigerated and galactosylated for 14 days 1) maintained their ability to aggregate when exposed to agonists in a standard aggregometry assay, 2) showed less pronounced changes in surface expression of GPIbalpha compared with room temperature (RT)-stored PLTs, 3) increased P-selectin expression, and 4) were poorly phagocytized by differentiated THP-1 cells in vitro. In addition, it is shown that refrigeration of PLTs does not affect their adhesive properties under in vitro flow conditions. CONCLUSION: It is shown that refrigerated human PLTs retain in vitro function better than RT PLTs during storage and demonstrate that galactosylation prevents recognition of stored refrigerated PLTs by macrophages in vitro.     

Anti-inflammatory Effects of Novel P2X4 Receptor Antagonists,NC-2600 and NP-1815-PX,in a Murine Model of Colitis

Inflammation - The pharmacological blockade of P2X4 receptors has shown potential benefits in the management of several immune/inflammatory diseases. However, data regarding the involvement of P2X4...     

Hematopoietic Progenitor Cell Rolling in Bone Marrow Microvessels: Parallel Contributions by Endothelial Selectins and Vascular Cell Adhesion Molecule 1

We have used intravital microscopy to study physiologically perfused microvessels in murine bone marrow (BM). BM sinusoids and venules, but not adjacent bone vessels, supported rolling interactions of hematopoietic progenitor cells. Rolling did not involve L-selectin, but was partially reduced in wild-type mice treated with antibodies to P- or E-selectin and in mice that were deficient in these two selectins. Selectin-independent rolling was mediated by α4 integrins, which interacted with endothelial vascular cell adhesion molecule (VCAM)-1. Parallel contribution of the endothelial selectins and VCAM-1 is not known to direct blood cell trafficking to other noninflamed tissues. This combination of constitutively expressed adhesion molecules may thus constitute a BM-specific recruitment pathway for progenitor cells analogous to the vascular addressins that direct selective lymphocyte homing to lymphoid organs.     

Changes in activity of non-specific esterases in cadmium treated Lymantria dispar larvae

Many biochemical, physiological and histological criteria have been used as indicators of exposures and effects of the contaminants. These changes can indicate the response of an organism to a specific environmental stressor. In the present paper, the effect of the acute and chronic exposure to cadmium as well as recovery from two cadmium concentrations (10 and 30 μgCd/g dry food) on gypsy moth (Lymantria dispar) midgut esterases was investigated. The influence of cadmium on trait plasticity was also examined. Esterases showed great sensitivity to low metal concentrations during acute and chronic treatments. Their activities during short-term exposure and after recovery significantly depended on cadmium concentrations. The esterases had greater index of plasticity during chronic treatments with 10 and 30 μgCd/dry food. Five esterase isoforms between 64 and 250 kDa were detected. Isoforms of esterases exposed to any of the two cadmium effects differed among several egg-masses. Isozymes were distinguished in one egg-mass during different cadmium treatments. We conclude that these enzymes could be considered potential and sensitive non-selective biomarkers for the presence of cadmium in food.     

Immunohistochemistry in Mohs Micrographic Surgery: A Review of the Literature

Mohs micrographic surgery has become the “gold standard” for surgical excision of nonmelanoma skin cancers for maximal preservation of normal tissue. Mohs micrographic surgery entails processing specimens in horizontal frozen sections with immediate examination under a light microscope. This technique offers the examination of lateral and deep margins in the same plane in contrast to wide local excision. Success with Mohs micrographic surgery depends on accurate mapping of the tumor, correct interpretation of the histopathological sections, and appreciation of aggressive tumor characteristics. The most common reason for recurrence of tumor after Mohs micrographic surgery is residual undetected tumor. Because hematoxylin and eosin stains may present difficulties in interpretation, immunohistochemistry techniques are being used to supplement these routine stains. Although immunohistochemistry is not being widely utilized by Mohs micrographic surgery surgeons, the many advantages of immunohistochemistry over routine staining of frozen sections in selected settings is of great value. Herein, the authors review the application of immunohistochemistry in Mohs micrographic surgery for a variety of neoplasms encountered most frequently by Mohs micrographic surgery surgeons. (J Clin Aesthetic Dermatol. 2009;2(7):37–42.)Mohs micrographic surgery (MMS) has become the “gold standard” for surgical excision of nonmelanoma skin cancers for maximal preservation of normal tissue. MMS entails processing specimens in horizontal frozen sections with immediate examination under a light microscope. This technique offers the examination of lateral and deep margins in the same plane in contrast to wide local excision (WLE). Standard histological examination of excision specimens demonstrates only 0.2 percent of the margins; whereas, MMS examines 100 percent of both deep and peripheral margins.¹Success with MMS depends on accurate mapping of the tumor, correct interpretation of the histopathological sections, and appreciation of aggressive tumor characteristics. Because hematoxylin and eosin (H&E) staining may present difficulties in interpretation of frozen sections, rapid immunohistochemistry (IHC) techniques are being used to supplement these routine stains. In a recent survey of 108 laboratories processing MMS surgery specimens, 87 percent used H&E stains to process sections. In this same survey, only 13 laboratories used IHC staining of frozen sections.²Adjunctive use of IHC in H&E frozen sections enhances tissue interpretation and spares resection of additional tissue. The most common reason for recurrence of tumor after MMS is residual undetected tumor. Polyclonal antibodies used in IHC offer greater sensitivity than routine H&E stains. Examination of frozen sections of aggressive cutaneous neoplasms, such as melanoma, has been facilitated by IHC. Advances in IHC have addressed issues of cost and time inefficiency in processing MMS frozen sections. IHC leads to facilitated surgical excision via MMS by reducing variable staining, high background or nonspecific staining, and turn-around time. Specifically, IHC is useful in clearly delineating malignant cells present in dense inflammation, identifying perineural invasion and pagetoid spread in carcinomas.^3–6While there are several advantages to utilizing IHC in MMS, there are some drawbacks as well. First, IHC stains were initially developed for permanent sections and not frozen sections. Consequently, there are problems with displacement of soluble antigens on frozen sections. Second, using polyclonal antibodies causes decreased specificity because some antigens identified by polyclonal antibodies may belong to normal tissue. Another concern is the incubation time required for each stain, which has been shortened by using higher antibody titers.^2,3As MMS is increasingly used for high-risk tumors, it is imperative that the dermatology community be familiar with the advances in MMS techniques, especially IHC. Although IHC is not being widely utilized by MMS surgeons, the many advantages of IHC over routine staining of frozen sections in selected settings is of great value. Herein, the authors review the application of IHC in MMS for a variety of neoplasms encountered most frequently by MMS surgeons (