Laboratory Investigation

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The degree of lung injury caused by prolonged inhalation of low levels of ozone (O3) is of interest since urban environmental levels periodically reach 0.2 to 0.3 ppm. Since the area of the junction of the conductive and respiratory regions of the lung has been reported as the major site of injury due to O3 inhalation, techniques were devised to specifically study alveolar tissue from this region. One-day-old or 6-week-old male rats were exposed to either 0.25 ppm O3 12 hours/day or to continuous room air for 6 weeks. An additional group of 6-week-old rats were exposed to 0.12 ppm O3 for the same time period. All lungs were fixed at the end of the exposure by intratracheal installation of buffered 2% glutaraldehyde. Cylinders of tissue containing a cross-section of a terminal bronchiole were punched out of lung tissue slices using a sharpened cannula. These tissue cylinders were oriented, embedded in Epon, serial sectioned until the first alveolar duct bifurcation was reached, and then thin sectioned for electron microscopy. Qualitative examination of the tissue revealed little observable damage to the proximal alveolar tissues. However, by ultrastructure morphometric analysis, significant changes occurred in the alveolar epithelium of the proximal alveolar region of all exposed animals. In the animals exposed to 0.25 ppm O3 from 1 day of age (juvenile animals), the number of type 1 epithelial cells doubled, their mean surface area decreased 38%, and their mean thickness increased 24%. The number of alveolar type 2 epithelial cells increased, and the number of alveolar macrophages doubled. Adult animals exposed to 0.25 ppm O3 showed similar patterns of changes in the epithelium of the proximal alveolar region and in addition had a doubling of interstitial macrophages, indicating a mild inflammatory stimulus in the interstitium. Adult animals exposed to 0.12 ppm O3 showed smaller, but statistically significant changes in the alveolar type 1 epithelium, suggesting a relatively linear concentration-response relationship. The change in number and size of type 1 cells in all exposed animals is consistent with an increased cell turnover rate due to prolonged O3 inhalation. These results suggest that low concentrations of O3 cause a chronic epithelial injury in the proximal alveolar region and that the extent of these changes occurs in a concentration-dependent manner, even at concentrations as low as 0.12 ppm. No statistically significant age-dependent effects were found.
 
Cathepsin B (CB) is involved in the turnover of proteins and has various roles in maintaining the normal metabolism of cells. In our recent study, CB is increased in the muscles of polymyositis/dermatomyositis (PM/DM). However, the role of CB in interstitial lung disease (ILD) has not been reported. ILD is a frequent complication of PM/DM, which is the leading cause of death in PM/DM. It carries high morbidity and mortality in connective tissue diseases, characterized by an overproduction of inflammatory cytokines and induced fibrosis, resulting in respiratory failure. The etiology and pathogenesis of ILD remain incompletely understood. This study investigated whether treatment with CA-074Me, a specific inhibitor of CB, attenuates ILD in PM. CB expression, inflammation, and fibrosis were analyzed in the lung tissues from patients with PM/DM. The animal model of PM was induced in guinea pigs with Coxsackie virus B1 (CVB1). CA-074Me was given 24 h after CVB1 injection for 7 consecutive days. At the end of the experiment, the animals were killed and lung tissues were collected for the following analysis. Inflammation, fibrosis and apoptosis cells, and cytokines were assessed by histological examinations and immunohistochemical analyses, western blot analysis and transferase-mediated dUTP nick-end labeling assay. In patients with PM/DM, the protein levels of CB were significantly elevated in lung tissues compared with healthy controls, which correlated with increases in inflammation and fibrosis. Similarly, the expression of CB, inflammation and fibrosis, CD8(+) T cell, CD68(+) cell, tumor necrosis factor-alpha, transforming growth factor-beta1 infiltrations, and apoptotic cell death were significantly increased in lung tissues of the guinea-pig model of CVB1-induced PM. These changes were attenuated by the administration of CA-074Me. In conclusion, this study demonstrates that PM/DM increases CB expression in lung tissues and inhibition of CB reduces ILD in a guinea-pig model of CVB1-induced PM. This finding suggests that CB may be a potential therapeutic target for ILD.Laboratory Investigation advance online publication, 10 November 2014; doi:10.1038/labinvest.2014.135.
 
Functional integrity of liver cell organelles in rats given the model abrupt cytotoxin 1,1-dichloroethylene (1,1-DCE) was examined by enzymatic histochemistry. Fasted 200-gm. male Sprague-Dawley rats were sacrificed 1, 2, 4, or 6 hours after an oral dose of 200 mg. of 1,1-DCE per kg. (in mineral oil) and 6 hours after 50, 100, or 150 mg. of 1,1-DCE per kg. Cubes of liver were quick frozen for histochemistry. Stage or degree of liver injury was assessed by histology and by measuring serum transaminase activities and liver ion levels. We found both early injury (2 hours following the 200-mg. per kg. dose) and slight injury (6 hours following the 50-mg. per kg. dose) characterized by: increases in liver sodium levels and striking decreases in the central area staining patterns of bile canaliculi membrane Mg++-ATPase, as well as of outer mitochondrial membrane monoamine oxidase and inner mitochondrial membrane succinate dehydrogenase and cytochrome oxidase. As injury progressed with time or increased in severity with dose, aberrations in the levels of other liver cell ions occurred, serum transaminase activities rose, and decreased staining of plasma membrane and mitochondrial membrane components were evident in progressively wider areas around the central vein. Glutathione depletion was panlobular. In contrast, only at later times (4 and 6 hours) and after the larger doses did alterations to functional components of the mitochondrial matrix, endoplasmic reticulum, lysosomes, and cytosol become evident in a narrow area around the central vein, which became necrotic. We consider these later appearing alterations secondary consequences of the midzonal necrosis and sinusoidal congestion produced by 1,1-DCE, whereas the plasma membranes and mitochondrial membranes appear to be primary foci of injury.
 
The long term (70 days) effects of administering ethane-1-hydroxy-1,1-diphosphonate (EHDP) (4 mg. per kg. per day) on parathyroid function was investigated in pregnant cows fed a low calcium diet. Serum calcium and phosphorus were significantly lower at parturition and postpartum in EHDP-treated cows compared to pregnant control cows fed the low calcium diet. Plasma immunoreactive parathyroid hormone levels were similar prepartum, at parturition, and postpartum in cows administered EHDP and control cows. Immediately available calcium reserves were greater preparation in control cows than in cows receiving EHDP as indicated by a more rapid rate of return of serum calcium toward normal levels following ethylenediaminetetraacetic acid (EDTA)-induced hypocalcemia approximately 10 days prepartum. EHDP-treated cows responded to the hypocalcemic challenge with similar changes in plasma immunoreactive parathyroid hormone levels as in control cows; however, urinary hydroxyproline excretion increased at certain intervals only in control cows. Ultrastructurally, chief cells in parathyroid glands of both groups of cows were in an active stage of the secretory cycle with well developed organelles concerned with hormonela synthesis. Chief cells in cows administered EHDP were degranulated and contained fewer secretory granules in response to the hypocalcemia than those in control cows. Chief cells in EHDP-treated cows often had prominent perinuclear accumulations of microfilaments, scattered vacuolated mitochondria, and lysosomal bodies in the cytoplasm. Thyroid C-cells were densely granulated and thyroid calcitonin content was similar in both groups of cows. The principal defect in calcium homeostasis of EHDP-treated cows appeared to be an impairment both in bone calcium mobilization and bone matrix catabolism in response to the secretion of parathyroid hormone. In vitro uptake of 45Ca by duodenal mucosa and urinary excretion of cyclic adenosine monophosphate were similar in both groups of cows. The ability of the parathyroid glands to synthesize and secrete parathyroid hormone in response to hypocalcemia induced either by EDTA or associated with parturition was not impaired by the administration of EHDP.
 
Male CF-1 mice (24 to 34 gm.) were exposed to either 250 p.p.m. or 1000 p.p.m. of 1,1,1-trichloroethane in air continuously for 14 weeks. Control mice were exposed to room air. Serial sacrifice of exposed and control mice from 1 to 14 weeks demonstrated significant changes in the centrilobular hepatocytes of animals in the 1000 p.p.m. group. Moderate liver triglyceride accumulation was evident in the 1000 p.p.m. group and peaked at 40 mg. per gm. of tissue (wet weight) after 7 weeks of exposure. Partial recovery was indicated by a decrease in the hepatic triglyceride level of 16 mg. per gm. by 14 weeks of exposure to 1000 p.p.m. Electron microscopic evaluation revealed that cytoplasmic altertions were most severe in centrilobular hepatocytes in the 1000 p.p.m. group and were mild to minimal in the 250 p.p.m. group. These alterations consisted of vesiculation of the rough endoplasmic reticulum, with loss of attached polyribosomes, increased smooth endoplasmic reticulum, microbodies, and triglyceride droplets. Some cells had ballooned cisternae of the rough endoplasmic reticulum. Necrosis of individual hepatocytes occurred in 40 per cent of the mice exposed to 1000 p.p.m. for 12 weeks. This necrosis was associated with an acute inflammatory infiltrate and hypertrophy of Kupffer cells. Comparison of these findings to the results obtained by other investigators studying dichloromethane indicates that the pathologic alterations observed with 1,1,1-trichloroethane were similar to those observed with dichloromethane, except for different time courses of the effects and different degrees of recovery. The toxic effects of 1,1,1-trichloroethane were of a type similar to those produced by carbon tetrachloride, but they appeared to be much less severe.
 
Studies were performed in vivo using 35S-(1,2-dichlorovinyl)-L-cysteine, a nephrotoxin that damages the S3 segment of the proximal tubule after metabolism to a reactive intermediate. Initiation of damage (35S covalent binding) was complete by 6 hour, and an early proliferative response was observed by 24 hour in the S2 or S3C segments. Necrosis in the S3M and increased blood urea nitrogen were maximal at 48 hours and were accompanied by an increase in proliferation of cells at the wound site. Regeneration was marked by the appearance of vimentin expressing cells that lacked brush border enzymes. The loss of differentiated character in the regenerative epithelium persisted after the proliferation (bromodeoxyuridine incorporation) had stopped; redifferentiation occurred between days 5 and 13. Much of the process was reproduced by culturing rat kidney proximal tubule epithelial cells in defined medium. As growth increased, the cells expressed vimentin and lost brush border marker enzymes. However, as the cells reached high density and stopped dividing there was an increase in brush border markers, as was seen in vivo. Vimentin expression did not decrease, however. The data support a mechanism for damage and nephrogenic repair composed of 1) interaction of the toxin with the target cells, 2) necrosis and exfoliation, 3) loss of differentiation and cell growth, 4) recovery of the damaged area and cessation of cell growth, and 5) differentiation of the quiescent cells. Nephrogenic repair may have similarities with the differentiation of the tubular epithelium during development.
 
Qualitative changes of glycoconjugates in luminal surface and goblet cell mucin from colon mucosa of 1,2-dimethylhydrazine (DMH)-treated rats were studied. Eight fluoresceinated lectins were used: Dolichos biflorus (DBA), Glycine max (SBA), Triticum vulgare (WGA), Limax flavus (LFA), Arachis hypogaea (PNA), Griffonia simplicifolia-I (GS-I), Ulex europaeus-I (UEA-I) and Canavalia ensiformis (Con A). The lectin-binding patterns were studied in tumors arising in proximal and distal portions of the colon, in transitional mucosa (TM) and in mucosa distant from tumors. Lectin reactivity observed in mucosa of DMH-treated rats was compared with that obtained in colon mucosa of control rats. In tumors and non-neoplastic mucosa of DMH-treated rats the reactivity of DBA, SBA, WGA, LFA, GS-I and Con A were similar to that in the mucosa of control rats. In contrast, important changes were observed in the reactivity of UEA-I and PNA. Contrary to the staining in the control mucosa, UEA-I bound intensely to all carcinomas and PNA to 50% and 60% of carcinomas arising in proximal and distal colon, respectively. Moreover, in TM and mucosa distant from tumors, UEA-I and PNA also differed in their binding patterns to that obtained in the colonic mucosa of the control rats. UEA-I- and PNA-binding to luminal surface and UEA-I-binding to the mucin of distal colonic mucosa from DMH-treated rats was similar to that observed in rat fetal colon suggesting a reappearance of a fetal-type pattern. Contrarily, PNA-reactivity in goblet cell and carcinoma mucin is a unique feature of colonic carcinogenesis not present during fetal development.
 
The purpose of this investigation was to evaluate, by static and dynamic histomorphometry, the comparative influence of dietary calcium on the effect of 1,25(OH)2D3 on bone. Young adult female rats were fed a diet containing 0.3% phosphorus and 0.05%, 0.5%, or 1.0% calcium. Rats in each group received placebo, or 27 ng or 135 ng of 1,25(OH)2D3, intraperitoneally, for 10 days. A dramatic osteosclerosis and hyperosteoidosis characterized by an increase in metaphyseal hard tissue percentage and percentage metaphyseal osteoid, a decrease in osteoclasts per millimeter of trabecular surface perimeter and longitudinal growth, and hypertrophy of osteoblasts developed in the proximal tibial metaphysis of rats fed 1.0% calcium diet and given 135 ng of 1,25(OH)2D3 compared with placebo-treated rats fed the same diets. Similar histomorphometric changes, with the exception of increased metaphyseal hard tissue percentage, occurred in rats fed 0.5% dietary calcium and treated with 135 ng of 1,25(OH)2D3. Metaphyseal hard tissue percentage, percentage metaphyseal osteoid, and osteoclasts per millimeter of trabecular surface perimeter were not affected by treatment with 135 ng of 1,25(OH)2D3 in rats fed a 0.05% calcium diet. Serum calcium was significantly elevated in all 1,25(OH)2D3-treated rats. Dietary calcium appears to modulate the effects of 1,25(OH)2D3 on bone. Only diets containing adequate dietary calcium permit exogenous pharmacologic levels of 1,25(OH)2D3 to produce a positive skeletal balance either by decreasing osteoclasis and/or increasing bone matrix synthesis.
 
Effects of 1,25(OH)2D3 on the binding activity of transcription factors to the promoter region of the TF gene. A probe containing AP-1 (a), NF-B (b), or Egr-1 sites (c) of the TF promoter was incubated with nuclear extracts isolated from THP-1 or U937 cells. Nuclear extracts were prepared from 5 106 cells. Cells were treated with (lane 3) or without (lane 2) 1,25(OH)2D3 (0.1 M) for 120 min. Before the addition of TNF (1 nM) for 60 min, cells were pretreated and maintained with (lane 5) or without (lane 4) 1,25(OH)2D3. Gel mobility shift assay was performed using the probe. Lane 1 corresponds to the DIG-labeled probe only. To demonstrate the specificity of protein binding, cold competitor oligonucleotides containing each site or the respective antibodies against transcription factors were used. A 100-fold molar excess of cold competitor oligonucleotides containing each site were included in control samples (lane 6). The respective antibodies (100 g/ml), anti-c-Jun, anti-p65, and anti-Egr-1, were included in TNF-treated samples (lane 7 indicated by the competitor with an asterisk). Data are representative of six independent experiments using each probe and confirmed by corrected densitometric quantification.
Effect of 1,25(OH)2D3 on TNF-induced activation of the TF gene. THP-1 cells were transfected with the pGL3 reporter vector containing p(AP-1)4SV40LUC (a), p(NF-B)4SV40LUC (b), or p(Egr-1)4SV40LUC (c). As a control vector, the pRL-SV40 vector was cotransfected, which expresses Renilla luciferase. pCMX-hVDR and pCI-RXR were cotransfected into cells to detect statistically significant differences in expression. After transfection for 60 h, cells were divided into four equal portions. Cells were preincubated with (column 2) or without (column 1) 1,25(OH)2D3 (0.1 M) for 6 h. Alternatively, cells were incubated with TNF (1 nM) for 5 h in the presence (column 4) or absence (column 3) of a 1-h pretreatment with 1,25(OH)2D3 (0.1 M). LUC activity was used to determine fold changes (meanss.d.) in six experiments.
Effect of 1,25(OH)2D3 and VDR complex on phosphorylation of c-Jun. THP-1 cells were preincubated with 1,25(OH)2D3 (0.1 M) (lanes 2 and 4) for 1 h, followed by stimulation with TNF (1 nM) for 15 min (lanes 3 and 4). Lane 1 shows untreated THP-1 cells as a control. After treatment, cells were lysed and immunoprecipitated, and the immunoprecipitates were immunoblotted using anti-c-Jun antibody as described in Materials and methods. Data are representative of three independent experiments.
An essential coagulation factor, tissue factor (TF), is rapidly expressed by human monocytes when exposed to a variety of agonists, such as lipopolysaccharide or tumor necrosis factor (TNF). We previously found that 1alpha,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) and its potent synthetic analogs downregulate TF and upregulate thrombomodulin expression on monocytic cells, counteracting the effects of TNF at the level of transcription. The human TF gene has characteristic binding sequences for activator protein-1 (AP-1) (c-Jun/c-Fos), nuclear factor-kappaB (NF-kappaB), Sp-1, and early growth response factor-1 (Egr-1). In this study, we investigated the regulatory mechanisms by which 1,25(OH)(2)D(3) inhibits TNF-induced TF expression in human monocytic cells. 1,25(OH)(2)D(3) reduced basal and TNF-induced TF activities. Gel-shift assay and luciferase assay with the respective reporter vectors showed that 1,25(OH)(2)D(3) reduced basal and TNF-induced activities of the nuclear proteins AP-1 and NF-kappaB, but not Egr-1. 1,25(OH)(2)D(3) inhibited TNF-induced phosphorylation of c-Jun without affecting phosphorylation of the other pathways. On the other hand, 1,25(OH)(2)D(3) directly inhibited nuclear binding and activities of NF-kappaB in the nucleus without affecting phosphorylation of the NF-kappaB activation pathway. These results indicate that 1,25(OH)(2)D(3) suppresses basal and TNF-induced TF expression in monocytic cells by inhibition of AP-1 and NF-kappaB activation pathways, but not of Egr-1. Our results may help to elucidate the regulatory mechanisms of 1,25(OH)(2)D(3) in TF induction, and may have physiological significance in the clinical challenge to use potential 1,25(OH)(2)D(3) analogs in antithrombotic therapy as well as immunomodulation and antineoplastic therapy of leukemia.
 
alpha1,4-N-acetylglucosaminyltransferase (alpha4GnT) is a glycosyltransferase that forms a unique glycan, GlcNAcalpha1-->4Galbeta-->R, specifically present in gastric gland mucous cell-type mucin. Recently, we molecularly cloned human alpha4GnT and showed that alpha4GnT is expressed in the mucous cells that secrete this particular mucin. In the present study, we first demonstrated that alpha4GnT was frequently expressed in gastric cancer cells but not in peripheral blood cells using immunohistochemistry. To detect gastric cancer cells circulating in the peripheral blood of gastric cancer patients, we quantitatively analyzed the expression level of alpha4GnT mRNA in the mononuclear cell fraction of peripheral blood using real-time reverse transcription polymerase chain reaction. The transcripts of alpha4GnT were detected in the mononuclear cell fraction isolated from 62.2% of 37 gastric cancer patients but not from any of 23 healthy individuals. Significant correlation was found in the expression levels of alpha4GnT mRNA in peripheral blood and alpha4GnT protein in gastric cancer cells. Surprisingly, alpha4GnT mRNA was detectable in 80% of five patients with an early stage of gastric cancer when the cancer cells were limited to the gastric mucosa, and the expression levels of alpha4GnT mRNA were increased in association with tumor progression. In three patients with gastric cancer, during postsurgical follow-up, the expression levels of alpha4GnT mRNA were decreased after surgical removal of gastric cancer. However, significant amounts of the alpha4GnT transcripts were again detected in two patients, who eventually developed to the recurrence of gastric cancer. Although alpha4GnT was detected in 33.3% of nine patients with Helicobacter pylori-infected chronic active gastritis as well as all of four patients with peptic ulcer, the mean expression level of alpha4GnT mRNA in these benign disorders was lower than that in gastric cancer. These results altogether indicate that the quantitative analysis of alpha4GnT mRNA expressed in the peripheral blood is useful for the detection and, possibly, monitoring of gastric cancer.
 
Many acute and chronic liver diseases are often associated with atypical ductular proliferation (ADP). These ADPs have gained increasing interest since a number of recent observations suggest that ADPs may represent progenies of the putative liver stem cell compartment. In this study, we show that feeding mice with 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) results in persistent proliferation of primitive ductules with poorly defined lumens. Similar to oval cell proliferation in other rodent models as well as in various human liver diseases, DDC-induced ADP originated from the portal tract, spread into the hepatic lobule, and was associated closely with appearance of hepatocytes harboring an antigen (A6), which normally is expressed in biliary epithelium. Furthermore, DDC treatment severely inhibited the regenerative capacity of mice after partial hepatectomy. The development of ADP was selectively blocked in DDC-fed TGF-beta1 transgenic mice producing active TGF-beta1 in the liver and no accumulation of new hepatocytes expressing the A6 antigen was observed. Moreover, the transforming growth factor beta1 (TGF-beta1) transgenic mice did not survive beyond 3 weeks from starting the DDC-containing diet. The results suggest that persistent activation of the hepatic stem cell compartment is essential for liver regeneration in the DDC model and that active TGF-beta1 may negatively control activation of stem cells in the liver. These data further emphasize the relevance of the DDC model as an experimental tool for studying chronic liver diseases.
 
Osteoclast activity is central to balanced bone turnover to maintain normal bone mass. A specialized osteoclast attachment to bone localizes acid secretion to remove bone mineral; in some cases, attachment is functionally impaired despite normal attachment proteins. The inositol-1,4,5-trisphosphate receptor-1 (IP3R1) is an intracellular calcium channel required for regulation of reversible osteoclast attachment by nitric oxide (NO), an important regulator of both normal and pathological bone degradation. In studies using human osteoclasts produced in vitro, we found that IP3R1 binds an endosomal isoform of the IP3R-associated cGMP-dependent kinase substrate (IRAG). IRAG is a substrate of cGMP-dependent kinase-1 (PKG1) and binds the PKG1 isoform PKG1β, which was the predominant form of PKG1 in human osteoclasts. Western blots of IRAG were consistent with NO-dependent serine phosphorylation of IRAG. An additional effect of PKG1β activity in osteoclasts was disassociation of IP3R1-IRAG complexes, as shown by analysis of IP3R1 complexes and by localization of the proteins within cells. IP3R1-IRAG complexes were stabilized by PKG or Src antagonists, Src activity being a requirement for IP3R1 calcium release downstream of PKG. IP3R1-mediated calcium release regulates cellular detachment in part through the calcium-dependent proteinase μ-calpain. In osteoclasts with IRAG suppressed by siRNA, activity of μ-calpain was increased relative to cells with normal IRAG, and regulation of μ-calpain by NO was lost. Furthermore, cells deficient in IRAG detached easily from substrate and had smaller attached diameters and randomly distributed podosomes, although IRAG knockdown did not affect cell viability. Our results indicate that IRAG is required for PKG1β-regulated cyclic calcium release during motility, and that disruption of the IP3R1-IRAG calcium regulation system is a novel cause of dysfunctional osteoclasts unrelated to defects in attachment proteins or acid secretion.
 
The present report describes the natural history of an experimental acute glomerulonephritis with massive proteinuria induced by a single intravenous injection of a (mouse) monoclonal anti-rat Thy 1.1 antibody into the rat. The disease is characterized by direct although transient binding of this monoclonal antibody to glomerular basement membrane and mesangium after injection as demonstrated by immunofluorescence microscopy. Immediate activation of complement occurs as shown by glomerular deposition of C3 and C9. Concomitant activation of the coagulation cascade is reflected by the presence of fibrinogen deposits in the affected glomeruli. One hour after injection mesangial alterations are prominent including condensation of mesangial cell chromatin, and lysis of mesangial cells as shown by light- and electron- microscopy, leading to the formation of aneurysms in the capillary tuft. Commencing on day 4 mesangial cell proliferation can be observed, accompanied by glomerular crescent formation at day 14, which decreases gradually 3 weeks after antibody administration, whereas mesangial hypercellularity can be observed up week 10 after intravenous injection of the antibody. The disease is clinically characterized by a massive transient proteinuria starting immediately after antibody injection, reaching mean values of 300 mg/24 hours at days 2 to 4, gradually decreasing to normal levels after 3 weeks. It is concluded that in this unique model of glomerulonephritis induced by a monoclonal antibody, recognition of Thy 1.1 epitopes as well as activation of complement including the C5-C9 membrane attack complex may play a major role in the pathogenesis of this experimental disease.
 
The phenotype of macrophages invading the mesangium and periglomerular region has not been described in experimental mesangial proliferative glomerulonephritis, although it has implications for the mechanism of entry of these cells into these locations and their function once there. We have, therefore, determined the phenotype of the periglomerular leukocytic infiltrate in the Thy 1.1 model of mesangial proliferative glomerulonephritis and compared it with that of cells invading the mesangium. The Thy 1.1 model was induced in Lewis rats, and sections were taken at 1 hour and at 1, 4, 9, 30, and 90 days postinduction. Immunohistochemistry was performed using monoclonal antibodies against macrophage markers (ED-1, CD4, RT1-B, and ED-2), chains of the beta 2 integrins (CD18, CD11a, and CD11b), T and B cell markers (CD8, T cell receptor, interleukin-2 receptor, and MRC OX33), and markers of mesangial cell proliferation (proliferating cell nuclear antigen, alpha-smooth muscle actin). Sections were compared with those obtained from control animals. In untreated rats, a striking resident periglomerular macrophage population (phenotype ED-1-ve ED-2-ve CD4+ve RT1-B+ve CD18+ve CD11a+ve CD11b+ve) was found, confirming a previous report. From 24 hours postinduction, this resident macrophage population was supplemented by a population whose predominant phenotype (ED-1+ve ED-2-ve CD4+ve RT1-B+ve CD18+ve CD11a+ve CD11b+ve) was identical to that of macrophages infiltrating the mesangium. Both infiltrates peaked at 4 days and returned to base-line levels by 1 to 3 months. There was no significant lymphocyte infiltrate within the glomerulus and only a minimal periglomerular T cell infiltrate. These data show, first, that disease limited to the mesangium can lead directly to periglomerular macrophage infiltration. Second, the presence of CR3 (i.e., CD11b and CD18) and LFA-1 (i.e., CD11a and CD18) on the macrophage infiltrate indicates that both ligands are important for cells to enter the mesangium and periglomerular areas. Third, the marked phenotypic and temporal similarities between the mesangial and periglomerular macrophage infiltrates suggests that a common factor(s) is involved in their pathogenesis. Finally, expression of RT1-B (Ia) but not ED-2 is reported to be typical of interstitial dendritic cells rather than tissue macrophages, suggesting a unique function for the glomerular and periglomerular macrophage infiltrate in this model.
 
Four PN/n mice between 12 and 17 months of age had fibrinoid necrosis of the renal arteries, but 32 mice up to the age of 17 months showed no consistent change in the media or intima of the renal arteries although perivascular cuffing with round cells was prominent from 9 months of age. Glomerulitis, indicated by increase in the mesangium on light microscopy, affected most mice over 7 months of age, and dense deposits on electron microscopy were present in most mice from 7 months of age. These changes occurred significantly more frequently than in CBA/MAC mice; the 101/MAC showed an intermediate frequency, and the pattern in a small sample of NZB/BL mice resembled that of the PN/n mice. Basement membrane thickening increased with age from 3 weeks in all of the strains but more often included dense deposits in PN/n mice. Splitting of the glomerular basement membrane in very young mice in all strains was not considered relevant and may have been developmental. Failure consistently to detect glomerulitis earlier by electron microscopy than by light microscopy was attributed to the greater sampling error of the former. Unidentified dense bodies in glomerular epithelial cells occurred at all ages in all strains, so the suggestion that they were virus particles relevant to the disease was not sustained. Intraobserver variation studies showed satisfactory repeatability for light microscopy and for dense deposits and basement membrane thickening, but the dense bodies were not detected with acceptable reproducibility, a difficulty partly due to technical variation.
 
Loss of the epithelial phenotype and disruption of adhesion molecules is a hallmark in the epithelial-mesenchymal transition (EMT) reported in several types of cancer. Most of the studies about the relevance of adhesion and junction molecules in lung cancer have been performed using established tumors or in vitro models. The sequential molecular events leading to EMT during lung cancer progression are still not well understood. We have used a rat model for multistep lung carcinogenesis to study the status of adherens and tight junction proteins and mesenchymal markers during EMT. After silica-induced chronic inflammation, rats sequentially develop epithelial hyperplasia, preneoplastic lesions, and tumors such as adenocarcinomas and squamous cell carcinomas. In comparison with normal and hyperplastic bronchiolar epithelium and with hyperplastic alveolar type II cells, the expression levels of E-cadherin, alpha-catenin and beta-catenin were significantly reduced in adenomatoid preneoplastic lesions and in late tumors. The loss of E-cadherin in tumors was associated with its promoter hypermethylation. alpha- and beta-catenin dysregulation lead to cytoplasmic accumulation in some carcinomas. No nuclear beta-catenin localization was found at any stage of any preneoplastic or neoplastic lesion. Zonula occludens protein-1 was markedly decreased in 66% of adenocarcinomas and in 100% squamous cell carcinomas. The mesenchymal-associated proteins N-cadherin and vimentin were analyzed as markers for EMT. N-cadherin was de novo expressed in 32% of adenocarcinomas and 33% of squamous cell carcinomas. Vimentin-positive tumor cells were found in 35% of adenocarcinomas and 88% of squamous cell carcinomas. Mesenchymal markers were absent in precursor lesions, both hyperplastic and adenomatoid. The present results show that silica-induced rat lung carcinogenesis is a good model to study EMT in vivo, and also provide in vivo evidence suggesting that the changes in cell-cell adhesion molecules are an early event in lung carcinogenesis, while EMT occurs at a later stage.
 
Ank is a 492-amino acid multipass transmembrane protein involved in the regulation of extracellular inorganic pyrophosphate levels and the control of tissue calcification. Previous Northern blot hybridization experiments revealed that Ank mRNA was expressed in the brain, but there have been no reports describing the anatomical sites or specific cell types in the brain that express Ank protein. In this study, we demonstrate that Ank is expressed primarily in human brain neurons, with the highest levels of expression observed in the thalamus, the III and V cortical layers, the Purkinje cells of the cerebellum, clusters of cells in the dorsal portion of the pons and midbrain, and neurons of the anterior horn of the spinal cord. In primary mouse neuronal cell cultures, Ank is detected on both the cell body and on cell extensions, mainly dendrites. In the rat brain, Ank mRNA is expressed at relatively high levels in the thalamus, midbrain, and spinal cord, and the Ank protein expression pattern is similar to that observed in the human brain. Finally, we observed a significant increase in Ank immunoreactivity in the rat amygdala, the CA-2 and CA-3 layers of the hippocampus, and the cerebral cortex after the induction of seizure activity. Ank regulation of ATP and/or inorganic pyrophosphate release from neurons may function to modulate the membrane excitability and cell death associated with seizure activity.
 
Expression of the DNA repair protein O6-alkylguanine-DNA-alkyltransferase (AGT), encoded by the O6-methylguanine (O6-mG) -DNA-methyltransferase (MGMT) DNA repair gene, results in resistance to alkylating agents, and hypermethylation of the MGMT promoter is associated with chemosensitivity as it prevents AGT expression. As the interpretation of the results of immunohistochemistry to evaluate AGT expression proved to be difficult, the aim of our present study is to establish a feasible, reliable, and robust method for MGMT promoter hypermethylation testing that can be easily implemented in a diagnostic setting and is applicable to routinely processed tissue. MGMT hypermethylation analysis using methylation-specific (MS-) multiplex ligation-dependent probe amplification (MLPA) was performed on 62 glioma samples of 55 individual tumors (including 12 cell lines) and compared to the more conventionally used, but improved, MS-polymerase chain reaction (PCR). In contrast to MS-PCR, MS-MLPA (i) is not based on bisulfite conversion of unmethylated cytosines (a somewhat troublesome step in MS-PCR), (ii) provided methylation status of all samples, (iii) proved to be semiquantitative, (iv) can be used to evaluate methylation status of multiple sequences (CpG dinucleotides) simultaneously, and (v) allows for a combined copy number detection and methylation specific analysis. The potential therapeutic value of MGMT hypermethylation evaluation using MS-MLPA was shown in a group of 20 glioblastoma patients receiving temozolomide chemotherapy. We conclude that MS-MLPA is a robust and reliable method that can be easily applied to differently processed tissues, including those fixed in formalin and embedded in paraffin. The semiquantitative aspect of MS-MLPA may prove to be of great value, especially in predicting response to alkylating agents, not only for gliomas as evaluated in this study but also for tumors in general.
 
Neprilysin (EC 3.4.24.11) (NEP), a membrane metallopeptidase, is identical with common acute lymphoblastic leukemia antigen or cluster differentiation antigen 10. This antigen is present in blast cells in acute lymphoblastic leukemias and is implicated in differentiation of B lymphocytes. NEP cleaves a variety of peptides including bradykinin, substance P, bombesin, enkephalins, and atrial natriuretic peptide. We investigated its expression in several variants of rat hepatomas and a human hepatocellular carcinoma cell line. Normal rat and human livers were used as controls. The expression of NEP (common acute lymphoblastic leukemia antigen) was determined with: (a) enzyme assays; (b) high performance liquid chromatography analysis of bradykinin metabolism; (c) immunoprecipitation; and (d) mRNA characterization. NEP activity increased by 2 to 3 orders of magnitude in all rat hepatomas and in the human SK-HEP1 cell line, compared with normal tissues. Antiserum against rat NEP precipitated 93% of endopeptidase activity in rat hepatomas, whereas monoclonal antibody to common acute lymphoblastic leukemia antigen immunoprecipitated 99% of that in human hepatocarcinoma cells. Solubilized rat hepatoma membranes cleaved bradykinin to a hepta- and dipeptide; the reaction was inhibited by an NEP inhibitor. Activity of three other membrane peptidases did not increase in rat hepatomas. Northern hybridization revealed the presence of NEP mRNA in rat hepatoma, but not in normal liver. Reverse transcriptase-polymerase chain reaction showed that hepatomas have higher amounts of NEP mRNA than normal liver of the same strain. Rat hepatomas and a human hepatocarcinoma cell line express high amounts of NEP, in contrast to normal rat and human livers, which have very little. The increase in NEP activity could be due to increased transcription by tumor cells and may signal malignant transformation of liver cells.
 
The aim of this study was to investigate the extracellular degrading proteolytic cascade proteins referred to as matrix metalloproteinase-1 (MMP-1), MMP-2, MMP-9, membrane-type matrix metalloproteinase-1 (MT1-MMP), tissue inhibitors of matrix metalloproteinase-1 (TIMP-1), TIMP-2, neutrophil elastase, and alpha1-antitrypsin in human pulmonary emphysema. Localization of MMP-1, MMP-2, MMP-8, MMP-9, MT1-MMP, TIMP-1, and TIMP-2 was verified by immunohistochemical analysis. The results of our study indicated that the immunoreactivity of MMP-1, MMP-8, MMP-9, and TIMP-1 was absent, whereas MT1-MMP and MMP-2 were mainly observed in pneumocytes, fibroblasts, and alveolar macrophages. Although MT1-MMP and MMP-2 were observed both in emphysematous and normal lung tissue, these immunoreactivities were intense in the emphysematous samples. The presence of MMP-1, MMP-2, MMP-9, TIMP-1, and TIMP-2 was confirmed at mRNA level by reverse transcription-PCR analysis and enzyme immunoassay (EIA). However, the only statistical difference that was observed was in MMP-2 and MMP-9 (MMP-2: emphysematous samples, 19.1+/-2.1 versus control samples, 5.2+/-0.60 microg/g protein, p < 0.05; MMP-9: emphysematous samples, 18.4+/-5.6 versus control samples, 8.1+/-2.7 microg/g protein, p < 0.05). Results of the neutrophil elastase as analyzed by EIA, and alpha1-antitrypsin levels as detected by laser nephelometric immunoassay, indicated no statistical difference between the emphysematous and control groups. In addition to the presence of mRNA levels, the level of MT1-MMP according to immunoblot analysis increased in the emphysematous samples. Gelatin zymographic analysis confirmed the presence of both pro and active forms of MMP-2, and the increased ratio of the active form of MMP-2 in emphysematous samples (25.9%+/-2.0% versus 11.2%+/-3.3%, p < 0.05), indicated in situ activation of MMP-2 by MT1-MMP. Elastin zymographic analysis showed elastolytic activity by MMP-2 and MMP-9 but not the reported band of macrophage metalloelastase (MMP-12). The data suggest that the MT1-MMP/MMP-2/TIMP-2 system plays a significant role in the MMP-mediated extracellular matrix degradation and tissue remodeling of emphysematous lungs, and thus may contribute to the weakening of lung parenchyma and lead to the formation of emphysema.
 
The effects of transforming growth factor-beta 1 (TGF-beta 1) on the regulation of basement membrane gene expression were studied in human fibrosarcoma HT-1080 cell cultures. Treatment of cells with TGF-beta 1 resulted in a time- and dose-dependent enhancement of type IV collagen and fibronectin gene expression, as detected both at the mRNA level by Northern hybridizations and at the protein level by semi-quantitative indirect immunofluorescence analyses. These changes were accompanied by profoundly altered morphology of the cell cultures. In contrast, laminin B1 and B2 chain, and nidogen mRNA levels remained unaltered by TGF-beta 1 in the same cultures, indicating uncoordinate modulation of the expression of basement membrane components by TGF-beta 1. Cycloheximide experiments provided evidence that the TGF-beta 1-elicited upregulation of the expression of the fibronectin gene is, but that of type IV collagen is not, entirely dependent on protein synthesis, suggesting two different mechanisms for enhancement of gene expression. Incubation of HT-1080 cells with TGF-beta 1 also resulted in a slightly enhanced expression of beta 1 and alpha 5 integrin mRNAs, as well as beta 1 integrin epitopes. Furthermore, incubation of the cells with anti-beta 1 integrin antibodies partially counteracted the TGF-beta 1-induced morphologic alterations. These studies provide evidence for the role of beta 1 integrins in TGF-beta 1 elicited alterations in HT-1080 cell culture morphology, possibly mediated by the expression of ligand proteins for these integrins, such as type IV collagen and fibronectin.
 
Utilizing a cDNA expression library established from human prostate PC-3ML tumor cells, we have cloned a truncated flt-4 gene, termed flt-4t(Delta773-1081). We have then utilized RNase protection and ELISA to measure the relative levels of VEGF B, C, D and flt-1, KDR, flt-4 and flt-4t(Delta773-1081) expression in freshly isolated benign prostatic hyperplasia or BPH tissue (n=21), primary prostate cancers (n=82) and matching sentinel lymph node metastases from stage T2a-T2b/T3 tumors (n=52). Comparisons of the primary tumors with BPH showed that there was a significant upregulation of VEGF-B (P=0.003), VEGF D (P=0.005), flt-1 (P=0.003), KDR (P=0.002), flt-4 (P=0.007), and flt-4t(Delta773-1081) (P=0.001), but not VEGF-C (P=0.543). There was no correlation between VEGF-B and its receptor flt-1 (P=0.545), or VEGF-C and flt-4 (P=0.16) and KDR (P=0.23) receptor expression in tumor specimens. Conversely, there was no significant relationship between VEGF-D and the flt-4t(Delta773-1081) receptor (P=0.516) expression. Statistical analysis further showed that there was no significant correlation between VEGF-B, VEGF-C, VEGF-D, flt-1, KDR, flt-4 and flt-4t(Delta773-1081) with patient age (P>0.10), stage (P>0.10), PSA value (P>0.15) or tumor size (P>0.15). Likewise, there was no significant correlation between VEGF-B, VEGF-C, flt-1, KDR, and flt-4 with Gleason score (P>0.15). In comparison, flt-4t(Delta773-1081) levels clearly increased significantly in Gleason score 7 and Gleason score 8-10 tumors as well as in stage T2a-T2b/T3 tumors. The studies were extended to compare gene expression profiles in T2a-T2b and T3 tumors with (n=26) and without (n=26) matching sentinel lymph node metastases. The data showed that VEGF D and flt-4t(Delta773-1081) expression levels were significantly elevated in primary tumors with sentinel lymph node involvement compared to those lacking lymph node involvement (P>0.0022 and 0.006, respectively). These data suggest that targeting VEGF D and flt-4t(Delta773-1081) receptors may be particularly effective in the prevention of lymph node metastases.
 
Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors of gastrointestinal tract. GISTs range from benign indolent neoplasms to highly malignant sarcomas. Gain-of-function mutations of tyrosine kinase receptors, KIT or PDGFRA, have been identified in most GISTs. In this study, we report 36 GIST patients whose tumors had homozygous KIT exon 11 mutations detected by direct sequencing of PCR products. Loss of heterozygosity in KIT locus and other chromosome 4 loci were documented in majority of these tumors. However, fluorescence in situ hybridization with KIT locus-specific probe and chromosome 4 centromeric enumeration probe showed no evidence of KIT hemizygosity in a majority of analyzed cases. These findings are consistent with duplication of chromosome 4 with KIT mutant allele. Homozygous KIT exon 11 mutations were found in 33 primary tumors and 7 metastatic lesions. In two cases, shift from heterozygosity to homozygosity was documented during tumor progression being present in metastases, but not in primary tumors. Among primary GISTs, there were 16 gastric, 18 intestinal and 2 from unknown locations. An average primary tumor size was 12 cm and average mitotic activity 32/50 HPFs. Out of 32 tumors 29 (90.6%) with complete clinicopathologic data were diagnosed as sarcomas with more than 50% risk of metastatic disease, and 26 of 29 patients with follow-up had metastases or died of disease. An average survival time among pre-imatinib patients, who died of the disease was 33.4 months. Based on these findings, we conclude that presence of homozygous KIT exon 11 mutations is associated with malignant course of disease and should be considered an adverse prognostic marker in GISTs.
 
Data on p53 alterations in human cancers are mainly based on studies restricted to the core domain (exons 5-9), because mutations outside this region are assumed to be rare. To test this assumption, we studied 25 consecutive patients with primary, untreated head and neck squamous cell carcinoma (HNSCC) with a p53 mutation analysis strategy that consists of sequencing all 11 p53 exons and the complete p53 mRNA. With this method, we encountered p53 mutations in 91% of patients; 33% of these were located outside the core domain. Overexpression of the p53 protein was assessed with staining with antibody Bp 53-12-1. Protein overexpression was found in 64%. In one case, p53 overexpression occurred without p53 gene mutations. Analysis of tumor tissue from two autopsied patients with multiple lesions in the head and neck and at distant sites allowed analysis of the clonal relationship of the different tumor foci. In one patient, the head and neck lesion had a mutation different from the one observed at the distant sites, suggesting two different primary tumors, one of them leading to widespread metastastic disease. In all lesions from the second patient, the same mutation was found, suggesting one primary that had metastatized. It appears that sequencing of all exons of the p53 gene is vital for assessment of the real incidence of p53 mutations in HNSCC, because 33% of all mutations are located outside the core domain, leading to a mutation frequency of almost 100% in HNSCC. In 96% of cases, either presence or absence of p53 protein expression could be explained by the type of p53 gene mutation. When only analyzing the p53 core domain, the incidence of p53 mutations in HNSCC is underestimated.
 
Previous studies have shown that acute injury to rodent small intestinal mucosa as a result of either chemotherapy or radiation is caused by a combination of high-frequency cell death caused by apoptosis, continued migration of epithelial cells from the intestinal crypts toward the villi, and absence of adequate compensatory mitotic activity in the crypt bases. Recently, we have shown that IL-11, a novel multifunctional bone marrow stromal-derived growth factor, could stimulate rapid repair of small intestinal villous structures in mice treated with combined radiation and chemotherapy by increasing the mitotic index of crypt cells. To further clarify the biological mechanism responsible for its protective action, we used a similar experimental model to evaluate whether IL-11 could reduce the high frequency of apoptosis observed after cytoablative treatment. In the present study, 78 C3H/HeJ mice received 5-fluorouracil at 150 mg/kg body weight intraperitoneal injection 3 days before 7.0-Gy total body irradiation. The animals received IL-11 250 microg/kg body weight/day divided into two equal doses or vehicle control by subcutaneous injections beginning on the same day of irradiation within 1 hour of the end of the dose. The mice killed on Days 1, 2, and 5 after cytoablative treatment were autopsied, the small intestine was processed for histologic examination, and the mitotic index and other parameters were measured, including the expression of the proliferating cell nuclear antigen and of the p53 protein. Apoptosis was detected by a nonisotopic in situ DNA end-labeling technique applied to the same histologic sections. The cytoablative treatment caused marked degenerative changes in the small intestinal mucosa, including shortening of the villi and damage to the crypt cells. The degenerative changes, which included nuclear fragmentation with formation of apoptotic bodies, increased expression of proliferating cell nuclear antigen, and strong expression of p53 was seen in mice killed on Days 1 and 2 after cytotoxic treatment. IL-11 administration resulted in a partial suppression of apoptosis, as shown by a reduced number of crypt cells stained with in situ DNA end-labeling for fragmented DNA. In addition, IL-11 treatment was associated with an increase in the frequency of mitosis and proliferating cell nuclear antigen expression in crypt cells as compared with the vehicle treated mice. Morphometric analysis of intestinal villi and crypt depth showed increased villus length and decreased crypt to villus ratio after IL-11 treatment. These results indicate that IL-11 can exert a potent effect on the recovery of the small intestinal mucosa of mice by its combined effects on proliferation and apoptosis of crypt cells. IL-11 may thus have potential clinical applications in limiting the intestinal toxicity that is associated with cytotoxic therapies.
 
Renal failure was induced in 15 normal Beagle dogs by ligation of approximately 5/6 of the renal arteries of the left kidney and contralateral nephrectomy in order to determine how: (a) 11/12 reduction in total renal mass influences urine protein excretion and renal morphology in dogs, and (b) dietary protein intake influences renal function, urine protein excretion, and renal morphology in canine renal failure. Dogs were fed a reduced protein diet for 12 weeks after induction of renal failure, while compensatory renal hypertrophy developed. Renal function was then evaluated and dogs were distributed into 2 groups with approximately equal degrees of renal dysfunction. One group was fed a high protein diet (42% protein) and a second group was fed moderately restricted protein diets (18% protein). After 8 weeks, renal function, magnitude of proteinuria, and renal morphology were re-evaluated. Inulin clearance increased in all dogs fed the 42% protein diet and 3 of 10 dogs fed the 18% protein diets. Proteinuria was significantly greater in dogs fed the high protein diet than dogs fed the reduced protein diets. Compared with previously nephrectomized contralateral control kidneys, glomerular sclerosis and renal interstitial lesions had developed in all dogs, regardless of severity of renal dysfunction or diet fed. Although reduced dietary protein intake did not prevent development of renal lesions, renal lesions were significantly more severe in the 5 dogs fed the 42% protein diet and 3 dogs fed the 18% protein diets in which inulin clearance increased, than in 7 dogs fed the reduced protein diets in which inulin clearance did not increase.
 
Natural killer (NK) cell subpopulations, identified by anti-Leu-7 and/or anti-Leu-11 monoclonal antibodies, have been investigated in immunoelectron microscopy by using a peroxidase-colloidal gold double labeling system. More than 90% of non-E rosetting/non-adherent Leu-11+ cells and approximately 60% of E rosetting Leu-11+ cells displayed phagocytic capacity for 2-aminoethylisothiouronium bromide hydrobromide treated sheep red blood cells, whereas Leu-7+ -Leu-11- cells consistently failed to ingest 2-aminoethylisothiouronium bromide hydrobromide treated sheep red blood cells. The phagocytic activity of Leu-11+ cells was not dependent on the Leu-7 antigen coexpression. Moreover, the NK capability of Leu-11+ cells has been verified. In both E rosetting and non-E rosetting/non-adherent cell populations, Leu-11+ enriched subsets displayed significantly higher NK capability when compared with Leu-11- cells. Previous reports provided evidence for different cytotoxic capability, recombinant human interleukin 2 mediated activation, and ultrastructural features of Leu-11+ cells in comparison to Leu-7+-Leu-11- cell subset. The data from the present investigation indicate that 2-aminoethylisothiouronium bromide hydrobromide treated sheep red blood cells phagocyting NK cells are confined to the Leu-11+ subset and give added strength to different function capabilities of Leu-11+ and Leu-7+-Leu-11- cells.
 
Recombinant human interleukin-11 (rhIL-11) is a pleiotropic cytokine with effects on multiple cell types. In addition to thrombopoietic activity, rhIL-11 has demonstrated anti-inflammatory activity in vitro and in vivo. rhIL-11 treatment reduces clinical signs and histologic lesions of colitis in transgenic rats expressing the human major histocompatibility complex (MHC) Class I allele, HLA-B27. We have investigated the effects of rhIL-11 at the molecular and cellular level in this model of inflammatory bowel disease. RT-PCR analysis of colonic RNA revealed that treatment with rhIL-11 down-regulated expression of proinflammatory cytokines including TNF-alpha, IL-1beta, and IFN-gamma. rhIL-11 also reduced the level of myeloperoxidase activity in the cecum indicating reduced inflammation. After stimulation in vitro with anti-CD3 antibody, spleen cell cultures derived from rhIL-11-treated rats produced less IFN-gamma, TNF-alpha, and IL-2 than cultures derived from vehicle-treated rats. These molecular and cellular effects correlated with amelioration of disease as measured by stool character and histologic lesion scores. These findings suggest that rhIL-11 acts to reduce inflammation through modulation of multiple proinflammatory mediators including products of activated T cells. This study has identified pharmacodynamic markers of rhIL-11 anti-inflammatory activity in vivo and supports rhIL-11 therapy to treat inflammatory bowel disease.
 
Detection and quantification of mutant alleles by LAPP. ( a ) Amplification of KIT exon 11 was performed on genomic DNA of a GIST as described 
Laboratory Investigation is the official journal of the United States and Canadian Academy of Pathology. Laboratory Investigation is available as both in print and online journal and is dedicated to the publication of original research that significantly advances the understanding of human and experimental disease including basic biology papers with a relevance to experimental or clinical aspects of disease. The journal also publishes papers detailing relevant advances in technical methodology.
 
Changes in transmural potential difference (PD) in isolated mucosal sheets from the jejunum of control rats (basal PD = -3.2 0.2 mV, n = 7 rats). Each data point represents the effects measured at their maximum (within 8 to 10 minutes) and expressed as mean sem from 6 to 12 experiments. * p < 0.05 vs vehicle control. A, Serosal application of increasing concentrations of human recombinant interleukin 11 (rhIL-11) causes a concentration-dependent reduction in basal PD, whereas an inactive rhIL-11 mutant molecule or the vehicle (0.1% BSA in saline) has no significant effect. B, Effects of increasing-concentrations of rhIL-11 on jejunal PD in tissues pretreated with 1 M tetrodotoxin (TTX) or with corresponding volumes of its vehicle (citrate buffer, pH 4.3) added to the serosal bathing solution 15 minutes before rhIL-11 addition.
Changes in transmucosal short-circuit current (Isc) in isolated mucosal sheets from the colon of control rats (basal Isc = 37 3 A/cm2, n = 7 rats). Each data point represents the effects measured at their maximum (within 8 to 10 minutes) and expressed as mean sem from 6 to 9 experiments. * p < 0.05 vs vehicle control. A, Serosal application of increasing concentrations of rhIL-11 causes a concentration-dependent reduction in basal Isc, whereas an inactive rhIL-11 mutant molecule or the vehicle (0.1% BSA in saline) has no significant effect. B, Effects of increasing concentrations of rhIL-11 on colonic Isc in tissues pretreated with TTX (1 M) or with corresponding volumes of its vehicle (citrate buffer, pH 4.3) added to the serosal bathing solution 15 minutes before rhIL-11 application.
Photomicrographs of jejunum (A) and colon (B) of rats receiving rhIL-11 for two weeks exhibit fewer inflammatory infiltrates and an intact luminal border. In contrast, in vehicle-treated animals, note the marked inflammatory infiltrates and shortened villi in the jejunum (C) and the uneven luminal border and submucosal inflammatory infiltrates in the colon (D). Hematoxylin and eosin stain. Original magnification, 100.
Myeloperoxidase (MPO) activity (A), basal values of transmural PD (B), and maximal changes in basal PD induced by serosal application of rhIL-11 (C) in the jejunum of naïve HLA-B27 transgenic rats (filled black bars) and HLA-B27 rats treated with rhIL-11 (33 g/kg sc at 48 hours intervals) during 1 or 2 weeks (hatched bars). Respective values obtained in jejunal tissue from control rats are also presented (open bars). Data for MPO activity are mean se calculated for 7 control or naive HLA-B27 rats and 4 HLA-B27 rats in each of the rhIL-11–treated groups. Data for PD are mean se from 8 to 12 experiments for each group. * p < 0.05 vs naïve HLA-B27 transgenic rats.
Myeloperoxidase (MPO) activity (A), basal values of Isc (B), and maximal changes in Isc induced by serosal application of rhIL-11 (C) in the colon isolated from naïve HLA-B27 transgenic rats (filled black bars) and HLA-B27 rats treated with rhIL-11 (33 g/kg sc at 48 hours intervals) during 1 or 2 weeks (hatched bars). Respective values obtained in jejunal tissue from control rats are also presented (open bars). Data for MPO activity are mean se from 7 control or naive HLA-B27 rats and 4 HLA-B27 rats in each of the rhIL-11–treated groups. Isc data are mean se from 8 to 9 experiments for each group. * p < 0.05 vs naïve HLA-B27 transgenic rats.
Human recombinant interleukin 11 (rhIL-11) is a cytokine that suppresses the clinical signs of colitis in animal models of inflammatory bowel disease (IBD) and may be an effective therapeutic agent in the treatment of IBD. The objective of the current study was to investigate whether rhIL-11 was capable of reversing abnormalities in secretomotor function associated with gut inflammation. We investigated the effects of rhIL-11 on epithelial electrogenic ion transport in the jejunum and colon. Application of rhIL-11 (10 to 10,000 ng/ml) at either the luminal or serosal side of mucosal sheets isolated from control rats induced a concentration-dependent reduction of transmural potential difference (PD) in the jejunum and decreased the short-circuit current (Isc), representative of active electrogenic transport, in the colon. To investigate the effect of rhIL-11 on an inflamed gut, we isolated jejunal and colonic tissue from HLA-B27 transgenic rats with active inflammation of the bowel that represents an animal model of IBD. In jejunum and colon isolated from HLA-B27 transgenic rats, basal electrogenic ion transport was significantly attenuated and, under these conditions, rhIL-11 caused no changes in either transmural PD or Isc. However, in HLA-B27 rats, pretreatment with subcutaneous doses of rhIL-11 suppressed the symptoms of diarrhea, normalized myeloperoxidase activity in the jejunum and colon and healed mucosal injury. In the jejunum from HLA-B27 rats, healing of the intestinal inflammatory response enhanced basal transmural PD and the rhIL-11-duced changes in mucosal ion transport resembled those seen in uninflamed controls. Conversely, in the colon, healing of the mucosa did not normalize basal active ion transport nor did it reverse the inhibition of rhIL-11-induced changes in colonic Isc. Our results suggest that endogenous IL-11 may act as a modulator of epithelial transport under physiologic conditions and may act as a potent anti-inflammatory cytokine during active intestinal inflammation.
 
Interleukin-11 (IL-11) reduces injury both in vivo and in vitro, but the mechanisms are unknown. Stimulation of serum- and growth factor-deprived HUVEC with IL-11 increased survivin mRNA and protein expression levels in a dose-dependent manner, with maximal induction at 50 to 100 ng/ml of IL-11. Survivin mRNA expression peaked after 3 to 6 hours of IL-11 treatment and decreased by 24 hours. Survivin protein expression was maximal at 6 hours of treatment and remained elevated through 24 hours. Survivin induction may be mediated by activation of protein kinase B/Akt, but IL-11 failed to activate this pathway in HUVEC. IL-11 did activate signal transducer and activator of transcription (STAT)-3 and IL-11 failed to induce survivin expression in HUVEC transduced with a dominant-negative STAT3 mutant, whereas control-transduced HUVEC responded normally. An IL-11 transgene caused increased survivin mRNA expression in mice compared with control littermates. Intradermal injection of IL-11 (500 ng) into human skin xenografts on immunodeficient mice up-regulated survivin protein in microvascular endothelium and epithelial keratinocytes. We conclude that IL-11 induces expression of survivin, an antiapoptotic protein, in vitro and in vivo, and identify STAT3 as a critical mediator of this response.
 
Context: Quantitative clinical measurement of heterogeneity in immunohistochemistry staining would be useful in both evaluating patient therapeutic response and identifying underlying issues in histopathology laboratory quality control. Objective: To create a heterogeneity scoring approach (HetMap) that allows the visualization of an individual patient's IHC heterogeneity in the context of a population. Design: We combined HER2 semi-quantitative analysis with the use of ecology diversity statistics to evaluate cell-level heterogeneity (consistency of protein expression within neighboring cells in a tumor nest) and tumor-level heterogeneity (differences of protein expression across a tumor as represented by a tissue section). We evaluated the approach on HER2 immunohistochemistry stained breast cancer samples, using 200 specimens across two different CLIA laboratories, with three pathologists at each laboratory each outlining regions of tumor for scoring by automatic cell-based image analysis. HetMap was evaluated using three different scoring schemes: HER2 scoring according to ASCO/CAP guidelines, H-Score and a new continuous HER2 score (HER2cont). Results: Two definitions of heterogeneity, cell-level and tumor-level, provided useful independent measures of heterogeneity. Cell-level heterogeneity, reported either as an average or the maximum area of heterogeneity across a slide, had low levels of dependency on the pathologist choice of region (coefficient of variation of 15%). Tumor-level heterogeneity measurements had more dependence on the pathologist choice of regions (coefficient of variation of 25%). Results were highly similar between the two laboratories, which is encouraging for HER2 standardization, as the labs involved different pathologists, different specimens, and different standardization procedures, although both complied with CLIA/CAP guidelines for HER2 testing. Conclusions: HetMap is a measure of heterogeneity, by which pathologists, oncologists, and drug development organizations can view cell-level and tumor-level heterogeneity for a patient for a given marker in the context of an entire patient cohort. Heterogeneity analysis can be a useful means to identify tumors with higher degrees of heterogeneity, or to highlight slides that should be rechecked for QC issues. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P5-11-17.
 
The origin of amyloid A and amyloid A-related proteins during induction of amyloidosis in C3H mice has been investigated using a quantitative immunoassay for the amyloid A fibril protein (AA). The response of the amyloid resistant A/J strain of mice has been monitored to serve as a control for the nonamyloidogenic effects of the amyloid-inducing agent, complete Freund's adjuvant with added Mycobacterium butyricum. The primary or preamyloid phase in C3H mice is characterized by an acute phase response to the amyloid-inducing agent in which the concentration of serum amyloid A is increased several 100-fold, and by a lag period in which serum amyloid A concentration is diminished as compared with acute phase levels, but still significantly elevated with respect to base line concentrations. The profile of serum amyloid A concentrations in A/J mice after treatment with the amyloid-inducing agent is similar to that of the C3H mice in which amyloidosis is induced. The amount of AA cross-reacting material in both plasma and liver begins to increase between 1 and 2 hours after intraperitoneal injection of C3H mice with bacterial lipopolysaccharide. The concentration of AA-related antigens in spleen and other tissues begins to increase to a lesser degree after 4 hours. The data are consistent with the interpretation that the liver is the site of acute phase serum amyloid A synthesis. The secondary, or amyloidotic, phase in C3H mice is marked by a 1000-fold increase in the concentration of AA-related proteins in the spleen. AA proteins are found in the spleens of A/J mice much later and then only in those instances where amyloidosis occurs. These findings are consistent with earlier morphologic and histochemical observations that have given rise to the concept of the biphasic development of amyloidosis. The data also suggest that in the spleen, where the first amyloid deposits are detected, amyloid fibrils may result from local cellular secretion.
 
The lack of effective therapies for end-stage lung disease validates the need for stem cell-based therapeutic approaches as alternative treatment options. In contrast with exogenous stem cell sources, the use of resident progenitor cells is advantageous considering the fact that the lung milieu is an ideal and familiar environment, thereby promoting the engraftment and differentiation of transplanted cells. Recent studies have shown the presence of multipotent 'mesenchymal stem cells' in the adult lung. The majority of these reports are, however, limited to animal models, and to date, there has been no report of a similar cell population in adult human lung parenchyma. Here, we show the identification of a population of primary human lung parenchyma (pHLP) mesenchymal stromal cells (MSCs) derived from intraoperative normal lung parenchyma biopsies. Surface and intracellular immunophenotyping by flow cytometry revealed that cultures do not contain alveolar type I epithelial cells or Clara cells, and are devoid of the following hematopoietic markers: CD34, CD45 and CXCR4. Cells show an expression pattern of surface antigens characteristic of MSCs, including CD73, CD166, CD105, CD90 and STRO-1. As per bone marrow MSCs, our pHLP cells have the ability to differentiate along the adipogenic, osteogenic and chondrogenic mesodermal lineages when cultured in the appropriate conditions. In addition, when placed in small airway growth media, pHLP cell cultures depict the expression of aquaporin 5 and Clara cell secretory protein, which is identified with that of alveolar type I epithelial cells and Clara cells, respectively, thereby exhibiting the capacity to potentially differentiate into airway epithelial cells. Further investigation of these resident cells may elucidate a therapeutic cell population capable of lung repair and/or regeneration.
 
Angiogenesis is a critical factor in the growth, progression, and metastatic spread of solid tumors. Furthermore, angiogenesis has been correlated with prognosis in patients with ovarian cancer. The pathogenesis of the angiogenic events in ovarian cancer, however, are not well defined. Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) is a multifunctional cytokine that has been shown to be an important regulator of tumor angiogenesis. The purpose of the present study was to define the expression of VPF/VEGF and its receptors flt-1 and KDR in ovarian tumors. Four specimens of normal ovarian cortex and 41 specimens of benign (4), borderline (8), and malignant (29) ovarian tumors were studied by in situ hybridization, and in some cases by immunohistochemical analysis. VPF/VEGF protein was also determined by an immunofluorometric assay in cyst fluids obtained from 11 patients, including 7 benign, 2 borderline, and 2 malignant tumors. VPF/VEGF mRNA and protein were expressed by the neoplastic cells in all of the malignant tumors evaluated, with the majority of tumors (28 of 29) showing strong expression of mRNA. Serous borderline tumors had variable VPF/VEGF mRNA expression, with two of six cases showing focal strong expression and four showing low-level expression. No definite expression of VPF/VEGF was seen in two cases of mucinous borderline tumors. No strong expression of VPF/VEGF mRNA was observed in normal ovarian cortex, including surface epithelium, or benign tumors. Substantially higher VPF protein concentrations were detected in cyst fluids of the two malignant (60, 440 pM) and two borderline tumors (210, 590 pM) than in the seven benign serous cysts (mean, 10 +/- 3 pM). In addition, microvascular endothelial cells strongly expressed mRNA of the VPF/VEGF receptors flt-1 and KDR and immunostained for VPF/VEGF protein in the majority of malignant and borderline tumors examined. These findings suggest that VPF/VEGF plays an important role in the angiogenesis associated with ovarian neoplasms.
 
Injury to endothelial cells appears to be an important initial event in the pathogenesis of many diseases such as acute lung injury, venous and arterial thromboembolism, and atherosclerosis. Different methods for detecting damage to cultured endothelial cells have been described. However, their relative sensitivity as markers of endothelial cell damage has not been adequately determined. We compared the loss of 51Chromium (51Cr), the cytoplasmic enzyme lactate dehydrogenase (LDH), and 111Indium (111In) from endothelial cells upon exposure to several injurious agents. Cultured bovine pulmonary artery endothelial cells in confluent monolayers were labeled with 51Cr or 111Inoxine and exposed to increasing concentrations of the nonionic detergent, Triton X-100 (0.2 to 1%), hydrogen peroxide (1 to 500 microM), or neutrophils stimulated with phorbol myristate acetate. With all forms of injury, loss of 51Cr occurred earlier and to a greater extent than LDH loss which in turn was greater than loss of 111In. Substantial loss of 51Cr was observed in the absence of appreciable ultrastructural damage to endothelial cell external membranes. The findings may reflect the relative ease with which small molecules such as adenine nucleotides (51Cr-labeled) escape whereas larger molecules such as LDH and proteins binding 111In are retained intracellularly. Thus, 51Cr loss appears to be a more sensitive indicator of sublytic endothelial cell injury than either 111In or LDH release.
 
The ability of tocainide (a primary amine derivative of lidocaine, 2-amino 2, 6-propionoxylidide) to inhibit leukocyte adhesion to and invasion of canine jugular veins was investigated. Leukocyte adhesion and migration were quantitated by use of 111indium-labeled leukocytes, and the morphologic characteristics of leukocytes and vessel lumen were studied by scanning electron microscopy. The morphologic characteristics of adhering and migrating 111indium-labeled leukocytes were similar to leukocytes that had not been manipulated, thus establishing their suitability for use as a marker for flammation. Exposure of leukocytes to 200 micrograms. per ml. of tocainide in autologous plasma in vitro inhibited adhesion and migration by 68 per cent. When labeled leukocytes were returned to the donor and exposed to intravenously infused tocainide the extent of reduction in adhesion and migration depended on whether tocainide infusion was started before or after neck dissection. Inhibition was only 46 per cent when dissection and injection of 111indium-labeled leukocytes preceded the start of infusion of tocainide but was 87 per cent when tocainide infusion was started before dissection and injection of leukocytes. The plasma level ranged from 25 to 47 microM over the 3 hour, 20 minute-infusion period, being 35 to 40 microM for the last hour. Migration of leukocytes across interendothelial junctions and their accumulation between the endothelial sheet and the basement membrane caused extensive damage to the endothelial lining of these veins. This was reduced when leukocyte migration was reduced. These observations suggest that the leukocyte-induced damage occurring in some sterile inflammations might be reduced by the use of local anesthetic drugs.
 
Analysis of Her-2/neu gene expression in laser-microdissected osteosarcoma cells derived from paraffin-embedded tissue. Transcripts were quantified by real-time quantitative RT-PCR using an external standard curve. Values are expressed as x-fold expression compared to the housekeeping gene PBGD. (a): Her-2/neu expression was analyzed in two different areas of individual samples. Data are presented as meanstandard deviation. Tumor regression was assessed histologically according to the six-grade scale of Salzer-Kuntschik et al. where grade 1 denotes no viable tumor cells; grade 2, solitary viable cells or one islet of less than 0.5 cm; grade 3, less than 10%; grade 4, 10–50%; grade 5, more than 50% and grade 6, no effect of chemotherapy. (b): Her-2/neu expression compared to therapy outcome. Good response was defined as <10% viable tumor cells (regression grade 1–3). The white lines indicate the medians, the lower boundary of the box the 25th percentile and the upper boundary of the box the 75th percentile. The whiskers indicate the highest and lowest values. (**P=0.005, determined by Mann–Whitney U- test)
Immunohistochemical analysis of Her-2/neu in osteosarcoma biopsies. Representative stainings from samples with different tumor regression are shown using DAB as substrate (brown) and hematoxylin as counterstain (blue): (a) grade 2, (b) grade 3, (c) grade 4, and (d) grade 5 according to the six-grade scale of Salzer-Kuntschik.
Histologic response to chemotherapy is currently the strongest prognostic factor in high-grade osteosarcoma, but it can only be assessed after several weeks of therapy. Thus, detection of chemosensitivity at the time of diagnosis would be of great clinical importance. The expression of the proto-oncogene Her-2/neu has been shown to be of predictive value in breast cancer and has also been considered as prognostic marker for osteosarcomas, but reports of mainly immunohistochemical studies are controversial. Therefore, the aim of this study was to investigate Her-2/neu gene expression in laser-microdissected osteosarcoma cells. Laser microdissection enables the precise isolation of morphological defined cells from archival tissue specimens and is in combination with the highly sensitive real-time RT-PCR technique a valuable tool for cell-specific analysis of gene expression. Through optimization of current protocols, we could show that this technique can be successfully applied on formalin-fixed, paraffin-embedded and decalcified osteosarcoma tissue with high sensitivity and reproducibility. In all 17 osteosarcoma biopsies analyzed, we could detect Her-2/neu gene expression. Expression correlated significantly with the response to preoperative chemotherapy, which was assessed histologically according to the six-grade scale of Salzer-Kuntschik. Risk assessment on the basis of increased Her-2/neu gene expression matched the histologic findings in 16 out of 17 cases (94%). These data demonstrate the reliability of laser microdissection in the analysis of gene expression and suggest a possible role of Her-2/neu as prognostic marker for therapy outcome in osteosarcomas.
 
Gene amplifications of c-myc, K-sam, and c-met were examined in cancer nuclei isolated from 154 primary gastric adenocarcinomas by fluorescence in situ hybridization (FISH) using cosmid probes for 8q24 (c-myc locus) and 7q31 (c-met), as well as a DNA probe for K-sam synthesized by PCR. The results were compared with those of Southern blot analysis. Dual-color FISH using gene locus and chromosome-specific probes detected gene amplifications of c-myc in 24 tumors (15.5%), c-met in 6 tumors (3.9%), and K-sam in 3 tumors (2.9%). The six tumors with c-myc amplification had also been found to have amplified c-erbB-2 in our previous study, and coamplification of c-myc and c-met was found in two other tumors. This technique also differentiated the amplified genes on the homogeneous staining region (HSR) and on double minute chromosomes (DMs) in metaphase spreads and interphase nuclei of cell lines established from poorly differentiated adenocarcinomas, KATO III, SNU 16, and HSC 39. Examination of FISH images of these cell lines suggested that the high-level amplifications of c-myc found in primary tumors occurred mainly on DM in four tumors and on HSR in one, and those of K-sam occured on DM in two tumors and on HSR in one. No high-level amplification of c-met was found. These high-level amplifications were also detected in formalin-fixed, paraffin-embedded tissues from primary gastric tumors and metastatic lymph nodes, in some of which heterogeneity of gene amplification was demonstrated within the same tumor. We conclude that FISH is an important tool for examining the proto-oncogene aberrations in intact cells in solid tumors.
 
Androgen-dependent Shionogi carcinoma 115 (SC115) is an undifferentiated medullary carcinoma showing a compact cell pattern. When SC115 was inoculated into castrated DS mice, slowly growing tumors containing cartilage-like tissue developed. The cartilage-like tissue showed close similarities in electron microscopical and histochemical features to normal cartilage tissue. The origin of the cartilage-like cells was investigated by using the electrophoretic pattern of 3-phosphoglycerate kinase (PGK, EC 2.7.2.3) as a marker. The original DS strain had B-type PGK, but we developed a congenic strain with A-type PGK. The SC115 tumor, which had B-type PGK, was grafted into castrated congenic mice with A-type PGK. The cartilage-like tissue that developed was divided into two parts; one part was used for PGK analysis and the other for histological examination. All histologically confirmed cartilage-like tissues had only B-type PGK, suggesting that SC115 cells may change into cartilage-like cells in androgen-depleted conditions.
 
Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) is an angiogenic cytokine with potential for the treatment of tissue ischemia. To investigate the properties of the new blood vessels induced by VPF/VEGF, we injected an adenoviral vector engineered to express murine VPF/VEGF164 into several normal tissues of adult nude mice or rats. A dose-dependent angiogenic response was induced in all tissues studied but was more intense and persisted longer (months) in skin and fat than in heart or skeletal muscle (< or =3 weeks). The initial response (within 18 hours) was identical in all tissues studied and was characterized by microvascular hyperpermeability, edema, deposition of an extravascular fibrin gel, and the formation of enlarged, thin-walled pericyte-poor vessels ("mother" vessels). Mother vessels developed from preexisting microvessels after pericyte detachment and basement membrane degradation. Mother vessels were transient structures that evolved variably in different tissues into smaller daughter vessels, disorganized vessel tangles (glomeruloid bodies), and medium-sized muscular arteries and veins. Vascular structures closely resembling mother vessels and each mother vessel derivative have been observed in benign and malignant tumors, in other examples of pathological and physiological angiogenesis, and in vascular malformations. Together these data suggest that VPF/VEGF has a role in the pathogenesis of these entities. They also indicate that the angiogenic response induced by VPF/VEGF is heterogeneous and tissue specific. Finally, the muscular vessels that developed from mother vessels in skin and perimuscle fat have the structure of collaterals and could be useful clinically in the relief of tissue ischemia.
 
We report here that rings of rat aorta embedded in gels of fibrin or collagen and cultured in MCDB 131, an optimized growth medium for microvascular endothelial cells, generate branching microvessels in the absence of serum or other soluble protein supplements. The angiogenic response is self-limited and can be quantitated by counting the newly formed microvessels daily in the living cultures. The microvascular growth curves are characteristic for each gel. Growth of microvessels in collagen gel peaks at the end of the 1st week and is followed by a rapid regression in the 2nd week. Fibrin gels, as compared with collagen, stimulate angiogenesis by 170%, support growth during the 2nd week, and protect the newly formed microvessels from early regression. Angiogenesis is inhibited by adding hydrocortisone to the culture medium. Conversely, a 230% stimulation of angiogenesis is obtained when aortic rings are cultured in collagen gels floating in serum-free medium conditioned by sarcoma 180 cells. Our results demonstrate that: (a) angiogenesis can be obtained reproducibly in serum-free culture; (b) serum-free culture is a sensitive method for testing the inhibitory or stimulatory effects of soluble or matrix factors on angiogenesis; (c) the aortic ring model can be used as a quantitative assay for the study of angiogenesis under chemically defined culture conditions.
 
Top-cited authors
Feng Jiang
  • Zhengzhou University
Vamsidhar Velcheti
  • New York University
Y-P Mei
  • University of Maryland, Baltimore
Kyeung Min Joo
  • Sungkyunkwan University
William Lewis
  • Emory University