Charalambos C. Solomides

Immunotargeting of catalase to the pulmonary endothelium alleviates oxidative stress and reduces acute lung transplantation injury

Vascular immunotargeting may facilitate the rapid and specific delivery of therapeutic agents to endothelial cells. We investigated whether targeting of an antioxidant enzyme, catalase, to the pulmonary endothelium alleviates oxidative stress in an in vivo model of lung transplantation. Intravenously injected enzymes, conjugated with an antibody to platelet-endothelial cell adhesion molecule-1, accumulate in the pulmonary vasculature and retain their activity during prolonged cold storage and transplantation. Immunotargeting of catalase to donor rats augments the antioxidant capacity of the pulmonary endothelium, reduces oxidative stress, ameliorates ischemia-reperfusion injury, prolongs the acceptable cold ischemia period of lung grafts, and improves the function of transplanted lung grafts. These findings validate the therapeutic potential of vascular immunotargeting as a drug delivery strategy to reduce endothelial injury. Potential applications of this strategy include improving the outcome of clinical lung transplantation and treating a wide variety of endothelial disorders.

Recombinant plasma gelsolin diminishes the acute inflammatory response to hyperoxia in Mice

Background The acute respiratory distress syndrome remains a common and poorly understood complication of a variety of insults. Ventilation with high concentrations of inspired oxygen may further damage already compromised lungs. By scavenging extracellular actin and modulating the effects of lysophosphatidic acid, plasma gelsolin could serve a critical protective role against oxidant injury. Methods Mice exposed to >95% O2 for a total of 72 hours were treated with gelsolin or albumin after 24 and 48 hours. Results Neutrophil counts in bronchoalveolar fluid rose (P=0.0002) and gelsolin levels dropped (P<0.00001) in mice with acute hyperoxic lung injury. The acute inflammatory response to hyperoxia was significantly reduced in the gelsolin-compared with the bovine serum albumin-treated mice (P=0.03). Conclusions These data imply that i) gelsolin depletion contributes to the pathogenesis of oxygen toxicity and ii) repletion of gelsolin can partially abrogate the resultant exudative response.

Diagnostic value of hepatocyte paraffin 1 antibody to discriminate hepatocellular carcinoma from metastatic carcinoma in fine‐needle aspiration biopsies of the liver

Diagnosing liver tumors by fine‐needle aspiration biopsy is safe and accurate. However, there are cases that prove diagnostically difficult. Traditionally, immunostains for α‐fetoprotein and polyclonal carcinoembryonic antigen have been used to distinguish adenocarcinomas from hepatocellular carcinomas (HCCs). In poorly differentiated tumors, these immunostains have limitations in both sensitivity and specificity. An hepatocyte‐specific immunostain has been described in the surgical pathology literature. To the authors knowledge, this hepatocyte antibody has not been studied in liver fine‐needle aspiration biopsies. The authors examined the Hepatocyte Paraffin 1 (HP1) antibody for its diagnostic utility in this cytologic setting.

Changes in plasma gelsolin concentration during acute oxidant lung injury in mice.

AbstractOxidant stress may contribute to acute lung injury under some circumstances. The rapid depletion of plasma gelsolin following major trauma in patients who subsequently develop respiratory distress suggests that this actin-scavenging protein might protect against delayed pulmonary complications. The specific aim of these experiments was to explore the temporal and quantitative relationship between gelsolin levels and lung damage. Gelsolin levels were measured in three murine models of oxidant injury: immunotargeting of pulmonary endothelium with an H2O2-generating enzyme; continuous exposure to >95% O2; and single high-dose thoracic radiation. The degree of lung injury was inversely related to gelsolin levels in mice treated with glucose oxidase-conjugated antibodies against platelet endothelial cell adhesion molecule-1 (p <0.0001). By 60–72 hours of hyperoxic exposure, gelsolin levels had dropped precipitously in all mice who sustained major lung damage (p <0.0001), establishing a quantitative association between gelsolin concentration and hyperoxic lung injury (r = -0.72; 95% confidence interval: ?0.81 to ?0.59). Gelsolin levels modestly but progressively fell in irradiated mice over the 3 days following treatment (p = 0.012) despite the development of only microscopic lung damage during this timeframe. These findings are consistent with the hypothesis that gelsolin depletion is involved in the pathogenesis of acute oxidant lung injury.n

Vascular Immunotargeting of Glucose Oxidase to the Endothelial Antigens Induces Distinct Forms of Oxidant Acute Lung Injury: Targeting to Thrombomodulin, But Not to PECAM-1, Causes Pulmonary Thrombosis and Neutrophil Transmigration

Oxidative endothelial stress, leukocyte transmigration, and pulmonary thrombosis are important pathological factors in acute lung injury/acute respiratory distress syndrome (ALI/ARDS). Vascular immunotargeting of the H(2)O(2)-generating enzyme glucose oxidase (GOX) to the pulmonary endothelium causes an acute oxidative lung injury in mice.(1) In the present study we compared the pulmonary thrombosis and leukocyte transmigration caused by GOX targeting to the endothelial antigens platelet-endothelial cell adhesion molecule (PECAM) and thrombomodulin (TM). Both anti-PECAM and anti-TM delivered similar amounts of (125)I-GOX to the lungs and caused a dose-dependent, tissue-selective lung injury manifested within 2 to 4 hours by high lethality, vascular congestion, polymorphonuclear neutrophil (PMN) sequestration in the pulmonary vasculature, severe pulmonary edema, and tissue oxidation, yet at an equal dose, anti-TM/GOX inflicted more severe lung injury than anti-PECAM/GOX. Moreover, anti-TM/GOX-induced injury was accompanied by PMN transmigration in the alveolar space, whereas anti-PECAM/GOX-induced injury was accompanied by PMN degranulation within vascular lumen without PMN transmigration, likely because of PECAM blockage. Anti-TM/GOX caused markedly more severe pulmonary thrombosis than anti-PECAM/GOX, likely because of TM inhibition. These results indicate that blocking of specific endothelial antigens by GOX immunotargeting modulates important pathological features of the lung injury initiated by local generation of H(2)O(2) and that this approach provides specific and robust models of diverse variants of human ALI/ARDS in mice. In particular, anti-TM/GOX causes lung injury combining oxidative, prothrombotic, and inflammatory components characteristic of the complex pathological picture seen in human ALI/ARDS.

Lymphoid Follicle Cells in Chronic Obstructive Pulmonary Disease Overexpress the Chemokine Receptor CXCR3

RATIONALEnThe mechanisms underlying formation of lung lymphoid follicles (LF) in chronic obstructive pulmonary disease (COPD) are unknown. The chemokine receptor CXCR3 regulates immune responses in secondary lymphoid structures elsewhere in the body and is highly expressed by Th1 lymphocytes in the airway in COPD. Because chemokine receptors control inflammatory cell homing to inflamed tissue, we reasoned that CXCR3 may contribute to LF formation in COPD.nnnOBJECTIVESnWe assessed the expression of CXCR3 and its ligands (IP-10/CXCL10, Mig/CXCL9, and ITAC/CXCL11) by LF cells in never-smokers, smokers without COPD, and subjects with COPD.nnnMETHODSnCXCR3, IP-10, Mig, and ITAC expression were assessed in lung sections from 46 subjects (never-smokers, smokers without COPD [S], and subjects with COPD in GOLD stages 1-4) by immunohistochemistry.nnnMEASUREMENTS AND MAIN RESULTSnCXCR3-expressing T cells (CD8+ or CD4+) and B cells (CD20+) were topographically distributed at the follicle periphery and center, respectively. The percentage of immunohistochemically identified CXCR3+ cells increased progressively while proceeding from S through GOLD 3-4 (P < 0.01 for GOLD 3-4 vs. S). Moreover, the number of CXCR3+ follicular cells correlated inversely with FEV(1) (r = 0.60). The CXCR3 ligands IP-10 and Mig were expressed by several cell types in and around the follicle, including CD68+ dendritic cells/ macrophages, airway epithelial cells, endothelial cells, and T and B cells.nnnCONCLUSIONSnThese results suggest that LF form in the COPD lung by recruitment and/or retention of CXCR3-expressing T and B lymphocytes, which are attracted to the region through production of CXCR3 ligands IP-10 and Mig by lung structural and follicular cells.

Diagnostic utility of Glut-1 and CA 15-3 in discriminating adenocarcinoma from hepatocellular carcinoma in liver tumors biopsied by fine-needle aspiration.

Diagnosing liver tumors can be difficult in the setting of a poorly differentiated tumor or tumors with no known prior malignancy. Frequently, α‐fetoprotein, carcinoembryonic antigen, factor VIII, and mucicarmine have been employed to distinguish hepatocellular carcinoma (HCC) from adenocarcinoma. However, these stains have their limitations. CA 15‐3 and Glut‐1 are expressed in a variety of carcinomas. To the authors knowledge, their expression in HCC has not been studied extensively. The authors examined the clinical utility of CA 15‐3 and Glut‐1 in the setting of fine‐needle aspiration biopsy samples from the liver.

Small Airway Mucous Metaplasia and Inflammation in Chronic Obstructive Pulmonary Disease

Mucous metaplasia is an important determinant of small airway obstruction in COPD. Its relationship to small airway inflammation is poorly defined. We analyzed 4 to 6 small airways in 19 COPD patients, GOLD stages 0–4, from lobectomy or lung volume reduction surgery tissue samples. To identify intracellular mucin, periodic acid fluorescent Schiffs (PAFS) stained slides were imaged by fluorescence microscopy. PAFS+ staining area, basement membrane length (LBM), epithelial height and area were measured. Mucin was expressed as a percentage of epithelial area. Mucin volume density (MVD) was calculated as PAFS+ area divided by the product of LBM and 4/π. Airways were Giemsa stained for eosinophils and immunostained with antibodies against CD3, CD4, CD8, CD68, and neutrophil elastase (NE), and the number of positively stained cells/mm2 was quantified in the airway wall. Mucin percent correlated with CD3+ cell density (r = 0.553, P < 0.0001), and MVD correlated with CD3+ (r = 0.570, P < 0.0001) and CD8+ cell density (r = 0.279, P = 0.016). There were weak negative correlations between mucin percent as well as MVD and CD68+ cell density (r = −0.270, P = 0.02 and r = −0.245, P = 0.036). There was no relationship between epithelial mucin content and CD4+, NE+, or eosinophil cell density. CD3+ and CD8+ lymphocytic inflammation is related to small airway mucous metaplasia in COPD and may play a causative role in its development.

Abdominal Aortic Aneurysm Is a Specific Antigen‐Driven T Cell Disease

Abstract:u2002 To determine whether monoclonal/oligoclonal T cells are present in abdominal aortic aneurysm (AAA) lesions, we amplified β‐chain T cell receptor (TCR) transcripts from these lesions by the nonpalindromic adaptor (NPA)‐polymerase chain reaction (PCR)/V‐β‐specific PCR followed by cloning and sequencing. Sequence analysis revealed the presence of substantial proportions of identical β‐chain TCR transcripts in AAA lesions in 9 of 10 patients examined, strongly suggesting the presence of oligoclonal populations of αβ TCR+ T cells. We have also shown the presence of oligoclonal populations of γδ TCR+ T cells in AAA lesions. Sequence analysis after appropriate PCR amplification and cloning revealed the presence of substantial proportions of identical VγI and VγII TCR transcripts in 15 of 15 patients examined, and of Vδ1 and Vδ2 TCR transcripts in 12 of 12 patients. These clonal expansions were very strong. All these clonal expansions were statistically significant by the binomial distribution. In other studies, we determined that mononuclear cells infiltrating AAA lesions express early‐ (CD69), intermediate‐ (CD25, CD38), and late‐ (CD45RO, HLA class II) activation antigens. These findings suggest that active ongoing inflammation is present in the aortic wall of patients with AAA. These results demonstrate that oligoclonal αβ TCR+ and γδ TCR+T cells are present in AAA lesions. These oligoclonal T cells have been clonally expanded in vivo in response to yet unidentified antigens. Although the antigenic specificity of these T cells remains to be determined, these T cells may play a significant role in the initiation and/or the propagation of the AAA. It appears that AAA is a specific antigen‐driven T cell disease.

Resistance to platinum-based chemotherapy in lung cancer cell lines.

PurposeA series of six lung cancer cell lines of different cell origin (including small cell and mesothelioma) were characterized immunohistochemically and the role of a series of protein candidates previously implicated in drug resistance were investigated.MethodsThese include colony-forming and cell growth assays, immunohistochemistry, siRNA knockouts, real-time PCR and western blots.ResultsNo correlation was found with AKT, HO-1, HO-2, GRP78, 14-3-3zeta and ERCC1 levels and cisplatin nor oxaliplatin cytotoxicity, but an association was observed with levels of the enzyme, dihydrodiol dehydrogenase (DDH); an enzyme previously implicated in the development of platinum resistance. The relationship appeared to hold true for those cell lines derived from lung epithelial primary tumors but not for the neuroendocrine/small-cell and mesothelioma cell lines. siRNA knockouts to DDH-1 and DDH-2 were prepared with the cell line exhibiting the greatest resistance to cisplatin (A549) resulting in marked decreases in the DDH isoforms as assessed by real-time PCR, western blot and enzymatic activity. The DDH-1 knockout was far more sensitive to cisplatin than the DDH-2 knockout.ConclusionThus, sensitivity to cisplatin appeared to be associated with DDH levels in epithelial lung cancer cell lines with the DDH-1 isoform producing the greatest effect. Results in keeping with transfection experiments with ovarian and other cell lines.