Molecular biomarker identification for esophageal adenocarcinoma using endoscopic brushing and magnified endoscopy.

BACKGROUND: Barrett's esophagus (BE) is a predisposing factor for esophageal adenocarcinoma (EAC); however, the precise mechanism underlying this association remains unclear. The identification of biomarkers that are associated with an increased risk of BE progression to EAC would facilitate diagnosis and early treatment. Toward this goal, we aimed to identify biomarkers associated with BE and EAC in patients. METHODS: In conjunction with high-resolution magnified endoscopy with narrow-band imaging (ME-NBI), we obtained brushing samples from the long-segment BE (LSBE) or short-segment BE (SSBE) of patients with EAC or without EAC (control). To identify candidate biomarker genes, microarray analysis was performed for a training set of 28 American samples. To confirm the microarray results, expression levels of the 16 candidate biomarkers were evaluated by real-time polymerase chain reaction analysis, using samples collected from an additional 53 American patients. In addition, we also performed a functional analysis for these genes using Gene Ontology (GO) enrichment analysis. RESULTS: Among the 16 genes identified as differentially expressed by microarray analysis, the GO analysis indicated matrix metalloproteinase (MMP) family associated with 'collagen metabolic process' and 'multicellular organismal macromolecule metabolic process' as the two top biological processes. Brushing samples of patients with EAC showed up-regulated expression of decay-accelerating factors (DAF and CD55) and topoisomerase type Iiα (TOP2A), and down-regulated expression of the sodium channel epithelial 1 beta subunit (SCNN1B). CONCLUSIONS: The up-regulation of CD55 and TOP2A, and the down-regulation of SCNN1B were common to the brushing samples and might serve as molecular biomarkers for identifying EAC in patients with SSBE. TRIAL REGISTRATION: University Hospital Medical Information Network (UMIN) (000004004).

Transportation of Temperature-sensitive Medications in an Air Medical Setting.

INTRODUCTION: This article describes an effective system for the transportation of temperature-sensitive medications within acceptable temperature ranges in the air medical setting. METHOD: A temperature audit using data logging thermometers of drug bags used to transport temperature-sensitive medications revealed that temperature excursions above the accepted maximums (8°C) occurred frequently. An experimental methodology was developed using a commercially available shipping container that was subject to a rapid conditioning regimen. Through a series of experimental trials, it was determined that with the devised conditioning regimen the system would maintain a consistent 2°C to 8°C. This system was implemented, and data were collected over a series of air medical missions (5) over a 90-day period. RESULTS: The average mission duration was 10 hours with temperature-sensitive medications spending an average of 15.3 hours out of the pharmacy fridge. Temperature data showed temperature-sensitive medications remained within the 2°C to 8°C range for the duration of all missions in which the shipping container was prepared appropriately. CONCLUSION: This proof of concept study showed a system that can maintain acceptable storage conditions for temperature-sensitive medications.

Role of EUS in patients with suspected Barrett's esophagus with high-grade dysplasia or early esophageal adenocarcinoma: impact on endoscopic therapy.

BACKGROUND AND AIMS: Endoscopic therapy is the standard treatment for high-grade dysplasia and some cases of T1a esophageal adenocarcinoma (EAC), but it is not appropriate for deeply invasive disease. Data on the value of EUS for patient selection for endoscopic or surgical resection are conflicting. We investigated the outcome of esophageal EUS for the staging and treatment selection of patients with treatment-naive, premalignant Barrett's esophagus (BE) and suspected superficial EAC. METHODS: We retrospectively reviewed consecutive patients who underwent EUS for staging of treatment-naive, suspected premalignant BE and superficial EAC from January 2006 to June 2014. All patients referred for endoscopic therapy routinely underwent EUS. Patients with esophageal masses, squamous cell cancers, previous neoadjuvant therapy, or unrelated pathologies were excluded. Each patient's final diagnosis was verified by EMR, esophagectomy, or forceps biopsy sampling. Test characteristics of EUS were calculated. RESULTS: Three hundred thirty-five patients (mean age, 68 years; 86% male) with BE, a Prague C mean of 2.8 cm, and a Prague M mean of 4.5 cm were staged (pT0, 78% [6% nondysplastic, 24% low-grade dysplasia, 42% high-grade dysplasia]; pT1a, 14%; pT1b, 7%; and pT2, 1%). Sensitivity, specificity, positive predictive value, negative predictive value, and accuracy for patient selection to endoscopic (T1aN0 or less) or surgical therapy with EUS TN staging were 50%, 93%, 40%, 95%, and 90%, respectively. Comparable rates were achieved for patients with nodular BE. Overstaging occurred in 7% of patients, and EUS selected 11% for incorrect treatment modalities compared with pathologic staging. CONCLUSIONS: This study confirms the limited value of EUS suggested in the latest American College of Gastroenterology guidelines for BE management.

Axial stereocontrol in tropos dibenz[c,e]azepines: the individual and cooperative effects of alkyl substituents.

6,7-Dihydro-5H-dibenz[c,e]azepines, a class of secondary amine incorporating a centre-axis chirality relay, can be prepared from N-(2-bromobenzyl)-N-(1-arylalkyl)methanesulfonamides via Pd-catalysed intramolecular direct arylation, and methylated at C(7) via the 5,7-trans diastereoselective addition of methylmagnesium bromide to the derived N-benzylazepinium tetraphenylborate. Using these methods, the 4,5-dimethylated and 4,5,7-trimethylated homologues 13 and 14 were obtained and shown by 1H NMR spectroscopy to be axially biased in opposite senses, as defined by the respective pseudoaxial or pseudoequatorial orientation of the 5-substituent in the preferred conformers, while retaining their tropos nature (the Arrhenius activation energy, EA, for the conformational exchange process in 14 was estimated to be 57 kJ mol-1 using 2D-EXSY NMR spectroscopy at 233-248 K). These results serve to illustrate how substituent effects might be exploited in new designs of bridged biaryl ligand in which tropos dynamics operate in combination with a pre-existing axial stereochemical bias.

Tubulin-binding dibenz[c,e]oxepines: Part 2. Structural variation and biological evaluation as tumour vasculature disrupting agents.

5,7-Dihydro-3,9,10,11-tetramethoxybenz[c,e]oxepin-4-ol 1, prepared from a dibenzyl ether precursor via Pd-catalysed intramolecular direct arylation, possesses broad-spectrum in vitro cytotoxicity towards various tumour cell lines, and induces vascular shutdown, necrosis and growth delay in tumour xenografts in mice at sub-toxic doses. The biological properties of 1 and related compounds can be attributed to their ability to inhibit microtubule assembly at the micromolar level, by binding reversibly to the same site of the tubulin αß-heterodimer as colchicine 2 and the allocolchinol, N-acetylcolchinol 4.

Biocatalytic Routes to Enantiomerically Enriched Dibenz[c,e]azepines.

Biocatalytic retrosynthetic analysis of dibenz[c,e]azepines has highlighted the use of imine reductase (IRED) and ω-transaminase (ω-TA) biocatalysts to establish the key stereocentres of these molecules. Several enantiocomplementary IREDs were identified for the synthesis of (R)- and (S)-5-methyl-6,7-dihydro-5H-dibenz[c,e]azepine with excellent enantioselectivity, by reduction of the parent imines. Crystallographic evidence suggests that IREDs may be able to bind one conformer of the imine substrate such that, upon reduction, the major product conformer is generated directly. ω-TA biocatalysts were also successfully employed for the production of enantiopure 1-(2-bromophenyl)ethan-1-amine, thus enabling an orthogonal route for the installation of chirality into dibenz[c,e]azepine framework.

"Wet suction technique (WEST)": a novel way to enhance the quality of EUS-FNA aspirate. Results of a prospective, single-blind, randomized, controlled trial using a 22-gauge needle for EUS-FNA of solid lesions.

BACKGROUND: Contemporary EUS-guided FNA techniques involve the use of a needle, with an air column within the lumen, with or without suction. We describe a novel technique with an aim to improve the quality of the aspirate. OBJECTIVE: To compare a novel "wet suction" technique (WEST) with the conventional FNA technique (CFNAT) of EUS-guided FNA using a 22-gauge FNA needle. DESIGN: Prospective, single-blind, and randomized trial. SETTING: Two large tertiary-care hospitals. PATIENTS: All consecutive adult patients presenting for EUS with possible FNA of solid lesions were offered the chance to participate in the study. METHODS: All lesions were sampled with the same needle by using alternating techniques. Patients were randomized to the WEST versus the CFNAT for the first pass. If the first pass was made with the WEST, the second pass was made with the CFNAT, and subsequent passes were made in an alternating manner by using the same sequence. All FNAs were performed using 22-gauge needles. MAIN OUTCOME MEASUREMENTS: Specimen adequacy, cellularity, and blood contamination of EUS-guided FNA aspirates graded on a predefined scale. RESULTS: The WEST yielded significantly higher cellularity in a cell block compared with the CFNAT, with a mean cellularity score of 1.82±0.76 versus 1.45±0.768 (P<.0003). The WEST cell block resulted in a significantly better specimen adequacy of 85.5% versus 75.2% (P<.035). There was no difference in the amount of blood contamination between the 2 techniques. LIMITATIONS: Lack of cross check and grading by a second cytopathologist. CONCLUSION: The novel WEST resulted in significantly better cellularity and specimen adequacy in cell blocks of EUS-guided FNA aspirate of solid lesions than the CFNAT.

Imagine a world without cancer.

BACKGROUND: Since the "War on Cancer" was declared in 1971, the United States alone has expended some $300 billion on research, with a heavy focus on the role of genomics in anticancer therapy. Voluminous data have been collected and analyzed. However, in hindsight, any achievements made have not been realized in clinical practice in terms of overall survival or quality of life extended. This might be justified because cancer is not one disease but a conglomeration of multiple diseases, with widespread heterogeneity even within a single tumor type. DISCUSSION: Only a few types of cancer have been described that are associated with one major signaling pathway. This enabled the initial successful deployment of targeted therapy for such cancers. However, soon after this targeted approach was initiated, it was subverted as cancer cells learned and reacted to the initial treatments, oftentimes rendering the treatment less effective or even completely ineffective. During the past 30 plus years, the cancer classification used had, as its primary aim, the facilitation of communication and the exchange of information amongst those caring for cancer patients with the end goal of establishing a standardized approach for the diagnosis and treatment of cancers. This approach should be modified based on the recent research to affect a change from a service-based to an outcome-based approach. The vision of achieving long-term control and/or eradicating or curing cancer is far from being realized, but not impossible. In order to meet the challenges in getting there, any newly proposed anticancer strategy must integrate a personalized treatment outcome approach. This concept is predicated on tumor- and patient-associated variables, combined with an individualized response assessment strategy for therapy modification as suggested by the patient's own results. As combined strategies may be outcome-orientated and integrate tumor-, patient- as well as cancer-preventive variables, this approach is likely to result in an optimized anticancer strategy. SUMMARY: Herein, we introduce such an anticancer strategy for all cancer patients, experts, and organizations: Imagine a World without Cancer.

Tubulin-binding dibenz[c,e]oxepines as colchinol analogues for targeting tumour vasculature.

Various methoxy- and hydroxy-substituted dibenz[c,e]oxepines were prepared via the copper(I)-induced coupling of ether-tethered arylstannanes or the dehydrative cyclisation of 1,1'-biphenyl-2,2'-dimethanols, assembled using the Ullmann cross-coupling of ortho-bromoaryl carbonyl compounds. The dibenzoxepines were screened for their ability to inhibit tubulin polymerisation and the in vitro growth of K562 human chronic myelogenous leukemia cells. The most active was 5,7-dihydro-3,9,10,11-tetramethoxydibenz[c,e]oxepin-4-ol, whose tubulin inhibitory and cytotoxicity (IC(50)) values were 1 µM and 40 nM, respectively.

Atroposelective formation of dibenz[c,e]azepines via intramolecular direct arylation with centre-axis chirality transfer.

5-Substituted 6,7-dihydrodibenz[c,e]azepines, a class of secondary amine incorporating a centre-axis chirality relay, are accessible from 1-substituted N-(2-bromobenzyl)-1-phenylmethanamines via N-acylation and ring-closing intramolecular direct arylation. The ring closure proceeds with high atropodiastereoselectivity due to strain effects that are induced by trigonalisation of the nitrogen atom, as predicted using molecular mechanics calculations.

How does CT image noise affect 3D deformable image registration for image-guided radiotherapy planning?

PURPOSE: To measure the sensitivity of deformable image registration to image noise. Deformable image registration can be used to map organ contours and other treatment planning data from one CT to another. These CT studies can be acquired with either conventional fan-beam CT systems or more novel cone-beam CT techniques. However, cone-beam CT images can have higher noise levels than fan-beam CT, which might reduce registration accuracy. We have investigated the effect of image quality differences on the deformable registration of fan-beam CTs and CTs with simulated cone-beam noise. METHOD: Our study used three CT studies for each of five prostate patients. Each CT was contoured by three experienced radiation oncologists. For each patient, one CT was designated the source image and the other two were target images. A deformable image registration process was used to register each source CT to each target CT and then transfer the manually drawn treatment planning contours from the source CT to the target CTs. The accuracy of the automatically transferred contours (and thus of the deformable registration process) was assessed by comparing them to the manual contours on the target CTs, with the differences evaluated with respect to interobserver variability in the manual contours. Then each of the target CTs was modified to include increased noise characteristic of cone-beam CT and the tests were repeated. Changes in registration accuracy due to increased noise were detected by monitoring changes in the automatically transferred contours. RESULTS: We found that the additional noise caused no significant loss of registration accuracy at magnitudes that exceeded what would normally be found in an actual cone-beam CT. SUMMARY: We conclude that noise levels in cone-beam CTs that might reduce manual contouring accuracy do not reduce image registration and automatic contouring accuracy.

Arterial thromboembolic disease: a single-centre case series study.

AIM: Paediatric venous thromboembolic disease has been reported with increased frequency during the last decade. In contrast, the pathophysiology of arterial thromboembolic disease in infants and children has not been adequately explored. The aim of this study was to determine the prevalence, aetiology, diagnostic criteria, management and outcome of arterial thromboembolism (TE) in a tertiary paediatric centre. METHODS: A prospective, single-centre registry was established at an Australian tertiary paediatric centre in order to address the aim of this study. RESULTS: One-hundred-and-two arterial thrombotic events occurred in 98 patients during 48 months. Infants were most likely to have a lower limb arterial TE (n = 22) whilst children were most likely to have a central nervous system arterial TE (n = 26). Surgery was a frequent predisposing factor in both infants and children. Doppler ultrasonography, computerized tomography and magnetic resonance imaging were the most commonly used diagnostic modalities. Unfractionated heparin was the most frequently used treatment in both age groups. At discharge, 25 infants and twelve children had complete resolution of their arterial TE. Direct thrombosis-related mortality was 4% in infants and 9% in children. Duration of follow-up ranged from 1 to 900 days, with thirteen infants and 32 children never achieving complete resolution. Forty-nine percent of post-discharge survivors had significant long term sequelae directly attributable to their arterial TE. CONCLUSION: Arterial TE occurred as frequently as venous TE in our tertiary paediatric population. The clinical outcome and long term sequelae of such events are significant.

Secretory carcinoma: the eastern Canadian experience and literature review.

BACKGROUND: Secretory Carcinoma (SC) is a recently described malignancy affecting salivary glands of the head and neck, with a paucity of evidence regarding the natural history, morbidity, and mortality. This study aimed to investigate the current treatment options utilized for SC, as well as its presentation and outcomes. METHODS: This study is a retrospective case series and includes patients diagnosed with SC at four Maritime Canadian institutions. Literature review of patient outcomes following treatment of SC is also included. RESULTS: Thirteen patients were identified. Parotid was the most common subsite (69%), followed by minor salivary gland (23%) and submandibular gland (8%). All patients were S100 positive and had at least one additional positive confirmatory stain, including mammaglobin, CK7, or vimentin. Two patients had N2b disease. All patients were treated with primary surgery, and four were offered adjuvant radiotherapy. There was one instance of locoregional recurrence, and one of metastasis. Three patients displayed perineural invasion on pathology, and one patient displayed lymphovascular invasion. CONCLUSION: Secretory Carcinoma remains understudied regarding its natural history, presentation, and treatment options. This study is the largest single case series in Canada, and highlights the young age and possible aggressiveness of SC. As well, we provide the most comprehensive literature review to date, with a focus on treatment and outcomes for this disease entity.

Technical Feasibility of Tissue Microarray (TMA) Analysis of Tumor-Associated Immune Response in Prostate Cancer.

Introduction: The androgen receptor (AR) regulates immune-related epithelial-to-mesenchymal transition (EMT), and prostate cancer (PCa) metastasis. Primary tumor-infiltrating lymphocytes (TILs) [CD3+, CD4+, and CD8+ TILs] are potential prognostic indicators in PCa, and variations may contribute to racial disparities in tumor biology and PCa outcomes. Aim: To assess the technical feasibility of tumor microarray (TMA)-based methods to perform multi-marker TIL profiling in primary resected PCa. Methods: Paraffin-embedded tissue cores of histopathologically-confirmed primary PCa (n = 40; 1 TMA tissue specimen loss) were arrayed in triplicate on TMAs. Expression profiles of AR, CD3+, CD4+, and CD8+ TILs in normal prostate, and the center and periphery of both the tumor-dominant nodule and highest Gleason grade were detected by IHC and associated with clinical and pathological data using standard statistical methodology. An independent pathologist, blinded to the clinical data, scored all samples (percent and intensity of positive cells). Results: TMAs were constructed from 21 (53.8%) Black and 18 (46.2%) White males with completely-resected, primarily pT2 stage PCa [pT2a (n = 3; 7.7%); pT2b (n = 2; 5.1%); pT2c (n = 27; 69.2%); pT3a (n = 5; 12.8%); mean pre-op PSA = 8.17 ng/ml]. The CD3, CD4, CD8, and CD8/CD3 cellular protein expression differed from normal in the periphery of the dominant nodule, the center of the highest Gleason grade, and the periphery of the highest Gleason grade (P < 0.05). Correlations between TIL expression in the center and periphery of the dominant nodule, with corresponding center and periphery of the highest Gleason grade, respectively, were robust, and the magnitude of these correlations differed markedly by race (P < 0.05). Conclusions: Multi-marker (AR, CD3, CD4, CD8) profiling with IHC analysis of TMAs consisting of primary, non-metastatic resected prostate cancer is technically feasible in this pilot study. Future studies will evaluate primary tumor immunoscore using semi-quantitative, IHC-based methodology to assess differences in the spectrum, quantity, and/or localization of TILs, and to gain insights into racial disparities in PCa tumor biology and clinical outcomes.

Myeloid sarcoma presenting as an isolated nodule in a patient with acute myelogenous leukemia.

We report a case of an elderly female in remission from acute myelogenous leukemia that presented with a nonhealing enlarging asymptomatic nodule on her right thigh. A wide excision of the nodule and histological examination revealed myeloid sarcoma without evidence or overlap of leukemia cutis, which had been suspected from nodules that had developed early in the course of the disease. The patient subsequently underwent radiation therapy to the area with sustained clearance.

Hypoxia-inducible factor-1-dependent regulation of the multidrug resistance (MDR1) gene.

The microenvironment of rapidly growing tumors is associated with increased energy demand and diminished vascular supply, resulting in focal areas of prominent hypoxia. A number of hypoxia-responsive genes have been associated with growing tumors, and here we demonstrate that the multidrug resistance (MDR1) gene product P-glycoprotein, a Mr approximately 170,000 transmembrane protein associated with tumor resistance to chemotherapeutics, is induced by ambient hypoxia. Initial studies using quantitative microarray analysis of RNA revealed an approximately 7-fold increase in MDR in epithelial cells exposed to hypoxia (pO(2) 20 torr, 18 h). These findings were further confirmed at the mRNA and protein level. P-Glycoprotein function was studied by analysis of verapamil-inhibitable efflux of digoxin and rhodamine 123 in intact T84 cells and revealed that hypoxia enhances P-glycoprotein function by as much as 7 +/- 0.4-fold over normoxia. Subsequent studies confirmed hypoxia-elicited MDR1 gene induction and increased P-glycoprotein expression in nontransformed, primary cultures of human microvascular endothelial cells, and analysis of multicellular spheroids subjected to hypoxia revealed increased resistance to doxorubicin. Examination of the MDR1 gene identified a binding site for hypoxia inducible factor-1 (HIF-1), and inhibition of HIF-1 expression by antisense oligonucleotides resulted in significant inhibition of hypoxia-inducible MDR1 expression and a nearly complete loss of basal MDR1 expression. Studies using luciferase promoter constructs revealed a significant increase in activity in cells subjected to hypoxia, and such hypoxia inducibility was lost in truncated constructs lacking the HIF-1 site and in HIF-1 binding site mutants. Extensions of these studies also identified a role for Sp1 in this hypoxia response. Taken together, these data indicate that the MDR1 gene is hypoxia responsive, and such results may identify hypoxia-elicited P-glycoprotein expression as a pathway for resistance of some tumors to chemotherapeutics.

Genomics, microRNA, epigenetics, and proteomics for future diagnosis, treatment and monitoring response in upper GI cancers.

One major objective for our evolving understanding in the treatment of cancers will be to address how a combination of diagnosis and treatment strategies can be used to integrate patient and tumor variables with an outcome-oriented approach. Such an approach, in a multimodal therapy setting, could identify those patients (1) who should undergo a defined treatment (personalized therapy) (2) in whom modifications of the multimodal therapy due to observed responses might lead to an improvement of the response and/or prognosis (individualized therapy), (3) who might not benefit from a particular toxic treatment regimen, and (4) who could be identified early on and thereby be spared the morbidity associated with such treatments. These strategies could lead in the direction of precision medicine and there is hope of integrating translational molecular data to improve cancer classifications. In order to achieve these goals, it is necessary to understand the key issues in different aspects of biotechnology to anticipate future directions of personalized and individualized diagnosis and multimodal treatment strategies. Providing an overview of translational data in cancers proved to be a challenge as different methods and techniques used to obtain molecular data are used and studies are based on different tumor entities with different tumor biology and prognoses as well as vastly different therapeutic approaches. The pros and cons of the available methodologies and the potential response data in genomics, microRNA, epigenetics and proteomics with a focus on upper gastrointestinal cancers are considered herein to allow for an understanding of where these technologies stand with respect to cancer diagnosis, prognosis and treatment.

Functionalized cyclobutenes via multicomponent thermal [2 + 2] cycloaddition reactions.

[reaction: see text] Enamine [2 + 2] cycloadditions can be achieved in useful yields simply by stirring a mixture of an aldehyde, diethylamine, a dialkyl fumarate, and potassium carbonate in acetonitrile at 25 degrees C, conditions that are compatible with the presence of a potential leaving group on the beta-position of the intermediate enamine. Methylation and elimination of the product cyclobutanes completes a mild nonphotochemical route to functionalized cyclobutenes.

Distribution of the H+/peptide transporter PepT1 in human intestine: up-regulated expression in the colonic mucosa of patients with short-bowel syndrome.

BACKGROUND: Intestinal adaptation after massive bowel resection in animal models is characterized by increased gut-mucosal growth and expression of nutrient transporters. Few data about these indexes exist in humans with short-bowel syndrome (SBS). OBJECTIVE: The objective was to compare small-bowel and colonic mucosal growth and expression of the peptide transporter PepT1 in adults with or without SBS. DESIGN: Mucosal biopsy specimens were obtained from the small bowel and colon of 33 control subjects with intact intestine and from 13 SBS patients dependent on parenteral nutrition because of chronic malabsorption. Gut-mucosal crypt depth, villus height, and villus width were measured, and expression of PepT1 was determined by Northern blotting, in situ hybridization, and immunohistochemistry. RESULTS: The indexes of small-bowel and colonic mucosal growth were not significantly different between the 2 groups. PepT1 expression was high in the apical region of duodenal, jejunal, and ileal villus epithelial cells; low in absorptive colonocytes; and not significantly different in the distal small intestine of the 2 groups. However, the abundance of PepT1 mRNA in the colon of SBS patients was more than 5-fold that in control subjects (P < 0.01). CONCLUSIONS: Gut adaptation in SBS patients does not appear to involve an increase in gut-mucosal crypt depth or villus size. PepT1 is abundant along the small-bowel brush border in humans; expression in the colon indicates that the large intestine has a mechanism for luminal di- and tripeptide transport. Up-regulation of colonic PepT1 in SBS may adaptively improve accrual of malabsorbed di- and tripeptides, independent of changes in the mucosal surface area.

Macrophage and retinal pigment epithelium expression of angiogenic cytokines in choroidal neovascularization.

PURPOSE: To determine the expression of angiogenic cytokines in macrophages and retinal pigment epithelium cells in choroidal neovascularization (CNV). METHODS: Ten surgically-excised subfoveal CNV specimens and ten eye bank eyes with subfoveal CNV were routinely processed, serially sectioned, and immunostained for factor VIII (F8), CD68 (KP1), cytokeratin 18 (CK18), vascular endothelial growth factor (VEGF), tissue factor (TF), and monocyte chemotactic protein (MCP). The CNV was classified as "inflammatory active" (more inflammation than fibrosis) or "inflammatory inactive" (morefibrosis than inflammation). The immunostaining was graded as none, mild (+), moderate (++), or heavy (+++). Five additional surgically-excised CNV specimens were dual labeled with CK18/MCP or CD68/TF and confocal scanning laser microscopy was performed. RESULTS: Vascular endothelium, macrophages, and RPE expressed F8, KP1, and CK18 respectively. Macrophages expressed + to ++ VEGF and ++ to +++ TF; RPE expressed ++ to +++ VEGF and ++ to +++ MCP. Staining for angiogenic cytokines was stronger in inflammatory active versus inflammatory inactive CNV. RPE dual labeled for CK18/MCP and macrophages dual labeled for CD68/TF. CONCLUSIONS: This study shows that RPE cells express MCP, a cytokine involved with macrophage recruitment, and that macrophages express TF in CNV. Macrophages and RPE express VEGF, thus perpetuating angiogenesis. TF is involved with fibrin formation and provides a scaffold effect for growth of the CNV complex. CNV likely represents a dynamic process with inflammatory active and inflammatory inactive (involutional) stages.