A Phase IIa Multicenter, Randomized, Vehicle-Controlled, Dose Escalating Study to Evaluate the Safety, Efficacy, and Pharmacokinetics of CBT-001 Ophthalmic Solution in Patients With Primary or Recurrent Pterygium.

Purpose: To evaluate the safety and efficacy of CBT-001, a multitarget tyrosine kinase inhibitor eyedrop, for pterygia. Design: Phase II clinical trial. Stage 1 was a single center, open-labeled, vehicle-controlled study. Stage 2 was a multicenter, randomized, double-masked, vehicle-controlled trial. Participants: Patients with primary or recurrent pterygia. Main Outcome Measures: The primary efficacy end point was lesion vascularity based on masked grading of photographs by an independent reading center. Other end points included dimensions of pterygia and safety. Methods: In stage 1, 24 eyes of 24 patients received 1 drop of CBT-001 in a dose escalation fashion (0.02%, 0.05%, and 0.2%) to determine the maximally tolerated dose based on adverse events (AEs) and blood drug levels. In stage 2, subjects were randomly assigned to receive the maximally tolerated dose of CBT-001 or vehicle dosed 3 times a day for 4 weeks with a 20-week follow-up. Results: In stage 1, the plasma maximum concentration values for all doses of CBT-001 were at or below the limit of detection (0.01 ng/ml). The most commonly reported AEs were mild foreign body sensation and irritation. CBT-001 0.2% was evaluated in stage 2. Baseline demographic characteristics were similar between patients receiving CBT-001 (n = 25) and vehicle (n = 23). After 4 weeks of dosing, the mean change from baseline in pterygium vascularity scores was -0.8 ± 0.7 (mean ± standard deviation) in subjects receiving CBT-001 0.2% and 0.0 ± 0.5 in subjects receiving vehicle (P < 0.001; 95% confidence interval: -1.12, -0.40). Pterygium vascularity scores remained significantly decreased, after the 4-week dosing period, at weeks 8 and 16, but not at week 24. The mean changes from baseline in the length of the pterygia were also significantly lower in subjects receiving CBT-001 compared with vehicle at weeks 2, 4, and 8 (P ≤ 0.014). The most commonly reported AEs were ocular, mild in severity, resolved after therapy, and did not result in discontinuation. Conclusions: CBT-001 0.2% decreased pterygia vascularity and lesion length after 4 weeks of dosing with a prolonged effect after dosing. The drug was well tolerated with minimal detected systemic drug levels. Financial Disclosures: Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

The expression and significance of AKR1B10 in laryngeal squamous cell carcinoma.

Aldosterone reductase family 1 member B10 (AKR1B10) is a nicotinamide adenine dinucleotide phosphate (reduced coenzyme II)-dependent oxidoreductase, and its biological functions include carbonyl detoxification, hormone metabolism, osmotic adjustment, and lipid synthesis. Studies suggested that AKR1B10 is a new biomarker for cancer based on its overexpression in epithelial tumors, such as breast cancer, cervical cancer, and lung cancer. At present, studies on the expression of AKR1B10 in laryngeal cancer have not been reported. However, we found that AKR1B10 is upregulated in laryngeal carcinoma, and its expression was negatively correlated with the degree of differentiation. In addition, AKR1B10 expression was positively correlated with tumor size; lymph node metastasis; alcohol use; and Ki-67, mutant p53, and matrix metalloproteinase 2 expression. AKR1B10 was overexpressed in Hep-2 laryngeal carcinoma cells. Oleanolic acid inhibited AKR1B10 activity and expression in Hep-2 cells and suppressed Hep-2 cell proliferation, migration, and invasion. Therefore, AKR1B10 may be related to the development of laryngeal carcinoma, suggesting its use as a prognostic indicator for laryngeal cancer.

Case Report: Bilateral Renal Cell Carcinoma With Different Histological and Morphological Features, Clear Cell and Cystic Thyroid-Like Follicular Subtype.

Thyroid-like follicular renal cell carcinoma is a rare subtype of renal cell carcinoma that has only been recently recognized, as most cases involve a solid tumor in one kidney. In this study, we report a rare case of bilateral renal cell carcinoma wherein the tumor in the left kidney was diagnosed as clear cell carcinoma, while the tumor in right kidney as thyroid-like follicular renal cell carcinoma. The difference between this case and the ones described in previous reports is that thyroid-like follicular renal cell carcinoma showed cystic changes on imaging. This suggests that when renal cystic lesions are encountered, we should consider the possibility of such rare tumors.

PD-L1 Expression in Chinese Patients with Advanced Non-Small Cell Lung Cancer (NSCLC): A Multi-Center Retrospective Observational Study.

Objective: This study aimed to investigate the prevalence of tumor programmed death-ligand 1 (PD-L1) expression in Chinese patients with advanced Non-Small Cell Lung Cancer (NSCLC). Methods: Tumor tissues with histologically confirmed stage IIIB/IV NSCLC were retrospectively obtained from 10 centers in China. PD-L1 expression was determined using the PD-L1 IHC 22C3 pharmDx kit (Agilent, Santa Clara, CA, USA) and the samples were repetitively assayed with the PD-L1 IHC 22C3 Ab concentrate (Agilent, Santa Clara, CA, USA). Results: Out of 901 patients who met the inclusion criteria, 879 (97.6%) had evaluable PD-L1 data. The number of patients with a PD-L1 tumor proportion score (TPS) < 1%, 1-49%, and ≥ 50% (corresponding to PD-L1 non-expression, low expression, and high expression) was 424 (48.2%), 266 (30.3%), and 189 (21.5%), respectively. PD-L1 expression was more likely to be found in patients younger than 75 years, men, current or former smokers, those with good performance status (PS) scores, and those with a wild-type epidermal growth factor receptor (EGFR). PD-L1 TPS ≥ 50% and ≥ 1% were respectively 28.0% and 50.2% among patients negative for both EGFR mutation and anaplastic lymphoma kinase (ALK) rearrangement. PD-L1 expression determined using the 22C3 antibody concentrate and pharmDx kit had comparable results. Conclusions: The prevalence of PD­L1 expression in advanced NSCLC was consistent with that reported in the global EXPRESS study. Age, gender, smoking history, PS scores, and EGFR/ALK mutation status affected PD-L1 expression. The 22C3 antibody concentrate appears to be an alternative reagent for the PD-L1 assay.

Association between androgen receptor gene polymorphisms and testicular germ cell tumor: A systematic review and meta-analysis.

OBJECTIVE: To estimate association between androgen receptor (AR) gene polymorphisms and testicular germ cell tumor (TGCT) susceptibility. MATERIALS AND METHODS: Systematic search of studies on the association between AR gene polymorphisms and TGCT susceptibility was conducted. Odds ratios and 95% confidence intervals were used to pool effect size. RESULTS: For CAG repeat, no evidence was found for association between (>25 vs. ≤25), (>25 vs. 21-25), (<21 vs. 21-25), (others vs. 21-25), (>23 vs. ≤23), (<21 vs. ≥21), (<21 vs. ≥21)'s some subgroups and TGCT susceptibility, which showed stability. In (>24 vs. ≤24), (>24 vs. 21-24), (<21 vs. 21-24), and (others vs. 21-24) and almost all of their subgroups, increased TGCT risk was found without sensitivity analysis. For GGN, no statistical change of TGCT risk was found in (<23 vs. ≥23), (<23 vs. 23), which showed stability. For single nucleotide polymorphism (SNP) rs6152 G > A, rs1204038 G > A and rs2361634 A > G, no statistical change was found without sensitivity analysis. CONCLUSIONS: GGN repeat number <23 may not be associated with TGCTs susceptibility. However, there was insufficient data to fully confirm association in GGN repeat number >23, CAG repeat number, SNP rs6152, rs1204038, and rs2361634.

Spontaneous peripheral nerve palsy with hourglass-like fascicular constriction in the upper extremity.

OBJECTIVE: Spontaneous paralysis from hourglass-like fascicular constriction of peripheral nerves is rare, its clinical manifestations are not well documented, and its pathogenesis remains unknown. The unclear origin of this disorder and difficulty in diagnosis result in its uncertain management. The authors sought to gain a more thorough understanding of this condition through describing the anatomy, clinical features, etiology, and treatment of hourglass-like constriction. METHODS: The authors retrospectively reviewed 20 patients (22 nerves) with hourglass-like constriction. The patients' clinical information was reviewed. Preoperative sonographic assessment and electrophysiological examination of involved nerves were performed. Surgical treatments included interfascicular neurolysis and neurorrhaphy. Samples of tissue subjected to resected constriction were sent for pathological analysis. The patients had regular face-to-face follow-up visits. RESULTS: Acute pain was always the first symptom and was followed by paralysis. Paralysis progression was rapid and serious. Surgical exploration indicated an hourglass-like constricted segment completely unrelated to the compressive structures. Electrophysiological analysis showed severe denervation, and histopathological examination showed inflammatory cell infiltration, demyelination, and reduction of nerve fibers. CONCLUSIONS: Hourglass-like fascicular constrictive neuropathy has an integrative effect from multiple different mechanisms. Surgical intervention is beneficial for selected patients who do not recover in a timely fashion and have hourglass-like lesions confirmed by preoperative ultrasound imaging. The authors recommend that early surgical intervention of the nerve be offered to patients who do not show any signs of recovery 3 months after onset. Both interfascicular neurolysis and neurorrhaphy are effective treatment methods. Mild to moderate constriction can usually be treated successfully by interfascicular neurolysis alone, whereas more advanced lesions with loss of fascicle continuity (severe constriction) may be best treated with resection and direct neurorrhaphy.

DNA demethylation of claudin-4 suppresses migration and invasion in laryngeal squamous carcinoma cells.

Claudin-4 (CLDN4) is a member of the claudin transmembrane protein family, which consists of integral membrane proteins that are components of the epithelial cell tight junctions; these tight junctions regulate movement of solutes and ions through the paracellular space. CLDN4 is also a differentiation marker and is believed to indicate an epithelial phenotype. However, the role of CLDN4 in laryngeal squamous carcinoma is still unclear. Here, we showed that CLDN4 expression was down-regulated in laryngeal squamous carcinoma tissues and negatively correlated with methyl-CpG-binding protein 2. In addition, CLDN4 was hypermethylated in HEp-2 cells. DNA demethylation of CLDN4 by 5-aza-2'-deoxycytidine suppressed migration and invasion of HEp-2 cells, whereas CLDN4 silencing restored the migration and invasion of HEp-2 cells. Therefore, CLDN4 plays a key role in laryngeal squamous carcinoma progression.

Correlation of low expression of hMOF with clinicopathological features of colorectal carcinoma, gastric cancer and renal cell carcinoma.

Human MOF (males absent on the first), as a histone acetyltransferase, is responsible for histone H4K16 acetylation in human cells. Recent studies have shown that the abnormal gene expression of hMOF is involved in certain primary cancers. Here, we first report the involvement of hMOF expression in clinically diagnosed primary colorectal carcinoma (CRC) and gastric cancer. Simultaneously, the correlation of hMOF expression and clinicopathological features in CRC, gastric cancer and renal cell carcinoma (RCC) was analyzed. The hMOF mRNA expression was assessed in 44 CRC, 16 gastric cancer and 47 RCC human tissue samples by quantitative PCR (qPCR). Statistical analysis of qPCR data revealed a significant reduction (>2-fold decrease) of hMOF gene expression in CRC, 57% (25/44), 94% (15/16) in gastric cancer and 74% (35/47) in RCC tissues of the patients. In patients with CRC, lymph node metastasis and tumor stage were associated with hMOF expression patterns. However, no significant association between hMOF expression and tumor types emerged (p>0.05). Interestingly, in patients with gastric cancer, although no statistically significant difference was found between adjacent (<2 cm away from the cancer tissue) and normal tissues (>5 cm away from the cancer tissue), >2-fold reduction of hMOF expression in adjacent tissues had already appeared in 35% of patients. In addition, low expression of hMOF was strongly correlated with tumor differentiation (p<0.05) and survival of patients with gastric cancer (p<0.001). While in patients with RCC, downregulation of hMOF was connected to ccRCC and tissues with T1 tumor status. Our results suggest that downregulation of hMOF may be common in cancer tissues, and may represent a novel biomarker for tumor diagnosis.

A higher frequency of circulating IL-22(+)CD4(+) T cells in Chinese patients with newly diagnosed Hashimoto's thyroiditis.

BACKGROUND: IL-22 and IL-17A are implicated in the pathogenesis of autoimmune diseases. However, the role of IL-22(+) and IL-17A(+) CD4(+) T cells in the pathogenesis of Hashimoto's thyroiditis (HT) is not fully understood. This study investigates serum IL-22 and IL-17A levels and determines the frequency of circulating IL-22(+) CD4(+) T cells in HT patients to understand their roles in the pathogenesis of HT. METHODS: The levels of serum IL-22, IL-17A and IFN-γ and the frequency of circulating IL-22(+)CD4(+) and IL-17A(+)CD4(+) T cells in 17 HT patients and 17 healthy controls (HC) were determined by enzyme-linked immunosorbent assay (ELISA) and flow cytometry. The levels of serum free triiodothyronine (FT4), free thyroxine (FT3), thyroid stimulating hormone (TSH), anti-thyroid peroxidase (TPO) and anti-thyroglobulin antibodies (TgAb) by chemiluminescent enzyme immunoassay and radioimmunoassay. RESULTS: The percentages of circulating IL-22(+)CD4(+) and IL-17(+)CD4(+) T cells (p<0.0001, p<0.0001) and the levels of serum IL-22, IL-17A and IFN-γ (p<0.0001, p<0.0001, p = 0.0210) in the HT patients were significantly higher than that in the HC. The percentages of IL-22(+)CD4(+) T cells were positively correlated with Th17 cells (r = 0.8815, p<0.0001) and IL-17A(+)IL-22(+)CD4(+) T cells (r = 0.8914, p<0.0001), but were negatively correlated with Th1 cells (r = -0.6110, p<0.0092) in the HT patients. The percentages of Th22 cells, Th17 cells and IL-17A(+)IL-22(+)CD4(+) T cells were negatively correlated with the levels of serum TSH in the HT patients (r = -0.8402, p<0.0001; r = -0.8589, p<0.0001; r = -0.8289 p<0.0001, respectively). CONCLUSIONS: A higher frequency of circulating IL-22(+)CD4(+) and IL-17A(+)CD4(+) T cells may be associated with the development of HT in Chinese patients.

Differential BCCIP gene expression in primary human ovarian cancer, renal cell carcinoma and colorectal cancer tissues.

Human BCCIP, a protein which interacts with BRCA2 and CDKN1A (Cip1, p21), has been implicated in many cellular processes including cell cycle regulation, DNA recombination and damage repair, telomere maintenance, embryonic development and genomic stability. BCCIP gene expression, which is an important BRCA2 cofactor in tumor suppression, has been identified in some primary cancers. Thus, we investigated the role of BCCIP expression in a large sample of clinically diagnosed primary ovarian cancer, renal cell carcinoma (RCC) and colorectal cancer (CRC) tissues. Using clinically diagnosed frozen primary cancer tissues, quantitative PCR (qPCR), western blot analysis (WB) and immunohistochemical staining (IHC) approaches were used to detect and measure gene expression. Reduced BCCIP gene expression in ovarian cancer, RCC and CRC tissues occurred in 74, 89 and 75% of tissue samples, respectively. qPCR analysis of mRNA expression in 54 ovarian cancer, 50 RCC and 44 CRC samples revealed significant (>2-fold decreased) BCCIP downregulation in 56, 70 and 46% of tissue samples, respectively. Although BCCIP expression in three different tumor tissues decreased, the relationship between BCCIP expression and clinicopathological features of each cancer was distinct. Compared to normal tissues, BCCIP expression in ovarian cancers was significantly downregulated in serous, endometrioid and mucinous carcinomas. Downregulation of BCCIP expression was strongly associated with clear cell RCC (ccRCC) and Fuhrman tumor grading, but significant differences in BCCIP expression between CRC and matched normal tissues occurred only in male CRC tissues (p<0.05) and in tissue with a T4 tumor stage (p<0.01). Thus, BCCIP protein was chiefly reduced in ovarian cancer and RCC tissue samples (p<0.05). BCCIP gene expression was downregulated in human ovarian cancer, RCC and CRC tissues, suggesting a role for the gene in the pathogenesis of these cancers.

A potential diagnostic marker for ovarian cancer: Involvement of the histone acetyltransferase, human males absent on the first.

Human males absent on the first (hMOF), a human ortholog of the Drosophila MOF protein, is responsible for histone H4 lysine 16 (H4K16) acetylation in human cells. The depletion of hMOF leads to a global reduction in histone H4K16 acetylation in human cells, genomic instability, cell cycle defects, reduced transcription of certain genes, defective DNA damage repair and early embryonic lethality. Studies have shown that abnormal hMOF gene expression is involved in a number of primary cancers. The present study examined the involvement of hMOF expression and histone H4K16 acetylation in clinically diagnosed primary ovarian cancer tissues. Clinically diagnosed frozen primary ovarian cancer tissues were used for polymerase chain reaction (PCR), quantitative PCR (qPCR), western blotting and immunohistochemical staining approaches. A PCR analysis of mRNA expression in 47 samples revealed a downregulation of hMOF mRNA in 81% of patients, whereas only 13% of patients demonstrated upregulation. qPCR was used to validate the frequent downregulation of hMOF expression in the primary ovarian cancer tissues. As expected, the analysis of hMOF expression in 57 samples revealed that hMOF mRNA expression was significantly downregulated (>2-fold decrease) in 65% of patients, while a <2-fold reduction of hMOF was observed in 10.5% of patients. Furthermore, the expression of hMOF-regulated human leukocyte antigen (HLA) complex 5, (HCP5), was also found to be downregulated in >87% of patients with a decrease in hMOF. hMOF and its regulated gene, HCP5, are frequently downregulated in human ovarian cancer, suggesting that hMOF may be involved in the pathogenesis of the disease.

Overexpression of Aldo-keto reductase family 1 B10 protein in ductal carcinoma in situ of the breast correlates with HER2 positivity.

BACKGROUND: Ductal carcinoma in situ of the breast constitutes the early stage of breast cancer when cancer cells are confined by the intact myoepithelial cell layer. Transition from DCIS to invasive carcinoma is a process yet poorly understood. MATERIALS AND METHODS: By liquid chromatography (LC) and mass spectrometry (MS/MS) methods, we analyzed this early event using paired samples of micro-dissected cells overlaid with focally disrupted myoepithelial layers and their adjacent counterparts within the intact duct from formalin-fixed paraffin-embedded blocks. RESULTS: AKR1B10, a member of Aldo-keto reductase family, was shown to be abundantly located in the filtering cells among a catalog of proteins. Moreover, strong correlation between AKR1B10 and HER2 positivity was found in an independent cohort of DCIS samples. CONCLUSION: AKR1B10 could become a potential diagnosis and therapeutic marker for early breast cancers with HER2 overexpression and poor prognosis.

Epidermal growth factor and gastrin on PDX1 expression in experimental type 1 diabetic rats.

INTRODUCTION: The aim of this study was to investigate whether combined epidermal growth factor (EGF) and gastrin can correct the hyperglycemia induced by streptozotocin (STZ) in rats and to determine the involvement of the transcription factor pancreatic and duodenal homeobox 1 (PDX1) in this process. METHODS: Rat diabetes was induced by intraperitoneal injection of STZ. The mRNA and protein levels of insulin and PDX1 were determined by real-time reverse transcriptase polymerase chain reaction and immunohistochemistry. Serum levels of C-peptide and insulin were analyzed using radioimmunoassay kits. RESULTS: The combined administration of EGF and gastrin efficiently reversed the hyperglycemia induced by STZ. Elevated insulin concentration was detected in diabetic rats treated with EGF plus gastrin. The authors also found that both insulin and PDX1 expression were reduced in STZ-treated rats. Interestingly, the combination treatment also significantly enhanced the mRNA levels of insulin and PDX1, and that of their protein products. CONCLUSIONS: Therapy with EGF plus gastrin corrected hyperglycemia and maintained insulin content in STZ-induced diabetic rats via up-regulation of PDX1 expression, suggesting that this combination treatment may provide a valuable approach for pancreatic islet neogenesis in vivo.

Effects of Combined Epidermal Growth Factor and Gastrin on PDX1 Expression in Experimental Type 1 Diabetic Rats.

INTRODUCTION:: The aim of this study was to investigate whether combined epidermal growth factor (EGF) and gastrin can correct the hyperglycemia induced by streptozotocin (STZ) in rats and to determine the involvement of the transcription factor pancreatic and duodenal homeobox 1 (PDX1) in this process. METHODS:: Rat diabetes was induced by intraperitoneal injection of STZ. The mRNA and protein levels of insulin and PDX1 were determined by real-time reverse transcriptase polymerase chain reaction and immunohistochemistry. Serum levels of C-peptide and insulin were analyzed using radioimmunoassay kits. RESULTS:: The combined administration of EGF and gastrin efficiently reversed the hyperglycemia induced by STZ. Elevated insulin concentration was detected in diabetic rats treated with EGF plus gastrin. The authors also found that both insulin and PDX1 expression were reduced in STZ-treated rats. Interestingly, the combination treatment also significantly enhanced the mRNA levels of insulin and PDX1, and that of their protein products. CONCLUSIONS:: Therapy with EGF plus gastrin corrected hyperglycemia and maintained insulin content in STZ-induced diabetic rats via up-regulation of PDX1 expression, suggesting that this combination treatment may provide a valuable approach for pancreatic islet neogenesis in vivo.

Cell budding from pre-invasive tumors: Intrinsic precursor of invasive breast lesions?

Our previous studies showed that in patients with ductal carcinoma in situ (DCIS) of the breast, the tumor cells that overlie focal myoepithelial cell layer disruptions (FMCLDs) are generally arranged as finger-like projections that bud into the stroma. These budding cells have significantly more genetic instability and invasion-related gene expression, and less estrogen receptor (ER) expression, than their epithelial cell counterparts. This study aimed to assess these cells for potential molecular markers that are uniquely associated with cell adhesion and motility. Seventeen ER-positive DCIS cases were screened by immunostaining for ER, and 7 cases which harbored FMCLD lesions were used to examine the expression of the potential markers. Two cases with both DCIS and invasive lesions were selected for comparing the differences in molecular expression between these lesion types. The results showed that expression levels of talin, E-cadherin and focal adhesion kinase (FAK) in tumor cells overlying FMCLDs were higher than those within the corresponding duct. Integrin ß1 staining was detected only in a small number of the tumor cells overlying the FMCLDs. Vinculin staining was weak (18%) or not detected (82%), and no expression was found in the tumor cells within the corresponding duct or in the pure isolated DCIS. By contrast, the expression levels of talin, vinculin and integrin ß1 in the invasive tumors were distinctly higher than those in DCIS, and the expression of FAK and E-cadherin was lower. Using electron microscopy, we found that the tight junctions between tumor cells overlying the FMCLDs were reduced compared to the adjacent tumor cells in the lumen. These results indicate that the tumor cells overlying FMCLDs are likely to represent the specific precursors of invasive breast lesions. Our findings may also facilitate the identification of specific targets for further molecular profiling, which will more completely characterize this important cell population.

Sensitivity and proportionality assessment of metabolites from microdose to high dose in rats using LC-MS/MS.

BACKGROUND: The objective of this study was to evaluate the sensitivity requirement for LC-MS/MS as an analytical tool to characterize metabolites in plasma and urine at microdoses in rats and to investigate proportionality of metabolite exposure from a microdose of 1.67 µg/kg to a high dose of 5000 µg/kg for atorvastatin, ofloxacin, omeprazole and tamoxifen. RESULTS: Only the glucuronide metabolite of ofloxacin, the hydroxylation metabolite of omeprazole and the hydration metabolite of tamoxifen were characterized in rat plasma at microdose by LC-MS/MS. The exposure of detected metabolites of omeprazole and tamoxifen appeared to increase in a nonproportional manner with increasing doses. Exposure of ortho- and para-hydroxyatorvastatin, but not atorvastatin and lactone, increased proportionally with increasing doses. CONCLUSION: LC-MS/MS has demonstrated its usefulness for detecting and characterizing the major metabolites in plasma and urine at microdosing levels in rats. The exposure of metabolites at microdose could not simply be used to predict their exposure at higher doses.

Atypical E-cadherin expression in cell clusters overlying focally disrupted mammary myoepithelial cell layers: implications for tumor cell motility and invasion.

Our recent studies showed that cell clusters overlying focal myoepithelial cell layer disruptions (FMCLD) had a significantly higher rate of ER negativity, genetic instabilities, and expression of invasion-related genes than adjacent cells within the same duct. This study attempted to determine if these cells would show aberrant E-cadherin expression, which imparts greater propensity for cell motility and invasion. Consecutive sections from breast tumors with a high frequency of FMCLD were double-immunostained for E-cadherin and a panel of related markers. The E-cadherin mRNA levels in cells overlying FMCLD and adjacent cells within the same duct were compared using real-time PCR. Nearly all the cell clusters overlying FMCLD were strongly immunoreactive for E-cadherin, whereas their adjacent counterparts within the same duct were largely negative. Cell clusters overlying FMCLD were generally arranged as tongue-like projections, "puncturing" deep into the stroma or tube-like structures that often contained red blood cells. The sub-cellular localization of E-cadherin in the above structures, however, was primarily cytoplasmic. The mRNA level of E-cadherin in cell clusters overlying FMCLD was significantly higher than that in adjacent cells within the same duct. These findings suggest that aberrant expression of E-cadherin may contribute to cell motility and invasion.

Aberrant c-erbB2 expression in cell clusters overlying focally disrupted breast myoepithelial cell layers: a trigger or sign for emergence of more aggressive cell clones?

Our recent studies revealed that cell clusters overlying focal myoepithelial cell layer disruption (FMCLD) had a significantly higher frequency of genetic instabilities and expression of invasion-related genes than their adjacent counterparts within the same duct. Our current study attempted to assess whether these cell clusters would also have elevated c-erbB2 expression. Human breast tumors (n=50) with a high frequency of FMCLD were analyzed with double immunohistochemistry, real-time RT-PCR, and chromogenic in situ hybridization for c-erbB2 protein and gene expression. Of 448 FMCLD detected, 404 (90.2%) were associated with cell clusters that had intense c-erbB2 immunoreactivities primarily in their cytoplasm, in contrast to their adjacent counterparts within the same duct, which had no or barely detectable c-erbB2 expression. These c-erbB2 positive cells were arranged as tongue-like projections, "puncturing" into the stroma, and about 20% of them were in direct continuity with tube-like structures that resembled blood vessels. Aberrant c-erbB2 expression was also seen in clusters of architecturally normal-appearing ducts that had distinct cytological abnormalities in both ME and epithelial cells, whereas not in their clear-cut normal counterparts. Molecular assays detected markedly higher c-erbB2 mRNA and gene amplification in cell clusters associated with FMCLD than in those associated with non-disrupted ME cell layers. Our findings suggest that cell clusters overlying FMCLD may represent the precursors of pending invasive lesions, and that aberrant c-erbB2 expression may trigger or signify the emergence of biologically more aggressive cell clones.

Xanthine oxidase interaction with vascular endothelial growth factor in human endothelial cell angiogenesis.

OBJECTIVES: Reduced capillary density occurs early in cardiovascular diseases. Oxidant stress is implicated in endothelial apoptosis. We investigated the effects of xanthine oxidase (XO) on endothelial survival signaling: protein kinase B/Akt, its cross-talk with p38 MAPK and apoptosis pathways, and its effect on vascular tube formation in vascular endothelial growth factor (VEGF)-simulated human umbilical vein cells. METHODS: We studied primary cultured human endothelial cells from the umbilical cord. Reactive oxygen species (ROS) production was detected by dihydroethidium staining, cell-signaling pathways by western blots, cell survival by western blots, and nuclear chromatin and angiogenesis response by MTT proliferation assay and three-dimensional Matrigel cultures. RESULTS: Exogenous XO increased cellular ROS production and caused superoxide-dependent inhibition of Akt phosphorylation and enhancement of p38 MAPK phosphorylation in a time-and dose-dependent manner. In contrast, application of the XO inhibitor oxypurinol or allopurinol inhibited VEGF-stimulated Akt phosphorylation, indicating that endogenous XO promotes VEGF-induced endothelial cell (EC) survival signaling. Exogenous XO induced activation of caspase-3 and reduced expression of the anti-apoptosis protein Bcl-2. Exogenous XO also reduced EC viability, proliferation, and vascular tube formation by p38 MAPK-dependent, phosphoinositide 3-kinase (PI3-K) reversible mechanisms; whereas VEGF promoted EC survival by PI3-K-dependent, p38 MAPK-independent effects. CONCLUSIONS: Exogenous XO activity is an important contributor to endothelial mechanisms for microvascular rarefaction, by modulation of cell survival signaling pathways; however, endogenous XO is necessary for maintaining EC survival.

Microdosing assessment to evaluate pharmacokinetics and drug metabolism in rats using liquid chromatography-tandem mass spectrometry.

PURPOSE: To evaluate the sensitivity requirement for LC-MS/MS as an analytical tool to support human microdosing study with sub-pharmacological dose, investigate proportionality of pharmacokinetics from the microdose to therapeutic human equivalent doses in rats and characterize circulating metabolites in rats administered with the microdose. MATERIALS AND METHODS: Five drugs of antipyrine, metoprolol, carbamazepine, digoxin and atenolol were administered orally to male Sprague-Dawley rats at 0.167, 1.67, 16.7, 167 and 1,670 microg/kg doses. Plasma samples were extracted using either solid phase extraction or liquid-liquid extraction, and analyzed using LC-MS/MS. RESULTS: Using 100 microl of plasma sample, the lower limit of quantitation for antipyrine (10 pg/ml), carbamazepine (1 pg/ml), metoprolol (5 pg/ml), atenolol (20 pg/ml), and digoxin (5 pg/ml) were achieved using an API 5000. Proportional pharmacokinetics were observed from 0.167 microg/kg to 1,670 microg/kg for antipyrine and carbamazepine and from 1.67 to 1,670 microg/kg for atenolol and digoxin, while metoprolol exhibited a non-proportional pharmacokinetics relationship. Several metabolites of carbamazepine were characterized in plasma from rats dosed at 1.67 mug/kg using LC-MS/MS. CONCLUSIONS: This study has shown the promise of sensitive LC-MS/MS method to support microdose pharmacokinetics and drug metabolism studies in human.