TSH-Mediated TNFα Production in Human Fibrocytes Is Inhibited by Teprotumumab, an IGF-1R Antagonist.

PURPOSE: Fibrocytes (FC) are bone marrow-derived progenitor cells that are more abundant and infiltrate the thyroid and orbit in Graves orbitopathy (GO). FCs express high levels of thyrotropin receptor (TSHR) and insulin-like growth factor-1 receptor (IGF-1R). These receptors are physically and functionally associated, but their role in GO pathogenesis is not fully delineated. Treatment of FCs with thyroid stimulating hormone (TSH) or M22 (activating antibody to TSHR) induces the production of numerous cytokines, including tumor necrosis factor α (TNFα). Teprotumumab (TMB) is a human monoclonal IGF-1R blocking antibody currently in clinical trial for GO and inhibits TSHR-mediated actions in FCs. AIM: To characterize the molecular mechanisms underlying TSH-induced TNFα production by FCs, and the role of IGF-1R blockade by TMB. DESIGN: FCs from healthy and GD patients were treated with combinations of TSH, M22, MG132 and AKTi (inhibitors of NF-κB and Akt, respectively), and TMB. TNFα protein production was measured by Luminex and flow cytometry. Messenger RNA expression was quantified by real time PCR. RESULTS: Treatment with TSH/M22 induced TNFα protein and mRNA production by FCs, both of which were reduced when FCs were pretreated with MG132 and AKTi (p<0.0001). TMB decreased TSH-induced TNFα protein production in circulating FCs from mean fluorescent index (MFI) value of 2.92 to 1.91, and mRNA expression in cultured FCs from 141- to 52-fold expression (p<0.0001). TMB also decreased M22-induced TNFα protein production from MFI of 1.67 to 1.12, and mRNA expression from 6- to 3-fold expression (p<0.0001). CONCLUSION: TSH/M22 stimulates FC production of TNFα mRNA and protein. This process involves the transcription factor NF-κB and its regulator Akt. Blocking IGF-1R attenuates TSH/M22-induced TNFα production. This further delineates the interaction of TSHR and IGF1-R signaling pathways. By modulating the proinflammatory properties of FCs such as TNFα production, TMB may be a promising therapeutic agent for GO.

The pathophysiology of thyroid eye disease.

The pathophysiology of thyroid eye disease (TED) is complex and incompletely understood. Orbital fibroblasts (OFs) seem to be the key effector cells that are responsible for the characteristic soft tissue enlargement seen in TED. They express potentially pathogenic autoantigens, such as thyrotropin receptor and insulin-like growth factor-1 receptor. An intricate interplay between these autoantigens and the autoantibodies found in Graves disease may lead to the activation of OFs, which then leads to increased hyaluronan production, proinflammatory cytokine synthesis, and enhanced differentiation into either myofibroblasts or adipocytes. Some of the OFs in TED patients seem to be derived from infiltrating fibrocytes. These cells originate from the bone marrow and exhibit both fibroblast and myeloid phenotype. In the TED orbit, they may mediate the orbital expansion and inflammatory infiltration. Last, lymphocytes and cytokines are intimately involved in the initiation, amplification, and maintenance of the autoimmune process in TED.

Chronic hypotony associated with the Boston type 1 keratoprosthesis.

PURPOSE: To report the occurrence of chronic hypotony as a complication following Boston type 1 keratoprosthesis (KPro) implantation and to determine associated risk factors. DESIGN: Retrospective case series. METHODS: The medical records of all patients undergoing KPro implantation between January 2004 and November 2010 at the Wilmer Eye Institute were reviewed. Patients who developed chronic hypotony following Boston type 1 KPro implantation not attributable to anatomic problems (eg, retinal detachment, overfiltering glaucoma tube shunts, tissue necrosis with aqueous leak) or other causes were identified. Demographics and preoperative, intraoperative, and postoperative clinical findings were analyzed. Light microscopic examination of an enucleated globe as well as fibrous retroprosthetic membranes excised at the time of vitrectomy from another patient was performed. RESULTS: Sixty-eight eyes received KPro implantation over the study period. Six eyes experienced chronic hypotony, with a median time of 18.5 months between KPro implantation and the onset of hypotony. The incidence of chronic hypotony in this series was calculated to be 3.7% at 1 year (95% CI = 0.9%-14.0%) and 13.3% at 2 years (95% CI = 5.5%-30.0%). Of the 6 eyes that developed chronic hypotony, 5 had a previous history of glaucoma or ocular hypertension, but only 3 had a glaucoma drainage implant. All eyes progressing to chronic hypotony were noted to have a retroprosthetic membrane prior to the onset of hypotony. Cox regression modeling demonstrated an increased risk of chronic hypotony in eyes with retroprosthetic membranes (P < .01) but no increase in risk for older patients (P > .1), eyes with glaucoma drainage implants (P > .5), or a previous history of multiple donor corneal transplants (P > .5). CONCLUSION: Chronic hypotony can be a significant complication of KPro implantation and should be assessed carefully in eyes with retroprosthetic membranes. Further study of an association between hypotony and retroprosthetic membrane formation may be useful to gain insight into the mechanism of this complication and potential prevention or treatment options.

Reluctance to undergo follow-up screening for head and neck cancer is associated with income, gender, and tobacco use.

AIM: To determine the factors associated with reluctance to undergo head and neck cancer follow-up screening. METHODS: We surveyed 813 individuals for their medical history, income, behavior habits, and willingness to participate in phone or physical examination follow-up screening for head and neck cancer. Association of reluctance to undergo follow-up screening with the other aforementioned factors was assessed. RESULTS: Overall, 10.9% (95% CI: 8.9-13.3%) of participants were reluctant to undergo follow-up screening. Patients with a history of cigar/pipe use (OR = 1.86, 95% CI: 1.1-3.3, p = 0.03) or low income (under USD 30,000; OR = 1.71, 95% CI: 1.0-2.9, p = 0.04) were more reluctant to undergo phone follow-up. Males (OR = 2.0, 95% CI: 1.0-4.1, p = 0.05) and those with low income (OR = 2.1, 95% CI: 1.1-4.0, p = 0.02) were more reluctant to undergo physical examination follow-up. CONCLUSION: Lower income, male gender, and tobacco use are associated with reluctance to undergo follow-up screening for head and neck cancer.

Asymptomatic cor triatriatum: utility of 64-slice multidetector computed tomography with 3-dimensional volume rendering.

The fast scanning time and exceptional resolution of 64-slice multidetector computed tomography have expanded the role of imaging of congenital cardiac anomalies. These disorders involve complex cardiac configurations and require thorough evaluation of the entire cardiopulmonary system. Interactive interpretation of the multidetector computed tomographic volume with multiplanar postprocessing tools is essential for interpretation and display. This case report describes the utility of 3-dimensional volume rendering in the assessment of a patient with asymptomatic cor triatriatum.

MDCT for suspected appendicitis: effect of reconstruction section thickness on diagnostic accuracy, rate of appendiceal visualization, and reader confidence using axial images.

OBJECTIVE: The purpose of this study was to evaluate interpretative performance with different MDCT reconstruction parameters in adult patients with suspected appendicitis. MATERIALS AND METHODS: MDCT scans of 212 adult patients obtained in an emergency department with a 64-MDCT scanner were prospectively collected. Acquisition technique included 24 x 1.2 mm detector configuration and IV contrast administration with or without oral contrast administration. Data sets were reconstructed with three techniques: 5 x 5 mm, 3 x 3 mm, and 2 x 1 mm (section thickness x interval). Each of the 212 sets of images (grouped by reconstruction technique) was reviewed retrospectively using axial sections by two independent readers blinded to diagnosis. Medical record review was conducted to identify patients with appendicitis. Visualization of the appendix, confidence in visualization, confidence for presence or absence of specific CT findings, diagnostic accuracy, and diagnostic confidence were compared across reconstruction techniques. Data were analyzed with simple and ordinal logistic regression with adjustment for multiple observations derived from each patient and for reader differences. RESULTS: Progressively thinner reconstruction section thickness was associated with a significant increase in the rate of visualization of the appendix (p < 0.001 for 5 x 5 vs 3 x 3; p = 0.03 for 3 x 3 vs 2 x 1), visualization confidence (p < 0.001 for 5 x 5 vs 3 x 3 and 3 x 3 vs 2 x 1), and confidence for presence or absence of findings. Seventeen subjects (8%) had appendicitis. Correctness of diagnosis was not significantly associated with reconstruction method. However, for correctly diagnosed cases interpreted as normal, impression confidence increased with progressively thinner section thickness (p < 0.001 for 5 x 5 vs 3 x 3 and 3 x 3 vs 2 x 1). CONCLUSION: In this investigation of contrast-enhanced MDCT of the appendix, visual ization of the appendix and confidence in interpretation of axial images progressively improved with use of thinner reconstruction sections.

MicroRNA alterations in head and neck squamous cell carcinoma.

MicroRNAs (mirs) are small noncoding RNA molecules (~22 nucleotides) that regulate posttranscriptional gene expression. Currently, there has not been a comprehensive study of their role in primary head and neck squamous cell carcinoma (HNSCC). To determine the role of mirs in HNSCC, we screened for altered microRNA expression in HNSCC primary tissue and cell lines. We then further tested the functional impact of alterations of specific mirs. An initial screening of 4 primary HNSCC, 4 normal mucosal controls and 4 HNSCC cell lines was analyzed for mature microRNA expression by microarray. Significance was determined using significance analysis of microarrays (SAM). Nine microRNAs were found by SAM to be upregulated or downregulated in tumor tissue including mir-21, let-7, 18, 29c, 142-3p, 155, 146b (overexpressed) and 494 (underexpressed). Mir-21 was validated by qRT-PCR. Functional validation by growth assays was performed, further validating mir-21. Transfection of mir-21 into JHU-011 and JHU-012 cell lines showed a 39% increase in cell growth at 72 hr relative to controls (p < 0.05). Transfection of the inhibitor into JHU-O12 cell lines showed a 92% decrease in cell growth relative to controls at 72 hr (p < 0.05). In addition, flow cytometry analysis of JHU-012 cells 48 hr after mir-21 inhibitor transfection showed a statistically significant increase in cytochrome c release and increased apoptosis. These differentially expressed microRNAs may be of interest as potential novel oncogenes and tumor suppressor genes in HNSCC. Mir-21 is a putative oncogenic microRNA in head and neck cancer.

Impact of cytogenetic and genomic aberrations of the kallikrein locus in ovarian cancer.

The tissue kallikrein (KLK) genes are a new source for biomarkers in ovarian cancer. However, there has been no systematic analysis of copy number and structural rearrangements related to their protein expression. Chromosomal rearrangements and copy number changes of the KLK region were studied by FISH with protein levels measured by ELISA. Ovarian cancer and cell lines revealed the KLK region was subject to copy number imbalances or involved in unbalanced translocations and were associated with increased protein expression of KLKs 5, 6, 7, 8, 9, 10 and 11. In this initial study, we introduce the potential for long-range chromosomal effects and copy number as a mechanism for the previously reported aberrant expression of many KLK genes in ovarian cancers.

A multiparametric panel for ovarian cancer diagnosis, prognosis, and response to chemotherapy.

PURPOSE: Our goal was to examine a panel of 11 biochemical variables, measured in cytosolic extracts of ovarian tissues (normal, benign, and malignant) by quantitative ELISAs for their ability to diagnose, prognose, and predict response to chemotherapy of ovarian cancer patients. EXPERIMENTAL DESIGN: Eleven proteins were measured (9 kallikreins, B7-H4, and CA125) in cytosolic extracts of 259 ovarian tumor tissues, 50 tissues from benign conditions, 35 normal tissues, and 44 tissues from nonovarian tumors that metastasized to the ovary. Odds ratios and hazard ratios and their 95% confidence interval were calculated. Time-dependent receiver operating characteristic curves for censored survival data were used to evaluate the performance of the biomarkers. Resampling was used to validate the performance. RESULTS: Most biomarkers effectively separated cancer from noncancer groups. A composite marker provided an area under the curve of 0.97 (95% confidence interval, 0.95-0.99) for discriminating normal and cancer groups. Univariately, hK5 and hK6 were positively associated with progression. After adjusting for clinical variables in multivariate analysis, both hK10 and hK11 significantly predicted time to progression. Increasing levels of hK13 were associated with chemotherapy response, and the predictive power of hK13 to chemotherapy response was improved by a panel of five biomarkers. CONCLUSIONS: The evidence shows that a group of kallikreins and multiparametric combinations with other biomarkers and clinical variables can significantly assist with ovarian cancer classification, prognosis, and response to platinum-based chemotherapy. In particular, we developed a multiparametric strategy for predicting ovarian cancer response to chemotherapy, comprising several biomarkers and clinical features.

Proteomics analysis of human amniotic fluid.

Amniotic fluid is a dynamic and complex mixture that reflects the physiological status of the developing fetus. In this study, the human amniotic fluid (AF) proteome of a 16-18-week normal pregnancy was profiled and analyzed to investigate the composition and functions of this fluid. Due to the complexity of AF, we utilized three different fractionation strategies to provide greater coverage. Two types of two-dimensional LC/MS/MS as well as an LC-SDS-PAGE-LC-MS/MS platform were used. A total of 16 AF samples between gestational ages of 16 and 18 weeks from women carrying chromosomally normal fetuses were analyzed by one of the three fractionation methods followed by a common reverse phase LC-MS/MS step. Mascot and The Global Proteome Machine engines were used to search the International Protein Index human database for peptide sequence identification. The list of proteins was generated by combining the results of both engines through the PeptideProphet of Scaffold software. All identified proteins were combined to generate the AF proteome comprising 1,026 unique gene matches or 842 non-redundant proteins. This list includes most of the currently used biomarkers for pregnancy-associated pathologic conditions such as preterm delivery, intra-amniotic infection, and chromosomal anomalies of the fetus. The subcellular localization, tissue expression, functions, and networks of the AF proteome were analyzed by various bioinformatic tools. These data will contribute to the better understanding of amniotic fluid function and to the discovery of novel biomarkers for prenatal diagnosis of fetal abnormalities.

Unfavorable prognostic value of human kallikrein 7 quantified by ELISA in ovarian cancer cytosols.

BACKGROUND: Human tissue kallikrein 7 (gene, KLK7; protein, hK7) is a member of the kallikrein family of secreted serine proteases. Reports indicate that in ovarian cancer, KLK7 is significantly up-regulated at the mRNA level. The aim of this study was to determine whether hK7, measured quantitatively by ELISA in ovarian cancer cytosols, is a prognostic biomarker for ovarian cancer. METHODS: We used a newly developed ELISA with 2 monoclonal antibodies to quantify hK7 production in 260 ovarian tumor cytosols and correlated these data with various clinicopathologic variables and patient outcomes [progression-free survival (PFS) and overall survival (OS)] over a median follow-up period of 52 months. RESULTS: Median (range) hK7 concentration in ovarian tumor cytosols was 2.84 (0-32.8) ng/mg of total protein. Compared with healthy and benign ovarian tissues and nonovarian tumors that metastasized to the ovary, malignant ovarian tumor cytosols highly overproduced hK7 (P <0.001). We used the median value as the cutoff value to categorize tumors as hK7-positive and hK7-negative. Women with hK7-positive tumors most frequently had advanced-stage disease, higher tumor grade (G3), suboptimal debulking, and serous or undifferentiated histotype (P <0.001). Univariate analysis showed that hK7 positivity was associated with significantly shorter PFS (P = 0.01) but not OS. Kaplan-Meier survival curves confirmed an increased risk of relapse in women with hK7-positive tumors (P = 0.009). In multivariate analysis, hK7 was not significantly associated with either PFS or OS. CONCLUSIONS: hK7 is associated with other unfavorable characteristics of ovarian cancer, but it is not an independent prognosticator for ovarian cancer.

Purification and characterization of human kallikrein 11, a candidate prostate and ovarian cancer biomarker, from seminal plasma.

PURPOSE: Preliminary data suggest that hK11 is a novel serum biomarker for prostate and ovarian cancer. To examine the enzymatic characteristics of hK11, we purified and functionally characterized native hK11 from seminal plasma. EXPERIMENTAL DESIGN: hK11 was purified from seminal plasma by immunoaffinity chromatography and characterized by kinetic analysis, electrophoresis, Western blots, and mass spectrometry. RESULTS: hK11 is present in seminal plasma at concentrations ranging from 2 to 37 microg/mL. Using immunoaffinity chromatography and reverse-phase high-performance liquid chromatography, we purified hK11 to homogeneity. In seminal plasma, hK11 is present as a free enzyme of approximately 40 kDa. About 40% of hK11 is enzymatically active, whereas the rest is inactivated by internal cleavage after Arg156 (Genbank accession no. AF164623), which generates two peptides of approximately 20 kDa, connected by internal disulfide bonds. Purified hK11 possesses trypsin-like activity and cleaves synthetic peptides after arginine but not lysine residues. It does not cleave chymotrypsin substrates. Antithrombin, alpha1-antichymotrypsin, alpha2-antiplasmin, and alpha1-antitrypsin have no effect on hK11 activity and do not form complexes with hK11 in vitro. The strongest inhibitor, APMSF, completely inhibited hK11 activity at a concentration of 2.5 mmol/L. Aprotinin and an hK11-specific monoclonal antibody inhibited hK11 activity up to 40%. Plasmin is a strong candidate for cleaving hK11 at Arg156. CONCLUSION: This is the first report on purification and characterization of native hK11. We speculate that hK11, along with other kallikreins, proteases, and inhibitors, participates in a cascade enzymatic pathway responsible for semen liquefaction after ejaculation.

Human kallikrein 4: quantitative study in tissues and evidence for its secretion into biological fluids.

BACKGROUND: Human kallikrein 4 (hK4) is a proteolytic enzyme belonging to the tissue kallikrein family of serine proteases. Previous tissue expression studies have demonstrated highest KLK4 mRNA expression in prostatic tissue, but there has been only limited evidence for the presence of hK4 protein in prostate and other tissues and in corresponding biological secretions. METHODS: To investigate the concentrations of hK4 in tissues and biological fluids, we developed a new hK4-specific sandwich-type immunoassay using a monoclonal antibody as the capture reagent. RESULTS: The assay has a detection limit of 0.02 microg/L and <0.1% cross-reactivity toward any of the other 14 human kallikreins. Twelve of 40 tissue extracts prepared from various human tissues contained detectable hK4 concentrations (0.68-7143 ng/g of total protein), with healthy prostate tissue containing the highest amount of hK4. Examination of 16 malignant and 18 benign prostate tissues revealed no significant differences in hK4 protein content, and the tissues contained a wide range of values (benign, <0.02 to 801 ng/g; malignant, <0.02 to 824 ng/g). Among the biological fluids tested, seminal plasma and urine contained widely varying amounts of hK4; concentrations in 54 urine samples were <0.02 to 2.6 microg/L, whereas concentrations in 58 seminal plasma samples were 0.2-202 microg/L. Affinity purification of hK4 from seminal plasma and subsequent mass spectrometry demonstrated the secreted nature of hK4 in seminal plasma. CONCLUSIONS: hK4 is found primarily in prostate tissue and is secreted in seminal plasma. Its value as a novel prostatic biomarker needs to be defined further.