Tyramide Amplification Research Articles

Magnetic Nanoagent Coated with an Activated Macrophage Membrane for Colorimetric Detection of Bacteria.

The construction of cell mimics replicating the surface landscape and biological functions of the cell membrane offers promising prospects for biomedical research and applications. Inspired by the inherent recognition capability of immune cells toward pathogens, we have fabricated activated macrophage membrane-coated magnetic silicon nanoparticles (aM-MSNPs) in this work as an isolation and recognition tool for enhanced bacterial analysis. Specifically, the natural protein receptors on the activated macrophage membrane endow the MSNPs with a broad-spectrum binding capacity to different pathogen species. By further incorporation of a tyramide amplification strategy, direct naked-eye analysis of specific bacteria with a detection limit of 10 CFU/mL can be achieved. Moreover, application to the diagnosis of urinary tract infections has also been validated, and positive samples spiked with bacteria can be clearly distinguished with an accuracy of 100%. This work may enrich cell membrane-based architectures and provide an experimental paradigm for point-of-care testing (POCT) detection of bacteria.

CD30 expression is frequently decreased in relapsed classic Hodgkin lymphoma after anti-CD30 CAR T-cell therapy.

Chimeric antigen receptor (CAR) T-cells anti-CD30 is an innovative therapeutic option that has been used to treat cases of refractory/relapsed (R/R) classic Hodgkin lymphoma (CHL). Limited data are available regarding the CD30 expression status of patients who relapsed after this therapy. This is the first study to show decreased CD30 expression in R/R CHL in patients (n = 5) who underwent CAR T-cell therapy in our institution between 2018 and 2022. Although conventional immunohistochemical assays showed decreased CD30 expression in neoplastic cells in all cases (8/8) the tyramide amplification assay and RNAScope in situ hybridisation detected CD30 expression at different levels in 100% (n = 8/8) and 75% (n = 3/4), respectively. Hence, our findings document that certain levels of CD30 expression are retained by the neoplastic cells. This is not only of biological interest but also diagnostically important, as detection of CD30 is an essential factor in establishing a diagnosis of CHL.

Aquaporin (AQP) channels in the spiny dogfish, Squalus acanthias I: Characterization of AQP3 and AQP15 function and expression, and localization of the proteins in gill and spiral valve intestine.

Complementary DNAs (cDNAs) for two aquaporin water channel genes (AQP3 and AQP15) were amplified cloned and sequenced to initiate this study. Northern blot analysis was carried out to confirm the mRNA sizes of these AQP genes with AQP3 mRNA bands exhibiting sizes of 1.2 and 1.6k bases and AQP15 had a mRNA band of 2.1k bases. Northern blot analysis was also performed on kidney and esophagus total RNA samples from fish acclimated to 75%, 100% or 120% seawater (SW). The level of AQP15 mRNA expression was shown to significantly decrease following salinity acclimation from 100 to 120% SW. An opposite but non-significantly different trend was observed for AQP3 mRNA levels. Full length cDNAs were then used to generate AQP3 and AQP15 mRNAs for microinjection into Xenopus oocytes. Both AQP3- and AQP15- microinjected oocytes exhibited significantly elevated apparent water permeability compared to control oocytes at neutral pH. The apparent water permeability was mercury-inhibitable, significantly so in the case of AQP3. AQP3 microinjected oocytes showed pH sensitivity in their apparent water permeability, showing a lack of permeability at acidic pH values. The Carboxyl-terminal derived amino acid sequences of AQP3 and AQP15 were used to generate rabbit affinity-purified polyclonal antibodies. Western blots with the antibodies showed a band of 31.3kDa for AQP3 in the kidney, with minor bands at 26, 24 and 21kDa. For AQP15 a band of 26kDa was seen in gill and kidney. Fainter bands at 28 and 24kDa were also seen in the kidney. There was also some higher molecular weight banding. None of the bands were seen when the antibodies were pre- blocked with their peptide antigens. Immunohistochemical localization studies were also performed in the gill and spiral valve intestine. In the gill, AQP15 antibody staining was seen sporadically in the membranes of surface epithelial cells of the secondary lamellae. Tyramide amplification of signals was employed in the spiral valve intestine. Tyramide-amplified AQP3 antibody staining was observed in the basal membrane of the invaginated epithelial cell layer of secondary intestinal folds in luminal surface of either the side wall of the spiral valve intestine or in internal valve tissue 'flaps'. For the AQP15 antibody, tyramide-amplified staining was instead found on the apical and to a lesser extent the lateral membranes of the same invaginated epithelial cell layer. The localization of AQP3 and AQP15 in the spiral valve intestine suggests that a trans-cellular water absorption pathway may exist in this tissue.

E-cadherin and aquaporin 1 co-expression analysis in hepatocellular carcinoma: a pilot study

Hepatocellular carcinoma (HCC) is the main primary liver malignancy, being associated with both health and economic burden worldwide. Recently, novel molecular markers and possible therapeutic targets were identified. Different adhesion molecules, as well as possible angiogenesis-associated targets can be prime candidates when investigating novel therapies. Considering these premises, our goal was to study the co-existence of E-cadherin and aquaporin 1 (AQP1) in a series of HCC diagnosed patients. Utilizing archived tissue fragments from 17 patients diagnosed with well-to-moderate and poorly differentiated HCC, as well as four samples of normal liver tissue and using a highly specific biotin-free tyramide amplification technique, we have assessed here the expression of E-cadherin and AQP1 during HCC carcinogenesis. Moreover, as we have observed that some of the AQP1 expression seems membrane-bound, we have sought to evaluate their co-localization. Our data showed, as expected, that E-cadherin decreases from control tissue to low-grade and respectively, high-grade HCC. AQP1 was expressed, also as already known, at the level of endothelial blood vessels and bile ducts epithelia, however, we have showed here for the first time that this water pore is also expressed in the cytoplasm and membranes of hepatocytes, both in control and HCC tissue. Moreover, AQP1 expression parallels the decrease of E-cadherin expression during carcinogenesis, but together with this downregulation, we have also found a spatial decrease in the colocalization of the two proteins. Altogether, utilizing a biotin-free tyramide signal amplification technique, this study shows for the first time that AQP1 is expressed at the level of liver epithelia, in both control and HCC tissue.

Somatostatin receptor profile in pituitary thyrotroph adenomas

ObjectivesThyrotroph adenomas are the most infrequent adenohypophysial tumors. Somatostatin (SST) inhibits hormone secretion and suppresses cell proliferation. SST receptors (sstr) belong to a family of 5 types of G-coupled membrane proteins, which show high binding affinity to SST. Currently, SST analogs used to treat pituitary adenomas, have a preferential binding activity to sstr2 and sstr5. The aim of this study was to evaluate the status of all active sstrs on cell membrane of thyrotroph adenomas. PatientsNine cases of thyrotroph adenomas were studied for all types of sstrs. All patients were clinically associated with hyperthyroidism. The adenomas were initially diagnosed and classified by histology and immunohistochemistry for all pituitary hormones and two of them were examined by electron microscopy. MethodsFor sstr immunohistochemistry, antisera against all sst types (1, 2A, 2B, 3, 4 and 5) were used. To enhance sensitivity, the tyramide amplification technique was applied. This is the first report investigating the full spectrum of sstrs in thyrotroph adenomas by immunohistochemistry. ResultsAll tumors were immunoreactive for β-subunit of thyroid-stimulating hormone and for α-subunit of glycoprotein hormones. The sst2A, sst2B and sstr5 were co-expressed in all adenomas. The sstr1 and sstr3 were noted in 8 and sstr4 in 7 adenomas respectively. High scores 2+ and 3+ were prominent in sstr2A, sstr2B, sstr3 and sstr5. High score 3+ for sstr4 was also noted in one tumor, while score 3+ for sstr1 was not observed. ConclusionsKnowledge of the sstr status may contribute to a better selection of patients, anticipating benefit from treatment with SST analogs. Given that multiligand SST analogs have a broader ability to bind other sstrs, such as sstr1 and sstr3, patients with thyrotroph adenomas expressing these receptors may benefit from novel sstr targeting therapy.

Abstract 5230: Development of a multi-parameter immunofluorescence assay for simultaneous detection of androgen receptor and androgen receptor variant 7 in prostate cancer circulating tumor cells

Abstract Background. Androgen receptor (AR) signaling is the primary driver of prostate cancer. Expression of the AR splice variant AR-V7, is predictive of resistance to anti-androgen therapies enzalutimide and abiratirone. There is great interest to non-invasively identify circulating tumor cells (CTCs) and investigate their expression of AR and AR-V7 and monitor them over time. RareCyte has developed a platform for automated visual identification of CTCs by immunofluorescence (IF) that allows up to 6-parameter assessment. We have developed a set of prototype assays using tyramide amplification to detect AR and AR-V7 individually and in combination with prostate membrane-specific antigen (PSMA). Using a novel method, we also developed an assay to identify AR and AR-V7 simultaneously. Methods. Normal human whole blood samples were spiked with prostate cancer lines serving as model CTCs (mCTCs). Prostate cancer blood from a patient with advanced and known high CTC count were collected under an IRB-approved protocol. Blood was processed onto microscope slides and stained on an automated stainer with CTC detection assays incorporating 4 core parameters (nuclear dye, CD45, pan-cytokeratin, EpCAM) plus the following markers: (1) AR; (2) AR-V7; (3) AR and PSMA; (4) AR-V7 and PSMA; (5) AR and AR-V7. Each assay was applied to the cancer cell lines PC3, LNCaP, and 22RV1 and to the patient sample. An antibody denaturing process was used to sequentially amplify two rabbit monoclonals for the assay containing both AR and AR-V7. No-primary (diluent only) staining controls were run to confirm success of the denaturing step. Results. Staining of spike-in CTC models confirmed the reported AR and AR-V7 phenotype of the cell lines, supporting the specificity of the assays. When applied to an equivalent volume of the patient sample, the assays identified a mean (SD) of 36 (11) CTCs. In the assays staining AR, the mean percent of AR+ CTCs was 70% (range 61 - 86) and for the assays staining AR-V7, the percent of AR-V7+ CTCs was 30% (range 26 - 39). In the assay staining both AR and AR-V7, 10 of 38 CTCs were AR-V7+ (26%); all of these were AR+; 15 CTCs were only AR+ (39%). Conclusions. We have developed multi-parameter IF assays for detection of AR and AR-V7 in prostate CTCs, including the first assay we are aware of to simultaneously visualize AR and AR-V7. It has been validated in prostate cancer CTC models. The percentages of AR and AR-V7-positive CTCs identified in a patient sample with the assay are consistent with the percentages observed when AR and AR-V7 are detected in independent assays, supporting the accuracy of the simultaneous detection assay. All AR-V7-positive CTCs were also AR-positive, supporting specificity of AR-V7 staining. Citation Format: Yao Sun, Daniel Campton, Arturo Ramirez, Tanisha Mojica, Alisa Clein, Celestia Higano, Daniel Sabath, Stephen Plymate, Eric Kaldjian. Development of a multi-parameter immunofluorescence assay for simultaneous detection of androgen receptor and androgen receptor variant 7 in prostate cancer circulating tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5230.

Detection of tissue factor-positive extracellular vesicles by laser scanning confocal microscopy

IntroductionIncreased levels of tissue factor-positive extracellular vesicles (TF+EVs) have been detected in the plasma of patients with various diseases, including cancer and endotoxemia. Levels of TF+EVs in plasma samples can be measured by antigen and activity assays. The aim of the present study was to visualize TF+EVs by laser scanning confocal microscopy (LSCM). MethodsEVs were isolated from the supernatant of two cultured human pancreatic cancer cell lines (Panc-1 and BxPc-3), from untreated or lipopolysaccharide (LPS) treated whole blood, and from plasma of pancreatic cancer patients. EV-TF activity was determined using an in-house assay. The EVs were labeled with 5(6)-carboxyfluorescein diacetate N-succinimidyl ester, which is converted to the impermeant green fluorescent molecule carboxyfluorescein inside the EVs. EVs were either captured using annexin V and detected using a fluorescent-labeled anti-TF antibody, or captured using an anti-TF antibody and detected using fluorescent-labeled annexin V. EVs were visualized by LSCM. ResultsTF+EVs were easily detected from high TF-expressing BxPc-3 cells using annexin V capture, whereas the addition of tyramide amplification was required to detect TF+EVs from low TF-expressing Panc-1 cells. Visualization of TF+EVs in plasma from LPS treated whole human blood and in plasma from pancreatic cancer patients required either capture with annexin V and detection with a fluorescent-labeled anti-TF antibody with tyramide signal amplification, or capture with an anti-TF antibody and detection with a fluorescent-labeled annexin V. ConclusionLSCM enables visualization of TF+EVs in the supernatant from cultured cells and in clinical samples.

Dual signal amplification for highly sensitive hybridization microassays on chemically activated surfaces

A highly sensitive hybridization assay is developed for detection of specific sequences of genomic DNA without thermal or isothermal chain reaction cycling. The assays in microarray format were performed on polymeric surfaces derived from the audio-video disc industry. In order to achieve this challenge, a photo-chemical activation method for the covalent immobilization of DNA oligomers is developed. In addition, dual signal amplification approach, by integrating tyramide with silver-assisted enhancement for the detection of DNA at low pg/μL level, is presented.The methodology was two orders of magnitude more sensitive in comparison with the non-tyramide amplified hybridization assay. To demonstrate the feasibility of this approach, complementary oligomers and PCR products as a model targets were employed, achieving a quantification limit of 0.30pM and 0.2pg/μL, respectively. The reproducibility was good (RSD <18%) and the hybridization assay took 65min.As a proof of concept, the detection of genomic DNA of Salmonella spp., as food-borne pathogen model, was addressed. The principle of assay is based on massive biotinylation and fragmentation of genomic DNA prior the hybridization step. The biotinylated fragments, ranging from 400 to 750bp hybridize with the covalently immobilized specific probes (38 mer long), generating an analytical signal after tyramide amplification proportional to the concentration of DNA. The signal is quantified with a disc drive as detector. The results showed that the methodology is a promising alternative for DNA sensing without thermal or isothermal cycling based amplification.

Mo1692 Vascular Endothelial Transient Receptor Potential Vanilloid 4 Links Colonic Inflammation in Dextran Sulfate Sodium-Induced Colitis Model Mice

BACKGROUND&AIMS: Transient receptor potential vanilloid 4 (TRPV4) is a nonselective cation channel, and involved in physical sensing in various types of tissues. TRPV4 is expressed in sensory neuron and vascular endothelial cells in various organs. In gastrointestinal tracts, TRPV4 activation in epithelial cells increases in intracellular calcium concentrations and induces colitis. However, the localization and the role of the TRPV4 channel expressed in vascular endothelial cells in colitis are still poorly defined. In the present study, we investigated the alteration of TRPV4 expression in the colonic vascular endothelial cells during dextran sulfate sodium (DSS)-induced colitis. METHODS: Eight week-old wild type (WT) and TRPV4 knock-out (TRPV4 KO) mice were used. Colitis was induced by 2% DSS solution given as drinking water for 7 days. The disease activity index (DAI) was evaluated during the treatment, while colon length, histological damage, and myeloperoxidase (MPO) activity were examined post-DSS treatment. Immunohistochemical analysis was performed in distal colon. TRPV4-immunoreactivity was detected by using immunohistochemical staining with fluorescein-conjugated tyramide amplification. CD31, CD105, CD11b, CD68, Ly6B.2, and VEGF receptor-2 were detected by indirect staining with their specific antibodies. Vascular leakage was investigated using Evans blue dye extrusion assay. RESULTS: TRPV4 KO mice protected against DSS-induced weight loss, neutrophil (MPO), colon shortening, diarrhea, occult-fecal blood, and histological damage. TRPV4 immunoreactivities were detected mostly in epithelial cells of normal distal colon. DSS treatment drastically increased TRPV4 expression in vascular endothelial cells of mucosa and submucosal layer which colocalized with CD31, CD105, and VEGF receptor-2, but not epithelial cells. Repeated intrarectal and intravenous administration of TRPV4 agonist GSK1016790A exacerbated the severity of DSS colitis. Increased vascular permeability was observed following single intravenous administration of GSK1016790A in DSS colitis and this effect was abolished with TRPV4 antagonist RN1734. CD11b, CD68, Ly6B.2 immunopositive cells were detected around TRPV4 positive vascular endothelial cells in mucosa and submucosal layer in DSS colitis. CONCLUSION: These findings indicate that the alteration of TRPV4 in vascular endothelial cells may contribute to progression of colonic inflammation via increasing vascular permeability.

Mo1693 Activation of Bone Marrow-Derived TRPV4-Expressing M2 Macrophages Promotes Intestinal Contraction

BACKGROUND&AIMS: Transient receptor potential vanilloid 4 (TRPV4) is a nonselective cation channel, and involved in physical sensing in various types of tissues. TRPV4 is expressed in sensory neuron and vascular endothelial cells in various organs. In gastrointestinal tracts, TRPV4 activation in epithelial cells increases in intracellular calcium concentrations and induces colitis. However, the localization and the role of the TRPV4 channel expressed in vascular endothelial cells in colitis are still poorly defined. In the present study, we investigated the alteration of TRPV4 expression in the colonic vascular endothelial cells during dextran sulfate sodium (DSS)-induced colitis. METHODS: Eight week-old wild type (WT) and TRPV4 knock-out (TRPV4 KO) mice were used. Colitis was induced by 2% DSS solution given as drinking water for 7 days. The disease activity index (DAI) was evaluated during the treatment, while colon length, histological damage, and myeloperoxidase (MPO) activity were examined post-DSS treatment. Immunohistochemical analysis was performed in distal colon. TRPV4-immunoreactivity was detected by using immunohistochemical staining with fluorescein-conjugated tyramide amplification. CD31, CD105, CD11b, CD68, Ly6B.2, and VEGF receptor-2 were detected by indirect staining with their specific antibodies. Vascular leakage was investigated using Evans blue dye extrusion assay. RESULTS: TRPV4 KO mice protected against DSS-induced weight loss, neutrophil (MPO), colon shortening, diarrhea, occult-fecal blood, and histological damage. TRPV4 immunoreactivities were detected mostly in epithelial cells of normal distal colon. DSS treatment drastically increased TRPV4 expression in vascular endothelial cells of mucosa and submucosal layer which colocalized with CD31, CD105, and VEGF receptor-2, but not epithelial cells. Repeated intrarectal and intravenous administration of TRPV4 agonist GSK1016790A exacerbated the severity of DSS colitis. Increased vascular permeability was observed following single intravenous administration of GSK1016790A in DSS colitis and this effect was abolished with TRPV4 antagonist RN1734. CD11b, CD68, Ly6B.2 immunopositive cells were detected around TRPV4 positive vascular endothelial cells in mucosa and submucosal layer in DSS colitis. CONCLUSION: These findings indicate that the alteration of TRPV4 in vascular endothelial cells may contribute to progression of colonic inflammation via increasing vascular permeability.

TRPM8 has a key role in experimental colitis-induced visceral hyperalgesia in mice.

Transient receptor potential channel melastatin 8 (TRPM8) is activated by cold temperatures and cooling agents (menthol and icilin). Recent studies showed TRPM8 is expressed in visceral organs and peripheral sensory pathways. However, the role of TRPM8 in visceral hyperalgesia is poorly understood in pathological states such as inflammatory bowel disease. Hence, we investigated the distribution of TRPM8 and its involvement in visceral hyperalgesia in experimental colitis mice. TRPM8 immunoreactivity was detected using immunohistochemical staining with fluorescein-conjugated tyramide amplification. Visceral hyperalgesia was measured by the intracolonic administration of TRPM8 agonist, WS-12, in control and dextran sodium sulfate (DSS)-induced colitis mice. TRPM8 immunoreactivity in the distal colon was much higher than in the transverse and proximal colon under physiological conditions. TRPM8 immunoreactivity markedly increased in the distal colon mucosa of DSS-induced colitis mice compared with control mice. The number of TRPM8 nerve fibers in mucosa of DSS- or 2,4,6-trinitrobenzene sulfonic acid-induced colitis model mice drastically increased compared with control mice. TRPM8 immunoreactivities colocalized with the calcitonin gene-related peptide- and substance P-immunoreactive nerve fibers in the mucosa. Intracolonic administration of WS-12 induced behavioral visceral pain-like responses. The numbers of these responses in the colitis model mice were 3 times higher than in control mice, and were decreased by pretreatment with the TRPM8 channel blocker AMTB. Increased expression of TRPM8 may contribute to the visceral hyperalgesia of experimental colitis.

New horizons in diagnosis and management of endocrine tumors

Endocrine tumors were considered relatively infrequent neoplasms. However, during the last decades, their frequency gradually increased. The use of imaging techniques, guided FNA biopsy, an endoscope camera in the investigation of endocrine lesions, permits early diagnosis. At the histological level, new applications such as non-biotin containing immunohistochemical detection systems, tyramide amplification method, in situ hybridization, FISH, CGH, and other molecular techniques have provided better knowledge on the protein and molecular background. The investigation of somatostatin and dopamine receptors assists targeted therapy of endocrine tumors. Novel treatment modalities have emerged for the management of pituitary and gastroenteropancreatic tumors respectively. Despite this progress, in some instances, the morphological diagnosis remains questionable. Similarities among normal elements, hyperplastic conditions and benign or malignant lesions can make separation difficult. The "gray zones" representing the overlapping in the sequence of normal parenchyma/ hyperplasia/ adenoma/ carcinoma signify a difficult and controversial diagnostic task, which merits special attention. Furthermore, in most endocrine tumors, the diagnosis of carcinoma is justified only in the presence of local or distant metastases. More precise guidelines are needed, by improving the currently available criteria, to minimize the "gray zones", leading to a more accurate separation of such endocrine lesions.

Antigen retrieval, blocking, detection and visualisation systems in immunohistochemistry: A review and practical evaluation of tyramide and rolling circle amplification systems

To achieve specificity and sensitivity using immunohistochemistry it is necessary to combine the application of validated primary antibodies with optimised pre-treatment, detection and visualisation steps. The influence of these surrounding procedures is reviewed. A practical evaluation of tyramide signal amplification and rolling circle amplification detection methods is provided in which formalin fixed paraffin embedded sections of adenocarcinomas of breast, colon and lung together with squamous metaplasia of lung were immunostained with CD20 and CK19 primary antibodies. The results indicate that the detection systems are of comparable sensitivity and specificity.

Aquaporins 6-12 in the human eye

Aquaporins (AQPs) are widely expressed and have diverse distribution patterns in the eye. AQPs 0-5 have been localized at the cellular level in human eyes. We investigated the presence of the more recently discovered AQPs 6-12 in the human eye. RT-PCR was performed on fresh tissue from two human eyes divided into the cornea, corneal limbus, ciliary body and iris, lens, choroid, optic nerve, retina and sclera. Each structure was examined to detect the mRNA of AQPs 6-12. Twenty-one human eyes were examined using immunohistochemical and immunofluorescence techniques to determine the topographical localization of AQPs 6-12. mRNA transcripts of AQP7, AQP9 and AQP11 were found in the ciliary body, corneo-limbal tissue, optic nerve, retina and sclera. AQP9 and AQP11 mRNA was also detected in the choroid. No mRNA of AQP6, AQP8, AQP10 or AQP12 was detected. Anti-AQP7 immunolabelling was detected in the corneal epithelium, corneal endothelium, trabecular meshwork endothelium, ciliary epithelia, lens epithelium, the inner and outer limiting membrane of the retina, the retinal pigment epithelium and the capillary endothelium of all parts of the eye. AQP9 immunolabelling was detected in the nonpigmented ciliary epithelium and retinal ganglion cells. AQP11 immunolabelling was detected in the corneo-limbal epithelium, nonpigmented ciliary epithelium and inner limiting membrane of the retina. Selective expression of AQP7, AQP9 and AQP11 was found within various structures of the human eye. The detection of these aquaporins in the eye implies a role that may be related not only to water transport but also to the transport of glycerol, lactate and ammonia, with importance for metabolism, especially in the retina.

Comparison of the sensitivity and specificity of real-time PCR and &lt;i&gt;in situ&lt;/i&gt; hybridization in HPV16 and 18 detection in archival cervical cancer specimens

The aim of this study was to analyze the correlation between real-time PCR (RT-PCR) treated as a reference method and in situ hybridization with tyramide amplification system (ISH-TSA) in the detection of HPV16 and 18 infection and the assessment of viral genome status. The study was performed on cervical cancer biopsies fixed in 10% neutral buffered formalin and embedded in paraffin obtained from 85 women. TaqMan-based 5'exonuclease RT-PCR with type-specific primers was used to assess HPV16 and 18 infections and genome status. Viral infection and genome status was also assessed by ISH-TSA. RT-PCR revealed 76 (89.4%), and ISH-TSA 81 (95.3%) cancers with HPV16 and 18 infections. The ISH-TSA sensitivity and specificity were: 96.1% and 11.1% compared to RT-PCR. The difference between these techniques in HPV detection was significant (p = 0.000). Among 76 HPV16/18 positive cancers in RT-PCR, there were 30 (39.5%) with integrated and 46 (60.5%) with mixed viral genome form. According to ISH-TSA, there were 39 (51.3%) samples with integrated and 37 with mixed form (48.7%). The sensitivity and specificity of ISH-TSA in genome status assessment were 70.0% and 60.9%, respectively. The difference between RT-PCR and ISH-TSA in genome state detection was not statistically significant (p = 0.391). These results suggest that ISH-TSA shows insufficient specificity in HPV detection for use in clinical practice. However, this assay could be applied for viral genome status assessment.

Experimental colitis alters expression of 5-HT receptors and transient receptor potential vanilloid 1 leading to visceral hypersensitivity in mice

Abnormalities of primary afferent nerve fibers are strongly associated with the visceral hypersensitivity state in inflammatory bowel disease. Hypersensitivity of afferent fibers occurs during inflammation. Therefore, to gain an insight into the alterations to receptors and channels expressed in primary afferent neurons, the current study aimed to investigate the time-dependent dynamic changes in levels of 5-hydroxytryptamine (5-HT)3 receptors, 5-HT4 receptors, transient receptor potential vanilloid type 1 (TRPV1) channels, and 5-HT regulatory factors in dextran sulfate sodium (DSS)-induced colitis model mice. 5-HT signaling molecules were detected by indirect staining with specific antibodies. TRPV1-immunoreactivity was detected by staining with fluorescein-conjugated tyramide amplification. To assess nociception, visceromotor responses (VMRs) to colorectal distension were measured by electromyography of abdominal muscles. Immunohistochemical analysis and VMRs to colorectal distention were measured during induction of DSS colitis (days 4 and 7). Inflammation led to downregulation of serotonin transporter immunoreactivities with concomitant increases in 5-HT and tryptophan hydroxylase-1-positive cell numbers. TRPV1-expressing nerve fibers gradually increased during DSS treatment. Abundant nonneuronal TRPV1-immunopositive cell-like structures were observed on day 7 of DSS treatment but not on day 4. The number of 5-HT3 receptor-expressing nerve fibers in the mucosa was increased on day 7. On the other hand, the number of 5-HT4 receptor-expressing nerve fibers in the mucosa decreased on day 7. We made the novel observation of increased expression of neuronal/nonneuronal TRPV1 channels and 5-HT3 receptors, and decreased expression of 5-HT4 receptors in the mucosa in a DSS-induced colitis model. Visceral hyperalgesia was observed on day 7 but not on day 4. A TRPV1 antagonist and a 5-HT3 receptor antagonist attenuated the visceral hyperalgesia to the control level. The alterations of 5-HT signaling via 5-HT3 receptors and of TRPV1 channels in mucosa may contribute to the visceral hypersensitivity in colitis model mice.

Tangle evolution linked to differential 3- and 4-repeat tau isoform deposition: a double immunofluorolabeling study using two monoclonal antibodies

Double immunofluorolabeling for 3-repeat (3R) and 4-repeat (4R) tau was performed with two monoclonal antibodies, RD3 and RD4, after an additional pretreatment with potassium permanganate and oxalic acid to eliminate nonspecific 3R tau cytoplasmic staining. This method involves hyperdilution of one of the primary monoclonal antibodies (≥100-fold), making it undetectable by usual secondary antibodies. The hyperdiluted primary antibody can then only be detected after tyramide amplification. Subsequent application of the other monoclonal antibody at its usual concentration allows double immunofluorolabeling without cross-reaction. This novel method revealed that tau immunoreactivity (IR) in the hippocampal pyramidal neurons of Alzheimer's disease (AD) brains is heterogeneous in that pretangle neurons exhibit 4R-selective (3R-/4R+) IR, ghost tangles exhibit 3R-selective (3R+/4R-) IR, and neurofibrillary tangles exhibit both 3R and 4R (3R+/4R+) IR. Some nigral neurons exhibited RD3 IR in both AD and corticobasal degeneration/progressive supranuclear palsy (CBD/PSP) brains. However, in CBD/PSP cases, 3R IR was always superimposed on 4R IR, while 3R-selective neurons were present in AD cases. These differential isoform profiles may provide a pivotal molecular reference, closely related to the morphological evolution of tau-positive neurons, which may be variable according to disease (CBD/PSP vs. AD), lesion site (cerebral cortex and substantia nigra), or the stage of evolution (from pretangles to ghost tangles). These findings should provide a more comprehensive understanding of the histological differentiation of various tau deposits in human neurodegenerative disease.

Role of Neuronal and Non-Neuronal Transient Receptor Potential Vanilloid Type 1 and Sensory Neuropeptides in DSS-Induced Peripheral Inflammatory Changes

BACKGROUND & AIMS: The activation of transient receptor potential vanilloid type 1 (TRPV1) leads to release of sensory neuropeptides such as substance P (SP), neurokinin A (NKA) and calcitonin gene-related peptide (CGRP) from the nerve endings, and these neuropeptides in turn initiate the biochemical cascade known as neurogenic inflammation. TRPV1 channels and the sensory neuropeptides are expressed not only in sensory nerve fibers but also in non-neuronal cells in the mucosa of large intestine. The aim of this study was to investigate alteration of the sensory neuropeptides and TRPV1 channels in mucosa of DSS-induced colitis mice and to characterize their non-neuronal cells. METHODS: Colitis was induced by 3% dextran sulfate sodium (DSS) solution given as drinking water for 7 days in C57BL/6 mice. To evaluate visceral nociception, colorectal distension was performed in mice treated with vehicle or BCTC on day 7 of DSS treatment. Immunohistochemical analysis was performed using rectal tissues of mice. SP, NKA, CGRP, 5-HT, F4/80, keratin, TNF-α, CD11c, and CD4 were detected by indirect staining with their specific antibodies. TRPV1-immunoreactivity was detected by using immunohistochemical staining with fluorescein-conjugated tyramide amplification. RESULTS: TRPV1 antagonist BCTC significantly attenuated the visceral hyperalgesia to control level in DSS-induced colitis model. We compared the number of the nerve fibers and non-neuronal cells expressing TRPV1 channels and sensory neuropeptides in normal and DSS-induced colitis model mice. The number of TRPV1and CGRP-expressing nerve fibers is significantly increased in colitis model. The non-neuronal TRPV1 cells were markedly increased but non-neuronal CGRP-expressing cells were not observed in colitis model. No significant alteration in the number of SP and NKA positive nerve fibers was detected. On the other hand, SP and NKA positive cells were drastically increased in colitis model. Next, double labeling studies were carried out to characterize TRPV1-, SP-, and NKA-expressing cells under inflammatory state. We found two types of non-neuronal TRPV1-expressing cells in mucosa of colitis model. One is keratin positive cells located upper part of mucosa. Another is TNF-α and/or F4/80 positive cells located middle part of the mucosa. Both types of TRPV1-expressing cells did not colocalize with CD4 and CD11c. SP and NKA positive cells almost completely colocalized with 5-HT in experimental colitis model mice. CONCLUSION: These results suggest that TRPV1immunopositive epithelial cells and macrophage are associated with visceral hyperalgesia in colitis.

Unique Clinicopathologic Features Characterize ALK-Rearranged Lung Adenocarcinoma in the Western Population

The anaplastic large cell kinase gene (ALK) is rearranged in approximately 5% of lung adenocarcinomas within the Asian population. We evaluated the incidence and the characteristics of ALK-rearranged lung adenocarcinomas within the western population and the optimal diagnostic modality to detect ALK rearrangements in routine clinical practice. We tested 358 lung adenocarcinomas from three institutions for ALK rearrangements by fluorescent in situ hybridization (FISH) and immunohistochemistry with and without tyramide amplification. The clinicopathologic characteristics of tumors with and without ALK rearrangements were compared. We identified 20 (5.6%) lung adenocarcinomas with ALK rearrangements within our cohort of western patients. ALK rearrangement was associated with younger age (P = 0.0002), never smoking (P < 0.0001), advanced clinical stage (P = 0.0001), and a solid histology with signet-ring cells (P < 0.0001). ALK rearrangement was identified by FISH in 95% of cases and immunohistochemistry with and without tyramide amplification in 80% and 40% of cases, respectively, but neither FISH nor immunohistochemistry alone detected all cases with ALK rearrangement on initial screening. None of the ALK-rearranged tumors harbored coexisting EGFR mutations. Lung adenocarcinomas with ALK rearrangements are uncommon in the western population and represent a distinct entity of carcinomas with unique characteristics. For suspected cases, dual diagnostic testing, with FISH and immunohistochemistry, should be considered to accurately identify lung adenocarcinomas with ALK rearrangement.

T1668 Mast Cell Proteases Stimulate Neurogenic and Non-Neurogenic Mucosal Chloride Secretion in Guinea Pig Distal Ileum

G A A b st ra ct s and involved in the various gut function. However little is known about the subpopulations of TRPV1-expressing nerves and sensory peptidergic nerves in gut. We previously demonstrated that TRPV1-positive nerve fibers in rectum were most abundant among the regions of mouse large intestine. In the present study, we investigated the immunohistochemical distribution of TRPV1 channels and sensory neuropeptides and their co-localization in mouse rectum. METHODS: Extensive TRPV1-immunoreactivity was detected by using immunohistochemical staining with fluorescein-conjugated tyramide amplification. Protein gene product 9.5 (PGP), CGRP, substance P (SP) and neurokinin A (NKA)-immunoreactivity was also detected by indirect staining with their specific antibodies. The longitudinal change in smooth muscle tone was isotonically measured using Magnus apparatus. RESULTS: The localization of TRPV1 was studied at both transverse and horizontal planes of sections in mouse rectum. Numerous TRPV1-immunoreactive nerve fibers were clearly detected in the mucosal, submucosal layer and myenteric plexus. In contrast, TRPV1 nerve fibers were sparsely distributed in circular and longitudinal muscle layers. Next, double labeling studies were carried out. In the mucosal layer, all the TRPV1 nerve fibers were found to colocalize with PGP and CGRP. SP and NKA positive nerve fibers were not observed. TRPV1 nerve fibers containing both CGRP and SP were observed running around blood vessels in the submucosal layer. In the myenteric plexus, most of the TRPV1 axons were found to colocalize with PGP and CGRP. Relatively small numbers of TRPV1 axons were immunopositive for SP. NKA and SP-immunoreactive axons were observed in the circular and longitudinal muscle, and the cell bodies were detected in the myenteric plexus. NKA and SP were almost colocalized at the axons and cell bodies in muscle layer. In the Magnus experiment, capsaicininduced contraction was significantly inhibited by NK1 and NK2 receptor antagonists. DISCUSION: The present results suggest that TRPV1 nerve fibers in mouse rectum are extrinsic primary afferents. TRPV1 fibers containing CGRP are distributed in mucosal, submucosal layer and myenteric plexus and play various roles in rectal function. TRPV1 fibers containing SP and/or NKA are distributed in myenteric plexus and involved in rectal motor function by stimulating intrinsic excitatory motor neurons.