Indirect Immunohistochemical Staining Research Articles

Cortisol as a cerebral cortex neurons apoptosis regulator in acute phase of ischemic stroke (clinical and pathological study)

In response to ischemic stroke (IS) a natural activation of the stress-realizing system occurs. The features of this activation influence the outcome of the acute period and the prognosis of recovery and can be adjusted. At the same time, the role of the stress-realizing system in the pathogenesis of IS is still unexplored.Objective: to investigate the effect of peripheral blood cortisol concentration on the regulation of apoptosis of neurons of the cerebral cortex in the acute phase of IS.Material and methods. A prospective clinical and pathological study was performed. It included 9 patients with IS in the left middle cerebral artery territory who were admitted to hospital and died in the hyperacute phase of IS and had no infectious complications, allergic reactions or oncological diseases and who did not undergo thrombolysis. The cerebral cortex was examined. Neuron-specific enolase (NSE), protein 53 (p53), caspase 3, caspase 8, Fas receptor (CD95), and Fas apoptotic inhibitory molecule 2 (FAIM2) were determined on the slices using an indirect immunoperoxidase immunohistochemical staining method. A total of 567 microscopic fields were analysed for the group of patients with IS and 63 fields for the control group (three people). Before death, the blood concentrations of sFas, sFasL, cortisol, adrenocorticotropic hormone, adrenaline and norepinephrine were determined by enzyme immunoassay (the control group consisted of 28 people).Results. Significant correlation was found between the proportion of casp3-positive neurons and the concentration of cortisol in peripheral blood in zones 2 (r=0.263; p<0.01) and 3 (r=0.383; p<0.01). In the 2nd zone, significant negative correlation was found with the concentrations of sFas (r=-0.177; p<0.05) and sFasL (r=-0.164; p<0.05); in the 3rd zone, significant positive correlation was found with the ratio of the concentrations of sFasL and sFas (r=0.240; p<0.01). The proportion of Fas-positive neurons in the cerebral cortex correlated significantly with the concentration of the soluble form of this molecule (for the 1st zone – r=0.222, for the 2nd zone – r=0.438, for the 3rd zone – r=0.289; p<0.01) and the ratio of the concentrations of sFasL and sFas (respectively: r=0.231, r=0.266 and r=0.281; p<0.01) in the peripheral blood.Conclusion. Peripheral blood cortisol concentration is a factor that determines the regulation of apoptosis of neurons in the cerebral cortex in the acute phase of IS.

Potential participation of CTRP6, a complement regulator, in the pathology of age related macular degeneration.

To investigate the localized expression of C1q/tumor necrosis factor related protein (CTRP) 6 in human age-related macular degeneration (AMD) retinal tissues. 4 AMD and 3 non-AMD whole eyes of Caucasian donors were used. Eyecups were excised at Eye Bank CorneaGen, Inc. To elucidate the effects of CTRP6, C3b was measured by an enzyme-linked immunosorbent-like assay. CFB versus CTRP6 competitive binding assay was applied to clarify the inhibition by CTRP6 of C3bBb complex formation. The cornea, iris, lens, and vitreous were removed and the eyes were cut into a posterior eye-cup including the retina, choroid, and sclera. Six-µm-thick serial sections of frozen samples underwent hematoxylin-eosin (HE) staining and indirect immunohistochemical staining using primary antibodies, anti-CTRP6, -CTRP5, -CTRP10, -Complement factor H (CFH) and -Clusterin (CLU). Results The two in vitro studies confirmed that CTRP6 has an inhibitory effect on alternative pathways of complement (APC) function and that the molecular target of CTRP6 is the inhibition of the formation of C3bBb. Localized expression for CTRP6 and CFH was found in the drusen of the AMD eyes, both associated with APC inhibition, CLU associated with membrane-attack complex (MAC) inhibition, and CTRP5 associated with retinal degeneration. The localized expression of CTRP6 in the drusen of AMD eyes may open a new insight into the possible involvement of APC regulatory factors in the pathogenesis of AMD, together with the known CFH so far analyzed solely as an APC inhibitor.

Production and application of mouse monoclonal antibodies targeting linear epitopes in pB602L of African swine fever virus

African swine fever (ASF) is an acute hemorrhagic disease of domestic pigs. The causative agent of ASF, ASF virus (ASFV), is a double-stranded DNA virus, the sole member in the family Asfarviridae. The non-structural protein pB602L of ASFV is a molecular chaperone of the major capsid protein p72 and plays a key role in icosahedral capsid assembly. This protein is antigenic and is a target for developing diagnostic tools for ASF. To generate monoclonal antibodies (mAbs) against pB602L, a prokaryotically expressed recombinant pB602L protein was produced, purified, and used as an antigen to immunize mice. A total of eight mouse mAbs were obtained, and their binding epitopes were screened by Western blot using an overlapping set of polypeptides from pB602L. Three linear epitopes were identified and designated epitope 1 (366ANRERYNY373), epitope 2 (415GPDAPGLSI423), and epitope 3 (498EMLNVPDD505). Based on the epitope recognized, the eight mAbs were placed into three groups: group 1 (B2A1, B2F1, and B2D10), group 2 (B2H10, B2B2, B2D8, and B2A3), and group 3 (B2E12). The mAbs B2A1, B2H10, and B2E12, each representing one of the groups, were used to detect pB602L in ASFV-infected porcine alveolar macrophages (PAMs) and pig tissues, using an indirect fluorescence assay (IFA) and immunohistochemical staining, respectively. The results showed that pB602L was detectable with all three mAbs in immunohistochemical staining, but only B2H10 was suitable for detecting the proteins in ASFV-infected PAMs by IFA. In summary, we developed eight anti-pB602L mouse mAbs recognizing three linear epitopes in the protein, which can be used as reagents for basic and applied research on ASFV.

MP60-10 LOCAL PHARMACOLOGICAL REGULATORY CHANGES IN ISOLATED BLADDERS OF ALZHEIMER MICE

You have accessJournal of UrologyUrodynamics/Lower Urinary Tract Dysfunction/Female Pelvic Medicine: Basic Research & Pathophysiology I1 Apr 2016MP60-10 LOCAL PHARMACOLOGICAL REGULATORY CHANGES IN ISOLATED BLADDERS OF ALZHEIMER MICE Aryo Zare, Mohammad Sajjad Rahnamai, Gommert A. van Koeveringe, Celine Meriaux, and Ramona Hohnen Aryo ZareAryo Zare More articles by this author , Mohammad Sajjad RahnamaiMohammad Sajjad Rahnamai More articles by this author , Gommert A. van KoeveringeGommert A. van Koeveringe More articles by this author , Celine MeriauxCeline Meriaux More articles by this author , and Ramona HohnenRamona Hohnen More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.863AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Autonomic dysfunction in Alzheimer′s disease (AD) results in incontinence and voiding dysfunction. So far these dysfunctions have been attributed to either cognitive decline or damage to micturition control centers in the central nervous system. Pharmacological therapy for AD often involves cholinergic agonists whereas overactive bladder symptoms need a counteracting drug treatment. To solve this therapeutic dilemma, specific knowledge on local bladder regulatory systems is necessary. In this study, we investigated the effect of muscarinic stimulation and complete depolarisation on bladders from transgenic AD mice, as well as the presence of amyloid-β aggregation within the bladder wall. METHODS Bladders of 16 transgenic AD mice of the APPSL/PS1M146L model and nine wild type (WT) mice were dissected. Twelve AD and seven WT bladders were catheterized transurethrally and placed in an organ bath containing carboxygenated physiological solution. After filling of the bladder and stimulation with 1 µM of carbachol the organ bath was washed. Subsequently the bladder was stimulated with 0.1 M KCl. The area under the curve (AUC) was analysed from the beginning of the contraction until the maximum pressure after addition of either carbachol or KCl was reached. Indirect immunohistochemical staining was performed on four AD mice and two WT mice using a primary antibody against amyloid precursor protein (APP) and amyloid-β and the appropriate secondary antibody. RESULTS A lower frequency of asynchronized microcontractions was recorded during the baseline state in AD mice. The AUC is lower in AD mice compared to WT mice after KCl administration. The bladder muscular layer of WT mice showed no staining to APP or amyloid-β in contrast to AD mice. Immunoreactivity was detected in the urothelial and suburothelial layers in bladders of WT mice. In contrast, mild urothelial staining was present in bladders of WT mice. CONCLUSIONS We hypothesize that in this model of Alzheimer′s disease a decrease of baseline microcontractile activity and of stimulated potassium depolarisation induced contractile activity are most probably related to amyloid-β induced alterations in neuromuscular membrane activity and more specifically the calcium or potassium channels at the bladder level. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e798 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Aryo Zare More articles by this author Mohammad Sajjad Rahnamai More articles by this author Gommert A. van Koeveringe More articles by this author Celine Meriaux More articles by this author Ramona Hohnen More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Multiple Myeloma and Bone Marrow Microenvironment Immunohistochemical Study of the Expression of 15 Proteins Related to Myeloma Bone Disease

Objective: Neoplastic milieu is an integral part of all malignant diseases including multiple myeloma and plays variable role in their development, retention/adhesivity, resistency or sensitivity to therapeutic approach, homing and also paraneoplastic manifestations. Relatively genetically stable milieu may play an important role in new specific molecular therapeutic approaches and therefore should be contextually studied with neoplastic cells as complex neoplastic tissues. The expressions of 15 proteins with close relation to the development of myeloma bone disease (MBD) were analysed in consecutive multiple myeloma specimens.Methods: Bone marrow trephine biopsy specimens (n=57) with multiple myeloma were included in our prospective study. FFPE tissues were processed in app. 5microm sections and placed on charged slides. The indirect immunohistochemical staining was applicated after antigen retrieval and commercial primary antibodies were used for the detection of observed proteins. Standard secondary antibody and ABC method were included in visualisation. We analysed the expressions of MIP1alfa, Annexin A2, TRAP, DKK-1, RANK, RANKL, OPG, Sclerostin, Activin A, NFkappaB proteins (p50, p52, p65), p62 (sequestosome 1), MMP9 and RUNX2.Results: Bone marrow multiple myeloma specimens showed variable positivity of MIP1alfa in 60% (cut-off point 20%), Annexin A2 in 42% (myeloma cells, cut-off point 30%) and in 74% (stromal cells, cut-off point 5%), TRAP in 28% (cut-off point 5%), DKK-1 in 23% (cut-off point 30%), RANK in 53% (cut-off point 30%), RANKL in 70%, OPG in 39% (cut-off point 5%), Sclerostin in 95% (cut-off point 90%), Activin A in 35% (cut-off point 30%), cytoplasmic positivity of p50 in 5% (cut-off point 10%), p52 in 86% (cut-off point 10%), p62 in 91% (cut-off point 10%), p65 in 89% (cut-off point 10%), positivity of MMP9 in 22% (cut-off point 30%) and positivity of RUNX2 in 56% (cut-off point 30%).Conclusion: Our study showed variable expression of proteins related to MBD in multiple myeloma and its bone marrow microenvironment that imply biological heterogeneity, different development and stromal plasticity in this complex hemato-oncological disease. The exact and contextual knowledge of the engaged signaling pathways may suggest more specific or tailored therapeutic approaches (e.g. anti-RANKL, anti-DKK-1, anti-Sclerostin, anti-Activin A).Supported by the grant NT 14393. DisclosuresNo relevant conflicts of interest to declare.

Selective expression of myosin IC Isoform A in mouse and human cell lines and mouse prostate cancer tissues.

Myosin IC is a single headed member of the myosin superfamily. We recently identified a novel isoform and showed that the MYOIC gene in mammalian cells encodes three isoforms (isoforms A, B, and C). Furthermore, we demonstrated that myosin IC isoform A but not isoform B exhibits a tissue specific expression pattern. In this study, we extended our analysis of myosin IC isoform expression patterns by analyzing the protein and mRNA expression in various mammalian cell lines and in various prostate specimens and tumor tissues from the transgenic mouse prostate (TRAMP) model by immunoblotting, qRT-PCR, and by indirect immunohistochemical staining of paraffin embedded prostate specimen. Analysis of a panel of mammalian cell lines showed an increased mRNA and protein expression of specifically myosin IC isoform A in a panel of human and mouse prostate cancer cell lines but not in non-cancer prostate or other (non-prostate-) cancer cell lines. Furthermore, we demonstrate that myosin IC isoform A expression is significantly increased in TRAMP mouse prostate samples with prostatic intraepithelial neoplasia (PIN) lesions and in distant site metastases in lung and liver when compared to matched normal tissues. Our observations demonstrate specific changes in the expression of myosin IC isoform A that are concurrent with the occurrence of prostate cancer in the TRAMP mouse prostate cancer model that closely mimics clinical prostate cancer. These data suggest that elevated levels of myosin IC isoform A may be a potential marker for the detection of prostate cancer.

Fare Embriyonik Kök Hücrelerden Nöronal Hücrelerin Farklılaşması

With new technologies emerging today, the importance of stem cells in the cell therapy of nervous system diseases is supported by recent studies. Therefore, the development of neuronal cell differentiation protocols from stem cells is of great importance. In our study, the differentiation of neuronal and neuroglial cells from mouse embryonic stem (ES) cell line and their analysis with neuronal cell markers are aimed. Mouse ES cells were differentiated to neurogenic series cells by adding N2 and bFGF to the culture medium on coated Fibronectin dishes. For the identification of differentiated cells, they were evaluated by light microscopy using immunhistochemistry techniques and by electron microscopy. Indirect immunohistochemical staining method was performed with SSEA-1 (mouse embriyonic stem cells marker), Nestin (neural precursor cells marker), βIII-Tubulin (neuronal cells marker), MAP-2 (neuronal cells marker), GFAP (astrocyte marker), and O4 (oligodendrocyte marker). After 1 week of differentiation of cells, immunoreactivities of SSEA-1 and Nestin were detected to be negative and moderate, respectively. After 2 weeks culture time, the differentiation was still continuing and especially positive immunoreactivities of β-III Tubulin and MAP-2 and weak immunoreactivities of O4 and GFAP were supported neuronal differentiation. In conclusion, our results suggest that neuronal cell derived from mouse ES cells were differentiated particularly to neuron using N2+bFGF+fibronectin culture condition. Therefore, these differentiated cells may be used as a treatment method in degenerative diseases of the nervous system.

The effect of di-n-butyl phthalate on testis and the potential protective effects of resveratrol

This study aimed to observe the possible protective effects of resveratrol (RSV) against the damage of di-n-butyl phthalate (DBP) on the testis. The study was conducted in 6 groups of rats with 6 animals in each group aged 20 days. The groups include group 1: control group; group 2: solvent (carboxymethylcellulose (CMC), 10 ml/kg); group 3: 500 mg/kg/day DBP; group 4: 500 mg/kg/day DBP + 20 mg/kg/day RSV; group 5: 1000 mg/kg/day DBP; and group 6: 1000 mg/kg/day DBP + 20 mg/kg/day RSV. Groups were treated by gavage for 30 days. Indirect immunohistochemical staining was performed with c-kit, AT1, and ER-α antibodies. The terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick-end labeling (TUNEL) method was used for apoptosis. It was found in the DBP-applied groups the C-kit immunostaining, which is parallel to increasing dose, decreased in comparison with the control. C-kit reactivity was similar to that of the control group in the group applied with 500 mg/kg/day + RSV; however, the reactivity was not same in the 1000 mg/kg/day DBP-applied group. It was observed that the reactivity of AT1 increased in the DBP-applied groups. RSV reversed these changes with its protective effects. While there was not much difference between the groups in terms of estrogen receptor reactivity, it was observed that the high dose of DBP reduced the level of estrogen receptor and the resveratrol was not at enough levels in all doses. In TUNEL analysis, high doses of DBP increased the apoptosis in all types of cells; nevertheless, the resveratrol application decreased the apoptosis in the low-level DBP dose. In the statistical analysis, while the length of epithelium and the diameter of seminiferous tubules decreased for all the other groups, it reverted to its original state in the RSV-applied groups. In conclusion, DBP (with increasing dose) administration caused cycle and hormonal changes in testis, resveratrol were recovered the cyclic changes but in hormonal changes, RSV is efficient too but inadequate.

Quantification of hepatic FOXP3+ T‐lymphocytes in HIV/hepatitis C coinfection

Coinfection with HIV adversely impacts every stage of hepatitis C (HCV) infection. Liver damage in HCV infection results from host antiviral responses rather than direct viral pathogenesis. Despite depressed cellular immunity, coinfected patients show accelerated hepatic fibrosis compared with HCV monoinfected patients. This paradox is poorly understood. T-regulatory (Treg) cells (CD4+ and FOXP3+) are hypothesized to limit hepatic damage in HCV. Our hypothesis was that reduced frequency of hepatic Treg in HIV/HCV coinfection compared with HCV monoinfection may explain poorer outcomes. We quantified FOXP3+, CD4+, CD8+ and CD20+ cells in liver biopsies of 35 male subjects matched by age and ISHAK fibrosis score, 12 HIV monoinfected, 11 HCV monoinfected and 12 HIV/HCV coinfected. Cell counts were performed using indirect immunohistochemical staining and light microscopy. HIV/HCV coinfected subjects had fewer hepatic FOXP3+ (P=0.031) and CD4+ cells (P=0.001) than HCV monoinfected subjects. Coinfected subjects had more hepatic CD8+ cells compared with HCV monoinfected (P=0.023), and a lower ratio of FOXP3+ to CD8+ cells (0.08 vs 0.27, P<0.001). Multivariate analysis showed number of CD4+ cells controlled for differences in number of FOXP3+ cells. Fewer hepatic FOXP3+ and CD4+ cells in HIV/HCV coinfection compared with HCV monoinfection suggests lower Treg activity, driven by an overall loss of CD4+ cells. Higher number of CD8+ cells in HIV/HCV coinfection suggests higher cytotoxic activity. This may explain poorer outcomes in HIV/HCV coinfected patients and suggests a potential mechanism by which highly active antiretroviral therapy may benefit these patients.

Dual regulation of Myc by Abl

The tyrosine kinase c-Abl (or Abl) and the prolyl-isomerase Pin1 cooperatively activate the transcription factor p73 by enhancing recruitment of the acetyltransferase p300. As the transcription factor c-Myc (or Myc) is a known target of Pin1 and p300, we hypothesized that it might be regulated in a similar manner. Consistent with this hypothesis, overexpression of Pin1 augmented the interaction of Myc with p300 and transcriptional activity. The action of Abl, however, was more complex than predicted. On one hand, Abl indirectly enhanced phosphorylation of Myc on Ser 62 and Thr 58, its association with Pin1 and p300 and its acetylation by p300. These effects of Abl were exerted through phosphorylation of substrate(s) other than Myc itself. On the other hand, Abl interacted with the C-terminal domain of Myc and phosphorylated up to five tyrosine residues in its N-terminus, the principal of which was Y74. Indirect immunofluorescence or immunohistochemical staining suggested that the Y74-phosphorylated form of Myc (Myc-pY74) localized to the cytoplasm and coexisted either with active Abl in a subset of mammary carcinomas or with Bcr-Abl in chronic myeloid leukemia. In all instances, Myc-pY74 constituted a minor fraction of the cellular Myc protein. Thus, our data unravel two potential effects of Abl on Myc: first, Abl signaling can indirectly augment acetylation of Myc by p300, and most likely also its transcriptional activity in the nucleus; second, Abl can directly phosphorylate Myc on tyrosine: the resulting form of Myc appears to be cytoplasmic, and its presence correlates with Abl activation in cancer.

Development of two potential diagnostic monoclonal antibodies against human cytomegalovirus glycoprotein B

Human cytomegalovirus glycoprotein B (gB) represents a target for diagnosis and treatment in view of the role it plays in virus entry and spread. Nevertheless, to our knowledge, rare detection of a gB antigen has been reported in transplant patients and limited information is available about diagnostic gB monoclonal antibodies (mAbs). Our aim was to develop gB mAbs with diagnostic potential. Hydrophilic gB peptides (ST: amino acids 27-40, SH: amino acids 81-94) of favorable immunogenicity were synthesized and used to immunize BALB/c mice. Two mAbs, named ZJU-FH6 and ZJU-FE6, were generated by the hybridoma technique and limited serial dilution and then characterized by indirect ELISA, Western blotting, immunoprecipitation, and immunohistochemical staining. The mAbs displayed high titers of specific binding affinities for the ST and SH synthetic peptides at an mAb dilution of 1:60,000 and 1:240,000, respectively. Western blotting and immunoprecipitation indicated that these mAbs recognized both denatured and native gB of the Towne and AD169 strains. The mAbs, when used as the primary antibody, showed positive staining in cells infected with both Towne and AD169 strains. The mAbs were then tested on patients submitted to allogeneic hematopoietic stem cell transplantation. The gB antigen positivity rates of the patients tested using ZJU-FH6 and ZJU-FE6 were 62.0 and 63.0%, respectively. The gB antigen showed a significant correlation with the level of pp65 antigen in peripheral blood leukocytes. In conclusion, two potential diagnostic gB mAbs were developed and were shown to be capable of recognizing gB in peripheral blood leukocytes in a reliable manner.

Nestin expression in Bowen's disease and Bowen's carcinoma associated with human papillomavirus

Human papillomaviruses (HPVs) have been detected in lesions of Bowen's disease (BD) and Bowen's carcinoma (BC); the invasive tumor retains the cytological characteristics of BD. Previous reports suggest that nestin-expressing hair follicle stem cells are undifferentiated and pluripotent, and nestin expression in some tumors indicates poor differentiation and high grade of malignancy. We identified HPV-DNA in BD (n=25) and BC (n=23) by in situ hybridization (ISH) analysis with INFORM(®) HPV III (Ventana Medical Systems. AZ, USA) and determined nestin expression by indirect immunohistochemical staining with anti-nestin polyclonal antibody (IBL, Gunma, Japan). We detected HPV-DNA in 68% of BD and in 87% of BC. In BD, 13 cases demonstrated the punctuate pattern, and four showed nestin expression. In BC, 19 cases showed the punctuate pattern and 16 showed nestin expression. HPV-DNA integrates into the host genome, and this is observed as the punctuate pattern on ISH. The nestin expression was statistically high in group of BC than BD (P<0.01). These results therefore suggest that HPV-DNA integrated in the genome of tumor cells of these diseases and contributed to malignant alteration. From the standpoint of tumorigenesis, BC might represent one type of poorly differentiated, high-grade squamous cell carcinoma.

Abstract 4885: Targeted multi-modal protein microspheres for cancer imaging

Abstract Optical coherence tomography (OCT) is a novel technology that has been developed for various clinical applications from ophthalmology to oncology. OCT is analogous to ultrasound but with micron-scale resolution by using light waves instead of sound waves to provide detailed structural information at the cellular level. The development of contrast agents has been critical to the development of OCT and its functional modalities such as magneto-motive OCT (MM-OCT). MM-OCT is a modality of OCT in which a small external magnetic field is modulated on and off during imaging, providing an added dimension of contrast from the magnetic particle responses. Protein microspheres consisting of a hydrophobic oil core and a hydrophilic BSA protein shell provide the vehicle for a multi-modal contrast agent. The microspheres encapsulate iron oxide nanoparticles in the oil core, providing magnetic signal contrast, and dyes such as Nile Red and DiR iodide, providing fluorescence contrast. The outer surface is functionalized using a layer-by-layer adhesion process to attach RGD peptide sequences to target integrin receptors. Using dynamic light scattering, we found the size distribution of the microspheres to be between 1-5 µm. Under SEM and TEM, we were able to visualize the various layers and coatings, such as silica and RGD peptides, of the microsphere. The microspheres were optimized to maximize the magnetic contrast under MM-OCT and MRI, and the fluorescent contrast under a dark box fluorescence imaging system, and fluorescence microscopy. These studies validated the use of MM-OCT as a method for quantifying the relative amount of iron oxide and the relative number of microspheres in the samples. To address the binding specificity and sensitivity of the RGD coated microspheres to the integrin receptors, the microspheres were incubated with cell lines of varying expression levels of the alpha(v)beta(3) integrin receptor and visualized under fluorescence microscopy. The cell lines used in this study included a normal epithelial cell line: hTERT-HME1, and several human breast cancer cell lines: HCC38, SK-BR-3, MCF-7, ZR-75-1, MDA-MB-231, and MDA-MB-435S. These results were externally validated by quantification of the receptors using indirect immunohistochemical staining and flow cytometry. Preliminary results, using the multi-spectral dark box fluorescence imaging system, demonstrate the localization of the microspheres to the vasculature surrounding the tumor and to lymph nodes. This is highly suggestive of the microsphere's selective binding to the vasculature. By combining the benefits of these various imaging modalities in a single agent, it becomes possible to use a wide-field imaging method such as MRI or small animal fluorescence imaging to initially locate the agents in-vivo, to use MM-OCT to provide micron scale resolution structural images in-vivo, and to use fluorescence microcopy to confirm the localization of these particles ex-vivo. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4885. doi:10.1158/1538-7445.AM2011-4885

IDENTIFICATION OF CD133+/NESTIN+ PUTATIVE CANCER STEM CELLS IN NON-SMALL CELL LUNG CANCER

No effective treatment for lung cancer exists currently. One reason for this, is the development of drug resistance, assumed to be associated with cancer stem cell (CSCs) emergence within the tumour. This pilot study aimed to identify CSCs in 121 non-small cell lung cancer (NSCLC) patient samples via detection of the expression of stem cell markers - CD133 and nestin. Archived paraffin blocks of 121 patient samples were prepared as Tissue Microarrays (TMA). Indirect immunohistochemical staining was used to determine the level of expression of CD133 and nestin. Double immunofluorescence staining was used to investigate the co-expression of these two markers. To determine the correlation between expression of nestin and CD133 with the length of asymptomatic period and overall patient survival we used the Kaplan-Meyer analysis. CD133 expression was detected in 22 (19%), nestin in the epithelium in 74 (66%) and vasculature in 78 (70%) of patients. Co-expression of these two markers was found in 21 (17%) patients in less than 1% of positive cells without impact on disease free or overall survival. We identified CD133(+)/nestin(+) cells as novel potential markers of lung cancer CSCs.

A Role for Intestinal Alkaline Phosphatase in the Maintenance of Local Gut Immunity

Intestinal alkaline phosphatase (IAP) is a gut mucosal defense factor known to dephosphorylate lipopolysaccharide (LPS); however, the role of IAP in the gut response to luminal bacteria remains poorly defined. We investigated immune responses of wild-type (WT) and IAP-knockout (IAP-KO) mice to LPS and Salmonella typhimurium challenges. Cryostat sectioning and standard indirect immunohistochemical staining for major histocompatibility complex (MHC) class II molecules were performed on liver tissue from WT and IAP-KO mice. WT and IAP-KO mice were orally gavaged with S. typhimurium; bacterial translocation to mesenteric nodes, liver, and spleen was determined by tissue homogenization and plating. In other experiments, WT and IAP-KO mice received intraperitoneal injections of LPS, with subsequent quantification of complete blood counts and serum interleukin (IL)-6 by enzyme-linked immunosorbent assay (ELISA). WT and IAP-KO whole blood were plated and stimulated with LPS and Pam-3-Cys, followed by cytokine assays. Immunohistologic liver examinations showed increased expression of MHC class II molecules in IAP-KO mice. Following S. typhimurium challenge, WT mice appeared moribund compared with IAP-KO mice, with increased bacterial translocation. WT mice had >50% decrease (P<.005) in platelets and 1.8-fold (P<.05) increased serum IL-6 compared with IAP-KO mice in response to LPS injections. IAP-KO whole-blood stimulation with LPS and Pam-3-Cys resulted in increased IL-6 and tumor necrosis factor (TNF)-alpha secretion compared with WT. IAP-KO mice exhibit characteristics consistent with local LPS tolerance. Whole-blood response of IAP-KO mice did not reflect systemic tolerance. These data suggest that IAP is a local immunomodulating factor, perhaps regulating LPS-toll-like receptor 4 (TLR4) interaction between commensal microflora and intestinal epithelium.

A Role for Intestinal Alkaline Phosphatase in the Maintenance of Local Gut Immunity

Background and Aims: Intestinal alkaline phosphatase (IAP) is a gut mucosal defense factor known to dephosphorylate lipopolysaccharide (LPS); however, the role of IAP in the gut response to luminal bacteria remains undefined. We investigated immune responses of wildtype (WT) and IAP-knockout (IAP-KO) mice to LPS and Salmonella typhimurium challenges. Methods: Cryotstat sectioning and standard indirect immunohistochemical staining for MHC Class II molecules were performed on liver tissue from WT and IAP-KO mice.

In Situ Deposition of Complement in Human Acute Brain Ischaemia

Experimental animal models indicate that complement contributes to tissue damage during brain ischaemia and stroke, but limited data are available for a role of the complement in human stroke. We, therefore, evaluated whether acute ischaemia leads to complement activation in human brain. Indirect immunohistochemical staining was performed on paraffin-embedded, formalin-fixed human brain from 10 patients and 10 controls. Complement components C1q, C3c and C4d were detected in all ischaemic lesions, suggesting activation via the classical pathway. C9, C-reactive protein and IgM were detected in necrotic zones. Marked CD59 and weak CD55 expression were found in normal brains, but these complement regulators were virtually absent in ischaemic lesions. Modest amounts of mannose-binding lectin (MBL), MBL-associated serine protease-2 and factor B were found in both ischaemic lesions and controls. These data suggest that increased deposition of complement components combined with decreased expression of complement regulators is a possible mechanism of tissue damage during ischaemia in human brain.

Development and evaluation of an indirect in situ polymerase chain reaction for the detection of porcine circovirus type 2 in formalin-fixed and paraffin-embedded tissue specimens

Taking advantage of the high sensitivity of polymerase chain reaction (PCR) and the cell-localizing ability of in situ hybridization (ISH), an indirect in situ PCR (ISPCR) method was developed for detecting the distribution of porcine circovirus type 2 (PCV2) in formalin-fixed and paraffin-embedded inguinal lymph nodes obtained from clinically healthy PCV2-carrier pigs and postweaning multisystemic wasting syndrome (PMWS)-affected pigs. Comparisons of the relative sensitivity of indirect ISPCR with other routinely used diagnostic methods for PCV2 indicated that nested PCR was the most sensitive method followed by indirect ISPCR, conventional PCR, ISH, and immunohistochemical (IHC) staining. Although indirect ISPCR, ISH, and IHC staining all revealed a similar signal distribution pattern of PCV2, using indirect ISPCR allowed specific amplification and detection of previously uneasily detected PCV2 signal than by routine ISH or IHC staining, particularly in those cells within the germinal center in clinically healthy PCV2-carrier pigs. Furthermore, six different PCV2 signal expression patterns in conjunction with the correlated lymphoid lesion stages were classified to describe the tissue morphological changes and viral infection. The result indicates that indirect ISPCR is a more effective, cell-based diagnostic tool with good specificity to detect limited PCV2 infection in formalin-fixed and paraffin-embedded tissue specimens and it would be a useful tool for further exploring the pathogenesis of PCV2 infection.

S187 – Autoimmune Indicators in Idiopathic Subglottic Stenosis

Objectives Present new data regarding the role of autoimmune disease in Idiopathic Subglottic Stenosis. Methods Patients with ISS identified between April 2007 and February 2008 were included. Directed subglottic biopsies and serum samples were collected after IRB approval and appropriate consent. Direct immunohistochemical staining was conducted on biopsy samples and indirect immunohistochemical staining was performed on serum samples. Results 35 females were identified with ISS. The mean age was 51 (range 23–81). 12 patients underwent direct Immunofluorescence staining with biopsies taken from the stenotic region. Only 2 biopsies had adequate epithelial surface area to perform direct immunofluorescence, which was negative in both cases. Indirect immunofluorescence stains were performed on 18 samples using a monkey esophagus model and human salt split skin to identify autoimmune antibodies to epithelial and basement membrane components. For the same specimens, ELISA tests were performed for Desmoglein 1, Desmoglein 3, BP 180, and BP230 markers that are positive in various autoimmune mucocutaneous disorders. Only 2 serum specimens were weakly positive for BP180. However, 13 of 18 specimens (72%) displayed nonspecific nuclear staining indicative of a nuclear autoantibody component, suggesting a connective tissue disorder. Conclusions This work does not demonstrate clear evidence of an autoimmune mucocutaneous disorder involved in the pathogenesis of ISS; however, the non-specific antinuclear staining may indicate an association with a connective tissue disorder. Further studies are required to identify the exact role and pathogenesis that the immune system plays in ISS for future targeted therapy.

Identification of progenitor cancer stem cell inlentigo malignamelanoma

The potential role of stem cells in neoplasia has aroused considerable interest over the past few years. A number of known biologic characteristics of melanomas support the theory that they may originate in a mutated stem cell. Melanocytic stem cell markers have been described recently. Moreover, the CD133 cells that show surface markers for CD34 are stem cells primitive. These stem cells are capable of differentiating into neurons, glia, keratinocytes, smooth muscle cells, and melanocytes in vitro. The identification of cancer stem/initiating cells with a crucial role in tumor formation may open up new pharmacologic perspectives. The purpose of this study is to detect the expression of CD133 and CD34, two putative markers of cancer stem cells in the lentigo maligna melanoma. Thirty cases of lentigo maligna melanoma were analyzed using indirect immunohistochemical staining. The vast majority of the samples analyzed showed the presence of rare cells, which were clearly positive for CD133 and CD34. Strong CD133 and CD34 staining was found in the outer root sheath of the mid-lower hair follicles, intermixed with atypical melanocytes extending along layers of the hair follicles. A number of these staminal cells were adjacent and intermixed with melanoma cells. This study supports the stem cell origin of this tumor and suggests that the precursor of the melanoma in question is a stem-like cell rather than the primitive melanoblast committed to be exclusively involved in melanocytic differentiation.