Streptavidin-biotin Peroxidase Complex Method Research Articles

Immunohistochemical Detection of Pro-Inflammatory and Anti-Inflammatory Interleukins in the Lungs of Sheep with Jaagsiekte

Objective: In this study, it was aimed to evaluate the levels of interleukins such as IL-1β, IL-6, IL-10 and IL-12β in Jaagsiekte sheep by immunohistochemical methods. In this way, it will be revealed whether interleukins are effective in the progression of Jaagsiekte and how useful they are in the diagnosis of the disease. 
 Material-Method: The material of current study consisted of lung tissues of 26 sheep (Control, n=6 and Jaagsiekte, n=20) brought to Department of Pathology for routine histopathological diagnosis. Tissue samples taken were fixed in 10% buffered formaldehyde solution. 5 µm-thick sections were taken from the paraffin blocks prepared after routine tissue follow-up procedures. Hematoxylin & Eosin staining was applied to the sections in order to detect histopathological changes. Sections were examined and photographed under a light microscope. The routine streptavidin–biotin peroxidase complex method was used. 
 Results: In sheep with Jaagsiekte, tumoral foci with large and small acinar or papillary growths were observed in the alveolar and bronchiole lumens. The control group was negative for IL-1β, IL-6, IL-10 and IL-12β immunoreactivity. IL 1β-6-10 and 12β levels were dramatically increased in the Jaagsiekte group compared to the control group. 
 Conclusion: It was determined that interleukins were produced from tumoral cells and tumor microenvironment elements, and these interleukins showed pro-inflammatory effects, except for IL-10. Data from the current study show that interleukins are very useful markers for the diagnosis of Jaagsiekte.

Immunohistochemical distribution of platelet-derived growth factor-C and platelet-derived growth factor receptor-α in small intestine of rats treated with capsaicin

The purpose of this study was to examine the effect of capsaicin administration immunohistochemically on platelet-derived growth factor (PDGF)-C and platelet-derived growth factor receptor (PDGFR)-\alpha release in the small intestine tissues of rats. Rats were divided into three groups: group 1 (subcutaneous drug administration), group 2 (oral drug administration), and group 3 (subcutaneous sham administration). Histometric measurements and counts were performed in order to determine whether there was a difference between groups in terms of height of villi, depth of crypts, and goblet cell count in small intestine. The small intestine was examined by Crossman?s triple staining method and streptavidin-biotin peroxidase complex method. As a result of statistical analysis, it was found that body weight increased in group 3, while it decreased in groups 1 and 2. Capsaicin administration increased height of villi, depth of crypts, and count of goblet cells located on villi and crypts. Immunohistochemical examinations revealed that capsaicin administration increased PDGF-C and PDGFR-? release. It was concluded that capsaicin has a stimulatory effect on growth and is an enhancer for PDGF-C and PDGFR-? release.

Immunohistochemical examination of effects of kefir, koumiss and commercial probiotic capsules on platelet derived growth factor-c and platelet derived growth factor receptor-alpha expression in mouse liver and kidney

We investigated using immunohistochemistry the effects of kefir, koumiss and commercial probiotic capsules on the expression of platelet derived growth factor-c (PDGF-C) and platelet derived growth factor receptor-alpha (PDGFR-α) in mouse liver and kidney. Mice were assigned to four groups: group 1 was given commercial probiotic capsules, group 2 was given kefir, group 3 was given koumiss and group 4 was untreated. After oral administration for 15 days, body weights were recorded and liver and kidney tissue samples were obtained. Hematoxylin and eosin staining was used to examine histology. PDGF-C and PDGFR-α in liver and kidney were localized using the streptavidin-biotin peroxidase complex method (ABC). We found that the weights of the mice in the kefir, koumiss and commercial probiotic capsules groups increased compared to the control group. No differences in liver and kidney histology were observed in any of the experimental groups. Kefir, koumiss and the commercial probiotic preparation increased PDGF-C and PDGFR-α expression.

Increased number of Langerhans cells in oral lichen planus and oral lichenoid lesions

The aim of this study was to quantify the presence of Langerhans cells (LC) in oral lichen planus (OLP) and oral lichenoid lesions (OLL), comparing them with normal epithelium. Thirty-six patients with biopsy-proven OLP or OLL were selected for the study, as well as 23 control subjects free of inflammatory conditions. Immunohistochemical reactions were performed using the streptavidin-biotin peroxidase complex method with CD1a and CD83 primary antibodies. Densities were compared between groups and correlated with microscopic findings. Patients with lichenoid conditions (OLP + OLL) presented higher densities of CD1a(+) cells than the control subjects (P = .03). Higher densities of CD1a were associated with a thinner layer of inflammatory cells (P = .02). This study indicates that OLP and OLL are characterized by the recruitment of LC, which may play a significant role on its pathogenesis.

Immunohistochemical Expression of Pituitary Tumor Transforming Gene (PTTG) in Pituitary Adenomas: A Correlative Study of Tumor Subtypes

We investigated the correlation between immunohistochemical expression of the pituitary tumor transforming gene (PTTG) and pituitary adenoma subtype. Pituitary adenomas (n = 89) were stained for PTTG using the streptavidin-biotin-peroxidase complex method and a monoclonal PTTG antibody. PTTG staining was found to be cytoplasmic with a pronounced paranuclear expression pattern. Reactivity was highest in growth hormone (GH) adenomas as compared with other tumors, including prolactin (PRL), follicle-stimulating hormone/luteinizing hormone/alpha subunit, as well as adrenocorticotrophic hormone-secreting adenomas. PRL adenomas exhibited the lowest expression levels. Among GH adenomas, untreated tumors demonstrated significantly higher PTTG levels than octreotide-treated examples. Although dopamine agonist-treated PRL adenomas tended to show lower expression levels, statistical significance was not reached. Our finding that PTTG was differentially expressed in pituitary adenoma subtypes suggests a cell-specific function for PTTG. Moreover, treatment of GH adenomas with somatostatin analogues lowered PTTG expression. Further investigation into mechanisms mediating cell-specific expression of PTTG is warranted.

EBV Infection and Altered Control of Apoptotic Pathways in Posttransplant Lymphoproliferative Disorders (PTLD).

Abstract 3926Poster Board III-862 IntroductionPTLD represent a spectrum of lymphoid diseases potentially complicating the clinical course of transplant recipients. Most PTLD are EBV associated with latency III of the virus. The interaction between EBV and the control of apoptosis is not precisely known. In fact, deregulation of Bcl-2 family proteins which has been implicated in the development of many malignancies can be divided into three groups: anti-apoptotic members (including Bcl-2, Bcl-XL and Mcl-1), pro-apoptotic proteins (including Bax and Bak) and the so-called BH3-only proteins. Among these BH3-only proteins, Bim plays a major role in the control of apoptosis in immune cells. To gain advantage in EBV linked tumorigenesis we undertook our investigations on PTLD. We carried out an immunohistochemical investigation concerning the expression of pro-apoptotic and anti-apoptotic molecules, possibly targeted by virus products. We have tested in a series of adult and pediatric PTLD: Bcl-2, Bax, Bak, Mcl-1, Bcl-xl, Bim, Bid, Bad, Puma and the apoptosis effector PARP. Materials and methods62 PTLD were studied (44 EBV positive PTLD and 18 EBV negative PTLD). These cases were retrieved from four institutions: Bicetre/Paul Brousse University hospitals : 25 cases, Zurich University hospital : 29, Pavia University hospital : 8. This study focused exclusively on B-cell PTLD. Hematoxylin and eosin (H&E) stained paraffin sections were reviewed by four Hematopathologists, and classified according to WHO classification: 53 DLBCL, 7 Polymorphic-PTLD and 2 MNI-like lesions. Paraffin sections immunostainings were performed with streptavidin-biotin peroxidase complex method and antigen retrieval if needed. Expression of each marker was considered positive when more than 20% of neoplastic cells positive. ResultsThe expression of anti-apoptotic proteins (Bcl2, Mcl1, BclxL) did not display any correlation with the presence or not of EBV in PTLD (p>0.05) as well as the two pro-apoptotic proteins Bax and Bak. In constrast with the results without any significant differences between EBV positive and negative PTLD, for the BH3 only proteins Puma, Bad and Bid, the expression of Bim was significantly lower in EBV positive PTLD :4 out of 44 EBV positive PTLD (9%) were Bim positive while 13 cases out of 18 EBV negative PTLD (72%) expressed Bim (p<0.005). Moreover, the number of cases having more than 20% of PARP positive cells was significantly lower in EBV positive PTLD group (6/44 PARP+, 13%) than in EBV negative PTLD group 12/18 PARP+ (67%), (p<0,005). ConclusionThe capacity of Bim to bind with all pro-survival Bcl-2 family proteins and its potential regulation via EBV latent proteins are known. In fact, our results strongly suggest, a major role of Bim in the disruption of apoptosis in EBV positive PTLD Disclosures:No relevant conflicts of interest to declare.

Immunohistochemical Expression of SNAP-25 Protein in Adenomas of the Human Pituitary

SNAP-25, a synaptosome-associated exocytosis protein of 25 kd mw, plays an important role in the secretory activity of several endocrine cells. In the present study, we investigated surgically removed pituitary adenomas including 40 prolactin (PRL), 31 growth hormone, 5 adrenocorticotropic hormone, 5 thyroid-stimulating hormone, 14 follicle-stimulating hormone/luteinizing hormone/alpha-subunit-producing tumors, and 5 null cell adenomas. Among the 40 patients with PRL-producing pituitary adenoma, 16 had been preoperatively treated with the dopamine agonist bromocriptine. Similarly, of the 31 patients with GH-producing pituitary adenomas, 15 had been treated with the long-acting somatostatin analog, octreotide. All tumors were subjected to transsphenoidal surgery, formalin-fixed, routinely processed, and paraffin-embedded. Sections of 4 to 6-microm thickness were stained with hematoxylin and eosin and the periodic acid-Schiff as well as the Gordon-Sweet silver methods. Immunostaining for SNAP-25 (streptavidin-biotin peroxidase complex method) showed that 10 PRL-producing adenomas were strongly immunoreactive. Immunopositivity was mainly cell membrane in distribution but several cells showed mild cytoplasmic staining. Nuclei were immunonegative. Preoperative bromocriptine treatment markedly decreased SNAP-25 immunopositivity. Among GH-producing adenomas, SNAP-25 was seen in 5 cases; reactivity being mild-to-moderate, membrane-bound, and cytoplasmic. Octreotide caused no significant reduction in immunopositivity. Other adenoma types were virtually immunonegative. Six autopsy-derived human pituitaries and 4 surgically obtained nontumorous pituitaries were also immunonegative for SNAP-25. It is conceivable that SNAP-25 plays an important role in PRL release and is involved in the bromocriptine-induced suppression of PRL secretion from PRL-producing adenoma cells.

Prevention of retinoic acid-induced early craniofacial abnormalities by folinic acid and expression of endothelin-1/dHAND in the branchial arches in mouse.

Prevention of retinoic acid-induced craniofacial abnormalities by folinic acid, and endothelin-1 (ET-1)/dHAND protein and mRNA expression were investigated in mouse embryos using the whole embryo culture, streptavidin-biotin peroxidase complex method, and whole-mount in situ hybridization. In the whole embryo culture, 1.0 and 0.1 mm-folinic acid dose dependently prevented branchial region malformations and decreased defects by 93 % and 77 %, respectively. Folinic acid at concentrations of 1.0 and 0.1 mm significantly increased ET-1 and dHAND protein expression levels compared to retinoic acid-exposed values in embryonic branchial areas. Folinic acid also increased ET-1 and dHAND mRNA levels in the same region. The present results suggest that folinic acid may prevent retinoic acid-induced craniofacial abnormalities via increasing ET-1 and dHAND levels in the branchial region during the organogenic period.

Prognostic value of VEGF expression in primary esophageal squamous cell carcinoma

Objective: To study the expression of vascular endothelial growth factor (VEGF) and microvessel density (MVD) in esophageal squamous cell carcinoma (ESCC) and clarify the association of VEGF expression with the angiogenesis and prognostic value of this disease. Methods: Eighty-two cases with primary ESCC treated with radical operation in Department of Surgery from Jan 1981 through May 1994 were enrolled. VEGF expression and MVD value were examined by immunohistochemical staining, the streptavidin-biotin peroxidase complex method (SP method), using anti-VEGF polyclonal antibody and anti-Factor-VIII antibody, respectively. We also analyzed the relationship between VEGF expression and MVD value and postoperative survival rate of patients. Results: Of the 82 cases, 63.4% cases showed positive for VEGF in tumor cells and the median of MVD in tumor was 37 (9–150) ·mm−2. There was a close correlation between MVD and VEGF (P=0.001). The 5-year survival rate of patients with low and high MVD was 34.1% and 12.2%, respectively. The 5-year survival rate was 46.7% in patients with VEGF-negative tumor and 11.5% in patients with VEGF-positive tumor. These differences were statistically significant (P=0.017 and P<0.001, respectively). Conclusion: In ESCC, angiogenesis is mediated mainly by VEGF and VEGF may be associated with tumor progression and increased malignancy via angiogenesis.

Expression of inducible nitric oxide synthase and heat shock proteins in periapical inflammatory lesions.

The mechanisms responsible for activation and proliferation of lining epithelium involved in inflammatory processes in periapical inflammatory lesions remain unclear. In this study, the expression and distribution of inducible nitric oxide synthase (iNOS) and heat shock proteins (HSPs) were immunohistochemically investigated in periapical inflammatory lesions. Control specimens of periodontal ligaments including Malassez epithelial rests from seven teeth and periapical inflammatory lesions (15 apical granulomas (AGs), 16 radicular cysts (RCs), and 10 residual radicular cysts (RRCs)) were prepared and examined by the standard streptavidin-biotin peroxidase complex method using anti-iNOS rabbit polyclonal antiserum, and anti-HSP27, -HSP60, -HSP70 mouse monoclonal antibodies. Immunoreactivity for iNOS was detected in macrophages, lymphocytes, and endothelial cells of granulation tissue and in lining epithelium of periapical inflammatory lesions. Malassez epithelial rests showed no or slight staining for iNOS. The epithelial staining intensity of iNOS in RCs was greater than that in Malassez epithelial rests and RRCs. Immunoreactivity for HSP27 was recognized in inflammatory cells, endothelial cells and lining epithelium of periapical inflammatory lesions and in Malassez epithelial rests. HSP60 was detected in some lymphocytes of granulation tissue and in lining epithelium of periapical inflammatory lesions, whereas Malassez epithelial rests showed no staining for HSP60. Epithelial HSP60 reactivity was more intense in RCs than in RRCs. HSP70 was expressed in lymphocytes, endothelial cells and lining epithelium of periapical inflammatory lesions and in Malassez epithelial rests. The staining intensity of HSP70 in Malassez epithelial rests was slightly lower than that in lining epithelium of RCs and RRCs. These data demonstrate that the expressions of iNOS, HSP60, and HSP70 are involved in inflammatory processes and might play a role in the activation and proliferation of lining epithelium, leading to progression of periapical inflammatory lesions.

Comparative Expression of E-cadherin, .ALPHA. and .BETA. Catenin in Salivary Gland Tumors.

To study the expression and role of adhesion molecules in normal salivary glands and their tumors, we have studied simultaneously the expression of E-cadherin, and associated cytoplasmic proteins, α-catenin and β-catenin. Paraffin sections were evaluated using the streptavidin biotin peroxidase complex (SBPC) method for E-cadherin, and the SBPC method with tyramide signal amplification (TCA) for α- and β-catenin.Normal submandibular (n=10), parotid (n=4) and oral minor glands (n=4) showed cell membrane staining for E-cadherin and a positive reaction in intercalated and striated ducts for α - and β -catenin. Pleomorphic adenoma (n = 17) showed cell membrane staining for E-cadherin and α-catenin in luminal and non-luminal cells, but were negative for β-catenin. E-cadherin was also focally or irregularly positive in modified myoepithelial cells. Warthin's tumors (n = 4) expressed Ecadherin only and were negative for a- and β-catenins. Papillary adenocarcinoma (n = 4) showed predominant staining for E-cadherin, with no expression in the other tumor cells. Adenoid cystic carcinoma (n = 4) showed only cell membrane positivity for E-cadherin, with an absence of α- and β-catenins in the luminal cells. Non-luminal tumor cells of adenoid cystic carcinoma were negative for E-cadherin and positive for α- and β-catenins. The results were concluded as follows, 1) there was uniform cell membrane staining for E-cadherin in normal and benign tumor epithelium, but α- and β-catenins showed different expression and heterogeneous localization, 2) loss of E-cadherin reactivity was seen in malignant lesions, 3) there was no correlation between the distribution of Ecadherin, α-catenin and β-catenin in benign or malignant tumors of salivary gland origins. The results show variability and heterogeneous expression for E-cadherin, α- and β-catenins in salivary gland tumors.

Immunohistochemical examination of c-Met protein expression in astrocytic tumors.

Hepatocyte growth factor/scatter factor (HGF/SF), which has various physiological functions, and its receptor c-Met, the human c-met proto-oncogene product, are thought to be determinant in the pathological processes of various malignancies. To investigate the possible role of HGF/SF in the progression of development of astrocytic tumors, we examined the expression of c-Met in these tumors. Immunohistochemistry using the streptavidin-biotin peroxidase complex method and immunofluorescence double staining with anti-c-Met polyclonal and anti-glial fibrillary acidic protein monoclonal antibodies were performed. Positive c-Met expression was detected in 31 of the 42 astrocytic tumors and some of the control cases analyzed. c-Met-positive cells showed morphological characteristics of astrocytes. Especially in the cases of high-grade tumors, c-Met positivity was abundant in cells in both vascular-rich and peripheral regions of the tumors but not in the cells with distinctly malignant features. Immunofluorescence double staining revealed that the c-Met-positive cells were in part of astrocytic origin. We suggest that c-Met-positive cells are affected by some factors in the lesions where the pathological processes are in a state of development. Our studies indicated that c-Met expression might take part in glioma invasion but not in the development of malignancy.

Quantitative Analysis of Calcitonin Gene-Related Peptide- and Neuropeptide Y-Immunoreactive Nerve Fibers in Mesenteric Blood Vessels of Rats Irradiated with Cobalt-60 Gamma Rays

The effects of 60Co gamma rays on calcitonin gene-related peptide (CGRP)-immunoreactive nerve fibers and neuropeptide Y (NPY)-immunoreactive nerve fibers were examined in mesenteric blood vessels of rats. Using a free-floating immunostaining streptavidin-biotin peroxidase complex method combined with a nickel-enhancement technique, we found that the distribution pattern of these two peptidergic nerve fibers in superior mesenteric arteries and superior mesenteric veins did not change, while the densities of CGRP-immunoreactive nerve fibers and NPY-immunoreactive nerve fibers in superior mesenteric arteries and veins varied with the time after irradiation. The results suggested that CGRP and NPY may be important in the development and elimination of radiation-induced injury.

Expression of p53 in oesophageal squamous epithelium from surgical specimens resected for squamous cell carcinoma of the oesophagus, with special reference to uninvolved mucosa.

Accumulation of p53 protein has been considered an intermediate biomarker in multistage oesophageal carcinogenesis. The aim of the present study was to investigate p53 expression by immunohistochemistry in 13 thoroughly sampled oesophagectomy specimens from a geographical area with a high oesophageal cancer incidence (Basse Normandie, France). Expression of p53 was looked for in tissue samples of cancer, intraepithelial neoplasia, and uninvolved mucosa. The streptavidin biotin peroxidase complex method was used for p53 immunostaining. p53 expression was found in invasive squamous cell carcinoma in 8 out of 11 cases and in intraepithelial neoplasia in 10 out of 11 cases. In all 13 cases, in uninvolved oesophageal mucosa, expression of p53 was focally present in areas of chronic oesophagitis. Chronic oesophagitis has been regarded by epidemiologists as a precursor lesion for squamous cell carcinoma of the oesophagus. Since oesophageal carcinogenesis is a multistage process, the study of precursor lesions could provide information on the timing of p53 gene abnormalities during oesophageal carcinogenesis. These preliminary data require to be confirmed by molecular analysis of the p53 gene.

Distribution of parasympathetic nerve terminals in the rat submandibular gland.

I examined the distribution of cholinergic neuron terminals in the rat submandibular gland. Immunohistochemical procedures with a monoclonal antibody against choline acetyltransferase (ChAT) were used to determine the ultrastructural features of the putatively cholinergic axons. Either the streptavidin biotin-peroxidase complex method or the post-embedding staining with protein A-colloidal gold method was used for immuno-staining. The reaction was too weak to detect using conventional methods for ChAT. Interleukin 3 (IL-3) was used to enhance immunohisto-staining of peripheral cholinergic axons. Good results were obtained with IL-3 treatment. The submandibular ganglions and nerve bundles were both close to the main duct, and both were immuno-reactive to ChAT.Although a number of ChAT positive fibers were seen within the acini and intercalated ducts of the submandibular parenchyma, they were not in other duct cells. ChAT positive terminals of adrenergic axons were detected by prior treatment with 5-hydroxydopamine (5-OHDA). Axons containing vesicles, not only aminergic but also occasionally cholinergic, were found adjacent to the endothelial cells of blood vessels. Although axons containing dense cores in their vesicles were noted at the basement membrane of acinar and duct cells, ChAT positive terminals were seen in both acinar and intercalated duct cells.These findings suggest that immunocytochemical staining using ChAT monoclonal antibodies with IL-3 treatment is a useful technique for detection of cholinergic peripheral nerve fibers in the submandibular gland.

INDUCTION OF RESISTANCE-PROTEINS AND ONCOPROTEINS IN MURINE AND HUMAN TUMOR-CELL LINES AFTER IRRADIATION

The aim of this investigation was to analyze whether or not ionizing radiation can induce the expression of resistance- and oncoproteins in murine sarcoma 180 cells and several human carcinoma cell lines. For assessment of the proteins the streptavidin-biotin peroxidase-complex method was performed using specific antibodies. Expression of glutathione S-transferase-pi and thymidylate-synthase was increased after a single dose of irradiation which was accompanied by a lower sensitivity against cisplatin and 5-fluorouracil. No differences in protein expression could be detected for P-glycoprotein 170 and topoisomerase II. Differences in the expression of the oncoproteins c-Fos and c-ErbB1 were also found after irradiation. In contrast, a decreased expression of topoisomerase II was found after fractionated irradiation. This was confirmed by mRNA analysis.