Avidin-biotin Complex Research Articles

Reliability of Conjunctival Biopsy for Diagnosis of Ocular Mucous Membrane Pemphigoid: Redetermination of the Standard for Diagnosis and Outcomes of Previously Biopsy-Negative Patients

ABSTRACT Purpose: To demonstrate the reliability of conjunctival biopsy analyzed by direct immunofluorescence (DIF) and supplemented with avidin-biotin complex immunoperoxidase (ABC) in diagnosing oMMP, and report therapy response in biopsy-positive patients, particularly when previously biopsy-negative elsewhere. Methods: Retrospective outcomes review of 136 consecutive patients after conjunctival biopsy for suspected oMMP. Results: Among 136 patients, 66% were diagnosed with oMMP by DIF and 13% via supplemental ABC immunoperoxidase. Sensitivity increased from 79.6% with DIF to 95.6% with supplemental ABC. Among 57 biopsy-positive patients, 77% were in remission at 1-year follow-up and 88% after 2 years. Of 34 previous biopsy-negative but now biopsy-positive patients with a 2-year follow-up, 91% achieved remission, including all 16 diagnosed via DIF and ABC. Conclusion: Conjunctival biopsy analyzed by histopathology and DIF supplemented by ABC has high reliability for diagnosing oMMP and is a useful tool to use before starting long-term immunomodulatory therapy in a patient with suspected oMMP.

An immunohistochemical study of endocrine cells in the digestive tract of Varanus salvator (Reptile: Varanidae).

Aim:The aim of the study was to identify the distribution pattern and frequency of endocrine cell types in the digestive tract of Varanus salvator.Materials and Methods:The presence of endocrine cells (glucagon, somatostatin, and serotonin) in the digestive tract (esophagus, stomach, and intestine) was detected using the avidin-biotin complex (ABC) method.Results:Three types of endocrine cells immunoreactive to antisera glucagon, serotonin, and somatostatin were found in the caudal portion of the small and large intestines but were not observed in the esophagus, stomach, and caput and medial sections of the small intestine. Endocrine cells distributed in the digestive tract of V. salvator vary in color intensity, from weak to sharp, in response to the primer antibody.Conclusion:Endocrine cells in the digestive tract that is immunoreactive to glucagon, somatostatin, and serotonin are those found in the caudal portion of the small and large intestines. They are varied in distribution pattern, frequency, and color intensity.

Immunohistochemical expression of CD44v9 and 8-OHdG in ovarian endometrioma and the benign endometriotic lesions adjacent to clear cell carcinoma.

Expression of CD44 variant isoforms (CD44v) promotes the synthesis of reduced glutathione and contributes to reactive oxygen species defense through up-regulation of the intracellular antioxidant. The aim of the study was to investigate the expression of CD44v9 and oxidative DNA damage marker, 8-OHdG, in benign ovarian endometrioma (OE) and OE harboring clear cell carcinomas (CCC). A retrospective study was performed at the Department of Gynecology, Nara Medical University hospital from January 2006 to December 2012. Patients with histologically confirmed benign OE (n = 27) and OE harboring areas of CCC (n = 8) were selected. Tissue samples were immunohistochemically analyzed for the presence of CD44v9 and 8-OHdG using avidin-biotin complex method. CD44v9 was located on the cell membrane of endometriotic epithelial cells and expressed in 88.9% (24/27) of benign OE tissues. Only 25.0% (2/8) of benign endometriotic lesions adjacent to CCC was found to stain weakly for CD44v9. Percentage of CD44v9 positive cells was 68.5 ± 20.2% (mean ± standard deviation) of benign OE, 16.7 ± 16.5% of CCC endometriotic tissue (P < 0.001). Compared to benign OE, CCC endometriotic tissue showed a significant increase in the proportion of 8-OHdG expression (77.3 ± 22.5% vs 94.9 ± 3.0%, P = 0.049). A significant negative correlation was observed between CD44v9 status and 8-OHdG nuclear expression (r = -0.458, P = 0.006). Alterations in CD44v9 and 8-OHdG may be associated with malignant transformation of benign OE.

Weak Cytotoxic T Cells Activation Predicts Low-Grade Dysplasia Persistence in Ulcerative Colitis.

INTRODUCTION:In patients with ulcerative colitis (UC), dysplasia develops in 10%–20% of cases. The persistence of low-grade dysplasia (LGD) in UC in 2 consecutive observations is still an indication for restorative proctocolectomy. Our hypothesis is that in the case of weak cytotoxic activation, dysplasia persists. We aimed to identify possible immunological markers of LGD presence and persistence.METHODS:We prospectively enrolled 112 UC patients who underwent screening colonoscopy (T0) who had biopsies taken from their sigmoid colon. Ninety of them had at least a second colonoscopy (T1) with biopsies taken in the sigmoid colon and 8 patients had dysplasia in both examinations suggesting a persistence of LGD in their colon. Immunohistochemistry and real time polymerase chain reaction for CD4, CD69, CD107, and CD8β messenger RNA (mRNA) expression and flow cytometry for epithelial cells expressing CD80 or HLA avidin-biotin complex were performed. Non-parametric statistics, receiver operating characteristic curves analysis, and logistic multiple regression analysis were used.RESULTS:Thirteen patients had LGD diagnosed at T0. The mucosal mRNA expression of CD4, CD69, and CD8β was significantly lower than in patients without dysplasia (P = 0.033, P = 0.046 and P = 0.007, respectively). A second colonoscopy was performed in 90 patients after a median follow-up of 17 (12–25) months and 14 of the patients were diagnosed with LGD. In these patients, CD8β mRNA expression at T0 was significantly lower in patients without dysplasia (P = 0.004). A multivariate survival analysis in a model including CD8β mRNA levels and age >50 demonstrated that both items were independent predictors of dysplasia at follow-up (hazard ratio [HR] = 0.47 [95% confidence interval [CI]: 0.26–0.86], P = 0.014, and HR = 13.32 [95% CI: 1.72–102.92], P = 0.013).DISCUSSION:These data suggest a low cytotoxic T cell activation in the colonic mucosa of UC patients who do not manage to clear dysplasia. Thus, low level of CD8β mRNA expression in non-dysplastic colonic mucosa might be considered in future studies about the decision making of management of LGD in UC.

The effect of endoplasmic reticulum stress on detrusor muscle apoptosis in diabetic cystopathy

Objective To observe the effect of Endoplasmic Reticulum Stress (ERS) on detrusor muscle apoptosis in Streptozotocin (STZ)-induced diabetic rats. Methods At 4, 8, 12, and 16 weeks after induction of diabetic rat models, the sections of detrusor muscle tissue were made and were stained with Van Gieson solution, the collagen fibers of each group were observed and their proportions were calculated; the sub-cellular ultrastructure was observed by Transmission Electron Microscopy (TEM). Proliferating Cell Nuclear Antigen (PCNA) assay kit and Strept Avidin-Biotin Complex (SABC) staining were used for the evaluation of Detrusor Smooth Muscle (DSM) cell proliferation. Moreover, the expression of three hallmarks of ERS-associated apoptosis, including Glucose-regulated protein 78 (GRP78), CCAAT/enhancer-binding protein homologous protein (CHOP), and cysteinyl aspartate-specific protease (Caspase)-12, was detected by Real-time quantitative polymerase chain reaction (RT-qPCR) and Western blotting. Results The proportion of collagen fibers showed a significant climbing trend [(13.38±2.65)%, (18.14±1.93)%, (19.71±3.05)% vs. (7.86±0.72)%, P<0.05] since DM8w group, except DM4w group [(8.02±0.43)%]. With the time prolonged, the swelled and fused cisternaes started to show; the mild particle fusion and degranulation also could be seen in the zone of ER; the distorted or pyknotic nucleus contained clusters of unidentified dark-stained granules that could be observed. Compared with the control group, the increased PCNA-positive percentages [(20.34±4.55)%, (46.88±7.12)%, (39.78±1.93)%, (22.15±5.64)% vs. (16.26±2.43)%, P<0.05] and apoptosis indexes [(17.12±2.78)%, (28.12±4.09)%, (28.12±4.09)%, (43.39±6.82)% vs. (6.32±1.23)%, P<0.05] can be seen in model groups. Elevated expression of GRP78, CHOP, and Caspase-12 at both protein and mRNA levels in a time-dependent fashion were detected (P<0.05). Conclusion Morphological and ultrastructural damages to the DSM as well as the cell proliferation and apoptosis indicate the time-dependent alterations. The occurrence of ERS may be involved in the development of diabetic cystopathy (DCP) and may contribute to the diabetic detrusor muscle apoptosis. Key words: Endoplasmic reticulum stress; Diabetic cystopathy; Detrusor; Apoptosis

Frequent and Specific Involvement of Changes of the p16-RB Pathway in Esophageal Neuroendocrine Carcinoma.

This study investigated the immunohistochemical expression of retinoblastoma (RB) protein and p16 protein in 10 neuroendocrine carcinomas (NECs), in comparison to two mixed-type NECs; 28 squamous cell carcinomas (SCCs), and 12 carcinosarcomas (CSs) from patients with esophageal cancer. Immunohistochemical staining was performed using the avidin-biotin complex detection method. The staining was evaluated as diffusely positive, heterogeneous (in 5-95% of tumor cells), or diffusely negative. The combination of a diffuse loss of RB and the diffuse overexpression of p16, which is found in highly aggressive malignant tumors and is considered to convincingly suggest changes in the p16-RB pathway, was found in all NECs (10/10). In contrast no mixed-type NECs, one SCC and one CS showed this finding. Coexisting intraepithelial carcinoma was detected in seven NECs and only one lesion showed the combination of diffuse RB loss and p16 overexpression. These data suggest that changes in the p16-RB pathway were universally and specifically involved in the development and invasion of esophageal NECs and that it may be a useful diagnostic marker and a potential therapeutic target.

Immunocytochemistry of mesenteric lymph node fine-needle aspirates in the diagnosis of feline infectious peritonitis.

Immunohistochemistry (IHC) of tissue samples is considered the gold standard for diagnosing feline infectious peritonitis (FIP), and, in cats without body cavity effusion, IHC is the only method available to establish definitive antemortem diagnosis. However, IHC requires invasive tissue sample collection. We evaluated sensitivity and specificity of an immunocytochemical assay of fine-needle aspirates (FNAs) of mesenteric lymph nodes that can be obtained noninvasively by ultrasound-guided aspiration to diagnose FIP. FNAs of mesenteric lymph nodes were obtained postmortem from 41 cats suspected of having FIP based on clinical and/or laboratory findings. FIP was confirmed immunohistochemically in 30 cats. In the other 11 cats, a disease other than FIP, which explained the clinical signs, was diagnosed histopathologically. Immunocytochemistry (ICC) was performed as an avidin-biotin complex method using a monoclonal anti-FCoV IgG 2A. Sensitivity, specificity, negative and positive predictive values (NPV, PPV, respectively) including 95% confidence intervals (95% CIs) were determined. ICC was positive in 17 of 30 cats with FIP, but also in 1 of 11 control cats that was diagnosed with lymphoma. Sensitivity of ICC was 53% (95% CI: 34-72); specificity 91% (95% CI: 59-100); NPV 42% (95% CI: 22-63); and PPV 94% (95% CI: 71-100). In a lethal disease such as FIP, specificity is most important in order to avoid euthanasia of unaffected cats. Given that a false-positive result occurred and FIP was correctly detected in only approximately half of the cases of FIP, ICC of mesenteric lymph node FNA alone cannot reliably confirm or exclude FIP, but can be a helpful test in conjunction with other diagnostic measures.

Evaluation of Immunohistochemical Reactivity with Avidin-Biotin Complex (ABC) Method

Immunohistochemistry is any technique that can detect cellular and extracellular components in situ by means of specific antibodies, using enzymatic detection systems. Among immunohistochemical methods, the technique of avidin - biotin complex (ABC) is widely used because of its high sensitivity. The aim of this study was to evaluate the immunohistochemical reactivity of the 4C4.9 antibody for detection of S-100 protein using the ABC method. For the evaluation of immunohistochemical reactivity 2 biopsies of human skin were used with histopathological diagnosis of ulcerated malignant melanoma and melanocytic intradermal nevi from the Research Laboratory on Animal Biotechnology of the Universidad de La Frontera, Chile. The Kit VECTASTAIN® was used as detection method, the dilution the 4C4.9 antibody was 1/250 and incubation temperature was at 4 ° C or 37 °C for 18 hours. To validate the technique, a positive control and a negative for 4C4.9 was performed. The results of immunohistochemical staining by the method of ABC complex showed positive staining for protein S-100 both in ulcerated malignant melanoma and melanocytic intradermal nevi, incubated for 18 hours at 4 ° C or 37 ° C. However, immunostaining was more intense when the primary antibody was incubated at 37° C. For a correct interpretation of the results, it is necessary to take into consideration that the antigen-antibody reaction is influenced by various factors such as the concentration of antibody, time and temperature of incubation. In conclusion, our results suggest incubating the samples with the first antibody (4C4.9) at 1/250 dilution in distilled water, incubating for 18 h at 37 oC. However, immunostaining was more intense when the primary antibody was incubated at 37° C. For a correct interpretation of the results, it is necessary to take into consideration that antigen- antibody reaction is influenced by various factors such as the concentration of antibody, time and temperature of incubation. In conclusion, our results suggest incubating the samples with the first antibody (4C4.9) at 1/250 dilution in distilled water, incubating for 18 h at 37 oC. The use of the antibody 4C4.9 is recommended to support the diagnosis and differential diagnosis.

Comparison of renal lesions in cats and dogs using pathomorphological and ımmunohistochemical methods

ABSTRACTWe investigated the pathogenesis of chronic renal fibrosis in cats and dogs using immunohistochemistry. We used the avidin-biotin complex peroxidase (ABC-P) method with antibodies against transforming growth factor-β1, cytokeratin, E-cadherin, S100A4, alpha-smooth muscle actin, vimentin and nestin to determine whether tubule epithelial cells had undergone epithelial-mesenchymal transformation (EMT) that resulted in loss of epithelial cells and an increased number of mesenchymal cells. Although nephrosis was more common in dogs, nephritis was more common in cats; these pathologies developed in both kidneys. We found that EMT participated in the pathogenesis of renal fibrosis in both dogs and cats.

Stable Surface Modification of Mesenchymal Stem Cells Using the Avidin-Biotin Complex Technique.

Mesenchymal stem cells (MSCs) hold promise in cell-based therapies because of their strong tissue repair ability and immunosuppressive effects; however, the therapeutic efficacy of transplanted MSCs is limited due to low survival rates and short-term functioning after transplantation. While the functionalization of MSCs is an ideal way to solve these problems, conventional cell functionalization methods have disadvantages such as cell damage, changes in cellular characteristics, and short-term modification. This unit describes a technique for MSC functionalization by surface modification via the avidin-biotin complex (ABC). This technique provides long-term modification MSC surfaces with biotinylated compounds. This easy method of MSC functionalization will support effective MSC-based therapy. © 2018 by John Wiley & Sons, Inc.

Cultural and immunohistochemical evaluation of bacterial agents in caprine pneumonia in Nigeria

Goats are important protein-producing animals but pneumonia, in spite of the huge investments in small ruminant production, remains a major limitation. This study evaluates the prevalence of important bacterial pathogens associated with caprine pneumonia in Nigeria using cultural and immunohistochemical techniques. One hundred and fifty goat lungs were randomly examined from macroscopic, cultural isolation and microscopic changes using standard techniques. The common bacteria identified were formalin inactivated for bacterine and polyclonal antibody production. Immunohistochemical staining was performed against the bacteria antigens using the avidin-biotin peroxidase complex technique. The data were presented in frequencies and percentages, and compared using non-parametric statistics at α = 0.05. The pathology in the caprine lungs included broncho-interstitial pneumonia (41), interstitial pneumonia (27), and bronchopneumonia (72). Ten of the lungs were normal. Bacterial isolation yielded Mannheimia haemolytica (40%), Pasteurella multocida (20%), Escherichia coli (13%), Klebsiella pneumoniae (10%), Streptococcus sp. (8%), Baccillus sp. (5%), and Pseudomonas sp. (3%). Mannheimia haemolytica (Mh) and Pasturella multocida (Pm) were the most common isolated bacteria. The bacterial antigens were detected on the surface of respiratory bronchiolar, and alveolar epithelia, within inflammatory exudate and in cytoplasm of alveolar macrophages. One hundred twenty (80%) of the caprine lungs were positive for the Mh and Pm antigens; 47 for Mh, 59 for Pm, 14 for both, and 30 negative for either antigens. Significant differences were observed in the staining intensities of the antigens. This study validated Mannheimia haemolytica and Pasteurella multocida as most important bacterial pathogens in caprine pneumonia in Nigeria. Thus, adequate control of caprine pneumonia should consider bacterine or multivalent vaccines incorporating these important bacteria to curb morbidity and mortality of goats.

Immunohistochemical expression of Mum-1, Oct-2 and Bcl-6 in systemic anaplastic large cell lymphomas.

Several transcription factors predominantly used for B-cell lineage identification are also expressed in a small percentage of T cells within germinal centers and interfollicular areas. The aim of the study was to evaluate the expression of Mum-1, Oct-2 and Bcl-6 in systemic anaplastic large cell lymphoma. Thirty cases of anaplastic large cell lymphoma were retrieved from our archives and tissue microarray constructed. Immunohistochemistry was carried out using an avidin-biotin peroxidase complex method. A predominance of nuclear staining was observed for all transcription factors. Mum-1 was positive in all but one case (96.7%). Half of the cases displayed Oct-2 expression (15/30 cases). A considerable number of cases also had Bcl-6 expression (9/30). Bcl-6 staining was noted to be more common in ALK positive cases. Our findings emphasize that these markers are not restricted to B-cell lineage and that extensive expression can be observed in anaplastic large cell lymphoma of T/null cell phenotype.

Rabbit Sertoli Cells: Immunohistochemical Profile from Neonatal to Adult Age.

Immunohistochemistry (IHC) on formalin-fixed paraffin-embedded tissue specimens is a widely used laboratory technique that allows the demonstration and the direct microscopical observation of antigens within tissue sections by means of specific antibodies. IHC is one the best tools to define the immunophenotype of a cell during maturation or neoplastic transformation.To investigate the immunophenotype of postnatal rabbit Sertoli cell, from neonatal to adult age, in this chapter, the immunohistochemical protocol described is one of the most common: the avidin-biotin complex (ABC) method.

Loop-mediated isothermal amplification for visual detection of Vibrio parahaemolyticus using gold nanoparticles.

Loop-mediated isothermal amplification (LAMP) eradicates the need of thermocycler in DNA amplification. Signals are usually obtained via fluorometry or turbidimetry, but such methods need improvement in order to become more effortless and reliable. The authors describe a set of six specific primers targeting the species-specific tlh gene of Vibrio parahaemolyticus which were used in accelerated LAMP reaction. Gold nanoparticles (AuNPs) were functionalized with streptavidin (Avidin-AuNPs), and engineered to signal the LAMP reaction. Two of the loop primers for LAMP were biotinylated and then can produce a DNA that can cause clusterization of Avidin-AuNPs based on the formation of avidin-biotin complex. This leads to a color change of the solution from red to blue. Amplification is completed within 30min and can be visually detected within 5min. The detection limit of the method is found to be 8.6cfu per reaction. This visual detection scheme does not require any fluorescent reagents and detection instruments. Conceivably, the method has a wide scope because such Avidin-AuNPs can be used as nanoprobes for a variety of other LAMP products. This rapid and universal strategy holds promise in point of care testing and food testing, particularly in resource-limited regions. Graphical abstract Six specific primers (two of them are biotinylated) were used to realize the accelerated Loop-Mediated Isothermal Amplification. Streptavidin modified gold nanoparticles (Avidin-AuNPs) cluster on the DNA products, leading to the apparent change of color from red to blue, which is readily identified even by unaided eye.

Long-term drug modification to the surface of mesenchymal stem cells by the avidin-biotin complex method

Mesenchymal stem cells (MSCs) have various functions, making a significant contribution to tissue repair. On the other hand, the viability and function of MSCs are not lasting after an in vivo transplant, and the therapeutic effects of MSCs are limited. Although various chemical modification methods have been applied to MSCs to improve their viability and function, most of conventional drug modification methods are short-term and unstable and cause cytotoxicity. In this study, we developed a method for long-term drug modification to C3H10T1/2 cells, murine mesenchymal stem cells, without any damage, using the avidin-biotin complex method (ABC method). The modification of NanoLuc luciferase (Nluc), a reporter protein, to C3H10T1/2 cells by the ABC method lasted for at least 14 days in vitro without major effects on the cellular characteristics (cell viability, cell proliferation, migration ability, and differentiation ability). Moreover, in vivo, the surface Nluc modification to C3H10T1/2 cells by the ABC method lasted for at least 7 days. Therefore, these results indicate that the ABC method may be useful for long-term surface modification of drugs and for effective MSC-based therapy.

Substance P and Calcitonin Gene-Related Peptide in the Glands of External Auditory Canal Skin.

ObjectivesThe earwax (cerumen) that covers external auditory canal (EAC) skin contains a mixture of ceruminous and sebaceous gland substances, such as lipids, peptides, and proteins. The components secreted from the ceruminous gland that is a modified sweat gland form cerumen and contain several antimicrobial factors. Since substance P (SP) and calcitonin gene-related peptide (CGRP), known as a secretagogue, have been found in sweat glands, our purpose was to determine the expression of SP and CGRP in the glands of EAC skin.Methods Sections of normal human EAC skins were immunostained for the presence of SP and CGRP using polyclonal antibodies. Immunoreactivity was detected using an avidin-biotin peroxidase complex method.Results SP staining was found in ceruminous gland acini and myoepithelial cells. But the SP staining was not found in the sebaceous glands and epidermal region. CGRP was strongly stained in the ceruminous gland and weakly in the sebaceous gland cells. Interestingly, most prominent staining of SP and CGRP was noted in the myoepithelial cells of the ceruminous gland.Conclusion The findings in this study suggest that SP and CGRP are expressed in the glands of the EAC skin and secreted in the process of ceruminous gland secretion.

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OBJECTIVES/SPECIFIC AIMS: The goal of this research is to use circulating tumor cells (CTC) enumeration and characterization to monitor anticancer treatment response. Emerging evidence strongly suggests the implications that epithelial-to-mesenchymal transition may have in cancer metastasis. Consequently, we hope to elucidate the significance of mesenchymal and stem-like CTCs in the peripheral blood of metastatic pancreatic cancer patients by analyzing the prevalence and frequency trends of CD133+ CTCs, as they relate to clinical events. We also hope to determine if there is a correlation between EpCAM+ CTCs and CD133+ CTCs numbers with tumor size, disease stage, and patient clinical outcome. METHODS/STUDY POPULATION: Blood samples of patients with metastatic pancreatic cancer (stage IV) were obtained from the University of Florida Health Cancer Center after informed consent through an IRB-approved protocol. CTC capture, characterization, and enumeration was performed on the blood of these cancer patients during their anticancer treatment. Patients had blood drawn for this purpose at time points aligned with clinical phlebotomy (every 2 weeks). CTC capture was performed by introducing treated patient blood samples into antibody-functionalized microdevices. The PDMS devices were functionalized by immobilizing either anti-EpCAM or anti-CD133, through an avidin-biotin complex. After capture, cells were fixated and permeabilized with 4% paraformaldehyde and 0.2% Triton X-100, respectively. Three-color immunocytochemistry (anti-cytokeratin-FITC, anti-CD45-PE, and DAPI) was performed to identify CTCs from nonspecifically captured blood cells. To be counted as a CTC, based on the FDA-approved technical definition, a cell with the appropriate cell size and morphology must be nucleated (DAPI+), express cytokeratin (CK+), and lack the leukocytic CD45 marker (CD45−). RESULTS/ANTICIPATED RESULTS: We tested the clinical utility of the device for monitoring the response of patients with advanced pancreatic cancer to a chemotherapy treatment consisting of anticancer drugs including 5-fluorouracil, leucovorin, oxaliplatin, and dasatinib. We have detected EpCAM+ CTCs in 47/47 (100%) and CD133+ CTCs in 41/47 (87.2%) of blood samples, coming from a cohort of 16 patients. We studied the correlation between the CTC numbers and the clinical result of patients in the study. We found that the size and changes in the size of the primary tumor (confirmed by CT scans) correlated with the frequency and increase/decrease trends in the number of CTCs detected. We expect to find some relationship between the number of detected CD133+ CTCs, or rather stem-like CTCs, and the clinical outcome of patients (eg, disease progression leading to withdrawal from study). DISCUSSION/SIGNIFICANCE OF IMPACT: Enumeration of patient CTCs and stem-like CTCs at different stages of a patient’s treatment may correlate with disease stage and prognosis, and prove useful in monitoring early recurrence, patient-specific treatment response, and newly acquired resistances; all of which would aid in providing guidance for the next step in clinical intervention. This type of liquid biopsy technology has great potential to make an impact in the future of personalized medicine and point-of-care diagnostics, as well as become a sturdy tool for translational research.

HER-2/neu overexpression in correlation to Vascular Endothelial Growth Factor ,grade and stage of Non other wised specified Invasive ductal carcinoma

This study aimed to assess the significance of human epidermal growth factor (HER-2/neu) protein overexpression and its possible correlation with vascular endothelial growth factor (VEGF),grade and stage in human non otherwise specified breast cancer. The present investigation was performed over a period starting from November 2008 to January 2009 .Formalin fixed, paraffin-embedded blocks from 30 patients with breast cancer were included in this study. A group of 15 patients with benign breast lesions (fibroadenoma) was included as a comparative group and 15 normal breast tissue sections were included as control group. Labeled Streptavidin-Biotin Complex (LSAB) method was employed for immunohistochemical detection of HER-2/neu. Avidin-Biotin Complex (ABC) method was employed for immunohistochemical detection of VEGF . A total of 30 malignant cases were included.HER2/neu was considered as positive in(18 cases)60% of non otherwise specified invasive ductal carcinoma and VEGF was considered as positive in (21cases)70% with concomitant positivity of both markers in more than half (16 cases)53.3% out of 30 malignant cases . No overexpression of both markers have been noticed in normal or benign (fibroadenoma) breast tissue sections with significant difference from that of malignant cases(P&lt;0.05).We did not find any significant difference between overexpression of both(HER-2/neu and VEGF) in relation to age ,tumor size ,tumor stage and positive or negative lymph node breast cancer cases (P&gt;0.05). However there was positive relation between HER-2/neu overexpression and the grade of tumor (P&lt;0.05) ,while there was no significant difference between VEGF overexpression and the tumor grade(P&gt;0.05).HER-2/neu overexpression was positively correlated with VEGF immunostaining in relation to grade and stage (P&lt;0.05).&#x0D; Based upon the findings of this study, it can be concluded that both HER-2/neu and VEGF play an important role in the pathogenesis of non other wised specified breast cancer and supports the evidence of its role in evolution ,angiogenesis and cell survival of this aggressive tumor . This study recommended that the blocking of both HER-2/neu and VEGF may be a target for blocking the evolution and angiogenesis and hence improving the efficacy of anti cancer therapy against this aggressive type of breast cancer.

COMPARATIVE STUDIES FOR DETECTION OF BRUCELLA MICRO-ORGANISM IN ABORTED BOVINE FOETI USING CONVENTIONAL, IMMUNOHISTOCHEMICAL AND MOLECULAR METHODS

The present study was carried out on a total number of 78 bovine (cows48- Buffaloes 30) The obtained sera were investigated for detection of brucella-antibody titer using serological tests Rose Bengal plate Test (RBPT), Tube agglutination test (TAT), indirect ELISA (iELISA) and Complement Fixation Test (CFT). Abomasal content were collected from 78 aborted fetuses for bacteriological and Polymerase Chain Reaction (PCR) assays. The results of serological analysis revealed that the positive reactors were 60 (76.92 %), 55 (70.51 %), 58 (74.36 %) and 57 (73.08 %) using RBPT, TAT, iELISA and CFT, respectively. RBPT and iELISA tests showed the highest seropositivity. Meanwhile, the lowest ones were obtained by TAT and CFT tests. Brucellaem elitensis biovar3 was isolated from 31 out of 48cows (abomasal content) aborted fetuses and21 out of 30buffaloes (abomasal content) aborted fetuses. PCR assay for detection of Brucella in aborted fetuses were 33 out of 48 in cow and 22 out of 30 in buffaloes. Pathological examination in organs of aborted fetuses infected by Brucella melitensis showed significantly pathological lesions in lungs, liver, spleen and placenta. Immunohistochemistry revealed the presence of positive immunostaining brucella antigen in formalin-fixed, paraffin imbedded tissue sections of lung, liver, spleen, and placenta by using avidin–biotin complex peroxidase technique. Electron microscopical finding of lung, liver and spleen showed that the cytoplasm of neutrophil and macrophages containing dark bodies of cocobacilli. TEM of placenta revealed that the Trophoblasts filled with degenerated organisms.

Technical note: EnVision™ FLEX improves the detectability of depletions of myoglobin and troponin T in forensic cases of myocardial ischemia/infarction.

Immunohistochemistry is a well-established technique used in many research laboratories as well as in clinical diagnostics. The method allows to visualize the expression of proteins in biological tissues, as well as to evaluate this expression semi-quantitatively. For diagnosis, an optimal staining, based on a straightforward protocol, is crucial. In many sudden cardiac death cases, immunohistochemistry is the only tool enabling the diagnosis of myocardial ischemia/infarction, thus the diagnosis of the cause of death. Improvements in immunoreactions are actually possible thanks to optimized detection systems. The recently introduced detection system EnVision Flex™ by Dako allows to dramatically improve (in terms of intensity of the signal and practically absence of background) the visualization of antigens in formalin-fixed paraffin-embedded (FFPE) tissue sections. We tested this method for the detection of myoglobin and troponin T in human postmortem cases of myocardial infarction, as the results obtained by using the « classical » ABC (avidin-biotin complex) method have proven to be sub-optimal, thus rendering any interpretation very difficult, if not impossible.