Peroxidase Technique Research Articles

Presença dos biomarcadores c-MET e ABCB5 no adenocarcinoma de próstata e sua associação com fatores prognósticos

Introduction: Prostate adenocarcinoma is one of the most prevalent malignancies worldwide. Despite often having a slow evolution, it still represents an important cause of death in oncology. Since its clinical results are very heterogeneous, there is a need to identify prognostic markers. Biomarkers are tools and technologies that can aid in this understanding. Objective: To evaluate the presence of c-MET and ABCB5 in prostate adenocarcinoma and to analyze whether their expression correlates with prognostic factors. Method: 170 cases were selected through an active search in electronic medical records and review of physical records and anatomopathological reports. Immunohistochemistry was performed for the biomarkers c-MET and ABCB5 in all samples. Retrospective clinical data were collected and plotted in tables. Through TMA (tissue microarray) the tissues were submitted to immunohistochemistry by the peroxidase technique. Clinical and epidemiological information was cross-referenced with the result obtained by immunostaining and its statistical analysis. Result: Forty-seven men with a mean age of 61.6±6.9 (48-75), with a mean PSA of 11±9.7 (2.4-60.5) were included in this study. 2% had a Gleason score of 5 or 6 and 29, 1%, 7 or 8. Regarding tumor classification, 6 cases had T3a and 2 T3b staging. None had a tumor greater than T3b and there was 1 case of metastasis. With regard to biomarkers, there was positive labeling of c-MET in 32 cases and of ABCB5 in 5. Positive labeling had no statistically significant association with any of the prognostic factors evaluated. Conclusion: There was expression of c-MET and ABCB5 in the prostate cancer, but without association of them with prognostic factors.

Immunohistochemical Expression of Caspase1 and Epidermal Growth Factor Receptor in Invasive Breast Carcinoma and Their Biological and Prognostic Associations.

Despite advances in breast carcinoma therapies, drug resistance mechanisms as anti-apoptosis and anti-pyroptosis limit the application of these therapies. This work assesses the immunohistochemical (IHC) expression of Caspase1 and EGFR in breast carcinoma and analyzes their clinicopathological associations as prognostic markers and potential therapeutic targets. Caspase1/EGFR expression patterns are utilized to specify breast carcinoma patients who may benefit from these therapies. After reviewing the hematoxylin and eosin-stained slides and the routine breast carcinoma IHC stains (estrogen receptors, progesterone receptors, HER2/NEU, Ki-67) by two pathologists and preparation of tissue microarray blocks, anti-Caspase-1 and EGFR IHC staining was performed using Horseradish Peroxidase (HRP) technique. Intensity and percentage-based scoring was applied dividing the 153 included breast carcinomas into Caspase1-negative and positive expression groups; and EGFR low and overexpression groups. Groups were statistically analyzed in relation to age, tumor size, histological and molecular subtype, grade, nodal status, metastasis/recurrence, TNM stage and Ki-67 proliferation index. Kaplan-Meier's analysis was used to compare disease-free survival (DFS) and overall survival (OS). Combined patterns based on Caspase1 and EGFR expression status were created to stratify patients into prognostic groups. Caspase1 was positive in 54.2% of breast carcinomas and its positivity was significantly associated with smaller tumor size, absence of metastasis/recurrence, luminal A and B molecular subtypes and longer OS (p<0.05). EGFR overexpression was detected in 32.7% of carcinomas and was significantly associated with larger tumor size, TNBLBC and a shorter OS (p<0.05). Caspase1-negative/EGFR-overexpression pattern comprised 14.4% of carcinomas and had the worst prognostic associations including larger tumor size, metastasis/recurrence, TNBLBC subtype and shortest OS (p=0.002, 0.002, 0.004 and ≤0.001 respectively). Conclusions: Combined Caspase1/EGFR IHC expression may provide a tool for selection of patients who benefit from combined EGFR-inhibitors with miR-155-5p down-regulators or photodynamic therapy via induction of apoptosis/pyroptosis in EGFR-overexpression carcinomas through enhanced Caspase1 signaling.

Immunohistochemical evaluation of IFN-γ levels in sheep verminous pneumonia

Lungworms, a group of parasitic nematodes, are recognized as one of the major and most common parasitic pneumonia agents in ruminants worldwide. In this study, the expressions of interferon gamma (IFN-γ), which is an important pro-inflammatory cytokine, were evaluated by immunohistochemical methods in order to evaluate the immune response against parasitic agents in sheep naturally infected with different types of lungworms. The material for this study consisted of lung tissue samples obtained from 40 dead sheep brought for routine histopathological diagnosis to the Department of Pathology. In order to reveal the histopathological changes in the tissues, Hematoxylin and Eosin (H&amp;E) staining was applied to the sections. Lung tissues were stained with IFN-γ commercial antibody using the Avidin-Biotin Peroxidase Technique (ABC) following the procedures of the manufacturer. Subpleural multifocal nodules of several mm in diameter were detected in the dorsal regions of the lung, especially in the caudal lobes. In the histopathological examination of the lungs, it was observed that the alveoli, bronchi, and bronchiole lumens were filled with adult forms, larvae, and eggs of the parasitic agents. Compared to the control group, the expressions of IFN-γ were significantly increased in the verminous pneumonia group. Overall, the study suggests that the Th1 response, as represented by increased IFN-γ expression, appears to play an active role in the immunity developed against lungworms in ruminants.

Cell proliferation, apoptosis and inflammation response of Melissa officinalis and Thymus vulgaris in SW480 colon cancer cells

The creation of novel therapeutic approaches and more potent chemotherapeutic agents is the current emphasis of research into cancer therapy. About 60% of drugs used for cancer treatment are isolated from natural products, and the plant kingdom has been the most important resource. The study aimed to evaluate the cytotoxicity of Melissa officinalis L. (MO) and Thymus vulgaris L. (TV) extracts against SW480 cell lines, to elucidate cell death mechanisms and to determine their effect on inflammation. The effect of MO and TV on cell viability was evaluated using an MTT assay in SW480 cell line. After MO and TV treatment, apoptosis was evaluated by Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. An indirect peroxidase technique for immunocytochemical analysis of related proteins such as IL-6, TNF-α, and NF-kB was done. Significant differences in cell viability were observed 24 hours after MO and TV application in the cell line. In terms of TUNEL+ apoptotic cells, there was a statistically significant difference between the control group and the IC50 group in SW480 cell lines treated with both MO and TV. IL-6 and TNF-α, expression levels were found to be significantly lower after MO and TV administration in a concentration-dependent manner, whereas NF-kB expression was upregulated. Our findings show that it is effective on inflammation and signaling pathways that control the cell cycle and apoptosis, but more studies are needed to determine what mechanistic effect the active components of these plants have.

Differential expression of "A Disintegrin and Metalloproteinase 10" in hepatocellular carcinoma and the noncancerous hepatic tissues: Contribution to HCV-promoted hepatocarcinogenesis.

A disintegrin and metalloproteinases (ADAMs) have emerged as therapeutic targets in many cancers. ADAM10 was particularly studied in hepatocellular carcinoma (HCC) for its potential role in hepatocarcinogenesis and HCC progression. To investigate the immunohistochemical (IHC) expression of ADAM10 in HCCs and the adjacent noncancerous tissues from 70 HCC patients, attempting to elucidate any association between ADAM10 and HCC development and/or progression. IHC staining for anti-ADAM10 was performed using horseradish peroxidase technique. An extent and intensity-dependent scoring was applied dividing samples into high- and low-expression groups. HCCs were statistically compared in relation with gender, age, cirrhosis, hepatitis C virus (HCV) status, alpha-fetoprotein (AFP) serum level, tumor size, multiplicity, encapsulation/invasion, grade, histological pattern and variant, mitosis, necrosis, vascular emboli, portal thrombosis, stage, recurrence, and mortality. Kaplan-Meier's method was used to analyze disease-free and overall survival (DFS and OS). ADAM10 was expressed in 77.1% of HCCs compared with 42.9% of noncancerous tissues. Differential expression showed significant statistical difference (P = 0.02), as 38.6% of HCCs showed high expression, whereas 92.8% of noncancerous samples showed low expression. No significant differences were observed when high- and low-ADAM10 expression HCCs were compared with respect to all tested prognostic parameters except the HCV status. Patients whose tumors showed high-ADAM10 expression had relatively longer DFS and OS times, but with insignificant log-rank differences. ADAM10 is frequently expressed in HCCs compared with noncancerous hepatic tissues suggesting its role in hepatocarcinogenesis, especially in association with HCV. It has no association with HCC progression or survival. Further studies should be sought to investigate its validity as a therapeutic target.

Rodlet cells in kidney of goldfish (Carassius auratus, Linnaeus 1758): A light and confocal microscopy study

Rodlet cells (RCs) have always been an enigma for scientists. RCs have been given a variety of activities over the years, including ion transport, osmoregulation, and sensory function. These cells, presumably as members of the granulocyte line, are present only in teleosts and play a role in the innate immune response. RCs are migratory cells found in a variety of organs, including skin, vascular, digestive, uropoietic, reproductive, and respiratory systems, and present distinct physical properties that make them easily recognizable in tissues and organs. The development of RCs can be divided into four stages: granular, transitional, mature, and ruptured, having different morphological characteristics. Our study aims to characterize the different stages of these cells by histomorphological and histochemical techniques. Furthermore, we characterized these cells at all stages with peroxidase and fluorescence immunohistochemical techniques using different antibodies: S100, tubulin, α-SMA, piscidin, and for the first time TLR-2. From our results, the immunoreactivity of these cells to the antibodies performed may confirm that RCs play a role in fish defense mechanisms, helping to expand the state of the art on immunology and immune cells of teleosts.

The study of immunohistochemical expression of vascular endothelial growth factor in renal-cell carcinoma

ObjectiveTo study immunohistochemical expression of vascular endothelial growth factor (VEGF) renal-cell carcinoma (RCC) and correlate its expression with the studied clinicopathologic parameters.BackgroundRCC is the sixth diagnosed cancer in males and the tenth in females accounting for 5 and 3% of cancer incidence, respectively. VEGF is a peptide with angiogenic properties. It is thought to have an important role in RCC development and progression.Patients and methodsThis retrospective study was performed on 58 cases of RCC and 31 cases of nonneoplastic kidney tissue. The slides were subjected to VEGF immunohistochemical staining using a streptavidin–biotin–peroxidase technique. The relationships between VEGF expression in RCC cases and nonneoplastic cases, as well as correlation between VEGF expression and clinicopathological parameters in RCC cases, were statistically analyzed.ResultsVEGF showed low expression in 27.6% and high expression in 72.4% of RCC cases versus 48.4 and 51.6% in nonneoplastic cases, respectively. Higher VEGF expression was correlated with female sex, left-sided tumors, low tumor-infiltrating lymphocytes, and higher-grade tumors.ConclusionHigher VEGF expression is correlated with RCC development and may be an indicator of poor outcome in RCC patients.

The relationship between Her 2 neu and N-cadherin expression in hepatocellular carcinoma of Egyptian patients: Immunohistochemical study.

Background: Despite advances in diagnosis and surgery, p atients with hepatocellular carcinoma (HCC)show bad prognosis and high recurrence rate. Therefore, it is important to search for new targeted therapy and prognostic markers. Aim: To evaluate the expression levels of Her2 neu and N-cadherin in HCC and whether they could be used as prognostic markers. Materials and Methods: This retrospective study included 40 HCC specimens retrieved from the archival cases of Pathology Department, National liver institute, Menoufia University, Egypt, spanning the period between January 2010 to December 2017. Overall survival time was available for 28 patients only. All specimens were stained by Her2 neu and N-cadherin antibodies using the streptavidinbiotin- peroxidase technique. Results: There was significant association between high Her2 neu expression and good prognostic parameters as absent lymphovascular invasion, low grade (P value = 0.028 and 0.049 respectively) and also was associated with low pathological stage but the results didn't reach the level of significance. However there was significant association between low N-cadherin score of expression and good prognostic parameters as low grade (p value = 0.022). Also, there is inverse association between Her 2 neu and N- Cadherin expression in HCC (P value = 0.000). Conclusion: The anti-Her 2 target therapy might not be valuable for HCC patients and also larger studies are recommended to explore potential alternative targeted Her2 enhancer or promoter as well as to understand the exact biological role of Her 2 neu in HCC.

Evaluation of BRCA1 and BRCA2 Protein Dysfunction in Breast Cancer Cells among Women in Osun State, Southwest Nigeria

Abstract Introduction/Objective Breast cancer among Nigeria women had been found to occur at a much younger age compared with their Caucasian age groups. BRCA1 and BRCA2 were suspected to responsible for breast cancers at a young age, therefore this work examined the BRCA1 and BRCA2 dysfunction among women suffering from breast cancer in Osun State, Nigeria. Methods This cross-sectional study was carried out at the Obafemi Awolowo University Teaching Hospitals Complex, Ile-Ife and Ladoke Akintola University Teaching Hospital, Osogbo, Nigeria. The request cards and tissue blocks were sorted from the year 2014 to 2017. The breast tissue blocks were sectioned, stained with H&amp;E. A representative tissue block was selected for each patient. Sections obtained from the blocks were stained with BRCA 1 and BRCA 2 antibody using a diaminobenzidine horseradish peroxidase technique. The cells were semi-quantitatively scored as percentage of tumour cells stained brown. Score 0-5% were taken as negative as proposed by Karuna et al., 2013. The value obtained was tabulated against the age of the patient. Results Out of 240 breast cancer patients sampled 16(6.7%), 32(13.3%), 85(35.4%), 43(17.9%), 64(26.7%) patients were between ages 21-30, 31-40, 41-50, 51-60 and 60+ years respectively. 54(22.5%) showed loss of BRCA 1 staining with only 1(0.4%) patient between age 21-40 years while 18(7.5%), 16(6.67), 19(7.9) patients between age 41-50, 51-60 and 60+ years respectively. 82(34.2%) showed loss of BRCA 2 staining; 7(2.9%), 12(5%), 31(12.9%), 15(6.25%),17(5.8%) patients for age between 21-30, 31-40, 41-50, 51-60 and 60+ years respectively. Among women aged 50 years and below, BRCA 1 and BRCA 2 dysfunctions were responsible for 14.3% and 37.6% of breast cancers respectively. Conclusion Increase in age increases the rate of BRCA 1 dysfunction while BRCA 2 dysfunction is not more associated to with any age. BRCA1 and BRCA2 are responsible for more than thirty percent of breast cancers among women less than 50 years of age in Osun state.

Neutrophil extracellular traps-associated protein peptidyl arginine deaminase 4 immunohistochemical expression in ulcerative colitis and its association with the prognostic predictors

Neutrophil extracellular traps (NETs) are incriminated in several immune and inflammatory diseases including ulcerative colitis (UC). Analysis of colonic tissues for NETs-related markers in UC carries prognostic and therapeutic implications. This work aims to evaluate the immunohistochemical (IHC) expression of NETs-associated-protein arginine deaminase 4 (PAD4) in colonic biopsies from UC patients in comparison to normal colon (NC). Association between PAD4 expression level and histopathologic grade, patient’s therapeutic response and other clinicopathological prognostic predictors in UC are determined. This cohort study included biopsies from 42 UC patients and 11 NC controls. Clinicopathological data including patient's age at diagnosis, gender, presenting symptoms, anatomical disease extent, extra-intestinal manifestations, type and response to therapy and surgical interventions were recorded and tabulated. Histopathological grading of disease activity and associated epithelial changes were assessed. PAD4 immunostaining was conducted using Horseradish Peroxidase technique and scored semiquantitatively considering intensity and percentage of nuclear staining of lamina propria inflammatory cells. Appropriate statistical tests were applied. Anti-PAD4 was localized mainly in the nuclei of lamina propria infiltrating neutrophils. It was expressed more significantly in UC (95.2 %) compared to NC (p 0.001). Increased PAD4 expression level was significantly associated with increasing histopathologic grade, anatomical disease extent, lacking response to therapy and subjection to radical surgery (p:0.001, = 0.038, 0.046, 0.046 respectively). Age, gender, presenting symptoms, extra-intestinal manifestations and epithelial changes showed insignificant associations. This study characterizes a subset of UC patients with high histopathological grade of activity, pancolonic involvement, strong/moderate PAD4 expression levels and who are unresponsive to routine medical therapeutic regimens rendering them candidates for radical surgery. In conjunction with histopathological grading, IHC evaluation of PAD4 in UC is recommended to guide patient’s selection for targeted therapy using the novel-discovered selective PAD4 inhibitors.

Uterine Patterning, Endometrial Gland Development, and Implantation Failure in Mice Exposed Neonatally to Genistein.

Background:Embryo implantation relies on precise hormonal regulation, associated gene expression changes, and appropriate female reproductive tract tissue architecture. Female mice exposed neonatally to the phytoestrogen genistein (GEN) at doses similar to those in infants consuming soy-based infant formulas are infertile due in part to uterine implantation defects.Objectives:Our goal was to determine the mechanisms by which neonatal GEN exposure causes implantation defects.Methods:Female mice were exposed to GEN on postnatal days (PND)1–5 and uterine tissues collected on PND5, PND22–26, and during pregnancy. Analysis of tissue weights, morphology, and gene expression was performed using standard histology, confocal imaging with three-dimensional analysis, real-time reverse transcription polymerase chain reaction (real-time RT-PCR), and microarrays. The response of ovariectomized adults to (E2) and artificial decidualization were measured. Leukemia inhibitory factor (LIF) injections were given intraperitoneally and implantation sites visualized. Gene expression patterns were compared with curated data sets to identify upstream regulators.Results:GEN-exposed mice exhibited reduced uterine weight gain in response to E2 treatment or artificial decidualization compared with controls; however, expression of select hormone responsive genes remained similar between the two groups. Uteri from pregnant GEN-exposed mice were posteriorized and had reduced glandular epithelium. Implantation failure was not rescued by LIF administration. Microarray analysis of GEN-exposed uteri during early pregnancy revealed significant overlap with several conditional uterine knockout mouse models, including Foxa2, Wnt4, and Sox17. These models exhibit reduced endometrial glands, features of posteriorization and implantation failure. Expression of Foxa2, Wnt4, and Sox17, as well as genes important for neonatal uterine differentiation (Wnt7a, Hoxa10, and Msx2), were severely disrupted on PND5 in GEN-exposed mice.Discussion:Our findings suggest that neonatal GEN exposure in mice disrupts expression of genes important for uterine development, causing posteriorization and diminished gland function during pregnancy that contribute to implantation failure. These findings could have implications for women who consumed soy-based formulas as infants. https://doi.org/10.1289/EHP6336

Evaluation of the Proliferation of Breast Cancer Cells Among Women in Osun State, Southwest Nigeria

Abstract Objectives Nigeria has one of the highest morbidity rates from breast cancer; therefore, this work examined the proliferation rate of breast cancer and the hospital presentation time lag among women in Osun State, Nigeria. Methods This cross-sectional study was carried out in Obafemi Awolowo University Teaching Hospital Complex, Ile-Ife and Ladoke Akintola University Teaching Hospital, Oshogbo, Nigeria. The request cards and tissue blocks were sorted from the year 2014 to 2017. The time lag it took the patients to report at the hospitals was retrieved from the request cards. The breast tissue blocks were sectioned, stained with H&amp;E, and graded. A representative tissue block was selected for each patient. Sections obtained from the blocks were stained with Mki67 antibody using a diaminobenzidine horseradish peroxidase technique. The cells were semiquantitatively scored as percentage of tumor cells stained brown. The mean ± SD was calculated. Results Out of 240 breast cancer patients sampled, 6 (2.5%), 97 (40.4), and 137 (57.1%), respectively, were grades 1, 2, and 3. The mean value of MKi67 was 23.90 ± 20.38, median 16.00. The mean value for grade 2 cancer was 15.31 ± 14.34; grade 3 cancer was 37.95 ± 22.47. Only 21.9% of breast cancer patients came to clinic within 3 months of lumps awareness in their breasts while 64.5% attended clinic after 6 months. Conclusions Breast cancer among the Osun State women was not very aggressive when comparing the Mki67 values obtained in this study with the values obtained in the developed world. Greater percentages of women with breast cancer waited too long before presenting themselves at the hospital. Thus, a high morbidity rate was recorded.

Cytokeratin expression is unrelated to hormone receptor expression in breast carcinoma

Cytokeratin expression is unrelated to hormone receptor expression in breast carcinoma - IJPO- Print ISSN No: - 2394-6784 Online ISSN No:- 2394-6792 Article DOI No:- 10.18231/2394-6792.2019.0007, Indian Journal of Pathology and Oncology-Indian J Pathol Oncol

Abstract 3860: Rodent multispecies multiplex immunohistochemistry using digoxigenin and polymer detection methods in mouse tissues

Abstract When mouse, rat, and rabbit antibodies are used, consideration of the target tissue is of critical importance. Endogenous IgGs present in tissues pose significant challenges to interpretation. When a species conflict exists for the antibody and the target tissue, it is likely to generate undesired background staining due to the detection system. In the past, mouse-on-mouse and rat-on-mouse utilized either a biotinylated primary antibody or a biotinylation approach to label the antibody. Tissues rich in endogenous biotin such as kidney or liver were difficult to use in these systems as streptavidin-horseradish peroxidase (HRP) or anti-biotin techniques were used as the detection complement. Attempts to negate endogenous biotin were minimally effective as avidin-biotin blocking steps were unlikely to work acceptably in tissues such as kidney and liver. Using anti-digoxigenin (dig) and polymer technology to generate multi-species multiplex stains may provide a solution. Materials and Methods: Mouse tissues were fixed for 24 hours and embedded in paraffin. Sections were cut at 5 microns and dried on slides before deparaffinizing and peroxidase blocking. Slides were then heated at 110°C for 15 minutes (spleen, liver, kidney) or 80°C for 60 minutes (brain) in a citrate-based buffer (pH 6.2) prior to application of antibodies. A sequential approach of two applications of dig-labeled mouse-on-mouse (MM) detection was performed with PAX8 (M) and CD10 (M) as well as with Neurofilament (M) and GFAP (M). Anti-rat alkaline phosphatase and anti-rat HRP polymer sequential applications were also executed with rat monoclonals CD4 and CD8. Then, a multispecies sequential multiplex stain using MM anti-dig along with anti-rabbit polymer was achieved with mouse hepatocyte specific antigen and rabbit CD3. Finally, a four-step cocktailed multispecies multiplex was developed using a rat monoclonal F480 combined with a rabbit CD3 in conjunction with a cocktail of anti-rat polymer and anti-rabbit polymer. Visualization in each assay was achieved by the use of two chromogenic end-points. Slides were counterstained in either hematoxylin or methyl green prior to dehydration and mounting. Results: Rodent multiplex stains were easily achieved with dig-based MM and cocktailed methods that use mouse/rabbit or rat/rabbit approaches. Specific, clean, and background-free staining on spleen, liver, kidney and brain mouse tissues was achieved with excellent signal-to-noise in both sequential and cocktailed formats. Conclusion: Using mouse-on-mouse anti-digoxigenin technology in multiplex staining is a superior approach to streptavidin or biotin strategies in rodent systems. Additional multiplexing techniques with rat or rabbit antibodies using polymer-based detections generates excellent staining with a significant reduction of background and an exceptional signal-to-noise ratio in mouse tissues. Citation Format: Joseph Vargas, David Tacha, Sara Figueroa, Cristin Douglas. Rodent multispecies multiplex immunohistochemistry using digoxigenin and polymer detection methods in mouse tissues [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3860.

Expression of Lymphangiogenesis Markers and Correlation with Lymph Nodal Metastasis in Breast cancer

AimTo study expression of VEGF and D II40 in breast cancer and correlate with lymph nodal metastasis.Materials &amp; MethodsFormalin fixed paraffin embedded blocks of breast cancer were retrieved and 4 mu thick sections obtained&amp; stained with routine ‘H&amp;E’. Sections were cut on APES (3‐Triethoxysilylpropylamine) coated slides &amp; immunohistochemistry (IHC) was performed using Streptavidin Biotin Peroxidase technique using monoclonal antibodies against VEGF and DII 40 on coated sections of cases &amp; controls. IHC stains were reviewed in conjunction with H&amp;E. Intensity of VEGF expression &amp; its distribution in the tumour were observed under high power magnification &amp; semi‐quantitatively analyzed. Brown granules in the cytoplasm/membrane of the cells were considered as +ve for VEGF expression when the proportion of immunoreactive cells was ≥5%. ; 5–25% staining, weakly +ve (+) 26–50% staining,+ve (++); &gt;50% staining, strongly +ve (+++). For statistical analysis, samples with 0/+ staining considered in low expression group, while ++/+++ expression were considered as high expression. For DII40 well demarcated vessels showing positivity in endothelial cells were counted per 10 hpf after selecting hot spots at scanner view. For statistical analysis, 0 was considered as no expression and 1/10HPF was considered as high expression.Results48 tissue specimens were included in the study. High VEGF expression (Fig 1) was seen 33/43 patients (76.74%%; 95% CI 61.37%–88.24%) and high DII 40 expression (Fig 2) in 30/44 (68.18%; 95% CI 52.42%–81.39%). Variation in expression of VEGF and DII 40 in relation to each other is given in figure 1. Of the 33 patients with high VEGF expression, DII 40 was expressed in 26 of them (78.79%). There is a strong association between VEGF expression and DII 40 expression (p&lt;0.01). The scatter diagram of VEGF and DII 40 expression showed a significant trend (Figure 3 ) indicating that as VEGF expression increases so does the DII 40 expression. The variance explained by VEGF level in the variance of DII 40 as measured by the correlation (R2) was 0.1265 (p=0.02). When VEGF expression was compared with LN status, low expression of VEGF was found in 38.47 % (5/13) of SLN positive and 16.67 % (5/30) of negative senitnel node patients. High expression of VEGF was in 61.53% of positive patients as compared to 83.33% of negative sentinel node patients. Results of VEGF expression and sentinel node status revealed no statistically significant relation between expression of VEGF and LN involvement. No D II 40 expression of was found in 38.46% (5/13) % of LN +ve patients as compared to 29.03% (9/31) in node negative. High D II 40 expression was found 61.54% LN+ve patients and 70.97% of snode negative. Results of DII 40 expression and LN status revealed no statistically significant relation between expression of DII 40 and node involvement. When the expression of VEGF and D II 40 was compared with tumour characteristics there was no statistically significant relation with tumour size, tumour type, grade, ER/PR staus and Her2neu status.ConclusionsLympahngiogenesis marker (VEGF) expression is high in most tumours and there is significant association between lymph vessels detection as studied by D II 40 expression and lymphangiogenesis marker expression as studied VEGF expresssion. However, no association was found between expression of VEGF and D II 40 with neither sentinel / non‐sentinel nodal status nor any of the primary tumour characteristics.Support or Funding InformationnoneThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Hemangiossarcoma primário intrauterino em um macaco aranha de cara vermelha (Ateles paniscus)

Hemangiosarcoma is a common neoplasm in dogs and less frequently seen in cats. In nonhuman primates, this tumor is rarely reported. A 17 year-old female spider monkey ( Ateles paniscus) was submitted an ultrasound exam due to gestation suspicion, which was seen a circular mass intra-uterine measuring 1.3 cm. New exams shown increase of the mass to 4.4 x 3.0 cm associated with a viable fetus. Was realized cesarean with ovariohysterectomy and excision of the mass; however the animal died in less than 24 hours after the surgery. In the necropsy, severe hemoabdomen was evidenced, although the surgical stumps were properly ligated and the complete sutures. Macroscopically, the uterine mass was soft, dark heterogeneous and measuring 5.0 cm in diameter. Histologically was visualized proliferation of spindle cells that form vascular channels replete of erythrocytes a nd some with thrombus, marked pleomorphism, nucleolus evident, binucleated cells and mitotic figures were rare. The immunohistochemistry (IHC) was performed, using streptavidin-biotin peroxidase technique (Dako Cytomation, USA) with the use of antibodies CD31 (clone JC/70A), CD34 (clone QB-END/10). The IHC showed a specific antigen-antibody reaction for CD31. According to localization, morphology and IHC, the present study reports a primary uterine hemangiosarcoma in a spider monkey that caused hemostatic abnormalities and consequent death of the animal.

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.

Can Toluidine Blue and Lectins become New Histochemical Markers for Early Recurrent Pregnancy Miscarriage?

Objective: The purpose of the study was to investigate morphological changes, lectin specificity features and toluidine blue staining specifics of the structural components of human embryos chorionic villi (CV), died out as a result of the first trimester miscarriage.Methods: Histological material included 27 4-13-week human embryos CV tissue samples, obtained after sporadic miscarriage (SM), recurrent miscarriage (RM) and 23 control group CV samples. They were stained with hematoxylin and eosin and toluidine blue. Lectin peroxidase technique was applied, using 4 lectins (SBA, HPA, CNFA (CNL), STA).Results: Destructive changes, edema and a probable decrease of synthetic activity were observed in RM chorionic villi samples. With the help of lectin peroxidase technique, we have identified two most active lectins – CNFA (CNL) and STA.Conclusion: One of the mechanisms of a regular early pregnancy loss can be a reduced expression of LacdiNAc and its component – GlucNAc and, as a result, unmasking an embryo against negative external factors, including such of immune nature. The other probable mechanism can be a reduced synthetic activity of RM chorionic villi, which can result in a decreased invasion activity and growth.

Clinicopathological and cell proliferation evaluation of ameloblastomas and keratocystic odontogenic tumors: a 10year retrospective study.

Odontogenic tumors (OTs) are important lesions of the gnathic bones due to their clinicopathological heterogeneity and variable biological behavior; therefore, epidemiological studies are needed to outline the incidence and behavior of these tumors. To evaluate the incidence and epidemiological profile of ameloblastoma (AMB) and keratocystic odontogenic tumor (KCOT) from an oral pathology service, and correlate morphological findings of these tumors with the immunoexpression of a cellular proliferation marker (Ki-67), a retrospective study (2002-2012) was conducted to characterize demographic, clinical, radiological, and morphological data of AMBs and KCOTs. Then, a representative sample composed of 49 cases of each tumor was selected to perform immunohistochemical (IHC) analysis of Ki-67 through the streptavidin biotin peroxidase technique. For statistical analysis, we used Fisher's exact test (p<0.05). A total of 279 OTs were found in the service, in which 91 (32.6%) were AMB and 98 (35%) were KCOT. Most cases occurred in white women, and the average age of patients with AMB and KCOT was 32 and 33 years, respectively. The maxilla-mandible ratio was 1:6 and 1:3.6 for AMB and KCOT, respectively. Regarding IHC analysis, AMB and KCOT had similar levels of cellular proliferation. However, KCOTs with intense inflammation showed higher Ki-67 expression (p<0.001). Recurrent cases had similar Ki-67 immunoexpression. The demographic profile of the studied tumors corroborates with data reported in the literature, and the levels of cellular proliferation were similar in both tumors, although the inflammation seems to induce a differential proliferative behavior in KCOT.

MIRAgel: the immunohistochemical expression of CD3, CD34, and CD68 in the surrounding capsule.

PurposeTo study the immunohistochemical features of the capsule tissue surrounding MIRAgel episcleral buckles.Patients and methodsThis Institutional interventional clinical cohort study examined a consecutive series of 21 referred patients who required MIRAgel removal from July 2009 to July 2013. All patients with hydrated and fragmented MIRAgel episcleral buckles were included in this study. Capsule biopsies from MIRAgel episcleral buckles were obtained from all patients. Capsule specimens of seven patients with extruded silicone bands were processed as controls. Paraffin-embedded specimens were examined using light microscopy and immunohistochemistry (via the PAP horseradish peroxidase technique) to detect the expression of CD3, CD20, CD34 and CD68, and S-100 protein.ResultsInflammation with granuloma, which was primarily related to sutures, was found in all (n=36) of the MIRAgel specimens and foreign body granulomas with multinucleated giant cells, histiocytes, and macrophages (CD68+ cells) surrounded the MIRAgel fragments. Average number of CD68+ cells was higher (P<0.001) for MIRAgel than for silicone rubber. The lymphocytic inflammatory infiltrate related to the MIRAgel fragments was CD3+ and CD20- (delayed T cell-mediated immune response). Moderate neoangiogenesis was indicated by the presence of CD34+ cells.ConclusionsThe immunohistochemical analysis revealed that the immune system is able to identify the fragments of MIRAgel (after its hydrolytic degradation) as a foreign body during a delayed T cell-mediated immune response. The phagocytosis by macrophages likely triggers and perpetuates local disease. Removal of MIRAgel explants before hydrolysis should be considered.