Avidin-biotin Complex Immunoperoxidase Research Articles

Tumor Associated Macrophages and Topoisomerase II Alpha As Prognostic Factors in Advanced Stage Hodgkin's Lymphoma

Background : Classic Hodgkin's lymphoma (cHL) is associated with a high cure rate, but approximately 20% of patients are refractory to initial treatment and 10-30% of patients will relapse after achieving complete remission (CR) , the search of new prognostic factors to identify these high risk patients remains challenging in daily practice, the aim of this study is to evaluate CD68 positive Tumor associated macrophages(TAM) and the cell proliferation marker topoisomerase II alpha as prognostic factor in advanced stage (cHL). Patient and Methods: This study included 82 patients with Advanced stage classic Hodgkin lymphoma presented to Medical Oncology Departments of the National Cancer Institute, Cairo University and Zagazig University hospitals in the period from 2012 to 2017. All patients received ABVD regimen as induction. Immunohistochemical (IHC) analysis was performed for all cases using Tissue microarrays (TMAs) sections and stained with anti‐ CD68 (clones KP1 and PGM1, Dako, Glostrup,Denmark). For Topo II testing, IHC staining was performed on deparaffinized sections using the avidin-biotin complex immunoperoxidase technique with separate antibodies recognizing CD30 and the isoform of Topo II. The percentage of cells expressing cytoplasmic CD30 and nuclear Topo II staining simultaneously was determined for each case. We examined four cutoff points of CD68 expression (<= 20% versus >20%), <5% low (score 1), 5-15% intermediate (score 2) & > 15% high (score 3), <5% low (score 1), 5-25% intermediate (score 2) & > 25% high (score 3) and <= 50% versus > 50%. For Topo II expression, two cut off points were analyzed (<= 30% versus >30%) and (<= 75% versus >75%). Results: The study included 41 males and 41 females with a ratio of 1: 1. The median age was 30.5 years (range: 18-60 years). CRwas achieved in 64.6% (53/82 of patients) and 35.4% of all patients (29/82) were refractory. Relapse was reported in17% of the CR cases (9/53). The significant statistical difference in disease-free survival (DFS) for CD68+was observed at a cut off value of 50%, where 60 patients had CD68 <= 50% and 22 patients had CD68 >50%. The mean DFS for patients with CD68 <= 50% was 49.33 months ± 2.18 months (confidence interval: 45.05 to 53.60 months) vs. 35.255 ± 4.94 months (confidence interval: 25.55 to 44.95 months) in patients with CD68 > 50% (p = 0.015). The low CD68 group included more patients in CR (67.9%, n=36 vs. 32.1%, n=17) , male to female ratio was equal(1:1) in both groups (<= 50% ,n=30:30) and (> 50% ,n=11:11), B symptoms also was equal in both groups (<= 50% , 81% ,n=49) and (> 50% 81% ,n=18), patients with CD68<= 50% had more extra nodal disease (22%, n=13 vs. 18%, n=4) and also more bulky disease (20%, n=12 vs. 4.5% n=1) compared with high (CD68>50%) group. The overall survival (OS) was not significant for all CD68 cutoff points. Higher CD68 is associated with decreased OS: 48 vs. 52 month (CI: 42 to 53 month, p=0.42) at cut off 50%. Topo II expression using the two cut off points was not statistically significant in relation to both DFS and OS , the higher topoII group included more patients in CR (80% ,n=42 topo II >30% vs. 20%, n=10 topo II<=30%) also,(67%, n=53 Topo II >= 75% vs. 33%, n=17 Topo II < 75%). Higher Topo II is associated with prolonged DFS (45 months vs. 36 month) confidence intervals: 40 to 50 months vs 27 to 46. Months) at cut off 30% (p=0.8). At cutoff 75%, the DFS was 46 vs. 38 months (Confidence intervals: 41.73 to 52.10 months vs. 30 to 47.7 months, p= 0.36). Higher Topo II is associated with decreased OS: 50 vs.54 months (CI: 46 to 54 month) at cutoff 75% (p=0.19). CD68 was only statistically significant in correlation to patient age (p = 0.05), and non significant to serum albumin ,hemoglobin level , white blood cells count ,international prognostic score (IPS). Topo II was not statistically significant with the previous patient characteristics. Conclusion : High level of CD68 expression in patients with advanced stage HL (>50%) was significantly associated with inferior DFS but not OS, Topo II does not have significant prognostic value in relation to both DFS or OS of advanced HL cases. DisclosuresNo relevant conflicts of interest to declare.

Distribution of Antigens Defined by OKB Monoclonal Antibodies on Benign and Malignant Lymphoid Cells and on Nonlymphoid Tissues

AbstractMonoclonal antibodies OKB1, OKB2, OKB4 and OKB7 have been previously shown to detect distinctive antigens displayed on B, but not on T, lymphocytes. Benign and malignant lymphoid cells were investigated for their reactivity with these antibodies in cell suspension by indirect immunofluorescence and in cryostat tissue sections by the avidin-biotin complex immunoperoxidase technique. Fetal liver pre-B cells and pre-B and common type acute lymphoblastic leukemia cells isolated from 15 patients were OKB1− OKB2 OKB4+ OKB7−. All mature lymphoid tissue B cells and the neoplastic cell surface immunoglobulin-positive (SIg+) B cells isolated from each of 47 B cell neoplasms were OKB2+. OKB1 and OKB7 were expressed by interfollicular, follicular center, and many, but not all, mantle zone B cells. OKB4 was expressed by follicular center cells, but not by mantle zone or interfollicular B cells. The neoplastic SIg+ B cells isolated from 45 of 47 B cell malignancies were OKB1+ OKB4+, and those isolated from 45 of 46 B cell malignancies were OKB7+. The neoplastic B cells of one mantle zone lymphoma were OKB1−, of one small lymphocytic cell lymphoma were OKB7−, of one large cell lymphoma were OKB4−, and of one small lymphocytic cell lymphoma with a monoclonal gammopathy were OKB1− OKB4−. Normal and myeloma plasma cells were OKB−. The malignant T cells isolated from 12 T cell neoplasms were OKB2− OKB4−, but were OKB1+ and/ or OKB7+ in 3 cases. Thus, the OKB antibodies appear to detect distinctive antigens that may be expressed at different stages of B cell differentiation. In addition, OKB4 reacted with selected renal and respiratory epithelium, and OKB2 reacted with a wide range of epithelial tissues. The OKB antibodies should prove useful in the investigation of B cell differentiation and may aid in the identification and characterization of lymphoproliferative malignancies with significant therapeutic and prognostic differences not identifiable by conventional histopathologic and immunologic methods.

Myointimoma of the penis

Myointimoma is an uncommon, benign soft-tissue tumor derived from the intimal cells of blood vessels. Since little is known about this rare tumor entity, our aim is to describe an additional case and to perform the first literature review on this topic. A 49-year-old Caucasian man presented with a 12-month history of a palpable, firm, solitary lesion involving the glans penis. On physical examination, there was a 1 cm palpable, endophytic well-circumscribed nodule located to the left side of glans penis, close to the coronal sulcus, with disease-free external urethral orifice. The patient was submitted to complete excisional biopsy. A skin rhombus measuring 1.1 × 0.8 × 0.3 cm was removed and the biopsy sample, fixed in 10% formaldehyde, sent to Pathology. At the 18-month follow-up visit, the patient was clinically disease free. Histopathology revealed a multinodular intravascular proliferation of the corpus spongiosum. This myointimal proliferation comprised bland predominantly spindle cells in an abundant fibromyxoid stroma. Immunostains for smooth muscle actin (1A4), cytokeratins (AE1/AE3, CAM5.2), and CD34 were carried out using the avidin-biotin complex (ABC) immunoperoxidase method. Lesional cells displayed positivity for smooth muscle actin and negativity for cytokeratins and CD34. Myointimoma is confirmed to be a penile benign lesion that may be adequately treated with excisional biopsy. Even after incomplete or marginal removal, the penile lesion has been shown to remain stable overtime or regress. Differential diagnosis is essential to exclude similar histologic entities that could be more aggressive or have possible systemic implications.

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.

Bcl-2, active caspase-9 and inactive caspase-9 levels as markers of apoptosis in the serum and tissue of vitiligo patients

Background Vitiligo is the most common depigmentation disorder of the skin. It is considered a multifactorial disorder. However, its exact etiology is unknown. As the acute inflammatory response is not a general finding in vitiligo, it was hypothesized that melanocytes may die in a controlled manner via apoptosis.Aim This study was done to evaluate Bcl-2, active caspase-9, and inactive caspase-9 levels in vitiligo in order to establish the role of apoptosis in the etiopathogenesis of vitiligo.Patients and methods We examined Bcl-2 (serum and tissue), active caspase-9 (serum) and inactive caspase-9 (tissue) levels of 48 vitiligo patients and 30 healthy volunteers by ELISA using commercial kits and immunohistochemically using the avidin–biotin immunoperoxidase complex technique. The collected data were analyzed by SPSS version 16.Results The mean age of vitiligo patients was 32.6±12.4 years. Of these, 65% of patients were women and 33% of patients have a positive family history. The mean vitiligo area scoring index was 5.84±6.57. Bcl-2 levels in the serum and tissues of patients were significantly lower than in controls. Bcl-2 expression in lesional skin was significantly lower than in the perilesional and unaffected skin. On the other hand, active caspase-9 level in patients’ serum was significantly higher than in controls. Inactive caspase-9 expression in lesional skin was significantly lower than in controls. Inactive caspase-9 expression in lesional skin was insignificantly lower than in the perilesional and unaffected skin.Conclusion Our data strongly support apoptosis rather than necrosis as a mechanism for melanocytes’ destruction in vitiligo.

Interaction between Esophageal Squamous Cell Carcinoma and Adipose Tissue in Vitro

Esophageal squamous cell carcinoma (ESCC) develops within the squamous epithelial layer and invades the submucosa to the subadventitia that has adipose tissue (AT). AT seems critical to ESCC progression, but the underlying mechanism is unknown. We aimed to address the association between ESCC and AT invitro. ESCC cells were cultured on rat or human subcutaneous AT-embedded or -non-embedded collagen gel. AT promoted the growth of ESCC cells and inhibited their apoptosis. AT promoted the expression of the squamous differentiation marker involucrin in ESCC cells. AT accelerated the expression of invasion-related factors in poorly differentiated ESCC cells only. AT promoted the expression of phosphorylated-insulin-like growth factor-1 receptor in ESCC cells, whereas it inhibited that of the human epidermal growth factor receptor 2. Insulin-like growth factor-1, but not leptin, adiponectin, or resistin, promoted and inhibited the growth and apoptosis of ESCC cells, respectively. In turn, ESCC cells decreased the production of these adipokines in AT and the number of preadipocytes and mesenchymal stem cell-like cells, which developed from AT. These results suggest that i) AT may influence the progression of ESCC with increased growth or invasion and decreased apoptosis through insulin-like growth factor-1/insulin-like growth factor-1 receptor signaling, ii) AT may affect human epidermal growth factor receptor 2-targeted therapy; and iii) the cancer cells may affect adipokine production in AT.

The expression of caspase-3, caspase-7, caspase-9 and cytokeratin AE1/AE3 in goats with enzootic nasal adenocarcinoma: an immunohistochemical study

The aim of this study was to examine the expression of caspase-3, caspase-7, caspase-9 and cytokeratin AE-1/AE-3 using the avidin-biotin complex (ABC) immunoperoxidase technique in 20 goats with enzootic nasal adenocarcinoma (ENA). Clinically, dyspnoea and nasal discharge were observed in all cases. Macroscopically, polypoid and sessile masses were seen in the ethmoidal area. At the histopathological examination, tubular, papillary and mixed patterns of ENA were diagnosed. Immunohistochemically, strong positive reactions were generally seen for caspase-3, while strong to moderate and slight reactions were observed for caspase-7 and caspase-9 in the cytoplasm of the tumour cells. Positive reactions for cytokeratin AE-1/AE-3 were only seen in epithelial cells. In addition, the causative agent of ENA, retrovirus, was detected immunohistochemically in tumour cells. &amp;nbsp;

A clinicopathological study of diagnostically challenging meningioma mimics

The occurrence of neoplastic and nonneoplastic dural based masses that mimic meningiomas is infrequent and may not be considered during radiological and intraoperative analysis. We describe single institute study of 20 such rare cases. This study included total of 20 cases of meningioma mimics. The clinical, radiological and histopathological findings were evaluated. Tissue fixed in 10% formalin was routinely processed and 5 μ thick sections were cut and stained with hematoxylin & eosin. Histochemistry and immunohistochemistry using avidin-biotin complex immunoperoxidase method was done wherever indicated. In the present study group, 15 were male and 5 female with a male: female ratio of 3:1. The age ranged from 14 to 78 years. Radiologically all these lesions were extra-axial in location, predominantly hypointense on T2W, isointense on T1W images and showed intense homogenous enhancement on contrast administration. Four cases were in pediatric age group with histopathological diagnosis of Rosai Dorfman disease, medulloblastoma, hemangiopericytoma and malignant melanoma. In the adult population, the histopathological diagnoses were hemangiopericytoma, undifferentiated sarcoma, extraskeletal osteosarcoma, Rosai Dorfman disease, medulloblastoma, and metastases from systemic malignancies. Of the total 6 cases of metastases 1 was nonseminomatous germ cell tumor from a primary in testis, 1 was adenocarcinoma from an unknown primary, and 4 were adenocarcinoma from lung. There was a single case of dural based frontal lobe malignant melanoma with congenital hairy nevi on anterior chest wall, scalp, anterior abdominal wall and inguinal region. As the management and biologic behaviour of many of the MM are different, it is essential to familiarize ourselves to them.

Tissue Tropism of African Horsesickness Virus in the Chicken Embryo Demonstrated With the Avidin–Biotin Complex Immunoperoxidase Method

In horses, African horsesickness virus (AHSV) exhibits marked tropism for certain microvascular endothelia and components of the mononuclear phagocyte system. In this study, the tropism of a field isolate of AHSV serotype 5 was studied in 24 chicken embryos. Histopathology on embryonic tissues harvested with 12 hour intervals revealed progressive changes associated with endothelial damage. Immunolabeling demonstrated viral antigens in the microvascular endothelium of the spleen, lungs, and the mesenchymal connective tissue at the base of the neck, from 24 hours post inoculation. Subsequently, specific immunolabeling increased steadily in endothelia of these and other tissues such as skeletal and cardiac muscle, gastrointestinal smooth muscle, mesonephric glomeruli, liver, subcutis and feathers. Positive immunolabeling was also occasionally observed in circulating mononuclear cells and in Kupffer cells in the liver. It was concluded, that this isolate of AHSV displayed similar tissue tropism in the chicken embryo as in the horse.

Can Helicobacter pylori Invade Human Gastric Mucosa?

BACKGROUND-GOALS: We used transmission electron microscopy and immunohistochemistry (IHC) to investigate how Helicobacter pylori affects the gastric mucosa of humans. Gastric biopsy specimens were obtained from 15 patients with gastric discomfort. The samples were processed using both microscopic examinations and a real-time polymerase chain reaction to detect H. pylori DNA. IHC staining was performed with an avidin-biotin complex immunoperoxidase kit for paraffin-embedded tissue sections. Polyclonal rabbit anti-H. pylori was used as a primary antibody. IHC-applied slides with brown-stained spiral bacteria on the luminal surface and in the intercellular spaces of the gastric epithelium; electron-dense spiral H. pylori of approximately 200 to 300 nm in diameter both in the gastric lumen and between the gastric epithelial cells; coccoid or ellipsoid H. pylori attached to the epithelial cells through egg-cup-like pedestals; coccoid H. pylori within the endocytotic vesicles in the apical cytoplasmic part of the epithelial cells, thus suggesting their internalization by phagocytosis; electron-dense spiral H. pylori within the membrane-bounded vacuoles of both the gastric epithelial cells, and the lamina propria; a prominent vacuolization of gastric epithelial cells invaded by H. pylori; and swollen and lytic gastric epithelial cells that suggest a mucosal erosion and may lead to peptic ulcer. All of these microscopic findings were not present in the H. pylori DNA-negative specimens that were used as the control group. This is the first histomicrobiologic study to show gastric cells invaded by H. pylori in patients with H. pylori infection confirmed by real-time polymerase chain reaction.

T cell receptor heterodimer (alpha beta/gamma delta) expressions in diffuse panbronchiolitis

Several reports have indicated that T cells bearing gamma delta T cell receptor (TCR) are major effector cells in mucosal immunity of the murine intestinal tract. In humans, abnormal distribution of gamma delta + T cells has also been found in inflammatory bowel diseases. We studied TCR heterodimer expressions in lymphocytes infiltrating the peripheral airways in patients with diffuse panbronchiolitis (DPB), because DPB is histologically characterized by chronic inflammation in bronchioles and could be a model for investigation of the significance of gamma delta + T cells in persistent inflammation of the human respiratory tract. Open lung biopsy specimens were obtained from 7 patients and peripheral blood lymphocytes (PBL) from 9 patients. Immunohistochemically, we detected alpha beta and gamma delta TCR by avidin-biotin complex immunoperoxidase method, utilizing BMA 031 and TCR delta 1 monoclonal antibody, respectively, PBL were analyzed by flow cytometry. Histological analysis revealed that DPB lesions mainly involved the bronchiolar walls with marked infiltration of small round cells. Many T cells were distributed in the submucosal area, although few T cells infiltrated the epithelium. Most of the T cells in the bronchioles expressed alpha beta TCR (98.7%), whereas only 1.3% expressed gamma delta TCR. There was no significant difference in the percentage of gamma delta + T cells in PBL between DPB and age- and sex-matched controls (6.7% vs. 9.1%). These results suggest that the increase of T cells bearing alpha beta TCR played a major role. However, the possibility that the decrease of T cells bearing gamma delta TCR caused reduced mucosal defense against external antigens, leading to persistent inflammation of the lower respiratory tracts in DPB, is not excluded.

Correlation of Confocal Endomicroscopy Imaging with Immunohistochemical Localization Patterns of Fluorescein in Biopsies of Colon Polyps and Cancers

Background: Confocal endomicroscopy (CEM) is a newly developed system that uses a laser confocal system together with a conventional endoscope to provide additional digital information about the tissue surface, subsurface morphology and microstructure. In our previous study, we reported that the localization of injected fluorescein was consistent with the signal in the CEM images and the CEM might be an important device for virtual biopsy. In this study, we evaluated CEM as a tool for conducting virtual biopsies of the colon by comparing structure and architecture in CEM images with biopsy histopathology of neoplastic lesions including hyperplastic polyps, adenomatous polyps and colon cancers. Methods: The large intestines of 51 patients (35 males and 16 females), with an average age of 64 years (range, 37 to 76 years) were examined using an intravenous injection of fluorescein during CEM to facilitate correlation of images taken in vivo with biopsied samples of each lesion. Six hyperplastic polyps, 12 adenomous polyps, and 13 colonic cancers were evaluated. CEM images were compared with conventional histopathology of biopsies. For correlation of biopsy sites corresponding to CEM images, fluorescein injected at endoscopy was localized immunohistochemically with the avidin-biotin complex (ABC) immunoperoxidase technique using mouse monoclonal antibody to fluorescein as the primary antibody. Results: All CEM images were concordant histopathologically with biopsy observations. Fluorescein immunohistochemical staining of both hyperplastic and adenomous polyps was the same as normal colon tissue. That is, staining is localized in the interstitium, capillary walls and at the mucosal surface of crypts. The mucus of goblet cells and the cytoplasm of mucosal surface enterocytes were stained, but not their nuclei. These immunohistochemical patterns were consistent with the fluorescein localization patterns in the CEM images. The stain distribution within colon cancers was similar to that in polyps, but stain localization was very irregular in pattern and intensity. Conclusions: All CEM images were consistent with their histopathological counterparts. In the hyperplastic and adenomous lesions the fluorescein localization corresponded to the CEM images and the distribution was the same as in normal colon tissue. The fluorescein localization in cancer showed irregular pattern and variable intensity, though the basic staining distribution was the same as in the other lesions. These observations further validate the utility of CEM for in vivo assessment of colonic lesions.

Immunohistochemical determination of the expression of endothelin receptors in bronchial smooth muscle and epithelium of healthy horses and horses affected by summer pasture-associated obstructive pulmonary disease

To immunohistochemically determine the expression of endothelin (ET) receptors in bronchial smooth muscle and epithelium of healthy horses and horses affected by summer pasture-associated obstructive pulmonary disease (SPAOPD). Tissue specimens obtained from 8 healthy and 8 SPAOPD-affected horses. Horses were examined and assigned to healthy and SPAOPD groups. Horses were then euthanatized, and tissue specimens containing bronchi of approximately 4 to 8 mm in diameter were immediately collected from all lung lobes, fixed in zinc-formalin solution for 12 hours, and embedded in paraffin. Polyclonal primary antibodies against ET-A or ET-B receptors at a dilution of 1:200 and biotinylated IgG secondary antibodies were applied to tissue sections, followed by the addition of an avidin-biotin immunoperoxidase complex. Photographs of the stained slides were digitally recorded and analyzed by use of image analysis software to determine the intensity of staining. Two-way ANOVA was used for statistical analysis. The left diaphragmatic lung lobe of SPAOPD-affected horses had a significantly greater area of bronchial smooth muscle that immunostained for ET-A, compared with that for healthy horses. All lung lobes of SPAOPD-affected horses, except for the right diaphragmatic lobe, had significantly greater staining for ET-B receptors in bronchial smooth muscle, compared with results for healthy horses. This study revealed overexpression of ET-A and, in particular, ETB receptors in the bronchial smooth muscle of SPAOPD-affected horses, which suggested upregulation of these receptors. These findings improve our understanding of the role of ET-1 in the pathogenesis of SPAOPD.

Prognostic impact of Deoxyribonucleic acid (DNA) image analysis cytometry and immunohistochemical expression of Ki67 in surgically resected non-small cell lung cancers

Background: The aim of the present study was to evaluate the prognostic significance of DNA ploidy and Ki67 expression in non-small cell lung carcinoma (NSCLC). Methods: This prospective study included 96 patients with stages I–IIIA NSCLC who underwent surgical excision. DNA image analysis cytometry was applied on imprints. Calculation of the DNA index (DI) and the 5c exceeding rate (5cER) was performed and the histograms were classified as peridiploid, peritetraploid, and x-ploid–multiploid. The Ki67 immunoreactivity was determined according to the avidin–biotin complex immunoperoxidase method. Results: DNA histogram classification disclosed 30 peridiploid cases, 15 peritetraploid and 51 x-ploid–multiploid. Forty-eight cases (50%) had 5cER > 5%. The Ki67 immunoreactivity was below 25% in 53 tumors (62.4%) and above 25% in 32 (32.6%). Our results revealed the existence of a statistically significant relationship of DNA ploidy with nodal status ( p = 0.042) and grade ( p = 0.005). Adenocarcinomas and large cell carcinomas were more frequently encountered in x-ploid–multiploid tumors as compared to squamous cell carcinomas, which were more frequently peridiploid ( p = 0.003). 5cER showed statistically significant association with nodal status ( p = 0.037). Univariate analysis with respect to survival revealed significant association with stage ( p < 0.001), nodal status ( p < 0.001), tumor status ( p < 0.001), DNA ploidy ( p = 0.008) and 5cER ( p = 0.0124). Multivariate analysis revealed stage and ploidy status as independent factors: peridiploid tumors were associated with better survival as compared to x-ploid–multiploid tumors ( p = 0.022). Conclusion: Our results suggest that DNA ploidy, as determined by image analysis, provides an independent prognostic parameter for patients with NSCLC and thus, could be used to identify a subset of patients with more aggressive tumors.

Effect of Lysophosphatidylcholine on Vasomotor Functions of Porcine Coronary Arteries 1,2

Lysophosphatidylcholine (LPC) is a product of phosphatidylcholine hydrolysis by phospholipase A(2) and a mediator of the lipid-induced atherosclerotic changes. In this study, we determined the effects of LPC on vasomotor functions, oxidative stress, and endothelial nitric oxide synthase (eNOS) expression in porcine coronary arteries. Porcine coronary arteries were cut into 5-mm rings and were treated with LPC or antioxidant selenomethionine (SeMet). For the vasomotor studies, we used a myograph tension system. Levels of superoxide anion (O(2)(-)) were detected by the lucigenin-enhanced chemiluminescence method. The eNOS protein level was studied by immunohistochemistry with avidin-biotin complex immunoperoxidase procedure. Endothelium-dependent relaxation in response to bradykinin was reduced by 36% and 81% for the rings treated with 12.5 and 25 mum of LPC, respectively, as compared with controls (P < 0.05). Endothelium-independent relaxation in response to sodium nitroprusside also was reduced by 63% after treatment with 25 mum LPC (P < 0.05). The O(2)(-) level was increased in the porcine arteries treated with 25 mum of LPC by 41% as compared with controls (P < 0.05). The antioxidant SeMet reversed the effects of LPC on vascular relaxation and O(2)(-) production. Immunoreactivity of eNOS in LPC-treated vessel rings also was reduced substantially. LPC impairs endothelium-dependent and endothelium-independent vasorelaxation. This effect is associated with increased superoxide radical production and decreased eNOS activity and is practically reversed with the use of the antioxidant SeMet.

PKC beta II Expression in Lymphoid Malignancies.

Protein Kinase C (PKC) is an important enzyme family. Twelve different isozymes have been described, which are implicated in diverse cell responses. Recently, studies have begun to define isoform-specific functions of PKC, and PKC beta II seems to play an antiapoptotic role. PKC beta II expression was analyzed by immunohistochemistry in 119 tumor samples of patients with lymphoid malignancies. The median age of the group was 55 years (range 14–85 y). Immunohistochemical staining was performed on formalin-fixed paraffin-embedded tissue, using the labeled avidin-biotin immuno-peroxidase complex method on the Ventana Benchmark automated immunostainer (Ventana Medical Systems, S.A., Illkirch, France). The sections were incubated with primary antibody (polyclonal antibody cPKC ßII (C-1): sc-210, Santa Cruz Biotechnology, Inc.) for 32 min at 1/100 dilution. The intensity of cytoplasmic and membrane immunostaining was assessed semi-quantitatively by two pathologists independently. Cases were considered “negative” when <10% of cells were stained, and “positive” when ≥10% of tumor cells showed cytoplasmic or membrane staining. Diffuse or heterogeneous staining was also specified.Intense and diffuse staining was observed in all mantle cell lymphomas (13 cases, 100%), and chronic lymphocytic lymphomas (CCL) (6 cases, 100%). PKC beta II was positive too in most follicular lymphomas (10 cases out of 11, 91%), although the pattern and the intensity of the staining were variable in accordance with the degree of lymphomatous infiltration. Indeed, in the same lymph node, the follicular lymphomatous areas were constantly labelled, whereas residual germinal centers remained negative. A majority of angioimmunoblastic T-cell lymphomas, lymphoblastic T-cell lymphomas and Marginal Zone/MALT lymphomas were labelled with anti-PKC beta II antibody, but the pattern of expression was more heterogeneous in these subtypes. A minority of diffuse large B-cell lymphomas expressed PKC beta II (8 out of 22 cases, including 4 with weak staining). Nearly all plasma cell neoplasms were negative. Nevertheless, 2 cases out of 16 (13%) displayed a diffuse moderate staining. In peripheral T-cell lymphomas, staining for PKC beta II was less frequent, as well as in primary cutaneous B and T lymphomas and pseudo-lymphomas. None of the cases of Hodgkin's disease (13 cases) and anaplastic large cell lymphoma (5 cases) expressed PKC beta II. However, most of the numerous reactive cells were strongly stained.In conclusion, the intensity and the staining pattern of anti-PKC beta II antibody varied greatly according to the type lymphoid malignancy. These results highlight a significant correlation between PKC beta II expression and some particular type of lymphomas. In our series, the highest level of expression was found in mantle cell lymphomas and CLL.

Myoepithelial markers are expressed in at least 29% of oestrogen receptor negative invasive breast carcinoma

Around 20% of invasive breast carcinoma are oestrogen receptor alpha (ER) negative. Theoretically, this negativity could be either due to the result of downregulation of ER expression in the tumour cells, or the result of the tumour being derived from or differentiating towards cells which normally lack that expression. Normal basal, including myoepithelial, cells of the breast are ERnegative. CD10, smooth muscle actin and S100 are markers of these basal cells that can be used for their demonstration in routinely processed sections. This study was aimed at comparing the incidence of positivity for three myoepithelial markers in ER-negative and ER-positive invasive breast carcinoma. We have examined sections of 117 cases of breast carcinoma, including 77 ER-negative and 40 ER-positive cases, for the expression of CD10, smooth muscle actin and S100, using the avidin–biotin complex immunoperoxidase technique. A tumour was considered positive if more than 10% of the tumour cells were positively stained. In all, 36 (47%) ER-negative tumours were positive for one or more of these myoepithelial markers. The percentage of positively stained tumour cells varied between 30 and 100%. Of the 40 ER-positive tumours, only three (8%) were positive; two for S100 and one for actin, with none being positive for CD10. If cases stained only with S100 are excluded, as some of these may represent luminal differentiation, definite myoepithelial differentiation seems to be present in 29% (22/77) of ER-negative tumours as compared with 2.5% (1/40) of ER-positive tumours; a difference which is highly significant (P<0.001). It is suggested that at least 29% of ER-negative invasive breast carcinomas may be derived from or differentiating along the direction of basal nonconventional luminal epithelial breast cells that normally lack the expression of ER but totally or partially express various myoepithelial markers. Such tumours might need a different therapeutic approach.

Hypermethylation of an E-Cadherin ( CDH 1) Promoter Region in High Grade Transitional Cell Carcinoma of the Bladder Comprising Carcinoma In Situ

We elucidated the role of methylation in the promoter region of the 1 gene in bladder carcinogenesis, particularly in those comprising carcinoma in situ. A total of 49 cases of transitional cell carcinoma of the bladder obtained from transurethral resection were examined. Methylation status of the 1 promoter region was analyzed by methylation specific polymerase chain reaction from chemically modified DNA after Na-bisulfite treatment. Loss of heterozygosity on 16q was examined by blunt end single strand DNA conformation polymorphism using 4 tetranucleotide repeat microsatellite markers assigned on 16q13 to 22.1. E-cadherin expression was evaluated by immunostaining on formalin fixed, paraffin embedded tissue sections using anti E-cadherin murine monoclonal antibody, HECD1 and standard avidin-biotin immunoperoxidase complex technique. Analysis of the 49 bladder transitional cell carcinoma samples showed 1 promoter methylation in 23 (47%). Methylation of the 1 gene did not correlate with tumor stage (p = 0.2097) but with high grade transitional cell carcinoma (p = 0.0416). 1 promoter methylation was observed at a significantly higher frequency in the carcinoma in situ positive group than in the carcinoma in situ negative group (16 of 18 cases or 89% versus 7 of 31 or 23%, p <0.0001) and it strongly correlated with abnormal E-cadherin expression (p <0.0001). We found 16q loss of heterozygosity in 16 of 47 cases (34%), which correlated with higher histological grade (p = 0.0069) but not with the presence of the carcinoma in situ component (p = 0.1235). This study showed that 1 gene promoter methylation is strongly associated with bladder transitional cell carcinoma comprising carcinoma in situ.

Demonstration of Listeria monocytogenes by immunohistochemistry in formalin-fixed brain tissues from natural cases of ovine and bovine encephalitis.

In the present work, evidence of Listeria monocytogenes antigens based on the avidin-biotin complex (ABC) immunoperoxidase technique was performed on formalin-fixed central nervous system tissues (CNS) from a total of 23 natural cases of encephalitis (four ovine and 19 bovine). Listeria monocytogenes serotype 4 was isolated from 10 of 17 cultured specimens. Meningoencephalitis characterized by focal necrosis, microabscesses, perivascular cuffing, and gliosis with presence of macrophages and/or neutrophils was observed at histological examination. Positive L. monocytogenes antigens were successfully identified by immunohistochemistry (IHC) in the CNS of all 23 cases. Paraffin-embedded tissues assayed were stored up for 17 years. Morbidity of the outbreaks was between 0.3-3% and 0.1-1% for ovine and bovine cases, respectively. In all the ovine cases, flocks involved were under extensive grazing conditions. In nine of the 19 bovine cases (47.3%), supplementation with corn silage was used. The ABC test can help as a practical tool for the diagnosis of natural cases of L. monocytogenes encephalitis on formalin-fixed specimens from ovine and bovine.

CD9 immunohistochemical staining of breast carcinoma: unlikely to provide useful prognostic information for routine use.

CD9, a cell membrane glycoprotein, is found in a variety of tumour cells and is believed to regulate cell motility and possibly cell growth. It has been reported that the absence of CD9 is associated with increased aggressiveness of breast carcinoma, but no detailed studies of the distribution of CD9 in normal and abnormal breast tissue are available. This investigation was aimed at studying the distribution of CD9 in a wide variety of breast biopsies including normal, benign, and malignant cases, and assessing its usefulness as a prognostic marker in breast cancer. Sections of 113 breast biopsies from female and male patients including 10 normal, 23 benign, and 80 malignant cases were examined. The monoclonal antibody CD9 (Novacastra Ltd, Newcastle-upon-Tyne, UK) was used with the avidin-biotin complex immunoperoxidase technique. The results were assessed semiquantitatively using a four scale system of 3+, 2+, 1+ and negative. All normal and benign epithelial cells were strongly stained (3+). In female breast carcinomas, 40% were 3+, 49% were 2+, and 11% were 1+. Both cases of male breast carcinomas scored 3+. For female breast cancers, the results were then correlated to tumour grade, the presence or absence of lymph node metastases, and oestrogen and progesterone receptor status. No significant statistical correlation was found with any of these parameters. We then examined 11 axillary lymph nodes with metastases from some of the above cases. Three of these cases had a CD9 score of 3+, seven were 2+, and one was 1+. The metastatic tumours in all 11 cases were strongly stained (3+). Immunostaining for CD9 is unlikely to provide any useful additional prognostic information for clinical purposes.