Indirect Peroxidase Research Articles

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.

Light-Induced Functional Pinealectomy: Expression of MT2 Receptors in Liver Cells of C57BL/6 Mice after Melatonin Treatment.

We performed an immunohistochemical study of MT2 melatonin receptor expression in the liver of C57BL/6 mice with modeled light-induced functional pinealectomy and after melatonin administration by the indirect avidin-biotin peroxidase ABC method. The animals were kept for 14 days under constant lighting. Intragastric administration of melatonin in physiological doses (1 mg/kg body weight for 14 days) to mice with light-induced functional pinealectomy resulted in a 2-fold increase in the relative expression area of MT2 receptors in liver cells in comparison with that in animals kept under standard lighting conditions, 24-h lighting for 14 days, or 24-h lighting receiving placebo (intragastric administration of 200 ml distilled water). Melatonin treatment had practically no effect on MT2 staining intensity. Our results attest to the important role of MT2 receptors in melatonin synthesis disorders and can serve as the basis for the development of therapeutic strategies aimed at melatonin receptors.

MORPHOLOGICAL AND IMMUNOHISTOCHEMICAL FEATURES OF PLACENTAL DAMAGE DUE TO THE INCORPORATION OF 137Cs.

to investigate the morphological and immunohistochemical features of placental damage due to theincorporation of 137Cs depending on the scenario of pregnancy completion. The study material consisted of placentas from 60 women with reproductive losses inanamnesis and signs of termination of the current pregnancy (first group) and placental samples from 30 women with an uncomplicated gestation and an unencumbered anamnesis (control group). The detailed study required the distribution of placental samples from the first group into subgroups. Subgroup 1a included 38 placentas from women who gave birth at 37-40 weeks, despite signs of termination of the current pregnancy. Subgroup 1b - placentas of 13 women who gave birth at a gestation period of 28-36 weeks + 6 days. Subgroup 1c - 9 placental samples from women who gave birth at a gestation period of 22-27 weeks + 6 days. The volumetric activity of the 137Cs in the placentas was measured using β-spectrometer. The histology of the placenta was studied using a standard technique. The following expressions were studied in placenta: CD31 / PECAM-1, CD45 / T200 / LCA, CD56 / NCAM-1, CEA / CD66e Ab-2, Vimentin, using indirect streptavidin peroxidase detection method. Placentas accumulate 137Cs. The different volumetric activity of the isotope correlates with scenarios of pregnancy. Due to the action of incorporated 137Cs with a specific mass of more than 1.1 Bq/kg, placental dysfunction develops. The consequences of placental dysfunction depend on the volumetric activity of the 137Cs and the preservation of adaptive and compensatory reactions in the placenta. Morphological and immunohistochemical features of placental damage to incorporated 137Cs were established, depending on the scenario of completion of pregnancy. A marker of unfavorable completion of pregnancy is the expression of a carcinoembryonic antigen (CEA) in the placenta. Premature termination of pregnancy (PTP) is a multifactorial pathology associated with pathological changes in immune and neuroendocrine regulation and hereditary, infectious, and environmental factors that disrupt the adaptation mechanisms in the mother-placenta-fetus system. Intraplacental irradiation of 137Cs is one of the factors in the multifactorial nature of reproductive losses. As a result of intraplacental irradiation of 137Cs, the architecture of the placenta is disturbed, the activity of pro-inflammatory cytokines CD45 and CD56 increases, and the coagulation cascade is activated. Extreme effects depend on the volumetric activity of the isotope incorporated in the placenta and the organ's compensatory capacity. Accumulation of up to 1.0 Bq/kg 137Cs does not affect the course of gestation. Internal irradiation with an activity of 4.5-10.4 Bq/kg 137Cs triggers late preterm labor. The nature of the damages corresponds to the category of «lesion of the maternal stroma» of the placenta. The volumetric activity of 137Cs over 10.4 Bq/kg is a probable cause of early preterm labor and antenatal fetal death. At the same time, the maternal and fetal structures of the placenta suffer damage. Expression of vimentin is a marker of placental destruction due to internal irradiation of 137Cs with a specific gravity of more than 4.5 Bq/kg. Expression of CEA in the structures of the placenta of women with PTP is a unique find and marker of premature birth and antenatal fetal death with intraplacental irradiation of 137Cs with an activity of more than 4.5 Bq/kg.

The expression of apoptosis-regulating proteins Bcl-2 and Bad in liver cells of C57Bl/6 mice under light-induced functional pinealectomy and after correction with melatonin.

The presence of humans and animals under long-term continuous lighting leads to a suppression of melatonin synthesis, that is, to light-induced functional pinealectomy (LIFP), and the development of desynchronosis. To create LIFP, C57Bl/6 mice were kept under 24-hour lighting (24hL) for 14 days. The animals in the control group were kept under standard lighting conditions. In the next series of experiments, mice with LIFP received daily intragastrically either melatonin (1 mg/kg body weight in 200 μl of distilled water) or 200 μl of water as a placebo. The comparison group consisted of intact animals that received placebo under standard lighting conditions. Immunohistochemical analysis (using an indirect avidin-biotin peroxidase method) revealed the expression of the antiapoptotic protein Bcl-2 and the proapoptotic protein Bad in sinusoid liver cells (a heterogeneous population consisting of the endotheliocytes, Kupffer cells, Ito cells, and Pit cells) and in individual hepatocytes. The Bad expression area in the liver of LIFP mice increased 4 times against a background of the unchanged Bcl-2 expression area. Changes in the brightness (a parameter inversely proportional to the marker concentration) of Bad and Bcl-2 areas did not reach significance. Our results indicate a weakening of the antiapoptotic protection of liver cells of LIFP animals, which creates conditions for activation of the “mitochondrial branch” of apoptosis. Melatonin treatment of LIFP mice resulted in a 3.3-fold increase in Bcl-2 expression area and a 2.7 % decrease in Bcl-2 region brightness compared with the experimental untreated group. Bad protein parameters were unreliable. Thus, melatonin treatment of animals cancels the effect of LIFP, restoring the Bcl-2 expression area and increasing this protein concentration, which indicates an increase in antiapoptotic protection and creates conditions for blocking the development of the “mitochondrial branch” of apoptosis in liver cells.

Revisiting Tumors and the Cardiovascular System: Mechanistic Intersections and Divergences in Ferroptosis.

Ferroptosis was recently identified as an iron-dependent regulatory necrosis process mediated by polyunsaturated fatty acid (PUFA) peroxidation. The pivotal events related to oxidative stress in ferroptosis include direct or indirect glutathione peroxidase 4 (GPX4) inhibition, ferrous iron overload, and lipid peroxidation. The links between ferroptosis and multiple pathological processes including tumor and cardiovascular system disease have become increasingly apparent, and the mechanisms and compounds involved in ferroptosis, such as reduction of coenzyme Q10 (ubiquinone/CoQ10), are gradually emerging. Current reports have revealed crossroads between ferroptosis and other multiple responses. This overview of the current research illuminates the mechanisms involving ferroptosis-related compounds and emphasizes the crosstalk between ferroptosis and other responses, including mitochondrial damage, endoplasmic reticulum stress (ER stress), autophagy, and the release of damage-associated molecular patterns (DAMPs), to reveal the intersections of regulatory mechanisms. This review also outlines the discovery, characterization, and pathological relevance of ferroptosis and notes controversial elements in ferroptosis-related mechanisms, such as nuclear factor E2-related factor 2 (Nrf2), sequestosome 1 (p62/SQSTM1), and heat shock protein family A member 5 (HSPA5). We hope our inferences will supply a partial reference for disorder prevention and treatment.

Quercetin-Mediated Apoptosis and Cellular Senescence in Human Colon Cancer.

Quercetin is a flavonol from the flavonoid group of polyphenols, which positively affects human health due to its anti-cancer, anti-inflammatory, anti-microbial and cardioprotective effects. The effects of phenolic compounds, including quercetin, on programmed cell death and cellular senescence, have been the subject of research in recent years. In this study, we aimed to investigate the effects of quercetin on cell viability, apoptosis and cellular senescence in primary (Colo-320) and metastatic (Colo-741) colon adenocarcinoma cell lines. Cytotoxicity was analyzed via MTT assay in Colo-320 and Colo-741 cell lines. After quercetin treatment, cell ularsenescence and apoptosis were evaluated by TUNEL staining, X-Gal staining and indirect peroxidase technique for immunocytochemical analysis of related proteins such as Bax, Bcl-2, caspase-3, Hsp27, Lamin B1, p16, cyclin B1. The effective dose for inhibition of cell growth in both cell lines was determined to be 25μg/ml quercetin for 48 hours. Increased Baximmunoreactivityfollowingquercetin treatment was significant in both Colo-320 and Colo-741 cell lines, but decreased Bcl-2 immunoreactivitywas significant only in theColo-320 primary cell line. In addition, after quercetin administration, the number of TUNEL positive cells and, immunoreactivities for p16, Lamin B1 and cyclin B1 in both Colo-320 and Colo-741 cells increased. Our results suggest that quercetin may only induce apoptosis in primary colon cancer cells. Furthermore, quercetin also triggered senescence in colon cancer cells, but some cells remained alive, suggesting that colon cancer cells might have escaped from senescence.

Apoptosis in the liver of male <em>db/db</em> mice during the development of obesity and type 2 diabetes

Obesity and diabetes mellitus are known to lead to the development of metabolic syndrome and non-alcoholic fatty liver disease (NAFLD). The mechanisms of programmed cell death are actively involved in maintaining cellular homeostasis along development of NAFLD. Proteins of the BCL-2 family are key regulators of physiological and pathological apoptosis. Homozygous males of BKS.Cg-Dock7mLeprdb/+/+/J mice (db/db mice) are characterized by progressive obesity and the development of type 2 diabetes mellitus (DM2) with severe hyperglycemia at 4–8 weeks and organ lesions at 8–10 weeks of age. The aim of this research was to study the expression of molecular cell regulators of apoptosis in liver cells of db/db mice males at different stages of obesity and diabetes development (at the age of 10 and 18 weeks). Immunohistochemical analysis (using the indirect avidin-biotin peroxidase method) and morphometric evaluation of the expression of the antiapoptotic protein Bcl-2 and the proapoptotic protein Bad in liver cells of studied animals at different stages of obesity and DM2 were carried out. An excess of the value of the Bcl-2 protein staining area over the Bad protein staining area was revealed in the liver of 10-week-old animals. The Bcl-2/Bad expression area ratio in 10-week-old animals was twice as high as in 18-week-old animals, which indicates the presence of conditions for blocking apoptosis in the liver of younger db/ db mice. At the 18th week of life, db/db mice displayed an almost threefold increase in the expression area of the Bad protein against the background of an unchanged expression of the Bcl-2 protein. The decrease in the Bcl-2/Bad staining area ratio in 18-week-old animals was due to the increase in the Bad expression area, which indicates the absence of antiapoptotic cell protection and creates conditions for activation of the mitochondrial pathway of apoptosis in the liver of male db/db mice with pronounced signs of obesity and DM2.

Composition of immunocompetent gingival epithelial cell in patients with chronic obstructive pulmonary disease

The influence of somatic pathology on oral cavity diseases particulary periodontal tissues is of great interest of dentists due to difficulty of diagnosis, frequent recurrences and lack of treatment standards. Chronic obstructive pulmonary disease (COPD) and coronary heart disease (CHD) are higly often comorbid with lesions of periodontal complex. These somatic diseases are not only the most leading causes of disability in patients but they are also characterized by development of systemic hypoxia, which supresses regenerative and reparative processes in periodontal tissues.Objective — to study immunocompetent gum cells in patients with cardiopulmonary pathology.Materials and methods. We examined 239 patients with verified diagnosis of COPD and CHD: the main group — 130 patients with COPD comorbid with CHD, comparison group N 1 — 38 patients with COPD, and comparison group N 2 — 71 patients diagnosed with CHD. We performed clinical dental examination and scraping from a surface of gum epithelium to study the cellular composition (neutrophils, monocytes, lymphocytes). А relative amount of immunocompetent cells with CD4+, CD8+, CD4+/CD8+, CD16+, CD54+, CD95+ markers has been indentified using the indirect immunocytochemical peroxidase method.Results and discussion. COPD, CHD and periodontitis are mutually complicating diseases. Typing of gum leukocytes on the expression of differentiation clusters (CD markers) on their surface made it possible to establish that the percentage of CD4+ cells (T helper-inducers) — 44,00 [39,00; 48,00] and CD8+ cells (cytotoxic T-lymphocytes) — 27,00 [23,00; 31,25], in all study groups, the specific weight of natural killers (CD16+) was increased. Typical feature of patients with COPD and COPD in combination with CHD is high level of leukocyte infiltration of the gingival epithelium and high expression of immune cells.Сonclusions. The changes in periodontal tissues change the ratio of T-­cell populations with predomi­nance of T-­killers. A detailed study of the components of pathogenesis in this category of patients will allow us to develop required correction methods and stabilize the progression of pathological changes in the periodontium.

IMMUNOMORPHOLOGICAL ASPECTS OF CHANGES OF PERIODONTIUM IN PATIENTS WITH CARDIOPULMONARY PATHOLOGY

The protective properties of the gum depend on the general condition of the body, the presence of diseases of internal organs and systems. Numerous data have been accumulated on the relationship of diseases of internal organs with dystrophic-inflammatory lesions of periodontium. It is known that impaired control of cell death and proliferation leads to disruption of cellular homeostasis and the progression of chronic diseases.Objective - to determine the parameters of the ratio of the processes of proliferation and apoptosis in patients with COPD in combination with CHD.Materials and methods. A cytological, morphological and immunohistochemical study of gingival epitheliocytes is conducted in 130 patients with COPD in combination with CHD, 38 patients with COPD, and 71 patients with CHD. Control group included 20 somatically healthy patients. Cytological examination was carried out on scrapings from the surface of the epithelium of the attached gingiva, followed by staining and study under light microscope Micros (Austria). Indirect immunocytochemical peroxidase method was used to study a relative amount of immunocompetent cells with CD95 + markers. To study the proliferation marker Ki-67 we used indirect immunocytochemical method with monoclonal antibodies of a biopsy gum material. Statistical processing was carried out using the software "SPSS 13" with the calculation of the median and interquartile range.Results: Main group showed increase in the percentage of cells at the final stages of differentiation compared with other groups, increased number of cells carrying the receptor for programmed cell death. However, in patients with ischemic heart disease, the proliferative activity of gingival epitheliocytes is increased by 3.7 times compared with the value in a group of practically healthy individuals and is characterized by delayed apoptosis.Conclusion: Cellular renewal of epithelial cells in this category of patients is characterized by a progressive delay of apoptosis of the epitheliocytes of the oral mucosa from the rate of proliferative processes.

MPTH-11ANAPLASTIC MENINGIOMAS WHO GRADE III LACK OF SOMATIC AKT1-MUTATIONS AND SHOW AN OVEREXPRESSION OF EGF-RECEPTORS

BACKGROUND: The AKT1 mutation was newly described in a subset of meningiomas and inhibitors of this mutation have shown promise in clinical trials in multiple cancer types. We sought to determine the frequency of the AKT1 mutation as well as the expression level of multiple growth factor receptors in a large series of patients with Anaplastic meningiomas (AM) WHO °III. METHODS: Patients with AM °III were tested for the AKT1 (E17K) mutation using PCR technique. Additionally, the expression level of the epidermal growth factor receptor (EGFR), the platelet derived growth factor receptor 1alpha (PDGFR) and the vascular endothelial growth factor receptor (VEGFR) was detected by immunohistochemistry (IHC) using the indirect peroxidase technique on tissue multi arrays (TMA) of paraffin-embedded specimens. Staining was evaluated using a semi-quantitative scoring system. Chi-square test was used for statistical evaluation. RESULTS: We identified 22 AM °III patients with 45 tumors (median follow-up of 8 years). None of the examined 45 tumor samples in this cohort showed an AKT1 mutation (0%). Regarding the IHC, recurrent AM showed increasing proliferation with each recurrence. Overexpression of EGFR was associated with malignancy (p < 0.01) and increased with recurrence (median score 2.3). The median overall survival rate for patients with EGFR overexpression was 2.2 years. In contrast, the overall survival of patients with low EGFR expression was not reached yet (p < 0.05). While PDGFR and VEGFR scores were high in malignant tumors, the difference in comparison with benign meningiomas was not significant. A further finding is a marked survival benefit in our patients cohort in respect to the extent of resection when repeated operations were performed (p = 0.025). CONCLUSIONS: Somatic AKT1 mutations are absent in anaplastic meningiomas WHO grade III. Additionally, our data demonstrate that the overexpression of EGFR in AM might make this receptor valuable as a therapeutic target for their treatment.

The Expressions of pAkt and PTEN in Lung Cancer Patients 24 Hours After the Cisplatin-Based Chemotherapy: A Prospective Pilot Study

Akt/PKB is a protooncogen while PTEN is a tumor suppressor gene. Their expressions are of immense importance in the development of lung cancer. However, little is known about their relations to anti-cancer treatments. Therefore, we aimed to elucidate how are these parameters affected by the treatment. Expression of phosphorylated Akt (pAkt) and PTEN have been analysed on tissues of 32 patients (stage III and IV) with lung cancer. In addition, the expression of these variables in 14 out of 32 patients have furtherly been analysed in terms of their response to cisplatin-based chemotherapy in vivo. Prior to and 24 h after the treatment, tumor tissues were obtained via broncoscopy and then evaluated immunohistochemically by indirect streptavidin-biotin peroxidase method. Immunoreactivity for pAkt was detected in 29 of 32 cases (91%). pAkt was observed to localize in the nucleus of positively stained cells. However, PTEN expression was found in 27 of 32 cases (84%). In contrast to the localization of pAkt that is nucleus, PTEN was however localized in the cytoplasm of positively stained cells. pAkt and PTEN expression levels of 14 post-chemotherapy patients were compared to those before chemotherapy. There was no statistically significant differences (p>0.05). Although these results do not imply any possible roles of pAkt or PTEN in the late stage lung cancer patients as a biomarker for the prediction of early response to treatment in vivo, this conclusion needs to be analyzed further at later time points in a larger cohort.

Expression of Master Regulators of Helper T-Cell Differentiation in Peripheral T-Cell Lymphoma, Not Otherwise Specified by Immunohistochemical Analysis.

Abstract 5004 BackgroundPeripheral T-cell lymphoma, not otherwise specified (PTCL-NOS) comprises nodal and extranodal mature T-cell lymphomas, which do not correspond to any of the specifically defined entities of mature T-cell lymphoma in the current classification.During T-cell maturation, naïve T-cells include two distinct cell types, CD4-positive helper T-cells (Th) and CD8-positive cytotoxic T-cells. Following antigen stimulation, naïve Th cells differentiate into several subtypes of effector Th cells, including helper T-cells type1 (Th1), helper T-cells type 2 (Th2), follicular helper T-cells (TFH), regulatory T-cells (Treg), IL-17 producing T-cells (Th17), central memory T-cells (TCM), and effector memory T-cells (TEM).Previously, these subtypes of effector Th cells have been classified according to their pattern of cytokine secretion and expression of surface antigens, including chemokine receptors. More recent immunological researches have identified several transcription factors responsible for T-cell differentiation. The master regulators associated with Th, Th1, Th2, Th17, and Treg differentiation are reported to be Th-POK, T-Bet, GATA3, RORγt, and FOXP3, respectively.The origin of the neoplastic cells of PTCL-NOS is thought to be effector Th cells because the tumor cells usually express specific surface antigens such as pan-T-cell markers (CD2 and CD3), an effector T-cell marker (CD45RO), and a Th marker (CD4). However, the normal cellular derivation of PTCL-NOS remains unclear because the immunophenotypic profiles of PTCL-NOS are heterogeneous and not entirely disease specific.To presume the normal counterparts of PTCL-NOS, we used immunohistochemical analysis to examine the expression of the master regulators of T-cell differentiation and surface antigens, including chemokine receptors associated with the subtypes of Th cells. Patients and MethodsThe study population included ten Japanese patients who were diagnosed histopathologically with PTCL-NOS at the hospital of Kyoto Prefectural University of Medicine from 1993 to 2008. There were six male and four female patients who ranged in age from 26 to 78 years (mean 56 years) at diagnosis.Immunohistochemical staining was applied to 4-μm-thick paraffin-embedded sections of diagnostic lymph node, skin or liver tumor biopsy specimens. Sections were stained using a standard indirect avidin-biotin horseradish peroxidase method and diaminobenzidine (DAB) color development. Cases were considered positive if 25% or more of the tumor cells were stained with an antibody. For negative and positive staining controls, we used paraffin-embedded tissue sections of tonsillitis, nonspecific lymphadenitis, AILT, follicular lymphoma and diffuse large B-cell lymphoma. ResultsFirst, we examined the expression of the master regulator of helper T-cells (Th-POK) and the marker of effector T-cells (CD45RO), and all cases were positive for both.Next, we examined the expression of the master regulators of differentiation to subpopulations within Th cells (Th1, Th2, Treg and Th17). Three cases each were positive for T-Bet, and GATA3, but none of the cases showed FOXP3 or RORγt.Among the four cases not expressing these master regulators, two cases were positive for the surface antigens of central memory (TCM) (CCR7 and CD62L), and one case was positive for follicular helper T-cell (TFH) phenotype (BCL6, CXCL13 and PD-1). The remaining case was negative for all markers of effector Th subtypes. ConclusionsAll ten PTCL-NOS cases exhibited CD45RO and Th-POK, which characterized effector helper T-cells. Furthermore, the lymphoma cells of nine of the ten cases exhibited the expression of master regulators or surface antigens which characterized Th1, Th2, TCM and TFH. DisclosuresNo relevant conflicts of interest to declare.

Lack of Immunohistochemical Detection of VEGF in Prostate Carcinoma

Vascular endothelial growth factor (VEGF) has been implicated in tumor angiogenesis and is a potential therapeutic target in prostatic adenocarcinoma (PrCa). Immunohistochemical (IHC) analysis has been used to demonstrate VEGF expression in PrCa, and in various other tumors including breast carcinoma, renal cell carcinoma, hepatocellular carcinoma, and gliomas. Prior studies have reported markedly varied VEGF expression in benign prostatic hyperplasia (0% to 100%) and PrCa (40% to 100%). The objective of this study was to measure VEGF expression in PrCa specimens, using IHC analysis with antibodies from different manufacturers and different antigen retrieval techniques. Cases were identified from an ongoing study analyzing 1270 cases of PrCa diagnosed at various Veterans Health Administration hospitals in the New England region during 1991 to 1995. From this study population, 50 cases (including 40 biopsies, 8 transurethral resections of prostate, and 2 radical resections) were selected. In all cases, tissues were fixed in 10% formalin and embedded in paraffin. Four different antibodies were used for IHC using indirect peroxidase method. For each antibody, different dilutions and antigen retrieval methods (steam with ethylene diamine tetra-acetate, steam with low pH, water bath with target unmasking fluid, trypsin, proteinase K) were tested. EnVision+ system was used to overcome nonspecific staining. Appropriate positive and negative controls were used. Using different antibodies, positive staining of varying intensity was seen in benign glands, malignant glands, endothelial cells, and fibromuscular stroma. Some cases showed cytoplasmic and granular staining in prostatic glands. However, the staining disappeared in all cases when EnVision+ system was used to block nonspecific staining except for focal and minimal staining in the endothelial cells. Our results show that when nonspecific staining is blocked, no staining is found for VEGF within the prostate, in either benign or malignant glands. The reasons for the granular and nonspecific staining are unclear at present. Our study may help to explain variable results reported in previous studies, and suggests caution in interpreting VEGF expression in studies of PrCa and benign glands.

Immunohistochemical Demonstration of Nitrosative Tissue Damage in Copper Induced Liver Toxicity in Japanese Quails

Summary Excess copper may cause liver toxicosis in various species. In this study, nitrosative tissue damage was investigated in an experimental chronic copper toxicity model in Japanese quails by immuno-histopathological means. Four groups (control, 1 st , 2 nd and 3 rd ), each comprised of 10 one-day-old chicks, were fed for 6 weeks with rations containing none, 100, 250 and 500 ppm copper sulfate, respectively. At the end of the feeding period, animals were euthanized, liver tissues were collected and processed routinely for histopathologic applications. Immunohistochemical staining was performed for inducible nitric oxide synthase (iNOS) and nitrotyrosine antibodies with indirect avidin-biotin peroxidase technique. Apoptotic cell death was investigated with in situ TUNEL staining. Microscopically, there were only mild and moderate degenerative changes in the liver of the chicks of 2 nd and 3 rd groups, respectively. In immunohistochemical staining, little immunoreactivity was detected in the control group for both antibodies. Immunoreactivities were gradually stronger with the increasing copper concentration. A few apoptotic hepatocytes were present only in the 3 rd group. These findings suggest that nitric oxide play a role in the oxidative

Expression and distribution of MMPs and TIMPs in human uveal melanoma

Matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) are involved in tumour invasion, metastasis and angiogenesis, and have been implicated as progression markers in uveal melanoma, although their topographical expression has not been fully described. In this study we compared the distribution and specificity of several classes of MMPs (MMP-1, -2, -9, -19, and MT1-MMP) and physiological MMP inhibitors (TIMP-2 and -3) in different regions of the tumour microenvironment and adjacent choroid in a series of primary uveal melanomas. Paraffin sections of untreated uveal melanomas ( n = 18, 3/18 spindle; 11/18 mixed, and 4/18 epithelioid) were examined for MMP-1 (collagenase 1), MMP-2 and MMP-9 (gelatinases A and B), MT1-MMP (membrane-type 1-MMP), MMP-19, TIMP-2 and TIMP-3 (tissue inhibitors of MMPs), using indirect peroxidase immunohistochemistry. The distribution and intensity of immunolabelling was graded semi-quantitatively (0–3) by 2 independent observers. Non-parametric analyses were used to test for associations between tumour cell type, and the average grade of MMP or TIMP expression. Immunostaining for MMP-1, -9, -19 and MT1-MMP was ≥Grade 2 in more than 70% of specimens, and a heterogeneous pattern of MMP-1, -9, MT1-MMP and TIMP-3 expression was observed. At the tumour-scleral interface (TSI), melanoma cells had a flattened morphology and a much reduced MMP and TIMP expression, with a high expression in tumour areas adjacent to the TSI. Tumour vasculature and stromal cells strongly expressed MMP-2. We also observed heterogeneous immunostaining of the vasculature by MMP-1, -9, MT1-MMP and TIMP-2 antibodies, and of the extravascular matrix by MMP-9 antibody. The distinct immunostaining patterns observed for MMPs and TIMPs within uveal melanoma are consistent with their involvement in tumour growth and angiogenesis. In particular, the heterogeneous expression within regions of the tumours, and the localized expression in vasculature and stromal cells emphasises the importance of the tumour microenvironment in the pathogenesis of uveal melanoma (and other tumours).

Prevalence of border disease virus in Spanish lambs

The prevalence of border disease virus (BDV) viraemia in Spanish lambs was determined from 2089 sera randomly collected at two slaughterhouses in 2001 and 2003, as well as in 126 sera obtained in 2004 from a fattening unit with an acute disease problem. BDV was detected with an indirect peroxidase monolayer assay (IPMA), and for the fattening unit sera also by an antigen ELISA. A subset of sera was additionally tested for BDV antibodies. The BDV prevalence in the slaughterhouse sera was 0.24%, whereas 7.1% of randomly selected and 38.6% of sera from clinically affected lambs in the fattening unit were virus positive. Pestivirus antibodies were found in 17.6% of the slaughterhouse sera and 28.6% of those from randomly selected lambs in the fattening unit. In total, 33 virus isolates and 3 antigen positive samples were identified. Genetic typing of 5′-UTR sequences classified all 36 pestiviruses as of BDV type 4. This shows that from a low BDV prevalence in apparently healthy lambs in the entire sheep population, clinical problems associated with BDV can develop when viraemic sheep are brought into intense rearing units.

The eyes as a window into disease prevention

<b>Objectives:</b> To determine if T cells in skin lesions in systemic sclerosis (SSc) express the early activation antigen CD69 that participates in cell-cell interactions. <b>Methods:</b> Skin biopsy specimens from 17 patients with SSc were analysed by immunohistochemistry using the indirect peroxidase method and monoclonal antibodies for CD3 (T cell marker), CD69 (early T cell activation marker), and CD68 (macrophage marker). <b>Results:</b> Mononuclear cells, containing mostly T cells and macrophages, were increased in SSc skin lesions and were present in perivascular areas. CD69 was expressed in these mononuclear cells. There was no correlation between the number of CD3+, CD69+, or CD68+ cells and the Rodnan skin score or disease duration. <b>Conclusions:</b> The expression of early T cell activation antigen CD69 in skin lesions suggests that T cells may actively participate in cell-cell contact with fibroblasts to promote fibrosis.

Genetic typing and prevalence of Border disease virus (BDV) in small ruminant flocks in Spain

Between 2001 and 2002, samples from 1413 animals in 21 Spanish small ruminant flocks, most of them with animals showing clinical signs compatible with Border disease (BD), were screened for the presence of Pestivirus antigen and antibodies by an indirect peroxidase monolayer assay (IPMA) and the virus neutralization test (VNT), respectively. Although all flocks harboured seropositive animals, virus could only be isolated from animals in five of the flocks. Between 4 and 11 months later all animals older than 6 months in three of the flocks were resampled. At this time, 51–83% of them had neutralizing antibodies. The prevalence of persistently infected (PI) animals within two of the flocks was 0.3 and 0.6%, respectively. The third flock presumably had eliminated all the PI animals. Fourteen virus isolates were obtained. The 5′ untranslated region (5′UTR) was amplified by RT-PCR and directly sequenced. Phylogenetic analyses classified them as a group of Border disease viruses (BDV), separated from BDV-1, but showing a relatively low bootstrap value. Three of the 14 isolates were in the same subgroup as a set of formerly characterised Spanish isolates from the Basque Country, which were allocated to subgroup BDV-C. In addition, they were in the group with an isolate from chamois, which is currently allocated in group BDV-4. Because of its close relation to the chamois isolate, these isolates were tentatively reallocated in a subgroup BDV-4a. The remaining isolates generated a new subgroup, related but not in the same cluster as the chamois isolate, and was therefore tentatively assigned to a new subgroup BDV-4b. Our results show that classification and nomenclature of BDV needs to be harmonised.

Clinical and etiological analysis of 177 patients with hypouricemia

目的 对177例低尿酸血症患者进行临床病因分析,探讨其降低的可能原因,提高其诊治水平.方法采用尿酸酶-过氧化物酶偶联法检测177例患者血尿酸浓度.结果 177例患者血尿酸浓度均低于119 μmol/L,各疾病组血尿酸浓度差异无显著意义(P＞0.05).结论低尿酸血症多见于肝肾疾病、颅脑疾病、外伤和中毒等,临床上应引起重视。

Morphological and Immunohistochemical Study of the Zebrafish (Danio rerio) Oral Cavity

The oral cavity in vertebrates plays a very important role in food intake mechanics and also contains chemical sensors for food and environmental water. Comparative studies revealed morphological and functional inter‐specific differences that can be correlated to evolutionary events and might represent the adaptation to environmental conditions present in different habitats (Iwasaki, 2002). Particularly these events are strictly dependent on the food intake mechanisms determining the success of the adaptation of vertebrates to their environment and therefore their survival (Roth and Wake, 1989). Since data about the morphology of the zebrafish oral cavity are scarce the aim of the present research was to study it by light, scanning and transmission electron microscopy and immunohistochemistry for some growth factor receptors, i.e. trkA and trkB. The zebrafish was chosen because as a vertebrate it has valuable features as a model for study of vertebrate development (Withfield, 2002). Adult zebrafishes were anesthesized with MS222 and the samples were taken and routinely processed for light and electron microscopy and for indirect peroxidase immunohistochemistry. The topographical and ultrastructural study demonstrated the presence of three different zones. The outer zone is characterized orally by the presence of a papilla and by an epithelium with numerous taste buds that are chemosensory endorgans consisting of modified epithelial cells. Their function is to sample potential food and selecting it according to its edibility (Hansen et al., 2002). Both in upper and lower intermediate zones, the presence of internal valves with a semi‐lunar shape was evident, probably able to regulate the mouth closure. The most internal lower zone corresponds to the tongue that in zebrafish is only a thickening of the pavement mucosa with a stratified epithelium. Immunohistochemistry demonstrated both in upper and lower zones the presence of numerous taste buds that were immunoreactive to TrkA and TrkB‐like neurotrophin receptors, confirming the role of these receptors in taste buds sensory cells (Germanà et al., 2004). The present results give a contribution to the morphological knowledge of the zebrafish oral cavity, an animal model commonly used for developmental studies of vertebrates.