Indirect Peroxidase Technique 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.

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.

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

P2 receptor agonists stimulate insulin release from human pancreatic islets.

Although P2 receptors for adenosine 5'-triphosphate (ATP) and/or adenosine 5'-diphosphate (ADP) have been characterized in mammalian pancreatic beta cells, no evidence for an insulin-secreting effect of P2 receptor agonists has been reported as yet in humans. The present study aimed at investigating whether P2 receptor agonists could stimulate insulin release in human pancreatic islets obtained from brain-dead organ donors. Experiments were performed using different glucose concentrations and insulin was measured by radioimmunoassay. When the glucose concentration (8.3 mmol/L) was slightly stimulating for insulin release, alpha,beta-methylene ATP (200 micromol/L) and ADPbetaS (50 micromol/L) similarly amplified insulin secretion: both compounds induced a threefold increase in insulin response. In the presence of a nonstimulating glucose concentration (3.0 mmol/L), only alpha,beta-methylene ATP could induce a significant 1.4-fold increase in insulin release, ADPbetaS being completely ineffective. These results give evidence that P2 receptor agonists are effective in stimulating insulin release in humans, the effect of the P2Y agonist being essentially glucose dependent.

Immunochemical localization of the small leucine-rich proteoglycan lumican in human predentine and dentine

The immunocalization of the small interstitial leucine-rich keratan sulphate proteoglycan lumican was studied in human teeth using a polyclonal antibody towards lumican and a standard indirect peroxidase technique. There was intense labelling for lumican in predentine, with immunopositivity also observed in and around the tubules, around the odontoblasts and in the pulp with a fibrillar distribution. The previous reported ability of lumican to control collagen fibrillogenesis and it putative function in collagen spacing suggest potential roles for lumican in predentine in relation to mineralization.

Immunohistochemical localization of phospholipase A 2 in human and bovine male reproductive organs

Phospholipase A 2(PLA 2) taken from human seminal plasma and purified about 1450-fold was injected into rabbits to prepare polyclonal antibodies. The antiserum produced and the IgG purified from the antiserum were used to compare the immunohistochemical localization of PLA 2 in the human male reproductive system with that in the bull. In Western blot analyses, the polyclonal antibodies cross-reacted with purified PLA 2s from human seminal plasma, bovine Cowper's glands and with partly purified PLA 2 from bovine seminal vesicle fluid. On the other hand, purified PLA 2s from Crotalus adamanteus venom, bovine pancreas or from bovine prostate were not recognized by the polyclonal antibodies. With an indirect peroxidase technique, PLA 2 was localized in the epithelial cells of the human prostate and bovine seminal vesicles as well as in the fibrous connective tissue of bovine Cowper's gland. Indirect peroxidase staining also gave an immunoreaction in the enlarged acrosomes of round spermatids in the human testis. Using an indirect immunofluorescence method, ejaculated human spermatozoa revealed an immunoreaction which was not uniform, and the reaction was restricted to the middlepiece and the acrosomal and postacrosomal regions. In conclusion, human seminal plasma, spermatozoa and prostate PLA 2s were immunohistochemically related to those from bovine Cowper's gland, seminal vesicles and seminal fluid.

EXPRESSION OF GLUTATHIONE-S-TRANSFERASE ISOTYPES IN RELATION TO DOXORUBICIN SENSITIVITY AND PROGNOSIS IN PATIENTS WITH RENAL-CARCINOMA

Specimens were obtained from the tumour and adjacent normal kidney in 29 patients with renal carcinoma. Expression of the alpha, pi and mu isoforms of gluthatione-S-transferase (GST) was estimated in formalin fixed paraffin embedded sections of the normal and neoplastic kidney following exposure to the appropriate polyclonal antibodies. binding of which was recognised by the indirect peroxidase technique. Expression of alpha, pi and mu GST in each tumour specimen was compared with the in vitro sensitivity of tumour cells to doxorubicin determined by drug inhibition of [Se-75] selenomethionine uptake by the appropriate tumour cells in culture. Expression of pi GST was associated with tumour cell resistance to doxorubicin. The level of alpha, pi and mu GST was not related to patient survival.

Expression of the alpha 6 beta 4 integrin by squamous cell carcinomas and basal cell carcinomas: possible relation to invasive potential?

We have studied the expression of alpha 6 beta 4 integrin, a carcinoma laminin receptor in ten squamous cell carcinomas (SCCs) and ten basal cell carcinomas (BCCs) of the skin in order to examine whether changes in alpha 6 beta 4 integrin expression may be related to invasive and metastatic potential. Monoclonal antibodies specific for each subunit were applied on cryosections, using a three step indirect peroxidase technique. In normal epidermis the basal cells expressed both the alpha 6 and the beta 4 subunits, and the expression was polarized against the basement membrane. In SCCs the expression of the alpha 6 and the beta 4 subunits paralleled each other, showing an increased intensity and loss of polarity. The BCCs, however, showed consistently decreased expression of both the alpha 6 and the beta 4 subunits. The results of our study, as well as those of other studies, support the assumption that the increase and depolarization of alpha 6 beta 4 integrin that occurs in SCCs might be related to the invasive properties of this tumour type.

Nitric oxide synthase-immunoreactive axons innervating the guinea-pig lingual artery: an ultrastructural immunohistochemical study using elastic brightfield imaging.

The ultrastructure of nitric oxide synthase-immunoreactive (NOS-IR) axons innervating the guinea-pig lingual artery was investigated by means of pre-embedding immunohistochemistry using an indirect peroxidase technique and diaminobenzidine. Sections ranging in thickness from 60 to 500 nm were ultrastructurally evaluated in elastic brightfield imaging mode. Thick sections (optimum at 300 nm) were advantageous for enhancement of the labelling intensity, whilst some subcellular details were better revealed by thin sections. NOS-IR axon terminals often contained aggregations of large, dense-cored vesicles, consistent with a previous light microscopical report on colocalization of NOS and vasoactive intestinal peptide-immunoreactivity in these fibres. NOS-IR axons formed direct neuro-muscular junctions (width less than 50 nm) at the outer surface of the tunica media, thus providing a structural basis for "nitrergic" vasodilation. In addition, NOS-IR axons made direct contacts with non-varicose and varicose segments of non-reactive axons, suggesting interneuronal communication between these elements.

Immunohistochemical study of phospholipase A2 in boyine prostate

Approximately 1,100-fold purified phospholipase A2 (PLA2) from bovine prostate was injected into rabbit to prepare polyclonal antibodies. Antibodies produced showed specific immunoprecipitation only with the purified enzyme, as well as with homogenate of bovine prostatic tissue. By Western blot analysis or by immunodiffusion test, no cross-reactivity with PLA2 purified from human seminal plasma, bovine pancreas, Crotalus adamanteus venom, or with partially purified PLA2 from bovine seminal vesicle fluid or Cowper's glands was observed. Using the indirect peroxidase technique, PLA2 was localized in the cytoplasm of bovine prostatic epithelial cells. By immunogold microscopy, this enzyme was directly visualized inside the lysosomes, as well as in the endoplasmic reticulum of the glandular epithelial cells. Enzyme activity was localized in two principal subcellular sites: the mitochondria and lysosome-enriched fraction, and in the microsomal fraction.

Immunohistochemical localization of phospholipase A 2 in the bovine seminal vesicle and on the surface of the ejaculated spermatozoa

1. 1. Approximately 150-fold purified phospholipase A 2 (PLA 2) from bovine seminal vesicle fluid was injected into rabbit to prepare antibodies. 2. 2. Produced antisera blocked PLA 2 activity in bovine seminal plasma, seminal vesicles and its fluid and it gave single precipitation lines with the same samples. No cross-reactivity was detected with other reproductive tissues of bull as well as human seminal plasma. 3. 3. Using indirect peroxidase technique PLA 2 was localized in the apical part of epithelia cells of the bull seminal vesicle and also some minor immunohistochemical reactions were observed in the tubular lumen. Indirect peroxidase staining gave weak or no reaction at all to seminal vesicles of immature bulls. This suggests that the enzyme may be under hormonal control. 4. 4. By indirect immunofluorescence method ejaculated spermatozoa of bull revealed immunoreaction which was not uniform and it was restricted to the middle piece, acrosome as well as postacrosomal region, but no specific immunostaining could be found on the surface of the epididymal spermatozoa. 5. 5. Enzyme visualization by immunoelectron microscopic labelling showed a predominant localization in membrane particles inside the lumen of bovine seminal vesicle but some gold particles were also seen in granules, larger vacuoles and in cytoplasm of epithelia cells.

Lysozyme in abnormal dermal elastic fibers of cutaneous aging, solar elastosis and pseudoxanthoma elasticum

Staining of elastic fibres with antilysozyme antibodies has been noted previously. In this study, we examined the staining pattern of dermal elastic fibres in aging, solar elastosis, and lesional skin of pseudoxanthoma elasticum (PXE) using an antibody to lysozyme and the indirect-peroxidase technique. To assess the effects of aging, sun-protected skin (buttock) from a younger and an older group of patients was used. Sun damage was studied in skin specimens from varying sun-exposed body regions (trunk; head and neck). No staining was seen in sun-protected skin from younger individuals, whereas sun-protected skin from older persons had scattered positive fibres. Solar elastotic material was intensely positive and the number of positive fibres appeared to correlate with the amount of sun damage. Abnormal elastic fibres in PXE also stained positively, but less intensely, than fibres in solar elastosis. This study shows that changes in the elastic fibres due to degenerative processes or genetic factors results in altered antigenic expression of the fibres. This may be an epiphenomenon secondary to changes in proteoglycans, which are known to occur with solar elastosis and PXE, or may represent an adaptive phenomenon to maintain the elastic properties of the altered fibres or to decrease their antigenicity.

The detection of human cytomegalovirus immediate early antigen in peripheral blood leucocytes

Recently, Van der Bij et al. (1988) reported that active human cytomegalovirus (HCMV) infection could be diagnosed by the detection of HCMV immediate early antigen (IEA) directly in the peripheral blood leucocytes of renal transplant recipients. However, the indirect peroxidase technique used resulted in high background staining due to endogenous peroxidase activity and thus the detection of HCMV-IEA positive leucocytes, which are sometimes present in extremely low numbers, was not always reliable. In an attempt to solve this problem, we have evaluated the alkaline phosphatase-anti-alkaline phosphatase (APAAP) technique, immunogold-silver staining (IGSS), and several fixatives. Fixation with acetone: methanol 1:1 in conjunction with the APAAP technique proved to be the most successful method. In 155 blood samples obtained from 44 patients following renal transplantation and from three AIDS patients, the number of positive cells ranged between 1 and 700 out of 400,000 (median 2). In 23 samples from 11 patients (one AIDS patient) at least one positive cell was found. In this series there were no problems with the evaluation since strong positive signals were obtained without any background staining. We therefore recommend the use of this protocol for the rapid and reliable detection of HCMV-IEA in peripheral blood leucocytes.

Major histocompatibility complex class II antigen (HLA-DR) expression by ileal epithelial cells in patients with seronegative spondylarthropathy.

Major histocompatibility complex molecules act as non-specific receptors for antigenic proteins and present them to T-cells. Presented antigen together with class II molecules activates antigen specific T-helper cells and may trigger a cellular immune response. The expression of HLA-DR antigens by epithelial cells was examined with an indirect peroxidase technique in ileal biopsies from 38 patients with seronegative spondylarthropathy and features of acute or chronic gut inflammation on biopsy, 14 patients with chronic inflammatory bowel disease, 10 rheumatic and 10 non-rheumatic controls. In acute ileitis, there was more HLA-DR expression in villous and crypt epithelial cells than in non-inflamed controls (p less than 0.01). In chronic inflammation and in chronic inflammatory bowel disease, class II antigens were more expressed in villus (p less than 0.02) and crypt epithelium (p less than 0.01). Strong HLA-DR expression in crypt epithelial cells was connected with active inflammation (p less than 0.02). These findings suggest binding of unknown enterobacterial or nutritional luminal antigens to HLA-DR antigens normally present in enterocytes. The enterocytes act as antigen presenting cells causing a local increase of targets for activated T-cells and trigger the gut inflammation responsible for the clinical symptoms of the seronegative spondylarthropathy.

Expression of cytokeratin in the epithelium of dentigerous cysts and odontogenic keratocysts: an aid to diagnosis.

Sections of tissue embedded in paraffin wax from 18 selected odontogenic cysts were studied both histologically and immunohistochemically with antibodies to cytokeratins using the indirect peroxidase technique. The cysts were divided on a clinical and histological basis into two equal groups comprising dentigerous cysts and odontogenic keratocysts. It was possible to differentiate the two cyst types in every case by the pattern of staining using the monoclonal antibody LP34 for cytokeratins of intermediate molecular weight. The monoclonal antibody CAM 5.2 for cytokeratins of low molecular weight was not discriminatory. Such a clear distinction may prove useful diagnostically in distinguishing between two cysts of similar appearance but very different behaviour.

Studies on immune response and protective capacity of rabies in indian dogs.

Summary: Mongrel dogs were inoculated once daily for seven days with 2 ml of a vaccine prepared from 5% suspension of sheep brain inactivated with propiolactone. Their humoral immune response was monitored by enzyme immunoassay at intervals during the year following vaccination. IgMwas formed during the early immune response, which lasted for up to 6 weeks, but subsequently IgG became dominant. A local strain of rabies street virus was used for challenge infection by inoculation ints he masseter muscle. All vaccinated dogs withstood challenge infection one year after vaccination, while all unvaccinated dogs died from rabies. The fluorescent antibody test, rapid rabies enzyme immunodiagnosis technique, indirect peroxidase technique and counter immunoelectrophoresis all proved to be highly specific and reliable. The correlation between immune response and protection is discussed.

Detection of Renibacterium salmoninarum in tissue sections by different methods - A comparative study with special regard to the indirect immunohistochemical peroxidase technique.

Various staining methods (the Gram and periodic acid stains;the indirect fluorescence antibody technique and the indirect peroxidase procedure) were compared for their ability to detect the kidney disease bacterium (Renibacterium salmoninarum) in the tissues of rainbow trout (Salmo gairdneri) fixed by various methods (fresh frozen tissue, frozen formalin-fixed tissue, formalin-or Bouin's-fixed paraffin-embedded tissue). The results demonstrated that only the indirect peroxidase technique gave positive results regardless of the fixation method used to prepare the tissue. The other methods were either less sensitive or yielded negative results after some tissue fixation methods.

CD30(Ki-1) Antigen Expression in a Subset of Gastric Mucosal Plasma Cells and in a Primary Gastric Plasmacytoma

With the use of frozen sections, 24 different monoclonal antibodies (MoAbs), and an indirect peroxidase technique, the antigenic profile of a well-differentiated primary gastric plasmacytoma was obtained. Corresponding to the normal plasma cell, tumor cells were devoid of HLA-D products and B-cell-restricted differentiation antigens CD19, CD20, CD21, CD22, and CD23 while expressing plasma cell-associated CD24 and CD38 molecules and IgA/lambda. The plasmacytoma furthermore reacted with CD30(Ki-1), a reactivity that for this tumor type has not been reported before. Subsequently, normal gastric mucosae affected by all degrees of chronic gastritis were examined. On the basis of serial sections, 5-15% of CD38(OKT10) reactive mucosal plasma cells were observed to also react with CD30(Ki-1). The authors conclude that the CD30 antigen is not only an activation antigen but is also expressed on a subset of terminally differentiated plasma cells.

Abl oncogene expression in non-Hodgkin lymphomas: correlation to histological differentiation and clinical status.

Eight reactive lymphatic tissues, 166 cases of non-Hodgkin lymphomas (NHL) and 11 cases of multiple myeloma were investigated for expression of the c-abl protein using the poly-clonal anti-abl antibody 4411 and an indirect peroxidase technique. In selected cases the results were compared to those obtained with a second polyclonal and 2 monoclonal anti-abl antibodies. In 7 cases, Northern blot analysis of abl-mRNA was performed in parallel. In reactive lymphatic tissues, cells positive for the 4411 antibody were confined to the B-cell areas, i.e., to the mantle zone and parts of the germinal center. In NHL, a positive staining of the cell membrane was predominantly detectable in lymphomas putatively originating in the germinal center or mantle zone (in particular in centrocytic NHL), independent of their proliferative activity. Clinically, 7 out of 8 abl-positive cases of chronic lymphocytic leukemia (CLL) had a more aggressive course of disease, whereas "progressive disease" occurred in only 7 out of 19 c-abl antigen-negative cases. When the clinical status of 78 patients with NHL and 11 patients with multiple myeloma was related to c-abl expression, c-abl-positivity was mostly confined to patients in advanced tumor stages [p less than 0.001 (NHL)].

Histological pattern and changes in extracellular matrix in aortic dissections.

Samples from 34 patients were studied both histologically and immunocytochemically by the indirect biotin-avidin peroxidase technique to analyse the distribution of the extracellular matrix components (type IV collagen, fibronectin, types I and III collagens) in dissection of the aorta. Most showed defects in type IV collagen around medial smooth muscle cells. Defects in smooth muscle cell basement membrane were found throughout the media in cystic medial degeneration and in medionecrosis, whereas in atherosclerosis such unlabelled areas were found only above advanced atherosclerotic plaques. In aortitis other defects in the smooth muscle cell basement membrane were found in areas of inflammatory infiltrates. In all of these conditions similar defects in fibronectin expression were also found. No defects in the expression of interstitial collagens type I and III were seen in the dissecting aortas. Moreover, cystic medial degeneration, medionecrosis, and atherosclerosis were characterised by intense staining of these interstitial matrix components. In the pathogenesis of the aortic dissection local changes in the basement membranes of the medial layer may be important.