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Results for “Staining and Labeling”

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PubMed1952

Fate of mumps virus in the embryonated egg as determined by specific staining with fluorescein-labelled immune serum.

Specific staining with fluorescein-labelled immune serum was used to study the progress of mumps virus infection in a series of embryos harvested at daily intervals. The results were compared with the hemagglutinin and infectivity titers of the corresponding extraembryonic fluids. The evidence obtained indicated that the multiplication of the virus was restricted to those cells which came into surface contact with infected fluid. Following intraamniotic inoculation into 8 day old embryos, these included the cells lining the amniotic membrane and the epidermal and pharyngeal epithelium. Depending apparently on the extent of the contamination of the allantoic cavity and of the extraembryonic celom in the course of inoculation, varying amounts of virus were also present in the cells lining the chorioallantoic membrane and occasionally in the peritoneum. During the later stages of the infection, staining which was principally if not entirely extracellular was seen in the gastrointestinal tract, and, after the dissolution of the tracheal plug, in the respiratory tract. The virus was first detected 1 to 2 days after inoculation as brightly fluorescent intracytoplasmic granules of varying size. As these granules increased in size and number they gradually filled the cytoplasm of the outer one or two layers of cells lining the adjoining cavity. The extent and brightness of the staining reached a peak 4 to 6 days after inoculation and thereafter decreased progressively. The staining of the tissues closely paralleled the rise and fall in the infectivity of the extraembryonic fluids. The development of hemagglutinins, on the other hand, provided a less sensitive measure of virus multiplication than did the staining. No differences were detected in the mortality of the infected and control series of embryos nor was there evidence of any pathological changes. In contrast to the 8 day old embryos, no virus multiplication was detected following the inoculation of 16 day old embryos.

Allantois↗

PubMed2003

Effects of ACE inhibition on myocardial apoptosis in an ischemia-reperfusion rat heart model.

Myocardial ischemia-reperfusion injury involves necrosis and apoptosis. The inhibition of angiotensin-converting enzyme (ACE) has been reported to suppress infarct size. In this study, it was investigated whether an ACE inhibitor affected myocardial apoptosis and apoptosis-related proteins in rats with experimental myocardial infarction. Anesthetized Sprague-Dawley rats were divided into four groups. Group I underwent 30 minutes of left coronary artery occlusion followed by 24 hours of reperfusion (control group); Group II underwent oral administration of the ACE inhibitor quinapril (10 mg/kg/day) before coronary occlusion (quinapril group); Group III underwent administration of the bradykinin B(2)-receptor antagonist Hoe 140 (250 microg/kg/day, subcutaneously) with quinapril (quinapril + Hoe 140 group); and Group IV underwent administration of Hoe 140 alone (Hoe 140 group). After reperfusion, myocardial infarct size was determined by triphenyltetrazolium chloride staining. Myocardial apoptosis was detected immunohistologically using terminal deoxynucleotidyl transferase-mediated nick end labeling staining and DNA electrophoresis. Myocardial caspase-3 activation was analyzed by Western blot and the expressions of Bcl-xL and Bax proteins were detected immunohistochemically. Quinapril significantly reduced the ratio of myocardial infarct size in the ischemic area at risk. In addition, quinapril significantly suppressed the incidence of apoptotic myocytes around the necrotic region (from 18.9 +/- 0.8% to 8.6 +/- 1.0%; P < 0.0001), the intensity of DNA ladder formation, and the activation of caspase-3. Hoe 140 attenuated these protective effects of quinapril. In the immunohistochemical study, Bax and Bcl-xL were expressed in myocytes, and ischemia-reperfusion abolished both proteins in the center region of ischemia. The Bax staining was equally observed among all groups. However, Bcl-xL staining remained in the ischemic area widely after quinapril treatment. In addition, Hoe 140 also depleted this effect of quinapril. These results suggest that inhibition of ACE reduces myocardial infarction and apoptosis via the bradykinin B(2) receptor in part. The antiapoptotic effect of the ACE inhibitor is attributed to the changing expression of Bcl-xL.

Angiotensin-Converting Enzyme Inhibitors↗

PubMed1999

Regulation of a novel pathway for cell death by lysosomal aspartic and cysteine proteinases.

PC12 cells undergo apoptosis when cultured under conditions of serum deprivation. In this situation, the activity of caspase-3-like proteinases was elevated, and the survival rate could be maintained by treatment with acetyl-DEVD-cho, a specific inhibitor of caspase-3. In a culture of PC12 cells treated with acetyl-DEVD-cho, where caspase-3-like proteinases are not activated, CA074, a specific inhibitor of cathepsin B induced active death of the cells. Cathepsin B antisense oligonucleotides showed a similar effect to CA074 on the induction of active cell death. By double staining of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling and activated caspase-3, the dying cells treated with CA074 were positive for terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling staining but negative for activated caspase-3. Ultrastructurally, the cells were relatively large and had nuclei with chromatin condensation. The initiation of cell death by CA074 or the cathepsin B antisense were inhibited by the addition of pepstatin A, a lysosomal aspartic proteinase inhibitor, or by cathepsin D antisense. To examine whether this cell death pathway was present in cell types other than PC12 cells, we analysed dorsal root ganglion neurons obtained from rat embryos on the 15th gestational day, a time when they require nerve growth factor for survival and differentiation in culture. When cultured in the absence of nerve growth factor, the neurons survived in the presence of acetyl-DEVD-cho or acetyl-YVAD-cho. Under these conditions, CA074 reduced the survival rate of the neurons, which was subsequently restored by the further addition of pepstain A. These results suggest that a novel pathway for initiating cell death exists which is regulated by lysosomal cathepsins, and in which cathepsin D acts as a death factor. We speculate that this death-inducing activity is normally suppressed by cathepsin B.

Animals↗

PubMed2000

Expression of BCL-2, BAX and BAK in the trophoblast layer of the term human placenta: a unique model of apoptosis within a syncytium.

The regulation of apoptosis in the syncytiotrophoblast is of particular interest because this is the only true syncytial epithelium in human cell biology. Nuclei characteristic of apoptotic cells have been localized to this syncytium especially in association with fibrin-containing fibrinoid deposits. The factors responsible for regulating cell death-like features in the trophoblast syncytium are unknown. We tested the hypothesis that fibrin was required for trophoblast apoptosis. TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP end-labelling) staining to detect DNA fragmentation typical of apoptosis was performed in term human placentae revealing labelled nuclei associated with fibrin-type fibrinoid, as well as labelled nuclei in discrete areas of syncytiotrophoblast without fibrin. We also hypothesized that members of the BCL-2 family of apoptosis-associated proteins contribute to the regulation of syncytiotrophoblast apoptosis. To identify members of this protein family that might regulate trophoblast apoptosis, we assessed expression of three important members of the bcl-2 gene family. We used immunohistochemistry with monoclonal antisera against human BCL-2 and polyclonal antisera against human BAX and BAK to study paraffin-embedded sections of human term placentae (n=5) from uncomplicated pregnancies. The anti-apoptotic BCL-2 protein was expressed throughout the syncytium of normal villi with much less staining in cytotrophoblast. Staining was also seen adjacent to fibrin deposits and in syncytium overlying fibrin deposits. Expression of the pro-apoptotic BAX protein was undetectable in the syncytiotrophoblast, was expressed in rare cytotrophoblast and was prominent in connective tissue and perivascular cells within the villous core. Localization of a second pro-apoptotic protein, BAK, revealed immunoreactivity in isolated areas of intact syncytium of normal villi. Additionally, fibrin deposits were associated with intense BAK staining in both syncytiotrophoblast and cytotrophoblast. From these data, we speculate that modulation of BAK expression is one factor regulating apoptosis in human trophoblast.

Adult↗

PubMed1991

The extracellular matrix in human myocardium: Part I: Collagens I, III, IV, and VI.

The composition of the extracellular matrix in normal human myocardium obtained at open-heart surgery was investigated using monoclonal antibodies against the collagens I, III, IV, and VI, and fluorescence microscopy. The aim of the study was to provide information on normal myocardium that could be used in the evaluation of pathological changes. Porcine myocardium was used for comparison, and both tissues showed a perfect agreement of the results, apart from collagen IV. This was negative in pig myocardium, due to the species specificity of the antibody. Collagens I and III were localized in the extracellular space as either coarse or fine fibrillar structures; the cellular elements of the interstitium, except for the endothelial cells, were also stained. Labeling for collagen VI was much finer than for the other collagens, and was present throughout the interstitium. Collagen IV stained the basement membranes of myocytes and capillary endothelial cells, and also labeled the T-tubular system in the myocytes. The second part of this communication will describe the localization of fibronectin, laminin and vimentin in normal human myocardium.

Animals↗

PubMed1999

Early effects of castration on the vascular system of the rat ventral prostate gland.

Recent studies have found that blood flow to the rat ventral prostate gland is drastically reduced at an early time after castration. These observations caused us to reevaluate the effects of castration on the various cell populations of the ventral prostate, especially those in the prostatic vascular system. Sections of ventral prostate glands obtained at different times after castration were analyzed using the TUNEL (terminal deoxynucleotide transferase-mediated dUTP nick END labeling) staining method to quantify apoptosis in different cell types. The results of this analysis showed a significant increase in TUNEL staining of prostate endothelial and (nonendothelial) stromal cells as early as 12 h postcastration that continued to 24 h after castration. In contrast, TUNEL labeling of prostate epithelial cells was not significantly increased compared with control values until 72 h after castration. The use of dual immunohistochemical staining procedures (anti-CD31 for endothelial cells or antismooth muscle actin for smooth muscle cells combined with TUNEL labeling) allowed us to confirm that the TUNEL-positive vascular cells at these early times after castration were endothelial in nature, whereas smooth muscle cells surrounding the prostate glands or portions of the afferent vascular endothelium were rarely TUNEL labeled. Electron microscopic evaluation of ventral prostate tissues at 48 h after castration provided further morphological evidence for the occurrence of apoptosis in prostate endothelial cells. Finally, the Lendrum-Fraser histochemical procedure used to identify fibrin leakage in tissues with vascular damage was applied to sections of the ventral prostate gland. This stain revealed diffuse fibrin accumulation in periglandular areas outside the capillaries and blood vessels in prostates from 24-h castrated rats, but not in prostates of sham-operated rats. Our results confirm an early effect of castration on the vascular system of the rat ventral prostate identified by increased apoptosis of endothelial cells and vascular leakiness. As these changes temporally precede the loss of epithelial cells, we propose that they may be causal rather than incidental to regression of the rat ventral prostate after castration.

Actins↗

PubMed2001

Filamentous actin is concentrated in specific subpopulations of neuronal and glial structures in rat central nervous system.

This paper is the second in a series of studies on the light and electron microscopic distribution of filamentous actin (F-actin) in the rat central nervous system (CNS) using phalloidin tagged with the fluorophore eosin followed by fluorescence photooxidation. A previous report described the selective localization of high concentrations of F-actin in subpopulations of dendritic spines in hippocampus, cerebellum and neostriatum. Dendritic spines were the most intensely stained structures in the CNS, but several other structures were notable for their consistent staining for F-actin. Although the majority of cell bodies, axons and large dendrites were unlabeled, mossy fibers and Schaffer collaterals in the hippocampal formation, basket cell axons in the cerebellar pinceau, and granule cell dendrites in the glomeruli of the cerebellar cortex routinely showed strong F-actin labeling. Staining was observed in all three glial cell types. Labeling was consistently observed in the astrocytic processes surrounding the Purkinje cell soma and primary dendrite. Intense but sporadic staining was observed in the perinodal glia of the Node of Ranvier. A few examples of labeled oligodendrocyte processes were also seen in the neostriatum. Labeling was observed in microglia in every brain region examined, although the labeling was present in the lumen of the endoplasmic reticulum and the nuclear membrane, leading to questions about its specificity. Perycites apposed to the blood vessels also showed very consistent labeling. Our results suggest that selected structures in the adult CNS in addition to dendritic spines are enriched in F-actin.

Actins↗

PubMed2001

In situ characterization of genetically targeted (green fluorescent) single cells and their microenvironment in an adoptive host.

Stable expression of transgene-encoded enhanced green fluorescence protein (eGFP) was used as a sensitive and specific marker to detect in situ donor cells engrafted into different tissues of adoptive hosts. eGFP(+) lymphoid or myeloid cells (eg, CD4(+) T cells or bone marrow-derived dendritic cells) from eGFP-transgenic C57BL/6 donor mice were injected into congenic, immunodeficient RAG1(-/-) C57/BL6 hosts. eGFP(+) cells were detected in the adoptive host from 2 days to 4 weeks after transfer using an optimized method of fixed cryopreservation to process the tissue. This allowed the simple, sensitive, and specific detection of eGFP(+) donor cells in histological sections of transplanted hosts. We further demonstrate that this technique can be combined with other established labeling methods such as 1) immunofluorescent labeling to characterize the host cells interacting with engrafted cells and to determine the phenotype of the engrafted cells in situ; 2) terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining to detect apoptotic death of engrafted and autochthonous cell populations; and 3) fluorescent antibody labeling of incorporated bromodeoxyuridine to measure the fraction of proliferating cells in the graft.

Adoptive Transfer↗

PubMed2002

The mouse interferon-inducible gene Ifi204 product interacts with the Tpr protein, a component of the nuclear pore complex.

We have used yeast two-hybrid screening to isolate cDNA-encoding proteins interacting with the protein encoded by the interferon (IFN)-inducible gene Ifi204. Four independent overlapping clones were isolated from an NIH3T3 cDNA library. The largest clone encoded a protein (1203 amino acids in length) sharing 94% identity with the C-terminal portion of the human translocated promoter region (Tpr) protein. Northern blot analysis revealed a 7.5-kilobase mRNA present in both mouse and human cell lines. In addition, in vivo interaction was demonstrated by coimmunoprecipitation experiments. Anti-Tpr polyclonal monospecific antibodies (Ab) used for immunofluorescence staining labeled the nuclear envelope (NE) in a punctate pattern characteristic of nucleoporins and also yielded staining throughout the nuclear interior. The intranuclear Tpr occurred in apparently discrete foci. When superimposed on optical sections obtained with anti-p204 Abs, these colocalized, with the sole exception of the nucleolar compartment stained by the anti-p204 Abs only. Although the specific function of Tpr is not defined, it appears to mediate p204 translocation from the cytoplasmic to the nuclear compartment following IFN treatment.

3T3 Cells↗

PubMed2003

Suppression of protein kinase Calpha triggers apoptosis through down-regulation of Bcl-xL in a rat hepatic epithelial cell line.

Inactivation of protein kinase C (PKC)alpha plays an important role in modulating hepatic failure and/or apoptosis during sepsis. To determine whether and how PKCalpha inactivation mediates the apoptosis, PKCalpha was suppressed by antisense treatment or transiently transfection in Clone-9 rat hepatic epithelial cell line. Apoptosis was evaluated by cell survival rate, poly-adenyl ribonuclease polymerase (PARP) cleavage, and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-digoxigenin nick end labeling stain. The expressions of PKCalpha and Bcl-xL were quantified by Western blot analysis after antisense treatment. In the transfection studies, cells were co-transfected with green fluorescent protein cDNA as a transfection marker. The expressions of PKCalpha and Bcl-xL were detected by immunohistochemical staining with second antibody conjugated with Texas red. Apoptosis was evaluated by tetramethyl-rhodamine labeling of DNA strand breaks and immunostaining of 85-kDa fragment of PARP. The results showed that cytosolic and membrane-associated PKCalpha were decreased by 54.5% and 41.4%, respectively, after PKCalpha antisense treatment. The apoptotic incidence and percentage of PARP cleavage were significantly increased, whereas protein expression of Bcl-xL was decreased after PKCalpha-antisense treatment. In the transfection studies, the results showed that most of the cells expressing green fluorescent protein revealed less PKCalpha and Bcl-xL protein contents and more in situ PARP cleavage and DNA strand breaks. These findings indicated that decrease of PKCalpha declines the Bcl-xL content and leads to the vulnerability of apoptosis in hepatic epithelial cells. Taken together, our data provide evidence that suppression of PKCalpha plays a critical role in triggering caspase-dependent apoptosis, which may act through modulating the Bcl-xL expression.

Animals↗

PubMed1996

[Immunohistochemical double labelling studies on liver tissues superinfected with hepatitis C virus and hepatitis B virus].

OBJECTIVE: To explore the relatiship between hepatitis C virus (HCV) and hepatitis B virus (HBV) replication in liver tissue of patients superinfected with HCV and HBV. METHODS: The expresion and distribution of HCVAg and HBVAg in paraffin-embeded liver tissue from 25 autopsy cases were studied with immunohistochemical double labelling techniques using monoclonal anti-HCV NS3 and anti-HCV NS5 as well as polyclonal anti-HBs and anti-HBc. RESULTS: HBsAg and HCV NS3 or NS5 antigen were detected at the same section in 11 and 12 cases, and HBcAg and HCV NS3 or NS5 antigen in each 10 cases, respectively. Nearly all of the specimens with single labelling stained positive tissue for HC-VAg or HBVAg were also those positive with double labelling studies for HCVAg and HBVAg. The distribution of HCV and HBV infected hepatocytes was characterized as diffuse and single or cluster scattered in liver lobular. There were no differences in expression related to replication such as membranous, cytoplasmic type of HBsAg or cytoplasmic type of HBcAg and the cases positive for HCV NS3Ag or HCV NS5Ag between the group of HCV superinfected with HBV and the group infected with single HCV or HBV (chi 2 = 0.154 and 0.198, P > 0.05). CONCLUSION: The results suggested that there was no interference or suppression each other in liver tissues superinfected with HCV and HBV.

Hepacivirus↗

PubMedDate not supplied

Flow cytometric measurement of mitochondrial mass and function: a novel method for assessing chemoresistance.

BACKGROUND: Chemotherapeutic agents induce apoptosis in cancer cells. Drugs failing to induce apoptosis are likely to have decreased clinical efficacy. We hypothesize that (1) chemotherapeutic agents induce mitochondrial changes and apoptosis through mechanisms associated with reactive oxidant species production; (2) the anti-apoptotic protein Bcl-2 prevents drug-induced mitochondrial changes, reactive oxygen species (ROS) production, and apoptosis; and (3) the assay of drug-induced mitochondrial changes can reflect drug-specific chemoresistance in a given cancer cell line. METHODS: A stable Bcl-2 transfectant of the Bcl-2 negative breast cancer cell line SKBr3 was created (SKBr3/Bcl2-2). Both SKBr3 and SKBr3/Bcl2-2 cells were treated with Herbimycin A (300 ng/mL) or vehicle (1% DMSO). Cell cycle changes were assessed by BRDU staining. Apoptosis was determined by electron microscopy, TUNEL (TdT-mediated dUTP-biotin nick end labeling) staining, and diphenylamine assay of DNA fragmentation. Changes in mitochondrial mass and transmembrane potential (deltapsi(m)) were assessed by flow cytometric assessment of JC-1 fluorescence. Reactive oxygen species production was measured by 2',7'-dichlorodihydrofluorescein diacetate (DCFH) fluorescence. RESULTS: Both SKBr3 and SKBr3/Bcl2-2 cells show cell cycle arrest after Herbimycin treatment. However, SKBr3 cells, but not SKBr3/Bcl2-2 cells, undergo apoptosis. Herbimycin-treated SKBr3 cells show increased mitochondrial mass (JC-1 green fluorescence), with no corresponding increase in deltapsi(m) (JC-1 red fluorescence). By contrast, Herbimycin-treated SKBr3/Bcl2-2 cells show no change in mitochondrial mass or deltapsi(m). Similarly, drug-treated SKBr3 cells, but not SKBr3/Bcl2-2 cells, demonstrate increased reactive oxygen species (ROS) production concomitant with the development of apoptosis. CONCLUSION: SKBr3 cells undergoing apoptosis demonstrate mitochondrial changes associated with ROS production. Bcl-2 transfection prevents these changes because it prevents apoptosis and induces chemoresistance to Herbimycin in SKBr3. Flow cytometric measurement of drug induced mitochondrial changes and ROS production may facilitate in vitro assessment of chemosensitivity or chemoresistance in breast cancer.

Antibiotics, Antineoplastic↗

PubMedDate not supplied

Preclinical investigation of internal limiting membrane staining and peeling using intravitreal brilliant blue G.

PURPOSE: To investigate the effects of intravitreal brilliant blue G (BBG) on the morphology and functions of the retina and its possible use for staining and peeling of the internal limiting membrane (ILM). METHODS: Rat eyes (n = 78) underwent gas compression vitrectomy. BBG solution was then injected into the vitreous cavity. The eyes were enucleated at 2 weeks and 2 months. Light as well as electron microscopy, terminal nick-end labeling staining, and electroretinography (ERG) were used to investigate retinal damage and function. To test the clinical potential of BBG, ILM staining was evaluated in primate eyes after pars plana vitrectomy followed by ILM peeling. RESULTS: In the rat eyes, no pathologic changes were observed with light microscopy. Electron microscopy revealed that high doses of BBG induced vacuolization in the inner retinal cells, but apoptosis was not detected. There was no reduction in the amplitude of the ERG waves. In the primate eyes, the ILM was clearly visualized after the intravitreous injection of BBG and was peeled off easily from the retina. CONCLUSIONS: These results demonstrate that BBG, which has low potential for toxicity, high staining ability, and ease of handling, is a good candidate dye for ILM peeling.

Animals↗

PubMed2002

In vivo antitumor activity of Sindbis viral vectors.

BACKGROUND: Sindbis virus, a blood-borne virus transmitted by mosquitoes, has been used as a vector to efficiently express exogenous genes in vitro and in vivo and to induce apoptosis. Because Sindbis virus infects mammalian cells by interacting with the high-affinity laminin receptors, which are expressed at higher levels in several human cancers than in normal cells, we determined whether a Sindbis viral vector could be used to target cancers in vivo. METHODS: C.B-17-SCID mice with established xenografts were given daily intraperitoneal injections of the Sindbis viral vector SinRep/LacZ containing the bacterial beta-galactosidase gene. Control mice were untreated or received injections with phosphate-buffered saline. Tumor size was measured daily. Expression of beta-galactosidase and Factor VIII (a marker for endothelial cells) was determined by immunohistochemical staining of tumor sections. Apoptosis was analyzed by TUNEL (terminal deoxynucleotidyl transferase [TdT]-mediated dUTP nick end labeling) staining. C.B-17-SCID beige mice, which lack natural killer (NK) cells, were used to assess the importance of NK cells in antitumor efficacy of Sindbis viral vectors. RESULTS: Tumors from mice treated with SinRep/LacZ were statistically significantly smaller than tumors from control mice. This effect was observed for tumor xenografts derived from BHK (kidney, hamster), LS174T (colon, human), HT29 (colon, human), and CFPAC (pancreas, human) cells. Expression of beta-galactosidase co-localized with that of Factor VIII in tumor sections. Tumors from SinRep/LacZ-treated mice contained more apoptotic cells than tumors from control mice. Complete tumor regression was observed in three of five C.B-17-SCID mice but in none of five C.B-17-SCID beige mice treated with SinRep/LacZ. CONCLUSION: Sindbis viral vectors efficiently targeted tumors in vivo, were apparently delivered through the circulation, and were more effective in the presence of NK cells.

Animals↗

PubMed1990

[Detection of coagulation factor XIII in the vitreous body and periretinal membranes in proliferative retinal diseases].

The human blood coagulation factor catalyses the cross-linking of fibrin monomers at the end of the coagulation cascade. Additional functions are the coupling of fibronectin and collagen to each other and fibrin. Therefore we tried to investigate the significance of factor XIII in the development of intraocular membranes. Using gel electrophoresis and western blotting, both subunits (A and B) of factor XIII could be detected in vitreous aspirates from patients with "idiopathic" proliferative vitreoretinopathy (PVR) (n = 5), traumatic PVR (n = 5), and proliferative diabetic retinopathy (n = 5). In contrast the vitreous of five human "normal" post mortem eyes did not contain the subunits of factor XIII. Furthermore, we observed immunofluorescence staining for both subunits of factor XIII in 20 surgically obtained periretinal membranes. In early cellular as opposed to late hypocellular membranes we observed stronger labeling for both subunits of factor XIII. With double label staining techniques, the fibroblastic cells recognized by vimentin staining did not contain factor XIII. About 50% of the macrophages stained positive for the A-subunit of factor XIII. We observed no labeling for the B-subunit in macrophages. Therefore, we hypothesize that factor XIII in proliferative vitreoretinal disorders (PVR, PDR) is derived from the exudation of plasma and platelets through disrupted blood-retinal barriers.

Diabetic Retinopathy↗

PubMed2001

Phosphorylation of cyclic adenosine monophosphate response element binding protein in oligodendrocytes in the corpus callosum after focal cerebral ischemia in the rat.

Phosphorylation of cyclic adenosine monophosphate (AMP) response element binding protein (CREB) was examined immunohistochemically in the corpus callosum of the rat brain at various time points after 90-minute focal cerebral ischemia. Focal ischemia was induced by occlusion of the middle cerebral artery (MCA) using the intraluminal suture method. Sham animals showed that numerous oligodendrocytes (OLGs) constitutively express unphosphorylated CREB. Local cerebral blood flow (lCBF) measured by the 14C-iodoantipyrine method was reduced from 44.2 +/- 15.4 (mL 100 g(-1) min(-1)) to 18.4 +/- 3.8 and from 53.9 +/- 14.4 to 4.8 +/- 4.5 in the medial and the lateral regions of the corpus callosum, respectively, during MCA occlusion (MCAO). After release of the MCAO, lCBF recovered to the control level in each region. The medial region of the corpus callosum showed a marked increase in phosphorylated CREB-positive OLGs at 3.5 hours of recirculation, and it remained increased until 2 weeks of recirculation as it gradually declined. The activation of CREB phosphorylation in the OLGs was accompanied by expression of antiapoptotic protein bcl-2, normal staining with cresyl violet, and negative TUNEL (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling) staining. Myelination detected by immunostaining with anti-myelin basic protein (MBP) antibody and anti-myelin associated glycoprotein (MAG) antibody remained normal in the medial region of the corpus callosum. The lateral region of the corpus callosum showed a significant but only transient increase in phosphorylated CREB-positive OLGs at 3.5 hours of recirculation, which was followed by a rapid decrease during the subsequent recirculation period. Expression of bcl-2 was suppressed in this region, and demyelination became apparent. These findings suggest that signal transduction through CREB phosphorylation may be closely associated with survival of OLGs and maintenance of myelination in the corpus callosum after cerebral ischemia.

Adenomatous Polyposis Coli Protein↗

PubMed2000

Pathologic significance of tumor progression in locally recurrent rectal cancer: different nature from primary cancer.

PURPOSE: It has recently been demonstrated that the tumor growth rate is a stronger determinant of survival than the extent of the growth in local recurrence of rectal cancer. We studied which factors controlled the tumor growth rate using modern immunohistochemical methods. METHODS: In 51 patients who underwent extended resection for this condition, paraffin-embedded specimens were examined for 1) tumor angiogenesis by CD31 staining and microvessel counting, 2) apoptosis by terminal deoxynucleotide transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling staining, and 3) cellular proliferative activity using anti-proliferative cell nuclear antigen antibody. The results were compared with carcinoembryonic antigen doubling time and survival. RESULTS: The five-year survival rate was 20 percent. The postoperative carcinoembryonic antigen doubling time, which was the strongest predictor of survival, correlated highly with proliferative cell nuclear antigen labeling index, but did not correlate with the apoptotic index or microvessel counts. CONCLUSION: Our study shows that cancer cell proliferation rather than apoptosis or angiogenesis is a major determinant of tumor growth rate and survival in patients with locally recurrent rectal cancer.

Adult↗

PubMed1990

A reliable method for simultaneous demonstration of two antigens using a novel combination of immunogold-silver staining and immunoenzymatic labeling.

We have developed a reliable and sensitive immunohistochemical staining technique which allows the simultaneous demonstration of two different antigens expressed in or on the same cell (referred to as mixed labeling), together with the evaluation of the general histopathological appearance of the tissue. The staining procedure combines a three-step (streptavidin-biotin) immunogold-silver staining (IGSS) with a three-step immunoenzymatic labeling. For this purpose, we investigated the compatibility of IGSS with various substrates of peroxidase or alkaline phosphatase (AP). Highly reliable and discernible mixed labeling was achieved only after initial labeling with IGSS followed by AP labeling using the substrates naphthol AS-MX phosphate/Fast Blue or naphthol AS-BI phosphate/New Fuchsin, respectively. To ensure utmost specificity, we applied FITC-conjugated mouse monoclonal antibodies and rabbit anti-FITC immunoglobulins visualized by AP-labeled immunoglobulins and the respective substrate in a final step. This novel approach provides an excellent means for demonstration of immunocompetent cells and unequivocal determination of the percentage of specific cell subsets in infiltrated tissue. The advantages of this method, as compared with double immunofluorescence or double immunoenzymatic labeling, were investigated and are discussed.

Antibodies, Anti-Idiotypic↗