Type 1 and type 2 cytokine regulation of macrophage endocytosis: differential activation by IL-4/IL-13 as opposed to IFN-gamma or IL-10. (1/138)

Cytokine regulation of endocytic activity in primary human macrophages was studied to define ultrastructural changes and mechanisms of pinocytic regulation associated with cytokines secreted by activated T cells. The effects of IFN-gamma (type 1) and IL-4/IL-13 and IL-10 (type 2) cytokines on fluid phase and mannose receptor-mediated endocytosis were assessed by horseradish peroxidase and colloidal gold-BSA uptake and computer-assisted morphometric analysis. IL-4 and IL-13 enhanced fluid phase pinocytosis and mannose receptor-mediated uptake by activation of phosphatidylinositol 3-kinase. Inhibition of actin assembly showed that both cytokines exerted actin-dependent and -independent effects. Ultrastructurally, IL-4 and IL-13 increased tubular vesicle formation underneath the plasma membrane and at pericentriolar sites, concurrent with decreased particle sorting to lysosomes. By contrast, IL-10 or IFN-gamma decreased both fluid phase pinocytosis and mannose receptor-mediated uptake. IFN-gamma stimulated increased particle sorting to perinuclear lysosomes, while IL-10 decreased this activity. In summary, our data document differential effects on macrophage endocytic functions by type 1 or type 2 cytokines associated with induction and effector pathways in immunity.  (+info)

Ultrastructural and ultracytochemical features of secretory granules in the ampullary epithelium of the hamster oviduct. (2/138)

The epithelium of mammalian oviducts consists mainly of ciliated and non-ciliated secretory cells. In some mammals, secretory products originating from oviductal secretory cells have been shown to bind to the surface of, or accumulate within, ovulated eggs and/or developing embryos. These findings suggest that the secretions of the oviductal epithelial cells may play an important role in reproductive and developmental events that occur in the oviduct. In the present study, ultrastructural and cytochemical features of secretory cells in the hamster ampullary epithelium were shown by routine electron microscopy, lectin-gold cytochemistry and both conventional freeze-fracture and rapid-freezing techniques with special reference to the organizational aspects of their secretory granules. The use of ferrocyanide-reduced osmium tetroxide as a post-fixative in the Epon embedment of ampullary tissue samples also proved to be advantageous especially in revealing the carbohydrate contents of certain cellular compartments. The most conspicuous characteristic of the secretory cells, based on their staining property, was the presence of two types of secretory granules: those with a homogeneous electron-dense matrix and those with an electron-lucent matrix. Under favorable conditions, distinct features of the organizational arrangement of a crystalline lattice inside the secretory granules were also revealed. This well organized crystalline lattice shown in sections of Epon-embedded oviductal tissue was confirmed by examination of replicas of freeze-fractured oviducts prepared by the rapid-freezing technique. We also demonstrated with high resolution lectin-gold cytochemistry the intracellular distribution of lectin-binding glycoconjugates in the secretory cells of the hamster oviductal ampulla often in a linear array following the crystalline lattice. The results obtained in this study, taken together, provide insight into a possible link of the internal topographical features of oviductal secretory granules along with the cytochemical properties of their contents to the anticipated regulatory mechanism underlying their process of secretions.  (+info)

Reciprocal connections between the red nucleus and the trigeminal nuclei: a retrograde and anterograde tracing study. (3/138)

An anterograde biocytin and a retrograde WGA-colloidal gold study in the rat can provide information about reciprocal communication pathways between the red nucleus and the trigeminal sensory complex. No terminals were found within the trigeminal motor nucleus, in contrast with the facial motor nucleus. A dense terminal field was observed in the parvicellular reticular formation ventrally to the trigeminal motor nucleus. The parvicellular area may be important for the control of jaw movements by rubrotrigeminal inputs. On the other hand, the contralateral rostral parvicellular part of the red nucleus receives terminals from the same zone in the rostral part of the trigeminal sensory complex, where retrogradely labelled neurones were found after tracer injections into the red nucleus. Such relationships could be part of a control loop for somatosensory information from the orofacial area.  (+info)

Nuclear localization of gold labeled-hydrocortisone-bovine serum albumin conjugate injected intravenously into the hormone-target cells of rat. (4/138)

We have suggested in a previous study using 2-nm colloidal gold labeled-testosterone-bovine serum albumin (testosterone-BSA-gold) that 2-nm gold labeled-steroid hormone-BSA conjugates would be a useful tool for analyzing the mechanism of steroid hormone action (39). In this study, we examined whether hydrocortisone-BSA conjugate (hydrocortisone-BSA) showed a similar distribution to radiolabeled hydrocortisone in vivo, by injecting 2-nm colloidal gold labeled-hydrocortisone-BSA (hydrocortisone-BSA-gold) into the rat tail vein. The hydrocortisone-BSA-gold with silver enhancement became visible as silver deposits under electron microscopy in the nuclei of hepatocytes and hepatic stellate cells but not in Kupffer cells in the liver, and in the thymocytes and thymic reticuloepithelial cells in the thymus of a rat killed 2 h postinjection. The percentage of nuclei showing deposits in the non-target cells, the epithelial cells of the seminal vesicle, was similar to the value in the seminal vesicle of a control rat injected with BSA labeled with 2-nm colloidal gold as reported previously. In the hepatocytes and thymocytes of a control rat not injected, the percentages of nuclei showing deposits were similar to those in the rat injected with testosterone-BSA-gold or BSA-gold as reported previously, but lower than those in the rat injected with hydrocortisone-BSA-gold. These results suggest that hydrocortisone-BSA-gold is useful for the morphological study of hydrocortisone target cells, and imply that BSA conjugated with hydrocortisone can enter the target cell nuclei of the rat. The present study further indicates that the fate of gold labeled-steroid hormone-BSA conjugates may be decided at the cell membrane level.  (+info)

Detection of stimulants in hair by laser microscopy. (5/138)

In order to detect methamphetamine, a common stimulant, laser microscopy and immuno-histochemical staining, which uses anti-methamphetamine labeled with colloidal gold, were employed. The intensity of reflection of colloidal gold at a 488- and 514-nm line of Ar laser was measured with a laser microscope equipped with a computerized image processing system. Microtomed hair samples from five drug users who died from methamphetamine intoxication were used. The drug distribution in the hair was quite different in these five cases, but the levels of drug concentration in two different hair samples from the same abuser were correlated. The results from two hair samples with roots showed a correlation between drug concentration in hair roots and plasma samples. The proposed method needs no melanin bleaching and is simple and sensitive enough to estimate the drug concentration using only a segment of hair.  (+info)

Comparison of two rapid diagnostic assays for detection of immunoglobulin M antibodies to dengue virus. (6/138)

Two easy-to-use commercial diagnostic assays, a dipstick enzyme-linked immunosorbent assay (ELISA) (Integrated Diagnostics, Baltimore, Md.) and an immunochromatographic card assay (PanBio, Brisbane, Australia) were evaluated for detection of immunoglobulin M (IgM) antibody to dengue virus with an in-house IgM antibody capture microplate ELISA as a reference assay. The dipstick ELISA was based on the indirect-ELISA format using dengue 2 virus as the only antigen and enzyme-labeled goat anti-human IgM antibody as the detector. The total assay time was 75 min. The immunochromatographic card assay was based on the antibody capture format and separately measured both anti-dengue virus IgM and IgG in the same test. Colloidal-gold-labeled anti-dengue virus monoclonal antibody bound with dengue virus 1 to 4 antigen cocktail was the detector, and anti-human IgM and IgG were the capture antibodies. The total assay time was <10 min. Sera from 164 individuals classified as either anti-dengue virus IgM positive (94) or anti-dengue virus IgM negative (70) in the reference microplate ELISA with a dengue virus 1 to 4 antigen cocktail were tested in the two commercial assays. The dipstick ELISA missed 7 of 94 positive samples, for a sensitivity of 92.6%, while the immunochromatographic card assay missed two positive samples, for a sensitivity of 97.9%. Of the 70 negative samples, four were false positive by the dipstick ELISA and two were false positive in the immunochromatographic card assay, resulting in specificities of 94.3 and 97.1%, respectively. Both commercial assays provide sensitive and specific detection of anti-dengue virus IgM antibody and could prove useful in settings where the microplate ELISA is impractical.  (+info)

Single-target molecule detection with nonbleaching multicolor optical immunolabels. (7/138)

We introduce and demonstrate the use of colloidal silver plasmon-resonant particles (PRPs) as optical reporters in typical biological assays. PRPs are ultrabright, nanosized optical scatterers, which scatter light elastically and can be prepared with a scattering peak at any color in the visible spectrum. PRPs are readily observed individually with a microscope configured for dark-field microscopy, with white-light illumination of typical power. Here we illustrate the use of PRPs, surface coated with standard ligands, as target-specific labels in an in situ hybridization and an immunocytology assay. We propose that PRPs can replace or complement established labels, such as those based on radioactivity, fluorescence, chemiluminescence, or enzymatic/colorimetric detection that are used routinely in biochemistry, cell biology, and medical diagnostic applications. Moreover, because PRP labels are nonbleaching and bright enough to be rapidly identified and counted, an ultrasensitive assay format based on single-target molecule detection is now practical. We also present the results of a model sandwich immunoassay for goat anti-biotin antibody, in which the number of PRP labels counted in an image constitutes the measured signal.  (+info)

Influence of fibrin network conformation and fibrin fiber diameter on fibrinolysis speed: dynamic and structural approaches by confocal microscopy. (8/138)

Abnormal fibrin architecture is thought to be a determinant factor of hypofibrinolysis. However, because of the lack of structural knowledge of the process of fibrin digestion, relationships between fibrin architecture and hypofibrinolysis remain controversial. To elucidate further structural and dynamic changes occurring during fibrinolysis, cross-linked plasma fibrin was labeled with colloidal gold particles, and fibrinolysis was followed by confocal microscopy. Morphological changes were characterized at fibrin network and fiber levels. The observation of a progressive disaggregation of the fibrin fibers emphasizes that fibrinolysis proceeds by transverse cutting rather than by progressive cleavage uniformly around the fiber. Plasma fibrin clots with a tight fibrin conformation made of thin fibers were dissolved at a slower rate than those with a loose fibrin conformation made of thicker (coarse) fibers, although the overall fibrin content remained constant. Unexpectedly, thin fibers were cleaved at a faster rate than thick ones. A dynamic study of FITC-recombinant tissue plasminogen activator distribution within the fibrin matrix during the course of fibrinolysis showed that the binding front was broader in coarse fibrin clots and moved more rapidly than that of fine plasma fibrin clots. These dynamic and structural approaches to fibrin digestion at the network and the fiber levels reveal aspects of the physical process of clot lysis. Furthermore, these results provide a clear explanation for the hypofibrinolysis related to a defective fibrin architecture as described in venous thromboembolism and in premature coronary artery disease.  (+info)