Preparation and characterization of liposomes containing the Ca2+-activated photoprotein, obelin.

1. Liposomes bearing different net surface charges have been prepared and their ability to entrap the Ca2+-activated photoprotein, obelin, has been studied 2. Negatively-charged liposomes, composed of egg-yolk lecithin, cholesterol and phosphatidylserine, consistently produced the most homogeneous populations of liposomes after sonication, as shown by electron microscopy after negative staining. These consisted of a large proportion of uni- and bilamellar vesicles within the size range of 20--50 nm, external diameter. 3. Sonicated negatively-charged liposomes had a mean aqueous obelin space of 6.8 +/- 0.8 microliter/mumol of phospholipid compared to a mean space for inulin [14C]carboxylic acid of 5.1 +/- 0.8 microliter/mumol of phospholipid. 4. Sonication reduced the Ca2+ permeability of the negatively-charged liposomes, as measured by the utilization of entrapped obelin. 5. Preparations of uncharged (no phosphatidylserine) and positively-charged (stearylamine instead of phosphatidylserine), sonicated liposomes contained a greater proportion of larger vesicles, which were more permeable to Ca2+ than sonicated, negatively-charged liposomes. 6. Obelin, trapped within sonicated, negatively-charged liposomes, responded to increases in the free Ca2+ concentration within the liposomes caused by the bivalent-cation ionophore A23187 at concentrations as low as 19 nM. 7. The effect of A23187 was inhibited by Mg2+ at a low concentration of Ca2+ (10 muM), but not at 1 mM Ca2+. 8. It was concluded that obelin could be trapped in the aqueous compartment of sonicated liposomes which remained relatively impermeable to Ca2+. Furthermore, trapped obelin could respond to changes in the free Ca2+ concentration within these liposomes.

Role of complement in the aetiology of Pick's disease?

Complement in the postmortem brains of 15 cases of Pick's disease has been widely analyzed immunohistochemically and, in 2 cases, by immunoelectron microscopy. Astrocytes and the Pick bodies and cytoplasm of ballooned neurons were immunoreactive with antibodies to classical pathway components C1, C1q, C4, C2 and C3 and the terminal complex components C5, C6 and C8. In almost all cases, no immunostaining was obtained with antibodies against C9 and neoepitopes in the membrane attack complex (MAC), the complement complex responsible for cytotoxicity. However, unequivocal staining with antibodies to two soluble complement regulatory proteins, S-protein and clusterin, and to the membrane complement inhibitor CD59 was found, although three other membrane inhibitors, CR1(CD35), DAF (CD55), and MCP (CD46), were not detected. The complement immunoreactivity of astrocytes and neurons could be the result of complement biosynthesis or attack. Complement attack will be restricted by the expressed regulatory proteins. However, neurons may be the victims of attack since they show pathological change. The internalization of complement-attacked membrane, perhaps involving the genesis of Pick bodies and ballooning, may explain the intracellular immunolocalization of complement in damaged neurons. Immunoglobulins, as a possible source of complement activation, were observed in only two cases, leaving unresolved the trigger for complement activation in the other cases.

Analysis of antigen presenting cell derived exosomes, based on immuno-magnetic isolation and flow cytometry.

We present a simple yet powerful method for the isolation and analysis of exosomes released by antigen-presenting cells (APC). Exosomes are small vesicles (40-90 nm) released by APC, and may have an immuno-regulatory function in vivo. Such exosomes originate from MHC class II peptide loading compartments and, as such, express high levels of MHC Class II. We have utilised magnetic beads, coated with monoclonal antibodies specific for HLA DP, DQ, DR for the specific isolation of exosomes from cell-free supernatants. Beads coated with exosomes are subsequently stained with conjugated antibodies, and analysed by flow cytometry. Characterisation of exosomes by this method demonstrated that exosomes derived from B-lymphocytes express abundant MHC Class I and II molecules. Other immunologically important molecules detected included the co-stimulatory molecules B7.1 (CD80) and B7.2 (CD86). The adhesion molecule ICAM-1 (CD54) was also detected. These exosomes also expressed the B cell marker CD20, and the complement inhibitory protein CD59. The expression of CD63, a lysosomal marker, was variable, and there was no detectable expression of transferrin receptor (CD71). Monocyte derived dendritic cells (cultured for 7 days in GM-CSF/IL-4), demonstrated an immature phenotype, and secreted exosomes with a similar phenotype, with abundant MHC molecules. The expression of CD63 was consistently strong, and the MHC Class I-like molecule CD1a was also present, suggesting a possible function in the presentation of lipid antigens. Again CD59 was expressed suggesting a possible role for APC exosomes in complement regulation. There was no detectable CD71, CD40, CD14, CD20 or CD83. Modification of the extraction protocol allowed a comparative analysis of exosome secretion under various conditions. Treatment of cells with calcium ionophore, or phorbol ester resulted in apparent increases in exosome release, while the phosphatidyl inositol 3-kinase inhibitor, wortmannin, reduced exosome secretion. The immuno-magnetic isolation and analysis of exosomes is a versatile and rapid tool for the analysis of APC exosomes, and may prove a valuable tool for the study of exosome biology.

Dinitrophenyl (DNP) hapten sandwich staining (DHSS) procedure. A 10 year review of its principle reagents and applications.

Over the past 10 years an immunoperoxidase method using dinitrophenyl (DNP) hapten-labelled primary or secondary probes has been devised. Its widely successful application in research and diagnostic work has depended upon the development of certain key reagents. These include a novel non-deleterious DNP labelling compound, a unique multivalent monoclonal bridge antibody, and an efficient DNP hapten substituted or anti-DNP linked marker enzyme. In this article the development of these reagents and various modifications of the basic technique are reviewed in conjunction with the special applications accruing from their use.

Alpha-actinin in nemaline bodies in congenital nemaline myopathy: immunological confirmation by light and electron microscopy.

To elucidate the protein composition of the nemaline bodies present in the muscle fibres of patients with congenital nemaline myopathy (CNM), we studied muscle biopsies with monoclonal antibodies against alpha-actinin and desmin in combination with a modified Gomori trichrome method. Electron microscopy of immunolabelled resin embedded sections was used for cytochemical localisation of alpha-actinin and desmin. Light microscopy of sections immunolabelled for alpha-actinin showed a cross-striation of the muscle fibres corresponding to the Z band pattern, focal thickening of the Z bands and additional reactivity with a granular pattern corresponding to the presence of nemaline bodies. Labelling of desmin did not show a similar pattern. Electron microscopy confirmed the presence of alpha-actinin in the nemaline bodies and Z bands, whereas desmin was only found in intermediate filaments around the Z bands. Western blots showed single, sharp alpha-actinin bands indistinguishable from normal. Our results provide direct evidence for the presence of alpha-actinin in nemaline bodies and a lack of quantitative or qualitative differences between the alpha-actinin of normal and CNM muscle.

Modern acrylics for post-embedding immunostaining techniques.

We describe two methods for rapid processing of biological tissues into LR White acrylic plastic. Both methods make use of LR White's compatibility with small amounts of water, enabling non-osmicated tissue to be only partially dehydrated before infiltration with the plastic, a procedure that improves the sensitivity of post-embedding immunocytochemistry. In addition, both methods are designed to reduce the time for which tissue is exposed to the damaging influence of the plastic monomer, which can cause extraction and sudden shrinkage. The tissue example used in the first method is immersion-fixed, surgically removed human pituitary which, by virtue of its thorough fixation, can be processed quickly at 50 degrees C using catalytic polymerization at room temperature. The concentration of the catalyst is critically set to prevent the temperature rising above 60 degrees C in the tissue blocks. Penetration of immunoperoxidase reagents into 330-nm LR White sections is demonstrated and possible modes of action are discussed. When "lightly" fixed tissue is processed as above, serious polymerization artifacts can result from autocatalysis. A second method, based on the first but employing slower polymerization at 0 degrees C, has therefore been developed. The high level of fine structure that can be retained using this method is illustrated by the demonstration of the trans-tubular Golgi in perfusion-fixed kidney of rat. Biotinylated lectin is localized to cells of the kidney proximal tubule with streptavidin-colloidal gold, to illustrate tissue reactivity. In a second example, the structure of the bacterial cell envelope is shown to be similar in appearance after partial dehydration and LR White embedding to that seen after progressive lowering of temperature, dehydration, and Lowicryl embedding.

Multiple hormone storage by cells of the human pituitary.

While immunostaining serial semi-thin sections of acrylic resin-embedded normal human pituitary using antisera to human pituitary hormones, it became clear that several cells were stained by more than one antiserum. The tissue had been surgically excised from a patient with a prolactinoma. The tumor, which was immunoreactive only with antiprolactin antiserum, was distinctly different from the pieces of tissue under study which had normal pituitary architecture and demonstrated immunoreactivity with antisera against all six of the common pituitary hormones. A major immunoelectron microscopic investigation, using immunocolloidal gold and immunoperoxidase methods, revealed cells in which follicle-stimulating hormone (FSH), luteinizing hormone (LH), and prolactin (PRL) were co-localized to the same electron-dense granules. Some similar cells also possessed electron-lucent granules immunoreactive only for anti-PRL antiserum. Adrenocorticotrophic hormone (ACTH) and PRL were also found in the same cell but were very largely localized to separate, morphologically different populations of electron-dense and -lucent storage granules. By employing double immunolabeling, a few granules in the ACTH/PRL cells were shown to be immunoreactive to both anti-ACTH and anti-PRL antisera. The possibility that the multipotential stem cells is discussed.

Metal compound intensification of the electron-density of diaminobenzidine.

Diaminobenzidine (DAB), commonly used in immunocytochemistry as the substrate for peroxidase, has a low electron density. DAB has a known affinity for the salts of some metals and therefore an examination of the ability of six metal compounds (including osmium tetroxide) to increase the electron density associated with DAB deposits has been undertaken. Ultra-thin sections of unosmicated rat pituitary gland, embedded in L. R. White resin, were immunostained by a hapten sandwich immunoperoxidase method, using antibodies to ACTH and TSH. The unintensified electron density of the DAB polymer reaction product on the specific endocrine granules was compared with the electron density resulting from the use of each of the six metal compounds. Lead and silver nitrate gave unsatisfactory results, while phosphotungstic acid and uranyl acetate produced a limited increase in specific electron density under the conditions used. Gold chloride was found to give the highest electron density to the specific endocrine granules, followed closely by osmium tetroxide. Background staining was greater when osmium was used. We conclude that several metal compounds may be used to intensify the electron density of DAB, but of the ones tested, gold chloride, which is safer, more stable, and cheaper than osmium tetroxide, was clearly the best. This approach not only increases the electron density of the DAB reaction product, but allows of the possibility of quantitation using energy dispersive X-ray analysis.

An immunocytochemical method for localization of estrogen receptors in rat tissues using a dinitrophenyl (DNP)-labeled rat monoclonal primary antibody.

We have developed an immunocytochemical method to demonstrate estrogen receptor in hormone-sensitive tissues of the rat using a dinitrophenyl (DNP) hapten-labeled rat antihuman estrogen receptor monoclonal antibody (MAb), H222. Mouse IgM anti-DNP was used secondarily, followed by a DNP/peroxidase conjugate, diaminobenzidine/hydrogen peroxide chromogen, and silver intensification. This method was applied to tissues from intact female rats and showed that estrogen receptor was localized in the nuclei of the stromal and glandular components of the uterine endometrium. Reduced receptor staining was observed in the luminal epithelium, with minimal myometrial staining. Anterior pituitary glands showed heterogeneous immunostaining and ovaries expressed the receptor predominantly in the interstitial cells; fallopian tubes demonstrated substantial epithelial staining. Uteri from chemically castrated rats showed reduced estrogen receptor immunostaining in both stromal and luminal cells, whereas staining was enhanced in the glandular elements. Classical estrogen-unresponsive tissues (heart, lung, and spleen) were unstained. Antibody controls involved pre-blocking antibody recognition sites on the receptor with unlabeled antibodies to estrogen receptor (H222, H226, and D547), as well as use of an inappropriate DNP-labeled antibody to metallothionein. These controls illustrated the specific nature of the DNP-H222 binding.

Rat anterior pituitary cells maintained on artificial capillaries: responses of thyrotrophs and lactotrophs to depolarization, TRH and dopamine.

Rat anterior pituitary cells have been maintained over an 18-day period in a perfusion system designed around artificial capillaries. Using novel methodology the cells have been visualized by light microscopy and appear as aggregates, closely attached to and sometimes stretching around the capillaries. Their morphology is consistent with previous histology at the level of light microscopy. The techniques described are compatible with immunohistochemistry and electron microscopy. The functional integrity of thyrotrophs and lactotrophs maintained in the system has been examined by measuring the dynamics of TSH and PRL secretion in response to depolarization, TRH and dopamine (DA). TSH and PRL were significantly and reproducibly released by TRH over a 7-day period. On each day the release was dose-dependent with a threshold of at least 28 pg. Qualitatively the responses were rapid in onset (within minutes) for both hormones. Similar responses were measured in response to high K+ depolarization. Basal secretion of TSH and PRL was rapidly and significantly inhibited by DA in a dose-dependent manner (ED50 20 +/- 25 nM for TSH and 70 +/- 40 nM for PRL). Inhibition was dependent on the continued presence of DA and could be mimicked by bromocriptine and stereospecifically prevented by the active but not the inactive isomer of the DA receptor antagonist butaclamol. Simultaneous administration of 10(-6) M DA with 10(-8) M TRH prevented the release of TSH and PRL. The effect of DA was transient, subsequent TRH responses being qualitatively and quantitatively normal.(ABSTRACT TRUNCATED AT 250 WORDS)

Scanning electron microscopic examination of bacterial immobilisation in a carboxymethyl cellulose (AQUACEL) and alginate dressings.

Dressings have been applied to open wounds for centuries. Traditionally they have been absorbent, permeable materials, i.e. gauze that could adhere to desiccated wound surfaces, inducing trauma on removal. With the advent of modern wound care products many dressings are now capable of absorbing large volumes of exudate whilst still continuing to provide a moist wound healing environment. Equally important is their ability to lock exudate in the dressing (i.e. bacterial retention within the dressing matrix) such that upon removal from a wound surface bacterial dispersion is minimised. In these studies detailed scanning electron microscopy techniques have demonstrated the fluid controlling properties of alginate wound dressings and a carboxymethylated cellulose wound dressing (AQUACEL) Hydrofiber) dressing (CMCH)). It was demonstrated that following hydration of the latter wound dressing, the subsequent formation of a cohesive gel was effective in encapsulating large populations of potentially pathogenic bacteria such as Psuedomonas aeruginosa and Staphylococcus aureus under the gelled surface, as well as being immobilised within the swollen fibres. In contrast, hydrated alginate wound dressings did not form a uniform, cohesive gel structure, with the result that fewer bacteria were immobilised within the gel matrix. Many bacteria were trapped on individual, non-hydrated fibres. The unique absorbent gelling properties of the CMCH dressing appears to provide an ideal environment for immobilising bacteria.

Glandular duodenal carcinoid--a somatostatin rich tumour with neuroendocrine associations.

The clinical and pathological features of four cases of duodenal carcinoid tumour are presented. All four tumours showed a glandular pattern, and in three cases this was associated with psammoma bodies. In three tumours somatostatin was identified by immunocytochemistry in most tumour cells. In two cases the duodenal tumours were associated with von Recklinghausen's disease and phaeochromocytoma. The importance of these unusual features is discussed, and it is suggested that these glandular carcinoids are a specific subgroup of endocrine cell tumours which appear to have potentially important clinical and pathological associations.

Immunohistochemical identification of prolactin and 24K protein in secretory endometrium.

Inadequate endometrial differentiation is a cause of infertility and recurrent pregnancy loss. Diagnostic histologic dating criteria may be supplemented by the immunohistochemical identification of protein markers in late secretory endometrium. Late secretory endometrium has been shown to contain prolactin. The authors report the immunohistochemical localization of prolactin and 24K protein in late secretory endometrium using monoclonal antisera and the dinitrophenyl hapten sandwich-staining technique. The appearance of these proteins in decidualized stromal cells of late secretory endometrium may provide a more specific indicator of endometrial development and differentiation, if their late appearance or absence can be correlated with pregnancy wastage.

Morphologic analysis of a strand recovered from a prosthetic mitral valve: No evidence of fibrin.

We report the first morphologic analysis of a linear mobile structure (strand) detected by transesophageal echocardiography on a bioprosthetic mitral valve and then recovered at surgery. Electron microscopy showed it to consist of a sparsely cellular component, with extracellular amorphous or fibrillary areas. Collagen was largely responsible for the fibrillary appearance.

Receptor secretion hypothesis for regulation of hormone action.

It is well established that modulation of target cell responses to hormonal stimulation can occur through variation of the number of effective hormone receptors per cell. Recent experimental evidence suggests that an alternative or additional autoregulatory mechanism may operate via secretion by the cell of 'receptors' which can bind to, and thereby regulate, the concentration of free hormone in the extracellular space. We present here a brief general analysis of this 'receptor-secretion' model, together with an example which shows that it can readily account for the phenomenon of self-limiting growth seen in many tissues in response to sustained hormonal stimulation. The biological significance of the model is discussed, and the close analogy with B-lymphocyte regulation pointed out. Finally, two important implications are considered, for the pathogenesis of nodular hyperplasia and tumours in hormonally-controlled tissues.

Immunomicroscopy: resin techniques and on-section labelling with immunocolloidal gold or immunoperoxidase--planning a protocol.

On-section immunocytochemistry is divided into two parts: (i) processing of biological tissue for section microscopy and (ii) immunolabelling of sections. Many of the more successful microscopical methods employ delicate aldehyde fixation of biological tissue followed by "sympathetic" processing into an acrylic resin. Processing regimens do not have to be complicated. Simple and cost effective room temperature protocols utilising partial dehydration have been devised and they can be as effective as the more complex low temperature techniques in preserving both ultrastructure and antigenic reactivity. The embedded material can be investigated by either light or electron microscopy. Frozen sections can be cut and immunolabelled but only if the tissue is chemically fixed first, as in resin embedding. Fixation with low concentrations of aldehyde will normally better preserve tissue immunoreactivity but this may be at the expense of good ultrastructure with these protocols. If so, low temperature resin embedding methods or rapid freezing and cryosubstitution can be tried. The choice of processing protocol will determine which acrylic resin to use, as will the preference for subsequent immunolabelling with either colloidal gold or peroxidase/diaminobenzidine (DAB). Both types of labelling system offer advantages to localisation studies and can be used in combination for double or even triple labelling. Silver enhancement of the colloidal gold or DAB allows for improved observation by light microscopy.

Strategies for improving the cytochemical and immunocytochemical sensitivity of ultrastructurally well-preserved, resin embedded biological tissue for light and electron microscopy.

Many techniques for processing tissue into resin are available, varying from conventional room temperature to low temperature procedures. The problem is to choose an appropriate method to suit the biological specimen under study. Room temperature approaches with aldehyde and osmium fixation do not give optimal retention of immunoreactivity. Osmium can be removed from sections, but recovery of immunosensitivity is reduced. Osmium post-fixation can be omitted, but heat polymerization of resins causes tissue extraction and loss of immunoreactivity. Alternative techniques rely on the use of milder polymerization methods and avoid osmium. However, while providing an improvement, this alone is not sufficient to maximize tissue reactivity. Fixation with high concentrations of glutaraldehyde (greater than 1%) and processing into resin at either room or low temperature results in retention of similar levels of immunoreactivity. Low concentration glutaraldehyde (less than 0.2%) fixation for short periods of time (less than 60 minutes) produces improved tissue immunoreactivity and allows low concentrations of antigen at secondary sites to be detected. However, the tissue is now only minimally stabilized and is prone to extraction and conformational damage during processing. It can be partially protected by employing one of two strategies: processing at room temperature with partial dehydration (upto 70% solvent) and rapid embedding in LR White or Lowicryl K4M at 0 degrees C, or processing at progressively lower temperatures (PLT) and embedding in Lowicryl at -35/-50 degrees C. In a third strategy, specimens sensitive to very low fixative concentrations are cryo-immobilized, then resin embedded after substitution or freeze-drying (this latter method awaiting evaluation for inclusion in our strategical approach).

Silver development in microscopy and bioanalysis: past and present.

With the experience accumulated from more than a century of silver applications in biology and medicine, physical development has become a powerful bioanalytical tool for marker amplification in blotting procedures, in situ hybridization, immunocytochemistry, histochemistry, and cytochemistry. Early, empirical techniques of silver impregnation followed by development in a reducing solution (chemical developer), or a solution which contained both silver reducers and silver salts (physical developer) were often capricious and suffered from unwanted silver precipitation caused by light and self-nucleation. To accommodate the modern demand for accurate physical development, various strategies have been devised to counter these problems. One approach has been to introduce organic colloids into the developer to keep the silver ions and reducer molecules apart, whilst excluding light by using a dark-room or by covering the solution. Albumen, gelatin, and complex polysaccharides have all been tested, but gum arabic is preferred. In addition, further control can be achieved by slowing down the rate of development with low pH and by changing from silver nitrate to silver lactate, which dissociates more slowly. Effective colloid protection in a physical developer is also provided by the inclusion of tungsten salts which can delay light-catalysed silver reduction and keep the developer clear for many minutes. The same result has been achieved by complexing the silver salt in the physical developer with very large organic molecules, restricting ionization. 'Light insensitive' commercial designer products have resulted. Probably no single formulation can satisfy all conditions of use, but with increased understanding of the mechanisms of physical developers a more flexible, user-friendly approach is anticipated.