Colin Ockleford

Confocal immunofluorescence localization of collagen types I, III, IV, V and VI and their ultrastructural organization in term human fetal membranes

The distribution of collagen types I, III, IV, V and VI in term human fetal membranes was examined using conventional and confocal indirect immunofluorescence techniques. Collagens I and III were present in most of the layers of fetal membranes except in the trophoblast layer contrary to what has been previously reported. Although collagen IV is considered to be a basement membrane component our study, using monoclonal and polyclonal antibodies, showed its consistent presence in the spongy and reticular layers in high intensity. This was first report on the distribution of type V collagen in the chorion where it was found in the reticular and in the trophoblast layers. Type VI collagen was present mainly in the amnion and the reticular layer. The ultrastructural examination of the extracellular matrix showed that the main fibrous skeleton of the fetal membranes was formed of large banded fibres (Ultrastructurally identical to collagens types I and III) connected together and to the epithelial basement membranes by networks of unbanded filaments (collagen types V, VI and other components). The extensive and continuous networks formed by these collagens may be a major factor responsible for the mechanical integrity of the fetal membranes.

Propidium iodide as a nuclear marker in immunofluorescence. I. Use with tissue and cytoskeleton studies.

Some examples are given of immunofluorescence with tissue sections and microtubular cytoskeletons of cultured cells where the fluorescent dye propidium iodide (PI) has been used as marker of nuclei. The emission wave length of IP is longer than that of fluorescein, making it possible to use several different and commonly available filter combinations. The use of nuclei as positional indicators is often a more suitable method than phase microscopy combined with immunofluorescence because of low background illumination against which morphology is viewed, circumventing the need for often expensive phase optics.

Expression of tissue type and urokinase type plasminogen activators as well as plasminogen activator inhibitor type-1 and type-2 in human and rhesus monkey placenta.

The distribution of mRNAs and antigens of tissue type (t) and urokinase type (u) plasminogen activators (PA) plus their corresponding inhibitors, type‐1 (PAI‐1) and type‐2 (PAI‐2) were studied in human and rhesus monkey placentae by in situ hybridisation and immunocytochemistry. Specific monkey cRNA and antibodies against human tPA, uPA, PAI‐1 and PAI‐2 were used as probes. The following results were obtained. (1) All the molecules tPA, uPA, PAI‐1 and PAI‐2 and their mRNAs were identified in the majority of the extravillous cytotrophoblast cells of the decidual layer between Rohrs and Nitabuchs striae and in cytotrophoblast cells of the chorionic plate, basal plate, intercotyledonary septae and cytotrophoblast cells of the chorionic villous tree. (2) Expression of uPA and PAI‐2 was noted in villous trophoblast whereas tPA and PAI‐1 were mainly concentrated where detachment from maternal tissue occurs. (3) No expression of tPA, uPA, PAI‐1 and PAI‐2 was observed in the basal plate endometrial stromal cells, chorionic plate connective tissue cells, septal endometrial stromal cells or villous core mesenchyme. (4) The distribution of probes observed following in situ hybridisation is generally consistent with the immunofluorescence pattern of the corresponding antigens and no significant interspecies differences were noted. It is possible that both decidual and extravillous trophoblast cells of placentae of human and rhesus monkey are capable of producing tPA, uPA, PAI‐1 and PAI‐2 to differing extents. Coordinated expression of these genes in the tissue may play an essential role in the maintenance of normal placentation and parturition. The differences in distribution we observed are consistent with the suggestion that coordinated expression of tPA and its inhibitor PAI‐1 may play a key role in fibrinolytic activity in the early stages of placentation and separation of placenta from maternal tissue at term. On the other hand, uPA with its inhibitor PAI‐2 appears mainly to play a role in degradation of trophoblast cell‐associated extracellular matrix, and thus may be of greatest importance during early stages of placentation.

A Study of the Mechanism of Internalisation of Tetanus Toxin by Primary Mouse Spinal Cord Cultures

Abstract: The fate of tetanus toxin bound to neuronal cells at 0°C was followed using an anti‐toxin 125I‐protein A assay. About 50%; of surface‐bound toxin disappeared within 5 min of warming cells to 37°C. Experiments with 125I‐toxin showed that much of this loss was due to dissociation of bound toxin into the medium. Some toxin was however rapidly internalised, and could be detected only by permeabilising cells with Triton X‐100 prior to assay. To investigate the mechanism of internalisation, tetanus toxin was adsorbed to colloidal gold. Toxin‐gold was shown to be stable, and to recognise the same receptor(s) as free toxin. Quantitation of the distribution of toxin‐gold particles bound to the cell body at 4°C showed that it was concentrated in coated pits. After 5 min at 37°C, toxin‐gold appeared in coated vesicles, endosomes, and tubules. After 15 min, it was found largely in endosomes, and at 30 min in multivesicular bodies. The involvement of coated pits in internalisation of tetanus toxin, but not cholera toxin, was confirmed using the free toxins, anti‐toxins, and protein A‐gold. Toxin‐gold also entered nerve terminals and axons via coated pits, accumulating in synaptic vesicles and in‐traaxonal uncoated vesicles, respectively.

Supramolecular cytology of coated vesicles

Publisher Summary This chapter discusses the advent of thin sectioning techniques for electron microscopy in erythroblasts and during the next decade they were shown to be present in many different cells and tissues from animals and plants although several different names were used to describe them. It is likely that they constitute a ubiquitous and unique organelle in essentially all eukaryotic cells. A partially purified preparation has been isolated from guinea pig brain and demonstrated that the coat structure was a regular array of pentagons and hexagons producing an icosahedral structure. The closed lattice has been proposed that was formed from a hexagonal network by conversion of 12 hexagons to pentagons. The proposed lattice structure has been amply supported by Heusers rapid-freeze deep-etch morphological studies. The chapter discusses the role of coated vesicles in endocytosis in many cell types. Finally, it has been indicated that coated vesicles are involved in the genesis of lysosomes.

Growth, breakdown, repair, and rapid contraction of microtubular axopodia in the heliozoan Actinophrys sol.

Breakdown of microtubules takes place at the tips of tubules at the distal ends of axopodia when axopodial shortening is induced with colchicine. When small flagellates are captured for ingestion, rapid contraction of axopodia (100 μm in under a second) occurs which apparently involves breakdown of axonemal microtubules. Elongation of a single axopodium after rapid contraction, when all other axopodia are of normal length, proceeds at the same rate as it does when all the axopodia are growing out after microtubule-breakdown induced by cold treatment. In both cases the rate of elongation decreases as axopodia increase in length. Axopodia elastically resist mild bending along their longitudinal axes. They yield nonelastically when more severe bending is applied; bends are formed at certain points along their longitudinal axes. These bends move out along axopodia to their tips.

Characterization of an in vitro model for the study of the short and prolonged effects of myocardial ischaemia and reperfusion in man

The mechanisms underlying myocardial ischaemia and reperfusion-induced injury have been investigated, mainly by using animal experimental preparations in vitro and in vivo, but little is known of the process in human myocardium. The present studies characterize an in vitro model using human myocardium for the study of early and delayed effects of ischaemia and reperfusion. The right atrial appendage was manually sliced and incubated in buffer through which was bubbled O(2)/CO(2) (19:1, v/v) for various time periods. Lactate dehydrogenase (LDH) leakage, 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl-2H-tetrazolium bromide (MTT) reduction, oxygen consumption, nucleotide levels and tissue morphology were all investigated as markers of myocardial injury. The specimens remained stable and viable up to 24 h, but had significantly deteriorated by 48 h. The preparation responded to ischaemia in a time-related manner. Tissue viability was reduced by 25% after 30 min ischaemia, declined to 60% after 60 min ischaemia and to 75% after 120 min ischaemia. Interestingly, the tissue was more susceptible when ischaemia was induced after 24 h of aerobic incubation. The effects of the duration of reperfusion were investigated after a fixed 60 min ischaemic insult. The results of LDH leakage suggest that reperfusion injury is mainly sustained within the first 2 h of reperfusion. However, the results of MTT reduction show that there is a progressive decrease in tissue viability over the 24 h reperfusion period, possibly reflecting the occurrence of tissue necrosis and apoptosis at different reperfusion times. In conclusion, the data provide evidence that the incubation of human atrial tissue in vitro is stable, and slices are viable for at least 24 h, which permits the study of early and delayed consequences of ischaemia and reperfusion in the human myocardium.

Morphogenesis of human placental chorionic villi: cytoskeletal, syncytioskeletal and extracellular matrix proteins

Immunocytochemical and ultrastructural methods were used to investigate the distribution of a family of structural proteins in the human placenta near term. These reveal the distribution of cytoskeletal and ‘syncytioskeletal’ components that may account for some of the more obvious micromorphological features of placental structure. In the syncytiotrophoblast a potentially supporting structure ‘the syncytioskeletal layer’ is described. It is an apparently continuous and complex polymeric network covering the villous tree, a surface of the order of 10m2 in area in the full term placenta (Aherne & Dunnill 1966). It is suggested that this layer plays a part in morphogenesis of the villous tree.

Cytotrophoblast cells: a barrier to maternofetal transmission of passive immunity.

The human fetus receives passive immunity via the chorioallantoic placenta in the form of maternal immunoglobulin G (IgG) class antibodies. This provides protection against pathogens at a time when the fetus is immunologically naive. We localized endogenous human IgG using confocal laser scanning fluorescence microscopy and immunoelectron microscopy of frozen sections of chorionic villi from early and late gestation. With confocal microscopy we also investigated the distribution of a receptor for IgG (Fc gamma RIII; CD16) that is typically expressed on the surface of human leukocytes. Endogenous IgG was present in the syncytiotrophoblast that surrounds chorionic villi but underlying cytotrophoblast cells were devoid of endogenous antibody. Fc gamma RIII immunoreactivity was confined to the syncytiotrophoblast and was also absent from cytotrophoblast cells. We propose that cytotrophoblast cells present a barrier to the transmission of maternally derived IgG across the human placenta. This accounts for the paradox that there are low levels of transport in the first trimester when the syncytiotrophoblast is known to express receptors for IgG. Cytotrophoblast cells form an almost complete epithelial layer underlying the syncytiotrophoblast at this stage of gestation, but this becomes discontinuous as the placenta matures, thus removing the cellular impediment to IgG transmission.

Confocal and conventional immunofluorescent and immunogold electron microscopic localization of collagen types III and IV in human placenta

Confocal and conventional indirect immunofluorescence and immunogold electron microscopic methods were applied to examine the distribution of extracellular matrix constituents (collagens types III and ITq in the villi of immature and term human placentae. The immunofluorescence study revealed that collagen type III is more distinct in the villous stroma of terns placenta as compared with that of the first trimester. Collagen type IV was detected mainly in endothelial and epithelial basement membranes and interestingly also to a certain extent in the stroma. Results obtained using immunoelectron microscopy support the proposal that collagen types III and IV are characteristic of stromal and basement membranes, respectively. Stromal collagen type IV is apparently localized in association with the interstitial types of collagen (I and III), in the villous stroma of term placenta.