Medical decision support for remote general practitioners using telemedicine.

A telemedicine link was set up between the casualty department of a remote community hospital and the accident and emergency department of a large urban hospital. The telemedicine link comprised teleradiology, videoconferencing and telepresence. The system was connected by ISDN (128 kbit/s) and also by a satellite link (64 kbit/s). During a one-year clinical trial, 120 teleconsultations took place between the community hospital and the specialist trauma centre, 110 using ISDN and 10 using the satellite link. Teleradiology was used in 116 teleconsultations, videoconferencing in 76, and telepresence in four. Survey results indicated that both the general practitioners running the community hospital and accident and emergency consultants felt that teleconsultation had improved patient care. Communication between clinicians using the telemedicine link avoided the transfer of 70 patients, representing an estimated cost saving of Pounds 65,000.

Evidence for membrane differentiation in polarised leucocytes: the distribution of surface antigens analysed with Ig-gold labelling.

The distribution of a number of leucocyte surface antigens was studied on both round and polarised neutrophil or mononuclear leucocytes using Ig-gold conjugates with transmission electron microscopy. Thin sections of cells, which had been lightly fixed before antibody labelling, were analysed using a statistical method to determine: (1) whether the antigens had a non-random distribution or 'clustering' over the cell surface; and (2) whether there was any overall bias in labelling to give a polarised distribution. Comparison between the results of this analysis and cell morphology were made. The results indicated that with the antigens investigated here, CD45, CD15, HLA-DR and CR3, the majority of polarised cells had a calculated direction of overall asymmetry of gold particles that was aligned with the long axis of morphological polarity. Maximal asymmetry was seen in polarised cells labelled for CD45 and HLA-DR, with labelling ratios of up to 6:1 between the front and back of the cell. A number of round mononuclear cells demonstrated significant polarisation of gold particles but this had no apparent morphological correlation and, in general, round cells showed a low degree of asymmetry. However, there was evidence that both round and polarised cells had a non-random distribution or 'clustering' of gold particles, which was more marked in morphologically polarised cells and particularly significant in polarised neutrophil leucocytes labelled for CR3. The significance of these results for models of cell locomotion involving membrane flow is discussed.

Effects of phorbol esters on shape and locomotion of human blood lymphocytes.

The effects of phorbol esters on shape change and locomotion of human blood lymphocytes were studied both immediately after separating the cells from blood and after overnight culture. Phorbol myristate acetate (PMA), phorbol dibutyrate (PDB) and related esters produced complex shape changes in lymphocytes at both times. These shapes were analysed quantitatively using objective measurements derived from the moments of cell shapes. Immediately after removal from blood, many lymphocytes (20-60% of the total) protruded and retracted veils or spikes at more than one point on the cell surface. The morphology of these cells was not typical of locomotor cells. Usually, formation of a veil was not followed by a contraction wave moving down the cell, though some cells did show contraction waves, and some moved into collagen gels or filters. After overnight culture, a high proportion (70-80%) of cells had changed shape in PMA and PDB. Although the shapes were still atypical, they resembled classical locomotor morphology more closely; veils formed at one point on the cell surface tended to persist, and contraction waves and constriction rings were seen in many cells. These cells moved in large numbers into collagen gels or filters. Comparison of the paths traversed by PMA-treated lymphocytes in collagen gels suggested that cells cultured in PMA for 24 h pursued more persistent paths that those in short-term culture, but the difference was not marked. We suggest that phorbol esters induce immediate shape change without inducing the complete sequence of motor events necessary for efficient locomotion, whereas after prolonged culture in phorbol esters, locomotion is more efficient, possibly because phorbol esters, like other growth activators, stimulate events during the G1 phase of growth that are necessary for full expression of locomotor capacity.

F-actin distribution in polymorphonuclear leucocytes.

The polarization and amoeboid locomotion of neutrophil leucocytes is stimulated by chemotactic factors, which initiate waves of contraction in both adherent and non-adherent neutrophils. These cyclical contractile events have previously been analysed by time-lapse filming but the mechanisms involved in the coordination of the cytoskeleton during locomotion have not been elucidated, one reason being because of the problems involved in fixing motile cells. In this paper we show that improved fixation of motile neutrophils with low concentrations of glutaraldehyde followed by glycine quenching demonstrated significant differences in the pattern of staining with TRITC-phalloidin in neutrophils moving on different substrata. Previous film analysis had shown the basic features of locomotion to be similar on all substrata. A prominent feature of leucocyte locomotion on two-dimensional substrata (e.g. protein-coated glass), on three-dimensional collagen gels or in motile cells floating in suspension, is the wave of contraction that passes antero-posteriorly along the length of the cell. The organization of the cytoskeletal elements has not been demonstrated at contraction waves, but light fixation with glutaraldehyde followed by staining with TRITC-phalloidin demonstrated prominent bands of F-actin in neutrophils inside collagen gels. These bands were not present in neutrophils either in suspension or moving on a two-dimensional substratum. Although all motile neutrophils had brightly stained anterior lamellipodia, the cells moving on the two-dimensional substratum had very extensively ruffled leading lamellae stained very brightly with TRITC-phalloidin. The reasons for the absence of consistent bands of F-actin at contraction waves are discussed.

Signal transduction in human neutrophil leucocytes: effects of external Na+ and Ca2+ on cell polarity.

Stimulation of neutrophil leucocytes with chemotactic factors is known to result in membrane permeability changes, as evidenced by fluxes of Na+ and K+ across the cell membrane together with an increased uptake of Ca2+ from the medium. These fluxes have been implicated in the transduction mechanisms of various responses, including locomotion and subsequent chemotaxis. We have previously reported that exposure of unstimulated, round neutrophils held in suspension, to the chemotactic peptide fMet-Leu-Phe confers morphological polarity on the neutrophils by stimulating waves of contraction, which are also intimately connected with locomotion on an appropriate substratum. As the acquisition of polarity is the important first step in the chemotactic response we have investigated the effects of modifying the external ionic environment and of various ion channel blockers on the polarizing response of neutrophils held in suspension. Removal and chelation of both Ca2+ and Mg2+ from the external medium did not inhibit the acquisition of polarity and a variety of inorganic Ca2+ channel blockers together with the organic Ca2+ antagonists, verapamil and D600, were ineffective in inhibiting the response. Replacement of Na+ in the external medium with choline inhibited the polarizing response completely but tetrodotoxin, which blocks fast Na+ channels, and amiloride, which inhibits Na+/K+ exchange, had no effect. Inhibition of the Na+/K+-ATPase with ouabain and also tetraethylammonium ions, which block potassium channels, had no inhibiting effect on polarization. These results indicate that while Ca2+ and Mg2+ are not required in the external medium, Na+ is essential, and therefore Na+/K+ fluxes across the cell membrane play a role in initiating locomotion.

Neutrophil leucocyte chemotaxis: a simplified assay for measuring polarizing responses to chemotactic factors.

A method is described which greatly simplifies the screening of compounds which are potentially chemotactic for neutrophil leucocytes. Neutrophils isolated from blood by a standardised procedure are greater than 95% spherical in morphology. Addition of chemotactic factors in isotropic, non-gradient, concentrations induces the spherical shape to become polarized. The degree of polarity depends on the concentration of the factor used, as does the percentage of cells which become polarised. All compounds which induce a good response in assays measuring cell accumulation or orientation in gradients induced a consistent polarizing response in non-gradient conditions with the cells held in suspension. The advantage of this simplified assay over methods currently in use are discussed.

Behaviour of neutrophil leucocytes in uniform concentrations of chemotactic factors: contraction waves, cell polarity and persistence.

The essential component of any hypothesis of random or directed cell movement is the mechanism of cell polarity. In this paper we describe the polar behaviour of human neutrophil leucocytes in uniform concentrations of chemotactic factors both in suspension and while moving across surfaces. Neutrophils exposed to uniform concentrations of chemotactic factors in suspension around the dissociation constant (Kd) for the receptor rapidly become distinctly bipolar; neutrophils exposed to supraoptimal uniform concentrations (100-fold greater than Kd) of chemotactic factors in suspension, although morphologically active, never reached the same degree of polarity as cells in optimal concentrations. These differences in polarity were shown to be the direct result of equatorial contraction waves stimulated on the cell surface by interaction with chemotactic factors. In optimal concentrations of chemotactic factors, contraction waves were initiated from one region of the cell, whereas in supraoptimal concentrations of chemotactic factors contraction waves emanated from all areas of the cell surface. Asymmetry in the distribution of surface receptors for Fc and C3b were observed in neutrophils polarized in uniform concentrations of chemotactic factor. In neutrophils, motile but not well polarized (in 10(-6) M-N-formylmethionyl-leucyl-phenylalanine (fMLP), receptors were uniformly distributed. In neutrophils polarized in concentrations of fMLP near the Kd for the receptor (10(-8) M) receptors for C3b and Fc were localized in the anterior region of the moving cell. The link between contraction waves, cell polarity and receptor redistribution and their initiation by chemotactic peptides is discussed in the context of neutrophil locomotion and response to chemical signals.

Contraction waves in lymphocyte locomotion.

In this paper we propose that the constriction ring, a prominent feature of moving leucocytes, is a major source of locomotive force. Analysis of time-lapse films of lymphocytes in suspension and moving through three-dimensional collagen gels, demonstrated that the constriction ring was the morphological manifestation of a wave of circular contraction that moved antero-posteriorly. In lymphocytes in suspension the wave moved, although the cells could not. Analysis of lymphocytes moving through a collagen gel revealed that the waves remained stationary with respect to the external environment while the cell appeared to move forward through them. Passage of a single equatorial contraction wave resulted in cell lengthening: a shortening of the region posterior to the constriction was observed in cells moving through collagen gels, but not in lymphocytes held in suspension, suggesting that attachment of cells to the collagen network was necessary for longitudinal contraction. Lymphocyte attachment to collagen gels was mediated through the rapid extension of bleb-like structures into the collagen network. Transmission electron microscopy (TEM) failed to demonstrate any organized structure at the constriction ring. NBD-Phallacidin staining of lymphocytes together with TEM demonstrated that F-actin was distributed evenly throughout the length of the cell. Cell polarity was clearly recognizable by the distribution of coated vesicles, microvilli, and all organelles to the rear, and Thy 1-2 to the front, of motile cells, but polarity could be reversed by the passage of a single contraction wave starting at the rear of the cell, without prior redistribution of these structures.

Lymphocyte migration into collagen gels: role of lymph.

In the present investigation we found that the presence of lymph within a 3-D collagen gel potentiates the invasion of mature recirculating lymphocytes into the gel. Preferential accumulation of lymphocytes in lymph-containing gels follows the same rules that apply for both in vitro lymphocyte adhesion to high endothelial venules of frozen sections of the lymph nodes and in vivo lymph node entry of lymphocytes. Furthermore, the results obtained suggest that lymph is chemotactic for lymphocytes. This chemoattractant activity of lymph could be assigned mainly to a protein fraction precipitating in the range of 40-60% concentration of ammonium sulphate. The biological significance of these findings for the selective process of lymphocyte emigration from blood to lymph, across the parenchyma of the nodes, is discussed.

The orientation of fibroblasts and neutrophils on elastic substrata.

The reaction of fibroblasts to the elastic properties of the substratum was studied using elastic collagen films. These films were stretched in one axis to give a substratum which was anisotropic in its elasticity and deformability. Analysis of the orientation of fibroblasts cultured on these substrata showed that they oriented along the axis of stretch which was also the axis of fibre alignment. This orientation was significantly reduced when the films were made less elastic by attachment to a glass slide and chemical fixation. Neither of these procedures appeared to alter the surface shape of these films, which suggests that the elastic properties of the substratum markedly influence the orientation of fibroblasts. The orientation of locomotion of neutrophil leukocytes on elastic collagen films was also analysed and no bias along the axis of stretch was observed. This was compared with neutrophil locomotion in 3-D stretched collagen gels, in which a strong bias along the axis of stretch and of fibre alignment was observed. The possible reasons for the response of these two cell types is discussed.

Separation of mouse lymphoblasts by discontinuous density centrifugation on Percoll gradients.

Mouse lymphoblasts generated in vivo by a topical application of the contact sensitizer oxazolone or by the contents of the gut lumen were separated by discontinuous density centrifugation on Percoll gradients. A 3-step gradient was used to divide the cells into two subpopulations. For cells from oxazolone stimulated lymph nodes, the low density band contained 20-30% of the initial cell number applied to the gradient; 25-40% of this population were in S phase and nearly all the large and pyroninophilic cells were confined to this layer. The high density step cells (70-80% of initial cell number) were predominantly small lymphocytes with less than 0.5% in S phase. Similar results were obtained using cells from picryl chloride stimulated lymph nodes or from mesenteric lymph nodes.

Some determinants of the locomotory behaviour of phagocytes and lymphocytes in vitro.

In this review, we discuss some physical and chemical determinants of locomotor behaviour of phagocytic cells and lymphocytes as studied in visual assays, paying particular attention to the following points. (a) A distinction is made between chemokinesis and other forms of kinesis. We propose that the term chemokinesis be reserved for responses resulting from selective recognition of chemical substances. Many kineses in leucocytes may not involve such recognition, but may result from a variety of physical factors, such as those that alter the adhesiveness between cell and substratum. (b) Neutrophils moving in aligned gels of collagen or fibrin show contact guidance of locomotion, i.e. bidirectional movement along the axis of alignment of the fibres of the gel. Thus neutrophils show directional locomotion not only in chemotactic gradients but also in response to physical properties of the substratum. (c) Lymphocytes adhere poorly and move poorly on 2D protein coated substrata. However they move rapidly through 3D collagen gels. This locomotion may be independent of adhesion since lymphocytes may gain traction for locomotion by expanding pseudopods into gaps in the gel matrix, then using the pseudopod as an anchor for subsequent locomotion in any direction.

Lymphocyte locomotion and attachment on two-dimensional surfaces and in three-dimensional matrices.

The adhesion and locomotion of mouse peripheral lymph node lymphocytes on 2-D protein- coated substrata and in 3-D matrices were compared. Lymphocytes did not adhere to, or migrate on, 2-D substrata suck as serum- or fibronectin-coated glass. They did attach to and migrate in hydrated 3-D collagen lattices. When the collagen was dehydrated to form a 2-D surface, lymphocyte attachment to it was reduced. We propose that lymphocytes, which are poorly adhesive, are able to attach to and migrate in 3-D matrices by a nonadhesive mechanism such as the extension and expansion of pseudopodia through gaps in the matrix, which could provide purchase for movement in the absence of discrete intermolecular adhesions. This was supported by studies using serum-coated micropore filters, since lymphocytes attached to and migrated into filters with pore sizes large enough (3 or 8 mum) to allow pseudopod penetration but did not attach to filters made of an identical material (cellulose esters) but of narrow pore size (0.22 or 0.45 mum). Cinematographic studies of lymphocyte locomotion in collagen gels were also consistent with the above hypothesis, since lymphocytes showed a more variable morphology than is typically seen on plane surfaces, with formation of many small pseudopodia expanded to give a marked constriction between the cell and the pseudopod. These extensions often remained fixed with respect to the environment as the lymphocyte moved away from or past them. This suggests that the pseudopodia were inserted into gaps in the gel matrix and acted as anchorage points for locomotion.