Influence of specificity of reagents and of behaviour of cell surface Ig on the detection and enumeration of immunoglobulin-bearing lymphocytes in humans.

Some parameters likely to influence detection and classification of human B-lymphocytes using anti-immunoglobulin (Ig) sera have been investigated. Of 20 separate mono- and polyspecific native or conjugated anti-Ig sera analysed by a passive haemagglutination technique, 13 exhibited non-specific reactivity. This technique showed no consistent correlation between the titre of individual sera against Fab2 and whole IgG respectively. The indirect immunofluorescence (IF) method applied to detect surface Ig on blood lymphocytes seemed to detect Fc-bearing rather than Ig-bearing cells. The direct method generally yielded fewer reacting cells (5%) than the indirect (10-25%), suggesting that Fc-bearing cells are more numerous than Ig-bearing cells. The Ig-bearing blood lymphocytes seemed to belong preferentially to the IgM class. Passively adsorbed Ig did not appear to contribute significantly to the number of Ig-bearing cells detected. Anti-Ig sera induced redistribution and some endocytosis of surface Ig but this did not markedly affect detection of Ig-bearing cells.

Detection of immunoglobulin-bearing lymphocytes in humans with special reference to patients with contact dermatitis.

Factors likely to influence the detection of immunoglobulin-bearing lymphocytes by immunofluorescence (IF) have been studied. Several of the examined anti-immunoglobulin reagents reacted to other Ig-classes but the relevant as demonstrated by hemagglutination test. When the indirect IF-method was used most anti-Ig reagents reacted with a significantly larger population of lymphocytes. Neither cytophilic Ig, nor plasma membrane dynamics did seem to exert any critical effect on classification of human lymphocytes. With selected sera lymphocytes of 24 patients with contact dermatitis were studied. No difference in number of Ig-bearing cells (direct and indirect technique respectively) between the patients and normals was found. In most individuals there was a good correlation between EA-rosette-forming cells and number of Ig-bearing lymphocytes including IgD-bearing using the indirect technique. This indicates the detection of a Fc-positive rather than a Ig-bearing cell population.

Fiber-fortified feedings in immobile patients.

The purpose of this pilot study was to test methods to measure the effects of a fiber-fortified, enteral feeding (Jevity) on the bowel habits of an immobile, tube-fed group of patients. Three experimental patients received fiber-fortified feedings for seven weeks, whereas four control patients received their usual enteral feeding. Measurements of the number of stools, the consistency of stools, the formula volume delivered, the daily water volume, the body temperature, the urinary output, and the bowel medications were made during three phases--a baseline, an adjustment, and a treatment period. Patients who receive the fiber-fortified enteral feedings had more stools and better consistency of stools than did those patients who did not receive the fiber-fortified formula. Results indicated that fiber-fortified feedings should be added gradually to immobile, tube-fed patients' diets under close supervision. Although measuring the effects of a new feeding in immobile, tube-fed patients is labor-intensive, it can be accomplished successfully.

Coprolite assessment in nursing research and practice.

A reliable assessment of stools without the use of metabolic assays of stool content has not previously existed. The Wanger Stool Assessment Instrument (WSAI) was initially conceived as an efficacy outcome measure for a clinical investigation of enteral feedings. The WSAI is a descriptive tool that has three scales: a seven-category color scale, an ordinal scale that describes amount, and an ordinal scale that ranks consistency of stools. Instrument testing and development were conducted in stages. Nursing staff using the instrument reported that its use improved data collection as well as the patient information communicated among health care staff.

Frequency of N. gonorrhoeae, T. vaginalis, and C. albicans in female venereological patients. A one-year study.

The frequency of Neisseria gonorrhoeae, Trichomonas vaginalis, and Candida albicans has been studied over a period of one year in women attending a venereal diseases clinic. A total of 1,347 women were investigated, all coming from the same catchment area. Gonorrhoea was established at the first visit in 506 patients (38 per cent.), who constituted 97.5 per cent. of the total number of cases of gonorrhoea. Trichomonas vaginalis was found in 272 (20 per cent.) and Candida albicans in 233 (17 per cent.). 176 patients (13 per cent.) had more than one pathogen. Of the patients attending, 22 per cent. (292 women) were so-called "named contacts". The frequency of gonorrhoea established at the first visit in these patients (64 per cent.), was significantly higher, but the frequency of symptoms did not differ from that in other gonorrhoea patients. The number of asymptomatic cases was so large that a single compulsory examination is undoubtedly very useful from the epidemiological point of view, but the value of repeated specimen collections for gonorrhoea is debatable. Complications of gonorrhoea were observed in 29 patients (6 per cent.) at the first visit.

Treatment of trichomoniasis in females with and without gonorrhoea.

46 women with trichomoniasis and gonorrhea were treated with nimorazole (300 mg twice daily for 7 days) and a trichomonal cure rate of 90-2 per cent. was noted. In addition, 53 of 112 patients with trichomoniasis but without gonorrhoea, were treated with metronidazole (200 mg three times a day for 7 days) and 59 with nimorazole (300 mg twice daily for 7 days) in a randomized trial. The cure rates were 96 and 90-5 per cent. respectively. There was no significant difference in the results noted in the two groups. Neither the trichomonal infection itself nor the result of antitrichomonal therapy was affected by antigonococcal therapy (one-day treatment with ampicillin or single-dose therapy with doxycycline). No increase was noted in the frequency of candidiasis after antitrichomonal therapy (in patients without gonorrhoea) or after antigonococcal therapy, but there was substantial variation between consecutive specimens in the prevalence of C. albicans.

Erysipelas and necrotizing fasciitis.

The clinical course of necrotizing fasciitis in 8 patients is compared with observations on 22 other patients with erysipelas. In necrotizing fasciitis the early erythematous areas turn into a dusky blue colour with later vesiculation and formation of bullae. An important finding is a non-pitting oedema extending outside the erythematous patches. The disease often progresses and involves further skin areas proximal to the initial ones. Gangrene tends to follow in multiple sites after the 1st week of illness. Group A streptococci in conjunction with widespread thrombosis and vascular necrosis of the involved skin are two major factors in the pathogenesis of the gangrene. Early debridement and excision of necrotic tissue in combination with large doses of penicillin and cloxacillin are confirmed as mandatory to remove toxaemia and inhibit further necrosis of the skin. In 3 of the 8 patients with necrotizing fasciitis the syndrome of disseminated intravascular coagulation complicated the course of the disease. A promising therapeutic result was seen in 2 further patients exhibiting alarming signs and symptoms of early necrotizing fasciitis; the combination of heparin, given intravenously in therapeutic doses guided by activated partial thromboplastin time studies, and of systemic antibiotics alleviated the symptoms, which vanished within 10 days of the start of treatment.

Expression of lymphocyte activation markers in benign cutaneous T cell infiltrates. Discoid lupus erythematosus versus lichen ruber planus.

The expression of lymphocyte activation markers (IL2 receptors, transferrin receptors and HLA-DR) was examined in cutaneous lymphoid infiltrates of 12 patients with lichen ruber planus (LP) and 10 individuals with discoid lupus erythematosus (DLE). The cell infiltrates in both conditions were generally of considerable size. The vast majority of the infiltrating cells were T cells. The reactivity of the anti-IL2 receptor antibody used was confined to lymphocytes. In patients with LP 26 +/- 17% of the infiltrating cells were IL2 receptor positive, 20 +/- 8% carried transferrin receptors and greater than 90% HLA-DR. In patients with DLE less than 1% were IL2 receptor positive, less than 5% carried transferrin receptors and greater than 90% were HLA-DR positive. These data indicate that IL2 receptor expression distinguishes the infiltrating T-lymphocytes in LP and DLE, although in both conditions the vast majority of the infiltrating cells were activated as revealed by their expression of HLA-DR.

Membrane-associated actin in psoriatic epidermis.

With immunofluorescence technique by using specific antibodies, polymerized actin (F-actin) was found to be present in the peripheral parts of the cells in all layers of epidermis from lesional skin taken from twelve psoriatics, whereas normal epidermis from the same individuals showed no such reactivity. This finding might have some bearing on the induction of these lesions.

Anchorage and lymphocyte function. Contact-induced augmentation of T-cell activation.

Beads of polyacrylamide, latex or DEAE-Sephadex markedly augmented the stimulation of unfractionated or T-enriched lymphocytes by concanavalin A (Con A) or phytohaemagglutinin (PHA). The beads were not mitogenic in the absence of Con A or PHA. A prerequisite for the bead-induced augmentation was that the stimulated lymphocytes had been depleted of phagocytic and/or adherent accessory cells. The enhancing effect of beads was most pronounced during the initial 12 h after the beginning of lymphocyte stimulation, but not limited to this early phase of the growth period. The stimulation of lymphocytes in petri dishes of adhesive tissue culture plastic and non-adhesive bacterial plastic were compared. The magnitude of the stimulation on the non-adhesive surface was 10--50% lower than on the adhesive one, this difference being most pronounced at hyperoptimal mitogen concentrations. These results indicate that contact between some cell type and a solid surface can improve lymphocyte stimulation under experimental conditions when the number of phagocytic and adherent accessory cells is a limiting factor. The fact that cultivation on bacterial plastic, where adhesion and spreading were abolished, produced substantial stimulation (albeit reduced) demonstrates that substrate contact may be important, but is not a prerequisite, for lymphocyte activation.

Anchorage and lymphocyte function II. Contact with non-cellular surfaces, cell density and T-cell activation.

Varying numbers of human blood lymphocytes were stimulated by concanavalin A (Con-A) in a constant medium volume. At 'low' cell densities DNA synthesis was proportional to the number of cells in the cultures whereas at 'high' densities DNA synthesis was considerably lower than expected. In the presence of non-mitogenic microcarrier beads, (diameter 195 micrometer) to which the cells attached, DNA synthesis was proportional or nearly proportional to the cell number in the cultures also at 'high' cell densities. The potentiating effect of beads on lymphocyte stimulation was particularly noteworthy in individuals showing a weak mitogen response. Another approach that yielded proportionality between DNA synthesis and cell number both at 'low' and 'high' cell densities was the use of culture vessels with a larger bottom area. Under such conditions the presence of beads did not augment DNA synthesis. These results suggest that the availability of non-cellular adhesive surface is a major limiting factor and cell density a major regulating factor in the control of lymphocyte activation. Anchorage of the cells to a surface may modulate the density dependent 'growth control mechanism' indirectly via an influence on cell-cell interaction. An alternative less-likely interpretation is that the contact with non-cellular surfaces directly gives rise to regulatory responses in lymphocytes or accessory cells.

Permissive effect of substratum contact on DNA synthesis in concanavalin A-treated lymphocytes.

The effects of contact with a solid surface (in the form of adhesive microcarrier beads (Cytodex)) on the induction of proliferation of human blood lymphocytes after exposure to concanavalin A (Con A) were investigated. The duration of the lag phase preceding DNA synthesis was identical in the presence and absence of beads. The kinetic course of initiation of DNA synthesis was exponential but with different rate constants both in the presence and in the absence of beads during the first 4 days of the culture period. Cytodex exerted its effect by increasing the proportion of cells initiating DNA synthesis in response to Con A. To elucidate whether lymphocytes responded to Con A exposure in a dose-dependent manner, cells were seeded at different concentrations and exposed to Con A for different times. At 36 and 48 h after start of stimulation the proportion of cells synthesizing DNA increased with increasing cell number in the cultures. In contrast, the portion of cells synthesizing DNA was markedly decreased at high cell densities 60, 72 and 96 h after start of stimulation. In the presence of microcarrier beads, however, the proportion of cells synthesizing DNA was proportional to the cell number in the cultures also after 60, 72 and 96 h.

Anchorage and lymphocyte function. Acquisition of spontaneous motile behaviour by human blood lymphocytes and its modulation by concanavalin A.

The majority of splenic lymphocytes were motile, showing lamellipodial activity almost immediately after purification. In contrast, fresh blood lymphocytes were non-motile and maintained their spherical suspension morphology. The number of motile blood lymphocytes increased markedly during a 2-day in vitro culture period. This increase was enhanced by high cell density and required a metabolically active cell with protein synthesis but not exogenous mitogens. The spontaneous development of motility in different subpopulations of blood lymphocytes was analysed by means of monoclonal antibodies. The results indicated that cells which were motile immediately after purification were almost exclusively non-T lymphocytes. Lymphocytes which became motile during in vitro culture included both T and non-T cells. Substrate adhesion mediated by concanavalin A (Con A) changed the morphology of motile T lymphocytes and instead of being polar, the cells flattened over the substratum and acquired a non-polar shape. Furthermore, the morphogenetic response induced by Con A-mediated substrate adhesion appeared to distinguish T and non-T lymphocytes. Thus, the length of the cell perimeter showing lamellar activity was greater in T than in non-T lymphocytes, and the degree of polarity was greater in non-T (with and without B-cell markers) than in T lymphocytes.

Cytochalasins distinguish by their action resting human T lymphocytes from activated T-cell blasts.

In the majority of resting human peripheral T lymphocytes obtained from separate individuals cytochalasin B (CB) and D (CD) cause a disappearance of microvilli and induce a rapid formation of prominent sac and bleb-like projections with a length of 1-10 microns randomly distributed over the cell surface. During mitogen stimulation the cells lose the tendency to develop such projections when subsequently exposed to CB and CD. By contrast, in activated T lymphocytes the cytochalasins provoke an asymmetric localization of microvilli including cell surface antigens and actin to a prominent protuberance often separated from the cell body by a constriction. This protuberance is distinct from conventional spontaneous uropods formed by conA-stimulated lymphocytes in relation to contact with other cells and with non-cellular surfaces. The cytochalasins therefore in their action distinguish resting small lymphocytes from activated T-cell blasts.

Anchorage and lymphocyte function. Antibodies as adhesion and spreading factors for human T lymphocytes.

When attached to a solid surface coated with protein A various antibodies reacting with lymphocyte membrane antigens (anti-beta 2m, OKT3, OKT8, Leu2, 3, 4 and certain patient sera) catalyse the formation of peripheral lamellar activity, i.e. an active spreading process in human T lymphocytes. In contrast, binding only of the same antibodies to the cells or allowing antibody-coated cells to settle and bind to a protein A-coated surface did not induce spreading although the number of cells attached to the solid surface was virtually the same as in the former case. The peripheral lamellar activity markedly facilitated short-range lymphocyte interactions and appeared to constitute the region of the lymphocyte that actively contacts other cells. These results show that antibodies can act as spreading factors, and indicate that this function is critically dependent on the presentation of the inducing ligand. The asymmetry in the induction of active cell edges may influence functional lymphocyte interactions with environmental surfaces.

Permissive effect of cytochalasin B on DNA synthesis in concanavalin-A-treated lymphocytes.

Unfractionated or T-cell-enriched human lymphocytes can be stimulated to undergo DNA synthesis and mitosis by the addition of polyclonal cell activators such as the plant lectins phytohaemagglutinin and concanavalin A (ConA). Under conventional culture conditions stimulated cells cease proliferating only a few days after the first cells have initiated DNA synthesis. Cytochalasin B (CB), which is non-mitogenic per se, causes a prolongation of the period during which ConA stimulates DNA synthesis from normally 3-5 days to more than 3 weeks. The CB-induced prolongation of cell proliferation is clearly stage-specific in the sense that the CB effects are exerted after an initial period of 24 h and do not come into effect until 48 h after onset of ConA stimulation. In contrast, CB exerts a slight suppressive action on DNA synthesis between 24 h (when activated cells initiate DNA synthesis) and 48 h after onset of stimulation. These two separate effects of CB, i.e. augmentation of lymphocyte stimulation 48 h after stimulation, and suppression of stimulation before this point of time, are relatively independent of the concentration of CB.