Measurement of skin swelling in the tuberculin test by ultrasonography.

A new method is described for non-invasive measurement of the thickness of the skin at the site of a skin test from the echo pattern of a pulsed ultrasonic (15 MHz) A-mode scanner. In tuberculin tests on normal volunteers, the skin thickness increased rapidly during days 1 and 2 and was usually greatest at day 4. The echogram measurements can be used to calculate the increase in skin volume and this is disproportionately greater than would be expected from measurements of the diameters of erythema and induration.

A simple method for determining the extent of cellular contamination in peripheral blood lymphocyte preparations.

A method is described for calculating, from the size distribution of the cells, the extent of cellular contamination of lymphocyte concentrates prepared from venous blood. The precision of this method was checked by direct comparison with differential leucocyte counts obtained by direct microscopy on 29 samples chosen because they showed a wide range of intensity of contamination. By virtue of its technical simplicity, the method has proved useful in a diagnostic service laboratory for checking the purity of lymphocyte preparations either before performing lymphocyte function tests or after a period of 24 h in tissue culture.

A new method of testing for mitogen-induced lymphocyte stimulation: measurement of the percentage of growing cells and of some aspects of their cell kinetics with an electronic particle counter.

Human lymphocytes were cultured with or without added PHA for periods not exceeding one day so that none of the cells had entered the first mitotic division. The size distribution of the cells was measured with an electronic particle counter and the results were collected in a multichannel analyser. An iterative stochastic model was developed to estimate the proportion of responding cells and their growth characteristics. This mathematical model was based on a few simple assumptions about the pattern of cell growth and was considered to fulfill basic requirements of plausibility and parsimony. The technique described in this paper makes measurement of the absolute percentage of responding cells and their average growth rate in mitogen-induced lymphocyte stimulation tests possible in routine diagnostic laboratories for the first time.

The sensitivity of peripheral blood lymphocytes to growth inhibition by hydrocortisone is not determined by their OKT4:OKT8 ratio.

Normal adult human subjects show considerable variation in sensitivity to depression of the activation phase of mitogen-induced lymphocyte growth by the natural glucocorticoid, hydrocortisone. In a study of 21 subjects, the slope of the log-dose response to hydrocortisone was unrelated to the relative numbers of cells in the two major T-cell subpopulations stained by the OKT4 and OKT8 monoclonal antibodies. It is concluded that the extent of the glucocorticoid-induced suppression of early mitogen-stimulated lymphocyte growth is probably not determined by relative numbers of the various immunoregulatory lymphocytes.

Histometric studies on cellular infiltrates of tuberculin tests in patients with sarcoidosis.

The density and microanatomical location of CD4 and CD8 lymphocytes and of monocytes/macrophages at the site of a tuberculin test were measured in 13 patients with sarcoidosis, and the results were compared with those seen in a group of healthy controls. The cellular infiltrate was significantly reduced in the sarcoid subjects compared with the controls for all cell phenotypes studied; the ratio of CD4 positive:CD8 positive lymphocytes was significantly increased in the sarcoid group. Clinically negative reactions showed substantial numbers of infiltrating mononuclear cells, although not as great as in clinically apparent reactions. A clinically negative tuberculin reaction does not necessarily imply anergy to the test substance and should not be termed "negative".

Histometric studies on cellular infiltrates of tuberculin tests in patients with haemophilia.

The number of microanatomical location of CD4 and CD8 lymphocytes, of cells bearing receptors for I12 and transferrin, and of monocyte/macrophages in the dermis at the site of a tuberculin test were measured in 13 patients with haemophilia (10 seronegative and three seropositive for human immunodeficiency virus (HIV]. The overall density of lymphocytes in the perivascular and diffuse parts of the infiltrate was similar to that reported in other groups of subjects without evidence of immunosuppression. The CD4:CD8 ratio of the infiltrating lymphocytes throughout the section showed an inverse relation with clotting factor consumption. There was no significant change in the CD4:CD8 ratio in the diffuse infiltrate at various levels into the dermis in tuberculin reactions in patients with haemophilia, unlike healthy controls and other groups with no evidence of immunosuppression, who have previously been shown to have increasing CD4:CD8 ratio with increasing depth into the dermis. The number of cells bearing receptors for I12 and transferrin and of monocyte/macrophages was related to total lymphocyte density in the infiltrate. There was no evidence of serious impairment of the cell mediated response to a long term recall antigen, but the relatively low preponderance of CD4 lymphocytes in the diffuse infiltrate, particularly in the deeper dermis, may be the earliest indicator of impending immunosuppression.

Skin test responsiveness to four new tuberculins in patients with chronic obstructive airways disease receiving short term high doses of, or long term maintenance treatment with, prednisolone: clinical appearances and histometric studies.

The response to skin testing with tuberculins extracted from various species of mycobacteria was studied in 49 patients from Dundee with chronic obstructive airways disease. Seventeen had never been treated with steroids (group 1), 17 were receiving short term high doses of prednisolone (group 2) and did not have impaired Synacthen tests; 15 were receiving long term maintenance treatment and did have impaired Synacthen tests (group 3). Erythematous and indurated reactions were seen in a few patients, more commonly to antigens from Mycobacterium tuberculosis than to the other species: neither of the latter treatment groups showed appreciable reduction in reactivity compared with that of the group 1 patients. The number and microanatomical distribution of the T4 and T8 lymphocytes and the M3 bearing monocytes and macrophages was studied immunocytochemically in cryostat sections of biopsy specimens from the antigen injection sites. The density of these cells was significantly less in clinically negative reactions than in those with erythema or induration, but was unrelated to the presence or absence of a history of treatment with prednisolone. The T4:T8 ratio in the section as a whole was similar to that of the peripheral blood, but T8 cells were relatively more common in the perivascular and periappendicular foci, and T4 lymphocytes were predominant in the diffuse component of the infiltrate. I12 receptor bearing lymphocytes were uncommon: such cells were least common in the clinically negative reactions, but the number and distribution were apparently unrelated to the presence or absence of prednisolone treatment. It was concluded that currently accepted regimens of treatment with prednisolone did not reduce the effector arm of type IV (delayed type hypersensitivity) responses and so are unlikely to compromise this aspect of protective immunity.

Histometric study of the localisation of lymphocyte subsets and accessory cells in human Mantoux reactions.

Intradermal injection of purified protein derivative produced typical delayed type hypersensitivity reactions in five healthy human subjects. The major subpopulations of lymphocytes and certain accessory cells were located in frozen sections of biopsies of the lesions with monoclonal antibodies and immunohistochemical staining. The densities (expressed as number/unit area for comparison) of the different types of cells were counted at various microanatomical locations in the tissue. The inflammatory cells were concentrated in narrow zones, initially (24 h) only surrounding small blood vessels but later (48-96 h) also around sweat ducts. Lymphocytes were the predominant cell type at these sites with T4 and T8 cells randomly intermixed at a ratio similar to that in the mononuclear cell fraction of the peripheral blood samples removed at the time of biopsy. There was also a scanty diffuse infiltrate in the intervening dermis, but here the T4:T8 ratio was significantly lower than that in the peripheral blood or perivascular cuffs. There was considerable intersubject variation in the relative preponderance of T8 cells in the diffuse infiltrate. The results suggest that there is no subset selection in the initial emigration of lymphocytes through vascular endothelium in the delayed hypersensitivity reaction, but that the subsets behave differently during the subsequent migration through the tissues. It remains to be determined whether the extent to which T8 cells migrate more rapidly than T4 cells through the tissues may influence the reaction at the site of entry of organisms or antigens into the body by altering the balance of the immunoregulatory lymphocyte subsets. This may underlie some of the differences in susceptibility to infection between subjects and determine the type of granuloma that develops in a particular patient.

Early delayed hypersensitivity responses in tuberculin skin tests after heavy occupational exposure to tuberculosis.

The early (six hours) reaction to tuberculin skin testing was studied in 33 Indonesian hospital workers with frequent occupational exposure to M tuberculosis and compared with responses maximal at the usual time (48 hours) in factory workers, from the same locality but with only occasional occupational exposure, to determine the nature of the early reaction. The early reaction had the same general histopathological appearance as that seen in the conventional (48 hour) reaction, and both had an infiltrate consisting largely of T lymphocytes and macrophages. The cell densities were lower in the six hour reactions, but the relative concentration of macrophages was greater in the earlier response. These histometric measurements suggested that the six hour reaction was an accelerated delayed hypersensitivity reaction. Moreover, the absence of a specific IgE response or of particulate masses of Ig or complement, made it unlikely an anaphylactoid or Arthus-type reaction could have been responsible. It is concluded that those with frequent occupational exposure to M tuberculosis have larger numbers of circulating T cells reactive with mycobacterial antigens, so that the development of the skin test response to tuberculin is less dependent on "by-stander" cell infiltration to mediate the delayed hypersensitivity reaction than the reactions in those with less intense and less frequent natural exposure. The skin test response maximal at six hours is probably a hyperimmune reaction to an antigen recognised by T cells.

Effect of hyperoxia at 1 and 2 ATA on hypoxia and hypercapnia in human skin during experimental inflammation.

Transcutaneous PO2 and PCO2 measurements and estimates of skin respiration were monitored at different levels of inspired PO2 in 20 healthy adults during the first 4 days of the tuberculin reaction, a convenient model of acute inflammation. Hyperoxia at 1 and 2 ATA significantly increased transcutaneous PO2 levels in undisturbed and in inflamed skin but did not fully correct the relative hypoxia at the site of inflammation. Hypercapnia was reduced with O2 breathing at 2 ATA. The apparent rate of O2 consumption at the reaction site was raised during hyperoxia, most prominently at 2 ATA. The most intense reactions showed a central relative slowing of laser-Doppler blood flow indicative of microcirculatory impairment. The extent of the relative hypoxia and hypercapnia was greatest in these strongest reactions. The density of lymphocytes and monocytes in biopsies of 48-h reactions was loosely related to the corresponding transcutaneous PO2 measurements. The present study provides evidence that diffusion barriers, in addition to increased local respiration, can contribute to the apparent hypoxia and hypercapnia of this inflammatory model.

Enzyme reaction rates and the stochastic theory of kinetics.

The kinetics of an elementary reaction step are discussed from the viewpoint of the stochastic theory of chemical kinetics. The general form of the rate constant found in the stochastic approach is described and compared with the expression from transition state theory. Whereas the stochastic theory predicts a rate enhancement in cases which are not adiabatic (in the quantum mechanical sense), transition state theory, which is essentially an adiabatic theory of reaction rates, does not permit inclusion of the effect. This effect can be expected to be of greater importance in cases of catalysis by structures, such as enzymes, containing large numbers of vibrational degrees of freedom (particularly low frequency ones) than in cases lacking such structures. The stochastic theory is more general than the transition state theory, the rate constant expression given by the latter being obtainable from the former when restrictive assumptions, including that of adiabaticity, are made. Interpretations of enzyme catalysis based on the transition state theory must thus be viewed as speculative.

Drip breast milk: it's composition, collection and pasteurization.

"Drip breast milk" is that milk which spontaneously drips from the contralateral breask during the suckling of an infant. Biochemically and immunologically, pooled drip milk resembled pooled mature expressed breast milk, although it has a lower fat concentration. About 15% of lactating women are capable of producing drip milk; volumes produced are up to 188 ml/donor/day. A milk bank is described which processes 1400 liters of drip milk/yr. Heat treatment of this milk with a semi-automated holder pasteurizer caused a 21% reduction in IgA concentration and a 36% reduction in lysozyme activity, as well as a decrease in the ability of the milk to inhibit the growth of E. coli. In comparison with boiling, pasteurization was as effective in reducing total bacterial content provided the milk initially contained fewer than 10(6) bacteria/ml.

Lymphocyte subsets and Langerhans cells in allergic and irritant patch test reactions: histometric studies.

This study has attempted to distinguish between allergic and irritant reactions to patch tests by semiquantitative histological methods. The extent of perivascular chronic inflammatory infiltrate at 72 h in irritant patch test reactions to sodium lauryl sulphate was shown to be small and very consistent, whereas in allergic reactions to nickel sulphate it was generally larger and more variable in size (p less than 0.02). The two major lymphocyte subsets (T4 and T8) were randomly intermixed in both types of reaction and formed the major component of both the perivascular and diffuse dermal infiltrate, without any evidence of selective migration. The T4:T8 ratios were similar in focal and diffuse infiltrates. The number of T6 dendritic (putative Langerhans) cells in the epidermis (per mm inner epidermal length) was usually greatly reduced in irritant reactions (5-16 mm-1, mean 10 mm-1) but remained within normal limits in allergic reactions (6-33 mm-1, mean 21 mm-1) (p less than 0.001). Comparable results were seen with other irritants (mercuric chloride and benzalkonium chloride) and other allergens (neomycin sulphate, ethylene diamine and potassium dichromate). In additional experiments, pairs of biopsies were taken from the reaction and from adjacent unaffected skin. The T6 cell density in the epidermis did not significantly differ between allergic reactions and control skin. By contrast, the irritant reactions had fewer T6 cells than the control skin (p less than 0.001).

The effect of culture temperature on the kinetics of human lymphocyte stimulation by phytohaemagglutinin.

The effect of varying the culture temperature on the growth kinetics of human lymphocytes stimulated by phytohaemagglutinin (PHA) has been studied by a new method using a Coulter Channelyzer. Each of three subjects had a distinctive percentage of responding cells (P less than 0.01), greater after 22 hr culture than after 15 hr (P less than 0.05) due to continued recruitment, but these values were unmodified by variation of the culture temperature in the range 35 degrees C--39 degrees C. The incremental growth rate is the major descriptor of cellular growth and, not unexpectedly, each subject has a distinctive value (P less than 0.01) which is greater after culture for 22 hr than after 15 hr (P less than 0.05). We attribute the increase to the effect of recruitment in modifying the mean value of this parameter. These growth rates were similar at 35 degrees C and 37 degrees C (P less than 0.01), but higher at 39 degrees C (P less than 0.01). Lymphocytes cultured at 41 degrees C failed to grow satisfactorily in either the control or PHA-stimulated wells and it was obvious that cells were damaged at this temperature. Our results establish that PHA-stimulated lymphocytes grow more quickly at 39 degrees C than at 37 degrees C in the early stages of the first cell cycle, but that there is no increase in the number of responding cells at the higher temperature. The results suggest that moderate fever might have a beneficial effect on lymphocyte responses to infection.

Measurements of blood flow and histometry of the cellular infiltrate in tuberculin skin test responses of the typical Koch type and the non-turgid variant form (Listeria-type) in pulmonary tuberculosis patients and apparently healthy controls.

The typical turgid Koch type and the non-turgid variant form (Listeria-type) of the tuberculin skin test responses were studied in 76 newly diagnosed pulmonary tuberculosis patients and 29 apparently healthy factory worker controls from Surabaya in Indonesia; in general, the patients had more intense responses than the controls. The blood flow velocity (RBCflux) at the centre of the reaction was similar in all groups, but central relative slowing (a presumed forme fruste of severe ischaemia) was much more common in the Koch-type reactions in tuberculosis patients. In both groups of subjects, the overall density of cellular infiltrate (and the major populations of inflammatory cells) was greater in the typical Koch-type reactions than in the non-turgid variant reactions. Thus the Koch-type reactions were indubitably more intense in inflammatory terms than the non-turgid variant form, but the results of this study do not exclude the possibility that there were underlying qualitative differences in pathogenesis between reactions of the two types as well as the obvious difference in severity.

Circulatory adaptation to the increased metabolism in the skin at the site of the tuberculin reaction.

The sequence of changes at the site of a positive tuberculin test response were studied in 19 healthy young adults who had been immunised with BCG in childhood. The development of erythema preceded that of induration and both were most intense at 48-72 h. The strongest reactions showed higher laser Doppler (LD) flux at the periphery than at the center (central relative slowing). All showed a substantial reduction in steady-state (ss) tcpO2 from 24 h onwards and the oxygen consumption rate (mlO2.kg-1.min-1), calculated from the rate of fall in tcpO2 during temporary cuff occlusion of arterial input, was raised (greater than two-fold) throughout the period of study (to 96 h). The density of lymphocytes and macrophages in the inflammatory infiltrate in the dermis was related to the fall in tcpO2.ss and to the extent of thickening of the dermis. These experiments showed that the previously healthy dermal microcirculation can adapt to temporary increase in metabolic demands of leucocytes emigrated from the circulation into the tissue: in intense delayed hypersensitivity (DHS) reactions there is considerable hypoxia and respiratory debt, but maintenance of viability in the short-term. It is likely that similar adaptations occur in the period of establishment of microbial infection.

Mechanisms of phytohaemagglutinin (PHA) stimulation of normal human lymphocytes: 'trigger' 'push' or both?

The growth in volume of human peripheral blood lymphocytes after stimulation with various concentrations of PHA was measured with an electronic particle counter. The percentage of growing cells and averaged values describing their growth rates during the elapsed period of culture were estimated by fitting to the observed data the volume distributions derived from a mathematical model. With sub-optimal doses, the percentage of cells stimulated , and their incremental growth rate, increased with increasing dose of PHA, but the time-course of recruitment into the G1-phase was similar with all PHA concentrations studied. The results provide strong support for the 'trigger' hypothesis that there is a distribution of stimulation thresholds within the lymphocyte population: consequently, increasing mitogen concentration will be expected to result in increased numbers of responding cells within the suboptimal concentration range.