Mast cells in leprosy skin lesions.

The variability of mast cell density within and between leprosy skin lesions was examined as a basis for future studies, and whether the number of mast cells in the lesion was determined by local or systemic factors was evaluated. The mast cell density in the granuloma, skin appendages, and intervening dermis was assessed by counting mast cells in glycol methacylate sections stained with Giemsa stain and relating these counts to area measurements obtained by planimetry. In biopsy specimens taken from the edge of established lesions the density of mast cells within the granulomata was considerably higher than that in the intervening dermis and was comparable with that found in the appendages. No major differences in mast cell density were found between unaffected skin and the centre or edge of individual lesions. Mast cell densities in biopsy specimens from the edge of different lesions on the same patient were also similar, suggesting that the mast cell density within the granulomata is independent of the site of the lesion and is determined systemically.

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 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.

Observer variability in histopathological reporting of malignant bronchial biopsy specimens.

AIMS: To evaluate the ability of histopathologists to classify lung carcinomas on bronchial biopsy material using the current World Health Organisation (WHO) classification. METHODS: Eleven histopathologists each reviewed 100 randomly selected bronchial biopsy specimens which had originally been reported as showing lung carcinoma. A single haematoxylin and eosin stained section from each case was circulated and a standard proforma completed. These were analysed using kappa statistics. RESULTS: The histopathologists were excellent at distinguishing between small cell and non-small-cell carcinoma kappa = 0.86), but not so good at subclassifying the non-small cell carcinoma group kappa = 0.25). CONCLUSIONS: The clinically important distinction between small cell and non-small cell carcinoma of the lung is reliably made by competent histopathologists even on limited material.

The variations in thyroid function tests compatible with clinical euthyroidism after medical and surgical treatment for Graves' disease.

Eight widely used tests of thyroid function (clinical and biochemical) were performed simultaneously on 46 thyrotoxicosis patients who were clinically well at a standardised interval after medical or surgical treatment. The aim was to establish the range of thyroid function compatible with clinical normality. While clinical scoring systems gave broadly similar results between surgically and medically-treated patients, thyroid biochemistry showed major differences. Thus while 87% of medically-treated and 74% of surgically-treated patients were classified clinically euthyroid by the clinical indices devised by Crooks and Billewicz, the TRH test was exaggerated in 77% of the latter group but in only 13% of the former. On the other hand, half of the 26% of surgical patients with a clinical index suggesting hyperthyroidism had normal thyroid hormone levels and exaggerated TRH tests. No patient was categorised as clinically hypothyroid despite basal TSH levels as high as 21 mU/L (N : less than 5 mU/L) and T4 levels as low as 42 nmol/L (N: 70.150 nmol/L). Substitution of FT4 index for total T4 did not significantly alter the classification or ranking of patients in either treatment group. The early (20-minute) 132I uptake was normal in all but one case. We conclude that abnormal laboratory tests of thyroid function are found frequently after treatment in thyrotoxicosis patients with few or no clinical features of thyroid disturbance.

Patterns of blood flow in the microcirculation of the skin during the course of the tuberculin reaction in normal human subjects.

Digital thermographic imaging and laser-Doppler velocimetry techniques were used to study the sequence of microcirculatory changes in typical Type IV (DHS-type) skin reactions to intradermal injection of PPD into seven healthy tuberculin-positive subjects. Thermography has shown that there is very little change in thermal status at 6 hr, that there is an area of raised temperature roughly corresponding to that of erythema around the site of antigen injection at 24 hr, and that the area of 'hot' skin starts to exceed that of the clinically observed reaction at 48 hr and remains extensive even at 96 hr. The area of the increased RBC flux detected by the laser-Doppler technique corresponded generally to that of erythema for the first 48 hr, but thereafter became smaller than the clinically observable reaction. Cuff occlusion experiments showed that the phenomenon of reactive hyperaemia is abolished at 24 and 48 hr in conditions of maximal hyperaemia, but that this response is recovering by 96 hr. Vasomotion was significantly exaggerated between 48 hr and 96 hr. These studies indicate that there is maximal arteriolar vasodilatation during the first 2 days of the reaction with rapid blood flow in the congested capillaries of the dermal papillae, and that engorgement of deeper venous plexus of the dermis is the predominant vascular change in the resolving response at 72-96 hr.

Measurement by Quantimet 720 of the proportion of actively growing cells in tissue cultures of human lymphocytes.

The size-distribution of normal human lymphocytes growing in tissue culture was measured with a Quantimet 720 image-analysing computer. The proportion of cells undergoing volumetric growth after phytohaemagglutinin (PHA) stimulation was dervived from a simple mathematical model describing the growth patterns of the cells up to the first mitotic division. The approach may have general application in biology as a method for measuring the proportion of growing cells by microscopy.

Influence of H-2 genes on growth of Mycobacterium tuberculosis in the lungs of chronically infected mice.

Mice infected by intraperitoneal injection of Mycobacterium tuberculosis were studied over a 23-week period. They showed progressive infection in the lung (with increasing microbial count and granuloma size) whereas viable bacillary counts remained largely stationary in the spleen and in the liver. The influence of H-2 genes on the progression of the lung infection was studied in four congenic strains of animals with B10 and three congenic strains of animals with BALB backgrounds. H-2k mice had significantly higher bacterial counts in the lung than H-2b mice on both B10 and BALB backgrounds, BALB. K (H-2k) mice were also more susceptible than BALB/c (H-2d) mice. Results with recombinant strains showed that bacillary counts and granulomatous infiltration were lower in the B10 (KbAbE-Db) compared with B10.A(3R) (KbAbEbDd) strain and in B10.A(4R) (KkAkE-Db) compared with B10.BR (KkAkEkDk) mice. This resistance to the late expansion of tuberculous infection in the lungs may be associated with the lack of an expressed I-E molecular or with the expression of the Db molecule.

The relationships between surgical histometry, outcome and pre-treatment in Graves' disease.

Quantitative histometric methods were used to established the relationships between the extent of thyroid lymphocytic infiltration at operation, and outcome exactly 18 months later in 50 surgically-treated Graves' disease patients prepared by carbimazole and triiodothyronine. Periods of pre-operative treatment, surgical technique, histometric analysis and diagnostic criteria were all standardised. Controls (107) were obtained from the forensic laboratory. Thirty-seven patients became euthyroid, but there was no relationship between outcome and epithelial or lymphoid content of the thyroid gland. Neither was there any correlation between the size of lymphoid infiltrate and epithelial mass of the resected thyroids, suggesting that simple lymphocyte infiltrations do not replace thyroid tissue as once thought. The variation in thyroid epithelial content was nearly 3-fold, so that a surgeon, even if able accurately to judge the anatomical mass of the remnant, would still have little or no idea of its functional mass. The scatter of epithelial content was even greater in glands from patients prepared for surgery by propranolol alone (38 glands, variation X 5.5) or propranolol and iodide (32 glands, variation X 5.9). Outcome after sub-total thyroidectomy for Graves' disease seems unrelated to the lymphocyte content of the gland and it is questionable to what extent the surgeon can either predict or control the outcome of thyroidectomy in individual Graves' disease patients.

Assessment of the respiratory metabolism in the skin from transcutaneous measurements of pO2 and pCO2: potential for non-invasive monitoring of response to tuberculin skin testing.

A method is described for non-invasive transcutaneous (tc) measurement of tissue respiratory gas tensions in the skin on the forearm for study of delayed hypersensitivity reactions in man. Steady state values for tcpO2 and tcpCO2 were measured, and the skin respiratory rate (oxygen consumption) and the tissue pH were estimated from the changes in tcpO2 and tcpCO2 observed after interruption of the arterial circulation by cuff occlusion for 4 minutes. The extent of within-experiment and between subject variation in the steady-state measurements was not great (coefficient of variation 10%): tcpCO2.ss (steady state) was higher in men and tcpO2.ss was higher in women, but the extent of these sex differences was also small. Reference ranges have been established for tc measurements and calculated indices of tissue respiration in the undisturbed forearm skin of normal volunteers, against which the changes induced by tuberculin testing can be assessed. Severe changes, indicative of profound hypoxia and acidosis, are seen in intense delayed hypersensitivity reactions. Similar, but less severe changes were seen at the site of skin tests on BCG-vaccinated subjects who were 'negative' by conventional criteria of measurement of dermal induration and they became greatly exaggerated after successful re-vaccination. Intradermal injection of saline did not induce hypoxia or local acidosis. These new methods are very sensitive indicators of the tissue response in the DHS reaction.

The relation between cutaneous blood flow and cell content in the tuberculin reaction.

The relationship of velocity of blood flow to density and microanatomical distribution of inflammatory cells in the dermis was studied in 20 human tuberculin tests. Most positive reactions showed maximal blood flow velocities (measured as red blood cell (RBC) flux) at the centre of the reaction, but the two most intense responses showed 'central relative slowing' (CRS) with higher RBC flux at the periphery. Two of the four clinically negative reactions showed a considerable acceleration of blood flow, but the other two showed no such acceleration. The packing density of lymphocytes/monocytes in the perivascular zone was greater in the stronger positives than in the weaker reactions. The density of cells in the intervening dermis was markedly lower than in the foci: the lesions with CRS had the highest density of cells in the diffuse infiltrate of the reticular dermis. At the centre of the reaction, blood flow velocity was generally related to density of cellular infiltrate, except in those with CRS, which had a disproportionately lower blood flow velocity. The finding that the circulatory adaptation to a delayed hypersensitivity reaction can be inadequate may explain the dermal acidosis previously observed in intense skin test reactions, and may be the underlying mechanism of necrosis in hypersensitivity reactions.