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Adult primary myxoedema is usually due to an autoimmune thryoiditis characterized by progressive shrinking of the thyroid gland, loss of epithelium, dense infiltration by sensitized lymphocytes and plasma cells with final replacement of the gland by a fibrous scar. Antibodies directed against thyroglobulin (TgHA) and microsomal antibodies (McHA), detectable years before the onset of hormonal failure, and cell-mediated immune mechanisms contribute to the pathogenesis. When 90% of the gland is destroyed, the secretion of thyroid hormones (T3/T4) falls below normal needs and pituitary thyrotrophs react by producing over 100 times the normal amount of thyroid-stimulating hormone (TSH) which should normally lead to regrowth of the gland as happens in goitrous Hashimoto thyroiditis. The inability of the thyroid in primary myxoedema to respond to the trophic action of TSH suggested that blocking antibodies exist in this disease which compete with TSH for its receptors. The trophic effect of TSH can be assayed in vitro by measuring DNA-synthesis by Feulgen cytophotometry; elevated pentose-shunt oxidative activity correlates with DNA synthesis. Here we report that immunoglobulins from patients with adult, primary myxoedema block the trophic effect of TSH, as assessed by this in vitro system. This is in marked contrast to the growth stimulation induced by immunoglobulins from patients with thyrotoxic Graves' disease with prominent goitres.

THE increased activity of pentose-shunt dehydrogenases, which had been demonstrated histochemically in cancers1, has been partly confirmed by biochemical investigations in carcinoma of the cervix in that the cells of the vaginal secretion gave high values for 6-phosphogluconate dehydrogenase2. Since this is said to be an oxidative pathway3, attempts were made to test if a concomitant rise in cytochrome oxidase activity could be demonstrated. This was done histochemically, to avoid dilution of active cancer tissue by inactive or necrotic tissue and by stroma. A more intense and permanent histochemical procedure for cytochrome oxidase4 was applied to sections from eight apparently normal cervices, three apparently normal endometria, ten primary carcinomata of the cervix and seven other carcinomata of the genital tract. The tissues were removed at operation and were frozen and sectioned by a controlled temperature freeze-sectioning method4,5. The cytochrome oxidase-levels were not elevated in the cancers; in some they seemed somewhat depressed.

RECENT biochemical studies on succinic dehydrogenase in livers of rats fed with the liver carcinogen p-dimethylaminoazobenzene (DAB) for periods of up to 150 days have confirmed the well-established phenomenon that the level of this enzyme generally falls as feeding progresses (Table 1). Samples of tissue from each liver lobe were taken for histological and histochemical investigations, and the remainder was homogenized. Succinic dehydrogenase was estimated to within an accuracy of ± 10 per cent by the anaerobic methylene blue technique, basing the QMB values on dry weight. For histochemical purposes the tissue was frozen1, stored for not longer than a week, and cut at − 20° C. in a refrigerated microtome. Succinic dehydrogenase was detected higtochemically by incubating frozen sections in a neotetrazolium-succinate buffer system2; in addition, the periodic acid–Schiff method3 was used to stain glycogen, while phospholipid was stained by the acid haematin method4.

THE dilemma of enzyme histochemistry is that if the tissue is not fixed, to avoid denaturation, the enzymes may dissolve out of the section into the incubation medium but, conversely, if the tissue is fixed to stop such loss of soluble enzymes, the fixation will inhibit activity and cause generalized denaturation. Thus, for most enzymes, it is argued that too much of the ‘soluble’ or lyo-form of otherwise bound enzymes1 will be lost from unfixed sections during incubation in an aqueous medium; consequently it is considered advisable to fix the tissue prior to such incubation1–3. Although it is usually conceded that the histochemistry of dehydrogenases depends on the use of unfixed sections, some4 have argued that even these should be studied in fixed tissue. Consequently it follows that the scientific basis of enzyme histochemistry must remain uncertain as long as a considerable loss of the activity to be investigated must be expected, whether this is caused by solution into the medium from unfixed sections, or from inhibition of activity produced by the preparative procedures.

XANTHINE oxidase (xanthine: oxygen oxidoreductase, EC 1.2.3.2) catalyses the oxidation of hypoxanthine to xanthine and of xanthine to uric acid. The increased excretion of hypoxanthine and xanthine, and the very low concentrations of uric acid in blood and urine which are characteristic of xanthinuria, were first reported by Dent and Philpot1 and by Dickinson and Smellie2. Gross deficiency of jejunal and hepatic xanthine oxidase was reported by Watts et al.3 and by Engelman et al.4. We report here the occurrence of crystalline material resembling xanthine and hypoxanthine in the striped muscle of two xanthinuric patients.

THE cytotoxic effect on mouse ascites tumour cells treated in vitro with various antibodies has been shown to require the presence of complement1,2. In the absence of complement the cells appear to be normal when viewed by phase contrast microscopy3; they retain their capacity to exclude trypan blue2 and to metabolize glucose4. The only changes which have been reported are slight alterations in the fine structure of the cell surface, which were thought to favour agglutination by rabbit antibody directed against ascites cells3.

IN a series of detailed publications1–5, Fine et al. have maintained that the probable cause of death in traumatic shock is the irretrievable collapse of the circulation caused by a bacterial toxin which intensifies vasoconstrictor activity so as to injure the splanchnic viscera, with resulting decline in vascular reactivity to catechol amines and functional damage in key organs, especially the liver and intestine. In the absence of any obvious systemic or local infection, these workers have considered that the most likely source of the toxin is an endotoxin derived from the intestinal flora. The difference between that type of shock which responds to resuscitative measures, that is, ‘reversible shock’, and the irreversible form would appear to depend on the ability of the reticulo-endothelial system, particularly in the liver and spleen, to inactivate this toxin. This ability declines if the animal is in shock for 2 h or longer. There are indications that this detoxication process is concurrent with an esterase-like protein in spleen and liver that releases phosphate. Abolition of vasoconstrictor activity in the splanchnic area by cœliac blockade preserves this process as well as the vascular integrity of the portal and systemic circulation. Under these circumstances the animals are able to survive an otherwise lethal exposure to shock. It is noteworthy, however, that while functional differences in the spleens and livers of normal and shocked animals have been observed, no histological abnormalities have been detected.

IT is believed generally that the normal complement of deoxyribonucleic acid (DNA) occurs exclusively on the chromosomes. However, that this is true of all plant cells has been contested. Thus ultra-violet absorption microscopy was used to show that it occurred predominantly in the cytoplasm of the root meristem cells of Vicia faba which had been treated with pectinase1. These results were supported by results obtained from fractionated homogenates of roots of V. faba and Allium cepa2. It was claimed that this localization was dependent on the lifelike preservation of the cytoplasmic particles, and that if these were damaged in any way the DNA would diffuse to the nuclei. However, these results were inadequate, partly because of the extreme variability of the commercial pectinase samples that were available1, and partly because it was just as reasonable to postulate that homogenization caused the DNA to diffuse from the nuclei instead of to the nuclei3.

THE uptake of 3,4-benzpyrene by cells has been of interest to many workers, particularly in view of the carcinogenic properties of this substance. Moreover, it is a fluorescent compound and has been used as a fluorochrome for lipids into which it will dissolve from a hydrotropic solution ; caffeine is usually used for dispersing it finely in water, since alone benzpyrene appears to be almost completely hydrophobic. Berg1 has used this method in a detailed and extensive study of the distribution of lipids in a wide range of normal and cancerous animal cells and in the root tips of Allium cepa. In order that the lipids should not be affected by his preparatory procedures, he fixed the tissues in formalin which had been stored over powdered chalk ; chalk seems to have the same effect as calcium chloride, which is frequently added with the intention of maintaining a high concentration of calcium ions to immobilize lipids2. This method of fixation seems to be much favoured by histologists. The tissue was sectioned with a freezing microtome, and the sections were then stained with benzpyrene. After a very careful investigation, Berg was forced to conclude that in animal and plant nuclei lipids are not present except in the nuclear membrane, and that biochemical demonstrations of these compounds in extracted nuclei were due to contamination by the cytoplasm. Indeed, he found that the nucleus was the one region in cells that never showed uptake of benzpyrene.

BIOCHEMICAL and physiological data from plant material are reported frequently as the amount or activity of matter per ceil, yet remarkably few such studies are made directly on the cells. Thus, for example, in a large number of publications on the chemistry and physiology of cell differentiation, very few have included any cytochemical investigations. That this is a general phenomenon is due, very largely, to the fact that no method exists by which plant cells may be investigated both for structure and for chemical activity, without the introduction of gross artefacts1. Animal cytology has advanced more rapidly because of the technique which involves freezing and drying the tissues, which are then embedded directly in paraffin wax and sectioned2. The wax is removed with xylol and the cells are fixed in absolute alcohol. This procedure has failed when applied to plant cells; moreover, the use of fat solvents is to be avoided at any stage.

IT has often been suggested that the erosion of articular cartilage in rheumatoid diarthrodial joints could be caused by lysosomal proteolytic enzymes derived from abnormal lysosomes either in the cells in the synovial fluid or in the synovial cells themselves1,2. Biochemical and histological studies have shown that proteolytic enzymes are released from the lysosomes of chondrocytes in chick limb rudiments which have been treated with an excess of retinol3 and that, in these conditions, there is proteolytic degradation of the cartilage in vitro. In general the critical factor seems to be whether the lysosomal membranes in cells close to the cartilage have been modified, for example, by retinol, in such a way that the normally latent proteolytic activity can become freely manifested. The difficulty in testing this hypothesis in human synovial matter is the many different cell types—each potentially capable of expressing its individual biochemistry—that make up either the synovial fluid or the synovial tissue. It has been shown recently, however4, that suitably prepared tissue sections can be used as if they were the more conventional tissue slices and that the enzyme activity of selected cells or small groups of cells in such sections can be measured directly5,6 if a scanning and integrating microdensitometer7,8 is used in rigorously defined conditions. Moreover, lysosomal proteolytic activity can be measured by the cleavage of the peptide bond of leucyl-β-naphthylamide in suitable conditions9–12; the colour produced in the presence of an azo-dye coupling agent is proportional to the enzyme activity5. Usually, however, not all of the lysosomal enzyme is freely available to the reaction-substrate, but it can be made available provided that the lysosomal membrane is altered by prior treatment at an acidic pH (ref. 13). It is thus possible to measure first the freely available lysosomal proteolytic activity and then, by pretreatment in 0.05 M acetate buffer at pH 5, to test the total proteolytic activity without the interference of the lysosomal membrane12. The difference between these values gives some measure of the “bound” activity. The ratio between freely available and bound activity indicates the functional state of the lysosomal membrane and it is this which we have wanted to test.

IN a series of histochemical studies on tissues of the human and of rats and mice, the activity of the enzyme 6-phosphogluconate dehydrogenase was found to be remarkably increased in proliferating cells, whether they were benign or malignant. This enzyme is part of the respiratory mechanism known as the pentose pathway, or ‘shunt’, which may provide an alternative glycolytic pathway from glucose to pyruvate; it may also be implicated in the synthesis of the nucleic acids1. Because of the possible significance of the results obtained in diverse experiments during the past two years, it was considered advisable to communicate these preliminary findings.

Enzyme Histochemistry and Its Application in the Study of Neoplasms By M. S. Burstone. Pp. xii + 621 (Figures). (New York and London: Academic Press, 1962.) 160s. 6d.

A RECENT histological study1 of the changes that occur in human bronchial epithelium has confirmed in part earlier American findings2 and shown that considerable proliferation may occur in the cells of the basal layer without any other histological abnormality. As it seemed possible that some of these changes might be related to the development of bronchial cancer, a histochemical investigation has been begun. Since this work was not concerned with the distribution of free lipids, formalin-fixed tissue embedded in paraffin was used. The most striking result has been the very marked increase in the content or availability of lipid, probably bound phospholipid3, as shown by colouring with sudan black and staining by the acid haematein method4. This effect was strongest in the nuclei of the basal cells and was particularly well seen in the condition known as basal cell hyperplasia. In contrast, scarcely any lipid could be demonstrated in the cells of foci of established squamous metaplasia.