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Summary Human rheumatoid and non-rheumatoid synovial lining cells have been studied by scanning and integrating microdensitometry. The ferric ferrocyanide reaction, controlled by blockade with mercury or maleimide, has been used for investigating sulphydryl groups; glucose 6-phosphate dehydrogenase and NADPH-diaphorase activities have been studied by quantitative tetrazolium cytochemistry. It has been shown that a greater proportion of sulphur-containing proteins in the rheumatoid cells are in the reduced (sulphydryl) state than in the non-rheumatoid cells. This supports the suggestion that the redox balance of human rheumatoid synovial lining cells is displaced towards the more reductive condition. Further support is given by the finding that, in the rheumatoid cells, the rate of production of NADPH-hydrogen by glucose-6-phosphate dehydrogenase activity is about 4 times greater than in the equivalent non-rheumatoid cells; there is little change in the rate of oxidation of the reduced NADP, so indicating the possibility of a metabolic basis to the reductive imbalance in the rheumatoid cells.

Crystals of hypoxanthine and xanthine and of oxipurinol (a metabolite of allopurinol) have been found in skeletal muscle biopsies from gout patients who are being treated with the xanthine oxidase inhibitor, allopurinol. These patients have no clinical manifestations of muscle disease. Biopsies from normal control subjects contained no more than two hypoxanthine crystals per standard size section, and no other crystals. Untreated gout patients' biopsies usually contained crystals of one or more of the following: anhydrous uric acid, uric acid dihydrate, sodium urate monohydrate, and no other crystals. The crystals were identified by: their size and shape, their extinction angle, their sign of birefringence, their refractive index, and the degree of asymmetry of the birefringence which was measured by positive and negative compensation in media of different refractive index. The latter method is described in the Appendix to this paper. The implications of these results for the treatment of gout, their relationship to the findings in congenital xanthine oxidase deficiency (xanthinuria) and the possible metabolic origin of the crystals are discussed.

NO methods are at present available for the detection of subnormal levels of pituitary hormones in serum or plasma. Even the most sensitive bioassays, radioreceptor assays and radioimmunoassays can detect only picogram per ml. concentrations. A hew biological assay has been described recently capable of determining femtogram (10−15 g) amounts of ACTH in diluent1,2. This report concerns the validation of this assay in a comparative study with the Lipscomb–Nelson bioassay and two radioimmunoassays (directed against different parts of the ACTH molecule). Subnormal levels of circulating ACTH following the i.v. administration of Cortisol to a normal subject have been measured for the first time, and the half-time for disappearance of endogenous hormone calculated.

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.

Specimens of myocardial tissue, taken from the apex of the left ventricle at biopsy and or at necropsy from seven patients with congestive cardiomyopathy, have been examined by a variety of histological and biological techniques. Sections of myocardial tissue taken from all seven patients contained peculiar structures; and these structures were seen in only one out of a hundred and twenty specimens taken from patients undergoing surgery for other lesions. The structures were recognised by their metachromtic staining, and were pleomorphic. The larger structures, termed "mark bodies", frequently appeared as a halo around swollen nuclei. Biochemical tests indicated that the structures were living but foreign to the myocardial tissue. The agent could not be grown by conventional bacteriological procedures, but did grow when specimens of tissue were placed in maintenance culture, with or without certain additives, and at high oxygen tensions. Injections of cardiomyopathic tissue proved fatal to mice and metachromatic particles could subsequently be identified in these animals. Thus Koch's four postulates are satisfied and it seems that there may be an aetiological infective agent in human congestive cardiomyopathy.

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.

An inhibitor of renal sodium-potassium-dependent ATPase was demonstrated in human plasma by a cytochemical technique. Circulating levels of this activity were 25 times greater in plasma taken when five healthy subjects were on a high-salt diet than in plasma obtained when they were on a low-salt diet. These results are consistent with the proposal that urinary sodium excretion is in part controlled by a circulating natriuretic substance.

Despite the high circulating levels of immunoreactive PTH in patients with pseudohypoparathyroidism type I (PSPI) the levels of bioactive PTH (bioPTH) have been found to be close to the normal range. To elucidate this dissociation, we have studied the recovery of the biological activity of bovine PTH added to the plasma of patients with either PSPI, or with hypoparathyroidism (PTX), primary hyperparathyroidism (HPT) or of normal subjects. In PSPI (n = 10) the recovery of biological activity was 5.6% +/- 3.6 (mean +/- SEM) whereas in PTX (n = 7), in HPT (n = 4) and in normal subjects (n = 8) it was 79% +/- 5, 75% +/- 9 and 68% +/- 4, respectively. In another PSPI patient, who had undergone total parathyroidectomy, bioPTH was undetectable but the recovery from the plasma of added PTH was 84%. Thus we have found inhibition of PTH bioactivity by plasma of PSPI patients which was absent after parathyroidectomy.

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.

Infra-red spectra have been obtained from small areas of tissue sections by the use of areflecting microscop in conjunction with an infra-red spectrometer. Absorption bands characteristic of both types of nucleic acid are obsered. The possibility of using these absortions for their separate detetion and estimation is discussesd.

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.