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Summary The dysfunction of lysosomal membranes has been investigated in a model system of inflammation in which human skin, maintained in vitro in non-proliferative maintenance culture, is subjected to the effect of histamine. The effect of histamine is time and dose related; 10 -7M histamine acting for 5–10 min. has been shown to have a significant effect on the permeability of lysosomal membranes in the epidermis. A comparison has been made between aspirin, salicylate and 4-acetamido-2-acetoxy-benzoate (benorylate) prepared by esterification of N-acetyl-p-aminophenol (paracetamol) with acetyl salicylic acid (aspirin). It has been shown that, in specimens from some subjects, aspirin alone has an activating effect whereas it has a stabilizing effect in other specimens. But in all cases aspirin retarded the effect of histamine on the lysosomal membranes. Benorylate had only a stabilizing effect and markedly inhibited the labilizing influence of histamine. This was not due to the properties of aspirin and paracetamol acting alone, but was a property of the benorylate molecule. It is suggested that the stabilizing effect may be related to the lipophilia of this molecule and its direct influence on the lysosomal membrane. Such studies, on the response of lysosomes in intact tissue, under the influence of a chemical mediator of inflammation, may clarify the controversial question of the effect of aspirin when studied on isolated lysosomes. The stabilizing influence of benorylate on lysosomal membranes found in the model experiments on human skin has been confirmed in studies on human rheumatoid synovial lining cells.

Summary This study has confirmed that the lysosomal membranes in the synovial lining cells of human rheumatoid synovial tissue are abnormally labile; that the cytoplasmic redox balance, as measured by the proportion of dithiol to disulphide groups, is exceptionally reductive; and that there is an excessive rate of generation of reduced NADP despite little change in its rate of oxidation. These results suggested that the increased reductive conditions in the cytoplasm might contribute to the state of the lysosomes. To test this, human synovial tissue was maintained in vitro , in adult maintenance culture, and hydrogendonating or hydrogen-accepting molecules were added to the culture medium. It was shown that ascorbate acts as a hydrogen-donor. In non-rheumatoid cells it increased the proportion of cytoplasmic sulphydryl groups (which indicates a more reductive redox balance) and also increased the lability of the lysosomal membranes. Hydrogen-accepting molecules, such as dehydroascorbate and particularly menadione, produced the reverse effect. Similar effects occurred in rheumatoid synovial lining cells, in which menadione also diminished the rate of generation of NADPH. It seems likely, therefore, that redox systems in the cytoplasm and in the extra-cellular space, can influence the cytoplasmic dithiol: disulphide balance and the functional state of lysosomal membranes in synovial lining cells. In rheumatoid synovial tissue, menadione diminishes the excessively reductive conditions in these cells and stabilizes lysosomal membranes.

It has been found that the heterochromatic region of interphase nuclei of plant roots and calf thymus, fixed in formaldehyde solution, do not stain for phospholipid as they do after Lewitsky's fixative. It has been shown that this is probably due to the extraction of a protein-phospholipid complex. Some lipid-like material has been purified from the used formalin and shown to contain glycerophosphate. The nature of the association between the lipid and protein is discussed.

Plant roots have been homogenized by a new method and the cell organelles were fractionated by differential centrifugation. Nuclei isolated by ideal conditions did not appear to contain DNA when examined cytochemically, although they did contain DNA if the roots were crushed or ground prior to, or during, homogenization. Biochemical estimation of the DNA showed that most of it was associated with the cytoplasmic fractions if the roots were unfixed, or treated with polyvinyl alcohol, but it was mainly nuclear if the roots were pretreated with an acetate solution of low pH or high ionic concentration. The significance of these, and of previous cytochemical observations on intact cells, is discussed.

A study of the activity of lysosomal enzymes in the retina and the structural integrity of lysosomes in the pigment epithelium of the normal and dystrophic rat eye was carried out using standard biochemical and cytochemical techniques respectively. The experiments were performed on eyes from rats aged 1–12 weeks which covers the developmental period of the retina and progressive stages of the degeneration. Measurement of total and free lysosomal enzyme activity in the retina was achieved by the use of acid protease as the lysosomal enzyme marker. Increases in both total and “free” activities above those found in the normal retina were first observed in the dystrophic retina at 4 weeks; these differences became more apparent as the degeneration progressed. The stability of lysosomes in the pigment epithelium was studied by measuring the accessibility of the lysosomal enzymes naphthylamidase and acid phosphatase to the respective substrates. It was found that, as early as 1 week of age, the lysosomes in the dystrophic pigment epithelium are less stable than those in the normal tissue. This instability became more pronounced as the animals aged. The results support the hypothesis that the degeneration of the visual cells in inherited retinal dystrophy is initiated by the infiltration of lysosomal enzymes from the pigment epithelium.

Summary 1. In previous studies three histochemical tests have been useful in detecting myocardial dysfunction. 2. These have been applied to left ventricular biopsies from 37 patients who underwent prolonged open-heart surgery. The immediate objective was to investigate whether these tests could be used as a guide to prognosis. 3. There was good correlation between the graded response to the histochemical tests and the clinical assessment of the myocardium. The tests over-warned in 3 cases out of 15, and did not warn of myocardial dysfunction in 2 cases out of 22 which showed clinical dysfunction. 4. One of the most difficult aspects of the pre-operative assessment of left heart lesions is the evaluation of the state of the myocardium. The development of cardiac biopsy methods that can be performed at the time of catheterisation would allow such histochemical analyses of myocardial function to be more widely used.

Burstone's procedure for the histochemical demonstration of cytochrome oxidase has been studied and applied to sections prepared by freezing in hexane and cutting by the controlled-temperature freezing-sectioning technique. The method has been modified by the inclusion of a treatment with Lugol's iodine to yield a stronger colour, which is stable for at least several weeks and which localizes the enzyme activity more precisely. The variants of the reaction have been compared in their effect on cardiac muscle, liver, and kidney of the rat.

Adenosine triphosphatase has been demonstrated histochemically in rat and human myocardium. To obtain its precise localization in discrete bands, apparently corresponding to the concentration of myosin, it was necessary to modify the existing technique to obtain better preservation of unfixed tissue and maximal enzymatic activity. Thus it was necessary to increase the concentration of calcium and to effect the reaction at pH 9.4 after treatment with 2:4-dinitrophenol. The specificity of the reaction was shown by these factors, by testing with phosphate esters other than adenosine triphosphate, and by the inhibitory effect of magnesium.

It has been shown that when methyl green is used to stain DNA, as by Kurnick's method, it binds firmly and cannot be washed off by immersion in a buffer solution. When tissue is stained with methyl green for a short time, however, either alone or mixed with pyronin, no strong binding is produced between the methyl green and the DNA. It is suggested that although Kurnick's method may be selective for DNA, the differential staining of cell structures produced by short treatment with methyl green-pyronin is due to competition.

The livers from rats fed either on a normal diet containing 20% of protein or on a diet which facilitated liver carcinogenesis and which contained only 5% of protein supplemented with methionine appeared histologically normal. Histochemical investigation of such livers showed that in the livers of animals fed on the protein-deficient diet there was a large increase in lipid staining. Biochemical investigation of these livers demonstrated that this increase in lipid staining was not due to a significant increase in total lipids nor to a significant decrease in total protein. These findings are discussed in terms of the ‘unmasking’ of the structural cytoplasmic lipids of the liver cells as the consequence of the feeding of an apparently protein-deficient diet.

The fixed nucleus of Vicia faba has been investigated in squash preparations and in sections and it has been shown to consist of separate regions: the peripheral zone, in which lie the chromosomes; the middle zone, which is a protein region, the size of which varies with the mitotic activity of the root and in which lie the nucleoli; the nucleoli, each of which contains an inner core of protein material surrounded by an outer zone of material which probably contains nucleoprotein. The nucleolus also bears the nucleolar organizer, which is connected to the peripheral zone by a fine chromosomal strand. It is suggested that the apparent expansion of the middle zone during the period of maximum mitotic activity of the root is due to hydration of the protein. Similar zonation of the nucleus and nucleolus has been found in fixed root tips of Allium Cepa and Zea mays . Nuclei of living staminal hair cells of Tradescantia bracteata have been studied with ultraviolet microscopy and have been shown to possess the same structure as was found in the fixed nuclei of Vicia faba . This suggests that, at least in these cells, the middle zone and nucleolar core are not artifacts.

A new bioassay for A.C.T.H. has been devised utilising non-proliferative organ culture of segments of guinea pig adrenal. The cultured adrenal is exposed to standard or unknown A.C.T.H. and sections stained for the presence of reducing groups. The intensity of staining in the zona reticularis is measured by scanning and integrating microdensitometry and is inversely proportional to the logarithm of the concentration of A.C.T.H. in the culture medium over the range 0.0027–27 pg/ml. Preliminary results using the assay, which greatly exceeds others in sensitivity, on human plasma are similar to those obtained with standard techniques.

1. 1. After the fixation of roots and anthers in Lewitsky's fluid the chromosomes in mitosis and meiosis show a cyclic staining behaviour in a mixture of orange G and aniline blue. In mitosis the change occurs before the dissolution of the nucleoli and in meiosis when the dissolution of the nucleoli is almost complete. 2. 2. In root tips of seven species the euchromatic parts of chromosomes stain yellow from about mid-prophase to the end of anaphase but blue at telophase, interphase and early prophase. Heterochromatic regions, however, in species where they are detectable, stain yellow at all times except in early prophase. 3. 3. A deviation from this staining behaviour is found in root tips in a narrow region of peripheral cells and in cells adjacent to the root cap. In these cells the chromosomes at all times stain yellow and the cytoplasm yellowish. A study of the early development of lateral roots in Vicia faba indicates that this is not an artefact. 4. 4. Examination of pollen grains in two diploid species of Tradescantia show that during mitosis the chromosomes undergo changes in staining similar to those found in meristematic cells. The vegetative and generative nuclei which arise from this mitosis have different staining qualities. The vegetative nucleus stains blue for a long period of pollen development but ceases to stain at all just before anthesis; the generative nucleus, however, stains blue for a brief period and yellow thereafter. 5. 5. In pollen-mother cells the chromosomes stain blue at leptotene and pachytene but yellow during diakinesis. At the second division the chromosomes stain as in mitosis. 6. 6. Under these conditions of fixation and staining, nucleoli always stain yellow, the cytoplasm stains greyish, the spindle blue but the nuclear sap remains colourless. The cytoplasm in the “vegetative cell” in pollen grains has an increased tendency to bind orange G. 7. 7. The staining behaviour of the chromosomes and nucleoli are briefly discussed in relation to earlier ideas on the chromosomal “matrix”.

1. The total mass M of substances other than water (the dry mass) in the living cell can be obtained from the expression M = φA/χ, where φ is the optical path difference (o.p.d.) due to the cell and A its projected area. The method makes use of the fact that the refractive increments α(χ = ioocα) of most substances in cells are approximately the same, and independent of concentration. Values for χ have been tabulated. Inaccuracies in the measurement of dry mass due to variations in χ (using λ average = 0.18) will be less than ± 10 per cent, in cells containing nucleic acids, proteins, and lipoproteins. When appreciable quantities of other substances are present the inaccuracy may be somewhat greater. When the total dry mass of living cells is determined in a medium other than water (e.g. isotonic solution), a correction term involving the thickness must be determined; this correction is often small. 3. The total dry masses and, in some cases, the concentrations of dry substance in a variety of biological objects including Amoebae, pollen grains at various stages of development, nuclei of cells in tissue culture, and sperm heads have been determined. In Tradescantia bracteata, during development from the microspore to the mature pollen grains, the dry mass increases by about tenfold. The dry masses of mature pollen grains were measured before and after successive digestion with ribonuclease, which removed about 4 to 14 per cent, of the dry mass, and with trypsin, after which about 40 per cent, of the original dry mass remained. In living ram sperm heads the ratio of deoxyribose nucleic acid to total dry mass determined by ultra-violet and interference microscopy respectively is 40 per cent. This is in good agreement with the value 45 per cent, obtained by bulk biochemical methods. 4. The interference microscope has been used to measure the refractive index of cells and, hence, the concentrations of dry substances in them, by immersing them in media of different known refractive indices. The application of this method to fixed cells is discussed theoretically. In experiments on fixed ram sperm heads the expected linear relationship between o.p.d. and refractive index of the immersion medium was obtained. Data on the average concentration of dry substance in ram sperm heads, the localized refractive index, and concentration in the denatured submicroscopic particles in the head, the percentage of the head volume occupied by them, and the geometrical thickness of the head were obtained. 5. Factors affecting the accuracy of the measurements of o.p.d., such as glare in the microscope, light scatter or absorption by the object, &c, are outlined.

A Histochemical Study of the Early Stages of Carcinogenesis in Rat Liver: Localization of Fluorescent Carcinogen and Changes in Succinic Dehydrogenase Activity

Plant tissues may be embedded in half the time taken for paraffin embedding if acetone is used as the dehydrating and infiltrating agent, and the tissue is embedded in ester wax. This procedure retains appreciably more lipid material inside the cells than does the more usual technique, even when the tissue has been fixed in osmium tetroxide or in La Cour's 2BE.

A study has been made of the concentration and location of phosphorylated compounds in serial myocardial biopsies taken from the left ventricle of dogs during and after brief coronary occlusion. Ischemia reduced the amount of phosphorylated substances, such as ATP and phosphocreatine. During reactive hyperemia these substances were lost into the intimate coronary circulation around the fibers. This raises the possibility that the leakage of such vasodilator substances from the myocardium into the vascular bed, either themselves or through their hydrolytic products, may account for the vasodilation of reactive hyperemia.

Specimens of human rheumatoid and nonrheumatoid synovial lining were maintained in nonproliferative organ culture for 20 hours. The culture fluids were then assayed for prostaglandin E(2) (PGE(2)), thromboxane B(2) (TXB(2)), and 6-keto-prostaglandin F(1alpha) (6-keto-PGF(1alpha)) by specific radioimmunoassay. The presence of each of these substances was confirmed by gas chromatography and mass spectrometry. Rheumatoid tissue produced significantly more of each cyclo-oxygenase product than nonrheumatoid tissue.

Fresh sections of left ventricular drill biopsies, taken at the beginning and end of cardiopulmonary bypass in 164 patients undergoing aortic valve replacement, have been analyzed for myocardial function. This has been assessed by changes in biochemical function, measured by semi-quantitative histochemical methods, and by changes in birefringence, measured quantitatively by polarization microscopy. It has been shown that continuous perfusion at 32 degrees C with the heart beating provided better preservation than did the other three perfusion procedures, in all of which the heart was fibrillating. More recently, in a small number of cases, cardioplegic arrest has been found to given even better preservation.

The development of the highly sensitive cytochemical bioassay for ACTH has permitted the measurement of plasma ACTH levels during the insulin hypoglycaemia test (I.H.T.) in patients treated with corticosteroids and corticotrophin. The ACTH, corticosteroid, and growth hormone (GH) responses in the I.H.T. were measured in three groups of 12 rheumatoid arthritis patients. One group was receiving long-term corticotrophin treatment, the second was undergoing long-term corticosteroid treatment, and the third had never received systemic hormone therapy. The increments in plasma ACTH, corticosteroids, and GH were diminished in the corticosteroid-treated group, as were increments in plasma GH and ACTH in the corticotrophin-treated group; but in this group the corticosteroid increment was normal. Examination of the area under the curve of the ACTH response showed that the total amount of ACTH secreted was normal though the rate of secretion was reduced. In the corticosteroid-treated group both rate and total secretion were diminished.

SUMMARY It has been shown previously that both thyrotrophin (TSH), and also immunoglobulins (Ig) derived from patients with goitrous Graves’disease, stimulate DNA-synthesis in guinea-pig thyroid tissue maintained in vitro. Here we describe the use of the same in vitro system and methods of quantitative cytochemistry to test the effect of these substances on the generation of NADPH, which is another indicator of the potential for growth. As could be predicted by its trophic action, TSH stimulated the generation of NADPH by glucose 6-phosphate dehydrogenase. The Ig-fraction from normal subjects depressed this activity. The Ig-fraction from Graves’disease patients with goitres stimulated the generation of NADPH, whereas the Ig from patients with Graves’disease but with minimal enlargement of the thyroid gland behaved like normal Ig. A similar lack of stimulation was found with Ig from patients with Pendred's syndrome, other dyshormonogenetic goitres, and autonomous single adenomas. In all specimens tested, there was good correlation between the amount of DNA-synthesis, measured by Feulgen cytophotometry, and the activity of glucose 6-phosphate dehydrogenase activity that generated NADPH. These results support the concept that there is a distinct type of autoantibody that influences thyroid growth.

Publisher Summary This chapter focuses on quantitative chemistry, used in cytochemical bioassays. The methods of quantitative cytochemistry depend on chilling the tissue, and sectioning it at low temperature, without producing any observable ice artifact. The aim of quantitative cytochemistry is to precipitate the colored reaction product in the cell in which the chemical activity resides. The greatest advantage of the cytochemical bioassay system is its use in defining the mode of action of hormones. The cytochemical approach allows the measurement of metabolic changes solely in the target cells. The metabolic consequences attendant on exposure of the target cells to the hormone can be measured sequentially. The flexibility of the cytochemical bioassay system makes it ideal for studying the interaction among possible agonists; the effects of other components of the plasma; and for investigating the possible presence of antibodies or other agents against the receptor.

Parietal cell antibodies (PCA) are found in up to 90% of sera from pernicious anaemia patients but it is often stated that they could represent an epiphenomenon without being directly responsible for the achlorhydria. In the present studies a direct effect of these antibodies on the secretory function of gastric acid-secreting cells has been demonstrated in two different experimental systems. In one set of experiments IgGs containing PCA activity were shown to inhibit acid secretion specifically in the living gastric mucosa of the bull frog suspended as a diaphragm between two chambers. The other system demonstrated their inhibition of carbonic anhydrase activity in a cytochemical bioassay for human G17-gastrin, suggesting a blocking effect on the gastrin receptors in the canalicular microvilli or the cell membrane. These experiments suggest a direct pathogenic role for PCA in autoimmune fundal gastritis and in pernicious anaemia.

There has been doubt as to whether elevated levels of parathyroid hormone, reported previously by radioimmunoassay, reflect increased concentrations of the biologically active hormone. The application of a recently developed, highly sensitive bioassay has shown considerable disparity between bioactivity and immunoreactivity in 5 rheumatic conditions and in normal subjects. Six patients with chondrocalcinosis had elevated levels; 3 of these did not have hypercalcaemia or any obvious cause other than possible subclinical hyperparathyroidism. One patient, assayed during an acute episode, had an elevated concentration of the hormone which reverted to normal when she was asymptomatic. Most patients with osteoarthrosis (13 our of 15) had low normal levels; 2 showed unexplained slightly elevated concentrations. Of 6 patients with haemochromatosis 3 had elevated levels, though this may have been related to the associated presence of diabetes mellitus. A third of patients with ankylosing spondylitis (10 out of 30) showed elevated parathyroid hormone levels but without hypercalcaemia. A number of spondylitic patients also showed anomalous results in this assay, possibly due to the presence of an antagonist. This would be consistent with the absence of clinical or biochemical evidence of hyperparathyroidism.

The efficacy of cold cardioplegic arrest as a method of myocardial preservation has been evaluated by cytochemical and biophysical assessments made on needle biopsies taken from 150 patients undergoing open-heart surgery (e.g., aortic valve replacement, aortic and mitral valve replacement, mitral valve replacement, coronary artery bypass graft, repair of atrial or ventricular septal defects). Comparison of endo- and epicardial preservation showed improved endocardial preservation with cardioplegia compared with that achieved with the previous method used—continuous coronary perfusion at 32°C; however, care had to be taken to ensure adequate cooling of the epicardium. Biopsies also showed the need for repeated infusions of cardioplegie solution if the aorta was occluded for more than 70 min. Preservation of right and left ventricle has also been compared.

A new method of measuring vasopressin activity is described. It depends on the finding that the Na+-K+-ATPase activity, measured cytochemically, in the thick ascending limb of the loop of Henle in rat renal tissue maintained in vitro, responded to increasing concentrations of synthetic arginine vasopressin in a log-dose related fashion. The limit of sensitivity was 0.002 pg/ml (2 x 10(-15) mol/l). The dose-responses were reproducible; the inter-assay coefficient of variation was 6.4% at a vasopressin concentration of 0.02 pg/ml. Normal plasma stimulated this Na+-K+-ATPase activity, the stimulation being reduced by 98% when the plasma had been treated with an antiserum specific for vasopressin. Measured in this system, the circulating levels of plasma vasopressin, in healthy adults after 18h dehydration, was 4.0 +/- 0.3 pg/ml (mean +/- SEM; n = 4) and fell to 0.6 +/- 0.1 pg/ml following a water load. Absolute plasma vasopressin values obtained by the cytochemical bioassay were comparable to those measured by radioimmunoassay (r = +0.97, p less than 0.001).

The efficacy of cold cardioplegic arrest as a method of myocardial preservation has been evaluated by cytochemical and biophysical assessments made on needle biopsies taken from 150 patients undergoing open-heart surgery (e.g., aortic valve replacement, aortic and mitral valve replacement, mitral valve replacement, coronary artery bypass graft, repair of atrial or ventricular septal defects). Comparison of endo- and epicardial preservation showed improved endocardial preservation with cardioplegia compared with that achieved with the previous method used--continuous coronary perfusion at 32 degrees C; however, care had to be taken to ensure adequate cooling of the epicardium. Biopsies also showed the need for repeated infusions of cardioplegic solution if the aorta was occluded for more than 70 min. Preservation of right and left ventricle has also been compared.

In assessing the possible efficacy of drugs for the treatment of osteoarthritis (OA), it may be helpful to have a model, in animals, of the early development of the disease prior to the expression of secondary phenomena. It is also necessary that such effects are quantifiable. To this end, the natural development of OA in the STR/ORT mouse hase been investigated. It has been shown that very early events in the development of the disease are disturbances in the activity of chondrocytic glucose 6-phosphate dehydrogenase, the initial step in the pentose-phosphate pathway, and in the orientation of the proteoglycans of the matrix of the articular cartilage. The study has been done by reference to the effect of diclofenac sodium, which previously has been reported to retard the destruction of articular cartilage. The results appear to indicate that these markers may provide quantitative measures for assessing potential therapeutic agents.