Diamine and polyamine oxidase activities in phytohaemagglutinin-induced growth of rat small intestine.

The activities of diamine and polyamine oxidases, two enzymes of polyamine catabolism, were studied in hyperplastic growth of rat small intestine induced by phytohaemagglutinin. This growth, evaluated by the elongation of Lieberkühn's crypts, was more extensive in the proximal than in the distal parts of the gut. The activity of diamine oxidase was significantly reduced in the proximal (70%), medial (45%) and the distal (25%) parts. The activity of polyamine oxidase was doubled. The concentrations of putrescine, cadaverine and spermidine were significantly elevated in the three intestinal parts studied, whereas those of histamine and spermine were unchanged. It appears that changes in the activities of diamine and polyamine oxidases may contribute to the increased putrescine content, which is necessary to maintain active polyamine turnover for sustaining growth of the gut.

The inflammatory and cytotoxic effects of a nitric oxide releasing cream on normal skin.

We describe the pro-inflammatory and cytotoxic effects of nitric oxide in vivo in human skin. Nitrite and ascorbic acid were mixed on the skin of 12 normal volunteers, three times daily, to release nitric oxide. Exposure to nitric oxide was varied by randomizing the concentration of nitrite and duration of application. Nitric oxide treated skin showed significant increases in cells expressing CD3, CD4, CD8, CD68, neutrophil elastase, ICAM-1, VCAM-1, nitrosotyrosine, p53, and apoptotic cells compared with skin treated with ascorbic acid alone. There was no significant increase in mast cells. Following application of nitric oxide there were significantly fewer CD1a positive Langerhans cells in the epidermis. These appeared to lose dendritic morphology and migrate from the epidermis. There was no significant difference in staining for Ki-67, a marker related to proliferating cell nuclear antigen, between active and control skin but staining was greater after exposure to higher dose nitric oxide than the low dose. Apoptosis, cytotoxicity, and p53 staining were relatively greater after 48 h exposure than after 24 h. These results suggest that nitric oxide is pro-inflammatory and is toxic to DNA, leading to the accumulation of p53 and subsequent apoptosis. High-dose nitric oxide paradoxically led to a smaller increase in macrophages and T cells than low dose suggesting an immunosuppressive effect of higher levels.

Tomato lectin resists digestion in the mammalian alimentary canal and binds to intestinal villi without deleterious effects.

Experiments were designed to investigate whether orally consumed tomato lectin could resist the digestive process and function as a lectin within the alimentary canal. Rats fed on a tomato lectin-rich diet passed faeces containing serologically detectable tomato lectin, and the lectin could be shown by immunoperoxidase staining bound to intestinal villi. Moreover, radioactivity was mainly recovered from the alimentary canal 3h after 125I-labelled tomato lectin administration with only traces in the circulation or internal organs. Radioactivity absorbed into the human circulation after consumption of 125I-labelled tomato lectin was also less than that expected for a digestable protein.

Expression of cytochrome P450 CYP1B1 in breast cancer.

The expression of CYP1B1 has been identified in breast cancer using the reverse transcriptase-polymerase chain reaction and immunoblotting. CYP1B1 mRNA was expressed in the majority of breast tumours and immunoblotting of breast tumours identified a single protein band of molecular weight 60 kDa corresponding to the predicted molecular weight of human CYP1B1. This is the first study to identify CYP1B1 expression in a tumour where it may represent a previously unknown pathway for the metabolism of oestradiol and chemotherapeutic drugs.

Zinc absorption in celiac disease and dermatitis herpetiformis: a test of small intestinal function.

The increments in plasma zinc concentrations after an oral dose of elemental zinc (50 mg) as the sulphate were used to assess the intestinal absorption of the metal in 11 patients with dermatitis herpetiformis (DH) before starting a gluten-free diet, 12 patients with newly diagnosed celiac disease (CD), 10 patients known to have CD, and 15 healthy volunteers. The areas under the plasma zinc increment curve plotted against time were determined for 3 (AUC3) and 6 (AUC6) h. The AUC3 in healthy volunteers was 401 +/- 48 mumol 1(-1) 3 h (mean +/- SD); it was reduced in newly diagnosed CD 187 +/- 76 mumol 1(-1) 3 h (p less than 0.001), and in dermatitis herpetiformis 206 +/- 87 mumol 1(-1) 3 h (p less than 0.01); but it was normal in the known CD 396 +/- 204 mumol 1(-1) 3 h, the wide variation reflecting the variable compliance with a previously instituted gluten-free diet. The AUC6 was similarly affected, healthy volunteers 700 +/- 111 mumol 1(-1) 6 h, new CD 380 +/- 169 mumol 1(-1) 6 h (p less than 0.01); dermatitis herpetiformis 471 +/- 107 mumol 1(-1) 6 h (p less than 0.01); known CD 725 +/- 380 mumol 1(-1) 6 h. The AUC3 was more consistently abnormal than conventional tests of small intestinal function. In a prospective study the AUC3 and AUC6 improved and reflected compliance with a gluten-free diet.

A new fluorescence method for alkaline phosphatase histochemistry.

We have developed a new fluorescence method for the histochemical localization of alkaline phosphatase activity. Calcium phosphate deposited at the sites of alkaline phosphatase activity in a Gomori-type reaction are identified by calcium binding fluorochromes. The calcium binding fluorochromes calcein, calcein blue, and xylenol orange were investigated, with each fluorochrome being included in the alkaline phosphatase incubating medium and used in a single-step procedure. Alkaline phosphatase activity was studied in freeze-substituted, resin-embedded human liver and jejunal biopsies, and each fluorochrome produced intense fluorescence of different colors at sites of alkaline phosphatase activity. Calcein, calcein blue, and xylenol orange produced green, blue, and red fluorescence, respectively. Sites of enzyme activity were accurately localized without evidence of diffusion, and there was an absence of non-enzyme-catalyzed binding of any of the fluorochromes to tissue. This fluorescence method, which is particularly suited to investigating the localization and distribution of the activity of different enzymes in the same section, was used to investigate the distribution and co-localization of alkaline phosphatase and aminopeptidase M in human liver and jejunum.

Enzyme histochemistry on freeze-substituted glycol methacrylate-embedded tissue.

We developed a method for histochemical demonstration of a wide range of enzymes in freeze-substituted glycol methacrylate-embedded tissue. Tissue specimens were freeze-substituted in acetone and then embedded at low temperature in glycol methacrylate resin. All enzymes studied (oxidoreductases, hydrolases) were readily demonstrated. The enzymes displayed high activity and were accurately localized without diffusion when tissue sections were incubated in aqueous media, addition of colloid stabilizers to the incubating media not being required. Freeze-substitution combined with low-temperature glycol methacrylate embedding permits the demonstration of a wide range of enzymes with accurate enzyme localization, maintenance of enzyme activity, and excellent tissue morphology.

Enzyme histochemical demonstration of NADH dehydrogenase on resin-embedded tissue.

We describe a method for enzyme histochemical demonstration of NADH dehydrogenase in cold (4 degrees C)-processed resin-embedded tissue. The effects on NADH dehydrogenase activity of processing tissue through a variety of dehydrating agents and embedding in three different acrylic resins were evaluated. The optimal procedure to maintain NADH dehydrogenase activity used a short (3-hr) fixation in 1% paraformaldehyde solution, followed by dehydration in acetone and embedding in glycol methacrylate resin. Embedding of tissue in resin combined preservation and accurate localization of NADH dehydrogenase activity with good tissue morphology. Blocks of the resin-embedded tissue could be stored at room temperature for at least 6 months without loss of NADH dehydrogenase activity.

Enzyme histochemistry on freeze-dried, resin-embedded tissue.

We have developed a method for histochemical demonstration of a wide range of enzymes in freeze-dried, resin-embedded tissue. Freeze-dried tissue specimens were embedded without fixation at low temperature (4 degrees C or -20 degrees C) in glycol methacrylate resin or LR Gold resin. Enzyme activity was optimally preserved by embedding the freeze-dried tissue in glycol methacrylate resin. All enzymes studied (oxidoreductases, esterases, peptidases, and phosphatases), except for glucose-6-phosphatase, were readily demonstrated. The enzymes displayed high activity and were accurately localized without diffusion when tissue sections were incubated in aqueous media, addition of colloid stabilizers to the incubating media not being required. Freeze-drying combined with low-temperature resin embedding permits the demonstration of a wide range of enzymes with accurate enzyme localization, high enzyme activity, and excellent tissue morphology.

A novel tetrazolium method for peroxidase histochemistry and immunohistochemistry.

We developed a new method for the histochemical demonstration of peroxidase. This method, which has a novel reaction mechanism, is based on the oxidation of phenol by peroxidase and coupling of this reaction to the reduction of a tetrazolium salt, with the deposition of an insoluble formazan at sites of enzyme activity. This new method was compared with an established diaminobenzidine (DAB) technique for peroxidase histochemistry and immunohistochemistry. Although both methods identified peroxidase activity in myeloid cells of bone marrow biopsy specimens, there was no interference from red cell pseudoperoxidase activity with the phenol-tetrazolium method, in contrast to the diaminobenzidine method. The detection of cytokeratin using an indirect immunoperoxidase technique was compared with both methods for demonstrating peroxidase activity. The phenol-tetrazolium method gave results similar to that obtained with DAB and appeared to be at least as sensitive as DAB in detecting low amounts of antigen. In addition, the production of a formazan as the final reaction product means that the phenol-tetrazolium method is ideally suited for quantitative peroxidase histochemistry. Therefore, the phenol-tetrazolium method represents a useful alternative method to DAB and for certain applications offers significant advantages over DAB.

Localization of microsomal epoxide hydrolase in normal and neoplastic human kidney.

Microsomal epoxide hydrolase is a xenobiotic metabolizing enzyme that catalyzes the conversion of toxic and carcinogenic epoxides to less toxic dihydrodiols. The cellular localization and distribution of microsomal epoxide hydrolase were investigated for the first time in normal and neoplastic human kidney. Light microscopic immunohistochemical studies using an alkaline phosphatase-anti-alkaline phosphatase technique showed that in normal kidney there was a wide distribution of epoxide hydrolase immunoreactivity. The main localization of epoxide hydrolase immunoreactivity was to the proximal and distal tubule epithelial cells. Strong epoxide hydrolase immunoreactivity was also identified in epithelium of the collecting ducts. In addition, epoxide hydrolase immunoreactivity was present in vascular endothelial cells, including endothelial cells lining glomerular capillaries. Epoxide hydrolase immunoreactivity was identified in all the renal tumors, and in each tumor immunoreactivity for epoxide hydrolase was localized to tumor cells. Immunoblotting of both normal kidney and tumor microsomes confirmed the presence of a single protein band of molecular weight 49 KD corresponding to the molecular weight of human hepatic microsomal epoxide hydrolase.

A combination of dietary protein depletion and PHA-induced gut growth reduce the mass of a murine non-Hodgkin lymphoma.

The results presented in this study show that a switch from a non-protein diet (NPD) to one of a normal protein content (LA) on the day of subcutaneous injection of non-Hodgkin lymphoma tumour cells greatly favoured the development and growth of the tumour. Interestingly, however, inclusion of the plant lectin phytohaemagglutinin (PHA) in the LA diet appeared to compete with the effect of switch to the protein-rich diet, resulting in decreased tumour size and an increased incidence of necrosis. PHA was shown to induce hyperplasia of the gut even in the presence of the growing tumour. This observation together with the fact that gut hyperplasia also occurred in animals which were fed NPD supplemented with PHA, indicated the strength of PHA as a growth signal. It would seem likely that this 'normal' growth is able to compete with the tumour for important growth factors and nutrients, including polyamines, effectively starving the tumour for these molecules and resulting in its decreased rate of proliferation.

The growth of an established murine non-Hodgkin lymphoma tumour is limited by switching to a phytohaemagglutinin-containing diet.

The growth of a non-Hodgkin lymphoma, developing subcutaneously as a solid tumour in NMRI mice, is markedly diminished by including phytohaemagglutinin (PHA), a lectin present in raw kidney bean (Phaseolus vulgaris), in the diet. In the experiment described in this communication the effect of first allowing tumours to develop for 5 days before switching the mice to a diet containing PHA at different concentrations was tested to establish whether or not feeding the lectin at late times also resulted in reducing tumour growth. This switch of diet indeed proved to be effective in slowing down growth of the lymphoma tumour. The reduced rate of growth occurs in a dose-dependent manner. We have suggested that a competition between the gut epithelium undergoing PHA-stimulated hyperplasia and the developing tumour may occur for polyamines and other nutrients from a common body pool and this could be an important contributory factor with regard to the observed low level of tumour growth following the feeding of PHA-containing diet. Recent data which showed that the level of hyperplasia of the small bowel in response to feeding the PHA diets was higher in non-injected mice compared to those which had been injected with tumour cells substantiated the concept of competition between gut and tumour for nutrients and other requirements for growth.

A novel method for optimum biopsy specimen preservation for histochemical and immunohistochemical analysis.

A novel method has been developed for optimally processing biopsy specimens combining freeze-substitution with low-temperature plastic embedding. Immunohistochemistry and conventional histochemical stains were all readily performed on tissue displaying high-quality morphologic preservation. Labile antigens, especially lymphoid cell surface antigens, were well preserved. This new method avoids the need for tissue fixation and combines the superior morphologic preservation of fixed embedded tissue with the reactivity of cryostat sections. This method ensures that diagnostic information from even the smallest biopsy specimen is maximized because a wide range of phenotypic markers can be applied and evaluated in relation to high-quality morphologic preservation of tissue. Biopsy specimens are stored at room temperature without loss of tissue-specific characteristics during storage.

The induction of gut hyperplasia by phytohaemagglutinin in the diet and limitation of tumour growth.

The growth of a transplantable murine non-Hodgkin lymphoma tumour, developing either intraperitoneally as an ascites tumour or subcutaneously as a solid tumour, has been shown to be markedly diminished by including phytohaemagglutinin (PHA), a lectin present in raw kidney bean (Phaseolus vulgaris) in the diet. In NMRI mice fed PHA within the range 0.45-7.0 mg/g diet, tumours which developed during a 10 day period after subcutaneous injection of cells were about 35% of the dry weight of those in lactalbumin-fed (control) animals. The reduced rate of growth occurred in a dose-dependent manner within the range 0.45-3.5 mg/g diet. Based on these observations it has been suggested that a competition between the gut epithelium undergoing hyperplasia and the developing tumour may occur for nutrients from a common body pool, and this may be an important factor with regard to the observed initial low level of tumour growth following the feeding of a PHA-containing diet. Observations which showed that the level of hyperplasia of the small bowel in response to feeding the PHA diets was higher in non-injected mice compared to those which had been injected with tumour cells substantiated the concept of competition between gut and tumour for nutrients etc. required for growth. Experiments with a second murine tumour cell line (a plasmacytoma) in Balb/c mice gave similar results indicating that the effect of PHA was not restricted to a single tumour system.

Dietary mistletoe lectin supplementation and reduced growth of a murine non-Hodgkin lymphoma.

The growth of a murine non-Hodgkin lymphoma (NHL) tumour has been shown to be reduced by incorporating mistletoe lectin (ML-1) into the diet. The morphological characteristics of NHL tumours in mice fed ML-1-supplemented diets were different from those in LA (control)-fed mice. The degree of mitotic activity was lower and nuclear area reduced. The degree of lymphocyte infiltration was increased in tumours from ML-1 fed mice and this was accompanied by a high incidence of apoptotic bodies. Visual observation of NHL tumours from individuals fed ML-1 diet showed a poorly developed blood supply in contrast to control-fed mice. A major reduction in number of blood capillaries in NHL tumours was confirmed by microscopic evaluation of tumour sections. The results suggested an anti-angiogenic response in ML-1-fed mice. The feeding of ML-1 compared to control diet thus provided several identifiable changes in the morphology of NHL tumours which were consistent with the observed reduction in tumour weight. There was no longer histological evidence of viable tumour in 25% mice fed the ML-1 diet for 11 days. Morphological studies of the small bowel indicated (a) that the lectin induces hyperplasia, and (b) that the lectin binds avidly to lymphoid tissue of Peyer's patches. There was evidence of limited endocytosis of the lectin. An experiment where ML-3 was added to the diet of mice three days after inoculation of tumour cells showed that the lectin was able to slow down further growth of an established tumour. The results show that ML lectins induce powerful anti-cancer effects when provided by the oral route.

A new approach to enzyme histochemical analysis of biopsy specimens.

A novel technique combining the freeze drying and embedding in glycol methacrylate at low temperature of tissue permitted the histochemical demonstration of a variety of enzymes, showing maintenance of enzyme activity, accurate enzyme localisation without apparent diffusion, and excellent morphological detail. The results obtained with this new approach were superior to standard techniques used for both enzyme histochemical and morphological studies. Moreover, blocks of the embedded tissue were stored for at least one year at room temperature without loss of enzyme activity. This method should find a wide range of applications in histopathology.

Amyloid in normal and pathological parathyroid glands.

Two groups of parathyroid glands have been examined for intrafollicular amyloid. In glands removed surgically it was found in nine cases (16%); in glands removed at necropsy it was found in 23 cases (46%). Also, two postmortem cases of systemic amyloidosis showed involvement of parathyroid glands with an entirely different distribution of amyloid. Compared with other amyloids the tinctorial properties of the intrafollicular amyloid were uniform in reactions with Congo red and Thioflavin T, but the intensity of the staining reaction for amino acids tryptophan and tyrosine was not constant for the follicle amyloid. This variation may be a feature of a maturation process but the derivation of this amyloid remains uncertain.

In situ hybridisation of albumin mRNA in normal liver and hepatocellular carcinoma with a digoxigenin labelled oligonucleotide probe.

AIMS: To study the localisation and distribution of albumin mRNA in normal liver and hepatocellular carcinoma by in situ hybridisation with an oligonucleotide probe. METHODS: A 51 base oligonucleotide was synthesised from a sequence at the 5' end of the human albumin gene and the probe was labelled at its 3' end with digoxigenin 11-dUTP. Formalin fixed, wax embedded sections of liver biopsy specimens were used to study the localisation and distribution of albumin mRNA. After in situ hybridisation the bound probe was visualised using a digoxigenin antibody conjugated with alkaline phosphatase. RESULTS: In normal liver albumin mRNA was detected in hepatocytes and no positive signal was observed in biliary epithelium, vascular endothelium, or Kupffer cells. In 75% (9/12) of the hepatocellular carcinomas studied a positive hybridisation signal was observed in tumour cells. CONCLUSIONS: Albumin mRNA can be detected in sections of formalin fixed, wax embedded liver, a digoxigenin labelled probe is ideally suited for in situ hybridisation of liver because there is no background from the detection system. The identification of albumin mRNA may be a useful marker of hepatocellular carcinoma, and the demonstration of albumin mRNA by in situ hybridisation overcomes the potential background problem associated with albumin immunohistochemistry.

Distribution of plasminogen activator inhibitor in normal liver, cirrhotic liver, and liver with metastases.

AIMS: To examine the distribution of PAI-1 antigen in normal and cirrhotic liver and liver with metastases. METHODS: Sections of normal and cirrhotic liver and liver with metastases were stained using the alkaline phosphatase antialkaline phosphatase (APAAP) technique and monoclonal antibody specific for plasminogen activator inhibitor (PAI-1). RESULTS: PAI-1 antigen was identified as discrete granules in the cytoplasm of hepatocytes in normal liver, particularly around portal tracts and central veins of the liver lobule. In cirrhotic liver a striking reduction of PAI-1 antigen was noted. In liver with metastases increased amounts of PAI-1 antigen were concentrated in hepatocytes around the margins of malignant deposits. CONCLUSIONS: Cirrhotic liver contains considerably less PAI-1 antigen than does normal liver, despite raised plasma concentrations of PAI-1. This may reflect release of hepatic PAI-1 into the circulation or decreased clearance of PAI-1 from the plasma. Secondary malignant deposits in the liver seem to stimulate production of PAI-1 in adjacent hepatocytes. This may influence the invasive process and may contribute to the thrombotic tendency associated with malignancy.