Inhibition of tumor cell line alkaline phosphatase by human peripheral blood monocytes. Correlation with growth inhibitory activity

Alkaline phosphatase (AP) activity in intact Ag8 cells was measured by a colorimetric assay in which spectrophotometer readings were obtained directly through wells of microtiter plates using a Multiskan ELISA reader. The wide difference between enzymatic activity observed in cells of this cell line and human peripheral blood monocytes permitted an assessment of inhibition of cell line growth in the presence of plastic-adherent peripheral blood cells. Comparison at 24 h of incubation of enzymatic measurements with those obtained using 3H-thymidine uptake showed comparable results, although values obtained with the enzyme were generally lower. Similarly, direct comparison of results obtained by the enzyme-based method showed good correlation with the number of viable tumor cells remaining in culture. Inhibition of AP activity was observed earlier than inhibition of 3H-thymidine incorporation, reaching its peak after 10 h of incubation. This technique has potential application for other enzyme systems and provides a new tool for analyzing mechanisms whereby effector cells are able to control proliferation of transformed cells.     

Dephosphorylation of endotoxin by alkaline phosphatase in vivo.

Natural substrates for alkaline phosphatase (AP) are at present not identified despite extensive investigations. Difficulties in imagining a possible physiological function involve its extremely high pH optimum for the usual exogenous substrates and its localization as an ecto-enzyme. As endotoxin is a substance that contains phosphate groups and is usually present in the extracellular space, we studied whether AP is able to dephosphorylate this bacterial product at physiological pH levels. We tested this in intestinal cryostat sections using histochemical methods with endotoxin from Escherichia coli and Salmonella minnesota R595 as substrate. Results show that dephosphorylation of both preparations occurs at pH 7.5 by AP activity. As phosphate residues in the lipid A moiety determine the toxicity of the molecule, we examined the effect of the AP inhibitor levamisole in vivo using a septicemia model in the rat. The results show that inhibition of endogenous AP by levamisole significantly reduces survival of rats intraperitoneally injected with E. coli bacteria, whereas this drug does not influence survival of rats receiving a sublethal dose of the gram-positive bacteria Staphylococcus aureus. In view of the endotoxin-dephosphorylating properties of AP demonstrated in vitro, we propose a crucial role for this enzyme in host defense. The effects of levamisole during gram-negative bacterial infections and the localization of AP as an ecto-enzyme in most organs as well as the induction of enzyme activity during inflammatory reactions and cholestasis is in accordance with such a protective role.     

Origins of serum alkaline phosphatase

A very rapid method of agar gel electrophoresis on glass slides, together with a superior visualization technique employing simultaneous coupling of a hydrolysed naphthol substrate, have been developed for the identification of the tissues of origin of serum alkaline phosphatase. Combined with L-phenylalanine inhibition, specific for the intestinal enzyme, and heat inactivation, specific for the placental enzyme, the heterogeneity of serum alkaline phosphatase has been demonstrated. Normal adult serum contains predominantly liver-type alkaline phosphatase with a small but variable quantity of intestinal enzyme, and little or no bone enzyme.     

Inhibition of suppressor cell function by cimetidine in a murine model

Immunomodulatory effects of cimetidine, an H2 histamine receptor antagonist, have been reported in humans and animals. To define these effects more clearly, the action of cimetidine on suppressor cell function was studied utilizing a murine model of contact hypersensitivity. Intravenous inoculation of BALB/c mice with DNP-coupled syngeneic spleen cells induced the production of DNP-specific suppressor cells which could easily be demonstrated by a reduction in ear swelling after contact sensitization with 1-fluoro-2,4-dinitrobenzene (DNFB) following transfer of spleen and lymph node cells to naive syngeneic recipients. Cimetidine treatment of animals in which suppressor cells were induced resulted in an inability of these mice to transfer cellular suppression as measured by development of a normal immunologic response in the recipient mice. The effect of cimetidine was both dose and time related. While all groups receiving cimetidine showed some loss of suppressor cell function, the maximum effect (up to 100% inhibition) was seen when 50 mg/kg of cimetidine was administered intraperitoneally 2 days before or on the day of suppressor cell induction. Some restoration also occurred when cimetidine was given after the day of induction. It has been shown that suppressor cells possess histamine receptors which may be involved in suppressor cell activation. The results indicate that cimetidine may inhibit the functioning of these receptors.     

Neutrophil alkaline phosphatase in pregnancy

Neutrophil alkaline phosphatase levels have been studied in a group of 194 pregnant women, using a modified technique which permits more rapid counting of the required number of neutrophils with a degree of consistency comparable with standard methods.No correlation was found between neutrophil alkaline phosphatase levels and the period of gestation; in contrast to previous findings, no differences were observed between the means for tests done in early pregnancy and those performed later in pregnancy. There was no demonstrable relationship between neutrophil alkaline phosphatase levels and age, parity, and white cell count. Elevated levels were also observed in women taking oral contraceptives. Thus, although the neutrophil alkaline phosphatase has diagnostic value in certain other disease states, the above findings suggest that it is of little or no value in pregnancy.The possibility of hormonal control of the elevated level of neutrophil alkaline phosphatase seen in pregnancy and simulated pregnancy is discussed, as is the possible relationship to increases in serum alkaline phosphatase in pregnancy.     

Rapid diagnosis of viral infections by an alkaline phosphatase immunocytochemical method

The use of alkaline phosphatase immunocytochemical staining was explored for the rapid diagnosis of poliovirus, adenovirus, herpes simplex virus and cytomegalovirus infections in cell cultures. In this test, viral antigens treated with their relative antibody were incubated with alkaline phosphatase-labelled antisera. The enzyme label was developed with a naphthol salt in the presence of a diazonium salt (Fast Blue) in order to obtain a blue coloured precipitate at the site of the enzyme. It is suggested that this immunocytochemical technique is valuable in the detection of viral infections and would be an appropriate test to use when rapid diagnosis is required.