Identification of proliferating lymphocyte subpopulations by combined alkaline phosphatase anti-alkaline phosphatase (APAAP) staining and autoradiography

An improvement in the classification of proliferating ([³H]thymidine incorporating) lymphocyte subpopulations in mitogen- or antigen-stimulated microcultures is described. The binding of subset-specific monoclonal antibodies is detected by the alkaline phosphatase anti-alkaline phosphatase method (APAAP). There are two advantages compared to the peroxidase anti-peroxidase (PAP) method; (1) endogenous enzyme (peroxidase)_activity exhibited by some cells causes no interference, and (2) the red alkaline phosphatase staining obtained with new fuchsin provides a far superior contrast to silver grains than conventional peroxidase staining.     

Comparative study of alkaline phosphatase and prostate specific antigen in prostate cancer

The study comprised between healthy and benign controls and proven 62 cases of prostate cancer at different clinical stages. The mean value of increasing level of PSA in group III was found to be highly significant when compared with group I. Whereas, the mean value of elevated levels of ALP in stage III and IV from group III was found to be highly significant when compared with group I. Though the mean value of ALP was increased in stage I and II from group III when compared with group I, it was not found highly significant. Hence the elevated levels of ALP were significantly correlated with advanced stages of prostate cancer. Whereas, increased levels of PSA contribute to the diagnosis of potentially increased volume of the prostate cancer.     

Enhancement of mitogen-induced lymphocyte proliferation by some inhibitors of alkaline phosphatase and diamine oxidase

We selected various compounds [bromolevamisole, levamisole, cimetidine, L-homoarginine, 2,3,5,6-tetrahydroimidazo-(2,1-b)thiazole (IT), imidazole, theophylline] previously reported as inhibitors of alkaline phosphatase (ALP) and/or diamine oxidase (DAO) and studied their activity on concanavalin A (ConA)-induced mouse spleen cell lymphocyte proliferation. According to the Ki values, the decreasing order of potency for ALP inhibition was: bromolevamisole, levamisole, theophylline, cimetidine, IT, imidazole and L-homoarginine. The order of potency was different for DAO inhibition. Cimetidine was the most potent inhibitor of DAO, followed by bromolevamisole, levamisole, IT, imidazole and L-homoarginine. Theophylline had no inhibitory effect on DAO. We show that these compounds, except theophylline, enhance ConA-induced lymphocyte proliferation. Similarly, all the compounds except imidazole and theophylline, significantly inhibited ALP at concentrations which enhanced lymphocyte proliferation as measured by (3H)-thymidine uptake. DAO inhibition correlated with DNA synthesis only for IT and cimetidine. These observations suggest that ALP and DAO play a negative role in the proliferation process; however, the degree of enhancement of ConA-induced proliferation did not correlate strictly with the degree of ALP and DAO inhibition.     

Knockdown of tissue nonspecific alkaline phosphatase impairs neural stem cell proliferation and differentiation

In the adult mammalian brain the subependymal layer of the lateral ventricles houses neural stem cells giving rise to young neurons migrating towards the olfactory bulb. The molecular cues controlling essential functions within the neurogenesis pathway such as proliferation, short and long distance migration, differentiation and functional integration are poorly understood. Neural progenitors in situ express the tissue nonspecific form of alkaline phosphatase (TNAP), a cell surface-located nonspecific phosphomonoesterase capable of hydrolyzing extracellular nucleotides. To gain insight into the functional role of TNAP in cultured multipotent neural stem cells we applied a knockdown protocol using RNA interference with shRNA and retroviral infection. We show that TNAP knockdown reduces cell proliferation and differentiation into neurons or oligodendrocytes. This effect is abrogated by addition of alkaline phosphatase to the culture medium. Our results suggest that TNAP is essential for NSC proliferation and differentiation in vitro and possibly also in vivo.     

Measurement of lymphocyte cytotoxicity by assessing endogenous alkaline phosphatase activity of the target cells

A simple, objective, non-isotopic method for testing lymphocyte cytotoxicity is described. The evaluation of the test is based on photometric measurement of endogenous alkaline phosphatese activity of the residual adherent target cells.     

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.     

A modified alkaline phosphatase enzyme amplification system and its application in an HIV antigen ELISA.

Alkaline phosphatase amplification systems increase the sensitivity of enzyme-linked immunoassays (ELISAs). Here we describe a modified method by which levamisole is a component of the substrate buffer. This increases sensitivity by reducing the signal from non-specific alkaline phosphatases. The modified procedure was applied to an 'in house' HIV-1 p24 antigen capture assay and was shown to increase the sensitivity 4-fold as compared to the unmodified system. The performance of the modified amplification system is comparable to that of commercially available systems.     

Identity of the neoplastic alkaline phosphatase as revealed with monoclonal antibodies to the placental form of the enzyme

A panel of six monoclonal antibodies and a conventional polyclonal antibody raised against human placental alkaline phosphatase (ALP) were used to characterize the alkaline phosphatase detected by means of histochemistry on tumors of breast, ovary, lung, gastrointestinal tract, and kidney. Complete antigenic identity between the tumor ALP and the placental ALP was found only in one lung tumor. However, ten tumors reacted with the polyclonal antibody and some monoclonal antibodies, thus exhibiting partial identity with the placental ALP.     

Bile canalicular alkaline phosphatase and disease.

The alkaline phosphatase reaction is normally absent in human bile canaliculi, but was found in 79 patients. In search for a common causal factor, these patients were further examined. Thirty-seven were autopsied. The conditions most ocmmonly associated with the phenomenon were malignant tumours with or without involvement of the liver, collagen diseases, long-standing partial obstruction of the common bile duct, and genetic variants of alpha-1-antitrypsin. No clinical or laboratory facts were common to all the patients.     

Discontinuous expression of a membrane antigen (HB-7) during B lymphocyte differentiation

We have examined the expression of a cell surface antigen by B lineage cells in human fetuses, newborns and adults using a newly produced monoclonal antibody, HB-7. The HB-7 antigen was found to be a protease sensitive 45,000 MW molecule that appeared to be the same molecule recognized by the OKT-10 antibody. The HB-7 reactive molecule was expressed by all fetal pre-B and B cells, and 50% of newborn blood and adult bone marrow B cells. In contrast, only a small minority of B cells (2–12%) from blood, spleen and tonsil of adults were weakly HB-7⁺. The pokeweed mitogen-responsive B cell precursors of plasma cells were also HB-7⁻, but the HB-7 antigen was re-expressed during the plasma cell stage. We conclude that this antigen is unique among known B cell differentiation antigens in its intermittent pattern of expression during B cell development. The reactivity of the HB-7 antibody with immature, but not mature, B cells makes it well suited for studies of B cell ontogeny.     

An improved method of antigen detection on nitrocellulose: in situ staining of alkaline phosphatase conjugated antibody

A biological stain for alkaline phosphatase was applied to detect immune complexes immobilized on nitrocellulose. This method was sensitive and useful for in situ staining of antigens, in dot-immunobinding assays and immunoblots. In addition to its sensitivity, the staining of alkaline phosphatase conjugated antibody was also highly stable, and allowed a permanent record of original immunochemical data. A rapid and simple method is also described using this technique for screening translation products of recombinant clones.     

The role of SLP-76 and LAT in lymphocyte development

SLP-76 and LAT are two recently identified adapter proteins that are involved in the signal transduction cascade initiated by engagement of the TCR. The role of these two molecules in thymocyte development has become clearer following studies of gene targeted mice. The data indicate that SLP-76 and LAT are each critical for the expansion and differentiation of double-negative thymocytes and that SLP-76 is essential for allelic exclusion at the TCRbeta locus.     

Anion-stimulated phosphohydrolase activity of intestinal alkaline phosphatase

Phosphohydrolase activity of a highly enriched commercial preparation of calf intestinal alkaline phosphatase was stimulated in the presence of HCO3. SO4, Cl, SCN, and acetate did not stimulate hydrolysis, whereas SO3 exhibited a bimodal effect, stimulating at low (25mM) concentration but inhibiting at high (100 mM) concentration. The pH optimum of this stimulation by HCO3 or SO3 was 8.5--9.0 and was maximal at a Mg concentration of 0.5 mM. HCO3 increased the Vmax of the reaction without changing the Km for ATP. ATP, GTP, UTP, and xanthosine triphosphate were equally effective as substrates, whereas AMP and p-nitrophenyl phosphate were much less effective. Alkaline phosphatase activity was inhibited by L-cysteine and L-phenylalanine, compounds that also inhibited the HCO3-ATPase activity of the preparation. Passage of the commercial preparation through an anion-exchange column yielded a fraction with enriched alkaline phosphatase and HCO3-ATPase activities; this fraction proved to be a single protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, on isoelectric focusing, and by immunologic techniques. These studies strongly suggest that alkaline phosphatase and anion-stimulated phosphohydrolase activities are properties of the same protein in small intestine. It is possible that alkaline phosphatase may function as a HCO3-ATPase involved in intestinal absorption and secretion.