Diagnostic significance of levamisole-resistant alkaline phosphatase in cytochemistry and immunocytochemistry

The authors have used two immunoalkaline phosphatase methods to study nonhematopoietic tumor tissues of four patients, one each with alveolar cell carcinoma of the lung, renal cell carcinoma, gastric adenocarcinoma, and colon carcinoma. They found, regardless of specific antibodies used, definite enzyme activity in the tumor cells of these four patients. Although it was possible to determine that the tumor cells were epithelial in origin because of their intense staining with antibodies to epithelial cell antigens, control slides labeled with nonimmune mouse ascites also contained cells with definite enzyme activity. In two of these cases, unlabeled smears were stained for alkaline phosphatase and showed that the tumor cells contained endogenous levamisole-resistant enzyme activity. This endogenous enzyme activity is not demonstrable in either the benign cells of these cases or the benign or malignant cells of other control cases. The findings suggest that the immunoalkaline phosphatase methods also have their inherent endogenous enzymic problems. They also suggest that cytochemical demonstration of levamisole-resistant alkaline phosphatase may be a useful cell marker for the identification of tumor cells in serous effusions.     

Statins and tissue mineralization: putative involvement of alkaline phosphatase

Many clinical trials have clearly demonstrated the benefit of statins in the prevention of coronary heart disease. Although other effects have subsequently been described, the so-called pleiotropic effects of statins, there is a tendency to relate all those effects, more or less directly, to inhibition of 3-hydroxy-3-methylglutaryl coenzyme A. Several clinical and laboratory studies show an association between statins and increased bone formation and/or density, but no clear explanation for that statin effect has emerged. We therefore hypothetized that statins may have an effect on alkaline phosphatase activity (ALP), an enzyme with an important role in bone mineralization and that may also contribute to pathological mineralization in other tissues, such as vascular calcification. Our experience with drug effects on ALP lead us to admit the possibility of finding a statin with an ALP increasing effect on bone but not on vascular tissue, or with a more marked effect upon one of the ALP isoforms or isoenzymes. That information would allow the design of clinical trials to confirm the suitability of a specific statin to a specific clinical condition.     

Ultrastructure and hydrolase cytochemistry of the developing marmoset yolk sac

Summary Yolk sacs from Callithrix jacchus were investigated light and electron microscopically as well as by qualitative light microscopic enzyme histochemistry on days 35 to 126 of gestation. The thin yolk sac wall of the early stages (day 35–41) consists of the cuboid, endodermal epithelium, the mesothelium of the exocoelom and some interposed blood vessels. The inner endodermal surface is rather smooth. At later stages, the epithelium becomes highly prismatic and forms folds which are lined by a mesenchyme and blood vessels. Microvilli and a small number of endocytotic vesicles are observed at the apices of the epithelial cells, which are interconnected by gap junctions, desmosomes and interdigitations. The cytoplasm of the epithelial cells is characterized by a well-developed rough endoplasmie reticulum, a large Golgi apparatus and glycogen deposits. Four different membrane-bordered types of inclusions can be distinguished in the cytoplasm of the epithelial cells: The type I and II inclusions are considered as secretion granules. Their increase and their localization in the cavities of the endoplasmic reticulum at later stages are ascribed to an inhibition of the intracellular transport at the onset of involution. The type III and IV inclusions may represent lysosomes and related organelles. Bile capillary-like spaces exist between the epithelial cells. The basement membrane is incomplete below the epithelium and absent around the capillaries, the endothelium of which is porous in certain areas. Aminopeptidase M is highly active in the plasmalemma and the bile capillary-like structures of the epithelium, dipeptidylpeptidase IV in the mesothelium and alkaline phosphatase in the blood vessel endothelium. Other membrane hydrolases are absent. Acid proteases, glycosidases, non-specific phosphatases and non-specific esterases can be detected stage-dependently with moderate to high activities in the yolk sac epithelium. Compared with other organs, the yolk sac structure and hydrolase equipment are similar to those of the liver and may, therefore, have similar functions, e.g. synthesis and secretion of proteins. In addition, however, the yolk sac epithelium might also be involved in resorptive processes of material from the lumen followed by lysosomal digestion. The Callithrix jacchus yolk sac starts involution on day 80 of gestation by disintegration of the cells. On day 100, this process is completed. the stage of involution which is late in comparison with other primates, e.g. man and Rhesus monkey, is ascribed to the strongly delayed development of Callithrix jacchus.Supported by the BMFT (Project CMT 35)     

Ultrastructure and cytochemistry of thyroid lysosomes following subtotal thyroidectomy

Following subtotal thyroidectomy, the amount of circulating thyroid hormone decreases and causes an increase in the secretion of thyrotropin (TSH) by the anterior pituitary gland. Serum levels of circulating TSH remain elevated until thyroid secretion returns to normal. In this study we have analyzed the effects of such chronic stimulation of thyroid cells by TSH, with particular emphasis on ultrastructural and cytochemical changes in the lysosomes. Weanling Sprague-Dawley rats underwent subtotal thyroidectomy and 6 weeks later the residual thyroid tissue was removed and processed for ultrastructural and cytochemical analysis. There were obvious ultrastructural signs of hyperactivity. The cells were hypertrophied and there were colloid droplets in the cells as well as extremely abundant oddly shaped lysosomes. The lysosomes reacted positively for acid phosphatase and for glycoproteins, suggesting that they are secondary lysosomes, ones which have complexed with thyroglobulin prior to release of thyroid hormones from the cells. This tremendous increase in the number of these structures in the cells is similar to that observed under normal conditions during the aging process and suggests a slowdown in the proteolytic degradation of thyroglobulin during long periods of chronic stimulation by TSH.     

Induction of glomerular alkaline phosphatase after challenge with lipopolysaccharide

Alkaline phosphatase (AP) can be considered as a host defence molecule since this enzyme is able to detoxify bacterial endotoxin at physiological pH. The question emerged whether this anti-endotoxin principle is inducible in the glomerulus and if so, which glomerular cells might be involved in the expression of ectoAP after stimulation with pro-inflammatory agents. Therefore kidneys of rats treated with either lipopolysaccharide (LPS), E. coli bacteria or non-toxic monophosphoryl lipid A (MPLA) were examined for AP activity 6 or 24 h after challenge. In addition cultures of endothelial cells or mesangial cells were evaluated for AP activity after stimulation with either LPS, TNFalpha or IL-6, and mRNA for AP was studied in TNFalpha-stimulated and control mesangial cells. The results show significant up-regulation of glomerular AP in LPS- or E. coli-injected rats compared to rats injected with MPLA. Endothelial and mesangial cells in vitro showed significant up-regulation of AP activity following stimulation with LPS, TNFalpha or IL-6, whereas increased mRNA for AP was observed in mesangial cells after TNFalpha stimulation compared to non-stimulated control cells. Since it appeared that hydrolysis occurred when endotoxin was used as a substrate in the histochemical staining, we concluded that inducible glomerular ectoAP may reflect a local endotoxin detoxifying principle of the kidney.     

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.     

Distribution of alkaline phosphatase activity in healthy and diseased human liver tissue.

Histochemical demonstration of the alkaline phosphatase activity with an azo-dye method in human livers showed that the activity, which is normally localized to endothelial cells of portal and central veins as well as of sinusoids around the central veins and to a lesser extent of sinusoids around the portal connective tissue, is increased in various liver diseases. Appreciable canalicular activity is rare, and when it occurs it is more prominent in parenchyma involved by malignant tumours.     

Cytochemical investigation of the alkaline phosphatase activity of the leukocytes after injection of pyrogenal

Summary A cytochemical method of azocombination was used to study the activity of alkaline phosphatase of human leukocytes after injection of pyrogenal (bacterial lipopolysaccharide). Intravenous injection of pyrogenal at first causes a decline and then an increase of the phosphatase activity of neutrophils attaining a maximum on the 3rd–4th day, after which the activity returns to normal. No direct relationship has been discovered between the phosphatase activity of neutrophils and the number of these cells in the peripheral blood. It is presumed that the growth of the phosphatase activity of neutrophils of the peripheral blood is connected with supply of cells rich in alkaline phosphatase from the bone marrow.(Presented by Active Member of the Academy of Medical Sciences of the USSR V. V. Parin). Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 62, No. 10, pp. 44–47, October, 1966.     

Immobilization of alkaline phosphatase on microporous nanofibrous fibrin scaffolds for bone tissue engineering

Alkaline phosphatase (ALP) promotes bone formation by degrading inorganic pyrophosphate (PP_i), an inhibitor of hydroxyapatite formation, and generating inorganic phosphate (P_i), an inducer of hydroxyapatite formation. P_i is a crucial molecule in differentiation and mineralization of osteoblasts. In this study, a method to immobilize ALP on fibrin scaffolds with tightly controllable pore size and pore interconnection was developed, and the biological properties of these scaffolds were characterized both in vitro and in vivo. Microporous, nanofibrous fibrin scaffolds (FS) were fabricated using a sphere-templating method. ALP was covalently immobilized on the fibrin scaffolds using 1-ethyl-3-(dimethylaminopropyl)carbodiimide hydrochloride (EDC). Scanning electron microscopic observation (SEM) showed that mineral was deposited on immobilized alkaline phosphatase fibrin scaffolds (immobilized ALP/FS) when incubated in medium supplemented with β-glycerophosphate, suggesting that the immobilized ALP was active. Primary calvarial cells attached, spread and formed multiple layers on the surface of the scaffolds. Mineral deposition was also observed when calvarial cells were seeded on immobilized ALP/FS. Furthermore, cells seeded on immobilized ALP/FS exhibited higher osteoblast marker gene expression compared to control FS. Upon implantation in mouse calvarial defects, both the immobilized ALP/FS and FS alone treated group had higher bone volume in the defect compared to the empty defect control. Furthermore, bone formation in the immobilized ALP/FS treated group was statistically significant compared to FS alone group. However, the response was not robust.     

Isoforms of alkaline phosphatase from mouse internal organs after bilateral adrenalectomy

The activity of alkaline phosphatase of female CBA and BALB/c mice is studied after bilateral adrenalectomy. Interstrain differences in enzyme activity are revealed in some organs of the control and experimental animals. The expression of new isoforms of alkaline phosphatase in hypocorticoidism is demonstrated. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 121, N ^o 3 pp. 262–264, March, 1996 Presented by V. P. Kaznacheev, Member of the Russian Academy of Medical Sciences     

Denaturing gradient gel electrophoresis analysis of the tissue nonspecific alkaline phosphatase isoenzyme gene in hypophosphatasia

Hypophosphatasia, a heritable form of rickets/osteomalacia, was first described in 1948. The biochemical hallmark, subnormal alkaline phosphatase (ALP) activity in serum, reflects a generalized disturbance involving the tissue-nonspecific isoenzyme of ALP (TNSALP). Deactivating mutations in the gene that encodes TNSALP have been reported in patients worldwide. Nevertheless, hypophosphatasia manifests an extraordinary range of clinical severity spanning death in utero to merely premature loss of adult teeth. There is no known medical treatment. To delineate the molecular pathology which explains the disease variability and to clarify the pattern(s) of inheritance for mild cases of hypophosphatasia, we developed comprehensive mutational analysis of TNSALP. High efficiency of mutation detection was possible by denaturing gradient gel electrophoresis (DGGE). Primers and conditions were established for all TNSALP coding exons (2-12) and adjacent splice sites so that the amplicons incorporated a GC clamp on one end. For each amplicon, the optimum percentage denaturant was determined by perpendicular DGGE. In 19 severely affected pediatric subjects (having perinatal or infantile hypophosphatasia or early presentation during childhood) from among our large patient population, we detected 2 TNSALP mutations each in 16 patients (84%) as expected for autosomal recessive disease. For 2 patients (11%), only 1 TNSALP mutation was detected by DGGE. However, one subject (who died from perinatal hypophosphatasia) had a large deletion as the second mutation. In the other (with infantile hypophosphatasia), no additional mutation was detected by DNA sequencing of all protein-coding exons. Possibly, she too has a deletion. For the final patient, with unclassifiable hypophosphatasia (5%), we detected only a single mutation which has been reported to cause relatively mild autosomal dominant disease; the other allele appeared to be intact. Hence, DGGE analysis was 100% efficient in detecting mutations in the coding exons and adjacent splice sites of TNSALP in this group of severely affected patients but, as expected, failed to detect a large deletion. To date, at least 78 different TNSALP mutations (in about 70 hypophosphatasia patients) have been reported globally. In our large subset of severely affected patients, we identified 8 novel TNSALP mutations (Ala34Ser, Val111Met, Delta G392, Thr117His, Arg206Gln, Gly322Arg, Leu397Met, and Gly409Asp) and 1 new TNSALP polymorphism (Arg135His) furthering the considerable genotypic variability of hypophosphatasia.     

Increase in postsynaptic density material in optic target neurons of the rat suprachiasmatic nucleus after bilateral enucleation

During and after degeneration of the optic nerve afferents in the rat suprachiasmatic nucleus (SCN) postsynaptic density (PD) material is added to existing symmetrical and asymmetrical appositions of non-optic synapses on optic target neurons. If the correlation between thickness of the postsynaptic density and excitatory or inhibitory function of a synapse holds in this nucleus, the change in morphology of synapses could mean that some inhibitory synapses turn into excitatory contacts.     

Metabolic changes in the nucleus of the optic tract after monocular enucleation as revealed by cytochrome oxidase histochemistry

The histochemistry for the mitochondrial enzyme cytochrome oxidase (CO) was used to evaluate the levels of metabolic activity in neurons of the nucleus of the optic tract (NOT) and dorsal terminal nucleus (DTN) in the opossum (Didelphis aurita). The observations were performed in four groups: normal juveniles (4 months old), monocularly enucleated juveniles analysed when adults, normal adults (8 to 18 months old) and monocularly enucleated adults. CO labeled cells were observed to have a similar distribution along the NOT-DTN anteroposterior axis in both juvenile and adult normal animals. Monocular enucleation performed in adults produced a significant reduction of the reactive neuropil but not of the number of CO labeled cells in the deafferented NOT-DTN: the number of labeled neurons per section in the deafferented side matched those of the ipsilateral complex. In juveniles, however, this procedure caused a systematic reduction of the number of CO labeled cells in the contralateral NOT-DTN in comparison to the spared complex. The lack of reduction in the number of neurons found on the deafferented side of the NOT-DTN of monocularly enucleated adult opossums compared with the ipsilateral side might result from the presence of compensatory inputs to maintain their metabolic equivalence. However, when the monocular enucleation was performed in juvenile opossums, a statistically significant asymmetry of CO neurons in the NOT-DTN was observed. In other words, the compensatory mechanisms proposed for the adults were either absent or insufficient to achieve symmetry in juveniles, suggesting a more heavily reliance in the retinal input.     

Hypophosphatasia: the mutations in the tissue-nonspecific alkaline phosphatase gene

Hypophosphatasia is an inborn error of metabolism caused by a deficiency of liver-, bone- or kidney-type alkaline phosphatase due to mutations in the tissue-nonspecific alkaline phosphatase (ALPL) gene. Most of the 65 distinct mutations described to date are missense mutations, a result which must be correlated with the great variability of clinical expression ranging from stillbirth without mineralized bone to pathologic fractures developing only late in adulthood. Correlations of genotype and phenotype have been established on the basis of clinical data exhibited by the patients, transfection studies, computer-assisted modeling, and examination of biochemical properties of ALP in cultured fibroblasts of patients. Screening for mutations in the TNSALP gene allows genetic counseling and prenatal diagnosis of the disease in families with severe forms of hypophosphatasia, and screening may also be helpful in confirming diagnosis of hypophosphatasia when biochemical and clinical data are not clear. Screening is also the necessary first step in further studies to elucidate dominant transmission of the disease and of liver-, bone- and kidney-type alkaline phosphatase activity mechanism.     

Agarose gel patterns of alkaline phosphatase isoenzymes before and after treatment with neuraminidase

The clinical value of alkaline phosphatase isoenzyme analysis is limited by the inability of most electrophoretic methods to resolve the liver and bone isoenzymes. The authors attacked this problem by treating serum samples with neuraminidase, then running treated and untreated samples side-by-side on specially prepared agarose gels. Each isoenzyme showed a characteristic mobility before and after neuraminidase treatment that allowed its identification. The mobility of the bone isoenzyme was most affected, whereas the intestinal isoenzyme was resistant to the action of neuraminidase. In samples with both liver and bone isoenzymes, pretreatment with neuraminidase clearly distinguished the bands, allowing quantitation by densitometry. Using this method, the authors discovered 22 liver isoenzymes in 54 samples that were interpreted as only bone isoenzyme before neuraminidase treatment. They also detected two bone isoenzymes in 35 samples that appeared to contain only liver +/- biliary isoenzymes. In addition, this procedure enabled them to characterize several unusual isoenzymes with respect to mobility, thus avoiding confusion with the other isoenzymes.