The distribution and localisation of acid trimetaphosphatase in developing heterophils and eosinophils in the bone marrow of the fowl and the duck

Summary The distribution and localisation of acid trimetaphosphatase was investigated in developing heterophils and eosinophils from fowl and duck. In the heterophils of both species, trimetaphosphatase activity progressively increased in concentration from a thin peripheral band in the round immature primary granules to a fairly dense uniform reaction product in most of the mature specific spindle-shaped granules. Fowl and duck primary eosinophil granules had a similar distribution of reaction product as heterophils. In duck specific eosinophil granules the crystalline interna or externa, or both regions, contained strong activity whereas in the fowl, the activity of the specific granules was strongly-uniform in appearance.     

Ultrastructural features of the developing eosinophils in bone marrow and spleen of the musk shrew, Suncus murinus

Eosinophilopoiesis in the musk shrew, Suncus murinus, a representative of the order Insectivora, was studied by light and electron microscopy. To examine biochemical features of cytoplasmic granules, extraction with proteolytic enzymes was carried out on ultrathin sections of bone marrow. In this species, eosinophils are produced in the same manner in both spleen and bone marrow. Developing eosinophils were distinguished as belonging to four stages, recognized by ultrastructural changes in cytoplasmic organelles as well as the eosinophilic granules during maturation. Granulogenesis began by budding of vacuoles containing flocculent material from the concave face of the Golgi apparatus, in the promyelocyte to myelocyte stage. The matrix of developing granules transformed into a finely granular structure, and the large spherical granules of mature eosinophils were homogeneous without crystalline cores. It was shown by proteolytic enzyme extraction that the proteinaceous cores of mature granules were uniformly removed; there was no evidence that they contained crystalloid inclusions. These results indicate that shrew eosinophils can be regarded as cells that retain a prototype of eosinophil granules, probably like those of ancestral mammals rather than those of higher living Mammalia.     

Differentiation potential of bone marrow mesenchymal stem cells in duck

The bone marrow mesenchymal stem cells （MSCs） are multipotent stem cells which can differentiate into mesenchymal cells in vitro. In this study, MSCs in duck were isolated from bone marrow by density gradient centrifuge separation, purified and expanded in the me- dium. The primary MSCs were expanded for 11 passages. The different-passage MSCs were induced to differentiate into osteoblasts and neuron-like cells. Karyotype analysis indicated that MSCs kept diploid condition and the hereditary feature was stable. The different- passage MSCs expressed CD44, ICAM-1 and SSEA-4, but not CD34, CD45 and SSEA-1 when detected by immunofluorescence staining There was no significant difference among the positive rates of passages 2, 6 and 8 （P 〉 0.05）, but a significant difference existed among those of passages 2, 6, 8 and 11 （P 〈 0.05）. After the osteogenic inducement was added, the induced different-passage MSCs expressed high-level alkaline phosphatase （ALP）, and are positive for tetracycline staining, Alizarin Red staining and Von Kossa staining. After the neural inducement was added, about 70% cells exhibited typical neuron-like phenotype, the induced different-passage MSCs expressed Nestin, neuron-specific enolase （NSE） and glial fibrillary acidic protein （GFAP） when detected by immunofluorescence staining. There was no significant difference among the positive rates of passages 3, 4 and 6 （P〉0.05）, but a significant difference existed among those of passages 3, 4, 6 and 8 （P〈0.05）. These results suggest that MSCs in duck were capable of differentiating into osteoblasts and neuron-like cells in vitro.     

Alkaline phosphatase cytochemistry in confocal scanning light microscopy for imaging the bone marrow stroma

This paper reports on the use of alkaline phosphatase cytochemistry and combined conventional and confocal reflection and fluorescence scanning light microscopic modes in the study of human marrow stroma. It was found that the end product of the enzyme reaction using Napthol AS phosphate as substrate and Fast Blue BB as coupler reflected the 633 nm (red) light from a Helium-Neon laser. Serial optical sections suitable for 3-D reconstruction and selectively depicting the marrow reticulum cells could be obtained from thick glycol methacrylate sections reacted for Alkaline phosphatase. Furthermore, the yellow background of uncoupled diazonium salt over cytochemically unreactive structures in the same specimens and fields was used for imaging haemopoietic cell mass by operating the microscope at 488 nm (argon ion laser, blue-green). These methods may offer advantages in the investigation of the bone marrow stroma and its interplay with haemopoiesis and osteogenesis in normal and disease conditions.     

Electron microscopic demonstration of acid phosphatase activity in the developing and mature heterophils of the chicken

Summary As shown by electron microscopic histochemistry using a modified Gomori lead salt technique, acid phosphatase is present in large dense granules and the Golgi apparatus —but not the light granules—in both immature and mature heterophils in the chicken. The large dense granules appear to form by budding from the Golgi cisternae while the light granules appear to be unassociated with the Golgi apparatus. The findings indicate that the large, dense granules are the lysosomes of the heterophils in the chicken.     

Light microscope and electron microscope alkaline phosphatase cytochemistry of rat bone marrow leukocytes.

An enzyme cytochemical method yielding an osmiophilic reaction product, visible at both the light and electron microscope levels, has been applied to the study of alkaline phosphatase in rat bone marrow cells. The enzyme is present in both eosinophils and, in much smaller amounts, in neutrophils. In both cases it is present on the plasma membrane, and in eosinophils intracellular aggregations of reaction product are also seen. The specific granules in both cell types fail to react and the enzyme is first detectable at the promyelocyte stage. Thus the enzyme is demonstrable before specific granule formation begins in the neutrophil, indicating that they are not a significant site of alkaline phosphatase activity in the rat.     

Action of compounds which predominantly affect the peripheral M-cholinoreactive systems on the bone marrow eosinophils

Experiments on rats have shown that the blockade of M-cholinoreceptors with atropine (50,100 and 150 mg/kg) delays, while the stimulation of the M-cholinoreceptors with aceclidine (5, 10 and 15 mg/kg) intensifies the release of mature eosinophils from the bone marrow to the circulating blood. It is concluded that the peripheral M-cholinoreactive systems participate in the control of the release of the mature bone marrow eosinophils into the blood.     

Enzyme cytochemistry of unfixed leukocytes and bone marrow cells using polyvinyl alcohol for the diagnosis of leukemia

Cytochemical methods for the demonstration of enzyme activities in blood and bone marrow cells were systematically improved by the addition of an inert polymer, polyvinyl alcohol (PVA), to the incubation medium and by using optimized reaction media. The methods investigated were tetrazolium salt methods for lactate, glucose-6-phosphate, succinate and glutamate dehydrogenase, the indoxyl-tetrazolium salt method for alkaline phosphatase, the diaminobenzidine method for peroxidase, and diazonium salt methods for chloroacetate esterase, beta-glucosaminidase, beta-glucuronidase, acid phosphatase, and dipeptidylpeptidase II and IV. PVA in the media preserved the morphology of cells very well and prevented leakage of large molecules such as enzymes from the cells. Therefore, fixation or long periods of air-drying prior to incubation leading to substantial loss of enzyme activity could be avoided. A brief period of drying (2 min at 37 degrees C) of the cell preparations just before the incubation was sufficient for making the cells permeable. Localization of enzyme activities was very precise and precipitation of the final reaction product was confined to sites which are known to contain the enzyme under study (granules, mitochondria, lysosomes). These advantages advocate the use of PVA in haematological enzyme cytochemistry and especially for diagnosis of leukemia.     

Enzyme cytochemistry of unfixed leukocytes and bone marrow cells using polyvinyl alcohol for the diagnosis of leukemia

Summary Cytochemical methods for the demonstration of enzyme activities in blood and bone marrow cells were systematically improved by the addition of an inert polymer, polyvinyl alcohol (PVA), to the incubation medium and by using optimized reaction media. The methods investigated were tetrazolium salt methods for lactate, glucose-6-phosphate, succinate and glutamate dehydrogenase, the indoxyl-tetrazolium salt method for alkaline phosphatase, the diaminobenzidine method for peroxidase, and diazonium salt methods for chloroacetate esterase, -glucosaminidase, -glucuronidase, acid phosphatase, and dipeptidylpeptidase II and IV. PVA in the media preserved the morphology of cells very well and prevented leakage of large molecules such as enzymes from the cells. Therefore, fixation or long periods of air-drying prior to incubation leading to substantial loss of enzyme activity could be avoided. A brief period of drying (2 min at 37° C) of the cell preparations just before the incubation was sufficient for making the cells permeable. Localization of enzyme activities was very precise and precipitation of the final reaction product was confined to sites which are known to contain the enzyme under study (granules, mitochondria, lysosomes). These advantages advocate the use of PVA in haematological enzyme cytochemistry and especially for diagnosis of leukemia.     

Electron cytochemistry of calcium uptake in the fragmented sarcoplasmic reticulum

Summary Ultrastructural aspects and cytochemical localisation of Ca-uptake in the fragmented sarcoplasmic reticulum (FSR) have been studied with positive and various negative staining techniques.Effect of fixation, staining and other preparative procedures for electron microscopy on the result of the cytochemical reaction have been investigated.Calcium outflow from loaded vesicles occurs in various degree depending on the method used so that no quantitative estimation of Ca-uptake appears possible by ultrastructural studies alone.Ultrastructure and proportion of calcium containing vesicles also vary considerably and no certain correlations can be made between their size and shape and localisation of Ca-uptake. Certain informations can be obtained only by comparison of results with different methods, taking into account also the limits of each method applied.     

Hypothalamic control of the generation of mature natural killer lymphocytes in bone marrow and spleen of the mouse

Following previous work showing that electrothermocoagulation of the median region of the hypothalamus (MH) caused a marked and permanent decrease in the cytotoxicity of natural killer (NK) cells and in the number of large granular lymphocytes, a study was made of the effect of such lesions on the generation of NK cells in the bone marrow (BM) and spleen of C57BL/6 mice. Fresh spleen and BM cells from MH-lesioned and sham-operated mice were cultured with 40 U of recombinant interleukin-2 (rIL-2). NK activity was significantly higher in BM of lesioned mice, whereas spleen NK activity was greater in the sham-operated controls. NK cells matured by culture with rIL-2 were characterized by assay with fluorescent monoclonal antibodies and found to display the typical NK phenotype. These results show that the number of NK precursors is greater in BM of MH-lesioned mice and that their migration into other organs is probably partially impeded. It can also be concluded that intactness of both BM and the hypothalamus is essential for the physiological generation of NK cells.