Marvin R. Alvarez

Factors affecting in vitro phagocytosis by oyster hemocytes

Abstract Hemocytes withdrawn from the pericardial cavity of the American oyster, Crassostrea virginica , were settled onto monolayers of immobilized fluorescent polystyrene microspheres (beads) in culture flasks. Phagocytosis was quantitated by fluorescence microscopy and flow cytometry after enzymatic detachment of the cells from unphagocytized beads and fixation of the cells. Unattached beads were removed by settling the mixture through a discontinuous sucrose gradient. Internalization of the beads following this procedure was verified by transmission electron microscopy. Phagocytosis was evident within 15 min after addition of the cells to the monolayer. Flow cytometry showed distinct cell populations corresponding to cells containing one to five or more beads indicating that multiple particle phagocytosis occurs rapidly after contact between cells and particles. Hemocytes incubated with beads at various temperatures showed maximum phagocytic activity over a wide range (10°–37°C) with inhibition occurring below 8°C. Inhibition of cell attachment to the bead monolayer was evident at 0° and 4°C and is probably a major cause of the decreased amount of phagocytosis observed at low temperatures. Treatment of the cells with cytochalasin B prior to incubation inhibited phagocytosis. Incubation of the cells with beads under atmospheres of pure nitrogen or helium produced no inhibition of phagocytosis. These data suggest that the hemocytes of the American oyster are able to phagocytize particles rapidly at extremes of the range of biological temperatures and under completely anaerobic conditions.

Nuclear size and DNA content in rat cardiac myocytes during growth, maturation and aging

Changes in nuclear volume and DNA content were examined in cardiac myocytes isolated from 21-day-old (weanling, W), 3-month-old (adult, A), and 2-year-old (old, O) rats to document normal parameters for nuclear growth and DNA content. Nuclear volume was calculated from direct measurements of isolated myocyte nuclear profiles and DNA content was measured from DAPI-stained nuclei using an image analysis microdensitometry system. Myocyte volume was measured with a Coulter Channelyzer system. Nuclear volume increased 79% from W to A as a result of an increase in nuclear length. Nuclear width was unchanged. Nuclear volume was not changed from A to O. Approximately 98% of the left ventricular myocytes from all three rat groups contained a diploid DNA content with the remainder of nuclei being tetraploid. The degree of polyploidy increased slightly, but significantly, in right ventricular myocytes from O. Due to the substantially greater increase in myocyte volume relative to nuclear volume, nuclear volume percentage decreased from 3.65 +/- 0.28 to 1.64 +/- 0.13 from W to A but was unchanged from A to O. To summarize: (1) nuclear volume of rat cardiac myocytes increases significantly during normal physiological growth (W to A) but the rate of nuclear growth is less than that of cell volume; (2) the increase in nuclear size from W to A is not due to an increase in DNA content; (3) cardiac myocytes from Sprague-Dawley rats are predominantly diploid; and (4) there is little change in DNA content of cardiac myocytes from rats of this strain during growth, maturation and aging.

Cytometric investigations on hemocytes of the American oyster, Crassostrea virginica

Pericardial hemolymph was obtained from American Oysters (Crassostrea virginica) and the hemocytes characterized by flow cytometry. The cells were found to have a broad unimodal size distribution with a median diameter of 7 micrometers. Total protein measured by flow cytometric fluorescence of dansylated cells also revealed a broad unimodal distribution similar to that obtained for size. The proportion of hemocytes in each stage of the cell cycle was measured using DNA-specific DAPI fluorescence. Histograms showed a single peak representing the G(0)/G(1) population. There was no evidence of S or G(2)+M phases of the cell cycle, nor was polyploidy seen. The forward and orthogonal light scatter of fixed hemocytes showed no evidence of sub-populations on the basis of cytoplasmic granularity. Thus, in terms of these parameters, oyster hemocytes appear to represent a single population exhibiting graded cellular differences.

Uptake and tissue distribution of abiotic particles from the alimentary tract of the American oyster: A simulation of intracellular parasitism☆

Abstract Windows were cut in the left valves of American oysters, Crassostrea virginica, to access the mouth. A suspension of 2.16-μm-diameter fluorescent polystyrene beads were introduced into their stomachs by intubation and the windows were sealed. Stomach contents were sampled and vibratome cross sections were made at various time intervals postintubation. The locations of the beads in the tissues were mapped using an image analysis system. Hemocytes containing phagocytized beads were found in the lumen of the stomach shortly after intubation. Hemocytes with internalized beads were clustered in the tissues around the gut, in the mantle cavity, and in the palps at 2 hr postintubation. At 2 and 5 days after intubation the intracellular beads were randomly scattered in the tissues and not necessarily associated with the digestive tract. The number of beads present in the tissues diminished with time but some remained in the tissues 10 days after intubation. The polystyrene beads simulate to some extent parasites, which are phagocytized but are not destroyed by the hemocytes. These data show that indigestible particles carried in the phagosomes of hemocytes can enter the tissues from the stomach. The phagocytized particles are gradually eliminated from the tissues, but a number remain residual in the tissues within hemocytes for more than 10 days.

Oxidant production by hemocytes of the eastern oyster, Crassostrea virginica (Gmelin)

Abstract In investigations of the oxygen metabolism of hemocytes of the eastern oyster, Crassostrea virginica , oxygen uptake was recorded from cell suspensions. Additionally, hydrogen peroxide production by hemocytes was measured using a sensitive spectrofluorometric method. Additions of Concanavalin A or Zymosan significantly increased oxidant production over an endogenous level, whereas an increase with Phorbol Myristate Acetate was insignificant. Using a diaminobenzidine method, cytoplasmic deposits thought to be associated with peroxidase activity were observed. The results suggest a hydroperoxide metabolism capable of enhancing microbiocidal activities of oyster phagocytes.

CYTOMETRIC STUDIES ON MERCENARIA HEMOCYTES

ABSTRACT: Hemocytes of two species of Mercenaria, M. campechiensis and M. mercenaria , were compared relative to size, total cellular protein, and nuclear DNA content by employing flow cytometry. No significant differences were found in these characteristics. Cytometric profiles of nuclear DNA appear superposable for Mercenaria and easily distinguished from Crassostrea, Lymnaea , and the guinea fowl, Numida.

FACTORS AFFECTING IN VITRO PHAGOCYTOSIS BY HEMOCYTES OF THE AMERICAN OYSTER

ABSTRACT: Proposed herein is a new method for assaying phagocytosis of polystyrene beads by hemocytes of Crassostrea virginica involving flow cytometry. In addition, it has been demonstrated that low temperature and cytochalasin B inhibit internalization. Anaerobic conditions did not inhibit bead phagocytosis.