Medusa cells: cytostructure and cytochemistry of amoeboid eosinophils with pseudopod-like processes

Summary Eosinophils having one or more pseudopod-like processes of various lengths are observed in bone marrow, peripheral blood, sputum, nasal smears, and in other exfoliative cytology and tissue specimens after fixation and staining for histochemical study; they are called medusa cells. Although the conformations and lengths of the processes vary, they resemble protozoal pseudopodia. The form which first called our attention to these cells is a conical determinate projection that tapers to a fine tip, much like a protozoal axopod. A basal specialization in the form of a vesicle or thickening may be frequently observed where the process protrudes from, or appears attached to, the cell body. The processes of these eosinophil variants appear morphologically specialized to interact with other cellular elements of the blood and are occasionally seen in contact with, or engulfing, erythrocytes, platelets or other leukocytes.Two hydroperoxidases have been elucidated in eosinophils and medusa cells by virtue of different substrate specificities, subcellular localizations and inhibitor sensitivities. One of these hydroperoxidases is shown by 3,3-diaminobenzidine, is cyanide resistant, and is never observed in granules or rods in the medusa cell processes; it is frequently polarized to the sites of contact of medusa cells with other cellular elements of the blood. The other hydroperoxidase is revealed byp-phenylenediamine-pyrocatechol, is sensitive to cyanide and is frequently observed in granules and rods in medusa cell processes as well as in a population of larger granules in the cell bodies; the granules in the processes appear to be precursors to the rods, which may be related to Charcot-Leyden crystals.The extrusion of medusa cell processes is facilitated by the divalent cations calcium and magnesium and is inhibited by anions which sequester them such as phosphate, EDTA, citrate and oxalate. Medusa cells have been observed in samples from both rodents and humans and can be very prominent when eosinophilia accompanies allergy, parasitosis and malignancy.     

Pseudopod-like basal cell processes in intestinal cholecystokinin cells

Cholecystokinin (CCK) is secreted by neuroendocrine cells comprising 0.1%-0.5% of the mucosal cells in the upper small intestine. Using CCK promoter-driven green fluorescent protein (GFP) expression in transgenic mice, we have applied immunofluorescence techniques to analyze the morphology of CCK cells. GFP and CCK colocalize in neuroendocrine cells with little aberrant GFP expression. CCK-containing cells are either flask- or spindle-shaped, and in some cells, we have found dendritic processes similar to pseudopods demonstrated for gut somatostatin-containing D cells. Most pseudopods are short, the longest process visualized extending across three cells. Pseudopods usually extend to adjacent cells but some weave between neighboring cells. Dual processes have also been observed. Three-dimensional reconstructions suggest that processes are not unidirectional and thus are unlikely to be involved in migration of CCK cells from the crypt up the villus. Abundant CCK immunostaining is present in the pseudopods, suggesting that they release CCK onto the target cell. In order to identify the type of cells being targeted, we have co-stained sections with antibodies to chromogranin A, trefoil factor-3, and sucrase-isomaltase. CCK cell processes almost exclusively extend to sucrase-isomaltase-positive enterocytes. Thus, CCK cells have cellular processes possibly involved in paracrine secretion.     

Characterization of basal lysosomes in exocrine acinar cells

Exocrine acinar cells possess a unique system of basally located lysosomes. Cytochemically, these lysosomes do not contain acid phosphatase, but react positively for trimetaphosphatase (C Oliver: J Histochem Cytochem 28:78, 1980). The present study extends the morphological and cytochemical characterization of these lysosomes in pancreatic, parotid, and exorbital lacrimal acinar cells from Sprague-Dawley rats and National Institutes of Health Swiss mice. The basal lysosomes are highly pleomoric in nature, and frequently appear as a system of anastomosing tubules of varying width. The lysosomes have a close morphological relationship with both the rough endoplasmic reticulum and mitochondria. In addition to trimetaphosphatase activity, the lysosomes are reactive for aryl sulfatase B, thiolacetic acid esterase, and cholinesterase. Since the cholinesterase activity could not be inhibited by specific inhibitors, this activity is most likely due to the presence of nonspecific esterases. The results of this study confirm the lysosomal nature of the basal lysosomes and underscore the necessity of using multiple enzyme activities to identify and characterize lysosomes.     

Characterization of basal nitric oxide production in living cells

Nitric oxide (NO) is an important modulator of immune, endocrine and neuronal functions; however, measuring physiological levels of NO in cell cultures is generally difficult because of the lack of suitable methodologies. We have selected three cell lines from different origins: the neuroblastoma-derived Neuro2A (N2A), the cholinergic SN56 and the non-neuronal COS-1. We first demonstrated the presence of NADPH-diaphoretic activity, a potential marker of the NO-synthesizing (NOS) enzyme. By immunocytochemistry, using specific antibodies for each NOS subtype, we observed that subtype I was present in all cell lines and that subtype II was present in COS-1 and N2A cell lines. The presence of these NOS subtypes was further verified by Western blot analysis. Control cells treated with DAF-2 DA exhibited significant fluorescent levels corresponding to basal NO production. The subcellular distribution of the synthesizing enzyme was consistent with the NO-fluorescence signal; whereas, fixation affected the subcellular pattern of NO fluorescence signal. Addition of NOS inhibitors or NO scavengers to the incubation medium reduced the intensity of the NO fluorescence signal in a concentration-dependent manner. Conversely, increasing concentrations of a NO donor, or incident light, increased the fluorescence intensity. Our observation of NO production and distribution using the DAF-2 method has a direct impact on studies using these cell lines.     

Evaluation through literature data on standardized ileal digestibility and basal ileal endogenous loss of amino acids associated with barley in pigs

This meta-analysis aimed to estimate the standardized ileal digestibility (SID) and the basal ileal endogenous amino acid losses (IAA_end) in barley for growing pigs. In total, 38 different barley treatments published in 26 peer-reviewed papers were used for the meta-analysis containing information on dietary composition including amino acid (AA) contents of the assay diets, and (or) barley samples, as well as apparent ileal digestibility (AID) of AA in barley. The SID of AA was determined by either correcting AID of AA for their IAA_end or by regression analysis between the apparent ileal digestible and total dietary AA contents. The SID values obtained by correcting the AID values for their IAA_end amounted to 70%, 77%, 74% and 63% for Lys, Met, Thr and Trp, whereas those based on regression analysis method were 82%, 82%, 69% and 55%, respectively. Estimates of basal ileal endogenous loss of CP in ileal digesta varied considerably and averaged 11.84 g/kg dry matter intake (DMI), whereas IAA_end for indispensable AA ranged from 0.05 g/kg DMI for Trp to 1.90 g/kg DMI for Leu. In most cases, these estimates were considerably higher than previously reported values for IAA_end. The results of the present regression analysis indicate for most AA higher SID values compared with SID of most AA that were obtained by correcting AID values for IAA_end. In view of the observed high variations in IAA_end and the low CP content of the barley samples, estimating SID of AA based on literature data by means of the regression method may improve accuracy of SID coefficients for barley. In contrast, transformation of AID values into their corresponding SID values by using a constant correction factor for IAA_end adds an additional source of error, thereby reducing the precision in estimating SID of AA.     

Stress-induced inhibition of the ileal, caecal, and colonic contractile activity in rabbits]

In alert rabbits, immobilisation stress decreased the spike burst rate and amplitude in ileum, caecum, and colon for 20 min. Following beta-adrenoceptor blockade, the contractile activity suppression was aggravated. The stress seems to lead to suppression of the ileal, caecal, and colonic contractile activity for up to 40 min which is unrelated to adrenergic inhibition of the smooth muscle activity mediated by beta-adrenoceptors.     

Role of increased basal expression of heat shock protein 72 in colonic epithelial c2BBE adenocarcinoma cells.

Although the expression of heat shock proteins (hsps) can be induced by a variety of stressful stimuli, certain neoplasms, including human intestinal T84, HT-29, and Caco2 adenocarcinoma cell lines, express constitutively high levels even under nonstress conditions. In this study, we examine the functional significance of increased hsp72 in spontaneously differentiating Caco2bbe (C2) cells. The expression of hsp72 in these cells was specifically inhibited by hsp72 antisense transfection. The loss of hsp72 expression did not affect growth rate, contact inhibition, morphological development, or functional differentiation. In contrast, these cells were significantly more sensitive to the injurious effects of oxidants and tumor necrosis factor (TNF) but not doxorubicin. To investigate potential mechanisms of action, a number of steps in the TNF-mediated cell death was measured. Antisense reduction of hsp72 did not alter activation of IkappaB. In contrast, mitochondrial cytochrome c release and activation of caspase 9 were significantly delayed in hsp72 antisense cells stimulated either with TNF or monochloramine. In conclusion, high endogenous expression of hsp72 by intestinal adenocarcinoma cells appears to confer selective survival advantage but does not affect their growth and differentiation.     

Gastric protective effect of peripheral PYY through PYY preferring receptors in anesthetized rats

The influence of intravenous peptide YY (PYY) on the gastric injury induced by 45% ethanol was investigated in urethane-anesthetized rats. PYY (25, 75, 125, and 250 pmol x kg(-1) x h(-1)) significantly reduced gastric lesions by 36, 59, 40, and 38%, respectively. Antibody against ratPYY (2 mg/rat) injected intravenously completely prevented the gastroprotective effect of intravenous PYY (75 pmol x kg(-1) x h(-1)), whereas injected intracisternally (460 microg/20 microl), it significantly prevented intracisternal PYY (24 pmol/rat)-induced 58% reduction of ethanol lesions but not that induced by intravenous PYY. Vagotomy did not influence the gastroprotective effect of intravenous PYY. The Y(1)/"PYY-preferring" receptor agonist [Pro(34)]PYY (75 pmol x kg(-1) x h(-1) iv) significantly decreased ethanol-induced gastric lesions by 82%, whereas [Leu(31), Pro(34)]NPY, a Y(1)/Y(3) agonist, and PYY-(3-36), a Y(2) agonist, had no effect. These data indicate that PYY-infused intravenously at doses reported to mimic postprandial peak blood levels prevents ethanol-induced gastric injury through vagal independent pathways and PYY-preferring receptors.     

Characterization of Na+/K+/Cl- cotransport in cultured HT29 human colonic adenocarcinoma cells

A Na+/K+/Cl- cotransport pathway has been examined in the HT29 human colonic adenocarcinoma cell line using 86Rb as the K congener. Ouabain-resistant bumetanide-sensitive (OR-BS) K+ influx in attached HT29 cells was 17.9 +/- 0.9 nmol/min per mg protein at 25 degrees C. The identity of this pathway as a Na+/K+/Cl- cotransporter has been deduced from the following findings: (a) OR-BS K+ influx ceased if the external Cl- (Cl-o) was replaced by NO3- or the external Na+ (Na+o) by choline; (b) neither OR-BS 24Na+ nor 36Cl- influx was detectable in the absence of external K+ (K+o); and (c) concomitant measurements of 86Rb+, 22Na+, and 36Cl- influx indicated that the stoichiometry of the cotransport system approached a ratio of 1N+:1K+:2Cl-. In addition, OR-BS K+ influx was exquisitely sensitive to cellular ATP levels. Depletion of the normal ATP content of 35-40 nmol/mg protein to 10-15 nmol/mg protein, a concentration at which the ouabain-sensitive K+ influx was unaffected, completely abolished K+ cotransport. OR-BS K+ influx was slightly reduced by the divalent cations Ca2+, Ba2+, Mg2+ and Mn2+. Although changes in cell volume, whether shrinking or swelling, did not influence OR-BS K+ influx, ouabain-sensitive K+ influx was activated by cell swelling. As in T84 cells, we found that the OR-BS K+ influx in HT29 cells was stimulated by exogenous cyclic AMP analogues and by augmented cyclic AMP content in response to vasoactive intestinal peptide, forskolin, norepinephrine and forskolin or prostaglandin E1.     

Characterization of rat colonic epithelial cell populations

Colonic epithelial cells from male Sprague-Dawley rats were isolated by incubating everted colon with hyaluronidase suspended in Puck's saline F with an average cell yield of 120 x 10(6). These cells were fractionated by discontinuous Ficoll gradient and by short-term cell culture techniques. Centrifugation of isolated cells on discontinuous Ficoll gradient (15-35%) yielded populations differing in their proliferative activity. Additionally, a short-term cell culture technique was standardized to fractionate these cells according to their proliferating activity as judged by their DNA synthesis and thymidine kinase activity. Viability of these cells were judged by trypan blue exclusion, capacity to oxidize glucose and incorporation of precursors into protein DNA, RNA and glycoproteins. These fractionated cells were examined and identified by cytological studies. Cells showing proliferative activity sedimented at heavier regions of the Ficoll gradient, and the majority of these cells attached to the surface under conditions of short-term culture. Columnar mature absorptive cells and mucus-secreting goblet cells that showed very little proliferative activity sedimented at lighter zones of the Ficoll gradient and a major portion of these cells failed to attach by the cell culture method.     

Peptide YY containing enteroendocrine cells and peripheral tissue sensitivity to PYY and PYY(3-36) are maintained in diet-induced obese and diet-resistant rats

Peptide YY (PYY) is a gastrointestinal hormone, localized in enteroendocrine L-cells. Its hydrolyzed form PYY(3-36) is a satiety factor. The aim of this study was to identify if intestinal PYY enteroendocrine cells or content correlate with the diet-induced obese (DIO) or diet-resistant (DR) phenotypes. We also examined intestinal sensitivity to PYY and PYY(3-36) in DIO and DR rats. Animals were maintained on a medium-high fat diet and split into DIO and DR groups based on weight gain. PYY immunoreactive cells were unaltered in DIO intestine and stomach compared to DR rats. PYY content and circulating levels were also unchanged in DIO rats. Intestinal PYY and PYY(3-36) responses were enhanced in fasted rats, and equipotent in both DIO and DR jejunum. We conclude that PYY cell number, tissue content and peripheral sensitivity are maintained in DIO rats. Our data suggests that neither PYY nor PYY(3-36) contribute to the maintenance of either the DIO or DR phenotype, and that peripheral resistance to PYY and PYY(3-36) does not accompany DIO.     

Characterization of inhibitory innervation in porcine colonic circular muscle

Effects of stimulation of intramural nerves in the circular smooth muscle layer of the porcine colon (Sus scrofa domestica) were studied using the sucrose-gap technique. Electrical field stimulation of the preparation, superfused with Krebs solution at 21 degrees C, induced a transient hyperpolarization of the smooth muscle cell membrane. This hyperpolarization was an inhibitory junction potential (IJP). The responses obtained from circular muscle originating from either the centripetal or centrifugal gyri of the ascending colon did not differ significantly. The IJP was characterized as being mediated by intramural, nonadrenergic, noncholinergic (NANC) nerves. The amplitude and latency of the IJP changed linearly with temperature (15-25 degrees C: +1 mV and -0.1 s per degree Celsius, respectively) reflecting a temperature-dependent synchronization of transmitter release. The membrane resistance decreased during the IJP. The IJP amplitude decreased or increased during conditioning hyperpolarizations or depolarizations, respectively, and reversed at membrane potentials about 30 mV more negative than the resting membrane potential. Potassium conductance blocking agents, barium (1 mM), tetraethylammonium chloride (TEA, 20 mM), 4-aminopyridine (4-AP, 5 mM), apamin (1 microM), and aminacrine (10(-4) M) added to the superfusion medium increased the membrane resistance. Only barium, TEA, and apamin depolarized the smooth muscle cell membrane. The IJP amplitude decreased in the presence of aminacrine and apamin to 75 and 35%, respectively, suggesting that apamin-sensitive Ca2+-activated K+ channels are involved in this response. ATP, adenosine, and related adenine nucleotides in concentrations up to 10(-3) M did not mimic the IJP. Superfusion with ATP for 15 min revealed a gradually increasing attenuation by up to 20% of the IJP. This might suggest that the release of neurotransmitter from intramural NANC nerves is modulated presynaptically via purinoceptors. Exogenously applied vasoactive intestinal polypeptide (VIP) in concentrations of 10(-9) to 10(-4) M did not affect the preparation. Also at elevated temperatures (up to 35 degrees C), VIP (10(-7) to 10(-4) M) did not cause measurable effects. It is concluded that the inhibitory mediator of the intramural NANC nerves present in the circular muscle layers of the porcine colon is neither a purine nor VIP.     

Characterization of uptake and release processes ford- andl-aspartate in primary cultures of astrocytes and cerebellar granule cells

The uptake ofl-andd-aspartate was studied in astrocytes cultured from prefrontal cortex and in granule cells cultured from cerebellum. A high affinity uptake system forl- andd-aspartate was found in both cell types, and the two stereoisomers exhibited essentially the sameK _m - andV _max -values in bouth astrocytes (l-aspartate:K _m 77 μM;V _max 11.8 nmol×min^?1×mg^?1;d-aspartate:K _m 83 μM;V _max 14.0 nmol×min^?1×mg^?1) and granule cells (l-aspartate:K _m 32 μM;V _max 2.8 nmol ×min^?1×mg^?1;d-aspartate:K _m 26 μM;V _max 3.0 nmol×min^?1×mg^?1). To investigate whetherl-glutamate,l-aspartate andd-aspartate use the same uptake system a detailed kenetic analysis was performed. The uptake kinetics of each one of the three amino acids was studied in the presence of the two other amino acids, and no essential differences between the uptake characteristics of the amino acids were found. In addition to the uptake studies the release ofD-aspartate from cerebellar granule cells was investigated and compared withl-glutamate release. A Ca²⁺-dependent, K⁺-induced release was found for both amino acids.     

A new molecular form of PYY: structural characterization of human PYY(3-36) and PYY(1-36)

A radioimmunoassay was developed using an antibody raised in rabbits against synthetic porcine PYY. This radioimmunoassay was used to detect PYY immunoreactivity in human intestinal extracts. Human colonic mucosa was extracted with acid, centrifuged and the supernatant concentrated by low pressure preparative reverse phase chromatography. A subsequent C-18 reverse phase HPLC step separated two peaks of PYY immunoreactivity. Each peak was purified by sequential steps of ion-exchange FPLC and reverse phase HPLC. In the final purification step single absorbance peaks were associated with PYY immunoreactivity. Microsequence, amino acid, and mass spectral analysis of the intact and tryptic fragments of the two peptides were consistent with the structures: YPIKPEAPGEDASPEELNRYYASLRHYLNLVTRQRY-amide [human PYY(1-36)] and--IKPEAPGEDASPEELNRYYASLRHYLNLVTRQRY-amide [human PYY(3-36)]. Human PYY(1-36) differs from porcine PYY only at position 3, with Ile instead of Ala, and position 18, with Asn instead of Ser. PYY(3-36) may differ in its biological activity from the intact peptide. Its high proportions in the colon suggest that it is released into the circulation where it could act as a partial antagonist of PYY(1-36).     

Immunocytochemical identification of polypeptide YY (PYY) cells in the human gastrointestinal tract

Various parts of the human gastrointestinal tract were investigated immunocytochemically for the occurrence of polypeptide YY (PYY) and pancreatic polypeptide (PP). PYY-immunoreactive cells were observed in the lower part of the ileum, in the colon and in the rectum, and PP-immunoreactive cells were found in the colon and rectum. Both cell types were of the open type, i.e. they extended from the basal lamina to the gut lumen. PYY-immunoreactive cells were seen to emit cytoplasmic processes to the neighbouring goblet cells. This latter observation suggests that PYY cells may exert a paracrine action on the mucus-secreting goblet cells. Staining of consecutive thin plastic sections and staining of the same section simultaneously for two peptides showed that PYY-immunoreactivity did not occur in PP- or enteroglucagon-immunoreactive cells. On the ultrastructural level PYY-immunoreactivity was localized in basal granulated endocrine cells. These cells contained round or slightly oval electron dense granules with a mean diameter of 150 nm (range 100-300 nm).     

Secretagogue response of goblet cells and columnar cells in human colonic crypts

Crypts of Lieberkühn were isolated fromhuman colon, and differential interference contrast microscopydistinguished goblet and columnar cells. Activation with carbachol(CCh, 100 µM) or histamine (10 µM) released contents from gobletgranules. Stimulation with prostaglandinE₂(PGE₂, 5 µM) or adenosine (10 µM) did not release goblet granules but caused the apical margin ofcolumnar cells to recede. Goblet volume was lost during stimulationwith CCh or histamine (~160 fl/cell), but not withPGE₂ or adenosine. Three-quartersof goblet cells were responsive to CCh but released only 30% of gobletvolume. Half-time for goblet volume release was 3.7 min.PGE₂ stimulated a prolonged fluidsecretion that attained a rate of ~350 pl/min. Columnar cells lost~50% of apical volume during maximalPGE₂ stimulation, with a half-timeof 3.3 min. In crypts from individuals with ulcerative colitis, goblet cells were hypersensitive to CCh for release of goblet volume. Theseresults support separate regulation for mucus secretions from gobletcells and from columnar cells, with control mechanisms restrictingtotal release of mucus stores.

     

Secretagogue response of goblet cells and columnar cells in human colonic crypts

Crypts of Lieberkühn were isolated from human colon, and differential interference contrast microscopy distinguished goblet and columnar cells. Activation with carbachol (CCh, 100 microM) or histamine (10 microM) released contents from goblet granules. Stimulation with prostaglandin E(2) (PGE(2), 5 microM) or adenosine (10 microM) did not release goblet granules but caused the apical margin of columnar cells to recede. Goblet volume was lost during stimulation with CCh or histamine ( approximately 160 fl/cell), but not with PGE(2) or adenosine. Three-quarters of goblet cells were responsive to CCh but released only 30% of goblet volume. Half-time for goblet volume release was 3.7 min. PGE(2) stimulated a prolonged fluid secretion that attained a rate of approximately 350 pl/min. Columnar cells lost approximately 50% of apical volume during maximal PGE(2) stimulation, with a half-time of 3.3 min. In crypts from individuals with ulcerative colitis, goblet cells were hypersensitive to CCh for release of goblet volume. These results support separate regulation for mucus secretions from goblet cells and from columnar cells, with control mechanisms restricting total release of mucus stores.     

Characterization of 5-fluorouracil microspheres for colonic delivery

The purpose of this investigation was to prepare and evaluate the colon-specific microspheres of 5-fluorouracil for the treatment of colon cancer. Core microspheres of alginate were prepared by the modified emulsification method in liquid paraffin and by cross-linking with calcium chloride. The core microspheres were coated with Eudragit S-100 by the solvent evaporation technique to prevent drug release in the stomach and small intestine. The microspheres were characterized by shape, size, surface morphology, size distribution, incorporation efficiency, and in vitro drug release studies. The outer surfaces of the core and coated microspheres, which were spherical in shape, were rough and smooth, respectively. The size of the core microspheres ranged from 22 to 55 μm, and the size of the coated microspheres ranged from 103 to 185 μm. The core microspheres sustained the drug release for 10 hours. The release studies of coated microspheres were performed in a pH progression medium mimicking the conditions of the gastrointestinal tract. Release was sustained for up to 20 hours in formulations with core microspheres to a Eudragit S-100 coat ratio of 1∶7, and there were no changes in the size, shape, drug content, differential scanning calorimetry thermogram, and in vitro drug release after storage at 40°C/75% relative humidity for 6 months.