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PubMed2001

Uterine tumor resembling ovarian sex-cord tumor--a case report and review of the literature.

A uterine tumor resembling an ovarian sex-cord tumor (UTROSCT) is a very rare lesion with only 38 cases reported in the literature so far. Here, we show an additional case of a pure UTROSCT with a DNA stemline at 1c in a 49-year-old woman presenting with abnormal vaginal bleeding. Problems in differential diagnosis arise mainly due to the variable histological picture of UTROSCT. Immunohistochemically, these tumors express cytokeratin, epithelial membrane antigen, vimentin, and smooth muscle actin. Moreover, in some cases, CD99 and alpha-inhibin are detectable. Although 36% of UTROSCT have infiltrative margins, almost all of them behave benignly. It is thus questionable whether the same prognostic criteria apply for these tumors as for endometrial stromal sarcomas. However, in the so-called mixed UTROSCT, the endometrial stromal sarcoma component determines the outcome.

Antigens, Neoplasm↗
PubMed1998

Quantitative analysis of small intestinal microcirculation in the mouse.

Impairment of intestinal nutritive perfusion and accumulation of inflammatory cells in the intestinal microvasculature are well-known sequelae of mesenteric ischemia/reperfusion, sepsis, and shock. However, the molecular mechanisms underlying these alterations are still not fully understood. The mouse is particularly suitable for the study of these mechanisms since in this species the involvement of, for example, adhesion receptors or pro-/anti-adhesive mediators can be selectively investigated by the use of monoclonal antibodies or gene-targeted strains. The aim of our present study was, therefore, to establish a model to investigate the microcirculation in the mouse small intestine. Under anesthesia by inhalation of isoflurane-N2O, Balb/c mice (n = 16) were laparotomized, and a segment of the jejunum was exteriorized for intrvital fluorescence microscopy. Using FITC-dextran (MW 150,000) as a plasma marker, functional capillary density (FCD) of both the intestinal mucosa and muscle layer was analyzed. Nutritive perfusion was homogeneous in both compartments with values for FCD of 512 +/- 15 cm-1 in mucosa and 226 +/- 21 cm-1 in the muscle layer. No significant changes were observed throughout the observation period of 2 h (FCD values at the end of the observation period: 524 +/- 31 cm-1 and 207 +/- 7 cm-1 in mucosa and muscle, respectively). Besides capillary perfusion, leukocyte-endothelial cell interaction was analyzed in postcapillary venules of the intestinal submucosa using rhodamine-6G as an in vivo leukocyte stain. Under physiological conditions only a few white blood cells were found rolling along or firmly adherent to the microvascular endothelium (number of rolling leukocytes 1 +/- 0.2 cells/mm per second; number of adherent leukocytes: 18 +/- 7 cells/mm2). In a separate group rhodamine-6G-labeled syngeneic platelets were infused to analyze platelet-endothelial cell interactions quantitatively in vivo. Platelets rolled along or attached to the endothelium in a manner similar to leukocytes. However, in contrast to leukocytes the interactions were not restricted to venules, but were also observed in small arterioles. The newly established model allows for the visualization and quantitative assessment of both nutritive perfusion and platelet/leukocytendothelial cell interactions within the distinct layers of the mouse small intestine. Using this model in combination with gene-targeted mice or monoclonal antibodies it is possible to investigate the molecular mechanisms of intestinal inflammation reactions.

Analysis of Variance↗
PubMed1998

Impact of ischemia-reperfusion injury on dimensional changes of hepatic microvessels.

Dimensional alteration of hepatic microvessels was demonstrated during reperfusion after normothermic hepatic ischemia. Using a specially designed cover glass, it was possible to relocate selected sites of observation and microvessels repeatedly throughout the whole reperfusion time. Twenty minutes of hepatic ischemia resulted in a decrease of sinusoidal diameter (mean +/- SEM; 10.0 +/- 0.3 microns at baseline, 8.2 +/- 0.2 microns after ischemia) and diameter of postsinusoidal venules (26.4 +/- 1.2 at baseline, 23.0 +/- 1.0 after ischemia). In the control group (no ischemia induced) no changes of these parameters were observed. Thus, the reduction of hepatic microvascular cross section was present during the early phase of reperfusion. Hepatic dysfunction was characterized by increased serum activity of liver enzymes and reduction of bile flow in the ischemia-exposed animals. It has been suggested that postischemic dimensional microvascular changes are involved in postischemic liver dysfunction.

Animals↗
PubMed1999

Localization and ultrastructure of pulmonary dendritic cells during delayed-type hypersensitivity reactions in mice.

Dendritic cells (DCs) are widely distributed in the airways and can serve as potent antigen-presenting cells. To clarify their involvement in the cell-mediated immune responses of the lung, we immunohistochemically investigated their distribution and kinetics during pulmonary delayed-type hypersensitivity (DTH) reactions induced in sensitized mice by intratracheal instillation of hapten. Cellular infiltrate appeared around the bronchiole and its accompanying blood vessel at 12 h after elicitation and progressively expanded by 48 h. As quantitated by computer-assisted morphometry, I-A(+) DCs and CD4(+) Th cells significantly increased in number around the bronchiole to a maximum at 24 h, whereas F4/80(+) macrophages were predominantly accumulated around the accompanying vessel with a peak at 48 h. Serial-section analysis revealed that DCs were colocalized with Th cells in the inflamed peribronchiolar tissue. Immunoelectron microscopy demonstrated that DCs found inside and around the capillaries and venules of peribronchiolar interstitium displayed round forms, indicating their emigration from here, while those situated far from the microvessels were elongated, often in close apposition to the lymphocytes. Mitosis of DCs was rarely seen. The present results suggest that peribronchiolar accumulation of DCs resulting from accelerated influx of blood-borne immature DCs and the interaction with T cells at the application site may play inducing roles in the development of pulmonary DTH reactions by enhancing the recruitment of macrophages.

Animals↗
PubMed1999

Ultrastructural, immunocytochemical and morphometric characterization of liver peroxisomes in gray mullet, Mugil cephalus.

Peroxisomes of the hepatocytes of gray mullets, Mugil cephalus, were characterized cytochemically and immunocytochemically using antibodies against the peroxisomal proteins catalase and palmitoyl-coenzyme A (CoA) oxidase. In addition, morphometric parameters of peroxisomes were investigated depending on the hepatic zonation, the age of the animals and the sampling season. Mullet liver peroxisomes were reactive for diaminobenzidine, but presented a marked heterogeneity in staining intensity. Most of the peroxisomes were spherical or oval in shape, although irregular forms were also observed. Their size was heterogeneous, with profile diameters ranging from 0.2 to 3 microm. Peroxisomes tended to occur in clusters, usually near the mitochondria and lipid droplets. They also showed a very close topographical relationship to the smooth endoplasmic reticulum. Mullet liver peroxisomes did not contain cores or nucleoids as rodent liver peroxisomes, but internal substructures were observed in the matrix, consisting of small tubules about 60 nm in diameter and larger semicircles 120 nm in diameter. The volume density of peroxisomes was higher in periportal hepatocytes of mullets sampled in summer than in pericentral hepatocytes, indicating that mullet peroxisomes vary depending on physiological and environmental conditions. By immunoblotting, the mammalian antibodies cross-react with the corresponding proteins in whole homogenates of mullet liver. Paraffin sections immunostained with the antibodies against catalase and palmitoyl-CoA oxidase showed a positive reaction corresponding to peroxisomes localized in the hepatocyte cytoplasm. In agreement, the ultrastructural study revealed that catalase and palmitoyl-CoA oxidase are exclusively localized in the peroxisomal matrix in fish hepatocytes, showing a dense gold labeling. The presence of the peroxisomal beta-oxidation enzyme palmitoyl-CoA oxidase in peroxisomes indicated that these organelles play a key role in the lipid metabolism of fish liver.

Animals↗
PubMed2006

Fitting experimental data to models that use morphological data from public databases.

Ideally detailed neuron models should make use of morphological and electrophysiological data from the same cell. However, this rarely happens. Typically a modeler will choose a cell morphology from a public database, assign standard values for Ra, Cm, and other parameters and then do the modeling study. The assumption is that the model will produce results representative of what might be obtained experimentally. To test this assumption we developed models of CA1 hippocampal pyramidal neurons using 4 different morphologies obtained from 3 public databases. The multiple run fitter in NEURON was used to fit parameter values in each of the 4 morphological models to match experimental data recorded from 19 CA1 pyramidal cells. Fits with fixed standard parameter values produced results that were generally not representative of our experimental data. However, when parameter values were allowed to vary, excellent fits were obtained in almost all cases, but the fitted parameter values were very different among the 4 reconstructions and did not match standard values. The differences in fitted values can be explained by very different diameters, total lengths, membrane areas and volumes among the reconstructed cells, reflecting either cell heterogeneity or issues with the reconstruction data. The fitted values compensated for these differences to make the database cells and experimental cells more similar electrotonically. We conclude that models using fully reconstructed morphologies need to be calibrated with experimental data (even when morphological and electrophysiological data come from the same cell), model results should be generated with multiple reconstructions, morphological and experimental cells should come from the same strain of animal at the same age, and blind use of standard parameter values in models that use reconstruction data may not produce representative experimental results.

Action Potentials↗
PubMed2006

The calcium binding proteins calbindin, parvalbumin, and calretinin have specific patterns of expression in the gray matter of cat spinal cord.

Calcium binding proteins (CBPs) regulate intracellular levels of calcium (Ca(2+)) ions. CBPs are particularly interesting from a morphological standpoint, because they are differentially expressed in certain sub-populations of cells in the nervous system of various species of vertebrate animals. However, knowledge on the cellular regulation governing such cell-specific CBP expression is still incomplete. In this work on the L7 segment of the cat spinal cord, we analyzed the localization and morphology of neurons expressing the CBPs calbindin-28 KD (CB), parvalbumin (PV), and calretinin (CR), and co-expressing CB and PV, CB and CR, and PV and CR. Single CBP-positive ((+)) neurons showed specific distributions: (1) CB was present in small neurons localized in laminae I, II, III and X, in small to medium size neurons in laminae III-VI, and in medium to large neurons in laminae VI-VIII; (2) PV was present in small size neurons in laminae III and IV and in medial portions of laminae V and VI, medium neurons and in lamina X at the border with lamina VII, in medium to large neurons in laminae VII and VIII; (3) CR labeling was detected in small size neurons in laminae I, II, III and VIII, in medium to large size neurons in laminae I and III-VII, and in small to medium size neurons in lamina X. Double labeled neurons were a small minority of the CBP(+) cells. Co-expression of CB and PV was seen in 1 to 2% of the CBP(+) cells, and they were detected in the ventral and intermediate portions of lamina VII and in lamina X. Co-localization of CB and CR was present in 0.3% of the cells and these cells were localized in lamina II. Double labeling for PV and CR occurred in 6% of the cells, and the cells were localized in ventral part of lamina VII and in lamina VIII. Overall, these results revealed distinct and reproducible patterns of localization of the neurons expressing single CBPs and co-expressing two of them. Distinct differences of CBP expression between cat and other species are discussed. Possible relations between the cat L7 neurons expressing different CBPs with the neurons previously analyzed in cat and other animals are suggested.

Animals↗
PubMed2004

Nephrogenic adenoma of the urinary bladder.

INTRODUCTION: The nephrogenic adenoma (NA) is a benign metaplastic lesion of the urothelium and is attributed to chronic irritation of the mucosa, by injury, infection, stone disease or intravesical instrumentations. We present our experience on this morbid entity, its clinical appearance in the urinary bladder, its frequency and relapses. Furthermore we reviewed the related recent literature and focused on its potential to neoplastic degeneration and the value of the new diagnostic modalities. PATIENTS AND METHODS: Four patients with NA of the urinary bladder are presented. The papilloid or polypoid formations observed by the cystoscopy were identified after the TUR, as NA of the urinary bladder. Their mean follow-up was 3.5 years. RESULTS: Remission of the symptoms was observed after TUR in all patients. Three out of four patients presented 1-7 relapses, while in one case, after seven NA relapses, a urothelial carcinoma of the bladder was diagnosed. CONCLUSIONS: Unlike histological features, the clinical--endoscopic characteristics of NA are non-specific. Even if it is not definitely considered like a premalignant condition, NA has to be followed up frequently and long lasting, because of its high recurrence rate. The combination of Cytology, Flow cytometry, DNA image analysis and Fluorescence in situ hybridisation of bladder washings or voided urine, are of high value in monitoring NA of the urothelium.

Adenoma↗
PubMed1995

Detection of interleukin-1 beta mRNA-expressing cells in human gingival crevicular fluid by in situ hybridization.

Interleukin-1 beta (IL-1 beta) mRNA-expressing cells in human gingival crevicular washings (GCW) obtained from patients with periodontitis and healthy controls were examined by in situ hybridization. GCW was done at 15 diseased sites [Gingival Index > 1; pocket depth > or = 5 mm] from five patients with adult periodontitis and eight clinically periodontal healthy sites from three volunteers GI < or = 1; PD < or = 3 mm), and then the samples were cytocentrifuged. In situ hybridization using digoxigenin-labelled oligonucleotide probe complementary to human IL-1 beta mRNA showed IL-1 transcripts in both polymorphonuclear leucocytes and mononuclear cells but not in epithelial cells in all GCW samples from diseased and healthy sites. Polymorphs were the predominant leucocytes in diseased and healthy sites, averaging 91.7 +/- 4.6 and 77.0 +/- 10.3%, respectively. The percentages of IL-1 beta mRNA-positive polymorphonuclear leucocytes in GCW samples from diseased and healthy sites were 92.3 +/- 4.7 and 80.9 +/- 10.3%, respectively. The IL-1 beta gene signals in individual cells were quantified in five samples (two healthy and three diseased sites). The mean amounts of IL-1 beta mRNA expression in polymorphonuclear leucocytes was higher than that of mononuclear cells in all samples and there was heterogeneity within the populations of polymorphonuclear and mononuclears cells in their ability to express the IL-1 beta gene. These findings indicate that IL-1 beta may be predominantly produced by polymorphonuclear leucocytes in the gingival crevice of patients with adult periodontitis and periodontally healthy controls.

Adult↗
PubMed1995

Light transmittance as an index of cell volume in hippocampal slices: optical differences of interfaced and submerged positions.

Light transmittance (T) in the CA1 region of hippocampal slices was measured during exposure to media of various osmolarities to determine the utility of optical measurements as an index of changes in cell volume. In slices positioned at the gas-liquid interface, hypo-osmotic medium consistently produced a decrease in T and hyperosmotic medium produced an increase in T. The magnitude of deltaT was graded as a function of the strength of osmotic change. All changes in T were reversible upon return to isosmotic medium. In contrast, osmotically induced changes in T in submerged slices were consistently opposite in direction to those observed in slices at the interface. The magnitude and direction of deltaT could be altered by systematic variation of the level of the bathing medium within the same chamber, indicating that both extrinsic optical properties of various interfaces, such as refraction and reflection, and intrinsic optical properties of the tissue contribute to the observed T. Spectral measurements eliminated the possibility that osmotically induced deltaT was the result of changes in light absorbance by intrinsic chromophores such as cytochromes or hemoglobin. The results show that measurements of deltaT can be a useful index of changes in cell volume in brain slices, provided that the level of the bath remains constant.

Animals↗
PubMed1996

Synergistic interactions between noradrenaline and glutamate in cytosolic calcium influx in cultured visual cortical neurons.

In primary neuronal cultures derived from the visual cortex of embryonic day 16-18 rats, intracellular free calcium concentration, [Ca2+]i, was increased by bath application of glutamate in a dose-dependent manner. Noradrenaline applied alone had relatively small effects. However, when glutamate concentrations eliciting modest increases in [Ca2+]i were applied together with 1 microM noradrenaline, the increase in [Ca2+]i could be enhanced by a factor of up to eight. The synergistic effect was seen in 147 neurons out of a total of 215 cells observed in 54 experiments. The observed enhancement was much more obvious at low doses of glutamate than with higher doses, augmenting all submaximal calcium responses to similar asymptotic levels. 2-Amino-5-phosphonovalerate (APV), the NMDA receptor antagonist, completely blocked the adrenergic enhancing effect (29/29 cells in 8 experiments). Among the antagonists specific to alpha 1, alpha 2 and beta subtypes of adrenoceptors, the beta antagonist propranolol most completely blocked the enhancing effect (13/14 cells in 4 experiments, reducing the effect by an amplitude of 90%). The involvement of the beta receptor pathway was further supported by the ability of a cAMP analog to mimic the enhancing effect of noradrenaline. On the other hand, an alpha 1 blocker showed no effect and an alpha 2 blocker showed only a relatively small effect. These results suggest that receptors for noradrenaline and glutamate colocalize on postsynaptic cortical cells and that adrenergic modulation of glutamate induced calcium influx most likely operate through the beta receptor pathway. It is further postulated that cortical ocular dominance plasticity may be at least partially implemented via a calcium dependent cascade.

2-Amino-5-phosphonovalerate↗
PubMed1995

Imaging of L-type Ca2+ channels in olfactory bulb neurones using fluorescent dihydropyridine and a styryl dye.

We have imaged the fluorescence of dihydropyridine-Bodipy (fDHP) in cultured olfactory bulb neurones in order to investigate the subcellular distribution of L-type calcium channels in these neurones. The neurones were stained with both fDHP and the voltage-sensitive styryl dye RH414. The fluorescence emission maxima of these dyes were in the green and red ranges of the spectrum, respectively, and were recorded by the 2 photomultiplier channels of a laser scanning microscope. The fDHP images were ratioed with the RH414 images taken simultaneously. The resulting ratio images revealed the spatial distribution of the surface density of L-type calcium channels. This density was highest on somata, in particular at the base of dendrites, and decreased with the distance from this maximum. Two classes of dendritic L-type channel distributions were observed: one with a homogeneously low density and another one with a characteristic gradient of the L-type channel density along proximal dendrites. This subcellular localization of L-type calcium channels is discussed in the light of specific functional roles.

Animals↗
PubMed1995

Apoptotic DNA fragmentation in the rat cerebral cortex induced by permanent middle cerebral artery occlusion.

Recent investigations have demonstrated internucleosomal DNA fragmentation in ischemic neuronal tissue. This type of fragmentation is characteristic of programmed cell death or apoptosis and suggests that neuronal death in stroke may be more complex than simple necrotic death. The present experiments provide a detailed examination of the regional localization and time course for apoptotic DNA fragmentation in the cerebral cortex following focal cerebral ischemia. Spontaneously hypertensive rats were subjected to permanent right middle cerebral artery occlusion and the cerebral cortices were examined for evidence of DNA fragmentation using electrophoretic, flow cytometric, and histological approaches. An electrophoretic examination of cortical DNA at 24 h after the occlusion indicated that the majority of nucleosomal ladders were in the transition zone or penumbra and the core of the infarction, with no fragmentation apparent in the contralateral normal cortex. A flow cytometric analysis of DNA fragmentation in intact cells revealed a similar pattern, with increased fragmentation observed in ischemic cortex vs. the contralateral cortex. Saggital sections taken 1.5 mm lateral to midline were collected from animals at 1, 4, and 24 h after the infarction and DNA fragmentation was examined histologically by terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labeling (TUNEL) staining. Quantitative analysis of these sections indicated that DNA fragmentation can be observed in the anterior and central area of the infarctions as soon as 1 h after the occlusion and that the extent and magnitude of the fragmentation increases at 4 and 24 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Cytological and DNA characteristics of hyperplasia/inflammation and cancer of the prostate.

A study has been made of the correlation between objectively measured nuclear DNA content (by image analysis cytometry) and visual pathologic/cytologic diagnoses in hyperplasia/inflammation and well, moderately and poorly differentiated carcinomas of the prostate. The DNA measurement data were described by four statistical description methods--namely mean deviation from 2C, percentage greater than 4.25 C, DNA index and entropy. In 36% (5/14) of well differentiated and 13% (1/8) of moderately differentiated cancer it was not possible by objective measurement methods to demonstrate any difference between hyperplasia/inflammation and cancer. On the other hand one or more of the statistical descriptors was significantly different from hyperplasia/inflammation in all of those cancers visually diagnosed as poorly differentiated. The results underline the need for improvement of diagnostic criteria of prostate cancer--in particular well differentiated--in order to correctly understand the epidemiology (true incidence), survival and effects of treatment on the disease.

DNA↗
PubMed1995

Propagation of intercellular calcium waves in PC12 cells overexpressing a carboxy-terminal fragment of amyloid precursor protein.

We have recently found that PC12 cells overexpressing a C-terminal 97 amino acid fragment containing the beta/A4 amyloid peptide of amyloid precursor protein (APP) exhibit an induced production of the gap junctional protein connexin43 (Cx43) and an induction of gap junctional communication as assessed by dye-transfer. In studies of two beta/A4-transfected PC12 clones, we show here that these cells, unlike normal PC12 cells or those transfected with vector alone, have the capacity for intercellular transmission of calcium waves as determined by imaging of calcium with fura-2. Intercellular wave propagation occurred in the absence of extracellular calcium and was blocked by known inhibitors of gap junctional communication (octanol and 12-O-tetradecanoyl-phorbol-13-acetate), suggesting mediation by gap junctions. The results indicate a disruptive influence of the C-terminal region of APP or of beta/A4 amyloid peptide on intercellular signaling via gap junctions, which may be relevant to the normal functions of APP or to pathology in Alzheimer's disease.

Amyloid beta-Protein Precursor↗
PubMed1995

Do caged-Ca2+ compounds mimic the physiological stimulus for secretion?

We measured the exocytotic response induced by a rapid and global increase in the concentration of Ca2+ or GTP gamma S (achieved by flash photolysis of caged compounds) in mast cells and chromaffin cells. Secretion was measured by following both cell membrane capacitance and amperometry. When Ca2+ was used to trigger secretion, we observed an immediate increase in capacitance, however, the first amperometric spike observed was delayed with respect to the change in capacitance (approximately 5 s in mast cells and approximately 0.6 s in chromaffin cells). In contrast, when GTP gamma S was used to trigger secretion, no such discrepancy was seen, both measurements reported a secretory response that was similarly delayed with respect to the stimulus. These data suggest that the capacitance increase, when triggered by a large Ca2+ step, reports events that do not result in the fusion of vesicles containing oxidizable substances. These results contrast with the smaller secretory responses evoked by the transient opening of Ca2+ channels. Under these more physiological conditions, pulsed-laser imaging studies revealed that the Ca2+ influx was localized to discrete areas at the plasma membrane. Thus the Ca2+ stimulus triggered by DM-nitrophen fails to reproduce the small localized changes seen during a physiological stimulation and creates an artifactual non-secretory capacitive response.

Animals↗
PubMed1995

Time resolved calcium microdomains and synaptic transmission.

The time course for the calcium entry that triggers release was studied at the squid giant synapse by imaging light emission from n-aequorin-J intracellularly injected into the presynaptic terminal. The imaging utilized a video system capable of acquiring 4000 frames per second. The results indicate that the calcium entry triggered by action potentials reaches a peak within 200 microseconds and has an overall duration of close to 800 microseconds, closely matching the duration of the presynaptic calcium current determined by voltage clamp results under similar conditions.

Aequorin↗
PubMed1996

An image analysis study of DNA content in early colorectal cancer.

The DNA content of 168 consecutive T3,N0,M0 (Dukes' B, Astler-Coller B2) colorectal cancers was studied using image analysis on formalin-fixed paraffin-embedded tissues. 72 cases (43%) were classified as diploid and the remaining 96 (57%) as non-diploid. After a median follow-up period of 6.7 years, a significant survival advantage was found for diploid compared with non-diploid cases (logrank test; P = 0.008). The long-term (8 year) survival rate was 70% for diploid and 46% for non-diploid tumours. Subgroup analysis showed that the survival advantage conferred by tumour diploidy was greatest in large (> or = 5 cm) cancers and was found both in colonic and rectal cancer cases. These data indicate that tumour ploidy status measured by image analysis might be useful in determining risk of colorectal cancer recurrence and death in patients following resection of early colorectal cancer.

Adult↗