Redistribution of mannose-6-phosphate receptors induced by tunicamycin and chloroquine.

The distribution of Man-6-P receptors was determined by immunoperoxidase cytochemistry in Clone 9 hepatocytes cultured in the presence or absence of tunicamycin and chloroquine, agents that perturb lysosomal enzyme sorting and lead to their secretion. In control (untreated) cells, receptors were localized in cis Golgi cisternae, coated vesicles, and in endosomes or lysosomes. After tunicamycin treatment, receptors were found in coated vesicles lined up along the cis cisternae but were not detected in endosomes or lysosomes. After chloroquine treatment, receptors were localized in large vacuolated endosomes or lysosomes but were not usually detected in Golgi cisternae or in coated vesicles. These results demonstrate a redistribution of receptors along the normal Man-6-P-dependent sorting pathway after these treatments. In ligand-deficient (tunicamycin-treated) cells, immunoreactive receptors accumulate at the presumptive sorting site in the cis Golgi and are depleted from endosomes and lysosomes. When the intralysosomal pH is increased (by chloroquine treatment) preventing ligand-receptor dissociation, receptors accumulate at the presumptive delivery site (lysosomes and endosomes) and are depleted from the cis Golgi region. The findings also suggest that (a) ligand binding triggers movement of the receptor to endosomes or lysosomes, and (b) ligand dissociation triggers their movement back to the cis Golgi region.

Recent Developments in Tele-Ultrasonography.

A long-standing trend that will continue to grow in healthcare is providing high quality services for all the patient, no matter the distance and no matter the place. One approach currently being used to increase population access to healthcare services is telemedicine. This narrative review presents one branch of e-health, in particular the use of teleultrasonography (TUS) in clinical practice, the challenges and barriers encountered. Current advances in ultrasound technology, including the growth of portable and small ultrasound devices have increased the range of applications of TUS, from traumatic patients in emergency medicine, maternal ultrasound and even for monitoring and screening for chronic illnesses. Even though some barriers are still looking for a solution, like standardized training and protocols, errors in data acquisition, the lack of trained professionals to operate in remote areas, TUS has the potential to redesign future health care systems.

Immunoelectron-microscopic localization of the terminal C5b-9 complement complex in human atherosclerotic fibrous plaque.

The assembly of the terminal C5b-9 complement complex is a prime mechanism of complement-induced membrane damage followed by inflammatory response mediation and subsequent extensive tissue damage. In the assembly process the terminal complement components expose neoantigenic determinants which can be recognized by specific antibodies. Using such a specific antibody, affinity-purified rabbit IgG and by means of immunoelectron microscopy, the C5b-9 neoantigens were localized on the structures of the human fibrous plaque from 3 iliac and 3 femoral arteries obtained at surgery. The immunoelectron-dense deposits were localized on the cell debris, enmeshed in the connective tissue matrix, consisting of irregular particles that frequently had the shape and size of intracellular organelles or vesicles with concentric osmiophilic lamellae. No deposits could be found on the intact cells, on the connective tissue matrix or on cholesterol and lipid deposits. The presence of C5b-9 neoantigens deposits in the fibrous plaques frequently associated with other immune-related proteins indicates that complement activation has occurred in situ and could be related to the chronic progression of the atherosclerotic lesion.

Establishment of a pure vascular endothelial cell line from human placenta.

UNLABELLED: Endothelial cells (EC) from various sectors of the circulatory system have distinct characteristics, some of which have only been identified in cultures upon their isolation from specific organs or tissues. Cultured vascular EC, derived from the human placenta (HPEC), may be helpful for studying their specific function in the fetoplacental unit, such as in the control of maternofetal traffic. In this paper we report an improved method for isolation, purification and culture of HPEC, that implies an enzymatic perfusion of the term placenta, followed by separation of resulting cells on a Percoll density gradient. The inoculated starting suspension was purified by a two-step selection procedure, based on differential trypsinization, leading to a pure population of about 8x10(7)cells/placenta, with 2.7-3.4 population doublings. The average population doubling time during eight passages was 60-65 h and the life span of HPEC was approximately 45-50 population doublings. The cell morphology at optical and electron microscopical level revealed a good differentiation of HPEC, which were endowed with numerous plasmalemmal vesicles (caveolae) and Weibel-Palade bodies. The transendothelial electrical resistance of the HPEC monolayer varied between 22 and 52 Ohm/cm(2). The cultures were mycoplasma free, as revealed by fluorescence microscopy using DNA dyes and the polymerase chain reaction (PCR). The negative immunofluorescent reaction for keratin confirmed that the HPEC were not contaminated with either type of placenta cells, as syncytiotrophoblast. Cultured HPEC demonstrated a strong reaction for von Willebrand factor antigen (by fluorescence microscopy), took up AcLDL-DiI and expressed active angiotensin converting enzyme. These characteristics substantiate the endothelial nature of cultured cells. The interactions with different lectins (BS-I, SBA, RCA, UEA and WGA) assessed by fluorescence microscopy and blotting reveal a strong reaction of HPEC with UEA and a negligible reaction with BS-I lectin. WGA lectin displayed a marked fluorescence staining in subconfluent HPEC, and at the level of intracellular clefts in post-confluent cultures. IN CONCLUSION: (i) we have obtained a pure line of cultured EC originating from the human placental venous side of the circulatory tree; (ii) the cells have the general characteristics and markers ascribed to EC; (iii) as opposed to large human placental vessels, HPEC do not react to BS-I lectin and, unlike human umbilical vein EC, have a much higher proliferation rate and a long lifespan; (iv) HPEC expressed a characteristic glycosylated coat particularly rich in alpha- L -fucose and beta-GlcNAc containing glycocompounds.

An ex vivo model to study the monocyte-endothelial cell interaction in the prelesional stage of experimentally-induced atherogenesis in hamster.

We imagined an ex vivo atherogenic model consisting in hypercholesterolemic versus normocholesterolemic aortic arch rings or sigmoid valves incubated in cell culture conditions with human monocytes. Normal tissues did not show any attached monocytes. On the atherogenic aortic arch three different aspects were observed: a) there were no monocytes attached on a normal zone; b) many monocytes adhered to the endothelium on a thickened area, with intimal smooth muscle cells infiltration, and c) the greatest number of attached monocytes was seen in mechanically injured zones of the aortic arch, where the subendothelial area was totally exposed. Immunohisto- and immunocytochemistry for LFA-1 revealed the presence of this leukocyte integrin on monocyte plasma membrane. The labelled monocyte had an activated shape, with pseudopodes extended over the endothelial cells and the anti-LFA-1 antibody coupled with colloidal gold decorating areas apposing to a morphologically modified endothelium. In conclusion, the ex vivo model reproduced the in vivo situations where the monocytes adhere to the modified endothelium covering the thickened areas of hypercholesterolemic aortic wall; they express at least one of the adhesion integrins, namely LFA-1. This study intended to contribute, at least in part, to the understanding of some mechanisms governing the monocyte-endothelial cell interactions in hypercholesterolemia.

Exposure to hypercholesterolemic serum modifies the expression of cytoskeletal proteins in cultured endothelia.

Arterial endothelial layer dysfunction is considered to be one of the most important events which initiate the development of the atherosclerotic plaque and the cell cytoskeleton plays an essential role in maintaining the integrity of the endothelium exposed continuously to haemodynamic forces. The aim of this work was to study the modifications of the cytoskeletal proteins in the vascular endothelium exposed to atherogenic conditions. A hamster aortic endothelial cell line (HAEC) grown on glass coverslips was exposed for 24 h to hypercholesterolemic or normal homologous serum. Upon staining with Oil Red O and examination by phase contrast and fluorescence microscopy, HAEC incubated with hypercholesterolemic serum appeared heavily loaded with lipid droplets that showed a yellow autofluorescence in UV light and the general aspect of a foam cell. HAEC were incubated with: a) anti-actin serum; b) anti-vinculin monoclonal antibody (MoAb); c) anti-alpha actinin MoAb, and d) anti-talin MoAb, followed by appropriate secondary antibodies coupled with FITC or rhodamine. As compared to normal HAEC, the cells exposed to hypercholesterolemic serum showed a modified pattern for actin and vinculin localization. Actin appeared as a weakly stained network around the nuclear zone whereas vinculin was distributed as small granules throughout the cell cytoplasm. These experimental data suggest that in advanced atherosclerosis, some of the endothelial cytoskeletal proteins undergo modifications which could represent one of the important factors involved in further development of the atheromatous plaque. In addition they indicate that HAEC exposed to hypercholesterolemic serum could represent an in vitro working model for studying the events occurring in the endothelium at advanced stages of atherosclerosis.

Diabetes-induced structural changes of venous and arterial endothelium and smooth muscle cells.

The structural alterations of endothelium and smooth muscle cells of the hind limb and heart veins and arteries were investigated in Golden Syrian hamsters subjected to streptozotocin induced diabetes. Animals were examined at 5, 10, and 15 weeks after induction of diabetes. At each time point body weight and plasma glucose concentrations were recorded. Anesthetised animals were washed out of blood, fixed in situ, and the femoral vein and artery, saphenous vein and artery, and heart veins and coronaries were dissected out, and processed for electron microscopical examination. Anionic sites of the endothelial plasmalemma were visualized by in situ perfusion of cationized ferritin. The endothelial localization of von Willebrand factor was carried out by immunocytochemistry. The results showed that induction of experimental diabetes generated morphological changes of the endothelium and smooth muscle cells of both hind limb and heart vessels. The common alterations developed in endothelial cells of venous and arterial origin consisted in: 1) the development of a secretory phenotype, enriched in biosynthetic and degradative organelles; 2) the abundance of cytoskeletal elements, especially intermediary filaments; 3) the increase in number of fused plasmalemmal vesicles and transendothelial channels, and 4) the hyperplasia of the basal lamina. In contradistinction to the arterial endothelium, the peculiarities of the venous endothelium in the diabetic hamsters examined were: 1) the uniform distribution of the anionic sites exposed on the luminal plasma-lemma (as in normal animals), and 2) the increased number of copies of Weibel-Palade bodies (up to 13 copies per endothelial cell in the hind limb). Von Willebrand factor was immunodetected in Weibel-Palade bodies, Golgi cisternae and some vesicles of normal and diabetic hamsters. With time, and especially pronounced at 15 weeks of diabetes, the smooth muscle cells of veins and arteries examined exhibited a characteristic secretory phenotype, and were surrounded by a reticulated basal lamina and a hyperplasic extracellular matrix (especially pronounced in arteries). These data indicate that diabetes affects both heart and hind limb veins and arteries, producing structural changes of the endothelium and smooth muscle cells which may account, at least in part, for the specific vascular complications.

Endothelial cell-derived foam cells fail to express adhesion molecules (ICAM-1 and VCAM-1) for monocytes.

The purpose of this study was to assess the expression of cell adhesion molecules ICAM-1 (intercellular adhesion molecule-1) and VCAM-1 (vascular cell adhesion molecule-1) in endothelial cell-derived foam cells. Hamster aortic endothelial cells (HAEC) in culture were exposed to hypercholesterolemic or normal homologous serum for 24 h. At the end of the incubation period, HAEC exposed to hypercholesterolemic serum exhibited numerous lipid droplets and had a general aspect of foam cells. When examined for the expression of ICAM-1 and VCAM-1 (by indirect immunofluorescence) normal HAEC expressed constitutively (to low level) on their surface these adhesion molecules; however HAEC-derived foam cells failed to display any labeling. To further assess these results, HAEC were first incubated with normal or hypercholesterolemic sera (as above) and then exposed to freshly isolated normal hamster blood monocytes. These experiments showed that monocytes adhered in small number to normal cells and failed to adhere to the surface of HAEC-derived foam cells. Together these data indicate that endothelial cell-derived foam cells: a) do not express ICAM-1 and VCAM-1 on their surface; b) have low or no adhesion properties for monocytes and c) may represent an appropriate experimental model to study the cellular alterations that take place in the advanced stages of atherosclerosis.

[Clinico-statistical observations on malignant ocular tumors surgically resolved at the Cluj-Napoca Ophthalmological Clinic between 1970 and 1990]. / Consideratii clinico-statistice asupra tumorilor maligne oculare rezolvate chirurgicale în Clinica oftalmologica din Cluj-Napoca între anii 1970-1990.

During the period mentioned above there ware hospitalized 54 patients having epibulbar tumours. The anatomopathological exam which was effectuated for 33 patients hes shown Bowen's disease for 14 cases, spinocellular carcinoma for 15 patients and basocellular for 3 patients. There were 4 cases for epibulbar precancerous. There was retinoblastoma for 19 cases; it had been practiced the enucleation of ocular globe for 17 patients and for two cases it had been practiced the exenteration of the eye socket. At the same period there were 91 uveal melanosis 6 of which having iridial localization, 10 cases of ciliary body [correction of cylar corpus], 75 cases of choroid. The hepatic scintigraphy was practiced for 56 patients and had shown for 8 cases hepatic metastasis. The histopathological exam of the melanomata of ciliary and choroidal corpus had shown type A fusiform cells for 22 cases, type B for 43 cases, epithelioid cells for 6 cases and mixed type cells for 13 cases. The hepatic metastasis were epithelioid cells for all the cases and, for 2 cases there were with mixed type cells, It is necessary an active discovering of intraocular and episcleral malign tumours, for increasing the chance of an earlier diagnosis during a stadium that would be able to be treated as conservator as possible be.

Proliferation, differentiation and characterization of osteoblasts from human BM mesenchymal cells.

BACKGROUND: The objective of this study was to isolate osteoprogenitor cells (OPC) from BM mesenchymal stromal cells (MSC) and test their capacity to proliferate and differentiate into osteoblasts. METHODS: Human MSC were separated on a Percoll gradient and cultured in DMEM supplemented with 15% human serum, and characterized by flow cytometric analyzes for CD34, CD13, CD90, CD105 and CD117. To induce differentiation, cultured cells were exposed to 10(-7) m dexamethasone (dexa) and/or 10(-3) m sodium beta-glycerophosphate (beta-GlyP) and 1,25-dihydroxyvitamin D3 (calcitriol) or 9-cis-retinoic acid (9-RA). RESULTS: alkaline phosphatase (AP) activity was detected in cells irrespective of the dexa and/or beta-GlyP treatment. Antigenic phenotypes of MSC were CD34- (more than 99%) and CD13+ CD90+ CD105+ CD117+ (c. 50%). The treatment induced extracellular calcium deposition and gene and protein expression of osteonectin (ON) and bone sialoprotein (BSP): beta-GlyP induced an increase (c. 2.2-fold) of the ON gene and dexa augmented (c. 2.7-fold) the gene expression of BSP II. Gene expression of BSP I reached a maximum at 3 weeks of combined treatment. Osteocalcin gene expression was induced only after additional treatment with calcitriol or 9-RA. Ultrastructural analysis revealed the secretory phenotype of OPC. DISCUSSION: Under appropriate treatment, MSC can give rise to OPC that have the capacity to differentiate into osteoblasts characterized by the expression of osteogenic markers, osteoblastic properties and stromal BM cells phenotypes. These cells may represent a promising material to be utilized in orthopedic cellular therapy.

Statistical probability applied in the study of the thyroid of immunized rats.

The study of the statistical significance was applied in the investigation of several analogous lots, in the thyroid of rats during the experimentally conditioned immunity. The method is described and some examples of calculus are presented with the indication of parameters with the highest and the lowest statistical significance.

A new freeze-drying device for platinum replica studies of cell surface and cytoskeleton: an example using immunogold-labeled human erythrocytes.

We designed and built a freeze-drying device that ensures the protection of the specimens against contaminants during mounting on the cold stage of the freeze-fracture machine, transferring into the vacuum chamber and deep etching. The device consists of a copper cap that covers the specimen and a thermal connection that ensures thermal transfer between the microtome arm and the copper cap. This device was used to study the ultrastructural features of the erythrocyte membrane skeleton and the immunocytochemical localization of spectrin in an "in situ" approach, by freeze drying and platinum rotary shadowing. Human erythrocytes adhered to polylysine-coated coverslips and were broken by a stream of buffer that mimics the intracellular ionic environment ("inside buffer"). The samples were prefixed in periodate-lysine-paraformaldehyde fixative, labeled with antispectrin 5-nm gold particles, fixed in glutaraldehyde, mordanted in tannic acid, postfixed in OsO4, repeatedly washed in water, rinsed quickly in 30% ethanol, freeze-dried, and rotary-shadowed. Electron microscopic examination of the replicas revealed the skeletal network on the inner surface of the erythrocyte membrane. Immunocytochemical labeling proved that spectrin represents a fibrillar component of the network. Our data confirm the speculative model of the molecular organization of the erythrocyte skeleton, based on studies on in vitro association of proteic constituents. Both the technique and the device developed by us may lead to a deeper understanding of the spatial organization of the cytoskeletal network of more complex cell types.

White matter changes in old age dementia.

This is the clinico-morphological study of 70 patients above age 60 with the clinical diagnosis of dementia made on clinico-psychometric criteria for the assessment of the deterioration-dementia state, and in some cases, using the Hachinski scale. For morphological macro- and microscopic examinations of the brain, the classic neuropathologic techniques were used. Although no case selection was carried out, the number of cases was uniformly distributed between the ages of 60-74 years. The sample was also relatively uniform with regard to the patients' sex. Morphologically, our patient group included cases with vascular dementia (VD-33%), mixed dementia (MD-14.3%), Alzheimer-type dementia (ATD-20%), isolated SAE (17%), other cases (15.7%). Myelinic pallors and rarefactions were present in 41.4% of all cases of which: as a single lesion in 41.4%, associated with VD in 34.5%, with MD in 17.2% and with ATD in 6.9%. Microscopic background of myelinic changes was represented by acute (perivascular and pericellular edema) and chronic (myelinic destruction, gliosis, perivascular hematic pigment) edematous lesions. In 10.3% of cases with myelinic changes, marked dilation and blood stasis in large periventricular and/or subcortical vessels with subsequent cerebral edema, generally overlapping critical zones of venous circulation could be observed. The size and severity of the myelinic lesions were not clearly correlated to the intra- and extraparenchymatous vascular changes. However, the myelinic involvement was more in cases with lesions, mainly atherosclerotic, of the vessel walls. The possible intervention of the venous factor in the development of subcortical arteriosclerotic encephalopathy (SAE) is discussed among other etiopathogenic factors.

Immunological detection of an analogue of the erythroid protein 4.1 in endothelial cells.

Endothelial cells (EC) of arterial and venous origin were investigated by indirect immunofluorescence and immunoautoradiography for the presence of red cell membrane 4.1-like protein. By immunofluorescence, EC exhibited a relatively uniform fluorescent staining sometimes of a reticular pattern, distributed over the entire cell. All controls were negative. Immunoblot analysis of EC revealed a cross reactive band of a molecular weight comparable to that of the erythrocyte band 4.1. These findings indicate that endothelial cells of arterial and venous origin express a polypeptide immunologically related to the erythrocyte protein 4.1, which may play an important role in membrane-cytoskeleton interactions.

Endothelial cells express a spectrin-like cytoskeletal protein.

Vascular endothelium was investigated by indirect immunofluorescence and immunoautoradiography for the possible presence of spectrin-like molecules. Antibodies were raised against electrophoretically purified rat, rabbit, and bovine red blood cell spectrin and against rabbit brain fodrin. Antibody specificity was assessed by immunoblotting and double-diffusion technique. Homogenates of endothelial cells freshly isolated from heart microvasculature or aorta, as well as cultured aortic endothelial cells, were analyzed by gel electrophoresis. Immunoautoradiograms of gels incubated with spectrin specific antibody, followed by radio-labeled protein A, revealed two bands of electrophoretic mobility similar to that of the alpha- and beta-subunits of spectrin. Indirect immunofluorescence of endothelial cells, both in situ and in vitro, showed the existence of a protein which cross-reacted with the antibodies against spectrin and fodrin. Controls, in which endothelial cells were exposed to spectrin antibody absorbed with pure spectrin or preimmune serum, were negative. These findings indicate that endothelial cells express a protein antigenically related to the spectrin family; both spectrin- and fodrin-like molecules, in various proportions, may coexist. In the endothelial cell, these proteins may play an important role in modulation of the cytoskeleton in response to various stimuli, and in maintaining the biochemically differentiated microdomains of plasmalemma.

[Possible relation between viruses and oromaxillofacial tumors. IV. Presence of the herpes antigen and anti-herpes antibodies in patients with tumors of the parotid gland]. / Relations possibles entre virus et tumeurs bucco-maxillo-faciales. Note IV. Présence de l'antigène herpétique et des anticorps anti-herpétiques chez des malades porteurs de tumeurs de la glande parotide.

The presence of herpesvirus antigen and of antiherpesvirus antibodies was detected in 46% and in 36.3%, respectively, of the patients bearing parotid gland tumors. Very high titers of antiherpesvirus antibodies were found in 70.6% of the patients with nasopharyngeal carcinoma.