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PubMedDate not supplied

Biochemical and microscopic urinalysis: time and cost in a nephrology laboratory.

BACKGROUND: Urinalysis is a fundamental test in internal medicine and nephrology. Figures for costs are available in the general laboratory, where biochemical and microscopic urinalysis are commonly performed as semiautomated screening tests. Information on costs is lacking in the nephrology laboratory, where a time-consuming morphological analysis is usually preferred. This study analyses the costs of urinalysis in a nephrology laboratory. METHODS: In the nephrology laboratory at the University of Turin - Italy, biochemical urinalysis consists of multi-property strip and proteinuria/creatininuria, done by laboratory technicians. Phase-contrast microscopy is done by a nephrologist or biologist. Time dedicated to the tests was recorded by the same operator over 20 working days, during which 350 urine samples were processed (median 19/day, range 842). The production costs were calculated with the logic bottom-up technique. RESULTS: Overall time needed was 11.9 minutes/sample. Biochemical urinalysis required 6.6 minutes/sample; time required and samples processed were inversely related (< or =19 samples: 7.95 minutes/sample; >19 samples: 5.6 minutes/sample, p=0.01). Microscopic urinalysis took 5.3 minutes/slide; the best time-to-samples ratio was at 18-22 samples per day (with peak efficiency at 21 samples: 4.6 minutes). Cost of reagents and disposables was Euro1.06/sample. Time accounted for euro 5.32/sample (technicians, nephrologist-biologist), with total direct cost of euro 6.38/sample. CONCLUSION: In a nephrology laboratory, microscopic urinalysis is a time-consuming, expensive test. Analysis of cost and modalities may be useful, in a time of budget constrains, to maintain a role for this precious semeiotic art.

Chemistry, Clinical↗

PubMed2003

Differentiation of rat embryonic neural stem cells promoted by co-cultured Schwann cells.

OBJECTIVE: To explore the factors which induce differentiation of embryonic neural stem cells. METHODS: Rat embryonic neural stem cells were co-cultured with newborn rat Schwann cells in serum-free medium. The phenotype and specific-markers including tubulin-beta, glial fibrillary acidic protein (GFAP) and galactorcerebroside (GalC), were demonstrated by phase contrast microscopy and double immunofluorescence staining. RESULTS: Overall, 80% +/- 5% of neural stem cells protruded several elongated processes and expressed tubulin-beta antigen at high levels, while 20 +/- 3% of them protruded several short processes and were GalC or GFAP positive. CONCLUSION: The factors secreted by Schwann cells could induce rat embryonic neural stem cell to differentiate.

Animals↗

PubMed1992

Red blood cell and platelet induced differentiation of endothelial cells into capillary-like structure.

We cultivated endothelial cells of human umbilical vein origin in the presence of red blood cells and platelet-rich plasma, and observed the phenomena which occurred in the petri dish under phase contrast microscopy. Many small particles were observed after an overnight incubation. We washed the dish two times, then added thrombin to the dish. The network of thread-like strands appeared within 10 to 20 minutes of the addition, and at the same time the small particles adhered on the surface of the strands, swelled and fused gradually to cover the surface of the strands completely. Within 30 to 60 minutes the network of the strands changed into a capillary-like structure. These phenomena were not observed if we omitted red blood cells or platelet-rich plasma. Studies by transmission electron microscopy revealed that the inner surface of the lumen of the structure was covered with cells. The cells isolated from the lumen by trypsin grew to confluence in the conventional culture medium, and showed vWF antigen on their surface. These observations indicated that the method described is useful for in vitro study of angiogenesis.

Blood Platelets↗

PubMed1992

Cytotoxicity of ophthalmic preservatives on human corneal epithelium.

Because the corneal epithelium invariably encounters the full concentration of the preservative that is contained in multi-dose topical ophthalmic preparations, we investigated the cytotoxicity of several of these agents by using a sensitive model of human corneal epithelial cells in vitro. Primary cultures of epithelial cells were prepared from freshly enucleated globes. At confluence, all experimental cultures received a single dose of preservative at the concentration present in marketed formulations. The serum in the culture medium simulated the possible neutralizing effect of proteins present in the tear film in vivo. The cells were observed continuously by phase-contrast microscopy and time-lapse videomicrography for 24 hrs. Benzalkonium chloride at a concentration of 0.01% and chlorobutanol at 0.5% caused immediate cell retraction, as well as cessation of normal cytokinesis, cell movement, and mitotic activity; the epithelial cells degenerated within 2 hrs and 8 hrs, respectively. Cultures treated with chlorobutanol developed conspicuous blebs on the cell surface after 3 to 5 hrs of exposure. Thimerosal (0.001%) caused cell retraction, cessation of mitotic activity, and total cell destruction within 9 hrs. Sorbic acid (0.1% and 0.2%) greatly reduced cell movement and suppressed mitotic activity, but no cell death occurred. At concentrations of 50 ppm and 30 ppm, H2O2 instantaneously caused a marked retraction of the cells, followed by cessation of cytokinesis, cell movement, and mitosis. Retraction and death of the epithelial cells occurred within 12-24 hrs after exposure to 1 ppm H2O2 in serum-free medium. Polyquaternium ammonium chloride (0.001%) and polyaminopropyl biguanide (0.00005%) had no discernible effects on cytokinetic movement or on the mitotic activity of the epithelial cells. We relate our findings in vitro to those reported in vivo and discuss the mechanism of cytotoxicity of the various preservatives.

Cell Death↗

PubMed2003

FGF-2 induced reorganization and disruption of actin cytoskeleton through PI 3-kinase, Rho, and Cdc42 in corneal endothelial cells.

PURPOSE: Corneal endothelial cells (CECs) undergo endothelial to mesenchymal transformation (EMT) in response to FGF-2 stimulation. One phenotypic change that occurs during EMT is a change in cell shape from polygonal to elongated fibroblast-like cells. We investigated whether FGF-2 plays a role in this morphogenetic pathway by reorganizing actin cytoskeleton through the actions of phosphatidylinositol (PI) 3-kinase and the Ras related Rho family of small guanosine triphosphatases (GTPases). METHODS: Cell morphology was analyzed using phase contrast microscopy, and the organization of actin cytoskeleton and focal adhesions were analyzed by immunofluorescent staining. Expression of vinculin and beta-actin was determined by immunoblot analysis. Pharmacologic inhibitors (LY294002, C3 exoenzyme, Y27632, or PD98059) or neutralizing antibody to FGF-2, respectively, were used to block PI 3-kinase, Rho, Rho associated kinase, extracellular signal regulated kinase, or FGF-2 pathways. RESULTS: CECs treated with FGF-2 became smaller and lost their characteristic polygonal cell morphology. Such cell shape change was completely blocked by treatment with LY294002. CECs in culture have abundant stress fibers that are oriented radially across the cell. However, FGF-2 caused a loss of these stress fibers and focal adhesions. The modulated cells contained a cortical actin ring while LY294002 completely abolished this action of FGF-2 on actin cytoskeleton. Treatment of cells with C3 exoenzyme or Y27632 in the presence of FGF-2 induced spindle shaped cells with prominent pseudopodia which were rapidly formed upon exposure to the inhibitor. The expression level of vinculin was found to be similar in all experimental conditions but vinculin was mostly translocated to the cytoplasm in response to FGF-2 stimulation. CECs plated on Matrigel matrix demonstrated findings similar to those from cells plated on the conventional culture dishes, except that Matrigel facilitated the formation of pseudopodia. We further investigated in vivo actin organization using organ cultures of corneal endothelium (CE) on Descemet's membrane. The contact inhibited endothelial monolayer demonstrated a circumferential actin ring, and no stress fibers were observed. When CE was treated with FGF-2, a half population of CE lost its characteristic contact inhibited cobblestone morphology. Actin cortex was greatly disrupted in these modulated cells. Both neutralizing antibody to FGF-2 and LY294002 completely impeded the modulating activity of FGF-2 on the endothelial monolayer. When CE was simultaneously treated with FGF-2 and Y27632, the circumferential actin cortex was greatly disrupted and the endothelial monolayer was transformed into multi-layers of fibroblastic cells containing pseudopodia. Both LY294002 and neutralizing antibody to FGF-2 antagonized the actions of FGF-2 and Y27632. CONCLUSIONS: These data indicate that CECs in culture have constitutively active Rho activity as evidenced by stress fiber formation and that PI 3-kinase negatively regulates the formation of stress fibers and focal adhesions, perhaps antagonizing the Rho pathways. Formation of pseudopodia in response to FGF-2 and Y27632 may suggest that the Rho/ROCK pathway negatively regulates Cdc42.

Actins↗

PubMedDate not supplied

Cold shock response of yeast cells: induction of a 33 kDa protein and protection against freezing injury.

Cold shock (10 degrees C) treatment to Saccharomyces cerevisiae cells normally grown at 30 degrees C resulted in splitting of vacuoles and retarded membrane fluidity as detected by phase contrast microscopy and in vivo nuclear magnetic resonance (NMR) studies, respectively. The treatment was found to impart protection against subsequent freezing as studied by cell viability and colony forming efficiency. We have earlier reported similar protection and retarded membrane fluidity as a result of heat shock treatment to these cells (Obuchi et al., 1990). This suggests that cold shock and heat shock treatments to yeast cells evoke some analogous responses. However, biochemically a new 33 kDa protein (CSP 33) was detected upon cold shock treatment which is distinct from heat shock induced family of proteins (Kaul et al., 1992). We present here the first report of this kind and its practical implications for protection against freezing.

Cold Temperature↗

PubMed1992

Effects of vitamin A on retinal pigment epithelial cells in vitro.

Human retinal pigment epithelial (RPE) cell monolayers were used in an in vitro wound-healing experiment. Wound closure were studied over time in the presence of various concentrations of all-trans retinol (vitamin A). It was found that 5-20 micrograms/ml (17.5-70 mumol/l) of vitamin A significantly inhibited wound closure by inhibiting cell migration and proliferation. In a collagen gel-contraction model, similar doses of vitamin A partially inhibited cell contractility. Cellular morphology, as assessed by phase-contrast microscopy, changed from a regular polygonal shape to an elongated stellate shape. Indirect immunofluorescence patterns of fibronectin, actin, tubulin, and vimentin were altered after exposure to vitamin A. All these effects were reversible after removal of the drug from the medium. Modulation of RPE growth and cell-mediated contraction may be useful in the treatment of ocular proliferative diseases.

Cell Count↗

PubMed2004

[Influence of substrates coated on cultured dorsal root ganglion cells].

Dorsal root ganglia from rat embryos were digested with trypsin to form single cell suspension. The cells were seeded on culture plates coated respectingly with poly-L-lysin (PLL), laminin (LN), PLL combined with LN, or collagen type I (CoI), then cultured in NB1 media. The cell survival and neurite outgrowth of cultured dorsal root ganglion cells were observed by phase-contrast microscopy. The results showed that the dorsal root ganglion neurons grown on PLL combined with LN were dispersively distributed at high survival rate, and those cells grown on CoI were clustered with thicker and longer neurites. It is suggested that the cell growth pattern could be influenced by different substances coated on the plates, and PLL combined with LN may provide a better substrum to culture and study single neuron soma and neurite.

Animals↗

PubMed2005

[Effects of hepatocyte growth factor on IL-1alpha triggered tubular epithelial-myofibroblast transdifferentiation and fibronectin secretion in vitro].

OBJECTIVE: To observe the effects of hepatocyte growth factor (HGF) on tubular epithelial-myofibroblast transdifferentiation (TEMT) triggered by IL-1alpha and the fibronectin secretion of TEMT. METHODS: The normal rat kidney tubular epithelial cell line (NRK52E) was cultured for six days on plastic or collagen type I-coated plates in the presence or absence of HGF or IL-1alpha. The morphology of transdifferentiation tubular cells was observed by scanning electron microscopy (SEM) and phase-contrast microscopy. The number of alpha-SMA+ cells, the percentage of alpha-SMA+ cells and the mean channel fluorescence (MCF) were assessed by immunohistochemistry and flowcytometry. The level of fibronectin in supernatant was measured by ELISA. RESULTS: The NRK52E cells triggered by IL-1alpha became fibroblast-like morphologically, and strong alpha-SMA immunostaining of those cells was seen. The level of FN in the culture supernatant, the percentage of alpha-SMA+ cells and the MCF of cells triggered by IL-1alpha were obviously higher than those of blank control group (P<0.05). In the groups with IL-1alpha and different doses of HGF, the transdifferentiation of NRK52E cells was inhibited. With the increase of HGF dose, the percentage of alpha-SMA+ cells and the level of FN showed a tendency to decrease. There was no significant difference between the groups treated with only HGF at different dose levels and the blank control group (P>0.05). CONCLUSION: IL-1alpha can induce tubular epithelial cell to transdifferentiate to myofibroblast and increase the secretion of FN. These results suggest that TEMT may play an important role in the pathogenesis of renal fibrosis. HGF could block the transdifferentiation of tubular epithelial cell and inhibit the secretion of FN. These would provide a novel therapeutic strategy for the treatment of renal interstitial fibrosis and end stage renal disease.

Actins↗

PubMed2003

[The preparation of fructose-modified chitosan microcarrier and culture of primary rat hepatocyte].

Crosslinked chitosan microcarries were prepared by the reaction of glutaraldehyde with fructose-modified chitosan. Various factors that influence the preparation were studied and the reaction conditions were optimized. Morphology of rat hepatocyte cultured on chitosan microcarriers was observed using phase contrast microscopy and scanning electron microscope, the metabolic activity was measured. Rat hepatocytes cultured on chitosan microcarrier retained the spherical shape as they have in vivo and had high metabolic activity. Fructose can enhance the metabolic activity of hepatocytes and fructose-modified chitosan microcarrier is a promising scaffold for hepatocytes attachment, which can be used in bioartificial liver support system.

Animals↗

PubMed1992

[The characteristics of hematuria in chronic glomerulonephritis and nonglomerular diseases of the kidneys and urinary tract].

Upon the analysis of qualitative and quantitative characteristics of hematuria in renal and urinary tract affections it was established that whatever the red blood cell count in the urine, hematuria is primarily glomerular in contrast to nonglomerular in chronic glomerulonephritis and nonglomerular diseases of the kidneys and urinary tracts, respectively. Morphologic studies of urinary red blood cells using phase-contrast microscopy proved a valuable tool for identification of hematuria origin.

Chronic Disease↗

PubMed1992

[Changes in the form and ultrastructure of the smooth-muscle cells in the human aorta in prenatal ontogeny].

The shape of smooth muscle cells (SMC) was analysed using the phase contrast microscopy of cell suspensions obtained by alcohol-alkali dissociation, as well as the semithin sections prepared in perpendicular planes. The phenotype of SMC was analysed using transmission electron microscopy. The shape of SMC changes from preferentially round to preferentially spindle-like and stellate one during development. The differentiation of SMC is accompanied with the increase in the contractile apparatus content and in the decrease in the content of synthetic organelles.

Actin Cytoskeleton↗

PubMed2006

Apoptotic activity of a novel synthetic cantharidin analogue on hepatoma cell lines.

Cantharidin isolated from Mylabris caraganae and other insects is used traditionally as an anti-cancer drug. However, its toxicity on the renal system and suppression effect on bone marrow limits its clinical usage. Recently, we have synthesized two cantharidin analogues, CAN 029 (compound 2) and CAN 030 (compound 3). Although both showed an apoptotic induction ability on cancer cells, they were still relatively toxic towards non-malignant haematological disordered bone marrow. Based on the principle structure of cantharidin, we have further chemically synthesized another analogue, CAN 032. The cytotoxic activity of this analogue was screened on both Hep3B hepatocellular carcinoma and SK-Hep-1 liver adenocarcinoma cell lines by [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] (MTS) assay. Morphological changes of hepatoma cell lines were recorded under an inverted microscope. The possible tolerance of these analogues was further investigated using non-malignant haematological bone marrow primary culture. CAN 032 showed a significant cytotoxic response on both hepatoma cell lines in which the potencies were comparable to that of cantharidin. Further screening on the bone marrow tolerance revealed that compound CAN 032 showed a relatively less toxic effect. Phase contrast microscopy demonstrated that cell shrinkage, rounding, loss of adherent property and loss of colony-formation ability were induced. The dose-dependence of the response of CAN 032 on Hep3B was further assayed by DNA fragmentation gel electrophoresis. The G1 peak of Hep3B cells was reduced. Chemically synthesized CAN 032 may provide an improved therapeutic advantage over traditional cantharidin.

Adult↗

PubMed1991

Megakaryocyte maturation in long-term marrow culture.

Evidence has been sought for megakaryocyte maturation in long-term cultures of mouse bone marrow. Cultures up to 14 weeks of age were examined for the presence of megakaryocytes with processes, that is, resembling the morphological appearance seen in vivo prior to platelet liberation. Such cells were found floating just above the adherent stromal layer using low magnification phase contrast microscopy. It was rare to observe as many as 20 of these cells per 25-cm2 flask. At higher magnification, processes were seen to be attenuated with constrictions at intervals along their length. Time-lapse photography was used to follow the development and behavior of the processes. Direct evidence of rupture was very rare; generally the megakaryocytes retracted their processes within 48 h. Careful searching of cultures occasionally revealed the presence of several process fragments, and sometimes individual platelets were found. Ultrastructurally, the processes were seen to contain organelles that are usually associated with platelets. The observations applied to both Dexter and Whitlock-Witte cultures. It is concluded that maturation of megakaryocytes occurs in long-term marrow culture to the point where platelet release appears imminent. Final rupture is rare and may require shearing forces, which in vivo would be provided by blood flow.

Animals↗

PubMed1991

[The neural apparatus of the small intestine of piglets after administration of a growth stimulator kormogrisein].

By means of classical neurohistological techniques, phase contrast microscopy and morphometry, a comparative investigation has been performed concerning the development of the intramural nervous apparatus in the small intestine, normal and at application of cormogrisine. The structural peculiarities of morphogenesis are considered together with signs of activation and inhibition of the neurons growth in tissue culture. A number of morphological criteria, demonstrating an increased extrusive activity and enhancing potensity of the neurons growth have been revealed. The number of nervous processes becomes greater; degree of their ramification increases; a part of neurons of Dogiel II type turns into multiprocessive neurons with some signs of Dogiel I type cells; growth cones and arcadian structures are present; giant processes appear; thick nervous fasciculi are formed; volume of the neuron bodies increases more intensively. After application of cormogrisine for 2 months a definite neurostimulatory effect is revealed; it demonstrates a more intensive morphogenesis of the small intestine nervous of Physiology, USSR, Academy of Medical Sciences, Leningrad.

Animal Feed↗

PubMedDate not supplied

Cardiomyopathy in vitro.

Phase contrast microscopy of cultured embryonic heart cells showed the beating frequency decreased more rapidly and the regularity the rhythm of of the beating cells was lost sooner in heart cells from cardiomyopathic hamsters than from the control hamsters. Studies of cultured heart cells by differential interference contrast (with Nomarski's prism) and by electron microscopy revealed a significant impediment in the maturation of the sarcomeric units in the diseased animals compared to controls. The incorporation of [14C] leucine into acid-insoluble fractions was studied, and no significant difference in incorporation between the two groups was found. An analysis of polyacrylamide gel electrophoresis revealed the possible existence of a quantitative difference in one of the composing proteins of the erythrocyte membrane between the two groups. The protein kinase activity of ghosts from the control group was more sensitive to cAMP than that from the diseased animals. In addition, the binding of [3H] cAMP to the ghost was almost identical between the two. The morphological and biochemical observations lead one to the plausible supposition that there are some differences in the interaction of the so-called catalytic and regulatory subunits between the two groups and that there is an impairment of the higher arrangement of myofibrils from their building blocks in the diseased hamster. The significance of the existence of abundant corpuscles resembling neurosecretory granules was not established by this study. They may have an etiological significance or they may be related to a disturbed function in the cultured cells of the cardiomyopathic hamster.

Animals↗

PubMed1990

Effect of a triclosan/copolymer/fluoride dentifrice on the oral microflora.

Eighty-one human subjects completed a double-blind study which examined the effects of a 0.3% triclosan/2% Gantrez copolymer/0.243% sodium fluoride dentifrice on the microflora of supragingival dental plaque. Subjects were divided into an experimental group which performed normal oral hygiene with the triclosan/copolymer/fluoride dentifrice and a control group which also performed normal oral hygiene with the same dentifrice minus the triclosan/copolymer. At baseline, 10 weeks, and 28 weeks, supragingival dental plaque was collected from buccal and lingual surfaces of the four first molar teeth and assayed for: 1) bacterial morphotypes by phase contrast microscopy, 2) Actinobacillus actinomycetemcomitans, Actinomyces species, Bacteroides forsythus, Bacteroides gingivalis, Bacteroides intermedius, Streptococcus mutans, Streptococcus sanguis, and Wolinella recta by immunofluorescence microscopy, and 3) Lactobacillus, yeast, enterics, Staphylococcus, aerobes and anaerobes by bacterial culture. After 28 weeks' use of their respective dentifrices, changes in the supragingival plaque microflora of the subjects were similar between the triclosan/copolymer/fluoride dentifrice group and the control dentifrice group, except for statistically significant reductions in fusiforms, spirochetes and staphylococci and significant increases in S. sanguis in the triclosan/copolymer/fluoride dentifrice group, as compared to the control dentifrice group. The subject population was unusual in the presence of enteric species and anaerobes found in supragingival plaque sites. This study indicates that the use of a dentifrice containing 0.3% triclosan and 2% Gantrez copolymer over an extended period of time (28 weeks) does not result in shifts in the microflora of supragingival plaque favoring the growth of either opportunistic or pathogenic bacterial species.

Adult↗

PubMed1990

[In vitro and in vivo effect of thyroid hormones on the growth of neuroblastoma cells. I. The effect of triiodothyronine in vitro].

The effect of triiodothyronine (T3) on the differentiation of cultured neuroblastoma (NB) cells was studied after 9 days of treatment with a dose of 10(-4) M/10(6) cells per day. Using phase contrast microscopy, 30-50% of NB cells showed formation of neurites as a morphological sign of cellular differentiation. The initial rise of the mitosis rate was followed by a plateau. Changes in cyclic nucleotide content, in the triphosphates and in the activity of the enzyme ornithine decarboxylase (ODC) were assessed in 2 human and 2 murine cell lines to serve as biochemical parameters of the cell differentiation induced by T3. Whereas the cAMP level increased significantly (3 to 7 fold compared with its initial value), the cGMP value dropped to 30 to 50% of that of the control group. ATP and GTP increased about 200%, the ODC showed a decrease of about 50%. The present studies show a biphasic effect of T3 on neuroblastoma cells: the initial rise of mitotic activity is followed by increased cell differentiation starting from day 4 of the treatment.

Adenosine Triphosphate↗